Note: Descriptions are shown in the official language in which they were submitted.
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
In Vivo Polynucleotide Delivery Conjugates Having Enzyme Sensitive Linkages
=
BACKGROUND OF THE INVENTION
The delivery of polynucleotide and other substantially cell membrane
impermeable
compounds into a living cell is highly restricted by the complex membrane
system of the cell.
Drugs used in antisense, RNAi, and gene therapies are relatively large
hydrophilic polymers
and are frequently highly negatively charged. Both of these physical
characteristics severely
restrict their direct diffusion across the cell membrane. For this reason, the
major barrier to
polynucleotide delivery is the delivery of the polynucleotide across a cell
membrane to the
cell cytoplasm or nucleus.
One means that has been used to deliver small nucleic acid in vivo has been to
attach the
nucleic acid to either a small targeting molecule or a lipid or sterol. While
some delivery and
activity has been observed with these conjugates, the nucleic acid dose
required with these
methods has been prohibitively large.
Numerous transfection reagents have also been developed that achieve
reasonably efficient
delivery of polynucleotides to cells in vitro. However, in vivo delivery of
polynucleotides
using these same transfection reagents is complicated and rendered ineffective
by in vivo
toxicity, adverse serum interactions, and poor targeting. Transfection
reagents that work well
in vitro, cationic polymers and lipids, typically form large cationic
electrostatic particles and
destabilize cell membranes. The positive charge of in vitro transfection
reagents facilitates
association with nucleic acid via charge-charge (electrostatic) interactions
thus forming the
nucleic acid/transfection reagent complex. Positive charge is also beneficial
for nonspecific
binding of the vehicle to the cell and for membrahe fusion, destabilization,
or disruption.
Destabilization of membranes facilitates delivery of the substantially cell
membrane
impermeable polynucleotide across a cell membrane. While these properties
facilitate nucleic
acid transfer in vitro, they cause toxicity and ineffective targeting in vivo.
Cationic charge
results in interaction with serum components, which causes destabilization of
the
.. polynucleotide-transfection reagent interaction, poor bioavailability, and
poor targeting.
Membrane activity of transfection reagents, which can be effective in vitro,
often leads to
toxicity in vivo.
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
For in vivo delivery, the vehicle (nucleic acid and associated delivery agent)
should be small,
less than 100 nm in diameter, and preferably less than 50 nm. Even smaller
complexes, less
that 20 nm or less than 10 nm would be more useful yet. Delivery vehicles
larger than 100
nm have very little access to cells other than blood vessel cells in vivo.
Complexes formed by
electrostatic interactions tend to aggregate or fall apart when exposed to
physiological salt "
concentrations or serum components. Further, cationic charge on in vivo
delivery vehicles
leads to adverse serum interactions and therefore poor bioavailability.
Interestingly, high
negative charge can also inhibit targeted in vivo delivery by interfering with
interactions
necessary for targeting, i.e. binding of targeting ligands to cellular
receptors. Thus, near
neutral vehicles are desired for in vivo distribution and targeting. Without
careful regulation,
membrane disruption or destabilization activities are toxic when used in vivo.
Balancing
vehicle toxicity with nucleic acid delivery is more easily attained in vitro
than in vivo.
Rozema et al., in U.S. Patent Publication 20080152661, provided a means to
reversibly
regulate membrane disruptive activity of a membrane active polyamine using
disubstituted
maleic anhydride modification. Maleamate linkages, formed by reaction of a
maleic
. anhydride with an amine are pH labile in a pH range suitable for in vivo
delivery. This
process allowed membrane active polymers to be used for in vivo delivery or
nucleic acid.
We now provide modified membrane active polymers having dipeptide-amidobenzyl-
carbamate linkages. The dipeptide-amidobenzyl-carbamate linkages are
reversible and
physiologically responsive. Unlike pH-labile maleamate linkages from by
modification with
disubstituted maleic anhydride, the polymer modification agents linkage
described herein
generate enzymatically cleavable linkages that are more stable in in vivo
circulation.
SUMMARY OF THE INVENTION
In a preferred embodiment we describe masking agents for reversibly modifying
and
inhibiting membrane activity of an amphipathic membrane active polyamine
comprising: a
steric stabilizer or targeting ligand attached to a dipeptide-amidobenzyl-
carbonate, referred to
herein as dipeptide masking agents or protease cleavable masking agents. The
dipeptide
masking agents have the general form:
R¨A' A2-amidobenzyl-carbonate.
wherein R is a steric stabilizer or targeting ligand, Al is an amino acid, and
A2 is an amino
acid. Reaction of the masking agent carbonate with a polymer amine yields a
carbamate
2
CA 02816041 2013-04-24
WO 2012/092373
PCMTS2011/067588
linkage. The masking agent is stable until the dipeptide is cleaved in vivo by
an endogenous
protease, thus cleaving the steric stabilizer or targeting ligand from the
polyamine. Following
enzymatic cleavage after the dipeptide (between A2 and the amidobenzyl), the
amidobenzyl-
carbamatc undergoes a spontaneous rearrangement which results in regeneration
of the
.. polymer amine. Preferably R is neutral. More preferably, R is uncharged. A
preferred steric
stabilizer is a polyethylene glycol (PEG). A targeting ligand may be selected
from the list
comprising haptens such as digoxigenin, vitamin such as biotin, antibody,
monoclonal
antibody, and cell surface receptor ligand. A targeting ligand may be linked
to the dipeptide
via a linker such as a PEG linker. A preferred cell surface receptor ligand is
an
asialoglycoprotein receptor (ASGPr) ligand. A preferred ASGPR ligand is an N-
Acetylgalactosamine (NAG). A preferred dipeptide consists of a hydrophobic
amino acid
linked to a hydrophilic uncharged amino acid via an amide bond. A preferred
amidobenzyl
group is a p-amidobenzyl group. A preferred carbonate is an activated amine
reactive
carbonate.
In a preferred embodiment, the invention features a composition for delivering
an RNA
interference (RNAi) polynucleotide to a cell in vivo comprising: a masked
amphipathic
membrane active polyamine (delivery polymer) wherein the polyamine is masked
by
reversible modification with the dipeptide masking agents described herein and
an RNAi
.. polynucleotide. The delivery polymer can be covalently linked to the RNAi
polynucleotide.
A preferred linkage for covalent attachment of the delivery polymer to the
RNAi
polynucleotide is a physiologically labile linkage. In one embodiment, this
linkage is
orthogonal to the dipeptide masking agent linkage. Alternatively, the delivery
polymer is not
covalently linked to the RNAi polynucleotide and the RNAi polynucleotide is
covalently
.. linked to a targeting molecule.
In a preferred embodiment, we describe a composition comprising: an
aMphipathie
membrane active polyamine covalently linked to: a) a plurality of targeting
ligands or steric
stabilizers via dipeptide-amidobenzyl-carbamate linkages; and, b) one or more
polynucleotides via one or more reversible linkages. In one embodiment,
dipeptide-
amidobenzyl-carbamate linkage is orthogonal to the polynucleotide reversible
covalent
linkage. The polynucleotide-polymer conjugate is administered to a mammal in a
pharmaceutically acceptable carrier or diluent.
3
CA 02816041 2013-04-24
WO 2012/092373
PCMTS2011/067588
In a preferred embodiment, we describe a composition comprising: a) an
amphipathic
membrane active polyamine covalently linked to a plurality of targeting
ligands or steric
stabilizers via dipeptide-amidobenzyl-carbamate linkages; and, b) an RNAi
polynucleotide
covalently linked to a targeting group selected from the list consisting of: a
hydrophobic
group having 20 or more carbons atoms and a galactose cluster. In this
embodiment, the
RNAi polynucleotide is not covalently linked to the modified amphipathic
membrane active
polyamine. The modified polyamine and the RNAi polynucleotide targeting group
conjugate
are synthesized separately and may be supplied in separate containers or a
single container.
The modified polyamine and RNAi polynucleotide-targeting group conjugate are
administered together or separately to a mammal in pharmaceutically acceptable
carriers or
diluents.
A preferred dipeptide masking agent comprises a protease (peptidase) cleavable
dipeptide-p-
amidobenzyl amine-reactive carbonate derivative. Protease cleavable masking
agents of the
invention employ a dipeptide connected to an amidobenzyl activated carbonate
moiety. A
targeting ligand or steric stabilizer is attached to the amino terminus of a
dipeptide. The
amidobenzyl activated carbonate moiety is attached at the carboxy terminus of
the dipeptide.
Protease cleavable linkers suitable for use with the invention have the
general structure:
R1 H 0
"3
11-1 R2
wherein R4 comprises a targeting ligand or steric stabilizer, R3 comprises an
amine reactive
carbonate moiety, and RI and R2 are amino acid side chains. A preferred
activated carbonate
is a para-nitrophenol. However, other amine reactive carbonates known in the
art are readily
substituted for the para-nitrophenol. Reaction of the activated carbonate with
an amine
connects the targeting ligand or steric stabilizer to the membrane active
polyamine via a
peptidase cleavable dipeptide-amidobenzyl carbamate linkage. Enzyme cleavage
of the
dipeptide, between the amino acid and the amidobenzyl group removes R4 from
the polymer
and triggers an elimination reaction which results in regeneration of the
polymer amine.
The dipeptide masking agents of the invention are useful for reversible
modification/inhibition of amphipathic membrane active polyamines. A covalent
bond is
4
created by the reaction of the activated carbonate of the dipeptide masking
agent with a
polymer amine, particularly a primary amine. group. Hence provided herein is a
conjugate
comprising the dipeptide-amidobenzyl-carbonate masking agent described herein
and an
amphipathic membrane active polyamine:
R1 0
0
polyamine
0 R2 0
In a further embodiment, described herein is a compound comprising a targeting
ligand
covalently linked to a dipeptide-amidobenzyl-carbonate having the structure
represented by
R1 0 R5
R4 (TI")
¨>R6
0 R2
wherein
X is -NH-,
Y is -NH-,
RI is -CH3,
R2 is -(CH2)3-NH-C(0)-NH2,
R4 is uncharged and comprises said targeting ligand,
R5 is at position 2, 4, or 6 and is -CH2-0-C(0)-Z wherein Z is
0
/ 0
'N
_
0 0 ,or F ;and
R6 is hydrogen.
In a further embodiment, described herein is a compound comprising:
R-A1A2-amidobenzyl-carbonate
wherein
R comprises a steric stabilizer,
A1 is a hydrophobic amino acid,
5
CA 2816041 2018-04-10
A2 is a hydrophilic uncharged amino acid linked to Al via an amide bond,
wherein
said hydrophilic uncharged amino acid is uncharged at neutral pH, and wherein
A2 is not glycine.
In a further embodiment, described herein is a compound having the structure
represented by:
R5
0
Ri
R6n
0 R2
wherein
R comprises a steric stabilizer,
RI is a side chain of a hydrophobic amino acid,
R2 is a side chain of a hydrophilic uncharged amino acid, wherein said
hydrophilic
uncharged amino acid is uncharged at neutral pH, and wherein said hydrophilic
uncharged amino acid is not glycine,
¨Y¨ is ¨NH¨ or ¨0¨,
R5 is at position 2, 4, or 6 and is ¨CH2-0¨C(0)¨Z wherein ¨Z is
0 0 0
0
0 0
\N
or ;and
R6 is independently hydrogen, an alkyl group, ¨(CH2)in¨CH3, ¨(CH2)¨(CH3)2, or
a
halide at each of positions 2, 3, 4, 5, or 6 except for the position occupied
by R5
and n and m are independently integers from 0 to 4.
In a further embodiment, described herein is a delivery polymer comprising:
Mly¨P¨M2z
wherein:
5a
CA 2816041 2018-04-10
P is an amphipathic membrane active polyamine,
MI comprises a targeting ligand linked to P via a dipeptide-amidobenzyl-
carbamate
linkage,
M2 comprises a steric stabilizer linked to P via a dipeptide-amidobenzyl-
carbamate
linkage,
y and z are each integers greater than or equal to zero,
y + z has a value greater than 50% of the primary amines on polyamine P as
determined by the quantity of amines on P in the absence of any masking
agents,
and
the dipeptide-amidobenzyl-carbamate linkage has the structure represented by:
R¨Al A2-am idobenzyl-c arbamate¨P
wherein
R comprises the targeting ligand of MI or the steric stabilizer of M2,
AI is a hydrophobic amino acid,
A2 is a hydrophilic uncharged amino acid linked to A' via an amide bond,
wherein said hydrophilic uncharged amino acid is uncharged at neutral pH,
and
P is the amphipathic membrane active polymer.
In a further embodiment, described herein is a use of a compound described
herein for reversibly
modifying an amphipathic membrane active polyamine.
In a further embodiment, described herein is a use of a compound described
herein for the
manufacture of a medicament for reversibly modifying an amphipathic membrane
active
polyamine.
In a further embodiment, described herein is a compound as described herein
for use in
reversibly modifying an amphipathic membrane active polyamine.
In a further embodiment, described herein is a use of a delivery polymer as
described herein for
delivering a polynucleotide to a cell in vivo.
In a further embodiment, described herein is a use of a delivery polymer as
described herein for
the manufacture of a medicament for delivering a polynucleotide to a cell in
vivo.
5b
CA 2816041 2018-04-10
In a further embodiment, described herein is a delivery polymer as described
herein, for use in
delivering a polynucleotide to a cell in vivo.
The compounds according to the present invention can be generally obtained
using methods
known to the person of ordinary skill in the art of organic or medicinal
chemistry. Further
objects, features, and advantages of the invention will be apparent from the
following
detailed description when taken in conjunction with the accompanying drawings.
BRIEF DESCRIPTION OF THE FIGURES.
FIG. 1. Illustration showing the structure of a dipeptide masking agent
wherein:
RI and R2 are the R groups of amino acids,
R4 is a targeting ligand of a steric stabilizer,
X is ¨NH¨, ¨0¨, or ¨CH2¨,
Y is ¨NH¨ or ¨0¨
R5 is at position 2, 4, or 6 and is ¨CH2-0¨C(0)-0¨Z wherein Z carbonate, and
R6 is independently hydrogen, alkyl, or halide at each of positions 2, 3, 4,
5, or 6
except for the position occupied by R5.
FIG. 2. Illustration showing the structure of a dipeptide masking agent linked
to a polyamine
wherein: R1 and R2 are the R groups of amino acids, R4 is a targeting ligand
of a
steric stabilizer, X is ¨NH¨, ¨0¨, or ¨CH2¨, and Y is ¨NH¨ or ¨0¨.
FIG. 3. Illustration shown the structures of various dipeptide masking agents.
FIG. 4. Graph illustrating circulation times of polymers modified with a
dipeptide masking
agent vs. two different maleic anhydride based masking agents.
DETAILED DESCRIPTION OF THE INVENTION
Described are masking agents useful for reversibly modification and inhibition
of
amphipathic membrane active polyamines and the delivery polymers formed by
modification
5c
CA 2816041 2018-04-10
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
of the polyamine by the dipeptide masking agents. The peptidase cleavable
linkages are
stable to hydrolysis in absence of enzyme, electrically neutral and provide
extended DPC
stability in storage and in in vivo circulation. Improved (longer) half-life
in circulation
facilitates widening of the window of opportunity for ligand-mediated
accumulation in tissue,
such as tumor tissue. The delivery polymers are particularly useful for in
vivo delivery of
RNAi polynucleotides. In vivo delivery of RNAi polynucleotides is useful for
therapeutic
inhibition (knockdown) of gene expression.
The dipeptide masking agents have the general form:
I 2
R-A A -amidobenzyl-carbonate.
wherein R is a steric stabilizer or targeting ligand, Ai is an amino acid, A2
is an amino acid,
and carbonate is an activated amine-reactive carbonate. R is preferably
uncharged. Reaction
of the masking agent carbonate with a polymer amine yields a carbamate
linkage. The
masking agent is stable until the dipeptide is cleaved in vivo by an
endogenous protease, thus
cleaving the steric stabilizer or targeting ligand from the polyamine.
Following enzymatic
cleavage after the dipeptide (between A2 and the amidobenzyl), the amidobenzyl-
carbamate
undergoes a spontaneous rearrangement which results in regeneration of the
polymer amine.
A preferred steric stabilizer is a polyethylene glycol (PEG). A preferred
targeting ligand for
liver delivery is an ASGPr ligand. A preferred ASGPr ligand is an N-
Acetylgalactosamine
(NAG). A preferred amidobenzyl group is a p-amidobenzyl group.
Dipeptides of the dipeptide masking agents, represented as A1A2 (or AA), are
dimers of
amino acids connected via amide bonds. Amino acids, including a and 13 amino
acids are well
known in biology and chemistry and are molecules containing an amine group, a
carboxylic
acid group and a side-chain that varies between different amino acids. A
preferred amino acid
is an a-amino acid having the generic formula H2NCHRCOOH, where R is an
organic
substituent. A preferred a amino acid is an uncharged naturally occurring
amino acid. In a
preferred dipeptide, Al is a hydrophobic amino acid and A2 is an uncharged
hydrophilic
amino acid. A preferred hydrophobic amino acid is phenylalanine, valine,
isoleucine, leucine,
__ alanine, or tryptophan. A preferred uncharged hydrophilic amino acid is
asparagine,
glutamine, or citrulline. A more preferred hydrophobic amino acid is
phenylalanine or valine.
A more preferred uncharged hydrophilic amino acid is citrulline. While
dipeptides are
preferred, it is possible to insert additional amino acids between Ai and R.
It is also possible
6
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
to use a single amino acid instead of a dipeptide by eliminating amino acid
Al. Any natural
amino acids used in the present invention are referred to herein by their
common
abbreviations. While charged amino acids can be used, it is preferred that the
masking agent
be uncharged.
= 5
In a preferred embodiment, an amphipathic membrane active polyamine is
reversibly
modified by reaction with a dipeptide-amidobenzyl-carbonate masking agent of
the invention
to yield a membrane inactive delivery polymer. The dipeptide masking agents
can shield the
polymer from non-specific interactions, increase circulation time, enhance
specific
interactions, inhibit toxicity, or alter the charge of the polymer.
Reversibly masked polymers of the invention comprise the structure:
R1 H 0
I
0
""-poiyamine
R2 0
wherein:
X is NH-, -0-, or -CH2--
Y is -NH- or -0-
R1 is preferably
-(CH2)k-phenyl (k is 1, 2, 3, 4, 5, 6; k = 1 phenylalanine),
-CH-(CH3)2 (vat me),
-CH2-CH-(CH3)2 (leucine),
-CH(CH3)-CH2-CH3 (isoleucine),
-CH3 (alanine),
-(CH2)2-COOH (glutamic acid),
or
N
(tryptophan);
R2 is preferably
7
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
hydrogen (glycine)
¨(CH2)3¨NH¨C(0)¨NH2 (citruIline),
¨(CH2)4¨N¨(CH3)2 (lysine(CH3)2),
¨(CH2)k ¨C(0)¨NH2; (k is I, 2, 3,4, 5, 6),
¨CH2¨C(0)¨NH2 (asparagine),
¨(CH2)2¨C(0)¨NH2 (glutamine),
¨CH2¨C(0)¨NR1R2 (aspartic acid amide),
¨(CH2)2¨C(0)¨NRIR2 (glutamic acid amide),
¨CH2¨C(0)-013.1 (aspartic acid ester), or
¨(0-12)2¨C(0)-0R1 (glutamic acid ester),
RI and R2 are alkyl groups
R4 comprises a polyethylene glycol or targeting ligand; and
the polyamine is an amphipathic membrane active polyamine.
While the structure above indicates a single dipeptide masking agent linked to
the polymer, in .
practice of the invention, 50% to 90% or more of polymer amines are modified
by dipeptide
masking agents.
In a preferred embodiment, reversibly masked polymer of the invention comprise
the
structure:
R1 0
0 N,
ypoIyamine
0 R2 H 0
wherein RI, R2, R4 and polyamine as described above.
Reversibly masked polymers of the invention are formed by reaction of
dipeptide masking
agents of the invention with amines on the polymer. Dipeptide masking agents
of the
invention have the structure:
0 R5
¨ 6
0 R2
8
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
wherein:
X, Y, RI, R2, and R4 are as described above
R5 is at position 2, 4, or 6 and is ¨CH2-0¨C(0)-0¨Z wherein Z is
¨Halide,
0
ilk NO2
, or
N 40
¨o
; and
R6 is independently hydrogen, alkyl, ¨(CH2)n¨CH3 (wherein n = 0-4),
¨(CH2)¨(Cl3)2, or
halide at each of positions 2, 3, 4, 5, or 6 except for the position occupied
by R5
In a preferred embodiment, X is ¨NH¨, Y is ¨NH¨, R4 is uncharged, R5 is at
position 4, and
R6 is hydrogen as shown by:
R1 0
3
HI
0 R2
In another embodiment, R4 is:
R¨(0¨CH2¨CH2)s¨O¨Y1¨, wherein
9
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
R is hydrogen, methyl, or ethyl; and s is= an integer from 1 to 150,
and Y1 is a linker selected from the list comprising:
¨0¨Y2¨NH¨C(0)¨(CH2)2¨C(0)¨, wherein Y2 is
¨(CH2)3-
¨C(0)¨N¨(CH2-0-12-0)p¨CF12¨CF12¨ (p is an integer from I to 20), and
A targeting ligand may be selected from the list comprising hapten, vitamin,
antibody,
monoclonal antibody, and cell surface receptor ligand. A targeting ligand may
be linked to
the dipeptide via a linker such as a PEG linker.
Non-limiting examples of membrane active polymers suitable for use with the
invention have
been previously described in US Patent Publications 20080152661, 20090023890,
20080287630, and 20110207799. Suitable amphipathic membrane active polyamine
can also
be small peptides such as a melittin peptide.
Polymer amines were reversibly modified using the enzyme cleavable linkers
described
herein. An amine is reversibly modified if cleavage of the modifying group
results in
regeneration of the amine. Reaction of the activated carbonate of the masking
agent with a
polymer amine connects a targeting ligand or steric stabilizer to the polymer
via a peptidase
cleavable dipeptide-amidobenzyl carbamate linkage as shown:
0
R'-AA-NH Z + H2N - R2 RI- AA-NH 10 CH2- 0)( NH- R2
RI comprises an targeting ligand (either with or without protecting groups) or
a PEG,
R2 is an amphipathic membrane active polyamine,
AA is a dipeptide (either with or without protecting groups), and
Z is an amine-reactive carbonate.
Protecting groups may be used during synthesis of the dipeptide masking
agents. If present,
protecting groups may be removed prior to or after modification of the
amphipathic
membrane active polyamine.
I0
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
Reversible modification of a sufficient percentage of the polymer amines with
the dipeptide
masking agents inhibits membrane activity of the membrane active polyamine.
Modification
of polymer amines with the dipeptide masking agents also preferably
neutralizes charge of
the amine. The dipeptide-amidobenzyl-carbamate linkage is susceptible to
protease (or
peptidase) cleavage. In presence of protease, the anilide bond is cleaved,
resulting in an
intermediate which immediately undergoes a 1,6 elimination reaction to release
free polymer:
0
R'¨ AA¨NH III CH2-0 NH¨R2 RI¨AA¨OH
+ H2N 1111.-4 CH2- 0)1' NH-R2
1,6-elimination
H2N¨R2
In the above reaction scheme, AA is a dipeptide, RI comprises a targeting
ligand or a steric
stabilizer, and R2 is an amphipathic membrane active polyamine. Importantly,
the free
polymer is unmodified and so membrane activity it restored.
In the masked state, the reversibly masked membrane active polyamine does not
exhibit
membrane disruptive activity. Reversible modification of more than 50%, more
than 55%,
more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, or
more
than 90% of the amines on the polyamine with dipeptide masking agents may be
required to
inhibit membrane activity and provide cell targeting function, i.e. form a
reversibly masked
membrane active polymer (delivery polymer).
The present invention also provides a method for delivery of a biologically
active substance,
into cells. More specifically, the present invention is directed to compounds,
compositions,
and methods useful for delivering RNAi polynucleotides mammalian cells in
vivo.
In one embodiment, the RNAi polynucleotide is linked to the delivery polymer
of the
invention via a physiologically labile covalent linkage. By using a
physiologically labile
linkage, the polynucleotide can be cleaved from the polymer, releasing the
polynucleotide to
engage in functional interactions with cell components.
11
CA 02816041 2013-04-24
WO 2012/092373
PCT/1JS2011/067588
The invention includes conjugate delivery systems of the general structure:
MI
N¨L ¨P
M2z,
wherein N is a RNAi polynucleotide, LI is a physiologically labile linkage, P
is an
amphipathic membrane active polyamine, MI is an targeting ligand linked to P
via a
dipeptide-amidobenzyl-carbamate linkage, and M2 is a steric stabilizer linked
to P via a
dipeptide-amidobenzyl-carbamate linkage. y and z are each integers greater
than or equal to
zero provided y + z has a value greater than 50%, greater than 60%, greater
than 70%, greater
than 80% or greater than 90% of the primary amines on polyamine P, as
determined by the
quantity of amines on P in the absence of any masking agents. In its
unmodified state, P is a
membrane active polyamine. Delivery polymer Mly¨P--M2 is not membrane active.
Reversible modification of P primary amines, by attachment of MI and/or M2,
reversibly
inhibits or inactivates membrane activity of P. It is noted that some small
amphipathic
membrane active polyamine, such as melittin peptide, contain as few as 3-5
primary amines.
Modification of a percentage of amines is meant to reflect the modification of
a percentage of
amines in a population of polymers. Upon cleavage of MI and M2, amines of the
polyamine
are regenerated thereby reverting P to its unmodified, membrane active state.
In another embodiment, the RNAi polynucleotide is co-administered in vivo with
a delivery
polymer of the invention. Thus, the invention includes compositions of the
general structure:
MI,¨P¨M2y plus N¨T,
wherein N is a RNAi polynucleotide, T is a targeting group, P is an
amphipathic membrane
active polyamine, MI is a targeting ligand linked to P via a dipeptide-
amidobenzyl-carbamate
linkage, and M2 is a steric stabilizer linked to P via a dipeptide-amidobenzyl-
carbamate
linkage. y and z are each integers greater than or equal to zero provided y +
z has a value
greater than 50%, greater than 60%, greater than 70%, greater than 80% or
greater than 90%
of the primary amines on polyamine P. as determined by the quantity of amines
on P in the
absence of any masking agents. In its unmodified state, P is a membrane active
polyamine.
Delivery polymer M')¨P¨M2z is not membrane active. Reversible modification of
P primary
amines, by attachment of MI and/or M2, reversibly inhibits or inactivates
membrane activity
of P. It is noted that some small amphipathic membrane active polyamine, such
as melittin
peptide, contain as few as 3-5 primary amines. Therefore, modification of a
percentage of
amines is meant to reflect the modification of a percentage of amines in a
population of
12
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
polymers. Upon cleavage of MI and M2, amines of the polyamine are regenerated
thereby
reverting P to its unmodified, membrane active state. N is linked to T via a
covalent bond to
form a RNAi polynucleotide-targeting group conjugate using methods standard in
the art. A
preferred covalent bond is a physiologically labile bond. N¨T. The delivery
polymer and
N¨T are synthesized or manufactured separately. Neither T nor N are covalently
linked
directly or indirectly to P, MI or M2. Electrostatic or hydrophobic
association of the
polynucleotide or the polynucleotide-conjugate with the masked or unmasked
polymer is not
required for in vivo liver delivery of the polynucleotide. The masked polymer
and the
polynucleotide conjugate can be supplied in the same container or in separate
containers.
They may be combined prior to administration, co-administered, or administered
sequentially.
For hepatocyte delivery, whether the RNAi polynucleotide is linked to the
delivery polymer
via a covalent bond or co-administered with the delivery polymer, y has a
value greater than
50% and up to 100% of the primary amines on polymer P. z therefore has a value
greater or
equal to zero percent (0%) but less than 50% of the primary amines on polymer
P.
For delivery to liver tumor cells, z may have a value greater up to 100% of
the primary
amines on polymer P. In a preferred embodiment, for delivery to tumor cells, z
has a value
greater than 50%, greater than 60%, greater than 70%, greater than 80% or
greater than 90%
of the primary amines on polyamine P and y is zero.
Membrane active polyamines are capable of disrupting plasma membranes or
lysosomal/endocytic membranes. This membrane activity is an essential feature
for cellular
delivery of the polynucleotide. Membrane activity, however, leads to toxicity
when the
polymer is administered in vivo. Polyamines also interact readily with many
anionic
components in vivo, leading to undesired bio-distribution. Therefore,
reversible masking of
membrane activity of the polyamine is necessary for in vivo use.
Masking is accomplished through reversible attachment of the described
dipeptide masking
agents to the membrane active polyamine to form a reversibly masked membrane
active
polymer, i.e. a delivery polymer. In addition to inhibiting membrane activity,
the masking
agents shield the polymer from non-specific interactions, reduce serum
interactions,
13
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
neutralize the polyamine to reduce positive charge and form a near neutral
charge polymer,
increase circulation time, and/or provide cell-specific interactions, i.e.
targeting.
It is an essential feature of the masking agents that, in aggregate, they
inhibit membrane
activity of the polymer. Masking agents may shield the polymer from non-
specific
interactions (reduce serum interactions, increase circulation time). The
membrane active
polyamine is membrane active in the unmodified (unmasked) state and not
membrane active
(inactivated) in the modified (masked) state. A sufficient number of masking
agents are
linked to the polymer to achieve the desired level of inactivation. The
desired level of
modification of a polymer by attachment of masking agent(s) is readily
determined using
appropriate polymer activity assays. For example, if the polymer possesses
membrane
activity in a given assay, a sufficient level of masking agent is linked to
the polymer to
achieve the desired level of inhibition of membrane activity in that assay.
Masking requires
modification of >50%, >60%, >70%, >80% or >90% of the primary amine groups on
a
population of polymer, as determined by the quantity of primary amines on the
polymer in
the absence of any masking agents. It is also a preferred characteristic of
masking agents that
their attachment to the polymer reduces positive charge of the polymer, thus
forming a more
neutral delivery polymer. It is desirable that the masked polymer retain
aqueous solubility.
The membrane active polyamine can be conjugated to masking agents in the
presence of an
excess of masking agents. The excess masking agent may be removed from the
conjugated
delivery polymer prior to administration of the delivery polymer.
As used herein, a "steric stabilizer" is a non-ionic hydrophilic polymer
(either natural,
synthetic, or non-natural) that prevents or inhibits intramolecular or
intermolecular
interactions of a polymer to which it is attached relative to the polymer
containing no steric
stabilizer. A steric stabilizer hinders a polymer to which it is attached from
engaging in
electrostatic interactions. Electrostatic interaction is the non-covalent
association of two or
more substances due to attractive forces between positive and negative
charges. Steric
stabilizers can inhibit interaction with blood components and therefore
opsonization,
phagocytosis, and uptake by the reticuloendothelial system. Steric stabilizers
can thus
increase circulation time of molecules to which they are attached. Steric
stabilizers can also
inhibit aggregation of a polymer. A preferred steric stabilizer is a
polyethylene glycol (PEG)
or PEG derivative. As used herein, a preferred PEG can have about 1-500
ethylene glycol
14
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
monomers, or 2-25. As used herein, a preferred PEG can also have a molecular
weight
average of about 85-20,000 Daltons (Da), about 85-1000 Da. As used herein,
steric stabilizers
prevent or inhibit intramolecular or intermolecular interactions of a polymer
to which it is
attached relative to the polymer containing no steric stabilizer in aqueous
solution.
''Targeting ligands" enhance the pharmacokinetic or biodistribution properties
of a conjugate
to which they are attached to improve cell- or tissue-specific distribution
and cell-specific
uptake of the conjugate. As used herein, for clarity, the term 'targeting
ligand' is used to
denote a targeting ligand that is attached to a dipeptide masking agent, and
'targeting group'
is a targeting ligand that is linked to an RNAi polynucleotide in an RNAi
polynucleotide-
targeting group conjugate. Targeting ligands enhance the association of
molecules with a
target cell. Thus, targeting ligands can enhance the pharmacokinetic or
biodistribution
properties of a conjugate to which they are attached to improve cellular
distribution and
cellular uptake of the conjugate. Binding of a targeting ligand to a cell or
cell receptor may
initiate endocytosis. Targeting ligands may be monovalent, divalent,
trivalent, tetravalent, or
have higher valency. Targeting ligands may be selected from the group
comprising:
compounds with affinity to cell surface molecule, cell receptor ligands,
antibodies,
monoclonal antibodies, antibody fragments, and antibody mimics with affinity
to cell surface
molecules. A preferred targeting ligand comprises a cell receptor ligand. A
variety of ligands
have been used to target drugs and genes to cells and to specific cellular
receptors. Cell
receptor ligands may be selected from the group comprising: carbohydrates,
glycans,
saccharides (including, but not limited to: galactose, galactose derivatives,
mannose, and
mannose derivatives), vitamins, folate, biotin, aptamers, and peptides
(including, but not
limited to: RGD-containing peptides, insulin, EGF, and transferrin).
For liver hepatocyte targeting, a preferred targeting ligand is a saccharide
having affinity for
the asialoglycoprotein receptor (ASGPr). Galactose and galactose derivates
have been used to
target molecules to hepatocytes in vivo through their binding to the ASGPr
expressed on the
surface of hepatocytes. As used herein, a "ASGPr targeting ligand" comprises a
galactose and
galactose derivative having affinity for the ASGPr equal to or greater than
that of galactose.
Binding of galactose targeting ligand to the ASGPr(s) facilitates cell-
specific targeting of the
delivery polymer to hepatocytes and endocytosis of the delivery polymer into
hepatocytes.
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
ASGPr targeting ligands may be selected from the group comprising: lactose,
galactose, N-
acetylgalactosamine (NAG), galactosamine, N-formylgalactosamine, N-acetyl-
galactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-
butanoyl-
galactosamine (lobst, S.T. and Drickamer, K. J.B.C. 1996, 271, 6686). ASGPr
targeting
moieties can be monomeric (e.g., having a single galactosamine) or multimeric
(e.g., having
multiple galactosamines).
In one embodiment, the membrane active polyamine is reversibly masked by
attachment of
ASGPr targeting ligand masking agents to 2:50%, >60%, 270%, 280%, or 290% of
primary
amines on the polyamine. In another embodiment, the membrane active polyamine
is
reversibly masked by attachment of ASGPr targeting ligand masking agents and
PEG
masking agents to>50%, 260%, 2:70%, 280%, or 2:90% of primary amines on the
polymer.
When both ASGPr targeting ligand masking agents and PEG masking agents, a
ratio of PEG
to ASGPr targeting ligand is about 0-4:1, more preferably about 0.5-2:1.
"Amphipathic", or amphiphilic, polymers are well known and recognized in the
art and have
both hydrophilic (polar, water-soluble) and hydrophobic (non-polar,
lipophilic, water-
insoluble) groups or parts.
"Hydrophilic groups" indicate in qualitative terms that the chemical moiety is
water-
preferring. Typically, such chemical groups are water soluble, and are
hydrogen bond donors
or acceptors with water. A hydrophilic group can be charged or uncharged.
Charged groups
can be positively charged (anionic) or negatively charged (cationic) or both
(zwitterionic).
Examples of hydrophilic groups include the following chemical moieties:
carbohydrates,
polyoxyethylene, certain peptides, oligonucleotides, amines, amides, alkoxy
amides,
carboxylic acids, sulfurs, and hydroxyls.
''Hydrophobic groups" indicate in qualitative terms that the chemical moiety
is water-
avoiding. Typically, such chemical groups are not water soluble, and tend not
to form
hydrogen bonds. Lipophilic groups dissolve in fats, oils, lipids, and non-
polar solvents and
have little to no capacity to form hydrogen bonds. Hydrocarbons containing two
(2) or more
carbon atoms, certain substituted hydrocarbons, cholesterol, and cholesterol
derivatives are
examples of hydrophobic groups and compounds.
16
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Hydrophobic groups are preferably hydrocarbons, containing only carbon and
hydrogen
atoms. However, non-polar substitutions or non-polar heteroatoms which
maintain
hydrophobicity, and include, for example fluorine, may be permitted. The term
includes
aliphatic groups, aromatic groups, acyl groups, alkyl groups, alkenyl groups,
alkynyl groups,
aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of
which may be
linear, branched, or cyclic. The term hydrophobic group also includes:
sterols, steroids,
cholesterol, and steroid and cholesterol derivatives.
As used herein, with respect to amphipathic polymers, a part is defined as a
molecule derived
when one covalent bond is broken and replaced by hydrogen. For example, in
butyl amine, a
breakage between the carbon and nitrogen bonds, and replacement with
hydrogens, results in
ammonia (hydrophilic) and butane (hydrophobic). If 1,4-diaminobutane is
cleaved at
nitrogen-carbon bonds, and replaced with hydrogens, the resulting molecules
are again
ammonia (2x) and butane. However, 1,4,-diaminobutane is not considered
amphipathic
because formation of the hydrophobic part requires breakage of two bonds.
As used herein, a surface active polymer lowers the surface tension of water
and/or the
interfacial tension with other phases, and, accordingly, is positively
adsorbed at the
liquid/vapor interface. The property of surface activity is usually due to the
fact that the
molecules of the substance are amphipathic or amphiphilic.
As used herein, "membrane active" polymers are surface active, amphipathic
polymers that
are able to induce one or more of the following effects upon a biological
membrane: an
alteration or disruption of the membrane that allows non-membrane permeable
molecules to
enter a cell or cross the membrane, pore formation in the membrane, fission of
membranes, or
disruption or dissolving of the membrane. As used herein, a membrane, or cell
membrane,
comprises a lipid bilayer. The alteration or disruption of the membrane can be
functionally
defined by the polymer's activity in at least one the following assays: red
blood cell lysis
(hemolysis), liposome leakage, liposome fusion, cell fusion, cell lysis, and
endosomal
release. Membrane active polymers that can cause lysis of cell membranes are
also termed
membrane lytic polymers. Polymers that preferentially cause disruption of
endosomes or
lysosomes over plasma membrane are considered endosomolytic. The effect of
membrane
active polymers on a cell membrane may be transient. Membrane active possess
affinity for
17
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
the membrane and cause a denaturation or deformation of bilayer structures.
Membrane
active polymers may be synthetic or non-natural amphipathic polymers.
As used herein, membrane active polymers are distinct from a class of polymers
termed cell
.. penetrating peptides or polymers represented by compounds such as the
arginine-rich peptide
derived from the HIV TAT protein, the antennapedia peptide, VP22 peptide,
transportan,
arginine-rich artificial peptides, small guanidinium-rich artificial polymers
and the like.
While cell penetrating compounds appear to transport some molecules across a
membrane,
from one side of a lipid bilayer to other side of the lipid bilayer,
apparently without requiring
.. endocytosis and without disturbing the integrity of the membrane, their
mechanism is not
understood.
Delivery of a polynucleotide to a cell is mediated by the membrane active
polymer disrupting
or destabilizing the plasma membrane or an internal vesicle membrane (such as
an endosome
or lysosome), including forming a pore in the membrane, or disrupting
endosomal or
lysosomal vesicles thereby permitting release of the contents of the vesicle
into the cell
cytoplasm.
Amphipathic membrane active polyamine copolymers of the invention are the
product of
copolymerization of two or more monomer species. In one embodiment,
amphipathic
membrane active heteropolymers of the invention have the general structure:
¨(A)a¨(B)b¨
wherein, A contains a pendent primary or secondary amine functional group and
B contains a
pendant hydrophobic group. a and b are integers >0. The polymers may be
random, block, or
alternating. The incorporation of additional monomers is permissible.
"Endosomolytic polymers" are polymers that, in response to an endosomal-
specific
environmental factors, such as the presence of lytic enzymes, are able to
cause disruption or
lysis of an endosome or provide for release of a normally cell membrane
impermeable
.. compound, such as a polynucleotide, from a cellular internal membrane-
enclosed vesicle,
such as an endosome or lysosome. Endosomolytic polymers undergo a shift in
their physico-
chemical properties in the endosome. This shift can be a change in the
polymer's solubility or
ability to interact with other compounds or membranes as a result in a shift
in charge,
18
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
hydrophobicity, or hydrophilicity. A reversibly masked membrane active
polymamine of the
invention are considered to be endosomolytic polymers.
"Melittin" is a small amphipathic membrane active peptide which naturally
occurs in bee
venom. Melittin can be isolated from a biological source or it can be
synthetic. A synthetic
polymer is formulated or manufactured by a chemical process "by man" and is
not created by
a naturally occurring biological process. As used herein, melittin encompasses
the naturally
occurring bee venom peptides of the melittin family that can be found in, for
example, venom
of the species: Apis mellifera, Apis cerana, Vespula maculifrons, Vespa
magnifica, Vespa
velutina nigrithorax, Polistes sp. HQL-2001, Apis florae, Apis dorsata, Apis
cerana cerana,
Polistes hebraeus. As used herein, melittin also encompasses synthetic
peptides having amino
acid sequence identical to or similar to naturally occurring melittin
peptides. Specifically,
melittin amino acid sequence encompass those shown in Table 1. Synthetic
melittin peptides
can contain naturally occurring L form amino acids or the enantiomeric D form
amino acids
(inverso). However, a melittin peptide should either contain essentially all L
form or all D
form amino acids but may have amino acids of the opposite stereocenter
appended at either
the amino or carboxy termini. The melittin amino acid sequence can also be
reversed
(reverso). Revers .melittin can have L form amino acids or D form amino acids
(retroinverso). Two melittin peptides can also be covalently linked to form a
melittin dimer.
Melittin can have modifying groups, other than masking agents, that enhance
tissue targeting
or facilitate in vivo circulation attached to either the amino terminal or
carboxy terminal ends.
A linkage or "linker" is a connection between two atoms that links one
chemical group or
segment of interest to another chemical group or segment of interest via one
or more covalent
bonds. For example, a linkage can connect a masking agent or polynucleotide to
a polymer. A
labile linkage contains a labile bond. A linkage may optionally include a
spacer that increases
the distance between the two joined atoms. A spacer may further add
flexibility and/or length
to the linkage. Spacers may include, but are not be limited to, alkyl groups,
alkenyl groups,
alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, aralkynyl
groups; each of
which can contain one or more heteroatoms, heterocycles, amino acids,
nucleotides, and
saccharides. Spacer groups are well known in the art and the preceding list is
not meant to
limit the scope of the invention.
19
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
A "labile bond" is a covalent bond other than a covalent bond to a hydrogen
atom that is
capable of being selectively broken or cleaved under conditions that will not
break or cleave
other covalent bonds in the same molecule. More specifically, a labile bond is
a covalent
bond that is less stable (thermodynamically) or more rapidly broken
(kinetically) under
appropriate conditions than other non-labile covalent bonds in the same
molecule. Cleavage
of a labile bond within a molecule may result in the formation of two
molecules. For those
skilled in the art, cleavage or lability of a bond is generally discussed in
terms of half-life (t)
of bond cleavage (the time required for half of the bonds to cleave). Thus,
labile bonds
encompass bonds that can be selectively cleaved more rapidly than other bonds
a molecule.
As used herein, a "physiologically labile bond" is a labile bond that is
cleavable under
conditions normally encountered or analogous to those encountered within a
mammalian
body. Physiologically labile linkage groups are selected such that they
undergo a chemical
transformation (e.g., cleavage) when present in certain physiological
conditions.
As used herein, a cellular physiologically labile bond is a labile bond that
is cleavable under
mammalian intracellular conditions. Mammalian intracellular conditions include
chemical
conditions such as pH, temperature, oxidative or reductive conditions or
agents, and salt
concentration found in or analogous to those encountered in mammalian cells.
Mammalian
intracellular conditions also include the presence of enzymatic activity
normally present in a
mammalian cell such as from proteolytic or hydrolytic enzymes. A cellular
physiologically
labile bond may also be cleaved in response to administration of a
pharmaceutically
acceptable exogenous agent.
RNAi interference-targeting group conjugate: A targeting group may be linked
to the 3' or the
5' end of the RNAi polynucleotide. For siRNA polynucleotides, the targeting
moiety may be
linked to either the sense strand or the antisense strand, though the sense
strand is preferred.
In one embodiment, the targeting group consists of a hydrophobic group More
specifically,
.. the targeting group consists of a hydrophobic group having at least 20
carbon atoms.
Hydrophobic groups used as polynucleotide targeting moieties are herein
referred to as
hydrophobic targeting moieties. Exemplary suitable hydrophobic groups may be
selected
from the group comprising: cholesterol, dicholesterol, tocopherol,
ditocopherol, didecyl,
didodecyl, dioctadecyl, didodecyl, dioctadecyl, isoprenoid, and choleamide.
Hydrophobic
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
groups having 6 or fewer carbon atoms are not effective as polynucleotide
targeting moieties,
while hydrophobic groups having 8 to 18 carbon atoms provide increasing
polynucleotide
delivery with increasing size of the hydrophobic group (i.e. increasing number
of carbon
atoms). Attachment of a hydrophobic targeting group to an RNAi polynucleotide
does not
provide efficient functional in vivo delivery of the RNAi polynucleotide in
the absence of co-
administration of the delivery polymer, While siRNA-cholesterol conjugates
have been
reported by others to deliver siRNA (siRNA-cholesterol) to liver cells in
vivo, in the absence
of any additional delivery vehicle, high concentrations of siRNA are required
and delivery
efficacy is poor. When combined with the delivery polymers described herein,
delivery of the
polynucleotide is greatly improved. By providing the siRNA-cholesterol
together with a
delivery polymer of the invention, efficacy of siRNA-cholesterol is increased
about 100 fold.
Hydrophobic groups useful as polynucleotide targeting moieties may be selected
from the
group consisting of: alkyl group, alkenyl group, alkynyl group, aryl group,
aralkyl group,
.. aralkenyl group, and aralkynyl group, each of which may be linear,
branched, or cyclic,
cholesterol, cholesterol derivative, sterol, steroid, and steroid derivative.
Hydrophobic
targeting groups are preferably hydrocarbons, containing only carbon and
hydrogen atoms.
However, substitutions or heteroatoms which maintain hydrophobicity, for
example fluorine,
may be permitted. The hydrophobic targeting group may be attached to the 3' or
5' end of the
.. RNAi polynucleotide using methods known in the art. For RNAi
polynucleotides having 2
strands, such as siRNA, the hydrophobic group may be attached to either
strand.
In another embodiment, the targeting group comprises a galactose cluster
(galactose cluster
targeting moiety). As used herein, a "galactose cluster" comprises a molecule
having two to
four terminal galactose derivatives. As used herein, the term galactose
derivative includes
both galactose and derivatives of galactose having affinity for the ASGPr
equal to or greater
than that of galactose. A terminal galactose derivative is attached to a
molecule through its
C-1 carbon. A preferred galactose cluster has three terminal galactosamines or
galactosamine
derivatives each having affinity for the asialoglycoprotein receptor. A more
preferred
galactose cluster has three terminal N-acetylgalactosamines. Other terms
common in the art
include tri-antennary galactose, tri-valent galactose and galactose trimer. It
is known that tri-
antennary galactose derivative clusters bind to the ASGPr with greater
affinity than bi-
antennary or mono-antennary galactose derivative structures (Baenziger and
Fiete, 1980,
Cell, 22, 611-620; Connolly et at., 1982, J. Biol. Chem., 257, 939-945).
Multivalency is
21
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
required to achieve nM affinity. The attachment of a single galactose
derivative having
affinity for the asialoglycoprotein receptor does not enable functional
delivery of the RNAi
polynucleotide to hepatocytes in vivo when co-administered with the delivery
polymer.
A galactose cluster contains two-four, preferably three, galactose derivatives
each linked to a
central branch point. The galactose derivatives are attached to the central
branch point
through the C-1 carbons of the saccharides. The galactose derivative is
preferably linked to
the branch point via linkers or spacers. A preferred spacer is a flexible
hydrophilic spacer
(U.S. Patent 5885968; Biessen et al. J. Med. Chem. 1995 Vol. 39 p. 1538-1546).
A preferred
flexible hydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3
spacer. The
branch point can be any small molecule which permits attachment of the three
galactose
derivatives and further permits attachment of the branch point to the RNAi
polynucleotide.
An exemplary branch point group is a di-lysine. A di-lysine molecule contains
three amine
groups through which three galactose derivatives may be attached and a
carboxyl reactive
group through which the di-lysine may be attached to the RNAi polynucleotide.
Attachment
of the branch point to the RNAi polynucleotide may occur through a linker or
spacer. A
preferred spacer is a flexible hydrophilic spacer. A preferred flexible
hydrophilic spacer is a
PEG spacer. A preferred PEG spacer is a PEG3 -spacer (three ethylene units).
The galactose
cluster may be attached to the 3' or 5' end of the RNAi polynucleotide using
methods known
in the art. For RNAi polynucleotides having 2 strands, such as siRNA, the
galactose cluster
may be attached to either strand. Suitable galactose clusters are described in
US Patent
Publication 20110207799.
The term "polynucleotide", or nucleic acid or polynucleic acid, is a term of
art that refers to a
polymer containing at least two nucleotides. Nucleotides are the monomeric
units of
polynucleotide polymers. Polynucleotides with less than 120 monomeric units
are often
called oligonucleotides. Natural nucleic acids have a deoxyribose- or ribose-
phosphate
backbone. A non-natural or synthetic polynucleotide is a polynucleotide that
is polymerized
in vitro or in a cell free system and contains the same or similar bases but
may contain a
backbone of a type other than the natural ribose or deoxyribose-phosphate
backbone.
Polynucleotides can be synthesized using any known technique in the art.
Polynucleotide
backbones known in the art include: PNAs (peptide nucleic acids),
phosphorothioates,
phosphorodiamidates, morpholinos, and other variants of the phosphate backbone
of native
nucleic acids. Bases include purines and pyrimidines, which further include
the natural
22
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural
analogs.
Synthetic derivatives of purines and pyrimidines include, but are not limited
to, modifications
which place new reactive groups on the nucleotide such as, but not limited to,
amines,
alcohols, thiols, carboxylates, and alkylhalides. The term base encompasses
any of the known
base analogs of DNA and RNA. A polynucleotide may contain ribonucleotides,
deoxyribonucleotides, synthetic nucleotides, or any suitable combination.
Polynucleotides
may be polymerized in vitro, they may be recombinant, contain chimeric
sequences, or
derivatives of these groups. A polynucleotide may include a terminal cap
moiety at the 5' -
end, the 3' -end, or both the 5' and 3' ends. The cap moiety can be, but is
not limited to, an
inverted deoxy abasic moiety, an inverted deoxy thymidine moiety, a thymidine
moiety, or 3'
glyceryl modification.
An "RNA interference (RNAi) polynucleotide" is a molecule capable of inducing
RNA
interference through interaction with the RNA interference pathway machinery
of
mammalian cells to degrade or inhibit translation of messenger RNA (mRNA)
transcripts of a
transgene in a sequence specific manner. Two primary RNAi polynucleotides are
small (or
short) interfering RNAs (siRNAs) and micro RNAs (miRNAs). RNAi polynucleotides
may
be selected from the group comprising: siRNA, microRNA, double-strand RNA
(dsRNA),
short hairpin RNA (shRNA), and expression cassettes encoding RNA capable of
inducing
RNA interference. siRNA comprises a double stranded structure typically
containing 15-50
base pairs and preferably 21-25 base pairs and having a nucleotide sequence
identical
(perfectly complementary) or nearly identical (partially complementary) to a
coding sequence
in an expressed target gene or RNA within the cell. An siRNA may have
dinucleotide 3'
overhangs. An siRNA may be composed of two annealed polynucleotides or a
single
polynucleotide that forms a hairpin structure. An siRNA molecule of the
invention comprises
a sense region and an antisense region. In one embodiment, the siRNA of the
conjugate is
assembled from two oligonucleotide fragments wherein one fragment comprises
the
nucleotide sequence of the antisense strand of the siRNA molecule and a second
fragment
comprises nucleotide sequence of the sense region of the siRNA molecule. In
another
embodiment, the sense strand is connected to the antisense strand via a linker
molecule, such
as a polynucleotide linker or a non-nucleotide linker. MicroRNAs (miRNAs) are
small
noncoding RNA gene products about 22 nucleotides long that direct destruction
or
translational repression of their mRNA targets. If the complementarity between
the miRNA
and the target mRNA is partial, translation of the target mRNA is repressed.
If
23
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
complementarity is extensive, the target mRNA is cleaved. For miRNAs, the
complex binds
to target sites usually located in the 3' UTR of mRNAs that typically share
only partial
homology with the miRNA. A "seed region" ¨ a stretch of about seven (7)
consecutive
nucleotides on the 5' end of the miRNA that forms perfect base pairing with
its target ¨plays
a key role in miRNA specificity. Binding of the RISC/miRNA complex to the mRNA
can
lead to either the repression of protein translation or cleavage and
degradation of the mRNA.
Recent data indicate that mRNA cleavage happens preferentially if there is
perfect homology
along the whole length of the miRNA and its target instead of showing perfect
base-pairing
only in the seed region (Pillai et al. 2007).
RNAi polynucleotide expression cassettes can be transcribed in the cell to
produce small
hairpin RNAs that can function as siRNA, separate sense and anti-sense strand
linear
siRNAs, or miRNA. RNA polymerase III transcribed DNAs contain promoters
selected from
the list comprising: U6 promoters, HI promoters, and tRNA promoters. RNA
polymerase II
promoters include Ul, U2, U4, and U5 promoters, snRNA promoters, microRNA
promoters,
and mRNA promoters.
Lists of known miRNA sequences can be found in databases maintained by
research
organizations such as Wellcome Trust Sanger Institute, Penn Center for
Bioinformatics,
Memorial Sloan Kettering Cancer Center, and European Molecule Biology
Laboratory,
among others. Known effective siRNA sequences and cognate binding sites are
also well
represented in the relevant literature. RNAi molecules are readily designed
and produced by
technologies known in the art. In addition, there are computational tools that
increase the
chance of finding effective and specific sequence motifs (Pei et al. 2006,
Reynolds et al.
2004, Khvorova et al. 2003, Schwarz et al. 2003, Ui-Tei et al. 2004, Heale et
al. 2005, Chalk
et al. 2004, Amarzguioui et al. 2004).
The polynucleotides of the invention can be chemically modified. Non-limiting
examples of
such chemical modifications include: phosphorothioate internucleotide
linkages, 2'-0-methyl
.. ribonucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy
ribonucleotides, "universal
base" nucleotides, 5-C-methyl nucleotides, and inverted deoxyabasic residue
incorporation.
These chemical modifications, when used in various polynucleotide constructs,
are shown to
preserve polynucleotide activity in cells while at the same time increasing
the serum stability
24
CA 02816041 2013-04-24
WO 2012/092373
PCT/1JS2011/067588
of these compounds. Chemically modified siRNA can also minimize the
possibility of
activating interferon activity in humans.
In one embodiment, a chemically-modified RNAi polynucleotide of the invention
comprises
a duplex having two strands, one or both of which can be chemically-modified,
wherein each
strand is about 19 to about 29 nucleotides. In one embodiment, an RNAi
polynucleotide of
the invention comprises one or more modified nucleotides while maintaining the
ability' to
mediate RNAi inside a cell or reconstituted in vitro system. An RNAi
polynucleotide can be
modified wherein the chemical modification comprises one or more (e.g., about
1, 2, 3, 4, 5,
6, 7, 8, 9, 10, or more) of the nucleotides. An RNAi polynucleotide of the
invention can
comprise modified nucleotides as a percentage of the total number of
nucleotides present in
the RNAi polynucleotide. As such, an RNAi polynucleotide of the invention can
generally
comprise modified nucleotides from about 5 to about 100% of the nucleotide
positions (e.g.,
5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%, 90%, 95% or 100% of the nucleotide positions). The actual percentage of
modified
nucleotides present in a given RNAi polynucleotide depends on the total number
of
nucleotides present in the RNAi polynucleotide. If the RNAi polynucleotide is
single
stranded, the percent modification can be based upon the total number of
nucleotides present
in the single stranded RNAi polynucleotide. Likewise, if the RNAi
polynucleotide is double
stranded, the percent modification can be based upon the total number of
nucleotides present
in the sense strand, antisense strand, or both the sense and antisense
strands. In addition, the
actual percentage of modified nucleotides present in a given RNAi
polynucleotide can also
depend on the total number of purine and pyrimidine nucleotides present in the
RNAi
polynucleotide. For example, wherein all pyrimidine nucleotides and/or all
purine nucleotides
present in the RNAi polynucleotide are modified.
An RNAi polynucleotide modulates expression of RNA encoded by a gene. Because
multiple
genes can share some degree of sequence homology with each other, an RNAi
polynucleotide
can be designed to target a class of genes with sufficient sequence homology.
Thus, an RNAi
polynucleotide can contain a sequence that has complementarity to sequences
that are shared
amongst different gene targets or are unique for a specific gene target.
Therefore, the RNAi
polynucleotide can be designed to target conserved regions of an RNA sequence
having
homology between several genes thereby targeting several genes in a gene
family (e.g.,
different gene isoforms, splice variants, mutant genes, etc.). In another
embodiment, the
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
RNAi polynucleotide can be designed to target a sequence that is unique to a
specific RNA
sequence of a single gene.
The term "complementarity" refers to the ability of a polynucleotide to form
hydrogen
bond(s) with another polynucleotide sequence by either traditional Watson-
Crick or other
non-traditional types. In reference to the polynucleotide molecules of the
present invention,
the binding free energy for a polynucleotide molecule with its target
(effector binding site) or
complementary sequence is sufficient to allow the relevant function of the
polynucleotide to
proceed, e.g., enzymatic mRNA cleavage or translation inhibition.
Determination of binding
free energies for nucleic acid molecules is well known in the art (Frier et
al. 1986, Turner et
al. 1987). A percent complementarity indicates the percentage of bases, in a
contiguous
strand, in a first polynucleotide molecule which can form hydrogen bonds
(e.g., Watson-
Crick base pairing) with a second polynucleotide sequence (e.g., 5, 6, 7, 8,
9, 10 out of 10
being 50%, 60%, 70%, 80%, 90%, and 100% complementary). Perfectly
complementary
means that all the bases in a contiguous strand of a polynucleotide sequence
will hydrogen
bond with the same number of contiguous bases in a second polynucleotide
sequence.
By inhibit, down-regulate, or knockdown gene expression, it is meant that the
expression of
the gene, as measured by the level of RNA transcribed from the gene or the
level of
polypeptide, protein or protein subunit translated from the RNA, is reduced
below that
observed in the absence of the blocking polynucleotide-conjugates of the
invention.
Inhibition, down-regulation, or knockdown of gene expression, with a
polynucleotide
delivered by the compositions of the invention, is preferably below that level
observed in the
presence of a control inactive nucleic acid, a nucleic acid with scrambled
sequence or with
inactivating mismatches, or in absence of conjugation of the polynucleotide to
the masked
polymer.
It was found that siRNA stabilization against degradation by endosomal /
lysosomal-localized
nucleases such as DNAse II strongly improves target knock down. Such
stabilization may
directly affect the amount of siRNA released into the cytoplasm where the
cellular RNAi
machinery is located. Only the siRNA portion available in the cytoplasm will
trigger the
RNAi effect.
26
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
In addition to poor pharmacokinetic characteristics, siRNAs are susceptible to
nucleases in
the biological environment when administered as such into the circulation
without a
protecting delivery vehicle. Accordingly, many siRNAs are rapidly degraded
either
extracellularly in the tissue and blood stream or after intracellular uptake
(endosome).
Nuclease cleavage can be inhibited by nucleotides lacking a 2'-OH group such
as 2`-deoxy,
2'-0-methyl (2'-0Me)or 2'-deoxy-2'-fluoro (2'-F) nucleotides and by
polynucleotides 5'-
terminal non-nucleotide moieties, like e.g. cholesterol, aminoalkyl-linker or
a phosphothioate
at the first intemucleotide linkage. Preferably, the RNAi polynucleotide lack
any 2'-OH
nucleotide within the strand, starting with a 2cOMe nucleotide at the 5'-end
connected by a
phosphorothioate (PTO) linkage to the second nucleotide.
siRNAs can be significantly stabilized when using the following design,
wherein an
oligonucleotide is provided with an antisense strand with the modification
pattern: 5'-(w)-
(Z1)-(Z2)-(Z3)na-31 and a sense strand with the modification pattern 5'-(Z3)ns-
3 , wherein
w is independently a 5'-phosphate or 5'-phosphothioate or H,
Z1 is independently a 2'-modified nuleoside.
Z2 is independently a 2'-deoxy nucleoside or 2'-Fluoro-modified nucleoside,
Z3 is independently a 2'-modified nucleoside,
na is 8-23 and ris is 8-25.
In one preferred embodiment an oligonucleotide is provided with an antisense
strand with the
modification pattern: 5`-(w)-(Z1)-(Z2)-(Z3)na-3' and a sense strand with the
modification
pattern 5'-(Z3)n5-3', wherein Z1 is a a 2'-Fluoro-modified nucleoside or a 2-
deoxy-nucleoside
and all remaining substituents as well as the variables n3 and ns have the
meaning given
above.
In one preferred embodiment an oligonucleotide is provided with an antisense
strand with the
modification pattern: 5'-(w)-(Z1)-(Z2)-(Z3)n.-3' and a sense strand with the
modification
pattern 5'-(Z3)n5-3', wherein Z3 is a 2'-0-Methyl modified nucleoside, a 2'-
Fluoro-modified
nucleoside or a 2-deoxy-nucleoside and all remaining substituents as well as
the variables na
and n, have the meaning given above.
In one preferred embodiment an oligonucleotide is provided with an antisense
strand with the
modification pattern: 5'-(w)-(Z1)-(Z2)-(Z3)na-3' and a sense strand with the
modification
27
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
pattern 5'-(Z3)ns-3' , wherein ZI is a a 2'-Fluoro-modified nucleoside or a 2-
deoxy-
nucleoside and Z3 is a 2'-0-Methyl modified nucleoside, a 2'-Fluoro-modified
nucleoside or
a 2-deoxy-nucleoside and all remaining substituents as well as the variables
na and Its have
the meaning given above.
The nucleosides in the nucleic acid sequence of the oligonucleotice with the
novel
modification pattern can either be linked by 5'-3' phosphodiesters or 5'-3'
phosphorothioates.
As used herein, the "anti-sense" strand is the siRNA strand that is
complementary to the
.. target mRNA and that will be binding to the mRNA once the siRNA is unwound.
The sense
strand of said siRNA comprising the novel modification pattern is
complimentary to the
antisense strand.
In principle a nuclease cleavage site, between the RNAi polynucleotide and the
targeting
moiety or delivery polymer to which it is covalently attached can be
introduced by 3'- or 5'-
overhangs containing at least one 2'-OH nucleotide at either the sense or the
antisense strand.
The final active siRNA species is generated by intracellular nuclease
processing. Also, the
use of defined cleavage sites implemented by 2'-OH nucleotides within the base
paired region
is possible. This can be done using at least one 2'-OH nucleotide
complementary to the
opposite strand or by introduction of either at least one mismatched 2'-OH
nucleotide or a
hairpin/bulge containing at least one 2'-OH nucleotide.
Linkage of polynucleotide to delivery polymer
In one embodiment, the RNAi polynucleotide is linked to the deliver), polymer
via a
physiologically labile bond or linker. The physiologically labile linker is
selected such that it
undergoes a chemical transformation (e.g., cleavage) when present in certain
physiological
conditions, (e.g., disulfide bond cleaved in the reducing environment of the
cell cytoplasm).
Release of the polynucleotide from the polymer, by cleavage of the
physiologically labile
linkage, facilitates interaction of the polynucleotide with the appropriate
cellular components
for activity.
The polynucleotide-polymer conjugate is formed by covalently linking the
polynucleotide to
the polymer. The polymer is polymerized or modified such that it contains a
reactive group
A. The polynucleotide is also polymerized or modified such that it contains a
reactive group
28
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
B. Reactive groups A and B are chosen such that they can be linked via a
reversible covalent
linkage using methods known in the art.
Conjugation of the polynucleotide to the polymer can be performed in the
presence of an
excess of polymer. Because the polynucleotide and the polymer may be of
opposite charge
during conjugation, the presence of excess polymer can reduce or eliminate
aggregation of
the conjugate. Alternatively, an excess of a carrier polymer, such as a
polycation, can be
used. The excess polymer can be removed from the conjugated polymer prior to
administration of the conjugate to the animal or cell culture. Alternatively,
the excess
polymer can be co-administered with the conjugate to the animal or cell
culture.
In Vivo Administration
In pharmacology and toxicology, a route of administration is the path by which
a drug, fluid,
poison, or other substance is brought into contact with the body. In general,
methods of
administering drugs and nucleic acids for treatment of a mammal are well known
in the art
and can be applied to administration of the compositions of the invention. The
compounds of
the present invention can be administered via any suitable route, most
preferably parenterally,
in a preparation appropriately tailored to that route. Thus, the compounds of
the present
invention can be administered by injection, for example, intravenously,
intramuscularly,
intracutaneously, subcutaneously, or intraperitoneally. Accordingly, the
present invention
also provides pharmaceutical compositions comprising a pharmaceutically
acceptable carrier
or excipient.
Parenteral routes of administration include intravascular (intravenous,
intraarterial),
intramuscular, intraparenchymal, intradermal, subdermal, subcutaneous,
intratumor,
intraperitoneal, intrathecal, subdural, epidural, and intralymphatic
injections that use a
syringe and a needle or catheter. Intravascular herein means within a tubular
structure called
a vessel that is connected to a tissue or organ within the body. Within the
cavity of the tubular
structure, a bodily fluid flows to or from the body part. Examples of bodily
fluid include
blood, cerebrospinal fluid (CSF), lymphatic fluid, or bile. Examples of
vessels include
arteries, arterioles, capillaries, venules, sinusoids, veins, lymphatics, bile
ducts, and ducts of
the salivary or other exocrine glands. The intravascular route includes
delivery through the
blood vessels such as an artery or a vein. The blood circulatory system
provides systemic
spread of the pharmaceutical.
29
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
The described compositions are injected in pharmaceutically acceptable carrier
solutions.
Pharmaceutically acceptable refers to those properties and/or substances which
are acceptable
to the mammal from a pharmacological/toxicological point of view. The phrase
pharmaceutically acceptable refers to molecular entities, compositions, and
properties that are
physiologically tolerable and do not typically produce an allergic or other
untoward or toxic
reaction when administered to a mammal. Preferably, as used herein, the term
pharmaceutically acceptable means approved by a regulatory agency of the
Federal or a state
government or listed in the U.S. Pharmacopeia or other generally recognized
pharmacopeia
.. for use in animals and more particularly in humans.
These carrier may also contain adjuvants such as preservatives, wetting
agents, emulsifying
agents and dispersing agents. Prevention of presence of microorganisms may be
ensured both
by sterilization procedures, supra, and by the inclusion of various
antibacterial and antifungal
agents, for example, paraben, chlorobutanol, phenol, sorbic acid, and the
like. It may also be
desirable to include isotonic agents, such as sugars, sodium chloride, and the
like into the
compositions. In addition, prolonged absorption of the injectable
pharmaceutical form may
be brought about by the inclusion of agents which delay absorption such as
aluminum
monostearate and gelatin.
In one embodiment, an RNAi polynucleotide-targeting 'group conjugate is co-
administered
with a delivery polymer of the invention. By co-administered it is meant that
the RNAi
polynucleotide conjugate and the delivery polymer are administered to the
mammal such that
both are present in the mammal at the same time. The RNAi polynucleotide-
targeting group
conjugate and the delivery polymer may be administered simultaneously or they
may be
delivered sequentially. For simultaneous administration, they may be mixed
prior to
administration. For sequential administration, either the RNAi polynucleotide-
targeting
moiety conjugate or the delivery polymer may be administered first.
Therapeutic Effect
RNAi polynucleotides may be delivered for research purposes or to produce a
change in a
cell that is therapeutic. In vivo delivery of RNAi polynucleotides is useful
for research
reagents and for a variety of therapeutic, diagnostic, target validation,
genomic discovery,
genetic engineering, and pharmacogenomic applications. We have disclosed RNAi
CA 02816041 2013-04-24
WO 2012/092373
PCT11TS2011/067588
polynucleotide delivery resulting in inhibition of endogenous gene expression
in hepatocytes.
Levels of a reporter (marker) gene expression measured following delivery of a
polynucleotide indicate a reasonable expectation of similar levels of gene
expression
following delivery of other polynucleotides. Levels of treatment considered
beneficial by a
person having ordinary skill in the art differ from disease to disease. For
example,
Hemophilia A and B are caused by deficiencies of the X-linked clotting factors
VIII and IX,
respectively. Their clinical course is greatly influenced by the percentage of
normal serum
levels of factor VIII or IX: <2%, severe; 2-5%, moderate; and 5-30% mild.
Thus, an increase
from 1% to 2% of the normal level of circulating factor in severe patients can
be considered
k 10 beneficial. Levels greater than 6% prevent spontaneous bleeds but not
those secondary to
surgery or injury. Similarly, inhibition of a gene need not be 100% to provide
a therapeutic
benefit. A person having ordinary skill in the art of gene therapy would
reasonably anticipate
beneficial levels of expression of a gene specific for a disease based upon
sufficient levels of
marker gene results. In the hemophilia example, if marker genes were expressed
to yield a
.. protein at a level comparable in volume to 2% of the normal level of factor
VIII, it can be
reasonably expected that the gene coding for factor VIII would also be
expressed at similar
levels. Thus, reporter or marker genes serve as useful paradigms for
expression of
intracellular proteins in general.
The liver is one of the most important target tissues for gene therapy given
its central role in
metabolism (e.g., lipoprotein metabolism in various hypercholesterolemias) and
the secretion
of circulating proteins (e.g., clotting factors in hemophilia). In addition,
acquired disorders
such as chronic hepatitis and cirrhosis are common and are also potentially
treated by
polynucleotide-based liver therapies. A number of diseases or conditions which
affect or are
affected by the liver are potentially treated through knockdown (inhibition)
of gene
expression in the liver. Such liver diseases and conditions may be selected
from the list
comprising: liver cancers (including hepatocellular carcinoma, HCC), viral
infections
(including hepatitis), metabolic disorders, (including hyperlipidemia and
diabetes), fibrosis,
and acute liver injury.
Actual dosage levels of the active ingredients in the pharmaceutical
compositions of the
present invention may be varied so as to obtain an amount of the active
ingredient which is
effective to achieve the desired therapeutic response for a particular
patient, composition, and
mode of administration, without being toxic to the patient. The selected
dosage level will
31
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
depend upon a variety of pharmacokinetic factors including the activity of the
particular
compositions of the present invention employed, the route of administration,
the time of
administration, the rate of excretion of the particular compound being
employed, the duration
of the treatment, other drugs, compounds and/or materials used in combination
with the
particular compositions employed, the age, sex, weight, condition, general
health and prior
medical history of the patient being treated, and like factors well known in
the medical arts.
The amount (dose) of delivery polymer, RNAi polynucleotide-targeting group
conjugate or
delivery polymer-RNAi polynucleotide conjugate that is to be administered can
be
determined empirically. We have shown effective knockdown of gene expression
using 0.1-
10 mg/kg animal weight of siRNA and 1.5-60 mg/kg animal weight delivery
polymer. A
preferred amount in mice is 0.25-2.5 mg/kg siRNA-conjugate and 10-40 mg/kg
delivery
polymer. More preferably, about 1.5-20 mg/kg delivery polymer is administered.
The amount
of RNAi polynucleotide-conjugate is easily increased because it is typically
not toxic in
larger doses.
As used herein, in vivo means that which takes place inside an organism and
more
specifically to a process performed in or on the living tissue of a whole,
living multicellular
organism (animal), such as a mammal, as opposed to a partial or dead one.
As used herein, "pharmaceutical composition" includes the conjugates of the
invention, a
pharmaceutical carrier or diluent and any other media or agent necessary for
formulation.
As used herein, "pharmaceutical carrier" includes any and all solvents,
dispersion media,
coatings, antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the
like that are physiologically compatible. Preferably, the carrier is suitable
for intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal administration
(e.g. by injection
or infusion).
EXAMPLES
Example 1. Synthesis ofprotease (peptidase) cleavable masking agents.
All reactions, except coupling of amino acids in aqueous NaHCO3 and silyl-
group
deprotection, were carried out in anhydrous conditions using fresh anhydrous
solvents.
32
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
Column purification was done on a silica gel using specified eluents. Mass-
spectra (MS) were
taken using electrospray ionization.
In preparation of active p-nitrophenyl-p-acylamidobenzyl carbonate derivatives
of NAG and
PEG (NAG-L-AA-PABC-PNP and PEG-AA-PABC-PNP) we utilized NHS ester of
respective PEG or NAG-containing derivatives to acylate amino terminus of
dipeptido-p-
acylaminobenzy alcohol precursor. In the following steps benzylic hydroxyl
group was
converted into p-nitrophenyl carbonate followed by removal of protective
groups from amino
acids and NAG moiety. In some applications, when paranitrophenol (PNP)-
carbonates were
used for modification of certain polymers, protective groups prior to polymer
modification.
o
+ H¨ A1¨ A2¨ NH 11 CH2OH --a. R¨ CO¨ Al¨ A2¨ CH2OH
0
H-A1-A2-PAPA R-A1-A2-PABA
(02N 41 0)-00 0
2 Deprotect ion
________________ . R¨ CO¨ A 1¨ A2¨ NH 40 CH201 = 41 NO2 __ I.
R-A'A2-PAI3C-PNP
R comprises an ASGPr ligand (protected or unprotected) or a PEG, and
Ai and A2 are amino acids (either protected or unprotected)
The synthesis starts from preparation of H-A1A2-PABA (Table I) derivatives.
These adducts
were obtained utilizing synthetic scheme described by Dubowchik at al.(2002)
with some .
modifications. Fmoc-protected amino acids, Fmoc-AI-OH, were activated by
conversion into
N-hydroxycuccin imide esters, Fmoc-A 1 -NHS, in reaction with d
icyclohexylcarbod iim ide
(DCC) and N-hydroxycuccinimide (NHS). These reactive NHS-esters were coupled
with
protected amino acids A2 in presence of aqueous NaHCO3 added to keep amino
group
reactive. For preparation of le and If (Table 1), instead of NHS esters,
commercially
available pentafluorophenyl esters (0Pfp) for were used for coupling.
33
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
Synthesis of Fmoc Dipeptides la-h.
a) NHS esters of AA were prepared from respective amino acids with NHS and DCC
and
used without additional purification.
(i)
Fmoc¨A¨OH Fmoc¨A¨NHS
Conditions: (i) N-hydroxysuccinimide (NHS), N-N'-dicyclohexylcarbodiimide
(DCC),
0-20 C.
For Fmoc-Ala-NHS, DCC (286 mg, 1.38 mmol) was added to an ice cold solution of
Fmoc-
Ala-OH (412 mg, 1.32 mmol) and NHS (160 mg, 1.38 mmol) in DCM (13 mL), stirred
for
30 min, and then at 20 C for 16 h. The solid dicyclohexylurea (DCU) was
filtered off and the
solvent was removed in vacuo.
For Fmoc-Asn(DMCP)-NHS, DCC (148 mg, 0.72 mmol) was added to an ice cold
solution
of Fmoc-Asn(DMCP)-OH (298 mg, 0.68 mmol) and NHS (83 mg, 0.72 mmol) in DCM
(13 mL), stirred for 30 min, and then at 20 C for 16 h. The solid DCU was
filtered off and the
solvent was removed in vacuo.
For Fmoc-Gly-NHS, Fmoc-Gly-OH (891 mg, 3 mmol) and NHS (380 mg, 3.3 mmol) were
stirred in THF (10 mL) at 0 C for 5 min and treated with a DCC solution (650
mg,
3.15 mmol) in THF (5 mL). The cooling bath was removed in 30 min and the
reaction
mixture was stirred at 20 C for 10 h. The solid DCU was filtered off, washed
with THF and
the solvent was removed on the rotovap. The product was weighed and dissolved
in DME to
make a 0.2 mM solution.
For Fmoc-Glu(0-2PhiPr)-NHS, DCC (217 mg, 1.05 mmol) was added to an ice cold
solution
of Fmoc-Glu(0-2PhiPr)-OH (487 mg, 1 mmol) and NHS (127 mg, 1.1 mmol) in THF
(5 mL), stirred for 15 min and then at 20 C for 10 h. The workup was done as
described for
Fmoc-Gly-NHS.
For Fmoc-Phe-NHS, DCC (1.181 g, 5.72 mmol) was .added to an ice cold solution
of Fmoc-
Phe-OH (2.11 g, 5.45 mmol) and NHS (664 mg, 5.77 mmol) in DCM (50 mL), stirred
for
min, and then at 20 C for 10 h. The solid DCU was filtered off and the solvent
was
removed in vacuo.
For Fmoc-Val-NHS, DCC (227 mg, 1.1 mmol) was added to an ice cold solution of
Fmoc-
30 Val-OH (339 mg, 1 mmol) and NHS (127 mg, 1.1 mmol) in DCM (13 mL), stirred
for
34
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
30 min, and then at 20 C for 16 h. The solid DCU was filtered off and the
solvent was
removed in vacuo.
b) Amino acids H-Asn(DMCP)-OH and H-Lys(MMT)-OH were prepared from available
Fmoc-protected derivatives
0 0
(i) H2Nyit,
Fmoc ________ r OH OH
0
0
Asn(DMCP), R= H2CAN\A lys(CH3)2, R= (H2C)4¨N(CH3)2
Frnoc
Conditions: (i) Triethylamine (Et3N) in dimethylformamide (DMF).
H-Asn(DMCP)-OH
Fmoc-Asn(DMCP)-OH (576 mg, 1.32 mmol) was stirred in DMF (9 mL) with Et3N (3.7
mL,
26.4 mmol) for 15h. All volatiles were removed on a rotovap at 40 C/oil pump
vacuum. The
residue was triturated with ether (30 mL) three times and dried in vacua.
Yield 271 mg
(96%). MS: 643.6 [3M-1-1]+; 451.3 [2M+Nar; 429.5 [2M-1-11+; 236.7 [M+Naj+;
215.3 [M+1]
4; 132.8 [M-DMCP+1] +.
H-Lys(MMT)-01-1. Fmoc-Lys(MMT)-OH (4.902 g, 7.65 mmol) was stirred in DMF
(100 mL) with Et3N (32 mL, 30 eq. 229.4 mmol) for 10h. All volatiles were
removed on a
rotovap at 40 C/oil pump vacuum. The residue was triturated with ether two
times and dried
in vacuo. Yield 3.1g (97%). MS (neg. mode): 455, 453.3 [M+C1]-; 417.8 [M-1].
c) Synthesis of Fmoc-A1A2-0H.
(i) (ii)
Fmoc¨A1¨NHS Fmoc¨A1¨A'OH Fmoc¨A1-0Pfp
1 a-h
A1-_ Ala, Phe
Ai= Gly, G1u(2PhiPr), Asn(DMCP), Phe, Ala, Val.
A2=Gly, Lys(MMT), Cit, Asn(DMCP), Lys(CH3)2.
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
Conditions: (i) H-A2-0H, NaHCO3, mixture of dimethoxyethane (DME),
tetrahydrofurane
(THF) and H20. (ii) H-A2-0H, NaHCO3, DME/THF/H20. (iii) H-Cit-OH, NaHCO3, THF
in
H20.
For Fmoc-GlyGly-OH la, Glycine (75 mg, I mmol) and NaHCO3 (100 mg, 1.2 mmol)
were
dissolved in H20 (10 mL) and dimethoxyethane (DME) (5 mL). Fmoc-Gly-NHS
solution in
DME (5 ml, 1 mmol) was added. THF (2.5 mL) was added, the mixture was
sonicated to
make it homogeneous and stirred for 20 h. All volatiles were removed on a
rotovap, the
residue was treated with Et0Ac.and 5% KHCO3 solution in H20. Product was
extracted four
times with Et0Ac, washed with brine at pH=3, dried (Na2SO4), concentrated and
dried in
vacua. Yield 321 mg (90%). MS: 775.0 [2M +2Na]; 377.4 [M+Na]; 355.1 [M+1]+.
For Fmoc-Glu(0-2PhiPr)Gly-OH lb, Glycine (75 mg, 1 mmol) and NaHCO3 (84 mg,
1 mmol) were dissolved in a mixture of H20 (2 mL), THF (4 mL) and DME(5mL).
Fmoc-
Glu(0-2PhiPr)-NHS solution in DME (5 mL, 1 mmol) was added and stirred for 10
h. All
volatiles was removed on a rotovap, 20 mL of 0.1M MES buffer (pH=5) was added
followed
by Et0Ac (25 mL). The reaction mixture was stirred on ice and acidified to
pH=5 with 5%
solution of KHSO4. Product was extracted four times with Et0Ac, rinsed with
brine at pH=5,
dried (Na2SO4), concentrated and dried in vacua. Yield 528 mg (96%). MS: 567
[M+Nar;
562 [M+NH.4]+; 545.0 [M+1]+; 427.1 [M-2PhiPr]+.
For Fmoc-Asn(DMCP)Gly-OH le was prepared from Fmoc-Asn(DMCP)-NHS and H-Gly-
OH as described above for lb. Yield 96%. MS: 987.4 [2M+I] +; 516.3 [M+Na] +;
494.4
[M+1]+; 412.2 [M-DMCP+1) +.
For Fmoc-PheLys(MMT)-OH Id was prepared from Frnoc-Phe-NHS and H-Lys(MMT)-OH
as described above for lb. Yield 94%. MS: 788.5 [M+11+, 273.1 [M-MMT+11+.
For Fmoc-PheCit-OH le:
i) To Fmoc-Phe-NHS (4.96 g, 10.26 mmol) in DME (40 mL) was added to a solution
containing L-citrulline (1.80 g, 10.26 mmol) and NaHCO3 (0.86 g, 10.26 mmol)
in a
mixture of H20 (40 mL) and THF (20 mL). The reaction was stirred for 15 h.
Residual
DCC from activation was filtered and the organic solvent was removed on a
rotovap. To the
residue was added H20 (100 mL) and iPrOH (10 mL). The suspension was acidified
to
pH=3 with 5% KHSO4, the product was extracted with an Et0Ac:iPrOH=9:1 solution
(3x,
500 mL), washed with a mixture of brine:iPrOH=9:1 (2x, 50 mL), dried (Na2SO4),
filtered
36
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
and concentrated, and dried with oil pump. Trituration with ether afforded the
pure product
le. Yield 3.84 g (68%). MS: 545.6 [M+Na]+; 528.5 [M-112014; 306.3 [M-Fmoc+H20]
+.
ii) A solution of Frnoc-Phe-OPfp (553 mg, 1 mmol) in THF (5 mL) was added to a
solution
of H-Cit-OH (184 mg, 1.05 mmol) and NaHCO3 (88.2 mg, 1.05 mmol) in H20 (2.6
mL).
THF (2 mL) was added to make the solution homogeneous and stirred for 10 h.
THF was
removed on a rotovap, the residue was diluted with H20 (10 mL) and iPrOH (1
mL) and
acidified to pH=1 with 3% HC1.. The product was extracted five times with an
Et0Ac:iPrOH=9:1 solution, rinsed with a mixture of brine:iPrOH=9:1, dried
(Na2SO4) and
concentrated in vacuo. Trituration with ether afforded 313 mg of pure product
le (57%).
Fmoc-AlaCit-OH if was prepared from Fmoe-Ala-NHS and H-Cit-OH as described
above
for le-(a). Yield 77%. MS: 959.8 [2M+Na]4'; 938.1 [2M+1]+; 491.4 [M+Nar; 469.9
[M+
Crude Fmoc-ValCit-OH lg was prepared from Fmoc-Val-NHS and H-Cit-OH as
described
above for lb. The final purification was done by trituration with ether. Total
yield 76%. MS:
1060.3 [2M+3Nar; 1015.7 [2M+Nar; 519.7 [M+Na]; 497.9 [M+1]'.
Fmoc-Ala-Asn(DMCP)-OH lh was prepared from Fmoc-Ala-NHS and H-Asn(DMCP)-OH
as described above for lb. Yield 95%. MS: 530.2 [M+Na]4'; 508.2 [M+1]; 426.0
[M-
DMCP+1].
Coupling with p-aminobenzyl alcohol, preparation of Fmoc-AA-PABA and Fmoc-A-
PABA
2a-,n.
Products la-h were coupled with p-aminobenzyl alcohol (PABA) in presence of 2-
ethoxy-1-
ethoxycarbony1-1,2-dihydroquinoline (EEDQ) to form 2a-h. Four representatives
3 j-1 with
only one amino acid attached to PABA moiety were also prepared.
37
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
(i)
Fmoc-A1-A2-0H ____________________ Fmoc-A1-A2-NH OH
1 a-h 2 a-h
A1= Gly, Glu(2PhiPr), Asn(DMCP), Phe, Ala, Val.
A2=Gly, Lys(MMT), Cit, Asn(DMCP), Lys(CH3)2
0)
Fmoc-AL-OH Fmoc-A1-NH 411 OH
1 I-I
21-1
A1=Lys(CH3)2, Leu, Asn(DMCP), Cit
Conditions: (i) PABA, EEDQ, THF
For Frnoc-GlyGly-PABA 2a, a solution of la (318 mg, 0.9 mmol) and PABA (220
mg,
1.8 mmol) in DCM (17 mL) and Me0H (6 mL) were stirred with EEDQ (444 mg, 1.8
mmol)
for 10 h. All volatiles were removed on a rotovap, the residue was triturated
with Et20 and
the product was filtered out and dried in vacuo. Yield 348 mg (84%).
For Fmoc-Glu(0-2PhiPr)Gly-PABA 2b, a solution of lb (524 mg, 0.96 mmol) and
PABA
(142 mg, 1.55 mmol) in DCM (10 mL) was stirred with EEDQ (357 mg, 1.44 mmol)
for 10
h. The workup was done as described above for 2a. Yield 462 mg (74%).
Fmoc-Asn(DMCP)Gly-PABA 2c, was prepared as described above for 2a. Yield 64%.
MS:
621.5 [M+22]; 599.3 [M 1]+.
Frnoc-PheLys(MMT)-PABA 2d, was prepared as described above for 2b. Yield 70 %.
For Fmoc-PheCit-PABA 2e, a solution of le (5.98 g, 10.97 mmol) and PABA (2.70
g,
21.95 mmol) in DCM (150 mL) and Me0H (50 mL) was treated with EEDQ (5.43 g,
21.95 mmol) and stirred for 15 h. The workup was done as described above for
2a. Yield
6.14 g(86%). MS: 650.7 [M+1]"; 527.3 [M-PABA+1]"'.
For Fmoc-AlaCit-PABA 2f, a solution of If (2.89 g, 6.17 mmol) and PABA (1.52
g,
12.34 mmol) in DCM (45 mL) and Me0H (15 mL) was treated with EEDQ (3.05 g,
12.34 minol) and stirred for 15 h. The workup was done as described above for
2a. Yield
4.56g (74%). MS (ES, neg. mode): 307.4 [M-263.6-1f; 349.9 [M-Fmoc-11-; 610,
608.4
[M+HC1-1]-.
38
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Fmoc-ValCit-PABA 2g was prepared as described above for 2b. (98%).
Fmoc-AlaAsn(DMCP)-PABA 2h was prepared as described above for 2a. Yield 59%.
MS:
613.2 [M+1]+; 531.4 [M-DMCP+1]+; 408.2 [M-205+1]+.
For Fmoc-Lys(CH3)2-PABA 21, Fmoo-Lys(CH3)2-0H HC1 salt (433 mg, Immo]) and
PABA
(246 mg, 2 mmol) were dissolved in DCM (10 mL) and Me0H (1.5 mL), cooled to 5
C and
EEDQ (495 mg, 2 mmol) was added. The cooling bath was removed and the mixture
was
stirred for 10 h at room temperature. All volatiles were removed on a rotovap,
the residue was
triturated with Et20, and the crude product was filtered off. It was
redissolved in a mixture of
DCM (2 mL) and Me0H (1 mL) and precipitated again by adding dropwise into Et20
(40 mL). Product was filtered and dried in vacuo. Yield 448 mg (83%).
For Fmoc-Leu-PABA 2j, a solution of Fmoc-Leu-OH (353 mg, I mmol), EEDQ (495
mg,
2 mmol) and PABA (222 mg, 1.8 mmol) in DCM (10 mL) was stirred for 10 h. All
volatiles
were removed on a rotovap, the residue was dissolved in Et20 (40 mL), chilled
on dry ice for
2h and the solid was separated by centrifugation. The obtained crude material
was purified on
a column, eluent gradient of Me0H (1-2%) in CHC13. Yield 444 mg (97%). MS:
459.4
[M+ I r.
Fmoc-Asn(DMCP)-PABA 2k was prepared as described for 2j. In workup instead of
column
purification after removing of DCM the residue was triturated with Et20,
chilled to 0 C and
the crude product was filtered off This treatment was repeated one more time
followed by
drying in vacuo. Yield 77%. MS: 542.5 [M+11+.
For Fmoc-Cit-PARA 21, a solution of Fmoc-Cit-OH (345.7 mg, 0.87 mmol) and PARA
(214 mg, 1.74 mmol) in DCM (10 mL) and Me0H (4 mL) was treated with EEDQ (430
mg,
1.74 mmol) and stirred for 15 h. The solid product was triturated three times
with ether, and
the product was filtered and dried. Yield 288 mg (67%). MS: 502.3 [M+1]+;
485.5 [M-
F120+1]+; 263 [M-Fmoc-H20+1]+; 179.0 [M-306+11+; 120.2 [M-365.3+1]+.
Product 2m was prepared using different scheme: coupling of H-Lys(CH3)2-PABA
derivative
3 with Frnoc-Phe-NHS.
39
CA 02816041 2013-04-24
WO 2012/092373 PCT/1JS2011/067588
(i) (ii)
2 i --1.- H¨Lys(CH3)2¨NH OH
3i
Fmoc¨Phe¨Lys(CH3)2¨NH II OH
2m
Conditions: (i) Triethy lam ine (Et3N) in DMF, 10h. (ii)
Fmoc-Phe-N HS,
disopropylethylamine (DIEA), DMF,
For Fmoc-PheLys(C13)-PABA 2m, Fmoc-Lys(CH3)2-PABA (2i) (448 mg, 0.83 mmol) was
Fmoc deprotected by stirring with Et3N (3.5 mL) in DMF (11mL) for 10h. All
volatiles were
removed on a rotovap at 40 C/oil pump vacuum to obtain the crude product 3i.
This product
was dissolved in DMF (7 mL), Fmoe-Phe-NHS (482 mg, 0.996 mmol) was added
followed
by DIEA (0.42 mL, 2.2 mmol) and the mixture was stirred for 10 h. The solvent
with DIEA
was removed on a rotovap at 40 C/oil pump vacuum to obtain crude 2m which was
used
without additional purification. MS: 549.4 [Mil].
Preparation of H-AA-PABA 3a-h, in and H-A-PABA 3j-1.
Fmoc¨ At - A2¨ i II OH H¨A1-_A2¨N . 0H
2 a-h 3 a-h
(i)
H¨Al¨N OH
Fmoc¨At¨N * OH
2 j-I 3j-I
Conditions: (i) Et3N in DMF, 10h.
Fmoc-derivatives 2a-h, j-I were treated with Et3N in DMF as described above
for 3i followed
by concentration and drying in vacuo. The crude products were dissolved in DMF
to make
' 0.1 M solution and used without additional purification.
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
Table I. Intermediates of H-AA-PABA (1-3)
Al A2
1, 2, 3a Gly Gly
1, 2, 3b Glu(2PhiPr) Gly
1, 2, 3c Asn(DMCP) Gly
1, 2, 3d Phe Lys(MMT)
1, 2, 3e Phe Cit
1,2, 3f Ala Cit
1, 2, 3g Val Cit
1, 2, 3h Ala Asn(DMCP)
1, 2, 3i Lys(CH3)2
1, 2, 3j Leu
1, 2, 3k Asn(DMCP)
1, 2, 31 Cit
2, 3m Phe Lys(CH3)2
o-
2PhiPr DMCP MMP
41
CA 02816041 2013-04-24
WO 2012/092373
PCT/1JS2011/067588
Preparation of protease cleavable NAG-masking reagents.
Preparation of NAG(RI,R2,R3)-L-AA-PABC-PNP (Tables 2, 3)
0
L1= N
R1 0
0 0
R2\R"--
L2= 00.N
H
NHAc
L3= /3
Preparation of NAG(RI,R2,R3)-L-AA-PABC-PNP where RI, R2 and R3 are protective
groups
and L is a linkage between galactosamine moiety (NAG) and dipeptide (AA)
starts from
preparation of NAG-L-CO2H acids 6, 10a,b,13 and 17 which, following conversion
into NHS
ester, were used to acylate H-AA-PABA 2. In carbonates 21a-f designed for base
sensitive
polymers protective groups had to be removed before polymer modification. For
this purpose
in preparation of 10a,b,13 and 17 Ac-protective groups in GAL moiety were
replaced with
labile triethylsily1 (TES) and tert-butyldimethylsilyl (TBDMS) groups. Those
groups can be
removed using a 70% solution of trifluoroacetic acid (TFA) in H20 at 0 C
without
compromising base sensitive PNP-carbonate moiety.
a) In preparation NAG-LI-CO2H 6 where RI=R2=R3=0Ac Z-protected NAG-
tetraacetate 4
.. [3-5.1 was Z-deprotected (H2, Pd/C(10%), Me0H, CHC13 (20%) to obtain NAO-
amine 5
which was than acylated with succinic anhydride. (succinic anhydride, Et3N,
DCM, 1h).
42
CA 02816041 2013-04-24
WO 2012/092373 PCT/1JS2011/067588
Ac0 Ac0
AcOs\---
OAc (I) Ac00Ac
NHAc
NHAc
4 5
Ac0
0 0
Z-
Ac0 0
OAc = II
0
NHAc
6
Conditions: (i) H2, Pd/C (10%), Me0H, CHCI3, (20%), (ii), Succinic anhydride,
Et3N, CDM,
I h.
NAG-amine 5: For preparation of 5 a solution of NAG 4 (6.74 g, 11.85 mmol) in
Me0H
(144 mL) and CHCI3 (36 mL) was hydrogenated in the presence of 10% Pd/C (674
mg) at
1 atm. for 10 h. The catalyst was filtered off through celite, the product was
concentrated and
dried in vacua. Yield 5.04 g (98%).
NAG-LI-OH 6: For preparation of 6 a solution of succinic anhydride (966 mg,
9.65 mmol) in
DCM (30 mL) was added to NAG-amine 5 (4g, 9.15 mmol) in DCM (50 mL) followed
by
Et3N (1.964 mL, 14 mmol). After 1 h the reaction mixture was concentrated and
dried in
vacua. The product was purified on a column, eluent gradient of Me0H (5-7%) in
CHCI3µ.
Yield 3.1 g (63%). MS: 535.3 [M+1]; 330.3 [product of deglycosylationr.
b) NAG derivatives with easily removable silyl ether protective groups were
prepared by
0-deacetylation of 4 in a mixture of triethylamine in aqueous methanol
followed by treatment
with trialkylsilyl chlorides.
=
43
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
I) NAG-LI-OH I0a,b. 10a: RI=OTES and OTBDMS, R2=0H, R3=0TES; 10b: RI=R3=
OTBDMS, R2.=0H.
Preparation of NAG 8a,b.
.
HO
H 8a
HO (id)
n
4 _______________ a (10
WHAc .õ1/48b
7
RI
R2\ __________ CI H
,.,,---
R3 `-.,../..""-,0./..\..,,,'N,,z 8a: R1=0TBDMS and
OTES,.R2=011, R3=0TES
8b: R1=R3=0TBDMS, R2=0H
NHAc
,
Conditions: (i) Et3N, Me0H, H20 (5:7:6) 10h. (ii) TBDMSC1 (1 eq.), imidazole,
lh followed
by TESCI (3 eq.), 10h in DMF. (iii) TBDMSCI (3 eq.), imidazole, 10h in DMF.
NAG derivative 7.
Preparation of 10a,b.
1,2'
R2 0 H
8a,b oi)
----1. . \Li,
0
NHAc ci-ac
9 a,b 10 a,b
9,10 a: R1=0TBEMS and OTES, R2f1, RpOTES
9, 10 b: 121=R3TIKAIS, R2#1
Conditions: (i) F12, Fd/C (10%), THF. (ii) succinic anhydride, Et3N, DCM, lb.
For preparation of 7 NAG 4 (2g, 3.52 mmol) was 0-deacetylated by stirring in a
solution of
Me0H (10 mL), H20 (32 mL), and Et3N (25 mL) for 10 h. All volatiles were
removed on a
rotovap at 40 C and the residue was dried by two evaporations of toluene from
the reaction
44
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
mixture. The product 7 was directly used in the following step. MS: 544.3
[M+Et3N+1r;
443.7 [M4-11+; 204 [product of deglycosylationr.
For preparation of 8a product 7 (1.76 mmol) in DMF (15 mL) was treated with
imidazole
(718 mg, 10.54 mmol) and TBDMSCI (265 mg, 1.76 mmol), stirred for 2 h and the
reaction
mixture was cooled to 0 C. TESCI (531 mg, 3.52 mmol) was added, stirred for 10
h,
concentrated and dried in vacua. The residue was taken in a mixture of Et0Ac
(110 mL) and
H20 (30 mL). The organic layer was separated, cooled to 5 C, washed with
citric acid (5%),
H20, NaHCO3 and dried (Na2SO4). The crude product was passed through a column,
eluent
Me0H 2% in CHCI3 to afford a mixture of TBDMS and TES di Si-protected NAG
derivatives 8a. Yield 575 mg (49%). MS: 672.0 [M+1]-; 432.5 [product of
deglycosylationr.
For preparation of 8b a solution of 7 (1.76 mmol) in DMF (15 mL) was stirred
with
imidazole (718 mg, 10.56 mmol) and TBDMSCI (1.061 g, 7 mmol) for 10 h. The
reaction
mixture was processed as described above for preparation of 8a. Yield after
column
purification 767 mg (65%). M: 672.0 [M+I]; 432.7 [product of deglycosylation].
Product 10a was prepared as a mixture of TBDMS and TES di Si-protected NAG
derivatives
following procedure described for 10b below.
For preparation of 10b compound 8b (920 mg, 1.37 mmol) was hydrogenated in THF
(20 mL) in presence of Pc/C 10% (150 mg) at I atm for 10 h. The catalyst was
filtered off
through celite, the product 9b was concentrated and dried in vacua.
The NAG-amine 9b without additional purification was dissolved in DCM (12 mL),
succinic
anhydride solution (140 mg, 1.40 mmol) in DCM (7 mL) was added followed by
Et3N
(0.236mL, 1.676 mmol) and stirred for 2 h. Solvent was removed on a rotovap
and product
was purified on a column, eluent 1% AcOH, 10% Me0H in CHCI3. Yield 614 mg
(72%).
ii) NAG-L2-0H 13. R1=R3=0TBDMS, R2=0H. NAG derivatives with longer PEG spacer.
For analogues with longer PEG spacers, the precursor 5 was first acetyl-
deprotected to yield
11 (Et3N, Me0H, H20 (5:7:6) 10h). 11 was then acylated with bis-dPEGshalf
benzyl half
NHS ester (Quanta product cat. #10237) to yield the benzyl ester 12 (NHS-PEG5-
0O2Bn,
Et3N, DCM). 12 was subsequently bis-silylated with TBDMSCI (TBDMSCI (3 eq.),
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
imidazole, 10h in DMF) and debenzylated by hydrogenation (H2, Pd/C (10%), THF)
to
obtain acid 13.
Preparation of NAG-derivative 12.
o 5
9 00 CINNcr7N82
H:14, H i
CH CtN/Nc("N/NN,'N.7
0 N-tAc
NHAc
11 12
Conditions: (i) Et3N, Me0H, H20 (5:7:6) 10h. (ii) NHS-PEG5-0O2Bn, Et3N, DCM.
Preparation of NAG-L2-0H 13.
TBDMSO
\ 12 --0.- 0.,,.;1 0 0Bnµ
OTBDe N --
5 (n)
HO
_________________________________________________________________ li
0 0
NHAc
. NAG-L2-0Bn
TBDMSO
H (/oH ,
5 o
NHAc
13
Conditions: TBDMSCI (3 eq.), imidazole, 10h in DMF. (vi) H2, Pd/C (10%), THF.
NAG-PFG8-SA hen7y1 ester 12 For preparation of NAG-amine 11 NAG-amine 5
(0.381 mmol) was 0-deacetylated as described for precursor 7 (procedure for
8a,b). Product
11 was dried by two evaporations of toluene on a rotovap and dissolved in DMF
(25 mL).
Bis-dPEG5half benzyl half NHS ester (200 mg, 0.381 mmol) was added to the
reaction
mixture followed by DIEA (0.079 mL, 0.457 mmol), stirred for 8 h and
concentrated on a
rotovap at 40 C/oil pump vacuum. Crude product 12 was used in the next step
without
additional purification. MS: 719.4 [M+1]+; 516.4 [product of deglycosylationr,
For preparation of NAG-L2-0Bn dry product 12 was dissolved in DMF (5 mL),
treated with
TBDMSCI (230 mg, 1.524 mmol) followed by imidazole (156 mg, 2.29 mmol). The
reaction
mixture was stirred for 10 h, all volatiles were removed on a rotovap at 40
C/oil pump
46
CA 02816041 2013-04-24
WO 2012/092373 PCT/US2011/067588
vacuum and the residue was taken in Et0Ac (85 mL) and washed with HC1 (1%),
H20. The
aqueous phases were combined and back extracted with Et0Ac. The combined
organic
solutions were dried (Na2SO4), concentrated and purified on a column, eluent
gradient of
Me0H (3-6%) in CHC13. Yield of the benzyl ester 291 mg (80 %). MS: 965.3
[M+NE14]+;948.0 [M+1]+; 516.4 [product of deglycosylationr.
For preparation of NAG-L2-0H 13 the ester NAG-L2-0Bn was hydrogenated as
described for
9b (procedure for 10b). Yield 98%. MS: 858.0 [M+1]t; 426.1 [product of
deglycosylation].
The product was used without additional purification.
iii) NAG-L3-0H 17. RI=R3=0TBDMS, R2=0H. NAG derivatives with longer PEG
spacer.
17 was prepared by glycosylation of pentaacetate 14 [3-5] with PEG4 mono-tBu
ester
(TMSOTf, DCE / HO-PEG4-0O2tBu, SnC14, DCM) to yield 15. 15 was hydrolyzed
(HCO2H,
10h) to obtain the acid 16, which was then 0-deacetylated (Et3N, Me0H, H20
(5:7:6) 10h)
and treated with TBDMSCI (TBDMSCI (3 eq.), imidazole, 10h in DMF) to obtain
bis-
silylated NAG-acid 17.
Preparation of ester NAG(0Ac)3-L3-0-t13u 15.
AcOs\r/v,
Ac0
0
OAc
Ac0 0 0
OAc
OAK AC Oikt
0
NI-lAc N NI1Ac 1
14 5
NAO-oxazoline
Conditions: (i) trimethylsilyl trifluoromethanesulfonate (TMSOTf),
dichloroethane (DCP.)
(ii) t-butyl 12-hydroxy-4,7,10-trioxadodecanoate (HO-PEG4-0O2tBu), Snat,
dichloromethane (DCM).
47 =
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
Aco\R___
Ho
(14
AGO
OAc H HO
OH
NHAc 16
NHAc
TESOMS0
0
(iii) HO
On
NHAc
17
Conditions:(i) HCO2H, 10h. (ii) Et3N, Me0H, H20 (5:7:6) 10h. (iii) TBDMSCI (3
eq.),
imidazole, 10h in DMF.
5 For ester
NAG(0Ac)3-L3-0-tBu 15 pentacetyl derivative of galactosamine 14 (10 g,
25.64 mmol) was dried by two evaporations from toluene. The resulting white
glass was
treated with TMSTf (5.18 mL, 28.6 mmol) in DCE (223 mL), and stirred at 60 C
for 16 h.
The reaction mixture was cooled to 0 C, quenched with TEA (2.6 mL), diluted
with CHC13
(300 mL) and washed twice with a NaHCO3 solution and with brine. The separated
organic
10 solution was
treated with MgSO4, concentrated, and dried in vacuo. The crude oxazoline
derivative was used without additional purification. Yield 8.14 g (96%). MS:
368.1 [M+Kr;
352.2 [M+Na]"; 330.2 [M+1]4.
To a stirring mixture of oxazoline derivative (5.28 g, 16 mmol), t-butyl 12-
hydroxy-4,7,10-
trioxadodecanoate (5.12g, 18.4 mmol) and CaSO4 (20g) in DCM (270 mL) was added
15 dropwise
SnC14 (0.84 mL, 0.84 mmol). The solution was stirred for 16 h, filtered,
diluted
with CFICI3 (250 mL), washed twice with NaHCO3 solution and brine. Product was
dried
with MgSO4, and concentrated. The crude was purified on a column, eluent
gradient of
Me0H (0-7%) in ethyl acetate. Yield 4.83 g (50%). MS: 630.8 [M+Na]; 625.5
[M+NH4];
608.4 [M+1]+; 552.6 EM-t-Bu+11+; 330.2 [product of deglycosylation]t
For NAG(0Ac)3-L3-0H 16, tert-butyl ester 15 (1.99 g, 3.27 mmol) was stirred in
neat formic
acid (54 mL) for 16 h and all volatiles were removed in vacuo followed by
three evaporations
of toluene. Product was dried with vacuum oil pump for 2 h and used without
additional
purification. Yield 1.77 g (98%). MS: 330.2 [product of deglycosylationr;
590.4 [M+Kr;
574.6 [M+Nar; 569.6 [M+NH4r; 552.6 [M+1r.
48
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
For NAG(RI,R2,R3)-L3-0H 17 (RI=R3=0TBDMS, R2=0H), product 16 was 0-
deacetylated,
treated with TBDMSC1 as in preparation of 8b and purified on a column, eluent
3% Me0H,
0.5% AcOH in CHCI3. Yield 18 %. MS: 1228.7 [Wi], 796.7 [product of
deglycosylation].
All five obtained acids 6, 10a,b, 13, 17 were converted into NHS ester 18a-e
in reaction with
NHS and DCC (NHS, DCC, DCM, 10h).
0
Ll=
0
(I) 0 0
R2 R3 r¨NHS , 6,10,13,17 --00-
5
NHAc
18 a-e L3=
3
0
18a R1=R2=R3=0Ac,
18b R1=0TBDMS and CITES, R2=0H, R3=TES, L1
18c RI=R3=OTBDMS, R2=0H,
16d R1=R3=0TBDMS, R2=0H, L2
18e R1=R3=0TBDMS, R2=0H, L3
Conditions: (i) NHS, DCC, DXCM, 10h.
For preparation of NAG-L-NHS 18a-e was used procedure described for 18c below.
For
product 18c, an ice cold solution of 10b (614 mg, 0.964 mmol) and NHS (122 mg,
1.061 mmol) in DCM (15 mL) was treated with DCC (219 mg, 1.061 mmol), stirred
for
30 min on ice and 8 h at 20 C. The reaction mixture was cooled to 0 C, DCU was
filtered
off, the residue was concentrated and dried in vacuo. The crude product was
dissolved in
DMF to make 0.05 M solution and used without additional purification.
Products 18b-e were prepared as described for 18a.
c) Formation of 20a-I, 3a-h were acylated with NHS ester of hydroxyl-protected
NAG-
derivatives 18a-e (D1EA, DMF, 5-10h) to provide 19a-1. Products 19a-1 were
than
treatedwith 5 equivalents of bis(p-nitrophenyl) carbonate ((PNP)2C0)
((PNP)2CO3 dioxane or
DCM, 40-50 C, 15-24h) to yield the 0-Acetyl protected PNP carbonate
derivatives 20a-1.
Products 20a-e were used directly for modification of peptides. The acetyl
groups and
49
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
protective groups 2PhiPr, DMCP, MMT from amino acids were removed post
modification
during consecutive treatment of DPC with TFA and Et3N.
ip OH
3 al 18 a.= p.
NaG4.-AiArReeek
19a4
0 NONI1/41)
1)
0 0
tbaG(0603-1r¨A,¨)52¨ # 0A = # NO2 NCRIR2Ra)-1--Ai¨A2¨ = # NO2
213 =-= 2014
Conditions: (i) DIEA, DMF, 5-10h. (ii) (PNP)2CO3 dioxane or DCM, 25-60 C, 16-
48h.
NAG(R I R2R3)-L-A A-PABA 19a-I.
For product 19a (RI=R2------R3=0Ac, L=LI, AA=GlyGly), an NAG-NHS ester 18a
solution in
DMF (0.05M) (0.282 mmol) was treated with 0.1 M of solution of 3a (0.282 mmol)
in DMF
and DIEA (59 AL, 0.338 mmol). In 3 h all volatiles were removed on a rotovap
at 40 C/oil
pump vacuum, triturated with Et20 and purified on a column, eluent: gradient
Et0Ac:CHC13:Me0H=8:7:5-8:7:6. Yield 114 mg (53%). MS: 754.4 [M+Ir.
Product 19b (RI=R2=R3=0Ac, L=LI, AA=Glu(2PhiPr)Gly) was prepared as described
for
19a and purified on a column, eluent Et0Ac:CHC13:Me0H=-8:7:3. Yield 64%. MS:
944.5
[M+ 1 r.
For product 19c (RI=R2=R3=0Ac, L=Li AA=Asn(DMCP)Gly), to a solution of 3c
(0.43 mmol) and DIEA (83 piL, 0.476 mmol) in DMF (2.15 iriL) was added a
solution of 18a
(0.43 mmol) in DMF (2.15 mL). The mixture was stirred for 16 h, filtered and
all volatiles
were removed on a rotovap at 40 C/oil pump vacuum .The crude product was
triturated with
Et20 and purified on a column, eluent CHC13:acetone:Me0H (5:5:1). Yield 242 mg
(62%).
MS: 915.3 [M+Na]"; 910.6 [M+NRs]4'; 893.6 [M+1]+.
Product 19d (RI=R2--R3=0Ac, L=L' AA=PheLys(MMT)) was prepared as described for
19a
and purified on a column, eluent gradient of Me0H (5-6%) in CHCI3. Yield 56%.
MS:
1187.9 [M+Ir.
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
For product 19e (RI =R2=R3=0Ac, L=L', AA=PheCit), to a solution -of 3e (0.57
mmol) and
DIEA (119 pt, 0.684 mmol) in DMF ,(3 mL) was added a solution of 18a (0.57
mmol) in
DMF (3 mL). The mixture was stirred for 16 h, filtered and all volatiles were
removed on a
rotovap at 40 C/oil pump vacuum. The crude product was precipitated into Et20
(45 mL)
from CHC13:Me0H (5 mL) and used without additional purification. Yield 392 mg
(73%).
MS: 966.8 [M+Na]; 944.7 [M+1]'; 926.8 [M-H20]*; 821.5 [M-PABA+In 615.6 [M-
NAcGal+I]; 492.3 [M-PABA-NAcGal+I].
Product 19f (R)=R3=0TBDMS, R2=0H, L=L', AA=AlaCit) was prepared as described
for
19e and used without additional purification. Yield 50%. MS: 993.2 [M+Nar;
971.0 [M+1r;
539.6 [product of deglycosylation].
Product 19g (RI=R3=0TBDMS, R2=0H L=L', AA=ValCit) was prepared as described
for
19f and used in the next step without additional purification. Yield 67%:
998.9 [M+1].
Product 19h (RI=R3=0TBDMS, R2=0H L=L', AA=Glu(2PhiPr)Gly) was prepared as
described for 19a and purified on a column, eluent solution of NH4OH 3% and
Me0H 7.5%
in DCM. Yield 15%. MS: 1047.2 [M+1]4, 615.7, 432.6 [product of
deglycosylationr.
Product 191 (RI=R3=0TBDMS, R2=0H, L=1.1, AA=PheCit) was prepared as described
for
preparation of 19e and used without additional purification. Yield 50%. MS:
1068.7
[M+Na]; 1047.3 [M+1r; 615.4 [product of deglycosylation]; 432.5 [product of
deglycosylation].
Product 19j (RI=OTBDMS and OTES, R2=0H, R3=0TES, L=LI, AA=PheCit) was prepared
from 3e and 18b as a mixture of C-3 and C-6 O-TBDMS and 0-TES protected NAG
derivatives as described for preparation of 19e and used in the next step
without additional
purification. Yield 76%. MS: 1047.4 [M+ I ], 615.8 [product of
deglYcosylationr.
Product 19k RI=R3=0TBDMS, R2=0H, L=L2, AA=PheCit was prepared as described for
19e and used in the next step without additional purification. Yield 67%:
1268.2 [M+1];
835.9 [product of deglycosylationr
Product 191 RI=R3=0TBDMS, R2=0H, L=L3, AA¨PheCit was prepared as described for
19e
and purified on a column, eluent 5% Me0H solution in CHCI3 Yield 60%. MS:
1064.0
[M+1]; 632.7 [product of deglycosylation].
51
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
NAG-AA-PABC-PNP 20a-1
For product 20a (12.1=R2=R3=0Ac, L=L', AA=GlyGly), a suspension of 19a (100
mg,
0.132 mmol), (PNP)2C0 (202 mg, 0.663 mmol) and DIEA (0.07 mL, 0.396 mmol) in
dioxane
(5 mL) was stirred for 8 h in the dark at 40 C. Another portion of (PNP)2C0
(121 mg,
0.397 mmol) and DIEA (0.04 mL, 0.226 mmol) were added and stirring was
continued
another 8 h at 40 C. All volatiles were removed on a rotovap and the product
was purified on
a column, eluent: CHC13:Et0Ac:Me0H=7:8:3. Yield 84 mg (69%).
For product 20b (RI=R2=R3=0Ac, L=L', AA=Glu(2PhiPr)Gly), a solution of 19b
(160 mg,
0.169 mmol), (PNP)2C0 (258 mg, 0.847 mmol) and DIEA (0.09 mL, 0.507 mmol) in
DCM
(10 mL) was stirred in the dark for 10 h, concentrated on a rotovap and the
product was
purified on a column, eluent: 5-6% Me0H solution in CHC13. Yield 174 mg (92%).
For product 20c (12.1=R2=R3=0Ac, L=L' AA=Asn(DMCP)Gly), a solution of 19c (127
mg,
0.142 mmol), (PNP)2C0 (216 mg, 0.710 mmol) and DIEA (74 ftL, 0.426 mmol) in
DCM
(5 mL) was stirred in the dark for 16 h, concentrated on a rotovap and the
product was
purified on a column, eluent: CHC13:Et0Ac:Me0H (7:2.2:0.8). Yield 110.6 mg
(74%). MS:
1080.9 [M+Nar; 1058.7 [M+1].
Product 20d (R1=R2=R3=0Ac, L=LI AA=PheLys(MMT)) was prepared as described for
20b
and purified on a column, eluent: CHC13:Et0Ac:Me0H= 9:7:1 Yield 76 mg (47%).
For product 20e (RI=R2=R3=0Ac, AA---PheCit), a solution of 19e (164 mg,
0.173 mmol), (PNP)2C0 (528 mg, 1.73 mmol) and DIEA (182 L, 1.04 mmol) in
dioxane
(17 mL) was stirred in the dark at 60 C for 16 h and all volatiles were
removed on a rotovap.
The residual DIEA was removed by two consecutive evaporations of DMF on a
rotovap at
40 C/oil pump vacuum and the product was purified on a column, eluent
CHC13:Et0Ac:Me0H (8:1.5:0.5) followed by CHC13:Me0H (7:1). Yield 85 mg (44%).
MS:
1132.0 [M+Na]; 1110.1 [M+1]*; 780.8 [product of deglycosylationr.
Product 20f (12.1=R3=0TBDMS, R2=0H, L'L', AA=AlaCit) was prepared as described
for
20e and purified on a column, eluent: CHC13:Et0Ac:Me01-1,-9:10:1. Yield 153 mg
(36%).
52
CA 02816041 2013-04-24
WO 2012/092373 PCT/US2011/067588
.
Product 20g (RI=R3=0TBDMS, R2=0H, L=L', AA=ValCit) was prepared as described
for
20e and purified on a column, eluent CHC13:Et0Ac:Me0H=16:3:1. Yield 44%. MS:
1164.5
[M+1].
Product 20h (R1=R3=OTBDMS, R2=0H, L=LI, AA=G1u(2PhiPr)Gly) was prepared as
described for 20b and purified on a column, eluent CHC13:Et0Ac:Me0H=8:7:1.
Yield 77%.
For product 201 (RI=R3=0TBDMS, R2=0H, L=L1, AA=PheCit), a solution of 191 (316
mg,
0.297 mmol), (PNP)2C0 (913mg, 2.97 mmol) and DIEA (310 )11,, 1.78 mmol) in
dioxane
(7 mL) was stirred in the dark at 65 C for 40h and all volatiles were removed
on a rotovap.
The residual DIEA was removed by two consecutive evaporations of DMF on a
rotovap at
40 C/oil pump vacuum and the product was purified on a column, eluent
CHC13:Et0Ac:Me0H (8:1.5:0.5) followed by CHC13:Me0H (92:08). Yield 297 mg
(81%).
MS: 1246.7 [M+NH4r; 1228.7 [M+1]; 797.6 [product of deglycosylation] +; 432.7
[product
of deglycosylation]t
Product 20j (11.1=0TBDMS and OTES, R2=0H, R3=0TES, L=L', AA=PheCit), product
20j
as a mixture of C-3 and C-6 O-TBDMS and 0-TES protected NAG derivatives was
prepared
as described for 20e and purified on a column, eluent CHC13:Et0Ac:Me0H=16:3:1
followed
by 10% Me0171 in CHC13. Yield 50%. MS: 1212.0 [M+1], 480.0 [product of
deglycosylation].
Product 20k (RI=R3=0TBDMS, R2=0H, L=L2, AA=PheCit) was prepared as described
for
20e and purified on a column, eluent gradient of Me0H (8-10%) in CHC13. Yield
85%.
Product was used directly in the next step.
For product 201 (RI=R3=OTBDMS, L=L3,
AA=PheCit), solution of 191 (316 mg,
= 297 mmol), (PNP)2C0 (912 mg, 3 mmol) and DIEA (0.31 mL, 1.78 mmol) in
dioxane
(8 mL) was stirred under Ar in the dark at 60 C for 48h. All volatiles were
removed on a
rotovap and the product was purified on a column, eluent:
CHC13:Et0Ac:Me0H=16:3:1.
Yield 297 mg (81 %).
NAG-L-AA-PABC-PNP 21a-f (RI=R2=R3=0H), deprotection of 20f-I
Formation of 21a-f. For modification of base sensitive polyacrylates, the
protective groups on
NAG and dipeptide AA were removed before attachment to polymer by treatment of
20f-I
53
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
with a mixture of TFA:H20=3:1(TFA/H20=3:1, 5 C, 2-3h) to provide NAG-dipeptide
masking reagents 21a-f.
20 f-1 NAG(ON)3-L--A1-A2---NH= OA*0 =
NO2
21a-f
Conditions: TFA/H20=3:1, 5 C, 2-3h
For product 21a (AA=AlaCit, LL') compound 20f (150 mg, 0.132 mmol) was stirred
in an
ice cold TFA:H203: I solution (2 mL) for 4 h and added dropwise to stirring
Et20 (20 mL).
The precipitate was separated and dried by evaporation of toluene on a
rotovap/30 C and then
in vacuo. Yield 112 mg (94%). MS: 907.2 [M+1]+; 704.4 [product of
deglyeosylationr.
For product 21b (AA=ValCit, L=L5 compound 20g (305 mg, 0.26 mmol) was stirred
in an
ice cold TFA:H20=3:1 solution (5 mL) for I h and added dropwise to stirring
Et20 (45 mL).
The solid product was separated and dried by evaporation of toluene on a
rotovap/30 C and
then in vacuo. Yield 193 mg (79%). MS: 935.8[M-Flr, 732.7 [product of
deglycosylationr.
Product 21c (AA=GluGly, L=L') was prepared as described for 21b. Yield 55 mg
(98 %).
.. MS: 865.5 [M+1r, 662.3 [product of deglycosylationr.
Product 21d (AA=PheCit, L=L') was prepared as described for 21b. MS: 983.7
[M+1]4
,
780.9 [product of deglycosylation]4.
For product 21e (AA=PheCit, L¨L2) compound 20k was stirred in an ice cold
TFA:H20=3:1
solution (5 mL) for 1.5h under conditions described for 21b. Yield 25%
counting from I9k.
MS: 1203.9 [M+1]', 1001.0 [product of deglycosylation]t.
Product 21f (AA=PheCit L=L3) was prepared from 201 using 3 h deprotection
under
conditions described for 21b. Yield 75%. MS: 1203.9 [M+1r, 1001.0 [product of
deglycosylationr.
54
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Table 2. Intermediates NAG-L-AA-PABA (19) and NAG-L-AA_PABC (20)
compound A1 A2 L RI R2 R3
19a Gly Gly Li OAc OAc , OAc
19b , Glu(2PhiPr) Gly L1 OAc _ OAc OAc
19c Asn(DMCP) Gly Li OAc OAc OAc
19d Phe Lys(MMT) Li OAc OAc OAc
19e Phe Cit L1 OAc OAc OAc
19, 201 Ala Cit L1 OTBDMS OH OTBDMS
19, 20g Val Cit Li OTBDMS OH OTBDMS
_
19, 20h Glu(2PhiPr) Gly Li OTBDMS OH OTBDMS
19,201 Phe Cit L1 OTBDMS
OH OTBDMS
19, 20j Phe Cit Li OTBDMS, OH OTES
TES
19, 20k Phe Cit L2 OTBDMS OH OTBDMS
19, 201 Phe Cit L3 OTBDMS OH OTBDMS
Table 3. Final NAG-L-A1A2-PABC used for DPC preparation. (20, 21)
compound A1 A2 L
20a Gly Gly L1
20b Glu Gly LI
20c Asn Gly Li
20d Phe Lys Li
20e Phe Cit Li
21a Ala Cit Li
21b Val Cit Li
2k Glu Gly Li
21d Phe Cit Li
21e Phe Cit L2
211 Phe Cit L3
Preparation of protease cleavable PEG-masking reagents.
The amino group of any of H-AA-PABA 3b,e,g,h,j,k-m was acylated with an NHS
ester of
PEG-acid (D1EA, DMF, 5-10h) to yield 22a-k. The hydroxyl group in product 22a-
k was
then converted into p-nitrophenyl carbonate ((PNP)2CO3 dioxane or THF, 40-60
C, 10h) to
yield 23a-k. For 23a,d,g, protective groups from Asn and Glu were removed by
treatment
with aqueous TFA (TFA/H20=3:1, 5 C, 2-3h) to obtain desired products 24a-c.
(Can you
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
retain consistency by converting 23a, d, g to 24a, d, g?) Also, is there
consistency between
the amino acids for each letter between NAG and PEG versions?
Preparation of PEGn-AA-PABA 22a-k.
(I)
+ 3 b,eigN-m
Su
OH
0
22 a-k
PEG-AA(Prot)-PABA
Conditions: (i) DIEA, DMF, 5-10h.
Product 22a (n=11, AA=GluGly). A 0.1M solution of 3b in DMF (3.5 mL, 0.35
mmol) was
stirred for 10 h with PEG] i-NHS ester (240 mg, 0.35 mmol) and DIEA (0.061 mL,
0.35 mmol). All volatiles were removed on a rotovap at 40 C/oil pump and the
product was
purified on a column, eluent: CHC13:MeOH:Ac01-1=38:2:1. Yield 274 mg (78%) MS:
1015.6
[M+Nfla], 998.7 [M+1].
Product 22b (n-11, AA=PheCit). To a solution of 3e (0.88 mmol) and DIEA (167
0.96 mmol) in DMF (3 mL) was added a solution of PEG' i-NHS ester (0.80 mmol)
in DMF
(3 mL). The mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap
at /10 C/oil pump vacuum. The crude was precipitated into Et20 (45 mL) from
CHC13:Me0H
(5 mL) and purified on a column, eluent a gradient of Me0H (10-16%) in CHCI3.
Yield
420 mg (53%). MS: 1015.9 [M+H2O]; 998.8 [M+ I r; 981.1 {M-H2O].
Product 22c (n=11, AA=ValCit). Product 22f was prepared from crude 3g
(obtained from
300 mg, 0.5 mmol of 2g), PEG11-NHS ester (298 mg, 0.435 mmol) and DIEA (0.09
mL,
0.522 mmol) as described for 22a. Following concentration on a rotovap at 40
C/oil pump
the product was suspended in a MeOH:DCM=1:1 mixture (6 mL), sonicated,
filtered and
precipitated into Et20 (50 mL). The solid was separated and the procedure
repeated again.
The residual solvents were removed in vacuo. Yield 283 mg (60%). MS: 951.5
[M+I]".
56
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Product 22d (n=11, AA=AlaAsn(DMCP)). To a solution of 3h (0.56 mmol) and DIEA
(116 i.tL, 0.67 mmol) in DMF (3 mL) was added a solution of PEGII-NHS ester
(0.56 mmol)
in DMF (3 mL). The mixture was stirred for 16 h, filtered and all volatiles
were removed on a
rotovap at 40 C/oil pump vacuum. The residue was dissolved in a CHC13:Me0H=1:1
mixture
(5 mL) and precipitated into chilled (0 C) Et20 (45 mL). The solid was
purified on a column,
eluent gradient of Me0H (3-14%) in DCM. Yield 261 mg (49%). MS: 983.7 [M+Nar;
979.1
[M+NH41; 961.8 [M+I]; 943.9 [M-H20+1]*.
Product 22e (n=11, AA=PheLys(Me2)). Product 22e was prepared as described for
22a.
Purification was done using HPLC column Nucleodur C-18, 250 x 4.6, eluent ACN-
H20
(0.1% TEA), ramp 15-30%. MS: 998.1 [M+1]+. The isolated product was desalted
on Dowex
I x8 resin, eluent H20. Yield 40%.
Product 22f (n=11, AA=Leu). Product 22f was prepared as described for 22a and
purified on
a column, eluent: CHC13:Et0Ac:MeOH:AcOH=9:7:2:0.04. Yield 48%. MS: 824.9
[M+NH4r.
Product 22g (n=11, AA=Asn(DMCP). Crude 3k (obtained from 419 mg, 0.77 mmol of
2k),
Pegi 'NHS ester (200 mg, 0.292 mmol) and DIEA (0.06 mL, 0.35 mmol) were
stirred in DCM
(5 mL) for 10 h. The solvent was removed on a rotovap and the product was
purified on a
column, eluent CHC13:Et0Ac:Me0H AcOH=4.5:3.5:1:0.02. Yield 254 mg (37%). MS:
891.1
[M+1]+.
Product 22h (n---I 1 AA=Cit). To a solution of 31(0.50 mmol) and DIEA (104 pL,
0.60 mmol)
in DMF (2.5 mL) was added a solution of PEGII-NHS ester (0.50 mmol) in DMF
(2.5 mL).
The mixture stirred for 16 h, filtered and all volatiles were removed on a
rotovap at 40 C/ oil
pump vacuum. The residue was dissolved in a CHC13:Me0H=1:1 mixture (5 mL) and
precipitated into Et20 (45 mL). Precipitation was repeated two more times and
the product
was used without additional purification. Yield 340 mg (80%). MS: 869.4
[M+NH4]'; 851.9
[M+1}+.
Product 221 (n=23, AA=PheCit). To a solution of 3e (0.72 mmol) and DIEA (130
'IL,
0.74 mmol) in DMF (3 mL) was added a solution of PEG23-NHS ester (0.60 mmol)
in DMF
(3 mL). The mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap
at 40 C/ oil pump vacuum. The residue was dissolved in a CHC13:Me0H=1:1
mixture (5 mL)
and precipitated into Et20 (45 mL). The solid product was purified on a
column, eluent
57
CA 02816041 2013-04-24
WO 2012/092373
PCMTS2011/067588
gradient of Me0H (7-12%) in Cl-1C13. Yield 487 mg (53%). MS: 1555.2 [M+Na]+;
1544.7
[M+NH4r;1527.7 [M+1]+.
Product 22j (PEG with average MW 1000. AA=PheCit). A mixture of mPEG-1000-
alcohol
(Fluka) (0.173g, 0.173 mmol), N,N-disuccinimidyl carbonate (62 mg, 0.242
mmol), and TEA
(0.101 mL, 0.726 mmol) were stirred in MeCN (1 mL) for 16 h. All volatiles
were removed
on a rotovap and the crude residue was dissolved in CHCI3 (10 mL). The organic
layer was
washed with H20 (1 mL, pH=5), then brine, dried over Na2SO4 and concentrated
to afford
PEG-1000-NHS carbonate. This product was stirred for 16 h with 3e (0.121 mmol)
and DIEA
(30 t.tL, 0.173 mmol) in DMF (1 mL), filtered and all volatiles were removed
on a rotovap at
40 C/oil pump vacuum. The residue was dissolved in a CHC13:Me0H--1:1 mixture
(5 mL)
and precipitated into Et20 (45 mL). Precipitation was repeated two more times
and the
product was used without additional purification. Yield 134 mg (79%).
Product 22k (rr-----23, AA=ValCit). To a solution of 3g (1.0 mmol) and DIEA
(183 1.1.1õ,
1.04 mmol) in DMF (4 mL) was added a solution of PEG23-NHS ester (0.87 mmol)
in DMF
(4 mL). The mixture was stirred for 16 h, filtered and all volatiles were
removed on a rotovap
at 40 C/ oil pump vacuum. The residue was dissolved in a CHC13:Me0H=1:1
mixture (5 mL)
and precipitated into Et20 (45 mL). Precipitation was repeated two more times
and the
product was used without additional purification. Yield 1.0 g (77%). MS:
1496.1
[M+NI-14]+;1479.3 (M+1]+.
PEG-AA-PABC-PNP 23a-k
0
(I) 11
22 a-k rHr
A-2-NH .µ0 NO2
0
23 a-k
PEG-AA(Prot)-PABC-PNP
Condition: (i) (PNP)2CO3 dioxane or THF, 40-60 C, 10h.
For product 23a (n=11, AA=Glu(2PhiPr)Gly), product 22a (274 mg, 0.274 mmol) in
DCM
(15 mL) was stirred in the dark with (PNP)2C0 (418 mg, 1.372 mmol) and DIEA
(0.143 mL,
0.823 mmol) for 15 h. The solvent was removed on a rotovap and the product was
purified on
58
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
a column, eluent 4% Me0H, 0.2%AcOH in CHCI3. Yield 260 mg (81%). MS: 1180.7
[M+NH4r,
For product 23b (n=11, AA=PheCit), a solution of 22b (419 mg, 0.42 mmol),
(PNP)2C0
(766 mg, 2.52 mmol) and DIEA (263 L, 1.52 mmol) in dioxane (4 mL) was stirred
in the
dark at 50 C for 15h and all volatiles were removed on a rotovap. The residual
DIEA was
removed by two consecutive evaporations of DMF on a rotovap at 40 C/oil pump
vacuum
and the product was purified on a column, eluent CHC13:Et0Ac:Me0H (4.5:5:0.5)
followed
by CHC13:Me0H (9:1). Yield 390 mg (80%). MS: 1181.2 [M+NH4],1164.2 [M+11+.
For product 23c (n=11, AA=ValCit), a solution of 22c (273 mg, 0.287 mmol),
(PNP)2C0
(874 mg, 2.88 mmol) and DIEA (0.3 mL, 1.72 mmol) in 1,4-dioxane (22 mL) was
stirred in
the dark for 24 h at 50 C. The solvent was removed on rotovap at 40 C/oil pump
and the
product was purified on a column, eluent: CHC13:Et0Ac:Me0H=16:3:1 followed by
12-15%
Me0H in CHCI3 Yield 163 mg (51%). MS: 1116.0 [M+1]+.
Product 23d (n=11, AA=AlaAsn(DMCP)) was prepared as described in the
preparation of
23b. The product was purified on a column, eluent CHC13:Et0Ac:Me0H (9:2:1).
Yield 77%.
MS: 1144.0 [M+NHa]4; 1127.3 [M+Ir.
Product 23e (n=11, AA=PheLys(Me)2) was prepared as described for 23a and
purified on a
column, eluent: 10% Me0H, 0.2%AcOH in CHC13. Yield 63%. MS: 1163.1 [M+13+
Product 23f (n=11, AA=Leu) was prepared as described for 23c using only 5
equivalents of
(PNP)2C0 and 3 equivalents of DIEA applying heat for 24h. The product was
purified on a
column, eluent gradient of Me0H (7-12%) in Cl-1C13. Yield 75%. MS: 972 [M+1]t
Product 23g (n=11, AA=Asn(DMCP)) was prepared as described for 23f and the
crude
product was used in the following step without additional purification. MS:
1073.4 [M+18]4.
For product 23h (n=11, AA=Cit), solution of 22h (340 mg, 0.40 mmol), (PNP)2C0
(608 mg,
2.00 mmol) and DIEA (208 ttL, 1.20 mmol) in DCM (4 mL) was stirred in the dark
at 30 C
for 15h and all volatiles were removed on a rotovap. The residual DIEA was
removed by two
consecutive evaporations of DMF on a rotovap at 40 C/oil pump vacuum and the
product
was purified on a column, eluent CHC13:Et0Ac:Me0H (7:2.5:0.5) followed by a
gradient of
Me0H (8-14%) in CHC13. Yield 390 mg (80%). MS: 1034.3 [M+NH4]; 1016.9 [M+1r.
59
CA 02816041 2013-04-24
WO 2012/092373 PCT/US2011/067588
Product 231 (n=23, AA=PheCit) was prepared as described in the preparation of
23b and
purified on a column, eluent CHC13:Et0Ac:Me0H (4.5:5:0.5) followed by a
gradient of
Me0H (6-12%) in CHC13. Yield 86%. MS: 1711.4 [M+NH4]+; 1694,4 [M+1r.
Product 23j (PEG 1000K AA=PheCit) was prepared as described in the preparation
of 23b
and purified on a column, eluent CHC13:Et0Ac:Me0H (4.5:5:0.5) followed by a
gradient of
Me0H (6-12%) in CHC13. Yield 72%.
Product 23k (n=23, AA=ValCit) was prepared as described in the preparation of
23b, and the
product purified with HPLC. Column: Luna (Phenomenex) 5u, C-8, 100 A. Mobile
phase:
ACN-1120 (F3CO2H 0.01%), ACN gradient 30-37%, 31 min. Yield: 530 mg (48%). MS:
1666.4 [M+Na];1644.2 [M+1]-.
PEG-AA-PABC-PNP 24a-c, AA deprotection.
(i) 23 a,d,g 0 0 ip NO2
0
24 a-c
PEG-AA-PABC-PNP
Conditions: (i) TFA/H20=3:1, 5 C, 2-3h.
Product 24a (n=11, AA=GluGly). Product 23a (250 mg, 0.215 mmol) was stirred in
a 3%
TFA solution of CHC13 (16 mL) for 35 min, concentrated on a rotovap and dried
in vacuo.
Yield 224 mg (100%) (MS: 1062.6 [M+NHa] ; 1045.9 [M+1]4
.
Product 24b (n=11, AA=AlaAsn-PABC-PNP). Compound 23d was stirred for 1.5 h in
a
mixture of TFA:DCM (3:1) and all volatiles were removed on a rotovap at 20 C.
The product
was purified on a column, eluent gradient of Me0H (6-12%) in CHC13. Yield 30%.
MS:
1066.7 [M+Nar, 1062.0 [M+NH4]; 1045.2 [M+1}.
Product 24c (n=11, AA=Asn). A reaction flask with 23g (160 mg, 0.143 mmol) was
chilled
to 0 C and a cold mixture of TFA:H20 (9:1) (12.5 mL) was added. The mixture
was stirred
for 1.5 h and was diluted with cold H20 (50 mL). The stirring was continued
for 20 min at
20 C. The precipitate was filtered off and rinsed with H20. All volatiles were
removed on a
rotovap at 40 C and the product was purified on a column, eluent
CHC13:Et0Ac:MeOH:AcOH= 4.5:3.5:1.2:0.02. Yield 43 mg (30%). MS: 974.0 [M+1]4.
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
=
Table 4. Final PEG-L-A1A2-PABC used for DPC preparation.
compound AA
size
PEGa-AA-PABA PEGõ-AA-(PNP) Ai A2
22 23a Glu(2PhiPr) Gly n 11
22 23b Phe Cit n 11
22 23c Val Cit n = 11
22 23d Ala Asn(DMCP) n = 11
22 23e Phe Lys(CH3)2 n = 11
22 23f Leu n = 11
22 23g Asn(DMCP) n = 11
22 23h Cit n 11
22 23i Phe Cit n = 23
22 23j Phe Cit 1 kDa
22. 23k Val Cit n = 23
24a Glu Gly n = 1 1
24b Ala Asn n = 11
24c Asn n = 11
Example 2. Linkage of protease cleavable masking agents to amine-containing
polymers ¨
formation ofp-acylamidobenzyl carbamate linkages.
A. ililadification of Melittin with protease cleavable masking agents. I x mg
of melittin
peptide and 10x mg HEPES base at 1-10 mg/mL peptide was masked by addition of
2-6x mg
of amine-reactive p-nitrophenyl carbonate or N-hydroxysuccinimide carbonate
derivatives of
the NAG-containing protease cleavable substrate. The solution was then
incubated at least 1.h
at room temperature (RI) before injection into animals.
B. Modification of polyamines with protease cleavable masking agents.
Activated (amine
reactive) carbonates of p-acylamidobenzyl alcohol derivatives are reacted with
amino groups
of amphipathic membrane active polyamines in H20 at pH>8 to yield a p-
acylamidobenzyl
carbamate.
0
RI-AA-NH Z + H2N-R2 _______ RI-AA-NH 40 CH2-0)(NH- R2
RI comprises an ASGPr ligand (either protected or unprotected) or a PEG,
= 61
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
R2 is an amphipathic membrane active polyamine,
AA is a dipeptide (either protected or unprotected), and
Z is an amine-reactive carbonate.
To x mg polymer was added 12x mg of HEPES free base in isotonic glucose. To
the buffered
polymer solution was added 2x to 16x mg 200 mg/ml dipeptide masking agent in
DMF. In
some applications, the polymer was modified with 2x mg dipeptide masking agent
followed
by attachment of siRNA. The polymer-siRNA conjugate was then further modified
with 6x to
8x mg dipeptide masking agent.
Example 3. siRWAs. siRNAs had the following sequences:
Factor VII siRNA
sense: (Chol)-5' GfcAfaAfgGfcGfuGfcCfaAfcUfcAf(invdT) 3' (Seq ID 1)
antisense: 5' pdTsGfaGfuUfgGfcAfcGfcCfuUfuGfcdTsdT 3' (Seq ID 2)
or
sense: 5' GGAUfCfAUfCfUfCfAAGUfCfUfUfACfdTsdT 3' (Seq ID 3)
antisense: 5' GUfAAGACfUtUfGAGAUfGAUfCfCfdTsdT 3' (Seq ID 4)
or
sense: (NH2C6)GfuUfgGfuGfaAfuGfgAfgCfuCfaGf(invdT) 3' (Seq ID 5)
antisense: pCfsUfgAfgCfuCfcAfuUfcAfcCfaAfcdTsdT 3' (Seq ID 6)
or
sense: 5'(NH2C6)uGuGfcAfaAfgGfcGfuGfcCfaAfclifcAf(invdT) 3' (Seq ID 23)
antisense: 5' pdTsGfaGfuUfgGfcAfeGfcCful/fuGfedTsdT 3' (Seq ID 24)
Factor VII siRNA(ptimate)
sense: (chol)-5' uuAGGfuUfgGfuGfaAfuGfgAfgCfuCfaGf(invdT) 3' (Seq ID 7)
antisense: 5' pCfsUfgAfgCfuCfcAfullfcAfcCfaAfcdTsdT 3' (Seq ID 8)
ApoB siRNA:
sense: (cholC6SSC6)-5' GGAAUCuuAuAuuuGAUCcAsA 3' (Seq ID 9)
antisense: 5' uuGGAUcAAAuAuAAGAuUCcscsU 3' (Seq ID 10)
Ahal siRNA:
sense: (NH2C6)GfgAfuGfaAfgUfgGfaGfaUfuAfgUf(invdT) 3' (Seq ID 11)
antisense: pdAsCfuAfaUfcUfcCfaCfuUfcAfuCfcdTsdT 3' (Seq ID 12)
=
62
CA 02816041 2013-04-24
WO 2012/092373
PCT/1JS2011/067588
= Luc siRNA
sense: (chol) 5'-uAuCfuUfaCfgCfuGfaGfuAfeUfuCfgAf(invdT)-3 (Seq ID 13)
antisense: 5'-UfcGfaAfgUfaCfuCfaGfcGfuAfaGfdTsdT-3' (Seq ID 14)
or
sense: (NH2C6)cuuAcGcuGAGuAcuucGAdTsdT 3' (Seq ID 15)
antisense: UCGAAGuACUcAGCGuAAGdTsdT 3' (Seq ID 16)
Eg5-KSP
sense: (NH2C6)UfcGfaGfaAfuCfuAfaAfcUfaAfcUf(invdT) 3' (Seq ID 17)
antisense: pAfGfullfaGfut1fuAfgAfuLlfclifcGfadTsdT 3' (Seq ID 18)
or
sense: AGUuAGUUuAGAUUCUCGAdTsdT 3' (Seq ID 19)
antisense: (NH2C6)ucGAGAAucuAAAcuAAcudTsdT 3' (Seq ID 20)
EGFP
sense: 5' (NH2C6)AuAucAuGGccGAcAAGcAdTsdT 3' (Seq ID 21)
antisense: 5' UGCUUGUCGGCcAUGAuAUdTsdT 3' (Seq ID 22)
lower case = 2'-0-CH3 substitution
s = phosphorothioate linkage
f after nucleotide = 2'-F substitution
d before nucleotide = 2'-deoxy
RNA synthesis was performed on solid phase by conventional phosphoramidite
chemistry on
an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany) and controlled pore
glass
(CPG) as solid support.
Example 4. Administration of RNAi polynucleotides in vivo, and delivery to
hepatocytes.
DPCs were prepared as described above. Six to eight week old mice (strain
C57BL/6 or !CR,
¨18-20 g each) were obtained from Harlan Sprague Dawley (Indianapolis IN).
Mice were
housed at least 2 days prior to injection. Feeding was performed ad libitum
with Harlan
Teklad Rodent Diet (Harlan, Madison WI). DPCs were synthesized as described
herein.
Conjugate solutions (0.4 mL) were injected by infusion into the tail vein. The
compositions
were soluble and nonaggregating in physiological conditions. Injection into
other vessels, e.g.
retro-orbital injection, are predicted to be equally effective. Wistar Han
rats, 175-200g were
obtained from Charles River (Wilmington, MA). Rats were housed at least 1 week
prior to
63
CA 02816041 2013-04-24
WO 2012/092373
PCMTS2011/067588
injection. Injection volume for rats was typically 1 ml. Unless indicated
otherwise, serum
samples were taken and/or liver samples were harvested 48 hours after
injection.
Serum ApoB levels determination. Mice were fasted for 4 h (16 h for rats)
before serum
.. collection by submandibular bleeding. For rats blood was collected from the
jugular vein.
Serum ApoB protein levels were determined by standard sandwich ELISA methods.
Briefly,
a polyclonal goat anti-mouse ApoB antibody and a rabbit anti-mouse ApoB
antibody
(Biodesign International) were used as capture and detection antibodies
respectively. An
HRP-conjugated goat anti-rabbit IgG antibody (Sigma) was applied afterwards to
bind the
ApoB/antibody complex. Absorbance of tetramethyl-benzidine (TMB, Sigma)
colorimetric
development was then measured by a Tecan Safire2 (Austria, Europe) microplate
reader at
450 nm.
Plasma Factor VII (F7) activity measurements. Plasma samples from animals were
prepared
by collecting blood (9 volumes) (by submandibular bleeding for mice or from
jugular vein for
rats) into microcentrifuge tubes containing 0.109 mol/L sodium citrate
anticoagulant
(1 volume) following standard procedures. F7 activity in plasma is measured
with a
chronnogenic method using a BIOPHEN VII kit (Hyphen BioMed/Aniara, Mason, 01-
1)
following manufacturer's recommendations. Absorbance of colorimetric
development was
measured using a Tecan Safire2 microplate reader at 405 nm.
Example S. Delivery of siRNA to liver cells in vivo using a amphipathic
membrane active
polyactylate polyamine reversibly modified with dipeptide cleavable masking
agents.
Polyacrylate Ant-41658-111 in 100 iiiM pH 7.5 HEPES buffer was modified 0.5
wt% with
the activated disulfide reagent succinimidyloxycarbonyl-alpha-methyl-alpha(2-
pyridyl-
dithio)toluene (SMPT) (Pierce) to provide thiol reactive groups for subsequent
attachment of
siRNA. The thiol-reactive polymer was then diluted to 5 mg/mL in 60 mg/mL
HEPES base.
To this solution was added 10 mg/mL various described enzyme cleavable masking
reagents.
This amount represented a molar ratio of 1 polymer amine to 2 masking
reagents. For the
polymer modification reaction, a preferred molar ratio of polymer amines to
masking
reagents is 1:1 to 1:5. A more preferred ratio is 1:2 to 1:4. A more preferred
ratio is 1:2, After
1 hour, acetate-protected thiol endogenous rodent factor VII siRNA (0.1 to 0.2
wt equivalents
relative to polymer) was added to polymer solution. After incubation
overnight, conjugates
were further modified by addition of an N-acetylgalactosamine derivative of
maleic
64
CA 02816041 2013-04-24
WO 2012/092373 PCMJS2011/067588
=
anhydride (NAG-CDM; Table 5). NAG-CDM was added to 25 mg/mL and incubated for
30
minutes to 4 hours.
For NAG-CDM polymer modification, NAG-CDM was lyophilized from a 0.1% aqueous
solution of glacial acetic acid. To the dried NAG-CDM was added a solution of
polymer.
Following complete dissolution of anhydride, the solution was incubated for at
least 30 min
at RT prior to animal administration. Reaction of d NAG-CDM with the polymer
yielded:
0 0
HN R1
0
wherein R is the polymer and RI comprises a ASGPr ligand (e.g. N-
acetylgalactosamine).
As shown in Table 5, Factor VII expression was reduced 49-85% in animals
treated with
dipeptide agent masked DPCs.
Table 5. Knockdown of Factor VII in vivo in mice treated with PEG-AA-p-
nitrophenyl-carbamate + NAG-CDM-DPCs.
Polymer
siRNA dose %
Dipeptide masking agent dose
(mg/kg) a activity
b
(mg/kg) a
PEG12-AlaAsn 15 2 32
PEG12-PheCit 15 2 15
PEG 12-AsnGly 15 2 51
PEG24-PheCit 1.5 0.25 23
PEG12-Asn 1.5 0.25 34
PEG24-ValCit 1.5 0.2$ 23
a mg polymer or siRNA per kg animal weight
b relative to naïve control
Example 6, siRNA in vivo delivery using NAG/PEG-AA-p-nitrophenyl-carbamate
poly(acrylate) DPCs.
A) PEG plus NAG nrodification. Polyacrylate Ant-41658-111 in 100 mM pH 7.5
HEPES
buffer was modified 0.5 wt% with the activated disulfide reagent
succinimidyloxycarbonyl-
alpha-methyl-alpha(2-pyridyldithio)toluene (SMPT) from Pierce. The thiol-
reactive polymer
was diluted to 5 mg/mL in 60 mg/mL HEPES base. To this solution was added 10
mg/mL
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
various PEG-AA-p-nitrophenyl-carbonate masking reagents. After 1 hour, acetate-
protected
thiol Factor VII siRNA was added to polymer solution at a polymer to siRNA
ratio range of
5-10 to I. After incubation overnight, NAG-AA-p-nitrophenyl-carbonate masking
reagents
were added to 40 mg/mL. After incubation for at least 30 minutes, but no
longer than 4 hours,
the DPC was injected into the tail vein of 20 g ICR mice. 48 hours after
injection, a sample of
serum was harvested and the levels of fV11 were measured.
B) NAG alone modification. Polyacrylate ANT-41658 111 in 100 mM pH 7.5 HEPES
buffer
was modified 0.5 wt% with the activated disulfide reagent
suceinimidyloxycarbonyl-alpha-
methyl-alpha(2-pyridyldithio)toluene (SMPT) from Pierce. The thiol-reactive
polymer was
diluted to 5 mg/mL in 60 mg/mL HEPES base. Acetate-protected thiol siRNA
factor VII was
added to polymer solution at a polymer to siRNA ratio range of 5-10 to 1.
After incubation
overnight, NAG-AA-p-nitrophenyl-carbonate masking reagents were added to 50
mg/mL.
After incubation for at least 30 minutes, but no longer than 4 hours the
polymer-conjugated
siRNA was injected into the tail vein of 20 gm ICR mice. 48 hours after
injection, a sample
of serum was harvested and the levels of fVII were measured.
Table 6. Knockdown of Factor VII in vivo in mice treated with PEG/NAG-AA-p-
nitrophenyl-carbamate DPCs
Polymer siRNA
% fVII
Masking Agent amount` dose dose
activity b
(mg/kg) a (mg/kg) a
12 unit PEG12-PheCit 2x
15 2 27
followed NAG-PEG4-PheCit 8x
24 unit PEG24-PheCit 2x
15 2 27
followed NAG-PEG4-PheCit 8x
PEG24-PheCit 2x
1.5 2.5 23
followed by NAG-PEG4-PheCit gx
NAG-PEG4-PheCit 10 x 15 2 44
NAG-PEG2-GluGly 10x 1.5 2.5 27
NAG-PEG2-PheCit 10x 1.5 2.5 72
a mg polymer or siRNA per kg animal weight
b relative to naïve control
weight equivalents
Example 7. siRNA in vivo delivery using NAG/PEG-AA-p-nitrophenyl-carbamate
poly(vinyl
ether) DPCs. Amphipathic membrane active poly(vinyl ether) polyamine DW1360
modified
with 10x wt equiv. dipeptide cleavable masking agents as described above for
Polyacrylate
66
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Ant-41658-111 except that the masking agents retained protected groups during
polymer
modification. After polymer modification, amino acid protecting groups were
removed by
TFA and NAG acetate protecting groups were removed by incubation in the
presence of a
solution of 30 volume % triethylamine, 50% methanol and 20% water. The acetate
deprotecting solution was removed by rotary evaporation. The masked polymer
was
coinjected into mice with cholesterol-ApoB siRNA conjugate.
Table 7. Knockdown of ApoB in vivo in mice co-injected with NAG-AA-p-
nitrophenyl-carbamate poly(vinyl ether) and cholesterol-ApoB siRNA conjugate.
Polymer dose siRNA dose
polymer masking agent (mg/kg) a (mg/kg)a 0/ b
0 ApoB
NAG-AsnGly 25 5 67
NAG-PheLys 25 5 70
NAG-GluGly 25 5 48
a mg polymer or siRNA per kg animal weight
relative to naïve control
Example 8. In vivo knockdown of endogenous ApoB levels following delivery of
ApoB siRNA
with melittin delivery peptide in mice, enzymatically cleavable masking
agents. Melittin was
reversibly modified with the indicated amount of enzymatically cleavable
masking agents as
described above. 200-300 u.g masked melittin was then co-injected with the 50-
100 tg ApoB
siRNA-cholesterol conjugate. Effect on ApoB levels were determined as
described above.
Peptidase cleavable dipeptide-amidobenzyl carbamate modified melittin was an
effective
siRNA delivery peptide. The use of D-from melittin petide is preferred in
combination with
the enzymatically cleavable masking agents. While more peptide was required
for the same
level of target gene knockdown, because the peptide masking was more stable,
the
therapeutic index was either not altered or improved (compared to masking of
the same
peptide with CDM-NAG).
67
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
Table 8. Inhibition of Factor VII activity in normal liver cells in mice
treated with
Factor VII-siRNA cholesterol conjugate and G1L-Melittin (D form) (Seq ID 25)
reversibly inhibited with the indicated enzymatically cleavable masking agent.
NAG-linkage jig jig percent
Peptide amount type peptide siRNA knockdown
5x CDM-NAG 200 100 97
5x NAG-AlaCit 200 50 96
5x NAG-GluGly 200 50 96
5x NAG-PEG4-PheC it 200 50 94
GIL d-Mel
5x NAG-PEG7-PheCit 200 50 86
(Seq ID 25)
5x CDM-NAG 300 50 _ 98
2x NAG-GluGly 300 50 95
4x NAG-GluGly 300 50 95
6x NAG-GluGly 300 50 82
a Amount of masking agent per Meliuin amine used in the masking reaction.
Example 9. Tumor Targeting with protease cleavable DPCs.
A) Target gene knockdown measurement. For all studies presented below an siRNA
specific
for the Ahal gene transcript, the target gene. An siRNA to the enhanced green
fluorescent
protein (EGFP) was used as an off-target control. The Ahal siRNA was
complementary to a
sequence motif in Ahal that is 100% homologous in both the human and mouse
gene.
Therefore, delivery of Ahal siRNA either into cells of the host or into tumor
cells in the
human xenograll results in mRNA cleavage and degradation. Using sequence
motifs different
in mouse and human Ahal genes, PCR primers were designed that enabled
quantitative
measurement of both human Ahal and mouse Ahal mRNA levels in tissue samples
that
contained a mixed population of cell types. At 24, 48 or 72 hours after siRNA
delivery,
tumors were harvested with some healthy mouse liver tissue attached and were
processed in
Tr-Reagent (lnvitrogen) for total RNA isolation. Both human and mouse Ahal
mRNA levels
were then measured by qPCR assays,. using human Cyc-A and mouse (3-actin as
internal
reference genes. Ahal mRNA levels in animals from mock-injected animals, or
mice that
received the off-target control GFP siRNA were considered 100%. Results are
expressed as
percent of Ahal mRNA level relative to control and are shown in Tables below.
B) Orthotopic hepatocellular carcinoma (HCC) tumor model mice. HegG2, Hep3B,
or HuH7
cells hepatocellular carcinoma were co-transfected with 2 expression vectors,
pMIR85 a
human placental secreted alkaline phosphatase (SEAP) vector and pM1R3 a
neomycinetkanamycin-resistance gene vector, to develop cell lines with stable
SEAP
68
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
expressionCell were grown DMEM supplemented with 10% FBS and 300 ug/ml G418),
collected, counted, and mixed with matrigel (BD Biosciences) (50% by volume).
Athymic
nude or Sold beige mice were anesthetized with ¨3% isoflourane and placed in a
sternal
recumbent position. A small, 1-2 cm, midline abdominal incision was made just
below the
xyphoid. Using a moist cotton swab, the left lobe of the liver was gently
exteriorized. The left
lobe of the liver was gently retracted and a syringe needle was inserted into
the middle of the
left lobe. The syringe needle was inserted with the bevel down about 0.5 cm
just under the
capsule of the liver. 10 I of cell/matrigel mixture, containing 100,000
cells, was injected into
the liver using a syringe pump. The needle was left in the liver for a few
moments (15-20
seconds) to ensure the injection was complete. SEAP-HepG2 cells were injected
into athymic
nude mice. SEAP-Hep3B and SEAP-HuH7 cells were injected into Scid beige mice.
The
syringe was then removed from the needle from the liver and a cotton swab was
placed over
the injection site to prevent leakage of the cells or bleeding. The
Matrigel/cells mixture
formed a mass that was visible and did not disappear after removal of the
needle. The liver
lobe was then gently placed back into the abdomen and the abdominal wall was
closed. Sera
were collected once per week after tumor implantation and subjected to SEAP
assay to
monitor tumor growth. For most studies, tumor mice were used 4-5 weeks after
implantation,
when tumor measurements are predicted to be around 4-8 mm based on SEAP
values.
C) Colorectal Metastatic Tumor model. HT29 cells were grown in McCoy's 5a
medium
supplemented with 10% FBS, collected, counted, and mixed with matrigel (BD
Biosciences)
(50% by volume). Athymic nude mice were anesthetized with ¨3% isoflourane and
placed in
a sternal recumbent position. A small, 1-2 cm, midline abdominal incision was
made just
below the xyphoid. Using a moist cotton swab, the left lobe of the liver was
gently
exteriorized. The left lobe of the liver was gently retracted and a syringe
needle was inserted
into the middle of the left lobe. The syringe needle was inserted with the
bevel down about
0.5 cm just under the capsule of the liver. 5 ill of cell/matrigel mixture,
containing ¨40,000
cells, was injected into the liver using a syringe pump. The needle was left
in the liver for a
few moments (15-20 seconds) to ensure the injection was complete. The syringe
was then
removed from the liver and a cotton swab was placed over the injection site to
prevent
leakage of the cells or bleeding. The Matrigel/cells mixture formed a mass
that was visible
and did not disappear after removal of the needle. The liver lobe was then
gently placed back
into the abdomen and the abdominal wall was closed. Tumor mice were used 4-5
weeks after
implantation.
69
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
Example 10. In vivo knockdown of target gene expression in HepG2-SEAP
orthotopic
hepatocellular carcinoma (HCC) model following PEG24-Val-Cit DPC
administration.
(2011062805) Ant-129-1 polymer DPCs were modified (masked) with either 18x
weight
excess PEG24-Phe-Cit masking agent (or PEG24-Val-Cit masking agent) or with 7x
PEG550-
CDM as described above. Ahal-siRNA (RD-09070) or GFP-siRNA (RD-05814; off-
target
control) was attached to the polymer as described above (4:1 weight ratio).
DPCs were not
purified by gel filtration prior to delivery, and no targeting ligand was
added. A 320 g
(polymer weight) DPC conjugate in 200 I isotonic glucose per animal was
administered by
tail vein injections (n=3 per group). After 24 hours, animals received second
injection of
320 ug (polymer weight) DPC conjugate in 200 gl isotonic glucose. 48 hours
after the second
injection, serum samples were collected to assess toxicity by measuring liver
enzyme (ALT
and AST) and blood urea nitrogen (BUN) levels, followed by tissue harvest, and
qPCR
analysis.
Using PE324-Val-Cit¨Ant-129-1¨siRNA DPCs to delivery Ahal siRNA, resulted 46%
knockdown of the Ahal gene in human tumor cells (Table 9). In contrast to
human Ahal
knockdown levels, mouse Ahal was knocked down 70% in response PEG24-Val-
Cit¨Ant-
129-1¨siRNA DPC administration (Table 1). Compared to similar DPCs made with
disustitited maleic anhydride masking agents (PEG550-CDM), endogenous
hepatocyte Ahal
knockdown was decreased. As indicated by ALT, AST and BUN levels, the PEG24-
Val-Cit
DPCs were well tolerated and did not exhibit toxicity (Table).
Table 9. Ahal knockdown in HepG2 liver tumor model by maleic anhydride
modified
vs. peptide cleavable modified Ahal siRNA DPCs.
PEG550-CDM DPC PEG24-Val-Cit DPC
control siRNA Ahal siRNA Ahal siRNA
human Ahal levels
100 8.0 54.4 7.3 54.9 7.6
(tumor)
mouse Ahal levels
100 9.2 7.5 0.9 30.4 3.1
(hepatocytes)
70
CA 02816041 2013-04-24
WO 2012/092373 PCT/US2011/067588
Table 10. Blood chemistry toxicity markers following administration of maleic
anhydride modified or peptide cleavable modified Ahal siRNA DPCs.
PEG550-CDIV1 DPC PEG24-Val-Cit DPC
control siRNA Ahal siRNA Ahal siRNA
ALT 44.3 5.1 58.0 37.5 35.0 11.3
AST 81.7 4.0 102.3 57.5
67.7 14.4
BUN 25.3 4.2 23.0 3.5 21.3 1.2
Example 11. Knockdown of targeting gene expression with bispecific antibody
(bsAb)-
targeted DPCs (2011090701). Ant-129-1 polymer was modified with 5x Dig-PheCit
(Dig-
FCit) masking agent as described above. siRNA was then attached to the
conjugate. Finally,
the Dig-FCit¨Ant-129-1¨siRNA conjugate was further modified with 8x (wt) PEGI2-
FCit.
Ahal-siRNA (RD-09070) or GFP siRNA (RD-05814) was attached at a 4:1
polymer:siRNA
weight ratio. PEG12-FCit DPCs were purified on Sephadex G50 spin columns to
remove
unbound reagents.
Cell targeting bispecific antibodies (bsAb) were made specific to heparan
sulfate
proteoglycan Glypican-3 (GPC3), a cell surface heparan sulfate proteoglycan
known to be
highly expressed in HepG2-SEAP cells, and digoxigenin (Dig). As a control,
bispecific
antibodies specific to the protein CD33 (marker of bone marrow-derived
hematopoietic stem
cells) and Dig were made. CD33 is not expressed by HepG2-SEAP cells. BsAbs
were =
complexed with modified DPCs at a 1.25:1 weight ratio to provide an estimated
1:1 molar
ratio. Complexes were formed in PBS at least 30 minutes prior to delivery.
DPCs were administered to HepG2-SEAP tumor bearing mice, either with or
without bsAb
targeting agent. Each animal (n=3 per group) received a single dose of 250 }tg
(polymer wt.)
DPCs. DPCs were injected into tail vein of mice in 200 gl sterile PBS. Serum
and tissue
samples were harvested 48 hours later and analyzed as described above. As
shown in Table
11, a single dose of bsAb targeting DPCs (250 ug polymer, 62.5 p.g siRNA)
resulted in target
gene knockdown of 21-32%.
71
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Table 11. Ahal knockdown in HepG2 liver tumor model by peptide cleavable
masking
agent modified Ahal siRNA DPCs targeted using bispecific antibodies.
Dig-FCit + PEG12-FCit masking agent
GPC-Dig bsAb CD33-Dig bsAb
control siRNA Ahal siRNA
human Ahal levels
100 10.1 78.6 11.5 72.7 2.4
68.1 6.4
(tumor)
mouse Ahal levels
100 9.5 65.1 8.1 73.7 10.5 91.3 8.3
(hepatocytes)
Example 12. Knockdown of targeting gene expression with bispecific antibody
(bsAb)-
targeted DPCs. DPCs were prepared as above except a) PEG24-FCit was used
instead of
PEG12-FCit and b) Dig-PEG12-NHS was used to attach Dig to the polymer. PEG24-
FCit DPCs
aggregated less than PEGI2-FCit DPCs and were smaller and more homogenous. In
addition
to being a non-labile linkage, Dig-PEGI2-NHS also contained a longer PEG. DPCs
were
complexes with bsAb and injected into animals as described above. Serum and
tissue harvest
was performed either at 24 or 48 hours post-injection. As shown in Table 12, a
single dose of
DPCs (250 jig polymer wt.) resulted in human Ahal knockdown of 46-56% 24 hours
post
injection.
Table 12. Ahal knockdown in HepG2 liver tumor model by Ahal siRNA DPCs
modified using peptide cleavable masking agents with increased PEG length.
Dig-PEG12-NHS + PEG24-FCit masking agent
GPC-Dig bsAb CD33-Dig bsAb
control siRNA Ahal siRNA
human Ahal levels
100 3.1 54.1 15.! 43.5 6,6
(tumor)
Example 13. Targeting DPCs to human colorectal adenocarcinoma metastatic liver
tumor
tissue by bsAb targeted DPCs. Ant-129-1 polymer was modified with 5x molar
excess Dig-
PEG12-NHS and 8x weight excess PEG24-FCit. Ahal siRNA or GFP siRNA was
attached to
the modified polymer at a 4:1 polymer:siRNA weight ratio. DPCs were purified
on a
Sephadex G50 spin column to remove unbound reagents. Dig-DPCs were complexed
with
equimolar amount of IGF1R-Dig bsAb or CD33-Dig bsAb or no bsAb in sterile PBS
at least
minutes prior to injections Animals containing HT29 tumor cells (human
colorectal
adenocarcinoma; ATCC Number HTB-38) were injected with the DPCs. HT29 cells
72
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
overexpress the insulin-like growth factor-1 receptor protein (IGF1R), and can
bind and
internalize an IGFIR-Dig bispecific antibody. Animals (n=3) received DPCs (320
lig
polymer). Injections were repeated after 24 hours. Serum and tissue samples
were collected
48 hours after the second dose. Knockdown of human Ahal in tumor cells was 26-
38%
(Table 13). Compared to CDM-DPCs, FCit-DPCs showed less off target liver Ahal
knockdown (78-83%compared to 24-36%). FCit-DPCs also showed diminished liver
accumulation compared to CDM-DPCs.
Table 13. Ahal knockdown in H129 colorectal adenocarcinoma metastatic liver
tumor
by dipeptide cleavable Ahal siRNA DPC.
Ant-129-1 polymer
Dig-PEG12-NHS + PEG24-FCit masking agent
IGF I R-Dig bsAb CD33-Dig bsAb
control siRNA Ahal siRNA
human Ahal levels
100 4.9 72.3 6.1 73.7 t 3.2
62.0 9.0
(tumor)
mouse Ahal levels
100.0 1 1 64.0 t 5.0 75.7 11
66.6 5.8
(liver)
Example 14. In vivo knockdown of endogenous Ahal in liver tumor. 400 pg
Lau41648-106
was modified with 8x (weight) PEG12-ValCit or 16x PEG24-PheCit. 100 lig Ahal
siRNA or
100 1.tg Eg5 control siRNA was attached to the modified polymer as described
above.
Animals containing Hep3B-SEAP tumor cells were injected with the DPCs. Serum
and tissue
samples were collected 48 hours after injection. Knockdown of human Ahal in
tumor cells
was 26-38%.
Table 14. Ahal knockdown in Hep3B-SEAP liver tumor by dipeptide cleavable
Ahal siRNA DPC.
Polymer siRNA
modification Ahal KD
(400 j.tg) (100 jig)
8x PEG12-ValCit Ahal 38 0.02 %
Lau41648-106 Ahal 41 0.07 %
16x PEG24-Phecit
EG5 13 0.11 %
73
CA 02816041 2013-04-24
WO 2012/092373 PCT/US2011/067588
Example 15. In vivo circulation and tissue targeting of masked polymer.
Lau24AB
polyacrylate (100 .tg) was treated with 125I-Bolton-Hunter (BH) reagent (50
uCi) in 50 mM
HEPES pH 8.0 buffer for 1 hr at RT. The labeled polymer was purified in 2 ml
Sephadex
QEA spin column in water. The solution of labeled polymer was stored at 4 C.
The unlabeled
polymer was supplemented with 1251-labeled polymer to inject approx. 1 mg
polymer having
0.2 uCi per 200 g rat. The mixture of labeled and unlabeled polymers
(calculated for ¨3.5
animals) was modified as described above with PEG24-FCit or PEG-CDM (2 mg/ml
polyacrylate, 14 mg/ml PEG-CDM reagent, 14 mg/ml NAG-PEG-CDM reagent, 16
mg,/m1
PEG24-FCit reagent). Incubation time 1 hr. The reaction mix was then diluted
with isotonic
glucose to yield the injection dose per animal in a volume of 1 ml. 3
animals/group were
injected. The animals were bled (0.1 - 0.2 ml) at the given times. The amount
of polymer
present in the samples was determined by counting in gamma counter. As shown
in FIG. 4,
polymer modified with the protease cleavable masking agent was cleared less
rapidly from
serum than polymer masked with the pH labile maleic anhydride masking agent.
Increase
circulation time is beneficial for targeting to non-liver tissue.
Example 16. Amphtpathic membrane active polymer syntheses.
A) Poly(vinylactylate)
i) RAFT copolymerization of N-Boc-ethylethoxy acrylate and propyl
methacrylate:
0
cH,
cH,
0 CH3 CH3
CH3
A
CH3
AIBN, Butyl Acetate, CN OH
80C, 16h
0
=
74
CA 02816041 2013-04-24
WO 2012/092373
PCT/1JS2011/067588
0
H3c
Ho
II3C CN
r-j
0 H30
0 0
H3C CH3
CH3
wherein: A is a boc protected ethyl-ethoxy amino acrylate
B is a propyl methacrylate
C is a RAFT agent CPCPA (4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid)
And n and m are integers.
Removal of the boc protecting group after synthesis yields the amine monomers.
For other membrane active polymers, A can be also beprotected ethyl, propyl,
or butyl amino
acrylate. B can be higher hydrophobic (10-24 carbon atoms, Cl 8 shown)
acrylate, lower
hydrophobic (1-6 carbon atoms, C4 shown) acrylate, or a combination of lower
an higher
hydrophobic acrylates.
Copolymers consisting of Amine acrylate/C3 methacrylate were synthesized as
follows. The
monomers and RAFT agent were weighed and brought up into butyl acetate at the
indicated
ratios. AIBN (azobis-isobutyronitrile) was added and nitrogen was bubbled
through the
reaction at RT for I h. The reaction mixture was then placed into an oil bath
at 80 C for 15h.
The polymer was then precipitated with hexane, and further fractionally
precipitated using a
DCM/Hexane solvent system (see below). The polymer was then dried under
reduced
pressure. The polymer was deprotected with 7m1 2M HCI in Acetic Acid for 30m1n
at RT.
After 30 min 15 ml of water was added to the reaction mixture, and the mixture
was
transferred into 3.5 kDa MWCO dialysis tubing. The polymer was dialyzed
overnight against
CA 02816041 2013-04-24
WO 2012/092373
PCMTS2011/067588
NaC1 and then another day against dH20. The water was then removed through
lyophilization, and the polymer was dissolved in dH20.
Monomer Synthesis for (Ant 41658-111). 2,2'-Azobis(2-methylpropionitrile)
(AIBN, radical
initiator), 4-Cyano-4-(phenylcarbonothioylthio) pentanoic acid (CPCPA, RAFT
Agent) and
butyl acetate were purchased from Sigma Aldrich. Propyl Methacrylate monomer
(Alfa
Aesar) was filtered to remove inhibitors.
In a 2L round-bottom flask equipped with a stir bar, 2-(2-aminoethoxy) ethanol
(21.1 g,
202.9 mmol, Sigma Aldrich) was dissolved in 350 mL dichloromethane. In a
separate IL
flask, BOC anhydride (36.6 g, 169.1 mmol) was dissolved in 660 mL
dichloromethane. The
2L round-bottom flask was fitted with an addition funnel and BOC anhydride
solution was
added to the flask over 6 h. The reaction was left to stir overnight. In a 2L
separatory funnel,
the product was washed with 300 ml each of 10% citric acid, 10% K2CO3, sat.
NaHCO3, and
sat. NaCI. The product, BOC protected 2-(2-aminoethoxy) ethanol, was dried
over Na2SO4,
gravity filtered, and DCM was evaporated using rotary evaporation and high
vacuum.
In a 500 ml round bottom flask equipped with a stir bar and flushed with
argon, BOC
protected 2-(2-aminoethoxy) ethanol (27.836 g, 135.8 mmol) was added, followed
by 240 mL
anhydrous dichloromethane. Diisopropylethyl amine (35.5 ml, 203.7 mmol) was
added, and
the system was placed in a dry ice/acetone bath. Acryloyl Chloride (12.1 ml,
149.4 mmol)
was diluted using 10 ml of dichloromethane, and added drop-wise to the argon
flushed
system. The system was kept under argon and left to come to room temperature
and stirred
overnight. The product was washed with 100 mL each of dH20, 10% citric acid,
10% K2CO3,
sat. NaHCO3, and saturated Naa. The product, BOC-amino ethyl ethoxy acrylate
(BAEEA),
was dried over Na2SO4, gravity filtered, and DCM was evaporated using rotary
evaporation.
The product was purified through column chromatography on 29 cm silica using a
7.5 cm
diameter column. The solvent system used was 30% ethyl acetate in hexane. RE
0.30.
Fractions were collected and solvent was removed using rotary evaporation and
high vacuum.
BAEEA, was obtained with 74% yield. BAEEA was stored in the freezer.
0
N/BOC
0
BAEEA
76
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Polymer Ant-41658-111: Solutions of AIBN (1.00 mg/mL) and RAFT agent (4-Cyano-
4(phenylcarbonothioylthio)pentanoic acid (CPCPA), 10.0 mg/mL) in butyl acetate
were
prepared. Monomer molar feed ratio was 75 BAEEA : 25 propyl methacrylate
(CAS:2210-
28-8) with 0.108 CPCPA RAFT agent and 0.016 AIBN catalyst (0.00562 total mol).
BAEEA (1.09 g, 4.21 mmol) (A), propyl meihacrylate (.180 g, 1.41 mmol) (13),
CPCPA
solution (.170 ml, .00609 mmol) (C), AIBN solution (.150 ml, .000915 mmol),
and butyl
acetate (5.68 ml) were added to a 20m1 glass vial with stirrer bar. The vial
was sealed with a
rubber cap and the solution was bubbled with nitrogen using a long syringe
needle with a
second short syringe needle as the outlet for 1 hour. The syringe needles were
removed and
the system was heated to 80 C for 15 h using an oil bath. The solution was
allowed to cool to
room temperature and transferred to a 50 ml centrifuge tube before hexane (35
ml) was added
to the solution. The solution was centrifuged for 2 min at 4,400 rpm. The
supernatant layer
was carefully decanted and the bottom (solid or gel-like) layer was rinsed
with hexane. The
bottom layer was then re-dissolved in DCM (7 mL), precipitated in hexane (35
mL) and
centrifuged once more. The supernatant was decanted and the bottom layer
rinsed with
hexane before the polymer was dried under reduced pressure for several hours.
Molecular
weight obtained through MALS: 73,000 (PD! 1.7); Polymer composition obtained
using
HINMR: 69:31 Amine:Alkyl.
Fractional Precipitation. The dried, precipitated product was dissolved in DCM
(100 mg/mL). Hexane was added until just after the cloud point was reached (-
20 ml). The
resulting milky solution was centrifuged. The bottom layer (thick liquid
representing ¨ 60%
of polymer) was extracted and fully precipitated into hexane. The remaining
upper solution
was also fully precipitated by further addition of hexane. Both fractions were
centrifuged,
after which the polymer was isolated and dried under vacuum. Fraction 1: Mw
87,000 (PDI
1.5); Fraction 2: Mw 52,000 (PD! 1.5-1.6).
MAU Analysis. Approximately 10 mg of the polymer was dissolved in 0.5 mL 89.8%
dichloromethane, 10% tetrahydrofuran, 0.2% triethylamine. The molecular weight
and
polydispersity (PD!) were measured using a Wyatt Helos II multiangle light
scattering
detector attached to a Shimadzu Prominence HPLC using a Jordi 5ji 7.8x300
Mixed Bed LS
DVB column. Crude Polymer: MW: 73,000 (PD! 1.7), Fraction 1: MW 87,000
(PD1:1.5),
Fraction 2: MW 52,000 (PD! 1.5-1.6)
77
CA 02816041 2013-04-24
WO 2012/092373
PCT/1TS2011/067588
The purified BOC-protected polymer was reacted 2M HCI in Acetic Acid (7 ml)
for 0.5 h to
remove the BOC protecting groups and produce the amines. 15 mL dH20 were added
to the
reaction, the solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against
high salt for 24 h, then against dH20 for 18 h. The contents were lyophilized,
then dissolved
in DI H20 at a concentration of 20 mg/ml . The polymer solution was stored at
2-8 C.
ii) Polymer Lau24B was prepared as above except the monomer feed ratio was
72.5 BAEEA :
27.5 propyl methacrylate.
iii) Am-129-1 was made as essentially as described above except the following
monomers
were used:
0
NHBoc
0
, and
ci'=/'y
0
=
=
78
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
Table 15. Ant-129-1 polymer synthesis reactants.
MW mass volume
reaction
mol% moles
(g/mol) (g) (ml) moles
Monomers
N-Boc-amino-propyl acrylate 229.27 70 3.94 x 10-3 0.9031
0.005627
butyl methacrylate 142.2 25 1.41 x 10-3 0.2000 0.224
0.005627
C I 8 methacrylate 338.54 5 2.81x10-4 0.0952 0.005627
ethylene glycol diacrylate 170.16 5 2.81x10-4 0.0479
0.44 0.005627
other reagents
CPCPA (RAFT reagent) 279.38 0.213 1.2 x10-5 0.0033
0.335 0.005627
AIBN (initiator) 164.21 0.032 1.8x10-6 0.0003
0.295 0.005627
butyl acetate 5.272
target molecular weight 100000
total units per CTA 469.56
%CTA 0.213
For N-Boc-Amino-Propyl-Acrylate (BAPA), In a 500 ml round bottom flask
equipped with a
stir bar and flushed with argon, 3-(B0C-amino)l-propanol (TCI) (135.8 mmol)
was added,
followed by 240mL anhydrous dichloromethane. Diisopropylethyl amine (203.7
mmol) was
added, and the system was placed in a dry ice/acetone bath. Acryloyl Chloride
(149.4 mmol)
was diluted using 10 ml of dichloromethane, and added drop-wise to the argon
flushed
system. The system was kept under argon and left to come to room temperature
and stirred
overnight. The product was washed with 100 mL each of dH20, 10% citric acid,
10% K2CO3,
sat. NaHCO3, and saturated NaCl. The product, BOC-amino propyl acrylate
(BAPA), was
dried over Na2SO4, gravity filtered, and DCM was evaporated using rotary
evaporation. The
product was purified through column chromatography on 29 cm silica using a 7.5
cm
diameter column. The solvent system used was 30% ethyl acetate in hexane. RE
0.30.
Fractions were collected and solvent was removed using rotary evaporation and
high vacuum.
BAPA was obtained with 74% yield. BAPA was stored in the freezer.
iv) Random copolymerization of N-Boc-ethylethoxy acrylcue and propyl
methacrylate.
Copolymers consisting of Amine acrylate/Cõ methacrylate were synthesized as
follows. The
monomers were weighed brought up into dioxane at the indicated ratios, AIBN
(azobis-
isobutyronitrile) was added and nitrogen was bubbled through the reaction at
RT for 1h. The
reaction mixture was then placed into an oil bath at 60 C for 3h. The polymer
was then dried
79
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
under reduced pressure. The polymer was purified by GPC. After which the
polymer
fractions were deprotected with 7m1 2M HC1 in Acetic Acid for 30 min at RT.
After 30 min,
15 ml of water was added to the reaction mixture, and the mixture was
transferred into 3.5
kDa MWCO dialysis tubing. The polymer was dialyzed overnight against NaCI and
then
another day against dH20. The water was then removed through lyophilization,
and the
polymer was dissolved in dH20.
Polymer Lau41648-106. Monomer molar feed ratio was 80 BAEEA : 20 propyl
methacrylate
(CAS:2210-28-8) and 3% AIBN catalyst based on total monomer moles. BAEEA (6.53
g,
25.2 mmol) (A), propyl methacrylate (0.808 g, 6.3 mmol) (B), AIBN (0.155 g,
0.945 mmol),
and dioxane (34.5 ml) were added to a 50m1 glass tube with stir bar. Compounds
A and B
were prepared described above in Example 16Ai. The reaction was set up in
triplicate. Each
solution was bubbled with nitrogen using a long pipette for 1 hour. The
pipette was removed
and each tube carefully capped. Then each solution was heated at 60 C for 3 h
using an oil
bath. Each solution was allowed to cool to room temperature and combined in a
round
bottom. The crude polymer was dried under reduced pressure. Molecular weight
obtained
through MALS: 55,000 (PDI 2.1); Polymer composition obtained using HINMR:
74:26
Am ine:A lky I.
H3c
=
=
0 H3C
HN./
oo
H3C CH3
CH3
Lau41648-106
GPC Fractionation. The dried crude polymer was brought up at 50 mg/ml in 75%
dichloromethane, 25% tetrahydrafuran, and 0.2% triethylamine. The polymer was
then
fractionated on a Jordi Gel DVB 104 A ¨ 500mm/22mm column using a flow rate of
CA 02816041 2013-04-24
WO 2012/092373
PCT/US2011/067588
ml/min and 10 ml injections. An earlier fraction was collected from 15-17
minutes, and a
later fraction was collected from 17-19 minutes. Fraction 15-17: Mw 138,000
(PDI 1.1);
Fraction 17-19: Mw 64,000 (PDI 1.2).
MALS Analysis. Approximately 10 mg of the polymer was dissolved in 0.5 mL
89.8%
5
dichloromethane, 10% tetrahydrofuran, 0.2% triethylarnine. The molecular
weight and
polydispersity (PDI) were measured using a Wyatt Helos II multiangle light
scattering
detector attached to a Shirnadzu Prominence HPLC using a Jordi 5 7.8x300
Mixed Bed LS
DVB column. Crude Polymer: MW: 55,000 (PDI 2.1), Fraction 15-17: MW 138,000
(PD1:1.1), Fraction 17-19: MW 64,000 (PDI 1.2)
The purified BOC-protected polymer was reacted 2M HCI in Acetic Acid (7 ml)
for 0.5 h to
remove the BOC protecting groups and produce the amines. 15 mL dH20 were added
to the
reaction, the solution was transferred to 3500 MW cutoff cellulose tubing,
dialyzed against
high salt for 24 h, then against dH20 for 18 h. The contents were lyophilized,
then dissolved
in DI H20 at a concentration of 20 mg/ml . The polymer solution was stored at
2-8 C.
v) Synthesis of water-soluble, amphipathic, membrane active poly(vinyl ether)
polyamine
terpolyiners. X mol% amine-protected vinylether (e.g., 2-Vinyloxy Ethyl
Phthalimide) is
added to an oven dried round bottom flask under a blanket of nitrogen in
anhydrous
dichloromethane. To this solution Y mol% lower hydrophobic group (e.g.,
propyl, butyl)
vinylether and optionally Z mol% higher hydrophobic group (e.g., dodecyl,
octadecyl)
vinylether are added (FIG. 1). The solution is placed in a ¨50 to ¨78 C bath,
and the
2-vinyloxy ethyl phthalimide is allowed to precipitate. To this solution 10
mol %
BF3-(OCH2CH3)2 is added and the reaction is allowed to proceed for 2-3 h at
¨50 to ¨78 C.
Polymerization is terminated by addition of ammonium hydroxide in methanol
solution. The
polymer is brought to dryness under reduced pressure and then brought up in
1,4-dioxane/methanol (2/1). 20 mol eq. of hydrazine per phthalimide is added
to remove the
protecting group from the amine. The solution is refluxed for 3 h and then
brought to dryness
under reduced pressure. The resulting solid is dissolved in 0.5 mol/L MCI and
refluxed for
15-min to form the hydrochloride salt of the polymer, diluted with distilled
water, and
refluxed for an additional hour. The solution is then neutralized with NaOH,
cooled to room
temperature (RI), transferred to molecular cellulose tubing, dialyzed against
distilled water,
and lyophilized. The polymer can be further purified using size exclusion or
other
81
CA 02816041 2013-04-24
WO 2012/092373
PCMJS2011/067588
chromatography. The molecular weight of the polymers is estimated using
columns according
to standard procedures, including analytical size-exclusion chromatography and
size-
exclusion chromatography with multi-angle light scattering (SEC-MALS).
Polymer DW1360. An amine/butyl/octadecyl poly(vinyl ether) terpolymer, was
synthesized
from 2-vinyloxy ethyl phthalimide (5 g, 23.02 mmol), butyl vinylether (0.665
g, 6.58 mmol),
and octadecyl vinylether (0.488 g, 1.64 mmol) monomers. 2-vinyloxy ethyl
phthalimide was
added to a 200 mL oven dried round bottom flask containing a magnetic stir bar
under a
blanket of Argon in 36 mL anhydrous dichloromethane. To this solution was
added butyl
vinyl ether and n-octadecyl vinyl ether. The monomers were fully dissolved at
room
temperature (RT) to obtain a clear, homogenous solution. The reaction vessel
containing the
clear solution was then placed into a ¨50 C bath generated by addition of dry
ice to a
1:1 solution of ACS grade denatured alcohol and ethylene glycol and a visible
precipitation
of phthalimide monomer was allowed to form. After cooling for about 1.5 min,
BF3.(OCH2CH3)2 (0.058 g, 0.411 mmol) was added to initiate the polymerization
reaction.
The phthalimide monomer dissolved upon initiation of polymerization. The
reaction was
allowed to proceed for 3 h at ¨50 C. The polymerization was stopped by the
addition of 5
mL of 1% ammonium hydroxide in methanol. The solvents were then removed by
rotary
evaporation.
The polymer was then dissolved in 30 mL of 1,4-dioxane/methanol (2/1). To this
solution
was added hydrazine (0.147 g, 46 mmol) and the mixture was heated to reflux
for 3 h. The
solvents were then removed by rotary evaporation and the resulting solid was
then brought up
in 20 mL of 0.5 mol/L HCI and refluxed for 15 minutes, diluted with 20 mL
distilled water,
and refluxed for an additional hour. This solution was then neutralized with
NaOH, cooled to
RT, transferred to 3,500 molecular weight cellulose tubing, dialyzed for 24 h
(2x2OL) against
distilled water, and lyophilized.
B)Melittin. All melittin peptides were made using peptide synthesis techniques
standard in
the art. Suitable melittin peptides can be all L-form amino acids, all D-form
amino acids
(inverso). Independently of L or D form, the melittin peptide sequence can be
reversed
(retro).
82
