Note: Descriptions are shown in the official language in which they were submitted.
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COMPRI~:ND PLUS D'UN TOME.
CECI EST ~.E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
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JUMBO APPLICATIONS / PATENTS
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THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
SITE-SPECIFIC CHEMICAL MODIFICATION OF HIV gp41-DERIVED PEPTIDES
FIELD OF INVENTION
The present invention relates to a method for the site-specific chemical
modification of an HIV gp41-derived peptide such that, during peptide
synthesis, added is
one or more amino acids having an amine group chemically protected with a
chemical
protecting agent, leaving one or more free amine groups of the synthetic
peptide chosen
to be unprotected so as to be chemically reactive ("free"). The resultant
synthetic peptide
may then be covalently coupled to an amine-reactive polymer in forming a
substantially
to homogeneous conjugate comprised of HIV gp41-derived peptide to which, in
selected
and specific sites) of the synthetic peptide, is covalently coupled the
polymer.
BACKGROUND OF THE INVENTION
It is now well known that cells can be infected by HIV through a process by
which
fusion occurs between the cellular membrane and the viral membrane. The
generally
accepted model of this process is that the viral envelope glycoprotein complex
(gp120/gp41 ) interacts with cell surface receptors on the membranes of the
target cells.
Following binding of gp120 to cellular receptors (e.g., CD4 in combination
with a
chemokine co-receptor such as CCR-5 or CXCR-4), induced is a conformational
change
2o in the gp120/gp41 complex that allows gp41 to insert into the membrane of
the target cell
and mediate membrane fusion.
The amino acid sequence of gp41, and its variation among different strains of
HIV,
is well known. FIG.1 is a schematic representation of the generally accepted
functional
domains of gp41 (note the amino acid sequence numbers may vary slightly
depending on
the HIV strain). The fusion peptide (fusogenic domain) is believed to be
involved in
insertion into and disruption of the target cell membrane. The transmembrane
domain,
containing the transmembrane anchor sequence, is located at the C-terminal end
of the
protein. Between the fusion peptide and transmembrane anchor are two distinct
regions,
known as heptad repeat (HR) regions, each region having a plurality of
heptads. The
3o amino acid sequence comprising the HR1 region and the amino acid sequence
comprising the HR2 region are each highly conserved regions in the HIV-1
envelope
protein. The HR1 region, nearer to the N-terminal end of the protein than the
HR2 region,
has been generally described as comprising amino acid residues of SEQ ID N0:1,
or
polymorphisms thereof (see, e.g., FIG. 2). The HR2 region has been generally
described
as comprising amino acid residues of SEQ ID N0:2, or polymorphisms thereof
(see, e.g.,
FIG. 3). As further shown in FIG.1, the HR regions have a plurality of 7 amino
acid
residue stretches or "heptads" (the 7 amino acids in each heptad designated
"a" through
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
"g"), with a predominance of hydrophobic residues at the first ("a") and
fourth ("d")
positions, charged residues frequently at the fifth ("e") and seventh ("g")
positions, and
with the amino acids in the "a" position and "d" position being primary
determinants that
influence the oligomeric state and strand orientation.
It was discovered that peptides derived from the native sequence of either the
HR1 region ("HR1 peptides") or HR2 region ("HR2 peptides") of HIV~gp41 inhibit
transmission of HIV to host cells both in in vitro assays and in in vivo
clinical studies. For
example, HR2 peptides, as exemplified by DP178 (also known as T20,
enfuvirtide, and
Fuzeon~ ; SEQ ID N0:3), T651 (SEQ ID N0:4), T649 (SEQ ID NO:S), blocked
infection
to of target cells with potencies of 0.5 ng/ml (EC50 against HIV-1 LAI)~ 5
ng/ml (IC50; HIV-1
IIIB), and 2 ng/ml (IC50; HIV-1 IIIB), respectively. Efforts have been made to
improve the
biological activity of HIV gp41-derived peptides, such as by trying to
stabilize the helical
structure of the peptide. Various efforts have been also been made to improve
the
pharmacological properties of HIV gp41-derived peptides.
15 Polymers have been used extensively to improve the pharmacokinetics and
pharmacodynamics (and hence, drug performance) of drugs such as peptides,
proteins,
and small molecules. The most widely used polymer for pharmaceutical
applications is
polyethylene glycol ("PEG"). "PEGylation" is the process by which the drug is
chemically
modified to result in the covalent attachment ("coupling") of one or more PEG
molecules
2o to the drug (depending on how many sites are available on the drug to
interact with, and
be conjugated to PEG). The improved pharmacological and biological properties
associated with PEGylation of drugs are well known in the pharmaceutical art.
For
example, PEGylation can increase therapeutic efficacy by means including, but
not
limited to, reducing degradation by proteolytic enzymes and thereby increasing
drug
25 concentration; increasing the size of the drug to which it is attached,
thereby improving
drug biodistribution; and shielding antigenic epitopes in reducing
immunogenicity where
desired. By increasing the therapeutic efficacy, reduced may be the frequency
of dosing
and/or the amount of drug need to achieve a therapeutic effect.
3o PEG, as a linear polyether, has a general structure of:
HO-(CH2-CH20)n-CHZCH2-OH where n can typically range from about 10 to
about 2000.
PEG, as a branched polyether, has a general structure of:
35 PEG-T-PEG
Z
wherein T is a linker or molecular bridge linking the PEG molecules, and Z is
the
2
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
functional group with chemically reactive moiety.
Many of the PEG modifications, in forming PEG derivatives (PEG and PEG
derivatives are known in the art as "PEG"), are directed to the end groups
("functionalities") in adding or varying their chemically reactive
functionalities to be used
to covalently attach the PEG molecule to a drug. Various PEG derivatives are
well known
in the art. To couple PEG to a drug, typically a functionality of the PEG
molecule needs
. to be activated so as to be chemically reactive. The type and specificity of
functionality is
based upon the choice of chemically reactive group on the drug to which the
PEG
molecule is to be coupled. Most commonly for proteins and peptides, the
chemically
to reactive group is present on an amino acid selected from the group
consisting of an
internal amino acid having a side chain with a free chemically reactive group
(e.g.,
including, but not limited to, lysine, cysteine, glutamic acid, serine,
threonine, and the
like), the N-terminal amino acid (having a N-terminal amine group, or a side
chain amine
group, as a free chemically reactive group), a C-terminal amino acid (ha'ving
a C-terminal
carboxylic acid, or side chain amine group, as a free chemically reactive
group), and a
combination thereof. Of the sites of a peptide to be coupled to PEG, most
frequently
chosen is the N-terminal amine group ("alpha amine") of the peptide's N-
terminal amino
acid, and the epsilon amine group ("epsilon amine") of a lysine (a lysine
found within the
amino acid sequence which is not the N-terminal amino acid or the C-terminal
amino acid
of the peptide) or an epsilon amine group of lysine when the lysine is present
in a peptide
as a N-terminal amino acid or as a C-terminal amino acid.
However, a problem arises with this standard strategy for PEGylation. Lysine
is
one of the most prevalent amino acids in proteins. As related to HIV gp41,
there are
multiple lysine residues in the amino acid sequences of the HR1 region and the
HR2
region (see, e.g., FIGs. 1-3). With respect to HIV gp41-derived peptide T20
(SEQ ID
N0:1 ), for example, there are two internal lysine residues in this 36 amino
acid residue
peptide. Thus, with a plurality of lysine residues in the amino acid sequence
(hence, a
plurality of side chain amines (epsilon amines) available to be reactive with
activated PEG
containing amine-reactive functionality) and an alpha amine, there exists
several sites to
3o which the activated PEG with an amine reactive functionality, can be
covalently coupled.
The result of standard PEGylation of such a peptide is a heterogeneous mixture
comprised of a population of several conjugates varying in the number of PEG
molecules
attached and in the sites of attachment. Heterogeneity of such a synthetic
peptide-
polymer conjugate is often an undesirable result. This is because the
pharmacological
and/or biological properties associated with PEGylation of peptides can be
dependent on
factors such as (a) the number of PEG molecules attached to the peptide, and
(b) the
location of the sites on the peptide to which PEG is coupled. For example, in
vitro
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
biological activity of FEGylated human growth hormone-releasing factor
depended on
both the site and degree of PEGylation. Further, from standard PEGylation, it
is very
difficult, if possible at all, to separate out the species of peptide-polymer
conjugate (with
the desired number of PEG molecules and desired sites) of attachment) from a
heterogeneous mixture using conventional separation techniques known in the
art. Such
separation attempts add to the expense, time, and reagents needed for
producing the
peptide-polymer conjugate of the desired species. Multiple lysine residues in
the amino
acid sequence of a peptide to be PEGylated are perceived as such a problem
that one
method of site-specific PEGylation was developed which involved replacing the
lysine
l0 residues with amino acids other than lysine, and which lack a side chain
having a free
amine.
Thus, in the formation of conjugates comprised of an HIV gp41-derived peptide
(containing one or more internal amino acid residues having a side chain amine
in its
amino acid sequence) and polymer, there is a need for a site-specific
modification of the
synthetic peptide so that produced is a synthetic peptide containing one or
more selected
amino acids having a side chain amine chemically protected with a chemical
protecting
agent, and one or more amino acids having an unprotected, free amine
available.
Accordingly, a polymer may be covalently coupled only to a specific site, or
specific sites,
on the synthetic peptide, as selected by a person performing the synthesis and
2o conjugation. Additionally, when the polymer to be conjugated is branched,
there is a
need for a site-specific modification of the synthetic peptide so as select
only one free
amine to be covalently coupled to polymer, in avoiding multiple branches of
the same
molecule of polymer conjugating to (and cross-linking of) the same molecule of
synthetic
peptide. More specifically, in an HIV gp41-derived peptide containing more
than one
chemically reactive ("free") amine group which is available for coupling to a
polymer
having amine-reactive functionality(s), it is desirable to chemically protect
one or more
selected amine groups, leaving the unprotected, free amine groups) available
for
covalently coupling to polymer. Additionally, it would be advantageous to
provide an HIV
gp41-derived peptide which has PEG coupled in a site-specific manner to one or
more
3o selected sites (i.e., in one or more selected amino acid positions) in the
synthetic peptide.
SUMMARY OF THE INVENTION
The present invention relates to a method for site-specific chemical
modification of
an HIV gp41-derived peptide during synthesis of the peptide, wherein the
synthesized
peptide has one or more amino acids having a side chain amine. The method
comprises
incorporating into the peptide, or a fragment thereof, during synthesis, at
least one amino
acid selected to have its side chain amine chemically reacted with a chemical
protecting
4
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
agent which protects the side chain amine from subsequent chemically
reactivity with an
amine-reactive functionality; and at least one amino acid having an amine
unprotected
and free for reacting with an amine-reactive functionality, wherein the free
amine is
selected from the group consisting of an N-terminal amine, a side chain amine,
and a
combination thereof. Also, produced from this method is an isolated HIV gp41-
derived
peptide having one or more amino acids containing a side chain amine, wherein
at least
one amino acid has its side chain amine chemically reacted with a chemical
protecting
agent which protects the side chain amine from subsequent chemically
reactivity with an
amine-reactive functionality; and at least one amino acid of the synthetic
peptide has an
to amine unprotected and free for reacting with an amine-reactive
functionality, wherein the
free amine is selected from the group consisting of an N-terminal amine, a
side chain
amine, and a combination thereof.
The present invention relates to a method for producing a substantially
homogeneous conjugate comprised of HIV gp41-derived peptide and polymer,
wherein
the HIV gp41-derived peptide has, incorporated into its amino acid sequence
during
synthesis, one or more amino acids having a side chain amine which.has been
selected
to be blocked by a chemical protecting agent, allowing only the desired
(selected)
unblocked free amine groups) of the synthetic peptide to be available for
reaction with a
polymer containing a amine-reactive functionality, in covalently coupling the
synthetic
2o peptide to the polymer at only specific sites) (amino acid positions) of
the synthetic
peptide) containing a free amine. The present invention also relates to a
substantially
homogeneous conjugate comprised of HIV gp41-derived peptide and polymer
produced
according to this method according to the present invention.
The present invention also provides a method for site-specific PEGylation of
an
HIV gp41-derived peptide, wherein PEG is covalently coupled in a site-specific
manner to
an HIV gp41-derived peptide. More particularly, the HIV gp41-derived peptide,
having
had incorporated into its amino acid sequence at selected amino acid positions
during
synthesis, one or more amine groups (e.g., one or more of: an alpha amine or
an epsilon
amine(s)) which is blocked with a chemical protecting agent from chemically
reactive with
amine-reactive functionality of PEG during PEGylation, thereby leaving
available for
PEGylation only the free amine groups) in selected amino acid positions
(through
chemical modification) of the synthetic peptide to be covalently coupled to
PEG. Using
the method of the present invention, provided is a substantially homogeneous
composition comprising a PEGylated HIV gp41-derived peptide containing one or
more
(as selected in performing the method of site-specific chemical modification)
amine
groups conjugated to PEG.
The methods of the present invention may further comprise removal of the
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
chemical protecting agent (in a "deprotection" step) in providing a
substantially
homogeneous conjugate comprised of an HIV gp41-derived peptide which is
conjugated
to a polymer at only specific sites) of the synthetic peptide, as selected in
performance of
the method of the present invention (e.g., through use of chemical
protection), wherein
such conjugate retains substantial anti-HIV activity (as compared to the anti-
HIV activity
of the synthetic peptide when not conjugated to polymer). The present
invention also
provides for a method of treating HIV infection (preferably, HIV-1 infection)
comprising
administering to an HIV-infected individual a pharmaceutical composition
comprising a
substantially homogeneous conjugate comprised of an HIV gp41-derived synthetic
1o peptide site-specifically coupled (e.g., conjugated) to polymer.
Preferably, the
pharmaceutical composition is in an amount effective to inhibit transmission
of HIV to a
target cell, and/or in an amount effective to inhibit gp41-mediated fusion of
HIV to a target
cell. Also provided is a method for inhibition of transmission of HIV to a
cell, comprising
contacting the virus in the presence of a cell with the substantially
homogeneous
is conjugate of polymer and synthetic peptide according to the present
invention in an
amount effective to inhibit infection of the cell by HIV. Additionally,
provided is a method
for inhibition of transmission of HIV to a cell, comprising adding to the
virus and the cell
an amount of the substantially homogeneous conjugate of polymer and synthetic
peptide
according to the present invention effective to inhibit infection of the cell
by HIV. Also
20 provided is a method for inhibiting HIV fusion (e.g., a process by which
HIV gp41
mediates fusion between the viral membrane and cell membrane during infection
by HIV
of a target cell), comprising contacting the virus in the presence of a cell
with an amount
of the substantially homogeneous conjugate of polymer and synthetic peptide
according
to the present invention effective to inhibit HIV fusion. These methods may be
used to
25 treat HIV-infected individuals.
The present invention also provides the use of a substantially homogeneous
conjugate of polymer and synthetic peptide, produced by the method according
to the
present invention, in the manufacture of a medicament for use with in therapy
of HIV
infection (e.g., used in a method of inhibiting transmission of HIV, a method
of inhibiting
3o HIV fusion, or a method of treating HIV infection), as described herein.
The medicament
is preferably in the form of a pharmaceutical composition comprising a
substantially
homogeneous conjugate of polymer and synthetic peptide according to the
present
invention together with a pharmaceutically acceptable carrier.
The above descriptions, features, and advantages of the present invention will
be
35 apparent in the following Detailed Description of the Invention when read
in conjunction
with accompanying drawings.
6
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WO 2005/089796 PCT/US2005/007486
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic of HIV-1 gp41 showing the heptad repeat 1 region (HR1 )
and
heptad repeat 2 region (HR2) along with other functional regions of gp41.
Exemplary
amino acid sequences corresponding to HR1 and HR2, and the amino acid position
numbering, are shown for purposes of illustration and in relation to gp160,
strain HIV,nB.
FIG. 2 shows a comparison of the sequences contained within the HR1 region of
HIV-1 gp41 for purposes of illustration, and not limitation, as determined
from various
laboratory strains and clinical isolates, wherein illustrated are some of the
variations in
amino acid sequence (e.g., polymorphisms), as indicated by the single letter
amino acid
to code.
FIG. 3 shows a comparison of the sequences contained within the HR2 region of
HIV-1 gp41 for purposes of illustration, and not limitation, as determined
from various
laboratory strains and clinical isolates, wherein illustrated are some of the
variations in
amino acid sequence (e.g., polymorphisms), as indicated by the single letter
amino acid
15 code.
FIG. 4 is a schematic showing synthesis of an HIV gp41-derived peptide using a
fragment condensation approach, wherein: the numbers represent respective
amino acid
positions relative to the synthesized HIV gp41-derived peptide; "K" represents
a lysine
internal to the sequence of the synthetic peptide, or to a fragment used in
the synthesis of
2o the synthetic peptide; "Ac" represents acetylation of the N-terminus; and
"NH" represents
amidation of the C-terminus.
FIG. 5 is a schematic showing synthesis of modified HIV gp41-derived peptide,
and a
conjugate of polymer and synthetic peptide, according to the present
invention, wherein:
the numbers represent respective amino acid positions relative to the
synthesized HIV
25 gp41-derived peptide; "K" represents a lysine internal to the sequence of
the synthetic
peptide, or to a fragment used in the synthesis of the synthetic peptide; "Ac"
represents
acetylation of the N-terminus; "NH" represents amidation of the C-terminus;
"X"
represents a chemical protecting agent which is coupled to an epsilon amine of
a
selected amino acid in selectively blocking the amino acid side chain from
further
3o chemical reactivity; and "I" represents a polymer which has specificity for
chemically
coupling with a free amine group, and becomes conjugated to a free amine of an
amino
acid which is not coupled to a chemical protecting agent in a site-specific
chemical
modification. Produced are: an isolated HIV gp41-derived peptide having at
least one
amine group (e.g., epsilon amine of the lysine residue at amino acid position
18)
35 chemically protected by a chemical protecting agent; and a substantially
homogeneous
conjugate comprised of HIV gp41-derived peptide and polymer, with polymer
conjugated
site-specifically to the synthetic peptide (e.g., at the epsilon amine of the
lysine residue at
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
amino acid position 28).
FIG. 6 is a schematic showing synthesis of modified HIV gp41-derived peptide,
and a
conjugate of polymer and synthetic peptide, according to the present
invention, wherein:
the numbers represent respective amino acid positions relative to the
synthesized HIV
gp41-derived peptide; "K" represents a lysine internal to the sequence of the
synthetic
peptide, or to the fragment used in .the synthesis of the synthetic peptide;
"Ac" represents
acetylation of the N-terminus; "NH" represents amidation of the C-terminus;
"X"
represents a chemical protecting agent which is coupled to an epsilon amine in
selectively blocking the amino acid side chain from further chemical
reactivity; and "I"
represents a polymer which is conjugated to an amine of an amino acid which is
not
coupled to a chemical protecting agent in a site-specific chemical
modification. Produced
are: an isolated HIV gp41-derived peptide having at least one amine group
(e.g., epsilon
amine of the lysine residue at amino acid position 28) chemically protected by
a chemical
protecting agent; and a substantially homogeneous conjugate comprised of HIV
gp41-
derived peptide and polymer, with polymer conjugated site-specifically to the
synthetic
peptide (e.g., at the epsilon amine of the lysine residue at amino acid
position 18).
DETAILED DESCRIPTION OF THE INVENTION
2o Definitions
The term "individual", when used herein for purposes of the specification and
claims, means a mammal, and preferably a human.
The term "target cell", when used herein for purposes of the specification and
claims, means a cell capable of being infected by HIV. Preferably, the cell is
a human
cell or are human cells; and more preferably, human cells capable of being
infected by
HIV via a process including membrane fusion.
The term "pharmaceutically acceptable carrier", when used herein for purposes
of
the specification and claims, means a carrier medium that does not
significantly alter the
biological activity of the active ingredient (e.g., a conjugate of polymer and
synthetic
3o peptide according to the present invention) to which it is added. A
pharmaceutically
acceptable carrier includes, but is not limited to, one or more of water,
buffered water,
saline, 0.3% glycine, aqueous alcohols, isotonic aqueous solution; and may
further
include one or more substances such as glycerol, oils, salts such as sodium,
potassium,
magnesium and ammonium, phosphonates, carbonate esters, fatty acids,
saccharides
(e.g., mannitol), polysaccharides, excipients, and preservatives and/or
stabilizers (to
increase shelf-life or as necessary and suitable for manufacture and
distribution of the
composition). Preferably, the pharmaceutically acceptable carrier is suitable
for
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WO 2005/089796 PCT/US2005/007486
intravenous, intramuscular, subcutaneous or parenteral administration.
By the term "amino acid" is meant, for purposes of the specification and
claims
and in reference to the synthetic peptides used in the present invention, to
refer to a
molecule that has at least one free amine group and at least one free carboxyl
group. The
amino acid may have more than one free amine group, or more than one free
carboxyl
group, or may further comprise one or more free chemical reactive groups other
than an
amine or a carboxyl group (e.g., a hydroxyl, a sulfhydryl, etc.). The amino
acid may be a
naturally occurring amino acid (e.g., L-amino acid), a non-naturally occurring
amino acid
(e.g., D-amino acid), a synthetic amino acid, a modified amino acid, an amino
acid
to derivative, an amino acid precursor, and a conservative substitution. One
skilled in the
art would know that the choice of amino acids incorporated into a peptide will
depend, in
part, on~the specific physical, chemical or biological characteristics
required of the
antiviral peptide. Such characteristics are determined, in part, by
determination of
structure and function (e.g., antiviral activity; as described herein in more
detail). For
example, the skilled artisan would know from the descriptions herein that
amino acids in a
synthetic peptide may be comprised of one or more of naturally occurring (L)-
amino acid
and non-naturally occurring (D)-amino acid. A preferred amino acid may be used
to the
exclusion of amino acids other than the preferred amino acid.
A "conservative substitution", in relation to amino acid sequence of a
synthetic
2o peptide used in the present invention, is a term used hereinafter for the
purposes of the
specification and claims to mean one or more amino acids substitution in the
sequence of
the synthetic peptide such that its biological activity is substantially
unchanged (e.g., if the
peptide inhibits HIV gp41-mediated fusion at a concentration in the nanomolar
range
before the substitution, after the substitution inhibition of HIV gp41-
mediated fusion is still
observed in the nanomolar range). As known in the art "conservative
substitution" is
defined by aforementioned function, and includes substitutions of amino acids
having
substantially the same charge, size, hydrophilicity, and/or aromaticity as the
amino acid
replaced. Such substitutions are known to those of ordinary skill in the art
to include, but
are not limited to, glycine-alanine-valine; isoleucine-leucine; tryptophan-
tyrosine; aspartic
3o acid-glutamic acid; arginine-lysine; asparagine-glutamine; and serine-
threonine. With
particular relevance to the present invention, a conserved substitution is
known in the art
to also include substituting lysine with ornithine, in providing a free amine
group (e.g.,
epsilon amine). For HIV gp41-derived peptides, such substitutions may also
comprise
polymorphisms at the various amino acid positions along the relevant HR region
(HR1 or
HR2) of gp41 found in any one or more of various Glades, laboratory strains,
or clinical
isolates of HIV, which are readily available from public databases and are
well known in
the art (see also, FIGs. 2 & 3, as illustrative examples).
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
The term "polymer" when used herein for purposes of the specification and
claims,
means a polymeric molecule which:(a) is employed in pharmaceutical
applications to
improve the pharmacological and/or biological properties when conjugated to a
drug (and
therefore is substantially nontoxic and substantially water soluble); (b) has
one or more
functionalities which by itself, and/or after activation to become chemically
reactive, can
be used to covalently couple to a free amine of the drug (e.g., a synthetic
peptide) in
forming a drug-polymer conjugate. Regarding the latter, the polymer preferably
has an
amine-reactive functionality for covalently coupling to synthetic peptide. A
polymer may
include, but is not limited to, polylysines or poly(D-L-alanine)-poly(L-
lysine)s, or polyols.
to A preferred polyol comprises a water- soluble poly(alkylene oxide) polymer,
and can have
a linear or branched chain. The term "polyol" is preferably a water-soluble,
polyalcohol
which may include, but is not limited to, polyethylene glycol ("PEG"),
polypropylene glycol
("PPG"), diethylene glycol, triethylene glycol, ethylene glycol, dipropylene
glycol,
copolymers comprising PPG (e.g., ethylene glycol/PPG), copolymers comprising
PEG
15 (e.g., PEG/PPG), mPEG (monomethoxy-polyethylene) glycol), and the like. A
polyol
encompasses both homopolymers and copolymers, and further may have a structure
comprising a branched structure or linear structure as known to those skilled
in the art.
Preferably, the polymer is substantially non-toxic when used for in vivo
applications in
individuals. In a preferred embodiment, the polymer has a molecular weight in
the range
2o between about 200 daltons to about 40,000 daltons; and in a more preferred
embodiment, the polymer has a molecular weight range between about 400 daltons
to
about 10,000 daltons. A preferred polymer for application in the present
invention
comprises a polyethylene glycol ("PEG"), and a more preferred polymer for
application in
the present invention comprises a polyethylene glycol having a molecular
weight range,
25 wherein the molecular weight range is no less than~about 400 daltons and is
no more
than about 20,000 daltons. As described previously herein, there are various
forms of
PEG that typically differ in the end groups or chemically reactive functional
groups to be
used to covalently attach the PEG molecule to a drug. Various PEGs are well
known in
the art. A preferred PEG, for use in coupling to one or more unprotected amine
groups of
3o the synthetic peptide in accordance with the present invention, has a
chemically reactive
group (e.g., "functionality") which can be used covalently couple PEG to the
to one or
more unprotected amine groups. PEG may include but is not limited to, PEG-
tresylate,
heterobifunctional PEG, PEG dichlorotriazine, PEG succinimidyl carbonate, PEG
benzotriazole carbonate, PEG p-nitrophenyl carbonate, PEG trichlorophenyl
carbonate,
35 PEG carbonylimidazole, PEG succinimidyl succinate, mPEG succinimidyl
propionate,
mPEG succinimidyl butanoate, PEG butyraldehyde, mPEG-propionaldehyde, PEG
aldehyde, PEG-acetaldehyde, PEG acetaldehyde diethyl acetal, PEG carboxylic
acid,
l0
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WO 2005/089796 PCT/US2005/007486
mPEG phenyl ether succinimidyl carbonates, mPEG benzamide succinimidyl
carbonates, PEG thioester, linear PEG, branched PEG, and linear forked PEG. A
preferred polymer may be applied to the present invention to the exclusion of
a polymer
other than the preferred polymer.
The terms "synthetic peptide" and "HIV gp41-derived peptide" are used
synonymously herein, in relation to a peptide employed in the present
invention, and for
the purposes of the specification and claims, to mean a peptide (a) comprising
an amino
acid sequence of no less than about 15 amino acids and no more than about 60
amino
acid residues in length, and comprises at least a portion of the amino acid
sequence
l0 (preferably, at least 9 contiguous amino acids) contained in either the HR1
region or HR2
region of gp41 of HIV (more preferably of HIV-1 ); and (b) capable of
inhibiting
transmission of HIV to a target cell (preferably, by complexing to an HR
region of HIV-1
gp41 and inhibiting fusion between HIV-1 and a target cell), as can be
determined by
assessing antiviral activity in vitro and/or in vivo, as will be described in
more detail
herein. More preferably, the synthetic peptide employed in the present
invention may
comprise a sequence of no less than 28 amino acids and no more than about 51
amino
acids in length, and even more preferably no less than about 36 amino acids
and no more
than about 51 amino acids in length. The term "isolated" when used in
reference to a
synthetic peptide means that it is substantially free of components which have
not
2o become part of the integral structure of the peptide itself; e.g., such as
substantially free
of chemical precursors or other chemicals when chemically synthesized,
produced, or
modified using biological, biochemical, or chemical processes. The synthetic
peptide
may comprise, in it's amino acid sequence, one or more conservative
substitutions and/or
one or polymorphisms found in the sequence of the relevant region of the HIV
gp41, or
may comprise one or more amino acid substitutions which are added to stabilize
helix
structure and/or affect oligomerization; provided that it retains substantial
antiviral activity
against HIV-1 (e.g., an IC50 in the picomolar to micromolar range). The
following are
illustrative examples of HIV gp41-derived peptides that can be site-
specifically conjugated
to polymer in accordance with the present invention. However, a preferred
synthetic
peptide may be used in the present invention to the exclusion of a synthetic
peptide other
than the preferred synthetic peptide. As apparent to one skilled in the art
and from the
teachings herein, a lysine iri the amino acid sequence of a synthetic peptide
may be
substituted with another amino acid (naturally occurring or not naturally
occurring) having
a side chain with a free amino group (e.g., epsilon amine). Ornithine is an
illustrative
example of such amino acid that may be used to substitute a lysine.
Preferably for use according to the present invention, for a synthetic peptide
comprising sequence derived from the HR1 region of HIV-1 gp41, the synthetic
peptide
11
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WO 2005/089796 PCT/US2005/007486
comprises a contiguous sequence of at least 15 amino acid residues in the
amino acid
sequence of SEQ ID N0:1, or polymorphisms thereof, as key determinants in this
portion
of the HR1 region (e.g., such as, noted by single letter amino acid
designation,
NNLLRAIEAQQHLLQLTVWG IKQLQARI LAVERYLKD which is amino acid residue 18 to
amino acid residue 54 of SEQ ID N0:1) have been found to influence structure,
and
biochemical and antiviral parameters described herein. Note that there are two
lysine
residues internal to this portion of the HR1 region, one or more of which may
be used for
site-specifically coupling to a polymer according to the present invention. A
preferred
example of a synthetic peptide derived from the HR-1 region of HIV gp41, and
as
containing the amino acids found in the native sequence of this region, is
illustrated as
having an amino acid sequence of SEQ ID N0:6. Other examples of a synthetic
peptide
derived from the HR-1 region of HIV gp41, and as containing the amino acids
found in the
native sequence of this region, are illustrated as having amino acid sequences
of SEQ ID
NOs:7-22, and may further comprise an amino acid sequence having at least 95%
identity, and more preferably having at least 90% identity, with any one or
more of SEQ
ID NOs:6-22. More preferably for use according to the present invention, a
synthetic
peptide derived from the HR1 region of HIV gp41 contains one or more amino
acid
substitutions (e.g., as compared to the amino acid sequence of SEQ ID NO:1 )
which
preferably enables the synthetic peptide to self-assemble into trimers (e.g.,
a trimer being
2o comprised of three molecules of synthetic peptide), as disclosed in more
detail in co-
pending application published as U.S. 20040076637. Examples of a synthetic
peptide
derived from the HR-1 region of HIV gp41 and which further comprises one or
more
amino acid substitutions which enable the synthetic peptide to self-assemble
into trimers
are illustrated as having amino acid sequences of SEQ ID NOs:23-36, and may
further
comprise an amino acid sequence having at least 95% identity, and more
preferably
having at least 90% identity, with any one or more of SEQ ID NOs: 23-36. Note
that such
synthetic peptides have one or more lysine residues internal to this portion
of the HR1
region, one or more of which may be chosen to be left unprotected (i.e., its
side chain
reactive group is not chosen to be coupled to the chemical protecting agent),
or may be
3o chosen to be chemically protected, in the site-specific chemical
modification according to
the present invention.
Preferably for use according to the present invention, for a synthetic peptide
comprising sequence derived from the HR2 region of HIV-1 gp41, the synthetic
peptide
comprises a contiguous sequence of at least amino acid residues 43 to 51 of
SEQ ID
N0:2 (e.g., QQEKNEQEL), or polymorphisms thereof, as key determinants in this
portion
of the HR2 region have been found to influence biochemical and antiviral
parameters
described herein. Note there is one internal lysine residue in this sequence.
Illustrative
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WO 2005/089796 PCT/US2005/007486
synthetic peptides derived from the HR2 region include, but are not limited to
peptides
having the amino acid sequences shown in SEQ ID NOs: 3, 4, 5, 37 to 63, and
175, and
may further comprise an amino acid sequence having at least 95% identity, and
more
preferably having at least 90% identity, with any one or more of SEQ ID NOs:3,
4, 5, 37 to
63, and 175. Note that such synthetic peptides have one or more internal
lysine residues
(and/or in the case of SEQ ID NOs. 34, 39, 48, and 175 at the carboxy
terminus), one or
more of which may be chosen to be left unprotected, or may be chosen to be
chemically
protected, in the site-specific chemical modification according to the present
invention.
More preferably for use according to the present invention, a synthetic
peptide derived
to from the HR2 region of HIV gp41 contains one or more amino acid
substitutions (e.g., as
compared to a relative portion of the amino acid sequence of SEQ ID N0:2)
which
preferably promotes the helicity and/or helix stability of the synthetic
peptide ("helix
stabilized peptide") in imparting improved biological activity, as disclosed
in more detail in
co-pending application PCT/US04/42918. Examples of such helix stabilized
synthetic
peptides are illustrated as having amino acid sequences of SEQ ID NOs:64-92,
and 113-
174, and may further comprise an amino acid sequence having at least 95%
identity, and
more preferably having at least 90% identity, with any one or more of SEQ ID
NOs: 64-92
and 113-174. Other examples of peptides designed for improved helicity and
derived
from the HR2 region of HIV gp41 may include SEQ ID NOs: 93-95. Note that such
helix
stabilized peptides have one or more internal lysine residues (and in some
cases
upwards to 25% of the amino acid sequence of the synthetic peptide), one or
more of
which may be chosen to be left unprotected, or chosen to be chemically
protected, in the
site-specific chemical modification according to the present invention.
In another preferred embodiment according to the present invention, the
synthetic
peptide may comprise a "hybrid" peptide comprising amino acid sequences
derived from
one or more of HIV-1, HIV-2, and SIV fusion proteins (see, e.g., U.S. Patent
No.
6,258,782). Examples of a hybrid synthetic peptide are illustrated as having
amino acid
sequences of SEQ ID NOs:96 to112, and may further comprise an amino acid
sequence
having at least 95% identity, and more preferably having at least 90%
identity, with any
one or more of SEQ ID NOs:96 to112. Note that such illustrated examples of
hybrid
synthetic peptides have at least two internal lysine residues, one or more of
which may be
chosen to be left unprotected, or chosen to be chemically protected, in the
site-specific
chemical modification according to the present invention.
The term "percent identity", when used herein for purposes of the
specification
and claims in reference to a sequence used in accordance with the present
invention,
means that the sequence is compared ("Compared Sequence") to a described or
13
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reference sequence ("Reference Sequence"); wherein a percent identity is
determined
according to the following formula:
percent identity= [1-(xC/yR)] x 100
wherein xC is the number of differences between the Reference Sequence and the
Compared Sequence over the length of alignment between the Compared Sequence
and
Reference Sequence wherein (a) each base or amino acid in the Reference
Sequence
that does not have a corresponding aligned base or amino acid compared to the
Compared Sequence, and (b) each gap in the Reference Sequence, and (c) each
aligned
base or amino acid in the Compared Sequence that is different from an aligned
base or
to amino acid in the Reference Sequence, constitutes a difference; and yR is
the number of
bases or amino acids in the Reference Sequence over the length of the Compared
Sequence with any gap created in the Reference Sequence as a result of
alignment also
being counted as a base or amino acid. Methods and software for alignment
between two
predetermined sequences are well know in the art. Thus, for example, a
Reference
Sequence may be a synthetic peptide according to any one of SEQ ID NOs: 1-175,
and a
Compared Sequence is an HIV gp41-derived peptide which is compared to the
Reference
Sequence, in determining an amino acid sequence having at least 95% identity
with any
one or more of the amino acid sequences of SEQ ID NOs: 1-175.
The term "chemical protecting agent", when used herein for purposes of the
specification and claims, means a chemical moiety that: (a) is chemically
reactive with a
free amine of an amino acid, thereby blocking ("chemically protecting") the
amine from
reacting with a polymer having a functionality that is amine-reactive; (b) can
withstand
(e.g., remains chemically reacted with the amine which it is chemically
protecting) a
deprotection step known to those skilled in the art for removing tBU (t-
butyl), Fmoc (9-
fluorenylmethoxycarbonyl), Boc (tent-butyloxycarbonyl), or trt
(triphenylmethyl(trityl)) from
an amino acid; and (c) can be subsequently removed from the amine of the amino
acid
to which it is chemically reacted, so that the amine becomes unprotected and
free for
chemical reactivity with an amine-reactive functionality. More particularly,
the chemical
protecting agent can withstand removal of Fmoc or Boc from a peptide by
reagents
3o typically used in the art for such deprotection, including, for example,
one or more of: 20%
piperdine, 2% DBU (1,8-diazabicyclo[5,4,0]undec-7-ene), 50% to 90%
trifluoroacetic acid,
a quartenary amine (such as tetrabutyl ammonium fluoride), or an inorganic
base such as
potassium carbonate. For example, as is discussed in more detail herein, the
chemical
protecting agent can remain stable (reacted with the amine group) until it is
desired to
remove the chemical protecting agent, and then the chemical protecting agent
is removed
in a subsequent, and separate deprotection step (e.g., using 2% hydrazine or
other
suitable reagent) in yielding a free amine. Such chemical protecting agents
are known in
14
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WO 2005/089796 PCT/US2005/007486
the art to include, but are not limited to, 1-(4,4-dimethyl-2,6-dioxocyclohex-
1-ylidene)ethyl
("Dde"), 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl (ivDde),
allyloxycarbonyl ("Alloc"), benzyloxycarbonyl ("Cbz"), and 2-
chlorobenzyloxycarbonyl ("2-
CI-Z"). Preferably, in a synthetic peptide, the free amine group reacting with
the chemical
protecting agent is an N-terminal amine of an N-terminal amino acid, or an
amine group
of a side chain (e.g., epsilon amine) of an amino acid (whether such amino
acid is the N-
terminal amino acid, a C-terminal amino acid, or an internal amino acid), or a
combination
thereof, as determined by the site-specific chemical modification. In a
preferred
embodiment, the chemical protecting agent is stable to amine bases to which
Fmoc, Boc,
l0 tBu, trt, or the like are labile.
The term "substantially homogeneous", when used herein for purposes of the
specification and claims and in reference to a conjugate comprised of an HIV
gp41-
derived peptide coupled to a polymer produced according to the present
invention,
means that at least 90%, and more preferably at least 95%, of the resultant
conjugate
15 produced contains the synthetic peptide site-specifically coupled to the
polymer as
intended (i.e., as a single species) by an orthogonal protection strategy
employed (as
described in more detail in Example 5 herein), according to the method of the
present
invention. The conjugate may be further purified using separation technology
including,
but not limited to, chromatographic techniques known in the art.
20 ____________________________________________________________
The present invention provides a method for site-specific chemical
modification of
an HIV gp41-derived peptide, and provides an isolated HIV gp41-derived peptide
containing at least one side chain amine chemically protected, and containing
at least one
amine (e.g., N-terminal amino acid alpha amine, one or more side chain
amines), or a
25 combination thereof) unprotected and free for reactivity with an amine-
reactive
functionality. The isolated HIV gp41-derived peptide may then be conjugated
(covalently
coupled) to a polymer at a site-specific location (i.e., at a particular amino
acid position, in
the synthetic peptide, having a free amine) which is selected (by
intentionally not
protecting it with a chemical protecting agent) to be coupled to the polymer.
Thus, the
3o coupling of the polymer to the synthetic peptide is via one or more free
amine groups of
the synthetic peptide available for chemical reaction with a polymer having a
functionality
which is amine-reactive. Accordingly, for example, one molecule of polymer is
covalently
coupled to an amino acid which is selected to have a free amine in producing a
substantially homogeneous conjugate comprised of polymer and HIV gp41-derived
35 peptide. For purposes of illustration, and not limitation, the following
schematics
demonstrate the methods according to the present invention, an isolated HIV
gp41-
derived peptide produced by a method according to the present invention, and a
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
substantially homogeneous conjugate comprised of polymer-synthetic peptide
according
to the present invention, by using an exemplary HIV gp41-derived peptide known
as T20
(SEQ ID N0:3).
Schematic 1: The synthetic peptide, being unmodified at the N-terminus after
synthesis,
has 3 free amine ("NH2") groups available for coupling to a polymer having an
amine
reactive functional group: the N-terminal amino acid alpha amine, and two
internal lysine
residues (labeled K~ and KZ for ease of description), each with a side chain
having an
epsilon amine.
to
Synthetic peptide: ~"N-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I ,
NH2 NH2
15 Schematic 2: The synthetic peptide shown in schematic 1 is conjugated to a
polymer ("I")
having an amine reactive functionality for coupling to the synthetic peptide.
A
heterogenous population of conjugates is possible from the conjugation
process, as
fol lows.
20 Conjugates: I-YTSLIHSLIEESQNQQE K~NEQELLELD K~WASLWNWF
I
I I
zHN-YTSLIHSLIEESQNQQE K~NEQELLELD K~WASLWNWF
2s
'I ~I
I -YTSLIHSLIEESQNQQE K~NEQELLELD K~WASLWNWF
3o
I NH2
~I -YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I
35 NH2 I
zHN-YTSLIHSLIEESQNQQE K~NEQELLELD KZWASLWNWF
I I
I NH2
zHN-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I
NH2
16
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WO 2005/089796 PCT/US2005/007486
I-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I
NH2 NH2
Schematic 3: For illustration purposes, only the free amine of internal lysine
residue "K~"
is selected to be free for chemical reactivity with an amine reactive
functionality. First, the
synthetic peptide is synthesized to incorporate amino acids, having amine
groups desired
to be blocked from reactivity with the polymer, protected by a chemical
protecting agent
to ("X") in forming an isolated HIV gp41-derived peptide having at least one
amino acid with
its side chain amine chemically protected (schematic 3A); polymer is then
conjugated to
such HIV gp41-derived peptide (schematic 3B) at the only amino acid having a
free
amine (lysine residue K~); and the chemical protecting agent is subsequently
removed
from amino acids to which it was chemically reacted, in yielding a
substantially
15 homogeneous conjugate (schematic 3C).
Schematic 3A: XZ"N-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I '
NH2 NH2X
20 Schematic 3BX2"N-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
I I
I NH2X
Schematic 3C: 2"N-YTSLIHSLIEESQNQQE K~NEQELLELD K2WASLWNWF
2s
I NH2
The following Examples illustrate the present invention, and should not be
construed as
limiting the present invention.
EXAMPLE 1
Synthetic peptides may typically be, and have been, synthesized by linear
synthesis on a peptide synthesizer using standard solid-phase synthesis
techniques and
using standard Fmoc peptide chemistry or other standard peptide chemistry.
Thus, as
shown in synthesis of a fragment illustrated herein, solid phase synthesis or
other
standard peptide chemistry may be used to synthesize the synthetic peptide,
wherein a
chemically protected amino acid (e.g., an amino acid having its side chain
amine
chemically protected as described herein) may be added in the desired amino
acid
position at the point in synthesis where such amino acid is incorporated into
the chain of
amino acids to produce the synthetic peptide (as illustrated in Example 6
herein).
However, in a preferred embodiment, the HIV gp41-derived peptide to undergo
site-
17
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WO 2005/089796 PCT/US2005/007486
specific chemical modification is synthesized using a fragment condensation
approach
(see, e.g., FIGs. 4-6), as described in more detail in Example 5 herein.
Briefly, 2 or more
fragments, each fragment containing amino acid sequence found in a respective
portion
of the synthetic peptide, is synthesized. In the synthesis of a fragment, if
desired,
incorporated may be an amino acid having its free amine (e.g., side chain
amine)
chemically protected by a chemical protecting agent. The fragments are then
assembled
(covalently coupled together in a manner and order) such that the synthetic
peptide is
produced (with the proper amino acid sequence). T20~ (SEQ ID N0:3) was
synthesized
by fragment condensation approach, as previously described in more detail
(see, e.g.,
l0 U.S. Patent No. 6,015,881). Briefly, and as summarized in FIG. 4, fragments
to be
assembled into the synthetic peptide are first synthesized. A fragment
comprising the
first 16 amino acids of SEQ ID N0:3 was synthesized by standard solid phase
synthesis
(using a super acid sensitive resin), with acetylation ("Ac") of the N-
terminus while having
a hydroxyl group (-OH) at the C-terminus. A fragment comprising amino acids 17-
26 of
SEQ ID N0:3 was synthesized by standard solid phase synthesis with Fmoc at the
N-
terminus, and -OH at the C-terminus. A fragment comprising amino acids 27-35
of SEQ
ID N0:3 was synthesized by standard solid phase synthesis with Fmoc at the N-
terminus,
and -OH at the C-terminus. As shown in FIG. 4, the fragment comprising amino
acids
27-35 of SEQ ID N0:3 is chemically coupled to amino acid 36 in solution phase
to result
2o in a fragment comprising amino acids 27-36 with amidation of the C-
terminus. The
fragment of amino acids.17-26 of SEQ ID N0:3 was chemically coupled with the
fragment of amino acids 27-36 of SEQ ID N0:3 (after removal of Fmoc from the N-
terminal amino acid 27). The resulting amino acid sequence having amino acids
17-36 of
SEQ ID N0:3 was chemically coupled with the fragment comprising amino acids 1-
16 of
SEQ ID N0:3 (after removal of Fmoc from the N-terminal amino acid 17) in
forming a
synthetic peptide comprising the amino acid sequence of SEQ ID N0:3. The
synthetic
peptide was deprotected/ decarboxylated (to remove tBU, trt, and Boc used in
the
synthesis of each fragment) with a deprotection step using a cocktail of
trifluoracetic
acid/dithiothrietol/water (volume percent:90/5/5) at 30 degrees C, for 5 to 6
hours with
3o stirring; and then purified using reverse-phase high performance liquid
chromatography.
Peptide identity was confirmed with electrospray mass spectrometry.
EXAMPLE 2
Illustrated is one embodiment of a method for site specific chemical
modification of
HIV gp41-derived peptide which may be used to produce (a) an isolated HIV gp41-
derived peptide having a side chain amine group of one or more of its internal
amino
acids chemically protected; and (b) a substantially homogeneous conjugate
comprised of
18
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WO 2005/089796 PCT/US2005/007486
HIV gp41-derived peptide and polymer. More specifically, incorporated into a
synthetic
peptide (or a fragment thereof if a fragment assembly approach is used) during
synthesis
are: one or more amino acids having its side chain amine blocked by a chemical
protecting agent from subsequent chemical reactivity with an amine-reactive
functionality;
and one or more amino acids having an amine (e.g., selected from the group
consisting
of N-terminal alpha amine, one or more epsilon amines, and a combination
thereof)
unprotected, and free for chemical reactivity with an amine-reactive
functionality. An
isolated synthetic peptide produced by this method for site-specific chemical
modification
can then be covalently coupled to polymer, in producing a substantially
homogeneous
1o conjugate, by chemically reacting the unprotected ("free") amine groups) of
the synthetic
peptide to the amine-reactive functionality of a polymer. In this illustrative
embodiment,
T20 (SEQ ID N0:3) was selected as the exemplary synthetic peptide, and the
lysine
residue at amino acid position 18 ("K18") (an amino acid with a side chain
amine) was
chosen to be chemically protected by a site-specifically chemical
modification, leaving
15 lysine residue at amino acid position 28 ("K28") as the internal amino acid
having a free
amine. The free amine may subsequently be chemically reacted with an amine-
reactive
functionality of a polymer, in covalently coupling the polymer to the
synthetic peptide via
the lysine at amino acid position 28 of the amino acid sequence of the
synthetic peptide.
In referring to FIGS, T20 (SEQ ID N0:3) was synthesized using the fragment
20 condensation approach previously described in Example 1 herein.
Briefly, and as summarized in FIG. 5, a fragment comprising the first 16 amino
acids
of SEQ ID NO:3 was synthesized by standard solid phase synthesis with the N-
terminal
amine of amino acid residue 1 ("Y") being acetylated ("Ac"). A fragment
comprising
amino acids 17-26 of SEQ ID N0:3 was synthesized by standard solid phase
synthesis
25 using Fmoc-Lys-(ivDde) as amino acid residue 18, so that the chemical
protecting agent
ivDde ("X" in FIG. 5) blocks the epsilon amine group of K18 from reacting
subsequently
with an amine reactive functionality. A fragment comprising amino acids 27-35
of SEQ ID
N0:3 was synthesized by standard solid phase synthesis, and chemically coupled
to
amino acid 36 in solution phase in forming a fragment comprising amino acids
27-36 of
3o SEQ ID N0:3. The fragment comprising amino acids 17-26 of SEQ ID N0:3 (with
the
ivDde protected K18) was chemically coupled with the fragment of amino acids
27-36 of
SEQ ID N0:3 (containing a lysine at position 28 ("K28") with a free epsilon
amine). The
resulting amino acid sequence comprising amino acids 17-36 of SEQ ID N0:3 was
combined with the fragment comprising amino acids 1-16 of SEQ ID N0:3 in
forming an
35 isolated HIV gp41-derived peptide having the amino acid sequence of SEQ ID
N0:3 and
containing at least one amino acid having its side chain amine group
chemically protected
(blocked from subsequent chemical reactivity with an amine-reactive
functionality) by a
19
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WO 2005/089796 PCT/US2005/007486
chemical protecting agent. The synthetic peptide (SEQ ID N0:3) was deprotected
to
remove protecting groups trt, Boc, and tBu used in standard solid phase
synthesis as
described in more detail in Example 1 herein (while K18 remains chemically
protected);
decarboxylated; and then purified using reverse-phase high performance liquid
chromatography. The isolated HIV gp41-derived peptide was then used for
coupling a
polymer site-specifically to the free epsilon amine of K28 of SEQ ID N0:3.
In producing a substantially homogeneous conjugate comprised of HIV gp41-
derived
peptide and polymer, mPEG succinimidyl propionate ("mPEG -SPA") was chosen as
the
exemplary polymer to conjugate to T20 (SEQ ID NO:3). T20 (SEQ ID N0:3) with
ivDde
to on epsilon amine group of K18 (9.0 mg, 2.O~mol) was dissolved in dimethyl
formamide
(DMF)(0.3 ml). Diisopropylethylamine (DIEA) (10p,1) was added to the reaction,
and then
added was mPEG-SPA (average molecular weight, 5000 daltons ("5K"); 20 mg,
4.O~mol)
in DMF (1 ml). The mixture was stirred at room temperature and the reaction is
monitored
by HPLC until the PEGylation was completed. To remove the chemical protecting
agent
15 ivDde from the epsilon amine group of K18, hydrazine (40p,1) was added to
the reaction to
reach 3% (v/v) of hydrazine in reaction mixture. The stirring continued for
another 30
minutes or until HPLC shows deprotection is completed. The reaction mixture
was diluted
by water (6.5m1) to make the final concentration of DMF at 20%, and then
filtered through
syringe filter (0.45p,m, 2ml). HPLC purification was carried out on
polystyrene/
2o divinylbenzene column (PRLP-S, 300A, 10~m, 250*21.2mm) with acetonitrile-
water-0.1
trifluoroacidic acid buffer as eluent. The collected fractions were checked by
HPLC with
both UV and ELS detectors. The pure fractions were pooled together and
lyophilized for
two days. The desired conjugate, a substantially homogeneous conjugate
comprised of
5K-PEG-T20 at K28, was obtained as fluffy white solid (5.5mg) after
lyophilization.
25 In another variation of this embodiment, the alpha amine of the N-terminal
amino
acid of SEQ ID N0:1 was not acetylated, but instead was protected with~an Fmoc
group.
The process of synthetic peptide synthesis and conjugation to polymer was
performed as
provided in this Example 2. Thus, the resultant substantially homogeneous
conjugate
comprised 5K-PEG-T20 at K28, except that the T20 (SEQ ID NO:3) of the
conjugate
30 contained a free alpha amine at the N-terminal amino acid (Y).
EXAMPLE 3
Illustrated is another embodiment of a method for site specific chemical
modification of HIV gp41-derived peptide which may be used to produce (a) an
isolated
35 HIV gp41-derived peptide having one or more internal amino acids with a
side chain
amine chemically protected; and (b) a substantially homogeneous conjugate
comprised of
HIV gp41-derived peptide and polymer. In this illustrative embodiment, T20
(SEQ ID
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WO 2005/089796 PCT/US2005/007486
N0:3) was selected as the exemplary synthetic peptide, and the lysine residue
at amino
acid position 28 ("K28") was chosen to be chemically protected by a site-
specific chemical
modification, leaving lysine residue at amino acid position 18 ("K18") as the
internal
amino acid to be free for subsequent coupling to a polymer via the lysine side
chain
amine and an amine-reactive functionality of a polymer. In referring to FIG.
6, T20 (SEQ
ID N0:3) was synthesized using the fragment condensation approach essentially
as
described in Example 1 herein. Briefly, and as summarized in FIG. 6, a
fragment of SEQ
ID N0:3 comprising the first 16 amino acids was synthesized by standard solid
phase
synthesis with the N-terminal amine of amino acid residue 1 (Tyr) being
acetylated ("Ac").
to A fragment of SEQ ID N0:3 comprising amino acids 17-26 was synthesized by
standard
solid phase synthesis. A fragment of SEQ ID N0:3 comprising amino acids 27-35
was
synthesized by standard solid phase synthesis using Fmoc-Lys-(ivDde) as amino
acid
residue 28 ("K28"), so that the chemical protecting agent ivDde ("X" in FIG.
6) blocks the
epsilon amine group of K28 from subsequent chemical reactivity with an amine
reactive
15 functional group. The latter fragment was coupled to amino acid 36 in
solution phase to
form a fragment having amino acids 27-36 of SEQ ID N0:3. The fragment having
amino
acids 17-26 (containing K18 with a free epsilon amine) was combined with the
fragment of
amino acids 27-36 (with the ivDde protected K28). The resulting amino acid
sequence
having amino acids 17-36 was combined with the fragment comprising amino acids
1-16
2o in forming a synthetic peptide comprising the amino acid sequence of SEQ ID
N0:3. The
synthetic peptide (SEQ ID N0:3) was deprotected to remove protecting groups
trt, Boc,
and tBu used in standard solid phase synthesis as described in more detail in
Example 1
herein (while K28 remains chemically protected); decarboxylated; and then
purified using
reverse-phase high performance liquid chromatography. The isolated HIV gp41-
derived
25 peptide was then used for coupling to a polymer site-specifically to the
free epsilon amine
of K18 of SEQ ID N0:3.
mPEG succinimidyl propionate ("mPEG -SPA") was chosen as the exemplary
polymer to conjugate to T20 (SEQ ID N0:3). T20 (SEQ ID N0:3) with ivDde on
epsilon
amine group of K28 (19.7 mg, 4.4pmol) was dissolved in DMF (0.5 ml). DIEA
(20p,1) was
3o added to the reaction, and then added was mPEG-SPA (average molecular
weight, 5000
daltons ("5K"); 50 mg, 10~,mol) in DMF (1ml). The mixture was stirred at room
temperature and the reaction is monitored by HPLC until the PEGylation was
completed.
To remove the chemical protecting agent ivDde from the epsilon amine group of
K28,
hydrazine (45p1) was added to the reaction to reach 3% (v/v) of hydrazine in
reaction
35 mixture. The stirring continued for another 30 minutes (or until HPLC shows
deprotection
is completed). The reaction mixture was diluted by water (6.5m1) to make the
final
concentration of DMF at 20%, then filtered through syringe filter (0.45~m,
2ml). HPLC
21
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WO 2005/089796 PCT/US2005/007486
purification was carried out on polystyrene/divinylbenzene column (PRLP-S,
300A, 10~m,
250*21.2mm) with acetonitrile-water-0.1 % trifluoroacidic acid buffer as
eluent. The
collected fractions were checked by HPLC with both UV and ELS detectors. The
pure
fractions were pooled together and lyophilized for taivo days. The desired
conjugate, a
substantially homogeneous conjugate comprised of 5K-PEG-T20 at K18 was
obtained as
fluffy white solid (10.4mg) after lyophilization.
In another variation of this embodiment, the alpha amine of the N-terminal
amino acid
of SEQ ID N0:1 was not acetylated, but instead was protected with an Fmoc
group. The
process of synthetic peptide synthesis and conjugation to polymer was
performed as
l0 provided in this Example 3. Thus, the resultant conjugate comprised a
substantially
homogeneous conjugate comprised of 5K-PEG-T20 at K18, except that the T20 (SEQ
ID
N0:3) of the conjugate contained a free alpha amine at the N-terminal amino
acid (Y).
EXAMPLE 4
15 Illustrated in this example are: (a) a method determining the antiviral
activity of
substantially homogeneous conjugates produced according to the present
invention; and
(b) the need for a site-specific chemical modification according to the
present invention to
produce a substantially homogeneous conjugate comprised of polymer covalently
coupled to HIV gp41-derived peptide. In using an in vitro assay for
demonstrating
2o antiviral potency, it is important to note that antiviral effect of
synthetic peptide
demonstrated in the in vitro assay has been correlated with the antiviral
effect of the
synthetic peptide in vivo. In determining antiviral activity (e.g., one
measure being the
ability to inhibit transmission of HIV to a target cell) of the synthetic
peptide-polymer
conjugates produced according to the present invention, used is an in vitro
assay which
25 has been shown, by data generated using synthetic peptides derived from
either of the
HR regions of HIV gp41, to be predictive of antiviral activity observed in
vivo. More
particularly, antiviral activity observed using an in vitro infectivity assay
("Magi-CCR5
infectivity assay"; see, e.g., U.S. Patent No. 6,258,782) has been shown to
reasonably
correlate to antiviral activity observed in vivo for the same HIV gp41-derived
peptides. To
3o further emphasize this point, T20 (SEQ ID N0:3) and T1249 (SEQ. ID N0:96)
each have
demonstrated potent antiviral activity against HIV in both the in vitro
infectivity assay and
human clinical trials.
The infectivity assays score for reduction of infectious virus titer employing
the
indicator cell lines MAGI or the CCRS expressing derivative cMAGI. Both cell
lines
35 exploit the ability of HIV-1 tat to transactivate the expression of a ~i-
galactosidase reporter
gene driven by the HIV-LTR. The ~-gal reporter has been modified to localize
in the
nucleus and can be detected with the X-gal substrate as intense nuclear
staining within a
22
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WO 2005/089796 PCT/US2005/007486
few days of infection. The number of stained nuclei can thus be interpreted as
equal to
the number of infectious virions in the challenge inoculum if there is only
one round of
infection prior to staining. Infected cells are enumerated using a CCD-imager
and both
primary and laboratory adapted isolates show a linear relationship between
virus input
and the number of infected cells visualized by the imager. In the MAGI and
cMAGI
assays, a 50% reduction in infectious titer (Vn/Vo = 0.5) is significant, and
provides the
primary cutoff value for assessing antiviral activity ("IC50" is defined as
the dilution
resulting in a 50% reduction in infectious virus titer). A secondary cutoff of
Vn/Vo = 0.1,
corresponding to a 90% reduction in infectious titer is also assessed
("IC90"). The
to substantially homogeneous conjugates tested for antiviral activity were
diluted into
various concentrations, and tested in duplicate or triplicate against an HIV
inoculum
adjusted to yield approXimately 1500-2000 infected cells/well of a 48 well
microtiter plate.
The substantially homogeneous conjugate (in the respective dilution) was added
to the
cMAGI or MAGI cells, followed by the virus inocula; and 24 hours later, an
inhibitor of
15 infection and cell-cell fusion (e.g., T20) is added to prevent secondary
rounds of HIV
infection and cell-cell virus spread. The cells were cultured for 2 more days,
and then
fixed and stained with the X-gal substrate to detect HIV-infected cells. The
number of
infected cells for each control and substantially homogeneous conjugate
dilution was
determined with the CCD-imager, and then the IC50 and IC90 is calculated
(typically
2o expressed in ~,g/ml).
In this example, several substantially homogeneous conjugates, separately
produced by the methods described herein, were analyzed for antiviral activity
as shown
in Table 1, and are identified as follows. "Conjugate A" is a synthetic
peptide having the
amino acid sequence of SEQ ID N0:3 having a 2K PEG site-specifically
conjugated to
25 the N-terminal amine (hence, the side chain amines of both K18 and K28 were
chemically
protected during synthesis). "Conjugate B" is a synthetic peptide having the
amino acid
sequence of SEQ ID N0:3 having a 2K PEG site-specifically conjugated to K18
(thus, the
N-terminal amine and the side chain amine of K28 were chemically protected
during
synthesis). "Conjugate C" is a synthetic peptide having the amino acid
sequence of SEQ
3o ID N0:3 having a 2K PEG site-specifically conjugated to K28 (thus, the N-
terminal amine
and the side chain amine of K18 were chemically protected during synthesis).
"Conjugate D" is a synthetic peptide having the amino acid sequence of SEQ ID
N0:3
having a 5K PEG site-specifically conjugated to the N-terminal amine (hence,
the side
chain amines of both K18 and K28 were chemically protected during synthesis).
35 "Conjugate E" is a synthetic peptide having the amino acid sequence of SEQ
ID N0:3
having a 5K PEG site-specifically conjugated to K18 (thus, the N-terminal
amine and the
side chain amine of K28 were chemically protected during synthesis).
"Conjugate F" is a
23
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WO 2005/089796 PCT/US2005/007486
synthetic peptide having the amino acid sequence of SEQ ID N0:3 having a 5K
PEG site-
specifically conjugated to K28 (thus, the N-terminal amine and the side chain
amine of
K18 were chemically protected during synthesis).
Table 1
Molecule tested Antiviral activity
IC50 /ml
S nthetic a tide SEQ ID N0:3< 0.01
Con'u ate A <0.02
Con'u ate B >0.05 <0.1
Con'u ate C >0.15
Con'u ate D >0.05 <0.1
Con'u ate E >0.3
Conjugate F >0.4
From this comparison, it is clear that antiviral activity of the synthetic
peptide is
best preserved when a polymer of average size of 2K (2,000 daltons) is
conjugated to the
N-terminal amine versus at K18 (with at least 5 fold less activity) or K28
(with at least 10
to fold less activity). Similarly, it is clear that antiviral activity of the
synthetic peptide is best
preserved when a 5K polymer is conjugated to the N-terminal amine versus at
K18 (about
fold less activity) or K28 (about 7 fold less activity). Further, it can be
concluded from
this example, that the method of site specific chemical modification according
to the
present invention may be used to site-specifically couple a polymer to a
selected amino
acid of the synthetic peptide in producing a substantially homogeneous
conjugate of
peptide-polymer conjugate having a desired level of biological activity (e.g.,
in this
example, desired antiviral activity is measured by having an IC50 of less than
0.02 pg/ml)
(e.g., "Conjugate A" of Table 1 ) while avoiding producing a synthetic peptide-
polymer
conjugate of multiple species that lacks the desired level of biological
activity using
2o standard PEGylation (e.g., a mixture of "Conjugate A" and "Conjugate B" and
"Conjugate
C" of Table 1 ).
EXAMPLE 5
In this example, illustrated are additional embodiments of the method for site-
specific chemical modification of an HIV gp41-derived peptide, wherein
incorporated into
a synthetic peptide during synthesis are: one or more amino acids having its
side chain
amine blocked by a chemical protecting agent from subsequent chemical
reactivity with
an amine-reactive functionality; and one or more amino acids having an amine
(e.g.,
selected from the group consisting of N-terminal alpha amine, one or more
epsilon
3o amines, and a combination thereof) unprotected, and free for chemical
reactivity with an
amine-reactive functionality. The resultant isolated HIV gp41-derived is then
conjugated
24
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WO 2005/089796 PCT/US2005/007486
to a polymer in producing a substantially homogeneous conjugate comprised of
polymer
and HIV gp-41 derived peptide.
Based on the teachings in Example 1 about a fragment condensation approach to
synthesizing an HIV gp41-derived peptide, it is apparent to one skilled in the
art that this
approach of fragment assembly can be used, and has been used for some of the
synthetic peptides having a sequence shown in SEQ. ID. NOs: 3-175, in the
methods
according to the present invention. Generally speaking, typically 3 fragments
are
synthesized (see, e.g., FIG. 3): an "N-terminal fragment" (usually comprised
of between
and 20 of the amino acids of the amino terminus of the synthetic peptide), a
"C-
to terminal fragment" (usually comprised of between 10 and 20 of the amino
acids of the
carboxy terminus of the synthetic peptide), and a "middle fragment" (usually
comprised of
between 10 and 20 of the amino acids found between the N-terminal fragment and
the C-
terminal fragment, in the synthetic peptide) which are then assembled to
produce the
complete synthetic 'peptide. However, depending on the length, the amino acid
sequence, the number and location of amino acids with side chain amines in the
amino
acid sequence of a particular synthetic peptide, anywhere from 2 to 4
fragments have
been synthesized, and then assembled to complete the synthesis of that
particular
synthetic peptide.
For example, T1249 (SEQ ID N0:96) was used in the method of site-specific
chemical modification according to the present invention. In this example, the
synthetic
peptide was synthesized by the fragment condensation approach using 3
fragments: an
N-terminal fragment comprising amino acids 1-12 and containing a lysine at
amino acid
position 7 ("K7"), and an acetylated N-terminal amino acid; a middle fragment
comprising
amino acids 13 to 26 and containing a lysine at amino acid position 21 ("K21
"); and a C-
terminal fragment comprising amino acids 27 to 36 and containing a lysine at
amino acid
position 28 ("K28") and a lysine at amino acid position 31 ("K31 ") (the amino
acid position
numbering corresponding to the respective positions in SEQ ID N0:96; i.e., in
the
assembled synthetic peptide). A number of isolated HIV gp41-derived peptides
according
to the present invention were separately produced: (a) a synthetic peptide
having the
3o amino acid sequence of SEQ ID N0:96 with a chemical protecting agent on K7,
K21,
K28, and K31 (leaving only the N-terminal amine free for subsequent
conjugation to
polymer); (b) a synthetic peptide having the amino acid sequence of SEQ ID
N0:96 with
a chemical protecting agent on the N-terminal amine, K21, K28, and K31
(leaving only K7
side chain amine free for subsequent conjugation to polymer); (c) a synthetic
peptide
having the amino acid sequence of SEQ ID N0:96 with a chemical protecting
agent on
the N-terminal amine, K7, K28, and K31 (leaving only K21 side chain amine free
for
subsequent conjugation to polymer); (d) a synthetic peptide having the amino
acid
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
sequence of SEQ ID NO:96 with a chemical protecting agent on the N-terminal
amine,
K7, K21, and K31 (leaving only K28 side chain amine free for subsequent
conjugation to
polymer); and (e) a synthetic peptide having the amino acid sequence of SEQ ID
N0:96
with a chemical protecting agent on the N-terminal amine, K7, K21, and K28
(leaving only
K31 side chain amine free for subsequent conjugation to polymer).
Several substantially homogeneous conjugates were separately produced from
these isolated HIV gp41- derived peptides having an amino acid sequence of SEQ
ID
N0:96 and the method for specific chemical modification described herein,
using different
sizes of PEG ranging from an average of 2K daltons ("2K") to an average of 20K
daltons
to ("20K"). Table 2 shows antiviral activity of some of these substantially
homogeneous
conjugates, identified as follows. "Conjugate A" is a synthetic peptide having
the amino
acid sequence of SEQ ID NO:96 having a 2K PEG site-specifically conjugated to
the N-
terminal amine (hence, the side chain amines of K7, K21, K28, and K31 were
chemically
protected during synthesis). "Conjugate B" is a synthetic peptide having the
amino acid
15 sequence of SEQ ID N0:96 having a 2K PEG site-specifically conjugated to K7
(thus, the
N-terminal amine, and the side chain amines of K21, K28, arid K31 were
chemically
protected during synthesis). "Conjugate C" is a synthetic peptide having the
amino acid
sequence of SEQ ID N0:96 having a 2K PEG site-specifically conjugated to K21
(thus,
the N-terminal amine, and the side chain amines of K7, K28, and K31 were
chemically
20 protected during synthesis). "Conjugate D" is a synthetic peptide having
the amino acid
sequence of SEQ ID N0:96 having a 2K PEG site-specifically conjugated to K28
(thus,
the N-terminal amine, and the side chain amines of K7, K21, and K31 were
chemically
protected during synthesis). "Conjugate E" is a synthetic peptide having the
amino acid
sequence of SEQ ID N0:96 having a 2K PEG site-specifically conjugated to K31
(thus,
25 the N-terminal amine, and the side chain amines of K7, K21, and K28 were
chemically
protected during synthesis). "Conjugate F" is a synthetic peptide having the
amino acid
sequence of SEQ ID N0:96 having a 5K PEG site-specifically conjugated to the N-
terminal amine. "Conjugate G" is a synthetic peptide having the amino acid
sequence of
SEQ ID N0:96 having a 5K PEG site-specifically conjugated to K7. "Conjugate H"
is a
30 synthetic peptide having the amino acid sequence of SEQ ID N0:96 having a
5K PEG
site-specifically conjugated to K21. "Conjugate I" is a synthetic peptide
having the amino
acid sequence of SEQ ID N0:96 having a 5K PEG site-specifically conjugated to
K28.
"Conjugate J" is a synthetic peptide having the amino acid sequence of SEQ ID
N0:96
having a 5K PEG site-specifically conjugated to K31.
26
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WO 2005/089796 PCT/US2005/007486
Table 2
Molecule tested Antiviral activity
IC50 /ml
S nthetic a tide SEQ ID N0:96< 0.01
Con'u ate A <0.01
Con'u ate B ~ <0.01
Con'u ate C >0.01
Con'u ate D <0.01
Con'u ate E >0.01
Con'u ate F >0.03
Con'u ate G >0.04
Con'u ate H >0.04
Con'u ate I >0.03
Con'u ate J >0.04
From this example, and in viewing the results using a PEG of average size of
2K
(2000 daltons), it can be concluded that the method of site-specific chemical
modification
according to the present invention may be used to site-specifically couple a
polymer to a
selected amino acid of the synthetic peptide in producing a substantially
homogeneous
conjugate having a desired level of biological activity (e.g., in this
example, the desired
antiviral activity is measured by having an IC50 of less than 0.01 pg/ml)
(e.g., "Conjugate
A", "Conjugate B", and "Conjugate D" of Table 2) while avoiding producing a
synthetic
l0 peptide-polymer conjugate of multiple species that lacks such desired level
of biological
activity using standard PEGylation (e.g., a mixture of the Conjugates A-E of
Table 2).
EXAMPLE 6
In another example, a synthetic peptide having an amino acid sequence of SEQ
ID N0:174 was used in the method of site-specific chemical modification
according to the
present invention. In this example, the synthetic peptide containing a lysine
at amino acid
position 30 ("K30") and a lysine at amino acid position 39 ("K39"; the C-
terminal amino
acid; the amino acid position numbering corresponding to the position in SEQ
ID N0:174)
was synthesized by linear synthesis with a chemical protecting agent on the N-
terminal
2o amine of the N-terminal amino acid, and on the side chain amine of K30
(leaving only the
K39 side chain amine free for subsequent conjugation to polymer).
Substantially
homogeneous conjugates were separately produced from this isolated HIV gp41-
derived
peptide using a polymer having an average size of 2K, a polymer having an
average size
of 5K, and a polymer having an average size of 20K. For example, the synthetic
peptide
alone (not conjugated to a polymer) has a desired level of biological activity
(e.g., antiviral
activity as measured by having an IC50 of less than or equal to 0.02 pglml),
whereas a
substantially homogeneous conjugate synthetic having a polymer of average size
of 2K
had biological activity about equal to 0.02 pg/ml. Substantially homogeneous
conjugates
27
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
having a polymer of average size of either 5K or 20K had biological activity
much greater
than 0.1 pg/ml (i.e., outside the range of desired biological activity for
this example).
EXAMPLE 7
The present invention provides for substantially homogeneous conjugates
comprised of an HIV gp41-derived peptide to which is coupled site-specifically
a polymer
("synthetic peptide-polymer conjugates"). Antiviral activity of such synthetic
peptide-
polymer conjugates can be utilized in a method for inhibiting transmission of
HIV to a
target cell, comprising adding to the virus and cell an amount of synthetic
peptide-polymer
conjugate according to the present invention effective to inhibit infection of
the cell by
HIV, and more preferably, to inhibit HIV-mediated fusion between the virus and
the target
cell. This method may be used to treat HIV-infected individuals
(therapeutically) or to
treat individuals newly exposed to or at high risk of exposure (e.g., through
drug usage or
high risk sexual behavior) to HIV (prophylactically). Thus, for example, in
the case of an
HIV-1 infected individual, an effective amount of synthetic peptide-polymer
conjugate
would be a dose sufFicient (by itself and/or in conjunction with a regimen of
doses) to
reduce HIV viral load in the individual being treated. As known to those
skilled in the art,
there are several standard methods for measuring HIV viral load which include,
but are
not limited to, by quantitative cultures of peripheral blood mononuclear cells
and by
plasma HIV RNA measurements. The synthetic peptide-polymer conjugates of the
invention can be administered in a single administration, intermittently,
periodically, or
continuously, as can be determined by a medical practitioner, such as by
monitoring viral
load. Depending on the formulation containing synthetic peptide-polymer
conjugate, and
such factors as the compositions of the polymer and synthetic peptide used in
forming the
synthetic peptide-polymer conjugate and whether or not further comprising a
pharmaceutically acceptable carrier and the nature of the pharmaceutically
acceptable
carrier, the synthetic peptide-polymer conjugate according to the present
invention may
be administered with a periodicity ranging from days to weeks or possibly
longer.
Further, a synthetic peptide-polymer conjugate according to the present
invention may be
3o used, in antiviral therapy, when used in combination or in a therapeutic
regimen (e.g.,
when used simultaneously, or in a cycling on with one drug and cycling off
with another)
with other antiviral drugs used for treatment of HIV (e.g., including, but not
limited to,
other HIV entry inhibitors (e.g., CCR5 inhibitors, retrocyclin, and the like),
HIV integrase
inhibitors, reverse transcriptase inhibitors (e.g., nucleoside or
nonnucleoside), protease
inhibitors, viral-specific transcription inhibitors, viral processing
inhibitors, HIV maturation
inhibitors, inhibitors of uridine phosphorylating enzyme, HIV vaccines, and
the like, as
well known in the art.
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CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
For example, in one preferred embodiment, one or more antiviral agents may be
combined in therapy with synthetic peptide-polymer conjugate according to the
present
invention, thus increasing the efficacy of the therapy, and lessening the
ability of the virus
to become resistant to the antiviral drugs. Such combinations may be prepared
from
effective amounts of antiviral agents (useful in treating of HIV infection)
currently
approved or approved in the future, which include, but are not limited to,
abacavir, AZT,
delaviridine, ddC, ddl, efavirenz, FTC, (+) and (-) FTC, Reverset, GS 840,
HBY097, 3TC,
nevirapine, d4T, FLT, emtricitabine, amprenivir, CGP-73547, CGP-61755, DMP-
450,
indinavir, nelfinavir, PNU-140690, ritonavir, saquinavir, telinavir,
tenofovir, adefovir,
to atazanavir, lopinavir, VX 478, PRO-542, and betulin and dihydrobetulin
derivatives (e.g.,
PA-457). Effective dosages of these illustrative antiviral agents, which may
be used in
combinations with synthetic peptide-polymer conjugate according to the present
invention, are known in the art. Such combinations may include a number of
antiviral
agents that can be administered by one or more routes, sequentially or
simultaneously,
depending on the route of administration and desired pharmacological effect,
as is
apparent to one skilled in the art.
Effective dosages of a synthetic peptide-polymer conjugate of the invention to
be
administered may be determined through procedures well known to those in the
art; e.g.,
by determining potency, biological half-life, bioavailability, and toxicity.
In a preferred
2o embodiment, an effective synthetic peptide-polymer conjugate dosage range
is
determined by one skilled in the art using data from routine in vitro and in
vivo studies
well know to those skilled in the art. For example, in vitro infectivity
assays of antiviral
activity, such as described herein, enables one skilled in the art to
determine the mean
inhibitory concentration (IC) of the synthetic peptide-polymer conjugate
necessary to
block some amount of viral infectivity (e.g., 50% inhibition, ICSO; or 90%
inhibition, IC9o).
Appropriate doses can then be selected by one skilled in the art using
pharmacokinetic
data from one or more standard animal models, so that a minimum plasma
concentration
(C[min]) of the synthetic peptide-polymer conjugate is obtained which is equal
to or
exceeds a predetermined IC value. While dosage ranges typically depend on the
route of
3o administration chosen and the formulation of the dosage, an exemplary
dosage range of
the synthetic peptide-polymer conjugate according to the present invention may
range
from no less than 0.1 ~g/kg body weight and no more than 10 mg/kg body weight;
preferably a dosage range of from about 0.1-100 ~g/kg body weight; and more
preferably,
a dosage of between from about 10 mg to about 250 mg of synthetic peptide-
polymer
conjugate.
A synthetic peptide-polymer conjugate of the present invention may be
administered to an individual by any means that enables the active agent to
reach the
29
CA 02556032 2006-08-14
WO 2005/089796 PCT/US2005/007486
target cells (cells that can be infected by HIV). Thus, the synthetic peptide-
polymer
conjugates of this invention may be administered by any suitable technique,
including
oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, or
subcutaneous
injection or infusion, intradermal, or implant), nasal, pulmonary, vaginal,
rectal, sublingual,
or topical routes of administration, and can be formulated in dosage forms
appropriate for
each route of administration. The specific route of administration will
depend, e.g., on the
medical history of the individual, including any perceived or anticipated side
effects from
such administration, and the formulation of conjugate being administered
(e.g., the nature
of the polymer and synthetic peptide of which the synthetic peptide-polymer
conjugate
1o comprises). Most preferably, the administration is by injection (using,
e.g., intravenous
or subcutaneous means), but could also be by continuous infusion (using, e.g.,
slow-
release devices or minipumps such as osmotic pumps, and the like). A synthetic
peptide-polymer conjugate according to the present invention may further
comprise a
pharmaceutically acceptable carrier; and may further depend on the formulation
desired,
site of delivery, the method of administration, the scheduling of
administration, and other
factors known to medical practitioners. a
The foregoing description of the specific embodiments of the present invention
have been described in detail for purposes of illustration. In view of the
descriptions and
illustrations, others skilled in the art can, by applying current knowledge,
readily modify
2o and/or adapt the present invention for various applications without
departing from the
basic concept; and thus, such modifications and/or adaptations are intended to
be within
the meaning and scope of the appended claims.
What is claimed is:
35
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPRI~:ND PLUS D'UN TOME.
CECI EST L,E TOME 1 DE 2
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des
Brevets.
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THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
THIS IS VOLUME 1 OF 2
NOTE: For additional valumes please contact the Canadian Patent Office.
