Note: Descriptions are shown in the official language in which they were submitted.
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HEPAROSAN POLYMERS AND METHODS OF MAKING AND USING SAME FOR THE
ENHANCEMENT OF THERAPEUTICS
BACKGROUND OF THE INVENTION
1. Field of the Invention
[0004] The presently claimed and disclosed invention(s) relates, in
general, to the
field of therapeutics and, more particularly but without limiting, to novel
compositions and
methods for making heparosan biomaterials that are suitable for conjugation to
therapeutics for the purpose of enhancing drug action and/or delivery as well
as bioreactive
agents for biotechnical applications.
2. Brief Description of the Related Art
[0005] Without limiting the scope of the presently claimed and disclosed
invention(s), the background of the related art is described in connection
with the use of
sugar polymers and, more particularly, heparosan as a therapeutic modifying
and/or
coupling agent.
[0006] The presently claimed and disclosed invention(s) relates generally
to the field
of therapeutics and, more particularly, to the development of enhanced
therapeutics
through the use of modifying and/or coupling agents and, in particular but
without
limitation, natural polysaccharides and oligosaccharides such as heparosan. A
wide range of
existing and near-term therapeutics has great potential, but many possess
drawbacks that
slow or prevent implementation for aiding human health. Fortunately, the
physical,
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chemical, and/or biological nature of a promising drug candidate may sometimes
be
assisted by modifying the parental drug. A widely used agent, poly[ethylene
glycol] (PEG)
has been approved by the Food & Drug Administration (FDA) for use with
therapeutic
"cargo" including small molecule drugs, polypeptides, and liposomes, for
example. The
process of adding PEG to a drug, i.e., "PEGylation," has been very successful,
as shown in
Table 1. The hydrophilic chains of PEG polymers increase the solubility of the
cargo in water,
protect the cargo when in the human body and prolong the therapeutic action of
the cargo.
Due to its artificial nature, its chemical synthesis, and its potential
harmful effects when
ingested in large quantities over long periods of time, the use of PEG has
significant
drawbacks and alternatives have been sought. The presently disclosed and
claimed
invention is directed to such alternative modifying and/or coupling agents.
SUMMARY OF THE INVENTION
Thus in one aspect, the present invention provides a pharmaceutical
composition for inducing a therapeutic effect in a mammalian patient, the
pharmaceutical
composition comprising: (a) a monodisperse or polydisperse biocompatible
heparosan polymer
having a mass in a range of from about 600 Da to 800 kDa; and (b) a
therapeutic drug covalently
or non-covalently conjugated to the heparosan polymer.
In another aspect, the present invention provides a pharmaceutical
composition for treating a mammalian patient, the pharmaceutical composition
comprising: (a)
a monodisperse or polydisperse biocompatible heparosan polymer having a mass
in a range of
from about 600 Da to 800 kDa; and (b) a therapeutic drug covalently or non-
covalently
conjugated to the heparosan polymer.
In another aspect, the present invention provides a method for preparing a
pharmaceutically active heparosan-therapeutic drug conjugate for
administration to a
mammalian patient, comprising the step of: reacting a therapeutic drug with an
activated
heparosan polymer under conditions sufficient to effect covalent or non-
covalent conjugation
of said therapeutic drug and said heparosan polymer to form a reaction mixture
containing said
therapeutic drug-heparosan conjugate.
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DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0007]
[0008] FIG. 1 graphically depicts the structures of heparosan and
polyethylene
glycol.
=
[0009] FIG. 2A is a graphical representation of the
pharmacokinetics (pK) of
radioactive heparosan conjugate in plasma in a rat model. Rats were injected
intravenously
with 125I-heparosan polymer (100 kDa mass) at 'Time 0', and at various times,
blood was
drawn, and the radioactivity in the plasma was measured. The data indicate
that 100 kDa
heparosan, the active molecule of HEPylation, has a long lifetime (half-life
of approximately
2 days) in the mammalian bloodstream.
[0010] Fig. 2B is a graphical representation of the
pharmacokinetics of radioactive
heparosan conjugate in plasma in a rat model. Rats were injected intravenously
with 125I-
heparosan polymer (60 kDa monodisperse polymer) at 'Time 0', and at various
times, blood
was drawn, and the radioactivity in the plasma was measured. The data indicate
that 60 kDa
heparosan, the active molecule of HEPylation, has a long lifetime (half-life
of approximately
15 hours) in the mammalian bloodstream. In addition, upon comparison of the
data in
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Figures 2A and 2B, the size of the polymer determines the plasma half-life,
thus allowing
tuning of drug-conjugate pharmacokinetics.
[0011] Fig. 3 is a graphical representation of the fate of a radioactive
heparosan
conjugate in a rat model. Rats were injected intravenously with 100 kDa 125I-
heparosan
polymer at 'Time 0', and at various times, the radioactivity in blood (Plasma
or red and
white blood cells, `R&W BC), organs (liver, kidney, spleen, heart, bladder,
brain), and
excreted waste (urine, feces) was measured. The data indicate that heparosan,
the active
molecule of HEPylation, circulated in the plasma of the mammalian blood
stream, did not
accumulate in major organs (note: the low signal present is due to blood
trapped in organs
based on saline-perfused controls), and was excreted via normal pathways
(i.e., urine,
feces).
[0012] Fig. 4 is a pictorial representation showing heparosan is very
stable in the
mammalian bloodstream. The 100 kDa 125I-heparosan conjugate was injected
intravenously
into rats, and at various times, blood was withdrawn, and the plasma was
isolated. The
samples were deproteinized and analyzed by agarose gel (1.5%) electrophoresis
and
autoradiography. The molecular weight of starting probe (lane H; arrow) and
the polymer in
plasma samples are equivalent even after approximately 1-2 days time. Over
time, the
polymer is removed from circulation within the mammal and then
metabolized/excreted.
[0013] Fig. 5 is graphical representation showing synthesis of
monodisperse
heparosan polymers. Three batches of heparosan polymer were analyzed on a 1.2%
agarose gel with Stains-all detection. The polymer size is readily controlled
(as indicated by
the three different size bands of 800 kDa, 380 kDa, and 100 kDa from top to
bottom). The
tight bands indicate that the products have a narrow size distribution
(polydispersity114,1Mn
= 1.06 to 1.18; for reference, the value of an ideal monodisperse polymer is
1). The size of
the polymer affects its half-life in the bloodstream; thus, HEPylation is a
means of tuning
therapeutic dosing profiles. In addition, the Food & Drug Administration (FDA)
regulatory
hurdles for production and approval of therapeutics are lower for a = more
defined,
monodisperse molecule in comparison to a less defined, polydisperse molecule.
[0014] Fig. 6 is a graphical representation of one strategy for
HEPylation reagent
preparation and utilization to form a therapeutic conjugate. Three or four
sequential
reactions are used to produce a HEPylated cargo in this embodiment, where
activated
heparosan vehicle is coupled to a cargo. I. An acceptor (a heparosan
tetrasaccharide, Hep4)
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is modified to add a reactive group (e.g., R=amino or hydrazide). II. Three
independent
reaction mixtures, each with a different ratio of UDP-sugar/reactive acceptor,
are elongated
with PmHS1 synthase via polymer grafting to yield a set of distinct reactive
monodisperse
heparosan polymers of three different sizes. III. The Cargo is modified
directly with any one
size polymer. III'. Alternatively (dotted line), an additional modification
step is used to alter
the reactivity of the heparosan reagent (e.g., add a R'=maleimide group onto
amino-
heparosan) allowing the next step, IV, modification of Cargo. Alternative
embodiments
include (a) activating heparosan produced by fermentation of bacteria and then
coupling to
cargo or (b) coupling the activated short acceptor to a cargo, then elongating
via polymer
grafting to a useful, desired size heparosan chain with heparosan synthase
PmHS1.
[0015] Fig. 7 are pictorial representations of SDS-PAGE gels
illustrating the
production of HEPylated BSA molecules (left panel) and degradation thereof
with heparosan
lyase (right panel).
[0016] Fig. 8 are pictorial representations of an SDS-PAGE gel (left
panel) and gel
filtration chromatography profile (right panel) illustrating production of a
series of higher
molecular weight products corresponding to a series of HEPylated BSA
molecules.
[0017] Fig. 9 is a pictorial representation of an SDS-PAGE gel
illustrating the
production of HEPylated IgG molecules (see arrow area).
[0018] Fig. 10 is a pictorial representation of a PAGE gel visualized
by virtue of
ultraviolet-induced fluorescence, demonstrating production of HEPylated
fluorescein
, molecules (see arrow). Unreacted FITC (fluorescein isothiocyanate) is
bracketed.
[0019] Fig. 11 is a graphical representation of thin layer
chromatography (TLC) of
short heparosan acceptor coupled to a radioactive cargo and its subsequent
elongated
product. This TLC shows the new radioactive acceptor formed by coupling BH and
amino-
Hep4 (middle lane) and its subsequent elongation by polymer grafting with
PmHS1 synthase
into a heparosan vehicle (left lane) suitable for prolonging residence time in
the mammalian
blood stream. (BH = 1251 Bolton-Hunter reagent; Hep4 = heparosan
tetrasaccharide; Poly =
BH conjugate of heparosan polymer of approximately 220 kDa).
[0020] Fig. 12 is a graphic depiction (right panel) of the production
of HEPylated
cargo by polymer grafting, and a pictorial representation (left panel) of an
SDS-PAGE gel that
demonstrates the use of said method to produce HEPylated BSA molecules (see
bracket
area).
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[0021] Fig. 13 is a pictorial representation of an SDS-PAGE gel
demonstrating the
production of HEPylated BSA molecules (see bracket area) utilizing naturally
occurring
heparosan obtained from in vivo microbial fermentation as the source of the
vehicle.
[0022] Fig. 14 is a graphical representation of an agarose gel analysis
of heparosan
coupled to radioactive cargo. This gel was stained with a sugar detection
reagent (Stains-all)
as well as exposed to X-ray film (Autorad; 2 exposure times - short or long)
to illustrate the
defined synthesis of a radioactive cargo coupled to approximately 220 kDa
heparosan (same
polymer as in the TLC of Fig. 11). The narrow size distribution
(monodispersity) is
demonstrated by loading of both a low (1x) and a high (10x) concentration of
HEPylated
probe as well as overexposure of the X-ray film.
[0023] Fig. 15 is a graphical representation of the total distribution of
the measured
radioactivity from Fig. 16 for i.m. dosing.
[0024] Fig. 16 is a graphical representation illustrating the curve-fit
for pK analysis of
the elimination of a radioactive heparosan compound from the plasma for i.m.
dosing.
[0025] Fig. 17 is a pictorial representation of an agarose gel
demonstrating the
stability of the heparosan vehicle utilized in Figs. 15-16 in the
extracellular compartments of
the mammalian body.
[0026] Fig. 18 is a graphical representation of TLC of Urine Metabolites.
A
radioactive conjugate of approximately 220 kDa heparosan/Bolton-Hunter reagent
was
injected into a rat. After 2 days, the radioactive breakdown products excreted
into urine
were analyzed by TLC (similar to Fig. 11). Small size fragments (equal or less
than 4 sugar
units or n=2) of the original probe are observed, indicating metabolic
breakdown of the
natural heparosan polymer after leaving the bloodstream (note: this
approximately 220 kDa
conjugate before injection into the rat would remain at the origin of the TLC
plate as in Fig.
11 and not run up the TLC plate as shown here).
[0027] Fig. 19 is a graphical representation of heparosan metabolite
excretion into
feces. A conjugate of approximately 220 kDa heparosan/Bolton-Hunter was
injected into a
rat. Over 2 days time, the radioactive breakdown products excreted into feces
were
measured in a gamma counter and plotted here as the percent fraction of the
entire initial
dose. Excretion into feces and urine accounts for the metabolized heparosan
vehicle
indicating that heparosan does not accumulate in a mammalian patient (as shown
in Fig.
3). The size of the radioactive polymers in the feces was less than 3 kDa (or
less than 15
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sugar units) as measured by ultrafiltration, thereby indicating that heparosan
is degraded
and then excreted over time.
DETAILED DESCRIPTION OF THE PRESENTLY
CLAIMED AND DISCLOSED INVENTION(S)
[0028]
Before explaining in detail at least one embodiment of the invention in detail
by way of exemplary drawings, figures, graphs, tables, and experimental
drafts, for example,
it is to be understood that the presently claimed and disclosed invention(s)
is not limited in
its application to the details of construction and the arrangement of the
components set
forth in the following description or illustrated in the drawings. Also, it is
to be understood that
the phraseology and terminology employed herein is for purpose of description
and should not
be regarded as limiting. While the making and using of various embodiments of
the presently
claimed and disclosed invention(s) are discussed in detail below, it should be
appreciated that
the scope of the claims should not be limited by the preferred embodiments set
forth in the
examples, but should be given the broadest interpretation consistent with the
description as a
whole.
[0029] The
needs of the presently claimed and disclosed invention(s) set forth above
as well as further and other needs and advantages of the present invention are
achieved by
the embodiments of the invention described herein below.
[0030] The
presently claimed and disclosed invention(s) provides for the
improvement and enhancement of therapeutics through the conjugation and use of
a novel
therapeutic modifying agent: heparosan, a natural polysaccharide related to
heparin.
Heparosan can be synthesized in a step-wise, reproducible, and defined manner
so as to
provide all of the advantages of PEG without its potential side effects.
Heparosan is soluble
in water, biocompatible, and bio-inert within the human body.
[0031] The
addition of heparosan (HEP) to a therapeutic cargo molecule, a process
termed herein as "HEPylation", is superior to PEGylation because: a) a larger
size range of
heparosan polymers is more readily synthesized than PEG; b) the size
distribution at longer
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chain lengths of heparosan can be controlled more carefully than PEG ; c)
heparosan has a
higher water solubility than PEG; d) as a naturally occurring polysaccharide,
heparosan's
degradation products are biocompatible; and e) heparosan is not immunogenic.
[0032] Several linear and branched PEGs having different molecular
weights have
been employed by those with skill in the art to improve the pharmacokinetic
behavior of
therapeutic drugs (i.e., the "cargo" carried by the PEG molecule). Several
distinct types of
reactive PEG polymers allow the synthesis of both reversible and irreversible
PEG-drug
conjugates. PEG-drug conjugates typically exhibit prolonged residence in vivo,
decreased
degradation by metabolic enzymes, and reduced immunogenicity. The therapeutic
cargo,
including proteins and peptides, small molecule drugs, and liposomes, have
been PEGylated
and evaluated successfully by the FDA (Table 1). Several PEGylated drugs have
been in use
for more than a decade, thus proving the general applicability and safety of
PEGylation. As
shown and claimed herein, therapeutic cargo that has been HEPylated (i.e.,
conjugated to
heparosan) retain all of the benefits of PEGylated cargo while minimizing the
negative and
undesirable attributes of PEG.
[0033] Various sized PEGs circulate and are cleared out of the
bloodstream of
mammals at different periods of times. As shown in Table 2, PEG polymers
having a
molecular weight of 6,000 Da or 6 kDa (PEG-6) have a shorter half-life in
blood serum than
PEG polymers having a molecular weight of 170,000 Da or 170 kDa (PEG-170). The
half-life
of PEG molecules in blood serum is directly dependent upon the size of the
polymer. Even
though PEGylation may lead to a loss in binding affinity due to steric
interference (due to
the PEG chain partially covering the drug surface and by conjugating with some
of the drugs
active sites) with the drug-target binding interaction, the loss in potency is
offset by the
longer half-life of the PEG drug conjugate circulating in the blood stream.
Certain drugs
have, therefore, been enabled for use by conjugating the drug to PEG and
thereby
increasing its half-life within a patient to be treated that otherwise could
not have been
developed. Much effort is currently ongoing with the goal of improving or re-
tooling
existing drugs by conjugating with PEG. The novel use of heparosan for a PEG
replacement
is, therefore, a significant step forward and is providing a highly
biocompatible and targeted
drug delivery device.
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Table 1. Currently Marketed PEGylated Drugs
(adapted from 'Pharmacotherapy (2003) 23 (8 pt 2):3S-8S)
Generic Name (Trade
Cargo of PEG Name) / Manufacturer Bioactivity of Native Main Effect of
Conjugate (FDA Approval Date) Agent Pegylation
Reason for Treatment
Pegademase (ADAGEN/ Enzyme Longer half-life, SCID
(severe
ADA (adenosine Enzon (March 1990) replacement, reduced immune
combined
deaminase) reverses symptoms response immunodeficiency
of ADA deficiency disease)
Asparaginase Pegasparagase Hydrolyzes Longer half-life In
combination
(ONCASPAR/ asparagine, on reduced immune
chemotherapy for
Enzon (February 1994) which leukemic response treatment of
acute
cells are dependent lymphoblastic
leukemia in patients
hypersensitive to L-
asparaginase
Granulocyte Pegfilgrastim (NEULASTA/ Stimulation of Longer half-life,
Prophylaxis against
colony- Amgen (January 2002) neutrophil self-regulating
severe neutropenia
stimulating production clearance and its
complications
factor during
myelosuppressive
chemotherapy
Interferon cab Peginterferon cab Antiviral
cytokine Slower clearance, Hepatitis C in patients
(PEGASYS/Roche sustained serum with
compensated
(October 2002) concentration liver disease
Stealth PEG Pegylated liposomal Antitumor Slower clearance,
Refractory ovarian
liposomes with doxorubicin (CAELYX anthracycline
greater distribution cancer, Kaposi's
doxorubicin DOXIL/Alza (June 1999) into tumors sarcoma
Table 2. Blood circulation of PEG (adapted from 'J. Phar. Pharm. Sci.' 2000
(3):125-136)
Parameter PEG-6 PEG-20 PEG-50 PEG-170
AUC 6.2 110 600 1110
t112, minutes 17.6 170 990 1390
AUC= area under the curve; t1/2= half life
[0034] The main pharmacokinetic outcomes of PEGylation are summarized as
changes occurring in the overall circulation life-span within blood serum,
tissue distribution
pattern, and elimination pathway of the drug PEG conjugate (Table 3). As with
PEG,
heparosan maintains all of the benefits of PEG while improving bio-
compatibility and the
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ability to selectively produce and target polymers of a desired predetermined
size (Sismey-
Ragatz et al., J. Biol. Chem, 2007). As with PEG conjugation, conjugation of a
drug or
therapeutic molecule with heparosan (1) increases retention of the drug in the
circulation
by protecting against enzymatic digestion, (2) slows filtration by the
kidneys, and (3) reduces
the generation of neutralizing antibodies. In all respects, HEPylation is a
clear substitute for
PEGylation and, as a naturally occurring polysaccharide, brings with it an
enhanced
biocompatibility and simpler sugar conjugation chemistry.
Table 3. Beneficial Features of Therapeutic Modifying Agents: PEGylation
versus HEPylation
PEG Heparosan HEPylation
a. Extend Cargo Half-life in Bloodstream? Yes Yes
(e.g., avoid renal clearance if larger molecular
weight)
b. Protect Cargo from Degradation? Yes Yes
(e.g., by proteases)
c. Shield Cargo from Immune Response? Yes Yes
(e.g., prevent antibody generation)
d. Trap Cargo in Cancerous Regions? Yes Yes
(e.g., due to altered tumor vasculature)
e. Enhance Solubility of Cargo? Yes
Yes, increased solubility potential
(e.g., especially hydrophobic chemotherapy than PEG due to its more
agents)? hydrophilic
nature
f. Variety of Cargo Coupling Chemistries? Yes Yes
(e.g., amine, sulfhydryl reactive)
g. Exhance Cargo Stability? Yes Yes
(e.g., prevent protein unfolding events)
h. Reduce Dosage and Maintain Constant Yes Yes
Blood Concentrations?
(e.g., avoid peaks and troughs; predictable
dosing plateau in desired range)
i. Suitability for a Range of Cargo:
(e.g., platform technology)
proteins, peptides? Yes Yes
small MW drugs? Yes Yes
liposomes? Yes Yes
hormones? Yes Yes
[0035]
First, the nature of degradation of artificial PEG may be a limiting factor
for
pharmaceuticals used at high doses and/or for long duration treatments.
Second, the
quality control of PEG polymer synthesis with respect to molecular weight
distribution is not
as great as desired.
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[0036] Certain carbohydrates play roles in forming and maintaining the
structures of
multicellular organisms in addition to more familiar roles as nutrients for
energy.
Glycosaminoglycans (GAGs) are long linear polysaccharides comprising
disaccharide repeats
that contain an amino sugar. GAGs are well known to be essential in
vertebrates.
[0037] The GAG structures possess a significant number of negative groups
and
hydroxyl groups and are, therefore, highly hydrophilic. Depending on the
tissue and cell
type, the GAGs are structural, adhesion, and/or signaling elements in humans.
A few
microbes also produce extracellular polysaccharide coatings called capsules
that are
composed of GAG chains and that serve as virulence factors. The capsule
assists in the
microbe's evasion of host defenses such as phagocytosis and complement. As the
microbial
polysaccharide is identical or very similar to the host GAG, the antibody
response to the
microbe is either very limited or non-existent.
[0038] In humans, polymers of heparosan (also called N-acetylheparosan or
unsulfated, unepimerized heparin; [4-GlcUA-beta-1,4-GIcNAc-alpha-1-],; shown
in Fig. 1)
only exist transiently, serving as a precursor to the more highly modified
final products of
heparan sulfate and heparin. The bacterial-derived enzymes used to produce
heparosan for
use in one embodiment of the presently claimed and disclosed invention(s)
synthesize
heparosan as their final product. A single polypeptide, the heparosan synthase
PmHS1 of
Pasteurella multocida Type D, polymerizes the heparosan sugar chain by
transferring both
GlcUA and GIcNAc. PmHS1 is a robust enzyme that efficiently makes polymers up
to ¨1
MDa (1,000 kDa or ¨5,000 monosaccharide units) in vitro. In Escherichia coli
K5, at least two
enzymes, KfiA, the alpha-GIcNAc transferase, and KfiC, the beta-GlcUA-
transferase, (and
perhaps KfiB, a protein of unknown function) work in concert to form the
disaccharide
repeat of heparosan. The E. coli enzyme complex is not as efficient as the
PmHS1 enzyme as
it is more difficult to produce the long polymer chains with the E. coli
enzyme complex.
However, for the purpose of the presently claimed and disclosed invention(s),
it is intended
and would be understood by one of skill in the art that any method which
produces
heparosan may be used. It is not the method of producing heparosan that is
determinative
¨ rather, it is the conjugation of heparosan from any source or method of
production (e.g.,
fermented heparosan produced by native or recombinant microbes, as well as
chemoenzymatic syntheses or organic chemical syntheses) to a target molecule
(i.e., the
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cargo) for increased solubility in water, bioavailability and dwell time
within the patient that
is presently disclosed and claimed.
[0039] A key advantage to using heparosan is that it has increased
biostability in the
extracellular matrix when compared to other GAGs such as hyaluronic acid and
chondroitin.
As with most compounds synthesized in the body, new molecules are typically
made, and
after serving their purpose, are broken down into smaller constituents for
recycling.
[0040] Heparin and heparan sulfate, for example, are degraded by a single
enzyme
known as heparanase. Experimental challenge of heparosan and N-sulfo-heparosan
with
heparanase, however, shows that since these polymers lack the 0-sulfation of
heparin and
heparan sulfate, heparosan and N-sulfo-heparosan are not sensitive to
enzymatic action in
vitro by heparanase. These findings indicate that heparosan is not fragmented
enzymatically in the body, thereby indicating that heparosan is a stable
biomaterial for use
as a drug conjugate.
[0041] However, if heparosan or any of its fragments (generated by
reactive oxygen
species, etc.) is internalized into the lysosome, then the molecules will be
degraded by
resident beta-glucosidase and beta-hexosaminidase enzymes (which remove one
sugar at a
time from the non-reducing termini of the GAG chain), similar to the
degradation of heparin
or hyaluronic acid. Therefore, the heparosan polymer is biodegradable and will
not
permanently reside in the body and thereby cause a lysosomal storage problem.
A key
advantage for therapeutic modification with heparosan polymer, HEPylation, is
that normal
monosaccharides, GIcNAc and GlcUA are the products of the eventual
degradation. In
contrast, PEG degrades into reactive artificial aldehydes and ketones which
are toxic above
certain levels. PEG also accumulates in the body, especially when present as
one or more
high molecular weight polymers.
[0042] The normal roles of heparin/heparan sulfate in vertebrates include
binding
coagulation factors (inhibiting blood clotting) and growth factors (signaling
cells to
proliferate or differentiate). The key structures of heparin/heparan sulfate
that are
recognized by these factors include a variety of 0-sulfation patterns and the
presence of
iduronic acid [IdoUA]; in general, polymers without these modifications do not
stimulate
clotting or cell growth. Heparosan-based materials which do not have such 0-
sulfation
patterns, therefore, do not provoke unwanted clotting or cellular
growth/modulation. As
such, HEPylated drug conjugates do not initiate clotting and/or cell growth
processes and
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remain solely bio-reactive as per the drug or cargo constituent -- the
heparosan is thus
termed or deemed to be biologically inert.
[0043]
Foreign or unnatural molecules stimulate the immune system. Heparosan
polymer exists transiently during heparan sulfate and heparin biosynthesis as
well as being
found in very short polymer structures within mature heparan sulfate or
heparin chains. In
the latter case, the N- and 0-sulfation reactions are not complete in mammals,
so traces of
the original heparosan remain; for example, approximately 1-5 unsulfated
disaccharide
repeats can be interspersed within the sulfated regions. Therefore, the body
treats
heparosan as 'self,' and does not mount an immune response. P. multocida Type
D and E.
coil K5 utilize heparosan coatings to ward off host defenses by acting as
molecular
camouflage. Indeed, scientists had to resort to using capsule-specific phages
or selective
GAG-degrading enzymes to type these heparosan-coated microbes since a
conventional
antibody or serum could not be generated - the heparosan is thus termed or
deemed non-
immunogenic or non-antigenic.
[0044] To
facilitate the understanding of the presently claimed and disclosed
invention(s), a number of terms are defined below. Terms defined herein have
meanings as
commonly understood by a person of ordinary skill in the areas relevant to the
presently
claimed and disclosed invention(s). Terms such as "a", "an" and "the" are not
intended to
refer to only a singular entity, but include the general class of which a
specific example may
be used for illustration. The terminology herein is used to describe specific
embodiments of
the invention, but their usage does not delimit the invention, except as
outlined in the
claims. Generally, all technical terms or phrases appearing herein (unless
defined explicitly
differently herein) are used as one skilled in the art would understand to be
their ordinary
meaning. It is contemplated that any embodiment discussed in this
specification can be
implemented with respect to any method, kit, reagent, or composition of the
presently
claimed and disclosed invention(s), and vice versa. Furthermore, compositions
of the
presently claimed and disclosed invention(s) can be used to achieve methods of
the
presently claimed and disclosed invention(s).
[0045] All
publications and patent applications mentioned in the specification are
indicative of the level of skill of those skilled in the art to which the
presently claimed and
disclosed invention(s) pertains.
CA 02773755 2015-11-12
13
[0046] The use of the word "a" or "an" when used in conjunction with the
term
"comprising" in the claims and/or the specification may mean "one," but it is
also consistent
with the meaning of "one or more," "at least one," and "one or more than one."
The use of
=
the term "or' in the claims is used to mean "and/or" unless explicitly
indicated to refer to
alternatives only or the alternatives are mutually exclusive, although the
disclosure supports
a definition that refers to only alternatives and "and/or." Throughout this
specification, the
term "about" is used to indicate that a value includes the inherent variation
of error for the
device, the method being employed to determine the value, or the variation
that exists
among the study subjects.
[0047] As used in this specification and claim(s), the words "comprising"
(and any
form of comprising, such as "comprise" and "comprises"), "having" (and any
form of having,
such as "have" and "has"), "including" (and any form of including, such as
"includes" and
"include") or "containing" (and any form of containing, such as "contains" and
"contain")
are inclusive or open-ended and do not exclude additional, unrecited elements
or method
steps.
[00481 The term "or combinations thereof' as used herein refers to all
permutations
and combinations of the listed items preceding the term. For example, "A, B,
C, or
combinations thereof" is intended to include at least one of: A, B, C, AB, AC,
BC, or ABC, and
if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB,
BAC, or CAB.
Continuing with this example, expressly included are combinations that contain
repeats of
one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB,
and so
forth. The skilled artisan will understand that typically there is no limit on
the number of
items or terms in any combination, unless otherwise apparent from the context.
All of the
compositions and/or methods disclosed and claimed herein can be made and
executed
without undue experimentation in light of the present disclosure. =
[00491 Heparosan is a sugar polymer of the formula --EGIcNAc-alpha4-GlcUA-
beta4b-
where n is from 2 to about 5,000. The term "oligosaccharide" generally denotes
n being
from about 1 to about 11 while the term "polysaccharide" denotes n being equal
to or
greater than 12. The term "conjugate" as used herein refers to a complex
created between
two or more compounds by covalent or weak bonds. The term "cargo" as used
herein
CA 02773755 2012-03-09
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14
refers to the drug, therapeutic or other biologically active component in the
conjugate,
while the term "vehicle" as used herein refers to the carrier of the cargo
(e.g., the
heparosan polymer) in the conjugate.
[0050] As used herein, the term "active agent(s)," "active
ingredient(s),"
"pharmaceutical ingredient(s)," "therapeutic," "medicant," "medicine,"
"biologically active
compound" and "bioactive agent(s)" are defined as drugs and/or
pharmaceutically active
ingredients. The presently claimed and disclosed invention(s) may be used to
encapsulate,
attach, bind or otherwise be used to affect the storage, stability, longevity
and/or release of
any of the following drugs as the pharmaceutically active agent in a
composition. One or
more of the following bioactive agents listed in (A)-(X) below may be combined
with one or
more carriers (however, said listing of agents provided in (A)-(X) is to be
understood to be
simply for illustration purposes, and is not to be construed as limiting):
[0051] (A) Analgesic anti-inflammatory agents such as, acetaminophen,
aspirin,
salicylic acid, methyl salicylate, choline salicylate, glycol salicylate, 1-
menthol, camphor,
mefenamic acid, fluphenamic acid, indomethacin, diclofenac, alclofenac,
ibuprofen,
ketoprofen, naproxene, pranoprofen, fenoprofen, sulindac, fenbufen, clidanac,
flurbiprofen,
indoprofen, protizidic acid, fentiazac, tolmetin, tiaprofenic acid, bendazac,
bufexamac,
piroxicam, phenylbutazone, oxyphenbutazone, clofezone, pentazocine,
mepirizole, and the
like.
[0052] (B) Drugs having an action on the central nervous system, for
example
sedatives, hypnotics, antianxiety agents, analgesics and anesthetics, such as,
chloral,
buprenorphine, naloxone, haloperidol, fluphenazine, pentobarbital,
phenobarbital,
secobarbital, amobarbital, cydobarbital, codeine, lidocaine, tetracaine,
dyclonine, dibucaine,
cocaine, procaine, mepivacaine, bupivacaine, etidocaine, prilocaine,
benzocaine, fentanyl,
nicotine, and the like. Local anesthetics such as, benzocaine, procaine,
dibucaine, lidocaine,
and the like.
[0053] (C) Antihistaminics or antiallergic agents such as,
diphenhydramine,
dimenhydrinate, perphenazine, triprolidine, pyrilamine, chlorcyclizine,
promethazine,
carbinoxamine, tripelennamine, brompheniramine, hydroxyzine, cyclizine,
meclizine,
clorprenaline, terfenadine, chlorpheniramine, and the like. Anti-allergenics
such as,
antazoline, methapyrilene, chlorpheniramine, pyrilamine, pheniramine, and the
like.
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Decongestants such as, phenylephrine, ephedrine, naphazoline,
tetrahydrozoline, and the
like.
[0054]
(D) Antipyretics such as, aspirin, salicylamide, non-steroidal anti-
inflammatory
agents, and the like. Antimigrane agents such as, dihydroergotamine,
pizotyline, and the
like. Acetonide anti-inflammatory agents, such as hydrocortisone, cortisone,
dexamethasone, fluocinolone, triamcinolone, medrysone, prednisolone,
flurandrenolide,
prednisone, halcinonide, methylprednisolone,
fludrocortisone, corticosterone,
paramethasone, betamethasone, ibuprophen, naproxen, fenoprofen, fenbufen,
flurbiprofen, indoprofen, ketoprofen, suprofen, indomethacin, piroxicam,
aspirin, salicylic
acid, diflunisal, methyl salicylate, phenylbutazone, sulindac, mefenamic acid,
meclofenamate sodium, tolmetin, and the like. Muscle relaxants such as,
tolperisone,
baclofen, dantrolene sodium, cyclobenzaprine.
[0055]
(E) Steroids such as, androgenic steriods, such as, testosterone,
methyltestosterone, fluoxymesterone, estrogens such as, conjugated estrogens,
esterified
estrogens, estropipate, 17-13 estradiol, 17-13 estradiol valerate, equilin,
mestranol, estrone,
estriol, 170 ethinyl estradiol, diethylstilbestrol, progestational agents,
such as,
progesterone, 19-norprogesterone, norethindrone, norethindrone acetate,
melengestrol,
chlormadinone, ethisterone, medroxyprogesterone acetate, hydroxyprogesterone
caproate,
ethynodiol diacetate, norethynodrel, 17-a hydroxyprogesterone, dydrogesterone,
dimethisterone, ethinylestrenol, norgestrel, demegestone, promegestone,
megestrol
acetate, and the like.
[0056]
(F) Respiratory agents such as, theophilline and 02 -adrenergic agonists, such
as, albuterol, terbutaline, metaproterenol, ritodrine, carbuterol, fenoterol,
quinterenol,
rimiterol, solmefamol, soterenol, tetroquinol, and the like. Sympathomimetics
such as,
dopamine, norepinephrine, phenylpropanolamine, phenylephrine, pseudoephedrine,
amphetamine, propylhexedrine, arecoline, and the like.
[0057]
(G) Antimicrobial agents including antibacterial agents, antifungal agents,
antimycotic agents and antiviral agents; tetracyclines such as,
oxytetracycline, penicillins,
such as, ampicillin, cephalosporins such as, cefalotin, anninoglycosides, such
as, kanamycin,
macrolides such as, erythromycin, chloramphenicol, iodides, nitrofrantoin,
nystatin,
amphotericin, fradiomycin, sulfonamides, purrolnitrin, clotrinnazole,
miconazole
chloramphenicol, sulfacetamide, sulfamethazine, sulfadiazine, sulfamerazine,
sulfamethizole
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16
and sulfisoxazole; antivirals, including idoxuridine; clarithromycin; and
other anti-infectives
including nitrofurazone, and the like.
[0058]
(H) Antihypertensive agents such as, clonidine, a-methyldopa, reserpine,
syrosingopine, rescinnamine, cinnarizine, hydrazine, prazosin, and the like.
Antihypertensive diuretics such as, chlorothiazide, hydrochlorothrazide,
bendoflumethazide,
trichlormethiazide, furosemide, tripamide, methylclothiazide, penfluzide,
hydrothiazide,
spironolactone, metolazone, and the like. Cardiotonics such as, digitalis,
ubidecarenone,
dopamine, and the like.
Coronary vasodilators such as, organic nitrates such as,
nitroglycerine, isosorbitol dinitrate, erythritol tetranitrate, and
pentaerythritol tetranitrate,
dipyridamole, dilazep, trapidil, trimetazidine, and the like. Vasoconstrictors
such as,
dihydroergotamine, dihydroergotoxine, and the like. 13-blockers or
antiarrhythmic agents
such as, timolol pindolol, propranolol, and the like.
Humoral agents such as, the
prostaglandins, natural and synthetic, for example PGE1, PGE2a, and PGF2a, and
the PGE1
analog misoprostol. Antispasmodics such as, atropine, methantheline,
papaverine,
cinnamedrine, methscopolamine, and the like.
[0059]
(l) Calcium antagonists and other circulatory organ agents, such as, aptopril,
diltiazem, nifedipine, nicardipine, verapamil, bencyclane, ifenprodil
tartarate, molsidomine,
clonidine, prazosin, and the like. Anti-convulsants such as, nitrazepam,
meprobamate,
phenytoin, and the like. Agents for dizziness such as, isoprenaline,
betahistine,
scopolamine, and the like. Tranquilizers such as, reserprine, chlorpromazine,
and
antianxiety benzodiazepines such as, alprazolam, chlordiazepoxide,
clorazeptate,
halazepam, oxazepam, prazepam, clonazepam, flurazepam, triazolam, lorazepam,
diazepam, and the like.
[0060]
(J) Antipsychotics such as, phenothiazines including thiopropazate,
chlorpromazine, triflupromazine, mesoridazine,
piperracetazine, thioridazine,
acetophenazine, fluphenazine, perphenazine, trifluoperazine, and other major
tranqulizers
such as, chlorprathixene, thiothixene, haloperidol, bromperidol, loxapine, and
molindone, as
well as those agents used at lower doses in the treatment of nausea, vomiting,
and the like.
[0061]
(K) Drugs for Parkinson's disease, spasticity, and acute muscle spasms such as
levodopa, carbidopa, amantadine, apomorphine, bromocriptine, selegiline
(deprenyl),
trihexyphenidyl hydrochloride, benztropine mesylate, procyclidine
hydrochloride, baclofen,
diazepam, dantrolene, and the like. Respiratory agents such as, codeine,
ephedrine,
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isoproterenol, dextromethorphan, orciprenaline, ipratropium bromide,
cromglycic acid, and
the like. Non-steroidal hormones or antihormones such as, corticotropin,
oxytocin,
vasopressin, salivary hormone, thyroid hormone, adrenal hormone, kallikrein,
insulin,
oxendolone, and the like.
[0062] (L) Vitamins such as, vitamins A, B, C, D, E and K and derivatives
thereof,
calciferols, mecobalamin, and the like for dermatologically use. Enzymes such
as, lysozyme,
urokinaze, and the like. Herbal medicaments or crude extracts such as, Aloe
vera, and the
like.
[0063] (M) Antitumor agents such as, 5-fluorouracil and derivatives
thereof, krestin,
picibanil, ancitabine, cytarabine, and the like. Anti-estrogen or anti-hormone
agents such
as, tamoxifen or human chorionic gonadotropin, and the like. Miotics such as
pilocarpine,
and the like.
[0064] (N) Cholinergic agonists such as, choline, acetylcholine,
methacholine,
carbachol, bethanechol, pilocarpine, muscarine, arecoline, and the like.
Antimuscarinic or
muscarinic cholinergic blocking agents such as, atropine, scopolamine,
homatropine,
methscopolamine, homatropine methylbromide, methantheline, cyclopentolate,
tropicamide, propantheline, anisotropine, dicyclomine, eucatropine, and the
like.
[0065] (0) Mydriatics such as, atropine, cyclopentolate, homatropine,
scopolamine,
tropicamide, eucatropine, hydroxyamphetamine, and the like. Psychic energizers
such as 3-
(2-aminopropy)indole, 3-(2-aminobutyl)indole, and the like.
[0066] (P) Antidepressant drugs such as, isocarboxazid, phenelzine,
tranylcypromine,
imipramine, amitriptyline, trimipramine, doxepin, desipramine, nortriptyline,
protriptyline,
amoxapine, maprotiline, trazodone, and the like.
[0067] (Q) Anti-diabetics such as, insulin, and anticancer drugs such as,
tamoxifen,
methotrexate, and the like.
[0068] (R) Anorectic drugs such as, dextroamphetamine, methamphetamine,
phenylpropanolamine, fenfluramine, diethylpropion, mazindol, phentermine, and
the like.
[0069] (S) Anti-malarials such as, the 4-aminoquinolines,
alphaaminoquinolines,
chloroquine, pyrimethamine, and the like.
[0070] (T) Protein therapeutics such as enzymes, cytokines, growth
factors,
hormones, receptors, antibodies, immune complexes, and the like. Also included
are
protein derivatives that enhance or block the activity of any of the naturally-
occurring or
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18
isolated molecules listed herein or interacting components in the biochemical
or cellular
pathways.
[0071] (U) Anti-ulcerative agents such as, misoprostol, omeprazole,
enprostil, and
the like. Antiulcer agents such as, allantoin, aldioxa, alcloxa, N-
methylscopolamine
methylsuflate, and the like. Antidiabetics such as insulin, and the like.
[0072] (V) Anti-cancer agents such as, cis-platin, actinomycin D,
doxorubicin,
vincristine, vinblastine, etoposide, amsacrine, mitoxantrone, tenipaside,
taxol, colchicine,
cyclosporin A, phenothiazines or thioxantheres.
[0073] (W) For use with vaccines, one or more antigens, such as, natural,
heat-killer,
inactivated, synthetic, peptides and even T cell epitopes (e.g., GADE, DAGE,
MAGE, etc.) and
the like.
[0074] (X) Example therapeutic or active agents also include water
soluble or poorly
soluble drugs of molecular weights from 40 to 1,100 including the following:
Hydrocodone,
Lexapro, Vicodin, Effexor, Paxil, Wellbutrin, Bextra, Neurontin, Lipitor,
Percocet, Oxycodone,
Valium, Naproxen, Tramadol, Ambien, Oxycontin, Celebrex, Prednisone, Celexa,
Ultracet,
Protonix, Soma, Atenolol, Lisinopril, Lortab, Darvocet, Cipro, Levaquin,
Ativan, Nexium,
Cyclobenzaprine, Ultram, Alprazolam, Trazodone, Norvasc, Biaxin, Codeine,
Clonazepam,
Toprol, Zithromax, Diovan, Skelaxin, Klonopin, Lorazepam, Depakote, Diazepam,
Albuterol,
Topamax, Seroquel, Annoxicillin, Ritalin, Methadone, Augmentin, Zetia,
Cephalexin, Prevacid,
Flexeril, Synthroid, Promethazine, Phentermine, Metformin, Doxycycline,
Aspirin, Remeron,
Metoprolol, Amitriptyline, Advair, Ibuprofen, Hydrochlorothiazide, Crestor,
Acetaminophen,
Concerto, Clonidine, Norco, Elavil, Abilify, Risperdal, Mobic, Ranitidine,
Lasix, Fluoxetine,
Coumadin, Diclofenac, Hydroxyzine, Phenergan, Lamictal, Verapamil,
Guaifenesin, Aciphex,
Furosemide, Entex, Metronidazole, Carisoprodol, Propoxyphene, Digoxin,
Zanaflex,
Clindamycin, Trileptal, Buspar, Keflex, Bactrim, Dilantin, Flomax, Benicar,
Baclofen, Endocet,
Avelox, Lotrel, lnderal, Provigil, Zantac, Fentanyl, Premarin, Penicillin,
Claritin, RegIan,
Enalapril, Tricor, Methotrexate, Pravachol, Amiodarone, Zelnorm, Erythromycin,
Tegretol,
Omeprazole, and Meclizine.
[0075] The drugs mentioned above may be used in combination as required.
Moreover, the above drugs may be used either in the free form or, if capable
of forming
salts, in the form of a salt with a suitable acid or base. If the drugs have a
carboxyl group,
their esters may be employed.
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19
[0076]
The "suitable acid" may be an organic acid, for example, methanesulfonic
acid, lactic acid, tartaric acid, fumaric acid, maleic acid, acetic acid, or
an inorganic acid, for
example, hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric
acid. The base
may be an organic base, for example, ammonia, triethylamine, or an inorganic
base, for
example, sodium hydroxide or potassium hydroxide. The esters may be alkyl
esters, aryl
esters, aralkyl esters, and the like.
Bioactive Delivery of the Heparosan Conjugate
[0077]
The heparosan conjugate may be administered parenterally,
intraperitoneally, intraspinally, intravenously,
intramuscularly, intravaginally,
subcutaneously, intranasally, rectally, or intracerebrally. Dispersions of the
heparosan
conjugate may be prepared in glycerol, liquid poly[ethylene glycols], and
mixtures thereof,
as well as in oils. Under ordinary conditions of storage and use, such
preparations of the
heparosan conjugate may also contain a preservative to prevent the growth of
microorganisms.
[0078]
Pharmaceutical compositions suitable for injection use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. In
all cases, the
composition must be sterile and must be fluid to the extent that easy
syringability exists. It
must be stable under the conditions of manufacture and storage and must be
preserved
against the contaminating action of microorganisms such as bacteria and fungi.
The
heparosan conjugate may be used in conjunction with a solvent or dispersion
medium
containing, for example, water, ethanol, poly-o1 (for example, glycerol,
propylene glycol, and
liquid poly[ethylene glycol], and the like), suitable mixtures thereof,
vegetable oils, and
combinations thereof.
[0079]
The proper fluidity of the heparosan conjugate may be maintained, for
example, by the use of a coating such as lecithin, by the maintenance of the
required
particle size in the case of dispersion, and/or by the use of surfactants.
Prevention of the
action of microorganisms may be achieved by various antibacterial and
antifungal agents,
for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and
the like. In
many cases, it will be preferable to include isotonic agents, for example,
sugars, sodium
chloride, or polyalcohols such as mannitol and sorbitol, in the composition.
Prolonged
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absorption of the injectable compositions may be brought about by including in
the
composition an agent that delays absorption, for example, aluminum
monostearate or
gelatin.
[0080] Sterile injectable solutions may be prepared by incorporating the
heparosan
conjugate in the required amount in an appropriate solvent with one =or a
combination of
ingredients enumerated above, as required, followed by filtered sterilization.
Generally,
dispersions are prepared by incorporating the heparosan conjugate into a
sterile carrier that
contains a basic dispersion medium and the required other ingredients from
those
enumerated above. In the case of sterile powders for the preparation of
sterile injectable
solutions, the methods of preparation may include vacuum drying, spray drying,
spray
freezing and freeze-drying that yields a powder of the active ingredient
(i.e., the heparosan
conjugate) plus any additional desired ingredient from a previously sterile-
filtered solution
thereof.
[0081] The heparosan conjugate may be orally administered, for example,
with an
inert diluent or an assimilable edible carrier. The heparosan conjugate and
other
ingredients may also be enclosed in a hard or soft shell gelatin capsule,
compressed into
tablets, or incorporated directly into the subject's diet. For oral
therapeutic administration,
the heparosan conjugate may be incorporated with excipients and used in the
form of
ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and
the like. The percentage of the heparosan conjugate in the compositions and
preparations
may, of course, be varied as will be known to the skilled artisan. The amount
of the
heparosan conjugate in such therapeutically useful compositions is such that a
suitable
dosage will be obtained.
[0082] It is especially advantageous to formulate parenteral compositions
in dosage
unit form for ease of administration and uniformity of dosage. Dosage unit
form as used
herein refers to physically discrete units suited as unitary dosages for the
subjects to be
treated; each unit containing a predetermined quantity of heparosan conjugate
calculated
to produce the desired therapeutic effect. The specification for the dosage
unit forms of the
presently claimed and disclosed invention(s) are dictated by and directly
dependent on (a)
the unique characteristics of the heparosan conjugate and the particular
therapeutic effect
to be achieved, and (b) the limitations inherent in the art of compounding
such a
therapeutic compound for the treatment of a selected condition in a subject.
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[0083] Aqueous compositions of the present invention comprise an
effective amount
of the nanoparticle, nanofibril or nanoshell or chemical composition of the
presently
claimed and disclosed invention(s) dissolved and/or dispersed in a
pharmaceutically
acceptable carrier and/or aqueous medium. The biological material should be
extensively
dialyzed to remove undesired small molecular weight molecules and/or
lyophilized for more
ready formulation into a desired vehicle, where appropriate. The active
compounds may
generally be formulated for parenteral administration, e.g., formulated for
injection via the
intravenous, intramuscular, sub-cutaneous, intralesional, and/or even
intraperitoneal
routes. The preparation of an aqueous composition that contains an effective
amount of the
nanoshell composition as an active component and/or ingredient will be known
to those of
skill in the art in light of the present disclosure. Typically, such
compositions may be
prepared as injectables, either as liquid solutions and/or suspensions; solid
forms suitable
for using to prepare solutions and/or suspensions upon the addition of a
liquid prior to
injection may also be prepared; and/or the preparations may also be
emulsified. Also, the
heparosan vehicle can be used to enhance a secondary vehicle (e.g., liposomes,
nanoparticles, etc.) that acts as a carrier or adjuvant for a drug.
[0084] Examples are provided hereinbelow. However, the present invention
is to be
understood to not be limited in its application to the specific
experimentation, results and
laboratory procedures. Rather, the Examples are simply provided as one of
various
embodiments and is meant to be exemplary, not exhaustive.
EXAMPLE 1
[0085] Defined GAG synthesis and heparosan synthesis in particular is
rather
versatile with respect to chemical functionality as well as size control. For
example, U.S.
Publication No. US 2008/0109236 A1 (US Patent Application No. 11/906,704 filed
October 3,
2007, entitled "PRODUCTION OF DEFINED MONODISPERSE HEPAROSAN POLYMERS AND
UNNATURAL POLYMERS WITH POLYSACCHARIDE SYNTHASES") discloses a methodology for
polymer grafting utilizing heparin/heparosan synthases from Pasteurella in
order to provide
heparosan polymers having a targeted size and that are substantially
monodisperse at the
desired size ranges. As disclosed in the '704 application, appropriate
reactive moieties may
be added to the heparosan polymer at the reducing or non-reducing termini or
throughout
the sugar chain. Having one reactive group/chain is preferable when
conjugating the
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22
heparosan polymer to its cargo. As such, the methodology of the '704
application can be
applied to produce heparosan polymers suitable for HEPylation with a cargo
molecule.
Table 4 lists different HEPylation polymer chemistries which are available
and/or suitable for
modifying the heparosan polymer to make it more acceptable or suitable for
conjugating
with specific cargo molecules. It is not the nature or manner of the
complexation or
conjugation between heparosan and the drug (by any covalent chemical or weak
bond) that
is controlling; rather, it is the particular use to which the heparosan will
be put.
Table 4. Heparosan Polymer Chemistries Available
Number
of Extra
Position on Typical
Functional Group Steps * Reactive with: Heparosan Cargo
Notes
1. aldehyde amines - Reducing Peptide, - Irreversible if
0
Protein NaCNBH3 coupling
2. malemide 2 sulfhydryls - Reducing
Peptide, - Irreversible
Protein
3. pyridylthio 2 sulfhydryls -
Reducing Peptide, - Reversible (disulfide)
Protein
4. Azido acetylenes - Non-
Various - Cu(I) Coupling
1 (tripleCC bond) Reducing - Irreversible
- Interior
5. Amino aldehydes - Reducing
Drugs - NaCNBH4 Coupling
- Non- - Irreversible
1
reducing -
Interior
6. N-hydroxy - Reducing
Peptide, - Irreversible
succimimide (NHS) 2 - Non- Protein
amines reducing
7. hydrazide aldehydes, - Reducing
Drugs - Irreversible if
1 ketones NaCNBH3
- Reversible otherwise
* beyond "normal heparosan" polymer
[0086] PmHS1 (SEQ ID NOS: 1 and 2, the amino acid and nucleotide
sequences,
respectively) was expressed as a carboxyl terminal fusion to maltose binding
protein (MBP)
using the pMAL-c2X vector (New England BioLabs). To facilitate extracting the
enzymes, the
expression host E. coli XJa (Zymo Research), which encodes a phage lysin
enzyme, was
employed and allowed for simple freeze/thaw lysis. Cultures were grown in
Superior Broth
(AthenaES) at 30*C with ampicillin (100 g/m1), and L-arabinose (3.25 mM). At
mid-log
phase, isopropyl 13-D-1-thiogalactopyranoside (IPTG) (0.2 mM final) was added
to induce
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23
fusion protein production. One hour after induction, the cultures were
supplemented with
fructose (12.8 mM final) and grown for approximately 5-12 hours before
harvesting by
centrifugation at 4*C. The bacteria were resuspended in 20 mM Tris, pH 7.2,
and protease
inhibitor cocktail on ice, then frozen and thawed twice, thus allowing lysin
to degrade the
cell walls. The lysates were clarified by centrifugation.
[0087] The synthase was affinity purified via the MBP unit using
amylose resin (New
England BioLabs). After washing extensively with column buffer (20 mM Tris, pH
7.2, 200
mM NaCI, 1 mM EDTA), the protein was eluted in column buffer containing 10 mM
maltose.
Protein concentration was quantitated by the Bradford assay (Pierce, Rockford,
IL) using a
bovine albumin serum standard. The purification was monitored by SDS-PAGE with
copper
negative staining (which adds comparable sensitivity as conventional silver
staining)
followed by Coomassie blue staining. The enzyme (approximately 90-95% pure;
yield ¨10
mg per liter of culture) may be used directly after buffer exchange into 50 mM
Tris, pH 7.2,
by ultrafiltration. Further purification by anion-exchange chromatography
provides an
approximately 95-99% pure PmHS1 enzyme.
= [0088] A heparosan polysaccharide (having a molecular weight of
approximately
200-300 kDa) derived from the spent fermentation broth of P. multocida Type D
cultures
was converted into heparosan tetrasaccharide (4-mer, having a molecular weight
of
approximately 700 Da), the starting material for the primers described later
herein. P.
multocida Type D cells were grown in a proprietary synthetic media at 37 C in
shake flasks
for approximately 24 hrs. Spent culture medium (the liquid part of culture
after microbial
cells were removed) was harvested (by centrifugation at 10,000 x g, 20 min)
and
deproteinized (solvent extraction with chloroform). The very large anionic
heparosan
polymer ("fermentation heparosan," having a molecular weight of approximately
200-300
kDa) was isolated via ultrafiltration (30 kDa molecular weight cut-off;
Amicon) and ion
exchange chromatography (NaCI gradient on Q-Sepharose; Pharmacia). Heparosan
from E.
coli K5 cultures can also be used, but the polymers are initially lower
molecular weight than
P. multocida Type D.
[0089] Heparosan oligosaccharides ((GlcUA-GIcNAc)n-(GlcUA-
anhydromannitol), n =
1, 2 or 3) were prepared by partial deacetylation of heparosan polysaccharide
with base,
nitrous acid hydrolysis, and reduction; these polymers contain intact non-
reducing termini,
but an anhydromannitol group at the reducing end. The fragments were purified
by gel
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24
filtration on a P2 column (BioRad, Hercules, CA) in 0.2 M ammonium formate,
followed by
normal phase thin layer chromatography (TLC) on silica plates (Whatman) with n-
butanol/acetic acid/water (1:1:1). The bands were detected by staining of side
lanes with
napthoresorcinol. The size and purity of oligosaccharides were verified by
matrix assisted
laser desorption ionization time of flight mass spectrometry (MALDI-ToF MS).
Alternatively,
acid hydrolysis or enzymatic cleavage yields oligosaccharides that can also be
employed for
use.
[0090] Amino-Hep4 was prepared by reductive amination of Hepa, the
heparosan
tetrasaccharide (n=2), with ammonium ion. The dry sugar was dissolved in
anhydrous
methanol (0.71 mg/ml w/v or 0.93 mM final) under sonication. After addition of
solid
ammonium acetate and NaBH3CN (final 1 M and 0.1 M, respectively), the mixture
was
heated to reflux (approximately 70-80 C) overnight. Thin layer chromatographic
analysis
(TLC - silica; BuOH/AcOH/H20 1:1:1 v:v:v with detection by napthoresorcinol
reagent) was
used to monitor consumption of the starting material. The reaction was
quenched by slow
addition of 20% AcOH. The solvent was evaporated in vacuo and the residue
dissolved in
0.2 M ammonium formate for desalting by gel filtration chromatography on a P-2
resin
column (Bio-Rad) in the same volatile buffer. The fractions containing the
target molecule
were pooled and lyophilized. The volatile salts were removed by two more
cycles of
dissolving in water and lyophilization. Flash silica gel column chromatography
(silica gel 60,
E. Merck; BuOH/AcOH/H20 1:1:1 v:v:v) was employed for further purification.
The structure
of the amino-Hep4 product was confirmed by matrix-assisted laser desorption
time-of-flight
mass spectrometry analysis. The derived amino-HEP4 primer was extended by the
PmHS1
enzyme as described in the '704 application to form amino-heparosan polymer
used as the
carrier portion of the heparosan conjugate.
[0091] The Amino-heparosan polymer may be further reacted with various
activated
bifunctional N-hydroxysuccinimide esters to thereby add desirable groups
including
maleimides, a sulfhydryl selective reagent, etc. The amino-heparosan polymer
was reacted
with approximately 10-fold molar excess of (Pierce) in 10% dimethylsulfoxide,
0.1 M
potassium phosphate, 0.15 M NaCI, pH 7.4, for 2 hrs at room temperature. The
target
compound in the reaction mixture was purified by gel filtration chromatography
on
Sephadex G-25 resin (PD-10, Pharmacia) as detailed above.
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[0092] The amino-HEP4 products may also be reductively aminated by
treatment
with adipic acid dihydrazide (30 eq) and sodium cyanoborohydride (100 eq) at
50-60 C in 1
M sodium phosphate buffer, pH 5.5. After desalting, the obtained hydrazide
amino-HEP4
may be further purified by strong anion exchange chromatography using
Sepharose Q
(Pharmacia) with an ammonium bicarbonate gradient elution. The hydrazide amino-
HEP4
primer may also be extended by the PmHS1 enzyme in order to produce polymers
having
varying sizes as described below.
[0093] Synchronized polymerization reactions were used to produce
monodisperse
polymers as previously described and disclosed in the '704 patent application.
The
formation of heparosan with narrow size distribution (i.e., monodisperse) is
dependent on
the ability of the PmHS1 enzyme to be primed by acceptors (thus avoiding a
slow de novo
initiation event yielding out of step elongation events) and efficiently
transfer
monosaccharides from UDP-sugars. Recombinant PmHS1 synthesizes heparosan
chains in
vitro if supplied with both required UDP-sugars according to the equation:
n UDP-GlcUA + n UDP-GIcNAc --> 2n UDP + [GlcUA-GIcNAc]n
[0094] However, if a heparosan-like oligosaccharide ([GIcUA-GIcNAch) is
also
supplied in vitro, then the overall incorporation rate is elevated up to
approximately 25-fold.
The rate of initiation of a new chain de novo is slower than the subsequent
elongation (i.e.,
repetitive addition of sugars to a nascent HA molecule). The observed
stimulation of
synthesis by exogenous acceptor primer appears to operate by bypassing the
kinetically
slower initiation step, allowing the elongation reaction to predominate as in
the following
equation:
n UDP-GlcUA + n UDP-GIcNAc + [GlcUA-GIcNAc] -->
2n UDP + [GlcUA-GIcNAch+n
[0095] If there are many termini (i.e., z is large), then a limited amount
of UDP-
sugars will be distributed among many molecules and thus result in many short
polymer
chain extensions. Conversely, if there are few termini (i.e., z is small),
then the limited
amount of UDP-sugars will be distributed among few molecules and thus result
in long
polymer chain extensions. Thus, by controlling the molar ratio of acceptor to
UDP-sugar, it
is possible to select the final polymer size desired. Typically, from about
50% to about 90%
of the starting UDP-sugars are consumed in the reactions on the basis of
polysaccharide
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26
recovery. Alternatively, if size control is not as critical, then
"fermentation heparosan" or its
fragments (generated by acid, base, enzyme or physical cleavage methods known
to those
of skill in the art) will suffice as the vehicle. Similarly, chemically
manufactured heparosan
may be utilized. As will be appreciated by one of ordinary skill in the art,
therefore, it is not
the source or manner in which the heparosan is made that is controlling;
rather, it is the
particular use to which the heparosan will be put. If
size is critical, recombinant
chemoenzymatic production is preferred. In situations where size is of a
secondary or lesser
importance, fermentation heparosan (or its derivatives) may be used. As such,
the use of
heparosan from any source or produced by any methodology is intended to be
within the
presently claimed and disclosed invention. Likewise, it is not the nature or
manner of the
complexation between heparosan and the drug (by any chemical or weak bond)
that is
controlling; rather it is the particular use to which the heparosan will be
put.
[0096]
The yield and molecular weight size distribution of the heparosan is checked
by (a) carbazole assays for uronic acid; and (b) agarose gel electrophoresis
(1X TAE buffer,
0.8-1.5% agarose) followed by Stains-All detection.
The carbazole assay is a
spectrophotometric chemical assay that measures the amount of uronic acid in
the sample
via production of a pink color; every other sugar in the heparosan chain is a
glucuronic acid
(GlcUA). The heparosan polymer size is determined by comparison to
monodisperse HA size
standards (HA Lo-Ladder, Hyalose, LLC) run on gels. The detection limit of the
carbazole and
the gel assays is approximately 5-15 micrograms of polymer. Any endotoxin is
removed by
passage through an immobilized polynnyxin column (Pierce); the material is
then tested with
a Limulus amoebacyte-based assay (www.Cambrex.com) to assure that the
heparosan
contains <0.05 endotoxin units/mg solid (based on USP guidelines).
[0097]
Examples of the productions of monodisperse heparosan are shown in Fig. 5
where providing various levels of primer yielded different Mw (weight average
molecular
mass) products with low polydispersity (Mw/Mn; Mn = number average molecular
weight).
For reference, the polydispersity value for an ideal monodisperse polymer
equals 1. The
parallel reaction without an acceptor (lane 0) resulted in a large product
that was
significantly polydisperse, i.e., it contains heparosan polymers of varying
size and length.
[0098]
The polymerization by synthases in the presence of an acceptor is a
synchronized process. Reactions without acceptor exhibit a lag period
interspersed with
numerous, out of step initiation events that yield a short heparosan
oligosaccharide. Once
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27
any chain is formed, the heparosan polymer is elongated rapidly. Other new
chains that
arise later during the lag period are also elongated rapidly, but the size of
these younger
chains never catches up to the older chains in a reaction with a finite amount
of UDP-sugars.
In contrast, in reactions containing an acceptor, all heparosan chains are
elongated in
parallel in a nonprocessive fashion resulting in a more homogenous final
polymer
population.
[0099] The enzymological properties of recombinant pmHS1 described above
also
allow for the control of heparosan polymer size in chemoenzymatic syntheses.
First, as
noted above, the rate-limiting step in vitro appears to be the chain
initiation step.
Therefore, PmHS1 transfers nnonosaccharides onto the existing heparosan
acceptor chains
before substantial de novo synthesis. Second, the enzyme polymerizes heparosan
in a rapid
nonprocessive fashion in vitro. Therefore, the amount of primer should affect
the final size
of the product when a finite amount of UDP-sugar is present. The synthase adds
all
available UDP-sugar precursors to the nonreducing termini of acceptors as in
the equation:
n UDP-GlcUA + n UDP-GIcNAc + z [GlcUA-GIcNAc] ¨> 2n UDP + z [GlcUA-
GIcNAc]x+(n/z)
[001001 Thus, by controlling the molar ratio of acceptor to UDP-sugar, it
is now
possible to select the final heparosan polymer size desired. Typically, from
about 50% to
about 90% of the starting UDP-sugars are consumed in the reactions on the
basis of
polysaccharide recovery.
[00101] The size distribution of the heparosan polymers produced was
determined by
high performance size exclusion chromatography-multi angle laser light
scattering (SEC-
MALLS). Polymers (2.5 to 12 lag mass; 50 .1 injection) were separated on PL
aquagel-OH 30
(8 ilm), -OH 40, -OH 50, -OH 60 (15 ptm ) columns (7.5 x 300 mm, Polymer
Laboratories) in
tandem or alone as required by the size range of the polymers to be analyzed.
The columns
were eluted with 50 mM sodium phosphate, 150 mM NaCI, pH 7 at 0.5 ml/min.
MALLS
analysis of the eluant was performed by a DAWN DSP Laser Photometer in series
with an
OPTILAB DSP Interferometric Refractometer (632.8 nm; Wyatt Technology). The
ASTRA
software package was used to determine the absolute average molecular mass
using a
dn/dc coefficient of 0.153 determined for HA, a polymer with the exact same
sugar
composition as heparosan, by Wyatt Technology. The Mw and polydispersity
values from at
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28
least two SEC-MALLS runs were averaged in order to obtain a final
approximation of the Mw
and polydispersity of the heparosan molecule.
[00102] Although there have been described many different types of
molecules that
can be conjugated with the heparosan polymer, two primary defined model
cargoes are of
particular interest and importance: (a) a chemotherapy agent, and (b) a
protein therapeutic.
For (a), doxorubicin and taxol are useful chemotherapy agents for treating
several cancers.
Taxol is only slightly soluble in water (i.e., approximately 0.4 micrograms
per mL), and such
solubility issues can be improved through conjugation with the hydrophilic
heparosan
polymer. The carbonyl groups found on the taxol or doxorubicin molecule allows
the drug
to couple monovalently to the heparosan polymer, thereby providing a heparosan
drug
conjugate. However, if desirable and to increase dosage of pharmaceutical
available for
pharmocological treatment, the drug molecule is also attachable to multiple
positions on
the heparosan polymer. A dihydrazide may also be added to the drug-heparosan
conjugate
in order to create a time-release formulation. The heparosan-doxorubicin or
taxol-
heparosan conjugate is water-soluble and nontoxic; as heparosan is slowly
degraded in
blood pH, the linkage releases free active doxorubicin or taxol in a specific
and controlled
manner.
[00103] For (b), protein targets include enzymes, cytokines, interferon,
antibodies,
receptors and growth factors as well as modified derivatives (e.g., with
either chemical or
molecular genetic changes). Bovine serum albumin, BSA, is a useful surrogate
for testing
and modeling a protein therapeutic conjugated with heparosan. The BSA protein
does not
have intrinsic glycosylation and facilitates analysis of the addition of one
or more heparosan
chains to the BSA-heparosan conjugate. The use of heparosan as vehicle for
drug
conjugation is also applicable to recombinant proteins with a bioengineered
extra cysteine
or an exposed sulfhydryl group, such as antibodies, resulting in an improved
strategy to
couple such cargo. The cysteine's sulfhydryl group is coupled to the
monovalent
heparosan-maleimide to provide the heparosan conjugate. Alternatively,
heparosan-
thiopyridyl may be used if protein release is desirable due to a reversible
disulfide linkage
between the sugar polymer (i.e., the heparosan polymer) and the cargo (i.e.,
recombinant
proteins, etc.). As with PEG, the aldehyde of heparosan chains may be coupled
to amines of
proteins via reductive amination with sodium cyanoborohydride; a useful
process for the
conjugation of growth factors and interferon.
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[00104] After the heparosan protein conjugation occurs, the molecule is
challenged
with (a) proteases and (b) antibodies (e.g., anti-BSA antibody). For protease
sensitivity,
samples of protein or protein-heparosan are treated with dilution series of
trypsin, an
aggressive serine protease, for 0-60 min, then run on the SDS-PAGE gel.
Relative resistance
to digestion occurs for protein-heparosan. For antigenicity, protein or
protein-heparosan
are incubated with anti-protein IgG beads (e.g., anti-BSA IgG beads from
Sigma) for 1 hour in
saline, then the supernatant analyzed by PAGE. Alternatively, soluble anti-
protein reagent
can be incubated with test samples and run on native gels (similar to standard
gels except
that sample buffer lacks reducing agent and will not be boiled). A higher
molecular weight
complex forms when the antibody builds to the protein-heparosan conjugate
causing a
"super-shift". The protein-heparosan conjugate is resistant to anti-protein if
the heparosan
blocks its epitope. The overall goal is to assess, and to optimize, as needed
the reaction
parameters to produce the heparosan-conjugate.
A Strategy for HEPvlation Reagent Preparation and Utilization
[00105] Three or four sequential reactions were used to produce a HEPylated
cargo in
this embodiment of drug-conjugate synthesis as in Fig 6. (1) An acceptor (a
heparosan
tetrasaccharide, Hep4) was modified to add a reactive group (R=amino or
hydrazide). (2)
Three independent reaction mixtures, each with a different ratio of UDP-
sugar/reactive
acceptor, were elongated with PmHS1 synthase to yield a set of distinct
reactive
monodisperse heparosan polymer preparations of three different sizes. (3) The
Cargo was
modified directly with any one size polymer. (3A) Alternatively (dotted line),
an additional
modification step was used to alter the reactivity of the heparosan reagent
(add a
R1=maleimide group onto amino-heparosan) allowing the next step, (4),
modification of the
cargo.
[00106] There are numerous possibilities for coupling the heparosan vehicle
and
therapeutic cargo involving various chemistries which include, but are not
limited to, the
examples listed previously in Table 4. In the examples that follow, two
proteins, BSA and
IgG antibody, and two small molecules, fluorescein and Bolton-Hunter reagent,
were used
as cargo for coupling various monodisperse or polydisperse heparosan polymers
produced
either via fermentation in vivo or chemoenzymatic synthesis in vivo.
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[00107]
Figs. 7-8 illustrate the production of HEPylated BSA molecules via
chemoenzymatic synthesis. In Fig. 7, radioactive bovine serum albumin [BSA]
(1251-Bolton-
Hunter labeled; migration marked with arrow) protein was reacted via reductive
amination
with sodium cyanoborohydride with two different reactive 20 kDa heparosan
polysaccharides, 'H' or 'N' (unmodified BSA starting material is lane '0').
Each reactive
heparosan was made by extending a short oligosaccharide acceptor into a longer
20 kDa
polymer with PmHS1 enzyme and UDP-sugars. The acceptors were derived from
heparosan
polysaccharide (-200-300 kDa) by two different methods: for H, a heparosan
tetrasaccharide formed by HCI cleavage with general structure [GlcUA-GIcNAc]2
was used
while for N, a heparosan tetrasaccharide formed by base treatment followed by
nitrous acid
cleavage with general structure [GlcUA-GIcNAc]-GlcUA-anhydromannitol was used.
As seen
by the SDS-PAGE gel visualized by autoradiography on the left, higher
molecular weight
products are observed in the N lane, corresponding to a series of HEPylated
BSA molecules
(see bracketed area). The H lane does not have the same pattern due to the
fact that the H
polymer must mutarotate to yield a free aldehyde that can react with the BSA
amine
groups, and thus has lower yields. On the other hand, the N polymer always has
a free
aldehyde, thus allowing better reaction. As a proof of the HEPylated BSA
structure, a
duplicate sample of the N material was treated with heparosan lyase, an enzyme
that
degrades the heparosan polymer but does not degrade other macromolecules such
as BSA.
As seen in the '+ lyase lane,' the BSA now runs at its original position,
demonstrating that
authentic heparosan chains were added to the protein cargo.
[00108] In
Fig. 8, bovine serum albumin [BSA] protein was reacted with Traut's
reagent (T; iminothiolane) to convert some of its amino groups (lysines and
amino termini)
into free sulfhydryl groups forming T-BSA; in this case, ¨1-3 residues on
average were
predicted to be modified based on the reaction stoichiometry employed and the
general
completeness of the reaction. This T-BSA material was incubated with a
reactive 75 kDa
maleimide heparosan. The reactive heparosan was made by (i) converting a
heparosan
tetrasaccharide ([GlcUA-GIcNAc]-GlcUA-anhydromannitol) into an amino
derivative using
reductive amination with sodium cyanoborohydride in the presence of ammonia,
(ii)
extending this sugar into a longer polymer with PmHS1 enzyme and UDP-sugars,
and (ii)
reacting the long amino-polymer with a N-hydroxysuccinimide ester of a
maleimide-
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containing compound. As seen by the SDS-PAGE gel visualized by Coomassie
staining, a
series of higher molecular weight products are observed in the 'Hep' lane
corresponding to
a series of HEPylated BSA molecules (untreated T-BSA control is in lane 0).
The gel filtration
chromatography profile (with absorbance at 280 nm detection) confirms that
higher
molecular weight polymers, HEPylated cargo, were formed. The production of
several
species is due to the nature of the chemically modified T-BSA (-1 to 3 T
reagents/BSA
molecule are formed in a rather uncontrolled chemical reaction); if a natural
protein or a
genetically engineered molecule (e.g., with an extra free cysteine residue)
contained only a
single sulfhydryl group, then a single mono-HEPylated species would result. In
addition, in
other embodiments, any sulfhydryl moiety could be used on the cargo (protein
or other
small molecule or secondary vehicles) as well as any alternative sulfhydryl-
reactive reagent
on the heparosan polymer including pyridylthiols or haloacids.
[00109] Immunoreactivity of many drugs is a serious issue, thus
necessitating
conjugation or humanization of the drug or the use of very low dosages and/or
short
treatments. If heparosan is attached to a therapeutic cargo, then it is
expected that the
cargo surface will be less accessible to antibody binding. The HEPylated BSA
material (BSA
with one to three 75 kDa heparosan chains/polypeptide) produced in experiments
depicted
in Fig. 8 was subjected to tests with an anti-BSA polyclonal antibody. To test
this hypothesis, the Fig. 8 material (either di-, tri-, or mono-HEPylated BSA -
all purified by
gel filtration) were compared to BSA and T-BSA (controls) in a radiometric
immune assay
(RIA) in a competition format (Table 5). First, a capture antibody coating was
placed on the
surface of a well of a 96-well plate. After blocking with ovalbumin, a
solution of one of the 4
test molecules above was added to the wells together with radioactive BSA
([1251] Bolton-
Hunter labeled). After extensive washing, the wells were counted to measure
radioactivity.
Each competitor protein was measured at two concentrations, 25 or 500
nanograms (ng).
The control wells did not have any BSA competitor, thus representing a
'maximal binding'
signal. BSA alone competed for binding with the radioactive probe to
immobilized antibody;
the signal was substantially reduced by 25 ng of BSA competitor and greatly
reduced with
500 nanograms of BSA. T-BSA without heparosan competed in a fashion similar to
normal
BSA. However, more HEPylated BSA was needed to partially inhibit the
radioactive signal,
indicating that the antibody did not recognize or bind to the HEPylated
molecules as well as
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32
BSA or T-BSA. Therefore, HEPylation will help shield cargo from the full brunt
of
immunological defenses in the mammalian body.
Table 5: Radiometric Immune Assay (RIA) comparing HEPylated BSA to BSA and T-
BSA
Sample ,125.1
cpm Bound
No competitor 1190
1270
BSA, 25 ng 750
760
BSA, 500 ng 140
100
T-BSA, 25 ng 770
830
T-BSA, 500 ng 140
190
Di,tri-HEP BSA, 25 ng 1050
Di,tri-HEP BSA, 500 ng 620
mono-HEP BSA, 25 ng 1220
mono-HEP BSA, 500 ng 630
[00110] Fig. 9 illustrates the production of HEPylated IgG molecules. A
preparation of
radioactive immunoglobulins [lgG] (1251-Bolton-Hunter labeled; migration
marked with
bracket) was oxidized with sodium periodate to create new aldehydes on the IgG
sugar
moieties on the Fc region. This oxidized glycoprotein was reacted via
reductive amination
with reactive hydrazide heparosan polysaccharide using sodium
cyanoborohydride. For
preparation of reactive heparosan, a short heparosan tetrasaccharide acceptor
(derived
from nitrous acid as described earlier) was first treated with adipic
dihydrazide (a compound
with 2 terminal hydrazide functional groups; one end couples with sugar and
the other end
remains free for reaction with cargo) and then extended into a longer ¨20 kDa
polymer with
PmHS1 enzyme and UDP-sugars. As seen by the SDS-PAGE gel visualized by
autoradiography, higher molecular weight products were observed in the 'Hep'
lane,
corresponding to HEPylated IgG molecules (see arrow area) (unmodified IgG
starting
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33
material is lane '0'; note that IgG preparations from serum contain multiple
species due to
the variety of heavy and light chains and there is also a small amount of
higher molecular
weight contaminant). This data demonstrates yet another chemistry for coupling
heparosan
vehicle to a therapeutic cargo such as a glycoprotein like an antibody.
[00111] Fig. 10 illustrates the production of HEPylated IgG molecules. To
produce
these molecules, a preparation of reactive hydrazide 75 kDa heparosan (similar
reagent as in
Fig. 9) was reacted with fluorescein isothiocyanate (FITC). As a control,
heparosan without
the reactive hydrazide group was also treated with the same FITC reagent (lane
'0'). As seen
by the PAGE gel visualized by virtue of ultraviolet-induced fluorescence, a
higher molecular
weight fluorescent product was observed in the 'Hy' lane, corresponding to
HEPylated
fluorescein molecules (see arrow) (unreacted FITC starting material is
bracketed). This data
demonstrates yet another example of coupling heparosan vehicle to a small
molecule that is
a proxy for a therapeutic cargo. In this case, the hydrazide linkage is meta-
stable at
physiological pH thus the HEPylated cargo will break down over time,
facilitating time-
release delivery of free small molecule. In the case of certain toxic
therapeutics such as
cancer chemotherapy drugs, this is a useful dosing feature.
[00112] Alternatively, the cargo can first be coupled to the reactive
acceptor, and
then the heparosan chain added by polymer grafting with PmHS1 (e.g., elongate
the
acceptor while coupled to cargo) due to the mild reaction conditions as shown
in the
example of Fig. 11. In Fig. 12, radioactive bovine serum albumin (1251-Bolton-
Hunter labeled
BSA) protein was reacted via reductive amination with sodium cyanoborohydride
with two
different reactive oligosaccharide acceptors derived from heparosan (same
acceptors as in
Fig. 7). For H, a heparosan tetrasaccharide formed by HCI cleavage with
general structure
[GlcUA-GIcNAc12 was used while for N, a heparosan tetrasaccharide formed by
base
treatment followed by nitrous acid cleavage with general structure [GlcUA-
GIcNAc]-GlcUA-
anhydromannitol was used. Then the short oligosaccharide acceptor covalently
attached
onto the BSA was extended via polymer grafting into a longer heparosan polymer
with
PmHS1 enzyme and UDP-sugars. As seen by the SDS-PAGE gel visualized by
autoradiography
on the left, higher molecular weight products were observed in the N lane
corresponding to
HEPylated BSA molecules (see bracketed area; the unmodified BSA starting
material is lane
'0' and is marked with arrow).
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34
[00113]
Therefore two different strategies can be used to add the heparosan vehicle
onto the therapeutic cargo: (I) a long reactive polymer is directly added to
the cargo as
shown in the schematic model of Fig. 6 and the data in Fig. 7 or (11) a short
reactive acceptor
is directly added to the cargo and then this conjugated molecule is elongated
via polymer
grafting with PmHS1 and UDP-sugars as in Fig. 12 (data shown here on the left
and
schematic model on the right).
[00114] In
another example, heparosan produced by bacteria in vivo can be purified
and (a) coupled via its reducing end aldehyde or (b) activated to couple to
cargo (this latter
approach with fermentation-derived heparosan results in functional, but more
heterogeneous final products with higher polydispersity). In Fig. 13,
radioactive bovine
serum albumin (1251-Bolton-Hunter labeled BSA; migration marked with arrow;
lane O=no
treatment; note that some contaminating minor bands are also present in all
lanes) protein
was reacted via reductive amination with sodium cyanoborohydride with
periodate-oxidized
reactive (contains new aldehyde groups) ¨200 kDa heparosan polysaccharide
formed by
fermentation of Pasteurella multocida Type F bacteria in vivo. Several
reaction pHs from pH
5-9 were tested. As seen by the SDS-PAGE gel visualized by autoradiography,
higher
molecular weight product was observed in the reaction lanes (pH 5, 7.2, or 9)
corresponding
to HEPylated BSA molecules (see bracketed area).
Therefore, in addition to
chemoenzymatic synthetically derived heparosan vehicles, naturally occurring
heparosan
from microbes may also be used as the source of vehicle. Such polymers
include, but are
not limited to, recombinant microbial hosts (e.g., Escherichia, Bacillus) with
heparosan
synthases such as PmHS1 or other native microbes that produce heparosan such
as
Escherichia coli K5.
[00115] In
order to monitor the half-life (t112) and persistence of heparosan in a
mouse or rat model, radioactive probes have been employed; however, ELISA or
NMR may
also be used to monitor the cargo. Typical elongation reactions contain: 50 mM
Tris, pH 7.2,
1 mM MnCl2, 1 to 50 mM UDP-sugars, 0.1 mg/ml PmHS1 enzyme and a primer; the
stoichiometric ratio of primer to UDP-sugars controls the heparosan chain
size.
[00116]
1251-Heparosan: A heparosan oligosaccharide primer with a 1251-Bolton-Hunter
reagent (a proxy for the therapeutic cargo) was elongated to any desired
length with the
PmHS1 enzyme. A series of polymers having distinct sizes, but equal
radiochemical specific
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activity (approximately 70 Ci/mmol) were generated; an example of one such
radioactive
conjugate is depicted in Fig. 14. The radioactive tetrasaccharide primer was
made from
heparosan polysaccharide by partial de-acetylation in base, nitrous acid
cleavage, reductive
amination with ammonia, then coupling to NHS ester of 1251-Bolton-Hunter
reagent (Perkin
Elmer NEN). The strong gamma-rays were readily detectable in samples of blood
or tissues
without additional processing. A series of 125I-heparosan probes were created
to monitor
the activity and functionality of the heparosan conjugate. Here, the rat was
used as the
model to track the fate of the heparosan conjugate after injection, but other
mammals such
as man are expected to behave similarly.
[00117] Three commonly employed modes of a therapeutic injection were
tested:
(a) intravenous (i.v.); (b) intraperitoneal (i.p.) and (c) intramuscular
(i.m.). On the order of 5-
50 uCi (approximately 10-100 x 106 dpm) radioactive heparosan probe in a small
volume of
saline per mouse or rat was injected at time zero into the tail vein or an
implanted jugular
port (i.v.) or abdominal cavity (i.p.) or the rear flank (i.m.). A group of
animals was injected
in parallel. The mice or rats were kept in special cages to facilitate urine
and feces
collection. At various times (typically 5, 30, 60, 120, minutes and 1 to 5
days), blood was
drawn from the animals. Duplicate animals were used for each time point. Some
animals
were sacrificed at early time points for harvesting organs that are known for
potentially
interacting with injected therapeutics (e.g., the kidney and the liver). The
fate of injected
heparosan conjugate in the mammalian body is shown in the example of Fig. 3.
[00118] For 125I-probes, the samples (equal volumes or weights per sample)
were
placed directly in test tubes and measured with a solid-state gamma
scintillation counter for
1 minute. For detecting low amounts of radioactivity, the counting interval
was extended to
5 or 10 minutes per sample (with a comparable blank value subtracted). The
amount of
radioactivity in the various samples overtime was used to calculate half-life
in various
compartments. Longer chains of heparosan and heparosan conjugates persist in
blood after
i.v. injection for long periods of time. Heparosan and heparosan conjugates
percolate
slowly out of the abdomen after i.p. injection, and thereafter appear in the
blood.
Heparosan and heparosan conjugates percolate slowly out of the muscle after
i.m. injection
and thereafter appear in the blood.
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36
Materials and Methods
[00119] Animals: Experiments were performed on male Sprague-Dawley rats
(270-345
g at time of experiment) purchased from Charles River (Wilmington, MA) with a
polyurethane catheter implanted into the right jugular vein. Rats were housed
under
controlled conditions (25 C, 12 h light/dark cycle) with free access to food
and water. Upon
arrival, each rat was placed into a cage and acclimated to the animal facility
for at least 7
days. The Institutional Animal Care and Use Committee of Oklahoma University
Health
Sciences Center approved the animal use for this protocol (#08-082R).
[00120] Test Compounds: The two test compounds (Fig. 2A - 100 kDa or Fig.
28 - 60
kDa polymer) were constructed in a radiolabeled form with 1-125 (70 Ci/mmol)
as decribed
previously. The activity of the radiolabel was set at 0.94 1.1Ci per 0.2 ml.
The use of 1-125 for
this study was reviewed and approved by the OUHSC Radiation Safety Office.
[00121] Dosing: Rats were anesthetized by isoflurane inhalation (5-2% to
effect)
before being administered 0.2 ml of the radiolabeled test compound by i.v.
infusion into the
right jugular vein. Following compound infusion, the i.v. catheter was flushed
with 0.2 ml of
sterile saline and then the catheter was locked with 0.2 ml of sterile
heparinized saline (1%
v/v, 10 Wm!). Rats were then placed into holding cages until being
reanesthetized just
before a terminal blood draw and organ collection.
[00122] The following groups of rats were dosed:
Fig. 2A ¨ 100 kDa
= Group 1: 0.5 hr post-dosing n=2 rats
= Group 2: 4 hr post-dosing n=2 rats
= Group 3: 8 hr post-dosing n=2 rats
= Group 4: 16 hr post-dosing n=2 rats
= Group 5: 24 hr post-dosing n=2 rats
= Group 6: 48 hr post-dosing n=2 rats
= Group 7: 72 hr post-dosing n=2 rats
Fig. 28 ¨ 60 kDa
= Group 1: 0.5 hr post-dosing n=2 rats
= Group 2: 4 hr post-dosing n=2 rats
= Group 3: 8 hr post-dosing n=2 rats
= Group 4: 16 hr post-dosing n=2 rats
= Group 5: 24 hr post-dosing n=2 rats
= Group 6: 30 hr post-dosing n=2 rats
= Group 7: 48 hr post-dosing n=2 rats
Perfusion Groups
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37
= Group 1: Probe 1, 24 hr post-dosing n=3 rats
= Group 2: Probe 2, 16 hr post-dosing n=3 rats
[00123] Sampling and Counting: At the post-dosing time-points listed above
rats were
euthanized via a terminal blood draw while under isoflurane anesthesia. Blood
was drawn
using a 10 cc syringe connected to the i.v. catheter. Median blood volume
withdrawn was
ml (3 ml, min ¨ 11.5 ml, max). For the perfusion groups, 1.5-3.8 ml of blood
was
withdrawn before perfusion. Blood was then transferred into 15 ml Falcon tubes
and
centrifuged (3000 rpm for 15 min. at 49 C, Beckman tabletop centrifuge) to
separate plasma
from blood cells. Once separated, 0.1 ml each of plasma and blood cells were
transferred
into plastic culture tubes for subsequent determination of radioactivity. In
addition, liver (3
tubes), spleen (1 tube), kidneys (1 tube/kidney), bladder (empty ¨ 1 tube),
heart (1 tube),
lungs (1 tube/lung), brain (perfusion groups only ¨ 2 tubes) and any urine (1
tube) or fresh
fecal pellets (1 tube) were collected and prepared for radioactive counting.
To determine
blood plasma half-life and relative distribution of the test compound, the
samples were
placed into a gamma counter were the total radioactivity was converted into
counts per
minute (CPM). Similar studies were done for i.m. and i.p. injection.
[00124] Transcardial Perfusion: At the post-dosing time-points listed
above, following
blood withdrawal under isoflurane anesthesia, rats were euthanized via cardiac
perfusion
with 200 ml of ice-cold sterile saline. For the perfusion, anesthetized rats
were placed on a
wire mesh cage top over a sink. Their fore limbs were then taped to the cage
top so that the
chest cavity was exposed. Using forceps to stabilize the skin, an initial cut
with scissors was
used to expose the musculature of the chest and upper abdomen. The caudal tip
of the
sternum was then grasped, and the diaphragm was rapidly punctured with the
scissors,
followed by cutting through the sternum to expose the heart. Additional cuts
to the ribs
were made with hemostats applied to act as retractors to provide an
unobstructed view of
the heart. The perfusion system consisted of a peristaltic pump (Masterflex,
Cole-Parmer,
Vernon Hills, IL) used at a setting of '7' with tubing connected to a 16 gauge
needle that was
inserted into the left ventricle of the heart which was gently held in place
with forceps to
deliver ice-cold sterile saline. The right atrium was then cut to allow the
blood and saline to
exit the circulatory system. The quality of the perfusion was dependent on the
amount of
time the heart remained beating following insertion of the perfusion needle
and could be
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38
monitored by the loss of color from the tail, hind limbs and liver. At the end
of the
perfusion, the rat had its organs removed and counted as previously listed.
[00125] Data Analysis: pK values: The blood plasma half-life (T112 or K10)
of the test
compound was determined with WinNonLin software (version 5.2.1) using a Gauss-
Newton
modeling algorithm (#7), as shown below. Additional derived pK values
including the area
under the curve, Cmax, and body clearance were calculated by the same software
package.
[00126] Calculation of total activity: Total activity was determined at
each time point
based on the averaged activity for each listed sample based on the following
calculations:
= Estimated total blood volume = [rat body weight (g)/100] x 6 ml
= Plasma ratio = ml of plasma/ml of total blood withdrawn
= Platelet ratio = ml of blood cells/ml of total blood withdrawn
= Estimated total plasma volume = plasma ratio x est. total blood volume
= Estimated total blood cell volume = platelet ratio x est. total blood
volume
= Total plasma CPM = plasma CPM/100 il x 1000 I/1 ml x est. total plasma
volume
= Total blood cell CPM = blood cell CPM/100 l x 1000 I/1 ml x est. total
blood
cell volume
= Total organ CPM = liver CPM + spleen CPM + kidney CPM + bladder CPM +
heart CPM + lung CPM
= Total Activity = total plasma + total blood cells+ total organ
= 0.5 hr Excreted CPM = 0.5 hr urine CPM + 0.5 hr fecal CPM
= Maximum Activity = 0.5 hr Total Activity + 0.5 hr Excreted CPM
= % Recovery = Total Activity/Maximum Activity
= Excreted CPM (all other time points) = Maximum Activity¨Total Activity
[00127] Calculation of % Relative Activity: For each time-point, the
average activity
for the sample (plasma, blood cells or individual organ) was divided by the
Total Activity for
that time-point. Thus, the total of all the Relative Activity = 100% for each
time-point, but
the Total Activity always decreased as the probe was excreted.
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39
[00128] Calculation of Perfusion Correction Factor: Data following
perfusion from
individual animals was compared to the values obtained from non-perfused
animals at the
same time-point. The average activity for each organ was expressed as a
percentage of the
non-perfused activity of the same organ ¨ this percentage was then used as the
Perfusion
Correction Factor and was applied to all time points for the same organ to
determine a
Corrected % Relative Activity (% relative activity x perfusion correction
factor = perfusion
corrected % relative activity). For both probes, following application of the
correction factor,
the activity that was removed from the organs was added to the % relative
activity for the
plasma and blood cells to keep the total at 100% per time-point.
Pharnnacokinetics (pK) Results
[00129] pK analysis Probe 1: Fig. 2A shows the curve-fit for the
elimination of the
radioactivity from the plasma for Probe 1. From the software analysis of the
data, the
T112 (half-life) or the elimination constant was 49.8 hours. The area under
the curve (AUC)
was 93.3 (hr)(pmol/m1) with a Cmax of 1.30 pmol/ml and a clearance of 0.014
ml/hr.
[00130] pK analysis Probe 2: Fig. 28 shows the curve-fit for the
elimination of the
radioactivity from the plasma for Probe 2. From the software analysis of the
data, the Ti/2
for the elimination constant was 15.4 hours. The area under the curve was 33.0
(hr)(pmol/m1) with a Cmax of 1.48 pmol/ml and a clearance of 0.040 ml/hr. In
addition to
long plasma half-life, the heparosan molecular weight is stable in the
mammalian
bloodstream as shown in Fig. 4.
[00131] Fig. 15 depicts an analysis of the blood plasma half-life and the
blood
absorption half-life of a radioactive heparosan compound following injection
thereof into
rats. Male Sprague-Dawley rats received two 0.1 ml injections of the
radioactive heparosan
compound (i.m.) in both hind limb calves. This test compound (100 kDa) was
radiolabeled
with 1251 (70 Ci/mmol). The activity of the radiolabel was set at 4.0 Ci per
0.2 ml, however
testing demonstrated that 10% of the probe was retained within the syringes,
thus the
effective dose was 3.6 O. At 1, 6, or 24 hr post-injection, 0.5 ml of whole
blood was
collected from the tail vein, while at 48, 72, 96 or 120 hr post-injection the
rats were
euthanized with blood and organs collected to determine distribution of the
probe. The
blood plasma half-life (T12 or K10) and the blood absorption half-life (K01)
of the test
compound was determined with WinNonlin software (version 5.2.1) using a Gauss-
Newton
modeling algorithm.
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[00132] Fig. 15 shows the total distribution of the measured radioactivity
for i.m.
dosing. As shown, the majority of the activity remains in the plasma with
relatively constant
activity in the red cells and organs. The amount of radioactivity in the
different organs
dissected from the rats was also measured. The activity in the liver was less
than 3.0% of
total activity at all time points. Total activity in the spleen was always
less than 0.3%. This
data indicates that i.m. dosing does not accumulate in these organs. Activity
in the kidneys
from remained about 2% of the total activity. Activity in the bladder was
minimal (<O.2%) at
all time points. The probe did not accumulate in either the heart or the lungs
as activity in
both organs was 1% or less throughout the study. Based on the perfusion tests
where
residual blood was washed out of organs, this small amount of radioactivity
was due to
trapped blood (for i.m. and i.p. studies, no perfusion was performed).
Finally, while the
injection site muscle already had a low % total activity (< 2%) at the first
measured time
point, the activity further decrease to less than 1% by the end of the
experiment. At all time-
points when samples were available, both the urine and the fecal pellets were
radioactive,
demonstrating that with i.m. dosing excretion began by 6 hr post-dosing and
continued
throughout the testing period. This radioactivity is due to the residual
Bolton-Hunter group
attached to a small sugar chain (less than 4 units). Finally, at 96 hr, the
brain accounted for
0.2% of the total activity and the testis had 0.3% of the total activity for
that rat.
[00133] Fig. 16 shows the curve-fit for the elimination of the
radioactivity from the
plasma for i.m. dosing. From the software analysis of the data, the T112 for
the absorption
constant was 6.8 hours and the Ti/2 for the elimination constant was 64.8
hours. The area
under the curve was 224.3 (hr)(pmol/rnI) with a Cmax of 1.70 pmol/ml (33.6% of
total dose)
at 21.9 hr and a clearance of 0.022 ml/hr.
[00134] In addition to intravenous and intramuscular injection,
radioactive heparosan
was administered to animals via an abdominal injection. Male Sprague-Dawley
rats
received a single 0.2 ml injection of the 100 kDa heparosan (i.p.) into the
peritoneum. At 1,
6, or 24 hr post-injection, 0.5 ml of whole blood was collected from the tail
vein, while at 48,
72, 96 or 120 hr post-injection the rats were euthanized with blood and organs
collected to
determine distribution of the probe in a similar to the i.m. study.
[00135] Similar to the i.m. dosing study, the Ti/2 was at least 2 days
when heparosan
was injected i.p. The activity in the liver was less than 3% all time-points.
Similar to the i.m.
dosing, total activity in the spleen was always less than 0.3%. The activity
in the kidneys was
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41
2% or less at all time-points. Activity in the bladder was minimal (<O.1%).
The probe did not
accumulate in either the heart or the lungs. This data indicates that i.p.
dosing does not
accumulate in these organs. At all time-points with measurements, both the
urine and the
fecal pellets were radioactive, demonstrating that i.p. dosing excretion began
within the
first hour post-dosing and continued throughout the testing period.
[00136] If the heparosan is not degraded in the extracellular compartments
of the
mammalian body and thus possesses stability, then the chain length or
molecular weight
should remain unchanged. The molecular weight of the heparosan samples from
the of i.m.
Study (M lanes) that generated the plot of Fig. 15 as well as the samples from
i.p. (P lanes)
study was examined by gel electrophoresis, as shown in Fig. 17. Basically,
equal amounts of
radioactivity in the plasma from various time points were concentrated into a
small volume
by ultrafiltration (Micron 10 kDa cut-off). The samples were loaded on an 1X
TAE agarose
gel (as in Fig. 4), electrophoresed, dried and exposed to X-ray film. As a
control, the original
starting radioactive heparosan (100 kDa) was also loaded (lane S). The data
show that the
heparosan migrates from the injection site through the various tissues and
compartments
and gets into blood stream in an intact form with the same molecular weight as
the starting
polymer. Even after 5 days (120 hours), the heparosan vehicle remains intact
thus
indicating its suitability as a useful vehicle for enhancing therapeutics.
Analyses of Metabolites after Leaving Bloodstream
[00137] Urine: Urine from various time points was extracted with
chloroform to
remove proteins and concentrated by ultrafiltration as above; the vast
majority of the
radioactivity passed through the 3 kDa membranes thus the heparosan fragments
that
passed through the kidneys was smaller than 3 kDa. This material was then
subjected to thin
layer chromatography (TLC) on silica plates developed with butanol:acetic
acid: water
solvent. The plate was then exposed to X-ray film; only small metabolized
fragments (4
sugar units and less or n=2 or less) of the original heparosan were observed
as shown in Fig.
18.
[00138] Feces: After injection into rats, heparosan metabolites were
excreted in feces
over time as shown in Fig. 19. Water extracts of feces from various time
points after
injection were subjected to ultrafiltration with various molecular weight cut-
off membranes
(3, 10, or 50 kDa; Amicon Microcon) and gamma counting of the retained and
eluted
fractions. The size of the radioactive metabolites were inferred by the
ability to penetrate
CA 02773755 2015-11-12
42
the pores of the membrane; the vast majority of the radioactivity passed
through the 3 kDa
membranes; thus, the heparosan fragments that passed through the intestines
was smaller
than 3 kDa.
Reductive Amination Drug-conjugate Synthesis
[00139] The aldehyde of heparosan polymers (e.g., either the natural
reducing end or
via periodate oxidation) is coupled directly to therapeutic proteins via its N-
terminal amino
groups using reductive amination with Na cyanoborohydride. For adding a single
heparosan
polymer per polypeptide chain, the reaction is done in 0.1 M Na acetate, pH 5,
at 4 to 37 C;
the buffer pH used de-selects the lysine groups with higher pKa (need an
unprotonated
amine for nucleophilic attack) in favor of the amino terminus. Lower
temperatures (e.g. 4
to 10 C) is used to preserve protein folding. Alternatively, reactions at pH 7-
9 (e.g., in
phosphate buffer) are used to add multple heparosan chains to the lysines as
well as the N-
terminus of the protein cargo. This methodology is therapeutically and
commercially
successful for proteins like interferons and GCSF (Neulasta). A wide variety
of chemistries
are useful for successful conjugate synthesis; the basic requirements are (a)
there is an
appropriate reactive or activated group on the heparosan polymer vehicle
(either short
acceptor or longer polymers) that will react or interact with a group on the
cargo (i.e.,
therapeutic agent or drug), or if desired, a secondary vehicle (e.g., liposome
or
nanoparticle), and (b) suitable mild reaction conditions that preserve the
integrity and
functionality of both the vehicle and the cargo.
[00140] Thus, in accordance with the presently disclosed and claimed
invention(s), there has been provided a methodology for HEPylation wherein a
heparosan
molecule servers as the vehicle for carrying a cargo in a heparosan-conjugate.
Although the
presently claimed and disclosed invention(s) has been described in conjunction
with the
specific drawings and language set forth above, the scope of the claims should
not be limited
by the preferred embodiments set forth in the examples, but should be given
the broadest
interpretation consistent with the description as a whole.
