Note: Descriptions are shown in the official language in which they were submitted.
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BOLAAMPHIPHILIC COMPOUNDS, COMPOSITIONS AND USES THEREOF
CROSS REFERENCES TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. Patent Application
14/328,419, filed on July
10, 2014, which is a continuation-in-part of International Application
PCT/U513/57956, filed on
September 4, 2013, which claims priority to U.S. Patent Application
61/696,789, filed on
September 4, 2012. U.S. Patent Application No. 14/328,419 also claims the
benefit of U.S.
Patent Application No. 61/845,185, filed on July 11, 2013, and U.S. Patent
Application No.
61/915,908, filed on December 13, 2013. This application also claims the
benefit of U.S. Patent
Application No. 62/148,511, filed on April 16, 2015, and U.S. Patent
Application No.
62/258,773, filed on November 23, 2015. The contents of each of the above-
referenced
applications are incorporated by reference herein.
FIELD
[0002] Provided herein are nanovesicles comprising bolaamphiphilic
compounds, and
complexes thereof with neurotrophins (NTFs), such as glial cell derived growth
factor (GDNF)
or nerve growth factor (NGF), and pharmaceutical compositions thereof Also
provided are
methods of delivering NTFs into the human brain and animal brain using the
compounds,
complexes and pharmaceutical compositions provided herein. In particular, the
present
disclosure is further directed to compounds, compositions, and method of the
treatement of
neurological diseases including, for illustrative purposes Parkinson's
disease, Alzheimers and
amyotrophic lateral sclerosis (ALS). Also provided are methods of delivering
NTFs into the
human brain and animal brain using the compounds, complexes and pharmaceutical
compositions provided herein.
BACKGROUND
[0003] Many studies using cell cultures and animal models of Parkinson's
disease (PD),
Alzeimer's disease (AD), or amyotrophic lateral scelerosis (ALS), and in some
cases human PD
and AD patients and human ALS patients, demonstrate that neurotrophins (NTFs),
such as glial
cell derived growth factor (GDNF) or nerve growth factor (NGF), have good
potential as
therapeutic agents in neurodegenerative diseases, including, e.g., PD or AD
treatment [1].
However, GDNF or NGF do not permeate through the blood-brain barrier (BBB),
thus they have
to be delivered directly into the brain in order to exert its therapeutic
action. Nevertheless,
attempts to deliver GDNF directly into the brain (e.g., intraputamenal
injection) had little benefit,
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most probably because its distribution within the brain was restricted to only
2-9% of the area
receiving the GDNF [2]. Also, convection-enhanced delivery of GDNF resulted in
a great deal of
variability in its distribution within the injected site [3]. The variability
in GDNF distribution,
and its limited diffusion throughout the brain, is most probably due to its
binding to the
extracellular matrix [4]. This implies that a delivery system which is capable
of distributing
GDNF uniformly within the brain, and not concentrating it at a small site
(that might cause
toxicity), should increase the probability that all affected neurons are
exposed to GDNF's
therapeutic activity and, thus, increase GDNF's efficacy in the treatment of
PD.
[0004] The brain capillary endothelial cells (BCECs) that form the BBB play
important role in
brain physiology by maintaining selective permeability and preventing passage
of various
compounds from the blood into the brain. One consequence of the highly
effective barrier
properties of the BBB is the limited penetration of therapeutic agents into
the brain, which makes
treatment of many brain diseases extremely challenging.
[0005] A delivery system that uses the intense capillary network that
supplies blood to the
brain, should deliver GDNF or NGF to a wide area within the brain, provided
that the delivery
system is capable of crossing the BBB and releasing the NTF there. Targeting
to specific sites
within the brain is abs greatly facilitated by an efficient penetration
through the BBB into the
brain after systemic administration.
[0006] Efforts to improve the permeation of GDNF across the BBB have been
attempted, but
have not proven therapeutically successful.
[0007] Efforts to improve the permeation of biologically active compounds
across the BBB
using amphphilic vesicles have been attempted.
[0008] For example, complexation of the anionic carboxyfluorescein (CF) (a
fluorescent
marker) with single headed amphiphiles of opposite charge in cationic
vesicles, formed by
mixing single-tailed cationic and anionic surfactants has been reported
(Danoff et al. 2007). In
addition to complexation, a certain portion of the CF is passively
encapsulated within the core of
the formed vesicles. The present disclosure employing bolaamphilies, includes
embodiments in
which a portion of the active agent may be complexed to the head groups of the
bolaamphiphiles
and another fraction of the active agents are encapsulated within the core of
the vesicles. In
many embodiments, the major portion of the active agent is encapsulated by
complexation with
the head groups.
[0009] Furthermore, WO 02/055011 and WO 03/047499, both of the same
applicant of the
present disclosure, disclose amphiphilic derivatives composed of at least one
fatty acid chain
derived from natural vegetable oils such as vernonia oil, lesquerella oil and
castor oil, in which
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functional groups such as epoxy, hydroxy and double bonds were modified into
polar and ionic
headgroups.
[0010] Additionally, WO 10/128504 reports a series of amphiphiles and
bolamphiphiles
(amphiphiles with two head groups) useful for targeted drug delivery of
insulin, insulin analogs,
TNF, GDNF, DNA, RNA (including siRNA), enkephalin class of analgesics, and
others.
[0011] These synthetic bolaamphiphiles (bolas) have recently been shown to
form
nanovesicles that interact with and encapsulate a variety of small and large
molecules including
peptides, proteins and plasmid DNAs delivering them across biological
membranes. These
bolaamphiphiles are a unique class of compounds that have two hydrophilic
headgroups placed at
each ends of a hydrophobic domain. Bolaamphiphiles can form vesicles that
consist of
monolayer membrane that surrounds an aqueous core. Vesicles made from natural
bolaamphiphiles, such as those extracted from archaebacteria (archaesomes),
are very stable and,
therefore, might be employed for targeted drug delivery. However,
bolaamphiphiles from
archaebacteria are heterogeneous and cannot be easily extracted or chemically
synthesized.
[0012] Thus, there remains a need to make new compositions and for novel
and optimized
methods to deliver NTF, such as glial cell derived growth factor (GDNF) or
nerve growth factor
(NGF), into the brain. The compounds, compositions, and methods described
herein are directed
toward this end.
SUMMARY OF THE IN
[0013] In certain aspects, provided herein are pharmaceutical compositions
comprising of a
bolaamphiphile complex.
[0014] In further aspects, provided herein are novel nano-sized vesicles
comprising of
bolaamphiphilic compounds.
[0015] In further aspects, provided herein are novel nano-sized vesicles
comprising of
bolaamphiphilic compounds which are capable of encapsulating NTF, GDNF or NU.
In
certain aspects, the vesicles comprise bolaamphiphilic compounds capable of
encapsulating a
neurotrophic factor selected from among Glial cell-derived neurotrophic factor
(GDNF), Nerve
Growth factor (NU), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3
(NT-3),
Neurotrophin-4/5 (NT-4/5), as well as combinations of two or more thereof.
[0016] In further aspects, provided herein are novel nano-sized bola
vesicles that encapsulate
GDNF or NGF and are capable of delivering the encapsulated material into the
brain. In other
aspects, the encapsulated neurotrophic factor is from among Glial cell-derived
neurotrophic
factor (GDNF), Nerve Growth factor (NGF), Brain-Derived Neurotrophic Factor
(BDNF),
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Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5), as well as combinations of
two or more
thereof
[0017] In further aspects, provided herein are novel nano-sized bola
vesicles that encapsulate
GDNF or NGF and are capable of delivering the encapsulated material to the
brain, specifically
to dopami.nergi.c neurons. In a further aspect there are submicron vesicles
with a monolayer
membrane or bilayer membrane encapsulating an inner core, with GDNF, NGF Brain-
Derived
Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5),
or
combination of two or more thereof
[0018] In certain embodiments, the present disclosure describes the use of
GDNF for the
treatment of amyotrophic lateral sclerosis (ALS) and for the treatment of
Alzheimer's disease in
a patient in need thereof.
[0019] In certain embodiments, the present disclosure describes treatment
of
neurodegenerative disease in a patient in need thereof comprising delivery of
one or more
neurotrophic factors (neurotrophins) using the vesicles and vesicle delivery
systems described
herein. In particular aspects of these embodiments, the neurotrophic factor is
selected from
among Glial cell-derived neurotrophic factor (GDNF), Nerve Growth factor
(NGF), Brain-
Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5
(NT-4/5), and
combinations of two or more thereof. In particular aspects of these
embodiments, the
neurodegenerative disease may be Alzheimer's disease, Parkinson's disease,
Amyotrophic lateral
sclerosis (ALS), Huntingdon's disease; neurodegeneration associated with
aging, and
combinations thereof.
[0020] In certain embodiments therefore, the present disclosure describes
vesicles and their
use as delivery systems for neurotropic factors that can be administered
systemically, e.g.,
intravenously and/or orally, that can pass intact through different biological
barriers, such as but
not limited to the blood brain barrier, and deliver their contents to targeted
to sites within the
brain and / or the peripheral nervous affected by the neurodegenerative
disease of a patient in
need of such treatment. Although such neurodegenerative diseases are currently
incurable and
involve debilitating conditions resulting from progressive degeneration and /
or death of nerve
cells affecting movement (ataxias), or mental functioning (dementias),
delivery systems and
bolavesicle carriers described herein can be used to ameliorate or reverse
these effects, to prevent
their occurrence; to mitigate the frequency and/or intensity of flare-ups, to
substantially arrest
progression of such effects, and/or to diminish the symptoms thereof
[0021] In further aspects, provided herein are novel nano-sized bola
vesicles that encapsulate
GDNF or NGF and are capable of delivering the encapsulated material into brain
regions affected
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in neurological disorders. In one particular embodiment, the neurological
disorder is Parkinson's
disease (PD) or Alzheimer's disease (AD).
[0022] In certain aspects, provided herein are novel bolaamphiphile
complexes comprising
bolaamphiphilic compounds and a compound active against PD. In one embodiment,
the
compound active against AD is GDNF.
[0023] In certain aspects, provided herein are novel bolaamphiphile
complexes comprising
bolaamphiphilic compounds and a compound active against AD. In one embodiment,
the
compound active against PD is NGF.
[0024] In other particular aspects, the present disclosure provides
bolaamphiphile complexes
comprising bolaamphiphilic compounds and active agents that are protein
neutrophic factors
(e.g., NTF), and neurotropins for the treatment of neurodegenerative diseases
such as Parkinson's
disease (PD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease, as
well as
Huntingdon's disease and neurodegeneration associated with aging.
[0025] In other embodiments, the present disclosure is directed to vesicles
containing
protein/peptide antibodies and the use thereof for the treatment of
Alzheimer's disease. These
methods can be employed for the prevention and treatment of Alzheimer's
disease, and can
exhibit improved pharmacokinetics with therapeutic amounts delivered to the
relevant sites in the
brain affected by Alzheimer's disease. The vesicles of the present invention
are drug delivery
systems that can overcome prior art limitations including poor
pharmacokinetics since the
proteinaceous agents are readily metabolized in vivo, have poor penetrability
through biological
barriers and have a large bio-distribution. In contrast, the vesicles
disclosed herein can be
administered via enteral administration (absorption of the drug through the
gastrointestinal tract)
or parenteral administration (generally injection, infusion, or implantation).
Topical applications
are also encompassed within the present disclosure.
[0026] In one aspect of these embodiments, the mode of administration for
many
applications is intravenous injections and/or oral administration, where the
antibody or antibody
fragment may be encapsulated with the vesicle's core and/or within the
encapsulating membrane
and the encapsulation may include complexation with the bolaamphiphiles or
additives
comprising the vesicles.
[0027] Thus the present disclosure provides delivery in the described
vesicles of protein
antibodies to the CNS for the treatment of Alzheimer's disease. In one
embodiment, the present
disclosure provides formulation of encapsulated anti-tau antibodies that can
strongly decrease tau
accumulation and/or prevent the accumulation of tau proteins as a therapy for
patients with
Alzheimer's disease and other neurodegenerative disorders.
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[0028] In another embodiment, the present disclosure provides vesicles with
a specific
antibody or antibody fragment against soluble aggregates of the AP peptide,
responsible for the
toxicity and cell death characteristic of Alzheimer's disease. In one
embodiment the whole
antibody does not have to be used; instead, an antibody fragment or a
recombinant antibody
consisting of the active part of the antibody responsible for the binding of
AP oligomers is
delivered to the target site. In one aspect of this embodiment, the present
disclosure provides
vesicles with both specific and nonspecific antibodies against AP-peptide and
their use in a
systemic treatment for patients with Alzheimer's disease.
[0029] In another embodiment, the present disclosure provides vesicles for
the delivery to
the CNS and sites affected by Alzheimer's disease with anti-A13 antibodies for
the removal of
brain AP peptide. In one aspect of this embodiment, the present disclosre
provides encapsulation
of bapineuzumab, which is composed of humanized anti-A13 monoclonal antibodies
that has
been shown to reduce AP burden in the brain of AD patients..
[0030] In still another embodiment for the treatment of Alzheimer, the
present disclosure
provides vesicles with encapsulated or complexed human immune globulin
intravenous (IGIV
[GAMMAGARD]) and their use in the treatment of Alzheimer's patients in need
thereof
[0031] In another embodiment, the present disclosure provides vesicles
comprising either
recombinant or naturally occurring antibodies directed against beta-amyloid
(Abeta and Abeta91-
42) and the use thereof for the treatment of Alzhiemer's disease via systemic
administration
which in illustrative example is IV administration or oral administration.
[0032] In further aspects, provided herein are novel formulations of GDNF
or NGF with
bolaamphiphilic compounds or with bolaamhphile vesicles.
[0033] In another aspect, provided here are methods of delivering GDNF or
NGF agents into
animal or human brain. In one embodiment, the method comprises the step of
administering to
the animal or human a pharmaceutical composition comprising of a
bolaamphiphile complex;
and wherein the bolaamphiphile complex comprises a bolaamphiphilic compound
and GDNF. In
another embodiment, the complex comprises bolaamphiphilic compound and NGF. In
other
aspects, the administered composition comprises a neurotrophic factor selected
from among Glial
cell-derived neurotrophic factor (GDNF), Nerve Growth factor (NU), Brain-
Derived
Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3), Neurotrophin-4/5 (NT-4/5),
as well as
combinations of two or more thereof
[0034] In a further embodiment, the present disclsosure provides
compositions and methods
for the delivery of the protein Activin to the CNS using bolaamphile vesicles
of the present
disclosure. In certain aspects of this embodiment, the Activin is at least one
of Activin A,
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Activin B, Activin AB, and combinations thereof In one specific aspect, the
Activin is
Activin A.
[0035] In one embodiment, the bolaamphiphilic compound consists of two
hydrophilic
headgroups linked through a long hydrophobic chain. In another embodiment, the
hydrophilic
headgroup is an amino containing group. In a specific embodiment, the
hydrophilic headgroup is
a tertiary or quaternary amino containing group.
[0036] In one particular embodiment, the bolaamphiphilic compound is a
compound
according to formula I:
HG2 ¨L1 ¨HG1
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof
wherein:
each HG1 and HG2 is independently a hydrophilic head group; and
Ll is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;
unsubstituted or
substituted with C,-C20 alkyl, hydroxyl, or oxo.
[0037] In one embodiment, the pharmaceutically acceptable salt is a
quaternary ammonium
salt.
[0038] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, the
bolaamphiphilic compound is a compound according to formula II, III, IV, V, or
VI:
r/Nn10 irz2õ
HG2 ___________________________________________ --)nl 1 __ HG1
0 0
0 0
)n10
HG2 ____________________ --)ng Z1 Z2 ¨)nl 1 __ HG1
III
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HG2-0R1 a R1 b
0 0
)n 1 0
R2a )n8 Z1 22 1( )12R2b
IV
HG2-0R1 a
0
)n10
R2a m(--')ng Z1 -HG1
V ,or
HG2-0R1 a
0
R2a (/)n--8 (=--)ng Z1
VI
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each HG' and HG2 is independently a hydrophilic head group;
each Z1 and Z2 is independently -C(R3)2-, -N(R3)- or ¨0-;
each Rla, Rib, ¨ 3,
K and R4 is independently H or Cl-C8 alkyl;
each R2a and R2b is independently H, C,-C8 alkyl, OH, alkoxy, or 0-HG' or 0-
HG2;
each n8, n9, n11, and n12 is independently an integer from 1-20;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[0039] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, or VI, each HG' and HG2 is independently selected from:
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0 0 0 R8
( it)
iml
Ml,
(.4n13 )ml µ( ')n13
0
)ml
(A R8
"(
and
i 401
in13 0 0
wherein:
X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted Ci-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted C1-C20 alkyl; each le
is independently
H, substituted or unsubstituted C1-C20 alkyl, alkoxy, or carboxy;
ml is 0 or 1; and
each n13, n14, and n15 is independently an integer from 1-20.
[0040] In certain embodiments, this disclosure provides novel monlayer
nanovescicles.
In particular aspects of these embodiments, the nanovesicles comprise
bolaamphiphilic
compounds with head groups faciliatating penetration of the bood brain
barrier. In other aspects,
the nanovesicles comprise bolaamphiphilic compounds with head groups that
facilitate targeting
to dopaminergic neurons in the brain. * In still another aspect the vesicles
formed from the
bolaamphiphiles contain additives that help to stabilize the vesicles, by
stabilizing the vesicle's
membranes, such as but not limited to cholesterol derivcatives such as
cholesteryl hemisuccinate
and cholesterol itself and combinations such as cholesteryl hemisuccinate and
cholesterol. In
another embodiment the vesicles comprise the bolaamphiphiles, vesicle membrane
stabilizing
additives, stearyl amine, and GDNF and NGF. In still another embodiments the
vesicles in
addition to these components have another addiitves which decorates the outer
vesicle
memrbanes with groups or pendants that enhance penetration though biological
barriers such as
the BBB and groups for targeting. A non limiting example of such additives may
be alkyl
conjugates of chitosan or bolaamphiphiles where one of the head groups is
chitiosan.
[0041] In certain embodiments, the present disclosure provides nanovesicles
that comprise
bolaamphiphilic compounds with chitosan head groups.
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[0042] In certain embodiments, the present disclosure provides nanovesicles
that comprise
bolaamphiphilic compounds with head groups that can function as ligands for
the dopamine
transporter.
[0043] In particular, embodiments, the present disclosure provides
nanovesicles that comprise
bolaamphiphilic compounds with head groups that can function as ligands for
the dopamine
transporter as well as with bolaamphiphilic compounds with that comprise
chitosan head groups.
[0044] In certain embodimetns, the present disclosure provides monlayer
nanovesicles
comprising the bolaamphiphilic compound designated herein as GLH-55a, the
bolaamphiphilic
compound designated herein as GLH-57, as well as encapsulated GDNF.
[0045] In other embodiments, the present disclosure provides a method of
treatment of a
neurotrophic disease comprising administration of an effective amount of
monlayer nanovesicles
of the disclosure comprising an encapsulated active agent. In particular
aspects of this
embodiment, the neutrophic disese is Parkinson's disease, and the administered
monlayer
nanovesicles comprise the bolaamphiphilic compound designated herein as GLH-
55a, the
bolaamphiphilic compound designated herein as GLH-57, as well as encapsulated
GDNF.
[0046] The present disclosure further provides compositions and methods for
controlling the
rate of release of vesicle-encapsulated materials by varying the length of
alkyl chains adjacent to
hydrolysable head groups of bolaamphiphilic vesicles. In one aspect of this
embodiment, the
head groups are acetylcholine head groups.
[0047] Other objects and advantages will become apparent to those skilled
in the art from a
consideration of the ensuing detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] Figure 1A: TEM micrograph of vesicles from GLH-20 and Figure 1B:
their size
distribution determined by DLS.
[0049] Figure 2A: Head group hydrolysis by AChE of GLH-19 (blue) and GLH-20
(red),
Figure 2B: release of CF from GLH-19 vesicles and Figure 2C: release of CF
from GLH-20
vesicles.
[0050] Figure 3A: CF accumulation in brain after i.v. injection of
encapsulated and non-
encapsulated CF. Only GLH-20 vesicles allow accumulation of CF in the brain.
Figure 3B: CS
improves GLH-20 vesicles' penetration into the brain.
[0051] Figure 4A: Analgesia after i.v. injection of enkephalin non-
encapsulated and
encapsulated in vesicles. Analgesia (compared with morphine, which was used as
a positive
control) is obtained only when enkephalin is encapsulated in GLH-20 vesicles,
the head groups
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of which are hydrolyzed by ChE. Figure 4B: The vesicles do not disrupt the BBB
since non-
encapsulated enkephalin co-injected with empty vesicles (extravesicular
enkephalin) did not
cause analgesia. **Significantly different from free leu-enkephalin (t-test,
P<0.01).
***Significantly different from free leu-enkephalin (t-test, P<0.001).
[0052] Figure 5A: Fluorescence in mouse cerebral cortex after i.v.
injection of albumin-FITC
(non-encapsulated); Figure 5B: Fluorescence in mouse cerebral cortex after
i.v. injection of
albumin-FITC encapsulated in GLH-20 vesicles.
[0053] Figure 6A: Mass spectra of GLH-20(A) and Figure 6B: Mass spectra of
GLH-19.
[0054] Figure 7: FT-IR spectra of original (CS) (spectrum a) and LMWCS
(spectrum b).
[0055] Figure 8: 1FINMR of the anhydroecgonine methyl ester, compound 4.
[0056] Figure 9A: HMQC of the anhydroecgonine methyl ester 4 and Figure 9B:
1I-1 COSY
NMR of the anhydroecgonine methyl ester 4.
[0057] Figure 10A depicts compound 5, (3-CFT. Figure 10B depicts the 1FINMR
spectra of
compound 5, (3-CFT.
[0058] Figure 11A: 1H-NMR spectra of the demethylated (3-CFT
fluoronortropane 7 and
Figure 11B: 13C-NMR spectra of the demethylated (3-CFT fluoronortropane 7.
[0059] Figure 12A: 1H-NMR spectrum of GLH-57, Figure 12B: enlargement of
the section
2.6-3.5 ppm of the 1H-NMR spectrum of GLH-57.
[0060] Figure 13: CryoTEM micrographs of vesicles made from the basic
bolas. Vesicles
were prepared by film hydration followed by probe sonication from a
formulation containing 10
mg/ml bola, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol.
(Panel A) empty
GLH-19 vesicles; (Panel B) GLH-19 vesicles loaded with 2 mg/ml trypsinogen;
(Panel C) empty
GLH-20 vesicles; (Panel D) GLH-20 vesicles loaded with 2 mg/ml trypsinogen;
(Panel E) GLH-
19 vesicles loaded with CF; (Panel F) GLH-20 vesicles loaded with CF
[0061] Figure 14: CryoTEM micrographs of vesicles made from a mixture of
GLH-19 and
GLH-20. Vesicles were prepared by film hydration followed by sonication from a
formulation
containing GLH-19 and GLH-20 at a ratio of 2:1, respectively (total of 10
mg/ml bolas),
cholesterol (1.6 mg/ml) and cholesteryl hemisuccinate (2.1 mg/ml). (Panel A)
empty vesicles;
(Panel B) vesicles loaded with 2 mg/ml trypsinogen.
[0062] Figure 15: CryoTEM of empty vesicles made from a mixture of 10 mg/ml
GLH-19
and GLH-20 (2:1) together with 1 mg/ml GLH-55a (Panel A), or 0.8 mg/ml GLH-57
(Panel B),
or 1 mg/ml GLH55a and 0.8 mg/ml GLH-57 (Panel C). (Bar=50nm)
[0063] Figure 16: CryoTEM of vesicles made from a mixture of GLH-19, GLH-
20, GLH-
55a, and GLH-57, as described in FIG. 15 (Panel C), and loaded with 40pg/m1
GDNF (Bar=50
nm)
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[0064] Figure 17A: Representative data from DLS measurements of size
distribution by
intensity for vesicles made from GLH-19; Figure 17B: Representative data from
DLS
measurements of size distribution by intensity for vesicles made from GLH-20;
and Figure 17C:
Representative data from DLS measurements of size distribution by intensity
for vesicles made
from a mixture of GLH-19 and GLH-20 at a ratio of 2:1. Vesicles were prepared
by film
hydration followed by sonication from 10 mg/ml bolas, 2.1 mg/ml cholesteryl
hemisuccinate and
1.6 mg/ml cholesterol. Each sample was measured by the DLS 3 times, and each
profile shows
the three measurements overlaid.
[0065] Figure 18: Size distribution of GLH-20 vesicles, with and without
encapsulated
trypsinogen. Vesicles were prepared by film hydration followed by sonication
from 10 mg/ml
GLH-20, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol, in
presence and
absence of trypsinogen. Size distribution was measured by DLS.
[0066] Figure 19: Stability of GLH-20 vesicles in storage. Encapsulation of
CF was
determined after diluting the vesicles to reduce the extravesicular CF
concentration, then, the
vesicles were disrupted by Triton X100 and the fluorescence of released CF was
measured at
various times as indicated. Encapsulation was normalized using encapsulation
at time 0 as 100%
[0067] Figure 20: Stability of bolaamphiphilic vesicles in whole serum.
Vesicles were
prepared from GLH-19 or GLH-20 or from mixtures of both bolas using two ratios
as shown.
Vesicles were added to the serum in a ratio of 1:10 (vesicles:serum). Percent
CF encapsulation
was determined by fluorescence measurements as described in FIG. 14.
Encapsulation was
normalized using encapsulation at time 0 as 100%
[0068] Figure 21: Release of CF from bolavesicles in response to AChE.
Vesicles were
prepared from either GLH-20 alone (plus the standard additives) (Panel A), or
a mixture of GLH-
19 and GLH-20 (plus the standard additives) (Panel B) and both loaded with CF.
The vesicles
were placed in a cuvette, and fluorescence was measured as a function of time
until stable
reading was achieved. Then, 2 units of AChE was added to each vesicle
preparation, and the
fluorescence measurement continued. The release of the encapsulated CF causes
increase in the
fluorescence. About 7 minutes after the addition of the AChE, triton X100 was
added (to a final
concentration of 0.15%), to fully disrupt the vesicles and to release the
remaining CF for the
determination of the total CF that was encapsulated.
[0069] Figure 22: Elution profile of a vesicle formulation that contained
encapsulated
(peak 1) and free trypsinogen (peak 2). The vesicles were applied on Sephadex
G50 column and
eluted with PBS.
[0070] Figure 23: Quantification of encapsulated trypsinogen using the data
obtained from
the experiment described in FIG. 17.
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[0071] Figure 24: Encapsulation of trypsinogen following vesicle
preparation by film
hydration and sonication or by extrusion. Upper graph (Panel C) shows the
overlap of the elution
profiles obtained running each vesicle preparation on the Sephadex G50 column.
The lower
graphs show the quantification of encapsulation for sonicated vesicles (Panel
A) and extruded
vesicles (Panel B).
[0072] Figure 25: Encapsulation of AlexaFluor0-488-labeled trypsinogen in
bolaamphiphilc
vesicles. Vesicles were made by film hydration followed by sonication from a
mixture of 10
mg/ml GLH-19 and GLH-20 (2:1) with 2.1 mg/ml choesteryl hemisuccinate and 1.6
mg/ml
cholesterol. Trypsinogen was labeled with AlexaFluor0-488, as described in the
method section,
and was included in the formulation at a concentration of 0.2 mg/ml. Vesicles
were placed on
Sephadex G50 column and eluted with either Tris buffer, pH=7.3 (Panel A); or
PBS, pH 7.3
(Panel B). Percent encapsulation was quantified for (Panel B) and is shown in
(Panel C). The
calculated percent encapsulation was 31%.
[0073] Figure 26A: Encapsulation efficiencies of trypsinogen and GDNF.
Vesicles were
prepared by film hydration followed by sonication from a mixture of GLH-19 and
GLH-20 at a
concentration of 10 mg/ml with 1.6 mg/ml cholesterol and 2.1 mg/ml cholesteryl
hemisuccinate.
The formulations contained 50 ug/m1trypsinogen, Figure 26B: Encapsulation
efficiencies of
trypsinogen and GDNF. Vesicles were prepared by film hydration followed by
sonication from a
mixture of GLH-19 and GLH-20 at a concentration of 10 mg/ml with 1.6 mg/ml
cholesterol and
2.1 mg/ml cholesteryl hemisuccinate. The formulations contained 100
ug/m1trypsinogen and
Figure 26C: Encapsulation efficiencies of trypsinogen and GDNF. Vesicles were
prepared by
film hydration followed by sonication from a mixture of GLH-19 and GLH-20 at a
concentration
of 10 mg/ml with 1.6 mg/ml cholesterol and 2.1 mg/ml cholesteryl
hemisuccinate. The
formulations contained 12.5 ug/m1 GDNF. All proteins were labeled with
AlexaFluor0-488.
After encapsulation, the vesicles were eluted from a Sephadex G50 column by
PBS and the
fluorescence of each fraction was determined.
[0074] Figure 27A: The effect of the encapsulation process on GDNF
integrity and activity.
Analysis of GDNF on PAGE, where lane 1 is empty vesicles; lane 2 is GDNF
encapsulated by
the method of film hydration followed by sonication; lane 3 is encapsulated
GDNF which was
incubated before the PAGE at 40oC for one hour; and lane 4 is free GDNF.
Figure 27B: Test of
GDNF activity using SH-SY5Y neuroblastoma cells where lane 1 is control
untreated cells; lane
2 is cells treated with free GDNF; lane 3 is cells treated with empty
vesicles; lane 4 is cells
treated with free GDNF added to empty vesicles; and lane 5 is cells treated
with GDNF
encapsulated in bolavesicles by the method of film hydration followed by
sonication
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[0075] Figure 28A: Uptake of CF-loaded vesicles into PC12 cells in culture.
Figure 28B:
Uptake of CF-loaded vesicles into SH-5Y5Y neuroblastoma cells in culture.
Figure 28C: Uptake
of CF-loaded vesicles into HeLa cells in culture. Vesicles were made from 10
mg/ml GLH-
19:GLH-20 (2:1) without (uncoated vesicles) and with 0.8 mg/ml GLH-57, a bola
that contains
DAT ligand as the head group (DAT-vesicles). Cells were incubated for 1 h with
the vesicles,
and tested by flow cytometry. A shift to the right of the peak indicates
fluorescent cells due to
uptake of the vesicles.
[0076] Figure 29: Accumulation of CF in the brain following i.v.
administration. Vesicles
were made by film hydration followed by sonication from a 10 mg/ml mixture of
GLH-19 and
GLH-20 (2:1), 1 mg/ml CS-fatty acid (vernolate) conjugate, 2.1 mg/ml
cholesteryl hemisuccinate
and 1.6 mg/ml cholesterol in absence (empty vesicles) and in presence of
0.2/m1 CF (CF-loaded
vesicles). Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine and 15
min afterward the
mice were injected i.v. with either free CF, or empty vesicles and then CF, or
CF-loaded vesicles.
The total amounts of the CF that were injected in each case were identical (10
mg/kg). 30 min
after the injection, the animals were sacrificed, perfused with 10 ml PBS and
the brains removed
and homogenized, deproteinized by 5% tricholoroacetic acid and fluorescence
determined in the
supernatants obtained following centrifugation. Each bar represents an average
value obtained
from 5 mice +/- SEM.
[0077] Figure 30: CF concentration in the brain after delivering it
encapsulated in vesicles
with CS surface groups. Vesicles were prepared as described in FIG. 24, except
that in one case,
1 mg/ml GLH-55a was used in the vesicle formulation to provide CS surface
groups (vesicles
with CS-bola), and in the other case, 1 mg/ml CS-fatty acid conjugate was
used. Conditions of
this experiment were similar to those presented in FIG. 24.
[0078] Figure 31: Distribution of CF in the brain after injecting CF-loaded
vesicles with
and without surface DAT ligand. Vesicles were prepared by film hydration
followed by
sonication from a 10 mg/ml mixture of GLH-19 and GLH-20 (2:1), 1 mg/ml GLH-55a
(a bola
with CS head group), 2.1 mg/ml cholesteryl hemisuccinate, 1.6 mg/ml
cholesterol, 0.2 mg/ml CF
and without (vesicle CS bola) or with GLH-57 (vesicles DAT CS bola). Mice were
pretreated
with 0.5 mg/kg (i.m.) pyridostigmine (to inhibit peripheral ChE) and 15 min
afterward the
vesicles were injected i.v. After 30 min the mice were sacrificed, perfused
with 10 ml PBS and
the brain removed and dissected into cortex, striatum and cerebellum. The
tissues were weighed,
homogenized and deproteinated by trichloroacetic acid, centrifuged and
fluorescence was
determined in the homogenates. The amount of the CF in each brain region
(cerebellum
(Panel A); cortex (Panel B); striatum (Panel C)) was calculated from a
calibration curve of CF,
taking into consideration the weight of the tissue and the dilution done
during the
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homogenization. Each bar represent an average value obtained from 5 mice +/-
SEM. The
comparative data are depicted in Panel D.
[0079] Figure 32A: Representative histofluorescence slides of brain tissue
of control
untreated mice; Figure 32B: Representative histofluorescence slide showing
AlxaFlour-488-
labeld trypsinogen in brain tissue of mice injected with 200 lig of free
trypsinogen labeled with
AlexaFluor0-488; Figure 32C: Representative histofluorescence slide showing
AlxaFlour-488-
labeld trypsinogen in brain tissue of mice injected with 200 pg of
encapsulated trypsinogen
labeled with AlexaFluor0-488; Figure 32D: Representative histofluorescence
slides of liver
tissue of control untreated mice; Figure 32E: Representative histofluorescence
slide showing
AlxaFlour-488-labeld trypsinogen in liver tissue of mice injected with 200 pg
of free trypsinogen
labeled with AlexaFluor0-488; Figure 32F: Representative histofluorescence
slide showing
AlxaFlour-488-labeld trypsinogen in liver tissue of mice injected with 200 pg
of encapsulated
trypsinogen labeled with AlexaFluor0-488; Figure 32G: Representative
histofluorescence slide
of kidney tissue of control untreated mice; Figure 32H: Representative
histofluorescence slide
showing AlxaFlour-488-labeld trypsinogen in kidney tissue of mice injected
with 200 pg of free
trypsinogen labeled with AlexaFluor0-488; Figure 321: Representative
histofluorescence slide
showing AlxaFlour-488-labeld trypsinogen in kidney tissue of mice injected
with 200 pg of
encapsulated trypsinogen labeled with AlexaFluor0-488.
[0080] Figure 33: Distribution of trypsinogen labeled with AlexaFluor0-488
in brain, kidney
and liver after the injection (i.v.) of the labeled protein in its free form
or encapsulated in
vesicles. For the quantification, data obtained in the experiment described in
FIG. 27 were used.
Each bar represent an average value of 5 mice +/- SEM
[0081] Figure 34: Representative brain sections stained for GDNF-biotin
with avidine-
AlexaFluor0-488. Mice were pretreated with 0.5 mg/kg (i.m.) pyridostigmine,
then injected i.v.
with vesicles coated with CS groups and DAT ligand with encapsulated GDNF-
biotin. After 30
min, animals were sacrificed, perfused with 10 ml PBS, brains removed and
striata, cortex and
cerebella were dissected out, frozen and cryosectioned. Brain sections from
these mice were
stained with DAPI (blue) and avidine-AlexaFluor0-488 (green) and observed
using confocal
microscopy at a magnification of 10X. (Panel A) Stiatum from a mouse treated
with PBS; (Panel
B) striatum from a mouse injected with GDNF-biotin encapsulated in vesicles;
(Panel C) cortex
from a mouse injected with PBS; (Panel D) cortex from a mouse injected with
GDNF-biotin
encapsulated in vesicles; (Panel E) cerebellum from a mouse injected with PBS;
(Panel F)
cerebellum from a mouse injected with GDNF-biotin encapsulated in vesicles
[0082] Figure 35: Distribution of exogenous GDNF-biotin in the brain after
delivering the
protein encapsulated in bolavesicles. These micrographs of high magnification,
(60X) were
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taken from brain sections obtained from the mice used in the experiment
described in FIG. 29.
The nuclei of the cells appear in blue, due to DAPI staining, and the GDNF-
biotin appears in
green, due to the binding of the avidine-AlexaFluor0-488. (Panel A) ) Stiatum
from a mouse
treated with PBS; (Panel B) striatum from a mouse injected with GDNF-biotin
encapsulated in
vesicles; (Panel C) cortex from a mouse injected with PBS; (Panel D) cortex
from a mouse
injected with GDNF-biotin encapsulated in vesicles; (Panel E) cerebellum from
a mouse injected
with PBS; (Panel F) cerebellum from a mouse injected with GDNF-biotin
encapsulated in
vesicles.
[0083] Figure 36: Chemical shifts of the chloromethylene (¨CH2C1) and
alkoxymethylene
(C(0)-0-CH2-) groups of comound 4.
[0084] Figure 37: Comparison of the NMR spectrum in CDC13 of the
dichloroacetate
intermediate 4 and the bolaamphiphile 5.
[0085] Figure 38A: TEM micrographs of particles formed from bolaamphiphile
GLH-20
and Figure 38B: TEM micrographs of particles formed from bolaamphiphile GLH-
32. Vesicles
were prepared by film-hydration-extrusion (FHE) using 200 nm and 100 nm
membranes,
consecutively
[0086] Figure 39: TEM micrographs of vesicles made from bolaamphiphile GLH-
20 (Panel
A) and bolaamphiphile GLH-32 (Panel B) formulated with CHOL and CHEMS at a
molar ratio
of 2:1:1. Vesicles were prepared by FHE using 200 nm and 100 nm membranes,
consecutively
[0087] Figure 40A: Vesicle stability determine by changes in vesicle size
and Figure 40B:
Vesicle stability determine by changes in percent encapsulation using vesicles
made from GLH-
20 and GLH-32 with CHOL and CHEMS at a ratio of 2:1:1.
[0088] Figure 41: Hydrolysis of the ACh head group of bioamphiphiles GLH-20
and GLH-32
by AChE. Hydrolysis was measured by determining the pH change after addition
of AChE to the
incubation medium and was converted to change in the proton concentration.
[0089] Figure 42A: Lineweaver-Burk plots of ATC hydrolysis by AChE in
presence of
several concentrations of GLH-20 and Figure 42B: Lineweaver-Burk plots of ATC
hydrolysis by
AChE in presence of several concentrations of GLH-32
[0090] Figure 43: The effect of AChE on the release of CF from vesicles made
from GLH-20
(Panel A) and GLH-32 (Panel B). The released CF was monitored by measuring the
fluorescence before and after the addition of X units of AChE dissolved in X
ill PBS. The
experiment was terminated by the addition of Triton X-100 to disrupt the
vesicles and release all
the encapsulated CF.
[0091] Figure 44: Percent release of encapsulated CF at different time after
exposing
bolaamphiphilic vesicles to AChE. Percent release was calculated from the
amount of CF that
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was released at a particular time point versus the total amout of encapsulated
CF, which was
determined after lysing the vesicles with Triton X100.
[0092] Figure 45: Depicts the 13C NMR spectra of the diester diglutarate
3 (Scheme
7), D-mannose and the bola GLH-64a in DMSO-d6.
[0093] Figure 46: Depicts the main fragmentations in ESI-MS (positive
mode) of
GLH-64a.
[0094] Figure 47: Depicts the HPLC chromatogram of GLH-64a, showing that
it was
obtained with a high purity.
[0095] Figure 48: Depicts the separation of methyl ricinoleate by liquid
¨liquid
extraction, where H = hexane; M = methanol; MR = methyl ricinoleate; and ME =
mixture of
methyl esters of castor oil.
[0096] Figure 49: Provides the cryo-TEM images of vesicles prepared from
a
formulation without GLH-64a (Panel A) in comparison to a formulation that
contained 5% GLH-
64a (Panel B). The bar represents 100 nm.
[0097] Figure 50: Depicts the ptake of fluorescent vesicles that contain
GLH-64a by
differentiated and non-differentiated J774 cells, as measured by FACS.
[0098] Figure 51: Depicts the uptake of fluorescent vesicles that
contain GLH-64a by
differentiated J774 cells in presence and absence of free mannose in the
bathing medium, as
measured by FACS.
[0099] Figure 52: Depicts the uptake of fluorescent vesicles that
contain GLH-64a
(Panel A) and GLH-64b (Panel B) by differentiated and non-differentiated J774
cells, as
measured by FACS.
[00100] Figure 53: Depicts the uptake of fluorescent vesicles (vesicles
with
encapsulated siRNA conjugated with AlexaFluor 546) with and without GLH-64a by
differentiated J774 cells, as measured by FACS.
[00101] Figure 54: Depicts the uptake of fluorescent vesicles that
contain GLH-64d by
differentiated and non-differentiated J774 cells, as measured by FACS.
[00102] Figure 55: Depicts the amount of CF encapsulation as a function
of time in
storage at 4 C in vesicles made from GLH-19 and GLH-20.
[00103] Figure 56: Depicts the amount of CF encapsulation as a function
of time in
storage at 4 C in vesicles made from GLH-19, GLH-20 and GLH-55b.
[00104] Figure 57: Depicts the amount of CF encapsulation as a function
of time in
storage at 4 C in vesicles made from GLH-19, GLH-20, GLH-55b and 1% GLH-64a.
[00105] Figure 58: Depicts the amount of CF encapsulation as a function
of time in
storage at 4 C in vesicles made from GLH-19, GLH-20, GLH-55b and 5% GLH-64a.
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[00106] Figure 59: Depicts the amount of CF encapsulation as a function
of time in
storage at 4 C in vesicles made from GLH-19, GLH-20, GLH-55b and 10% GLH-64a.
[00107] Figure 60: Depicts the stability in 4% albumin during storage at
4 C of
vesicles made from GLH-19, GLH-20 and GLH-55b.
[00108] Figure 61: Depicts the stability in 4% albumin during storage at
4 C of
vesicles made from GLH-19, GLH-20, GLH-55b and 1% GLH-64.
[00109] Figure 62: Depicts the stability in 4% albumin during storage at
25 C of
vesicles made from GLH-19, GLH-20 and GLH-55b.
[00110] Figure 63: Depicts the stability in 4% albumin during storage at
25 C of
vesicles made from GLH-19, GLH-20 and GLH-55b and 1% GLH-64a.
[00111] Figure 64: Depicts the effect of GLH-55b and GLH-64a on the
release of
encapsulated CF from vesicles. Panel A: Vesicles made of GLH-19 and GLH-20,
without GLH-
55b and GLH-64; Panel B ¨ Vesicles made of GLH-19, GLH-20, GLH-55b and 1% GLH-
64a;
Panel C ¨ Vesicles made of GLH-19, GLH-20, GLH-55b and 10% GLH-64a.
DEFINITIONS
Chemical Definitions
[00112] Definitions of specific functional groups and chemical terms are
described in more
detail below. The chemical elements are identified in accordance with the
Periodic Table of the
Elements, CAS version, Handbook of Chemistry and Physics, 75th
Ed., inside cover, and specific
functional groups are generally defined as described therein. Additionally,
general principles of
organic chemistry, as well as specific functional moieties and reactivity, are
described in Thomas
Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith
and March,
March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New
York, 2001;
Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York,
1989; and
Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge
University
Press, Cambridge, 1987.
[00113] Compounds described herein can comprise one or more asymmetric
centers, and thus
can exist in various isomeric forms, e.g., enantiomers and/or diastereomers.
For example, the
compounds described herein can be in the form of an individual enantiomer,
diastereomer or
geometric isomer, or can be in the form of a mixture of stereoisomers,
including racemic
mixtures and mixtures enriched in one or more stereoisomer. Isomers can be
isolated from
mixtures by methods known to those skilled in the art, including chiral high
pressure liquid
chromatography (HPLC) and the formation and crystallization of chiral salts;
or preferred
isomers can be prepared by asymmetric syntheses. See, for example, Jacques et
al.,
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Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981);
Wilen etal.,
Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds
(McGraw¨Hill, NY,
1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268
(EL. Eliel, Ed.,
Univ. of Notre Dame Press, Notre Dame, IN 1972). The invention additionally
encompasses
compounds described herein as individual isomers substantially free of other
isomers, and
alternatively, as mixtures of various isomers.
[00114] When a range of values is listed, it is intended to encompass each
value and sub¨range
within the range. For example "C1_6 alkyl" is intended to encompass, C1, C2,
C3, C4, C5, C6, C1-6,
C1_5, Ci_4, Ci_3, Ci_2, C2-6, C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5,
and C5_6 alkyl.
[00115] The following terms are intended to have the meanings presented
therewith below and
are useful in understanding the description and intended scope of the present
invention.
When describing the invention, which may include compounds, pharmaceutical
compositions
containing such compounds and methods of using such compounds and
compositions, the
following terms, if present, have the following meanings unless otherwise
indicated. It should
also be understood that when described herein any of the moieties defined
forth below may be
substituted with a variety of substituents, and that the respective
definitions are intended to
include such substituted moieties within their scope as set out below. Unless
otherwise stated, the
term "substituted" is to be defined as set out below. It should be further
understood that the
terms "groups" and "radicals" can be considered interchangeable when used
herein. The articles
"a" and "an" may be used herein to refer to one or to more than one (i.e. at
least one) of the
grammatical objects of the article. By way of example "an analogue" means one
analogue or
more than one analogue.
[00116] "Alkyl" refers to a radical of a straight¨chain or branched saturated
hydrocarbon group
having from 1 to 20 carbon atoms ("C1_20 alkyl"). In some embodiments, an
alkyl group has 1 to
12 carbon atoms ("C 1_12 alkyl"). In some embodiments, an alkyl group has 1 to
10 carbon atoms
("C1_10 alkyl"). In some embodiments, an alkyl group has 1 to 9 carbon atoms
("C1_9 alkyl"). In
some embodiments, an alkyl group has 1 to 8 carbon atoms ("C1_8 alkyl"). In
some
embodiments, an alkyl group has 1 to 7 carbon atoms ("C1_7 alkyl"). In some
embodiments, an
alkyl group has 1 to 6 carbon atoms ("C1_6 alkyl", also referred to herein as
"lower alkyl"). In
some embodiments, an alkyl group has 1 to 5 carbon atoms ("C1_5 alkyl"). In
some
embodiments, an alkyl group has 1 to 4 carbon atoms ("C1_4 alkyl"). In some
embodiments, an
alkyl group has 1 to 3 carbon atoms ("C1_3 alkyl"). In some embodiments, an
alkyl group has 1
to 2 carbon atoms ("C1_2 alkyl"). In some embodiments, an alkyl group has 1
carbon atom ("C1
alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2_6
alkyl"). Examples
of C1_6 alkyl groups include methyl (C1), ethyl (C2), n¨propyl (C3), isopropyl
(C3), n¨butyl (C4),
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tert¨butyl (C4), sec¨butyl (C4), iso¨butyl (C4), n¨pentyl (C5), 3¨pentanyl
(C5), amyl (C5),
neopentyl (C5), 3¨methyl-2¨butanyl (C5), tertiary amyl (C5), and n¨hexyl (C6).
Additional
examples of alkyl groups include n¨heptyl (C7), n¨octyl (C8) and the like.
Unless otherwise
specified, each instance of an alkyl group is independently optionally
substituted, i.e.,
unsubstituted (an "unsubstituted alkyl") or substituted (a "substituted
alkyl") with one or more
substituents; e.g., for instance from 1 to 5 substituents, 1 to 3
substituents, or 1 substituent. In
certain embodiments, the alkyl group is unsubstituted C1_10 alkyl (e.g.,
¨CH3). In certain
embodiments, the alkyl group is substituted C1_10 alkyl.
[00117] "Alkylene" refers to a substituted or unsubstituted alkyl group, as
defined above,
wherein two hydrogens are removed to provide a divalent radical. Exemplary
divalent alkylene
groups include, but are not limited to, methylene (-CH2-), ethylene (-CH2CH2-
), the propylene
isomers (e.g., -CH2CH2CH2- and -CH(CH3)CH2-) and the like.
[00118] "Alkenyl" refers to a radical of a straight¨chain or branched
hydrocarbon group having
from 2 to 20 carbon atoms, one or more carbon¨carbon double bonds, and no
triple bonds ("C2_20
alkenyl"). In some embodiments, an alkenyl group has 2 to 10 carbon atoms
("C2_10 alkenyl"). In
some embodiments, an alkenyl group has 2 to 9 carbon atoms ("C2_9 alkenyl").
In some
embodiments, an alkenyl group has 2 to 8 carbon atoms ("C2_8 alkenyl"). In
some embodiments,
an alkenyl group has 2 to 7 carbon atoms ("C2_7 alkenyl"). In some
embodiments, an alkenyl
group has 2 to 6 carbon atoms ("C2_6 alkenyl"). In some embodiments, an
alkenyl group has 2 to
carbon atoms ("C2_5 alkenyl"). In some embodiments, an alkenyl group has 2 to
4 carbon
atoms ("C2_4 alkenyl"). In some embodiments, an alkenyl group has 2 to 3
carbon atoms ("C2_3
alkenyl"). In some embodiments, an alkenyl group has 2 carbon atoms ("C2
alkenyl"). The
one or more carbon¨carbon double bonds can be internal (such as in 2¨butenyl)
or terminal (such
as in 1¨buteny1). Examples of C2_4 alkenyl groups include ethenyl (C2),
1¨propenyl (C3), 2¨
propenyl (C3), 1¨butenyl (C4), 2¨butenyl (C4), butadienyl (C4), and the like.
Examples of C2_6
alkenyl groups include the aforementioned C2_4 alkenyl groups as well as
pentenyl (C5),
pentadienyl (C5), hexenyl (C6), and the like. Additional examples of alkenyl
include heptenyl
(C7), octenyl (C8), octatrienyl (C8), and the like. Unless otherwise
specified, each instance of an
alkenyl group is independently optionally substituted, i.e., unsubstituted (an
"unsubstituted
alkenyl") or substituted (a "substituted alkenyl") with one or more
substituents e.g., for instance
from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent. In certain
embodiments, the alkenyl
group is unsubstituted C2_10 alkenyl. In certain embodiments, the alkenyl
group is substituted C2_
alkenyl.
[00119] "Alkenylene" refers a substituted or unsubstituted alkenyl group, as
defined above,
wherein two hydrogens are removed to provide a divalent radical. Exemplary
divalent
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alkenylene groups include, but are not limited to, ethenylene (-CH=CH-),
propenylenes (e.g., -
CH=CHCH2- and -C(CH3)=CH- and -CH=C(CH3)-) and the like.
[00120] "Alkynyl" refers to a radical of a straight-chain or branched
hydrocarbon group having
from 2 to 20 carbon atoms, one or more carbon-carbon triple bonds, and
optionally one or more
double bonds ("C2_20 alkynyl"). In some embodiments, an alkynyl group has 2 to
10 carbon
atoms ("C2_10 alkynyl"). In some embodiments, an alkynyl group has 2 to 9
carbon atoms ("C2-9
alkynyl"). In some embodiments, an alkynyl group has 2 to 8 carbon atoms
("C2_8 alkynyl"). In
some embodiments, an alkynyl group has 2 to 7 carbon atoms ("C2_7 alkynyl").
In some
embodiments, an alkynyl group has 2 to 6 carbon atoms ("C2_6 alkynyl"). In
some embodiments,
an alkynyl group has 2 to 5 carbon atoms ("C2_5 alkynyl"). In some
embodiments, an alkynyl
group has 2 to 4 carbon atoms ("C2_4 alkynyl"). In some embodiments, an
alkynyl group has 2 to
3 carbon atoms ("C2_3 alkynyl"). In some embodiments, an alkynyl group has 2
carbon atoms
("C2 alkynyl"). The one or more carbon-carbon triple bonds can be internal
(such as in 2-
butynyl) or terminal (such as in 1-butyny1). Examples of C2_4 alkynyl groups
include, without
limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-
butynyl (C4), and
the like. Examples of C2_6 alkenyl groups include the aforementioned C2_4
alkynyl groups as
well as pentynyl (C5), hexynyl (C6), and the like. Additional examples of
alkynyl include
heptynyl (C7), octynyl (C8), and the like. Unless otherwise specified, each
instance of an alkynyl
group is independently optionally substituted, i.e., unsubstituted (an
"unsubstituted alkynyl") or
substituted (a "substituted alkynyl") with one or more substituents; e.g., for
instance from 1 to 5
substituents, 1 to 3 substituents, or 1 substituent. In certain embodiments,
the alkynyl group is
unsubstituted C2_10 alkynyl. In certain embodiments, the alkynyl group is
substituted C2_10
alkynyl.
[00121] "Alkynylene" refers a substituted or unsubstituted alkynyl group, as
defined above,
wherein two hydrogens are removed to provide a divalent radical. Exemplary
divalent
alkynylene groups include, but are not limited to, ethynylene, propynylene,
and the like.
[00122] ¨Aryl" refers to a radical of a monocyclic or polycyclic (e.g.,
bicyclic or tricyclic)
4n+2 aromatic ring system (e.g., having 6, 10, or 14 7C electrons shared in a
cyclic array) having
6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring
system ("C6_14 aryl").
In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; e.g.,
phenyl). In some
embodiments, an aryl group has ten ring carbon atoms ("C10 aryl"; e.g.,
naphthyl such as 1-
naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring
carbon atoms
("C14 aryl"; e.g., anthracyl). "Aryl" also includes ring systems wherein the
aryl ring, as defined
above, is fused with one or more carbocyclyl or heterocyclyl groups wherein
the radical or point
of attachment is on the aryl ring, and in such instances, the number of carbon
atoms continue to
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designate the number of carbon atoms in the aryl ring system. Typical aryl
groups include, but
are not limited to, groups derived from aceanthrylene, acenaphthylene,
acephenanthrylene,
anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene,
hexacene, hexaphene,
hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene,
octaphene, octalene,
ovalene, penta-2,4-diene, pentacene, pentalene, pentaphene, perylene,
phenalene, phenanthrene,
picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, and
trinaphthalene. Particularly
aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Unless
otherwise
specified, each instance of an aryl group is independently optionally
substituted, i.e.,
unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl")
with one or more
substituents. In certain embodiments, the aryl group is unsubstituted C6_14
aryl. In certain
embodiments, the aryl group is substituted C6_14 aryl.
[00123] In certain embodiments, an aryl group substituted with one or more of
groups selected
from halo, C1-C8 alkyl, C1-C8 haloalkyl, cyano, hydroxy, C1-C8 alkoxy, and
amino.
[00124] Examples of representative substituted aryls include the following
R56 R56 R56
R57 , and
R57 R57 =
In these formulae one of R56 and R57 may be hydrogen and at least one of R56
and R57 is each
independently selected from C1-C8 alkyl, Ci-C8 haloalkyl, 4-10 membered
heterocyclyl,
alkanoyl, C1-C8 alkoxy, heteroaryloxy, alkylamino, arylamino, heteroarylamino,
NR58C0R59,
NR58S0R59NR58S02R59, COOalkyl, COOaryl, C0NR58R59, C0NR580R59, NR58R59,
S02NR58R59, S-alkyl, SOalkyl, SO2alkyl, Saryl, SOaryl, SO2aryl; or R56 and R57
may be joined to
form a cyclic ring (saturated or unsaturated) from 5 to 8 atoms, optionally
containing one or more
heteroatoms selected from the group N, 0, or S. R6 and R61 are independently
hydrogen, C1-C8
alkyl, Ci-C4 haloalkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, C6-C10
aryl, substituted
C6-Cio aryl, 5-10 membered heteroaryl, or substituted 5-10 membered
heteroaryl.
[00125] "Fused aryl" refers to an aryl having two of its ring carbon in common
with a second
aryl ring or with an aliphatic ring.
[00126] "Aralkyl" is a subset of alkyl and aryl, as defined herein, and refers
to an optionally
substituted alkyl group substituted by an optionally substituted aryl group.
[00127] "Heteroaryl" refers to a radical of a 5-10 membered monocyclic or
bicyclic 4n+2
aromatic ring system (e.g., having 6 or 10 7C electrons shared in a cyclic
array) having ring carbon
atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein
each heteroatom
is independently selected from nitrogen, oxygen and sulfur ("5-10 membered
heteroaryl"). In
heteroaryl groups that contain one or more nitrogen atoms, the point of
attachment can be a
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carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems
can include one or
more heteroatoms in one or both rings. "Heteroaryl" includes ring systems
wherein the
heteroaryl ring, as defined above, is fused with one or more carbocyclyl or
heterocyclyl groups
wherein the point of attachment is on the heteroaryl ring, and in such
instances, the number of
ring members continue to designate the number of ring members in the
heteroaryl ring system.
"Heteroaryl" also includes ring systems wherein the heteroaryl ring, as
defined above, is fused
with one or more aryl groups wherein the point of attachment is either on the
aryl or heteroaryl
ring, and in such instances, the number of ring members designates the number
of ring members
in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein
one ring does not
contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the
point of attachment
can be on either ring, i.e., either the ring bearing a heteroatom (e.g.,
2¨indoly1) or the ring that
does not contain a heteroatom (e.g., 5¨indoly1).
[00128] In some embodiments, a heteroaryl group is a 5-10 membered aromatic
ring system
having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic
ring system,
wherein each heteroatom is independently selected from nitrogen, oxygen, and
sulfur ("5-10
membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-8
membered aromatic
ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the
aromatic ring
system, wherein each heteroatom is independently selected from nitrogen,
oxygen, and sulfur
("5-8 membered heteroaryl"). In some embodiments, a heteroaryl group is a 5-6
membered
aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms
provided in the
aromatic ring system, wherein each heteroatom is independently selected from
nitrogen, oxygen,
and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered
heteroaryl
has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some
embodiments, the
5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,
oxygen, and sulfur.
In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom
selected from
nitrogen, oxygen, and sulfur. Unless otherwise specified, each instance of a
heteroaryl group is
independently optionally substituted, i.e., unsubstituted (an "unsubstituted
heteroaryl") or
substituted (a "substituted heteroaryl") with one or more substituents. In
certain embodiments,
the heteroaryl group is unsubstituted 5-14 membered heteroaryl. In certain
embodiments, the
heteroaryl group is substituted 5-14 membered heteroaryl.
[00129] Exemplary 5¨membered heteroaryl groups containing one heteroatom
include, without
limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5¨membered heteroaryl
groups
containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl,
oxazolyl,
isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5¨membered heteroaryl
groups containing
three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and
thiadiazolyl. Exemplary
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5¨membered heteroaryl groups containing four heteroatoms include, without
limitation,
tetrazolyl. Exemplary 6¨membered heteroaryl groups containing one heteroatom
include,
without limitation, pyridinyl. Exemplary 6¨membered heteroaryl groups
containing two
heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and
pyrazinyl. Exemplary 6¨
membered heteroaryl groups containing three or four heteroatoms include,
without limitation,
triazinyl and tetrazinyl, respectively. Exemplary 7¨membered heteroaryl groups
containing one
heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl.
Exemplary 5,6¨
bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl,
indazolyl,
benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,
benzoisofuranyl,
benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,
benzisothiazolyl,
benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6¨bicyclic heteroaryl
groups include,
without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,
cinnolinyl, quinoxalinyl,
phthalazinyl, and quinazolinyl.
[00130] Examples of representative heteroaryls include the following:
LJ
cs
jN
N
N\\N 40 \N \
Y
wherein each Y is selected from carbonyl, N, NR65, 0, and S; and R65 is
independently hydrogen,
C1-C8 alkyl, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, C6-C10 aryl, and 5-
10 membered
heteroaryl.
[00131] Examples of representative aryl having hetero atoms containing
substitution include
the following:
= W)
401
and Y
>
Y '
wherein each W is selected from C(R66)2, NR66, 0, and S; and each Y is
selected from carbonyl,
NR66, 0 and S; and R66 is independently hydrogen, C1-C8 alkyl, C3-C10
cycloalkyl, 4-10
membered heterocyclyl, C6-C10 aryl, and 5-10 membered heteroaryl.
[00132] "Heteroaralkyl" is a subset of alkyl and heteroaryl, as defined
herein, and refers to an
optionally substituted alkyl group substituted by an optionally substituted
heteroaryl group.
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[00133] "Carbocycly1" or "carbocyclic" refers to a radical of a non¨aromatic
cyclic
hydrocarbon group having from 3 to 10 ring carbon atoms ("C3_10 carbocyclyl")
and zero
heteroatoms in the non¨aromatic ring system. In some embodiments, a
carbocyclyl group has 3
to 8 ring carbon atoms ("C3_8 carbocyclyl"). In some embodiments, a
carbocyclyl group has 3 to
6 ring carbon atoms ("C3-6 carbocyclyl"). In some embodiments, a carbocyclyl
group has 3 to 6
ring carbon atoms ("C3_6 carbocyclyl"). In some embodiments, a carbocyclyl
group has 5 to 10
ring carbon atoms ("C5_10 carbocyclyl"). Exemplary C3_6 carbocyclyl groups
include, without
limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4),
cyclobutenyl (C4), cyclopentyl
(C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl
(C6), and the like.
Exemplary C3_8 carbocyclyl groups include, without limitation, the
aforementioned C3_6
carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7),
cycloheptadienyl (C7),
cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), bicyclo[2.2.1
lheptanyl (C7),
bicyclo[2.2.2loctanyl (C8), and the like. Exemplary C3_10 carbocyclyl groups
include, without
limitation, the aforementioned C3_8 carbocyclyl groups as well as cyclononyl
(C9), cyclononenyl
(C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H¨indenyl (C9),
decahydronaphthalenyl
(C10), spiro[4.51decanyl (C10), and the like. As the foregoing examples
illustrate, in certain
embodiments, the carbocyclyl group is either monocyclic ("monocyclic
carbocyclyl") or contain
a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic
carbocyclyl") and can
be saturated or can be partially unsaturated. "Carbocycly1" also includes ring
systems wherein the
carbocyclyl ring, as defined above, is fused with one or more aryl or
heteroaryl groups wherein
the point of attachment is on the carbocyclyl ring, and in such instances, the
number of carbons
continue to designate the number of carbons in the carbocyclic ring system.
Unless otherwise
specified, each instance of a carbocyclyl group is independently optionally
substituted, i.e.,
unsubstituted (an "unsubstituted carbocyclyl") or substituted (a "substituted
carbocyclyl") with
one or more substituents. In certain embodiments, the carbocyclyl group is
unsubstituted C3-10
carbocyclyl. In certain embodiments, the carbocyclyl group is a substituted
C3_10 carbocyclyl.
[00134] In some embodiments, "carbocyclyl" is a monocyclic, saturated
carbocyclyl group
having from 3 to 10 ring carbon atoms ("C3_10 cycloalkyl"). In some
embodiments, a cycloalkyl
group has 3 to 8 ring carbon atoms ("C3_8 cycloalkyl"). In some embodiments, a
cycloalkyl
group has 3 to 6 ring carbon atoms ("C3_6 cycloalkyl"). In some embodiments, a
cycloalkyl
group has 5 to 6 ring carbon atoms ("C5_6 cycloalkyl"). In some embodiments, a
cycloalkyl
group has 5 to 10 ring carbon atoms ("C5_10 cycloalkyl"). Examples of C5_6
cycloalkyl groups
include cyclopentyl (C5) and cyclohexyl (C5). Examples of C3_6 cycloalkyl
groups include the
aforementioned C5_6 cycloalkyl groups as well as cyclopropyl (C3) and
cyclobutyl (C4).
Examples of C3_8 cycloalkyl groups include the aforementioned C3_6 cycloalkyl
groups as well as
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cycloheptyl (C7) and cyclooctyl (C8). Unless otherwise specified, each
instance of a cycloalkyl
group is independently unsubstituted (an "unsubstituted cycloalkyl") or
substituted (a
"substituted cycloalkyl") with one or more substituents. In certain
embodiments, the cycloalkyl
group is unsubstituted C3_10 cycloalkyl. In certain embodiments, the
cycloalkyl group is
substituted C3_10 cycloalkyl.
1001351 "Heterocycly1" or "heterocyclic" refers to a radical of a 3¨ to
10¨membered non¨
aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms,
wherein each
heteroatom is independently selected from nitrogen, oxygen, sulfur, boron,
phosphorus, and
silicon ("3-10 membered heterocyclyl"). In heterocyclyl groups that contain
one or more
nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as
valency permits. A
heterocyclyl group can either be monocyclic ("monocyclic heterocyclyl") or a
fused, bridged or
spiro ring system such as a bicyclic system ("bicyclic heterocyclyl"), and can
be saturated or can
be partially unsaturated. Heterocyclyl bicyclic ring systems can include one
or more heteroatoms
in one or both rings. "Heterocycly1" also includes ring systems wherein the
heterocyclyl ring, as
defined above, is fused with one or more carbocyclyl groups wherein the point
of attachment is
either on the carbocyclyl or heterocyclyl ring, or ring systems wherein the
heterocyclyl ring, as
defined above, is fused with one or more aryl or heteroaryl groups, wherein
the point of
attachment is on the heterocyclyl ring, and in such instances, the number of
ring members
continue to designate the number of ring members in the heterocyclyl ring
system. Unless
otherwise specified, each instance of heterocyclyl is independently optionally
substituted, i.e.,
unsubstituted (an "unsubstituted heterocyclyl") or substituted (a "substituted
heterocyclyl") with
one or more substituents. In certain embodiments, the heterocyclyl group is
unsubstituted 3-10
membered heterocyclyl. In certain embodiments, the heterocyclyl group is
substituted 3-10
membered heterocyclyl.
[00136] In some embodiments, a heterocyclyl group is a 5-10 membered
non¨aromatic ring
system having ring carbon atoms and 1-4 ring heteroatoms, wherein each
heteroatom is
independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and
silicon ("5-10
membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8
membered non¨
aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms,
wherein each
heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8
membered
heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-6 membered
non¨aromatic
ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each
heteroatom is
independently selected from nitrogen, oxygen, and sulfur ("5-6 membered
heterocyclyl"). In
some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms
selected from
nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered
heterocyclyl has 1-2 ring
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heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments,
the 5-6
membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen,
and sulfur.
[00137] Exemplary 3¨membered heterocyclyl groups containing one heteroatom
include,
without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4¨membered
heterocyclyl groups
containing one heteroatom include, without limitation, azetidinyl, oxetanyl
and thietanyl.
Exemplary 5¨membered heterocyclyl groups containing one heteroatom include,
without
limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,
dihydrothiophenyl,
pyrrolidinyl, dihydropyrrolyl and pyrroly1-2,5¨dione. Exemplary 5¨membered
heterocyclyl
groups containing two heteroatoms include, without limitation, dioxolanyl,
oxasulfuranyl,
disulfuranyl, and oxazolidin-2-one. Exemplary 5¨membered heterocyclyl groups
containing three
heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and
thiadiazolinyl.
Exemplary 6¨membered heterocyclyl groups containing one heteroatom include,
without
limitation, piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl.
Exemplary 6¨
membered heterocyclyl groups containing two heteroatoms include, without
limitation,
piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6¨membered
heterocyclyl groups
containing two heteroatoms include, without limitation, triazinanyl. Exemplary
7¨membered
heterocyclyl groups containing one heteroatom include, without limitation,
azepanyl, oxepanyl
and thiepanyl. Exemplary 8¨membered heterocyclyl groups containing one
heteroatom include,
without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered
heterocyclyl
groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic
heterocyclic ring) include,
without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,
dihydrobenzothienyl,
benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused
to an aryl ring
(also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without
limitation,
tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
[00138] Particular examples of heterocyclyl groups are shown in the following
illustrative
examples:
\l)v `cN3=
vv,
_______________________ Yo- 40,,
[00139] wherein each W is selected from CR67, C(R67)2, NR67, 0, and S; and
each Y is selected
from NR67, 0, and S; and R67 is independently hydrogen, Ci-C8 alkyl, C3-Cio
cycloalkyl, 4-10
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membered heterocyclyl, C6-C10 aryl, 5-10 membered heteroaryl. These
heterocyclyl rings may be
optionally substituted with one or more substituents selected from the group
consisting of the
group consisting of acyl, acylamino, acyloxy, alkoxy, alkoxycarbonyl,
alkoxycarbonylamino,
amino, substituted amino, aminocarbonyl (carbamoyl or amido),
aminocarbonylamino,
aminosulfonyl, sulfonylamino, aryl, aryloxy, azido, carboxyl, cyano,
cycloalkyl, halogen,
hydroxy, keto, nitro, thiol, -S-alkyl, ¨S-aryl, -S(0)-alkyl,¨S(0)-aryl, ¨S(0)2-
alkyl, and -S(0)2-
aryl. Substituting groups include carbonyl or thiocarbonyl which provide, for
example, lactam
and urea derivatives.
[00140] "Hetero" when used to describe a compound or a group present on a
compound means
that one or more carbon atoms in the compound or group have been replaced by a
nitrogen,
oxygen, or sulfur heteroatom. Hetero may be applied to any of the hydrocarbyl
groups described
above such as alkyl, e.g., heteroalkyl, cycloalkyl, e.g., heterocyclyl, aryl,
e.g,. heteroaryl,
cycloalkenyl, e.g,. cycloheteroalkenyl, and the like having from 1 to 5, and
particularly from 1 to
3 heteroatoms.
[00141] "Acyl" refers to a radical -C(0)R20, where R2 is hydrogen,
substituted or unsubstitued
alkyl, substituted or unsubstitued alkenyl, substituted or unsubstitued
alkynyl, substituted or
unsubstitued carbocyclyl, substituted or unsubstituted heterocyclyl,
substituted or unsubstituted
aryl, or substituted or unsubstitued heteroaryl, as defined herein. "Alkanoyl"
is an acyl group
wherein R2 is a group other than hydrogen. Representative acyl groups
include, but are not
limited to, formyl (-CHO), acetyl (-C(=0)CH3), cyclohexylcarbonyl,
cyclohexylmethylcarbonyl,
benzoyl (-C(=0)Ph), benzylcarbonyl (-C(=0)CH2Ph), --C(0)-C1-C8 alkyl, ¨C(0)-
(CH2)(C6-Cio
aryl), ¨C(0)-(CH2)t(5-10 membered heteroaryl), ¨C(0)-(CH2)t(C3-C10
cycloalkyl), and ¨C(0)-
(CH2)t(4-10 membered heterocyclyl), wherein t is an integer from 0 to 4. In
certain
embodiments, R21- is Ci-C8 alkyl, substituted with halo or hydroxy; or C3-C10
cycloalkyl, 4-10
membered heterocyclyl, C6-C10 aryl, arylalkyl, 5-10 membered heteroaryl or
heteroarylalkyl,
each of which is substituted with unsubstituted Ci-C4 alkyl, halo,
unsubstituted Ci-C4 alkoxy,
unsubstituted Ci-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl, or
unsubstituted Ci-C4
haloalkoxy or hydroxy.
[00142] "Acylamino" refers to a radical -NR22C(0)R23, where each instance of
R22 and R23 is
independently hydrogen, substituted or unsubstitued alkyl, substituted or
unsubstitued alkenyl,
substituted or unsubstitued alkynyl, substituted or unsubstitued carbocyclyl,
substituted or
unsubstituted heterocyclyl, substituted or unsubstituted aryl, or substituted
or unsubstitued
heteroarylõ as defined herein, or R22 is an amino protecting group. Exemplary
"acylamino"
groups include, but are not limited to, formylamino, acetylamino,
cyclohexylcarbonylamino,
cyclohexylmethyl-carbonylamino, benzoylamino and benzylcarbonylamino.
Particular
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exemplary "acylamino" groups are ¨NR24C(0)-Ci-C8 alkyl, ¨NR24C(0)-(CH2)t(C6-
C10 aryl), ¨
NR24C(0)-(CH2)(5-1 0 membered heteroaryl), ¨NR24C(0)-(CH2)(C3-C10 cycloalkyl),
and ¨
NR24C(0)-(CH2)t(4-1 0 membered heterocyclyl), wherein t is an integer from 0
to 4, and each R24
independently represents H or C1-C8 alkyl. In certain embodiments, R25 is H,
C1-C8 alkyl,
substituted with halo or hydroxy; C3-C10 cycloalkyl, 4-10 membered
heterocyclyl, C6-C10 aryl,
arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is
substituted with
unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-
C4 haloalkyl,
unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or
hydroxy; and R26 is H,
C1-C8 alkyl, substituted with halo or hydroxy;
C3-C10 cycloalkyl, 4-10 membered heterocyclyl, C6-C10 aryl, arylalkyl, 5-10
membered
heteroaryl or heteroarylalkyl, each of which is substituted with unsubstituted
C1-C4 alkyl, halo,
unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted C1-C4
hydroxyalkyl, or
unsubstituted C1_C4 haloalkoxy or hydroxyl; provided that at least one of R25
and R26 is other than
H.
[00143] "Acyloxy" refers to a radical -0C(0)R27, where R27 is hydrogen,
substituted or
unsubstitued alkyl, substituted or unsubstitued alkenyl, substituted or
unsubstituted alkynyl,
substituted or unsubstituted carbocyclyl, substituted or unsubstituted
heterocyclyl, substituted or
unsubstituted aryl, or substituted or unsubstituted heteroaryl, as defined
herein. Representative
examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl,
cyclohexylmethylcarbonyl, benzoyl and benzylcarbonyl. In certain embodiments,
R28 is C1-C8
alkyl, substituted with halo or hydroxy; C3-C10 cycloalkyl, 4-10 membered
heterocyclyl, C6-C10
aryl, arylalkyl, 5-10 membered heteroaryl or heteroarylalkyl, each of which is
substituted with
unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-
C4 haloalkyl,
unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or
hydroxy.
[00144] "Alkoxy" refers to the group ¨0R29 where R29 is substituted or
unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or
unsubstituted carbocyclyl, substituted or unsubstituted heterocyclyl,
substituted or unsubstituted
aryl, or substituted or unsubstituted heteroaryl. Particular alkoxy groups are
methoxy, ethoxy, n-
propoxy, isopropoxy, n-butoxy, tert-butoxy, sec-butoxy, n-pentoxy, n-hexoxy,
and 1,2-
dimethylbutoxy. Particular alkoxy groups are lower alkoxy, i.e. with between 1
and 6 carbon
atoms. Further particular alkoxy groups have between 1 and 4 carbon atoms.
[00145] In certain embodiments, R29 is a group that has 1 or more
substituents, for instance,
from 1 to 5 substituents, and particularly from 1 to 3 substituents, in
particular 1 substituent,
selected from the group consisting of amino, substituted amino, C6-C10 aryl,
aryloxy, carboxyl,
cyano, C3-C10 cycloalkyl, 4-10 membered heterocyclyl, halogen, 5-10 membered
heteroaryl,
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hydroxyl, nitro, thioalkoxy, thioaryloxy, thiol, alkyl-S(0)-, aryl¨S(0)-,
alkyl¨S(0)2- and aryl-
S(0)2-. Exemplary 'substituted alkoxy' groups include, but are not limited to,
¨0-(CH2)(C6-C10
aryl), ¨0-(CH2)(5-10 membered heteroaryl), ¨0-(CH2)(C3-C10 cycloalkyl), and ¨0-
(CH2)t(4-10
membered heterocyclyl), wherein t is an integer from 0 to 4 and any aryl,
heteroaryl, cycloalkyl
or heterocyclyl groups present, may themselves be substituted by unsubstituted
Ci-C4 alkyl, halo,
unsubstituted C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted Ci-C4
hydroxyalkyl, or
unsubstituted C1-C4 haloalkoxy or hydroxy. Particular exemplary 'substituted
alkoxy' groups are
-0CF3, -OCH2CF3, -OCH2Ph, -OCH2-cyclopropyl, -OCH2CH2OH, and -OCH2CH2NMe2.
[00146] "Amino" refers to the radical -NI-12.
[00147] "Substituted amino" refers to an amino group of the formula -N(R38)2
wherein R38 is
hydrogen, substituted or unsubstituted alkyl, substituted or unsubstitued
alkenyl, substituted or
unsubstitued alkynyl, substituted or unsubstitued carbocyclyl, substituted or
unsubstituted
heterocyclyl, substituted or unsubstituted aryl, substituted or unsubstitued
heteroaryl, or an amino
protecting group, wherein at least one of R38 is not a hydrogen. In certain
embodiments,each R38
is independently selected from: hydrogen, C1-C8 alkyl, C3-C8 alkenyl, C3-C8
alkynyl, C6-C10 aryl,
5-10 membered heteroaryl, 4-10 membered heterocyclyl, or C3-C10 cycloalkyl; or
C1-C8 alkyl,
substituted with halo or hydroxy; C3-C8 alkenyl, substituted with halo or
hydroxy; C3-C8 alkynyl,
substituted with halo or hydroxy, or -(CH2)t(C6-C10 aryl), -(CH2)t(5-10
membered heteroaryl), -
(CH2)t(C3-C10 cycloalkyl), or -(CH2)(4-10 membered heterocyclyl), wherein t is
an integer
between 0 and 8, each of which is substituted by unsubstituted Ci-C4 alkyl,
halo, unsubstituted
C1-C4 alkoxy, unsubstituted C1-C4 haloalkyl, unsubstituted Ci-C4 hydroxyalkyl,
or unsubstituted
C1-C4 haloalkoxy or hydroxy; or both R38 groups are joined to form an alkylene
group.
[00148] Exemplary 'substituted amino' groups are ¨NR39-C1-C8 alkyl, ¨NR39-
(CH2)t(C6-Cio
aryl), ¨NR39-(CH2)t(5-10 membered heteroaryl), ¨NR39-(CH2)t(C3-C10
cycloalkyl), and ¨NR39-
(CH2)(4-10 membered heterocyclyl), wherein t is an integer from 0 to 4, for
instance 1 or 2, each
R39 independently represents H or C1-C8 alkyl, and any alkyl groups present,
may themselves be
substituted by halo, substituted or unsubstituted amino, or hydroxy; and any
aryl, heteroaryl,
cycloalkyl, or heterocyclyl groups present, may themselves be substituted by
unsubstituted Ci-C4
alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted Ci-C4 haloalkyl,
unsubstituted Ci-C4
hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or hydroxy. For the avoidance
of doubt the
term 'substituted amino' includes the groups alkylamino, substituted
alkylamino, alkylarylamino,
substituted alkylarylamino, arylamino, substituted arylamino, dialkylamino,
and substituted
dialkylamino as defined below. Substituted amino encompasses both
monosubstituted amino and
disubstituted amino groups.
[00149] "Azido" refers to the radical -N3.
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[00150] "Carbamoyl" or "amido" refers to the radical -C(0)NH2.
[00151] "Substituted carbamoyl" or "substituted amido" refers to the radical -
C(0)N(R62)2
wherein each R62 is independently hydrogen, substituted or unsubstituted
alkyl, substituted or
unsubstitued alkenyl, substituted or unsubstitued alkynyl, substituted or
unsubstitued carbocyclyl,
substituted or unsubstituted heterocyclyl, substituted or unsubstituted aryl,
substituted or
unsubstitued heteroaryl, or an amino protecting group, wherein at least one of
R62 is not a
hydrogen. In certain embodiments, R62 is selected from H, C1-C8 alkyl, C3-Cio
cycloalkyl, 4-10
membered heterocyclyl, C6-C10 aryl, aralkyl, 5-10 membered heteroaryl, and
heteroaralkyl; or
C1-C8 alkyl substituted with halo or hydroxy; or C3-Cio cycloalkyl, 4-10
membered heterocyclyl,
C6-Cio aryl, aralkyl, 5-10 membered heteroaryl, or heteroaralkyl, each of
which is substituted by
unsubstituted Ci-C4 alkyl, halo, unsubstituted Ci-C4 alkoxy, unsubstituted Ci-
C4 haloalkyl,
unsubstituted C1-C4 hydroxyalkyl, or unsubstituted C1-C4 haloalkoxy or
hydroxy; provided that
at least one R62 is other than H.
[00152] Exemplary 'substituted carbamoyl' groups include, but are not limited
to, ¨C(0) NR64-
Ci-C8 alkyl, ¨C(0)NR64-(CH2)t(C6-C10 aryl), ¨C(0)N64-(CH2)t(5-1 0 membered
heteroaryl), ¨
C(0)NR64-(CH2)t(C3-C10 cycloalkyl), and ¨C(0)NR64-(CH2)t(4-1 0 membered
heterocyclyl),
wherein t is an integer from 0 to 4, each R64 independently represents H or Ci-
C8 alkyl and any
aryl, heteroaryl, cycloalkyl or heterocyclyl groups present, may themselves be
substituted by
unsubstituted C1-C4 alkyl, halo, unsubstituted C1-C4 alkoxy, unsubstituted C1-
C4 haloalkyl,
unsubstituted C1-C4 hydroxyalkyl, or unsubstituted Ci-C4 haloalkoxy or
hydroxy.
[00153] `Carboxy' refers to the radical -C(0)0H.
[00154] "Cyano" refers to the radical -CN.
[00155] "Halo" or "halogen" refers to fluoro (F), chloro (Cl), bromo (Br), and
iodo (I). In
certain embodiments, the halo group is either fluoro or chloro. In further
embodiments, the halo
group is iodo.
[00156] "Hydroxy" refers to the radical -OH.
[00157] "Nitro" refers to the radical ¨NO2.
[00158] "Cycloalkylalkyl" refers to an alkyl radical in which the alkyl group
is substituted with
a cycloalkyl group. Typical cycloalkylalkyl groups include, but are not
limited to,
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, cyclohexylmethyl,
cycloheptylmethyl,
cyclooctylmethyl, cyclopropylethyl, cyclobutylethyl, cyclopentylethyl,
cyclohexylethyl,
cycloheptylethyl, and cyclooctylethyl, and the like.
[00159] "Heterocyclylalkyl" refers to an alkyl radical in which the alkyl
group is substituted
with a heterocyclyl group. Typical heterocyclylalkyl groups include, but are
not limited to,
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pyrrolidinylmethyl, piperidinylmethyl, piperazinylmethyl, morpholinylmethyl,
pyrrolidinylethyl,
piperidinylethyl, piperazinylethyl, morpholinylethyl, and the like.
[00160] "Cycloalkenyl" refers to substituted or unsubstituted carbocyclyl
group having from 3
to 10 carbon atoms and having a single cyclic ring or multiple condensed
rings, including fused
and bridged ring systems and having at least one and particularly from 1 to 2
sites of olefinic
unsaturation. Such cycloalkenyl groups include, by way of example, single ring
structures such
as cyclohexenyl, cyclopentenyl, cyclopropenyl, and the like.
[00161] "Fused cycloalkenyl" refers to a cycloalkenyl having two of its ring
carbon atoms in
common with a second aliphatic or aromatic ring and having its olefinic
unsaturation located to
impart aromaticity to the cycloalkenyl ring.
[00162] "Ethenyl" refers to substituted or unsubstituted ¨(C=C)-.
[00163] "Ethylene" refers to substituted or unsubstituted ¨(C-C)-.
[00164] "Ethynyl" refers to ¨(CC)-.
[00165] "Nitrogen-containing heterocyclyl" group means a 4- to 7- membered non-
aromatic
cyclic group containing at least one nitrogen atom, for example, but without
limitation,
morpholine, piperidine (e.g. 2-piperidinyl, 3-piperidinyl and 4-piperidinyl),
pyrrolidine (e.g. 2-
pyrrolidinyl and 3-pyrrolidinyl), azetidine, pyrrolidone, imidazoline,
imidazolidinone, 2-
pyrazoline, pyrazolidine, piperazine, and N-alkyl piperazines such as N-methyl
piperazine.
Particular examples include azetidine, piperidone and piperazone.
[00166] "Thioketo" refers to the group =S.
[00167] Alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and
heteroaryl groups, as
defined herein, are optionally substituted (e.g., "substituted" or
"unsubstituted" alkyl,
"substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted"
alkynyl, "substituted"
or "unsubstituted" carbocyclyl, "substituted" or "unsubstituted" heterocyclyl,
"substituted" or
"unsubstituted" aryl or "substituted" or "unsubstituted" heteroaryl group). In
general, the term
"substituted", whether preceded by the term "optionally" or not, means that at
least one hydrogen
present on a group (e.g., a carbon or nitrogen atom) is replaced with a
permissible substituent,
e.g., a substituent which upon substitution results in a stable compound,
e.g., a compound which
does not spontaneously undergo transformation such as by rearrangement,
cyclization,
elimination, or other reaction. Unless otherwise indicated, a "substituted"
group has a substituent
at one or more substitutable positions of the group, and when more than one
position in any given
structure is substituted, the substituent is either the same or different at
each position. The term
"substituted" is contemplated to include substitution with all permissible
substituents of organic
compounds, any of the substituents described herein that results in the
formation of a stable
compound. The present invention contemplates any and all such combinations in
order to arrive
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at a stable compound. For purposes of this invention, heteroatoms such as
nitrogen may have
hydrogen substituents and/or any suitable substituent as described herein
which satisfy the
valencies of the heteroatoms and results in the formation of a stable moiety.
[00168] Exemplary carbon atom substituents include, but are not limited to,
halogen, -CN, -
NO2, -N3, -S02H, -S 03H, -OH, -0R', -0N(Rbb)2, -
N(Rb), b.2 _ MR-hh-)3+X-, -N(OR)R',
-SR', -S CC,-C(=0)Raa, -C 02H, -CHO, -C(OR)2, -CO2Raa, -0 C(=0)Raa, -OC 02R", -
C(=0)N(R) bbµ2, -
OC(=0)N(Rbb)2, - h
NRb_ _ (_0)Raa, NRbbc 02Raa, NRbb
u( 0)N(Rbb)2, -
c (_NRbb)Raa, (_NRbK
b)o- aa,
0 C (=
NRbbµK aa,
) OC(=
NRbb)0Raa, (_NRbb)N(Rbb)2, _
OC(=
NRbb)N(Rbb)2, NRbb (_NRbb)N(Rbb)2, C(=0)NRbb s 02Raa, NRbb s 02-K aa,
SO2N(Rbb)2, -
SO2Raa, -S020Raa, -0S02Raa, -S(=0)Raa, -0S(=0)Raa, -S1(Raa)3, -0S1(R)3 -
C(=S)N(Rbb)2, -
C(=0)SRaa, -C(=S)SRaa, -S C(=S)SRaa, -SC(=0)SRaa, -0 C(=0)SRaa, -S C(=0)0Raa, -
SC(=0)Raa, p(_0)2Raa, op (_0)2Raa, p (_0)(R)aa, 2,
OP (=0)(Raa)2, -OP (=0)(ORcc)2,
(_0)2N(R) bb, 2
P (= )2N(Rbb)2, -P (-0)(NR OP (=0 )(NRbb)2, _ NRbb-
_t( 0)(ORcc)2, -
NRbbp(_0)(NRbb)2, p(RCC)2, p (RCCµ 3,
bb'
)
) -OP(R)2, -OP (Wc)3 , -B(Raa)2, -B(OR)2, -
BRaa(ORcc),
C 1_10 alkyl, Ci_10 perhaloalkyl, C2_10 alkenyl, C2_10 alkynyl, C3_10
carbocyclyl, 3-14 membered
heterocyclyl, C6_14 aryl, and 5-14 membered heteroaryl, wherein each alkyl,
alkenyl, alkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5
Rdd groups;
or two geminal hydrogens on a carbon atom are replaced with the group =0, =S,
=NN(R)2,
_NNRbb (_0)Raa, _NNRbbc (_0)0Raa, _NNRbb s(_0)2Raa, bb,
INK or =NOR;
each instance of Raa is, independently, selected from Ci_io alkyl, Ci_io
perhaloalkyl, C2-10
alkenyl, C2_10 alkynyl, C3_10 carbocyclyl, 3-14 membered heterocyclyl, C6_14
aryl, and 5-14
membered heteroaryl, or two Raa groups are joined to form a 3-14 membered
heterocyclyl or 5-
14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,
carbocyclyl, heterocyclyl,
aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd
groups;
each instance of Rbb is, independently, selected from hydrogen, -OH, -OR", -
N(R)2, -CN,
_cc,2, - 0R-
C(=0)Raa, -C(=0)N(R C SO2Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rcc)2, -S
02N(R)2,
-SO2Rcc, -S 02 0Rcc, -SORaa, -C(=S)N(Rcc)2, -C(=0)S Rcc, -C(=S)SRcc, -
P(=0)2Raa, -
P(=0)(Raa)2, -P(0)2N(R)2, -P (=0)(NRcc)2, C1_10 alkyl, C1_10 perhaloalkyl,
C2_10 alkenyl, C2_10
alkynyl, C3_10 carbocyclyl, 3-14 membered heterocyclyl, C6_14 aryl, and 5-14
membered
heteroaryl, or two Rbb groups are joined to form a 3-14 membered heterocyclyl
or 5-14
membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl, aryl,
and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd
groups;
each instance of Rcc is, independently, selected from hydrogen, C1_10 alkyl,
C1_10 perhaloalkyl,
C2_10 alkenyl, C2_10 alkynyl, C3_10 carbocyclyl, 3-14 membered heterocyclyl,
C6_14 aryl, and 5-14
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membered heteroaryl, or two Rcc groups are joined to form a 3-14 membered
heterocyclyl or 5-
14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,
carbocyclyl, heterocyclyl,
aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rdd
groups;
each instance of Rdd is, independently, selected from halogen, -CN, -NO2, -N3,
-S02H, -S03H,
-OH, -ON(R)2, -N(R)2, -N(R)3X, -N(OR)R, -SH, -SR, -
C(=0)Ree, -
CO2H, -CO2Ree, -0C(=0)Ree, -0CO2Ree, -C(=0)N(Rff)2, -0C(=0)N(Rff)2, -
NRffC(=0)Ree, -
NRffCO2Ree, -NRffC(=0)N(Rff)2, -C(=NRff)0Ree, -0C(=NRff)Ree, -0C(=NRff)0Ree, -
C(=NRff)N(Rff)2, -0C(=NRff)N(Rff)2, -NRffC(=NRff)N(Rff)2,-NRffS02Ree, -
SO2N(Rff)2, -
SO2Ree, -S020Ree, -0S02Ree, -S(=0)Ree, -Si(V)3, -0Si(Ree)3, -C(=S)N(Rff)2, -
C(=0)SRee, -
C(=S)SRee, -SC(=S)SRee, -P(=0)2Ree, -P(=0)(Ree)2, -0P(=0)(Ree)2, -
0P(=0)(0Ree)2, C1-6
alkyl, Ci_6 perhaloalkyl, C2_6 alkenyl, C2_6 alkynyl, C3_10 carbocyclyl, 3-10
membered
heterocyclyl, C6_10 aryl, 5-10 membered heteroaryl, wherein each alkyl,
alkenyl, alkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5
Rgg groups, or two geminal Rdd substituents can be joined to form =0 or =S;
each instance of Ree is, independently, selected from Ci_6 alkyl, Ci_6
perhaloalkyl, C2_6 alkenyl,
C2_6 alkynyl, C3-10 carbocyclyl, C6_10 aryl, 3-10 membered heterocyclyl, and 3-
10 membered
heteroaryl, wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,
aryl, and heteroaryl is
independently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;
each instance of Rff is, independently, selected from hydrogen, C1_6 alkyl,
C1_6 perhaloalkyl, C2-6
alkenyl, C2_6 alkynyl, C3_10 carbocyclyl, 3-10 membered heterocyclyl, C6_10
aryl and 5-10
membered heteroaryl, or two Rff groups are joined to form a 3-14 membered
heterocyclyl or 5-
14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,
carbocyclyl, heterocyclyl,
aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgg
groups; and
each instance of Rgg is, independently, halogen, -CN, -NO2, -N3, -S02H, -S03H,
-OH, -0C1-6
alkyl, -0N(C1_6 alky1)2, -N(C1_6 alky1)2, -N(C1_6 alky1)3+X-, -NH(C1_6
alky1)2+X-, -NH2(C1-6
alkyl) +X-, -NH3+X-, -N(0C1_6 alkyl)(Ci_6 alkyl), -N(OH)(C1_6 alkyl), -NH(OH),
-SH, -5C1-6
alkyl, -SS(C1_6 alkyl), -C(=0)(C1_6 alkyl), -CO2H, -0O2(C1_6 alkyl), -
0C(=0)(C1_6 alkyl), -
00O2(C1_6 alkyl), -C(=0)NH2, -C(=0)N(C1_6 alky1)2, -0C(=0)NH(C1_6 alkyl), -
NHC(=0)( C1-
6 alkyl), -N(C1_6 alkyl)C(=0)( C1_6 alkyl), -NHCO2(C1_6 alkyl), -NHC(=0)N(C1_6
alky02, -
NHC(=0)NH(C1_6 alkyl), -NHC(=0)NH2, -C(=NH)0(C1_6 alkyl),-0C(=NH)(C1_6 alkyl),
-
OC(=NH)0C1_6 alkyl, -C(=NH)N(C1_6 alky1)2, -C(=NH)NH(C1_6 alkyl), -C(=NH)NH2, -
0C(=NH)N(C1_6 alky1)2, -0C(NH)NH(C1_6 alkyl), -0C(NH)NH2, -NHC(NH)N(C1_6
alky1)2, -
NHC(=NH)NH2, -NH502(C1_6 alkyl), -5O2N(C1_6 alky1)2, -5O2NH(C1_6 alkyl), -
502NH2,-
502C1_6 alkyl, -5020C1_6 alkyl, -0502C1_6 alkyl, -50C1_6 alkyl, -5i(C1_6
alky1)3, -05i(C1-6
alky1)3 -C(=5)N(C1_6 alky1)2, C(=5)NH(C1_6 alkyl), C(=5)NH2, -C(=0)5(C1_6
alkyl), -
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C(=S)SC1-6 alkyl, -SC(=S)SC1-6 alkyl, -P(=0)2(C1_6 alkyl), -P(=0)(C1-6
alky1)2, -0P(=0)(C1-6
alky1)2, -OP(=0)(0C1_6 alky1)2, Ci_6 alkyl, C1_6 perhaloalkyl, C2_6 alkenyl,
C2_6 alkynyl, C3-10
carbocyclyl, C6_10 aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl;
or two geminal
Rgg substituents can be joined to form =0 or =S; wherein X- is a counterion.
[00169] A "counterion" or "anionic counterion" is a negatively charged group
associated with a
cationic quaternary amino group in order to maintain electronic neutrality.
Exemplary
counterions include halide ions (e.g., F, a-, Br-, 1-), NO3-, C104-, OW, H2PO4-
, HSO4-,
sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate, p-
toluenesulfonate,
benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate, naphthalene-l-
sulfonic
acid-5-sulfonate, ethan-l-sulfonic acid-2-sulfonate, and the like), and
carboxylate ions (e.g.,
acetate, ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,
glycolate, and the like).
[00170] Nitrogen atoms can be substituted or unsubstituted as valency permits,
and include
primary, secondary, tertiary, and quarternary nitrogen atoms. Exemplary
nitrogen atom
substitutents include, but are not limited to, hydrogen, -OH, -0R', -N(R)2, -
CN, -C(=0)Raa,
- (=0)N(Rcc)2, 02R', -S 02R', -C(=NRbb)Raa, -C(=NRcc)0Raa, -
C(=NRcc)N(Rcc)2, -
S 02N(Rcc)2, -S 02R, -S020Rcc, -s OR', (=S)N(Rcc)2, (=0)SRcc, (=S )S Rcc,
(=0)2Raa,
-P(=0)(Raa)2, -P(0)2N(R)2, -13(=0)(NRcc)2, C1-10 alkyl, C1_10 perhaloalkyl,
C2_10 alkenyl, C2-
alkynyl, C3_10 carbocyclyl, 3-14 membered heterocyclyl, C6_14 aryl, and 5-14
membered
heteroaryl, or two Rcc groups attached to a nitrogen atom are joined to form a
3-14 membered
heterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,
alkynyl,
carbocyclyl, heterocyclyl, aryl, and heteroaryl is independently substituted
with 0, 1, 2, 3, 4, or 5
R', Rcc an ,
Rdd groups, and wherein Raa, R a tc are as defined above.
[00171] In certain embodiments, the substituent present on a nitrogen atom is
a nitrogen
protecting group (also referred to as an amino protecting group). Nitrogen
protecting groups
include, but are not limited to, -OH, -OR', -N(R)2, -C(=0)Raa, -C(=0)N(Rcc)2, -
CO2Raa, -
SO2Raa, -C(=NRcc)Raa, -C(=NRcc)0Raa, -C(=NRcc)N(Rcc)2, -SO2N(Rcc)2, -SO2Rcc, -
S020Rcc, -
SORaa, -C(S)N(R)2, -C(0)SR, -C(=S)SRcc, C1_10 alkyl (e.g., aralkyl,
heteroaralkyl), C2_10
alkenyl, C2_10 alkynyl, C3_10 carbocyclyl, 3-14 membered heterocyclyl, C6_14
aryl, and 5-14
membered heteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,
heterocyclyl,
aralkyl, aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4,
or 5 Rdd groups, and
R', Rcc and R' Kd d
wherein Raa, R a are as defined herein. Nitrogen protecting groups are
well known in
the art and include those described in detail in Protecting Groups in Organic
Synthesis, T. W.
Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated
herein by
reference.
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[00172] For example, nitrogen protecting groups such as amide groups (e.g.,
¨C(=0)Raa)
include, but are not limited to, formamide, acetamide, chloroacetamide,
trichloroacetamide,
trifluoroacetamide, phenylacetamide, 3¨phenylpropanamide, picolinamide, 3¨
pyridylcarboxamide, N¨benzoylphenylalanyl derivative, benzamide,
p¨phenylbenzamide, o¨
nitophenylacetamide, o¨nitrophenoxyacetamide, acetoacetamide, (N'¨
dithiobenzyloxyacylamino)acetamide, 3¨(p¨hydroxyphenyl)propanamide, 3¨(o¨
nitrophenyl)propanamide, 2¨methyl-2¨(o¨nitrophenoxy)propanamide, 2¨methy1-
2¨(o¨
phenylazophenoxy)propanamide, 4¨chlorobutanamide, 3¨methyl-3¨nitrobutanamide,
o¨
nitrocinnamide, N¨acetylmethionine derivative, o¨nitrobenzamide and o¨
(benzoyloxymethyl)benzamide.
[00173] Nitrogen protecting groups such as carbamate groups (e.g., ¨C(=0)0Raa)
include, but
are not limited to, methyl carbamate, ethyl carbamante, 9¨fluorenylmethyl
carbamate (Fmoc), 9¨
(2¨sulfo)fluorenylmethyl carbamate, 9¨(2,7¨dibromo)fluoroenylmethyl carbamate,
2,7¨di¨t¨
butyl¨[9¨(1 0,1 0¨di oxo-1 0,1 0,1 0,1 0¨tetrahydrothi oxanthyl)] methyl
carbamate (DBD¨Tmoc), 4¨
methoxyphenacyl carbamate (Phenoc), 2,2,2¨trichloroethyl carbamate (Troc), 2¨
trimethylsilylethyl carbamate (Teoc), 2¨phenylethyl carbamate (hZ),
1¨(1¨adamanty1)-1¨
methylethyl carbamate (Adpoc), 1,1¨dimethy1-2¨haloethyl carbamate,
1,1¨dimethy1-2,2¨
dibromoethyl carbamate (DB¨t¨BOC), 1,1¨dimethy1-2,2,2¨trichloroethyl carbamate
(TCBOC),
1¨methy1-1¨(4¨biphenylypethyl carbamate (Bpoc), 1¨(3,5¨di¨t¨butylpheny1)-
1¨methylethyl
carbamate (t¨Bumeoc), 2¨(2'¨ and 4'¨pyridyl)ethyl carbamate (Pyoc), 2¨(NN¨
dicyclohexylcarboxamido)ethyl carbamate, t¨butyl carbamate (BOC), 1¨adamantyl
carbamate
(Adoc), vinyl carbamate (Voc), ally' carbamate (Alloc), 1¨isopropylally1
carbamate (Ipaoc),
cinnamyl carbamate (Coc), 4¨nitrocinnamyl carbamate (Noc), 8¨quinoly1
carbamate, N¨
hydroxypiperidinyl carbamate, alkyldithio carbamate, benzyl carbamate (Cbz),
p¨methoxybenzyl
carbamate (Moz), p¨nitobenzyl carbamate, p¨bromobenzyl carbamate,
p¨chlorobenzyl
carbamate, 2,4¨dichlorobenzyl carbamate, 4¨methylsulfinylbenzyl carbamate
(Msz), 9¨
anthrylmethyl carbamate, diphenylmethyl carbamate, 2¨methylthioethyl
carbamate, 2¨
methylsulfonylethyl carbamate, 2¨(p¨toluenesulfonypethyl carbamate,
[2¨(1,3¨dithianyOlmethyl
carbamate (Dmoc), 4¨methylthiophenyl carbamate (Mtpc), 2,4¨dimethylthiophenyl
carbamate
(Bmpc), 2¨phosphonioethyl carbamate (Peoc), 2¨triphenylphosphonioisopropyl
carbamate
(Ppoc), 1,1¨dimethy1-2¨cyanoethyl carbamate, m¨chloro¨p¨acyloxybenzyl
carbamate,p¨
(dihydroxyboryl)benzyl carbamate, 5¨benzisoxazolylmethyl carbamate,
2¨(trifluoromethyl)-6¨
chromonylmethyl carbamate (Tcroc), m¨nitrophenyl carbamate,
3,5¨dimethoxybenzyl
carbamate, o¨nitrobenzyl carbamate, 3,4¨dimethoxy-6¨nitrobenzyl carbamate,
phenyl(o¨
nitrophenyl)methyl carbamate, t¨amyl carbamate, S¨benzyl thiocarbamate,
p¨cyanobenzyl
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carbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentyl carbamate,
cyclopropylmethyl carbamate, p¨decyloxybenzyl carbamate,
2,2¨dimethoxyacylvinyl carbamate,
o¨(N,N¨dimethylcarboxamido)benzyl carbamate, 1,1¨dimethy1-34N,N¨
dimethylcarboxamido)propyl carbamate, 1,1¨dimethylpropynyl carbamate,
di(2¨pyridyl)methyl
carbamate, 2¨furanylmethyl carbamate, 2¨iodoethyl carbamate, isoborynl
carbamate, isobutyl
carbamate, isonicotinyl carbamate, p¨(p' ¨methoxyphenylazo)benzyl carbamate,
I¨
methylcyclobutyl carbamate, 1¨methylcyclohexyl carbamate,
1¨methyl¨l¨cyclopropylmethyl
carbamate, 1¨methyl-143,5¨dimethoxyphenyl)ethyl carbamate, 1¨methy1-14p¨
phenylazophenypethyl carbamate, 1¨methyl¨l¨phenylethyl carbamate, 1¨methy1-
144¨
pyridypethyl carbamate, phenyl carbamate,p¨(phenylazo)benzyl carbamate,
2,4,6¨tri¨t¨
butylphenyl carbamate, 4¨(trimethylammonium)benzyl carbamate, and
2,4,6¨trimethylbenzyl
carbamate.
[00174] Nitrogen protecting groups such as sulfonamide groups (e.g.,
¨S(=0)21e) include, but
are not limited to, p¨toluenesulfonamide (Ts), benzenesulfonamide,
2,3,6,¨trimethy1-4¨
methoxybenzenesulfonamide (Mtr), 2,4,6¨trimethoxybenzenesulfonamide (Mtb),
2,6¨dimethy1-
4¨methoxybenzenesulfonamide (Pme), 2,3,5,6¨tetramethy1-
4¨methoxybenzenesulfonamide
(Mte), 4¨methoxybenzenesulfonamide (Mbs), 2,4,6¨trimethylbenzenesulfonamide
(Mts), 2,6¨
dimethoxy-4¨methylbenzenesulfonamide (iMds), 2,2,5,7,8¨pentamethylchroman-6¨
sulfonamide (Pmc), methanesulfonamide (Ms), 0¨trimethylsilylethanesulfonamide
(SES), 9¨
anthracenesulfonamide, 4¨(4',8'¨dimethoxynaphthylmethyl)benzenesulfonamide
(DNMBS),
benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.
[00175] Other nitrogen protecting groups include, but are not limited to,
phenothiazinyl¨(10)¨
acyl derivative, N ' ¨p¨toluenesulfonylaminoacyl derivative, N '
¨phenylaminothioacyl derivative,
N¨benzoylphenylalanyl derivative, N¨acetylmethionine derivative, 4,5¨dipheny1-
3¨oxazolin-2¨
one, N¨phthalimide, N¨dithiasuccinimide (Dts), N-2,3¨diphenylmaleimide, N-2,5¨
dimethylpyrrole, N-1,1,4,4¨tetramethyldisilylazacyclopentane adduct (STABASE),
5¨
substituted 1,3¨dimethy1-1,3,5¨triazacyclohexan-2¨one, 5¨substituted
1,3¨dibenzy1-1,3,5¨
triazacyclohexan-2¨one, I¨substituted 3,5¨dinitro-4¨pyridone, N¨methylamine,
N¨allylamine,
N[2¨(trimethylsilypethoxylmethylamine (SEM), N-3¨acetoxypropylamine,
N¨(1¨isopropy1-4¨
nitro-2¨oxo-3¨pyroolin-3¨y0amine, quaternary ammonium salts, N¨benzylamine,
N¨di(4¨
methoxyphenyl)methylamine, N-5¨dibenzosuberylamine, N¨triphenylmethylamine
(Tr), N¨[(4¨
methoxyphenyl)diphenylmethyllamine (MMTr), N-9¨phenylfluorenylamine (PhF), N-
2,7¨
dichloro-9¨fluorenylmethyleneamine, N¨ferrocenylmethylamino (Fcm), N-
2¨picolylamino N'¨
oxide, N-1,1¨dimethylthiomethyleneamine, N¨benzylideneamine, N¨p¨
methoxybenzylideneamine, N¨diphenylmethyleneamine, N¨[(2-
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pyridyl)mesityllmethyleneamine, N-(N',N'-dimethylaminomethylene)amine, 1V,N'-
isopropylidenediamine, N-p-nitrobenzylideneamine, N-salicylideneamine, N-5-
chlorosalicylideneamine, N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine, N-
cy clohexylideneamine, N-(5,5-dimethy1-3-oxo-1-cyclohexenyDamine, N-borane
derivative,
N-diphenylborinic acid derivative, N-Iphenyl(pentaacylchromium- or
tungsten)acyllamine, N-
copper chelate, N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,
diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),
diphenylthiophosphinamide
(Ppt), dialkyl phosphoramidates, dibenzyl phosphoramidate, diphenyl
phosphoramidate,
benzenesulfenamide, o-nitrobenzenesulfenamide (Nps), 2,4-
dinitrobenzenesulfenamide,
pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,
triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).
[00176] In certain embodiments, the substituent present on an oxygen atom is
an oxygen
protecting group (also referred to as a hydroxyl protecting group). Oxygen
protecting groups
include, but are not limited to, -R", -N(Rbb)2, -C(=0)SR", -C(=0)R", -CO2Raa, -
C(=0)N(Rbb)2, -C(=NRbb)Raa, -C(=NRbb)0Raa, -C(=NRbb)N(Rbb)2, -S(=0)Raa, -
SO2Raa, -
Si(Raa)3, -P(R)2, -P(R)3, -P(=0)2Raa, -P(=0)(Raa)2, -P(=0)(ORcc)2, -1)
(=0)2N(Rbb)2, and -
P(=0)(NRbb)2, wherein R Rbb,
aa, and Rcc
are as defined herein. Oxygen protecting groups are
well known in the art and include those described in detail in Protecting
Groups in Organic
Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley & Sons,
1999, incorporated
herein by reference.
[00177] Exemplary oxygen protecting groups include, but are not limited to,
methyl,
methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,
(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM), p-
methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),
guaiacolmethyl
(GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM), siloxymethyl, 2-
methoxyethoxymethyl
(MEM), 2,2,2-trichloroethoxymethyl, bis(2-chloroethoxy)methyl, 2-
(trimethylsilyl)ethoxymethyl (SEMOR), tetrahydropyranyl (THP), 3-
bromotetrahydropyranyl,
tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl (MTHP),
4-
methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranyl S,S-dioxide, 1-
1(2-chloro-4-
methyl)pheny11-4-methoxypiperidin-4-y1 (CTMP), 1,4-dioxan-2-yl,
tetrahydrofuranyl,
tetrahydrothiofuranyl, 2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethy1-4,7-
methanobenzofuran-2-
yl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-l-methoxyethyl, 1-methyl-
l-
benzyloxyethyl, 1-methyl-l-benzyloxy-2-fluoroethyl, 2,2,2-trichloroethyl, 2-
trimethylsilylethyl, 2-(phenylselenyl)ethyl, t-butyl, allyl, p-chlorophenyl, p-
methoxyphenyl,
2,4-dinitrophenyl, benzyl (Bn),p-methoxybenzyl, 3,4-dimethoxybenzyl, o-
nitrobenzyl, p-
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nitrobenzyl, p¨halobenzyl, 2,6¨dichlorobenzyl, p¨cyanobenzyl, p¨phenylbenzyl,
2¨picolyl, 4¨
picolyl, 3¨methyl-2¨picoly1N¨oxido, diphenylmethyl, p,p '¨dinitrobenzhydryl,
5¨
dibenzosuberyl, triphenylmethyl, a¨naphthyldiphenylmethyl,
p¨methoxyphenyldiphenylmethyl,
di(p¨methoxyphenyl)phenylmethyl, tri(p¨methoxyphenyl)methyl, 4¨(4'¨
bromophenacyloxyphenyl)diphenylmethyl,
4,41,4"¨tris(4,5¨dichlorophthalimidophenyl)methyl,
4,41,4"¨tris(levulinoyloxyphenyl)methyl, 4,4',4"¨tris(benzoyloxyphenyl)methyl,
3¨(imidazol-1¨
yObis(41,4"¨dimethoxyphenyOmethyl, 1,1¨bis(4¨methoxypheny1)-1'¨pyrenylmethyl,
9¨anthryl,
9¨(9¨phenyl)xanthenyl, 949¨pheny1-10¨oxo)anthryl, 1,3¨benzodisulfuran-2¨yl,
benzisothiazolyl S,S¨dioxido, trimethylsilyl (TMS), triethylsilyl (TES),
triisopropylsilyl (TIPS),
dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),
dimethylthexylsilyl, t¨
butyldimethylsily1 (TBDMS), t¨butyldiphenylsilyl (TBDPS), tribenzylsilyl,
tri¨p¨xylylsilyl,
triphenylsilyl, diphenylmethylsilyl (DPMS), t¨butylmethoxyphenylsilyl (TBMPS),
formate,
benzoylformate, acetate, chloroacetate, dichloroacetate, trichloroacetate,
trifluoroacetate,
methoxyacetate, triphenylmethoxyacetate, phenoxyacetate,
p¨chlorophenoxyacetate, 3¨
phenylpropionate, 4¨oxopentanoate (levulinate), 4,44ethylenedithio)pentanoate
(levulinoyldithioacetal), pivaloate, adamantoate, crotonate,
4¨methoxycrotonate, benzoate, p¨
phenylbenzoate, 2,4,6¨trimethylbenzoate (mesitoate), alkyl methyl carbonate,
9¨fluorenylmethyl
carbonate (Fmoc), alkyl ethyl carbonate, alkyl 2,2,2¨trichloroethyl carbonate
(Troc), 2¨
(trimethylsilyl)ethyl carbonate (TMSEC), 2¨(phenylsulfonyl) ethyl carbonate
(Psec), 2¨
(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutyl carbonate, alkyl
vinyl carbonate
alkyl ally' carbonate, alkyl p¨nitrophenyl carbonate, alkyl benzyl carbonate,
alkyl p¨
methoxybenzyl carbonate, alkyl 3,4¨dimethoxybenzyl carbonate, alkyl
o¨nitrobenzyl carbonate,
alkyl p¨nitrobenzyl carbonate, alkyl S¨benzyl thiocarbonate, 4¨ethoxy-
1¨napththyl carbonate,
methyl dithiocarbonate, 2¨iodobenzoate, 4¨azidobutyrate, 4¨nitro-
4¨methylpentanoate, o¨
(dibromomethyl)benzoate, 2¨formylbenzenesulfonate, 2¨(methylthiomethoxy)ethyl,
4¨
(methylthiomethoxy)butyrate, 2¨(methylthiomethoxymethyl)benzoate, 2,6¨dichloro-
4¨
methylphenoxyacetate, 2,6¨dichloro-4¨(1,1,3,3¨tetramethylbutyl)phenoxyacetate,
2,4¨bis(1,1¨
dimethylpropyl)phenoxyacetate, chlorodiphenylacetate, isobutyrate,
monosuccinoate, (E)-2¨
methy1-2¨butenoate, o¨(methoxyacyl)benzoate, a¨naphthoate, nitrate, alkyl N
,1V ,N ',N '¨
tetramethylphosphorodiamidate, alkyl N¨phenylcarbamate, borate,
dimethylphosphinothioyl,
alkyl 2,4¨dinitrophenylsulfenate, sulfate, methanesulfonate (mesylate),
benzylsulfonate, and
tosylate (Ts).
[00178] In certain embodiments, the substituent present on an sulfur atom is
an sulfur
protecting group (also referred to as a thiol protecting group). Sulfur
protecting groups include,
but are not limited to, ¨R', N(Rb) b, 2,
C(=0)SRaa, ¨C(=0)Raa, ¨CO2Raa, ¨C(=0)N(Rbb)2, ¨
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C(=NRbb)Raa, ¨C(=NRbb)0Raa, ¨C(=NRbb)N(Rbb)2, ¨S(=0)Raa, ¨SO2Raa,
¨Si(Raa)3,¨P(Rcc)2, ¨
P(R)3, ¨P(=0)2Raa, ¨P(=0)(Raa)2, ¨P(=0)(ORcc)2, ¨P(=0)2N(Rbb)2, and
¨P(=0)(NRbb)2, wherein
Raa, K¨bb,
and Rcc are as defined herein. Sulfur protecting groups are well known in the
art and
include those described in detail in Protecting Groups in Organic Synthesis,
T. W. Greene and P.
G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein by
reference.
[00179] "Compounds of the present invention", and equivalent expressions, are
meant to
embrace the compounds as hereinbefore described, in particular compounds
according to any of
the Formula herein recited and/or described, which expression includes the
prodrugs, the
pharmaceutically acceptable salts, and the solvates, e.g., hydrates, where the
context so permits.
Similarly, reference to intermediates, whether or not they themselves are
claimed, is meant to
embrace their salts, and solvates, where the context so permits.
[00180] These and other exemplary substituents are described in more detail in
the Detailed
Description, Examples, and claims. The invention is not intended to be limited
in any manner by
the above exemplary listing of substituents.
Other definitions
[00181] "Pharmaceutically acceptable" means approved or approvable by a
regulatory agency
of the Federal or a state government or the corresponding agency in countries
other than the
United States, or that is listed in the U.S. Pharmacopoeia or other generally
recognized
pharmacopoeia for use in animals, and more particularly, in humans.
[00182] "Pharmaceutically acceptable salt" refers to a salt of a compound of
the invention that
is pharmaceutically acceptable and that possesses the desired pharmacological
activity of the
parent compound. In particular, such salts are non-toxic may be inorganic or
organic acid
addition salts and base addition salts. Specifically, such salts include: (1)
acid addition salts,
formed with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric
acid, phosphoric acid, and the like; or formed with organic acids such as
acetic acid, propionic
acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid,
lactic acid, malonic
acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid,
citric acid, benzoic acid,
3-(4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid,
methanesulfonic acid,
ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid,
benzenesulfonic
acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-
toluenesulfonic acid,
camphorsulfonic acid, 4-methylbicyclo[2.2.21-oct-2-ene-1-carboxylic acid,
glucoheptonic acid,
3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid,
lauryl sulfuric acid,
gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic
acid, muconic acid,
and the like; or (2) salts formed when an acidic proton present in the parent
compound either is
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replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or
an aluminum ion; or
coordinates with an organic base such as ethanolamine, diethanolamine,
triethanolamine, N-
methylglucamine and the like. Salts further include, by way of example only,
sodium,
potassium, calcium, magnesium, ammonium, tetraalkylammonium, and the like; and
when the
compound contains a basic functionality, salts of non toxic organic or
inorganic acids, such as
hydrochloride, hydrobromide, tartrate, mesylate, acetate, maleate, oxalate and
the like. The term
"pharmaceutically acceptable cation" refers to an acceptable cationic counter-
ion of an acidic
functional group. Such cations are exemplified by sodium, potassium, calcium,
magnesium,
ammonium, tetraalkylammonium cations, and the like (see, e.g., Berge, etal., I
Pharm. Sci.
66(1): 1-79 (Jan."77) .
[00183] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant,
excipient or carrier
with which a compound of the invention is administered.
[00184] "Pharmaceutically acceptable metabolically cleavable group" refers to
a group which
is cleaved in vivo to yield the parent molecule of the structural Formula
indicated herein.
Examples of metabolically cleavable groups include -COR, -COOR,-CONRR and
¨CH2OR
radicals, where R is selected independently at each occurrence from alkyl,
trialkylsilyl,
carbocyclic aryl or carbocyclic aryl substituted with one or more of alkyl,
halogen, hydroxy or
alkoxy. Specific examples of representative metabolically cleavable groups
include acetyl,
methoxycarbonyl, benzoyl, methoxymethyl and trimethylsilyl groups.
[00185] "Prodrugs" refers to compounds, including derivatives of the compounds
of the
invention,which have cleavable groups and become by solvolysis or under
physiological
conditions the compounds of the invention that are pharmaceutically active in
vivo. Such
examples include, but are not limited to, choline ester derivatives and the
like, N-
alkylmorpholine esters and the like. Other derivatives of the compounds of
this invention have
activity in both their acid and acid derivative forms, but in the acid
sensitive form often offers
advantages of solubility, tissue compatibility, or delayed release in the
mammalian organism
(see, Bundgard, H., Design of Prodrugs, pp. 7-9, 21-24, Elsevier, Amsterdam
1985). Prodrugs
include acid derivatives well know to practitioners of the art, such as, for
example, esters
prepared by reaction of the parent acid with a suitable alcohol, or amides
prepared by reaction of
the parent acid compound with a substituted or unsubstituted amine, or acid
anhydrides, or mixed
anhydrides. Simple aliphatic or aromatic esters, amides and anhydrides derived
from acidic
groups pendant on the compounds of this invention are particular prodrugs. In
some cases it is
desirable to prepare double ester type prodrugs such as (acyloxy)alkyl esters
or
((alkoxycarbonyl)oxy)alkylesters. Particularly the Ci to C8 alkyl, C2-C8
alkenyl, C2-C8 alkynyl,
aryl, C7-C12 substituted aryl, and C7-C12 arylalkyl esters of the compounds of
the invention.
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[00186] "Solvate" refers to forms of the compound that are associated with a
solvent or water
(also referred to as "hydrate"), usually by a solvolysis reaction. This
physical association
includes hydrogen bonding. Conventional solvents include water, ethanol,
acetic acid and the
like. The compounds of the invention may be prepared e.g. in crystalline form
and may be
solvated or hydrated. Suitable solvates include pharmaceutically acceptable
solvates, such as
hydrates, and further include both stoichiometric solvates and non-
stoichiometric solvates. In
certain instances the solvate will be capable of isolation, for example when
one or more solvent
molecules are incorporated in the crystal lattice of the crystalline solid.
"Solvate" encompasses
both solution-phase and isolable solvates. Representative solvates include
hydrates, ethanolates
and methanolates.
[00187] A "subject" to which administration is contemplated includes, but is
not limited to,
humans (i.e., a male or female of any age group, e.g., a pediatric subject
(e.g, infant, child,
adolescent) or adult subject (e.g., young adult, middle¨aged adult or senior
adult)) and/or a non-
human animal, e.g., a mammal such as primates (e.g., cynomolgus monkeys,
rhesus monkeys),
cattle, pigs, horses, sheep, goats, rodents, cats, and/or dogs. In certain
embodiments, the subject
is a human. In certain embodiments, the subject is a non-human animal. The
terms "human",
"patient" and "subject" are used interchangeably herein.
[00188] "Therapeutically effective amount" means the amount of a compound
that, when
administered to a subject for treating a disease, is sufficient to effect such
treatment for the
disease. The "therapeutically effective amount" can vary depending on the
compound, the
disease and its severity, and the age, weight, etc., of the subject to be
treated.
[00189] "Preventing" or "prevention" refers to a reduction in risk of
acquiring or developing a
disease or disorder (i.e., causing at least one of the clinical symptoms of
the disease not to
develop in a subject not yet exposed to a disease-causing agent, or
predisposed to the disease in
advance of disease onset.
[00190] The term "prophylaxis" is related to "prevention", and refers to a
measure or procedure
the purpose of which is to prevent, rather than to treat or cure a disease.
Non-limiting examples
of prophylactic measures may include the administration of vaccines; the
administration of low
molecular weight heparin to hospital patients at risk for thrombosis due, for
example, to
immobilization; and the administration of an anti-malarial agent such as
chloroquine, in advance
of a visit to a geographical region where malaria is endemic or the risk of
contracting malaria is
high.
[00191] "Treating" or "treatment" of any disease or disorder refers, in
certain embodiments, to
ameliorating the disease or disorder (i.e., arresting the disease or reducing
the manifestation,
extent or severity of at least one of the clinical symptoms thereof). In
another embodiment
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"treating" or "treatment" refers to ameliorating at least one physical
parameter, which may not be
discernible by the subject. In yet another embodiment, "treating" or
"treatment" refers to
modulating the disease or disorder, either physically, (e.g., stabilization of
a discernible
symptom), physiologically, (e.g., stabilization of a physical parameter), or
both. In a further
embodiment, "treating" or "treatment" relates to slowing the progression of
the disease.
[00192] As used herein, the term "isotopic variant" refers to a compound that
contains
unnatural proportions of isotopes at one or more of the atoms that constitute
such compound.
For example, an "isotopic variant" of a compound can contain one or more non-
radioactive
isotopes, such as for example, deuterium (2H or D), carbon-13 (13C), nitrogen-
15 (15N), or the
like. It will be understood that, in a compound where such isotopic
substitution is made, the
following atoms, where present, may vary, so that for example, any hydrogen
may be 2H/D, any
carbon may be 13C, or any nitrogen may be 15N, and that the presence and
placement of such
atoms may be determined within the skill of the art. Likewise, the invention
may include the
preparation of isotopic variants with radioisotopes, in the instance for
example, where the
resulting compounds may be used for drug and/or substrate tissue distribution
studies. The
radioactive isotopes tritium, i.e., 3H, and carbon-14, i.e., 14C, are
particularly useful for this
purpose in view of their ease of incorporation and ready means of detection.
Further, compounds
may be prepared that are substituted with positron emitting isotopes, such as
11C, 18F, 150 and
13N, and would be useful in Positron Emission Topography (PET) studies for
examining substrate
receptor occupancy. All isotopic variants of the compounds provided herein,
radioactive or not,
are intended to be encompassed within the scope of the invention.
[00193] It is also to be understood that compounds that have the same
molecular formula but
differ in the nature or sequence of bonding of their atoms or the arrangement
of their atoms in
space are termed "isomers". Isomers that differ in the arrangement of their
atoms in space are
termed "stereoisomers".
[00194] Stereoisomers that are not mirror images of one another are termed
"diastereomers"
and those that are non-superimposable mirror images of each other are termed
"enantiomers".
When a compound has an asymmetric center, for example, when it is bonded to
four different
groups, a pair of enantiomers is possible. An enantiomer can be characterized
by the absolute
configuration of its asymmetric center and is described by the R- and S-
sequencing rules of Cahn
and Prelog, or by the manner in which the molecule rotates the plane of
polarized light and
designated as dextrorotatory or levorotatory (i.e., as (+) or (-)-isomers
respectively). A chiral
compound can exist as either individual enantiomer or as a mixture thereof A
mixture
containing equal proportions of the enantiomers is called a "racemic mixture".
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[00195] "Tautomers" refer to compounds that are interchangeable forms of a
particular
compound structure, and that vary in the displacement of hydrogen atoms and
electrons. Thus,
two structures may be in equilibrium through the movement of n electrons and
an atom (usually
H). For example, enols and ketones are tautomers because they are rapidly
interconverted by
treatment with either acid or base. Another example of tautomerism is the aci-
and nitro- forms
of phenylnitromethane, which are likewise formed by treatment with acid or
base.
Tautomeric forms may be relevant to the attainment of the optimal chemical
reactivity and
biological activity of a compound of interest.
[00196] As used herein a pure enantiomeric compound is substantially free
from other
enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess).
In other words, an
"S" form of the compound is substantially free from the "R" form of the
compound and is, thus,
in enantiomeric excess of the "R" form. The term "enantiomerically pure" or
"pure enantiomer"
denotes that the compound comprises more than 75% by weight, more than 80% by
weight, more
than 85% by weight, more than 90% by weight, more than 91% by weight, more
than 92% by
weight, more than 93% by weight, more than 94% by weight, more than 95% by
weight, more
than 96% by weight, more than 97% by weight, more than 98% by weight, more
than 98.5% by
weight, more than 99% by weight, more than 99.2% by weight, more than 99.5% by
weight,
more than 99.6% by weight, more than 99.7% by weight, more than 99.8% by
weight or more
than 99.9% by weight, of the enantiomer. In certain embodiments, the weights
are based upon
total weight of all enantiomers or stereoisomers of the compound.
[00197] As used herein and unless otherwise indicated, the term
"enantiomerically pure R-
compound" refers to at least about 80% by weight R-compound and at most about
20% by
weight S-compound, at least about 90% by weight R-compound and at most about
10% by
weight S-compound, at least about 95% by weight R-compound and at most about
5% by weight
S-compound, at least about 99% by weight R-compound and at most about 1% by
weight 5-
compound, at least about 99.9% by weight R-compound or at most about 0.1% by
weight 5-
compound. In certain embodiments, the weights are based upon total weight of
compound.
[00198] As used herein and unless otherwise indicated, the term
"enantiomerically pure 5-
compound" or "S-compound" refers to at least about 80% by weight S-compound
and at most
about 20% by weight R-compound, at least about 90% by weight S-compound and at
most about
10% by weight R-compound, at least about 95% by weight S-compound and at most
about 5%
by weight R-compound, at least about 99% by weight S-compound and at most
about 1% by
weight R-compound or at least about 99.9% by weight S-compound and at most
about 0.1% by
weight R-compound. In certain embodiments, the weights are based upon total
weight of
compound.
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[00199] In the compositions provided herein, an enantiomerically pure compound
or a
pharmaceutically acceptable salt, solvate, hydrate or prodrug thereof can be
present with other
active or inactive ingredients. For example, a pharmaceutical composition
comprising
enantiomerically pure R-compound can comprise, for example, about 90%
excipient and about
10% enantiomerically pure R-compound. In certain embodiments, the
enantiomerically pure R-
compound in such compositions can, for example, comprise, at least about 95%
by weight R-
compound and at most about 5% by weight S-compound, by total weight of the
compound. For
example, a pharmaceutical composition comprising enantiomerically pure S-
compound can
comprise, for example, about 90% excipient and about 10% enantiomerically pure
S-compound.
In certain embodiments, the enantiomerically pure S-compound in such
compositions can, for
example, comprise, at least about 95% by weight S-compound and at most about
5% by weight
R-compound, by total weight of the compound. In certain embodiments, the
active ingredient
can be formulated with little or no excipient or carrier.
[00200] The compounds of this invention may possess one or more asymmetric
centers; such
compounds can therefore be produced as individual (R)- or (S)- stereoisomers
or as mixtures
thereof
[00201] Unless indicated otherwise, the description or naming of a particular
compound in the
specification and claims is intended to include both individual enantiomers
and mixtures, racemic
or otherwise, thereof The methods for the determination of stereochemistry and
the separation
of stereoisomers are well-known in the art.
[00202] One having ordinary skill in the art of organic synthesis will
recognize that the
maximum number of heteroatoms in a stable, chemically feasible heterocyclic
ring, whether it is
aromatic or non aromatic, is determined by the size of the ring, the degree of
unsaturation and the
valence of the heteroatoms. In general, a heterocyclic ring may have one to
four heteroatoms so
long as the heteroaromatic ring is chemically feasible and stable.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[00203] In certain aspects, provided herein are pharmaceutical compositions
comprising of a
bolaamphiphile complex.
[00204] In further aspects, provided herein are novel nano-sized vesicles
comprising of
bolaamphiphilic compounds.
[00205] In further aspects, provided herein are novel nano-sized vesicles
comprising of
bolaamphiphilic compounds which are capable of encapsulating NTF, GENF or NGF.
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[00206] In still another aspect the vesicles formed from the
bolaamphiphiles to
encapsulating NTF, GDNF or NGF, contain additives that help to stabilize the
vesicles, by
stabilizing the vesicle's membranes, such as but not limited to cholesterol
derivatives such as
cholesteryl hemisuccinate and cholesterol itself and combinations such as
cholesteryl
hetnisuccinate and cholesterol.
[00207] In still another embodiments the vesicles in addition to these
components have
another addiitves which decorates the outer vesicle memrbanes with groups or
pendants that
enhance penetration though biological barriers such as the BBB, or groups for
targeting to
specific sites such as dopaminergic neurons.
[00208] In a further embodiment the bolaamphiphile head groups that
comprise the
vesicles membranes can interact with the neuro active agents such as GDNF or
NDF to be
delivered in to the brain and brain sites ionic interactions to enhance the %
encapsulation via
complexation and well as passive encapsulation within the vesicles core.
Further the formuation
may contain other additives within the veicles membranes to futhjer enhance
the degree of
encapsulation of neuro active agents such as GDNF or NDF. It is understood by
one skilled in
the state of art that the pH in which the vesicle foi _______________ illation
and encapsulation of the neuro active
agent such as GDNF or NDF is such as to maximize the electrostatic or inonic
interactions
between the said agents and the sai bolaamphiphiles and or additives to
maximize the %
encapsulation.
[00209] In further aspects, provided herein are novel nano-sized bola vesicles
described above
that encapsulate GDNF or NGF and are capable of delivering the encapsulated
material into the
brain.
[00210] In further aspects, provided herein are novel nano-sized bola vesicles
that encapsulate
GDNF or NGF and are capable of delivering the encapsulated material to the
brain, specifically
to dopaminergic neurons.
[00211] In further aspects, provided herein are novel nano-sized bola vesicles
that encapsulate
GDNF or NGF and are capable of delivering the encapsulated material into brain
regions affected
in neurological disorders. In one particular embodiment, the neurological
disorder is Parkinson's
disease (PD) or Alzheimer's disease (AD).
[00212] In certain aspects, provided herein are novel bolaamphiphile complexes
comprising
bolaamphiphilic compounds and a compound active against PD. In one embodiment,
the
compound active against PD is GDNF.
[00213] In certain aspects, provided herein are novel bolaamphiphile complexes
comprising
bolaamphiphilic compounds and a compound active against AD. In one embodiment,
the
compound active against AD is NGF.
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[00214] In further aspects, provided herein are novel formulations of GDNF or
NGF with
bolaamphiphilic compounds or with bolaamhphile vesicles.
[00215] In another aspect, provided here are methods of delivering GDNF or NGF
agents into
animal or human brain. In one embodiment, the method comprises the step of
administering to
the animal or human a pharmaceutical composition comprising of a
bolaamphiphile complex;
and wherein the bolaamphiphile complex comprises a bolaamphiphilic compound
and GDNF. In
another embodiment, the complex comprises bolaamphiphilic compound and NGF.
[00216] In one embodiment, the bolaamphiphilic compound consists of two
hydrophilic
headgroups linked through a long hydrophobic chain. In another embodiment, the
hydrophilic
headgroup is an amino containing group. In a specific embodiment, the
hydrophilic headgroup is
a tertiary or quaternary amino containing group.
[00217] In one particular embodiment, the bolaamphiphilic compound is a
compound
according to formula I:
HG2 ¨L1 ¨HG1
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each HG' and HG2 is independently a hydrophilic head group; and
Ll is alkylene, alkenyl, heteroalkylene, or heteroalkenyl linker;
unsubstituted or
substituted with Cl-C20 alkyl, hydroxyl, or oxo.
[00218] In one embodiment, the pharmaceutically acceptable salt is a
quaternary ammonium
salt.
[00219] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, Ll is
heteroalkylene, or heteroalkenyl linker comprising C, N, and 0 atoms;
unsubstituted or
substituted with Cl-C20 alkyl, hydroxyl, or oxo.
[00220] In another embodiment, with respect to the bolaamphiphilic compound of
formula I, Ll
is
¨0-L2¨C(0)-0-(CH2)04-0-C(0)-L3-0-, or
¨0-L2¨C(0)-0-(CH2)05-0-C(0)-(CH2)06-,
and wherein each L2 and L3 is C4-C20 alkenyl linker; unsubstituted or
substituted with Cl-C8
alkyl or hydroxy;
and n4, n5, and n6 is independently an integer from 4-20.
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[00221] In one embodiment, each L2 and L3 is independently -C(R1)-C(OH)-CH2-
(CH=CH)-
(CH2)117-; Rl is C1-C8 alkyl, and n7 is independently an integer from 4-20.
[00222] In another embodiment, with respect to the bolaamphiphilic compound of
formula I, Ll
is -0-(CH2)111-0-C(0)-(CH2).2-C(0)-0-(CH2)113-0-.
[00223] In another embodiment, with respect to the bolaamphiphilic compound
of formula I,
Ll is
,Z1 /HMO Z2
________________________ (-)n9
0 0
Linker AA
0 0
J"
________________________ (¨)n9 Zi Z2 _______ (")n11
Linker BB
Rib 0 ______
0 0
R2a( )n8 ')n9 Zi Z2 ("-)n12 R2b
Linker CC
or
_________________ ORla
0
R2a
Nin8 ()n9 Z1`Z2 _________________________________________
Linker DD
wherein:
each Z1 and Z2 is independently -C(R3)2-, -N(R3)- or -0-;
each Rla, Rib, R3,
and R4 is independently H or Ci-C8 alkyl;
each R2a and R2b is independently H, Ci-C8 alkyl, OH, or alkoxy;
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each n8, n9, n11, and n12 is independently an integer from 1-20;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
and wherein each methylene carbon is unsubstituted or substituted with Ci-C4
alkyl; and each
nl, n2, and n3 is independently an integer from 4-20.
[00224] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, the
bolaamphiphilic compound is a compound according to formula II, III, IV, V, or
VI:
()n10 Z2
HG2 ___________________ (-)n9 --)nl 1 HG1
0 0
0 0
J"
HG2 ___________________ (-)n9 Z1 ()nl1 HG1
III
HG2-0R1 a R1 b
0 0
J" '')1110
R2a ( )118 =-=-)119 Z1 22 )nl 1( )12R2b
IV
HG2-0R1 a
0
R2a )n8 (--)ng Z1 ¨HG1
V ,or
HG2-0R1 a
0
R4
R2a ( )n8 (-')ng Z1
VI
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or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each HG' and HG2 is independently a hydrophilic head group;
each Z1 and Z2 is independently -C(R3)2-, -N(R3)- or ¨0-;
each Rla, Rib, tc-3,
and R4 is independently H or C,-C8 alkyl;
each R2a and R2b is independently H, C,-C8 alkyl, OH, alkoxy, or 0-HG' or 0-
HG2;
each n8, n9, n11, and n12 is independently an integer from 1-20;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[00225] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each n9 and n11 is independently an integer from 2-12. In
another embodiment, n9
and n11 is independently an integer from 4-8. In a particular embodiment, each
n9 and n11 is 7
or 11.
[00226] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each n8 and n12 is independently 1, 2, 3, or 4. In a particular
embodiment, each n8
and n12 is 1.
[00227] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each R2a and R2b is independently H, OH, or alkoxy. In another
embodiment, each
R2a and R2b is independently H, OH, or OMe. In another embodiment, each R2a
and R2b is
independently-0-HG1 or 0-HG2. In a particular embodiment, each R2a and R2b is
OH.
[00228] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each Ria and Rib is independently H, Me, Et, n-Pr, i-Pr, n-Bu, i-
Bu, sec-Bu, n-
pentyl, isopentyl, n-hexyl, n-heptyl, or n-octyl. In a particular embodiment,
each Ria and Rib is
independently n-pentyl.
[00229] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each dotted bond is a single bond. In another embodiment, each
dotted bond is a
double bond.
[00230] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, n10 is an integer from 2-16. In another embodiment, n10 is an
integer from 2-12. In
a particular embodiment, n10 is 2, 4, 6, 8, 10, 12, or 16.
[00231] In one embodiment, with respect to the bolaamphiphilic compound of
formula IV, R4
is H, Me, Et, n-Pr, i-Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl. In
another embodiment, R4 is
Me, or Et. In a particular embodiment, R4 is Me.
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[00232] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each Z1 and Z2 is independently C(R3)2-, or -N(R3)-. In another
embodiment, each
Z1 and Z2 is independently C(R3)2-, or -N(R3)-; and each R3 is independently
H, Me, Et, n-Pr,
Pr, n-Bu, i-Bu, sec-Bu, n-pentyl, or isopentyl. In a particular embodiment, R3
is H.
[00233] In one embodiment, with respect to the bolaamphiphilic compound of
formula II, III,
IV, V, or VI, each Z1 and Z2 is ¨0-.
[00234] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
or IV, each HG' and HG2 is independently selected from:
0 0 0 R8
(A)
/1111 (A)
/1111 (A)M1
µ( ')n13
0
(A) R8
/1111 )n14 0
(irml
( )n15
)X and
0 0
wherein:
X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted C,-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted C,-C20 alkyl; each R8
is independently
H, substituted or unsubstituted Cl-C20 alkyl, alkoxy, or carboxy;
ml is 0 or 1; and
each n13, n14, and n15 is independently an integer from 1-20.
[00235] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
or IV, HG' and HG2 are as defined above, and each ml is 0.
[00236] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
or IV, HG' and HG2 are as defined above, and each ml is 1.
[00237] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
or IV, HG' and HG2 are as defined above, and each n13 is 1 or 2.
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[00238] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
or IV, HG' and HG2 are as defined above, and each n14 and n15 is independently
1, 2, 3, 4, or 5.
In another embodiment, each n14 and n15 is independently 2 or 3.
[00239] In one particular embodiment, the bolaamphiphilic compound is a
compound
according to formula VIIa, VIIb, VIIc, or VIId:
0
Vila
X,rip 0 0
0 _ 0
N(=--)7 "N 0
Vllb
0 0
0 Nn10 0
0
N(===-)7 "N 0
VIIc
or
X X
0 0
VI Id
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted C,-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted Cl-C20 alkyl;;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[00240] In another particular embodiment, the bolaamphiphilic compound is a
compound
according to formula VIIIa, VIIIb, VIIIc, or VIIId:
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0 0
X
0
Villa
0
0 (^)3
)1,
0
VIllb
x..---...õõ0y(--)31(0õ 0 0
0 0 0
'-(1)
VIIIc
or
X
0 0
VII Id
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted C1-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted C1-C20 alkyl;;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[00241] In another particular embodiment, the bolaamphiphilic compound is a
compound
according to formula IXa, IXb, or IXc:
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0 0
0 )X
IXa
0
0 0
0 (inio
`N)..L(.-)310X
IXb
X' )r(N)31((:) 0 0 0
0 (inio
0
IXc
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted Ci-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted C1-C20 alkyl;;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[00242] In another particular embodiment, the bolaamphiphilic compound is a
compound
according to formula Xa, Xb, or Xc:
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x(C) 0 0
0 _ )nici X
(==-)7)0"
Xa
X=rC) 0 0 0
0 )"L
H0---N(.--)7 0" OX
Xb
X0y(31*((:) 0 0
0
0 (-)7)*LO" )ni'D'OA(..)3 LC)X
XC
or a pharmaceutically acceptable salt, solvate, hydrate, prodrug,
stereoisomer, tautomer, isotopic
variant, or N¨oxide thereof, or a combination thereof;
wherein:
each X is ¨NR5aR5b, or ¨N+R5aR5bR5c; each R5a, and R5b is independently H or
substituted or
unsubstituted Ci-C20 alkyl or R5a and R5b may join together to form an N
containing
substituted or unsubstituted heteroaryl, or substituted or unsubstituted
heterocyclyl;
each R5c is independently substituted or unsubstituted C1-C20 alkyl;;
n10 is an integer from 2-20; and
each dotted bond is independently a single or a double bond.
[00243] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each dotted bond is a single bond. In
another embodiment,
each dotted bond is a double bond.
[00244] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integer from 2-16.
[00245] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is an integer from 2-12.
[00246] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, n10 is 2, 4, 6, 8, 10, 12, or 16.
[00247] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R5a, R5b, and R5C is independently
substituted or
unsubstituted Ci-C20 alkyl.
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[00248] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, each R5a, R5b, and R5C is independently
unsubstituted C1-
C20 alkyl.
[00249] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R5a, R5b, and R5C is C1-C20 alkyl
substituted with ¨
OC(0)R6; and R6 is C1-C20 alkyl.
[00250] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R5a, R5b, and R5C are
independently C1-C20 alkyl
substituted with ¨0C(0)R6; and R6 is C1-C20 alkyl. In one embodiment, R6 is
Me, Et, n-Pr, i-Pr,
n-Bu, i-Bu, sec-Bu, n-pentyl, isopentyl, n-hexyl, n-heptyl, or n-octyl. In a
particular embodiment,
R6 is Me.
[00251] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, one of R5a, R5b, and R5C is C1-C20 alkyl
substituted with
amino, alkylamino or dialkylamino.
[00252] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, two of R5a, R5b, and R5C are
independently C1-C20 alkyl
substituted with amino, alkylamino or dialkylamino.
[00253] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R5a, and R5b together with the N they
are attached to form
substituted or unsubstituted heteroaryl.
[00254] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R5a, and R5b together with the N they
are attached to form
substituted or unsubstituted pyridyl.
[00255] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, R5a, and R5b together with the N they
are attached to form
substituted or unsubstituted monocyclic or bicyclic heterocyclyl.
[00256] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is substituted or unsubstituted
[00257] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is
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substituted with one or more groups selected from alkoxy, acetyl, and
substituted or
unsubstituted Ph.
[00258] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is
0
F
[00259] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is ¨NMe2 or ¨N+Me3.
[00260] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is ¨N(Me)-CH2CH2-0Ac or ¨N+(Me)2-
CH2CH2-0Ac.
[00261] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a chitosanyl group; and the
chitosanyl group is a
poly-(D)glucosaminyl group with MW of 3800 to 20,000 Daltons, and is attached
to the core via
N.
[00262] In one embodiment, the chitosanyl group is
0 H 0 H
H ¨ [ ¨04 ____________________ n
r 0
-L
p2
N H z N H
R 7 a
and wherein each p1 and p2 is independently an integer from 1-400; and each
R7a is H or acyl.
[00263] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a mannose group.
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[00264] In one embodiment, with respect to the bolaamphiphilic compound of
formula VIIa-
VIId, VIIIa-VIIId, IXa-IXc, or Xa-Xc, X is a maleimide group.
[00265] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is a
pharmaceutically acceptable salt.
[00266] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is in a
form of a quaternary salt.
[00267] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is in a
form of a quaternary salt with pharmaceutically acceptable alkyl halide or
alkyl tosylate.
[00268] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is any
one of the bolaambphilic compounds listed in Table 1.
[00269] In another specific aspect, provided herein are methods for
incorporating GDNF in the
bolavesicles. In one embodiment, the bolavesicle comporises one or more
bolaamphilic
compounds described herein.
[00270] In another specific aspect, provided herein are methods for brain-
targeted drug delivery
using the bolavesicles incorporated with GDNF.
[00271] In another specific aspect, provided herein are methods for delivering
GDNF to the
brain.
[00272] In another specific aspect, provided herein are nano-particles,
comprising one or more
bolaamphiphilic compounds and GDNF. In one embodiment, the bolaamphiphilic
compounds
and GDNF are encapsulated within the nano-particle.
[00273] In another specific aspect, provided herein are pharmaceutical
compositions,
comprising a nano-sized particle comprising one or more bolaamphiphilic
compounds and
GDNF; and a pharmaceutically acceptable carrier.
[00274] In another specific aspect, provided herein are methods for treatment
or diagnosis of
diseases or disorders selected from PD and related diseases using the nano-
particles,
pharmaceutical compositions or formulations of the present invention.
[00275] In another specific aspect, provided herein are methods for
incorporating NGF in the
bolavesicles. In one embodiment, the bolavesicle comporises one or more
bolaamphilic
compounds described herein.
[00276] In another specific aspect, provided herein are methods for brain-
targeted drug delivery
using the bolavesicles incorporated with NGF.
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[00277] In another specific aspect, provided herein are methods for delivering
NGF to the
brain.
[00278] In another specific aspect, provided herein are nano-particles,
comprising one or more
bolaamphiphilic compounds and NGF. In one embodiment, the bolaamphiphilic
compounds and
NGF are encapsulated within the nano-particle.
[00279] In another specific aspect, provided herein are pharmaceutical
compositions,
comprising a nano-sized particle comprising one or more bolaamphiphilic
compounds and NGF;
and a pharmaceutically acceptable carrier.
[00280] In another specific aspect, provided herein are methods for treatment
or diagnosis of
diseases or disorders selected from AD and related diseases using the nano-
particles,
pharmaceutical compositions or formulations of the present invention.
[00281] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is other
than Comound ID GLH-16, GLH-19, GLH-20, GLH-26, GLH-29, or GLH-41.
[00282] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is other
than Comound ID GLH-6, GLH-8, GLH-12, GLH-13, GLH-13a, or GLH-49 to GLH-54
(all can
be used as intermediates for bolaamphiphiles).
[00283] In another specific aspect, provided herein are composition of novel
bolaamphiphilic
compounds, wherein the bolaamphiphilic compound is selected from the
bolaambphilic
compounds listed in Table 1. In one embodiment, with respect to the
bolaamphiphilic compound,
the bolaamphiphilic compound is other than Comound ID GLH-16, GLH-19, GLH-20,
GLH-26,
GLH-29, or GLH-41. In another embodiment, with respect to the bolaamphiphilic
compound, the
compound is other than compound with ID GLH-3, GLH-4, GLH-5, or GLH-21.
[00284] In one particular embodiment, bolaamphiphilic compound is selected
from the
bolaambphilic compounds listed in Table 1, and the compound is compound with
ID GLH-7,
GLH-9, GLH-10, GLH-11, GLH-14, GLH-15, GLH-17, GLH-18, GLH-22, GLH-23, GLH-24,
GLH-25, GLH-27, GLH-28, GLH-30 to GLH-48, GLH-55, GLH-56, or GLH-57.
[00285] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is
Comound ID GLH-19, or GLH-20.
[00286] In one embodiment, with respect to the bolaamphiphilic compound of
formula I, II, III,
IV, V, VI, VIIa-VIIc, VIIIa-VIIIc, IXa-IXc and Xa-Xc, the bolaamphiphilic
compound is
Comound ID GLH-16, GLH-26, GLH-29, or GLH-41.
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[00287] The Derivatives and Precursors disclosed can be prepared as
illustrated in the
Schemes provided herein. The syntheses can involve initial construction of,
for example,
vernonia oil or direct functionalization of natural derivatives by organic
synthesis manipulations
such as, but not limiting to, epoxide ring opening. In those processes
involving oxiranyl ring
opening, the epoxy group is opened by the addition of reagents such as
carboxylic acids or
organic or inorganic nucleophiles. Such ring opening results in a mixture of
two products in
which the new group is introduced at either of the two carbon atoms of the
epoxide moiety. This
provides beta substituted alcohols in which the substitution position most
remote from the CO
group of the main aliphatic chain of the vernonia oil derivative is
arbitrarily assigned as position
1, while the neighboring substituted carbon position is designated position 2.
For simplicity
purposes only, the Derivatives and Precursors shown herein may indicate
structures with the
hydroxy group always at position 2 but the Derivatives and Precursors wherein
the hydroxy is at
position 1 are also encompassed by the invention. Thus, a radical of the
formula --CH(OH)--
CH(R)-- refers to the substitution of --OH at either the carbon closer to the
CO group, designated
position 2 or to the carbon at position 1. Moreover, with respect to the
preparation of
symmetrical bolaamphiphiles made via introducing the head groups through an
epoxy moiety
(e.g., as in vernolic acid) or a double bond (-C=C-) as in mono unsaturated
fatty acids (e.g., oleic
acid) a mixture of three different derivatives will be produced. In certain
embodiments, vesicles
are prepared using the mixture of unfractionated positional isomers. In one
aspect of this
embodiment, where one or more bolas are prepared from vernolic acid, and in
which a hydroxy
group as well as the head group introduced through an epoxy or a fatty acid
with the head group
introduced through a double bond (-C=C-), the bola used in vesicle preparation
can actually be a
mixture of three different positional isomers.
[00288] In other embodiments, the three different derivatives are isolated.
Accordingly,
the vesicles disclosed herein can be made from a mixture of the three isomers
of each derivative
or, in other embodiments, the individual isomers can be isolated and used for
preparation of
vesicles.
[00289] Symmetrical bolaamphiphiles can form relatively stable self
aggregate vesicle
structures by the use of additives such as cholesterol and cholesterol
derivatives (e.g., cholesterol
hemisuccinate, cholesterol ley' ether, anionic and cationic derivatives of
cholesterol and the
like), or other additives including single headed amphiphiles with one, two or
multiple aliphatic
chains such as phospholipids, zwitterionic, acidic, or cationic lipids.
Examples of zwitterionic
lipids are phosphatidylcholines, phosphatidylethanol amines and
sphingomyelins. Examples of
acidic amphiphilic lipids are phosphatidylglycerols, phosphatidylserines,
phosphatidylinositols,
and phosphatidic acids. Examples of cationic amphipathic lipids are diacyl
trimethylammonium
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propanes, diacyl dimethylammonium propanes, and stearylamines cationic
amphiphiles such as
spermine cholesterol carbamates, and the like, in optimum concentrations which
fill in the larger
spaces on the outer surfaces, and/or add additional hydrophilicity to the
particles. Such additives
may be added to the reaction mixture during formation of nanoparticles to
enhance stability of
the nanoparticles by filling in the void volumes of in the upper surface of
the vesicle membrane.
[00290] Stability of nano vesicles according to the present disclosure can
be demonstrated
by dynamic light scattering (DLS) and transmission electron microscopy (TEM).
For example,
suspensions of the vesicles can be left to stand for 1, 5, 10, and 30 days to
assess the stability of
the nanoparticle solution/suspension and then analyzed by DLS and TEM.
[00291] The vesicles disclosed herein may encapsulate within their core the
active agent,
which in particular embodiments is selected from peptides, proteins,
nucleotides and or non-
polymeric agents. In certain embodiments, the active agent is also associated
via one or more
non-covalent interactions to the vesicular membrane on the outer surface
and/or the inner surface,
optionally as pendant decorating the outer or inner surface, and may further
be incorporated into
the membrane surrounding the core. In certain aspects, biologically active
peptides, proteins,
nucleotides or non-polymeric agents that have a net electric charge, may
associate ionically with
oppositely charged headgroups on the vesicle surface and/or form salt
complexes therewith.
[00292] In particular aspects of these embodiments, additives which may be
bolaamphiphiles or single headed amphiphiles, comprise one or more branching
alkyl chains
bearing polar or ionic pendants, wherein the aliphatic portions act as anchors
into the vesicle's
membrane and the pendants (e.g., chitosan derivatives or polyamines or certain
peptides)
decorate the surface of the vesicle to enhance penetration through various
biological barriers such
as the intestinal tract and the BBB, and in some instances are also
selectively hydrolyzed at a
given site or within a given organ. The concentration of these additives is
readily adjusted
according to experimental determination.
[00293] In certain embodiments, the oral formulations of the present
disclosure comprise
agents that enhance penetration through the membranes of the GI tract and
enable passage of
intact nanoparticles containing the drug. These agents may be any of the
additives mentioned
above and, in particular aspects of these embodiment, include chitosan and
derivatives thereof,
serving as vehicle surface ligands, as decorations or pendants on the
vesicles, or the agents may
be excipients added to the formulation.
[00294] In other embodiments, the nanoparticles and vesicles disclosed
herein may
comprise the fluorescent marker carboxyfluorescein (CF) encapsulated therein
while in particular
aspects, the nanoparticle and vesicles of the present disclosure may be
decorated with one or
more of PEG, e.g. PEG2000-vernonia derivatives as pendants. For example, two
kinds of PEG-
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vernonia derivatives can be used: PEG-ether derivatives, wherein PEG is bound
via an ether
bond to the oxygen of the opened epoxy ring of, e.g., vernolic acid and PEG-
ester derivatives,
wherein PEG is bound via an ester bond to the carboxylic group of, e.g.,
vernolic acid.
[00295] In other embodiments, vesicles, made from synthetic amphiphiles, as
well as
liposomes, made from synthetic or natural phospholipids, substantially (or
totally) isolate the
therapeutic agent from the environment allowing each vesicle or liposome to
deliver many
molecules of the therapeutic agent. Moreover, the surface properties of the
vesicle or liposome
can be modified for biological stability, enhanced penetration through
biological barriers and
targeting, independent of the physico-chemical properties of the encapsulated
drug.
[00296] In still other embodiments, the headgroup is selected from: (i)
choline or
thiocholine, 0-alkyl, N-alkyl or ester derivatives thereof; (ii) non-aromatic
amino acids with
functional side chains such as glutamic acid, aspartic acid, lysine or
cysteine, or an aromatic
amino acid such as tyrosine, tryptophan, phenylalanine and derivatives thereof
such as levodopa
(3,4-dihydroxy-phenylalanine) and p-aminophenylalanine; (iii) a peptide or a
peptide derivative
that is specifically cleaved by an enzyme at a diseased site selected from
enkephalin, N-acetyl-
ala-ala, a peptide that constitutes a domain recognized by beta and gamma
secretases, and a
peptide that is recognized by stromelysins; (iv) saccharides such as glucose,
mannose and
ascorbic acid; and (v) other compounds such as nicotine, cytosine, lobeline,
polyethylene glycol,
a cannabinoid, or folic acid.
[00297] In further embodiments, nano-sized particle and vesicles disclosed
herein further
comprise at least one additive for one or more of targeting purposes,
enhancing permeability and
increasing the stability the vesicle or particle. Such additives, in
particular aspects, may selected
from from: (i) a single headed amphiphilic derivative comprising one, two or
multiple aliphatic
chains, preferably two aliphatic chains linked to a midsection/spacer region
such as --NH--
(CH2)2--N--(CH2)2--N--, or --0--(CH2)2--N--(CH2)2-0--, and a sole headgroup,
which may be a
selectively cleavable headgroup or one containing a polar or ionic selectively
cleavable group or
moiety, attached to the N atom in the middle of said midsection. In other
asepcts, the additive
can be selected from among cholesterol and cholesterol derivatives such as
cholesteryl
hemmisuccinate; phospholipids, zwitterionic, acidic, or cationic lipids;
chitosan and chitosan
derivatives, such as vernolic acid-chitosan conjugate, quaternized chitosan,
chitosan-
polyethylene glycol (PEG) conjugates, chitosan-polypropylene glycol (PPG)
conjugates, chitosan
N-conjugated with different amino acids, carboxyalkylated chitosan, sulfonyl
chitosan,
carbohydrate-branched N-(carboxymethylidene) chitosan and N-(carboxymethyl)
chitosan;
polyamines such as protamine, polylysine or polyarginine; ligands of specific
receptors at a target
site of a biological environment such as nicotine, cytisine, lobeline, 1-
glutamic acid MK801,
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morphine, enkephalins, benzodiazepines such as diazepam (valium) and librium,
dopamine
agonists, dopamine antagonists tricyclic antidepressants, muscarinic agonists,
muscarinic
antagonists, cannabinoids and arachidonyl ethanol amide; polycationic polymers
such as
polyethylene amine; peptides that enhance transport through the BBB such as OX
26,
transferrins, polybrene, histone, cationic dendrimer, synthetic peptides and
polymyxin B
nonapeptide (PMBN); monosaccharides such as glucose, mannose, ascorbic acid
and derivatives
thereof modified proteins or antibodies that undergo absorptive-mediated or
receptor-mediated
transcytosis through the blood-brain barrier, such as bradykinin B2 agonist
RMP-7 or
monoclonal antibody to the transferrin receptor; mucoadhesive polymers such as
glycerides and
steroidal detergents; and Ca2+ chelators. The aforementioned head groups on
the additives
designed for one or more of targeting purposes and enhancing permeability may
also be a head
group, preferably on an asymmetric bolaamphiphile wherein the other head group
is another
moiety, or the head group on both sides of a symmetrical bolaamphiphile.
[00298] In other embodiments, nano-sized particle and vesicles discloser
herein may
comprises at least one biologically active agent is selected from: (i) a
natural or synthetic peptide
or protein such as analgesics peptides from the enkephalin class, insulin,
insulin analogs,
oxytocin, calcitonin, tyrotropin releasing hormone, follicle stimulating
hormone, luteinizing
hormone, vasopressin and vasopressin analogs, catalase, interleukin-II,
interferon, colony
stimulating factor, tumor necrosis factor (TNF), melanocyte-stimulating
hormone, superoxide
dismutase, glial cell derived neurotrophic factor (GDNF) or the Gly-Leu-Phe
(GLF) families; (ii)
nucleosides and polynucleotides selected from DNA or RNA molecules such as
small interfering
RNA (siRNA) or a DNA plasmid; (iii) antiviral and antibacterial; (iv)
antineoplastic and
chemotherapy agents such as cyclosporin, doxorubicin, epirubicin, bleomycin,
cisplatin,
carboplatin, vinca alkaloids, e.g. vincristine, Podophyllotoxin, taxanes, e.g.
Taxol and Docetaxel,
and topoisomerase inhibitors, e.g. irinotecan, topotecan.
[00299] Additional embodiments within the scope provided herein are set forth
in non-limiting
fashion elsewhere herein and in the examples. It should be understood that
these examples are
for illustrative purposes only and are not to be construed as limiting in any
manner.
PHARMACEUTICAL COMPOSITIONS
[00300] In another aspect, the invention provides a pharmaceutical composition
comprising a
pharmaceutically acceptable carrier and a pharmaceutically effective amount of
a compound of
Formula I or a complex thereof
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[00301] When employed as pharmaceuticals, the compounds provided herein are
typically
administered in the form of a pharmaceutical composition. Such compositions
can be prepared
in a manner well known in the pharmaceutical art and comprise at least one
active compound.
[00302] In certain embodiments, with respect to the pharmaceutical
composition, the carrier is
a parenteral carrier, oral or topical carrier.
[00303] The present invention also relates to a compound or pharmaceutical
composition of
compound according to Formula I; or a pharmaceutically acceptable salt or
solvate thereof for
use as a pharmaceutical or a medicament.
[00304] Generally, the compounds provided herein are administered in a
therapeutically
effective amount. The amount of the compound actually administered will
typically be
determined by a physician, in the light of the relevant circumstances,
including the condition to
be treated, the chosen route of administration, the actual compound
administered, the age,
weight, and response of the individual patient, the severity of the patient's
symptoms, and the
like.
[00305] The pharmaceutical compositions provided herein can be administered by
a variety of
routes including oral, rectal, transdermal, subcutaneous, intravenous,
intramuscular, and
intranasal. Depending on the intended route of delivery, the compounds
provided herein are
preferably formulated as either injectable or oral compositions or as salves,
as lotions or as
patches all for transdermal administration.
[00306] The compositions for oral administration can take the form of bulk
liquid solutions or
suspensions, or bulk powders. More commonly, however, the compositions are
presented in unit
dosage forms to facilitate accurate dosing. The term "unit dosage forms"
refers to physically
discrete units suitable as unitary dosages for human subjects and other
mammals, each unit
containing a predetermined quantity of active material calculated to produce
the desired
therapeutic effect, in association with a suitable pharmaceutical excipient.
Typical unit dosage
forms include prefilled, premeasured ampules or syringes of the liquid
compositions or pills,
tablets, capsules or the like in the case of solid compositions. In such
compositions, the
compound is usually a minor component (from about 0.1 to about 50% by weight
or preferably
from about 1 to about 40% by weight) with the remainder being various vehicles
or carriers and
processing aids helpful for forming the desired dosing form.
[00307] Liquid forms suitable for oral administration may include a suitable
aqueous or
nonaqueous vehicle with buffers, suspending and dispensing agents, colorants,
flavors and the
like. Solid forms may include, for example, any of the following ingredients,
or compounds of a
similar nature: a binder such as microcrystalline cellulose, gum tragacanth or
gelatin; an
excipient such as starch or lactose, a disintegrating agent such as alginic
acid, Primogel, or corn
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starch; a lubricant such as magnesium stearate; a glidant such as colloidal
silicon dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint, methyl
salicylate, or orange flavoring.
[00308] Injectable compositions are typically based upon injectable sterile
saline or phosphate-
buffered saline or other injectable carriers known in the art. As before, the
active compound in
such compositions is typically a minor component, often being from about 0.05
to 10% by
weight with the remainder being the injectable carrier and the like.
[00309] Transdermal compositions are typically formulated as a topical
ointment or cream
containing the active ingredient(s), generally in an amount ranging from about
0.01 to about 20%
by weight, preferably from about 0.1 to about 20% by weight, preferably from
about 0.1 to about
10% by weight, and more preferably from about 0.5 to about 15% by weight. When
formulated
as a ointment, the active ingredients will typically be combined with either a
paraffinic or a
water-miscible ointment base. Alternatively, the active ingredients may be
formulated in a cream
with, for example an oil-in-water cream base. Such transdermal formulations
are well-known in
the art and generally include additional ingredients to enhance the dermal
penetration of stability
of the active ingredients or the formulation. All such known transdermal
formulations and
ingredients are included within the scope provided herein.
[00310] The compounds provided herein can also be administered by a
transdermal device.
Accordingly, transdermal administration can be accomplished using a patch
either of the
reservoir or porous membrane type, or of a solid matrix variety.
[00311] The above-described components for orally administrable, injectable or
topically
administrable compositions are merely representative. Other materials as well
as processing
techniques and the like are set forth in Part 8 of Remington's Pharmaceutical
Sciences, 17th
edition, 1985, Mack Publishing Company, Easton, Pennsylvania, which is
incorporated herein by
reference.
[00312] The above-described components for orally administrable, injectable,
or topically
administrable compositions are merely representative. Other materials as well
as processing
techniques and the like are set forth in Part 8 of Remington's The Science and
Practice of
Pharmacy, 21st edition, 2005, Publisher: Lippincott Williams & Wilkins, which
is incorporated
herein by reference.
[00313] The compounds of this invention can also be administered in sustained
release forms
or from sustained release drug delivery systems. A description of
representative sustained
release materials can be found in Remington's Pharmaceutical Sciences.
[00314] The present invention also relates to the pharmaceutically acceptable
formulations of
compounds of Formula I. In certain embodiments, the formulation comprises
water. In another
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embodiment, the formulation comprises a cyclodextrin derivative. In certain
embodiments, the
formulation comprises hexapropyl-P-cyclodextrin. In a more particular
embodiment, the
formulation comprises hexapropyl-P-cyclodextrin (10-50% in water).
1003151 The present invention also relates to the pharmaceutically acceptable
acid addition
salts of compounds of Formula I. The acids which are used to prepare the
pharmaceutically
acceptable salts are those which form non-toxic acid addition salts, i.e.
salts containing
pharmacologically acceptable aniovs such as the hydrochloride, hydroiodide,
hydrobromide,
nitrate, sulfate, bisulfate, phosphate, acetate, lactate, citrate, tartrate,
succinate, maleate, fumarate,
benzoate, para-toluenesulfonate, and the like.
[00316] The following formulation examples illustrate representative
pharmaceutical
compositions that may be prepared in accordance with this invention. The
present invention,
however, is not limited to the following pharmaceutical compositions.
Formulation 1 - Injection
[00317] A compound of the invention may be dissolved or suspended in a
buffered sterile
saline injectable aqueous medium to a concentration of approximately 5 mg/mL.
METHODS OF TREATMENT
[00318] The nano-sized stable vesicles [5,6,7,8,9] can be used to deliver GDNF
to the brain.
These nano-sized vesicles are made of novel bolaamphiphiles (bolas). The
vesicles that these
novel bolas form were shown to aggregate into vesicles or nano particleds that
cross the BBB and
deliver small molecules, peptides and proteins to the brain. Bolas are
promising building block
candidates for vesicles used as a drug delivery system targeted to the brain,
since they can form
vesicles with monolayer membranes, which are more stable than liposomes with
bilayer
membranes, due to the high energy barrier for lipid exchange that
characterizes bolas [10]. The
high stability of such vesicles allows them to circulate in the blood stream
until they reach the
brain, and then penetrate the BBB in their intact form. In addition, the
monolayer membrane is
thinner than a bilayer membrane, thus providing higher inner volume for
encapsulation as
compared to vesicles of the same size made of an encapsulating bilayer
membrane [11].
Moreover, a controlled release mechanism is more likely to be achieved with
vesicles made of
bolas that form monolayer membranes, as compared to classical liposomes made
of bilayer
encapsulating membranes, since monolayer membranes are known to rapidly change
their
morphology from vesicles to fibers and sheets upon small changes in their
surface groups [10]. A
controlled release mechanism should allow release of the encapsulated material
only after the
vesicles penetrate into the brain, thus preventing leakage in non relevant
tissues. Indeed, the
vesicles made from bolas do cross the BBB, transport encapsulated small
molecules, peptides
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and proteins into the brain and release them primarily there. This novel
delivery system can be an
effective delivery system for GDNF, and has the potential to be used in the
treatment of PD,
since it can distribute the NTF within a wide brain area and, thus, can
positively affect
degenerating neurons throughout the brain. The resulting bola-GDNF delivery
systems or
formulations may be capable of delivering other neurotrophic factors for the
treatment of several
neurodegenerative diseases, particularly PD.
[00319] Thus, various PD active drug molecueles, such as GDNF, can be
encapsulated in the
bolaamphiphilic vesicles and then delivered to the brain in sufficient
concentrations for
therapeutic use.
[00320] In ceratin embodiments, the active drug molecules, including a
neurotrophic factor
selected from among Glial cell-derived neurotrophic factor (GDNF), Nerve
Growth factor
(NGF), Brain-Derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3),
Neurotrophin-4/5
(NT-4/5), and combinations of two or more thereof can be encapsulated and
delivered to the
brain and/or periphperal nervous system in sufficient concentrations for
therapeutic use.
[00321] The vesicles formed from the bolaamphiphiles to encapsulating NTF,
GDNF or
NGF, may contain additives that help to stabilize the vesicles, by stabilizing
the vesicle's
membranes, such as but not limited to cholesterol derivatives such as
cholesteryl hemisuccinate
and cholesterol itself and combinations such as cholesteryl hemisuccinate and
cholesterol. The
bola vesicles in addition to these components have another addiitves which
decorates the outer
vesicle memrbanes with groups or pendants that enhance penetration though
biological barriers
such as the BBB, or groups for targeting to specific sites such as
dopaminergic neurons. In a
further embodiment the bolaamphiphile head groups that comprise the vesicles
membranes can
interact with the neuro active agents such as GDNF or NDF to be delivered in
to the brain and
brain sites ionic interactions to enhance the % encapsulation via complexation
and well as
passive encapsulation within the vesicles core. Further the formuation may
contain other
additives within the veicles membranes to futhjer enhance the degree of
encapsulation of neuro
active agents such as GDNF or NDF. It is understood by one skilled in the
state of art that the
pH in which the vesicle formation and encapsulation of the neuro active agent
such as GDNF or
NDF is such as to maximize the electrostatic or inonic interactions between
the said agents and
the sai bolaamphiphiles and or additives to maximize the % encapsulation.
[00322] The bolaamphiphile vesicles disclosed herein are capable of
penetrating the brain
blood barrier (BBB) and transporting their encapsulated compounds into the
brain. Those
encapsulated compounds include small molecules as well as nucleic acids and
proteins.
Examples of such proteins include trypsinogen (molecular weight ¨ 24 kDa), the
homodimeric
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protein - GDNF (molecular weight ¨ 30 kDa), horseradish peroxidase (molecular
weight ¨ 44
kDa) and albumin (molecular weight ¨ 60 kDa).
[00323] Biological activity of these bolaamphiphile-vesicle-encapsulated
therapeutic
proteins, e.g. in the brain, can be evaluated in SBE transgenic mice. In
particular, this animal
model system may be used as a tool to measure the activity of Activin A
following its delivery to
the brain. Activin A, a member of the TGF-r3 superfamily, is a homodimeric
protein with a
molecular weight of ¨ 25 kD.
[00324] Preparation of bolaamphiphile-vesicle-encapsulated Activins,
including Activin
A, is accomplished according to the methods disclosed herein. Similarly,
pharmaceutically
acceptable formulations for administration of bolaamphiphile-vesicle-
encapsulated Activins are
also prepared according to the presently-disclosed methods. In particular,
bolaamphiphile-
vesicle-encapsulated Activin, including bolaamphiphile-vesicle-encapsulated
Activin A, may be
used for regulation of FSH secretion from the pituitary, neuronal development
and spine
formation, neurogenesis, late-phase long-term potentiation, and maintenance of
long-term
memory. Activin receptors are highly expressed in neuronal cells and their
activation by Activin
A leads to a transduction cascade that involves the phosphorylation of Smad
proteins and their
translocation into the nucleus where they are used as transcription factors
and regulate gene
expression. The SBE transgenic mice contain a Smad-responsive luciferase
reporter that responds
to Smad activation. This Smad-dependent signaling can be assessed non-
invasively in the living
mouse by bioluminescence imaging. Accordingly, this system is used to
determine, in live
animals, the activity of Activin A, delivered by the bolaamphiphile vesicles
of the present
disclosure, in the brain.
[00325] Accordingly, in one aspect, the present disclosure is directed to
methods for
delivery of the protein Activin (in particular Activin A) to the CNS by
intravenvous or other
non-invasive methods of administration. Activin is a protein complex that can
be delivered into
the brain by intravenous, oral, intraperitoneal and other noninvasive
administration methods
using the bolaamphiphilic vesicles of the present disclosure. Activin can be
classified as either
Activin A, Activin B and Activin AB; Actvin A is a peptide dimer of two r3A
subunits, Actvin
AB is a peptide dimer of a r3A and a r3B subunit, and Activin B is a dimer of
two r3B subunits.
All these are deliverable into the CNS and other organs by the bolaamphiphilc
vesicles of the
present disclosure.
[00326] Activin can enhance the survival of neural cell and that activin
may act in vivo as
a neuronal rescue factor. Activin may act in different parts of the CNS, and
may be used for the
treatment of Huntington's disease; Activin A as well as the other Activins,
Activin B and AB
may be a useful alternative to NGF in treating those conditions in which NGF
therapy has shown
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promise, including peripheral neuropathy and Alzheimer's disease. Activin A
partially reverses
the phenotypic degeneration of striatal parvalbumin and NADPH intemeurons.
Activin A can
rescue both striatal intemeurons and striatal projection neurons from
excitotoxic lesioning with
quinolinic acid. Treatment with Activin A may help to prevent the degeneration
of vulnerable
striatal neuronal populations in Huntington's disease. Together with the
localization of activin
receptors to certain regions in the brain, specific central roles for activin
are now being
recognised. One of the first defined roles for activin in the brain was its
modulation of oxytocin
release and fluid regulation in the neurosecretory cells of the hypothalamus
and brain stem.
Activin may thus also be useful for mitigating or reversing autism. Given that
activin can
enhance the survival of neural cell lines and is neuroprotective for cultured
midbrain neurons
exposed to N-methyl-4-phenylpyridinium (MPP1) 42. Therefore, in one aspect of
this
embodiment, Activin may be administered (as disclosed herein) in vivo as a
neuronal rescue
factor and for treatement of diseases and conditions in need thereof
GENERAL SYNTHETIC PROCEDURES
[00327] The compounds provided herein can be purchased or prepared from
readily available
starting materials using the following general methods and procedures. See,
e.g., Synthetic
Schemes below. It will be appreciated that where typical or preferred process
conditions (i.e.,
reaction temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given, other
process conditions can also be used unless otherwise stated. Optimum reaction
conditions may
vary with the particular reactants or solvent used, but such conditions can be
determined by one
skilled in the art by routine optimization procedures.
[00328] Additionally, as will be apparent to those skilled in the art,
conventional protecting
groups may be necessary to prevent certain functional groups from undergoing
undesired
reactions. The choice of a suitable protecting group for a particular
functional group as well as
suitable conditions for protection and deprotection are well known in the art.
For example,
numerous protecting groups, and their introduction and removal, are described
in T. W. Greene
and P. G. M. Wuts, Protecting Groups in Organic Synthesis, Second Edition,
Wiley, New York,
1991, and references cited therein.
[00329] The compounds provided herein may be isolated and purified by known
standard
procedures. Such procedures include (but are not limited to)
recrystallization, column
chromatography or HPLC. The following schemes are presented with details as to
the
preparation of representative substituted biarylamides that have been listed
herein. The
compounds provided herein may be prepared from known or commercially available
starting
materials and reagents by one skilled in the art of organic synthesis.
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[00330] It is to be understood that the bolaamphiphile compounds may be
used as racemic
mixtures or mixtures of geometric isomors such as cis or trans, or as mixtures
of geometric
isomers unless otherwise specified as being enantiomerically pure compounds.
The
enantiomerically pure compounds thay may be provided herein may be prepared
according to
any techniques known to those of skill in the art. For instance, they may be
prepared by chiral or
asymmetric synthesis from a suitable optically pure precursor or obtained from
a racemate by any
conventional technique, for example, by chromatographic resolution using a
chiral column, TLC
or by the preparation of diastereoisomers, separation thereof and regeneration
of the desired
enantiomer. See, e.g., "Enantiomers, Racemates and Resolutions," by J.
Jacques, A. Collet, and
S.H. Wilen, (Wiley-Interscience, New York, 1981); S.H. Wilen, A. Collet, and
J. Jacques,
Tetrahedron, 2725 (1977); E.L. Eliel Stereochemistry of Carbon Compounds
(McGraw-Hill, NY,
1962); and S.H. Wilen Tables of Resolving Agents and Optical Resolutions 268
(EL. Eliel ed.,
Univ. of Notre Dame Press, Notre Dame, IN, 1972, Stereochemistry of Organic
Compounds,
Ernest L. Eliel, Samuel H. Wilen and Lewis N. Manda (1994 John Wiley & Sons,
Inc.), and
Stereoselective Synthesis A Practical Approach, Mihaly Nogradi (1995 VCH
Publishers, Inc.,
NY, NY).
[00331] In certain embodiments, an enantiomerically pure compound of formula
(1) may be
obtained by reaction of the racemate with a suitable optically active acid or
base. Suitable acids
or bases include those described in Bighley et al., 1995, Salt Forms of Drugs
and Adsorption, in
Encyclopedia of Pharmaceutical Technology, vol. 13, Swarbrick & Boylan, eds.,
Marcel Dekker,
New York; ten Hoeve & H. Wynberg, 1985, Journal of Organic Chemistry 50:4508-
4514; Dale
& Mosher, 1973, J. Am. Chem. Soc. 95:512; and CRC Handbook of Optical
Resolution via
Diastereomeric Salt Formation, the contents of which are hereby incorporated
by reference in
their entireties.
[00332] Enantiomerically pure compounds can also be recovered either from the
crystallized
diastereomer or from the mother liquor, depending on the solubility properties
of the particular
acid resolving agent employed and the particular acid enantiomer used. The
identity and optical
purity of the particular compound so recovered can be determined by
polarimetry or other
analytical methods known in the art. The diasteroisomers can then be
separated, for example, by
chromatography or fractional crystallization, and the desired enantiomer
regenerated by
treatment with an appropriate base or acid. The other enantiomer may be
obtained from the
racemate in a similar manner or worked up from the liquors of the first
separation.
[00333] In certain embodiments, enantiomerically pure compound can be
separated from
racemic compound by chiral chromatography. Various chiral columns and eluents
for use in the
separation of the enantiomers are available and suitable conditions for the
separation can be
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empirically determined by methods known to one of skill in the art. Exemplary
chiral columns
available for use in the separation of the enantiomers provided herein
include, but are not limited
to CHIRALCELO OB, CHIRALCELO OB-H, CHIRALCELO OD, CHIRALCELO OD-H,
CHIRALCELO OF, CHIRALCELO OG, CHIRALCELO OJ and CHIRALCELO OK.
[00334] ABBREVIATIONS
BBB, blood brain barrier
BCECs, brain capillary endothelial cells
CF, carboxyfluorescein
CHEMS, cholesteryl hemisuccinate
CHOL, cholesterol
Cryo-TEM, Cryo-transmission electron microscope
DAPI, 4,6- diamidino-2-phenylindole
DDS, drug delivery system
DLS, dynamic light scattering
DMPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine
DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine
DMPG,1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol)
EPR, electron paramagnetic resonance
FACS, fluorescence-activated cell sorting
FCR, fluorescence colorimetric response
GUVs, giant unilamellar vesicles
HPLC, high performance liquid chromatography
IR, infrared
MRI, magnetic resonance imaging
NMR, nuclear magnetic resonance
NPs, nanoparticles
PBS, phosphate buffered saline
PC, phosphatidylcholine
PDA, polydiacetylene.
TMA-DPH, 1-(4 trimethylammoniumpheny1)-6-phenyl-1,3,5-hexatriene
Example 1
Bolaamphiphile synthesis
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[00335] The boloamphiphles or bolaamphiphilic compounds of the invention can
be
synthesized following the procedures described previously (see below).
[00336] Briefly, the carboxylic group of methyl vemolate or vemolic acid was
interacted with
aliphatic diols to obtain bisvemolesters. Then the epoxy group of the vemolate
moiety, located
on C12 and C13 of the aliphatic chain of vemolic acid, was used to introduce
two ACh
headgroups on the two vicinal carbons obtained after the opening of the
oxirane ring. For GLH-
20 (Table 1), the ACh head group was attached to the vemolate skeleton through
the nitrogen
atom of the choline moiety. The bolaamphiphile was prepared in a two-stage
synthesis: First,
opening of the epoxy ring with a haloacetic acid and, second, quatemization
with the 1V,N-
dimethylamino ethyl acetate. For GLH-19 (Table 1) that contains an ACh head
group attached to
the vemolate skeleton through the acetyl group, the bolaamphiphile was
prepared in a three-stage
synthesis, including opening of the epoxy ring with glutaric acid, then
esterification of the free
carboxylic group with /V,N-dimethyl amino ethanol and the final product was
obtained by
quatemization of the head group, using methyl iodide followed by exchange of
the iodide ion by
chloride using an ion exchange resin.
[00337] Each bolaamphiphile was characterized by mass spectrometry, NMR and IR
spectroscopy. The purity of the two bolaamphiphiles was >97% as determined by
HPLC.
[00338] Materials. Iron(III) acetylacetonate (Fe(acac)3), diphenyl ether, 1,2-
hexadecanediol,
oleic acid, oleylamine, and carboxyfluorescein (CF) were purchased from Sigma
Aldrich
(Rehovot, Israel). Chloroform and ethanol were purchased from Bio-Lab Ltd.
Jerusalem, Israel.
1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DMPG), 1,2-dimyristoyl-
sn-glycero-3-
phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphocholine
(DMPC),
cholesterol (CHOL), cholesteryl hemisuccinate (CHEMS) were purchased from
Avanti Lipids
(Alabaster, AL, USA), The diacetylenic monomer 10,12- tricosadiynoic acid was
purchased from
Alfa Aesar (Karlsruhe, Germany), and purified by dissolving the powder in
chloroform, filtering
the resulting solution through a 0.45 pm nylon filter (Whatman Inc., Clifton,
NJ, USA), and
evaporation of the solvent. 1-(4 trimethylammoniumpheny1)-6-phenyl-1,3,5-
hexatriene (TMA-
DPH) was purchased from Molecular Probes Inc. (Eugene, OR, USA).
SYNTHESIS OF REPRESENTATIVE BOLAAMPHIPHILIC COMPOUNDS
[00339] The synthesis bolaamphiphilic compounds of this invention can be
carried out in
accordance with the methods described previously (Chemistry and Physics of
Lipids 2008, 153,
85-97; Journal of Liposome Research 2010, 20, 147-59; W02002/055011;
W02003/047499; or
W02010/128504) and using the appropriate reagents, starting materials, and
purification
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methods known to those skilled in the art. Table 1 lists the representative
bolaamphiphilic
compounds of the invention.
[00340] Table 1: Representative
Bolaamphiphiles
# Structure
Oa
GLH-3 ,CH2,16
- ..,..,
,
N,,e0õ04,(cH2)11 =0=IL i 1 )1=0(cH2)4f1".ke'N,-,
e.
...,
GLH-4
`.,,.====.õ P,1-Nz%=-'
es P A
H H
0
GLH-5
iic=a"AH - OH
H
HOrn,0 OH
GLH-6 a
- OH
H H
0
N. ,N,,,,a x,Nae,'Itrot
GLH-7
H H
j
GLH-8
rnro
/ N.,(02,...N
HO - cH - OH
H H
3D ..........
pt: )T (CH2)õ
40 ¨ N= %=1s1 ¨ OA
GLH-9 H H
0
8,",..."ro=""yi
GLH-10 / ,,,, (CH N.
HO
N.,2)õ,N
¨ OH
H H
Nkf2.HC;
HO 9-110-CH2-CH:2-tH-00001{
o
II I I
NH- C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
GLH-11 / ff-T2.1:-ICi
(CH 2)2
HO O-C:0-013.2 CH2 H COOOH
\ V I I
NH- C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
i..... .
GLH-12 a 0
r
-- \ =,
cH3-(cH2)4-CH - CH- CH2- CH=CH-(CH2)7- COOCH3 GLH-13 a CH 1- co-o-CHT-C
CH2-CO-HN OH
I I
CH3-(CH2)4-CH - CH- CH2- CH=CH-(CH2)7- COOCH3
- (-E-,
GLH-13 a A
caxo-o-ca,-(11{2-N- CH2-CO-HN 0-CO-CH2-N-CH2-C.F.12=0-CO-CH3
I I
CH3-(CH2)4-CH - CH- CH2- CH=CH-(CH2)7- COOCH3
GLH-14
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=11,'s.,,,,, 14,1,-\-4 (CH 2)2 0 oci
ö
H H 0 o
GLH-15
'k''''P"'''Nr'j=../ j (CH)io 0 ea n
_ i µ. ,
..:
H H o ..
o
0 (cHoi2 0 "(A
;...=
GLH-16
H H 0
GLH-1 7 'C''''-'`oriro=="yi r6.'lf c'''.6"1"
¨ OH
H H
0
GLH-18 ,,,,,,60,r10
0
a N = (CI12)12 N .11..........A.
H 0 H 0
H
NW".= )conco 0,,,,,,A,
GLH-19
¨ C)H
Ci9 CO
GLH-20 N'esks",,, '''µY):4o (cH2), z./%=''''Y'Nfi's=N'.=AY
o <0
c=.--.)i=1
p31.-, N-,-q
0 HI) r-Cfp&,,,\./...... ,2,_.
4 ,õ. 2
II
1C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
GLH-21 NH
I COOH
(CH 2)2
I
NH - 1-VCR -41 NH
0 Hy
ii
\ C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
:-`-
µ1-
HO O-CO-CH - N -CI-I--CI-I--0-('0-CH
I I
,-- C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
GLH-22 NH
(OH 2)12 .S1
I , - --
0 HO O-CO-CH2- N -CI-T2-CI-T2-0-W-CEI3
NH......... II I I
C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
GLH-23 o olols tr"eN FeANy/
o 0
H Olk")40 c"/N"""P cfrklIc2)H
N H2 H
a,
GLH-24
=i<:."==w40.***" ""===0 :::: '
,
GLH-25
Ctri '105)` 0., 2,ioso
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o pCOCH2 M -ca42-cf{,.Ø.co.ca;
GLH-26 C-(CH 2)7 -CH=CH-CH 2-CH-(CH 2)5-CH3
CH- Cr-is 0.
(CH 2)10
I 0 p-CO-CH2-
II
C-(CH 07 -CH=CH-CH 2-CH-(CH 2)5-CH3
,
\ /4-
0 H f)-CO-CH2- N
GLH-27 C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
(+I. ea, (CH 2)8
I 0 H9 0-CO-CH2-
O II I I
C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
C ;
O
Hi? 1)-CO-CH2- N -CH 2-CH2-0-CO-CH3
GLH-28 C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
(CH 2)8
I 0 Hy 0-00-042-N -042-0-12-0-00-03:,
O II
C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
(`-
'Qj.`=
0 HO 0-CO-CH2- N -CH7-CH2-0-CO-CTh
I I
GLH-29 C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH
3
0
(H2)10 CH- 1-1-13
0 cH,
II
\14-
C-(CH 2)11 -N
GLH-30
(CH 2)16
I 0
0 II - Br
C-(CH 2) 1 1 -N -CH-, -011,-O-00-011,
0 CH ('H
II
C-(CH 2)11
GLH-30
(CH2)16
I 0 CH% Cfti
0 II l+
C-(CH 2)ii -N
CH3 CH-3 cr
4/.4.
O HO 0-CO-CH2- N
GLH-31 I I
C-(CH 2)7 -CH=CH-CH 2-CH-CH-(CH 2)4-CH 3
(CH 2)12 CH, CH. cr
\ if+
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'Ny.-'-',..."Nlzj--,,,,,,'-' .,'''y'N'ic.'"w'N,0-=
GLH-32 s A 6 0 , =:' 1 '" 0....(cHoio....0
Ho oH
GLH-33
,.. 0
HO 0 "===0;:c,1:1µ,(0-0.4
=r;'-
sõ
0
GLH-34
HO
0..(cH2).,00
¨ 0H
0 -
GLH-35
HO =0 OH
.0--
0 -
y*."gry =.-N,...,µ-'= "
GLH-36
oõ(cH2)2.0
:.õ
GLH-37
f ~,00t
cH
r...ff'j=
= (2),0
No ¨ 0.
GLH-38
GLH-39a i-12N 41/4 -.NI s - OH o IL
o _ ¨(cHoi o¨o (cH2)12
(=-'--= rnr r)1 GLH-40 cl,ni,002zr,N3
cr
o
GLH-41 l'xi cr
_ C)114)IrlY
/ \
A
i-i,-) 0 0
s l''(-) 0
i-12Nv's= sN,*-' o¨(cH2)4¨o (cH2)12 0H .11,
GLH-42 a :.:
= , , OH
noik (-1-1
i-1.2N1k\s"W o¨,(cH2)12¨_
(_..2)112koi-i
GLH-43 a
GLH-44 ,='`40",',,' .,00=%i/\MI\A10=\/\~\/\. teN/N/= p(4'\\,=''N'W
0 0 ** 0
0 :.*1
, k-,i. crok,õ,:g .00t4{,........................... 0
tr........"..............)40-......."........- rr n......., - ws
GLH-45 cp. o 0 4' . 0
GLH-46
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o
O s,.Ø o o
,A0-N.N.emo=i~wtow. IewN~' IrR-N,pc'n,
2cle
o . o
2Ã::0
o o
:*.'s''-
µ0,,N*8..w.=========110%...."....="...0".."..140."......"."..."..0=004() n,
GLH-47 o ., s 0
Eise
o
GLH-48 Ho,NINw0i,.,40.0%.õ......."........,.".....4-\,..:DH
GLH-49 a õA -)s-s=Ntv,:-'1,....A0,0%.,õ"..",,,,,".,/===
1101..."..."..."...".,,"Koi-i
,o o
GLH-50a
- b -
o " o
v ,i LI
\-0,.N,õ6:õ.-0Ø.......w.,õ0".õctr......w.õ/*õ.......õ"................õAc)k
GLH-51 a cie o
O 0
GLH-52a H OH
H' HO 0
N 14
- 0.., m 0
HO 0-(CH2CH20),-H o
GLH-53 a _
0¨ c H3
H30
0 o
GLH-54 a
H3C
_
HO 4
GLH-55
H , \ C.f.) ."
0 0 0
,...' NH p /
"4::".\ ,..")...A10.=================="0140...."0"4~.
4
:.:4C)
GLH-56 0 / 4,0
\,':-. - 0.--(cH2p¨oi(cH .NEi 1.
s-A 2)mr'
GLH-57
..,
a - intermediate
Example 2
Vesicle formation and their optimization
[00341] The vesicles shown to be effective in delivering enkephalin and
albumin to the CNS
were made from the bola GLH-20, or a mixture of GLH-19 and GLH-20 [Table 11.
Both of these
bolas contain acetyl choline (ACh) head groups [8], but only GLH-20 is
hydrolyzed by choline
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esterases (ChE). The mixture of these two bolas enables extended release of
the encapsulated
material. Stability and release rates can be used as the criteria to get the
optimal ratio between
GLH-19 and GLH-20. Stability and release rates can be studied using
fluorescent measurements
of encapsulated CF as described by us previously [7, 81. Increasing the
proportion of GLH-19
(which is not hydrolyzed by AChE) is expected to result in a slower release
rate, thus prolonging
the duration of action of the encapsulated active compound. Then, vesicles
will be prepared by
the method of film hydration, followed by sonication [14].
[00342] Each of the vesicle formulations can be examined for vesicle size (by
dynamic light
scattering), morphology (by cryo-transmission electron microscopy), zeta
potential (by Zeta
Potential Analyzer) and stability (by fluorescence measurements of
encapsulated CF at various
incubation times before and after exposing the vesicles to AChE and then to a
detergent) [5,7,8].
Optimal formulations can be selected based on stability and ability to release
encapsulated.
material by AChE. Vesicle stability can be tested first in PBS and, then, if
stable, in whole serum
at 37 C in the presence and absence of pyridostigmine -- an AChE inhibitor.
Example 3
Bolavesicle preparation and characterization
[00343] Bolaamphiphiles, cholesterol, and CHMES (2:1:1 mole ratio) are
dissolved in
chloroform or a suitable solvent. 0.5 mg of the GDNF dispersed in chloroform
is added to the
mix. The solvents are evaporated under vacuum and the resultant thin films are
hydrated in 0.2
mg/mL CF solution in PBS and probe-sonicated (Vibra-Cell VCX130 sonicator,
Sonics and
Materials Inc., Newtown, CT, USA) with amplitude 20%, pulse on: 15 sec, pulse
off: 10 sec to
achieve homogenous vesicle dispersions. Vesicle size and zeta potential were
determined using a
Zetasizer Nano ZS (Malvern Instruments, UK).
Example 4
Measurement of the quality and activity of the encapsulated GDNF
[00344] The encapsulated GDNF can be run on acrylamide gel electrophoresis
(after release
from vesicles by a detergent) to confirm that it maintained its integrity
during the encapsulation
process. In addition, the activity of the GDNF affected by the encapsulation
process can be tested
by measuring the ability of the encapsulated material to induce tyrosine
hydroxylase (TH) gene
expression in comparison to free GDNF. SK-N-MC cells stably transfected with
expression
constructs of c-ret and with a luciferase reporter gene driven by 2 kb of the
rat TH gene promoter
region can be used. In the presence of GDNF, luciferase activity is expected
to increase [15].
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Spectral Characterization
Example 5
Cryogenic transmission electron microscopy (cryo-TEM)
[00345] Specimens studied by cryo-TEM were prepared. Sample solutions (4 [tL)
are deposited
on a glow discharged, 300 mesh, lacey carbon copper grids (Ted Pella, Redding,
CA, USA). The
excess liquid is blotted and the specimen was vitrified in a Leica EM GP
vitrification system in
which the temperature and relative humidity are controlled. The samples are
examined at -180 C
using a FEI Tecnai 12 G2 TWIN TEM equipped with a Gatan 626 cold stage, and
the images are
recorded (Gatan model 794 charge-coupled device camera) at 120 kV in low-dose
mode. Figure
1 shows TEM micrograph of vesicles from GLH-20 (A) and their size distribution
determined by
DLS (13).
Assays
Example 6
Lipid/polydiacetylene (PDA) assay
[00346] Lipid/polydiacetylene (PDA) vesicles (PDA/DMPC 3:2, mole ratio) are
prepared by
dissolving the lipid components in chloroform/ ethanol and drying together in
vacuo. Vesicles are
subsequently prepared in DDW by probe-sonication of the aqueous mixture at 70
C for 3 min.
The vesicle samples are then cooled at room temperature for an hour and kept
at 4 C overnight.
The vesicles are then polymerized using irradiation at 254 nm for 10-20 s,
with the resulting
emulsions exhibiting an intense blue appearance. PDA fluorescence is measured
in 96-well
microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) on a Fluoroscan
Ascent
fluorescence plate reader (Thermo Vantaa, Finland). All measurements are
performed at room
temperature at 485 nm excitation and 555 nm emission using LP filters with
normal slits.
Acquisition of data is automatically performed every 5 min for 60 min. Samples
comprised 30
[IL of DMPC/PDA vesicles and 54 bolaamphiphilic vesicles assembled with HIV
drug,
followed by addition of 30 [IL 50 mM Tris-base buffer (pH 8.0).
[00347] A quantitative value for the increasing of the fluorescence intensity
within the
PDA/PC-labeled vesicles is given by the fluorescence colorimetric response
(%FCR), which is
defined as follows27:
Eq. 1. %FCR = [(F1-F0)/F1001 = 100
[00348] Where F1 is the fluorescence emission of the lipid/PDA vesicles after
addition of the
tested membrane-active compounds, Fo is the fluorescence of the control sample
(without
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addition of the compounds), and F100 is the fluorescence of a sample heated to
produce the
highest fluorescence emission of the red PDA phase minus the fluorescence of
the control
sample.
Example 8
Cell culture
[00349] b.End3 immortalized mouse brain capillary endothelium cells are kindly
provided by
Prof Philip Lazarovici (Institute for Drug Research, School of Pharmacy, The
Hebrew
University of Jerusalem, Israel). The b.End3 cells were cultured in DMEM
medium
supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 100 IU/mL
penicillin and 100
pg/mL streptomycin (Biological Industries Ltd., Beit Haemek, Israel). The
cells are maintained
in an incubator at 37 C in a humidified atmosphere with 5% CO2.
Example 9
Internalization of CF by the cells in vitro
1003501 b.End3 cells are grown on 24-well plates or on coverslips (for FACS
and fluorescence
microscopy analysis, respectively). The medium is replaced with culture medium
without serum
and CF solution, or tested bolavesicles (equivalent to 0.5 pg/mL CF), or
equivalent volume of the
medium are added to the cells and incubated for 5 hr at 4 C or at 37 C. At the
end of the
incubation, cells are extensively washed with complete medium and with PBS,
and are either
detached from the plates using trypsin-EDTA solution (Biological Industries
Ltd., Beit Haemek,
Israel) and analyzed by FACS (FACSCalibur Flow Cytometer, BD Biosciences,
USA), or fixed
with 2.5% formaldehyde in PBS, washed twice with PBS, mounted on slides using
Mowiol-
based mounting solution and analyzed using a FV1000-IX81 confocal microscope
(Olympus,
Tokyo, Japan) equipped with 60x objective. All the images are acquired using
the same imaging
settings and are not corrected or modified. The Figure 2 shows head group
hydrolysis by AChE
(A) of GLH-19 (blue) and GLH-20 (red) and release of CF from GLH-19 vesicles
(B) and GLH-
20 vesicles (C). AChE causes the release of encapsulated material from GLH-20
vesicles, but not
from GLH-19 vesicles (Fig.2). The vesicles are capable of delivering small
molecules, such as
carboxyfluorescein (CF), into a mouse brain, but the fluorescent dye
accumulates only if it is
delivered in vesicles that release their encapsulated CF in presence of AChE,
namely, GLH-20
vesicles (Fig. 3A). These results suggest that the release is due to head
group hydrolysis by
AChE in the brain. Corroboration for this conclusion also comes from an
experiment showing
that when an analgesic peptide is delivered to the brain by the bola vesicles,
analgesia (which is
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caused when the encapsulated peptide is released in the brain) was observed
only with GLH-20
vesicles, but not by GLH-19 vesicles (Figure 4A).The vesicles do not break the
BBB, but rather
penetrate it in their intact form, as indicated by the finding that analgesia
is obtained only when
enkephalin is administered while encapsulated within the vesicles, but not
when free enkephalin
is administered together with empty vesicles (Figure 4B).
[00351] The ACh head groups also provide the vesicles with cationic surfaces,
which promote
penetration through the BBB [Lu et al, 20051 and transport of the encapsulated
material into the
brain. Toxicity studies showed that the dose which induced the first toxic
signs was 10-20 times
higher than the doses needed to obtain analgesia by encapsulated analgesic
peptides.
[00352] The addition of chitosan (CS) surface groups, by employing CS-
vernolate conjugates,
increased BBB permeability of the vesicles (Figure 4B), probably by increasing
transcytosis
[Newton, 20061. However, the CS groups, when added to the vesicles by
employing fatty acid-
CS conjugate (in this case, vernolic acid), are not stable in circulation as
surface groups because
of the low energy barrier for lipid exchange of such conjugates. Thus
bolaamphiphiles with
chitosan head groups were synthesized and used to form vesicles with better
penetration into the
brain through the BBB as shown in the examples provided below.
[00353] In addition to the peptide leu-enkephalin, and the small molecules:
CF, uranyl acetate,
kyotorphin and sucrose, the inventors have also successfully encapsulated in
these vesicles the
proteins albumin and trypsinogen and the polysaccharide Dextran-FITC (MW
9000). Albumin-
FITC, encapsulated, was delivered successfully to the brain (Fig. 5B), while
un-encapsulated
albumin-FITC showed little, if any, brain accumulation (Fig. 5A), indicating
that the vesicle
transported the protein into the brain through the BBB. These results strongly
suggest that the
vesicles can be made to encapsulate other molecules, such as agents agains
neurodegerative
diseases such as GDNF and NGF, and other agents agains other diseases such as
anti-retroviral
drugs, and deliver them into the brain without harming the BBB.
Example 11
Statistical analysis
[00354] The data are presented as mean and standard deviations (SD) or
standard errors of
mean (SEM). Statistical differences between the control and the studied
formulations are
analyzed using ANOVA followed by Dunnett post-test using InStat 3.0 software
(GraphPad
Software Inc., La Jolla, CA, USA). P values of less than 0.05 are defined as
statistically
significant.
Synthesis And Delivery Of Neurotrophin-Containing Nanovesicles To The Brain
Introduction
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[00355] The
experiments herein describe the preparation of vesicles of the invention for
the delivery of glial cell line-derived neurotrophic factor (GDNF)
systemically to the brain and
demonstrate the capability of these vesicles to target the delivered GDNF to
brain regions
affected in Parkinson's disease (PD). Delivering GDNF to brain regions
affected in PD, such as
the Substantia Nigra pars compacta (SNpc) and the striatum (STR), may be
beneficial in slowing
down the progression of PD and may even promote neurorestoration, thus
improving the status of
the PD patient [Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L,
McBride J,
Chen EY, Palfi S, Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD,
Carvey P, Ling
Z, Trono D, Hantraye P, Deglon N, Aebischer P. Neurodegeneration prevented by
lentiviral
vector delivery of GDNF in primate models of Parkinson's disease. Science.
2000;
290(5492):767-7731. However, GDNF does not permeate through the blood-brain
barrier (BBB)
and, to demonstrate efficacy, it has to be delivered directly into the brain
[Slevin JT, Gash DM,
Smith CD, Gerhardt GA, Kryscio R, Chebrolu H, Walton A, Wagner R, Young AB.
Unilateral
intraputamenal glial cell line-derived neurotrophic factor in patients with
Parkinson disease:
response to 1 year of treatment and 1 year of withdrawal. J Neurosurg. 2007;
106(4):614-6201.
Nevertheless, attempts to deliver GDNF directly into the brain by
intraputamenal injection, or
convection enhanced delivery (CED) were generally not successful, most
probably because of
poor distribution of the delivered GDNF within the brain, which was restricted
to only 2-9% of
the application area [Salvatore MF, Ai Y, Fischer B, Zhang AM, Grondin RC,
Zhang Z,
Gerhardt GA, Gash DM. Point source concentration of GDNF may explain failure
of phase II
clinical trial. Exp Neurol. 2006 ;202(2):497-505; Gash DM, Zhang Z, Ai Y,
Grondin R, Coffey
R, Gerhardt GA. Trophic factor distribution predicts functional recovery in
parkinsonian
monkeys. Ann Neurol. 2005; 58(2):224-2331. The limited GDNF diffusion
throughout the brain
was accounted for by its tight binding to the extracellular matrix [Hamilton
JF, Morrison PF,
Chen MY, Harvey-White J, Pernaute RS, Phillips H, Oldfield E, Bankiewicz KS.
Heparin
coinfusion during convection-enhanced delivery (CED) increases the
distribution of the glial-
derived neurotrophic factor (GDNF) ligand family in rat striatum and enhances
the
pharmacological activity of neurturin. Exp Neurol. 2001; 168(1):155-161]. This
implies that a
delivery system capable of transporting GDNF to a wide area within the brain
and targeting it to
brain regions which are affected in PD should increase the probability that
all affected neurons
are exposed to therapeutic concentrations of the neurotrophin and, thus,
increase its efficacy in
the treatment of PD.
[00356] The experiments herein describe the development of nano-sized stable
vesicles based
on the novel delivery system. These nano-sized vesicles are made of novel
bolas that are the
building block materials for nanoparticles used as a drug delivery system that
can self-assemble
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into vesicles with monolayer membranes. These nanoparticles are more stable
than liposomes
made of bilayer membranes, due to the higher energy barrier for lipid exchange
that characterizes
monolayer membranes made from bolas [Fuhrhop JH, Wang T. Bolaamphiphiles, Chem
Rev.
2004; 104:2901-29371. The high stability of such vesicles allows them to
circulate in the blood
stream until they reach the brain, and then penetrate the blood brain barrier
(BBB) to deliver their
cargo into the brain. In addition, the monolayer membrane is thinner than the
bilayer membrane,
which is an important parmater in nano-sized vesicles, since is provides a
higher inner volume
for encapsulating drugs and biologically active compounds, as compared to
liposomes of the
same size that are made of a bilayer membrane. The vesicles described herein
may also be
characterized as providing a controlled-release mechanism that enables the
release of the cargo
preferentially in the brain.
[00357] The experiments herein describe, for delivery of GDNF to brain regions
that are
affected in PD, provide vesicles two important components: a) a bola with a
chitosan (CS) head
group for increasing BBB permeability of the vesicles, and b) a bola with a
dopamine transporter
(DAT) ligand for targeting the vesicles to dopaminergic cells in the brain.
Materials And Methods
[00358] Chemicals:
[00359] Vernonia
oil that was used as the starting material for the synthesis of the bolas was
purchased from Ver-Tech, Inc., Bethesda, Maryland, USA. Chitosan-vernolate
conjugate was
synthesized in our lab. Pyridostigmine(3-(Dimethylamino-carbonyloxy)-1-
methylpyridiniumbromide); Carboxyfluorescein; Triton X-100 (t-Octylphenoxy-
polyethoxyethanol); Triton X-100 - Reduced form; Cholesterol (5-Cholesten-30-
ol); Cholesteryl
Hemisuccinate (5-Cholesten-30-ol 3-hemisuccinate); Sephadex G-50, 50-150
micron; Trizma
Base (Tris{hydroxymethyl} aminomethane) and its hydrochloride salt; Trichloro
acetic acid
(TCA); trypsinogen from bovine pancreas and chitosan; all were of analytical
grade and
purchased from Sigma Chemicals. Human GDNF (hGDNF) and hGDNF-sulfo-NHS-LC-
biotin
(GDNF-biotin) were purchased from Alomone Labs, Jerusalem. Cocaine that was
used for the
synthesis of the DAT ligand was obtained under license from the Chief
Pharmacist of the
Regional Health Office, Southern Region, Ministry of Health. AlexaFluor0-488
Protein Labeling
Kit (A10235) and AlexaFluor0-488 Microscale Protein Labeling Kit (A30006) were
bought
from Invitrogen. Other standard chemicals were all purchased from commercial
sources.
Solutions for inducing anastasia in animals (Ketamine HC1 100 mg/ml and
Xylazine 2%) were
obtained from the Ben Gurion University (BGU) animal facility.
[00360] Synthesis of bolaamphiphiles (bolas) and chitosan-fatty acid conjugate
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[00361] Vernonia oil was hydrolyzed to obtain vernolic acid or transesterified
with methanol to
obtain the methyl vernolate, both compounds were used as starting materials
for the synthesis of
bolaamphiphiles, described below. Vernolic acid has an epoxy group that
provides a reactive
moiety to which functional groups are conjugated.
[00362] Vernolate-Chitosan that was used for comparison to the bola with
the chitosan head
groups, as described below, was synthesized by attaching a low molecular
weight chitosan to N-
hydroxy succineimide vernolate.
[00363] Analysis of the synthesized bolas
[00364] Elemental analysis was outsourced to a commercial laboratory. FT-IR
analysis was
carried out on a Nicolet spectrometer. 1I-1 and 13C NMR (500 MHz) spectra were
recorded on a
Brucker WP-500 SY spectrometers, respectively, in CDC13 with TMS as the
internal standard or
d6DMS0 solutions. HPLC analysis was carried out on a C18RP column with an
evaporative
light scattering detector (evaporation temperature 46 C; mobile phase
methanol:water (9:1, v/v);
flow rate 0.5 ml/min). MS analysis was carried out on a Waters Micromass Q-TOF
Premier Mass
spectrometer (Waters-Micromass, Milford, MA, USA).
[00365] Vesicle Preparation
[00366] The basic components of the vesicles were the bolas GLH-19 and GLH-20.
In addition
to the bolas, the vesicle formulation contained the additives cholesterol and
cholesteryl
hemisuccinate and as indicated in the text, some formulations included also CS-
vernolate
conjugate or GLH-55a (a bola with CS head group) and/or GLH-57 (a bola with
DAT ligand
head groups). Unless otherwise stated, the ratio between GLH-19 and GLH-20 was
2:1. This
ratio was found to give stable vesicles that release their content in a
controlled manner (see
Results). Thus, the different formulations used in this project were: (a) GLH-
19+GLH-20
(2:1)/cholesterol/cholesteryl-hemisuccinate (10/1.6/2.1); (b) GLH-19+GLH-
20/cholesterol/cholesteryl-hemisuccinate/CS-conjugate (10/1.6/2.1/1); (c) GLH-
19+GLH-
20/cholesterol/cholesteryl hemisuccinate/GLH-55a (CS-bola) (10/1.6/2.1/1); (d)
GLH-19+GLH-
20/cholesterol/cholesteryl hemisuccinate/GLH-57 (bola DAT) (10/1.6/2.1/0.8);
(e) GLH-
19+GLH-20/cholesterol/cholesteryl hemisuccinate/CS conjugate or GLH-55a/GLH-57
(10/1.6/2.1/1/0.8).
[00367] Vesicles were prepared from the formulation described above by known
methods: (a)
Film Hydration followed by extrusion; or (b) Film hydration followed by
sonication. Vesicle
formation was conducted at room temperature (i.e. 25 C), which is above the
transition point of
the bolaamphiphilic compounds used in the present study. When the vesicles
were prepared by
extrusion, the bolas and the additives were dissolved in an organic solvent
(usually chloroform).
The solution was then placed in a vial and dried under stream of nitrogen. The
film that was
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formed in the vial was then placed under vacuum overnight to remove residual
solvent. Then, the
thin film was hydrated by adding an aqueous solution containing the material
to be encapsulated
in the appropriate buffer solution. Then the solution was vortexed and
extruded using a LipexTm
extruder (Northern Lipids Inc.) via 0.2 and then 0.1 um Polycarbonate
membranes until the
solution became transparent (approx. 8-10 times for each membrane). The
polycarbonate
membranes were manufactured by GE Water & Process Technologies, and purchased
from
Tamar Laboratory Supplies Ltd., Israel. When vesicles were prepared by film
hydration followed
by sonication, the first steps of film formation and then hydration are
similar to those described
for the extrusion method, then, the hydrated film was further sonicated, using
a probe sonicator
(Vibra Cell Model H540/CV54, Sonics and Materials USA). Probe sonication was
carried out at
4 C for 10-14 min (15 sec pulses and 10 sec rest).
[00368] Vesicle characterization
[00369] The vesicles were characterized with respect to morphology (by cryo-
transmission
electron microscopy - cryoTEM), size and size distribution (by dynamic light
scattering ¨ DLS),
surface charge (by Zeta potential analyzer) and stability (by fluorescent
measurements).
[00370] Cryo-Transmission Electron Microscope (Cryo-TEM): Samples of vesicles
(about 5 -
pt) were deposited on 300-mesh holey carbon cupper grids (Ted Pella, Inc.
Redding, CA). A
drop of 5 ul was applied to the grid and blotted with a filter paper to form a
thin liquid film of the
solution. Grids were rapidly plunged into a liquid ethane bath cooled with
liquid nitrogen and
maintained at a temperature of approximately ¨170 C using a cryo-holder. The
samples were
imaged at ¨180 C using a FEI Tecnai 12 G2 TEM, at 120kV with a Gatan cryo-
holder
maintained at -180 C. Images were recorded with the Digital Micrograph
software package, at
low dose conditions, to minimize electron beam radiation damage, at the Ilse
Katz Institute for
Nanoscale Science and Technology of Ben-Gurion University ("BGU").
[00371] Dynamic light scattering (DLS): Vesicle size and homogeneity was
determined by
DLS using HPPS-NIBS, light scattering apparatus (ALV-Laser, Langen, Germany)
with the laser
powered at 3mW HE-Ne laser line (632.8nm), at the Ilse Katz Institute for
Nanoscale Science
and Technology of BGU. Standardyesicle solutions were diluted 1:10 and loaded
into a cuvette
for light scattering measurements. The measurements were conducted at an angle
of 1730, during
30-180 seconds, from different positions of the cell in order to avoid
measurements of multiple
scattering.
[00372] Zeta-potential measurements: Particle size and zeta potential were
measured by using
zeta potential and Particle size analyzer, ZetaPlus, (Brookhaven Instruments
Corporation Ltd,
NY, USA), in the range of 10-1000 nm, in the Chemistry Department of BGU.
Vesicle solutions
were diluted 1:10 in appropriate buffers and loaded into a 4 ml cuvette for
light scattering
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measurements. The measurements were conducted at an angle of 900, at 10
repeated
measurements, and zeta potential was estimated as an average of 5 repeated
readings.
[00373] Vesicle stability: To determine vesicle stability, samples of
carboxyfluorescein (CF)-
loaded vesicles (see below method for loading vesicles with CF and
determination of percent
encapsulation) were incubated in PBS and percent encapsulation was determined
at different
times. For the measurement of vesicle decapsulation by acetylcholine esterase
(AChE), the
fluorescence of a sample of intact CF-loaded vesicles was monitored in a
quartz cuvette under
constant stirring for a few minutes, until a stable fluorescence reading was
obtained, and then,
AChE (2 ill containing 2 units) was added and the fluorescence measurement
continued for
additional 5 min. At this point Triton X100 (0.15% final concentration) was
added to break the
remaining vesicles and to obtain the total fluorescence of the encapsulated
CF.
[00374] To determine the stability of vesicles with encapsulated protein,
vesicle samples were
incubated in PBS and the percent encapsulation was determined at different
times. Stability was
also determined by changes in the vesicle size (by DLS, as was described in
previous sections) at
various time points after vesicle preparation.
[00375] Encapsulation experiments
[00376] Encapsulation was achieved by including the material to be
encapsulated in the
hydration buffer during the hydration stage of the vesicle preparation (see
above). After the
vesicles were formed and the material in the hydrating buffer was
encapsulated, non encapsulated
material was removed over a Sephadex G-50 column (for details see below).
Encapsulation
efficiency was determined initially with CF as described in Popov eta!
[10,13]. Briefly, the
encapsulation capacity of CF was assessed by measuring the fluorescence
intensity (at excitation
wavelength of 492 nm and emission wavelength of 517 nm) of CF-loaded vesicular
preparation
before and after disrupting the vesicular structure by Triton X100 at a final
concentration of
0.15%. The released CF is dequenched and emits a fluorescent signal, which is
quantified by
comparing to a calibration curve. The encapsulation efficiency was calculated
according to the
following equation:
R õ ¨ R,
¨ ____________________________ x 100 = %Encapsulation
RAf
where RB is the initial fluorescence reading and RAf is the fluorescence
reading after the addition
of Triton X-100.
[00377] The encapsulation efficiencies of trypsinogen and GDNF were assessed
by first
running the sample through a Sephadex G-50 column to remove non-encapsulated
protein from
the encapsulated protein. The fractions obtained from the column (generally
0.5 ml per fraction)
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were treated by Triton X-100 reduced form to avoid interference with the
absorbance reading of
the encapsulated material at 280 nm. The concentration of trypsinogen or GDNF
proteins was
measured by either UV absorbance at 280 nm, or when the quantities of the
proteins were low,
the proteins were labeled with AlexaFluor0-488 (see procedure for labeling the
proteins below)
and their quantities determined by fluorescence spectroscopy. Percent
encapsulation was
determined by dividing the area under the curve of the vesicle fractions by
the total area under
the curve, which was the sum of the area under the curve of the vesicle
preparation and the area
under the curve of the free protein. With the use of a calibration curve the
concentrations in each
were determined.
[00378] To encapsulate biotinylated GDNF (GDNF-biotin) in the vesicles, the
following
procedure was used for 100pg GDNF-biotin: The biotinylated GDNF is dissolved
in 1 ml
distilled water. Empty vesicles are prepared by film hydration followed by
sonication, using
formulation e, which is described in the section on vesicle preparation above.
The GDNF-biotin
solution is then added to 1 ml vesicle suspension and the solution is
sonicated on ice to form
vesicles made of 5 mg/ml of the basic bolaamphiphile with about 70pg of
encapsulated GDNF-
biotin (the encapsulation efficiency is about 70%).
[00379] Labeling of trypsinogen and GDNF for detection of low quantities
[00380] Since GDNF would be used at sub milligram range and its spectroscopic
absorption
could not be accurately measured at these concentrations, therefore,
fluorescent tagging was
investigated as a means of increasing the sensitivity of the determination of
low quantities of the
encapsulated protein. For labeling the protein, we used AlexaFluor0-488, which
emits a strong
and stable fluorescent signal. To label small quantities of the protein, we
used a microscale
Protein Labeling Kit (A30006) that was purchased from Invitrogen. For labeling
we dissolved 20
pg of the relevant protein (either trypsinogen or GDNF) in sterile 0.1M Na2CO3
(total volume of
20 pl to form a concentration of 1000 pg/ml protein). Then, we added the
reactive dye, which
reacted with the protein to form AlexaFluor0-488-protein conjugate. To purify
the labeled
protein, we centrifuged the product over a resin provided by the manufacturer
and the effluent
contained the purified labeled protein, which can be determined quantitatively
by fluorescent
measurement at an excitation wavelength of 492 nm and an emission wavelength
of 517 nm.
[00381] Purification of the vesicles from the non-encapsulated material
[00382] The vesicles were purified by size exclusion chromatography on
Sephadex G50
columns. The eluting buffer for the routine vesicle purification was 16 mM
NaCl in phosphate
buffer pH 7.3 but other eluting buffers were used as described in the Results
Section. The Flow
rate used for the elution was 15 ml/hr. Column dimensions were 20cm X 0.7cm
(length and
diameter, respectively). The volume of each fraction collected from the column
was 0.5 ml
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(equal to 1-2% of the total column volume). Optical density or fluorescence of
each fraction was
measured to determine the concentration of the eluted material.
[00383] Determination of GDNF activity
[00384] To determine the activity of GDNF, we measured the effect of
20ng/m1hGDNF on the
activation of the enzymes AKT and MAPK using SH-SY5Y neuroblastoma cells
(obtained from
the ATTC). For this measurement, we plated the cells in 12 well plates coated
with PEI at a
density range of 3x105- 6x105 cells/well 48h prior to the bioassay. Cells were
deprived of serum
for 2.5h, stimulated by the test material (plain medium as a control or free
GDNF or encapsulated
GDNF), washed once with ice-cold PBS and then lysed in 120W/well sample
buffer. The lysates
were boiled, sonicated and centrifuged and then loaded onto 10% acrylamide gel
for SDS-PAGE.
After the electrophoresis, the samples were immune-blotted using antibodies
against phospho-
MAPK44/42 and against phospho-AKT.
[00385] Determining the integrity of GDNF after encapsulation within the
vesicles
[00386] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
was used to
examine whether the encapsulation process affects the integrity of the
encapsulated GDNF. The
test samples were applied on a 5%-/15% SDS-polyacrylamide gel and
electrophoresis was
performed using a running buffer of 14.4 g glycine and 3 g Tris base per Liter
with 1% SDS. The
gels were then stained by Coomasie blue using the Bio-Safe Coomassie staining
protocol and
then destained for 30 min in water.
[00387] Determining targeting of the vesicles to cultured cells that express
the dopamine
transporter (DAT)
[00388] Three cell lines were used to test the ability of the surface DAT
ligand to target the
vesicles to the dopamine transporter: (a) HeLa cells ¨ human cervical cancer
cells that do not
express the dopamine transporter and were used as control cells. HeLa cells
were grown in
DMEM medium supplemented with 5% fetal bovine serum, 2 mM L-Glutamine, 100
IU/mL
penicillin and 100 pg/mL streptomycin at 37 C under humidified atmosphere with
5% CO2. (b)
PC-12 cells - derived from a rat pheochromocytoma and which highly express the
dopamine
transporter [19]. PC-12 cells were grown in RPMI-1640 medium, supplemented
with heat-
inactivated horse serum to a final concentration of 10%, fetal bovine serum to
a final
concentration of 5%, 2 mM L-Glutamine, 100 IU/mL penicillin and 100 pg/mL
streptomycin at
37 C under humidified atmosphere with 5% CO2. (c) SH-SY5Y human neuroblastoma
cells that
were reported to express the dopamine transporter [20]. SH-SY5Y cells were
grown in DMEM
medium supplemented with 5% fetal bovine serum, 2 mM L-Glutamine, 100 IU/mL
penicillin
and 100 pg/mL streptomycin at 37 C under humidified atmosphere with 5% CO2.
Vesicles are
taken up by these cells after the vesicles adhere to the cell surface.
Vesicles that contain DAT
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ligand on their surface will bind to cells that express the dopamine
transporter. The binding of the
vesicles to the cells facilitate the uptake and therefore, the extent of the
internalization of the
vesicles into the cells may be used as an index for targeting.
[00389] To measure uptake of the vesicles into the cells, vesicles were loaded
with
carboxyfluorescein (CF) and the cells were contacted with these CF-loaded
vesicles. Uptake of
the vesicles by the cells labeled the cells with the fluorescent marker
encapsulated in the vesicles
and the fluorescent cells identified by flow cytometry. Thus, to measure
uptake of CF-loaded
vesicles into the cells, cells were plated in 24 well plates at a density of
200,000 cells/well and
after 24 hours, the the medium was replaced with a culture medium without
serum and incubated
in this medium for 30 min. Encapsulated or non-encapsulated CF were then added
to the cells
(free 0.1 lig CF or the same amount of CF encapsulated in 5 lig vesicles) and
the cells were
incubated for 3-5 hours. At the end of the incubation, the cells were washed
with PBS and
detached from the bottom of the well by trypson-EDTA. The cell suspension was
analyzed by
flow cytometry (FACS).The fluorescence intensity of the treated cells was done
by the FlowJo
software.
[00390] In vivo studies
[00391] Animals: Eight-week-old male ICR or 10-week-old male C57BL/6 mice,
weighing
between 25-30g, were maintained on standard mice chow and tap water ad lib.
The mice were
kept in 12 hours light/dark cycles at a temperature of 25 3 C. All the animal
experiments were
performed according to the protocol approved by the Animal Care and Use
Committee of BGU,
according to an approved protocol (# IL-24-04-2008).
[00392] Injection of test material
[00393] Unless otherwise stated, animals were pretreated 15 min before the
injection of the test
material by pyridostigmine (o.5 mg/kg, i.m.) to inhibit peripheral ChE and
thus, reduce vesicle
decapsulation in the blood circulation before they enter the brain. The test
material was injected
i.v. into the tail vein in a volume of 100-200 1 per mouse.
[00394] Determination of CF concentrations in tissues after injecting CF-
loaded vesicles
into mice
[00395] The test material (either free CF or encapsulated CF) was injected
into the tail vein of
mice in a volume of up to 200 pl. The quantity of encapsulated CF was always
determined prior
to the administration and similar amounts of either encapsulated or free CF
were injected. At 30
minutes after the injection, mice were anesthetized by Xylazine-Ketamine and
blood was
withdrawn through cardiac puncture, the mouse was perfused with 10 ml PBS and
tissues were
dissected out. The specimens were homogenized in PBS, deproteinated by 5%
(final
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concentration) of tricholoro acetic acid (TCA), centrifuged and the
supernatants were used for
fluorescence measurements.
[00396] Tissue distribution of Trypsinogen AlexaFluor0-488 conjugate after
i.v. injection
[00397] Trypsinogen- AlexaFluor0-488 was injected i.v. in its free form or
encapsulated in the
bolavesicles. At 30 min after the injection, mice were anesthetized by
Xylazine-Ketamine
mixture and blood was withdrawn through cardiac puncture, the mouse was
perfused with 10 ml
PBS and tissues were dissected out. The dissected tissues were attached to
labeled paper stripes,
frozen in isopentane cooled over liquid nitrogen, and stored at -80 C. The
selected tissues were
cryosectioned and the fluorescence of the sections was analyzed using confocal
fluorescent
microscopy. All images were acquired using the same imaging settings and were
not corrected or
modified. Fluorescence of slices from different organs was quantified by
imaging software after
subtracting background fluorescence. In effect, for quantifying the
fluorescence in the tissue, the
average fluorescence obtained from the control mice, which were injected with
PBS instead of
the fluorescent material was subtracted from the fluorescence values obtained
from the same
tissue taken from animals that received Trypsinogen-AlexaFluor0-488. At least
4 slices from
each organ of each mouse were used for the quantitative analysis and each
group contained 4-5
mice.
[00398] Distribution of delivered biotinylated GDNF (GDNF-biotin) in the brain
[00399] Mice were injected with the test material that contained either non-
encapsulated
GDNF-biotin, or encapsulated GDNF-biotin following the procedure that was
described above
for the labeled trypsinogen, including the procedure for the collection of the
tissues. The frozen
brains were cryosectioned and then, stained with DAPI and with avidin-
AlexaFluor0-488. The
details of the staining are as follows: AlexaFluor0-488 was added to the
sections and after a few
minutes, the sections were washed 3 times with PBS. Then, 20 ill of DAPI
solution was placed
on each section and incubated at room temperature for 10 min. The sections
were then washed
twice with PBS and wiped gently around the tissue with a paper towel. At this
stage, the sections
were left to dry in the air. After the staining procedure, the sections were
mounted on slides using
Mowiol-based mounting solution and fluorescence of the images (3-4 images per
each section)
were taken, using the CF and the DAPI channels of the confocal microscope.
[00400] Statistical analysis
[00401] The significance of the differences between the experimental groups
was analyzed
using the Student t-test.
Example 12
Synthesis of bolaamphiphiles (bolas)
[00402] Synthesis of basic bolas
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[00403] Bolas GLH-19 and GLH-20 were synthesized and used as the basic
building blocks for
the preparation of vesicles. Briefly, the carboxylic group of vernolic acid
(compound 1 in
Scheme 1) was reacted with aliphatic diols to obtain the bisvernolester 2
(Scheme 1, below). This
bisvernolester is the skeleton for both bolas GLH-19 and GLH-20. Then, the
epoxy group of the
vernolate moiety, located on C12-C13 of the aliphatic chain of vernolic acid,
was used to
introduce two ACh headgroups on each side of the alkyl chain, via one of the
two vicinal carbons
obtained after opening of the oxirane ring. For GLH-20 (Scheme 2, below), the
ACh head group
was attached to the vernolate skeleton through the nitrogen atom of the
choline moiety. The
attachment of the head group was carried out in a two-stage synthesis: First,
the epoxy ring of the
bisvernolester was opened with an excess of chloroacetic acid in toluene at 85
C and, in the
second stage, we performed quaternization using a threefold excess of the /V,N-
dimethylamino
ethyl acetate. After removing the excess of the amine, the crude product was
purified by flash
chromatography with acetonitrile:water as the eluent. The mass spectrum, ESI-
MS calculated for
C62H114N2014C12 : ([M-2C11+/2 = 555.5, of the bola GLH-20 is shown in FIG 1A:
[00404] Scheme 1: Formation of the bola's skeleton
H
1
HO-(CH2r-OH lCiaasndeida antarctica
'1CIO 0 0
0=0 (CH 2)n
2
[00405] Scheme 2: Synthesis of GLH-20:
2
0
CI 0
OH
0 0 / 0 HO 0
FlOn
0 0
C I ...04k 0 OH 0 0 HO 0)C1
\/ \)¨<==\/ \ MA0.. IC n 0 /N/ %,==.?¨<M
>1%."0
Cle
0
GLH-20
=
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[00406] For GLH-19 (Scheme 3, below), a bola with an ACh head group attached
to the
vemolate skeleton through the acetyl group, the compound was prepared in a
three-stage
synthesis, including opening of the epoxy ring with glutaric acid, then
esterification of the free
carboxylic group with /V,N-dimethyl amino ethanol and the final product was
obtained by
quatemization of the tertiary amine, with methyl iodide followed by exchange
of the iodide ion
by chloride using an ion exchange resin. The mass spectrum for GLH-19
calculated for
C66H122N204C12, ESI¨MS/MS (positive mode) m/z: [M+]/2 = 583.6 (z = 2) is shown
in Fig. 1B.
[00407] Scheme 3: Synthesis of GLH-19:
2
HOOCICH233-COOH 0 0
0 0 0 HO 0)CLOH
/\/=õ,-1=õ/=\WA0.0(CF12)%0A,W/---0-<~=
0 0 0 0
HO/NAO OH 0 0 HO 0)CAOH
EDCI
DMAP
OH 0 0 HO A" \Ao"..= it =
========)-(================011Ø0 (C He = 0011-1==========
CH3X
0 0 \
HO - 0.0 (CHon
- OH
[00408] Synthesis of a bola with chitosan (CS) head group.
[00409] Depolymerization of chitosan as a first step for the synthesis of
bolaamphiphile with
chitosan head group. To enhance penetration of the vesicles into the brain,
they were
"decorated" on their surface with chitosan (CS) head groups. This was
accomplished by
incorporating into the vesicle formulation a bola with a novel, chitosan-
containing head group.
[00410] The synthesis of this new bolaamphiphile included the following: (1)
preparation of a
low molecular weight chitosan (LMWCS), (2) synthesis of an asymmetric bola
skeleton, and
(3) binding the head groups to the skeleton. The starting material for the
preparation of LMWCS
is commercial chitosan, which is of high-molecular weight, and is insoluble in
water and organic
solvents. The LMWCS, which has improved water solubility, could be obtained by
a
depolymerization reaction of the commercial high molecular weight chitosan,
using hydrogen
peroxide (H202), which is a strong oxidant that produces free radicals, which
can attack the (3-D-
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(1-4)-glycosidic bond and depolymerizes chitosan. Oxidative depolymerization
of chitosan by
heterogeneous treatment of commercial high-molecular chitosan (MW ¨ 50 kDa)
with hydrogen
peroxide, was accomplished by a dropwise addition of 30% hydrogen peroxide
solution to
chitosan dispersed in water at 60 C for 6 h. The filtrate, after separation
of insoluble fragments,
was evaporated and precipitated by ethanol to obtain LMWCS, providing, e.g., 2
g of the
LMWCS using the method described above.
[00411] The FT-IR spectrum of the LMWCS
[00412] The FT-IR spectrum of the LMWCS (FIG. 2) exhibited most of the
characteristic
absorption peaks of the original chitosan with some differences. The
vibrational band at 1154
-
cm1 , which corresponds to the ether bond between the pyranose rings, was
weakened, indicating
rupture of the P-glucosidic bonds in the molecular chain of chitosan. The band
at 1596 cm-1 in
LMWCS becomes stronger than that of the original chitosan, suggesting that the
content of
amino groups and correspondingly, the degree of deacetylation (DD) changed.
The decrease in
the NH2 (pH-potentiometric titration of amino groups) content in LMWCS may be
explained by
the presence of "other groups". These "other groups" may be titrated together
with the amino
groups, for example, carboxylic groups. The carboxylic groups of the LMWCS
were determined
by a direct titration with sodium hydroxide. In fact, LMWCS contained 1.05 ¨
1.15 mmol
carboxylic groups per gram of chitosan (Table 2), below:
Table 2: Chemical analysis of original and LMWCS
Chitosan %, C %, H %, N N/C COOH,
mmol/ g
Original 41.22 7.15 7.34 0.178 0
LMWCS 38.88 6.63 6.12 0.157 1.14
[00413] Table 2 also shows that the depolymerization of the commercial
chitosan led to a
decrease in nitrogen, carbon and hydrogen contents, suggesting an increase of
oxygen content.
The mass ratio N/C decreased, confirming the loss of nitrogen as the result of
the
depolymerization. Thus, the depolymerization of chitosan led not only to the
rupture of the P-
glucosidic bonds, but also to a change in the chemical structure of the
original chitosan and
possibly, to the formation of carboxylic groups. This result correlates with
data previously
presented in the literature [23,24] that show the effect of H202 treatment on
chitosan.
[00414] MALDI-TOF mass spectrometry of LMWCS
[00415] The composition of the LMWCS chitosan was also analyzed by MALDI-TOF
mass
spectrometry. The analysis of the mixture of oligomers obtained by the
depolymerization of the
original chitosan was performed in a positive-ion mode. Table 3 (below) shows
that the
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depolymerization of the original chitosan led to the formation of oligomers
with a degree of
polymerization (DP) between 3 and 8. The peaks correspond to oligomers
carrying fragments of
deacetylated (GLcN) and acetylated (GLc NAc) chitosan. Deacetylated chitosan
(G1cN) contains
a repeat unit of C6H1104N, with a MW of 161 Da and the acetylated chitosan
contains a repeat
unit of C8I-11305N, with a MW of 203 Da.
Table 3
Assigned ion composition of LMWCS, determined by MAILDI-TOF analysis
(Solvent CH3CN, H20)
m/z Types Ion Composition
524.2 [M + Nal+ (G1cN)3
539.6 [M +I(1+
566.1 [M + Nal+
(G1cN)2-(G1cNAc)
582.1 [M + Kl+
624.1 [M + Kl+ (G1cN)-(G1cNA02
685.1 [M + Nal+ (G1cN)4
727.3 [M + Nal+ (G1cN)3-(G1cNAc)
743.1 [M + Kl+
769.1 [M + Nal+ (G1cN)2-(G1cNA02
811.1 [M + Nal+ (G1cN)- (G1cNAc)3
846.2 [M + Nal+ (G1cN)5
888.2 [M + Nal+ (G1cN)4-(G1cNAc)
930.2 [M + Nal+
(G1cN)3-(G1cNA02
946.2 [M + Kl+
972.2 [M + Nal+
(G1cN)2-(G1cNA03
988.2 [M + Kl+
1014.1 [M + Nal+ (G1cN)- (G1cNA04
1091.2 [M + Nal+ (G1cN)4-(G1cNAc)2
1133.2 [M + Nal+ (G1cN)3-(G1cNAc)3
1175.3 [M + Nal+ (G1cN)2_(G1cNAc)4
1252.3 [M + Nal+ (G1cN)5-(G1cNA02
1294.3 [M + Nal+ (G1cN)4-(G1cNAc)3
1455.3 [M + Nal+ (G1cN)5-(G1cNAc)3
1497.8 [M + Nal+ (G1cN)4-(G1cNAc)4
1540.0 [M + Nal+ (G1cN)3-(G1cNAc)5
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1658.3 [M + Nal+ (G1cN)5-(G1cNAc)4
Example 13
Synthesis of an asymmetric bola with a chitosan head group
[00416] This example describes the synthesis of an asymmetric bola with an
acetylcholine
(ACh) head group on one side of the bola's skeleton and a CS head group on the
other side of the
bola's skeleton (bola-CS). The rationale behind such a bola comes from packing
parameters
considerations. The ACh head group on the bola-CS is smaller than the CS head
group and is
similar to the ACh head groups of the symmetrical bolaamphiphile GLH-20. Thus,
during
aggregation, the ACh head group of the bola-CS is expected to be situated on
the inner
membrane surface of the vesicle, together with one of the ACh head groups of
GLH-20 and
GLH-19 (which also has an ACh head group, but attached in a different way to
the hydrophobic
skeleton). The CS head group will thus become an outer surface moiety and will
be free to
interact with the endothelial cells of the BBB, thus enhancing BBB
permeability of the vesicles.
The synthesis of this asymmetric bola-CS is a multi stage process that is
depicted in Scheme 4,
below.
[00417] Scheme 4: Stages in the synthesis of bola-CS
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HOOH
1
C =N# 01-1
Zi 3 0
HO
l'IN/4%/AOH
0
0 0
0
0
0
1
6 <-00
0
0 H 0
=<,0140., C)1,/ \/\ ¨
7 0
1 o
0 0 0 0
CrNs, Nes,"0 \W/No C)1.4
ck.3 0
9
chitosan HoJrBC) 01;)41-9r,-
o o Rii V'=0 .10
N P
,A04`Nkv.õ.,INAE ."`....."....".."...".== ==1
CO'
GLH-55a
[00418] For the synthesis of symmetric bolas, such as GLH-19 and GLH-20,
the strategy
followed ws to to form the bolaskeleton first and then attach the head groups
to both ends of the
hydrophobic chain. This strategy was revised for the synthesis of an
asymmetric bola as follows:
[00419] Stage 1: For the asymmetric bola GLH-55a, the synthesis began with the
formation of
monochloroacetate of decanediol 3 through the esterification of 1,10-
decanediol 1 with
chloroacetic acid 2 at a molar ratio of 5:1 respectively (Scheme 4). The
reaction was carried out
in toluene by azeotropic distillation in the presence of Amberlyst 15 as a
heterogeneous acidic
catalyst that can be easily removed by filtration at the end of the reaction,
avoiding the tedious
work needed to neutralize the soluble acidic catalyst. 11-1NMR of the product
displayed the
characteristic bands attributed to the new chloroacetate moiety: a singlet at
4.05 ppm arising from
the chloromethylene protons (CH2C1) and a triplet at 4.17 ppm arising from the
methylenic
protons of the ester group (CH,-0-00). The corresponding chemical shifts in
13C-NMR
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PCT/US2016/027726
spectrum appeared at 40.95 ppm (LH2C1) and 66.42 ppm (H2-0-00). The carbonyl
signal
appeared at 167.45 ppm.
[00420] Stage 2: The second step of the synthesis (Scheme 4, above)
includes the
elongation of the hydrophobic chain. The intermediate 5 was obtained by
reacting the
monochloroacetate of decanediol 3 with a dicarboxylic acid 4 (1,10-
decanedicarboxylic acid) at a
molar ratio of 1:5, respectively. The reaction mixture was refluxed in toluene
with constant
removal of water by azeotropic distillation and was catalyzed by Amberlyst 15.
The crude
product was purified over a silica gel flash chromatography. The chloroacetate
intermediates
were then characterized by 1I-1 and 13C NMR spectroscopy (Table 4, below).
Table 4: 1I-1 and 13C NMR data for derivative 5
0
14 16 18 20 22 W 8 6 4 2 0 $0 H
12 - 9 7 5 3 1 II 15 17 19 21 23
0
Group 111 NMR 13C NMR
no.
6 II 6 c (ppm)
(ppm) Multiplicity
1 4.05 t 64.40
10 4.17 t 66.42
11 167.46
12 4.06k' s 40.95
13 174.10
14 2.34 t 33.62
23 2.29 t 34.40
24 179.23
a overlapping with the triplet at 4.05
[00421] Stage 3: The next step was the preparation of an active ester of
the carboxylic acid
with N-hydroxysuccinimide (NHS). The active N-hydroxy- succinimide of the
ester of the
carboxylic acid was synthesized by reacting intermediate 5 (Scheme 4) and N-
hydroxy-
succinimide in the presence of a coupling agent (dicyclohexylcarbo-diimide
DCC) at room
temperature by the method of [25]. The pure intermediate 7 was isolated by
flash
chromatography on silica gel with hexane ¨ ether as the eluent. The structure
of product was
confirmed by FT-IR and NMR spectroscopy. The chemical shift of the protons of
the methylene
group, CH,-CO-N, of the NHS in intermediate 7 appeared at 2.83 ppm and the
carbon at 25.60
ppm, respectively.
[00422] Stage 4: In order to attach the acetylcholine head group, the
chloro derivative
obtained in the previous stage was reacted with intermediate 7, that will
serve as the alkylating
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agent with a small excess of the tertiary amine, N,N-dimethylaminoethyl
acetate. The reaction
was carried out in acetone as the solvent at the reflux temperature for about
8 hours. The progress
of the reaction was followed by TLC and HPLC. The reaction mixture was washed
several times
with diethyl ether to remove the excess of the unreacted amine. The degree of
quaternization of
the amphiphile intermediate 9 was about 95-98%, as determined by argentometric
titration. The
molecular weight, as determined by electrospray ionization mass spectrometry
(ESI ¨MS), was
655.41 (690-C1-). In the FT-IR spectra, the absorption bands characteristic of
the chloroacetate
ester group disappeared, and a new absorption band appeared at 1237 cm-1; this
is the so-called
"acetate band".
[00423] Stage 5: This stage constitutes the conjugation of the low
molecular weight
chitosan (LMWCS) (prepared as described above) with the bolaskeleton 9 (Scheme
4) containing
already the cationic head group at one end, and the N-hydroxysuccinimide ester
at the other end.
The conjugation was performed by adding the solution of intermediate 9 in DMSO
to the
solution of LMWCS and triethylamine in DMSO. The molar ratio LMWCS to the
activated ester
9 was 10:1. The reaction mixture was stirred for 72 h at RT. The solution was
lyophilized. The
yellow powder obtained was triturated several times with ether and ethanol, to
remove the
unreacted intermediate 9, filtered and dried. The obtained product, GLH-55a,
is the
bolaamphiphile, having the chitosan head group on one side and the acetyl
choline head group on
the other side of the bolaskeleton, was soluble in DMSO and water.
[00424] When the FT-IR spectrum of bola GLH-55a is compared with the
spectrum of
intermediate 9, the disappearance of the absorption bands at 1784 and 1814 cm-
1, characteristic of
the N-hydroxysuccinimide group is noticeable. Also noticeable is the
appearance of new
absorption bands at 1564 cm-1, characteristic of the amide bond, which was
formed between the
amino group of chitosan and the active ester (NHS ester) of intermediate 9.
Additional
absorption bands, at 1740 cm-1 for the ester group, and 1247 cm-1 for the
acetate group, are also
the result of the conjugation.
[00425] Table 5 and Table 6 present the chemical shifts of the final bola
GLH-55a. As can
be seen, the chemical shifts of the original LMWCS could also be found in the
modified CS
(marked with a star). In 13C-NMR spectrum, the new signals at 52.56 ppm
[N+(CH3)21, 58.22
PPm1N+-CH2-CH2] 62.62 ppm [N+-CH2-CH21, 61.40 ppm [CO-CH2-N+1 indicate the
formation
of conjugation product.
[00426] In the 1H-NMR spectrum of the asymmetric bolaamphiphile with a
chitosan and
acetyl choline head group GLH-55a, we could again find the chemical shifts
characteristic of the
starting LMWCS (marked with a star). The new signals at 3.85 ppm [N+(CH3)21,
4.40 ppm [N+-
CH2-CH21, indicate the formation of conjugation product.
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Table 5: "C-NMR spectrum of GLH-55a (DMSO)
,YD-R CH,0õ
i:i*: - 0
*:.';:i
i? =4140'6NH ''j1.30'Ne%,\M=o ' 1,41-i 'P iiiiiiiiiiiiiiiiii ii
.g 1*
3 ce :. ,o, r=====:õ,_,...--1
::::::::::::::::::::: ::::::::::::.= ,,. '1
NH-CO-013
i!i!i!i!i!i!i!iii: =:::::::::::::::
Carbon in Carbon in
No 69 PPm No 69 PPm
the group the group
1* Cl 102,46 6 N+(CH3)2 52.56
2* C2 57.30 7 CH2-0 64.52
3* C3 70.45 8 CH2-0 66.27
4* C4 78.50 9 N+-CH2-CH2 62.62
5* C5 75.40 10 N+-CH2-CH2 58.22
6* C6 60.91 11 CO-CH2-N+ 61.40
19.02;21.03
7* CH3C0 12 CH2-CO-OCH2 174.63; 175.60
23.43
173.43
1 (CH2)11 29.30-25.0 13 CO-CH3
173.27
2 CH3-CO 19.78 16 0-CO-CH2-N+ 170.13; 169.0
3 NH-C 0-CH2 - 169.44
17 NH-CO
4 CH2-00-0 34.10 165.41
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Table 6: 111-NMR spectrum of GLH-55a
69 PPm
No Proton in the group
d6-DMS0 D20
1* H1 4.51 4.63; 4.55
2* H2
3*,4*,5*6* H3, H4,H5,H6 3.60-3.90
7* H7 2.0
1 (CH2)11 1.60-1.22
2 CH3-CO 2.07
3 CO-CFJ2 2.16
4 CI12,C0-0-CH2 2.25
6 N+(CH3)2 3.85
7 CFJ2-0 3.98
8 CFJ2-0 4.16
9 N+-CFJ2-CH2-0 4.40
[00427] MALDI-TOF mass spectrometry of the bolaamphiphile GLH-55a:
[00428] The structure of this bolaamphiphile having the quaternary acetyl
choline head group
on one side of the hydrophobic skeleton and the chitosan head group on the
other side of the
hydrophobic skeleton was investigated by MALDI-TOF mass spectrometry. Positive
ion
MALDI spectrum was acquired in a reflector mode, using DHB (2,5-
dihidroxybenzoic acid as
matrix). MALDI-TOF-MS was taken over a mass range of 500-2200. The analysis of
the
MALDI spectrum of this bola showed the appearance of new peaks between 1000-
2000 Da,
including the polymer unit of the MW 701 Da. This new polymeric unit, which
contains the bola
with the acetyl choline as one head group and the chitosan as the second head
group, has a MW
of 736 Da. The repeat unit in the MALDI spectrum was 701 Da (the polymeric
unit without the
chloride anion has a MW of 701 (736-C1=701) presented below:
_ CH2OH
0_1+_
0 0 0 Me Me
II \/F 0
R= kCH2)10-t0-(CH2)10-0-C-CH2-N-CH2-CH2-0-tCH3 C1-
dH \NH-R
C301-15507C1N MW 576
C361165011N2C1
MW=736
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and
I- ___________________________________________________
0
________________________________________ 0_
R' =(R-C1")
dH NH-R'
C361165011N2 MW=701(736-35)
[00429] These results are in agreement with the data published in the
literature for
quaternary ammonium salts with a low molecular weight.
[00430] Synthesis of the bolaamphiphile with DAT ligand head groups
[00431] These examples describe the at synthesis of a dopamine transporter,
r3-CFT (30-
(4-fluorophenyl)tropane-20-carboxylic acid methyl ester), also known as WIN
35, or 428, that is
one of the most potent congeners at [3H] cocaine binding sites in striatum
[26]. This has been
used for imaging in humans as a marker of the nigrostriatal pathway to assess
the severity of
Parkinson's disease (PD) [27]. This ligand was selected, in part, for its high
affinity to
dopaminergic cells, as an illustrative surface group for targeting the
vesicles to dopaminergic
neurons in the Substantia Nigra.
[00432] Commercially-available (100 mg) of r3-CFT was used as a reference
compound while,
for the purpose of synthesizing the bola-DAT, synth r3-CFT was synthesized as
described. The 13-
CFT ligand was attached to the bolaskeleton described herein by modifiying the
r3-CFT ligand to
perform the alkylation of the amino group of the ligand with the bromoacetate
derivative of the
bola skeleton. We started the synthesis of the modified r3-CFT ligand from
cocaine HC1 by
following the procedures of Clarke et al and Melzer et al. [28,29].
Example 14
Synthesis of the 13-CFT derivative
[00433] Cocaine.HC1 was neutralized with ammonium hydroxide, extracted with
diethyl ether,
and the solvent was removed under reduced pressure. GC-MS m/z [M]+ = 303 and
NMR
spectroscopy confirmed the structure of cocaine.
[00434] Stage 1 encmpasses the three steps ((a) ¨ (c)) described below.
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0 0 0
(R) 0.8N HC1 R
OCH3
OH + OH + CH3OH
(R) (R)
2 (s) 0 41 (s) OH
HC1 = ecgonine benzoic acid
0-cocaine. HC1
1 2
a)
[00435] The cocaine hydrochloride 1 was refluxed with dilute hydrochloric
acid. The progress
of the reaction was followed by TLC. After cooling, the aqueous solution was
extracted with
ether to remove benzoic acid. The aqueous phase was concentrated to dryness to
give ecgonine 2
as a viscose brown oil that gave only one spot on TLC (EtAc:MeOH:H20:25% NH4OH
85:10:3:1) and was used for the next stage without further purification
0
0
(R) b) s) OH POC13
OH (R)
HC1
(
(E)
2 3
[00436] Ecgonine (2) and POC13 were refluxed for lh. The excess of P0C13 was
removed
under reduced pressure. FT-IR spectrum of the residue 3 showed the appearance
of the double
bond at 3032 cm-1, and the acyl chloride C=0(C1) absorption peak at 1735 cmi.
0
0
CI 0430H
OC H3
(E) = HC1
) (E)
3 4
anhydroecgonine methyl ester
c)
[00437] The product (3) was cooled in a dry ice/acetone bath to -73 C and
esterified with
methanok.to obtain the crude anhydroecgonine methyl ester hydrochloride. The
procedure was
followed by TLC (the same eluent system as mentioned above). The excess of
methanol was
removed under reduced pressure, the product neutralized with 25% NH40Hand
purified by flash
chromatography, with a mixture of ethyl acetate: methanol 95:5 as the eluent.
The
anhydroecgonine methyl ester 4 was obtained in a 55.4% yield (from cocaine
hydrochloride).
The mass spectrum of the ester 4 calculated for the C10H15NO2, GC-MS m/z [M] =
181.
[00438] The FT-IR shows the presence of the ester group (COOCH3) at 1711 cm1.
The NMR
spectrum allowed the different protons to be distinguished (FIG. 8).
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0
8
Ha \
\ N 10
H6¨ 7 1 9
0/
4 N2-
Hb i ".1 i/1
H N
b Ha 3
[00439] The difference between the protons of the methylene groups 4, 6 and 7
was elucidated
with HMQC and 11-1 COSY NMR [30,31] (FIG. 9). For example, the H-6a and H-4a
overlap at
1.8-1.9 ppm and H-4b appears at 2.6 ppm as a doublet.
[00440] Stage 2 encompassed the following reaction:
(R)
OCH3 (R)
) (E) F Mg Br
(s) OCH3
4 5 (s)
[00441] This stage involved the Michael addition of the aromatic Grignard
reagent, (p-
fluorophenyl) magnesium bromide, to the anhydroecgonine methyl ester 4. The
methyl ester 4 in
anhydrous ether was added drop wise to a mixture of the Grignard reagent in
anhydrous ether at
-30 C under a stream of nitrogen.
[00442] The method of quenching can determine the relative distribution of the
a- and13-
carbomethoxy isomers. Since the a-isomer is biologically inactive, there was a
need to optimize
the yield of the (3-carbomethoxy compound. The improved quenching procedure
described
herein uses the ethereal solution of hydrochloric acid that is added to the
reaction mixture
followed by addition of ice. The aqueous layer was basified to pH = 10 with
ammonium
hydroxide and the product extracted with dichloromethane. A total yield of
52.5% of the a-and
13-isomer was obtained. When the reaction was performed at -70 , 81.0 % yield
of a-and 13-isomer
was obtained. After removing the solvent, the crude mixture was checked by TLC
and the GC-
MS showed that the mixture contained 9.7 % of the a- isomer and 53.8% of the
13-isomer. The
products were then separated by flash chromatography with a mixture of diethyl
ether ¨ triethyl
amine as the eluent. The 13-isomer (13-CFT) 5 was isolated in a 32.4-36% yield
(based on 4) with
87-91% purity (determined by GC).
[00443] The NMR spectrum of the 13-CFT 5 (FIG 10) shows the disappearance of
the double
bond proton at 6.79 ppm of the starting methyl ester 4 and the appearance of
the aromatic protons
at 6.93-7.26 ppm and their corresponding carbons at 114,63, 114.8, 138.44,
138.44, 160.10,
162.12. The carbonyl carbon was shifted from 166.53 in 4 to 172.04 in 5
[00444] Stage 3 encompassed the following reaction:
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0
0 0
N y CH3 CH20C0C1 0-13 N
0/
0'
(s)
6 (s)
(s)
(s)
0
NH (R)
0/
(S)
(S)
7
[00445] This reaction involves a demethylation reaction providing derivative 7
to be attached to
the bolaskeleton. The N-demethylation of the N-methyltropane analog 5 was
carried out by
using a-chloroethyl chloroformate (ACE-C1) [32,33]. The reaction of
chloroformates with
tertiary aliphatic amines provides a convenient method for promoting
dealkylation. Compound 5
was reacted with ACE-C1 to provide the a-chloroethyl carbamate intermediate 6.
The hydrolysis
was then carried out with methanol to obtain the crude compound 7 in a 68.4%
yield with a
purity of 83% (GC). The mixture still contained 11.8% (3-CFT and 4.2%
byproducts. Purification
by flash chromatography gave fluoro nortropane 7 in about 40% yield with a 98%
purity.
GC-MS m/z: [M]+= 263.
[00446] The 1I-1 (FIG. 11 A) and 13C-NMR spectra (FIG. 11 13) confirmed the
structure; the
peaks at 2.23 and 51.18 ppm in the proton and respectively, carbon NMR spectra
(CH3N)
disappeared.
[00447] Synthesis of the bolaskeleton
[00448] Synthesis of the bolaskeletons, GLH-19 and GLH-20 was described above.
Attachment
of the DAT ligand head group employed the decanedivernolate skeleton in which
the opening of
the epoxy ring was carried out with bromoacetic acid, as depicted in Scheme 5,
below.
[00449] Scheme 5 ¨ bolaskeleton preparation
vernolic acid
0 00,(cH21)n...0 0 0
decanedivernolate
Br_
0 k 0
Br.)ko
OH 0 0
HO 0 Br
/0--</=~)L0=".(cliori3O
8
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[00450] A mixture of decane divernolate with an excess of bromoacetic acid in
toluene was
heated for 24 h. The reaction mixture was washed several times with a
saturated solution of
sodium bicarbonate to remove the excess of bromoacetic acid, and the crude
product was purified
by flash chromatography, using a mixture of dichloroethane:acetone (50:1) to
yield 33% of the
dibromodiacetate of the divernolester 8, with 96.7% purity (HPLC). In the FT-
IR spectrum, we
observed a broad absorption peak at 1735 cm-1 characteristic of both ester
groups, contrary to the
two absorption peaks, one of the original ester and the second one of the
chloroacetate group, at
1735 and 1758 cm-1, respectively for the dichlorodiacetate of the
divernolester (Scheme 2,
above). In the 1H-NMR spectrum the bromomethylene protons (CH2-Br) appear at
3.69-3.93
ppm. CH-OH and CH-OC(0) groups obtained after opening the epoxy ring appear at
3.60 and
4.90 ppm, respectively. The presence of structural isomers, I and II,
,
obtained after opening of the epoxy ring, was observed in the 1I-1 and 13C NMR
spectra. 1H-NMR
showed two multiplets at 4.94-4.91 ppm and 4.89-4.86 ppm for the CH-O-CO
proton and two
overlapping triplets at 0.90-0.87 ppm for the terminal methyl group. 13C-NMR
showed two peaks
at 167.11 and 166.93 ppm for the carbonyl adjacent to the bromomethylene group
BrCH2C=0,
four peaks at 133.82, 133.60 and 124.08, 123.31 ppm for the double bond and
two peaks at 72.03
and 71.88 ppm for the CH-OH carbon
Example 15
Attachment of the fluor nortropane (7) to the bola skeleton
[00451] Conjugation of the fluoronortropane 7 to the bolaskeleton
dibromodiacetate 8 (Scheme
6), was carried out using the alkylation reaction described by Riss [34] with
a short chain alkyl
halide.
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[00452] Scheme 6 nortropane (7) attachment
o 0
Br=AO OH 0 0 HO 04,=Br
.."..."..)-4,00=%,"=====140,(C112)100011/400=00%."..."..)¨(M.
8
0
NH
0/
R 4
(s) \ 7 (s) R R (R
=
/ µ (S) N
N 0 0
4op OH 0 0 HO 041/4,
('-'-õ 2)100
[00453] The reaction was performed in acetonitrile as the solvent, in the
presence of a strong
base, proton sponge or Hilnig's base, using a molar ratio of 2:1 of the
reagents, respectively. The
reaction mixture was refltmed overnight. The progress of the reaction was
followed by TLC. The
hydrobromide of the base was filtered out, and the solvent removed under
reduced pressure. The
residue was dissolved in CHC13, and washed with water. The product, GLH-57,
from the reaction
with the Hfinig's base was obtained in a yield of 84.5% and was purer (97% by
HPLC) than that
obtained with proton sponge base. M/z[M/21+= 687.4504 (MALDO.
[00454] The NMR spectra (FIG. 12) shows the disappearance of the
bromomethylene protons
CH2-Br at 3.93 ppm and the appearance of the CH2-N protons obtained after
conjugation at 3.05-
3.25 ppm, as well as of the aromatic protons at 6.93-7.21 ppm
[00455] The ratio between the structural isomers (where the OH group is once
on the 41
carbon, isomer I, and once on the 42 carbon, isomer II)
n I p II
i .4:p /
:,..):.: AN ol
4F: 45
,..(.,..., z.
. ,
,4,::,,,, .,., , ,.õ, .=,4 ,e= im
\----\._(
= = w. ._
1 0 r i I
w, 1;
was studied through the double bond protons that appear at 5.35 ppm (one H)
and at 5.50 ppm
(three F).
[00456] The protons at position 38 have a characteristic peak at 5.5ppm in
both I and II
configurations. The proton at position 39 of isomer II, shifted from 5.35ppm
to 5.50 ppm
compared to isomer I and appears together with the protons at position 38. For
a ratio of 1:1 of
the isomers I and 11 (4 protons on the atoms 38 and 39), 3H appeared at 5.5ppm
(2 protons for
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isomers I and II on 38 atom and 1 proton from isomer I on the atom 39) and 1H
on the 39 atom
from isomer II appeared at 5.35ppm.
Example 15
Vesicle Formation And Characterization
[00457] Vesicle Morphology
[00458] Aggregation of the basic bolas, GLH-19 and GLH-20, nanoparticles was
studied using
cryo-transmission electron microscopy (cryoTEM). Both bolas, GLH-19 and GLH-
20, formed
spherical vesicles with a diameter range of 50-120 nm (FIG. 13 A and FIG. 13
B).
[00459] Trypsinogen wasas a model protein for GDNF in initial encapsulation
studies, since
(a) trypsinogen is considerably less expensive and is available in large
quantities for initial
exploratory studies, (b) both proteins have similar molecular weights and
isoelectric points
(molecular weights of 22KDa and 18KDa for trypsinogen [35] and glycosylated
GDNF [36],
respectively, and (c) have similar isoelectric points of 9.25-9.36 and 9.26
for trypsinogen [37]
and GDNF [36], respectively. Thus encapsulation, which is mostly affected by
molecular weight
and isoelectric point, is expected to be almost identical for both proteins.
When trypsinogen was
encapsulated at a concentration of 2 mg/ml in the vesicles, the spherical
morphology of the
vesicles remained similar to that of empty vesicles, with a slight increase in
vesicles size (FIG.13
C and FIG. 13 D) and with a somewhat wider size distribution, as indicated by
the dynamic light
scattering data, below. Trypsinogen at concentrations below 1 mg/ml did not
affect vesicle
morphology at all nor did it affect vesicle size or size distribution, as
indicated by the dynamic
light scattering data, below.
[00460] Vesicle stability and quantitative biodistribution studies used
trypsinogen, as well as
vesicles loaded with carboxyfluorescein, a self quenched fluorescent dye. As
an initial matter, it
was determined that CF-loaded vesicles maintained their spherical shape and
size, with no
apparent effect on any vesicle characteristics or morphology. (FIG 13 E and
FIG. 13 F).
[00461] Vesicles were also prepared from a mixture of GLH-19 and GLH-20. The
rationale
behind such vesicles was an attempt to obtain vesicles with higher stability,
lower toxicity
(preliminary studies indicate that vesicles from GLH-19 have lower toxicity
than vesicles made
from GLH-20), and which release their encapsulated content upon exposure to
choline esterases
(ChE). Notably, GLH-19 vesicles do not release their encapsulated content when
exposed to
ChE, whereas vesicles made from GLH-20 release their encapsulated content in
presence of ChE,
yet GLH-19 form more stable vesicles with lower toxicity than GLH-20. Without
wishing to be
held to this belief, it is our understanding that that in vesicles that are
made from a mixture of
GLH-19 and GLH-20, the former (GLH-19) will contribute to stability and to a
lower toxicity,
whereas the latter (GLH-20) will contribute head groups that are sensitive to
ChE, thus providing
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a controlled release mechanism once the vesicles enter into the brain. A
formulation of GLH-19
together with GLH-20 and cholesterol and cholesteryl hemisuccinate (the two
cholesterol
compounds are used in all vesicle formulations as membrane stabilizers)
yielded spherical
vesicles (FIG. 14 A) that were very similar in size and morphology to the
vesicles made from
either GLH-19 or GLH-20 alone (note that membrane stabilizers were added to
all the
formulations). Also in vesicles made from a mixture of GLH-19 and GLH-20, the
encapsulated
trypsinogen did not affect vesicle morphology and only slightly increased
their size (FIG. 14 B).
[00462] The formulations for preparing vesicles, expected to be
particularly useful for in
vivo studies, further contain small quantities (1 mg/ml) of a bolaamphiphile
with a CS head group
on one side of the hydrophobic skeleton and an ACh head group on the other
side of the
hydrophobic skeleton (GLH-55a), and also 0.8 mg /ml of a bolaamphiphile with
the DAT ligand
on each side of the hydrophobic skeleton (GLH-57). Data obtained indicate that
the
incorporation of GLH-55a and GLH-57 does not affect vesicle morphology and
other vesicle
characteristics. As can be seen from FIG. 15, the spherical shape and size of
the vesicles was
maintained after incorporating into the formulation GLH-55a (FIG. 15 A), GLH-
57 (FIG. 15 B)
or both GLH-55a and GLH-57 (FIG. 15 C); thus, vesicles that contained GLH-55a
and GLH-57
were similar to vesicles made from a mixture of GLH-19 and GLH-20 without the
CS and the
DAT ligand decoration.
[00463] Since trypsinogen at concentrations below 1 mg/ml did not affect
vesicle morphology
and the concentration of the GDNF was not expected to exceed 1 mg/ml, further
studies to
determine the effect of the encapsulated protein on vesicle characterization,
including vesicle
morphology, were done with the "real" protein - GDNF. As can be seen from FIG.
16,
encapsulated GDNF had no effect on vesicle morphology and vesicle size
[00464] Vesicle size and size distribution determine by dynamic light
scattering
[00465] Representative dynamic light scattering profiles are provided in FIG.
17, i.e., for empty
vesicles made from GLH-19 (FIG. 17 A), GLH-20 (FIG. 17 B), and a mixture of
GLH-19 and
GLH-20 (FIG. 17 C).
[00466] As can be seen, all vesicles preparations were fairly homogeneous,
showing one peak
in the DLS profile, with a vesicle diameter that averages about 100 nm. The
effect of
encapsulated trypsinogen on the size of the vesicles and their surface charge
(zeta potential) was
also tested. In general, it was observed that some increase in vesicle size,
and a somewhat wider
size distribution than empty vesicles, when we encapsulated trypsinogen in the
vesicles. A
representative DLS profile of GLH-20 vesicles, with and without encapsulated
trypsinogen, is
shown in FIG 18.
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[00467] The sizes of several types of vesicle preparations and their zeta
potentials measured by
DLS are summarized in Table 7.
[00468] Table 7: Vesicle size and zeta potential values measured by DLS
Type of vesicles and loaded Diameter in nm Zeta potential in mV
protein (mean SEM) (mean SEM)
GLH-19 (empty vesicles) 100.8 0.9 28.70 2.52
GLH-19 loaded with 4mg/m1 129.2 7.2 27.68 2.79
trypsinogen
GLH-20 (empty vesicles) 104.7 2.5 41.88 1.45
GLH-20 loaded with 4mg/m1 165.6 2.0 35.50 1.49
trypsinogen
GLH-19:GLH-20 (2:1) (empty 113.1 10.6 37.30 2.90
vesicles)
GLH-19:GLH-20 (2:1) loaded 133.2 6.2 33.05 2.69
with 4 mg/ml trypsinogen
GLH-19:GLH-20 (2:1) with CS 109.6 0.1 33.6 1.23
surface groups (empty vesicles)
GLH-19:GLH-20 (2:1) with 123.5 0.5 44.6 1.27
DAT ligand surface groups
(empty vesicles)
GLH-19:GLH-20 (2:1) with CS 115.2 1.3 34.7 3.96
and DAT ligand surface groups
(empty vesicles)
GLH-19:GLH-20 (2:1) with CS 114.3 1.5 36.0 3.16
and DAT ligand surface groups
(loaded with GDNF)
[00469] Vesicles were prepared by film hydration followed by sonication
from 10 mg/ml
bolas, 2.1 mg/ml cholesteryl hemisuccinate and 1.6 mg/ml cholesterol. Each
measurement was
done on at least 3 different vesicle preparations and the values are means
SEM.
[00470] As can be seen from Table 7, the average diameter of the empty
vesicles ranged
between 100-123 nm. The empty vesicles are positively charged (cationic
vesicles), with GLH-
20 vesicles having higher zeta potential than GLH-19 vesicles. Vesicles made
from a mixture of
GLH-19 and GLH-20 show zeta potential in between those of GLH-19 vesicles and
GLH-20
vesicles. Encapsulation of trypsinogen, at a concentration of 4 mg/ml,
consistently increased the
vesicle size and reduced somewhat the zeta potential in all the vesicle
preparations, indicating
that some protein binds to the vesicle surface and neutralizing the positively
charged groups.
Addition of either CS or DAT ligand alone or both of of them together, did not
change
significantly the vesicle size or zeta potential. Also, encapsulation of GDNF
did not change the
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vesicle size or zeta potential, probably because the concentration of the GDNF
was much smaller
than the trypsinogen concentration (the GDNF concentration was 40pg/m1).
[00471] Vesicle stability
[00472] Vesicle stability was determined by measuring the amount of
encapsulated fluorescent
dye (carboxyfluorescein (CF)) as a function of time, during either storage or
incubation in whole
serum. In general, vesicles made from the different formulations which were
shown above, were
very stable under storage. Even vesicles from GLH-20, which in whole serum
were less stable
than vesicles made from GLH-19, were stable in storage (FIG. 19).
[00473] Determinatio of vesicle stability in whole serum is complicated by the
presence of ChE
activity. It had been demonstrated that vesicles from GLH-20 release their
encapsulated
material when exposed to ChE, whereas vesicles made from GLH-19 were not
sensitive to ChE.
Vesicles made from a mixture of GLH-19 and GLH-20 are believed (without
wishing to be held
to that belief) to be potentially more effective for the purpose of delivering
GDNF to the brain
since the GLH-19 component should reduce toxicity. This inference is based on
preliminary
studies in mice, that suggested that GLH-19 is less toxic than GLH-20,
although the toxicity of
either bola is significantly below the dose expected to be used in vesicles
that will injected into
mice in vivo, as well as on the increased vesicle stability in which the GLH-
20 component will
contribute the controlled release mechanism. In fact, vesicles made from a
mixture of GLH-19
and GLH-20 were more stable in serum than vesicles made from GLH-20 alone and
increasing
the amount of the GLH-19 component within vesicles made from a mixture of both
bolas
increased vesicle stability in serum (FIG. 18).
[00474] That is, as can be seen from FIG. 18, the half life of vesicles made
from a mixture of
GLH-19 and GLH-20 is about 4-6 hours (depending on the ratio between GLH-19
and GLH-20),
compared with a half life of 2.5 hours for vesicles made from GLH-20 only.
[00475] In other experiments, it was observed that vesicles made from GLH-20
decapsulate
and release their content in presence of ChE. Since the vesicles are designed
to release their
content in the brain by the influence of the brain ChE, it was important to
confirm that the
relatively small amount of GLH-20 in the vesicles made from a mixture of GLH-
19 and GLH-20
at a ratio of 2:1, is sufficient to cause decapsulation when exposed to ChE.
Accordingly,
vesicles were prepared from GLH-20 and from a mixture of GLH-19 and GLH-20,
and loaded
with CF and exposed to ChE, the release of the fluorescent marker was measured
as a function of
time after exposing them to the enzyme. The results are shown in FIG. 20.
[00476] As can be seen, both vesicle preparations started to release their
content after the
addition of AChE. However, release from the vesicles made from GLH-20 was
somewhat faster
than the release from the vesicles made from a mixture of GLH-19 and GLH-20.
Thus, 5 min
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after the addition of AChE, the vesicles made from GLH-20 released 42% of
their content
whereas at this time point the vesicles made from the mixture of GLH-19 and
GLH-20 released
33% of the total CF that was encapsulated. Accordingly, vesicles made from a
mixture of GLH-
19 and GLH-20 would be predicted to release their content in the brain in
response to brain ChE
and therefore, these vesicles can be used to deliver compounds to the brain
and release their
cargo in the brain.
Example 16
Protein Encapsultion
[00477] Optimization of the encapsulation using a model protein (trypsinogen)
[00478] Initial studies on optimization of the encapsulation, relied on the
use of an inexpensive,
readily available, model protein (trypsinogen) with similar properties to
GDNF. Parameters that
may influence encapsulation include the molecular weight of the protein and
its isoelectric point.
Large proteins may affect vesicle properties in a different way than small
proteins. For example,
in our preliminary studies we observed that smaller proteins can be used at
higher concentrations
compared with larger proteins before the vesicles aggregate and form a turbid
suspension. The
isoelectric point may influence the binding of the protein to the positively
charged head groups of
the bolaamphiphiles. Trypsinogen, has a molecular weight (22KDa) comparable to
that of
GDNF (18 KDa of the glycosylated form), as well as a comparable isoelectric
point, with both
proteins having an isoelectric point near pH 9.
[00479] The concentraion of trypsinogen can be measured by UV absorbance at
280 nm. Since
the vesicles are prepared in media that contain trypsinogen, encapsulated
protein had to be
separated from non-encapsulated protein to determine encapsulation efficiency.
For example,
encapsulated trypsinogen could be separated from free protein by size
exclusion chromatography
on a Sephadex G50 column. As can be seen from FIG. 22, on a Sephadex G50
column, the
vesicles were eluted in the first 3-5 ml of the eluting buffer, while the free
trypsinogen was eluted
in the next 6-11 ml; thus, the encapsulated protein could be well separated
from the non-
encapsulated protein.
[00480] This method for the separation of the encapsulated trypsinogen from
the non-
encapsulated protein (free trypsinogen), allowed quantification of the
encapsulated protein. As
can be seen from FIG. 23, each peak that was eluted from the column could be
quantified by
determining the area under the curve (AUC), using the Prism Graph Pad software
that takes into
consideration overlaps between peaks, in case of overlaps.
[00481] Based on the above methods for separation of the encapsulated protein
and quantifying
percent encapsulation, the encapsulation process was optimized to provide
maximum
encapsulation efficiency. In the first stage of the optimization, several
concentrations of
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trypsinogen were used with a fixed amount of bolaamphiphiles (10 mg/ml of a
mixture of GLH-
19 and GLH-20 in the ratio of 2:1), using a trypsinogen concentration of 4
mg/ml. This approach
facilitated the accurate measurement of the amount of the protein in each
fraction collected from
the column, without interference by the small light diffraction of the
vesicles that were eluted
from the column. The parameters varied in the optimization included: a) the
ratio between the
bolas and the additives - cholesterol and cholesteryl hemisuccinate; b) the pH
in which the
vesicles were prepared; c) the pH of the eluting buffer; and d) the method for
vesicle
preparation and encapsulation. The data obtained from these initial
optimization studies are
summarized in Table 8.
[00482] Table 8: Encapsualtion Of Trypsinogen In Bolaamphiphilic Vesicles
Vesicle formulation Preparation Elution Percent
Bola (GLH-19:GLH-20): CHOL:CHEMS medium medium encapsulation
4(2:1):1:1 PBS TB (pH=7.3) 25
AB 8
2(2:1):1:1 PBS TB (pH=7.3) 27
AB 15
4(2:1):1:1 TB TB (pH=7.3) 36
AB 13
2(2:1):1:1 TB TB (pH=7.3) 30
AB 15
[00483] The formulations that were used for the vesicle preparations contained
10 mg/ml bolas
(the ratio between the bolas was always 2 parts of GLH-19 and 1 part of GLH-
20), and the ratios
between the bolas and the cholesterol (CHOL) and cholesteryl hemisuccinate
(CHEMS) were
varided as indicated in the Table 8. Vesicles were prepared in the media as
indicated in Table 8.
All media contained 4 mg/ml trypsinogen. PBS is phosphate buffered saline,
pH=7.4; TB is Tris
buffer 10 mM pH=9.5, except for cases when pH=7.3 is indicated; AB is acetate
buffer 10 mM,
pH=3.5.
[00484] As can be seen from Table 8, the highest trypsinogen encapsulation was
obtained when
the vesicles were prepared in high pH (9.5), possibly due to binding of the
protein, at pH above
its PI (the PI is about 9), to the positively charged head groups of the
bolas. Eluting the vesicles
with a buffer of physiological pH (7.3) did not reduce encapsulation even if
the vesicles were
prepared in high pH, but eluting the vesicles from the Sephadex G50 column
with a buffer of a
low pH (3.5) significantly reduced encapsulation, probably because at low pH
trypsinogen is
more positively charged (the PI of trypsinogen is around 9) and its
complexation with the
cationic head groups of the bolaamphiphiles is weakened. Increasing the ratio
of the bolas, in
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relation to cholesterol and cholesteryl hemisuccinate, did not affect the
encapsulation efficiency
very significantly.
[00485] The method of the vesicle preparation on encapsulation was also
examined, using two
different methods: a) film hydration followed by sonication and b) extrusion
via membrane with
a pore size of 100 nm. Since sonication may damage the protein, extrusion
methods would be
viable alternatives, although they require higher working volumes and for
scale up, would be
advantageous. The date of FIG. 24 demonstrate encapsulation obtained following
sonication
compared with extrusion.
[00486] As can be seen in FIG. 24, similar encapsulation efficiency was
achieved by both
methods. Accordingly, for small scale studies, film hydration followed by
sonication could be
used while, for scale-up for larger quantities of vesicles, the extrusion is
advantageous.
[00487] To increase the sensitivity for measurements of encapsulated protein,
that material was
labled with AlexaFluor0-488 and measured the protein concentration measured by
fluorescence.
Elution of the vesicles in Tris buffer does not exhbit adequate separation
between the
encapsulated protein and the free protein (FIG 25 A). Elution in PBS, however,
provided better
results and an adequate separation between encapsulated and non-encapsulated
trypsinogen (FIG.
25 B), with 30% encapsulation obtained under similar conditions to those used
for the larger
quantities of trypsinogen (FIG. 25 C).
[00488] Additional experiments, using several levels of trypsinogen within the
range expected
for GDNF,were carried out with several amounts of bolas to evaluate
encapsulation efficiency.
The results are summarized in Table 9.
[00489] Table 9: Percent Encapsulation Of AlexFluor0 488 Labled Tyrpsinogen
mg/m1 GLH-19: 5 mg/m1 GLH-19: 2.5 mg/m1 GLH-19:
Trypsinogen
GLH-20 (2:1) GLH-20 (2:1) GLH-20 (2:1)
17.5 [tg/m1 77 56 59
35 pg/m1 60 54 61
70 pg/m1 51 28 32
140 pg/m1 56
[00490] Vesicles were prepared by film hydration followed by sonication in
presence of
various amounts of AlexaFluor0-488-labeled trypsinogen and various
concentrations of the
bolas, as indicated. Values are percent encapsulation, calculated by using the
amount of the
labeled trypsinigen that was present during vesicle preparation as 100%.
[00491] As can be seen from the data of Table 9, higher encapsulation
efficiencies were
obtained with higher bolas/protein ratio.
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[00492] GDNF Encapsulation
[00493] In light of the data obtained for optimized encapsulation of
trypsinogen GDNF
encapsulation was carried out using the vesicle formulation that gave the
highest trypsinogen
encapsulation. In this experiment, GDNF was added at a concentration of 12.5
[tg/m1 for
preparation of vesicles for in vivo studies. In this experiment, the percent
encapsulation obtained
with GDNF was compared to that obtained with trypsinogen under similar
conditions.
[00494] For this experiment, vesicles were prepared by film hydration followed
by sonication
from a mixture of GLH-19 and GLH-20 at a concentration of 10 mg/ml with 1.6
mg/ml
cholesterol and 2.1 mg/ml cholesteryl hemisuccinate. The formulations
contained 50 [tg/m1
trypsinogen (A), 100 [tg/m1trypsinogen (l3) and 12.5 [tg/m1 GDNF (C). All
proteins were labeled
with AlexaFluor0-488. After encapsulation, the vesicles were eluted from a
Sephadex G50
column by PBS and the fluorescence of each fraction was determined. The
results are shown in
FIG. 26.
[00495] The percent encapsulation for each vesicle preparation was detrmined
and the data
presented in FIG. 26. The data reveal 42% and 54% encapsulation for 100 [tg/m1
and 50 [tg/m1
trypsinogen (FIG. 26 B and FIG. 26A, respectively), and 66% encapsulation for
the GDNF (FIG.
26 C).
Example 17
Determination of GDNF integrity and activity following encapsulation
[00496] Although sonication may affect the integrity and/or the activity of
the GDNF, film
hydration followed by sonication were to be employed for initial preparations
of vesicles for use
with in vivo studies, since this method is economical, does not require a high
volume of vesicles
compared with the extrusion method, and allows smaller amounts of GDNF.
Accordingly, the
integrity and the activity of naked GDNF to that of encapsulated GDNF, where
encapsulation
was achieved by the method of film hydration followed by sonication.
[00497] The integrity of GDNF was examined by polyacryl amide gel
electrophoresis (PAGE),
where we looked for possible changes in molecular weight that may suggest
fragmentation due to
sonication. The results that are shown in FIG. 27 and clearly suggest that
both the monomeric
form of the GDNF and its dimeric form were not changed after sonication, and
since no
additional bands appeared on the gel, indicating that no fragmentation of the
GDNF occurred
during the encapsulation process.
[00498] In particular, FIG. 27 depicts the effect of the encapsulation process
on GDNF
integrity and activity. (A) Analysis of GDNF on PAGE, where lane 1 is empty
vesicles; lane 2 is
GDNF encapsulated by the method of film hydration followed by sonication; lane
3 is
encapsulated GDNF which was incubated before the PAGE at 40 C for one hour;
and lane 4 is
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free GDNF. (B) Test of GDNF activity using SH-SY5Y neuroblastoma cells where
lane 1 is
control untreated cells; lane 2 is cells treated with free GDNF; lane 3 is
cells treated with empty
vesicles; lane 4 is cells treated with free GDNF added to empty vesicles; and
lane 5 is cells
treated with GDNF encapsulated in bolavesicles by the method of film hydration
followed by
sonication.
[00499] The effect of the encapsulation process on GDNF activity was also
examined by
treating neuroblastoma cells that respond to GDNF by activation of kinases,
particularly AKT
and MAPK. Notably, AKT activity regulates cell survival and this activity is
particularly
relevant to neuroprotection conferred by GDNF and to PD therapy. AKT and MAPK
are
activated when they are phosphorylated and GDNF induces phosphorylation of
these enzymes in
SH-SY5Y neuroblastoma cells. If GDNF activity is impaired, it will not
phosphorylate AKT and
MAPK to pAKT and pMAPK, respectively. pAKT and pMAPK can be detected by
specific
antibodies for the phosphorylated forms of the enzymes on a Western blot. As
can be seen in
FIG. 27 B, GDNF caused the same degree of phosphorylation in its free form as
in its
encapsulated state, or after it was added to empty vesicles. These results
suggest that neither the
bolaamphiphilic vesicles, nor the encapsulation process that included
sonication affected GDNF
activity.
Example 18
Targeting Of DAT Ligand-Coated Vesicles In Vitro
[00500] Vesicles GLH-57, were formed by additiona of bolas with DAT ligand
head groups to
the vesicle formulation of GLH 19/GLH 20 CHEMS/CHOL. When this bola is
included in the
formulation, the resulting vesicles are decorated on their surface with the
DAT ligand, intended
to target cells that express the dopamine transporter, namely, dopaminergic
cells. To test if the
DAT-ligand-coated vesicles have higher affinity to dopaminergic cells, the
vesicles were added
to three types of cells: a) PC12 cells that highly express DAT [19]; b) SH-
SY5Y neuroblastoma
cells that are known to express DAT [20]; and c) HeLa cells that do not
express DAT.
[00501] Vesicles were loaded with CF, and each cell type contacted with added
the
fluorescently labeled vesicles uptake of the fluorescent dye into the cell
measured by flow
cytometry. Vesicles were made from 10 mg/ml GLH-19:GLH-20 (2:1) without
(uncoated
vesicles) and with 0.8 mg/ml GLH-57, a bola that contains DAT ligand as the
head group (DAT-
vesicles). Cells were incubated for 1 h with the vesicles, and tested by flow
cytometry. A shift to
the right of the peak indicates fluorescent cells due to uptake of the
vesicles.
[00502] As can be seen from FIG. 28, significantly higher uptake of the
fluorescent vesicles
was seen when the vesicles were coated with DAT ligand and added to PC12 cells
(FIG. 28 A)
that highly express DAT compared to uncoated vesicles. Also, higher uptake of
the DAT ligand-
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coated vesicles, compared to uncoated vesicles, was observed in SH-SY5Y
neuroblastoma cells
(FIG. 28 B) that also express DAT. Yet, when the fluorescently labeled
vesicles were added to
HeLa cells that do not express DAT, no difference was observed in the uptake
between DAT
ligand-coated vesicles and uncoated vesicles (FIG. 28 C).
[00503] These results strongly suggest that vesicles coated with DAT ligand
are targeted to
cells that express DAT, and after penetrating the brain, such vesicles will
target dopaminergic
cells in brain regions such as the striatum and the Sunstantial Nigra pars
compacta.
Example 19
Biodistribution Of The Delivered GDNF Withi The Mouse Brain
[00504] Effect of CS surface groups on BBB permeability of the vesicles
[00505] Preliminary studies indicated that vesicles coated with CS surface
groups penetrate the
BBB better than uncoated vesicles. The CS coating in these preliminary
experiments, was
achieved by incorporating a CS-fatty acid conjugate into the vesicle
formulation. However, the
energy barrier for pulling the CS surface group out of the vesicle membrane,
which is anchored
to the membrane via fatty acid, is low, and therefore, some of the CS surface
groups may be lost
before the vesicle reaches the BBB. Pulling bolas out of a monolayer membrane
is much more
difficult since the hydrophilic head group has to pass through the hydrophobic
domain of the
monolayer membrane, and this takes more energy. Accordingly, bola with CS
attached
covalently to the bola's skeleton were sued instead of CS-fatty acid
conjugate. To verify that the
vesicles do not compromise the BBB, but rather crossed the BBB in their intact
form, the
experiment depicted in FIG. 29 was carried out.
[00506] This experiment mesure accumulation of CF in the brain following i.v.
administration.
Vesicles were made by film hydration followed by sonication from a 10 mg/ml
mixture of GLH-
19 and GLH-20 (2:1), 1 mg/ml CS-fatty acid (vernolate) conjugate, 2.1 mg/ml
cholesteryl
hemisuccinate and 1.6 mg/ml cholesterol in absence (empty vesicles) and in
presence of 0.2/m1
CF (CF-loaded vesicles). Mice were pretreated with 0.5 mg/kg (i.m.)
pyridostigmine and 15 min
afterward the mice were injected i.v. with either free CF, or empty vesicles
and then CF, or CF-
loaded vesicles. The total amounts of the CF that were injected in each case
were identical (10
mg/kg). 30 min after the injection, the animals were sacrificed, perfused with
10 ml PBS and the
brains removed and homogenized, deproteinized by 5% tricholoroacetic acid and
fluorescence
determined in the supernatants obtained following centrifugation. The data
obtained are
presented in FIG 29, where dach bar represents an average value obtained from
5 mice +/- SEM.
[00507] As can be seen in Fig. 29, free CF hardly entered the brain and a very
little amount of
CF was measured in brain homogenate taken from animals that received free CF.
By
comparison, 15 times more CF was found in the brain after the injection of
encapsulated CF.
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When free CF was injected immediately after the injection of empty vesicles,
about 3 times more
CF in the brain was obaserved as compared with the amount found after the
injection of free CF
alone. This increase may be attributed to binding of the negatively charged CF
to the positively
charged surface groups of the vesicles, before the vesicles entered the brain,
thus, some of the
dye was carried into the brain while being bound to the vesicles. Similar
results were obtained
when the CF was injected just before the injection of empty vesicles (not
shown). The profound
increase in the CF concentration on the brain obtained after the injection CF
loaded vesicles
compared to that obtained after the injection of empty vesicles and free CF
suggest that the
vesicles do not compromise the BBB, but rather enter into the brain in their
intact form and
release the encapsulated drug within the brain after their entry.
[00508] In the experiment shown in FIG. 29, a CS-fatty acid (vernolic acid)
conjugate to was
used to introduce the CS groups to the surface of the vesicles. BBB
permeability of the vesicles
with CS surface groups that are an integral part of the membrane structure
(using the bola with a
CS head group as synthesized herein) to vesicles with CS surface groups that
were introduced by
the CS-fatty acid conjugate (FIG. 30).
[00509] In this experiment, vesicles were prepared as described in FIG. 29,
except that in one
case 1 mg/ml GLH-55a was used in the vesicle formulation to provide CS surface
groups
(vesicles with CS-bola), and in the other case, 1 mg/ml CS-fatty acid
conjugate was used.
Conditions of this experiment were similar to those presented in FIG. 29.
[00510] As demonstrated by the data of FIG. 30, the presence of CS surface
groups increased
the amount of the CF that was measured in the brain. The amount of the CF that
was measured
in the brain after injecting the dye encapsulated in vesicles, was increased
by about 50% when
the CF was encapsulated in vesicles to which the CS surface groups were added
by using a CS-
fatty acid conjugate, compared to naked vesicles. By comparison, the amount of
the CF in the
brain was increased by about 100% when the CF was encapsulated in vesicles in
which the CS
surface group was an integral part of the membrane (by using the bola-CS - GLH-
55a). These
results indicate that bola-CS is better than fatty acid-CS for enhancing the
permeability of the
vesicles via the BBB.
Example 20
Targeting of vesicles coated with DAT ligand to the striatum
[00511] As demonstrated above, the vesicles described herein transport
their encapsulated
content through the BBB into the brain. This experiment was intended to
demonstrate that the
vesicles that are coated with DAT ligand will be targeted to brain regions
that contain
dopaminergic neurons. Vescicles were loaded with CF and injected into the tail
vein of mice and
30 min after the injection, the mice were sacrificed, the brain removed and
dissected into three
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brain regions: (1) the cortex; (2) the striatum; and (3) the cerebellum. Each
of these brain
regions was homogenized, deproteinized by trichloroacetic acid, and
fluorescence intensity was
measured in the supernatant that was obtained after centrifugation.
[00512] In particular, vesicles were prepared by film hydration followed by
sonication
from a 10 mg/ml mixture of GLH-19 and GLH-20 (2:1), 1 mg/ml GLH-55a (a bola
with CS head
group), 2.1 mg/ml cholesteryl hemisuccinate, 1.6 mg/ml cholesterol, 0.2 mg/ml
CF and without
(vesicle CS bola) or with GLH-57 (vesicles DAT CS bola). Mice were pretreated
with 0.5 mg/kg
(i.m.) pyridostigmine (to inhibit peripheral ChE) and 15 min afterward the
vesicles were injected
i.v. After 30 min the mice were sacrificed, perfused with 10 ml PBS and the
brain removed and
dissected into cortex, striatum and cerebellum. The tissues were weighed,
homogenized and
deproteinated by trichloroacetic acid, centrifuged and fluorescence was
determined in the
homogenates. The amount of the CF in each brain region was calculated from a
calibration curve
of CF, taking into consideration the weight of the tissue and the dilution
done during the
homogenization. Each bar represent an average value obtained from 5 mice +/-
SEM.
[00513] FIG. 31 depicts the results of this experiment. As can be seen from
FIG. 31, the
highest amount of CF was found in the striatum of animals that were injected
with DAT ligand-
coated vesicles. The largest difference in CF concentrations between uncoated
vesicles and
coated vesicles was observed in the striatum, then in the cortex and lastly in
the cerebellum,
where there was almost no difference between the amounts of the CF that were
measured in
animals that received uncoated vesicles versus those that received DAT ligand-
coated vesicles.
Free CF did not penetrate into the brain in significant amounts. These data
show that the vesicles
penetrate into the brain, and once in the brain, the vesicles that were coated
with DAT ligand
were targeted to brain regions that are known to have dopaminergic neurons.
Example 21
Delivery Of Labeled Trypsinogen By The Bolavesciles
[00514] Prior to delivering GDNF to the brain, pilot experiment, in vivo,
studies were
carried out with the model protein - trypsinogen, which was labeled for this
purpose with
AlexaFluor0-488. The experiment was intended to determine whether the labeled
protein can be
seen in brain sections directly by histofluorescence. Mice were pretreated
with pyridostigmine
15 min prior to vesicle injection (to inhibit peripheral ChE) and 30 min after
the injection of the
vesicles, the mice were sacrificed, perfused with 10 ml PBS and tissues were
dissected out,
frozen in isopentane that was cooled by liquid nitrogen, sectioned by
cryomicrotime and
fluorescence was observed by confocal microscopy. The fluorescence that was
seen in three
different tissues: a) brain; b) liver; c) kidney; is shown in FIG 32.
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[00515] FIG. 32, provides representative histofluorescence slides showing
AlxaFlour-488-
labeld trypsinogen in brain (A-C); liver (D-F) and kidney (G-I) of mice that
were injected with
the labeled protein encapsulated in CS-coated vesicles or with the free
protein. Panels A, D and
G are micrographs taken from control untreated mice. Panels B,E and H are
micrographs taken
from mice injected with 200 lig of free trypsinogen labeled with AlexaFluor0-
488 and C,F and I
are micrographs taken from mice that were injected with 200 lig of
encapsulated trypsinogen
labeled with AlexaFluor0-488.
[00516] As can be seen in FIG 32, the labeled trypsinogen is found in the
brain only when
it was injected encapsulated in the bolavesicles. Also, in the liver, the
injection of encapsulated
trypsinogen resulted in higher fluorescence than was obtained after injection
of the free labeled
protein. In the kidney, high fluorescence was observed also after injection of
the free labeled
protein. Quantification of these results was done by imaging software and is
shown in FIG. 33.
[00517] More specifically, FIG. 33 depicts that distribution of trypsinogen
labeled with
AlexaFluor0-488 in brain, kidney and liver after the injection (i.v.) of the
labeled protein in its
free form or encapsulated in vesicles. This figure presents quantification of
the data obtained in
the experiment described in FIG. 32. Each bar represent an average value of 5
mice +/- SEM.
[00518] The data of FIG 33 indicate that kidney was highly labeled with the
fluorescent
protein, due to the high penetration of the free labeled protein into this
organ. However, the
amount of the delivered protein, as estimated by the fluorescence, is similar
in the liver, which is
known to take up nanoparticles, and in the brain, to which nanoparticles do
not normally enter.
These results suggest that the vesicles enter the brain quite efficiently and
carry their protein
cargo into the brain.
[00519] The experiment described above was set to study whether a labeled
protein can be
detected in the brain, using a relatively high amount of the labeled
trypsinogen (200 lig per
mouse). These data suggested a more sensitive method would be advantageus for
detection and
visualization of lower levels of, e.g., GDNF.
Example 22
Delivery of GDNF to the brain
[00520] In view of the sensitivity issues noted above using the
fluorescently labeled
protein trypsinogen, this experiment was designed to test the use of GDNF-
biotin (Alomone Lab
Inc., Jerusalem, IL) a derivative protein that maintains all the properties of
GDNF, including full
GDNF activity. The GDNF-biotin was introduced inot vesicles that were made
from a
formulation that contained all the components described above, including GLH-
55a and GLH-57,
bolas that contain CS and DAT ligand head groups, and the GDNF-biotin-loaded
vesicles were
injected (iv.) into mice. Based on the above studies with CF and trypsinogen,
in which the
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labeled encapsulated material was seen in the brain 30 min after the
injection, this time point was
chosen for the initial detection of the GDNF-biotin in the brain. For the
detection of the
delivered GDNF-biotin in the brain, mice were sacrificed, perfused with 10 ml
PBS, to remove
the GDNF-biotin from blood vessels, and brains were removed and frozen in
isopentane
immersed in liquid nitrogen. The frozen brains were cryosectioned and the
sections were stained
with DAPI (to visualize the nuclei of the cells for orientation purposes).
Then, avidine-
AlexaFluor0-488 was added to the slides, which were then washed and observed
using a
confocal microscope. The avidine binds specifically to the GDNF-biotin and
only the sites in the
brain that contained the delivered GDNF-biotin showed fluoresce. To exclude
non specific
binding of the GDNF-biotin the avidine-AlexaFluor0-488 was added also to brain
sections taken
from mice that were injected with PBS.
[00521] Representative brain sections are shown in FIG. 34. In this
experiment, mice were
pretreated with 0.5 mg/kg (i.m.) pyridostigmine, then injected i.v. with
vesicles coated with CS
groups and DAT ligand with encapsulated GDNF-biotin. After 30 min, animals
were sacrificed,
perfused with 10 ml PBS, brains removed and striata, cortex and cerebella were
dissected out,
frozen and cryosectioned. Brain sections from these mice were stained with
DAPI (blue) and
avidine-AlexaFluor0-488 (green) and observed using confocal microscopy at a
magnification of
10X. (A) Stiatum from a mouse treated with PBS; (B) striatum from a mouse
injected with
GDNF-biotin encapsulated in vesicles; (C) cortex from a mouse injected with
PBS; (D) cortex
from a mouse injected with GDNF-biotin encapsulated in vesicles; (E)
cerebellum from a mouse
injected with PBS; (F) cerebellum from a mouse injected with GDNF-biotin
encapsulated in
vesicles.
[00522] As can be seen in FIG. 34, sections of the striatum from animals
that were injected
with vesicles with encapsulated GDNF-biotin, show a sharp focused fluorescence
arranged in a
circular shape around and within the striatum, whereas less fluorescence was
seen in the cortex
and even less fluorescence was seen in the cerebellum. The small amount of the
fluorescence
seen in the cortex was diffused and as focused as in the striatum. No
fluorescence was seen in
the control mice, indicating that the fluorescence which is seen in the brain
section is specific for
GDNF-biotin. Localization of the fluorescence in the brain section were also
examined under
higher magnification, and these results are shown in FIG. 35. It is clear from
FIG. 35 that the
GDNF-biotin is concentrated around many cells in the striatum and is found to
a lesser extent in
the cortex and the cerebellum. In particular, the the micrographs of high
magnification, (60X) of
FIG. 35 were taken from brain sections obtained from the mice used in the
experiment described
in FIG. 34. The nuclei of the cells appear in blue, due to DAPI staining, and
the GDNF-biotin
appears in green, due to the binding of the avidine-AlexaFluor0-488. (A) ).
Stiatum from a
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mouse treated with PBS; (B) striatum from a mouse injected with GDNF-biotin
encapsulated in
vesicles; (C) cortex from a mouse injected with PBS; (D) cortex from a mouse
injected with
GDNF-biotin encapsulated in vesicles; (E) cerebellum from a mouse injected
with PBS; (F)
cerebellum from a mouse injected with GDNF-biotin encapsulated in vesicles.
Whether all the
cells which stained for GDNF-biotin are dopaminegic neurons will be answered
from co-
localization studies that performed using antibodies against tyrosine
hydroxylase (TH) to stain
specifically the TH expressing cells.
[00523] In view
of all of the above, it is apparent that vesicles have been prepared from
bolas and coated with CS groups and DAT ligands. It is also apparent that
these vesicles are
capable of delivering GDNF to brain regions affected in Parkinson's disease.
In these studies,
building blocks (bolas) were designed and synethesized, and vesicles that were
made from these
building blocks were characterized. Further, GDNF encapsulation in these
vesicles was
optimized, and it has been demonstrated that they have a controlled release
mechanism enabling
the vesicles to release their content via the hydrolysis of the ACh head
groups by brain ChE. It
has also been demonstrated that the vesicles are targeted to dopamine
transporter expressing
cells, but not to cells that do not express the dopamine transporter. In
particular, these
experiments had demonstrated that the vesicles described herein are capable of
transporting
GDNF to the brain following systemic administration, and targeting the
neurotrophin to brain
regions that are affected in PD.
Example 23
Controlling the rate of drug release from bolaamphiphilic vesicles
[00524] The present disclosure further provides a method for controlling the
rate of drug
release from bolaamphiphilic vesicles with acetylcholine head groups by
changing the length of
an alkyl chain adjacent to the head group. Bolaamphiphilic compounds with
acetyl choline
(ACh) head groups with two different alkyl chains adjacent to the head groups
were investigated
for their ability to form vesicles that release the encapsulated material upon
the introduction of a
triggering event. One of these bolaamphiphiles, which was synthesized from
vernolic acid, has an
alkyl chain with 5 methylene groups adjacent to the ACh head group and the
other, which was
synthesized from oleic acid, has an alkyl chain with 8 methylene groups
adjacent to the ACh
head group. Both bolaamphiphiles formed stable vesicles with a diameter of
about 100 nm. The
ACh head groups of both bolaamphiphiles were hydrolyzed by acetylcholine
esterase (AChE),
however, the hydrolysis rate was significantly faster for the bolaamphiphile
with the shorter
aliphatic chain pendant. Likewise, when vesicles made from these
bolaamphiphiles were
subjected to AChE, those made from the bolaamphiphile with the shorter alkyl
chain near the
ACh head groups, released their encapsulated content faster than vesicles made
from the
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bolaamphiphile with the longer alkyl chain pendant. That is, the rate of drug
release from
bolaamphiphilic vesicles with acetylcholine head groups can be controlled by
varying the length
of the alkyl chain adjacent to the ACh head, and, therefore, this approach can
be used to design
vesicles that with different varied rates of drug release.
[00525] Synthesis of bolamphiphiles
[00526] The starting materials for bolaamphiphile synthesis are functional
vegetable oils and
their corresponding fatty acids. Vemolic acid, a naturally epoxidized fatty
acid (cis-12,13
epoxy, cis-9 octadecenoic acid) that constitutes the main constituent of
vemonia oil was used for
the synthesis of the bolaamphile GLH-20, noted above, which has a head group
hydrolyzed by
AChE. In order to compare the rate of the hydrolysis of a similar ACh head
group that contains
an adjacent longer alkyl chain, a second bolaamphiphile (GLH-32) was prepared
from oleic acid.
==,N0014.10/==.w.AOH
Vernolic acid
0
Oleic acid
0
e e
HO OH
GLH-20
CU,
N.,.. N:s.0"=PeeN.t>
0
0 - A
====.(cH2õ,0%... 0
HO
GLH-32 OH
[00527] For this synthesis the oleic acid was first epoxidized by a novel
approach that yielded
the corresponding C9-Cio epoxy stearic acid. The synthetic strategy included
two main steps: (a)
synthesis of the bolaamphiphile's skeleton by elongation of the corresponding
fatty acid through
its carboxylic group in an esterification reaction and (b) incorporation of
the head groups through
the functional groups on the fatty acid aliphatic chain, as depicted in Scheme
7, below.
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0 0
=/ws/1/%/%,""AocH3
."N\/.\\/\,/=/N).L0Fi
2
0 0 0 0
3 I 0
OH 0
0 OH 0 0 HO 0
W=/=>-4/W\Acr(C112),1.0)/
4
0
1 A(21./<
0 c9 0 0 e 0
\()( )01
AO/a) OH 0 .. 0 HO 0
CH
[00528] Bolaskeleton formation
[00529] To synthesize the skeleton 3 (Scheme 7a) of the bolaamphiphile, GLH-
32, the methyl
monoepoxy stearate 1 was used that was obtained by the epoxidation of methyl
oleate in the
presence of grafted titanium-containing silica materials as the catalyst. The
methyl monoepoxy
stearate 1 was hydrolyzed to obtain the monoepoxy stearic acid 2. MS at the
negative mode
showed m/z=296.8 [M-1]+. A peak at 1700 cm-1 appeared in the IR spectra,
indicating the
presence of the carbonylic carboxyl group. NMR spectra showed that the epoxy
group remained
untouched (2.93-2.91 ppm), and CH,-COOH was shifted at 2.37-2.34 ppm. The
monoepoxy
stearic acid 2 was reacted, using a chemo enzymatic reaction, with an
aliphatic diol, 1,10-decane
diol, in toluene, in stoichiometric amounts, in the presence of immobilized
Candida antarctica
lipase as the catalyst. The product, diepoxy distearate 3 is the skeleton of
the bolaamphiphilic
compound.
[00530] The FT-IR spectrum of the diester 3 showed the disappearance of the
absorption band
at 1700 cm-1, which is related to the carboxylic acid group, and the
appearance of the absorption
band at 1727 cm-1, characteristic of the new ester group. The new alkoxy
methylene group
CH2-0-C(0)- appeared at 4.04 ppm in the 1H-NMR spectrum and at 64 ppm in the
13C-NMR.
The epoxy group remained unchanged
[00531] Attachment of the head group
[00532] After obtaining the decane diepoxy distearate as the bolaamphiphile's
skeleton, the
head groups were attached in a two-stage reaction (Scheme 7b), involving (1)
opening the epoxy
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ring with chloroacetic acid to give the dichloroacetate, derivative 4, and (2)
quaternization stage
of N,N-dimethylaminoethyl acetate with 4 to give the bolaamphiphile 5 with two
acetylcholine
head groups bound to the hydrophobic chain through the nitrogen of the choline
moiety. The
diepoxy distearate 3 was reacted with an excess of chloroacetic acid in dry
toluene at 85 C for 48
h. The progress of the reaction was followed by TLC and HPLC. In order to
remove the excess
of chloroacetic acid, the reaction mixture was washed with a concentrated
solutionconcentrated
solution of NaHCO3, and the product was purified by column chromatography. The
FT-IR
spectrum of the dichloroacetate derivative 4 showed a new absorption band of
the chloroacetate
group at 1758 cm-1 and carboxylic ester absorption band at 1732 of the
starting diester. In the
11-1 NMR spectrum the following new signals appeared: a peak at 4.09 ppm of
the methylene
protons of the chloroacetate group (¨CH2C1), a peak at 4.87-4.91 ppm of the
proton of the new
ester group (CH¨O¨C(0)), and a peak at 3.60 ppm of the proton near the
hydroxyl group ¨CH¨
OH group ( FIG. 36). The corresponding chemical shifts in the 13C NMR spectrum
appeared at
40.96 ppm (¨CH2C1) for the chloroacetate group, at 72.32 ppm for the carbon
near the hydroxyl
group (¨CH¨OH), at 78.81 and 78.89 ppm for the carbon adjacent to the new
ester group (¨CH-
0¨CO-CH2C1) and at 167.2 ppm for the carbonyl carbon of this new ester group.
The formation
of structural isomers in 4 was expressed in the appearance of the multiplet of
the methylene
protons of chloroacetate group at 4.09 ppm in the 1FINMR spectrum and two
peaks at 71.70 and
71.83 ppm, of the carbon atom adjacent to the OH group (CH¨OH) and 78.81,
78.89 for CH-
OC(0) in the 13C NMR spectrum, confirming previously reported data regarding
the presence of
structural isomers for the chloroacetate of methyl vernolate. The presence of
structural isomers,
can also be followed from the terminal methyl group at 0.87 ppm and the a-
carbonyl group at
2.27 ppm; both appear as two triplets.
[00533] In Fig 36 it can be seen that due to the proximity of the chiral
carbon, the two protons
of the chloromethylene group, are diastereotopic hydrogens, and they split
each other. Two
doublets were obtained, one for the Ha proton and the second one for the Hb
proton, one of the
signals overlap with the triplet of the alkoxy methylene group of the original
ester. The different
intensities of the peaks at 4.056 and at 4.028 ppm are due to this secondary
phenomena.
[00534] The last
stage of the synthesis is the quaternization reaction of /V,N-dimethylamino
ethyl acetate with the dicholoro acetate 4 (scheme 7B) that yields the final
bolaamphiphile 5 with
two acetyl choline head groups. The reaction was carried out with a large
excess of the amine at
45 C for 6 h followed up by repeated washings with ether to remove the excess
of the tertiary
amine and the desired bolaamphiphile was obtained as a yellow viscous product.
[00535] The 1H-NMR of the bolaamphiphilic compound (Fig 4) can distinguish the
new peaks
of the ACh head group. The methyl (24) of the acetate CH3-C(0)-0 appear as a
singlet at 2.12
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ppm. The methylene group (22) N+-CH2-CH2-0- near the quaternary nitrogen
appeared at 4.25
ppm and the methylene group (23) N+-CH2-CH2-0-C(0)- near the oxygen appeared
at 4.60
ppm. The two methyl groups (21) of the quaternary nitrogen appeared as two
singlets at 3.61 and
3.62 ppm, while the two different protons of the methylene group (20) between
the quaternary
nitrogen and the carbonyl ¨0-C(0)-CH2-N+- appeared each one as a multiplet at
4.79 and 5.46
ppm.
[00536] Vesicle formation and characterization
[00537] Amphiphiles in general, and specifically bolaamphiphiles, can form
micelles,
multilayered sheets, vesicles, rings, or a variety of microstructures with
cylindrical geometry,
such as rods, tubules, ribbons, and helices. The morphology of the self-
aggregate structure is a
function of the molecular parameters of the specific bolaamphiphile. The
morphology of
aggregate structures formed by film formation-hydration and sonication of the
tested
bolaamphiphiles was studied by transmission electron microscopy (TEM), and
showed spherical
aggregate nanostructures for both bolaamphiphilic compounds (FIG. 38).
[00538] As can be seen from FIG. 38, the vesicles were fairly heterogeneous in
size with
diameters ranging between 50-300 nm. The size distribution was determined by
dynamic light
scattering (DLS) and the data are shown in Table 11. The average diameter of
the vesicles that
were made from GLH-20 was 368 nm, whereas the average diameter of vesicles
made from
GLH-32 was 345. However, vesicles made from GLH-32 were more heterogeneous in
size as
only 68% of the main peak was within the range of the average diameter. The
hydrodynamic
diameter of the vesicles, as determined by DLS, was higher than the size seen
in the TEM
because the DLS measurements are size average dependent and also measure the
size of the
hydrated particles, whereas the hydration layer is not seen in the TEM
micrographs.
Table 11: DLS measurements of vesicles from GLH-20 and GLH-32
Bolaamphiphile Diameter (nm) Weight
of Main peak (%)
GLH-20 368 97%
GLH-32 345 68%
[00539] GLH-20 and GLH-32 are symmetrical bolaamphiphiles forming monolayer
membranes. Due to differences in the void spaces between the bolaamphiphiles
at the inner
versus the outer surfaces of the vesicle's membrane, the aggregation of the
bolaamphiphiles into
a stable vesicle structure requires relatively large diameter vesicles to
reduce the relative large
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differences in surface areas the inner and the outer surfaces. One way of
stabilizing smaller
vesicles made of symmetrical bolaamphiphiles is by incorporating additives
that act as membrane
stabilizers that will be situated among the outer parts of the bolaamphiphiles
and thus, will be
used as spacers that stabilize a higher curvature between the bolaamphiphiles.
Membrane
stabilizers, such as cholesterol (CHOL) and cholesteryl hemysuccinate (CHEMS),
may be used
for this purpose. In addition to serving as spacers, such compounds raise the
order¨disorder
transition temperature and make the membrane more stable at higher
temperatures. Upon
incorporating CHOL and CHEMS, together with the symmetrical bolaamphiphiles,
into the
vesicle formulation, we observed that the bolaamphiphiles aggregated into
smaller vesicles (FIG.
39), which were also more homogeneous in size as compared to vesicles that
were made from the
bolaamphiphiles without the additives (Table 12).
Table 12: DLS measurement of vesicles made from GLH-20 and GLH32 formulated
with CHOL and CHEMS at a ratio of 2:1:1.
Diameter Weight MO
Bolaamphiphilic compond
(nm) main peak
GLH-20 120 100
GLH-32 134 100
[00540] Vesicle stability
[00541] Vesicle stability was evaluated by measuring both changes in the
concentration of
encapsulated carboxyfluorescein (CF) and vesicles size as a function of time
when incubated in
PBS at room temperature.
[00542] Preliminary studies showed that vesicles that were made from the
bolaamphiphiles
without CHOL and CHEMS tended to aggregate during time and form large
particles. Therefore,
the stability studies were performed with the vesicles that contained CHOL and
CHEMS.
Vesicles that were formulated with CHOL and CHEMS remained stable for at least
16 days (the
last time point measured), without changing their size (FIG. 40A) or the
amount of CF
encapsulation (FIG. 40 B).
[00543] Enzymatic Cleavage of the Head Group by AChE and release of
encapsulated CF
from the vesicles upon their exposure to the enzyme
[00544] When ACh head groups are covalently attached to the skeleton of
bolaamphiphiles via
the nitrogen atom of the choline moiety, the head groups are hydrolyzed by
AChE (data not
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shown). For this study, it was hypothesized that the length of the alkyl chain
which is adjacent to
the ACh head group may affect head group's hydrolysis rate by by affecting how
the ACh head
group sterically fits into the the enzyme's hydrolytic site. FIG. 41 shows
that indeed, the head
groups of both bolaamphiphiles are hydrolyzed by AChE, but the rate of the
hydrolysis of GLH-
20's head group is significantly faster than that of GLH-32's head group,
suggesting that a longer
alkyl chain near the ACh moiety retards the hydrolysis rate. By comparison,
the hydrolysis of
free acetylthiocholine (ATC - an analogue of ACh) is much faster than that of
the ACh head
groups of both bolaamphiphiles, corroborating our conclusion that an alkyl
chain adjacent to the
ACh head group affects the rate of the hydrolysis.
[00545] The finding that the head groups of both GLH-20 and GLH-32 are
hydrolyzed by
AChE suggests that both bolaamphiphiles bind to the enzyme and therefore, may
compete with
ATC for binding to the enzyme and inhibit its hydrolysis. In order to compare
between the
inhibitory potentials of the two bolaamphiphiles, the rate of ATC hydrolysis
was measured in
presence of three concentrations of the GLH compounds and assessed the results
by a
Lineweaver-Burk analysis. From FIG. 42 it can be seen that both GLH-20 and GLH-
32 acted as
competitive inhibitors, as increasing their concentrations affected the Icn
but not the Vrnax,. Yet,
the tested concentrations of GLH-20 (FIG. 42 A) affected the K11,
significantly more than the
same concentrations of GLH-32 (FIG. 42 B), suggesting that the affinity of GLH-
20's head
group to the enzyme is higher than those of GLH-32. This finding suggest that
GLH-20 is a
better substrate for AChE than GLH-32, explaining why the rate of the
hydrolysis of the GLH-
20's head groups is faster than the rate of hydrolysis of GLH-32's head
groups.
[00546] The hydrolysis of the surface groups on bolaamphiphilic vesicles
results in the
destabilization of the vesicular structure and the release of the encapsulated
material (data not
shown). The hydrolysis of the bolaamphiphile's head group was tested to see if
there were a
correlation between that rate and the rate of release of CF from vesicles
after exposing them to
AChE. Release of the encapsulated CF was measured by an increase in
fluorescence that occurs
when the released CF is diluted in the medium in which the vesicles are
incubated. The
encapsulated CF in the vesicles is quenched and when it is released into the
medium it is diluted
and dequenched, emitting a fluorescence signal.
[00547] As can be seen from FIG. 43, both vesicles started to release their
encapsulated
material immediately after the addition of AChE to the vesicle suspension. The
release rate was
biphasic with a more rapid release rate seen immediately after the addition of
the enzyme and
then, after about 20-50 seconds, the release stabilized at a slower but
constant rate. From FIG. 43
it can be seen that for both phases, the rate of release from GLH-20 vesicles
was more rapid than
the release rate from GLH-32 vesicles. To quantify the differences in the
release rates from both
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vesicle types, the percent release from each vesicle preparation was
calculated at 4 times after the
addition of AChE, which were taken during the second phase. The results of
this analysis (FIG.
44), show that the slope of the curve that represents the release as a
function of time is
significantly greater for GLH-20 vesicles, compared to GLH-32 vesicles.
[00548] The greater slope represents a faster release rate; indeed, at 400
seconds after the
exposure of the vesicles to the enzyme, GLH-20 vesicles released about 44% of
their content,
whereas GLH-32 vesicles released only about 20% of their content (FIG. 44).
[00549] Altogether, these results demonstrate that although both GLH-20 and
GLH-32 form
similar vesicles that release their content upon exposure to AChE but release
their encapsulated
material at a different rate.
Example 24
Compositions And Methods For Treatment Of ALS
[00550] In another embodiment, the present disclosure is directed to
compounds,
compositions, and method of the treatement of neurological diseases including,
for illustrative
purposes amyotrophic lateral sclerosis (ALS). In one aspect of this
embodiment, the present
disclosure is directed to testing demonstrate in a mouse model of ALS
beneficial effects of
systemically administered GDNF, encapsulated in novel nano-sized vesicles
provided herein.
[00551] The present disclosure provides vesicles that will be designed to
target sites in the
CNS where motor neurons degenerate and the encapsulated GDNF will be released
at these sites,
where the neurotrophin, upon its release, will induce its neuroprotective
effect and may also
cause neuronal regeneration. Targeting of the vesicles to sites in the CNS
where motor neurons
degenerate will be achieved by coating the vesicles with manose pendants that
will direct the
vesicles to activated microglia, which over express manose receptors.
Selective release of the
encapsulated GDNF is achieved by enzymatic hydrolysis of the head groups at
the sites where
the vesicles accumulate; in the case of the proposed vesicles ¨ in regions of
the CNS where
activated microglia accumulate due to motor neurons degeneration in the ALS
mouse. Specific
elements of this approach include: 1) synthesis of bolaamphiphiles (bola) -
the vesicle's building
blocks; 2) formation of vesicles coated with manose pendants and encapsulation
of GDNF in
these vesicles; 3) testing the nano-sized vesicles for brain delivery and for
targeting to activated
microglia pharmacokinetic (PK) studies); and 4) demonstrating the beneficial
effects of the
delivered GDNF in an ALS mouse model. The above was repeated using
neurotrophins such as
insulin-like growth factor 1 (IGF-1) instead of GDNF, demonstrating
significant beneficial
effects in an ALS mouse model.
[00552] The GDNF-loaded vesicle system disclosed herein may be a
breakthrough in the
treatment of ALS for which there is no effective treatment. Moreover,
developing the presently-
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disclosed nanovesicle platform for GDNF has wider implications for additional
neurotrophic
factors with the potential for ALS therapy as well as for other
neurodegenerative diseases that
may benefit from neurotrophic factors.
[00553] As demonstrated above, the present disclosure provides nano-sized
vesicles made
from bolaamphiphiles (bolas) that were designed drug delivery and were
synthesized as
described herein along with vesicles made of monolayer membrane that provides
stability (due to
high energy barrier for lipid exchange); high encapsulation capacity (due to
their thin membrane
that makes vesicles with a large inner volume), good brain penetrability (due
to surface pendants
that induce transcytosis via the brain microvessels endothelial cells) and an
efficient controlled
release mechanism (due to specific hydrolysis of the head groups at the target
site). These
vesicles have been used to deliver a variety of compounds into the brain,
including small
molecules, peptides, nucleic acids; proteins; and, as demonstrated above,
e.g., GDNF.
[00554] Similar vesicles can be coated with manose pendants that will
direct the vesicles
to sites in the brain where motor neurons degenerate, for use in the treatment
of ALS. The
targeting concept is based on the notion and findings that in brain regions in
which motor
neurons degenerate, there is an accumulation of activated microglia [Xiao et
al, 2007; Corcia et
al, 2012; Liao et al, 2012; Hoyden et al, 20131 that overexpress manose
receptors [Galea et al,
20051. Our success in targeting vesicles coated with dopamine transporter
ligand to dopaminergic
neurons, suggest that there is a high probability that vesicles coated with
manose pendants will be
targeted to activated microglia, which are abundant in regions of degenerating
motor neurons and
over express manose receptors.
[00555] For ALS treatment, GDNF will be encapsulated in the vesicles
described herein.
As demonstrated herein, conditions for encapsulation of GDNF in vesicles have
been worked
out. In one approach will be to coat the vesicles with manose pendants
followed by
demonstrating that the encapsulated GDNF is beneficial in the treatment of ALS
in an animal
model. This will allowd encapsulation of additional neurotrophins with the aim
of obtaining
synergism and will also provide a strong rationale for performing clinical
trials in human
subjects. In another embodiment, ALS can be treated by administration of IGF-1
encapsulated in
vesicles of the present disclosure, including, in one aspect vesicles coated
with mannose pendants
prepared according to the present disclosure. Therefore, in certain
embodiments of the present
disclosure ALS is treated by delivery of neurotropic factors including but not
limited to GDNF,
IGF-1, and combinations thereof, in vesicles of the disclosure, including
those that comprise
mannose surface groups.
[00556] Synthesis of bolaamphiphiles and the preparation and
characterization of vescicles
of the disclosure have been described above. In the present instance, vesicles
will be prepared
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that are coated with mannose pendants will include encapsulated GDNF and or
other
neurotrophins such as insulin-like growth factor 1 (IGF-1). That is, a bola
for the in vivo studies,
i.e., a bola with the manose head groups, will be added to the vesicle
formulation to coat the
vesicles with manose pendants for targeting to activated microglia. The
synthesis of this bola
will be based on the methods used with many other bolas described herein. In
various
approaches, at lest two different vegetable oils can be used as the starting
material (see below),
including castor oil and vernonia oil.
Formation of vesicles coated with manose pendants and encapsulate GDNF and or
other
neurotrophins in these vesicles.
[00557] In view of the data provided above, the manose head groups are
expected not to
affect vesicle properties except for targeting them to cells that express
manose receptors.
However, the proportions between the bolas within the vesicle formulation that
will yield stable
vesicles that are targeted to cells that express manose receptors will be
determined before
initiation of the in vivo studies.
[00558] In particular, vesicles made from GLH-19 are quite stable in whole
serum (GLH-
19 vesicles are not disrupted by choline esterase), whereas vesicles made of
GLH-20 release their
content in whole serum relatively quickly. In one approach, the ratio between
GLH-19 and
GLH-20 will be gradually adjusted to provide the most stable vesicles that
still release their
content upon exposure to choline esterase and this basic formulation will be
used for all future
studies. With the optimal formulation of GLH-19 and GLH-20 determined, the
bola GLH-55B
(an asymmetric bola with a CS head group on one side and acetylcholine head
group on the other
side) can be we incorporated into the formulation at the highest proportion
that will not change
vesicle stability. The last stage of optimizing the vesicle formulation will
be an introduction of
the bola with manose head groups into the vesicle formulation and test the
proportion of this bola
that does not affect vesicle properties. As a parameter for the targeting
efficiency, endocytosis of
vesicles with and without manose pendant into macrophage cell line that
express manose
receptors will be tested.
Testing the nano-sized vesicles for brain delivery and for
targeting to activated microglia pharmacokinetic (PK) studies)
[00559] Since GDNF is rather expensive, for testing targeting in vivo and
for the PK
studies, vesicles loaded with a fluorescent marker (carboxyfluorescein or FITC-
dextran) will be
used as a model system for initial experiments and the initial PK studies will
be carried out with
control mice. Mice will be injected with vesicles loaded with a fluorescent
marker (vesicles with
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and without manose pendants) and the amount of the fluorescent dye in the
brain will be
measured. The proportion of the manose-bola trying will be varied (along with
other parameters)
optimize targeting efficiency without losing penetration into the brain in
normal mice. In normal
mice biodistribution of the encapsulated fluorescent dye in various tissues
will also be tested.
Upon finalization of an optimal composition, PK studies will be carried with
ALS mice, using
vesicles loaded with GDNF. Mice will be injected with GDNF-loaded vesicles
with and without
manose pendant and the distribution and the quantities of the GDNF in the
brain will be
determined using ELISA and immunohistochemical techniques (for details see
method section).
Demonstrating beneficial effects of the delivered GDNF in an ALS mouse model
[00560] ALS mice were injected with the optimal vesicle formulation and
efficacy
parameters were assessed. The following experimental groups were used (10
animals per group):
1) Mice injected with empty vesicles as control; 2) Mice injected with optimal
vesicles
containing encapsulated GDNF; 3) Mice injected with free GDNF as a negative
control. The
mice received multiple injections of the test material during 45 days and the
intervals between
injections were determined in the PK studies (see above), whereas the criteria
for the intervals
were the time period that takes for the clearance of the GDNF from the brain.
During the
treatment period changes in body weight, test motor behavior, and performance
of
electromyographical analysis (see below) were measured. Life span of the
treated mice was
determined and used as one criterion for efficacy. In case of a life span
shorter than the planned
duration of the experiment, the treatment period was shortened accordingly and
the mice were
sacrificed at the end of the treatment period to test the effect of the
treatment on motor neurons
(see below).
[00561] Synthesis of bolas GLH-19, GLH-20, and GLH-55b are described above.
Synthesis of the bolaamphiphile with the mannose head groups is provided in
Scheme 7 (below).
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Scheme 7: the synthesis of bolaamphiphile with manose head groups
HOtr%=eN=%=NT-/-1 + HO-(CH2)11-0H
2
dicarboxylic acid
HO rOH
On
3
OH
HO
HO OH
mannose
n
Asymmetric bola compound
1E0 Olt
I I
Symmetric bola compound
o.õ
HO OH
OH
[00562] As indicated in this Scheme, ricinoleic acid (the main component of
castor oil,
>97%) was used as the starting material to form the hydrophobic skeleton of a
symmetric
bolaamphiphile. The diester 3 (see Scheme 7) was synthesized by the extension
of the ricinoleic
moiety in a chemoenzymatic esterification or transesterification reaction of
ricinoleic acid (1,
R=H) or methyl ricinoleate (1, R=CH3) with aliphatic diols of different chain
lengths using
Candida antarctica lipase as the catalyst.
[00563] The second stage was the esterification of the secondary hydroxyl
groups of the
ricinoleic moiety of the diricinoleate 3 with a dicarboxylic acid in the
presence of an acidic
catalyst under azeotropic conditions.
[00564] The attachment of the mannose head group was performed by a
chemoenzymatic
esterification, in order to obtain selective binding to the primary hydroxylic
position. This is a
consecutive nucleophilic substitution reaction, which yields a mixture of
monoester 4 and
diesters 5, allowing the formation of a symmetric and asymmetric bolacompounds
that were
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further separated by flash chromatography and their effect on vesicle
formation, vesicle stability
and targeting was investigated.
[00565] An alternative approach to this synthesis of a bola with the manose
head groups
was to use vernodiester 6 as the starting material similar to GLH-19 and GLH-
20. The synthesis
started by opening the epoxy group with a dicarboxylic acid. The attachment of
D-mannose to
the intermediate diester dicarboxylate was done by enzymatic esterification
with Candida
antarctica lipase.
[00566] Characterizatinn of the synthesized bolas, vesicle formation and
characterization,
GDNF encapsulation, determination of GDNF activity, determination of vesicle
stability, and
determination of GDNF release in vitro were carried out as described herein.
Investigation of targeting to manose receptors in vitro
[00567] Macrophages that express manose receptors on their surface were
grown in 24-
well plates the medium was replaced with culture medium without serum and
samples of
carboxyfluorescein-loaded vesicles with and without manose pendants or free
(non-encapsulated)
carboxyfluorescein (equivalent to the encapsulated carboxyfluorescein) were
added to the cells
and incubated for 1-5 h at 4 C or at 37 C. At the end of the incubation,
cells were washed and
either detached from the plates using cell detachment medium and analyzed by
FACS
(FACSCalibur Flow Cytometer, BD Biosciences, USA). The presence of manose
pendants on
the surface of the vesicles increased the uptake of the vesicles. Higher
uptake was expressed in a
shift of the peak in the flow cytometry profile and was indicative for
targeting in vitro. This
technique to measure targeting of vesicles that were coated with dopamine
transporter ligand to
cells that express dopamine transporter has been verified (data not shown).
Pharmacokinetic (PK) studies:
[00568] Since GDNF is expensive, until the vesicle formulation is fully
optimized
encapsulated fluorescent marker were used to follow the PK properties of the
vesicles. This
approach was employed successfully when the biodistribution of vesicles that
coated with
dopamine transporter were studied and were targeted in vivo to dopaminergic
cells in the brain
(see above). Initially vesicles with encapsulated carboxyfluorescein were
injected into normal
mice and fluorescence in various tissues (blood, liver, kidney, lung, spleen,
spinal cord and brain)
was measured at various times after the injection. Mice (5 per group) were
pretreated 15 min
prior to the injection of the vesicles with 0.5 mg/Kg pyridostigmine to
inhibit peripheral choline
esterase. For comparison, in parallel to the pyridostigmine-treated animals,
similar experiment
was performed with animals that did not receive pretreatment with
pyridostigmine. Tissues were
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removed at various times after the injection of the vesicles (1, 2, 4, 8, 12,
24, 48 hours),
homogenized and deproteinized by trichloroacetic acid or perchloric acid.
Fluorescence
intensities were determined in the supernatant of the tissue extracts after
centrifugation.
[00569] The next stage of the PK studies was done with SOD1 transgenic mice
as an
animal model for ALS. In this experiment vesicles coated with a targeting
ligand (manose) were
compared to vesicles without targeting ligand (naked vesicles). For this
experiment GDNF-
loaded vesicles were used and concentrations of GDNF in several regions of the
CNS (spinal
cord, cortex and cerebellum) were determined by ELISA, e.g., using
commercially-available kits
(e.g. Promega, Fitchburg WI). This testing was carried out using, e.g., 5
animals per group and
the time points were selected according to the results obtained in the first
stage of the PK studies
(see above).
[00570] Another set of PK studies with SOD1 transgenic mice was done with
vesicles
loaded with GDNF-biotin. In this set of experiments the distribution of the
injected GDNF in the
CNS was determined by histofluorescence technique. Vesicles with and without
targeting ligand
were loaded with GDNF-biotin and were injected to ALS mice (3-4 animals per
group). At
various times after the injection, the animals were deeply anasthetized,
perfused with PBS, brains
and spinal cords removed and frozen sections were prepared from these tissues.
The sections
were stained with DAPI to visualize the nuclei and identify the region of the
CNS according to
the morphology. Co-staining with avidine-AlexaFlour was performed on the same
sections to
visualize the GDNF-biotin and histochemical staining of nucleoside-
diphosphatase, an enzyme
specific to microglia were performed in order to see if the GDNF was
concentrated around the
microglia.
Efficacy studies
[00571] ALS mice were divided into three groups (10 animals per group) as
follows: 1)
Mice that were treated with empty vesicles; 2) Mice that were treated with
optimal vesicles
loaded with encapsulated GDNF; 3) Mice that were treated with free GDNF. The
treatment
regimen was determined according to the results from the PK studies and
continued for up to 45
days. The following parameters were used for the assessment of the efficacy of
the treatment: A)
Measurements of changes in body weight; B) Motor behavior: Several motor tests
were
performed in order to assess the condition of the ALS mice during the
treatment period. The
various tests were performed at different days. These tests were (1) Open
field test: The animal
was placed in the center of the open field apparatus and allowed to move
freely for 10 min. The
total distance moved, the frequency and duration of rearing (standing on the
hind legs) and the
time spent in the center area were recorded. The total distance moved was
evaluated as an index
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for locomotor behavior and rearing behavior and time spent in the center was
evaluated as an
index for exploratory behavior. This test was performed every 4 days from the
beginning of the
treatmen; (2) Rotarod test: Mice were tested on the rotarod for the assessment
of their motor
function. The rotarod consists of five textured drums of 1.25 cm diameter.
Total time that the
mouse was able to remain on the rotating drum was recorded. Training consisted
of habituation
during which the mice were acclimatized to the rotarod at 5 rpm for 180
seconds and training
during which they were allowed to remain on the rotarod at 10 and 15 rpm for
180 sec. On the
test day, all mice were tested at 15, 20, 25 and 30 rpm for 180 sec and 10 min
rest period were
allowed between each trial. This test was performed one day befor the
treatment and again before
sacrificing the mice; (3) Swimming tank test. To assess progression of motor
deficit, swimming
movements were monitored in a water-filled tank. The device consisted of a 90-
cm long, 6-cm
wide and 40-cm high tank filled to a depth of 20 cm with water at a
temperature of 24 C. A
visible escape platform was positioned at the end of the tank. As a starting
point for recording
swimming performance, a vertical black line was drawn on one side of the tank,
marking a
horizontal 70-cm distance to reach the platform. A high-resolution web camera
for video
recording of limb kicks during swimming was used. As a training procedure, the
mouse was
allowed to swim from the starting line to the platform for 2 consecutive days
with five trials per
day. The mice were given five consecutive trials on a 10-d basis. To avoid
artifacts and to always
obtain the fastest swimming performance for each animal, analysis of the
swimming latency was
based on the mean scores of the three shortest latencies. The number of
hindlimb kicks will be
video recorded once for each animal and for each session. For mice with late-
stage disease that
may not be able to climb onto the platform anymore, the timer was stopped once
the forepaws
touched the platform. The maximum swimming latency was set at 20 sec.
[00572] C) Electromyographical analysis. Evoked CMAP amplitudes and
spontaneous
fibrillation potentials (SFPs) were evaluated with an electromyogram
apparatus. Measurements
were repeated every 7 days. Mice were anesthetized with isoflurane and the
sciatic nerve was
stimulated at a paraspinal site by a single 0.1-ms, 1-Hz supramaximal pulse
through an unipolar
needle electrode and recorded CMAPs from the medial part of the gastrocnemius
with the same
type of electrode. SFPs were recorded through a concentric needle electrode
and only SFPs with
an amplitude ranging from 20 to 300 pAi were considered.
[00573] D) Histological analysis: At the end of the treatment session, mice
were
anesthetized and transcardially perfused with 50 ml of 4% paraformaldehyde in
phosphatebuffered saline (PBS). Brain and spinal cords were post-fixed in the
same fixative for 4
h and processed for either paraffin or cryoprotective embedding. For
immunohistochemistry the
tissue was stained using antibodies against EGFP, nonphosphorylated
neurofilament and NF-L.
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Cryostat sections of 16-pm thickness were incubated with primary antibodies
diluted in 4%
bovine serum albumin, 5% donkey serum in PBS containing 0.1% Triton-X100.
Immunoreactivity was visualized with AlexaFlour-conjugated secondary
antibodies diluted in the
same solution. For histopathological analysis, 8-pm deparaffinized sections
were stained with
cresyl violet and motor neurons were ccounted every five sections.
Example 25
Compositions and Methods Comprising Bolaamphiphiles with Mannose Head Groups
for
Specific Targeting of Vesicles to ALS Sites in the CNS
[00574]
Disclosed herein is a novel treatment for amyotrophic lateral sclerosis (ALS)
that is based upon the use of bolaamphiphile vesicles capable of targeting
degenerating motor
neurons in the central nervous system (CNS) of ALS patients and release
encapsulated GDNF
near the degenerating motor neurons, where the released GDNF will provide its
neurotrophic
activity, namely, promoting neuroprotection and neuroregeneration. The
targeting of these
vesicles is achieved by surface groups that have high affinity to mannose
receptors highly
expressed in activated microglia that accumulate near degenerating motor
neurons. The present
disclosure provides vesicles with surface targeting ligands and that have been
tested to
demonstrate the vesicles' capability to target cells that highly express
mannose receptors. .
[00575]
Described are vesicle compositions comprising the following components 1) the
bolaamphiphiles (bolas) GLH-19 and GLH-20, which contain acetyl choline (ACh)
head groups
for controlled release of encapsulated GDNF; 2) the bola GLH-55b, which
contains a chitosan
(CS) head group to enhance penetration of the vesicles via the blood-brain
barrier (BBB); 3)
several types of GLH-64 (GLH-64a-e), a bola family with mannose head groups
that target the
vesicles to mannose receptors.
[00576] As
demonstrated herein, vesicles that contain mannose moieties on their surface
(mannose surface groups were introduced by inclusion in the vesicle
formulation one of the
GLH-64 bola's family, particularly GLH-64a), provide efficient targeting of
GLH 64 with the
mannose head group. As can be seen from Figure 53 (below), the vesicles that
contained
mannose surface groups were taken up about 10 times more than vesicles that
did not contain
mannose groups on their surface. Inclusion of free mannose in the bathing
medium (10 mM)
completely abolished the effect of the mannose surface groups since it
competed with the
mannose surface groups for binding to the mannose receptors that were
expressed on the
membrane of the differentiated cells. Free (non-encapsulated) fluorescent
probe (siRNA
conjugated with alexaFluor 546) was not taken up by the cells at all and the
peak of the cells that
were exposed to the free fluorescent probe was identical to the peak of the
control cells that were
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not exposed to neither vesicles and fluorescent probe. The data below show
that the vesicles with
their encapsulated fluorescent probe, and not the free fluorescent probe, were
taken up by the
cells and that vesicles with mannose surface groups were taken up by the cells
much more than
vesicles without the targeting ligand on their surface. Altogether, these
results indicate that
vesicles with mannose surface groups target cells that express mannose
receptors.
[00577] Theresults obtained with GLH-64a are conclusive and show that a
bola which is
bound to the mannose moiety via the primary hydroxyl which is situated on
carbon 6 is capable
of providing efficient targeting.
[00578] The results obtained with GLH-64b showed that targeting can be
achieved with
this bola as well, although the uptake of the vesicles that contain GLH-64b
(uptake indicates
targeting) was somewhat smaller than that obtained with GLH-64a (see Figure
52). GLH-64b
contains a mixture of bolas where the mannose is bound to the bola skeleton
either via the
primary or the secondary hydroxyls. Therefore, it was interesting to see
whether a bola in which
the mannose moiety is bound only via the secondary hydroxyl is capable of
providing good
targeting. GLH-64d is such a bola in which the mannose moiety is bound via the
secondary
hydroxyl, which is situated on carbon number 1 of the mannose. The results of
the targeting
experiment with GLH-64d are described in Figure 54 (below) As can be seen from
Figure54,
vesicles that contain GLH-64d were taken up somewhat better by differentiated
cells than by
non-differentiated cells, but the shift was much smaller than that obtained
with GLH-64a. Based
upon all results of the targeting experiments described herein, targeting can
be achieved with
vesicles that contain mannose surface moieties, particularly when the mannose
is bound to the
bola's skeleton via the primary hydroxyl situated on carbon 6 of the mannose.
Synthesis of GLH 64a-e, a bola family with mannose head groups
[00579] This bola family contains D-mannose head groups for targeting of
the vesicles to
mannose receptors. When such bolas are included in vesicle formulation, the
mannose head
group is positioned on the vesicle's surface and provides a targeting moiety
that is expected to be
recognized by and bind to the mannose receptor. Binding of the mannose moiety
to the bola's
skeleton can be done via each of the hydroxyl groups of the mannose, Since it
was not clear
which hydroxyl group or groups are important for the recognition and binding
to the mannose
receptors, several species of GLH-64 (GLH-64a-e) were prepared that contain
both alpha and
beta configurations of the sugar and the binding was done via either the
primary hydroxyl, or via
one of the secondary hydroxyl groups of the mannose. The synthesis of these
bolas is described
herein.
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Synthesis of GLH-64a, a bola with mannose moiety bound to the bola's skeleton
via
the primary hydroxyl of the mannose
[00580] The starting material for the synthesis of this first species of
GLH-64 was
ricinoleic acid (the main component of castor oil, >97%), which was obtained
by the hydrolysis
of castor oil. The synthetic steps are shown in Scheme 7 (above). Ricinoleic
acid (compound 1 in
Scheme 7) was reacted with aliphatic diol to achieve extension of the
ricinoleic moiety by a
chemoenzymatic esterification of ricinoleic acid (1, R=F), using Candida
antarctica lipase as the
catalyst, providing compound 2 of Scheme 7..
[00581] The second stage was the synthesis of the bola's skeleton (compound
3 in Scheme
7) by esterification of the secondary hydroxyl groups of compound 2 (Scheme 7)
with a
dicarboxylic acid in the presence of an acidic catalyst under azeotropic
conditions. The
attachment of the mannose head group to the bola's skeleton was achieved by a
chemoenzymatic
esterification, in order to obtain selective binding to the primary hydroxylic
position. This
constituted a consecutive nucleophilic substitution reaction, which yielded a
mixture of the
monoester 4 and the diester 5 (GLH 64a). The mixture of the monoester D-
mannose
bolaamphiphile (compound 4, 36.4%) and the diester D-mannose bolaamphiphile
GLH-64a
(2.3%) were separated by flash column chromatography on silica gel, using
detection by
common spectroscopic methods.
Analysis of reaction products by FT-IR, 11-1 and 13C NMR as well as by ESI-MS.
[00582] FT-IR analysis showed the appearance of the broad absorption band
of O-H for
GLH-64a and compound 4 and the disappearance of the carbonyl band of the
carboxylic acid
(1712 cm-1) for GLH-64a.
[00583] 11-1 and 13C NMR spectra were performed in DMSO-d6 and it appeared
that both
compound 4 and GLH-64a showed the same 11-1 NMR signals, differentiated only
by the number
of their protons in the integration curves. In the 11-1 NMR spectrum of GLH-
64a, in addition to
the signals of the D-mannose moieties, we observed the presence of two
multiplets of the protons
from CH=CH groups (5.42 and 5.28 ppm), multiplet of the methine protons of the
ricinoleic
moieties (4.76 mppm), as well as the methylene protons of the glutarate unit
near the carbonyl
esters at 2.31 and 2.29 mppm. The chemical shifts of the methine and methylene
protons of D-
mannose groups were assigned by analyzing the two dimensions HMQC spectrum of
GLH-64a
in comparison with the HMQC spectrum of D-mannose in the same solvent. This
spectrum
showed the following direct correlations: Cl-H (94.49 and 4.86 ppm), C2-H
(71.75 and 3.54
ppm), C3-H (70.73 and 3.71 ppm), C4-H (67.47 and 3.37ppm), C5-H (73.36 and
4.78 ppm) and
C6-H2 (64.73 & 4.31). However, it is worth mentioning that the signals of the
methylene group
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of the primary alcohol (CH2-0H) appearing in the D-mannose HMQC spectrum at
62.01 ppm
and 3.61and 3.42 ppm were deshielded and shifted to lower magnetic fields for
GLH-64a (64.73
and 4.31 ppm). Figure 45 provides the 13C NMR spectra of the diester
diglutarate 3 (Scheme 7),
D-mannose and the bola GLH-64-a in DMSO-d6. Contrary to the 13C NMR spectrum
of GLH-
64a (Figure 45) that shows the disappearance of the peak of the carbonyl of
COOH groups of the
glutaric acid moieties, the 13C NMR spectrum of compound 4, showed the
presence of the
carbonyl of the carboxylic acid at 174.43 ppm and the appearance of a new peak
of carbonyl of
ester at 172.60 ppm (C00-mannose) in addition to the two other peaks of
carbonyl of ester
groups of the molecule. We also saw in the spectrum of compound 4 the presence
of the peak at
33.11 ppm for the methylene carbon of the glutaric acid unit adjacent to the
carboxylic acid
moiety (CH2-COOH), the peak at 33.41 ppm for the methylene carbon of the
glutaric acid
moiety attached to the mannose ester unit (CH2-000-mannose) and the signals of
the D-
mannose carbons at 94.54, 73.53, 71.75, 70.81, 67.61 and 64.87 ppm.
[00584] The formation of bolaamphiphiles GLH-64a and compound 4 was also
confirmed
byESI-MS analyses. In a negative mode, the mass spectrum of compound 4
(containing one
mannose head group and one carboxylic acid head group) showed the peak of the
molecular ion
at m/z 1124.3, matching with the molecular weight of its formula C62H108017.
The mass spectrum
of bola GLH-64a showed in a positive mode the peak of the molecular ion at m/z
1286.6,
corresponding to the molecular weight of the formula C68H118022, which is GLH-
64a. The
fragmentations of the molecular ions [M+Nal+ in positive mode of both
compounds showed the
presence of peaks at m/z 721.4 and 1015.4. The signal at m/z 721.4 corresponds
to the fragment
of their molecular ions without all the glutaric acid and D-mannose moieties.
The peak at 1015.4
in the case of compound 4, is the fragment of the molecular ion without one
glutaric acid moiety
and for GLH-64a, this signal represents the fragment of the molecular ion
without one glutaric
acid and unit of D- mannose, as shown in Figure 46 which presents the main
fragmentations of
GLH-64a in ESI-MS (positive mode).
[00585] Initial experiments provided about 50 mg of GLH-64a with high
purity as can be
seen from the HPLC chromatogram presented in Figure 47. This quantity was
sufficient for
product characterization and to perform initial studies with vesicles that
contain GLH-64a.
Described below is an alterntive, improved process for the synthesis of GLH-
64a.
Experiment 26
Alternative Proess For GLH-64a Synthesis
[00586] Improvements in the synthesis of GLH-64a were achieved by varying
several
parameters as follows:
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[00587] A) Previously, to obtain GLH-64a the starting material ricinoleic
acid or methyl
ricinoleate (see Scheme 7) was that obtained by hydrolysis of triricinolein,
or as a product of
transesterification of castor oil (Scheme 8).
OH 0
0
(7:11-1 OH
triricinolein
oH 0
methyl ricinoleate
Scheme 8: Transesterification of triricinolein
[00588] Castor oil contains, besides the triricinolein, other triglycerides
(about 10%) and
the metyl ricinoleate that was obtained from the transesterification of
triricinolein needed to be
purified, e.g., using flash chromatography to separate the methyl ricinoleate
as in the procedures
above, a procedure requiring expensive silica gel and substantial amounts of a
hexane-diethyl
ether (7%) mixture. It also involved the collection and analysis of many
fractions, a method
better suited for milligram to gram scale.
[00589] To improve the synthesis of GLH-64a, particularly at a larger
scale, a liquid-liquid
extraction of methyl ricinoleate was employed, without using the
chromatography separation
according to procedures described in the literature [Bordeaux et al., JAOCS
1997;74 (8):1011].
This liquid-liquid extraction procedure which enabled purification of large
amounts of methyl
ricinoleate, is presented schematically in Figure 48, and is described below:
[00590] Crude methyl ricinoleate (20g) was shaken with 120mL of hexane and
60mL of
90% aq. methanol in the first separating funnel. The layers were separated,
and the methanolic
phase was removed. The hexane phase was extracted with another 12 portions of
60mL of 90%
aq. methanol consequently one after another to yield 13 portions of methanolic
solution. Each
methanolic solution was sequentially passed through two more separating
funnels, each
containing 120mL of hexane. Each methanolic solution was examined by thin
layer
chromatography (TLC) using hexane:ether (1:1) to obtain pure methyl
ricinoleate. The solvent
was removed under reduced pressure. 35.8 g of the mixture methyl ricinoleate
and was used
without further purification.
[00591] The procedure was repeated using 76g of crude methyl ricinoleate.
The same
proportions of hexane and methanol were used. 702.6 g of hexane and 2065g of
methanol were
recovered. The hexane residue was examined and no methyl ricinoleate was found
in this
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fraction, but it contained about lOg (about 13% from total esters) of other
methyl esters that were
separated from the methyl ricinoleate.
[00592] B) In initial experiments for the synthesis of GLH-64a p-toluene
sulfonic acid was
used as a catalyst in the reaction of esterification of diester diricinoleate
with glutaric acid
(scheme 9)
0
0
0 0
H 0)C-)L0 H
0
0
H H
Diricinoleate Diglutarate
Scheme 9: The synthesis of diricinoleate diglutarate
[00593] Initial experiments required the presence of a soluble catalyst
that required
multiple washings with water at the end of the reaction. Then the solution
that contained the
product had to be dried out with MgSO4. In this alternative, improved
approach, Amberlyst 15
was used as an acidic solid catalyst. Since it is a solid, it is easy to
remove this catalyst by
filtration and the washings was saved.
[00594] C) In initial experiments, binding of the mannose moiety to
diricinoleate
diglutarate (scheme 10), provided only a low product yield, apparently because
only a small
amount of mannose could have been dissolved in most of the organic solvents
used in
esterification reaction. Accordingly, solvents that dissolve higher amounts of
mannose, such as
DMSO or pyridine, were tested but the enzyme lipase that was used in this
reaction was not
active in these solvents. Therefore, the reaction was performed in
heterogeneous conditions using
t-butanol as a solvent, but the yield was low. In an attempt to overcome this
problem, a
super-saturated solution of mannose in ionic liquid (1-butyl-1-
methylpyrolidinium
trifluoromethanesulfonate) was used, but it was difficult to remove the ionic
liquid. In an
attempted alternative approach, involved binding the mannose without using the
lipase.
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0
0
HO
HO
0 OH
HCYCY'YC)H
0 0 1 0 0 0
HO 0 0 0 OH
HOUOH
OH GLH 64
Scheme 10: Synthesis of GLH64a:
The reaction between diricinoleate diglutarate and D-mannose.
[00595] This reaction was performed in pyridine (in which mannose is
soluble in relatively
high quantities) as a solvent and EDCI* HC1 was used as a water scavenger. A
solution of
EDCI*HC1 in dry CHC13 was added drop-wise to a solution of diglutarate
diricinoleate in dry
CHC13 at -5 C (over ice with NaCl). Then the reaction was stirred overnight at
room temperature.
After the removal of the solvent under reduced pressure, the product was
purified by flash
chromatography using CHC13-CH3OH (7 -8%) as an eluent. Although the product
was obtained
in relatively high yield, it contained a mixture of bolas with the mannose
bound via both the
primary and secondary hydroxyls of the mannose (note that the lipase binds the
mannose
selectively via the primary hydroxyl and in the absence of the lipase the
binding was not
selective). Since the product was a mixture of bolas in which the mannose
moiety is bound via
both the primary and the secondary hydroxyls, it was essentially different
than GLH-64a and
therefore, it was named GLH-64b.
Example 27: Synthesis of Additional GLH-64 Species
[00596] To facilitate the synthesis of additional GLH-64 species (GLH-64 c-
e) with the
mannose bound specifically via the primary or the secondary hydroxyls of the
mannose, selective
binding of the mannose to the bola's skeleton via either the primary or one of
the secondary
hydroxyls of the sugar, was provided by the use of protected mannose compounds
as reagents.
[00597] A) The use of monoprotected mannose ¨ alfa D-1-benzyl-mannopyranose
to
prepare GLH-64c: The compound alfa D-1-benzyl-mannopyranose (a derivative of
mannose
with protection on the hydroxyl group of carbon number 1) is soluble in tert-
butanol, but not in
chloroform. Therefore, the reaction was performed in tert-butanol in the
presence of lipase
Novozym 435 as a catalyst (Scheme 11).
[00598] The reaction was performed at room temperature and at 60 C. The
products
contained a mixture of many materials. The target product (GLH-64c) was
obtained in less than
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5% yield by chromatography when the reaction was carried out at 60 C. The
reaction at room
tem perature gave almost no target product.
0 0
0
Ho
Bn0 OH
T'
HO OH
BnO-st,OH
OH
0
040 0
oBn
HO
OH
GLH64c with protecti on on the hydroxyl group on carbon 1
Scheme 11: Esterification of divernolate diglutarate with monoprotected
mannose.
[00599] B) Completely protected mannose was used to prepare other GLH-64
species
(GLH-64d-e): The protected mannose is soluble in organic solvents and the
reaction should be
selective. In this case specific binding of the mannose to the bola's skeleton
was obtained
without the use of lipase.
[00600] The protected mannose was synthesized using benzyl bromide [Lu et
al.
Carbohydrate Research 2005;340:1231 (Scheme 12)
HO Bn0
HO o Bn0
0
HO"
OH vv-. 0Bn
HO\-- Bn0--
Scheme 12: Etherification of mannose with benzyl bromide
[00601] Mannose was added to a suspension of powdered KOH in DMSO. The
suspension
was cooled in an ice bath and stirred with mechanical stirrer. Benzyl bromide
was added drop
wise. The temperature was allowed to reach room temperature and the reaction
was stirred
overnight. The product was extracted with diethyl ether, washed with water and
saturated NaCl
solution, dried with MgSO4, and the solvent was removed under reduced
pressure. A mixture of
hexane and ethyl acetate (8% ethyl acetate) was added and the solution was
kept in a freezer
overnight. Decantation and recrystallization from the hexane ethyl acetate
mixture were
performed. The precipitate was filtered out and TLC (hexane:ethyl acetate 8:2
was used as the
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running solvent) showed one spot. HPLC (CH3CN 100%, 97.6% purity), Mass
Spectrometry
(MS) m/z [M+231+ = 654 and NMR spectra of the product confirmed the identity
of the obtained
compound.
[00602] The next step was to replace the benzyl group on C6 of protected
mannose with
the acetate group (selective transesterification) , as depicted in Scheme 13:
Bn0 Ac0
Bn0 0 BflO0
w0Bn Bn0 w0Bn
Bn
Scheme 13: Selective transesterification of 1, 2, 3, 4õ 6, pentabenzyl mannose
[00603] This reaction was performed in two ways: The freshly prepared
solution of 1:1
TMSOTf - CH2C12 was added drop-wise to a solution of carbohydrate in freshly
distilled acetic
anhydride at -78 C (cooled in acetone bath with dry ice). Then, the reaction
was stirred at -78 C
for lh under nitrogen. The cold bath was removed and a saturated solution of
NaHCO3 and
CH2C12was added. The mixture was stirred for 0.5h. The organic layer was
separated. The
aqueous layer was extracted with CH2C12 and the organic layers were combined,
washed with
water, dried with Na2SO4 and the solvent was removed under reduced pressure.
Acetic anhydride
was still present in the mixture and MS showed the presence of the mixture of
products
containing 1, 2, and 3 acetic groups.
[00604] The other way of selective 6-0-debenzylation was to use ZnC12.
ZnC12 was
melted at 340 C for lh, cooled and a solution of 1:5 HOAc : Ac20 was added.
The carbohydrate
was added drop wise at -5 C for about lh. Then ice water was added. The
aqueous phase was
extracted with CHC13 and the combined organic phases were washed with
NaHCO3sat., H20,
NaClsat., dried over MgSO4 and the solvent was removed under reduced pressure.
MS showed
the presence of a mixture of products containing 1, 2, and 3 acetic groups,
but the main product
was the target compound with one acetate group, yet, acetic anhydride was
still present.
Nevertheless, this method provided advantages over that described above, in
that there was no
need to use very low temperatures (-78 C) and no need to use expensive reagent
(TMSOTf). The
results were also better in terms of yield.
[00605] The next step was the hydrolysis of the acetate 6-0 mannose group,
as depicted in
Scheme 14.
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AGO HO
6n0 6n0
0 0
Bno nArv.oBn ArvxpoBn
6n0
Bn Bn
Scheme 14: Transesterification or hydrolysis of acetate group on
6-0 of 1, 2, 3, 4 tetrabenzyl mannose.
[00606] Initially, the reaction was performed according the procedure
described in Lu et
al. Carbohydrate Research 2005;340:123. A solution of Na0Me in methanol was
added to a
suspension of crude material from the previous reaction in dry methanol. The
reaction was
stirred for 6h. No target product was observed. NaOH and water were then added
and the
mixture was stirred for 0.5h. Methanol was removed under reduced pressure.
Water and diethyl
ether were added, the phases were separated. The organic phase was washed with
water, NaClõt ,
dried over MgSO4 and the solvent was removed under reduced pressure. The
purification of the
product was performed using flash chromatography with hexane: ethyl acetate
8:2 as an eluent.
TLC (hexane:ethyl acetate 7:3), MS m/z [M+ 18]+ =558.07 and HPLC (CH3CN 100%)
confirmed the identity of the product.
[00607] C) Binding protected mannose to diglutarate diricinoleate through
secondary
hydroxyl on C-1 to obtain GLH-64d was carried out using commercially available
1-0H-2,3,4,6,
tetrabenzyl mannose that was reacted with diglutarate diricinoleate by the
addition of the solution
of EDCI*HC1 in dry CHC13 to a solution of diglutatate diricinoleate,
tetrabenzyl mannose and
DMAP in dry CHC13 and cooling with ice + NaCl. The reaction mixture was
allowed to reach
room temperature and was stirred overnight. TLC (hexane: ethyl acetate 7:3)
showed new spots
and no diglutarate was observed. Water and more chloroform were added. The
phases were
separated and the organic phase was washed with 2M HC1, NaCl õt, dried over
MgSO4 and the
solvent was removed under reduced pressure. The reaction is described in
Scheme 15.
0
0
0
\ OH
an
07....\===4no
oan
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GLH-64d with protection groups still on the mannose
Scheme 15: The reaction of diglutarate diricinoleate with 1-0H-2,3,4,6,
tetrabenzyl
mannose to obtain GLH-64d
[00608] Purification of the product was performed using flash
chromatography with
hexane: ethyl acetate 8:2 as an eluent. HPLC (CH3CN 100%) showed two main
fractions that
contained the target product that was characterized by MALDI m/z [M+Na] =
2030.
[00609] D) Binding of the protected mannose to diglutarate diricioleate via
the primary
hydroxyl on C-6 to obtain GLH-64e, was carried out using a reaction performed
similarly to the
reaction described above and is described in Scheme 16. MALDI m/z [M+Na]+ =
2030
confirmed the identity of the product.
0 0
HOA,,,sj\-0 0
¨ 0,...-\......./.../ ¨
HO HO
BnO,
Bn0
Bno oBn ,Aõt...
oBn ws0Bn
Bno Bn
0 0
ko 0 1
0
Bn0......1,
NAr0Bn
Bn0
Bn
GLH-64e with the protection groups still on the mannose
Scheme 16: The reaction of diglutarate diricinoleate with
6-0H-1,2,3,4, tetrabenzyl mannose
[00610] Removal of the protection groups to obtain GLH-64a with unprotected
mannose
bound to its skeleton, required removal of the protection from the mannose
moiety. Removal of
benzyl groups from the products of bola¨protected mannose was performed in the
ethyl
acetate:methanol mixture 1:3 with 10% Pd/C as a catalyst as described in
Scheme 17.
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Bno
BnO,A7.1:Bn
OBn
0 0
'========"...A0 0
HO 10% Pd/C
Vv=OBn
Bn0
OH Bn
0 V
%*===./\)L 0 0
o..=,\õ==-w.,s/.õs;D=r./"=.../\..="==.¨.==="
1()NO
HO
WOH
Scheme 17: Deprotection of bola¨protected mannose by hydrogenation
Example 28: Studies with the synthesized bolas
[00611] Toxicity of the newly-prepared GLH-64 bolas was examined to
determine the
levels to be used in vesicle formulation and testing. The newly-synthesized
bolas were also used
for vesicle formation and characterized the resulting vesicles. In addition, a
model protein was
encapsulated and the efficiency of that process determined. The model protein
used, trypsinogen,
resembles GDNF in both molecular weight and pI point (characteristics relevant
for
encapsulation). In particular, these studies were to determine the effect of
inclusion of GLH64
bola family members on vesicle formulation, encapsulation efficiency, as well
as other properties
of the vesicles that are needed for drug delivery, including but not limited
to stability and
controlled release.
[00612] The customized, stable vesicles obtained, which ere capable of
encapsulating a
protein similar to GDNF, were tested for their ability to target cultured
cells that express
mannose receptors. For these purposes siRNA conjugated with AlexaFluor 546 was
encapsulated since this fluorescent probe provided the strongest signal in the
FACS used to
separate fluorescent cells from non-fluorescent cells (the cells become
fluorescent after taking up
vesicles with encapsulated fluorescent probe and targeting of the vesicles to
mannose receptors
causes more vesicle uptake and therefore, more cells became fluorescent). The
results obtained
are described below:
[00613] Toxicity studies: a suspension of GLH-64 was injected intravenously
into the tail
vein of male mice, starting with a dose of 100 mg/kg. This dose was selected
as the initial dose
for the toxicity studies based on the preliminary estimation that with the
vesicle formulation
would include no more than 10% of a GLH-64 species and previous studies showed
that the
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maximal tolerated dose of a mixture of GLH-19, GLH-20 and GLH-55b (at a ratio
of 2:1:0.1)
was about 100 mg/kg. Therefore, if a GLH-64 species were to be found to be non-
toxic at 100
mg/kg, the use of this bola in the vesicle formulation is safe at any
potential vesicle formulation
comprising a mixture of GLH-19, GLH-20, GLH-55b and a GLH-64 species. Three
male mice
were injected with 100 mg/kg GLH-64a and 3 other male mice with a mixture of
bolas GLH-19,
GLH-20, GLH-55b and GLH-64a at a ratio of 2:1:0.1:0.1, respectively. No signs
of toxicity
were observed at a dose of 100 mg/kg for either GLH-64a alone, or with the
mixture of the bolas
as described above. Thus, GLH-64a does not seem to be toxic at the dose range
that will be used
for the PK and efficacy studies.
[00614] Vesicle formation and characterization: The bolaamphiphiles that
were
synthesized for this project were used to form vesicles (V-Smart vesicles)
vesicles) customized for
targeting to cells that express mannose receptors and release their
encapsulated compounds there.
The following sections describe the studies that were done with these vesicles
(V-Smarti'm
vesicles).
[00615] The effect of GLH-64a on vesicle shape, size and surface charge
(zeta potential):
In previous studies, spherical vesicles were routinely obtained with diameters
ranging between
50-150nm with a net positive surface charge (Zeta potential in the range of 30-
50 mV). Vesicles
with this range of size and surface charge showed good drug delivery
properties. To assess the
effect of GLH-64a on these vesicles' properties, we prepared vesicles with
different amounts of
GLH-64a in the vesicle formulation and investigated their properties in term
of shape (cryo-
TEM), size distribution (DLS) and zeta potential.
[00616] Vesicles were prepared by film hydration followed by sonication as
described
above. The tested formulations included: a) GLH-19 and GLH-20 at a molar ratio
of 2:1,
respectively. This formulation contains only the basic bolas that make up the
membrane matrix
of the vesicles; b) GLH-19:GLH-20:GLH-55b at molar ratios between the bolas of
2:1:0.1,
respectively. This formulation contains also a bola with CS (chitosan) head
groups and it
represents our standard formulation that showed, in previous experiments, the
capability of
delivering small molecules [Popov et al. (2012) Site-directed decapsulation of
bolaamphiphilic
vesicles with enzymatic cleavable surface groups. I Controlled Release, Jun
10;160(2):306-141,
peptides [Popov et al. Delivery of analgesic peptides to the brain by nano-
sized bolaamphiphilic
vesicles made of monolayer membranes. Eur J Pharm Biopharm. 2013 Nov; 85 (3 Pt
A): 381-91
and proteins [Dakwar et al. (2012) Delivery of proteins to the brain by
bolaamphiphilic nano-
sized vesicles. J. Controlled Release, Jun 10;160(2):315-211 into a mouse
brain, thus this
formulation was used as a control for comparison purposes; c) GLH-19:GLH-
20:GLH-55b:GLH-
64a at molar ratios of 2:1:0.1:0.01, respectively; d) GLH-19:GLH-20;GLH-
55b:GLH-64a at
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molar ratios of 2:1:0.1:0.05, respectively; e) GLH-19:GLH-20:GLH-55b:GLH64a at
molar ratios
of 2:1:0.1:0.1, respectively. All the formulations contained also CHOL
(cholesterol) and
CHEMS (cholesterol hemisuccinate) at a molar ratio of 2:1:1 (bolas:CHOL:CHEMS,
respectively).
[00617] Cryo-TEM of vesicles with and without the bola that contains
mannose head
groups (GLH-64a): Images of vesicles with and without mannose surface pendants
are shown in
Figure 49. As can be seen, spherical vesicles were obtained from formulation
without GLH-64a
(Figure 10, Panel A) and formulation with 5% GLH-64a (Figure 10, Panel B). The
size of the
vesicles ranged from about 50 nm to about 120 nm. A more quantitative analysis
of size
distribution was obtained by dynamic light scattering (DLS) measurements
[00618] Size distribution and zeta potential of vesicles without and with
GLH-64a bolas;
i.e., the effect of the bola with the mannose head groups (GLH-64a) on vesicle
size and surface
charge is shown in Table 14.
[00619] Table 14: Effect of GLH-64a bolas with mannose head groups on
vesicle size and
charge.
Percent of GLH-64a Without (-) or Vesicle size Zeta
in the vesicle with (+) CS determined by potrential
formulation (GLH-55b) in DLS
(mV)
the
(nM)
formulation
0 148.7 0.7 50.2 0.9
0 136.5 5.8 47.6 0.7
1 137.5 7.9 42.4 0.6
107.3 0.9 48.4 1.1
87.6 5.9 41.3 0.1
[00620] As demonstrated in Table 14, increasing the amount of GLH-64a in
the vesicle
formulation caused a gradual decrease of the vesicle size from a diameter of
136.5 nm, which
was measured for vesicles without GLH-64, to a diameter of 87.6 nm measured
for vesicles with
10% GLH-64a. Applicants believe, without wishing to be held to that belief,
that smaller
vesicles may accumulate more selectively in the brain due to less filtration
in the lung and better
permeability through the BBB.
[00621] Vesicle stability in storage: To study the effect of vesicle
composition on stability,
vesicleswith encapsulated CF (5,6-carboxyfluorescein) were prepared and the
percent CF still
encapsulated was measured as a function of time in storage. Vesicles of the
following
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formulations were prepared: a) A mixture of GLH-19 and GLH-20 (2:1) was used
as a control;
and b) A formulation similar to that described in 'a' above with the addition
of different amounts
of GLH 64a (1%, 5% and 10% of the total bolas in the vesicle formulation).
Each of the
formulations described in 'a' and 'b' above, was prepared with or without the
bola that contains
the chitosan head group (GLH-55b). All formulations contained also CHOL and
CHEMS at a
molar ratio of 1:1 as described.
[00622] Targeting of (V-Smart) vesicles to cultured macrophages that
express mannose
receptors: The ability of the customized vesicles (V-Smart vesicles) (vesicles
that were
customized to target to mannose receptors) to target cells that express
mannose receptors was
examined using J774 macrophage cell line. This cell line does not normally
express mannose
receptors, but it can be differentiated by dexamethasone to express
significant number of
mannose receptor [Fiani et al. Regulation of mannose receptor synthesis and
turnover in mouse
J774 macrophages. J Leukoc Biol. 1998;64(1):85-911. Vesicles with the
capability of binding to
mannose receptors are expected to bind preferentially to differentiated J774
cells, but not to non-
differentiated J774 cells [Dubey et al. Surface structured liposomes for site
specific delivery of
an antiviral agent-indinavir. J Drug Target. 2011;19(4):258-691. The vesicles
described herein
are designedt to target microglia that accumulate in the CNS near degenerating
motor neurons in
ALS. These activated microglia express mannose receptors. Accordingly, the
mannose-
receptor-expressing J774 macrophage cell line can be used a s model cells to
study targeting.
Binding of the vesicles to the cell surface results in the uptake of the
vesicles into the cells by
means of endocytosis [Dakwar et al. (2012) Delivery of proteins to the brain
by bolaamphiphilic
nano-sized vesicles. J. Controlled Release, Jun 10;160(2):315-211. When more
vesicles bind to
the cell's surface, more vesicle uptake will occur, therefore, targeting of
the vesicles to cells will
increase their uptake into the cells. To assess the amount of the uptake of
the vesicles by J774
cells, vesicles loaded with a fluorescent probe were used that when are taken
up by the cells
make them fluorescent. Fluorescent cells are separated from non-fluorescent
cells by FACS,
where the peak of the fluorescent cells is shifted to the right (see, e.g.
Figures 50-54) as
compared to non-fluorescent cells. The degree of the shift indicates the
degree of the binding and
is related to targeting.
[00623] For the assessment of the targeting, binding of the customized
vesicles (vesicles
that contain mannose on their surface) was compared to that of non-
differentiated and
differentiated J774 cells. Vsicles that contain mannose surface groups with
vesicles that do not
contain mannose surface groups were also compared for their ability to bind
differentiated J774
cells. To investigate the specificity of the targeting, the binding to
differentiated J774 cells of
vesicles with mannose surface groups in presence and absence of free glucose
in the bathing
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medium was also mesured. That is, if the binding of the mannose surface groups
to the mannose
receptor is specific, then free mannose will compete with the bound mannose
(the mannose on
the vesicle surface) and will reduce binding (thus will reduce uptake of the
encapsulated
fluorescent materials).
[00624] Figure 50 shows results from flow cytometry (FACS) obtained with
customized
vesicles carrying surface mannose groups (V-Smart), demonstrating the uptake
of fluorescent
vesicles that contain GLH-64a by differentiated and non-differentiated J774
cells. Cells were
differentiated by exposing them to 1 [tg/mL dexamethasone for 24 hours.
Untreated cells were
grown in parallel to the differentiated cells, but without dexamethasone.
Cells were incubated
with fluorescent vesicles that contained mannose moieties on their surface (by
the including 5%
GLH-64a in the vesicles formulation) for 4 hours and were examined by FACS. A
shift of the
peak to the right indicates higher uptake (namely higher binding)Binding of
the vesicles was
measured to non-differentiated cells (cells that do not express mannose
receptors) in comparison
to differentiated cells (cells that highly express mannose receptors). As can
be seen, the peak of
the fluorescent cells was shifted more to the right (about 8 times more) for
differentiated cells
compared to non-differentiated cells. These results indicate that the
customized vescicles bind 8
times more to cells that express mannose receptors compared to cells that do
not express
mannose receptors.
[00625] The specificity of the binding is demonstrated in Figure 51, which
depicts a
comparison between the bindings of the customized vesicles carrying surface
mannose groups
described herein to differentiated cells that was done in presence and absence
of free mannose in
the bathing medium. More specifically, Figure 51 depicts that uptake of
fluorescent vesicles
formulated with GLH-64a by differentiated J774 cells in presence and absence
of free mannose
in the bathing medium. Cells were differentiated by exposing them to 1 [tg/mL
dexamethasone
for 24 hours. Cells were incubated with fluorescent vesicles that contained
mannose moieties on
their surface (by the including 5% GLH-64a in the vesicles formulation) for 4
hours and were
examined by FACS. A shift of the peak to the right indicates higher uptake
(namely higher
binding. As can be seen, the presence of free mannose in the bathing medium
decreased the
uptake of the fluorescent vesicles, indicating that the free mannose competed
with the mannose
surface groups of the vesicles and interfered with the binding of the vesicles
to the cells.
[00626] Additional targeting experiments were done with vesicles that
contain higher
proportion of GLH-64a, to determin if increasing the surface density of
mannose groups would
improve targeting. In this experiment, the GLH-64a level in the formulation
was increased to
10% and the contributions of GLH-64a and GLH-64b in the formulation were
compared. The
mannose moiety in GLH-64a is bound via the primary hydroxyl on carbon number 6
of the
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mannose while GLH-64b is a mixture of bolas in which the mannose is bound via
either the
primary or any one of the secondary hydroxyls of the mannose as the reaction
was done without
lipase and the binding of the mannose was not site specific, as described
above. The advantage
of GLH-64b is that it can be obtained in high yield and its synthesis is
simpler than that of GLH-
64a. The results of the targeting experiment with vesicle formulations that
contained 10% GLH-
64a and GLH-64b in comparison are shown in Figure 52, which presents the
uptake of
fluorescent vesicles that contain GLH-64a (Panel A) and GLH-64b (Panel B) by
differentiated
and non-differentiated J774 cells. Cells were differentiated by exposing them
to 1 ug/mL
dexamethasone for 24 hours. Untreated cells were grown in parallel to the
differentiated cells, but
without dexamethasone. Cells were incubated with fluorescent vesicles that
contained mannose
moieties on their surface (by the including 10% GLH-64a (Panel A) or 10% GLH-
64b (Panel B)
in the vesicles formulation) for 4 hours and were examined by FACS. A shift of
the peak to the
right indicates higher uptake (namely higher binding).
[00627] As can be seen from Figure 52, vesicles that contained both GLH-64a
and GLH-
64b were taken up more by differentiated cells that express mannose receptors
than by non-
differentiated cells that do not express mannose receptors. However, the
signal of the fluorescent
cells was smaller than the signal obtained in the first experiment (compare
the data of Figures 50
and 51 to that of Figure 52). Although these results migh reflect a different
amount of the
fluorescent probe that was used in the second experiment, or less efficient
differentiation of the
cells that resulted in lower expression of mannose receptors by the
differentiated cells, the shifts
of the peaks of the differentiated cells that were exposed to the customized
vesicles are clear and
significant. These data therefore indicate that the vesicles with the mannose
surface group target
cells that express mannose receptors. To validate the conclusion that only the
vesicles that
contain mannose surface group target cells that express mannose receptors,
binding of vesicles
without mannose head groups (vesicles that were prepared from formulations
that did not contain
GLH-64) was compared to the binding of vesicles that contain mannose surface
groups (a
formulation with GLH-64a), using differentiated cells that express mannose
receptors. In this
experiment for the differentiation of the cells was obtained by contact with
10 ug/mL
dexamethasone (instead of 1 ug/mL that was used in earlier experiments) to
assure efficient
differentiation. The results of the experiment in which vesicles without
mannose surface groups
were compared to vesicles with mannose surface groups are shown in Figure 53,
which depicts
uptake of fluorescent vesicles with and without GLH-64a by differentiated J774
cells. Cells were
differentiated by exposing them to 10 ug/mL dexamethasone for 24 hours. Cells
were incubated
with fluorescent vesicles (vesicles with encapsulated siRNA conjugated with
AlexaFluor 546)
with mannose moieties on their surface (mannose surface groups were introduced
by the
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including 5% GLH-64a in the vesicles formulation) or with fluorescent vesicles
without mannose
surface groups (without GLH-64 in the vesicle formulation) for 4 hours and
were examined by
FACS. Cells were also incubated with non-encapsulated (free) siRNA conjugated
with
AlexaFluor 546, which was used as the fluorescent probe. A shift of the peak
to the right
indicates higher uptake (namely higher binding).
[00628] As can be seen from Figure 53, the vesicles that contained mannose
surface
groups were taken up about 10 times more than vesicles that did not contain
mannose groups on
their surface. Inclusion of free mannose in the bathing medium (10 mM)
completely abolished
the effect of the mannose surface groups since it competed with the mannose
surface groups for
binding to the mannose receptors that were expressed on the membrane of the
differentiated
cells. Free (non-encapsulated) fluorescent probe (siRNA conjugated with
alexaFluor 546) was
not taken up by the cells at all and the peak of the cells that were exposed
to the free fluorescent
probe was identical to the peak of the control cells that were not exposed to
neither vesicles and
fluorescent probe. These data show that the vesicles with their encapsulated
fluorescent probe,
and not the free fluorescent probe, were taken up by the cells and that
vesicles with mannose
surface groups were taken up by the cells much more than vesicles without the
targeting ligand
on their surface. Altogether, these results indicate that vesicles with
mannose surface groups
target cells that express mannose receptors.
[00629] More specifically, the results obtained with GLH-64a were
conclusive and
showed that a bola which is bound to the mannose moiety via the primary
hydroxyl which is
situated on carbon 6 is capable of providing efficient targeting.
[00630] The results obtained with GLH-64b showed that targeting can be
achieved with
this bola, although the uptake of the vesicles that contain GLH-64b (uptake
indicates targeting)
was somewhat less than that obtained with GLH-64a (see Figure 52). GLH-64b
contains a
mixture of bolas where the mannose is bound to the bola skeleton either via
the primary or the
secondary hydroxyls. Therefore, it was interesting to see whether a bola in
which the mannose
moiety is bound only via the secondary hydroxyl is capable of providing good
targeting. GLH-
64d is such a bola in which the mannose moiety is bound via the secondary
hydroxyl, which is
situated on carbon number 1 of the mannose. The results of the targeting
experiment with GLH-
64d are described in Figure 54, which depicts the uptake of fluorescent
vesicles that contain
GLH-64d by differentiated and non-differentiated J774 cells. Cells were
differentiated by
exposing them to 1 ug/mL dexamethasone for 24 hours. Untreated cells were
grown in parallel
to the differentiated cells, but without dexamethasone. Cells were incubated
with fluorescent
vesicles that contained mannose moieties on their surface (by the including 5%
GLH-64a in the
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vesicles formulation) for 4 hours and were examined by FACS. As noted above, a
shift of the
peak to the right indicates higher uptake (namely higher binding).
[00631] As can be seen from Figure 54, vesicles that contain GLH-64d were
taken up
somewhat better by differentiated cells than by non-differentiated cells, but
the shift was much
smaller than that obtained with GLH-64a. Again, it is not yet clear whether
this smaller shift is
due to non-efficient differentiation which did not cause enough expression of
mannose receptors,
or because mannose which is bound via its secondary hydroxyl is not recognized
by the mannose
receptor as well as a mannose moiety which is bound to the bola's skeleton via
the primary
hydroxyl. Nonehtheless, it is safe to conclude, based upon all the results of
the targeting
experiments described above, that targeting can be achieved with vesicle that
contain mannose
surface moieties, particularly where the mannose is bound to the bola's
skeleton via the primary
hydroxyl situated on carbon 6 of the mannose.
Example 29
Vesicle Stability During Storage
[00632] To study the effect of vesicle composition on stability, vesicles
were prepared
with encapsulated CF and studied the percent CF still encapsulated as a
function of time in
storage. Vesicles of the following formulations were prepared: a) a mixture of
GLH-19 and
GLH-20 (2:1) used as a control; and b) a formulation similar to that described
in 'a' above with
the addition of different amounts of GLH 64a (1%, 5% and 10% of the total
bolas in the vesicle
formulation). Each of the formulations, described in 'a' and 'b' above, was
prepared with or
without the bola that contains the chitosan head group (GLH-55b). All
formulations contained
also CHOL and CHEMS at a molar ratio of 1:1 as described.
[00633] Vesicles were prepared from each of the above formulations by the
method of
film hydration followed by sonication as described above, and percent CF
encapsulation was
determined at different times in storage at 4 C. The results of the vesicle
stability are shown in
Figures 55-59. Vesicles that were made from the basic bolas (GLH-19 and GLH-
20) were stable
and maintained the amount of their encapsulated material for at least 14 days,
which was the
maximum period studied in this project (Figure 55). Addition of CS surface
groups, by including
GLH-55b in the vesicle formulation, did not change vesicle stability and these
vesicles were
stable as well (Figure 56). Addition of 1% GLH-64 to vesicle formulation that
contained GLH-
19, GLH-20 and GLH-55b did not affect significantly the stability of the
resulting vesicles
(Figure 57), but 5% and 10% GLH-64 in the vesicle formulation reduced somewhat
the stability
of the resulting vesicles (25% less encapsulated CF was found after two weeks
in storage), as can
be seen from Figures 58 and 59, respectively. This reduction in the amount of
CF encapsulation
may be related to dissociation of negatively charged CF that was bound to the
positively charged
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surface of the vesicles and not to disintegration of the vesicular structure
or loss of encapsulated
CF from the interior. Notably, dissociation of CF is expected to increase when
more non-
charged mannose groups are present on the vesicle surface and interfere with
CF binding to the
positively charged ACh groups.
[00634] Vesicle
stability in presence of 4% albumin was also studied. It is well known
that proteins in the bathing medium may affect vesicle stability, and, since
albumin is the major
protein of the serum and, thus, when vesicles are injected into mice they will
first circulate in the
blood that contains serum proteins, we examined the effect of albumin on
vesicle stability. The
experiments employed 4% albumin, which is the concentration of this protein in
serum. As can
be seen in Figure 60, albumin reduced somewhat the stability of vesicles made
of GLH-19, GLH-
20 and GLH-55b. By comparison, addition of 1% GLH-64 increased somewhat
vesicle stability
(Figure 61). This was even more apparent when the vesicles were incubated at
25 C instead of
4 C (compare vesicle stability in Figure 62, that show stability of vesicles
without GLH-64 to
Figure 63 that shows stability of vesicles with GLH-64).
[00635] These
results suggest that GLH-64a may increase vesicle stability in the blood.
Previous data obtained upon IV administration of proteins, peptides and low
molecular weight
molecules, has also shown that the majority of the administered IV dose of
vesicles of the
disclosure delivers most of the encapsulated ingredients to the CNS within 2
hours after
administration. Thus long term blodd circulatory stability may not be
important. The reason
why GLH-64a reduced vesicle stability in buffer and increased stability in
buffer that contains
albumin, may be attributed to interference of the mannose surface groups with
the interaction of
the protein with the vesicle surface. If this is the case, then 5% and 10% GLH-
64a may even
further increase stability. Vescicle stability can be maximized by fine tuning
of the vesicle
formulation. For example, increased stability of vesicles that contain GDNF in
storage, may be
obtained using freeze-dried GDNF-loaded vesicles that are maintained as solids
in storage
followed by reconstitution of the vesicles before injection.
[00636]
Controlled release by AChE: The controlled release mechanism is based on the
hydrolysis of the acetylcholine head groups of the matrix bolas (particularly
GLH-20, the head
groups of which are hydrolyzed by AChE). The hydrolyzing enzyme, AChE, is
abundant in the
CNS and can be inhibited selectively in peripheral tissues, without affecting
its activity in the
CNS, by pyridostigmine [Grauer et al. Stress does not enable pyridostigmine to
inhibit brain
cholinesterase after parenteral administration. Toxicol Appl Pharmacol.
2000;164(3):301-3041, a
safe drug used in human for the treatment of myasthenia gravis [Bolourchian et
al. Prolonged
release matrix tablet of pyridostigmine bromide: formulation and optimization
using statistical
methods. Pak J Pharm Sci. 2012;25(3):607-6161. Since, in addition to GLH-19
and GLH-20,
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GLH-55b and GLH-64a are also present in the vesicle formulation, experiments
were carried out
to determine if the addition of GLH-55b and GLH-64a influences the enzymatic
hydrolysis of
GLH-20's head groups, which controls the release of the active agent in the
brain [Popov et al.
(2012) Site-directed decapsulation of bolaamphiphilic vesicles with enzymatic
cleavable surface
groups. J. Controlled Release, Jun 10;160(2):306-1461. To test this, vesicles
were prepared with
and without CS, and with different amounts of GLH-64a, and tested how vesicles
that were
prepared from these formulations release their encapsulated content upon
exposure to AChE.
The results that are shown in Figure 20 indicate that neither GLH-55b nor GLH-
64a inhibit the
release rates induced by AChE.
[00637] Figure 20 depicts the effect of GLH-55b and GLH-64 on the release
of
encapsulated CF from vesicles that contain encapsulated CF were incubated in
PBS while
monitoring their fluorescence. AChE dissolved in water (2 units/24), or water
(24) was added
while fluorescence was continuously monitored. Due to its high concentration
inside the vesicles,
the fluorescence of encapsulated CF is quenched. An increase in fluorescence
indicates release
of CF from the vesicles as the released drug is diluted in the bathing medium.
Triton X100 was
added at the end of the experiment to completely disrupt the vesicles and
obtain the total
fluorescence of the encapsulated CF. The graphs on the left side of Figure 20
show the slope of
the increase in fluorescence, representing the rate of the release. The graphs
in middle column of
Figurre 20 show the release induced by AChE and the graphs in the right column
of Figure 20
show the release induced by the vehicle (used as a control). Panel A: Vesicles
made of GLH-19
and GLH-20, without GLH-55b and GLH-64a); Panel B: Vesicles made of GLH-19,
GLH-20,
GLH-55b and 1% GLH-64; Panel C: Vesicles made of GLH-19, GLH-20, GLH-55b and
10%
GLH-64a.
[00638] Encapsulation studies: To learn how GLH-64a affects the
encapsulation capacity
of the vesicles, CF was used as the fluorescent probe and experiments compared
the amount of
CF encapsulation in vesicles without and with GLH-64a. The results are
summarized in Table
15.
Table 15: CF encapsulation in vesicles containing different amounts of GLH-64
% GLH-64 in the % CF % CF
vesicle encapsulation encapsulation 36 h
formulation immediately after after vesicle
vesicle preparation preparation
0 27.6 0.2 30.5 1.5
1 27.9 1.2 31.1 2.6
34.1 2.0 32.1 3.7
8 35.3 4.4 34.7 0.5
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[00639] As can be seen from Table 15, CF encapsulation ranged between 28-
35% in all
the vesicle formulations that were tested, with a tendency of increased
encapsulation capacity
with increased amount of GLH-64a in the vesicle formulation (the amount of GLH-
64a was
increased in the vesicle formulation from 1% to 8%). Yet, the difference in
percent encapsulation
among the various formulations was not significant and this led us to conclude
that GLH-64a
does not interfere with encapsulation, even though inclusion of GLH-64a in the
vesicle
formulation reduces somewhat vesicle size (see above). Therefore, from the
vesicle properties
and encapsulation points of view, relatively high concentrations of GLH-64 may
be prepared in
the vesicle formulation, to help ensure efficient targeting, since high
concentrations of GLH-64
in the vesicle formulation will produce high number of targeting ligands on
the vesicle's surface.
The maximum amount of GLH-64 that will not interfere with the properties of
the vesicles as
drug carriers will be established, and used in targeting studies in vitro and
in vivo.
[00640] Encapsulation of a protein similar in properties to GDNF was also
examined.
Initial encapsulation studies require relatively high amounts of protein and,
since GDNF is
expensive, initial studies were conducted with a model protein ¨ trypsinogen,
which has similar
molecular weight and isoelectric point to that of GDNF. Trypsinogen has a
similar isoelectric
point (about 9) and close molecular weight (about 24 KDa) to that of GDNF and
these two
properties are most important for encapsulation. The initial studies are done
with relatively large
amounts of trypsinogen, which were needed for the determination of its
concentration by UV
absorbance. Then, once initial conditions for encapsulation have been worked
out, it will be
possible to use smaller amounts of the protein, similar to those that will be
used with GDNF,
with detection of these small amounts facilitated using fluorescence
measurements. For this
purpose, trypsinogen was labeled with a fluorescent probe (Alexa FlourTM 488)
as described
above. Note that GDNF can be labeled in this manner as well.
[00641] The determination of encapsulation with the labeled trypsinogen was
carried out
in the following way: fluorescently-labeled trypsinogen was dissolved in
distilled water, at a
concentration of up to 100pg/ml. Then, empty vesicles were prepared by film
hydration followed
by sonication as described above. The trypsinogen solution was added to the
vesicle suspension,
and the mixture was sonicated on ice to form vesicles of 5 mg/ml of the bolas
with encapsulated
fluorescently-labeled protein. Then, non-encapsulated material was removed by
running the
vesicles over a Sephadex G-75 column. The fractions collected from the column
were treated by
Triton X-100 reduced form, and the fluorescence of each fraction was
determined by
fluorescence spectroscopy. Percent encapsulation was determined by dividing
the AUC of the
vesicle fractions by the total AUC, which is the sum of the AUC of the vesicle
fractions and the
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AUC of the free trypsinogen. This approach was used to determine how the
addition of the bola
with the mannose head groups (GLH-64a) affects encapsulation. The amount of
the encapsulated
protein was determined right after vesicle formation and again, after 24hrs.
[00642] Table 16 shows the percent encapsulation of labeled trypsinogen
obtained with
vesicles prepared from formulations without and with GLH-64a.
Percent encapsulation of trypsinogen conjugated with Alexa FlourTM-488 in
vesicles with
and without mannose surface groups
No GLH-64a 1% GLH-64a 5% GLH-64a 10% GLH-64a
Time after Without With Without With Without With Without With
vesicle GLH- GLH- GLH- GLH GLH- GLH GLH- GLH-
formation 55b 55b 55b -55b 55b -55b 55b 55b
Immediate 45.0 21.1 59.0 19.6 58.7 38.5 49.5
--
24h 43.4 42.2 23.4 49.1 34.2 37.5
[00643] As can be seen, the addition of chitosan bola (GLH-55b) to the
vesicle
formulation significantly reduced the amount of trypsinogen encapsulation.
However, the
inclusion of GLH-64a in the vesicle formulations (with GLH-55b) increased
trypsinogen
encapsulation (from 21.1% in the control to 34.2% and 37.5% in vesicles with
5% and 10%
GLH-64a, respectively), although not to the same value, which was observed in
vesicles without
GLH-55b (about 59%). In other words, GLH-64a partially reversed the drop in
encapsulation
caused by GLH-55b. The trypsinogen-loaded vesicles without CS surface groups
(no GLH-55b)
were not completely stable and lost about 16-28% of the encapsulated
trypsinogen within 24 h.
By comparison, vesicles that contained GLH-55b, although starting with less
trypsinogen
encapsulation, were more stable and lost only about 11% of the encapsulated
trypsinogen within
24 h.
[00644] As described herein, novel formulations of bolavesicles can be
produced through
co-assembly of GDNF with bolaamphiphile/lipid unilamellar vesicles. The
formulations can be
examined for their chemical and biophysical properties.
[00645] The incorporation of GDNF or NGF within the bolavesicles can be shown
to
significantly modulate interactions with membrane bilayers in model systems.
This observation is
important, suggesting that GDNF or NGF encapsulated in bolavesicles might be
excellent
candidates for targeting and transport of different molecular cargoes into the
brain.
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[00646] From the foregoing description, various modifications and changes in
the compositions
and methods provided herein will occur to those skilled in the art. All such
modifications
coming within the scope of the appended claims are intended to be included
therein.
[00647] All publications, including but not limited to patents and patent
applications, cited in
this specification are herein incorporated by reference as if each individual
publication were
specifically and individually indicated to be incorporated by reference herein
as though fully set
forth.
[00648] At least some of the chemical names of compounds of the invention as
given and set
forth in this application, may have been generated on an automated basis by
use of a
commercially available chemical naming software program, and have not been
independently
verified. Representative programs performing this function include the
Lexichem naming tool
sold by Open Eye Software, Inc. and the Autonom Software tool sold by MDL,
Inc. In the
instance where the indicated chemical name and the depicted structure differ,
the depicted
structure will control.
[00649] Chemical structures shown herein were prepared using ISIS /DRAW. Any
open
valency appearing on a carbon, oxygen or nitrogen atom in the structures
herein indicates the
presence of a hydrogen atom. Where a chiral center exists in a structure but
no specific
stereochemistry is shown for the chiral center, both enantiomers associated
with the chiral
structure are encompassed by the structure.
[00650] REFERENCES
*Abu Hammad I, Popov M, Linder C, Grinberg S, Heldman E, Stepensky D (2011)
Bolaamphiphilic nanovesicles for the delivery of proteins to the brain,
submitted to the
Journal of Controlled Release.
Agyare, EK, Kandimalla KK, Poduslo JF, Yu CC, Ramakrishnan M, Curran GL (2008)
Development of a smart nano-vesicle to target cerebrovascular amyloid deposits
and brain
parenchymal plaques observed in Alzheimer's disease and cerebral amyloid
angiopathy.
Pharm Res Nov; 25(11):2674-2684.
Ansorena E, Garbayo E, Lanciego JL, Aymerich MS, Blanco-Prieto MJ. Production
of highly
pure human glycosylated GDNF in a mammalian cell line. Int J Pharm. 2010;
385(1-2):6-11.
Clarke R.L, Daum S.J, Gambino A/J, Aceto MD, Pearl J, Levitt M, Cumiskey W.R.
and Bogado
E.F. Compounds Affecting the Central Nervous System. 4. 30-Phenyltropane-2-
carboxylic
Esters and Anologs. J. Med. Chem. 1973; 16:1260-1267.
- 159 -
CA 03018945 2018-09-25
WO 2016/168580 PCT/US2016/027726
Dakwar GR, Abu Hammad I, Popov M, Linder C, Grinberg S, Heldman E, Stepensky
D.
Delivery of proteins to the brain by bolaamphiphilic nano-sized vesicles. J
Control Release.
2012; 160(2):315-321.
Fazil M, Md S, Hague S, Kumar M, Baboota S, Sahni JK, Ali J. Development and
evaluation of
rivastigmine loaded chitosan nanoparticles for brain targeting. Eur J Pharm
Sci. 2012;
47(1):6-15
Fuhrhop J.H. and Wang T. (2004) Bolaamphiphiles, Chem. Rev. 104:2901-2937.
Gash DM, Zhang Z, Ai Y, Grondin R, Coffey R, Gerhardt GA. Trophic factor
distribution
predicts functional recovery in parkinsonian monkeys. Ann Neurol. 2005;
58(2):224-
233Gisslen M and Hagberg L and Hagberg (2001) Antiretroviral treatment of
central nervous
system HIV-linfection: a Review. HIV Medicine (2001) 2, 97-104.
G Gnanarajan, AK Gupta, V Juyal, P Kumar, PK Yadav, P Kailash "A validated
method for
development of tenofovir as API and tablet dosage forms by UV spectroscopy"
Pharm
Analysis 2009 Vol 1 Issue 4 pp 351-353.
*Grinberg S, C. Linder, E. Heldman, Z. Weizman, and V. Kolot: EP1360168, 2003-
11-12 and
W02002IL00043and 20020116, Filed by BG Negev "Amphiphilic Derivatives for the
Production of Vesicles, Micelles, Complexants, and Uses Thereoff' in 2003
*Grinberg S., Linder C., Kolot V., Waner T., Wiesman Z., Shaubi E., Heldman E.
(2005) Novel
cationic amphiphilic derivatives from vernonia oil: synthesis and self-
aggregation into bilayer
vesicles, nanoparticles, and DNA complexants. Langmuir. 21(17):7638-7645.
*Grinberg S., Kolot V., Linder C., Shaubi E., Kas'yanov V., Deckelbaum R.J.,
Heldman E.
(2008) Synthesis of novel cationic bolaamphiphiles from vernonia oil and their
aggregated
structures. Chem Phys Lipids 153(2):85-97.
*Grinberg, S., Kipnis, N., Linder, C., Kolot, V. and Heldman, E., (2010)
Assymetric
bolaamphiphiles from veronica oil designed for drug delivery. Eur. I Lipid
sci. Technol.,
112, 137-151.
Hamilton JF, Morrison PF, Chen MY, Harvey-White J, Pernaute RS, Phillips H,
Oldfield E,
Bankiewicz KS. Heparin coinfusion during convection-enhanced delivery (CED)
increases
the distribution of the glial-derived neurotrophic factor (GDNF) ligand family
in rat striatum
and enhances the pharmacological activity of neurturin. Exp Neurol. 2001;
168(1):155-161
*E. Heldman E, C. Linder, S. Grinberg Amphiphilic compounds and vesicles
liposomes for
organ-specified drug targeting" US patent Application 20060039962 + W003047499
- 2003-
06-12.
Highleyman, L (2009) HIV and the Brain BETA. 2009 Summer-Fall;21(4):16-29. C.
R.
- 160 -
CA 03018945 2018-09-25
WO 2016/168580 PCT/US2016/027726
Holmquist C.R, Keverline-Franz K.I, Abraham P, Boj a J.W, Kukar M.J, Carol!
F.I. 3a1pha-(4"-
Substituted Phenyl) Tropane-2beta-Carboxylic Acid Methyl Ester: Novel Ligands
with High
Affinity and Selectivity at the Doopamine Transporter. J. Med. Chem. 1996;
39:4139.
*Hutter T, Linder C, Heldman E, Grinberg S (2011) Interfacial and self-
assembly properties of
bolaamphiphilic compounds derived from a multifunctional oil, Journal of
Colloid and
Interface Science, 2012; 365(1):53-62.
Jiang H, Jiang Q, Feng J. Parkin increases dopamine uptake by enhancing the
cell surface
expression of dopamine transporter. J Biol Chem. 2004; 279(52):54380-543806.
Jonasdottir TJ, Fisher DR, Borrebaek J, Bruland OS, Larsen RH (2006) First in
vivo evaluation of
liposome-encapsulated 223Ra as a potential alpha-particle-emitting cancer
therapeutic agent.
Anticancer Res. 26(4B):2841-2848.
N. N. Kabal'nova, K. Yu. Murinov, I. R. Mullagaliev, N. N. Krasnogorskaya, V.
V.
Shereshovets, Yu. B. Monakov,G. E. Zaikov , Oxidative Destruction of Chitosan
Under the
Effect of Ozone and Hydrogen Peroxide. Journal of Applied Polymer Science.
2001; 81:875-
881.
T Kadota, T Yamaai, Y Saito, Y Akita, S Kawashima, K Moroi, N Inagaki and K
Kadota.
Expression of dopamine transporter at the tips of growing neurites of PC12
cells. J
Histochem Cytochem 1996; 44: 989-996.
Kiyohito Shimura, Wang Zhi, Hiroyuki Matsumoto and Ken-ichi Kasai. Accuracy in
the
Determination of Isoelectric Points of Some Proteins and a Peptide by
Capillary Isoelectric
Focusing: Utility of Synthetic Peptides as Isoelectric Point Markers. Anal.
Chem. 2000;
72:4747-4757.
Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen
EY, Palfi S,
Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling Z,
Trono D,
Hantraye P, Deglon N, Aebischer P. Neurodegeneration prevented by lentiviral
vector
delivery of GDNF in primate models of Parkinson's disease. Science. 2000;
290(5492):767-
773.
Lapidot Y., Rappaport S., Wolman Y. Use of esters of N-hydroxysuccinimide in
the synthesis of
N-acylamino acids. The Journal of Lipid Research. 1967; 8:142-145.
Letendre S, Marquie-Beck J, Capparelli E, Best B, Clifford D, Collier AC,
Gelman BB,
McArthur JC, McCutchan JA, Morgello S, Simpson D, Grant I, Ellis RJ; CHARTER
Group.(2008) Validation of the CNS Penetration-Effectiveness rank for
quantifying
antiretroviral penetration into the central nervous system. Arch Neurol. 2008
Jan;65(1):65-70.
*Linder C; Grinberg S; Heldman E "Nano-sized Particles Composing Multi-Headed
Amphiphiles for Targeted Drug-Delivery" WO 2010128504 (A2) 2010.
- 161 -
CA 03018945 2018-09-25
WO 2016/168580 PCT/US2016/027726
Lu W, Tan YZ, Hu KL and Jiang XG. (2005) Cationic albumin conjugated pegylated
nanoparticle with its transcytosis ability and little toxicity against blood-
brain barrier. Int J
Pharm. May 13;295 (1-2); 247-260.
Madras BK, Fahey MA, Bergman J, Canfield DR, Spealman RD. Effects of cocaine
and related
drugs in nonhuman primates. I. [3H]cocaine binding sites in caudate-putamen. J
Pharmacol
Exp Ther. . 1989; 251(1):131-141.
Melzer PC, Liang A.Y, Brownell A.L. and Madras B.K. Substituted 3-
Phenyltropane Analogs of
Cocaine; Synthesis, Inhibition of binding at Cocaine Recognition Sites, and
Positron
Emmission Tomography Imaging. J. Med. Chem. 1993; 36:855-862.
Millius R.A, Saha J.K, Madras B.K. and Neumeyer J.L. Synthesis and Receptor
Binding of N-
Substituted Tropane derivatives. High Affinity ligands for the Cocaine
Receptor. J. Med.
Chem. 1991; 34:1728-1731.
Myers A.L, Williams H.E, Kraner J.C.K. and P.S. Callery J. Identification of
Anhydroecgonine
Ethyl Ester in the Urine of a Drug Overdose Victim. Forensic Sci. 2005; 50:1-
5.
New R.R.C. (ed). (1997) Liposomes. A Practical Approach. IRL Press, Oxford.
Newton HB (2006) Advances in strategies to improve drug delivery to brain
tumors. Expert Rev
Neurother. 6(10):1495-509.
Pickrell AM, Pinto M, Hida A, Moraes CT. Striatal dysfunctions associated with
mitochondrial
DNA damage in dopaminergic neurons in a mouse model of Parkinson's disease. J
Neurosci.
2011; 31(48):17649-17658.
*Popov M., Linder C., Deckelbaum R.J., Grinberg S., Hansen I.H., Shaubi E.,
Waner T.,
Heldman E. (2009) Cationic vesicles from novel bolaamphiphilic compounds. J
Liposome
Res. 20(2):147-159.
*Popov M, Grinberg S, Linder C, Bachar Z, Waner T, Deckelbaum R, Heldman E.
(2011) Site-
directed decapsulation of bolaamphiphilic vesicles with enzymatic cleavable
surface groups
Journal of Controlled Release 2012; 160(2): 306-314.
*Puri, A., Loomis, K., Smith, B., Lee, J Yavlovich, A., Heldman, E. and
Blumenthal, R. (2009)
Lipid-Based Nanoparticles as Pharmaceutical Drug Carriers: From Concepts to
Clinic. Crit
Rev Ther Drug Carrier Syst, 26(6): 523-580.
Qin C.Q., Du Y.M., Xiao L. Effect of hydrogen peroxide treatment on the
molecular weight and
structure of chitosan. Polymer Degradation and Stability. 2002; 76:211-221.
Riss PJ, Hummerich R, Schloss P. Synthesis and monoamine uptake inhibition of
conformationally constrained 2beta-carbomethoxy-3beta-phenyl tropanes. Org
Biomol Chem.
2009; 7(13):2688-2698
- 162 -
CA 03018945 2018-09-25
WO 2016/168580 PCT/US2016/027726
Saiyed Z, Gandhi N, and Nairi M (2010)Magnetic Nanoformulation of
Azidothymidine 5'-
triphosphate for Targeted Delivery across the Blood¨Brain Barrier.
International Journal of
Nanomedicine 5 :157-166.
Salvatore MF, Ai Y, Fischer B, Zhang AM, Grondin RC, Zhang Z, Gerhardt GA,
Gash DM.
Point source concentration of GDNF may explain failure of phase II clinical
trial. Exp
Neurol. 2006 ;202(2):497-505.
Slevin JT, Gash DM, Smith CD, Gerhardt GA, Kryscio R, Chebrolu H, Walton A,
Wagner R,
Young AB. Unilateral intraputamenal glial cell line-derived neurotrophic
factor in patients
with Parkinson disease: response to 1 year of treatment and 1 year of
withdrawal. J
Neurosurg. 2007; 106(4):614-620
Songfiang Z and Lixiang W. (2009) Amyloid-Beta Associated with Chitosan Nano-
Carrier has
Favorable Immunogenicity and Permeates the BBB. ,LIAPSPharm Sci Tech,
10(3):900-905.
Spudich S and Antses B (2011) Central Nervous System Complications of HIV
Infection. Top.
Antiviral Med 19(2), 48-57.
Steiner JM, Medinger TL, Williams DA. Purification and partial
characterization of feline
trypsin. Comp Biochem Physiol B Biochem Mol Biol. 1997; 116(1):87-93.
Stepanov V, Schou M, Jary J, Halldin C. Synthesis of 3H-labeled N-(3-iodoprop-
2E-eny1)-2beta-
carbomethoxy-3beta-(4-methylphenyOnortropane (PE2I) and its interaction with
mice striatal
membrane fragments. Appl Radiat Isot. 2007; 65(3):293-300
Stern J, Freisleben HJ, Janku S, Ring K. (1992) Black lipid membranes of
tetraether lipids
from Thermoplasma acidophilum, Biochim Biophys Acta 1128:227-236.
Uchegbu I. The biodistribution of novel 200-nm palmitoyl muramic acid vesicles
International
Journal of Pharmaceutics. 1998; 162:19-27.
Varatharajan L and Thomas S. (2009)The transport of anti-HIV drugs across
blood¨CNS
interfaces: Summary of current knowledge and recommendations for further
Research
Antiviral Res. 2009 May; 82(2): A99¨A109.
*Wiesman Z., Dom N.B., Sharvit E., Grinberg S., Linder C., Heldman E., Zaccai
M. (2007)
Novel cationic vesicle platform derived from vernonia oil for efficient
delivery of DNA
through plant cuticle membranes. J Biotechnol. 130(1):85-94.
Wu Y., Zheng Y. Yang W. Wang C., Hu J., Fu S. Synthesis and characterization
of a novel
amphiphilic chitosan¨polylactide graft copolymer. Carbohydrate Polymers. 2005;
59:165-
171.
Yagi S, Yoshikawa E, Futatsubashi M, Yokokura M, Yoshihara Y, Torizuka T,
Ouchi Y.
Progression from unilateral to bilateral parkinsonism in early Parkinson
disease: implication
of mesocortical dopamine dysfunction by PET. J Nucl Med. 2010; 51(8):1250-1257
- 163 -
CA 03018945 2018-09-25
WO 2016/168580 PCT/US2016/027726
* Zabicky J; Linder C; Grinberg S; Heldman E "Nano - and Mesosized Particles
Comprising an
Inorganic Core, Process and Applications Thereof" US2009011002
- 164 -
