Title of Invention

A PEGylated VPAC2 RECEPTOR PEPTIDE AGONIST

Abstract The present invention encompasses peptides that selectively activate the VPAC2 receptor and are useful in the treatment of diabetes.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13)
SELECTIVE VPAC2 RECEPTOR PEPTIDE AGONISTS
ELI LILLY AND COMPANY
a corporation of the State of Indiana, U.S.A., having a principal place of business at Lilly Corporate Center, City of Indianapolis, State of Indiana 46285, United
States of America.
The following specification particularly describes the invention and the manner in which it is to be performed.

The present invention relates to selective VPAC2 receptor peptide agonists.
In particular, the present invention relates to selective VPAC2 receptor peptide agonists which are covalently attached to one or more molecules of polyethylene glycol or a derivative thereof.
Type 2 dianetes, or non-insulin dependent diabetes mellitus (NIDDM), is the most
common form of diabetes, affecting 909b' of people with diabetes. With NIDDM, patients
have impaired P-cell function resulting in insufficient insulin production and/or decreased
insulin sensitivity. If NIDDM is not controlled, excess glucose accumulates in the blood,
resulting in hyperglycemia. Over time, more serious complications may arise including
renal dysfunction, cardiovascular problems, visual loss, lower limb ulceration,
neuropathy, and ischemia. Treatments for NIDDM include improving diet, exercise, and
weight control as well as using a variety of oral medications. Individuals with NIDDM
can initially control their blood glucose levels by taking such oral medications. These
medications, however, do not slow the progressive loss of p-cell function that occurs in
NIDDM patients and, thus, are not sufficient to control blood glucose levels in the later
stages of the disease. Also, treatment with currently available medications exposes
NIDDM patients to potential side effects such as hypoglycemia, gastrointestinal
problems, fluid retention, oedema, and/or weight gain.
Pituitary adenylate cyclase-activating peptide (PACAP) and vasoactive intestinal v peptide (VIP) belong to the same family of peptides as secretin and glucagon. PACAP
and VIP work through three G-protein-coupled receptors that exert their action through
the cAMP-mediated and other Ca^-mediated signal transduction pathways. These
receptors are known as the PACAP-preferring type 1 (PAC1) receptor (Isobe, et al,
ReguL Pept, 110:213-217 (2003); Ogi, et al, Biochem. Biophys. Rex Commun.,
196:1511-1521 (1993)) and the two VIP-ahared type 2 receptors (VPAC1 and VPAC2)
(Sherwood et al, Endocr. Rev., 21:619-670 (2000); Hammar et al, Pharmacol Rev,
50:265-270 (1998); Couvineau, et aL, J. Biol Chan., 278:24759-24766 (2003);
Sreedharan, et aL, Biochem. Biophys. Res. Commun., 193:546-553 (1993); Lute, et al.,
FEBSLetL, 458:197-203 (1999); Adamou, era/., Biochem. Biophys. Res. Commun., 209:

385-392 (1995)). A series of PACAP analogues is disclosed in US 6,242,563 and WO 2000/05260.
PACAP has comparable activities towards all three receptors, whilst VIP selectively activates the two VPAC receptors (Tsutsumi et aL, Diabetes; 51:1453-1460 (2002)). Both VIP (Eriksson et aL, Peptides, 10:481-484 (1989)) and PACAP (Filipsson et al., JCEM, 82:3093-3098 (1997)) have been shown to not only stimulate insulin secretion in man when given intravenously but also increase glucagon secretion and hepatic glucose output As a consequence, PACAP or VIP stimulation generally does not result in a net improvement of glycemia. Activation of multiple receptors by PACAP or VIP also has broad physiological effects on nervous, endocrine, cardiovascular, reproductive, muscular, and immune systems (Gozes etal, Curr. Med. Chem, 6:1019-1034 (1999)). It appears that VTP-induced watery diarrhoea in rats is mediated by only one of the VPAC receptors, VPAC1 (Ito et al., Peptides, 22:1139-1151 (2001); Tsutsumi etal. Diabetes, 51:1453-1460 (2002)). The VPAC1 and PAC1 receptors are expressed on a-cells and hepatocytes and, thus, are most likely involved in the effects on hepatic glucose output.
Exendin-4 is found in the salivary excretions from the Gila Monster, Heloderma Suspectum, (Eng et aL, J.BioLCliem., 267(11):7402-7405 (1992)). It is a 39 amino acid peptide, which has glucose dependent insulin secretagogue activity. Particular PEGylated Exendin and Exendin agonist peptides are described in WO 2000/66629.
Recent studies have shown mat peptides selective for the VPAC2 receptor are able to stimulate insulin secretion from the pancreas without gastrointestinal (GI) side effects and without enhancing glucagon release and hepatic glucose output (Tsutsumi etal., Diabetes, 51:1453-1460 (2002)). Peptides selective for the VPAC2 receptor were initially identified by modifying VIP and/or PACAP. (See, for example, Xia et al;, / Pharmacol Exp Ther., 281:629-633 (1997); Tsutsumi etal, Diabetes, 51:1453-1460 (2002); WO 01/23420 and WO 2004/06839.)
Many of die VPAC2 receptor peptide agonists reported to date have, however, less than desirable potency, selectivity, and stability profiles, which could impede their clinical viability. In addition, many of these peptides are not suitable for commercial candidates as a result of stability issues associated with the polypeptides in formulation, as well as issues with the short half-life of these polypeptides in vivo. It has, furthermore,

been identified that some VPAC2 receptor peptide agonists are inactivated by dipeptidyl-peptidase (DPP-IV). A short serum half-life could hinder the use of these agonists as therapeutic agents. There is, therefore, a need for new therapies, which overcome the problems associated with current medications for NIDDM.
The present invention seeks to provide improved compounds that are selective for . the VPAC2 receptor and which induce insulin secretion from the pancreas only in the presence of high blood glucose levels. The compounds of the present invention are peptides, which are believed to also improve beta cell function. These peptides can have the physiological effect of inducing insulin secretion without GI side effects or a corresponding increase in hepatic glucose output and also generally have enhanced selectivity, potency, and/or in vivo stability of the peptide compared to known VPAC2 receptor peptide agonists.
The present invention also seeks to provide selective VPAC2 receptor peptide agonists, which have reduced clearance and improved in vivo stability.' It is desirable that the agonists of the present invention be administered a minimum number of times during a prolonged period of time.
According to a first aspect of the invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a sequence selected from:
SEQ ID NO: 17 HSDAVFTEQY(OMe)TRAibRAibQLAAAibOrnY(OMe)LQSIK
AibOrn;
SEQ ID NO: 18 HSDAVFTEK(CO(CH2)2SH)Y(OMe)TOrnLRAibQVAAAibOrn
YLQSIOrnOrn;
SEQ ID NO: 19 HSDAVFIEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrnK(W)
Orn;
SEQ ID NO: 20 HSDAVFl^QY(OMe)TOrnLRAibQVAAAibK(CO(CH2)2SH)YLQ
SlOrnOrn;
SEQ ID NO: 21 HSDAVFrEQY(OMe)TOrnLRAibQVAAK(CO(CH2)2SH)OmYLQ
SlOrnOrn;
SEQ ID NO: 22 HSDAVFTEQY(OMe)TOrnLRAibQVCAAibOrnYLQSIOrnOrn;
SEQ ID NO: 23 HSDAVFTEQY(OMe)TOrnLRCQVAAAibOrnYLQSIOrnOrn;
SEQ ED NO: 24 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQSIOrnOrn;

SEQ ID NO: 25 HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYAibQSIOmOrn; SEQ ID NO: 26 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYLQAibIOmOm; SEQ ID NO: 27 HSDAVFrEQY(OMe)TOmLRAibQVAAbuAibOmYLQAibIOmOm; SEQ ID NO: 28 HSDAVFTEQY(OMe)TOmLRAibQLAAAibOmYLQAibIOmOni; SEQ ID NO: 29 HSDAVFreQY(OMe)TC>rnIJRAibQI^AAibOmYAibQAibIOmOm; SEQ ID NO: 30 ' HSDAVFrEQY(OMe)TOmLRAibQLAAbuAibOmYAibQSIOmOm; SEQ ID NO: 31 HSDAVFTEQY(OMe)TOrnURAibQLAAbuAibOmYLQSIOmOm; SEQ ID NO: 32 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYAibQAibIOni
Om;
SEQ ID NO: 33 HSDAVFTEQY(OMe)TOmLRAibQLAAAibOmYAibQSIOmOm; SEQ ID NO: 34 HSDAVFTEQY(OMe)TbrnLRK(W)QVAAAibOmYLQSIOmOm; SEQ ID NO: 35 HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYLK(W)SIOmOni; SEQ ID NO: 36 HSDAVFTEQY(0Me)T0mLRAibQK(W)AAAib0mYLQSI0m0m; SEQ ID NO: 37 HSDAVFTEQY(OMe)TOmLRK(CO(CH2)2SH)QVAAAibOmYLQ
SlOrnOm;
SEQ ID NO: 38 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(W)YLQSIOmOrn; SEQ ID NO: 39 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibCYLQSIOmOrn;
SEQ ID NO: 40 HSDAVFTEQY(OMe)TOrnIJRAibQLAAbuAibOmYLQAibIOniOni; SEQ ID NO: 41 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYLQSK(W)OmOrn; SEQ ID NO: 42 HSDAVFTEQY(OMe)TOniLRAibQLAAbuAibOmYLQAibIOmC
Orn;
SEQ ID NO: 43 HSDAVFTEQY(OMe)T0mLRAlbQLAAbuAibOrtYLQAibC0m
Orn;
SEQ ID NO: 44 HSDAVFTEQY(0Me)T0rnLRAibQCAAbuAibOmYLQAibIOmOm; SEQ ID NO: 45 HSDAVFTEQY(OMe)TOrnIJlCQLAAbuAibOmYLQAibIOniC)m; SEQ ID NO: 94 HSDAVFTEQY(OMe)TOmLRAibQVK(CO(CH2)2SH)AAibOni
YLQSIOmOrn;
SEQ ID NO: 95 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSIOniCOm; SEQ ID NO: 96 HSDAVFTEQY(0Me)T0mIJRAibQLAAbiiAib0mYLQSC0m0m; SEQ ID NO: 97 HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOmYLQAibIOrn
K(CO(CH2)2SH)Orn;
SEQ ID NO: 98 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSIOm

K(CO(CH2)2SH)Om;
SEQ ID NO: 99 HSDAVFTEQY(OMe)TOrnLRK(W)QLAAbuAibOmYLQAibIOm
Orn;
SEQ ID NO: 100 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOmYLQSIOmOmC; SEQ ID NO: 101 HSDAVFIEQY(OMe)TC)mLJlAibQVAAAibOmYLQSIOmOrnC; SEQ ID NO: 102 HSDAVFTEQY(OMe)TOrnIJRAibQLAAbuAibOmYLQSIOmOrnC; SEQ ID NO: 103 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmY(OMe)LQAibI
OrnOrn;
SEQ ID NO: 104 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmY(OMe)LQAibI
OrnCOrn;
SEQEcfNO: 105 HSDAVFTEQY(OMe)TOmLRAibQCAAbuAibOmY(OMe)LQAibI
OmOrn;
SEQ ID NO: 106 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQAibIOm
OmC;
i
SEQ ED NO: 107 HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOrhY(OMe)LQSI
OmOm;
SEQ ID NO: 108 HSDAVFTEQY(OMe)TOniLRAibQCAAbuAibOmY(OMe)LQSI
OmOrn;
SEQ ED NO: 109 HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOmY(OMe)LQSI
OrnCOrn;
SEQ ID NO: 110 HSDAVFTEQY(OMe)TOmLRAibQLAbuAAibOmYLQSIOmOm; SEQ ID NO: 111 HSDAVFrEQY(OMe)TOrnLRAibQK(CO(CH2)2SH)AAbu
AibOrnYLQAiblOmOrn; and
SEQ ID NO: 112 HSDAVFTEQY(OMe)TOmLRAibQK(W)AAbuAibOmYLQ
AiblOrnOrn; and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide sequence and wherein the C-terminal extension comprises and amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaas-Xaas-Xaa7-Xaag-Xaa9-Xaaio-Xaai i-Xaau Formula 3 (SEQ ED NO: 3) wherein:

Xaaj is: Gly, Cys, or absent; Xaa2 is: Gly, Axg, 01 absent; Xaa3 is: Pro, Thr, or absent; Xaa4 is: Ser, or absent; Xaaj is: Ser, or absent; Xaae is: Gly, or absent; Xaa7 is: Ala, or absent; Xaag is: Pro, or absent; Xaa9 is: Pro, or absent; Xaaio is: Pro, or absent; Xaan is: Ser, Cys, or absent; and Xaau is: Cys, or absent;
wherein at least five of Xaai to Xaau of the C-terminal extension are present, and wherein if Xaai, Xaa2, Xaa3, Xaaj, Xaas, Xaag, Xaa7, Xaag, Xaag, Xaaio, or Xaau is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,
and wherein;
die peptide agonist comprises at least one Cys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one Lys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(W) which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(CO(CH2)2SH) which is covalently attached to a PEG molecule, or
the carboxy-terrninal amino acid of the peptide agonist is covalently attached to a PEG molecule, or
a combination thereof.
Preferably, at least six of Xaai to Xaai2 of the C-terminal extension of Formula 3 is present. More preferably, at least seven, eight, nine, ten, eleven, or all of Xaai to Xaat2 of the C-terminal extension are present.

More preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist is selected from:
SEQIDNO:5 IGGPSSGAPPPS SEQIDNO:6 GGPSSGAPPPS-NH2 SEQ ID NO: 7 GGPSSGAPPPC SEQIDNO:8 GGPSSGAPPPC-NH2 SEQ ID NO: 9 GRPSSGAPPPS SEQ ID NO: 10 GRPSSGAPPPS-NH2 SEQ ID NO: 11 GGPSSGAPPPCC SEQ ID NO: 12 GGPSSGAPPPCC-NH2
Even more preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist is SEQ ID NO: 11 or SEQ ID NO: 12.
A PEG molecules) may be covalently attached to any Lys, Cys, K(W) or K(CQ(CH2)2SH) residues at any position in the VPAC2 receptor peptide agonist according to the first aspect of the present invention.
Where the PEGylated VPAC2 receptor peptide agonist comprises a sequence selected from SEQ ID NO: 22,23,39,42,43,44,45,95,96,100,101,102,104,105, 106,108 and 109, it is preferred mat the cysteine residue is PEGylated.
Where the PEGylated VPAC2 receptor peptide agonist comprises a sequence selected from SEQ ID NO: 19,34,35,36,38,41,99 and 112, it is preferred mat the K(W) residue is PEGylated.
Where the PEGylated VPAC2 receptor peptide agonist comprises a sequence selected from SEQ ID NO: 18,20,21,37,94,97,98 and 111, it is preferred that the K(CO(CH2)2SH) residue is PEGylated.
Where the PEGylated YPAC2 receptor peptide agonist comprises a C-terminal extension, die PEG molecule(s) may be covalently attached to one or more Cys residues in said C-terminal extension. Where the sequence selected from SEQ ID NO: 17 to 45 and 94 to 112 comprises one or more Lys, Cys, K(W), or K(CO(CH2)2SH) residues and the C-terminal extension comprises one or more Cys residues, there may be one or more PEGylated residues in either or both sequences.

Preferably, there is at least one PEG molecule covalently attached to a residue in the C-terminal extension of the VPAC2 receptor peptide agonist.
Where there is more than one PEG molecule, there may be a combination of Lys,
Cys, K(CO(CH2)2SH), K(W) and carboxy-terminal amino acid PEGylation. For example,
if there are two PEG molecules, one may be attached to a Lys residue and one may be
attached to a Cys residue.
Preferably, the PEG molecule is branched. Alternatively, the PEG molecule may be linear.
Preferably, the PEG molecule is between 1,000'daltons and 100,000 daltons in molecular weight More preferably, the PEG molecule is selected from 10,000,20,000; 30,000,40,000,50,000 and 60,000 daltons. Even more preferably, it is selected from 20,000,30,000,40,000, or 60,000 daltons. Where there are two PEG molecules covalently attached to the peptide agonist of the present invention, each is 1,000 to 40,000 daltons and preferably, they have molecular weights of 20,000 and 20,000 daltons, 10,000 and 30,000 daltons, 30,000 and 30,000 daltons, or 20,000 and 40,000 daltons.
The PEGylated VPAC2 receptor peptide agonist sequence may further comprise a histidine residue at the N-terminus of the peptide before Xaai.
Preferably, the PEGylated VPAC2 receptor peptide agonist according to the first aspect of the present invention further comprises a N-terminal modification at the N-terminus of the peptide agonist wherein the N-terminal modification is selected from: (a) addition of D-histidine, isoleucine, methionine, or norleucine; (b)'addition of a peptide comprising the sequence Ser-Trp-Cys-Glu^Pro-Gly-Trp-Cys-
Arg (SEQ ID NO: 93) wherein the Arg is linked to the N-terminus of the peptide
agonist;
(c) addition of C1-C16 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3;
(d) addition of-CCOJR1 wherein R1 is a C1-C16 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen, -SH and -CFj; an aryl optionally substituted with one or more substituents independently selected from Ci-C
C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C16 alkoxy, -NH2,'-OH, halogen and "CF3; -NR2R3 wherein R2 and R3 are independently hydrogen, C1- alkyl, aryl or aryl C1-C4 alkyl; -OR4 wherein R4 is C2-C6 alkyl optionally substituted with one or more substituents independently selected from aryl, C2-C6 alkoxy, -NH2, -OH, halogen and -CF3, aryl optionally substituted with one or more substituents independently selected from C2-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 alkoxy, -NH2, -OH, halogen and -CF3, or aryl C2-C6 alkyl optionally substituted with one or more substituents independently selected from C2-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 alkoxy, -NH2, -OH, halogen and -CF3; or 5-pyrrolidin-2-one;
(e) addition of-SO^5 wherein R5 is aryl, aryl C1-C4 alkyl or C2-C6 alkyl;
(f) formation of a succinimide group optionally substituted with C2-C6 alkyl or -SR6, wherein R6 is hydrogen or C2-C6 alkyl;
(g) addition of methionine sulfoxide;
(h) addition of biotinyl-6-aminohexanoic acid (6-aminocaproic acid); and
(i) addition of-C(=NH)-NH2.
Preferably, the N-terminal modification is the addition of a group selected from:
acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-
phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-
mercaptopropionyl, biotinyl-6-aminohexanoic acid (6-aminocaproic acid), and -
C(=NH)-NH2. It is especially preferred that the N-terminal modification is the addition of
• acetyl or hexanoyl.
It will be appreciated by the person skilled in the art that PEGylated VPAC2
receptor peptide agonists comprising various combinations of peptide sequence selected
from SEQ ID NO: 17 to 45 and 94 to 112, C-terminal extensions and N-terminal
modifications as described herein, may be made based on the above disclosure.
It is preferred that the PEGylated VPAC2 receptor peptide agonist according to
the first aspect of the present invention comprises an amino acid sequence selected from:
Agonist I SEQ I Sequence
# ID
NO
P410 46 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(W-
J I PEG40K)YLQSIOrnOrnGGPSSGAPPPS-NH2

, , _,
HSDAVFTEQY(OMe)TRAibRAibQLAAAibOmY(OMe)LQ
SIKAibOmGGPSSGAPPPC(PEG40K)-NH2
P451 48 C6-
HSDAVFTEK(CO(CH2)2SPEG40K)Y(OMe)TOniLRAibQVA
AAib0mYLQSI0ni0mGGPSSGAPPPS-NH2
P454 49 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYLQSIOm
K(WPEG40K)OrnGGPSSGAPPPS-NH2
P460 50 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibC(PEG40K)YL
QSIOmOmGGPSSGAPPPS-NH2
~Wft 51 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibK(CO(CH2)2S
PEG40K)YLQSIOmOmGGPSSGAPPPS-NH2
P473 52 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibK(CO(CH2)2S
PEG20K)YLQSIOmOmGGPSSGAPPPS-NH2
P475 53 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAK(CO(CH2)2SPEG
40K)OmYLQSIOmOmGGPSSGAPPPS-NH2
P478 54 C6-
HSDAVFTEQY(OMe)TOmLRAibQVC(PEG40K)AAibOmY
LQSIOmOmGGPSSGAPPPS-NH2
P483 55 C6-
HSDAVFTEQY(OMe)TOmLRC(PEG40K)QVAAAibOmYL
QSIOmOmGGPSSGAPPPS-NH2
P485 56 C6-
HSDAVFTEQY(OMe)TOmLRK(CO(CH2)2SPEG40K)QVAA
AibOmYLQSIOmOmGGPSSGAPPPS-NH2
P507 57 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYAibQSI
OmOrnGGPSSGAPPPC(PEG40K)-NH2
P509 58 C6-
HSDAVFrEQY(OMe)TOrnLRAibQVAAAibOrnYLQAibI
OmOmGGPSSGAPPPC(PEG40K)-NH2
P511 59 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAbuAibOrnYLQAibI
OTnOmGGPSSGAPPPC(PEG40K)-NH2
P513 60 C6-
HSDAVFTEQY(OMe)TOmLRAibQLAAAibOmYAibQSI
OmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P515 61 C6-
HSDAVFTEQY(0Me)T0mLRAibQLAAAib0mYLQAibI
| |omOrnGGPSSGAPPPC(PEQ20K)C(PEG20K)-NH2

:[P517[62 [c£
HSDAVFTEQY(OMe)TOtnLEtAibQLAAbuAibOmYLQAibI
OmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
, P519 63 C6-
HSDAVFTEQY(OMc)TOrnLRAibQLAAAibOmYAibQAibI
: OmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P521 64 . C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYAibQSI
OmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P523 65 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSI
OmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P525 66 ■ ' 06-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYAibQ
AibIOmOmGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P529 67 C6-
HSDAVFTEQY(6Me)TOrnLElK(WPEG40K)QVAAAibOmY
LQSIOmOmGGPSSGAPPPS-NH2
P531 68 C6-HSDAVFTEQY(0Me)T0mLRAibQVAAAib0mYLK(W
PEG40K)SIOmOmGGPSSGAPPPS-NH2
P533 69 C6-HSDAVFTEQY(OMe)TOmLRAibQK(WPEG40K)AAAib
OmYLQSIOmOmGGPSSGAPPPS-NH2
P535 70 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOniYLQSK
•■ (WPEG40K)OmOmGGPSSGAPPPS-NH2 '
P537 71 C6-
HSDAVFtEQY(OMe)TOmLRAibQLAAbuAibOmYLQAibI
OmC(PEG40K)OmGGPSSGAPPPS-NH2
. P541 72 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQAibC
(PEG40K)OmOmGGPSSGAPPPS-NH2
P545 73 C6-HSDAVFTEQY(OMe)TOrnLRAibQC(PEG40K)AAbuAib
0roYLQAibI0m0mGGPSSGAPPPS-NH2
; P547 74 C6-
HSDAVFTEQY(OMe)TOrnLRC(PEG40K)QLAAbuAibOmY.
LQAibIQmOmGGPSSGAPPPS-NH2
P480 113 C6-
HSDAVPTEQY(OMe)TOrnLRAibQVK(CO(CH2)2SPEG40K)
AAibOmYLQSIQmOmGGPSSGAPPPS-NrH2
P481 114 C6-
HSDAVFTEQY(OMe)TOmLRAibQVK(CO(CH2)2SPEG20K)
AAibOmYLQSIOmOmGGPSSGAPPPS-NHa
P539 115 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSI
OrnC(PEG40K)DmGGPSSGAPPPS-NH2
P543 116 C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQS
I I Ca,EG40K)OmOrnGGPSSGAPPPS-NH2 '

.
P549 TTvi I C6-HSDAVFTEQY(0Me)T0niLRAibQLAAbuAib0mYL
QAibIOrnK(CO(CH2)2SPEG20K)OmGGPSSGAPPPC(PEG20
K)-NH2
P551 118 C6-HSDAVFreQY(0Me)T0niLRAibQLAAbuAib0niYL
QSIOmK(CO(CH2)2SPEG20K)OrnGGPSSGAPPPC(PEG20K
j^Sfe
P555 119 C6-
HSDAVFrEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSI
OrnC(PEG20K)QmGGPSSGAPPPC(PEG20K)-NH2
P557 120 G6-
HSDAVFTEQY(OMe)TOrnLRK(WPEG40KX2LAAbuAib
OmYLQAibIOmOmGGPSSGAPPPS-NH2
P560 121 G6-
HSDAVFTEQY(OMe)TOniLRAibQLAAAibOmYLQSIOni
OiDC(PEG40K)GGPSSGAPPPS-NHi
P562 122 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYLQSIOrn
OmC(PEG20K)GGPSSGAPPPC(PEG20K)-NH2
P564 123 C6-
HSDAVFTEQY(OMe)TOraLRAibQLAAbuAibOraYLQAibI
OmOmC(PEG40K)GGPSSGAPPPS-NH2
P566 124 C6-
HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOmYLQSI
OmOmC(PEG40K)GGPSSGAPPPS-NH2
P572 125 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQSI
OmOmC(PEG20K)GGPSSGAPPPC(PEG20K)-NHi
P574 126 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmY(OMe)L
QAibIOmQmGGPSSGAPPPC(PEG20K)C(PEG20K>NH2
P576 127 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmY(OMe)L
QAibIOmC(PEG40K)OmGGPSSGAPPPS-NHa
P578 128 C6-HSDAVFTEQY(OMe)TOraLRAibQC(PEG40K)AAbuAib
OmY(OMe)LQAibIOmQniGGPSSGAPPPS-NH2
P580 129 C6-
HSDAVFTEQY(OMe)TOinLRAibQLAAbuAibOmYLQAibI
OmOmC(PEG20KXKSPSSGAPPPC(PEG20K)-NH2
P582 130 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmY(OMe)L
QSIOmOmGGPSSGAPPPC(PEG20K)C(PEG20K>NH2
P584 131 C6-
HSDAVPTEQY(OMe)TOrnLRAibQLAAbuAibOniY(OMe)L
a QSIOrnC(PEG40K)OmGGPSSGAPPPS-NH2
P586 132 C6-HSDAVFTEQY(OMe)TOmLRAibQC(PEG40K)AAbuAib
J I OmY(OMe)LQSIOmOrnQGPSSGAPPPS-NH2


__ p_- _
HSDAVFTEQY(OMe)TQrnLRAibQLAAbuAibOmY(OMe)L
' QSIOmC(PEG20K)OmGGPSSGAPPPC(PEQ20K)-NH2
P590 134 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAbuAAibOmYLQSI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P597 135 C6-
HSDAVFrEQY(OMe)TOmLRAibQK(CO(CH2)2SPEG20K)A
AbuAibOmYLQAibIOtnOmGGPSSGAPPPC(PEG20K)-NH2
P599 136 . C6-
. HSDAVFTEQY(OMe)TOrnLRAibQK(CO(CH2)2SPEG40K)A
AbuAibOmYLQAiblOmOmGGPSSGAPPPS-NH:
P601 137 • C6-
HSDAVFTEQY(OMe)TOrnLRAibQK(WPEG40K)AAbuAib
OmYLQAiblOmOmGGPSSGAPPPS-NHa
P469 139 C6-
HSDAVFrEK(CO(CH2)2SPEG20K)Y(OMe)TOrnLRAibQVA
AAibOmYLQSIOmOmGGPSSGAPPPS-NH2
P486 140 C6-
HSDAVFTEQY(OMe)TOraLRK(CO(CH2)2SPEG20K)QVAA
; AibOrnYLQSIOmOrnQGPSSOAPPPS-NHb
P553 141 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQAibI
; OrnC(PEG20K)OrnGGPSSGAPPPC(PEG20K)-NH2
P570 144 . C6-
HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOmYLQAibI
OmOmGGPSSGAPPPC(PEG30K)C(PEG30K)-^fH2
P595 146 C6-HSDAVFTEQY(OMe)TOinLRAibQC(PEG20K)AAbuAib
OmYLQAibIOmOrnGGPSSGAPPPC(PEG20K)-NH2
P476 147 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAK(CO(CH2)2SPEG
20K)OmYLQSIQmOmGGPSSGAPPPS-NH2
P602 148 C6-
HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOrnYAibQSI
| . I OmOmGGPSSGAPPPC(PEG30K)C(PEG30K)-NH2
It is more preferred that the PEGylated VPAC2 receptor peptide agonist according to the first aspect of the present invention comprises an amino acid sequence selected from: SEQ ID NO: 47,64,66,115,119,122,126,130 and 144.
According to a second aspect of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence selected from:

__^ 2_
Agonist ISEQ I Sequence
# ID .
_NO
P470 75 C6-
HSDAVFTEQY(OMe)TOmK(CO(CH2)2SPEG20K)RAibQV
; AAAibOrnYLQSIOmOrnGQPSSGAPPPS-NH2
P490 76 C6-HSDAVFTEQY(OMe)TOtnLRAibQVAAAibOmYL
K(CO(CH2)2SPEG20K)SIOmOmGGPSSGAPPPC(PEG20K)-
Nfy ; .
P492 77 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAK(CO(CH2)2SPEG
20K)AibOmYLQSIOmOmGGPSSGAPPPC(PEG20K)-NH2
P49S 78 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYLQ
K(CO(CH2)2SPEG20K)IOmOmGGPSSGAPPPC(PEG20K>
m^ ; .
P497 79 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYLQS
K(CO(CH2)2SPEG20K)OmOinGGPSSGAPPPC(PEG20K)-
_NHg . 1
P499 80 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibOrnYLQSIOrn
K(CO(CH2)2SPEG20K)OmGGPSSGAPPPC(PEG20K)-NH2
P501 81 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLC(PEG
20K)SIOmOmGGPSSGAPPPC(PEG20K)-NH2
P503 82 C6-HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYLQSC
(PEG20K)OmOrnGGPSSGAPPPC(PEG20K>NH2
P505 83 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOmYLQSIOmC
(PEG20K)OmGGPSSGAPPPC(PEG20K)-NH2
P402 138 C6-
HSDAVFTEQY(OMe)TOmLRAibQVAAAibOmYLQSIOm
. OmGGPSSGAPPPK(W-PEG40K)-NH2
P558 142 C6-
HSDAVFTEQY(OMe)TOmLRAibQC(PEG20K)AAAibOmY
■ LQSIOmOrnGGPSSGAPPPS-NH2
P568 143 C6-
HSDAVFTEQY(OMe)TOmLRAibQC(PEG20K)AAAibOmY
LQSIOmOmGGPSSGAPPPC(PEG20K)-NH2
P593 145 C6-
HSDAVFTEQY(OMe)TOmLRAibQLAAbuAibOmYAibQ
| 1 AibIOmOrnGGPSSGAPPPK(WPEG40K)-NH2

According to a third aspect of the present invention, there is provided a PEOylated
VPAC2 receptor peptide agonist comprising an amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaaj-Xaa6-Thr-Xaa8-Xaa9-Xaaio-Thr-Xaai2-Xaai3-Xaai4-Xaai5-Xaai6-Xaan-Xaai8 -Abu-Xaa20-Xaa2i-Xaa22- Xaa23-Xaa24-Xaa25-Xaa2 Formula 4 (SEQ ID NO: 4)
wherein:
Xaai is: His, dH, or is absent;
Xaa2 is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib;
Xaa3 is: Asp or Glu;
Xaa4 is: Ala, lie, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or NMeA;
Xaa5 is: Val, Leu, Phe, lie, Thr, Trp, Tyr, dV, Aib, or NMeV;
Xaa« is: Phe, lie, Leu, Thr, Val, Trp, or Tyr,
Xaag is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr,
Xaas is: Asn, Gin, Asp, Glu, Ser, Cys, Lys, or K(CO(CH2)2SH);
Xaaib is: Tyr, Trp, Tyr(OMe), Ser, Cys, or Lys;
Xaau is: Arg, Lys, Glu, hR, Om, Lys (isopropyl), Aib, Cit, Ala, Leu, Gin, Phe, Ser, or
Cys;
Xaais is: Leu, Phe, Glu, Ala, Aib, Ser, Cys, Lys, or K(CO(CH2)2SH);
Xaau is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gin, Aib, Cit, Ser, or Cys;
Xaais is: Lys, Ala, Arg, Glu, Leu, hR, Orn, Lys (isopropyl), Phe, Gin, Aib, K(Ac), Cit,
; Ser, Cys, K(W), or K(CO(CH2)2SH);
Xaaie is: Gin, Lys, Glu, Ala, hR, Om, Lys (isopropyl), Cit, Ser, Cys, K(CO(CH2)2SH), or
K(W);
Xaai7 is: Val, Ala, Leu, lie, Met, Nle, Lys, Aib, Ser, Cys, K(CO(CH2)2SH), or K(W);
Xaais is: Ala, Ser, Cys, Lys, K(CO(CH2)2SH), K(WX Abu, or Nle;
Xaa2o is: Lys, Gin, hR, Arg, Ser, His, Om, Lys (isopropyl), Ala, Aib, Trp, Thr, Leu, lie,
Phe, Tyr, Val, K(Ac), Cit, Cys, K(CO(CH2)2SH), or K(W);
Xaa2i is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gin, Orn, hR, K(Ac), Cit, Ser, Cys, Val, Tyr,
lie, Thr, Trp, K(W), or K(CO(CH2)2SH);

Xaa22 is: Tyr, Trp, Phe, Thr, Leu, He, Val, Tyr(OMe), Ala, Aib, Ser, Cys, Lys, K(W), or
K(CO(CH2)2SH); .
Xaa23 is: Leu, Phe, He, Ala, Trp, Thr, Val, Aib, Ser, Cys, Lys, K(W)vor K(CO(CH2)2SH);
XaaM is: Gin, Glu, Asn, Ser, Cys, Lys, K(CO(CH2)2SH), or K(W);
Xaa2j is: Ser, Asp, Phe, lie, Leu, Thr, Val, Trp, Gin, Asn, Tyr, Aib, Glu, Cys, Lys,
K(CO(CH2)2SH), or K(W)^
Xaa26 is: lie, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, Lys, K(CO(CH2)2SH), or K(W);
Xaa27 is: Lys, hR, Arg, Gin, Ala, Asp, Glu, Phe, Gly, His, lie, Met, Asn, Pro, Ser, Thr,
Val, Trp, Tyr, Lys (isopropyl), Cys, Leu, Ora, dK, K(W), or KCCCKCHzfcSH);
Xaa2B is: Asn, Asp, Gin, Lys, Arg, Aib, 0m, hR, Cit, Pro, dK, Ser, Cys, K(CO(CH2)2SH),
orK(W);
Xaa29 is: Lys, Ser, Arg, Asn, hR, Ala, Asp, Glu, Phe, Gly, His, lie, Leu, Met, Pro, Gin,
Thr, Val, Trp, Tyr, Cys, Orn, Cit, Aib, K(W), K(CO(CH2)2SH), or is absent;
Xaa3o is: Arg, Lys, lie, Ala, Asp, Glu, Phe, Gly, His, Leu, Met, Asn, Pro, Gin, Ser, Thr,
Val, Trp, Tyr, Cys, hR, Cit, Aib, Orn, K(W), K(CO(CH2>2SH), or is absent;
Xaa3i is: Tyr, His, Phe, Thr, Cys, Ser, Lys, Gin, K(W), K(CO(CH2)2SH), or is absent;
Xaa32 is: Ser, Cys, Lys, or is absent;
Xaa33 is: Trp or is absent;
Xaa34 is: Cys or is absent;
Xaa3j is: Glu or is absent;
Xaa36 is: Pro or is absent;
Xaa37 is: Gly or is absent;
Xaa3s is: Trp or is absent;
Xaa39 is: Cys or is absent; and
XaaM is: Arg or is absent
wherein if Xaa29, Xaa3o, Xaa3i, Xaa32, Xaa33, XaaM, Xaa35, Xaase, Xaa37, Xaa3g, or Xaa39 is absent, the next amino acid present downstream is the next amino acid in the peptide agonist sequence,
and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 4 and wherein the C-terminal extension comprises an amino acid sequence of the formula:

Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaag-Xaa9-Xaaio-Xaai i -Xaaj2 Fonnula3(SEQIDNO:3) : wherein: Xaai is: Gly, Cys, or absent; Xaa2 is: Gly, Arg, or absent; Xaaa is: Pro, Thr, or absent; Xaa wherein at least five of Xaai to Xaai2 of the C-terminal extension are present and wherein if Xaai, Xaa2, Xaa3, Xaa4, Xaa5, Xaas, Xaa7, Xaag, Xaas, Xaaib, or Xaau is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,
and wherein;
the peptide agonist comprises at least one Cys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one Lys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(W) which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(CO(CH2>2SH) which is covalently attached to a PEG molecule, or
the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or
a combination thereof.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the present invention comprises a sequence of the Formula 4 (SEQ ID NO: 4) wherein Xaa3 is Asp or Glu, Xaag is Asp or Glu, Xaag is Asn or Gin, Xaaio is Tyr or Tyr(OMe), Xaai2 is Arg, hR, Lys, or Orn, Xa&u is Arg, Gin, Aib, hR, Orn, Cit, Lys, Ala, or Leu, Xaais is Lys, Aib, Orn, or Arg, Xaa is Gin or Lys, Xaan is Val, Leu, Ala, lie, Lys, or Nle, Xaaw is Lys, Val, Leu, Aib, Ala, Gin, or Arg, Xaa2i is Lys, Aib, Orn, Ala, Gin, or Arg, Xaaa is Leu or Aib, Xaau is Ser or Aib, Xaa27 is Lys, Orn, hR, or Arg, Xaajg is Asn, Gin, Lys, hR, Aib, Orn, or Pro and Xaa& is Lys, Orn, hR, or is absent
Preferably, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the'present invention comprises a sequence of the Formula 4 (SEQ ID NO: 4), wherein either Xaa23 or Xaa2s is Aib. Even more preferably, Xaa^ and Xaa2s are both Aib.
Preferably, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the present invention comprises a sequence of the Formula 4 wherein either Xaaw or Xaajs is Aib.
Alternatively, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the present invention comprises a sequence of the Formula 4 wherein either Xaa2o or Xaa2i is Aib.
More preferably, either Xaau or Xaau is Aib and either Xaaio or Xaa2i is Aib. It is especially preferred that Xaais is Aib and Xaa2o is Aib.
Preferably, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the present invention comprises a sequence of the Formula 4 wherein Xaais is Aib, Xaa2o is Aib, and Xaai2, Xaa2i, Xaa27 and Xaa28 are all Orn. More preferably, Xaais is Aib, Xaa2o is Aib, Xaai2, Xaa2j, Xaa27 and Xaa2g are all Orn, Xaag is Glu, Xaa$ is Gin and Xaaio is Tyr(OMe). Even more preferably, Xaais is Aib, Xaa2o is Aib, Xaan, Xaa2i, Xaa27 and Xaa2g are all Orn, Xaag is Glu, Xaag is Gin, Xaaio is Tyr(OMe), and Xaa23 and/or Xaa^ is Aib. Any one or more of Xaag, Xaag, Xaaio, Xaai2, Xaai 3> Xaa2o, Xaa2i, Xaa23, Xaa25, Xaa27 and Xaa2g may be a PEGylated Lys, Cys, K(CO(CH2)2SH) or K(W), whilst all the other positions have the preferred amino acid substitutions as described.
Preferably, at least six of Xaai to Xaai2 of the C-terminal extension of Formula 3 is present. More preferably, seven, eight, nine, ten, eleven, or all of Xaai to Xaa^of the C-terminal extension are present

Preferably, the C-terminal extension of the PEGyated VPAC2 receptor peptide agonist according to the third aspect of the present invention is selected from: SEQ ID NO: 5,6,7,8,9,10,11 and 12.
More preferably, the C-terminal extension of the PEGyated VPAC2 receptor peptide agonist according to the third aspect of the present invention is SEQ ID NO: 11 or SEQIDNO:12.
A PEG molecule(s) may be covalently attached to any Lys, Cys, K(W) or K(CO(CH2)2SH) residue at any position in the VPAC2 receptor peptide agonist according to the third aspect of the present invention. The C-terminal extension may comprise one or more Cys residues which may be PEGylated. Where the sequence according to Formula 4 comprises one or more Lys, Cys, K(W), or K(CO(CH2>2SH) residues and the C-terminal extension comprises one or more Cys residues, there may be one or more PEGylated residues in either or both Sequences.
Preferably, there is at least one PEG molecule covalently attached to a residue in Formula 4. More preferably, there is a PEG molecule covalently attached to a residue at one or more of the following positions of Formula 4:9,13, IS, 16,17,18,20,21,24,25, 26 and 28.
Preferably, there is at least one PEG molecule covalently attached to a residue in the C-terminal extension of the VPAC2 receptor peptide agonist.
Where there is more than one PEG molecule, there may be a combination of Lys, Cys, K(CO(CH2)2SH), K(W) and carboxy-terminal amino acid PEGylation. For example, if there are two PEG molecules, one may be attached to a Lys residue and one may be attached to a Cys residue.
Preferably, the PEG molecule is branched. Alternatively, the PEG molecule may be linear.
Preferably, the PEG molecule is between 1,000 daltons and 100,000 daltons in molecular weight. More preferably, the PEG molecule is selected from 10,000,20,000, 30,000,40,000,50,000 and 60,000 daltons. Even more preferably, it is selected from 20,000,30,000,40,000, or 60,000 daltons. Where there are two PEG molecules covalently attached to the peptide agonist of the present invention, each is 1,000 to 40,000 daltons and preferably, they have molecular weights of 20,000 and 20,000 daltons, 10,000 and 30,000 daltons, 30,000 and 30,000 daltons, or 20,000 and 40,000 daltons.

Preferably, the PEGylated VPAC2 receptor peptide agonist of the third aspect of the present invention further comprises a N-terminal modification at the N-terminus of the peptide agonist wherein the N-terminal modification is selected from:
(a) addition of D-histidine, isoleucine, methionine, or norleucine;
(b) addition of a peptide comprising the sequence Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg (SEQ ID NO: 93) wherein the Arg is linked to the N-terminus of the peptide agonist;
(c) addition of C1-C16 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3;
(d) addition of-CCOJR1 wherein Rl is a C1-C16 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen, -SH and -CF3; an aryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkvnyl, C1-C6 alkoxy,-NH2,-OH, halogen and-CF3; and aryl C1-C4 alkyl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3; -NR2R3, wherein R2 and R3 are independently hydrogen, Q-Ce alkyl, aryl or aryl C1-C4 alkyl; -OR4 wherein R4 is C1-C6 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3, aryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3 or aryl C1-C4 alkyl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C2-C6 alkoxy, -NH2, -OH, halogen and -CF3; or 5-pyrrolidin-2-one;
(e) addition of-S02Rs wherein R5 is aryl, aryl C1-C4 alkyl or C1-C6 alkyl;
(f) formation of a succinimide group optionally substituted with C1-C6 alkyl or -SR6, wherein R6 is hydrogen or C1-C6 alkyl;
(g) addition of methionine sulfoxide;
(h) addition of biotinyl-6-aminohexanoic acid (6-aminocaproic acid); and (i) addition of-C(=NH)-NH2-

Preferably, the N-terminal modification is the addition of a group selected from: acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-pheriylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid (6-aminocaproic acid), and -C(=NH)-NH2. It is especially preferred that the N-terminal modification is the addition of : acetyl or hexanoyl.
It will be appreciated by the person skilled in the art that PEGylated VPAC2 receptor peptide agonists comprising various combinations of peptide sequence according to Formula 4, C-terminal extensions and N-terminal modifications as described herein, may be made based on the above disclosure.
It is preferred that the PEGylated VPAC2 receptor peptide agonist according to the third aspect of the present invention comprises an amino acid sequence selected from: SEQ ID NO: 59,62, 64, 65,66,71,72,73,74,115,116,117,118,119,120,123,124, 125,126,127,128,129,130,131,132,133,135,136,137,141,144,146 and 148.
According to a fourth aspect of the present invention, there is provided a pharmaceutical composition comprising a PEGylated VPAC2 receptor peptide agonist of the present invention and one or more pharmaceutically acceptable diluents, carriers and/or excipients.
According to a fifth aspect of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist of the present invention for use as a medicament.
According to a sixth aspect of the present invention, there is provided the use of a PEGylated VPAC2 receptor peptide agonist of the present invention for the manufacture of a medicament for the treatment non-insulin-dependent diabetes.
According to a further aspect of the present invention, there is provided the use of a PEGylated VPAC2 receptor peptide agonist of the present invention for the manufacture of a medicament for the treatment insulin-dependent diabetes.
The present invention provides a method of treating diabetes in a patient in need thereof comprising administering a PEGylated VPAC2 receptor peptide agonist of the present invention, wherein the diabetes may be non-insulin dependent diabetes or may be insulin-dependent diabetes.

The present invention further provides a pharmaceutical composition containing a
PEGylated VPAC2 receptor peptide agonist of the present invention for treating non-insulin dependent diabetes or insulin-dependent diabetes.
According to an alternative embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a sequence selected from SEQ ID NO: 17 to 45 and 94 to 112;
and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide sequence and wherein the C-terminal extension comprises an amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaas-Xaas-Xaa7-Xaag-Xaa9-Xaaio-Xaan-Xaai2-Xaai3 Formula 1 (SEQ ID NO: 1) wherein:
Xaai is: Gly.Cys.Lys, K(W),K(CO(CH2)2SH), or absent;-Xaa2 is: Gly, Arg, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa3 is: Pro, Thr.Ser, Ala, Cys, Lys, K(W),K(CO(CH2)2SH), or absent; Xaa4 is: Ser, Pro, His, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa5 is: Ser, Arg, Thr, Trp, Lys, Cys, K(W), K(CO(CH2)2SH), or absent; Xaae is: Gly, Ser, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa7 is: Ala, Asp, Arg, Glu, Lys, Gly, Cys, K(W), K(CO(CH2)iSH), or absent; Xaag is: Pro, Ser, Ala, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaaj is: Pro, Ser, Ala, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaaio is: Pro, Ser, Ala, Arg, Lys, His, Cys, K(W), K(CO(CH2)2SH), or absent; Xaau is: Ser, Cys, His, Pro, Lys, Arg, K(W), K(CO(CH2)2SH)I or absent; Xaai2 is: His, Ser, Arg, Lys, Cys, K(W), KCCOCCH^SH), or absent; and Xaai3 is: His, Ser, Arg, Lys, Cys, K(W), KCCO(CH2)2SH)1 or absent; .
provided that if Xaai, Xaa2, Xaa3, Xaa4, Xaas, Xaa6, Xaa7, Xaag, Xaag, Xaaio, Xaau, or Xaai2 is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,
and wherein;
the peptide agonist comprises at least one Cys residue which is covalently attached to a PEG molecule, or

the peptide agonist comprises at least one Lys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(W) which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(CO(CH2>2SH) which is covalently attached to a PEG molecule, or
the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or
a combination thereof.
It is preferable that the C-terminal extension of Formula 1 has no more than three of any one of the following; Cys, Lys, K(W) or K(CO(CH2>2SH). It is more preferable that the C-terminal extension has no more than two of any of these residues. If there are two Cys residues in the C-terminal extension, it is preferred that the Cys residues are at the C-terminus. It is even more preferable that the C-terminal extension has no more than one of any of these residues. If there is only one Cys residue in the C-terminal extension, it is preferred that the Cys residue is at the C-terminus.
Preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist according to the above alternative embodiment comprises an amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaag-Xaa9-Xaaio-Xaau-Xaai2-Xaai3 Formula 2 (SEQ ID NO: 2) wherein:
Xaai is: Gly, Cys, Lys, or absent; Xaa2 is: Gly, Arg, Cys, Lys, or absent; Xaa3 is: Pro, Thr, Ser, Ala, Cys, Lys, or absent; Xaa4 is: Ser, Pro, His, Cys, Lys, or absent; Xaas is: Ser, Arg, Thr, Trp, Lys, Cys, or absent; Xaas is: Gly, Ser, Cys, Lys, or absent; Xaa7 is: Ala, Asp, Arg, Glu, Lys, Gly, Cys, or absent; Xaas is: Pro, Ser, Ala, Cys, Lys, or absent; Xaag is: Pro, Ser, Ala, Cys, Lys, or absent; Xaaio is: Pro, Ser, Ala, Arg, Lys, His, Cys, or absent;

Xaau is: Ser, Cys, His, Pro, Lys, Arg, or absent; Xaan is: His, Ser, Arg, Lys, Cys, or absent; and Xaai3 is: His, Ser, Arg, Lys, Cys, or absent;
provided that if Xaau Xaa2, Xaa3, Xaa4, Xaas, Xaa6, Xaa?, Xaag, Xaag, Xaaio, Xaau, or Xaai 2 is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated.
Preferably, at least one of Xaai to Xaau of the C-terminal extension of Formula 1 or 2 is present. More preferably, at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or all of Xaai to Xaai 3 of the C-terminal extension are present
More preferably, the C-terminal extension of the PEGylated VPAC2 receptor peptide agonist according to the above alternative embodiment comprises an amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaas-Xaa5-Xaa7-Xaa8-Xaa9-Xaaio-Xaai i-Xaan Formula 3 (SEQ ID NO: 3) wherein:
Xaai is: Gly, Cys, or absent; Xaa2 is: Gly, Arg, or absent; Xaa3 is: Pro, Thr, or absent; Xaa4 is: Ser, or absent; Xaas is: Ser, or absent; Xaae is: Gly, or absent; Xaa7 is: Ala, or absent; Xaag is: Pro, or absent; Xaas is: Pro, or absent; Xaaio is: Pro, or absent; Xaau is: Ser, Cys, or absent; and Xaai2 is: Cys, or absent;
provided that if Xaai, Xaa2, Xaa3, Xaa
Preferably, at least one of Xaai to Xaa)2 of the C-terminal extension of Formula 3 is present More preferably, at least two, three, four, five, six, seven, eight, nine, ten, eleven, or all of Xaai to Xaai2 of the C-terminal extension are present
An alternative C-terminal extension, which may be used in any of the aspects and embodiments of the present invention, comprises an amino acid sequence of the formula: Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaaio Formula 13 (SEQ ID NO: 13) wherein:
Xaai is: Ser, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa2 is: Arg, Ser, hR, Orn, His, Cys, Lys, K(W), K(CO(CH2)2SH), or absent, Xaa3 is: Thr, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa4 is: Ser, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaas is: Pro, Ser, Ala, Cys, Lys, K(W), KCCOCCH^SH), or absent; Xaa6 is: Pro, Ser, Ala, Arg, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaa7 is: Pro, Ser, Ala, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; Xaag is: Lys, K(W), Pro, Cys, K(CO(CH2)2SH), or absent; Xaa? is: K(E-C16), Ser, Cys, Lys, K(W), K(CO(CH2)2SH), or absent; and Xaaib is: Ser, Cys, Lys, K(W), K(CO(CH2)2SH), or absent
It is preferred that if Xaai, Xaa2, Xaa3, Xaa*, Xaa5, Xaag, Xaa7, Xaag or Xaag of Formula 13 is absent, the next amino acid downstream is the next amino acid in the C-terminal extension. The C-terminal amino acid may be amidated.
Preferably, at least one of Xaai to Xaaio of the C-terminal extension of Formula 13 is present More preferably, at least two, three, four, five, six, seven, eight, nine or all of Xaai to Xaaio of the C-terminal extension are present
More preferably, an alternative C-terminal extension, which may be used in any of
the aspects and embodiments of the present invention, is selected from:
SEQ ID NO: 85 I SRTSPPP
SEQ ID NO: 86 SRTSPPP-NH2
SEQ ID NO: 87 SSTSPRPPSS
SEQ ID NO: 88 SSTSPRPPSS-NH2
SEQ ID NO: 89 ~ SRTSPPPK(W)


SEQE>NO:90 I SRTSPPPK(W)-NH2
SEQIDN0:91 SRTSPPPC
SEQIDNO:92 '. SRTSPPPC-NH2
The VPAC2 receptor peptide agonists of the present invention have the advantage that they have enhanced selectivity, potency and/or stability over known VPAC2 receptor peptide agonists. In particular, the addition of the C-tenninal sequence of Exendin-4, or a variant of this C-teraiinal sequence, as the c-capping sequence surprisingly increased the VPAC2 receptor selectivity as well as increasing proteolytic stability.
The covalent attachment of one or more molecules of PEG to particular residues of a VPAC2 receptor peptide agonist results in a biologically active, PEGylated VPAC2 receptor peptide agonist with an extended half-life and reduced clearance when compared to that of non-PEGylated VPAC2 receptor peptide agonists.
The term "VPAC2" is used to refer to and in conjunction with the particular receptor (Lutz, et al., FEBS Lett., 458:197-203 (1999); Adamou, et al., Biochem. Biophys. Res. Commun., 209: 385-392 (1995)) that the agonists of the present invention activate. This term also is used to refer to and in conjunction with the agonists of the present invention. •
A "selective VPAC2 receptor peptide agonist" or a "VPAC2 receptor peptide agonist" of the present invention is a peptide that selectively activates the VPAC2 receptor to induce insulin secretion. Preferably, the sequence for a selective VPAC2 receptor peptide agonist of the present invention has twenty-eight to forty naturally occurring and/or non-naturally occurring amino acids and may or may not additionally comprise a C-terminal extension.
A "selective PEGylated VPAC2 receptor peptide agonist" or a "PEGylated VPAC2 receptor peptide agonist" is a selective VPAC2 receptor peptide agonist covalently attached to one or more molecules of polyethylene glycol (PEG), or a derivative thereof, wherein each PEG is attached to a cysteine or lysine amino acid, to a K(W) or K(CO(CH2)2SH), or to the carboxy terminus of a peptide.
Selective PEGylated VPAC2 receptor peptide agonists may have a C-terminal extension. The "C-terminal extension" of the present invention comprises a sequence having from one to thirteen naturally occurring or non-naturally occurring amino acids

linked to the C-terminus of the sequence at the N-terminus of the C-terminal extension via a peptide bond. Any Cys, Lys, K(W), or K(CO(CH2>2SH) residues in the C-terminal extension may be covalently attached to a PEG molecule, and/or the carboxy-terminal amino acid of the C-terminal extension may be covalently attached to a PEG molecule.
As used herein, the term "linked to" wim reference to the term C-terminal extension, includes the addition or attachment of amino acids or chemical groups directly to the C-terminus of the peptide sequence.
Optionally, the selective PEGylated VPAC2 receptor peptide agonist may also have an N-terminal modification. The term "N-terminal modification" as used herein includes die addition or attachment of amino acids or chemical groups directly to the N-terminus of a peptide and the formation of chemical groups, which incorporate the nitrogen at the N-terminus of a peptide.
The N-terminal modification may comprise the addition of one or more naturally occurring or non-naturally occurring amino acids to the VPAC2 receptor peptide agonist sequence, preferably there are not more than ten amino acids, with one amino acid being more preferred. Naturally occurring amino acids which may be added to the N-terminus include methionine and isoleucine. A modified amino acid added to the N-terminus may be D:histidine. Alternatively, the following amino acids may be added to the N-terminus: SEQ ID NO: 93 Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg, wherein.the Arg is linked to the N-terminus of the peptide agonist. Preferably, any amino acids added to the N-terminus are linked to the N-terminus by a peptide bond.
The term "linked to" as used herein, wim reference to the term N-terminal modification, includes the addition or attachment of amino acids or chemical groups directly to the N-terminus of the VPAC2 receptor agonist. The addition of the above N-terminal modifications may be achieved under normal coupling conditions for peptide bond formation.
The N-terminus of the peptide agonist may also be modified by the addition of an alkyl group (R), preferably a Ci-Cig alkyl group, to form (R)NH-.
Alternatively, the N-terminus of the peptide agonist may be modified by the addition of a group of the formula -C(0)Rl to form an amide of the formula R'C(0)NH-. The addition of a group of the formula -C(0)Rl may be achieved by reaction with an organic acid of the formula R'COOH. Modification of the N-terminus of an amino acid

sequence using acylation is demonstrated in the art (e.g. Gozes et at, J. Pharmacol Exp Ther, 273:161-167 (1995)). Addition of a group of the formula -C(0)R! may result in the formation of a urea group (see WO 01/23240, WO 2004/06839) or a carbamate group at the N-terminus. Also, the N-terminus may be modified by the addition of pyroglutamic acid, or 6-aminohexanoic acid.
The N-terminus of the peptide agonist may be modified by the addition of a group of the formula -SO2R5, to form a sulfonamide group at the N-terminus.
The N-terminus of the peptide agonist may also be modified by reacting with succinic anhydride to form a succinimide group at the N-terminus. The succinimide group incorporates the nitrogen at the N-terminus of the peptide.
The N-terminus may alternatively be modified by the addition of methionine sulfoxide, biotinyl-6-aminohexanoic acid, or -C(=NH)-NH2. The addition of -C(=NH)-NH2 is a guanidation modification, where the terminal NH2 of the N-terminal amino acid becomes -NH-C(=NH)-NH2.
Most of the sequences of die present invention, including the N-terminal modifications and the C-terrninal extensions contain the standard single letter or three letter codes for the twenty naturally occurring amino acids. The other codes used are defined as follows:,
C6 = hexanoyl
d = the D isoform (nonnaturally occurring) of the respective amino acid, e.g., dA = D-alanine, dS = D-serine, dK = D-lysine
hR = homoarginine
Aib = amino isobutyric acid
OMe = methoxy
Nle = Nor-leucine
NMe = N-methyl attached to the alpha amino group of an amino acid, e.g., NMeA = N-methyl alanine, NMeV « N-methyl valine
Om = ornithine'
K(CO(CH2)2SH) ■ e-(3'-mercaptopropionyl)-lysine
K(W) = e-(L-tryptophyl)-lysine
Abu = a-amino-n-butyric acid or 2-aminobutanoic acid
Cit = citrulline

K(Ac) = e-acetyl lysine PEG = polyethylene glycol PEG40K = 40,000 Dalton PEG molecule PEG30K = 30,000 Dalton PEG molecule PEG20K ss 20,000 Dalton PEG molecule
VIP naturally occurs as a single sequence having 28 amino acids. However, PACAP exists as either a 38 amino acid peptide (PACAP-38) or as a 27 amino acid ' peptide (PACAP-27) with an amidated carboxyl (Miyata, etaL, Biochem Biophys Res Commun, 170:643-648 (1990)). The sequences for VIP, PACAP-27, and PACAP-38 are as follows:
Peptide SeqJD Sequence
, J .
VIP SEQ ID HSDAVFTDNYTRLRKQMAVKKYLNSILN
NO: 14
PACAP-27 SEQ ID HSDGIFTDSYSRYRKQMAVKKYlAAVL-rNIfc
NO: 15
PACAP-38 SEQ ID HSDGn^TOSYSRYRKQMAVKKYLAAVLGKRYQRVKNK-
I NO: 16 | NH2
The term "naturally occurring amino acid" as used herein means the twenty amino acids coded for by the human genetic code (i.e. the twenty standard amino acids). These twenty amino acids are: Alanine, Arginine, Asparagine, Aspartic Acid, Cysteine, Glutamine, Glutamic Acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine.
Examples of "non-naturally occurring amino acids" include both synthetic amino acids and those modified by the body. These include D-amino acids, arginine-like amino acids (e.g., homoarginine), and other amino acids having an extra methylene in the side chain ("homo" amino acids), and modified amino acids (e.g norleucine, lysine (isopropyl) - wherein the side chain amine of lysine is modified by an isopropyl group). Also included are amino acids such as ornithine, amino isobutyric acid and 2-aminobutanoic acid.
"Selective" as used herein refers to a VPAC2 receptor peptide agonist with increased selectivity for the VPAC2 receptor compared to other known receptors. The


degree of selectivity is determined by a ratio of VPAC2 receptor binding affinity to VPAC1 receptor binding affinity or by a ratio of VPAC2 receptor binding affinity to PAC1 receptor binding affinity. Binding affinity is determined as described below in Example 4.
"Insulinotropic activity" refers to the ability to stimulate insulin secretion in response to elevated glucose levels, thereby causing glucose uptake by cells and decreased plasma glucose levels. Insulinotropic activity can be assessed by methods known in the art, including using experiments that measure VPAC2 receptor binding activity or receptor activation (e.g. insulin secretion by insulinoma cell lines or islets, intravenous glucose tolerance test (IVGTT), intraperitoneal glucose tolerance test (IPGTT), and oral glucose tolerance test (OGTT)). Insulinotropic activity is routinely measured in humans by measuring insulin levels or C-peptide levels. -Selective PEGylated VPAC2 receptor peptide agonists of the present invention have insulinotropic activity.
"In vitro potency" as used herein is the measure of the ability of a peptide to activate the VPAC2 receptor in a cell-based assay. In vitro potency is expressed as the "ECso" which is the effective concentration of compound that results in a 50% of maximum increase in activity in a single dose-response experiment For the purposes of the present invention, in vitro potency is determined using two different assays: DiscoveRx and Alpha Screen. See Examples 3 and 5 for further details of these assays. Whilst these assays are performed in different ways, the results demonstrate a general correlation between the two assays.
The term "plasma half-life" refers to the time in which half of the relevant molecules circulate in the plasma prior to being cleared. An alternatively used term is "elimination half-life." The term "extended" or "longer" used in the context of plasma half-life or elimination half-life indicates there is a statistically significant increase in the half-life of a PEGylated VPAC2 receptor peptide agonist relative to that of the reference molecule (e.g., the non-PEGylated form of the peptide or the native peptide) as determined under comparable conditions. The half-life reported herein is the elimination half-life; it is that which corresponds to the terminal log-linear rate of elimination. The person skilled in the art appreciates that half-life is a derived parameter mat changes as a function of both clearance and volume of distribution.

Clearance is the measure of the body's ability to eliminate a drug. As clearance decreases due, for example, to modifications to a drug, half-life would be expected to increase. However, this reciprocal relationship is exact only when mere is no change in the volume of distribution. A useful approximate relationship between the terminal log-linear half-life (t«), clearance (C), and volume of distribution (V) is given by the equation: t w« 0.693 (V/C). Clearance does not indicate how much drug is being removed but, rather, the volume of biological fluid such as blood or plasma that would have to be completely freed of drug to account for the elimination. Clearance is expressed as a volume per unit of time.
"Percent (%) sequence identity" as used herein is used to denote sequences which when aligned have similar (identical or conservatively replaced) amino acids in like positions or regions, where identical or conservatively replaced amino acids are those which do not alter the activity or function of the protein as compared to the starting protein. For example, two amino acid sequences with at least 85% identity to each other have at least 85% similar (identical or conservatively replaced residues) in a like position when aligned optimally allowing for up to 3 gaps, with the proviso mat in respect of the gaps a total of not more man 15 amino acid residues is affected.
The reference peptide used for the percentage sequence identity calculations
herein is:
"P487 I C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQSI
SEQIDNO:84 | OrnOrnGGPSSGAPPPS-NH2
Percent sequence identity may be calculated by determining the number of residues that differ between a peptide encompassed by the present invention and a reference peptide such as P487 (SEQ ID NO: 84), taking that number and dividing it by the number of amino acids in the reference peptide (e.g. 39 amino acids for P487), multiplying the result by 100, and subtracting that resulting number from 100. For example, a sequence having 39 amino acids with four amino acids that are different from P487 would have a percent (%) sequence identity of 90% (e.g. 100 - ((4 / 39) x 100)). For a sequence that is longer than 39 amino acids, the number of residues that differ from the P487 sequence will include the additional amino acids over 39 for purposes of the aforementioned calculation. For example, a sequence having 40 amino acids, with four

amino acids different from .the 39 amino acids in the P487 sequence and with one additional amino acid at the carboxy terminus which is not present in the P487 sequence, would have a total of five amino acids that differ from P487. Thus, this sequence would have a percent (%) sequence identity of 87% (e.g. 100 - ((5 / 39) x 100)). The degree of sequence identity may be determined using methods well known in the art (see, for example, Wilbur, W.J. and Lipman, D.J., Proc. Natl Acad. Set USA 80:726-730 (1983) and Myers E. and Miller W., Comput Appi Bioscu 4:11-17 (1988)). One program which may be used in determining the degree of similarity is the MegAlign Lipman-Pearson one pair method (using default parameters) which can be obtained from DNAstar Inc, 1128, Selfpark Street, Madison, Wisconsin, 53715, USA as part of the Lasergene system. Another program, which may be used, is Clustal W. This is a multiple sequence alignment package developed by Thompson et al (Nucleic Acids Research, 22(22):4673-4680(1994)) for DNA or protein sequences. This tool is useful for performing cross-species comparisons of related sequences and viewing sequence conservation. Clustal W is a general purpose multiple sequence alignment program for DNA or proteins. It produces biologically meaningful multiple sequence alignments of divergent sequences. It calculates the best match for the selected sequences, and lines them up so that the identities, similarities and differences can be seen. Evolutionary relationships can be seen via viewing Cladograms or Phylograms.
The sequence for a selective PEGylated VPAC2 receptor peptide agonist of the present invention is selective for the VPAC2 receptor and preferably has a sequence identity in the range of 60% to 70%, 60% to 65%, 65% to 70%, 70% to 80%, 70% to 75%, 75% to 80%, 80% to 90%, 80% to 85%, 85% to 90%, 90% to 97%, 90% to 95%, or 95% to 97%, with P487 (SEQ ID NO: 84). Preferably, the sequence has a sequence identity of greater than 82% with P487 (SEQ ID NO: 84). More preferably, the sequence has greater than 90% sequence identity with P487 (SEQ ID NO: 84). Even more preferably, the sequence has greater than 92% sequence identity with P487 (SEQ ID NO: 84). Yet more preferably, the sequence has greater than 95% sequence identity or 97% sequence identity with P487 (SEQ ID NO: 84).
The term "CI-CKS alkyl" as used herein means a monovalent saturated straight, branched or cyclic chain hydrocarbon radical having from 1 to 16 carbon atoms or when cyclic, having from 3 to 16 carbon atoms. Thus the term "C1-C16 alkyl" includes, for

: example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-heptyl, n-octyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The C1-C6 alkyl group may be optionally substituted with one or more substituents including, for example, aryl, C1-C6 alkoxy, -OH, halogen, -CF3 and -SH.
The term " C1-C6 alkyl" as used herein means a monovalent saturated straight, branched or cyclic chain hydrocarbon radical having from 1 to 6 carbon atoms or when cyclic, having from 3 to 6 carbon atoms. Thus the term " C1-C6 alkyl" includes, for example, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl. The C1-C6 alkyl group may be optionally substituted with one or more substituents.
The term "C2-C6 alkenyl" as used herein means a monovalent straight, branched or cyclic chain hydrocarbon radical having at least one double bond and having from 2 to 6 carbon atoms or when cyclic, having form 3 to 6 carbon atoms. Thus the term "C2-C6 alkenyl" includes vinyl, prop-2-enyl, but-3-enyl, pent-4-enyl and isopropenyl. The C2-C6 alkenyl group may be optionally substituted with one or more substituents.
The term " C2-C6 alkynyl" as used herein means a monovalent straight or branched chain hydrocarbon radical having at least one triple bond and having from 2 to 6 carbon atoms. Thus the term " C2-C6 alkynyl" includes prop-2-ynyl, but-3-ynyl and pent-4-ynyl. The C2-C6 alkynyl may be optionally substituted with one or more substituents.
The term " C1-C6alkoxy" as used herein means a monovalent unsubstituted saturated straight-chain or branched-chain hydrocarbon radical having from 1 to 6 carbon atoms linked to the point of substitution by a divalent O radical. Thus the term " C1-C6alkoxy" includes, for example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy and tert-butoxy. The C1-C6alkoxy group may be optionally substituted with one or more substituents.
The term "halo" or "halogen" means fluorine, chlorine, bromine or iodine.
The term "aryl" when used alone or as part of a group is a 5 to 10 numbered aromatic or heteroaromatic group including a phenyl group, a 5 or 6- membered monocyclic heteroaromatic group, each member of which may be optionally substituted with 1,2,3,4 or 5 substituents (depending upon the number of available substitution positions), a naphthyl group or an 8-, 9- or 10- membered bicyclic heteroaromatic group, each member of which may be optionally substituted with 1,2,3,4,5 or 6 substituents

(depending on the number of available substitution positions). Within this definition of aryl, suitable substitutions include C2-C6 alkyl, C2-C6 alkenyl, C2-C4 alkynyl, -NH2, -OH, halogen, -SH and CF3.
The term "aryl C1-C4 alkyl" as used herein means a C1-C4alkyl group substituted with an aryl. Thus the term "aryl C1-C4 alkyl" includes benzyl, 1-phenylethyl (o-methylbenzyl), 2-phenylethyl, 1-naphthalenemethyl or 2-naphthalenemethyl.
The term "naphthyl" includes 1-naphthyl, and 2-naphthyl. 1-naphthyl is preferred.
The term "benzyl" as used herein means a monovalent unsubstituted phenyl radical linked to the point of substitution by a-CH2-group.
The term "5- or 6-membered monocyclic heteroaromatic group" as used herein means a monocyclic aromatic group with a total of 5 or 6 atoms in the ring wherein from 1 to 4 of those atoms are each independently selected from N, O and S. Preferred groups have 1 or 2 atoms in the ring which are each independently selected from N, O and S. Examples of 5-membered monocyclic heteroaromatic groups include pyrrolyl (also called azolyl), furanyl, thienyl, pyrazolyl (also called lH-pyrazolyl and 1,2-diazolyl), imidazolyl, oxazolyl (also called 1,3-oxazolyl), isoxazolyl (also called 1,2-oxazolyl), thiazolyl (also called 1,3-thiazolyl), isothiazolyl (also called 1,2-thiazolyl), triazolyl, oxadiazolyl, thiadiazolyl, tetrazolyl, oxatriazolyl and thiatriazolyl. Examples of 6-membered monocyclic heteroaromatic groups include pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl and triazinyl.
The term "8-, 9- or 10-membered bicyclic heteroaromatic group" as used herein means a fused bicyclic aromatic group with a total of 8,9 or 10 atoms in the ring system wherein from 1 to 4 of those atoms are each independently selected from N, O and S. Preferred groups have from 1 to 3 atoms in the ring system which are each independently selected from N, O and S. Suitable 8-membered bicyclic heteroaromatic groups include imidazo[2,l-b][l,3]thiazolyl, thieno[3,2-b]thienyl, thieno[2,3-d][l,3]thiazolyl and thieno[2,3-d]imidazolyl. Suitable 9-membered bicyclic heteroaromatic groups include indolyl, isoindolyl, benzofuranyl (also called benzo[b]furanyl), isobenzofuranyl (also called benzo[c]furanyl), benzothienyl (also called benzo[b]thienyl), isobenzothienyl (also called benzo[c]thienyl), indazolyl, benzimidazolyl, 1,3-benzoxazolyl, 1,2-benzisoxazolyl, 2,lrbenzisoxazolyl, 1,3-benzothiazolyl, 1,2-benzoiso thiazolyl, 2,1-benzoisothiazolyl,

benzotriazolyl, 1,2,3-benzoxadiazolyl, 2,1,3-benzoxadiazolyl, 1,2,3-benzdthiadiazolyl, 2,1,3-benzothiadiazolyl, thienopyridinyl, purinyl and imidazo[l,2-a]pyridine. Suitable 10-membered bicyclic heteroaromatic groups include quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, quinoxalinyl, 1,5-naphthyridyl, 1,6-naphthyridyl, 1,7-naphthyridyl and 1,8-naphthyridyl.
The term "PEG" as used herein means a polyethylene glycol molecule. In its ' typical form, PEG is a linear polymer with terminal hydroxy! groups and has the formula HO-CH2CH2-(CH2CH20)n-CH2CH2-OH, where n is from about 8 to about 4000. The terminal hydrogen may be substituted with a protective group such as an alkyl or alkanol group. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this hydroxy group which is preferably activated to react with the peptide. There are many forms of PEG useful for the present invention. Numerous derivatives of PEG exist in the art and are suitable for use in the invention. (See, e.g., U.S. Patent Nos: 5,445,090; 5,900,461; 5,932,462; 6,436,386; 6,448,369; 6,437,025; 6,448,369; 6,495,659; 6,515,100 and 6,514,491 and Zalipsky, S. Bioconjugate Chent 6:150-165,1995). The PEG molecule covalently attached to VPAC2 receptor peptide agonists in the present invention is not intended to be limited to a particular type. The molecular weight of the PEG molecule is preferably from 500-100,000 daltons. PEG may be linear or branched. PEGylated VPAC2 receptor peptide agonists of the invention may have one, two or three PEG molecules attached to the peptide. It is more preferable that there be one or two PEG molecules per PEGylated VPAC2 receptor peptide agonist, however, when there is more than one PEG molecule per peptide molecule, it is preferred that there be no more than three. It is further contemplated that both ends of the PEG molecule may be homo- or hetero-functionalized for crosslinking two or more VPAC2 receptor peptide agonists together. Where there are two PEG molecules present, the PEG molecules will preferably each be 20,000 dalton PEG molecules or each be 30,000 dalton PEG molecules. However, PEG molecules having a different molecular weight may be used, for example, one 10,000 dalton PEG molecule and one 30,000 PEG molecule, or one 20,000 dalton PEG molecule and one 40,000 dalton PEG molecule.
In the present invention, a PEG molecule may be covalently attached to a Cys or Lys residue or to the C-terminal residue. The PEG molecule may also be covalently attached to a Trp residue which is coupled to the side chain of a Lys residue (K(W)).

Alternatively, a K(CO(CH2)2SH) group may be PEGylated to form K(CO(CH2)2S-PEG). Any Lys residue in the peptide agonist may be substituted for a K(W) or K(CO(CH2)2SH)l which may then be PEGylated. In addition, any Cys residue in the peptide agonist maybe substituted for a modified cysteine residue, for example, hC. The modified Cys residue may be covalently attached to a PEG molecule.
The term "PEGylation" as used herein means the covalent attachment of one or more PEG molecules as described above to the VPAC2 receptor peptide agonists of the present invention.
According to a preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a peptide sequence selected from SEQ ID NO: 17 to 45 and 94 to 112, and a C-terminal extension selected from: SEQ ID NO: 5, 6,7,8,9,10,11 and 12. It is especially preferred that the C-terminal extension is SEQ ID NO: 11 or SEQ ID NO: 12.
According to a more preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising a peptide sequence selected from: SEQ ID NO: 17 to 45 and 94 to 112, and a C-terminal extension selected from: SEQ ID NO: 5,6,7,8,9,10,11 and 12, and wherein the VPAC2 receptor peptide agonist further comprises a N-terminal modification, which modification is the addition of acetyl, propionyl,butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-arninohexanoic acid (6-aminocaproic acid), and -C(=NH>NH2- In this embodiment, it is more preferred mat the N-terminal modification is the addition of acetyl or hexanoyl.
According to a further preferred embodiment of the present invention, there is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid sequence of Formula 4 (SEQ ID NO: 4) and a C-terminal extension selected from: SEQ ID NO: 5,6,7,8,9,10,11 and 12, and wherein the PEGylated VPAC2 receptor peptide agonist further comprises a N-terminal modification, which modification is the addition of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid (6-aminocaproic acid), and -

C(=NH>NH2. In this embodiment, it is more preferred that the N-terminal modification is the addition of acetyl or hexanoyl.
According to a more preferred embodiment of the present invention, mere is provided a PEGylated VPAC2 receptor peptide agonist comprising an amino acid . sequence of Formula 4 (SEQ ID NO: 4), wherein Xaau is Aib, Xaazo is Aib, and Xaau, 1 Xaa2t, Xaa27 and Xaa28 are all Orn, and a C-terminal extension selected from: SEQ ID NO: 5,6,7,8,9,10,11 and 12, and wherein the PEGylated VPAC2 receptor peptide ' agonist further comprises a N-terminal modification, which modification is the addition of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid (6-aminocaproic acid), and -C(=NH)-NH2. In this embodiment, it is more preferred that Xaais is Aib, Xaa20 is Aib, Xaai2, Xaa2i, Xaa27 and Xaa2g are all Orn, Xaas is Glu, Xaa9 is Gin, and Xaaio is Tyr(OMe). It is especially preferred that Xaa^ is Aib, Xaa2o is Aib, Xaau, Xaa2i, Xaa27 and Xaa28 are all Orn, Xaag is Glu, Xaag is Gin, Xaato is Tyr(OMe), and Xaa23 and/or Xaazs is Aib
PEGylation of proteins may overcome many of the pharmacological and' toxicological/immunological problems associated with using peptides or proteins as therapeutics. However, for any individual peptide it is uncertain whether the PEGylated form of the peptide will have significant loss in bioactivity as compared to the unPEGylated form of the peptide.
Hie bioactivity of PEGylated proteins can be affected by factors such as: i) the size of die PEG molecule; ii) the particular sites of attachment; iii) the degree of modification; iv) adverse coupling conditions; v) whether a linker is used for attachment or whether the polymer is directly attached; vi) generation of harmful co-products; vii) damage inflicted by the activated polymer; or viii) retention of charge. Work performed on the PEGylation of cytokines, for example, shows the effect PEGylation may have. Depending on the coupling reaction used, polymer modification of cytokines has resulted in dramatic reductions in bioactivity [Francis, G.E., et al., (1998) PEGylation of cytokines and other therapeutic proteins and peptides: the importance of biological optimization of coupling techniques, Intl. J. Hem. 68:1-18], Maintaining the bioactivity of PEGylated peptides is even more problematic than for proteins. As peptides are smaller

than proteins, modification by PEGylation may potentially have a greater effect on bioactivity.
The VPAC2 receptor peptide agonists of the present invention are modified by the covalent attachment of one or more molecules of a PEG and generally have improved pharmacokinetic profiles due to slower proteolytic degradation and renal clearance. PEGylation will increase the apparent size of the VPAC2 receptor peptide agonists, thus reducing renal filtration and altering biodistribution. PEGylation can shield antigenic epitopes of the VPAC2 receptor peptide agonists, thus reducing reticuloendothelial clearance and recognition by the immune system and also reducing degradation by proteolytic enzymes, such as DPP-IV.
Covalent attachment of one or more molecules of PEG to a small, biologically active VPAC2 receptor peptide agonist poses the risk of adversely affecting the agonist, for example, by destabilising the inherent secondary structure and bioactive conformation and reducing bioactivity, so as to make the agonist unsuitable for use as a therapeutic. The present invention, however, is based on the finding that covalent attachment of one or more molecules of PEG to particular residues of a VPAC2 receptor, peptide agonist surprisingly results in a biologically active, PEGylated VPAC2 receptor peptide agonist with an extended half-life and reduced clearance when compared to that of non-PEGylated VPAC2 receptor peptide agonists. The compounds of the present invention include selective PEGylated VPAC2 receptor peptide agonists.
In order to determine the potential PEGylation sites in a VPAC2 receptor peptide agonist, serine scanning may be conducted. A Ser residue is substituted at a particular position in the peptide and the Ser-modified peptide is tested for potency, and selectivity. If the Ser substitution has minimal impact on potency and the Ser-modified peptide is selective for the VPAC2 receptor, the Ser residue is then substituted for a Cys or Lys residue, which serves as a direct or indirect PEGylation site. Indirect PEGylation of a residue is the PEGylation of a chemical group or residue which is bonded to the PEGylation site residue. Indirect PEGylation of Lys includes PEGylation of K(W) and K(CO(CH2)2SH).
The invention described herein provides PEGylated VPAC2 receptor peptide agonists. PEGylation can enhance the half-life of the selective VPAC2 receptor peptide agonists, resulting in PEGylated VPAC2 receptor peptide agonists with an elimination

half-life of at least one hour, preferably at least 3, 5, 1,10, IS, 20, or 24 hours and most preferably at least 48 hours. The PEGylated VPAC2 receptor peptide agonists of the present invention preferably have a clearance value of 200 ml/h/kg or less, more preferably 180, ISO, 120,100, 80,60 ml/h/kg or less and most preferably less than SO, 40 or 20 ml/h/kg.
The present invention encompasses the discovery that specific amino acids added to the C-terminus of a peptide sequence for a VPAC2 receptor peptide agonist may protect the peptide as well as may enhance activity, selectivity, and/or potency. For example, these C-terminal extensions may stabilize the helical structure of the peptide and stabilize sites located near to the C-terminus, which are prone to enzymatic cleavage. Furthermore, many of the C-terminally extended peptides disclosed herein may be more : selective for the VPAC2 receptor and can be more potent than VIP, PACAP, and other known VPAC2 receptor peptide agonists. An example of a preferred C-terminal extension is the extension peptide of Exendin-4 as the C-capping sequence. Exendin-4 is found in the salivary excretions from the Gila Monster, Helodenna Suspectum, (Eng et , al., J.Biol.Chem., 267(ll):74O2-7405 (1992)). Other examples of C-terminal extensions are the C-terminal sequences of helodermin and helospectin. Helodermin and helospectin are also found in the salivary excretions of the Gila Monster.
It has furthermore been discovered that modification of the N-terminus of the VPAC2 receptor peptide agonist may enhance potency and/or provide stability against DPP-IV cleavage.
VIP and some known VPAC2 receptor peptide agonists are susceptible to cleavage by various enzymes and, thus, have a short in vivo half-life. Various enzymatic cleavage sites in the VPAC2 receptor peptide agonists are discussed below. The cleavage sites are discussed relative to the amino acid positions in VIP (SEQ ID NO: 14), and are applicable to the sequences noted herein.
Cleavage of the peptide agonist by the enzyme dipeptidyl-peptidase-IV (DPP-IV) occurs between position 2 (serine in VIP) and position 3 (aspartic acid in VIP). The agonists of the present invention may be rendered more stable to DPP-IV cleavage in this region by the addition of a N-terminal modification. Examples of N-terminal modifications that may improve stability against DPP-IV cleavage include the addition of acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-


phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mercaptopropionyl, biotinyl-6-aminohexanoic acid, or -C(=NH2)-NH2. Preferably, the N-tennihal modification is the addition of acetyl or hexanoyl.
There are chymotrypsin cleavage sites in wild-type VIP between the amino acids 10 and 11 (tyrosine and threonine) and those at 22 and 23 (tyrosine and leucine). Making substitutions at position 10 and/or 11 and position 22 and/or 23 may increase the stability of the peptide at these sites. For example, substitution of tyrosine at position 10 and/or position 22 with Tyr(OMe) may increase stability.
There is a trypsin cleavage site between the amino acids at positions 12 and 13 of wild-type VIP. Certain amino acids render the peptide less susceptible to cleavage at this site, for example, ornithine at position 12 and amino isobutyric acid at position 13.
In wild-type VIP, and in numerous VPAC2 receptor peptide agonists known in the art, there are cleavage sites between the basic amino acids at positions 14 and 15 and between those at positions 20 and 21. The selective PEGylated VPAC2 receptor peptide agonists of the present invention may have improved proteolytic stability in-vlvo due to substitutions at these sites. The preferred substitutions at these sites are those which render the peptide less susceptible to cleavage by trypsin-like enzymes, including trypsin. For example, amino isobutyric acid at position IS, amino isobutyric acid at position 20, and ornithine at position 21 are all preferred substitutions which may lead to improved stability. The improved stability of a representative number of selective PEGylated VPAC2 receptor peptide agonists with resistance to peptidase cleavage and encompassed by the present invention is demonstrated in Example 7.
There is also a cleavage site between the amino acids at positions 25 and 26 of wild type VIP.
The region of the VPAC2 receptor peptide agonist encompassing the amino acids at positions 27,28,29,30 and 31 is also susceptible to enzyme cleavage. The addition of a C-terminal extension may render the peptide agonist more stable against neuroendopeptidase (SEP), it may also increase selectivity for the VPAC2 receptor. This region may also be attacked by trypsin-like enzymes. If that occurs, the peptide agonist may lose its C-terminal extension with the additional carboxypeptidase activity leading to an inactive form of the peptide. Preferred substitutions which may increase resistance to

cleavage in this region include ornithine at position 27, ornithine, or amino isobutyric acid at position 28 and ornithine at position 29.
In addition to selective PEGylated VPAC2 receptor peptide agonists with resistance to cleavage by various peptidases, the selective PEGylated VPAC2 peptide receptor agonists of the present invention may also encompass peptides with enhanced selectivity for the VPAC2 receptor, increased potency, and/or increased stability compared with some peptides known in the art. The increased potency and selectivity for various PEGylated VPAC2 receptor peptide agonists of the present invention is demonstrated in Examples 3,4 and 5.
Table 1 in Example 3 provides a list of selective PEGylated VPAC2 receptor peptide agonists and their corresponding in vitro potency results. Preferably, the selective PEGylated VPAC2 receptor peptide agonists of the present invention have an ECjo value less than 200 nM. More preferably, the ECJO value is less than 100 nM. Even more preferably, die EC30 value is less than SO nM. Still more preferably, the EC30 value is less. than 30 nM.
Table 2 in Example 4 provides a list of PEGylated VPAC2 receptor peptide agonists and their corresponding receptor binding results for human VPAC2, VPAC1 and PAC1. See Example 4 for further details of these assays. The degree of selectivity is determined by a ratio of VPAC2 receptor binding affinity to VPAC1 receptor binding affinity and by a ratio of VPAC2 receptor binding affinity to PAC1 receptor binding affinity. Preferably, the agonists of the present invention have a selectivity ratio where the affinity for the VPAC2 receptor is at least 50 times greater thanfor the VPAC1 and/or
for PAC1 receptors. More preferably, this affinity is at least 100 times greater for
1
VPAC2 than for VPAC1 and/or for PAC1. Even more preferably, the affinity is at least 200 times greater for VPAC2 than for VPAC1 and/or for PAC1. Still more preferably, the affinity is at least 500 times greater for VPAC2 than for VPAC1 and/or for PAC1. Yet more preferably, the ratio is at least 1000 times greater for VPAC2 than for VPAC1 and/or for PAC1.
As used herein, "selective VPAC2 receptor peptide agonists" also include pbarmaceutically acceptable salts of the agonists described herein. A selective VPAC2 receptor peptide agonist of this invention can possess a sufficiently acidic, a sufficiently basic, or both functional groups, and accordingly react with any of a number of inorganic


bases, and inorganic and organic acids, to form a salt. Acids commonly employed to form acid addition salts are inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, and the like, and organic acids such as p-toluenesulfonic acid, methanesulfonic acid, oxalic acid,p-bromophenyl-sulfonic acid, carbonic acid, succinic acid, citric acid, benzoic acid, acetic acid, trifluoroacetic acid, and the like. Examples of such salts include the sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caproate, heptanoate, propiolate, oxalate, fnalonate, succinate, suberate, sebacate, fumarate, maleate, butyne-l,4-dioate, hexyne-l,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, phthalate, sulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, gamma-hydroxybutyrate, glycolate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate, mandelate, and the like.
Base addition salts include those derived from inorganic bases, such as ammonium or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, and the like. Such bases useful in preparing the salts of this invention thus include sodium hydroxide, potassium hydroxide, ammonium hydroxide, potassium carbonate, and the like.
The selective PEOylated VPAC2 receptor peptide agonists of the present invention are preferably formulated as pharmaceutical compositions. Standard pharmaceutical formulation techniques may be employed such as those described in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be formulated for administration through the buccal, topical, oral, transdermal, nasal, or pulmonary route, or for parenteral administration.
Parenteral administration can include, for example, systemic administration, such as by intramuscular, intravenous, subcutaneous, intradermal, or intraperitoneal injection. The selective PEGylated VPAC2 receptor peptide agonists can be administered to the subject in conjunction with an acceptable pharmaceutical carrier, diluent, or excipient as part of a pharmaceutical composition for treating NIDDM, or

the disorders discussed below. The pharmaceutical composition can be a solution or, if administered parenterally, a suspension of the PEGylated VPAC2 receptor peptide agonist or a suspension of the PEGylated VPAC2 receptor peptide agonist complexed with a divalent metal cation such as zinc. Suitable pharmaceutical carriers may contain inert ingredients which do not interact with the peptide or peptide derivative. Suitable pharmaceutical carriers for parenteral administration include, for example, sterile water, physiological saline, bacteriostatic saline (saline containing about 0.9% . mg/ml benzyl alcohol); phosphate-buffered saline, Hank's solution, Ringer's-lactate and the like. Some examples of suitable excipients include lactose, dextrose, sucrose, trehalose, sorbitol, and mannitol.
The PEGylated VPAC2 receptor peptide agonists of the invention may be formulated for administration such that blood plasma levels are maintained in the efficacious range for extended time periods. The main barrier to effective oral peptide drug delivery is poor bioavailability due to degradation of peptides by acids and enzymes, poor absorption through epithelial membranes, and transition of peptides to an insoluble form after exposure to the acidic pH environment in the digestive tract. Oral delivery systems for peptides such as those encompassed by the present invention are known in the art. For example, PEGylated VPAC2 receptor peptide agonists can be encapsulated using microspheres and men delivered orally. For example, PEGylated VPAC2 receptor peptide agonists can be encapsulated into microspheres composed of a commercially available, biocompatible, biodegradable polymer, poly(lactide-co-glycolide)-COOH and olive oil as a filler (see Joseph, et al. Diabetologia 43:1319-1328 (2000)). Other types of microsphere technology is also available commercially such as Medisorb® and Prolease® biodegradable polymers from Alkermes. Medisorb® polymers can be produced with any of the lactide isomers. Lactide:glycolide ratios can be varied between 0:100 and 100:0 allowing for a broad range of polymer properties. This allows for the design of delivery systems and implantable devices with resorption times ranging from weeks to mondis. Emisphere has also published numerous articles discussing oral delivery technology for peptides and proteins. For example, see WO 95/28838 by Leone-bay et al. which discloses specific carriers comprised of modified amino acids to facilitate-absorption.

The selective PEGylated VPAC2 receptor peptide agonists described herein can be used to treat subjects with a wide variety of diseases and conditions. Agonists encompassed by the present invention exert their biological effects by acting at a receptor referred to as the VPAC2 receptor. Subjects with diseases and/or conditions that respond favourably to VPAC2 receptor stimulation or to the administration of VPAC2 receptor peptide agonists can therefore'be treated with the PEGylated VPAC2 agonists of the present invention. These subjects are said to "be in need of treatment with VPAC2 agonists" or "in need of VPAC2 receptor stimulation".
The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be employed to treat diabetes, including both type 1 and type 2 diabetes (non-insulin dependent diabetes mellitus or NIDDM). The agonists may also be used to treat subjects requiring prophylactic treatment with a VPAC2 receptor agonist, e.g., subjects at risk for developing NIDDM. Such treatment may also delay the onset of diabetes and diabetic complications. Additional subjects which may be treated with the agonists of the present invention include those with impaired glucose tolerance (IGT) (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5,1999), or impaired fasting glucose (IFG) (Charles, et al., Diabetes 40:796,1991), subjects whose body weight is about 25% above normal body weight for the subject's height and body build, subjects having one or more parents with NIDDM, subjects who have had gestational diabetes, and subjects with metabolic disorders such as those resulting from decreased endogenous insulin secretion. The selective PEGylated VPAC2 receptor peptide agonists may be used to prevent subjects with impaired glucose tolerance from proceeding to develop NIDDM, prevent pancreatic p-cell deterioration, induce P-cell proliferation, improve p-cell function, activate dormant P-cells, differentiate cells into P-cells, stimulate P-cell replication, and inhibit p-cell apoptosis. Other diseases and conditions that may be treated or prevented using agonists of the invention in methods of the. invention include: Maturity-Onset Diabetes of the Young (MODY) (Herman, et al., Diabetes 43:40,1994); Latent Autoimmune Diabetes Adult (LADA) (Zimmet, et al., Diabetes Med. 11:299,1994); gestational diabetes QAttzget, Diabetes, 40:197,1991); metabolic syndrome X, dyslipidemia, hyperglycemia, hyperinsulinemia, hypertriglyceridemia, and insulin resistance.

The selective PEGylated VPAC2 receptor peptide agonists of the invention may also be used to treat secondary causes of diabetes (Expert Committee on Classification of Diabetes Mellitus, Diabetes Care 22 (Supp. 1):S5,1999). Such secondary causes include glucocorticoid excess, growth hormone excess, pheochromocytoma, and drug-induced diabetes. Drugs that may induce diabetes include, but are not limited to, pyriminil, nicotinic acid, glucocorticoids, phenytoin, thyroid hormone, (^adrenergic agents, o> interferon and drugs used to treat HTV infection.
The selective PEGylated VPAC2 receptor peptide agonists of the present invention may be effective in the suppression of food intake and the treatment of obesity.
The selective PEGylated VPAC2 receptor peptide agonists of the present invention may also be effective in the prevention or treatment of such disorders as atherosclerotic disease, hyperlipidemia, hypercholesteremia, low HDL levels, hypertension, primary pulmonary hypertension, cardiovascular disease (including atherosclerosis, coronary heart disease and coronary artery disease), cerebrovascular . disease and peripheral vessel disease; and for the treatment of lupus, polycystic ovary syndrome, carcinogenesis, and hyperplasia, male and female reproduction problems, ' sexual disorders, ulcers, sleep disorders, disorders of lipid and carbohydrate metabolism, circadian dysfunction, growth disorders, disorders of energy homeostasis, immune diseases including autoimmune diseases (e.g., systemic lupus erythematosus), as well as acute and chronic inflammatory diseases, rheumatoid arthritis, and septic shock.
The selective PEGylated VPAC2 receptor peptide agonists of die present invention may also be useful for treating physiological disorders related to, for example, cell differentiation to produce lipid accumulating cells, regulation of insulin sensitivity and blood glucose levels, which are involved in, for example, abnormal pancreatic P-cell function, insulin secreting tumors and/or autoimmune hypoglycemia due to autoantibodies to insulin, autoantibodies to the insulin receptor, or autoantibodies that are stimulatory to pancreatic P -cells, macrophage differentiation which leads to the formation of atherosclerotic plaques, inflammatory response, carcinogenesis, hyperplasia, adipocyte gene expression, adipocyte differentiation, reduction in the pancreatic P -cell mass, insulin secretion, tissue sensitivity to insulin, liposarcoma cell growth, polycystic ovarian disease, chronic anovulation, hyperandrogenism, progesterone production, steroidogenesis, redox potential and oxidative stress in cells, nitric oxide synthase (NOS)

production, increased gamma glutamyl transpeptidase, catalase, plasma triglycerides, HDL, and LDL cholesterol levels, and the like.
In addition, the selective PEGylated VPAC2 receptor peptide agonists of the invention may be used for treatment of asthma (Bolin, et al., Biopolymer 37:57-66 (1995); U.S. Patent No. 5,677,419; showing that polypeptide R3PO is active in relaxing guinea pig tracheal smooth muscle); hypotension induction (VIP induces hypotension, tachycardia, and facial flushing in asthmatic patients (Morice, et al., Peptides 7:279-280 (1986); Morice, et al., Lancet 2:1225-1227 (1983)); for the treatment of male reproduction problems (Siow, et al., Arch. Androl. 43(1):67-71 (1999)); as an anti-apoptosis/neuroprotective agent (Brenneman, et al., Ann. N. Y. Acad. Sci. 865:207-12 (1998)); for cardioprotection during ischemic events ( Kalfin, et al., /. Pharmacol Exp. Ther. 1268(2):952-8 (1994); Das, et al., Ann. N. Y. Acad Sci. 865:297-308 (1998)); for manipulation of the circadian clock and its associated disorders (Hamar, et al., Cell 109:497-508 (2002); Shen, et al., Proc. Natl Acad. Sci. 97:11575-80, (2000)); and as an anti-ulcer agent (Tuncel, et al., Ann. N. Y. Acad Sci. 865:309-22, (1998)).
An "effective amount" of a selective PEGylated VPAC2 receptor peptide agonist is the quantity that results in a desired therapeutic and/or prophylactic effect without causing unacceptable side effects when administered to a subject in need of VPAC2 receptor stimulation. A "desired therapeutic effect" includes one or more of the following: 1) an amelioration of the symptom(s) associated with the disease or condition; 2) a delay in the onset of symptoms associated with the disease or condition; 3) increased longevity compared with the absence of the treatment; and 4) greater quality of life compared with the absence of the treatment. For example, an "effective amount" of a PEGylated VPAC2 agonist for the treatment of NIDDM is the quantity that would result in greater control of blood glucose concentration than in the absence of treatment, thereby resulting in a delay in the onset of diabetic complications such as retinopathy, neuropathy, or kidney disease. An "effective amount" of a selective PEGylated VPAC2 receptor peptide agonist for the prevention of NIDDM is the quantity that would delay, compared with the absence of treatment, the onset of elevated blood glucose levels that require treatment with anti-hypoglycemic drugs such as sulfonylureas, thiazolidinediones, insulin, and/or bisguanidines.

An "effective amount" of the selective PEGylated VPAC2 receptor peptide agonist administered to a subject will also depend on the type and severity of the disease and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The dose of selective PEGylated VPAC2 peptide receptor agonist ■ effective to normalize a patient's blood glucose will depend on a number of factors, among which are included, without limitation, the subject's sex, weight and age, the severity of inability to regulate blood glucose, the route of administration and bioavailability, the pharmacokinetic profile of the peptide, the potency, and the formulation.
A typical dose range for the selective PEGylated VPAC2 receptor peptide agonists of the present invention will range from about 1 ug per day to about 5000 ug per day. Preferably, the dose ranges from about 1 ug per day to about 2S00 |ig per day, more preferably from about 1 ug per day to about 1000 ug per day. Even more preferably, the dose ranges from about 5 ug per day to about 100 ug per day. A further preferred dose range is from about 10 ug per day to about SO ug per day. Most preferably, the dose is about 20 jig per day.
A "subject" is a mammal, preferably a human, but can also be an animal, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs,'and the like).
The selective PEGylated VPAC2 receptor peptide agonists of the present invention can-be prepared by using standard methods of solid-phase peptide synthesis techniques. Peptide synthesizers are commercially available from, for example, Rainin-: PTI Symphony Peptide Synthesizer (Tucson, AZ). Reagents for solid phase synthesis are commercially available, for example, from Glycopep (Chicago, JL). Solid phase peptide synthesizers can be used according to manufacturers instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, decoupling, and capping of unreacted amino acids.
Typically, an oc-JV-protected amino acid and the ^-terminal amino acid on the growing peptide chain on a resin is coupled at room temperature in an inert solvent such as dimethylformamide, N-methylpyrrolidone or methylene chloride in the presence of coupling agents such as dicyclohexylcarbodiimide and 1-hydroxybenzotriazole and a base

such as diisopropylethylamine. The a-JV-protecting group is removed from the resulting peptide resin using a reagent such as trifluoroacetic acid or piperidine, and the coupling reaction repeated with the next desired H-protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in die art and are described, for example, in Green and Wuts, "Protecting Groups in Organic Synthesis", John Wiley and Sons, 1991. Examples include t-butyloxycarbonyl (tBoc) and fluorenylmethoxycarbonyl (Fmoc).
The selective VPAC2 receptor peptide agonists may also be synthesized using standard automated solid-phase synthesis protocols using t-butoxycarbonyl- or fluorenylrnethoxycarbonyl-alpha-amino acids with appropriate side-chain protection. After completion of synthesis, modification of the N-terminus may be accomplished by reacting the a-amino group with, for example: (i) active esters (using similar protocols as described above for the introduction of an a-JV-protected amino acid); (ii) aldehydes in the presence of a reducing agent (reductive amination procedure); and (iii) guanidation reagents. Then, peptides are cleaved from the solid-phase support with simultaneous side-chain deprotection using standard hydrogen fluoride methods or trifluoroacetic acid (TFA). Crude peptides are then further purified using Reversed-Phase Chromatography on VYDAC C18 columns using acetonitrile gradients in 0.1% TFA. To remove acetonitrile, peptides are lyophilized from a solution containing 0.1 % TFA, acetonitrile and water. Purity can be verified by analytical reversed phase chromatography. Identity of peptides can be verified by mass spectrometry. Peptides can be solubilized in aqueous buffers at neutral pH.
The peptide agonists of the present invention may also be made by recombinant methods known in the art using bom eukaryotic and prokaryotic cellular hosts.
Once a peptide is prepared and purified, it is modified by covalently linking at least one PEG molecule to Cys or Lys residues, to K(W) or K(CO(CH2)2SH), or to die carboxy-terminal amino acid. A wide variety of methods have been described in the art to produce peptides covalently conjugated to PEG and the specific method used for the present invention is not intended to be limiting (for review article see, Roberts, M. et al. Advanced Drug Delivery Reviews, 54:459-476,2002).
An example of a PEG molecule which may be used is methoxy-PEG2-MAL-40K, a bifurcated PEG maleimide (Nektar, Huntsville, Alabama). Omer examples include, but

are not limited to bulk mPEG-SBA-20K (Nektar), mPEG2-ALD-40K (Nektar) and methoxy-PEG-MAL-30K (Dow).
Carboxy-terminal attachment of PEG may be attached via enzymatic coupling using recombinant VPAC2 receptor peptide agonist as a precursor or alternative methods known in the art and described, for example, in U.S. Patent 4,343,898 or Intl. J. Pept. & Prot. Res. 43:127-38 (1994).
One method for preparing the PEGylated VPAC2 receptor peptide agonists of the present invention involves the use of PEG-maleimide to directly attach PEG to a thiol group of the peptide. The introduction of a thiol functionality can be achieved by adding or inserting a Cys or hC residue onto or into the peptide at positions described above. A thiol functionality can also be introduced onto the side-chain of the peptide (e.g. acylation > of lysine e-amino group by a thiol-containing acid, such as mercaptopropionic acid). A PEGylation process of the present invention utilizes Michael addition to form a stable , thioether linker. The reaction is highly specific and takes place under mild conditions in the presence of other functional groups. PEG maleimide has been used as a reactive polymer for preparing well-defined, bioactive PEG-protein conjugates. It is preferable that the procedure uses a molar excess, preferably from 1 to 10 molar excess, of a thiol-containing VPAC2 receptor peptide agonist relative to PEG maleimide to drive the reaction to completion. The reactions are preferably performed between pH 4.0 and 9.0 at room temperature for 10 minutes to 40 hours. The excess of unPEGylated thiol-containing peptide is readily separated from the PEGylated product by conventional separation methods. The PEGylated VPAC2 receptor peptide agonist is preferably isolated using reverse-phase HPLC or size exclusion chromatography. Specific conditions required for PEGylation of VPAC2 receptor peptide agonists are set forth in Example 8. Cysteine PEGylation may be performed using PEG maleimide or bifurcated PEG maleimide.
An alternative method for preparing the PEGylated VPAC2 receptor peptide ' agonists of the invention, involves PEGylating a lysine residue using a PEG-succinimidyl derivative. In order to achieve site specific PEGylation, the Lys residues which are not used for PEGylation are substituted for Arg residues.
Another approach for PEGylation is via Pictet-Spengler reaction. A Tip residue with its free amine is needed to incorporate the PEG molecule onto a VPAC2 receptor

selective peptide. One approach to achieve this is to site specifically introduce a Tip residue onto the amine of a Lys sidechain via an amide bond during the solid phase synthesis (see Example 10).
Various preferred features and embodiments of the present invention will now be described only by way of the examples with reference to the following figure in which:-Figure 1 shows the enhancement of the insulin response to the i.v. glucose challenge in animals treated with P505 or P52S 24h prior to the glucose injection. The insulin respons Example 1 - Preparation of the Selective VPAC2 Receptor Peptide Agonists bv Solid Phase t-Boc Chemistry:
Approximately 0.5-0.6 grams (0.38-0.45 mmole) Boc Ser(Bzl)-PAM resin is placed in a standard 60 mL reaction vessel. Double couplings are run on an Applied Biosystems ABI430A peptide synthesizer.
The following side-chain protected amino acids (2 mmole cartridges of Boc amine acids) are obtained from Midwest Biotech (Fishers, IN) and are used in the synthesis:
Arg-Tosyl (TOS), Asp-5-cyclohexyl ester (OcHx), Glu-5-cycohexyl ester (OcHx), His-benzyloxymethyl(BOM), Lys-2-chlorobenzyloxycarbonyl (2CI-Z), Ser-O-benzyl ether (OBzl), Thr-O-benzyl ether (OBzl), Trp-formyl (CHO) and Tyr-2-bromobenzyloxycarbonyl (2Br-Z).
Trifluoroacetic acid (TFA), di-isopropylethylamine (DEEA), 0.5 M hydroxybenzotriazole (HOBt) in DMF and 0.5 M dicyclohexylcarbodiimide (DCC) in dichloromethane are purchased from PE-Applied Biosystems (Foster City, CA). Dimethylformamide (DMF-Burdick and Jackson) and dichloromethane (DCM-Mallinkrodt) is purchased from Mays Chemical Co. (Indianapolis, IN)- •
Standard double couplings are run using either symmetric anhydride or HOBt esters, both formed using DCC. At the completion of the syntheses, the N-terminal Boc group is removed and the peptidyl resins are treated with 20% piperidine in DMF to deformylate the Trp side chain if Trp is present in me sequence. For the N-terminal acylation, four-fold excess of symmetric anhydride of the corresponding acid is added onto the peptide resin. The symmetric anhydride is prepared by diisopropylcarbodiimde (DIC) activation in DCM. The reaction is allowed to proceed for 4 hours and monitored

by ninhydrin test. After washing with DCM, the resins are transferred to a TEFLON reaction vessel and are dried in vacuo.
Cleavages are done by attaching the reaction vessels to a HF (hydrofluoric acid) apparatus (Penninsula Laboratories). 1 mL m-cresol per gram/resin is added and 10 mL HF (purchased from AGA, Indianapolis, IN) is condensed into the pre-cooled vessel. 1 mL DMS per gram resin is added when methionine is present. The reactions are stirred one hour in an ice bath. The HF is removed in vacuo. The residues are suspended in ethyl ether. The solids are filtered and are washed with ether. Each peptide is extracted into aqueous acetic acid and either is freeze dried or is loaded directly onto a reverse- phase column.
Purifications are run on a 2.2 x 25cm VYDAC C18 column in buffer A (0.1% TFA in water). A gradient of 20% to 90% B (0.1% TFA in acetonitrile) is run on an HPLC (Waters) over 120 minutes at 10 mL/minute while monitoring the UV at 280 run (4.0 A) and collecting one minute fractions. Appropriate fractions are combined, frozen and lyophilizei Dried products are analyzed by HPLC (0.46 x 15 cm METASIL AQ C18) and MALDI mass spectrometry.
Example 2 - Preparation of the Selective VPAC2 Receptor Peptide Agonists by Solid Phase FMoc Chemistry:
Approximately 114 mg (50 mMole) FMOC Ser(tBu) WANG resin (purchased from GlycoPep, Chicago, IL) is placed in each reaction vessel. The synthesis is conducted on a Rainin Symphony Peptide Synthesizer. Analogs with a C-terminal amide are prepared using 75 mg (50 umole) Rink Amide AM resin (Rapp Polymere. Tuebingen, Germany).
The following FMOC amino acids are purchased from GlycoPep (Chicago, IL), and NovaBiochem (La Jolla, CA): Arg-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), Asn-trityl (Trt), Asp-p-t-Butyl ester (tBu), Glu-5-t-butyl ester (tBu), Gln-trityl (Trt), His-trityl (Trt), Lys-t-butyloxycarbonyl (Boc), Ser-t-butyl ether (OtBu), Thr-t-butyl ether (OtBu), Trp-t-butyloxycarbonyl (Boc), Tyr-'t-butyl ether (OtBu).

Solvents dimethylformamide (DMF-Burdick and Jackson), N-methyl pyrrolidone (NMP-Burdick and Jackson), dichloromethane (DCM-Mallinkrodt) are purchased from Mays Chemical Co. (Indianapolis, IN).
Hydroxybenzotrizole (HOBt), di-isopropylcarbodiimde (DIC), di-
isopropylethylamine (DIEA), and piperidine (Pip) are purchased from Aldrich Chemical
Co (Milwaukee, WI).
All amino acids, are dissolved in 0.3 M in DMF. Three hour DIC/HOBt activated couplings are run after 20 minutes deprotection using 20% Pip/DMF. Each resin is washed with DMF after deprotections and couplings. After the last coupling and deprotection, the peptidyl resins are washed with DCM and are dried in vacuo in the reaction vessel. For the N-terminal acylation, four-fold excess of symmetric anhydride of the corresponding acid is added onto the peptide resin. The symmetric anhydride is prepared by DIC activation in DCM. The reaction is allowed to proceed for 4 hours and monitored by ninhydrin test. The peptide resin is then washed with DCM and dried in vacuo.
The cleavage reaction is mixed for 2 hours with a cleavage cocktail consisting of 0.2 mL thioanisole, 0.2 mL methanol, 0.4 mL triisopropylsilane, per 10 mL TFA, all purchased from Aldrich Chemical Co., Milwaukee, WI. If Cys is present, in the sequence, 2% of ethanedithiol is added. The TFA filtrates are added to 40 mL ethyl ether. The precipitants are centrifuged 2 minutes at 2000 rpm. The supernatants are decanted. The pellets are resuspended in 40 mL ether, re-centrifuged, re-decanted, dried under nitrogen and then in vacuo.
0.3-0.6 mg of each product is dissolved in 1 mL 0.1% TFA/acetonitrile(ACN), with 20 uL being analyzed on HPLC [0.46 x 15cm METASIL AQ C18, lmL/min, 45°C, 214 nM (0.2A), A=0.1%TFA, B=0.1%TFA/50%ACN. Gradient = 50% B to 90% B over 30 minutes].
Purifications are run on a 2.2 x 25 cm VYDAC C18 column in buffer A (0.1% TFA in water). A gradient of 20% to 90% B (0.1 % TFA in acetonitrile) is run on an HPLC (Waters) over 120 minutes at 10 mL/minute while monitoring the UV at 280 nm (4.0A) and collecting 1 minute fractions. Appropriate fractions are combined, frozen and lyophilized. Dried products are analyzed by HPLC (0.46 x 15 cm METASIL AQ C18) and MALDI mass spectrometry.

Precursor to P521 (CfrHSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY AibQSIOrnOrnGGPSSGAPPPCC-NH2): Synthesis is carried out using the FMoc protocols described above. The peptide is characterised by analytical HPLC: to = 10.9 rain, HPLC conditions as described above, and MALDI-TOF: calculated m/z = 4290.0, measured m/z = 4290.8 [M+H+]. After purification using reversed-phase preparative HPLC, pure fractions are combined and lyophilised: 14.8 mg is obtained as a final lyophilised powder.
Precursor to P525 (C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQ AibIOrnOrnGGPSSGAPPPCC-NH2): As described for the precursor to P521. Analytical HPLC: IR = 11.0 min. MALDI-TOF: calculated m/z = 4288.0, measured m/z = 4288.8 [M+H4]. 18.7 mg is obtained as a final lyophilised powder. Precursor to P574 (C6-HSDAVFTEQY(OMe)TOniLRAibQLAAbuAibOrn Y(OMe)LQAibIOrnOrnGGPSSGAPPPCC-NH2): As described for the precursor to P521. Analytical HPLC: tR= 11.7 min. MALDI-TOF: calculated m/z = 4330.1, measured m/z = 4330.7 [M+H*]. 46.2 mg is obtained as a final lyophilised powder.
Example 3 - In-vitro Potency at Hiinyin VPAC2 Receptors:
Alpha screen: Cells are washed in the culture flask once with PBS. Then, the cells are rinsed with enzyme free dissociation buffer. The dissociated cells are removed. The cells are then spun down and washed in stimulation buffer. For each data point, 50,000 cells suspended in stimulation buffer are used. To this buffer. Alpha screen acceptor beads are added along with the stimuli. This mixture is incubated for 60 minutes. Lysis buffer and Alpha screen donor beads are added and are incubated for 60 to 120 minutes. The Alpha screen signal (indicative of intracellular cAMP levels) is read in a suitable instrument (e.g. AlphaQuest from Perkin-Elmer). Steps including Alpha screen donor and acceptor beads are performed in reduced light. The EC50 for cAMP generation is calculated from the raw signal or is based on absolute cAMP levels as determined by a standard curve performed on each plate.
Results for each agonist are, at minimum, from two analyses performed in a single run. For some agonists, the results are the mean of more than one run. The tested peptide concentrations are: 10000,1000,100,10,3,1,0.1,0.01,0.003,0.001,0.0001 and 0.00001 nM.

DiscoveRx: A CHO-S cell line stably expressing human VPAC2 receptor in a 96-well microtiter plate is seeded with 50,000 cells/well the day before the assay. The cells are allowed to attach for 24 hours in 200 uL culture medium. On the day of the experiment, the medium is removed. Also, the cells are washed twice. The cells are incubated in assay buffer plus BMX for IS minutes at room temperature. Afterwards, the stimuli are added and are dissolved in assay buffer. The stimuli are present for 30 minutes. Then, the assay buffer is gently removed. The cell lysis reagent of the DiscoveRx cAMP kit is added. Thereafter, the standard protocol for developing the cAMP signal as described by the manufacturer is used (DiscoveRx Inc., USA). EC50
values for cAMP generation are calculated from the raw signal or are based on absolute cAMP levels as determined by a standard curve performed on each plate.
Results for each agonist are the mean of two independent runs. The typically tested concentrations of peptide are: 1000,300,100,10,1,0.3,0.1,0.01,0.001,0.0001 andOnM.
The activity (EC50 (nM)) for the human VPAC2 receptor is reported in Table 1 for
the different assay formats.
Table 1. Peptide potency at human VPAC2 receptors
Agonist # Human VPAC2 Receptor; I Human VPAC2 Receptor
Alphascreen DiscoveRx
(EC5o;nM) (EC50;nM)
VIP tO 07
PACAP-27 ■ 2.3 OJ
P410 128JJ
P417 14J
P451 68;6
P4S4 59L9
P460 430.2
P470 833 .
P472 1233
P473 26J
P47S 1093
P478 4JW
P483 33J
P48S 44k5 ; .
P490 115^6
P492 509.6
P495 [ 116.7 I

, ,
P497 I 176.1
P499 57/7
P501 1309
P503 146JJ
PSOS 405 .
P507 2T1
PS09 13J) .
P511 T6
P513 28JJ
P517 7J
P519 30.3 .
PS21 22jl
PS23 5j$
PS25 25J)
PS29 1705
PS31 94^6
PS33 2382.0
P535 9JW
P537 32X)
PS41 45J
P54S 45.0
PS47 62X1
P539 107 473
P543 842 8£
P5S1 15J
PS53 15X1
P555 22J 8,1
P557 _. 1083
P560 13J 11.3
P562 302 24^2
P566 11.8 JhS
P568 128J>
P570 134 4.5
P572 1L2
P574 2L2 3JU
P576 55.0
P578 2053
P580 109
P582 203
P584 5L2
P586 1224 .
P588 54X>
P590 4J
P602 I 42.1 I 7.2

Example 4 - Selectivity:
Binding assays: Membrane prepared from a stable VPAC2 cell line (see Example
3) or from cells transiently transfected with human VPAC1 or PAC1 are used. A filter
binding assay is performed using 1251-labeled VIP for VPAC1 and VPAC2 and 1251-
labeled PACAP-27 for PAC1 as the tracers.
For this assay, the solutions and equipment include:
Presoak solution: 0.5 % Polyethyleneamine in Aqua dest
Buffer for flushing filter plates: 25 mM HEPES pH 7.4
Blocking buffer: 25 mM HEPES pH 7:4; 0.2 % protease free BSA"
Assay buffer 25 mM HEPES pH 7.4; 0.5 % protease free BSA
Dilution and assay plate: PS-Microplate, U form
Filtration Plate: Multiscreen FB Opaque Plate; 1.0 uM Type B Glasfiber filter
In order to prepare the filter plates, the presoak solution is aspirated by vacuum filtration. The plates are flushed twice with 200 uL flush buffer. 200 uL blocking buffer is added to the filter plate. The filter plate is then incubated with 200 uL presoak solution for 1 hour at room temperature.
The assay plate is filled with 25 uL assay buffer, 25 uL membranes (2.5 ug) suspended in assay buffer, 25 uL agonist in assay buffer, and 25 uL tracer (about 40000 cpm) in assay buffer. The filled plate is incubated for 1 hour with shaking.
The transfer from assay plate to filter plate is conducted. The blocking buffer is aspirated by vacuum filtration and washed two times with flush buffer. 90 uL is transferred from the assay plate to the filter plate. The 90 uL transferred from assay plate is aspirated and washed three times with 200 uL flush buffer. The plastic support is removed. It is dried for 1 hour at 60 °C. 30 uL Microscint is added. The count is performed.
The receptor binding (ICso) for human VPAC2, VPAC1 and PAC1 is reported in
Table 2.
Tabie 2.
Agonist # Human Human Human
1 VPAC2 | VPAC1 1 PAC1

,
Receptor Receptor Receptor
Binding Binding Binding
(IC50;nM) qC50;nM) (IC50;nM)
VIP SA. 3.3 >10Q0
PACAP-27 2.6 4.5 10.2
P410 264.4 >25000
P417 24.1 >250Q0
P451 595.7
P454 36.6 >25000
P460 914.5 >25000
P470 262.9
P473 256.4 >25000
P47S 436.0
P478 30.0 -
P483 37.3 >25000
P485 17.3
P490 13.2
P492 156.0
P495 142.1
P497 146.0
P499 34.0 >25000
P501 111.0
P503 41.5
P505 26.1 >25000 >25Q00
P509 11.2 >25000
P511 7.2 >25000 >25000
P513 50.5
P517 3.7 >25000
. P519 51.6
P521 67.1
\ P523 4.8
P525 41.8 >25000 >25000
P529 192.4 .
P531 69.7
P533 959.0
, P535 145.4 >25000
P537 17.2 >25000 >25000
P539 13.5
P541 59.8 >25000
■ P543 56.3
P545 79.9 >25000 >25000
P547 5.6
P551 16.6 >25000 >25000
P553 21.7 >25000 >25000
P555 15.4 >25000 >25000
P557 I 380.0 1 1

• , -jr
P560 I 11.6 I 1
P562 42.8 >25000 >25000
P566 8.2
P570 4.1 >25000 >25000
P572 14.9 >25000 >25000
P574 32.3 >25000 >25000
P580 20.7 >25000 >25000
P582 48.3 >2S000 >25000
P590 — 7.4' >2S000 >25000
P602 1 28.0 I >25000 | >25000
Example 5 - In vitro potency at rat VPAC1 and VPAC2 receptors:
DiscoveRx: CHO-PO cells are transiently transfected with rat VPACi or VPAC2 receptor DNA using commercially available transfection reagents (Lipofectamine from Invitrogen). The cells are seeded at a density of 10,000/well in a 96-well plate and are allowed to grow for 3 days in 200ml culture medium At day 3, the assay is performed.
On the day of the experiment, the medium is removed. Also, the cells are washed twice. The cells are incubated in assay buffer plus EBMX for IS minutes at room temperature. Afterwards, the stimuli are added and are dissolved in assay buffer. The stimuli are present for 30 minutes. Then, the assay buffer is gently removed. The cell lysis reagent of the DiscoveRx cAMP kit is added. Thereafter, the standard protocol for developing the cAMP signal as described by the manufacturer is used (DiscoveRx Inc., USA). ECjo values for cAMP generation are calculated from the raw signal or are based on absolute cAMP levels as determined by a standard curve performed on each plate.
Results for each agonist are die mean of two independent runs. Rat VPACI and VPAC2 results are only generated using the DiscoveRx assay. The typically tested concentrations of peptide are: 1000,300,100,10,1,0.3,0.1,0.01,0.001,0.0001 and 0 nM.
The activity (ECso(nM)) for the rat VPAC2 and VPACI receptors is reported in
Table 3.
Table 3
Agonist # I Rat I Rat VPACI VPAC2 Receptor Receptor. DiscoveRx: DiscoveRx:
(EC50;nM) (ECso;nM)
VIP [ 0.01 I 0-6

P410 I >100Q I 318.5
P417 232.3 8.7
P454 1229.5 79.6
P460 >1000 . 1163.1
P470 >1000 110.9
P472 >1000 284.6
P473 >,1000 25.5
P475 >1000 220.4
P478 2053.8 120.1
P483 223.9 128.4
P485 734.9 42.5
P490 >1000 191.8
P492 >4000 962.7
P497 >1000 344.7
P499 803.8 68.2
P501 >1000 272.1
P503 >4000 520.3
P505 639.4 42.6
P507 >1000 94.9
P509 328.4 28.2
P511 61.9 13.9
P517 92.4 10.0
P521 602.4 33.1
P523 20.4 5.3
P525 1137.3 44.0
P529 1182.9 199.3
P531 300.7 69.9
P535 >1000 317.6
P537 205.8 38.8
P539 32.6 21.0
P541 665.1 101.9
P543 355.0 101.1
P545 380.5 99.5
P547 76.2 149.8
P551 171.9 13.2
P553 261.8 22.0
P555 68.9 32.5
P557 581.1 397.2
P560 155.6 20.4
P562 393.4 52.5
P566 52.7 13.1
P568 >1000 250.3
P570 86.7 21.3
P572 94.5 10.8
P574 162.9 33.8
P576 | 460.9 | 67.4

PS78 I 269.4 I 305.6
P580 U 11.9
P582 137.4 31.0
P584 188.1 67.2
P586 501.6 186.8
P588 406.3 311.2
P590 245.0 6.9
P602 1 209.0 | 36.9
Example 6 - In vivo assays:
Intravenous glucose tolerance test (TVG7T): Normal Wistar rats are fasted overnight and are anesthetized prior to the experiment. A blood sampling catheter is inserted into the rats. The agonist is given subcutaneously, normally 24h prior to the glucose challenge. Blood samples are taken from the carotid artery. A blood sample is drawn immediately prior to the injection of glucose along with the agonist After the initial blood sample, glucose mixed is injected intravenously (i.v.). A glucose challenge of 0.5 g/kg body weight is given, injecting a total of 1.5 mL vehicle with glucose and agonist per kg body weight The peptide concentrations are varied to produce the desired dose in ug/kg. Blood samples are drawn at 2,4,6 and 10 minutes after giving glucose. The control group of animals receives the same vehicle along with glucose, but with no agonist added. In some instances, 20 and 30 minute post-glucose blood samples were drawn. Aprotinin is added to the blood sample (250-500 klU/ml blood). The plasma is then analyzed for glucose and insulin using standard methodologies.
The assay uses a formulated and calibrated peptide stock in PBS. Normally, this . stock is a prediluted 100 uM stock. However, a more concentrated stock with approximately 1 mg agonist per mL is used. The specific concentration is always known. Variability in the maximal response is mostly due to variability in the vehicle dose. Protocol details are as follows:
SPECIES/STRAIN/WEIGHT I Rat/Wistar Unilever/approximately 275 - 300 g
TREATMENT DURATION Single dose
DOSE VOLUME/ROUTE ~I.5mL/kg/iv ~
VEHICLE 8% PEG300,0.1% BSA in water
FOOD/WATER REGIMEN Rats are fasted overnight prior to surgery.
LIVE-PHASE PARAMETERS Animals are sacrificed, at the end of the test
IVGTT: Performed on rats (with two | Glucose PV bolus: 500 mg/kg as 10% solution


catheters, jugular vein and carotid (5 ml/kg) at time = 0.
artery) of each group, under Compound iv: 0 - 240 rain prior to glucose
pentobarbital anesthesia. Blood samplings (300 uL from carotid artery;
EDTA as anticoagulant; aprotinin and PMSF
as antiproteolytics; kept on ice): 0,2,4,6, and
10,20 and 30 minutes.
Parameters determined: Insulin + glucose
TOXICOKINETICS Plasma samples remaining after insulin
measurements are kept at -20°C and
compound levels are determined.
Table 4a
Agonist # Time % increase % increase % increase
between AUC:Dose AUC:Dose AUC:Dose
glucose & =0.09 =0.1mg/kg =0.3mg/kg
compound mg/kg
P505 24h 12 74
P5U 24h 38 59
P525 24h 72 208
PS70 24h 36
P602 1 24h 1 73 | 1 198
AUC = Area under curve (insulin, 0 -10 min after glucose)
Pharmacokinetic profiles of PEGylatedpeptides. Healthy Fisher 344 rats (3 animals per group) were injected with 100 ug agonist/kg (agonist amount based on peptide content and dissolved in PBS buffer). Blood samples were drawn 3,12,24,48,72,96 and 168 hour post dosing and the peptide content in plasma was analysed by a radio-immunoassay (RIA) directed against die N-terminus of the peptide. PK parameters were then calculated using a model-independent method (WinNonlin Pro, Pharsight Corp., Mountain View, CA.USA).
Table 4b. PK parameters of PEGylated peptide agonists. Mean and (SD) values for N=3.
. . .. Cmax Tmax AUCw*, T1/2 Cl/F Vd/F
Agom« # (ngflmL) (h) (ng*h/mL) (h) (mL/h/kg) (mL/kg)
P499 132. 24 6650 25 15 529
^ (9) ( P505 160 12 7006 22 13 425
(30) (
,1 P611 100 16 3067 NC NC* NC
' (16) (7) (374) NC NC NC
PS21 233 12 7633 19 13 380
(29) ( ' P525 133 16 4740 22 20 642
■ (21) (7) (486) (3) (2) (126)
P539 102 24 4013 15 25 540
(16) ( P545 139 72 12737 43 7 457
(48) (42) (2492) (5) (2) (66)
P555 102 16 4918 13 20 389
(9), (7) (597) (1) (3) (65)
P662 120 24 5456 25 19 673
(67) (21) (2157) 17) (6) (315)
P570 71 20 3891 25 25 877
(34) (7) (1309) (4) (7) (326)
P574 70 20 3408 18 2B 727
(6) (7) 012) (2) (2) (71)
*NC = not calculated due to insufficient data
Example 7 - Rat Serum Stability Studies:
, In order to determine the stability of VPAC2 receptor peptide agonists in rat serum, CHO-VPAC2 cells clone #6 (96 well plates/50,000 cells/well and 1 day culture), PBS IX (Gibco), the peptides for the analysis in a 100 uM stock solution, rat serum from a sacrificed normal Wistar rat, aprotinin, and a DiscoveRx assay kit are obtained. The rat serum is stored at 4°C until use and is used within two weeks.
On Day 0, two 100 uL aliquots of 10 uM peptide in rat serum are prepared by adding 10 uL peptide stock to 90 uL rat serum for each aliquot 2S0 klU aprotinin / mL is added to one of these aliquots. The aliquot is stored with aprotinin at 4°C. The aliquot is stored without aprotinin at 37°C. The aliquots are incubated for 24 or 72 hours.
On Day 1, after incubation of the aliquots prepared on day 0 for 24 or 72 hours, an incubation buffer containing PBS + 1.3 mM CaCk, 1.2 mM MgCfe, 2 mM glucose, and 0.25 mM IBMX is prepared. A plate with 11 serial 5X dilutions of peptide for the 4°C and 37°C aliquot is prepared for each peptide studied. 2000 nM is used as the maximal concentration if the peptide has an ECso above 1 nM and 1000 nM as maximal concentration if the peptide has an ECso below 1 nM from the primary screen (see Example 3). The plate(s) are washed with cells twice in incubation buffer. The plates are allowed to hold 50 uL incubation media per well for 15 minutes.' 50 uL solution per well

is transferred to the cells from the plate prepared with 11 serial SX dilutions of peptide for the 4°C and 37°C aliquot for each peptide studied, using the maximal concentrations that are indicated by the primary screen, in duplicate. This step dilutes the peptide
, concentration by a factor of two. The cells are incubated at room temperature for 30 minutes. The supernatant is removed. 40 uL/well of the DiscoveRx antibody/extraction buffer is added. The cells are incubated on the shaker (300 rpm) for 1 hour. Normal procedure with the DiscoveRx kit is followed. cAMP standards are included in column ~ 12. ECso values are determined from the cAMP assay'data. The remaining amount of
: active peptide is estimated by the formula ECso. 4°e/ECso, 37«c for each condition.
. Table 5 Estimated peptide stability after 24h or 72h in rat serum at 37°C
I Agonist # ]24hstab(%) |72hstab(%)
VIP 02
P417 265
P472 66
P473 86
P475 61
, P478 34
P483 22
P48S 65
P492 131_
1 P503 174
P505 133 161
P521 294
P523 281
P525 298 353
P529 119
F543 U9
P539 123
, P555 117
, • P5S7 131
P560 192
P562 213 .
P566 152 •
P574 288
P602 1 573 I

'.Values >100% may represent release of intact peptide from the PEG conjugate
Example 8 - PEGvlation of selective VPAC2 receptor peptide agonists usinp thiol-based chemistry:
In general, PEGylation reactions are run under conditions that permit the formation of a thioether bond. Specifically, the pH of the solution ranges from about 4 to 9 and the thiol-containing peptide concentrations range from 0.7 to 10 molar excess of PEG maleimide concentration. The PEGylation reactions are normally run at room • temperature. The PEGylated VPAC2 receptor peptide agonist is men isolated using reverse-phase HPLC or size exclusion chromatography (SEC). PEGylated peptide agonists are characterized using analytical RP-HPLC, HPLC-SEC, SDS-PAGE, and/or MALDI Mass Spectrometry.
Usually a thiol function is introduced into or onto a selective VPAC2 receptor peptide agonist by adding a cysteine or a homocysteine or a thiol-containing moiety at either or bom termini or by inserting a cysteine or a homocysteine or a thiol-containing moiety into the sequence. Thiol-containing VPAC2 receptor peptide agonists are reacted with 40 kDa, 30 kDa or 20 kDa PEG-maleimide to produce derivatives with PEG covalently attached via a thioether bond.
Synthesis of P505
19 mg of the peptide precursor (non-PEGylated P505) and 162 mg of methoxy-PEG-maleimide (NOF, Japan) with an average molecular weight of 20,000 Daltons are dissolved in 2 mL of 100 mM NH4AC buffer containing 10 mM EDTA (pH 6.8) and the reaction is allowed to proceed for 4 h. 97 mg of the product is obtained as a lyophilized powder after preparative RP-HPLC purificatioa The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro activity.
Synthesis of P525
18.7 mg of the peptide precursor (non-PEGylated P525) and 157 mg of methoxy-PEG-
maleimide with an average molecular weight of 20,000 Daltons are dissolved in 2 mL of 100 mM NH4AC buffer containing 10 mM EDTA (pH 6.8) and the reaction is allowed to proceed for 4 h. 114 mg of the product is obtained as a lyophilized powder after preparative RP-HPLC purification. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro activity.

Synthesis of P572
22.3 mg of the peptide precursor (non-PEGylated P572), and 177 mg of methoxy-PEG-maleimide with an average molecular weight of 20,000 Daltons are dissolved in 2 mL of 100 mM NH4AC buffer containing 10 mM EDTA (pH 6.8) and the reaction is allowed to proceed for 4 h. 137 mg of the product is obtained as a lyophilized powder after preparative RP-HPLC purification. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro activity. Synthesis of P574
31.9 mg of the peptide precursor (non-PEGylated P574) and 283 mg of methoxy-PEG-maleimide with an average molecular weight of 20,000 Daltons are dissolved in 3 mL of 100 mM NH4AC buffer containing 10 mM EDTA (pH 6.8) and the reaction is allowed to , proceed for 4 h. 171 mg of the product is obtained as a lyophilized powder after two runs , of preparative RP-HPLC purifications. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro activity. Synthesis of P602
20 mg of the peptide'precursor (non-PEGylated P602) and 223 mg of methoxy-poly(ethyleneglycol) maleimido-propioaamide (Chirotech Technology Ltd., UK) with an average molecular weight of 30,000 Daltons are dissolved in 2 mL of 100 mM NHUAc buffer containing 10 mM EDTA (pH 6.8) and the reaction is allowed to proceed for 4 h. 132 mg of the product is obtained as a lyophilized powder after preparatiye RP-HPLC purification. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro activity.
Example 9 - PEGvlation via acvlation on the sidechain of Lysine;
In order to achieve site-specific PEGylation of selective VPAC2 receptor peptide agonists, all the Lys residues are changed into Arg residues except for Lys residues where PEGylation is intended. A PEG molecule which may be used is mPEG-SBA-20K (Nektar, Lot #: PT-04E-11). The PEGylation reaction is preferably performed at room temperature for 2-3 hours. The peptide is purified by preparative HPLC.


Example 10 - PEGvlation via Pictet-Speneler reaction:
For PEGylation via Pictet-Spengler reaction to occur, a Trp residue with its free amine is needed to incorporate the PEG molecule onto die selective VPAC2 receptor peptide agonist To couple a Trp residue onto the sidechain of Lys residue. The extensive SAR indicates that this modification does not change the properties of the parent peptide in terms of its in vitro potency and selectivity.
PEG with a functional aldehyde, for example mPEG2-BUTYRALD-40K (Nektar, USA), is used for the reaction. The site specific PEGylation involves the formation a - tetracarboline ring between PEG and the peptide. PEGylation is conducted in glacial acetic acid at room temperature for 1 to 48 hours. A 1 to 10 molar excess of the PEG aldehyde is used in the reaction. After the removal of acetic acid, the PEGylated VPAC2 receptor peptide agonist is isolated by preparative RP-HPLC.
Synthesis of P535
27.7 mg of the peptide precursor (non-PEGylated P535) and 590 mg of mPEG2-BUTYRALD-40K are dissolved in 3 mL acetic acid and the reaction is allowed to proceed for 2 days. The product is isolated by preparative RP-HPLC to yield 94 mg of the PEGylated peptide agonist as a lyophilized powder. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro.
Synthesis of P557
23 mg of the peptide precursor (non-PEGylated P557) and 460 mg of mPEG2-BUTYRALD-40K are dissolved in 3 mL acetic acid and the reaction is allowed to proceed for 2 days. The product is isolated by preparative RP-HPLC to yield 50 mg of the PEGylated peptide agonist as a lyophilized powder. The PEGylated peptide agonist is characterized by RP-HPLC and size-exclusion HPLC, and tested for in vitro.
Other modifications of the present invention will be apparent to those skilled in the art without departing from the scope of the invention.

We claim
1. A PEGylated VPAC2 receptor peptide agonist comprising a sequence
selected from:
SEQ ID NO: 17 HSDAVFTEQY(OMe)TRAibRAibQLAAAibOrnY(OMe)LQSIK
AibOrn;
ID NO: 18 HSDAVFTEK(CO(CH2)2SH)Y(OMe)TOrnLRAibQVAAAibOrn
YLQSIOrnOrn;
SEQ ID NO: 19 HSDAVFTEQY(0Me)T0rnLRAibQVAAAib0rnYLQSI0rnK(W)
Orn;
SEQ ID NO: 20 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(CO(CH2)2SH)YLQ
SlOrnOrn;
SEQ ID NO: 21 HSDAVFTEQY(OMe)TOrnLRAibQVAAK(CO(CH2)2SH)OrnYLQ
SlOrnOrn;
SEQ ID NO: 22 HSDAVFTEQY(OMe)TOrnLRAibQVCAAibOrnYLQSIOrnOrn; SEQ ID NO: 23 HSDAVFTEQY(OMe)TOrnLRCQVAAAibOrnYLQSIOrnOrn;
SEQ ID NO: 24 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQSIOrnOrn; SEQ ID NO: 25 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYAibQSIOrnOrn; SEQ ID NO: 26 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQAibIOrnOrn; SEQ ID NO: 27 HSDAVFTEQY(OMe)TOrnLRAibQVAAbuAibOrnYLQAibIOrnOrn; SEQ ID NO: 28 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQAibIOrnOrn; SEQ ID NO: 29 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYAibQAibIOrnOrn; SEQ ID NO: 30 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQSIOrnOrn; SEQ ID NO: 31 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSIOrnOrn; SEQ ID NO: 32 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQAibIOrn
Orn;
SEQ ID NO: 33 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYAibQSIOrnOrn; SEQ ID NO: 34 HSDAVFTEQY(OMe)TOrnLRK(W)QVAAAibOrnYLQSIOrnOrn; SEQ ID NO: 35 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLK(W)SIOrnOrn; SEQ ID NO: 36 HSDAVFTEQY(OMe)TOrnLRAibQK(W)AAAibOrnYLQSIOrnOrn; SEQ ID NO: 37 HSDAVFTEQY(OMe)TOrnLRK(CO(CH2)2SH)QVAAAibOrnYLQ
SlOrnOrn;

SEQ ID NO: 38 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(W)YLQSIOrnOrn; SEQ ID NO: 39 HSDAVFTEQY(0Me)T0rnLRAibQVAAAibCYLQSI0rn0rn;
SEQ ID NO: 40 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibIOrnOrn; SEQ ID NO: 41 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSK(W)Orn
Orn;
SEQ ID NO: 42 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibIOrnC
Orn;
SEQ ID NO: 43 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibCOrn
Orn;
SEQ ID NO: 44 HSDAVFTEQY(OMe)TOrnLRAibQCAAbuAibOrnYLQAibIOrnOrn; SEQ ID NO: 45 HSDAVFTEQY(OMe)TOrnLRCQLAAbuAibOrnYLQAibIOrnOrn; SEQ ID NO: 94 HSDA VFTEQ Y(OMe)TOrnLRAibQVK(CO(CH2)2SH)AAibOrn
YLQSIOrnOrn;
SEQ ID NO: 95 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSIOrnCOrn; SEQ ID NO: 96 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSCOrnOrn; SEQ ID NO: 97 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibIOrn
K(CO(CH2)2SH)Orn;
SEQ ID NO: 98 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSIOrn
K(CO(CH2)2SH)Orn;
SEQ ID NO: 99 HSDAVFTEQY(OMe)TOrnLRK(W)QLAAbuAibOrnYLQAibIOrn
Orn;
SEQ ID NO: 100 HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQSIOrnOrnC; SEQ ID NO: 101 HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrnOrnC; SEQ ID NO: 102 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSIOrnOrnC; SEQ ID NO: 103 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)LQAibI
OrnOrn;
SEQ ID NO: 104 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)LQAibI
OrnCOrn;
SEQ ID NO: 105 HSDAVFTEQY(OMe)TOrnLRAibQCAAbuAibOrnY(OMe)LQAibI
OrnOrn;
SEQ ID NO: 106 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibIOrn

OrnC;
SEQIDNO: 107 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)LQSI
OrnOrn;
SEQIDNO: 108 HSDAVFTEQY(OMe)TOrnLRAibQCAAbuAibOrnY(OMe)LQSl
OrnOrn;
SEQIDNO: 109 HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)LQSI
OrnCOrn;
SEQIDNO: 110 HSDAVFTEQY(OMe)TOrnLRAibQLAbuAAibOrnYLQSIOrnOrn; SEQ ID NO: 111 HSDAVFTEQY(OMe)TOrnLRAibQK(CO(CH2)2SH)AAbu
AibOrnYLQAiblOrnOrn; and
SEQIDNO: 112 HSDAVFTEQY(OMe)TOrnLRAibQK(W)AAbuAibOrnYLQ
AiblOrnOrn; and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide sequence and wherein the C-terminal extension comprises an amino acid sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaag-Xaa9-Xaaio-Xaaii-Xaai2 Formula 3 (SEQ ID NO: 3) wherein:
Xaai is: Gly, Cys, or absent; Xaa2 is: Gly, Arg, or absent; Xaa3 is: Pro, Thr, or absent; Xaa4 is: Ser, or absent; Xaas is: Ser, or absent; Xaa6 is: Gly, or absent; Xaa7 is: Ala, or absent; Xaag is: Pro, or absent; Xaag is: Pro, or absent; Xaaio is: Pro, or absent; Xaai i is: Ser, Cys, or absent; and Xaai2 is: Cys, or absent;

wherein at least five of Xaai to Xaai2 of the C-terminal extension are present and wherein if Xaai, Xaa2, Xaa3, Xaa4, Xaa,, Xaa6, Xaa7, Xaag, Xaag, Xaaio, or Xaai i is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,
and wherein;
the peptide agonist comprises at least one Cys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one Lys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(W) which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(CO(CH2)2SH) which is covalently attached to a PEG molecule, or
the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or
a combination thereof.
2. A PEGylated VPAC2 receptor peptide agonist according to claim 1
wherein the C-terminal extension is selected from:
SEQ ID NO: 5 I GGPSSGAPPPS
SEQ ID NO: 6 GGPSSGAPPPS-NH2
SEQ ID NO: 7 GGPSSGAPPPC
SEQ ID NO: 8 GGPSSGAPPPC-NH2
SEQ ID NO: 9 GRPSSGAPPPS
SEQ ID NO: 10 GRPSSGAPPPS-NH2
SEQ ID NO: 11 GGPSSGAPPPCC
SEQ ID NO: 12 GGPSSGAPPPCC-NH2
3. A PEGylated VPAC2 receptor peptide agonist according to claim 2
wherein the C-terminal extension is SEQ ID NO: 11 or SEQ ID NO: 12.

4. A PEGylated VPAC2 receptor peptide agonist according to claim 1 wherein there is at least one PEG molecule covalently attached to a residue in the C-terminal extension.
5. A PEGylated VPAC2 receptor peptide agonist according to any one of the preceding claims wherein the PEG molecule is branched.
6. A PEGylated VPAC2 receptor peptide agonist according to any one of claims 1 to 4 wherein the PEG molecule is linear.
7. A PEGylated VPAC2 receptor peptide agonist according to any one of the preceding claims wherein each PEG molecule is 20,000, 30,000, 40,000 or 60,000 daltons in molecular weight.
8. A PEGylated VPAC2 receptor peptide agonist according to any one of the preceding claims further comprising a N-terminal modification at the N-terminus of the peptide agonist wherein the N-terminal modification is selected from:

(a) addition of D-histidine, isoleucine, methionine, or norleucine;
(b) addition of a peptide comprising the sequence Ser-Trp-Cys-Glu-Pro-Gly-Trp-Cys-Arg (SEQ ID NO: 93) wherein the Arg is linked to the N-terminus of the peptide agonist;
(c) addition of C1-C16, alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6alkoxy, -NH2, -OH, halogen and -CF3;
(d) addition of-C(0)R wherein R1 is a C1-C16 alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6alkoxy, -NII2, -OH, halogen, -SH and -CF3; an aryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C2-C6alkenyl, C2-C6alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3; an aryl C1-C4 alkyl optionally substituted with one or more substituents independently selected from C1-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3; -NR R wherein R and R are independently hydrogen, C1-C6alkyl, aryl or

aryl C1-C4 alkyl; -OR4 wherein R4 is C1-C6alkyl optionally substituted with one or more substituents independently selected from aryl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3, aryl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3 or aryl C1-C4 alkyl optionally substituted with one or more substituents independently selected from C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, -NH2, -OH, halogen and -CF3; or 5-pyrrolidin-2-one;
(e) addition of-S02R5 wherein RS is aryl, aryl C1-C4 alkyl or C1-C6 alkyl;
(f) formation of a succinimide group optionally substituted with C1-C6 alkyl or ~SR , wherein R6 is hydrogen or C1-C6 alkyl;
(g) addition of methionine sulfoxide;
(h) addition of biotinyl-6-aminohexanoic acid (6-aminocaproic acid); and (i) addition of-C(=NH)-NH2.
9. A PEGylated VPAC2 receptor peptide agonist according to claim 8 wherein the N-terminal modification is the addition of a group selected from: acetyl, propionyl, butyryl, pentanoyl, hexanoyl, methionine, methionine sulfoxide, 3-phenylpropionyl, phenylacetyl, benzoyl, norleucine, D-histidine, isoleucine, 3-mer^aptopropionyl, biotinyl-6-aminohexanoic acid (6-aminocaproic acid), and -C(=NH)-NH2.
10. A PEGylated VPAC2 receptor peptide agonist according to claim 9 wherein the N-terminal modification is the addition of acetyl or hexanoyl.
11. A PEGylated VPAC2 peptide receptor agonist according to claim 1
comprising an amino acid sequence selected from:
Agonist SEQ Sequence
# ID
NO
P410 46 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(W-
I I PEG40K)YLQSIOrnOrnGGPSSGAPPPS-NH2

P417 [41 [C6-
HSDAVFTEQY(OMe)TRAibRAibQLAAAibOrnY(OMe)LQ
SIKAibOrnGGPSSGAPPPC(PEG40K)-NH2
P451 48 C6-
HSDAVFTEK(CO(CH2)2SPEG40K)Y(OMe)TOrnLRAibQV
AAAibOrnYLQSIOrnOrnGGPSSGAPPPS-NHz
P454 49 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrn
K(WPEG40K)OrnGGPSSGAPPPS-NH2
P460 50 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibC(PEG40K)YL
QSIOrnOrnGGPSSGAPPPS-NH2
P472 51 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(CO(CH2)2S
PEG40K)YLQSIOrnOrnGGPSSGAPPPS-NH2
P473 52 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibK(CO(CH2)2S
PEG20K)YLQSIOrnOrnGGPSSGAPPPS-NH2
P475 53 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAK(CO(CH2)2SPEG
40K)OrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P478 54 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVC(PEG40K)AAibOrnY
LQSIQrnOrnGGPSSGAPPPS-NH2
P483 55 C6-
HSDAVFTEQY(OMe)TOrnLRC(PEG40K)QVAAAibOrnYL
QSIOrnOrnGGPSSGAPPPS-NHa
P485 56 C6-
HSDAVFTEQY(OMe)TOrnLRK(CO(CH2)2SPEG40K)QVAA
AibOrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P507 57 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYAibQSI
OrnOrnGGPSSGAPPPC(PEG40K)-NH2
P509 58 C6-
HSDAVFTEQY(0Me)T0rnLRAibQVAAAib()rnYLQAibI
OrnOrnGGPSSGAPPPC(PEG40K)-NH2
P511 59 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAbuAibOrnYLQAibI
OrnOrnGGPSSGAPPPC(PEG40K)-NH2
P513 60 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYAibQSI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P515 61 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYLQAibI
I | OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2

P517 [62 |~c£
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P519 63 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAAibOrnYAibQAibI
OrnQrnGGPSSGAPPPC(PEG2QK)C(PEG2QK)-NH2
P521 64 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQSI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG2QK)-NH2
P523 65 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P525 66 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQ
AibIOrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P529 67 C6-
HSDAVFTEQY(OMe)TOrnLRK(WPEG40K)QVAAAibOrn
YLQSIOrnOrnGGPSSGAPPPS-NHz
P531 68 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLK(W
PEG40K)SIOrnOrnGGPSSGAPPPS-NH2
P533 69 C6-
HSDAVFTEQY(OMe)TOrnLRAibQK(WPEG40K)AAAib
OrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P535 70 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSK "
(WPEG40K)OrnOrnGGPSSGAPPPS-NH2
P537 71 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibI
OrnC(PEG40K)OrnGGPSSGAPPPS-NH2
P541 72 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibC
(PEG40K)OrnOrnGGPSSGAPPPS-NH2
P545 73 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG40K)AAbuAib
OrnYLQAibIOrnOrnGGPSSGAPPPS-NH2
P547 74 C6-
HSDAVFTEQY(OMe)TOrnLRC(PEG40K)QLAAbuAibOrnY
LQAibIOrnOrnGGPSSGAPPPS-NH2
P480 113 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVK(CO(CH2)2SPEG40K
)AAibOrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P481 114 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVK(CO(CH2)2SPEG20K
)AAibOrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P539 115 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAib()rnYLQSI
| J OrnC(PEG4QK)OrnGGPSSGAPPPS-NH2

P543 [116 I C6-HSDAVFTEQY(0Me)T0rnLRAibQLAAbuAib0rnYLQS
C(PEG40K)OrnOrnGGPSSGAPPPS-NH2
P549 117 C6-HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYL
QAibIOrnK(CO(CH2)2SPEG20K)OrnGGPSSGAPPPC(PEG2
0K)-NH2
P551 118 C6-HSDAVFTEQY(0Me)T0rnLRAibQLAAbuAib0rnYL
QSIOrnK(CO(CH2)2SPEG20K)OrnGGPSSGAPPPC(PEG20K
)-NH2
P555 119 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSI
OrnC(PEG2QK)OrnGGPSSGAPPPC(PEG2QK)-NH2
P557 120 C6-
HSDAVFTEQY(OMe)TOrnLRK(WPEG40K)QLAAbuAib
OrnYLQAibIOrnOrnGGPSSGAPPPS-NH2
P560 121 C6-
HSDAVFTEQY(0Me)T0rnLRAibQLAAAib0rnYLQSI0rn
OrnC(PEG40K)GGPSSGAPPPS-NH2
P562 122 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrn
OrnC(PEG20K)GGPSSGAPPPC(PEG20K)-NH2
P564 123 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOmYLQAibI
OmOrnC(PEG4QK)GGPSSGAPPPS-NH2
P566 124 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSI
OrnOrnC(PEG40K)GGPSSGAPPPS-NH2
P572 125 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQSI
OrnOrnC(PEG20K)GGPSSGAPPPC(PEG20K)-NH2
P574 126 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)L
QAibIOrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P576 127 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)L
QAibIOrnC(PEG40K)OrnGGPSSGAPPPS-NH2
P578 128 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG40K)AAbuAib
OrnY(QMe)LQAibIOrnOrnGGPSSGAPPPS-NH2
P580 129 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibI
OrnOrnC(PEG20K)GGPSSGAPPPC(PEG20K)-NH2
P582 130 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)L
I | QSIOrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2

P584 [m |~c£
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)L
QSIOrnC(PEG40K)OrnGGPSSGAPPPS-NH2
P586 132 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG40K)AAbuAib
OrnY(OMe)LQSIOrnOrnGGPSSGAPPPS-NH2
P588 133 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnY(OMe)L
QSIOrnC(PEG20K)OrnGGPSSGAPPPC(PEG20K)-NH2
P590 134 C6-
HSDAVFTEQY(0Me)T0rnLRAibQLAbuAAib0mYLQSI
OrnOrnGGPSSGAPPPC(PEG20K)C(PEG20K)-NH2
P597 135 C6-
HSDAVFTEQY(OMe)TOrnLRAibQK(CO(CH2)2SPEG20K) AAbuAibOrnYLQAibIOrnOrnGGPSSGAPPPC(PEG20K)-
NH2
P599 136 C6-
HSDAVFTEQY(OMe)TOrnLRAibQK(CO(CH2)2SPEG40K)
AAbjAibOrnYLQAiblOrnOrnGGPSSGAPPPS-NH2
P601 137 C6-
HSDAVFTEQY(OMe)TOrnLRAibQK(WPEG40K)AAbuAib
OrnYLQAibIOrnOrnGGPSSGAPPPS-NH2
P469 139 C6-
HSDAVFTEK(CO(CH2)2SPEG20K)Y(OMe)TOrnLRAibQV
AAAibOrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P486 140 C6-
HSDAVFTEQY(OMe)TOrnLRK(CO(CH2)2SPEG20K)QVAA
AibOrnYLQS10rnOrnGGPSSGAPPPS-NH2
P553 141 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibI
OrnC(PEG20K)OrnGGPSSGAPPPC(PEG20K)-NH2
P570 144 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYLQAibI
OrnOrnGGPSSGAPPPC(PEG30K)C(PEG30K)-NH2
P595 146 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG20K)AAbuAib
OrnYLQAibIOrnOrnGGPSSGAPPPC(PEG20K)-NH2
P476 "147 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAK(CO(CH2)2SPEG
2QK)OrnYLQSIQrnOrnGGPSSGAPPPS-NH2
P602 148 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQSI
I I OrnOrnGGPSSGAPPPC(PEG30K)C(PEG30K)-NH2

12. A PEGylated VPAC2 peptide receptor agonist comprising an amino acid
sequence selected from:
Agonist SEQ Sequence
# ID
NO
P470 75 C6-
HSDAVFTEQY(OMe)TOrnK(CO(CH2)2SPEG20K)RAibQV
AAAibOrnYLQSIOrnOrnGGPSSGAPPPS-NH2
P490 76 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYL
K(CO(CH2)2SPEG20K)SIOrnOrnGGPSSGAPPPC(PEG20K)-
NH2
P492 77 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAK(CO(CH2)2SPEG
2QK)AibQrnYLQS10rnOrnGGPSSGAPPPC(PEG2QK)-NH2
P495 78 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQ
K(CO(CH2)2SPEG20K)IOrnOrnGGPSSGAPPPC(PEG20K)-
NH2
P497 79 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQS
K(CO(CH2)2SPEG20K)OrnOrnGGPSSGAPPPC(PEG20K)-
NH^
P499 80 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrn
K(CO(CH2)2SPEG20K)OrnGGPSSGAPPPC(PEG20K)-NH2
P501 81 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLC(PEG
20K)SIOrnOrnGGPSSGAPPPC(PEG20K)-NH2
P503 82 C6-HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSC
(PEG20K)OrnOrnGGPSSGAPPPC(PEG20K)-NH2
P505 83 C6-
HSDAVFTEQY(OMe)TOrnLRAibQVAAAibOrnYLQSIOrn
C(PEG20K)OrnGGPSSGAPPPC(PEG20K)-NH2
P402 138 C6-
HSDAVFTEQY(0Me)T0rnLRAibQVAAAib0rnYLQSI0rn
OrnGGPSSGAPPPK(W-PEG4QK)-NH2
P558 142 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG20K)AAAibOrnY
LQSIOrnOrnGGPSSGAPPPS-NH2
P568 143 C6-
HSDAVFTEQY(OMe)TOrnLRAibQC(PEG20K)AAAibOrnY
LQSIOrnOrnGGPSSGAPPPC(PEG20K)-NH2
P593 145 C6-
HSDAVFTEQY(OMe)TOrnLRAibQLAAbuAibOrnYAibQ
I | AibIOrnOrnGGPSSGAPPPK(WPEG40K)-NH2

13. A PEGylated VPAC2 receptor peptide agonist comprising a sequence of the formula:
Xaai-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Thr-Xaa8-Xaa9-Xaaio-Thr-Xaai2-Xaai3- Xaau-Xaai5-Xaau,-Xaai7-Xaai8 -Abu-Xaa2()-Xaa2i-Xaa22- Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa3o-Xaa3i-Xaa32-Xaa33-Xaa34-Xaa35-Xaa36-Xaa37-Xaa38-Xaa39-Xaa4o
Formula 4 (SEQ ID NO: 4)
wherein:
Xaai is: His, dH, or is absent;
Xaa2 is: dA, Ser, Val, Gly, Thr, Leu, dS, Pro, or Aib;
Xaa3 is: Asp or Glu;
Xaa4 is: Ala, He, Tyr, Phe, Val, Thr, Leu, Trp, Gly, dA, Aib, or NMeA;
Xaa5 is: Val, Leu, Phe, He, Thr, Trp, Tyr, dV, Aib, or NMeV;
Xaa6 is: Phe, He, Leu, Thr, Val, Trp, or Tyr;
Xaas is: Asp, Glu, Ala, Lys, Leu, Arg, or Tyr;
Xaa9 is: Asn, Gin, Asp, Glu, Ser, Cys, Lys, or K(CO(CH2)2SH);
Xaaio is: Tyr, Trp, Tyr(OMe), Ser, Cys, or Lys;
Xaai2 is: Arg, Lys, Glu, hR, Orn, Lys (isopropyl), Aib, Cit, Ala, Leu, Gin, Phe, Ser, or
Cys;
Xaan is: Leu, Phe, Glu, Ala, Aib, Ser, Cys, Lys, or K(CO(CH2)2SH);
Xaai4 is: Arg, Leu, Lys, Ala, hR, Orn, Lys (isopropyl), Phe, Gin, Aib, Cit, Ser, or Cys;
Xaa)5 is: Lys, Ala, Arg, Glu, Leu, hR, Orn, Lys (isopropyl), Phe, Gin, Aib, K(Ac), Cit,
Ser, Cys, K(W), or K(CO(CH2)2SH);
Xaa16 is: Gin, Lys, Glu, Ala, hR, Orn, Lys (isopropyl), Cit, Ser, Cys, K(CO(CH2)2SH), or
K(W);
Xaa,7 is: Val, Ala, Leu, He, Met, Nle, Lys, Aib, Ser, Cys, K(CO(CH2)2SH), or K(W);
Xaa,8 is: Ala, Ser, Cys, Lys, K(CO(CH2)2SH), K(W), Abu, or Nle;
Xaa2o is: Lys, Gin, hR, Arg, Ser, His, Orn, Lys (isopropyl), Ala, Aib, Trp, Thr, Leu, He,
Phe, Tyr, Val, K(Ac), Cit, Cys, K(CO(CH2)2SH), or K(W);

Xaa2i is: Lys, His, Arg, Ala, Phe, Aib, Leu, Gin, Orn, hR, K(Ac), Cit, Ser, Cys, Val, Tyr,
He, Thr, Trp, K(W), or K(CO(CH2)2SH);
Xaa22 is: Tyr, Trp, Phe, Thr, Leu, He, Val, Tyr(OMe), Ala, Aib, Ser, Cys, Lys, K(W), or
K(CO(CH2)2SH);
Xaa23 is: Leu, Phe, He, Ala, Trp, Thr, Val, Aib, Ser, Cys, Lys, K(W), or K(CO(CH2)2SH);
Xaa24 is: Gin, Glu, Asn, Ser, Cys, Lys, K(CO(CH2)2SH), or K(W);
Xaa2, is: Ser, Asp, Phe, He, Leu, Thr, Val, Trp, Gin, Asn, Tyr, Aib, Glu, Cys, Lys,
K(CO(CH2)2SH), or K(W);
Xaa% is: He, Leu, Thr, Val, Trp, Tyr, Phe, Aib, Ser, Cys, Lys, K(CO(CH2)2SH), or K(W);
Xaa27 is: Lys, hR, Arg, Gin, Ala, Asp, Glu, Phe, Gly, His, He, Met, Asn, Pro, Ser, Thr,
Val, Trp, Tyr, Lys (isopropyl), Cys, Leu, Orn, dK, K(W), or K(CO(CH2)2SH);
Xaa28 is: Asn, Asp, Gin, Lys, Arg, Aib, Orn, hR, Cit, Pro, dK, Ser, Cys, K(CO(CH2)2SH),
or K(W);
Xaa29 is: Lys, Ser, Arg, Asn, hR, Ala, Asp, Glu, Phe, Gly, His, He, Leu, Met, Pro, Gin,
Thr, Val, Trp, Tyr, Cys, Orn, Cit, Aib, K(W), K(CO(CH2)2SH), or is absent;
Xaa3o is: Arg, Lys, He, Ala, Asp, Glu, Phe, Gly, His, Leu, Met, Asn, Pro, Gin, Ser, Thr,
Val, Trp, Tyr, Cys, hR, Cit, Aib, Orn, K(W), K(CO(CH2)2SH), or is absent;
Xaa3, is: Tyr, His, Phe, Thr, Cys, Ser, Lys, Gin, K(W), K(CO(CH2)2SH), or is absent;
Xaa32 is: Ser, Cys, Lys, or is absent;
Xaa33 is: Trp or is absent;
Xaa34 is: Cys or is absent;
Xaa35 is: Glu or is absent;
Xaa36 is: Pro or is absent;
Xaa37 is: Gly or is absent;
Xaa38 is: Trp or is absent;
Xaa39 is: Cys or is absent; and
Xaa4o is: Arg or is absent
wherein if Xaa2g, Xaa30, Xaa3], Xaa32, Xaa33, Xaa34, Xaa35, Xaa36, Xaa37, Xaa38, or Xaa3g
is absent, the next amino acid present downstream is the next amino acid in the peptide
agonist sequence,

and a C-terminal extension wherein the N-terminus of the C-terminal extension is linked to the C-terminus of the peptide of Formula 4 and wherein the C-terminal extension comprises an amino acid sequence of the formula:
Xaai -Xaa2-Xaa3-Xaa4-Xaa5-Xaa6-Xaa7-Xaa8-Xaa9-Xaai o-Xaai i -Xaai2 Formula 3 (SEQ ID NO: 3) wherein:
Xaai is: Gly, Cys, or absent; Xaa2 is: Gly, Arg, or absent; Xaa3 is: Pro, Thr, or absent; Xaa4 is: Ser, or absent; Xaas is: Ser, or absent; Xaa6 is: Gly, or absent; Xaa7 is: Ala, or absent; Xaas is: Pro, or absent; Xaag is: Pro, or absent; Xaaio is: Pro, or absent; Xaai i is: Ser, Cys, or absent; and Xaai2 is: Cys, or absent;
wherein at least five of Xaai to Xaai2 of the C-terminal extension are present and wherein if Xaai, Xaa2, Xaa3, Xaa4, Xaas, Xaa6, Xaa?, Xaag, Xaa9, Xaaio, or Xaai i is absent, the next amino acid present downstream is the next amino acid in the C-terminal extension and wherein the C-terminal amino acid may be amidated,
and wherein;
the peptide agonist comprises at least one Cys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one Lys residue which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(W) which is covalently attached to a PEG molecule, or
the peptide agonist comprises at least one K(CO(CH2)2SH) which is covalently attached to a PEG molecule, or

the carboxy-terminal amino acid of the peptide agonist is covalently attached to a PEG molecule, or
a combination thereof.
14. A pharmaceutical composition comprising a PEGylated VPAC2 receptor peptide agonist according to any one of claims 1 to 13 and one or more pharmaceutically acceptable diluents, carriers or excipients.
15. A PEGylated VPAC2 receptor peptide agonist according to any one of claims 1 to 13 for use as a medicament.
16. The use of a PEGylated VPAC2 receptor peptide agonist according to any one of claims 1 to 13 for the manufacture of a medicament for the treatment non-insulin-dependent diabetes or insulin-dependent diabetes.
17. A method of treating non-insulin-dependent diabetes or insulin-dependent diabetes in a patient in need thereof comprising administering to said patient an effective amount of a PEGylated VPAC2 receptor peptide agonist according to any one of claims 1 to 13.

ABSTRACT
SELECTIVE VPAC2 RECEPTOR PEPTIDE AGONISTS
The present invention encompasses peptides that selectively activate the VPAC2 receptor and are useful in the treatment of diabetes.

Documents:

614-mumnp-2008-digram.doc

646-MUMNP-2008-ABSTRACT(24-1-2011).pdf

646-mumnp-2008-abstract(4-4-2008).pdf

646-mumnp-2008-abstract(amended)-(24-1-2011).pdf

646-mumnp-2008-abstract(granted)-(24-3-2011).pdf

646-mumnp-2008-abstract.doc

646-mumnp-2008-abstract.pdf

646-MUMNP-2008-ASSIGNMENT(12-1-2011).pdf

646-mumnp-2008-assignment(24-1-2011).pdf

646-mumnp-2008-cancelled pages(16-3-2011).pdf

646-MUMNP-2008-CANCELLED PAGES(24-1-2011).pdf

646-mumnp-2008-claims(4-4-2008).pdf

646-MUMNP-2008-CLAIMS(AMENDED)-(16-3-2011).pdf

646-MUMNP-2008-CLAIMS(AMENDED)-(24-1-2011).pdf

646-mumnp-2008-claims(granted)-(24-3-2011).pdf

646-MUMNP-2008-CLAIMS(MARKED COPY)-(16-3-2011).pdf

646-MUMNP-2008-CLAIMS(MARKED COPY)-(24-1-2011).pdf

646-mumnp-2008-claims.doc

646-mumnp-2008-claims.pdf

646-MUMNP-2008-CORRESPONDENCE 10-7-2008.pdf

646-MUMNP-2008-CORRESPONDENCE(12-1-2011).pdf

646-MUMNP-2008-CORRESPONDENCE(16-3-2011).pdf

646-MUMNP-2008-CORRESPONDENCE(20-10-2008).pdf

646-mumnp-2008-correspondence(24-1-2011).pdf

646-MUMNP-2008-CORRESPONDENCE(27-12-2010).pdf

646-mumnp-2008-correspondence(ipo)-(24-3-2011).pdf

646-mumnp-2008-correspondence-others.pdf

646-mumnp-2008-correspondence-received.pdf

646-mumnp-2008-description (complete).pdf

646-mumnp-2008-description(complete)-(4-4-2008).pdf

646-mumnp-2008-description(granted)-(24-3-2011).pdf

646-MUMNP-2008-DRAWING(24-1-2011).pdf

646-mumnp-2008-drawing(4-4-2008).pdf

646-mumnp-2008-drawing(amended)-(24-1-2011).pdf

646-mumnp-2008-drawing(granted)-(24-3-2011).pdf

646-mumnp-2008-drawings.pdf

646-MUMNP-2008-FORM 1(24-1-2011).pdf

646-mumnp-2008-form 1(4-4-2008).pdf

646-mumnp-2008-form 13(24-1-2011).pdf

646-MUMNP-2008-FORM 18 10-7-2008.pdf

646-MUMNP-2008-FORM 2(COMPLETE)-(4-4-2008).pdf

646-MUMNP-2008-FORM 2(GRANTED)-(24-3-2011).pdf

646-MUMNP-2008-FORM 2(TITLE PAGE)-(24-1-2011).pdf

646-mumnp-2008-form 2(title page)-(4-4-2008).pdf

646-mumnp-2008-form 2(title page)-(granted)-(24-3-2011).pdf

646-MUMNP-2008-FORM 26(24-1-2011).pdf

646-MUMNP-2008-FORM 26(27-12-2010).pdf

646-MUMNP-2008-FORM 3(20-10-2008).pdf

646-MUMNP-2008-FORM 3(24-1-2011).pdf

646-mumnp-2008-form 3(4-4-2008).pdf

646-MUMNP-2008-FORM PCT-ISA-210(24-1-2011).pdf

646-mumnp-2008-form-1.pdf

646-mumnp-2008-form-2.doc

646-mumnp-2008-form-2.pdf

646-mumnp-2008-form-3.pdf

646-mumnp-2008-form-5.pdf

646-mumnp-2008-form-pct-ib-304.pdf

646-mumnp-2008-form-pct-ib-311.pdf

646-mumnp-2008-form-pct-isa-220.pdf

646-mumnp-2008-form-pct-isa-237.pdf

646-mumnp-2008-form-pct-separate sheet-237.pdf

646-mumnp-2008-marked copy(24-1-2011).pdf

646-MUMNP-2008-OTHER DOCUMENT(24-1-2011).pdf

646-mumnp-2008-pct-search report.pdf

646-MUMNP-2008-PETITION UNDER RULE 137(1)-(24-1-2011).pdf

646-MUMNP-2008-PETITION UNDER RULE 137(2)-(24-1-2011).pdf

646-mumnp-2008-petition under rule 137(24-1-2011).pdf

646-MUMNP-2008-PETITION UNDER RULE 137(3)-(24-1-2011).pdf

646-MUMNP-2008-REPLY TO EXAMINATION REPORT(24-1-2011).pdf

646-MUMNP-2008-SEQUENCE LISTING(24-1-2011).pdf

646-mumnp-2008-sequence listing(24-3-2011).pdf

646-mumnp-2008-sequence listing(4-4-2008).pdf

646-mumnp-2008-specification(amended)-(24-1-2011).pdf

646-MUMNP-2008-WO INTERNATIONAL PUBLICATION REPORT A1 10-7-2008.pdf


Patent Number 247006
Indian Patent Application Number 646/MUMNP/2008
PG Journal Number 13/2011
Publication Date 01-Apr-2011
Grant Date 24-Mar-2011
Date of Filing 04-Apr-2008
Name of Patentee ELI LILLY AND COMPANY
Applicant Address LILLY CORPORATE CENTER, CITY OF INDIANAPOLIS, STATE OF INDIANA 46285,
Inventors:
# Inventor's Name Inventor's Address
1 BOKVIST, BENGT, KRISTER LILLY FORSCHUNG GMBH, ESSENER BOGEN 7, 22419 HAMBURG,
2 ALSINA-FERNANDEZ, JORGE 5463 NORTH CAPITAL AVENUE, INDIANAPOLIS, IN 46208,
3 ZHANG, LIANSHAN 13244 SNOW OWL DRIVE, CARMEL, IN 46033,
PCT International Classification Number A61K47/48
PCT International Application Number PCT/US2006/041550
PCT International Filing date 2006-10-24
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/730,291 2005-10-26 U.S.A.
2 60/740,342 2005-11-29 U.S.A.
3 60/743,364 2006-02-28 U.S.A.