Title of Invention

PEPTIDE-BASED COMPOUNDS COMPRISING A POLY (ETHYLENE GLYCOL) MOIETY AND PHARMACEUTICAL COMPOSITIONS COMPRISING SAID PEPTIDE-BASED COMPOUNDS

Abstract There is disclosed a peptide-based compound comprising a peptide moiety and at least one poly(ethylene glycol) moiety, wherein the peptide moiety is a peptide monomer or a peptide multimer comprising a plurality of peptide monomers, each peptide monomer of the peptide moiety comprising no more than 50 amino acids; and wherein the poly(ethylene glycol) moiety is linear and has a molecular weight of 20 KDaltons or more.
Full Text PEPTIDE-BASED COMPOUNDS COMPRISING A POLY(ETHYLENE "GLYCOL)
MOIETY AND PHARMACEUTICAL COMPOSITIONS COMPRISING SAID PEPTIDE-
BASED COMPOUNDS
1. CROSS-REFERENCE TO RELATED APPLICATIONS
Priority is claimed under 35 U.S.C.§119(e) to co-pending U.S. Provisional
Patent Application Serial No. 60/470,246 filed on May 12, 2003. The contents of this
priority application are hereby incorporated into the present disclosure by reference
and in their entirety.
1. FIELD OF THE INVENTION
The present invention relates to modification of peptide-based compounds
with poiy(ethylene glycol) or "PEG." In particular, the invention relates to peptide
monomers, diniers, and oligomers that are modified with PEG, preferably a linear
PEG moiety between 20 and 60 KDaltons. In addition, the invention relates to novel
therapeutic metliods using such PEG modified compounds.
2. BACKGROUND OF THE INVENTION
In recent years, with the development of research on proteins, a great number
of peptides having various actions have been found. With the progress of genetic
recombination techniques and organic synthetic methods of peptides, it has become
possible to obtain these physiologically active peptides and their structurally
analogous compounds in a large amount. Many of these peptides having special
activity are extremely useful as pharmaceuticals.
Examples of such peptides include peptides that bind to erythropoietin (EPO)
receptors (EPO-R). EPO is a glycoprotein hormone with 165 amino acids, 4
glycosylation sites on amino acid positions 24, 38, 83, and 126, and a molecular
weight of about 34,000. It stimulates mitotic di%dsion and tlie differentiation of
erythrocyte precursor cells and thus ensures the production of eiythrocytes. EPO is
essential in th^^gsFQcess of red blood cell formation, the honnone has potentially usefi,il

applications in both the diagnosis and the treatment of blood disorders characterized
by low or defective red blood cell production. A number of peptides^that interact with
the EPO-R have been discovered. (See e.g., U.S. Patent No. 5,773,569 to Wrighton et
al; U.S. Patent No. 5,830,851 to Wrighton et al; and WO 01/91780 to Smith-
Swintosky et al.
However, the clearance of peptides, particularly when administered in the
circulatory system, is generally very iiast: ¦ Therefore, it is desirable to improve tlie
durability of supli,peptides. =JXLaddition, when the peptides are obtained from different
species of animals, designed by peptide protein engineering, and/or having structures
different from those of tlie subject, there is a risk of causing serious symptoms due to
the production of antibodies. Hence, it is also desirable to improve the antigenicity of
such peptides. In order to use these peptides as pharmaceuticals, it is necessary to
have both improved antigenicity and durability.
Chemical modification of the peptides with macromolecular compounds such
as poly(ethylene glycol) has been shown to be effective to improve the antigenicity
and durability of various peptides. Thus, poly(ethylene glycol) and poly(ethylene
glycol) derivatives have been widely used as peptide-modifying macromolecular
reagents.
In its most common form, poly(ethylene glycol) has the following structure:
HO-(CH2CH20)nCH2CH2-OH
The above polymer, alpha-, omega-dihydi-oxyl poly(ethylene glycol) can be
represented in brief form as HO-PEG-OH where it is understood that the -PEG-
symbol represents the following structural unit:
-CH2CH20-(CH2CH20)„-CH2CH2-
Without being Umited to any particular theory or mechanism of action, the
long, chain-like PEG molecule or moiety is believed to be heavily hydrated and in
rapid motion when in an aqueous medium. This rapid motion is beheved to cause the
PEG to sweep out a large volume and prevents the approach and interference of other
molecules. As a result, when attached to another chemical Gntiiy (such as a peptide),
PEG polymer chains can protect such chemical entity from immune response and
other clearance mechanisms. As a result, PEGylation leads improved drug efficacy
and safety by optimizing pharmacokinetics, increasing bioavailability, and decreasing
iramunogenicity and dosing jfrequency.
For example, some active derivatives of PEG have been attached to proteins
and enzymes with beneficial results. PEG is soluble in organic solvents. PEG
attached to enzymes can result in PEG-enzyme conjugates that are soluble and active
in organic solvents. Attachment of PEG to protein can reduce the innnimogenicity
and rate of kidney clearance of the PEG-protein conjugate as compared to the
unmodified protein, which may result in dramatically increased blood circulation
lifetimes for the conjugate.
For example, covalent attachment of PEG to therapeutic proteins such as
interleulcins (Knauf, M. J. et al, J. Biol. Chem. 1988, 263,15,064; Tsutsumi, Y. et al,
J. Controlled Release 1995, 33, 447), interferons (Kita, Y. et al. Drug Des. DeUvery
1990, 6, 157), catalase (Abuchowski, A. et al, J. Biol. Chem. 1977, 252, 3, 582),
superoxide dismutase (Beauchamp, C. O. et al, Anal. Biochem. 1983, 131, 25), and
adenosine deaminase (Chen, R. et al, BiocMm. Biophy. Acta 1981, 660, 293), has
been reported to extend their half life in vivo, and/or reduce their immunogenicity and
antigenicity.
In addition, PEG attached to surfaces can reduce protein and cell adsorption to
the surface and alter the electrical properties of the surface. Similarly, PEG attached
to liposomes can result in a great increase in the blood circulation lifetime of these
particles and thereby possibly increase their utility for drug delivery. (J. M. Harris,
Ed., "Biomedical and Biotechnical Applications of Polyethylene Glycol Chemistry,"
Plenum, New York, 1992).
U.S. Patent 5,767,078 to Johnson et al. discloses dimerization of peptide
monomers which can bind to EPO-R. The dimerization is based on covalent linkage
of the monomers. PEG is the preferred linlcer to form tlie dimers. The PEGs
specifically used therein have a molecular weight of only 3400 or 5000.
WO 01/91780 to Smitli-Swintosky et al discloses dimers and multimers of
peptides that exhibit binding and signal initiation of growth factor-type receptors.
The linker disclosed is polyethylene glycol. The linker disclosed is polyethylene
glycol. Hov/ever, the reference offers no guidance for selecting the appropriate sizes
or classes (e.giAin.eai) of PEG.

U.S. Patent 6Sill,92>9 to Wei et al. discloses compositions consisting
essentially of a polypeptide and a water-soluble polymer covalentl}^. bound thereto at
the N-tenninal a-carbon atom via a hydrazone or reduced hydrazone bond, or an
oxime or reduced oxime bond. The molecular weight range of the water soluble
poljaner is in tlie range of 200 to 200K Daltons. PEG is disclosed as an example of
tlie water-soluble polymer. The molecular weight of the PEG is from only 700 to 20K
Daltons, and PEG moieties of only 5K Daltons are said to be preferred.
WO 01/38342 to Balu et al. discloses a dimer formed by a Ci-u linking moiety
linking two peptide chains. It indicates that the N-termini of the dimer may be
PEGlylated. However, the publication does not specify the molecular weight of the
PEG used or indicate whether it is linear or branched.
Saifer et al. (Adv. Exp. Med. Biol. (1994), 366:377-87) describe PEGylated
adduct of bovine and recombinant human Cu, Zn superoxide dismutase (SOD) in
which 1-9 strands of high molecular weight (35K - 120K Daltons) PEG are coupled of
SOD. Somack et al. (Free Rad. Res. Comms; (1991), 12-13:553-562) describe SOD
adducts containing 1 to 4 strands of high molecular weight (41K - 72K Daltons) PEG.
Neither of these two references teaches modifying a peptide with PEG. Moreover, it
is believed that the PEG moieties used in tliese compoimds were branched, as opposed
to hnear PEG.
Despite the advances made in the area of the PEG-modified peptide-based
compounds, there remains a need for novel PEG-modified compounds with improved
antieenicitv and durabiiitv.
The citation and/or discussion of a reference in this section, and throughout
this specification, shall not be construed as an admission that such reference is prior
art to the present invention.
SUINIMARY OF THE INVENTION
The present invention relates to a peptide-based compound comprising a
peptide moiety and a poly(ethylene glycol) moiety wherein the poly(ethylene glycol)
¦moietvis linftnr has n molecular weight of more than 20 KDaltons.

Preferably, the poly(ethyIene glycol) moiety has a moleculai- weight of jfrom
about 20 to 60 KDaltons. More preferably, the poly(ethylene glycol).moiety has a
molecular weight of from about 20 to 40 KDaltons. Most preferably, the PEG has a
molecular v/eight of about 20 KDaltons.
Preferably, the poly(ethylene glycol) moiety has a polydispersity value
(Mw/Mn) of less than 1.20, more preferably less than 1.1, and most preferably less
than 1.05.
Preferably, the peptide moiety is dimeric and comprises two monomeric
peptides linked by a linlcer moiety. Moreover, such dimers and other multimers may
be heterodimers or heteromultimers.
In one embodiment, the peptide moiety is selected from peptides which bind
to erythropoietin-receptors. Non-limiting examples of such EPO-R binding peptides
include those disclosed in published intemational applications PCT/USOO/32224
(publication no. WO 01/38342 A2), PCT/US96/09S10 (publication no. WO 96/40749)
and PCT/USOl/16654 (publication no. WO 01/91780 Al); U.S. Patents 5,767,078,
5,773,569, 5,830,851, 5,986,047. Still other exemplary EPO-R binding peptides
which may be used as the peptide moiety in the present invention are described in
U.S. Provisional Application Serial No. 60/479,245 filed May 12, 2003. Still other
exemplary EPO-R binding peptides which may be used as the peptide moiety in the
present invention are described, in U.S. Provisional Application Serial No. 60/469,993
filed May 12, 2003. Yet still other exemplary EPO-R binding peptides which may be
used as the peptide moiety in the present invention are described in U.S. Provisional
Application Serial No. 60/470,244 filed May 12, 2003.
In another embodiment, the peptide moiety is selected from peptides which
bind to thrombopoietin-receptors ("TPO-R"). Non-limiting examples of such TPO-R
binding peptides include those disclosed in U.S. Patents 6,552,008, 6,506,362,
6,498,155, 6,465,430, 6,333,031, 6,251,864, 6,121,238, 6,083,913, 5,932,546,
5,869,451, 5,683,983, 5,677,280, 5,668,110, and 5,654,276; and published U.S. Patent
Applications 2003/0083361, 2003/0009018,2002/0177166 and 2002/0160013.
Preferably, such peptide-based compound further comprises a spacer moiety
between the peptide moiety and the poly(eihylene glycol) moiety. More preferably,
the spacer moipty has the structure:

-NH-(CH2)a-[0-(CH2)pVOs-(CH2)s-Y-
wherein a, P, y, 5,and e are each integers whose values are independently selected.
Such spacer moiety is described in more details in U.S. provisional patent application
Serial No. 60/469,996, filed May 12, 2003, entitled "Novel Spacer Moiety For
Poly(ethylene Glycol) Modified Peptide-base Compomids".
In preferred embodiments,
a is an integer, 1 p is an integer, 1 £ is an integer, 1 5 is 0 or 1;
Y is an integer, 0 Y is either NH or CO.
Li certain preferred embodiments, P = 2 when y > 1.
hi one particularly preferred embodiment,
a = p = e = 2;
y = 5 = 1; and
YisNH.
hi other embodiinents,
y = 5 = 0;
2 Y is CO.
hi certain other embodiments,
y = 5 = 0;
a + 8 =5; and
YisCO.
The present invention fiirthei* relates to phannaceutical compositions
comprising o^ or more of the peptide-based compounds described above.

DETAILED DESCRIPTION
Definitions
Amino acid residues in peptides are abbreviated as follows: Phenylalanine is
Phe or F; Leucine is Leu or L: Isoleucine is He or I; Metliionine is Met-or M; Valine is
Val or V; Serine is Ser or S; Proline is Pro or P; Thieonine is Tlir or T; Alanine is Ala
or A; Tyrosine is Tyr or Y; Histidine is His or H; Glutamine is Gin or Q; Asparagine
is Asn or N; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or
E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and Glycine is
Gly or G. The unconventional amino acids in peptides are abbreviated as follows: 1-
naphthylalanine is 1-nal; 2-naphthylalanine is 2-nal; N-methylglj^cine (also laiown as
sarcosine) is MeG; acetylated glycme (N-acetylglycine) is AcG; homoserine
methylether is Hsm.
"Peptide" or "polypeptide" refers to a polymer in which the monomers are
alpha amino acids joined together through amide bonds. Peptides are two or often
more amino acid monomers long. Generally, when used in the art and in the context
of the present invention, the term "peptide" refers to a polypeptide that is only a few
amino acid residues in length. In particular, peptides of the present invention are
preferably no more than about 50 amino acid residues in length, and are more
preferably between about 5 and 40 amino acid residues in length, even more
preferably between about 17 and 40 amino acid residues in length. By contrast, a
polypeptide may comprise any ntmiber of amino acid residues. Hence, polypeptides
include peptides as well as longer sequences of amino acids, such as proteins which
can be hundreds of amino acid residues in length.
A peptide used in the present invention can be part of or "derived from" a
longer polypeptide sequence, such as the sequence of a protein.
As used herein, the phrase "phannaceutically acceptable" refers to molecular
entities and compositions that are "generally regarded as safe", e.g., that are
physiologically tolerable and do not typically produce an allergic or similar untoward
reaction, such as gastric upset, di22;iness and the lilce, when administered to a human.
Preferably, as;used herein, the term "phannaceutically acceptable" means approved by
a regulatory s^igeajcy 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. The term "carrier" refers to a' diluent, adjuvant,
excipient, or vehicle with which tlie compound is administered. Such pharmaceutical
carriers can be sterile liquids, such as water and oils, including those of petroleum,
animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. Water or aqueous solution saline solutions and aqueous
dextrose and glycerol solutions are preferably employed as carriers, particularly for
injectable solutions. Suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E.W. Martin.
As used herein the term "agonist" refers to a biologically active Ugand which
binds to its complementary biologically active receptor and activates the latter either
to cause a biological response in the receptor, or to enhance preexisting biological
activity of the receptor.
PEG Moiety
The PEG moiety used in the present invention is linear and has a molecular
weight of 20 KDaltons or more. Preferably, the PEG has a molecular weight of from
about 20 to 60 KDaltons. More preferably, the PEG has a molecular weight of from
about 20 to 40 KDaltons. Most preferably, the PEG has a molecular weight of 20
KDaltons. '
The PEG moiety is covalently attached to the compounds of the invention,
either directly to a peptide moiety, a linker moiety, or a spacer moiety. In one
embodiment, a PEG moiety is attached to at least one terminus (N-temiinus or C-
terminus) of a peptide monomer or dimer: for example, each N-terminus of a peptide
dimer may have an attached PEG moiety (for a total of two PEG moieties). In one
embodiment, PEG may serve as a linlcer that dimerizes two peptide monomers: for
example, a single PEG moiety may be simultaneously attached to both N-termini of a
peptide dimer. In another embodiment, PEG is attached to a spacer moiety of a
peptide monomer or dimer. In a preferred embodiment PEG is attached to the linlcer
moiety of a peptide dimer. In a highly preferred embodiment, PEG is attached to a
spacer moiety, where said spacer moiety is attached to the linlcer Lk. moiety of a
peptide dimer. Most preferably, PEG is attached to a spacer moiety, where said

spacer moiety is attached to a peptide dimer via the carbonyj carbon of a lysine linlcer
or the amide nitrogen of a lysine amide linlcer.
The peptide-based compounds of the present invention may compnse multiple
PEG moieties {e.g., 2, 3, 4, or more). In certain embodiments the PEG moiet>
comprises two linear monomeric PEG chains. Preferably the two linear PEG chains
are linked together through lysine residue or a lysine amide (a lysine residue wherein
the carboxyl group has been converted to an amide moiety-CONHa)- More
preferably, the two PEG chains are linked to lysine's alpha and epsilon amino groups
while the carboxylic group is activated as hydroxysuccinimidyl esters for binding to
the spacer moiety. For example, when a lysine amide hnks the two monomeric PEG
chains the dimer may be illustrated structurally as shown in Formula I, and
summarized as shown in Formula II:
In Formula I, N represents the nitrogen atom of lysine's g-amino group and
N' represents the nitrogen atom of lysine's a-amino group. In preferred
embodiments, the C-tenninal lysine of the two peptide monomers is L-lysine. In
alternative embodiments, one ore more lysine residues can be D-lysine.
Where the compound comprises more than one PEG moieties, the multiple
PEG moieties may be tlie same or different chemical moieties (e.g., PEGs of different
molecular weight). In some cases, the degree of PEGylation (the number of PEG
moieties attached to a peptide and/or the total number of peptides to which a PEG is
attached) may be influenced by the proportion of PEG molecules versus peptide
molecules in a PEGylation reaction, as well as by the total concentration if each in the
reaction mixture. In general, the optimum PEG versus peptide ratio (in terms of
reaction efficiency to provide for no excess unreacted peptides and/or PEG) will be
determined by factors such as the desired degree of PEGylation (kg., mono, di-, tri-,
etc.), the molecular weight of the polymer selected, whether the polymer is branched
or unbranched, and the reaction conditions for a particular attachment method.
There are a number of PEG attachment methods available to those skilled in
the art [see, e.g., Goodson, et al. (1990) Bio/Technology 8:343 (PEGylation of
interleuldn-2 at its glycosylation site after site-directed mutagenesis); EP 0 401 384
(coupling PEG to G-CSF); Malik, et al, (1992) Exp. Hematol. 20:1028-1035
(PEGylation of GM-CSF using tresyl chloride); PCT Pub. No. WO 90/12874
(PEGylation of er5lhropoietin containing a recombinantly introduced cysteme residue
using a cysteine-specific mPEG derivative); U.S. Pat. No. 5,757,078 (PEGylation of
EPO peptides); U.S. Pat. No. 5,612,460 (Active Carbonates of Polyalkylene Oxides
for Modification of Polypeptides), U.S. Pat. No. 5,672,662 (Poly(ethylene glycol) and
related polymers monosubstituted with propionic or butanoic acids and fimctional
derivatives thereof for biotechnical applications); U.S. Pat. No. 6,077,939
(PEGylation of an N-tenndnal a-carbon of a peptide); Veronese et al., (1985) Appl.
Biochem. Bioechnol 11:141-142 (PEGylation of an N-terminal a-carbon of a peptide
with PEG-nitrophenylcarbonate ("PEG-NPC") or PEG-trichlorophenylcarbonate); and
Veronese (2001) Biomaterials 22:405-417 (Review article on peptide and protein
PEGylation)].
For examjple, PEG may be covalently bound to amino acid residues via a
reactive group. Reactive groups are those to which an activated PEG molecule may
be bound {e.g., a fi:ee amino or carboxyl group). For example, N-tenninal amino acid
residues and lysine (K) residues have a free amino group; and C-tenninal amino acid
residues have a firee carboxyl group. Sulfhydryl groups (e.g., as found on cysteine
residues) may also be used as a reactive group for attaching PEG. In addition,
enzyme-assisted methods for introducing activated groups {e.g., hydrazide, aldehyde,
and aromatic-amino groups) specifically at the C-terminus of a polypeptide have been
described [Schwarz, et al. (1990) Methods Enzymol. 184:160; Rose, et al. (1991)
Bioconjugate Chem. 2:154; Gaertner, etal. (1994) J. Biol. Chem. 269:7224].
For example, PEG molecules may be attached to amino groups using
methoxylated PEG ("mPEG") having dijBferent reactive moieties. Non-limiting .
examples of &^^reactive moieties include succinimidyl succinate (SS), succinimidyl

carbonate (SC), mPEG-imidate, para-nitrophenylcarbonate (NPC), succinimidy]
propionate (SPA), and cyanuric chloride. Non-limiting examples of such mPEGs with
reactive moieties include mPEG-succininiidyl succinate (mPEG-SS), mPEG-
succinimidyl carbonate (raPEG-SC), mPEG-imidate, mPEG-para-
nitrophenylcarbonate (niPEG-NPC), niPEG-succininiidyl propionate (mPEG-SPA),
and mPEG-cyanuric chloride.
Wliere attachment of the PEG is non-speciiic and a peptide containing a
specific PEG attachment is desired, the desired PEGylated compomid may be purified
firom the mixture of PEGylated compounds. For example, if an N-terminally
PEGylated peptide is desired, the N-termiBally PEGylated form may be purified from
a population of randomly PEGylated peptides {i.e., separating this moiety from other
monoPEGylated moieties),
Li preferred embodiments, PEG is attached site-specifically to a peptide. Site-
specific PEGylation at the N-tenninus, side chain, and C-terminus of a potent analog
of growth hormone-releasing factor has been performed through solid-phase synthesis
[Felix, et al. (1995) Int. J. Peptide Protein Res. 46:253]. Another site-specific method
involves attaching a peptide to extremities of liposomal surface-grafted PEG chains in
a site-specific manner tlirough a reactive aldehyde group at the N-terminus generated
by sodium periodate oxidation of N-terminal threonine [Zaiipsky, et al. (1995)
Bioconj. Chem. 6:705]. However, this method is limited to polypeptides with N-
terminaJ serine or threonine residues.
hi one method, selective N-terminal PEGylation may be accomplished by
reductive allcylation which exploits differential reactivity of different types of primary
amino groups (lysine versus the N-terminal) available for derivatization in a particular
protein. Under the appropriate reaction conditions, a carbonyl group containing PEG
is selectively attached to the N-terminus of a peptide. For example, one may
seleciayely N-terminally PEGylate the protein by performing the reaction at a pH
which exploits the pKa differences between the s-amino groups of a lysine residue and
the a- amino group of tlie N-terminal residue of the peptide. By such selective
attachment, PEGylation takes place predominantly at the N-terminus of the protein,
with no significant modification of other reactive groups {e.g., lysine side chain amino
groups). Using reductive aUcylation, the PEG should have a single reactive aldehyde
for coupling toffee protein {e.g., PEGproprionaldehyde may be used).


Site-specific mutagenesis is a fiirther approach which may be used to prepare
peptides for site-specij5c polymer attachment. By this method,Nthe amino acid
sequence of a peptide is designed to incorporate an appropriate reactive group at the
desired position within the peptide. For example, WO 90/12874 describes the site-
directed PEGylation of proteins modified by the insertion of cysteine residues or the
substitution of other residues for cysteine residues. This publication also describes
the preparation of mPEG-erythropoietin ("mPEG-EPO") by reacting a cysteine-
specific mPEG derivative with a recombinantly introduced cysteine residue on EPO.
Where the PEG moiety is attached to a spacer moiety or linker moiety, similai"
attacliment methods may be used. In this case, the linlcer or spacer contains a reactive
, group and an activated PEG molecule containing the appropriate complementary
reactive group is used to effect covalent attaclmient. In preferred embodiments the
linlcer or spacer reactive group is a terminal reactive group {i.e., positioned at the
terminus of the linker or spacer).
Peptides, peptide dimers and other peptide-based molecules of the invention
can be attached to water-soluble polymers (e.g., PEG) using any of a variety of
chemistries to linlc the water-soluble polymer(s) to the receptor-binding portion of tlie
molecule (e.g., peptide + spacer). A typical embodiment employs a single attachment
junction for covalent attachment of the water soluble polymer(s) to the receptor-
binding portion, however in alternative embodiments multiple attachment junctions
may be used, including further vaiiations wherein diffeirent species of water-soluble
polymer are attached to the receptor-binding portion at distinct attachment junctions,
which may include covalent attachment junction(s) to the spacer and/or to one or both
peptide chains. In some embodiments, tlie duner or higher order multimer will
comprise distinct species of peptide chain (i.e., a heterodimer or other
heteromultimer). By way of example and not limitation, a dimer may comprise a first
peptide chain having a PEG attachment junction and the second peptide chain may
either lack a PEG attachment junction or utilize a different Unkage chemistry than the
first peptide chain and in some variations the spacer may contain or lack a PEG
attachment junction and said spacer, if PEGylated, may utilize a linlcage chemistry
different than that of the first and/or second peptide chains. An alternative
embodiment employs a PEG attached to the spacer portion of the receptor-binding

portion and a different water-soluble polymer (e.g., a carbohydrate) conjugated to a
side chain of one of the amino acids of the peptide portion of the molecule. ¦
A wide variety of polyethylene glycol (PEG) species may be used for
PEGylation of the receptor-binding portion (peptides + spacer). Substantially any
suitable reactive PEG reagent can be used. In preferred embodiments, the reactive
PEG reagent will result in formation of a carbamate or amide bond upon conjugation
to the receptor-binding portion. Suitable reactive PEG species include, but are not
limited to, those which are available for sale in the Drug Deliveiy Systems catalog
(2003) of NOF Corporation (Yebisu Garden Place Tower, 20-3 Ebisu 4-chome,
Shibuya-lcu, Tokyo 150-6019) and the Molecular Engineering catalog (2003) of
Nelctar Therapeutics (490 Discovery Drive, Huntsville, Alabama 35806). For example
and not limitation, the following PEG reagents are often preferred in various
embodiments: niPEG2-NHS, niPEGz-ALD, multi-Ami PEG, mPEG(MAL)2,
mPEG2(MAL), mPEG-NHa, mPEG-SPA, mPEG-SBA, mPEG-thioesters, mPEG-
Double Esters, mPEG-BTC, mPEG-ButyrALD, mPEG-ACET, heterofunctional PEGs
(NH2-PEG-C00H, Boc-PEG-NHS, Fmoc-PEG-NHS, NHS-PEG-VS, NHS-PEG-
MAL), PEG acrylates (ACRL-PEG-NHS), PEG-phospholipids (e.g., mPEG-DSPE),
multiamied PEGs of the SUNBRITE series including the GL series of glycerine-
based PEGs activated by a chemistry chosen by those skilled in the art, any of the
SUNBRITE activated PEGs (including but not limited to carboxyl-PEGs, p-NP-
PEGs, Tresyl-PEGs, aldehyde PEGs, acetal-PEGs, amino-PEGs, thiol-PEGs,
maleimido-PEGs, hydroxyl-PEG-amine, amino-PEG-COOH, hydroxyl-PEG-
aldehyde, carboxylic anhydride type-PEG, functionalized PEG-phospholipid, and
otlier similar and/or suitable reactive PEGs as selected by those skilled in the art for
their particular application and usage.
Peptide Moiety
Any peptides derived from various animals including humans,
microorganisms or plants and those produced by genetic engineering and by synthesis
may be employed as the peptide moiety. Examples include peptides that bind to
EPO-R and peptides that bind to TPO-R.
Preferably, the peptide moiety comprises one or more peptides, the length of
each peptide is less than 50 amino acids, more preferably between^about 10 and 25
amino acids, and most preferably between about 12-18 amino acids.
In one preferred embodiment, the peptide moiety is selected &om peptides that
bind to EPO-R such as those disclosed in (e.g. those disclosed in U.S. Pat. Nos.
5,773,569; 5,830,851; and 5,986,047 to Wrighton, et al; PCT Pub. No". WO 96/40749
to Wrighton, et al; U.S. Pat. No. 5,767,078 and PCT Pub. No. 96/40772 to Johnson
and Zivin; PCT Pub. No. WO 01/38342 to Balu; WO 01/91780 to Smith-Swintoslcy,
et al; U.S. Provisional Application Serial No. 60/479,245 filed May 12, 2003; U.S.
Provisional Application Serial No. 60/469,993 filed May 12, 2003^ and U.S.
Provisional Apphcation Serial No. 60/470,244 filed May 12, 2003.
In another preferred embodiment, the peptide moiety is selected firom peptides
wluch bind to thrombopoietin-receptors ("TPO-R"). Non-limiting examples of such
TPO-R binding peptides include those disclosed in U.S. Patents 6,552,008, 6,506,362,
6,498,155, 6,465,430, 6,333,031, 6,251,864, 6,121,238, 6,083,913, 5,932,546,
5,869,451, 5,683,983, 5,677,280, 5,668,110, and 5,654,276; and published U.S. Patent
Applications 2003/0083361, 2003/0009018, 2002/0177166 and 2002/0160013.
In one embodiment, the peptide moiety is a monomeric peptide of 10 to 40 or
more amino acid residues in length and having the sequence X3X4X5GPX6TWX7X8
where each amino acid is indicated by standard one letter abbreviation; X3 is C; X^s
R, H, L, or W; X5 is M, F, or I; Xg is independently selected fi-om any one of the 20
genetically coded L-amino acids; X7 is D, E, I, L, or V; and Xs is C, which bind and
activate the erythropoietin receptor (EPO-R) or otherwise act as an EPO agonist.
In another embodiment, the peptide moiety is a monomeric peptide of 17 to
about 40 amino acids in length that comprise the core amino acid sequence
]^ACHMGPITXj,y^QPLRj_ where each amino acid is indicated by standard one
"iettef abbreviation; and Xi is tryptophan (W), 1-naphthylalanine (1-nal), or 2-
naphthylalanine (2-nal).
In yet another embodiment, the peptide moiety comprises one or more TPO-R
binding peptides with sequence such as Ac-Ile-Glu-Gly-PEO-Thr-Leu-Arg--Qta=Nat(i)-
Leu-Ala-AJa-Arg-Sar, or . Ac-Ile-Glu-GIy-Pro-Thr-Leu-Arg-Ghi-Trp-Leu-Ala-Ala-

According to some embodiments of tliis invention, two or more, and
preferably between two to six amino acid residues, independently' selected from any
of the 20 genetically coded L-amino acids or the stereoisomeric D-amino acids, will
be coupled to either or both ends of the core sequences described above. For
example, the sequence GG will often be appended to either or both termini of the core
sequences for ease in synthesis of the peptides. The present invention also provides
conjugates of these peptides and derivatives and peptidomimetics of the peptides that
retain tlie property of EPO-R binding.
Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids,
minatural amino acids such as a,a-disubstituted amino acids, N-alkyl amino acids,
lactic acid, and otiier unconventional amino acids may also be suitable components
for compounds of the present invention. Examples of unconventional amino acids
include, but are not limited to: p-alanine, 3-pyridylalamne, 4-hydroxyproline, O-
phosphoserine, N-methylglycine, N-acetylserine, N-formylmethionine, 3-
methylhistidine, 5-hydroxylysine, nor-leucine, and other similar amino a.cids and
imino acids.
In preferred embodiments, the peptide moieties of tlie invention contain an
intraniolecular disulfide bond between tlie two cysteine residues of the core sequence.
For exaii)p|e: ^


Dimeric and Oligomeric Peptides
The preferred embodiment, the monomeric peptide moietiiss of the present
invention are dimerized or oligomerized to form dimers or oligomers.
In one embodiment, the peptide monomers of the invention may be
oligomerized using the biotin/streptavidin system. Biotinylated analogs of peptide
monomers may be synthesized by standard techniques. For example, the peptide
monomers may be C-terminally biotinylated. These biotinylated monomers are then
oUgomerized by incubation with streptavidin [e.g., at a 4:1 molar ratio at room
temperature in phosphate buffered saline (PBS) or HEPES-buffered RPMI medium
(Invitrogen) for 1 hour]. In a variation of this embodiment, biotinylated peptide
monomers may be ohgomerized by incubation with any one of a number of
commercially available anti-biotin antibodies [e.g., goat anti-biotin IgG from
Kirkegaard & Perry Laboratories, Inc. (Washington, DC)].
LiTikers
In preferred embodiments, the peptide monomers of the invention are
dimerized by covalent attachment to at least one linker moiety. The linlcer (Lk)
moiety is preferably, although not necessarily, a Ci-i2 linldng moiety optionally
terminated with one or two -NH- linlcages and optionally substituted at one or more
available carbon atoms with a lower alkyl substituent. Preferably the linlcer Lk
comprises -NH-R-NH- wherein R is a lower (Ci-e) linear hydrocarbon substituted
with a functional group such as a carboxyl group or an amino group that enables
binding to another molecular moiety (e.g., as may be present on the surface of a solid
support). Most preferably the linker is a lysine residue or a lysine amide (a lysine
residue wherein the carboxyl group has been converted to an amide moiety -CONH2).
In preferred embodiments, the linker bridges the C-termini of two peptide monomers,
by simultaneous attachment to the C-terminal amino acid of each monomer.
For example, when the C-terminal linlcer Lk is a lysine amide the dimer may
be illustrated structurally as shown in Formula I, and summarized as shown in
Formula 11:
In Formula I, N represents the nitrogen atom of lysine's s-amino group and N
represents the nitrogen atom of lysine's a-aniino group. The dimeric structure can be
written as [peptide]2Lys-ainide to denote a peptide bound to both the a and s amino
groups of lysine, or [Ac-peptideJaLys-amide to denote an N-teraiinally acetylated
peptide bound to both the a and s amino gi^oups of lysine, or [Ac-peptide,
disulfideJaLys-amide to denote an N-terminally acetylated peptide bound to both tlie
a and s amino groups of lysine with each peptide containing an intramolecular
disulfide loop, or [Ac-peptide, disulfideJaLys-spacer-PEG to denote an N-terminally
acetylated peptide bound to both the a and s amino groups of lysine with each peptide
containing an intramolecular disulfide loop and a spacer molecule forming a covalent
linkage between the C-termius of lysine and a PEG moiety.
Generally, although not necessarily, peptide dimers dimerized by a technique
other than formation of intemiolecular disulfide bonds, will also contain one or more
disulfide bonds between cysteine residues of the peptide monomers. For example, the
two monomers may be cross-linked by one or more intermoleculai' disulfide bonds.
Preferably, the two monomers contain at least one intramolecular disulfide bond.
Most preferably, both monomers of a peptide dimer contain an intramolecular
disulfide bond, such that each monomer contains a cyclic group.
Peptide Modification
One can also modify the amino and/or carboxy termini of the peptide
compounds of the invention to produce other compounds of the invention. Amino
terminus mo^qations include methylation {i.e., -NHCH3 or -N(CH3)2), acetylation

{e.g., with acetic acid or a halogenated derivative thereof such as a-chloroacetic acid,
a-bromoacetic acid, or a-iodoacetic acid), adding a benzyloxycarbcwjyl (Cbz) group,
or blocking the amino terminus with any blocking group containing a carboxylate
fonctionality defined by RCOO- or sulfonyl fiinctionality defined by R—SO2-, where
R is selected firom the group consisting of alkyl, aryl, heteroaryl, alkyl aryl, and the
like, and similar groups. One can also incorporate a desamino acid atlhe N-tenninus
(so that there is no N-terminal amino group) to decrease susceptibility to proteases or
to restrict the conformation of the peptide compound, hi preferred embodiments, the
N-temiinus is acetylated. In most prefen-ed embodiments an N-temiinal glycine is
acetylated to yield N-acetylglycine (AcG).
Carboxy terminus modifications include replacing the fi-ee acid with a
carboxaniide group or forming a cyclic lactam at the carboxy terminus to introduce
structural constraints. One can also cyclize the peptides of the invention, or
incorporate a desamino or descarboxy residue at the termini of the peptide, so that
there is no terminal amino or carboxyl group, to decrease susceptibility to proteases or
to restrict the conformation of the peptide. C-terminal flmctional groups of the
compoxmds of the present invention include amide, amide lower aUcyl, amide di(lower
alkyl), lower alkoxy, hydroxy, and carboxy, and the lower ester derivatives thereof,
and the pharmaceutically acceptable salts thereof
One can replace the naturally occurring side chains of the 20 genetically
encoded amino acids (or the stereoisomeric D amino acids) with other side chains, for
instance with groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7-membered aUcyl,
amide, amide lower allcyl, amide di(lower aJkyl), lower alkoxy, hydroxy, carboxy and
the lower ester derivatives thereof, and with 4-, 5-, 6-, to 7-membered heterocyclic.
hi particular, proline analogues in which the ring size of the proline residue is
changed from 5 members to 4, 6, or 7 members can be employed. Cychc groups can
be saturated or unsaturated, and if unsaturated, can be aromatic or non-aromatic.
Heterocychc groups preferably contain one or more nitrogen, oxygen, and/or sulfiir
heteroatoms. Examples of such groups include the furazanyl, furyl, imidazohdinyl,
imidazolyl, imidazolinyl, isothiazolyl, isoxazolyl, morpholinyl {e.g. morphohno),
oxazolyl, piperazinyl {e.g., 1-piperazinyl), piperidyl {e.g., 1-piperidyl, piperidino),
pyranyl, pj^azinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl,
pyrimidinyl,
thiazolyl, thienyl, thiomoipholinyl (e.g., thiomorpholino), and triazolyl. These
heterocyclic groups can be substituted or unsubstituted. Where a groiip is substituted,
the substituent can be allcyl, alkoxy, halogen, oxygen, or substituted oi" unsubstituted
phenyl.
One can also readily modify the peptide moieties by phosphorylation, and
other methods (e.g., as described in Hruby, et al (1990) Biochem J. 268:249-262).
The peptide moieties of the invention may also serve as structural models for
non-peptidic compounds with similar biological activity. Those of skill in the ai't
recognize that a variety of techniques are available for constructing compounds with
the same or similax desired biological activity as the lead peptide compound, but with
more favorable activity than the lead with respect to solubility, stability, and
susceptibility to hydrolysis and proteolysis [See, Morgan and Gainor (1989) Ann.
Rep. Med. Chem. 24:243-252]. These teclmiques include replacing the peptide
backbone with a backbone composed of phosphonates, amidates, carbamates,
sulfonamides, secondary amines, and N-methylamino acids.
The monomeric, dimeric or oligomeric peptide moieties may be attached
directly to the PEG moiety or it may be attached to via one or more spacer moieties.
Spacer Moiety
hi embodiments where tlie monomeric, dimeric, or ohgomeric peptide
moieties are attached to the PEG moiety via a spacer moiety, the spacer moiety may
be a moiety optionally terminated with -NH- linlcages or -C(0)0- groups. For
example, the spacer could be lower (C1.12) linear hydrocarbon optionally substituted
with a functional group such as a carboxyl group or an amino group tliat enables
binding to another molecular moiety, or one or more glycine (G) residues, or amino
hexanoic acids (Ahx) such as 6-amino hexanoic acid; or lysine (K) residues or a
lysine amide (K-NH2, a lysine residue wherein the carboxyl group has been converted
to an amide moiety -CONH2).
In preferred embodiments, the spacer moiety has the following structure:
->ffl-(CH2)a-[0-(CH2)pV05-(CH2)s-Y-
wherein a, P,/j^l^and s ai-e each integers whose values are independently selected.

In preferred embodiments,
a is an integer, 1 P is an integer, 1 e is an integer, 1 5 is 0 or 1;
Y is an integer, 0 Y is either NH or CO.
In certain preferred embodiments, p = 2 when y > 1 •
In one particularly preferred embodiment,
a = p = e = 2;
Y = 5 = 1; and
YisNH.
In other preferred embodiments,
Y = 5 = 0;
2 Y is CO.
In one embodiment,
Y = 5 = 0;
a + 8 =5; and
YisCO.
According to the invention, a water-soluble moiety (preferably PEG) is
attached to the NH terminus of the spacer. The water-soluble moiety may be attached
directly to the spacer or it may be attached indirectly, for example with an amide or
carbamate linkage. The peptide moiety is attached to the Y terminus of the spacer.
The spacer maybe attached to either the C-terminus or the N-tenninus of the peptide.
Hence, in embodiments where the spacer is attached to the C-tenninus of the peptide,
Y is NH. In embodiments where the spacer is attached to the N-terminus of the
peptide, Y is?^9. In preferred embodiments, a spacer of the invention is attached to a


peptide dimer, by a lysine linker described below. In such embodiment, the spacer is
preferably attached to the C-terminus of the linlcer moiety, and Y isJNH. hi another
preferred embodiment, the spacer of the invention is attached to a peptide as part of a
trifimctional linker (also described below). In that embodiment, and Y is CO and Y
forms an amide bond with an N atom of the trifimctional linker.
The spacer moiety may be incorporated into the peptide during peptide
synthesis. For example, where a spacer contains a free amino group and a second
functional group {e.g., a cai-boxyl group or an amino group) that enables binding to
another molecular moiety, the spacer may be conjugated to the solid support.
Thereafter, the peptide may be synthesized directly onto the spacer's free amino
group by standard sohd phase techniques.
In a preferred embodiment, a spacer containing two frmctional groups is first
coupled to the sohd support via a first fimctional group. When a dimer peptide is to
be synthesized, optionally a liiiker Lk moiety having two or more frinctional groups
capable of serving as initiation sites for peptide syntliesis and an additional functional
group (e.g., a carboxyl group or an amino group) that enables binding to another
molecular moiety is conjugated to the spacer via the spacer's second functional group
and the linker's third functional group. Thereafter, two peptide monomers may be
synthesized directly onto the two reactive nitrogen groups of the linker Lk moiety in a
variation of tlie soHd phase synthesis technique. For example, a solid support coupled
spacer with a free amine group may be reacted with a lysine linlcer via the linker's
free carboxyl group.
In alternate embodiments where the peptide moiety is attached to a spacer
moiety, said spacer may be conjugated to the peptide after peptide s>Tithesis. Such
conjugation may be acliieved by methods well established in the art. In one
embodiment, the linker contains at least one functional group suitable for attachment
to tiie target functional group of the synthesized peptide. For example, a spacer with a
free amine group may be reacted with a peptide's C-terminal carboxyl group. In
another example, a spacer with a free carboxyl group may be reacted with the free
amine group of a peptide's N-terminus or of a lysine residue. In yet another example,
a spacer containing a free sulfhydryl group may be conjugated to a cysteine residue of
a peptide by Oxidation to form a disulfide bond.
Pharmaceutical compositions
In another aspect of the present invention, pharmaceutical compositions of the
above PEG-modified peptide based compounds are provided. Conditions alleviated
or modulated by the administration of such compositions include those indicated
above. Such pharmaceutical compositions may be for administration by oral,
parenteral (intramuscular, intraperitoneal, intravenous (IV) or subcutaneous injection),
transdermal (either passively or using iontophoresis or electroporation), transmucosal
(nasal, vaginal, rectal, or sublingual) routes of administration or using bioerodible
inserts and can be formulated in dosage forms appropriate for each route of
administration, hi general, comprehended by the invention are pharmaceutical
compositions comprising effective amounts of a therapeutic peptide (e.g. peptides that
bind to EPO-R), witli pharmaceutically acceptable diluents, preservatives,
solubilizers, emulsifiers, adjuvants and/or carriers. Such compositions include
diluents of various buffer content {e.g., Tris-HCl, acetate, phosphate), pH and ionic
sti-ength; additives such as detergents and solubilizing agents (e.g., Tween 80,
Polysorbate SO), anti-oxidants {e.g., ascorbic acid, sodium metabisulfite),
preservatives {e.g., Thimersol, benzyl alcohol) and bulking substances {e.g., lactose,
mannitol); incorporation of the material into particulate preparations of polymeric
compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes.
Hylauronic acid may also be used. Such compositions may influence the physical
state, stability, rate of in vivo release, and rate of in vivo clearance of the present
proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed.
(1990, Mack Pubhsliing Co., Easton, PA 18042) pages 1435-1712 which are herein
incorporated by reference. The compositions may be prepared in liquid form, or may
be in dried powder {e.g., lyophilized) form.
OralDeliveiy
Contemplated for use herein are oral solid dosage forms, which are described
generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing
Co, Easton PA 18042) at Chapter 89, which is herein incorporated by reference.
Solid dosag^forms include tablets, capsules, pills, tioches or lozenges, cachets,

pellets, powders, or granules. Also, liposomal or proteinoid encapsulation may be
used to fonnulate the present compositions (as, for example, proteiiiQid microspheres
reported in U.S. Patent No. 4,925,673). Liposomal encapsulation may be used and
the liposomes may be derivatized with various polymers (e.g., U.S. Patent No.
5,013,556). A description of possible solid dosage forms for the therapeutic is given
by Marshall, K. In: Modem Pharmaceutics Edited by G.S. Banlcer and C.T. Rhodes
Chapter 10, 1979, herein incorporated by reference, hi general, the fonnulation will
include the EPO-R agonist peptides (or chemically modified forms thereof) and inert
ingredients which allow for protection against tlie stomach environment, and release
of the biologically active material in the intestine.
Also contemplated for use herein ai-e hquid dosage forms for oral
administration, including pharmaceutically acceptable emulsions, solutions,
suspensions, and syrups, which may contain other components including inert
diluents; adjuvants such as wetting agents, emulsifying and suspending agents; and
sweetening, flavoring, and perfuming agents.
The peptides may be chemically modified so that oral delivery of the
derivative is efficacious. Generally, the chemical modification contemplated is the
attachment of at least one moiety to the component molecule itself, where said moiety
permits (a) inhibition of proteolysis; and (b) uptake into the blood stream firom the
stomach or intestine. Also desired is the increase in overall stabihty of the component
or components and increase in circulation time in the body. As discussed above,
PEGylation is a preferred chemical modification for pharmaceutical usage. Other
moieties that may be used include: propylene glycol, copolymers of ethylene glycol
and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, polyproline, poly-l,3-dioxolane and poly-l,3,6-tioxocane [see, e.g.,
Abuchowsld and Davis (1981) "Soluble Polymer-Enzyme Adducts," in Enzymes as
Dings. Hocenberg and Roberts, eds. (Wiley-Interscience: New York, NY) pp. 367-
383; and Ne\vmark, etal (1982) J. Appl. Biochem. 4:185-189].
For oral formulations, the location of release may be the stomach, the small
intestine (the duodenum, the jejunem, or the ileum), or the large intestine. One skilled
in the art has available formulations which will not dissolve in the stomach, yet will
release the Material in the duodenum or elsewhere in the intestine. Preferably, the
release will^oid the deleterious effects of the stomach environment, either by

protection of the peptide (or derivative) or by release of the peptide (or derivative)
beyond the stomach environment, such as in the intestine.
To ensure full gastric resistance a coating impermeable to at least pH 5.0 is
essential. Examples of the more common inert ingredients that are used as enteric
coatings are cellulose acetate trimellitate (CAT), hydroxj'propylmethylcellulose
phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP),
Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S,
and Shellac. These coatings may be used as mixed j&lms.
A coating or mixture of coatings can also be used on tablets, which are not
intended for protection against the stomach. This can include sugar coatings, or
coatings which make the tablet, easier to swallow. Capsules may consist of a hard
shell (such as gelatin) for dehvery of dry therapeutic {i.e. powder), for liquid forms a
soft gelatin shell may be used. The shell material of cachets could be thick starch or
otlier edible paper. For pills, lozenges, molded tablets or tablet triturates, moist
massing techniques can be used.
The peptide (or derivative) can be included in the formulation as fine
multiparticulates in the form of granules or pellets of particle size about 1mm. The
formulation of the material for capsule administration could also be as a powder,
hghtly compressed plugs, or even as tablets. These therapeutics could be prepared by
compression.
Colorants and/or flavoring agents may also be included. For example, the
peptide (or derivative) may be formulated (such as by liposome or microsphere
encapsulation) and then further contained within an edible product, such as a
rejfrigerated beverage containing colorants and flavoring agents.
One may dilute or increase the volume of the peptide (or derivative) with an
inert material. These diluents could include carbohydrates, especially mannitol,
a-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch.
Certain inorganic salts may be also be used as fillers including calcium triphosphate,
magnesium carbonate and sodium chloride. Some commercially available diluents
are Fast-Flo, Eradex, STA-Rx 1500, Emcompress and Aviceil.
Disi&tegrants may be included in the formulation of the therapeutic into a solid
dosage form?^Materials used as disintegrates include but are not limited to starch,


including the commercial disintegrant based on starch, Explotab. Sodium starch
glycolate, Amberlite, sodium carboxymethylcellulose, ultramylapectin, sodium
alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and
bentonite may all be used. The disintegrants may also be insoluble cationic exchange
resins. Powdered gums may be used as disintegrants and as binders, and can include
powdered gums such as agar, Karaya or tragacanth. Alginic acid and "its sodium salt
ai-e also useful as disintegrants.
Binders may be used to hold the peptide (or derivative) agent together to form
a hard tablet and include materials from natural products such as acacia, tragacanth,
starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and
carboxymethyl cellulose (CMC). Polyvinyl pyrroUdone (PVP) and
hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to
granulate the peptide (or derivative).
An antifrictional agent may be included in the formulation of the peptide (or
derivative) to prevent sticking during the formulation process. Lubricants may be
used as a layer between the peptide (or derivative) and the die wall, and these can
include but are not limited to; stearic acid mcluding its magnesium and calcium salts,
polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble
lubricants may also be used such as sodiimi lauryl sulfate, magnesium lauryl sulfate,
polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.
Glidants that might improve the flow properties of the drug during formulation
and to aid rearrangement during compression might be added. The glidants may
include starch, talc, pyrogenic silica and hydrated silicoaluminate.
To aid dissolution of the peptide (or derivative) into the aqueous environment
a surfactant might be added as a wetting agent. Smfactants may include anionic
detergents such as sodium lauryl sulfate, dioctyl sodimn sulfosuccinate and dioctyl
sodium sulfonate. Cationic detergents might be used and could include benzallconiimi
chloride or benzethomium chloride. The list of potential nonionic detergents that
could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl
40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol
monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose
and carboxymethyl cellulose. These surfactants could be present in the formulation of
the protein or derivative either alone or as a mixture in different ratios,.
Additives which potentially enhance uptake of the peptide (or derivative) are
for instance the fatty acids oleic acid, linoleic acid and linolenic acid.
Controlled release oral formulations may be desirable. The peptide (or
derivative) could be mcorporated into an inert matrix which permits release by either
diffusion or leacliing mechanisms, e.g., gums. Slowly degenerating matrices may also
be incorporated into the formulation. Some enteric coatings also have a delayed
release effect. Another form of a controlled release is by a method based on the Oros
therapeutic system (Alza Corp.), i.e. the drug is enclosed in a semipenneable
membrane which allows water to enter and push drug out through a single small
opening due to osmotic effects.
Other coatings may be used for the formulation. These include a variety of
sugars which could be apphed in a coating pan. The peptide (or derivative) could also
be given in a film coated tablet and the materials used in this instance are divided into
2 groups. The first are the nonenteric materials and include methyl cellulose, ethyl
cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl
cellulose, hydroxypropyl-methyl cellulose, sodium carboxy-methyl cellulose,
providone and the polyethylene glycols. The second group consists of the enteric
materials that are conunonly esters of phthalic acid.
A mix of materials might be used to provide the optimum film coating. Film
coating may be carried out in a pan coater or in a fluidized bed or by compression
coating.
Parenteral delivety
Preparations according to this invention for parenteral administration include
sterile aqueous or non-aqueous solutions, suspensions, or emulsions. Examples of
non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable
oils, such as olive oil and com oil, gelatin, and injectable organic esters such as ethyl
oleate. Such dosage forms may also contain adjuvants such as preserving, wetting,
emulsifying,'and dispersing agents. They may be sterilized by, for example, filtration
through a IMt^a retaining filter, by incorporating sterihzing agents into the


compositions, by irradiating the compositions, or by heating the compositions. They
can also be manufactured using sterile water, or some other sterile injectable medium,
immediately before use.
Rectal or vaginal delivery
Compositions for rectal or vaginal administration are preferably suppositories
which may contain, in addition to the active substance, excipients such as cocoa butter
or a suppository wax. Compositions for nasal or sublingual administration are also
prepared with standard excipients well known in the art.

Pulmonary Delivery
Also contemplated herein is pulmonary delivery of the>EPO-R agonist
peptides (or derivatives thereof). The peptide (or derivative) is delivered to the lungs
of a mammal while inhaling and traverses across the lung epitlielial Hniug to the blood
stream [see, e.g., Adjei, et al. (1990) Pharmaceutical Reseai'ch 7:565-569; Adjei, et al.
(1990) Int. J. Pharmaceutics 63:135-144 (leuprolide acetate); Braquet^/* al (1989) J.
Cardiovascular Pharmacology 13(sup5): 143-146 (endothelin-1); Hubbard, et al.
(1989) Annals of Internal Medicine, Vol. m, pp. 206-212 (a 1-antitrypsin); Smith, et
al. (1989) J. Clin. Invest. 84:1145-1146 (a-1-proteinase); Oswein, et al. (1990)
"Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug
Delivery 11 Keystone, Colorado (recombinant human growth homione); Debs, et al.
(1988) J. himiunol. 140:3482-3488 (interferon-y and tumor necrosis factor a); and
U.S. Pat. No. 5,284,656 to Platz, et al. (granulocyte colony stimulating factor). A
method and composition for puhnonar}' delivery of drugs for systemic effect is
described in U.S. Pat. No. 5,451,569 to Wong, et al.
Contemplated for use in the practice of tliis invention are a wide range of
mechanical devices designed for pulmonary delivery of therapeutic products,
including but not limited to nebuHzers, metered dose inlialers, and powder inhalers,
all of which aie familiar to those skilled in the art. Some specific examples of
commercially available devices suitable for the practice of this invention are the
Ultravent nebulizer (IVialhnckrodt Inc., St. Louis, MO); the Acorn II nebuhzer
(Marquest Medical Products, Englewood, CO); the Ventolin metered dose inhaler
(Glaxo Inc., Research Triangle Park, NC); and the Spinhaler powder inhaler (Fisons
Corp., Bedford, MA).
All such devices require the use of formulations suitable for the dispensing of
peptide (or derivative). Typically, each formulation is specific to the type of device
employed and may involvb the use of an appropriate propellant material, in addition
to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of
liposomes, microcapsules or microspheres, inclusion complexes, or other types of
carriers is contemplated. Chemically modified peptides may also be prepared in
different formulations depending on tlie type of chemical modification or the type of
device employed.

Fomiulations suitable for use with a nebulizer, either jet or ultrasonic, will
typically comprise peptide (or derivative) dissolved in water at a concentration of
about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation
may also include a buffer and a simple sugar {e.g., for protein stabilization and
regulation of osmotic pressure). The nebuUzer formulation may also contain a
siu-factant, to reduce or prevent surface induced aggregation of the peptide (or
derivative) caused by atomization of the solution in forming the aerosol.
Formulations for use with a metered-dose iulialer device will generally
comprise a finely divided powder containing the peptide (or derivative) suspended in
a propellant with the aid of a surfactant. The propellant may be any conventional
material employed for this purpose, such as a chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including
trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and
1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include
sorbitan trioleate and soya lecithin. Oleic acid may also be useflxl as a surfactant.
Formulations for dispensing from a powder ualialer device will comprise a
finely divided dry powder containing peptide (or derivative) and may also include a
bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts wliich
facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the
formulation. The peptide (or derivative) should most advantageously be prepared in
particulate forin with an average particle size of less tlian 10 mm (or microns), most
preferably 0.5 to 5 mm, for most effective delivery to the distal lung.
Nasal Delivery
Nasal delivery of the EPO-R agonist peptides (or derivatives) is also
contemplated. Nasal delivery allows the passage of the peptide to the blood stream
directly after administering the therapeutic product to the nose, without the necessity
for deposition of the product in the limg. Formulations for nasal deUvery include
those with dexfran or cyclodextran.
Dosages
For all of the peptide compoxmds, as further studies"", are conducted,
information will emerge regarding appropriate dosage levels for treatment of various
conditions in various patients, and the ordinary sldlled worker, considering the
therapeutic context, age, and general health of the recipient, will be able to ascertain
proper dosing. The selected dosage depends upon the desired therapeutic effect, on
the route of administration, and on the duration of the treatment desired. Generally
dosage levels of between 0.001 to 10 mg/kg of body weight daily ai-e administered to
mammals. Generally, for inti^avenous injection or infusion dosage may be lower. The
dosing schedule may vary, depending on the circulation half-Ufe, and the formulation
used.
The peptides of the present invention (or their derivatives) may be
administered in conjunction with one or more additional active mgredients or
pharmaceutical compositions.
EXAMPLES
The following Examples illustrate the invention, but are not limiting.
EXAMPLE 1: Synthesis of H-TAP-Boc molecule
A solution of TAP (lOg, 67.47mmol, purchased from Aldrich Chemical Co.)
in anhydrous dichloromethane (DCM) (100 ml) was cooled to oV. A solution of
benzyl chloroformate (Cbz-Cl, Cbz = carboxybeuzyloxy) (4.82ml, 33.7imnol) in
anhydrous DCM (50ml) was added slowly to the TAP solution through a dropping
funnel over a period of 6-7 hrs while the temperature of the reaction mixture was
maintained aj 0°C throughout. The resulting mixture then allowed to warm to room
temperature Q^°C). After another 16 hrs, the DCM was removed under vacuum and


the residue was partitioned between 3N HCl and ether. The aqueous layers ^ere
collected and neutralized with 50% aq. NaOH to pH 8-9 and extracted -with ethyl
acetate. The ethyl acetate layer was dried over anhydrous Na2S04^ and then
concentrated imder vacuum to provide the crude mono-Cbz-TAP (5g, about 50%
yield). This compound was used for the reaction in Step B without further
piuification.

B0C2O (3.86g, 17.7mmol, Boc = tert-butoxycarbonyl) was added to a
vigorously stirred suspension of the Cbz-TAP (5g, 17.7imnol) in hexane (25ml).
Stirring continued at room temperature overnight. The reaction mixture was diluted
with DCM (25ml) and washed witli 10% aq. citric acid (2X), water (2X) and brine.
The organic layer was dried over anhydrous Na2S04 and concentrated under vacuimi.
The crude product (yield 5g) was used directly in the reaction in Step C.

The crude Cbz-TAP-Boc from Step B was dissolved in methanol (25ml) and
hydrogenated in presence of 5% Pd on Carbon (5% w/w) under balloon pressure for
16 hrs. The mixture was filtered, washed with methanol and the filtrate concentrated
under vacuum to provide the crude H-TAP-Boc product (yield 3.7g).
Tlie overall yield after Steps A-C is approximately 44% (calculated based on
the amount of Cbz-Cl used).
EXAIMPLE 2; Attaching Spacer to Peptide with C-Terniinus
EXAMPLE 6: Attaching Spacer and Synthesizing Peptide
The reaction scheme below illustrates how to attach, a spacer to on solid
support and synthesize a peptide on such solid support.
TentaGel bromide (2.5 g, 0.48 mmol/g, obtained from Rapp Polymere,
Germany), phenolic linlcer (5 equivalent), and K2CO3 (5 equivalent) were heated in 20
mL of N,N-dimethylformamide (DMF) to 70°C for 14 hrs. After cooling to room
temperature, the resin was washed (0.1 N HCl, water, Acetonitrile (ACN), DMF,
MeOH) and dried to give an amber-colored resin.
¦~o o
2.5 g of the resin from Step A above and H-TAP-Boc (1.5gms, 5 eq.) and
glacial AcOH (34 |j,l, 5 eq.) was taken in a mixture of 1:1
MeOH/Tetraliydrofiiran(THF) and shaken overnight. A IM solution of sodimn
cyanoborohydride (5 eq.) in THF was added to the mixtm^e and shalcen for another 7
hrs. The resin was filtered washed (DMF, THF, 0.1 N HCl, water, MeOH) and dried.
A • small amount of the resin was benzoylated with benzyl chloride and
diisopropylethylamine (DIEA) in DCM and cleaved with 70% trifluoroacetic acid
(TFA)-DCM and checked by LCMS and HPLC.
The resin firom Step B above was treated with an activated solution of Fmoc-
Lys(Fmoc)-OH (Fmoc = 9-Fluorenylmethoxycarbonyl, prepared from 5 eq. of amino
acid and 5 eq. of HATU (N,N,N',N'-Tetramethyl-0-(7-azabenzotriazol-l-yl)uronium
hexafluorophosphate) dissolved at 0.5 M in DMF, followed by tlie addition of 10 eq.
of DIEA) and gently shaken for 14 hrs. The resin was then washed (DMF, THF,
DCM, MeOH) and dried to yield the protected resin. Residual amine groups were
capped by treating the resin with a solution of 10% acetic anliydride, 20% pyridine in
DCM for 20 minutes, followed by washing as above. The Fmoc groups were
removed by gently shaking the resin in 30% piperideine in DMF for 20 minutes,
followed by washing (DMF, THF, DCM, MeOH) and drying.
The resin from Step C above was subjected to repeated cycles of Fmoc-amino
acid couplings with. HBTU/HOBt activation and Fmoc removal with piperidine to
build both peptide chains simultaneously. This was conveniently carried out on an
ABI433 automated peptide synthesizer available from Applied Biosystems, Inc.
After the final Fmoc removal, the terminal amine groups were acylated with acetic
anhydride (10 eq.) and DIEA (20 eq.) in DMF for 20 minutes, followed by washing as
above.
The resin from Step D above was suspended in a solution of TFA (82.5%),
phenol (5%), ethanedithiol (2.5%), water (5%o), and tliioanisole (5%) for 3 lirs at room
temperature. Alternative cleavage coclctails such as TFA (95%), water (2.5%), and
triisopropylsilane (2.5%)) can also be used. The TFA solution was cooled to 5°C and
poured into Et20 to precipitate tlie peptide. Filtration and drying under reduced
pressure gave the desired peptide dimer with spacer. Purification via preparative
HPLC with a C18 column yielded pure peptide dimer with spacer.
step F: Oxidation
Dimeric peptide (attached to spacer) with reduced cysteifie residues was
oxidized to yield dimeric peptide with disulfide bonds.
The dimeric peptide was dissolved in 20% DMSOAvater (1 mg dry weight
peptide/mL) and allowed to stand at room temperature for 36 hrs. The peptide was
purified by loading the reaction mixture onto a C18 HPLC column (Waters Delta-Pak
CIS, 15 micron particle size, 300 angstrom pore size, 40 mm x 200 mm length),
followed by a linear ACN/water/0.01% TFA gradiant firom 5 to 95% ACN over 40
minutes. Lyopholization of the fi-actions containing the desired peptide yielded a
fluffy white solid product.
The dimeric peptide attached to the spacer was mixed with an equal amount
(mole basis) of activated PEG species (mPEG-NPC manufactured by NOF Corp.,
Japan, available througli Nelctar Therapeutics, U.S., (formerly "Shearwater Corp.")) in
dry DMF to afford a clear solution. After 5 minutes, 4 eq. of DIEA was added to
above solution. The mixture was stirred at ambient temperature for 14 hrs, followed
by purification with CIS reverse phase HPLC. The stmcture of PEGylated peptide is
confirmed by Matrix-assisted-laser-desorption-ionization (MALDI) mass
spectrometry.
mPEG-NPC has the following stmcture:
EXAMPLE 9; PEGvlation of Peptide Dlmer with Spacer, with mPEG-
SPA
PEGylation of the peptide dimer with spacer can also by carried out with
mPEG-SPA. mPEG-SPA has the following structure.

mPEG-SPA
EXAMPLE 10: Ion Exchange Purification
___ sample
obtained in Example 8 was used to identify ion exchange supports suitable for
purifying peptide-Spacer-PEG conjugates.
The general procedure was as follows:
the ion exchange resin (2-3g) was loaded into a 1 cm column, followed by
conversion to the sodium form (0.2 N NaOH loaded onto column until elutant was at
pH 14), and then to the hydrogen form (eluted with either 0.1 N HCl or 0.1 M HO Ac
imtil elutant matched load pH), followed by washing with 25% ACN/water imtil pH
6. Either the peptide prior to conjugation or the peptide-PEG conjugate was dissolved
in 25% ACN/water (10 mg/mL) and the pH adjusted to below 3 with TFA, then
loaded onto the colunm in separate experiments. After washing with 2-3 column
volumes of 25% ACN/water and collecting 5 mL fractions, the peptide was released
from the column by elution wth 0.1 M NKUOAc in 25% ACN/water, again collecting
5 mL fractions. Analysis via HPLC revealed which fractions contained the desired
peptide. Analysis with an Evaporative Light-Scattering Detector (ESLD) indicated
that when the peptide was retained on the column and was eluted with the NH4OAC
solution (generally between fractions 4 and 10), no non-conjugated PEG was
observed as a contaminant. When the peptide eluted in the initial wash buffer
(generally the first 2 fractions), no separation of desired PEG-conjugate and excess
PEG was observed.
Ion exchange supports were chosen based their ability to separate the peptide-
PEG conjugate from unreacted (or hydrolyzed) PEG as well as their ability to retain
the starting dimferic peptides. Mono S HR 5/5 strong cation exchange pre-loaded


column (Amersham Biosciences), SE53 Cellulose microgranular strong cation
exchange support (Whatman), and SP Sepharose Fast Flow strong ^cation exchange
support (Amersham Biosciences) were identified as suitable ion exchange supports.
EXAMPLE 11: Synthesis of Trifunctional Molecules based on a-Amino
Acids:
Trifunctional molecules having the stmcture
m=l-5, n = 1-14, m and n are integei
wherein
EXAMPLE 12: Synthesis of Trifunctional Molecules Based on Tertiary
Amides
Trifunctional molecules having the structure:

EXAMPLE 14: C-Terminus Dimerization and PEGylation Using A
Trifunctional Molecule
A trifunctional molecule having the structure
was made according to Example 12.
This trifunctional molecule was used in C-terminus dimerization and
PEGylation according to the following reaction scheme:

0 °C was added DCC (10.5g, 50.9 mmol) over 5 min. A wliite precipitate formed
within 2 min. The reaction mixture was allowed to warm to room temperature and
was stirred for 24h. The urea was filtered off with a sintered filter (medium porosity)
and the solvent removed under reduced pressure. The residue was taken up in 500
niL of EtOAc (EtOAc = ethyl acetate), filtered as above, and ti-ansfen-ed to a
separatory funnel. The organic phase was washed (sat. NaHCOs, brine, 1 N HCl,
brine), dried (MgS04), filtered, and dried to yield a colorless oil. The oil solidified to
yield a white crystalline solid within 10 min.

The cmde diester was taken up in. 75 mL of THF (THF =
tetraliydrofiirane) and 75 mL of MeOH (MeOH = methanol) and 50 mL of water was
added. To this solution was added a solution of KOH (KOH = potassium hydroxide)
(8.6g, 153 mmol) in 25 mL of water. The reaction mixture turned light yellow in
color. After stirring for 12 h (pH was still ~12), the organic solvent was removed on a
rotary evaporator and die resultant slurry partitioned between Et20 (Et20 = Diethyl
ether)and sat. NaHCOa. The combined aq. phase was acidified to pH 1, saturated
with NaCl, and extracted with EtOAc. The EtOAc phase was washed (brine), dried
(MgS04), and concentrated to yield 13.97g of product as a white soUd (90.2% for 2
steps).
Notes: the yield dropped to 73% when the DCC reaction was
performed in ACN. When using DIC, the urea byproduct could not be removed from
the desired product without chromatography; the DCC urea can be quantitatively
removed without chromatography. The reaction also works well with water-soluble
carbodiimide.


To a solution of diacid (l.OOg, 3.29 mmol) and hydroxysuccinimide
(0.945g, 8.21 nunol) in 50 mL of ACN was added DCC (1.36g, 6.S9 nitnol) over 5
min. A white ppt formed inimediately. The reaction mixture was stirred 22h and was
filtered to remove the DCC urea. The solvent was removed under reduced pressure
and the residue talcen up in EtOAc (250 raL) and transfen-ed to a separatoiy funnel.
The organic phase was washed (sat. NaHCOs, brine, 1 N HCl, brine), dried (MgSO^),
and concentrated to afford a white solid. The solid was talcen up in 75 mL of ACN,
filtered, and concentrated to yield 1.28g of product as a white solid (78.2% yield).
Notes: the yields dropped to 31% in THF, 68Vo in DMF (with DIC
instead of DCC), and 57% in DCM/DMF. The starting diacid is soluble in ACN, so if
tliere is any material which has not dissolved before the DCC is added, it may be
filtered off and discarded.
This trifixnctional molecule was used in N-tenninus dimerization and
PEGylation according to the following reaction scheme:
EXAMPLES 16: Synthesis of mPEGj-Lysinol-NPC
Commercially available lysinol is treated with an excess of niPEG2 resulting
in the fomiation of niPEGi-Lysinol. Thereafter, inPEG2-Lysinol is treated with
excessive NPC forming PEGa-Lysinol-NPC
EXAMPLE!?: PEGvlation Using A Trifunctional Molecule (PEG Mf^iety
Comprises Two Linear PEG Chains)
A trifunctional molecular having the structure
Was made according to Example 15.
Step 1 - Coupling of the trifunctional linker to the peptide monomers:
For coupling to the linkei% 2 eq peptide is mixed with 1 eq of trifunctional linlcer in
diy DMF to give a clear solution, and 5eq of DIEA is added after 2 minutes. The mixture is
stirred at ambient temperature for 14h. The solvent is removed under reduced pressure and
the crude product is dissolved in 80% TFA in DCM for 30min to remove the Boc group,
followed by purification with C18 reverse phase HPLC. The structure of the dimei" is
confirmed by electrospray mass spectrometry. This coupling reaction attaches the linker to
the nitrogen atom of the s-amino group of the lysine residue of each monomer.
Step 4 - PEGylation of the peptide dimer:
PEGylation via a carbamate bond:
The peptide dimer and the PEG species (mPEGz-Lysinol-NPC) are mixed in a 1:2
molar ratio in dry DMF to afford a clear solution. After 5 minutes 4eq of DIEA is added to
above solution. The mixture is stirred at ambient temperature 14h, followed by purification
with CIS reverse phase HPLC, The structure of PEGylated peptide is confirmed by MALDI
mass. The purified peptide was also subjected to purification via cation ion exchange
chromatography as outlined below.
PEGylation via an amide bond:
The peptide dimer and PEG species [mPEGz-Lys-NHS] are mixed in a 1:2
molar ratio in dry DMF to afford a clear solution. niPEG2-Lys-NHS may be obtained
commercially, for example, from the Molecular Engineering catalog (2003) of Nektar
Therapeutics (490 Discovery Drive, Huntsville, Alabama 35806), item no.
2Z3X0T01. ..After 5 minutes lOeq of DIEA is added to above solution. The mixture
is stirred at ag^bient temperature 2h, followed by purification with CI8 reverse "phase

HPLC. The structure of PEGylated peptide was confiraied by MALDI mass. The
purified peptide was also subjected to purification via cation ion exchange
chromatography as outhned below.

The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications ofvthe invention in
addition to those described herein will become apparent to those skilled in the art
from the foregoing description and the accompanying figures. Such modifications are
intended to fall within the scope of the appended claims.
Numerous references, including patents, patent applications, protocols and
various publications, are cited and discussed in the description of this invention. The
citation and/or discussion of such references is provided merely to clarify the
description of the pi-esent invention and is not an admission that any such reference is
"prior art" to the invention described herein. All references cited and discussed in this
specification are incorporated herein by reference in their entirety and to the same
extent as if each reference was individually incorporated by reference.
WE CLAIM:
1. A peptide-based compound comprising:
(a) a peptide moiety that binds to an erythropoietin receptor, said peptide
moiety having an N-terminus and a C-terminus; and
(b) at least one poly(ethylene glycol) moiety attached to the C-terminus of
the peptide moiety,
wherein the peptide moiety is a peptide monomer or a peptide multimer comprising a
plurality of peptide monomers, each peptide monomer of the peptide moiety
comprising no more than 50 amino acids; and
wherein the poly(ethylene glycol) moiety is linear and has a molecular weight of 20
KDaltons or more.
2. The compound as claimed in claim 1, wherein the poly(ethylene glycol) moiety
has a molecular weight from 20 to 40 KDaltons.
3. The compound as claimed in claim 2, wherein the poly(ethylene glycol) moiety
has polydispersity value (Mw/M,,) of less than 1.20.
4. The compound as claimed in claim 1, wherein the peptide moiety is peptide
monomer comprising a single peptide.
5. The compound as claimed in claim 1, wherein the peptide moiety' is a peptide
dimer comprising two peptide monomers linked by a linker moiety.
6. The compound as claimed in claim 5, wherein each peptide monomer has a C-
terminus and an N-terminus, the linker moiety linking the C-terminus of each peptide
monomer.
7. The compound as claimed in claim 6, in which the poly (ethylene glycol) moiety
is attached to the linker moiety.
8. The compound as claimed in claim 7, further comprising a spacer moiety between
the poly (ethylene glycol) moiety and the linker moiety.
9. The compound as claimed in claim 4 or 5, wherein each peptide monomer
comprises between 10 and 25 amino acids.
10. The compound as claimed in claim 9, wherein each peptide monomer comprises
between 12 and 18 amino acids.
11. The compound as claimed in claim 1, further comprising a spacer moiety between
the peptide moiety and the poly(ethylene glycol) moiety.
12. The compound as claimed in claim 8 or 11, wherein the spacer moiety has the
structure:

wherein a, p, y, 5,_and s are each integers whose values are independently selected.
13. The compound as claimed in claim 12, wherein:
a is an integer, 1 P is an integer, 1 e is an integer, I 5 is 0 or 1;
y is an integer, 0 Y is either NH or CO.
14. The compound as claimed in claim 13, wherein 1 15. A pharmaceutical composition comprising:
(a) a peptide-based compound according to any of claims 1 to 14, and
(b) one or more pharmaceutically acceptable diluents, preservatives,
solubilizers, emulsifiers, adjuvants and/or carriers.


There is disclosed a peptide-based compound comprising a peptide moiety and at
least one poly(ethylene glycol) moiety, wherein the peptide moiety is a peptide monomer
or a peptide multimer comprising a plurality of peptide monomers, each peptide
monomer of the peptide moiety comprising no more than 50 amino acids; and wherein
the poly(ethylene glycol) moiety is linear and has a molecular weight of 20 KDaltons or
more.

Documents:

02500-kolnp-2005-abstract.pdf

02500-kolnp-2005-claims.pdf

02500-kolnp-2005-description complete.pdf

02500-kolnp-2005-form 1.pdf

02500-kolnp-2005-form 3.pdf

02500-kolnp-2005-form 5.pdf

02500-kolnp-2005-international publication.pdf

2500-KOLNP-2005-FORM-27.pdf

2500-kolnp-2005-granted-abstract.pdf

2500-kolnp-2005-granted-assignment.pdf

2500-kolnp-2005-granted-claims.pdf

2500-kolnp-2005-granted-correspondence.pdf

2500-kolnp-2005-granted-description (complete).pdf

2500-kolnp-2005-granted-examination report.pdf

2500-kolnp-2005-granted-form 1.pdf

2500-kolnp-2005-granted-form 13.pdf

2500-kolnp-2005-granted-form 18.pdf

2500-kolnp-2005-granted-form 3.pdf

2500-kolnp-2005-granted-form 5.pdf

2500-kolnp-2005-granted-gpa.pdf

2500-kolnp-2005-granted-others.pdf

2500-kolnp-2005-granted-reply to examination report.pdf

2500-kolnp-2005-granted-specification.pdf


Patent Number 239836
Indian Patent Application Number 2500/KOLNP/2005
PG Journal Number 15/2010
Publication Date 09-Apr-2010
Grant Date 06-Apr-2010
Date of Filing 06-Dec-2005
Name of Patentee AFFYMAX, INC.
Applicant Address 4001 MIRANDA AVENUE, PALO ALTO, CA
Inventors:
# Inventor's Name Inventor's Address
1 TUMELTY, DAVID 10417 WHITCOMB WAY # 105, SAN DIEGO, CA 92127
2 HOLMES, CHRISTOPHER,P. 13633 WESTOVER DRIVE, SARATOGA, CA 95070
3 YIN QUN 747 COASTLAND DRIVE, CA 94303
PCT International Classification Number C07K 1/107
PCT International Application Number PCT/US2004/014888
PCT International Filing date 2004-05-12
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/470,246 2003-05-12 U.S.A.