Peptide conjugates of GLP-1 receptor agonists and gastrin and their use

Abstract

The present invention relates, inter alia, to certain peptide conjugates, and to the use of the conjugates in the treatment of a variety of diseases or disorders, including diabetes (type 1 and/or type 2) and diabetes related diseases or disorders.

Claims

1. A peptide conjugate or pharmaceutically acceptable salt thereof, wherein the peptide conjugate has the sequence: Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 1) Exendin-4(1-39)-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 2) Exendin-4(1-39)-K-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 3) Exendin-4(1-39)-AAA-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 4) Exendin-4(1-39)-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 5) Exendin-4(1-39)-Peg3-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 6) Exendin-4(1-39)-8Aoc-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 7) Exendin-4(1-39)-DBF-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 8) Exendin-4(1-39)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 9) Exendin-4(1-28-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 19) Exendin-4(1-28)-K-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 20) Exendin-4(1-28)-AAA-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 21) Exendin-4(1-28)-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 22) Exendin-4(1-28)-Peg3-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 23) Exendin-4(1-28)-Peg3-Peg3-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 24) Exendin-4(1-28)-8Aoc-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 25) Exendin-4(1-28)-DBF-SKK-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 26) Exendin-4(1-28)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17, (SEQ ID NO: 27).

2. A pharmaceutical composition comprising a peptide conjugate, or pharmaceutically acceptable salt thereof, according to claim 1, in combination with one or more peptide conjugates or pharmaceutically acceptable salt thereof together with a pharmaceutically acceptable carrier, excipient or vehicle.

3. A device comprising at least one peptide conjugate, or pharmaceutically acceptable salt thereof, according to claim 1, for delivery of the peptide conjugate to a subject.

4. A kit comprising at least one peptide conjugate, or pharmaceutically acceptable salt thereof, according to claim 1, further comprising packaging or instructions for use.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A-1B. Mean plasma concentration versus time (log-linear) after i.v. and s.c. administration of 100 nmol/kg to mice. FIG. 1A: Compound 33, FIG. 1B: Compound 1. Data represent meanSD, n=3/datapoint.

(2) FIG. 2. Mean plasma concentration versus time (log-linear) after s.c. administration of 100 nmol compound/kg of Compound 33, 74, 76, 77, 78 and 80 to mice. n=2/datapoint.

(3) FIG. 3 presents data for total pancreatic insulin content (in g) in db/db diabetic mice following administration of (i) three concentrations (1, 10 and 50 nmol/kg) of Compound 1 of the invention [Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17; vide infra], (ii) three corresponding concentrations of a 1:1 additive combination of the Exendin-4(1-39) and h[Leu15]Gastrin17 peptides (1, 10 and 50 nmol/kg of each peptide) and (iii) vehicle.

(4) FIG. 4. -Blood glucose in mice. Data were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test,***p<0.001. Comparison of Compound 33, Exendin-4 a combination of Exendin-4(1-39) and h[Leu15]Gastrin17 and Vehicle respectively; n=16-19 per group.

(5) FIG. 5. -Plasma insulin in mice. Data were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test, ***p<0.001. Comparison of Compound 33, Exendin-4, a combination of Exendin-4(1-39) and h[Leu15]Gastrin17 and Vehicle respectively; n=16-19 per group.

(6) FIG. 6. Pancreatic insulin content in mice. Data were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test, **p<0.01. Comparison of Compound 33, Exendin-4, a combination of Exendin-4(1-39) and h[Leu15]Gastrin17 and Vehicle respectively; n=16-19 per group.

(7) FIG. 7. -HbA1c in mice. Data were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test, **p<0.01, ***p<0.001. Comparison of Compound 33, Exendin-4, a combination of Exendin-4(1-39) and h[Leu15]Gastrin17 and Vehicle respectively; n=16-19 per group.

(8) FIG. 8. -plasma C peptide in mice. Data were analyzed using Kruskal-Wallis test followed by Dunn's multiple comparison test, *p<0.05. Comparison of Compound 33, Exendin-4,a combination of Exendin-4(1-39) and h[Leu15]Gastrin17 and Vehicle respectively; n=16-19 per group.

(9) FIGS. 9A-9C. Effect of SC administration of Exendin-4, Liraglutide or Compound 33 on glucose tolerance as measured by the Area Under the Curve (AUC) following a glucose load in db/db mice. Three treatment regimens were applied: Prevention (FIG. 9A), Treatment (FIG. 9B), or Holiday (FIG. 9C). Data are given as mean with SEM (n=8-13/group). Statistic: Data was compared by 2-way ANOVA followed by Bonferroni's post-test: *p<0.05; **p<0.01; ***p<0.001 vs. vehicle.

(10) FIGS. 10A-10C. Effect of SC administration of Exendin-4, Liraglutide or Compound 33 on 8 h fasting blood glucose in db/db mice. Three treatment regimens were applied: Prevention (FIG. 10A), Treatment (FIG. 10B), or Holiday (FIG. 10C). Data are given as mean with SEM (n=8-13/group). Statistic: Data was compared by 2-way ANOVA followed by Bonferroni's post-test: *p<0.05; **p<0.01; ***p<0.001 vs. vehicle.

(11) FIGS. 11A-11C. Effect of SC administration of Exendin-4, Liraglutide or Compound 33 on terminal (day 93) values of plasma C-peptide (FIG. 11A), plasma insulin (FIG. 11B), or HbA1c (%) (FIG. 11C). Data are given as mean with SEM (n=8-13/group). Statistic: Data was compared by 1-way ANOVA Kruskal-Wallis test followed by Dunn's MC test: ***p<0.001, **p<0.01, *p<0.05 vs. vehicle.

DETAILED DESCRIPTION OF THE INVENTION

(12) As already indicated above, one aspect of the present invention relates to a peptide conjugate having the formula:
Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17,(1)
Exendin-4(1-39)-[Gln1,Leu15]Gastrin17,(2)
Exendin-4(1-39)-K-[Gln1,Leu15]Gastrin17,(3)
Exendin-4(1-39)-AAA-[Gln1,Leu15]Gastrin17,(4)
Exendin-4(1-39)-SKK-[Gln1,Leu15]Gastrin17,(5)
Exendin-4(1-39)-Peg3-SKK-[Gln1,Leu15]Gastrin17,(6)
Exendin-4(1-39)-8Aoc-SKK-[Gln1,Leu15]Gastrin17,(7)
Exendin-4(1-39)-DBF-SKK-[Gln1,Leu15]Gastrin17,(8)
Exendin-4(1-39)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17,(9)
Exendin-4(1-39)-[Leu4]Gastrin6,(10)
Exendin-4(1-39)-K-[Leu4]Gastrin6,(11)
Exendin-4(1-39)-AAA-[Leu4]Gastrin6,(12)
Exendin-4(1-39)-SKK-[Leu4]Gastrin6,(13)
Exendin-4(1-39)-Peg3-SKK-[Leu4]Gastrin6,(14)
Exendin-4(1-39)-Peg3-Peg3-[Leu4]Gastrin6,(15)
Exendin-4(1-39)-8Aoc-SKK-[Leu4]Gastrin6,(16)
Exendin-4(1-39)-DBF-SKK-[Leu4]Gastrin6,(17)
Exendin-4(1-39)-8Aoc-8Aoc-[Leu4]Gastrin6,(18)
Exendin-4(1-28)-[Gln1,Leu15]Gastrin17,(19)
Exendin-4(1-28)-K-[Gln1,Leu15]Gastrin17,(20)
Exendin-4(1-28)-AAA-[Gln1,Leu15]Gastrin17,(21)
Exendin-4(1-28)-SKK-[Gln1,Leu15]Gastrin17,(22)
Exendin-4(1-28)-Peg3-SKK-[Gln1,Leu15]Gastrin17,(23)
Exendin-4(1-28)-Peg3-Peg3-[Gln1,Leu15]Gastrin17,(24)
Exendin-4(1-28)-8Aoc-SKK-[Gln1,Leu15]Gastrin17,(25)
Exendin-4(1-28)-DBF-SKK-[Gln1,Leu15]Gastrin17,(26)
Exendin-4(1-28)-8Aoc-8Aoc-[Gln1, Leu15]Gastrin 17,(27)
Exendin-4(1-28)-[Leu4]Gastrin6,(28)
Exendin-4(1-28)-K-[Leu4]Gastrin6,(29)
Exendin-4(1-28)-AAA-[Leu4]Gastrin6,(30)
Exendin-4(1-28)-SKK-[Leu4]Gastrin6,(31)
Exendin-4(1-28)-Peg3-SKK-[Leu4]Gastrin6,(32)
Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6,(33)
Exendin-4(1-28)-8Aoc-SKK-[Leu4]Gastrin6,(34)
Exendin-4(1-28)-DBF-SKK-[Leu4]Gastrin6,(35)
Exendin-4(1-28)-8Aoc-8Aoc-[Leu4]Gastrin6,(36)
GLP-1(7-36)-[Gln1,Leu15]Gastrin17,(37)
GLP-1(7-36)-K-[Gln1,Leu15]Gastrin17,(38)
GLP-1(7-36)-AAA-[Gln1,Leu15]Gastrin17,(39)
GLP-1(7-36)-SKK-[Gln1,Leu15]Gastrin17,(40)
GLP-1(7-36)-Peg3-SKK-[Gln1,Leu15]Gastrin17,(41)
GLP-1(7-36)-Peg3-Peg3-[Gln1,Leu15]Gastrin17,(42)
GLP-1(7-36)-8Aoc-SKK-[Gln1,Leu15]Gastrin17,(43)
GLP-1(7-36)-DBF-SKK-[Gln1,Leu15]Gastrin17,(44)
GLP-1(7-36)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17,(45)
GLP-1(7-36)-[Leu4]Gastrin6,(46)
GLP-1(7-36)-K-[Leu4]Gastrin6,(47)
GLP-1(7-36)-AAA-[Leu4]Gastrin6,(48)
GLP-1(7-36)-SKK-[Leu4]Gastrin6,(49)
GLP-1(7-36)-Peg3-SKK-[Leu4]Gastrin6,(50)
GLP-1(7-36)-Peg3-Peg3-[Leu4]Gastrin6,(51)
GLP-1(7-36)-8Aoc-SKK-[Leu4]Gastrin6,(52)
GLP-1(7-36)-DBF-SKK-[Leu4]Gastrin6(53)
or
GLP-1(7-36)-8Aoc-8Aoc-[Leu4]Gastrin6,(54)
wherein each of the Exendin-4(1-39), the Exendin-4(1-28) and the GLP-1(7-36) peptide moieties is covalently attached to the remaining part of the respective conjugate molecule via its C-terminal, and each of the [Gln1,Leu15]Gastrin17 and [Leu4]Gastrin6 peptide moieties is covalently attached to the remaining part of the respective conjugate molecule via its N-terminal;
or a pharmaceutically acceptable salt or solvate thereof.

(13) In a further aspect, the present invention relates to a peptide conjugate having the formula:
Exendin-4(1-28)-Peg3-Peg3-[Leu3]Gastrin5(55)
Exendin-4(1-28)-Peg3-Peg3-[Ala1,Leu4]Gastrin6(56)
Exendin-4(1-28)-Peg3-Peg3-[Ala2,Leu4]Gastrin6(57)
Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(58)
Exendin-4(1-28)-Peg3-Peg3-[Leu2]Gastrin4(59)
[Leu14]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(60)
[Orn12]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(61)
[Orn27]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(62)
[Phe25]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(63)
[Asp28]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(64)
[Tyr13]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(65)
[Orn20]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(66)
Exendin-4(1-28)-Peg3-[Leu4]Gastrin6(67)
Exendin-4(1-28)-[Leu4]Gastrin6(68)
Exendin-4(1-27)-[Leu4]Gastrin11(69)
Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(70)
Exendin-4(1-27)-Peg3-[Leu3]Gastrin5(71)
Exendin-4(1-26)-Peg3-[Leu3]Gastrin5(72)
Exendin-4(1-27)-Peg3-[Leu2]Gastrin4(73)
[Tyr13,Leu14]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(74)
[Tyr13,Phe25]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(75)
[Leu14,Phe25]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(76)
[Tyr13,Leu14,Phe25]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(77)
Side chain-cyclo([Lys12,Glu16]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(78)
Side chain-cyclo([Glu16,Lys20]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(79)
Side chain-cyclo([Lys20,Glu24]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(80)
[Lys16]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6(81)
Exendin-4(1-28)-Peg3-K-Peg3-[Leu4]Gastrin6(82)
Exendin-4(1-28)-[Thr4]Gastrin6(83)
Exendin-4(1-28)-[Phe4]Gastrin6(84)
[Leu14]Exendin-4(1-28)-[1Nal3,Leu4]Gastrin6(85)
[Leu14]Exendin-4(1-28)-[NIe4]Gastrin6(86)
[Leu14]Exendin-4(1-28)-[Leu4,[3-(3-Pyridyl)-Ala]6]Gastrin6(87)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6(88)
[Leu14, Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3-[Leu4, Phe3]Gastrin6(89)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3-[Leu4,Phe3]Gastrin6(90)
[Arg27,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(91)
[Arg12,27,Leu14,Lys16,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(92)
[Arg12,27,Leu14,Lys20,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(93)
[Arg12,27,Leu14,Lys24,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(94)
[Arg12,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(95)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-[Leu2]Gastrin4(96)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu2]Gastrin4(97)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Peg3-[Leu2]Gastrin4(98)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Orn-[Leu2]Gastrin4(99)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Orn-[Leu2]Gastrin4(100)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-[Leu4]Gastrin6(101)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-[Leu4]Gastrin6(102)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Peg3-[Leu4]Gastrin6(103)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Orn-[Leu4]Gastrin6(104)
[Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Orn-[Leu4]Gastrin6(105)
[Lys(Hexadecanoyl-isoGlu)34]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(106)
[Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(107)
[Arg26,34,Lys(Hexadecanoyl-isoGlu)36]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(108)
[Lys(Hexadecanoyl-isoGlu)26]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(109)
[Arg26,34,Gly8,Lys(Hexadecanoyl-isoGlu)36]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(110)
[Aib8,Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(111)
[Aib8,Arg34]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(112)
[Arg34]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6(113)
or a pharmaceutically acceptable salt or solvate thereof.

(14) The above formulae for the peptide conjugates of the invention, which are written employing conventional and widely used abbreviations/designations for the various peptide moieties in question [i.e. Exendin-4(1-39), Exendin-4(1-28) and GLP-1(7-36)] may be written in conventional full amino acid sequence form as follows (with linker moieties highlighted in bold font):

(15) TABLE-US-00012 (SEQ ID NO: 1) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Peg3-Peg3- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 2) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSQGPWLEEEEEAYGWLDF- NH.sub.2 (no linker) (SEQ ID NO: 3) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-K- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 4) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-AAA- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 5) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-SKK- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 6) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Peg3-SKK- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 7) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-8Aoc-SKK- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 8) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-DBF-SKK- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 9) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-8Aoc-8Aoc- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 10) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPSYGWLDF-NH.sub.2 (no linker) (SEQ ID NO: 11) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-K-YGWLDF-NH.sub.2 (SEQ ID NO: 12) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-AAA-YGWLDF-NH.sub.2 (SEQ ID NO: 13) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 14) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Peg3-SKK-YGWLDF- NH.sub.2 (SEQ ID NO: 15) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-Peg3-Peg3-YGWLDF- NH.sub.2 (SEQ ID NO: 16) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-8Aoc-SKK-YGWLDF- NH.sub.2 (SEQ ID NO: 17) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-DBF-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 18) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-8Aoc-8Aoc-YGWLDF- NH.sub.2 (SEQ ID NO: 19) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNQGPWLEEEEEAYGWLDF-NH.sub.2 (no linker) (SEQ ID NO: 20) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-K-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 21) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-AAA-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 22) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-SKK-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 23) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-SKK-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 24) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 25) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Aoc-SKK-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 26) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-DBF-SKK-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 27) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Aoc-8Aoc-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 28) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-YGWLDF-NH.sub.2 (no linker) (SEQ ID NO: 29) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-K-YGWLDF-NH.sub.2 (SEQ ID NO: 30) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-AAA-YGWLDF-NH.sub.2 (SEQ ID NO: 31) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 32) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 33) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH.sub.2 (SEQ ID NO: 34) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Aoc-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 35) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-DBF-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 36) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-8Aoc-8Aoc-YGWLDF-NH.sub.2 (SEQ ID NO: 37) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRQGPWLEEEEEAYGWLDF-NH.sub.2 (no linker) (SEQ ID NO: 38) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-K-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 39) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-AAA-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 40) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-SKK-QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 41) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Peg3-SKK-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 42) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Peg3-Peg3- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 43) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-8Aoc-SKK- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 44) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-DBF-SKK-QGPWLEEEEEAYGWLDF- NH.sub.2 (SEQ ID NO: 45) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-8Aoc-8Aoc- QGPWLEEEEEAYGWLDF-NH.sub.2 (SEQ ID NO: 46) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRYGWLDF-NH2 (no linker) (SEQ ID NO: 47) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-K-YGWLDF-NH.sub.2 (SEQ ID NO: 48) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-AAA-YGWLDF-NH.sub.2 (SEQ ID NO: 49) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 50) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Peg3-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 51) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-Peg3-Peg3-YGWLDF-NH.sub.2 (SEQ ID NO: 52) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-8Aoc-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 53) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-DBF-SKK-YGWLDF-NH.sub.2 (SEQ ID NO: 54) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-8Aoc-8Aoc-YGWLDF-NH.sub.2 (SEQ ID NO: 55) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-GWLDF-NH2 (SEQ ID NO: 56) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-AGWLDF-NH2 (SEQ ID NO: 57) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-YAWLDF-NH2 (SEQ ID NO: 58) H-HGEGTFTSDLSKQMEEEAVRLFIEWLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 59) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-Peg3-WLDF-NH2 (SEQ ID NO: 60) H-HGEGTFTSDLSKQLEEEAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 61) H-HGEGTFTSDLS-Orn-QMEEEAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 62) H-HGEGTFTSDLSKQMEEEAVRLFIEWL-Orn-N-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 63) H-HGEGTFTSDLSKQMEEEAVRLFIEFLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 64) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKD-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 65) H-HGEGTFTSDLSKYMEEEAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 66) H-HGEGTFTSDLSKQMEEEAV-Orn-LFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 67) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-YGWLDF-NH2 (SEQ ID NO: 68) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNYGWLDF-NH2 (SEQ ID NO: 69) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKEEEEAYGWLDF-NH2 (SEQ ID NO: 70) H-HGEGTFTSDLSKQMEEEAVRLFIEWLK-Peg3-YGWLDF-NH2 (SEQ ID NO: 71) H-HGEGTFTSDLSKQMEEEAVRLFIEWLK-Peg3-GWLDF-NH2 (SEQ ID NO: 72) H-HGEGTFTSDLSKQMEEEAVRLFIEWL-Peg3-GWLDF-NH2 (SEQ ID NO: 73) H-HGEGTFTSDLSKQMEEEAVRLFIEWLK-Peg3-WLDF-NH2 (SEQ ID NO: 74) H-HGEGTFTSDLSKYLEEEAVRLFIEWLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 75) H-HGEGTFTSDLSKYMEEEAVRLFIEFLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 76) H-HGEGTFTSDLSKQLEEEAVRLFIEFLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 77) H-HGEGTFTSDLSKYLEEEAVRLFIEFLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 78) H-HGEGTFTSDLS-K( )-QME-E( )-EAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 79) H-HGEGTFTSDLSKQME-E( )-EAV-K( )-LFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 80) H-HGEGTFTSDLSKQMEEEAV-K( )-LFI-E( )-WLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 81) H-HGEGTFTSDLSKQMEKEAVRLFIEWLKN-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 82) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKN-Peg3-K-Peg3-YGWLDF-NH2 (SEQ ID NO: 83) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNYGWTDF-NH2 (SEQ ID NO: 84) H-HGEGTFTSDLSKQMEEEAVRLFIEWLKNYGWFDF-NH2 (SEQ ID NO: 85) H-HGEGTFTSDLSKQLEEEAVRLFIEWLKNYG-1Nal-LDF-NH2 (SEQ ID NO: 86) H-HGEGTFTSDLSKQLEEEAVRLFIEWLKNYGW-Nle-DF-NH2 (SEQ ID NO: 87) H-HGEGTFTSDLSKQLEEEAVRLFIEWLKNYGWLD-[3-(3-Pyridyl)-alanyl]-NH2 (SEQ ID NO: 88) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 89) H-HGEGTFTSDLSKYLEEEAVRLFIEFLK-Peg3-Peg3-YGFLDF-NH2 (SEQ ID NO: 90) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-Peg3-YGFLDF-NH2 (SEQ ID NO: 91) H-HGEGTFTSDLSKYLEEEAVRLFIEFLR-Peg3-YGWLDF-NH2 (SEQ ID NO: 92) H-HGEGTFTSDLSRYLEKEAVRLFIEFLR-Peg3-YGWLDF-NH2 (SEQ ID NO: 93) H-HGEGTFTSDLSRYLEEEAVKLFIEFLR-Peg3-YGWLDF-NH2 (SEQ ID NO: 94) H-HGEGTFTSDLSRYLEEEAVRLFIKFLR-Peg3-YGWLDF-NH2 (SEQ ID NO: 95) H-HGEGTFTSDLSRYLEEEAVRLFIEFLK-Peg3-YGWLDF-NH2 (SEQ ID NO: 96) H-HGEGTFTSELSKYLEEEAVRLFIEFLKWLDF-NH2 (SEQ ID NO: 97) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-WLDF-NH2 (SEQ ID NO: 98) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Orn-Peg3-WLDF-NH2 (SEQ ID NO: 99) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-Orn-WLDF-NH2 (SEQ ID NO: 100) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Orn-Orn-WLDF-NH2 (SEQ ID NO: 101) H-HGEGTFTSELSKYLEEEAVRLFIEFLKYGWLDF-NH2 (SEQ ID NO: 102) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-YGWLDF-NH2 (SEQ ID NO: 103) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Orn-Peg3-YGWLDF-NH2 (SEQ ID NO: 104) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Peg3-Orn-YGWLDF-NH2 (SEQ ID NO: 105) H-HGEGTFTSELSKYLEEEAVRLFIEFLK-Orn-Orn-YGWLDF-NH2 (SEQ ID NO: 106) H-HAEGTFTSDVSSYLEGQAAKEFIAWLV-K(Hexadecanoyl-isoGlu)-GRG-Peg3- Peg3-YGWLDF-NH2 (SEQ ID NO: 107) H-HAEGTFTSDVSSYLEGQAA-K(Hexadecanoyl-isoGlu)-EFIAWLVRGRG-Peg3- Peg3-YGWLDF-NH2 (SEQ ID NO: 108) H-HAEGTFTSDVSSYLEGQAAREFIAWLVRG-K(Hexadecanoyl-isoGlu)-G-Peg3- Peg3-YGWLDF-NH2 (SEQ ID NO: 109) H-HAEGTFTSDVSSYLEGQAA-K(Hexadecanoyl-isoGlu)-EFIAWLVKGRG-Peg3- Peg3-YGWLDF-NH2 (SEQ ID NO: 110) H-HGEGTFTSDVSSYLEGQAAREFIAWLVRG-K(Hexadecanoyl-isoGlu)-G-Peg3- Peg3-YGWLDF-NH2 (SEQ ID NO: 111) H-H-Aib-EGTFTSDVSSYLEGQAA-K(Hexadecanoyl-isoGlu)-EFIAWLVRGRG- Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 112) H-H-Aib-EGTFTSDVSSYLEGQAAKEFIAWLVRGRG-Peg3-Peg3-YGWLDF-NH2 (SEQ ID NO: 113) H-HAEGTFTSDVSSYLEGQAAKEFIAWLVRGRG-Peg3-Peg3-YGWLDF-NH2
wherein the abbreviations Peg3, 8Aoc,DBF, 1 Nal, bAla, Orn, DPR, Dbu, Gaba and Aib represent the following non-naturally occurring amino acid moieties: Peg3: NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2C(O) (derived from 8-amino-3,6-dioxaoctanoic acid); 8Aoc: NHCH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2C(O) (derived from 8-aminooctanoic acid)I; DBF:

(16) ##STR00001##
[derived from 4-(2-aminoethyl)-6-dibenzofuranpropanoic acid]; 1Nal: 1-napthylalanine bAla: beta-alanine Gaba: -aminobutanoic acid Aib: -Amino-isobutanoic acid Dbu: Diaminobutanoic acid DPR: Diaminopropionic acid Orn: Ornitine

(17) Thus, with regard to the orientation of the linker moiety in a peptide conjugate of the invention, the linker moiety -Peg3-Peg3-, for example, designates the chemical moiety
NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2C(O)NHCH.sub.2CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2C(O),
the NH. . . moiety to the left of the linker moiety in question being covalently attached to the Exendin-4- or GLP-1-derived moiety of the peptide conjugate in question, and the . . .C(O) moiety to the right of the linker moiety in question being attached to the Gastrin-derived moiety of the peptide conjugate in question.

(18) With regard to the remaining specified linker moieties, -K- designates a lysine amino acid residue, -AAA-designates a -Ala-Ala-Ala- tripeptide residue and -SKK- designates a -Ser-Lys-Lys- tripeptide residue. In some of the peptide conjugates of the invention, listed above, it is to be understood that the GLP-1(7-36) peptide sequence moiety is derived from the sequence of human GLP-1 (hGLP-1) sequence or is an analogue thereof.

(19) In some other of the peptide conjugates of the invention, listed above, it is to be understood that the Exendin-4(1-39) peptide sequence moiety is derived from the sequence of the Heloderma suspectum Exendin-4 sequence or is an analogue thereof.

(20) Likewise, the [Gln1,Leu15]Gastrin17 and [Leu4]Gastrin6 moieties in the conjugates are derived from human Gastrin.

(21) It is to be understood that SEQ ID NO: 1 equals Compound 1, SEQ ID NO: 2 equals Compound 2 etc.

(22) It is to be understood that each one of the above peptide conjugates 1-54 individually, i.e. compound 1 or compound 2 or compound 3. . . (etc., up to compound 54), and each one of the further peptide conjugates 55-113 disclosed below (See Table 2 and 3 in Example 2), individually, i.e. compound 55 or compound 56. . . (etc. up to compound 113), or a pharmaceutically acceptable salt or solvate thereof, constitutes a further, individual aspect of the present invention.

(23) In the context of the present invention, unless amino acids are referred to by their full name (e.g. alanine, arginine, etc.), they are designated by their conventional three-letter and/or single-letter abbreviations (e.g. Ala or A for alanine, Arg or R for arginine, etc.).

(24) The term peptide conjugate in the context of the present invention refers to a molecule in which a first peptide moiety is attached (i.e. coupled or linked), either directly or via a linking (i.e. bridging or spacing) chemical moiety, by means of covalent chemical bonding to a second peptide moiety. Compounds of the invention may carry one or more intramolecular lactam bridges within the peptide sequence. Each such bridge in compounds listed in Table 2 (referred to by the prefix side-chain-cyclo) is formed between a side chain containing a carboxylic acid and another side chain containing an amine. The two amino acid residues are typically separated by three amino acids in the linear sequence.

(25) In peptide conjugates of the invention, exendin-4 or Z.sub.a may have at least 75% identity to native exendin-4, eg. at least 80, 85, 90 or 95%.

(26) In the peptide conjugates of the invention, gastrin or Y.sub.a may have at least 70% identity to native gastrin, eg. at least 75, 80, 85, 90 or 95%. In the peptide conjugates of the invention, GLP-1 or X.sub.a may have at least 85% identity to native GLP-1, eg. at least 90 or 95%.

(27) In an embodiment, the polypeptide of the invention may comprise the amino acid sequence set forth in any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 and 113 or a functional fragment/variant thereof that is at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5% identical to one or more of the recited sequences, or functional fragments/variants thereof that have at most 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions compared to one or more of the recited sequences, with or without the signal sequence, with or without substitution of one or more cysteine residues with another residue, such as a serine, and contiguous segments thereof of at least 2 amino acids in length.

(28) In an embodiment, the polypeptide of the invention (i) shares at least 99% amino acid sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 and 113, or to a portion thereof; or (ii) comprises at least 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 contiguous amino acids of any one of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112 and 113; or (iii) both.

(29) The term pharmaceutically acceptable salt in the context of the present invention (pharmaceutically acceptable salt of a peptide conjugate of the invention) is intended to indicate a salt which is not harmful to a patient or subject to which the salt in question is administered. It may suitably be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts. Examples of basic salts include salts where the cation is selected among alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R.sup.1)(R.sup.2)(R.sup.3)(R.sup.4).sup.+, where R.sup.1, R.sup.2, R.sup.3 and R.sup.4 independently will typically designate hydrogen, optionally substituted C.sub.1-6-alkyl or optionally substituted C.sub.2-6-alkenyl. Examples of relevant C.sub.1-6-alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. Examples of C.sub.2-6-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in Remington's Pharmaceutical Sciences, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985 (and more recent editions thereof), in the Encyclopaedia of Pharmaceutical Technology, 3.sup.rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66:2 (1977).

(30) The term solvate in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a peptide conjugate or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water, ethanol or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

(31) Another aspect of the present invention relates to a peptide conjugate or pharmaceutically acceptable salt thereof according to the invention, for use as a medicament. In a further aspect, the medicament in question is a medicament for use in the treatment, in a subject in need thereof, of one or more of the following diseases or disorders and associated conditions: type 1 diabetes, type 2 diabetes, pre-diabetes, Insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, hyperglycemia, hypertension, atherogenic dyslipidemia, arteriosclerosis (e.g. atherosclerosis), coronary heart disease, peripheral artery disease, stroke, microvascular disease, gastric disease, metabolic syndrome, cancer (e.g. colon cancer), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).

(32) Further diseases or disorders of possible relevance in this connection include obesity, morbid obesity, obesity-linked inflammation, obesity-linked gall bladder disease and obesity-induced sleep apnea.

(33) In a still further aspect, the medicament in question is a medicament for use in inducing, in a subject in need thereof, pancreatic islet neogenesis (e.g. for promoting formation of new -cells in the islets of the pancreas).

(34) In a still further aspect, the medicament in question is a medicament for use in inducing, in a subject in need thereof, survival of -cells in the pancreatic islets (e.g. for preventing loss of -cells in the pancreatic islets).

(35) In yet another aspect, the medicament in question is a medicament for use in preventing, in a subject in need thereof, -cell apoptosis in the pancreatic islets (e.g. for preventing loss of -cells in the pancreatic islets).

(36) In a further aspect, the medicament in question is a medicament for use in reducing, in a subject in need thereof, haemoglobin b1Ac (glycosylated haemoglobin; HbA1c) levels in the blood.

(37) A further aspect of the invention relates to the use of a peptide conjugate of the invention in the manufacture of a medicament for the treatment, in a subject in need thereof, of one or more of the following conditions, diseases or disorders: type 1 diabetes, type 2 diabetes, pre-diabetes, Insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, hyperglycemia, hypertension, atherogenic dyslipidemia, arteriosclerosis (e.g. atherosclerosis), coronary heart disease, peripheral artery disease, stroke, microvascular disease, gastric disease, metabolic syndrome, cancer (e.g. colon cancer), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS).

(38) A peptide conjugate of the invention may further be used in: the manufacture of a medicament for inducing pancreatic islet neogenesis in a subject in need thereof; the manufacture of a medicament for preventing -cell apoptosis in the pancreatic islets in a subject in need thereof; or the manufacture of a medicament for reducing haemoglobin b1Ac (glycosylated haemoglobin; HbA1c) levels in the blood of a subject in need thereof.

(39) Among related, additional aspects of the invention are corresponding methods of treatment of conditions, diseases or disorders among those mentioned above. Thus, one such additional aspect of the invention relates to a method for treatment, in a subject in need thereof, of one or more of the following diseases or disorders:

(40) type 1 diabetes, type 2 diabetes, pre-diabetes, Insulin resistance syndrome, impaired glucose tolerance (IGT), disease states associated with elevated blood glucose levels, hyperglycemia, hypertension, atherogenic dyslipidemia, arteriosclerosis (e.g. atherosclerosis), coronary heart disease, peripheral artery disease, stroke, microvascular disease, gastric disease, metabolic syndrome, cancer (e.g. colon cancer), inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS), the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(41) Again, further conditions, diseases or disorders of possible relevance in the context of methods of treatment according to the invention include obesity, morbid obesity, obesity-linked inflammation, obesity-linked gall bladder disease and obesity-induced sleep apnea.

(42) A further aspect of the present invention relates to a method for inducing pancreatic islet neogenesis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(43) An additional aspect of the invention relates to a method for promoting -cell survival in the pancreatic islets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(44) An additional aspect of the invention relates to a method for reducing or preventing -cell apoptosis in the pancreatic islets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(45) Another aspect of the invention relates to a method for reducing haemoglobin b1Ac (glycosylated haemoglobin; HbA1c) levels in the blood of a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(46) Still further aspects of the present invention relate to the following: A method of treatment, in a subject in need thereof, of a disease state associated with elevated blood glucose levels; A method for lowering blood glucose levels in a subject in need thereof; A method of stimulating Insulin release in a subject in need thereof; A method for regulating gastric emptying in a subject in need thereof; and A method for lowering plasma lipid levels in a subject in need thereof.

(47) In each of the latter methods of the invention, the method comprises administering to the subject a therapeutically effective amount of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention.

(48) The term therapeutically effective amount as employed in the context of the above-described methods of treatment or other therapeutic intervention according to the invention refers to an amount that is sufficient to cure, ameliorate, alleviate or partially arrest the clinical manifestations of the particular disease, disorder or condition that is the object of the treatment or other therapeutic intervention in question. An amount adequate to accomplish this is defined as a therapeutically effective amount. The administered amount and the method of administration can be tailored to achieve optimal efficacy. An amount effective for a given purpose will depend, inter alia, on the severity of the disease, disorder or condition that is the object of the particular treatment or other therapeutic intervention, on the body weight and general condition of the subject in question, on diet, on possible concurrent medication, and on other factors well known to those skilled in the medical arts. Determination of an appropriate dosage size and dosing regimen most appropriate for administration of a peptide conjugate or pharmaceutically acceptable salt or solvate thereof according to the invention to a human may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials. An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are well known to the skilled person.

(49) The terms treatment and grammatical variants thereof (e.g. treated, treating, treat) as employed in the present context refer to an approach for obtaining beneficial or desired clinical results. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilization (i.e. not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treatment can also mean prolonging survival relative to expected survival time if not receiving treatment. Treatment in some embodiments may be an intervention performed with the intention of preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, treatment may refer both to therapeutic intervention or to prophylactic or preventive measures. A subject (e.g. a human) in need of treatment may thus be a subject already suffering from the disease or disorder in question, or a subject in which the disorder is to be prevented. The term treatment thus includes inhibition or reduction of an increase in severity of a pathological state or symptoms (e.g. weight gain or hyperglycemia) relative to the absence of treatment, and

(50) is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.

(51) The term agonist as employed in the context of the invention refers to a substance (ligand) that activates the receptor type in question.

(52) The term GLP-1 receptor agonist as employed in the context of the invention (sometimes termed elsewhere GLP-1 agonist) refers to a substance (ligand) that activates a GLP-1 receptor, such as the human GLP-1 receptor. Substances that activate the human GLP-1 receptor include the native GLP-1 peptide hormones GLP-1(7-37), GLP-1(7-36)amide, oxyntomodulin, exendin-3, exendin-4, glucagon, gastric inhibitory polypeptide (GIP), and functional peptide analogues and derivatives thereof.

(53) The term antagonist as employed in the context of the invention refers to a substance (ligand) that blocks, neutralizes or counteracts the effect of another substance (ligand) that functions as an agonist towards the receptor type in question.

(54) In the context of the invention, a subject in need of the particular treatment or other therapeutic intervention referred to in connection with the various aspects of the invention described above is preferably a mammal, and more particularly, is a human.

(55) An additional aspect of the invention relates to a pharmaceutical composition comprising a peptide conjugate, or pharmaceutically acceptable salt or solvate thereof, according to the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle.

(56) Synthesis of Peptide Conjugates

(57) The peptide conjugates of the invention may be manufactured by standard synthetic methods, by use of recombinant expression systems, or by any other suitable method. Thus, the conjugates may be synthesized in a number of ways, including, e.g., methods which comprise: (a) synthesizing the peptide conjugate by means of standard solid-phase or liquid-phase methodology, either stepwise or by fragment assembly, and isolating and purifying the final peptide conjugate product; (b) expressing a nucleic acid construct that encodes the peptide conjugate in a host cell and recovering the expression product from the host cell culture; or (c) effecting cell-free in vitro expression of a nucleic acid construct that encodes the peptide conjugate, and recovering the expression product; or by any combination of methods of (a), (b) or (c) to obtain fragments of the peptide conjugate, subsequently ligating the fragments to obtain the peptide conjugate, and recovering the peptide conjugate.

(58) It may often be preferable to synthesize the conjugates of the invention by means of solid-phase or liquid-phase peptide synthesis. In this connection, reference may be made to WO 98/11125 or, inter alia, Fields, G. B. et al., Principles and Practice of Solid-Phase Peptide Synthesis; in: Synthetic Peptides, Gregory A. Grant (ed.), Oxford University Press (2.sup.nd edition, 2002) and the synthesis examples herein.

(59) One or more of the amino acid side chains in the compound of the invention may be further conjugated to a lipophilic substituent. The lipophilic substituent may be covalently bonded to an atom in the amino acid side chain, or alternatively may be conjugated to the amino acid side chain by a spacer. The amino acid may be part of the peptide Z, or part of the peptide Y.

(60) Without wishing to be bound by theory, it is thought that the lipophilic substituent binds albumin in the blood stream, thus shielding the compounds of the invention from enzymatic degradation which can enhance the half-life of the compounds. The spacer, when present, is used to provide a spacing between the compound and the lipophilic substituent.

(61) The lipophilic substituent may be attached to the amino acid side chain or to the spacer via an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. Accordingly it will be understood that preferably the lipophilic substituent includes an acyl group, a sulphonyl group, an N atom, an O atom or an S atom which forms part of the ester, sulphonyl ester, thioester, amide or sulphonamide.

(62) Preferably, an acyl group in the lipophilic substituent forms part of an amide or ester with the amino acid side chain or the spacer.

(63) The lipophilic substituent may include a hydrocarbon chain having 4 to 30 C atoms. Preferably it has at least 8 or 12 C atoms, and preferably it has 24 C atoms or fewer, or 20 C atoms or fewer. The hydrocarbon chain may be linear or branched and may be saturated or unsaturated. It will be understood that the hydrocarbon chain is preferably substituted with a moiety which forms part of the attachment to the amino acid side chain or the spacer, for example an acyl group, a sulphonyl group, an N atom, an O atom or an S atom. Most preferably the hydrocarbon chain is substituted with acyl, and accordingly the hydrocarbon chain may be part of an alkanoyl group, for example palmitoyl, caproyl, lauroyl, myristoyl or stearoyl.

(64) Accordingly, the lipophilic substituent may have the formula shown below:

(65) ##STR00002##

(66) A may be, for example, an acyl group, a sulphonyl group, NH, N-alkyl, an O atom or an S atom, preferably acyl. n is an integer from 3 to 29, preferably at least 7 or at least 11, and preferably 23 or less, more preferably 19 or less.

(67) The hydrocarbon chain may be further substituted. For example, it may be further substituted with up to three substituents selected from NH.sub.2, OH and COOH. If the hydrocarbon chain is further substituted, preferably it is further substituted with only one substituent. Alternatively or additionally, the hydrocarbon chain may include a cycloalkane or heterocycloalkane, for example as shown below:

(68) ##STR00003##

(69) Preferably the cycloalkane or heterocycloalkane is a six-membered ring. Most preferably, it is piperidine.

(70) Alternatively, the lipophilic substituent may be based on a cyclopentanophenanthrene skeleton, which may be partially or fully unsaturated, or saturated. The carbon atoms in the skeleton each may be substituted with Me or OH. For example, the lipophilic substituent may be cholyl, deoxycholyl or lithocholyl.

(71) As mentioned above, the lipohphilic substituent may be conjugated to the amino acid side chain by a spacer. When present, the spacer is attached to the lipophilic substituent and to the amino acid side chain. The spacer may be attached to the lipophilic substituent and to the amino acid side chain independently by an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. Accordingly, it may include two moieties independently selected from acyl, sulphonyl, an N atom, an O atom or an S atom. The spacer may have the formula:

(72) ##STR00004##
wherein B and D are each independently selected from acyl, sulphonyl, NH, N-alkyl, an O atom or an S atom, preferably from acyl and NH. Preferably, n is an integer from 1 to 10, preferably from 1 to 5. The spacer may be further substituted with one or more substituents selected from C.sub.1-6 alkyl, C.sub.0-6 alkyl amine, C.sub.0-6 alkyl hydroxy and C.sub.0-6 alkyl carboxy.

(73) Alternatively, the spacer may have two or more repeat units of the formula above. B, D and n are each selected independently for each repeat unit. Adjacent repeat units may be covalently attached to each other via their respective B and D moieties. For example, the B and D moieties of the adjacent repeat units may together form an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide. The free B and D units at each end of the spacer are attached to the amino acid side chain and the lipophilic substituent as described above.

(74) Preferably the spacer has five or fewer, four or fewer or three or fewer repeat units. Most preferably the spacer has two repeat units, or is a single unit.

(75) The spacer (or one or more of the repeat units of the spacer, if it has repeat units) may be, for example, a natural or unnatural amino acid. It will be understood that for amino acids having functionalized side chains,

(76) B and/or D may be a moiety within the side chain of the amino acid. The spacer may be any naturally occurring or unnatural amino acid. For example, the spacer (or one or more of the repeat units of the spacer, if it has repeat units) may be Gly, Pro, Ala, Val, Leu, Ile, Met, Cys, Phe, Tyr, Trp, His, Lys, Arg, Gln, Asn, -Glu, -Glu, Asp, Ser Thr, Gaba, Aib, bAla, 5-aminopentanoyl, 6-aminohexanoyl, 7-aminoheptanoyl, 8-aminooctanoyl, 9-aminononanoyl or 10-aminodecanoyl.

(77) For example, the spacer may be a single amino acid selected from -Glu, Gaba, b-Ala and -Gly. The lipophilic substituent may be conjugated to any amino acid side chain in the compounds of the invention. Preferably, the amino acid side chain includes an carboxy, hydroxyl, thiol, amide or amine group, for forming an ester, a sulphonyl ester, a thioester, an amide or a sulphonamide with the spacer or lipophilic substituent. For example, the lipophilic substituent may be conjugated to Asn, Asp, Glu, Gln, His, Lys, Arg, Ser, Thr, Tyr, Trp, Cys or Dbu, Dpr or Orn. Preferably, the lipophilic substituent is conjugated to Lys or Cys. However, any amino acid shown as Lys in the formulae provided herein may be replaced by Dbu, Dpr or Orn where a lipophilic substituent is added.

(78) An example lipophilic substituent and spacer is shown in the formula below:

(79) ##STR00005##

(80) Here, a Lys from the compound of the present invention (e.g. from X) is covalently attached to -Glu (the spacer) by via an amide moiety. Palmitoyl is covalently attached to the -Glu spacer via an amide moiety.

(81) Alternatively or additionally, one or more amino acid side chains in the compound of the invention may be conjugated to a polymeric moiety, for example, in order to increase solubility and/or half-life in vivo (e.g. in plasma) and/or bioavailability. Such modification is also known to reduce clearance (e.g. renal clearance) of therapeutic proteins and peptides.

(82) The polymeric moiety is preferably water soluble (amphiphilic or hydrophilic), non-toxic, and pharmaceutically inert. Suitable polymeric moieties include polyethylene glycol (PEG), homo- or co-polymers of PEG, a monomethyl-substituted polymer of PEG (mPEG), or polyoxyethylene glycerol (POG). See, for example, Int. J. Hematology 68:1 (1998); Bioconjugate Chem. 6:150 (1995); and Crit. Rev. Therap. Drug Carrier Sys. 9:249 (1992).

(83) Other suitable polymeric moieties include poly-amino acids such as poly-lysine, poly-aspartic acid and poly-glutamic acid (see for example Gombotz, et al. (1995), Bioconjugate Chem., vol. 6: 332-351; Hudecz, et al. (1992), Bioconjugate Chem., vol. 3, 49-57; Tsukada, et al. (1984), J. Natl. Cancer Inst., vol 73; 721-729; and Pratesi, et al. (1985), Br. J. Cancer, vol. 52: 841-848).

(84) The polymeric moiety may be straight-chain or branched. It may have a molecular weight of 500-40,000 Da, for example 500-10,000 Da, 1000-5000 Da, 10,000-20,000 Da, or 20,000-40,000 Da.

(85) A compound may comprise two or more such moieties, in which case the total molecular weight of all such moieties will generally fall within the ranges provided above.

(86) The polymeric moiety may be coupled (by covalent linkage) to an amino, carboxyl or thiol group of an amino acid side chain. Preferred examples are the thiol group of Cys residues and the epsilon amino group of Lys residues, and the carboxyl groups of Asp and Glu residues may also be used.

(87) The skilled reader will be well aware of suitable techniques which can be used to perform the coupling reaction. For example, a PEG moiety carrying a methoxy group can be coupled to a Cys thiol group by a maleimido linkage using reagents commercially available from Nektar Therapeutics AL. See also WO 2008/101017, and the references cited above for details of suitable chemistry.

(88) Therapeutic Uses

(89) In the following, it will be understood that reference to the use of a peptide conjugate of the invention also encompasses use of a pharmaceutically acceptable salt or solvate thereof.

(90) The peptide conjugates of the invention may provide an attractive treatment option for metabolic diseases or disorders, including diabetes, in particular type 1 and/or type 2 diabetes, and possibly obesity.

(91) Diabetes comprises a group of metabolic diseases characterized by hyperglycemia resulting from defects in Insulin secretion, Insulin action, or both. Acute signs of diabetes include excessive urine production, resulting compensatory thirst and increased fluid intake, blurred vision, unexplained weight loss, lethargy, and changes in energy metabolism. The chronic hyperglycemia of diabetes is associated with macro- and microvascular complications that can lead to long-term damage, dysfunction, andin some casesultimately failure of various organs, particularly the eyes (notably in the form of diabetic retinopathy), kidneys (in the form of diabetic nephropathy), nerves (in the form of diabetic neuropathy), heart and blood vessels. Diabetes may be subdivided into three classes, viz. type 1 diabetes, type 2 diabetes and gestational diabetes, on the basis on pathogenetic characteristics.

(92) Type 1 diabetes accounts for 5-10% of all diabetes cases and is caused by auto-immune destruction of Insulin-secreting pancreatic -cells.

(93) Type 2 diabetes accounts for 90-95% of diabetes cases and is a result of a complex set of metabolic disorders. Type 2 diabetes is the consequence of endogenous Insulin production and/or whole-body Insulin sensitivity becoming insufficient to maintain plasma glucose levels below the diagnostic thresholds. Gestational diabetes refers to any degree of glucose intolerance identified during pregnancy.

(94) A condition known as pre-diabetes is also recognized. It includes, e.g., impaired fasting glucose levels and impaired glucose tolerance, and refers generally to those states that occur when blood glucose levels are elevated, but are below levels that are established for the clinical diagnosis for diabetes.

(95) A large proportion of subjects with type 2 diabetes and pre-diabetes are at increased risk of morbidity and mortality due to the high prevalence of additional metabolic risk factors, including abdominal obesity (excessive fat tissue around the abdominal internal organs), atherogenic dyslipidemia (blood fat disorders, including high triglyceride levels, low HDL cholesterol levels and/or high LDL cholesterol levels, which foster plaque build-up in artery walls), elevated blood pressure (hypertension), a prothrombotic state (e.g. high Fibrinogen or Plasminogen activator inhibitor-1 levels in the blood), and a proinflammatory state (e.g., elevated C-reactive protein levels in the blood).

(96) Conversely, obesity confers an increased risk of developing, for example, pre-diabetes, type 2 diabetes, certain types of cancer, obstructive sleep apnea and gall-bladder disease.

(97) Dyslipidemia is associated with increased risk of cardiovascular disease. High Density Lipoprotein (HDL) is of clinical importance since an inverse correlation exists between plasma HDL concentrations and risk of atherosclerotic disease. The major part of cholesterol stored in atherosclerotic plaques originates from Low Density Lipoproteins (LDL), and hence elevated concentrations of LDL are closely associated with atherosclerosis. The HDL/LDL ratio is a parameter employed is assessing clinical risk of atherosclerosis and coronary atherosclerosis in particular.

(98) Without being bound by any particular theory, it appears that the peptide conjugates of the invention may unexpectedly combine the physiological effects of GLP-1 receptor agonists with those of Gastrin peptides vide supra in a manner such that the observed activity may be significantly greater than that observed when employing a corresponding additive (non-conjugated) combination of the individual peptide components. It is consequently believed that the peptide conjugates of the invention may be of particular benefit in the treatment of pre-diabetes, diabetes (notably type 1 and/or type 2 diabetes) and diabetes-related conditions, diseases or disorders such as those discussed above, including treatment to promote pancreatic islet -cell formation (islet neogenesis), and thereby Insulin production, that will be beneficial with respect to regulation of blood glucose concentrations. Peptide conjugates of the invention may therefore be of value, inter alia, in limiting or arresting disease progression in type 1 and/or type 2 diabetes.

(99) The peptides of the present invention may further be useful for promoting survival and inhibiting apoptosis of -cells in the pancreatic islets. Effects of GLP-1 and Gastrin includes effects on -cell proliferation and maturation but also prevention of -cell apoptosis and enhanced neogenesis, thus the effects of the peptides of the invention may include such effects and thereof effects on improved insulin and glucose regulation.

(100) The peptide conjugates of the present invention may thus be useful as pharmaceutical agents for treatment of Insulin resistance, glucose intolerance, pre-diabetes, elevated fasting glucose levels, type 1 and/or type 2 diabetes, hypertension and/or dyslipidemia (or a combination of these metabolic risk factors), atherosclerosis, arteriosclerosis, coronary heart disease, peripheral artery disease and stroke. They may also be useful in preventing weight gain, promoting weight loss, reducing excess body weight and/or treating obesity (e.g. by control of appetite, feeding, food intake, calorie intake, and/or energy expenditure), including morbid obesity, as well as associated diseases, disorders and health conditions, including, but not limited to, obesity-linked inflammation, obesity-linked gallbladder disease and obesity-induced sleep apnea. Effects of the peptide conjugates of the invention on these conditions may be mediated in whole or in part via an effect on body weight, or may be independent thereof.

(101) Pharmaceutical Compositions

(102) In the following, it will be understood that reference to the inclusion of one or more of a peptide conjugate of the invention in a pharmaceutical composition also encompasses inclusion of a pharmaceutically acceptable salt or solvate of a peptide conjugate of the invention.

(103) The peptide conjugates of the present invention may be formulated as pharmaceutical compositions which are suited for administration with or without storage, and which typically comprise a therapeutically effective amount of at least one peptide conjugate of the invention, together with a pharmaceutically acceptable carrier, excipient or vehicle.

(104) The term pharmaceutically acceptable carrier includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, Pa., USA, 1985. For example, sterile saline and phosphate-buffered saline at slightly acidic or physiological pH may be used. Suitable pH-buffering agents may, e.g., be phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, arginine, lysine or acetate (e.g. as sodium acetate), or mixtures thereof. The term further encompasses any carrier agents listed in the US Pharmacopeia for use in animals, including humans.

(105) A pharmaceutical composition of the invention may be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component or components. The unit dosage form may be presented as a packaged preparation, the package containing discrete quantities of the preparation, for example, packaged tablets, capsules or powders in vials or ampoules. The unit dosage form may also be, e.g., a capsule, cachet or tablet in itself, or it may be an appropriate number of any of these packaged forms. A unit dosage form may also be provided in single-dose injectable form, for example in the form of a pen device containing a liquid-phase (typically aqueous) composition. Compositions may be formulated for any suitable route and means of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for e.g. oral, intraviteral, rectal, vaginal, nasal, topical, enteral or parenteral (including subcutaneous, intramuscular, intravenous, intradermal and transdermal) administration or administration by inhalation. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmaceutical formulation. Subcutaneous or transdermal modes of administration may be particularly suitable for the peptide conjugates of the invention.

(106) A further aspect of the invention relates to devices, dosage forms and packages used to deliver the pharmaceutical formulations of the present invention. Thus, at least one peptide conjugate or specified portion or variant in either the stable or preserved formulations or solutions described herein, can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.

(107) A still further aspect of the invention relates to oral formulations and administration. Formulations for oral may rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation. The active constituent compound of the solid-type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride. These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, alpha-tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.

(108) Dosages

(109) A typical dosage of a peptide conjugate of the invention as employed in the context of the present invention may be in the range from about 0.001 to about 100 mg/kg body weight per day, such as from about 0.01 to about 50 mg/kg body weight per day, e.g. from about 0.05 to about 10 mg/kg body weight per day, administered in one or more doses, such as from one to three doses. As already indicated to some extent above, the exact dosage employed will depend, inter alia, on: the nature and severity of the disease or disorder to be treated; the sex, age, body weight and general condition of the subject to be treated; possible other, concomitant disease or disorder that is undergoing or is to undergo treatment; as well as other factors that will be known to a medical practitioner of skill in the art.

(110) Combination Therapy

(111) As noted above, it will be understood that reference in the following to a peptide conjugate of the invention also extends to a pharmaceutically acceptable salt or solvate thereof as well as to a composition comprising more than one different peptide conjugate of the invention.

(112) A peptide conjugate of the invention may be administered as part of a combination therapy together with another active agent for the treatment of the disease or disorder in question, e.g. diabetes, obesity, metabolic syndrome, dyslipidemia or hypertension, and in such cases, the two active agents may be given together or separately, e.g. as constituents in the same pharmaceutical composition or formulation, or as separate formulations.

(113) Thus a peptide conjugate of the invention may be used in combination with an anti-diabetic agent of known type, including, but not limited to, Metformin, a sulfonylurea, a glinide, a DPP-IV inhibitor, a glitazone, or Insulin or an Insulin analogue. In a preferred embodiment, the peptide conjugate of the invention is administered in combination with Insulin or an analogue thereof, a DPP-IV inhibitor, sulfonylurea or Metformin, particularly sulfonylurea or Metformin, for achieving adequate glycemic control. In a more preferred embodiment, the peptide conjugate is administered in combination with Insulin or an Insulin analogue for achieving adequate glycemic control. Examples of appropriate Insulin analogues include, but are not limited to, Lantus, Novorapid, Humalog, Novomix, Actraphane HM, Levemir Degludec and Apidra. Other relevant anti-diabetic agents in this connection include GLP-1 receptor agonists, such as exenatide (Byetta; Exendin-4) and liraglutide (Victoza)

(114) A peptide conjugate of the invention may also be used in combination with an anti-obesity agent of known type, including, but not limited to, Peptide YY or an analogue thereof, Neuropeptide Y (NPY) or an analogue thereof, a cannabinoid receptor 1 antagonist, a lipase inhibitor, Human prolslet Peptide (HIP), a Melanocortin receptor 4 agonist or a Melanin concentrating hormone receptor 1 antagonist.

(115) A peptide conjugate of the invention may further be used in combination with an anti-hypertension agent of known type, including, but not limited to, an angiotensin-converting enzyme inhibitor, an Angiotensin II receptor blocker, a diuretic, a beta-blocker or a calcium channel blocker.

(116) A peptide conjugate of the invention may still further be used in combination with an anti-dyslipidemia agent of known type, including, but not limited to, a statin, a fibrate, a niacin and/or a cholesterol absorption inhibitor.

(117) A peptide conjugate of the invention may also be used in combination with a proton pump inhibitor (i.e. a pharmaceutical agent possessing pharmacological activity as an inhibitor H.sup.+/K.sup.+-ATPase) of known type, including, but not limited to, an agent of the benzimidazole derivative type or of the imidazopyridine derivative type, such as Omeprazole Lansoprazole, Dexlansoprazole, Esomeprazole, Pantoprazole Rabeprazole, Zolpidem, Alpidem, Saripidem or Necopidem.

(118) A peptide conjugate of the invention may, moreover, be used in combination with an anti-inflammatory agent of known type, including, but not limited to:

(119) steroids and corticosteroids, such as beclomethasone, methylprednisolone, betamethasone, prednisone, dexamethasone, and hydrocortisone; non-steroidal antiinflammatory agents (NSAIDs), such as propionic acid derivatives (e.g. alminoprofen, benoxaprofen, bucloxic acid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen, indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen, pranoprofen, suprofen, tiaprofenic acid and tioxaprofen); acetic acid derivatives (e.g. indomethacin, acemetacin, alclofenac, clidanac, diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac, isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin and zomepirac); fenamic acid derivatives (e.g. flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid); biphenylcarboxylic acid derivatives (e.g. diflunisal and flufenisal); oxicams (e.g. isoxicam, piroxicam, sudoxicam and tenoxicam); salicylates (e.g. acetylsalicylic acid and sulfasalazine); and pyrazolones (e.g. apazone, bezpiperylon, feprazone, mofebutazone, oxyphenbutazone and phenylbutazone);

(120) COX II inhibitors, such as rofecoxib and celecoxib; preparations of interferon beta (e.g. interferon beta-1a or interferon beta-1b);

(121) and certain other compounds, such as 5-aminosalicylic acid and prodrugs and pharmaceutically acceptable salts thereof.

(122) Metformin has also been demonstrated to have anti-inflammatory properties [see Haffner et al., Diabetes 54: 1566-1572 (2005)] and as such may also be useful in the present context

(123) Each of the cited publications and patent applications are incorporated herein by reference in its entirety The following examples demonstrate certain specific embodiments of the present invention. The following examples were carried out using standard techniques, which are well known and routine to those of skill in the art, except where otherwise described in detail. It is to be understood that these examples are for illustrative purposes only and do not purport to be wholly definitive as to conditions or scope of the invention. As such, they should not be construed in any way as limiting the scope of the present invention.

EXAMPLES

(124) Abbreviations employed in the examples include: NMP: N-methylpyrrolidone DCM: dichloromethane DMF: N,N-dimethylformamide

(125) HATU: 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate DIPEA: diisopropylethylamine EtOH: ethanol Et.sub.2O: diethyl ether Peg3: 8-amino-3,6-dioxaoctanoyl 8Aoc: 8-aminooctanoyl DBF: 4-(2-aminoethyl)-6-dibenzofuranpropanoyl TFA: trifluoroacetic acid MeCN: acetonitrile HPLC: high performance liquid chromatography MS: mass spectrometry IBMX: 3-isobutyl-1-methylxanthine BSA: bovine serum albumin cAMP: cyclic adenosine monophosphate DMEM: Dulbecco's Modified Eagle Medium FCS: fetal calf serum HEPES: N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid p-ERK: phosphorylated extracellular regulated kinase PBS: phosphate-buffered saline Boc: t-Butoxycarbonyl NEP: N-methylpyrrolidone Liraglutide: [Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP-1(7-37)

Example 1

Synthesis of Compounds and Peptide Properties

(126) Materials and Methods

(127) Unless otherwise specified, reagents and solvents employed in the following were available commercially in standard laboratory reagent or analytical grade, and were used without further purification.

(128) General Procedure for Synthesis of Peptide Conjugates of the Invention

(129) Solid phase peptide synthesis was performed on a CEM Liberty Peptide Synthesizer using standard Fmoc chemistry. TentaGel S Ram resin (1 g; 0.25 mmol/g) was swelled in NMP (10 ml) prior to use and transferred between tube and reaction vessel using DCM and NMP. Pseudoprolines, which are dipeptides employed to minimize aggregation during peptide synthesis, such as Fmoc-Phe-Thr(-Me,Me-Pro)-OH and Fmoc-Asp-Ser(-Me,Me-Pro)-OH, were used where appropriate, and the non-natural amino acids forming the Peg3, 8Aoc and DBF linker moieties (vide supra) were employed as Fmoc-protected amino acids (i.e. Fmoc-Peg3-OH, Fmoc-8Aoc-OH and Fmoc-DBF-OH, respectively), and without any changes to the general procedure.

(130) Coupling:

(131) An Fmoc-amino acid in NMP/DMF/DCM (1:1:1; 0.2 M; 5 ml) was added to the resin in a CEM Discover microwave unit together with HATU/NMP (0.5 M; 2 ml) and DIPEA/NMP (2.0 M; 1 ml). The coupling mixture was heated to 75 C. for 5 min while nitrogen was bubbled through the mixture. The resin was then washed with NMP (410 ml).

(132) Deprotection:

(133) Piperidine/NMP (20%; 10 ml) was added to the resin for initial deprotection, and the mixture was microwave-heated (40 C.; 30 sec.). The reaction vessel was drained and a second portion of piperidine/NMP (20%; 10 ml) was added and heated (75 C.; 3 min) again. The resin was then washed with NMP (610 ml).

(134) Cleavage:

(135) The resin was washed with EtOH (310 ml) and Et.sub.2O (310 ml) and dried to constant weight at room temperature (r.t.). The crude peptide was cleaved from the resin by treatment with TFA/ethanedithiol (95/5, 40 ml, 2 h; r.t.). Most of the TFA was removed under reduced pressure, and the crude peptide was precipitated and washed three times with Et.sub.2O and dried to constant weight at room temperature.

(136) Purification and Characterisation:

(137) The crude peptide was purified to greater than 90% purity by preparative reverse phase HPLC using a PerSeptive Biosystems VISION Workstation equipped with a suitable column and a fraction collector, and run with a gradient of buffer A (0.1% TFA, aq.) and buffer B (0.1% TFA, 90% MeCN, aq.). Fractions were analysed by analytical HPLC and MS, and relevant fractions were pooled and lyophilised. The final product was characterised by HPLC and MS.

(138) Synthesis Example

(139) Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17 (Compound 1) was synthesized on a CEM Liberty Peptide Synthesizer using TentaGel S Ram resin (0,67 g; 0.23 mmol/g) and Fmoc chemistry as described above. Fmoc-8-amino-3,6-dioxaoctanoic acid and pseudoprolines Fmoc-Phe-Thr(-Me,Me-Pro)-OH and Fmoc-Ser(tBu)-Ser(-Me,Me-Pro)-OH were employed.

(140) The peptide was cleaved from the resin as described above, and the purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 50% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (122 mg), which was analysed by analytical HPLC as being 58% pure.

(141) The product was purified again on a Luna column (125 cm; 5 m; C8) with a 4 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 50% buffer B over 47 min, and fractions (2 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to a give white powder (63 mg), which was analysed by analytical HPLC as being 82% pure.

(142) Another portion of Compound 1 was synthesized using TentaGel S Ram resin (0,70 g; 0.23 mmol/g) and otherwise the same conditions as described above for synthesis and cleavage.

(143) Purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 50% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (113 mg), which was analysed by analytical HPLC as being 57% pure.

(144) The product was purified again on a Luna column (125 cm; 5 m; C8) with a 4 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 55% buffer B over 47 min, and fractions (2 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (29 mg), which was analysed by analytical HPLC as being 77% pure.

(145) The products from the first synthesis (63 mg; 82%) and second synthesis (29 mg; 77%) were combined and purified once more on a Kromasil column (125 cm; 10 m; C8) with a 4 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 25% to 65% buffer B over 47 min, and fractions (2 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (33 mg), which was analysed by analytical HPLC as being 94% pure. The mass was 6553.39 Da as determined by MS (Calc. 6553.06 Da).

(146) Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 (Compound 33) was synthesized on a CEM Liberty Peptide Synthesizer using TentaGel S Ram resin (0,55 g; 0.23 mmol/g) and Fmoc chemistry as described above. Fmoc-8-amino-3,6-dioxaoctanoic acid and pseudoproline Fmoc-Phe-Thr(-Me,Me-Pro)-OH were employed.

(147) The peptide was cleaved from the resin as described above, and the purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 25% to 55% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (70 mg), which was analysed by analytical HPLC as being 90% pure. The mass was 4364.08 Da as determined by MS (Calc. 4364.11 Da).

(148) [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-[Leu4]Gastrin6 (Compound 101) was synthesized on a CEM Liberty Peptide Synthesizer using TentaGel S Ram resin (1,15 g; 0.25 mmol/g) and Fmoc chemistry as described above using Fmoc-Phe-Thr(-Me,Me-Pro)-OH. NEP was used instead of NMP during coupling and deprotection.

(149) The peptide was cleaved from the resin as described above, and the purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 20% to 50% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (50 mg), which was analysed by analytical HPLC as being 85% pure. The mass was 3952.00.08 Da as determined by MS (Calc. 3951.97 Da).

(150) GLP-1(7-36)-Peg3-Peg3-[Gln1,Leu15]Gastrin17 (Compound 42) was synthesized on a CEM Liberty Peptide Synthesizer using TentaGel S Ram resin (1,16 g; 0.23 mmol/g) and Fmoc chemistry as described above. Fmoc-8-amino-3,6-dioxaoctanoic acid and pseudoproline Fmoc-Ser(tBu)-Ser(Psi Me, Me pro)-OH were employed.

(151) The peptide was cleaved from the resin as described above, and the purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 25% to 45% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (172 mg), which was analysed by analytical HPLC as being 86% pure. The mass was 5664.72 Da as determined by MS (Calc. 5664.70 Da).

(152) [Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP-1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 (Compound 107) was synthesized on a CEM Liberty Peptide Synthesizer using TentaGel S Ram resin (1,30 g; 0.25 mmol/g) and Fmoc chemistry as described above. NEP was used instead of NMP during coupling and deprotection. Fmoc-8-amino-3,6-dioxaoctanoic acid and pseudoproline Fmoc-Phe-Thr(-Me,Me-Pro)-OH were employed as well as Fmoc-Lys(Dde)-OH at the point of attachment for the acylation.

(153) The N-terminal of the solid-phase attached peptide was Boc protected using Boc.sub.2O (330 mg) and DIPEA (54 l) in DCM. Then the Dde protection group was cleaved using hydrazine hydrate/NEP (4%; 215 min), and the resin was washed with NEP (82 min), DIEA/NEP (10%; 55 min) and NEP (82 min). The synthesis was completed on a CEM Liberty Peptide Synthesizer as described above using Fmoc-Glu-OtBu and hexa-decanoic acid. NEP was used instead of NMP during coupling and deprotection.

(154) The peptide was cleaved from the resin as described above, and the purification was performed on a Gemini-NX column (525 cm; 10 m; C18) with a 35 ml/min flow of a mixture of buffer A (0.1% TFA; aq.) and buffer B (0.1% TFA; 90% MeCN; aq.). The product was eluted with a linear gradient from 30% to 70% buffer B over 47 min, and fractions (9 ml) were collected with a fraction collector. Relevant fractions were analysed by analytical HPLC and MS, pooled and lyophilised to give a white powder (60 mg), which was analysed by analytical HPLC as being 88% pure. The mass was 4819.95 Da as determined by MS (Calc. 4819.45 Da).

Example 2

Activation (EC50) of GLP-1 Receptor and Gastrin CCK-B Receptor In Vitro by Peptide Conjugates of the Invention

(155) Materials and Methods

(156) Human GLP-1 Receptor (GLP-1 R) Efficacy Assay:

(157) In vitro effects of peptide conjugates of the invention were assessed by measuring the induction of cAMP following stimulation of the receptor by GLP-1(7-36), Exendin-4(1-39) or tested conjugates of the invention using the FlashPlate cAMP kit from Perkin-Elmer. Briefly, HEK293 cells expressing the human GLP-1 R (stable cell line generated through transfection of the cDNA for GLP-1 R and selection of stable clones) were seeded at 40,000 cells/well in 96-well microtiter plates coated with 0.01% poly-L-lysine, and grown for 1 day in culture in 100 l growth medium [DMEM, 10% FCS, Penicillin (100 IU/ml), Streptomycin (100 g/ml)]. On the day of analysis, growth medium was removed and the cells were washed once with 200 l Tyrode buffer [Tyrode's Salts (9.6 g/l), 10 mM HEPES, pH 7.4]. Cells were incubated in 100 l Tyrode buffer containing increasing concentrations of test compounds, 100 M IBMX, and 0.1% BSA for 15 min at 37 C. The reaction was stopped by addition of 25 l 0.5 M HCl and incubated on ice for 60 min. For further methodological details, see WO 2008/152403.

(158) CCK-B Receptor (CCK-B R) Efficacy Assay:

(159) To test binding and activation of CCK-B R we produced stable cell lines expressing one of the human or mouse CCK receptors in a manner similar to that for production of the hGLP-1 R cell lines were generated (vide supra). In brief, we used HEK293 cells for transfection of the cDNA for human or mouse CCK-A R or CCK-B R [hCCK-A R (gene identity: L19315), hCCK-B R (NM_176875), mCCK-A R (NM_009827) and mCCK-B R (NM_007627)] all cloned into the transfection plasmid pIRESneo2dNGFR. The cells were grown according to standard protocol in growth medium and transfected with the plasmids using Lipofectamin (Invitrogen). The cells stably expressing CCK receptors were selected using G418 in the growth medium (only cells that have taken up and incorporated the cDNA expression plasmid survive) and propagated. Stocks of cells were frozen for later use.

(160) In vitro effects of peptide conjugates of the invention were estimated by measuring p-ERK (using the AlphaScreen SureFire p-ERK assay) in HEK293 cells stably expressing the human and mouse CCK-B R (high-affinity Gastrin receptor), respectively. The Gastrin receptor efficacy assays (AlphaScreen SureFire p-ERK assay) were performed as follows:

(161) Day 1: Seeding of Cells

(162) The CCK-B R expressing cells in question were seeded at 20,000 cells/well in 100 l growth medium [DMEM, 10% FCS, Penicillin (100 IU/ml), Streptomycin (100 g/ml)] in a 96-well plate coated with poly-D-lysine. The cells were incubated in an incubator (37 C., 5% 002) for two days.

(163) Day 3: Change to Serum-free Medium

(164) The growth medium was changed to 80 l of serum-free medium [DMEM, Penicillin (100 IU/ml), Streptomycin (100 g/ml)] per well, and incubation of the cells was continued for 19 hours in the incubator (37 C., 5% CO.sub.2).

(165) Day 4: Peptide Conjugate Stimulation and AlphaScreen SureFire p-ERK Assay

(166) 1. After 19 hours, 20 l of serum-free medium containing one of 5 different concentrations of peptide conjugate was added (performed in triplicate for each concentration), and the cells were incubated for 5 min. at room temperature. 2. The stimulation medium was discarded by quickly turning the plate upside down, and 60 l 1 lysis buffer (from the SureFire assay kit) was added per well. 3. The plate was shaken on a plate-shaker for 5 min and then placed on ice. 4. SureFire P-ERK assay: 4 l of each supernatant was transferred to a 384 well proxiplate (Perkin Elmer). 5. 4 l of each of the two control lysates (unstimulated and stimulated) were added to the proxiplate in duplicate. 6. 60 parts reaction buffer, 10 parts activation buffer, 1 part acceptor beads and 1 part donor beads were mixed (reaction buffer+activation buffer+beads). 7 l of the latter reaction buffer+activation buffer+beads per well were added in the proxiplate, the mix being resuspended carefully before addition to the wells. 7. The plate was incubated for 2 hours in a dark box in a 22 C. incubator. 8. The plate was analyzed on an Envision light-emission plate reader (Perkin-Elmer) using the appropriate reading program (Perkin-Elmer).

(167) The peptide conjugates of the invention were tested in the above-described assays (i.e. human GLP-1 R activation efficacy, human CCK-B R activation efficacy and mouse CCK-B R activation efficacy). Human GLP-1(7-36) and Exendin-4(1-39) were used as positive controls in the human GLP-1 receptor (hGLP-1 R) activation efficacy assay, and h[Gln1,Leu15]Gastrin17 and CCK-8 (consisting of the C-terminal 8 active amino acid residues of CCK) were used as positive controls in the human CCK-B receptor (hCCK-B R) efficacy assay and the mouse CCK-B receptor (mCCK-B R) assay.

(168) In the present CCK receptor activation study, h[Gln1,Leu15]Gastrin17 (having the sequence H-QGPWLEEEEEAYGWLDF-NH.sub.2) was used as a control compound. The glutamine (Gln) residue may rear-range to some extent to PyroGlu, but without loss of receptor binding activity. The results (EC.sub.50 values, in mol/l) are summarized in Tables 1, 1a and 2, below.

(169) TABLE-US-00013 TABLE 1 in vitro efficacy (EC.sub.50, mol/l) of compounds (peptide conjugates) of the invention in activation of hGLP-1 R, hCCK-B R and mCCK-B R. Cpd hGLP-1 R hCCK-B R mCCK-B R No. Peptide conjugate EC.sub.50 EC.sub.50 EC.sub.50 1 Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17 5.68 10.sup.10 1.90 10.sup.8 5.40 10.sup.8 33 Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 7.74 10.sup.10 3.08 10.sup.8 7.15 10.sup.8 37 GLP-1(7-36)-[Gln1,Leu15]Gastrin17 1.45 10.sup.10 2.59 10.sup.8 7.31 10.sup.8 38 GLP-1(7-36)-K-[Gln1,Leu15]Gastrin17 8.42 10.sup.11 7.73 10.sup.8 1.14 10.sup.7 39 GLP-1(7-36)-AAA-[Gln1,Leu15]Gastrin17 1.14 10.sup.10 1.88 10.sup.8 5.41 10.sup.8 40 GLP-1(7-36)-SKK-[Gln1,Leu15]Gastrin17 1.16 10.sup.10 1.56 10.sup.8 5.86 10.sup.8 41 GLP-1(7-36)-Peg3-SKK-[Gln1,Leu15]Gastrin17 9.26 10.sup.11 8.76 10.sup.9 5.11 10.sup.8 42 GLP-1(7-36)-Peg3-Peg3-[Gln1,Leu15]Gastrin17 7.78 10.sup.11 8.20 10.sup.9 5.83 10.sup.8 43 GLP-1(7-36)-8Aoc-SKK-[Gln1,Leu15]Gastrin17 1.56 10.sup.10 5.19 10.sup.8 8.25 10.sup.8 44 GLP-1(7-36)-DBF-SKK-[Gln1,Leu15]Gastrin17 1.37 10.sup.10 2.57 10.sup.8 8.69 10.sup.8 45 GLP-1(7-36)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17 1.22 10.sup.10 1.28 10.sup.8 7.14 10.sup.8 Control GLP-1(7-36) 1.66 10.sup.11 Control Exendin-4(1-39) 1.63 10.sup.10 Control h[Gln1,Leu15]Gastrin17 4.69 10.sup.9 1.82 10.sup.8 Control CCK-8 6.1 10.sup.9 1.63 10.sup.7

(170) TABLE-US-00014 TABLE 1a In vitro efficacy (EC.sub.50, mol/l) of compounds (peptide conjugates) of the invention in activation of hGLP-1 R, hCCK-B R and mCCK-B R. Cpd. hGLP-1 R hCCK-B R mCCK-B R No. Peptide conjugate EC.sub.50 EC.sub.50 EC.sub.50 1 Exendin-4(1-39)-Peg3-Peg3- 2.7 10.sup.11 8.0 10.sup.9 5.4 10.sup.8 [Gln1,Leu15]Gastrin17 33 Exendin-4(1-28)-Peg3-Peg3- 2.8 10.sup.11 1.1 10.sup.8 7.1 10.sup.8 [Leu4]Gastrin6

(171) TABLE-US-00015 TABLE 2 In vitro efficacy (EC.sub.50, mol/l) of further compounds (peptide conjugates) of the invention in activation of hGLP-1 R and hCCK-B R. Cpd. hGLP-1 R hCCK-B R No. Peptide conjugate EC.sub.50 EC.sub.50 55 Exendin-4(1-28)-Peg3-Peg3-[Leu3]Gastrin5 4.2 10.sup.11 7.4 10.sup.9 56 Exendin-4(1-28)-Peg3-Peg3-[Ala1,Leu4]Gastrin6 2.2 10.sup.11 9.1 10.sup.9 57 Exendin-4(1-28)-Peg3-Peg3-[Ala2,Leu4]Gastrin6 3.3 10.sup.11 7.2 10.sup.9 58 Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6 3.3 10.sup.11 96 10.sup.9 59 Exendin-4(1-28)-Peg3-Peg3-[Leu2]Gastrin4 2.5 10.sup.11 9.00 10.sup.9 60 [Leu14]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 4.1 10.sup.11 6.3 10.sup.9 61 [Orn12]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 2.8 10.sup.11 5.8 10.sup.9 62 [Orn27]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 3.1 10.sup.11 5.6 10.sup.9 63 [Phe25]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 4.1 10.sup.11 8.5 10.sup.9 64 [Asp28]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 6.1 10.sup.11 3.3 10.sup.9 65 [Tyr13]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 4.6 10.sup.11 1.2 10.sup.8 66 [Orn20]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 3.8 10.sup.11 1.5 10.sup.8 67 Exendin-4(1-28)-Peg3-[Leu4]Gastrin6 5.0 10.sup.11 1.0 10.sup.8 68 Exendin-4(1-28)-[Leu4]Gastrin6 6.1 10.sup.11 1.4 10.sup.8 69 Exendin-4(1-27)-[Leu4]Gastrin11 1.0 10.sup.10 4.3 10.sup.9 70 Exendin-4(1-27)-Peg3-[Leu4]Gastrin6 1.4 10.sup.11 5.3 10.sup.9 71 Exendin-4(1-27)-Peg3-[Leu3]Gastrin5 2.9 10.sup.11 4.1 10.sup.9 72 Exendin-4(1-26)-Peg3-[Leu3]Gastrin5 1.4 10.sup.9 3.2 10.sup.9 73 Exendin-4(1-27)-Peg3-[Leu2]Gastrin4 4.1 10.sup.11 8.1 10.sup.9 74 [Tyr13,Leu14]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6 5.0 10.sup.11 8.4 10.sup.9 75 [Tyr13,Phe25]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6 3.5 10.sup.11 8.4 10.sup.9 76 [Leu14,Phe25]Exendin-4(1-27)-Peg3-Peg3-[Leu4]Gastrin6 2.6 10.sup.11 7.9 10.sup.9 77 [Tyr13,Leu14,Phe25]Exendin-4(1-27)-Peg3-Peg3- 4.4 10.sup.11 9.1 10.sup.9 [Leu4]Gastrin6 78 Side chain-cyclo([Lys12,Glu16]Exendin-4(1-28)-Peg3- 2.5 10.sup.11 5.9 10.sup.9 Peg3-[Leu4]Gastrin6 79 Side chain-cyclo([Glu16,Lys20]Exendin-4(1-28)-Peg3- 2.6 10.sup.11 5.3 10.sup.9 Peg3-[Leu4]Gastrin6 80 Side chain-cyclo([Lys20,Glu24]Exendin-4(1-28)-Peg3- 3.2 10.sup.11 4.0 10.sup.9 Peg3-[Leu4]Gastrin6 81 [Lys16]Exendin-4(1-28)-Peg3-Peg3-[Leu4]Gastrin6 3.2 10.sup.11 1.5 10.sup.8 82 Exendin-4(1-28)-Peg3-K-Peg3-[Leu4]Gastrin6 2.8 10.sup.11 2.14 10.sup.8 83 Exendin-4(1-28)-[Thr4]Gastrin6 4.4 10.sup.11 5.8 10.sup.8 84 Exendin-4(1-28)-[Phe4]Gastrin6 4.4 10.sup.11 1.4 10.sup.7 85 [Leu14]Exendin-4(1-28)-[1Nal3,Leu4]Gastrin6 4.0 10.sup.11 2.7 10.sup.7 86 [Leu14]Exendin-4(1-28)-[Nle4]Gastrin6 5.2 10.sup.11 2.4 10.sup.8 87 [Leu14]Exendin-4(1-28)-[Leu4,[3-(3-Pyridyl)- 2.8 10.sup.11 2.0 10.sup.8 Ala]6]Gastrin6 88 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3- 4.6 10.sup.11 2.0 10.sup.8 [Leu4]Gastrin6 89 [Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3- 2.3 10.sup.11 1.2 10.sup.6 [Leu4,Phe3]Gastrin6 90 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Peg3- 3.2 10.sup.11 1.3 10.sup.6 [Leu4,Phe3]Gastrin6 91 [Arg27,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3- 6.8 10.sup.11 1.8 10.sup.8 [Leu4]Gastrin6 92 [Arg12,27,Leu14,Lys16,Phe25,Tyr13]Exendin-4(1-27)- 1.7 10.sup.10 2.6 10.sup.8 Peg3-[Leu4]Gastrin6 93 [Arg12,27,Leu14,Lys20,Phe25,Tyr13]Exendin-4(1-27)- 5.8 10.sup.11 2.6 10.sup.8 Peg3-[Leu4]Gastrin6 94 [Arg12,27,Leu14,Lys24,Phe25,Tyr13]Exendin-4(1-27)- 1.3 10.sup.10 3.6 10.sup.8 Peg3-[Leu4]Gastrin6 95 [Arg12,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3- 7.9 10.sup.11 1.4 10.sup.8 [Leu4]Gastrin6 96 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)- 1.2 10.sup.9 2.11 10.sup.7 [Leu2]Gastrin4 97 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3- 6.0 10.sup.11 1.3 10.sup.8 [Leu2]Gastrin4 98 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Peg3- 4.6 10.sup.11 1.0 10.sup.8 [Leu2]Gastrin4 99 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Orn- 4.1 10.sup.11 5.1 10.sup.8 [Leu2]Gastrin4 100 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Orn- 1.1 10.sup.10 8.6 10.sup.7 [Leu2]Gastrin4 101 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)- 1.1 10.sup.10 3.0 10.sup.8 [Leu4]Gastrin6 102 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3- 9.1 10.sup.11 5.4 10.sup.8 [Leu4]Gastrin6 103 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Peg3- 1.2 10.sup.10 1.0 10.sup.7 [Leu4]Gastrin6 104 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Peg3-Orn- 8.7 10.sup.11 8.8 10.sup.9 [Leu4]Gastrin6 105 [Glu9,Leu14,Phe25,Tyr13]Exendin-4(1-27)-Orn-Orn- 1.1 10.sup.10 1.6 10.sup.8 [Leu4]Gastrin6

(172) TABLE-US-00016 TABLE 3 In vitro efficacy (EC.sub.50, mol/l) of GLP-1 compounds (peptide conjugates) of the invention in activation of hGLP-1 R, hCCK-B R and mCCK-B R. (Only included in the GLP-1 application) Cpd. hGLP-1 R hCCK-B R mCCK-B R No. Peptide conjugate EC50 EC50 EC50 37 GLP-1(7-36)-[Gln1,Leu15]Gastrin17 3.0 10.sup.11 2.6 10.sup.8 7.3 10.sup.8 38 GLP-1(7-36)-K-[Gln1,Leu15]Gastrin17 3.0 10.sup.11 7.7 10.sup.8 1.1 10.sup.7 39 GLP-1(7-36)-AAA-[Gln1,Leu15]Gastrin17 3.4 10.sup.11 1.9 10.sup.8 5.4 10.sup.8 40 GLP-1(7-36)-SKK-[Gln1,Leu15]Gastrin17 3.1 10.sup.11 1.6 10.sup.8 5.9 10.sup.8 41 GLP-1(7-36)-Peg3-SKK-[Gln1,Leu15]Gastrin17 2.3 10.sup.11 8.8 10.sup.9 5.1 10.sup.8 42 GLP-1(7-36)-Peg3-Peg3-[Gln1,Leu15]Gastrin17 2.8 10.sup.11 8.2 10.sup.9 5.8 10.sup.8 43 GLP-1(7-36)-8Aoc-SKK-[Gln1,Leu15]Gastrin17 4.9 10.sup.11 5.2 10.sup.8 8.3 10.sup.8 44 GLP-1(7-36)-DBF-SKK-[Gln1,Leu15]Gastrin17 5.3 10.sup.11 2.6 10.sup.8 8.7 10.sup.8 45 GLP-1(7-36)-8Aoc-8Aoc-[Gln1,Leu15]Gastrin17 4.2 10.sup.11 1.3 10.sup.8 7.1 10.sup.8 Control GLP-1(7-36) 1.7 10.sup.11 N.T. N.T. Control Exendin-4(1-39) 2.4 10.sup.11 N.T. N.T. Control h[Gln1,Leu15]Gastrin17 N.T 4.7 10.sup.9 1.8 10.sup.8 Control CCK-8 N.T 6.3 10.sup.9 1.6 10.sup.7 Control h[Leu15]Gastrin17 N.T 2.4 10.sup.9 N.T 106 [Lys(Hexadecanoyl-isoGlu)34]GLP-1(7-37)- 4.9 10.sup.11 1.8 10.sup.8 Peg3-Peg3-[Leu4]Gastrin6 107 [Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP- 1.4 10.sup.10 2.9 10.sup.8 1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 108 [Arg26,34,Lys(Hexadecanoyl-isoGlu)36]GLP- 1.1 10.sup.10 7.8 10.sup.8 1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 109 [Lys(Hexadecanoyl-isoGlu)26]GLP- 9.9 10.sup.11 2.6 10.sup.8 1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 110 [Arg26,34,Gly8,Lys(Hexadecanoyl-isoGlu)36]GLP- 1.2 10.sup.10 2.9 10.sup.8 1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 111 [Aib8,Arg34,Lys(Hexadecanoyl-isoGlu)26]GLP- 8.8 10.sup.11 5.9 10.sup.8 1(7-37)-Peg3-Peg3-[Leu4]Gastrin6 112 [Aib8,Arg34]GLP-1(7-37)-Peg3-Peg3- 4.3 10.sup.11 4.2 10.sup.8 [Leu4]Gastrin6 113 [Arg34]GLP-1(7-37)-Peg3-Peg3- 2.7 10.sup.11 2.4 10.sup.8 [Leu4]Gastrin6 N.T: Not tested
Results

(173) The results summarized in Tables 1, 1a, 2 and 3 above indicate that generally all of the peptide conjugates of the invention are potent agonists of all of the three receptors in question, and that they exhibit closely similar levels of efficacy.

Example 3

Screening of Compound 73 on 92 Selected Peptide GPCRs

(174) Compound 73 was selected for testing on a large selection of peptide receptors of the GPCR type, in order to discover any receptor promiscuity early on. The receptors are of the class A and B GPCR families and the assay was carried out at Millipore using their GPCR screening platform. Each GPCR was activated by its control peptide ligand (known activator of the respective receptors) or by Compound 73 at 100 nM concentration. Agonism on a receptor is given as % of the control peptide (that gives 100% activation by definition). Only the expected GLP-1 receptor and the CCKB (CCK2) receptor was activated significantly (100 and 95%, respectively) by Compound 73 showing that the peptide is specific for these two receptors.

Example 4

Pharmacokinetic (PK) of Compound 1 and 33 in Mice

(175) Method

(176) Three C57Bl mice were given 100 nmol of compound 1 or 33 per kg as i.v. or s.c. bolus, and plasma samples were collected up to 240 min post-dose. Samples were collected from three mice at each time point. The plasma samples were analyzed for the presence of compound 33 using LC/MS/MS (10-1000 nM).

(177) Results

(178) TABLE-US-00017 TABLE 4 PK parameters after i.v. and s.c. administration of 100 nmol/kg to mice Compound 33 Compound 1 Parameter Unit s.c. i.v. s.c. i.v. t.sub.1/2 hr 3.4 2.8 0.45 0.28 F % 100% 97%

(179) Both compounds exhibited an excellent bioavailability (F) after sc administration, with compound 33 showing a superior PK profile likely due to a longer half-life (FIG. 1A, 1B and Table 4).

Example 5

PK of Compound 33 and 74-80

(180) Method

(181) Two C57Bl mice were given a single subcutaneous dose of 100 nmol/kg of each peptide. Blood samples were taken after 5 and 30 min and after 1, 2, 4, 6, 16 and 24 hour. At each time point, samples from two mice were taken. Plasma samples were analyzed after solid phase extraction (SPE) by liquid chromatography mass spectrometry (LC-MS/MS).

(182) TABLE-US-00018 TABLE 5 T after s.c. administration of 100 nmol/kg to mice Compound t.sub.1/2 (h) 33 2.0 74 3.6 76 2.3 77 3.1 75 2.5 78 2.0 79 1.5 80 1.9

(183) The peptides show promising T profiles, with compound 74 and 77 being superior to the rest with respect to half-life and exposure (Table 5 and FIG. 2).

Example 6

In Vivo Activity of Peptide Conjugates of the Invention in Db/Db Mice

(184) Materials and Methods

(185) The db/db mouse model has previously been used to assess the -cell preserving effects of potential therapeutic candidates [Rolin, B. et al., Am. J. Physiol. Endocrinol. Metab. 283: E745-E752 (2002)]. Several studies have demonstrated a correlation between pancreatic Insulin content and -cell mass [Rolin, B. et al. (loc.cit.); Suarez-Pinzon, W. L. et al., Diabetes 54: 2596-2601 (2005); Suarez-Pinzon W. L. et al., Diabetes 57: 3281-3288 (2008)].

(186) In the present study, 6 week old db/db (BKS.Cg-m Lepr.sup.db/J) female mice (Taconic Europe A/S, Lille Skensved, Denmark) were acclimatized to their new environment and given ad libitum access to normal chow and water. Mice were housed in pairs in a light-, temperature- and humidity-controlled room. The progression of diabetes was followed for 2 weeks by monitoring blood glucose levels, and then before treatment the diabetic mice were randomized according to their blood glucose levels into treatment groups (n=10/group). Animals were then mock-injected subcutaneously (sc) with 100 l vehicle (once daily) for a period of three days to acclimatize the animals to handling and injections. Following randomization and mock injection, animals were then treated (sc, twice daily) for 16 days with combinations of h[Leu15]Gastrin17 (1, 10 and 50 nmol/kg) and Exendin-4(1-39) (1, 10 and 50 nmol/kg), or with Compound 1 (peptide conjugate of the invention) [i.e. Exendin-4(1-39)-Peg3-Peg3-[Gln1,Leu15]Gastrin17] (1, 10 and 50 nmol/kg), or with vehicle (PBS buffer; injection volume 5 ml/kg). Daily injections took place between 8:00 and 9:00 hours, and between 15:00 and 16:00 hours, with fresh solutions prepared immediately before dosing. Blood samples (200 l) were obtained from the orbital plexus and placed in EDTA coated tubes before dosing (day 1), and at day 8 and day 16 of the treatment. Each blood sample was centrifuged, and plasma (100 l) was stored at 80 C. for later analysis. Blood samples for blood glucose determinations were taken from the tail vein. Following the last day of dosing, all animals were sacrificed (day 16) by CO.sub.2 anesthesia, followed by cervical dislocation. The pancreas from each animal was immediately isolated, weighed, and stored for later analysis of Insulin content.

(187) Measurements

(188) Whole blood glucose concentration (mM) was determined by the immobilized glucose oxidase method (Elite Autoanalyser, Bayer, Denmark). Plasma C-peptide was determined using a rat C-peptide radioimmunoassay kit (Linco/Millipore, kit RCP-21 K). Pancreatic Insulin content was determined using a rat Insulin radioimmunoassay kit (Linco/Millipore, kit R1-13).

(189) Results

(190) As is clearly apparent from FIG. 3, a markedly higher content of pancreatic Insulin was seen in animals treated with the peptide conjugate of the invention (Compound 1) compared to that in animals treated with a simple combination of Exendin-4(1-39) and h[Leu15]Gastrin17 or treated with vehicle.

(191) It thus appears that the effect on pancreatic Insulin levels arising as a result of the covalent coupling or linkage (conjugation) of the Exendin-4 moiety and the Gastrin moiety in the peptide conjugate of the invention may be unexpectedly greater than that achieved when employing a corresponding, additive combination of the two, individual peptide components.

Example 7

Six Week Study

(192) Protocol

(193) 125 db/db (BKS.Cg-m+/+Lepr.sup.db/J) female mice (6 weeks at arrival) were obtained from Taconic Europe A/S.

(194) At day 4 blood was collected from semi-fasted animals for determination of baseline plasma C-peptide, plasma insulin, blood glucose, and HbA1c levels. Animals were then stratified into 5 treatment groups of n=20 based on baseline plasma C-peptide and HbA1c levels. Animals were injected s.c. with 100 l of vehicle twice daily for at least 3 days to acclimatize the animals to handling and experimental procedures.

(195) Then animals were injected s.c. twice daily with peptides or vehicle for a total of 42 days according to Table 6. The daily injections took place between 08:00-09:00 h and 15:00-16:00 h with freshly prepared solutions. The last day of dosing was day 42 in the morning.

(196) TABLE-US-00019 TABLE 6 Groups and doses Dose Groups Substance Route (nmol/kg/day) Group 1 Vehicle (PBS) SC twice Group 2 Exendin-4 daily 2 25 Group 3 Exendin-4 + 2 25 + 2 25 Gastrin17 Group 4 Compound 33 2 25

(197) The study was terminated on day 42. Animals were semi-fasted, and they received the final dose in the morning. Blood was sampled for determination of plasma C-peptide, plasma insulin, blood glucose, and HbA1c. After the blood sampling, animals were euthanized using CO.sub.2 followed by cervical dislocation. The pancreas was isolated, weighed, divided into 3 pieces, and transferred to tubes containing 2 ml of cold acidic alcohol and analyzed for insulin content.

(198) Compound 33 lowered blood glucose levels (FIG. 4) and elevated plasma insulin concentrations relative to vehicle in the db/db mice (FIG. 5). In addition, treatment with Compound 33 caused a statistically significant reduction in HbA1c levels compared to vehicle-treated and Gastin17+Exendin-4 treated animals (FIG. 6). These results suggest that Compound 33 improved glycemic control in the diabetic mice.

(199) Also, Compound 33 caused a statistically significant increase in pancreatic insulin content relative to vehicle (FIG. 7). In addition both Compound 33 and Exendin-4 caused a significant increase in the delta C-peptide level in plasma, indicating an improved pancreatic function in all groups (FIG. 8). Co-administration of Exendin-4 and Gastrin was not superior to Exendin-4 in improving glycemic control in the db/db mice. Thus, in the doses used in this study there was no synergistic effect of Exendin-4 and Gastrin on glycemia.

(200) Our data show that the peptide conjugate Compound 33 increases the pancreatic insulin content and improves glycemic control in the db/db mice significantly as evident by the decrease in HbA1c.

Example 8

Drug Holiday Study

(201) Protocol

(202) 150 male db/db mice were obtained at an age of 5-6 weeks. The animals were housed (5 mice/cage) under controlled conditions (20-22 C., 55-85% humidity) following a 12:12-hrs light/dark cycle with light on at 05.00 AM. The animals were fed ad libitum with standard Altromin No. 1324 diet and had free access to acidified tap water. At the time of study start the animals were 8-9 weeks old. All animals were acclimatized and handled daily minimum one week prior to the experiment.

(203) Blood Samples: Before treatment start, and on day 93 (before termination) in fasted mice (17 hrs) a blood sample (150 l) was obtained from orbital plexus with an EDTA coated micro-pipette. Blood samples were taken into EDTA coated tubes and kept on ice. The blood sample was centrifuged and the resulting plasma (at least 50 l) was stored (at 80 C.) for later analysis of C-peptide and insulin level. Also, on day 10/12 (before treatment start), and day 93 (before termination) a blood sample (50 l) obtained from orbital plexus was analyzed for BG (sticks) and HbA1c.

(204) Stratification

(205) On days 6 to 4 before the first drug dose, fasted animals (17 hrs) were subjected to an oral glucose tolerance test (OGTT, see below). The area under the blood glucose concentration curve obtained over a 240-minute period (AUC.sub.0-240; unit: mM*min) was used to stratify animals into 5 groups (A-E) of 26 animals each in order to obtain similar glucose tolerances in both groups. After the first 50 days of dosing (period 1) a second OGTT were performed. On the basis of this second OGTT test, each group of mice was stratified by AUC (as above) into two sub-groups displaying similar glucose tolerances.

(206) Dosing: The animals were given one daily (QD) subcutaneous (SC) dose of vehicle (2*n=26), Exendin-4 (n=26) or Compound 33 (n=26) and dosed according to Table 6 for a period of 50 days. Dosing was performed between 02.00 and 03.00 PM every day, with an injection volume of 5 ml/kg. After 50 days of dosing the animals were stratified into 7 groups as illustrated in Table 1. This dosing regimen was continued for 40 days until animals were sacrificed on day 93.

(207) Oral Glucose Tolerance Test (OGTT): OGTT was performed on days 6/4, 50, 65, 78 and 91 of the treatment period on animals fasted overnight (17 hours) after the last injection of vehicle or compound. Blood samples were taken from the tip of the tail and blood glucose measured. To prevent confounding food intake, the animals were kept fasted during all OGTTs. Immediately after the initial blood sample (t=0, fasting blood glucose level) an oral dose (1 g/kg) of glucose (Glucose-monohydrat, SAD 500 g/l), dissolved MQ water was given (5 ml/kg, 0.2 g/ml), and the animals were returned to their home cages (t=0). Then, BG levels were measured at t=15, 30, 60, 120, and 240 minutes.

(208) Fasting blood glucose: To further monitor the diabetic status of the animals, fasting blood glucose levels were measured after 8 hours of fasting on day 0, 37, 44, 58, 72, and 85. To minimize stress, animals were fasted during the day (from 06.00 AM when habitual consumption of food was low), and fasting blood glucose was determined at 02.00 PM.

(209) TABLE-US-00020 TABLE 6 Study groups Substance Substance Dose Period 1 Period 2 Route (nmol/kg/day) Vehicle Vehicle SC once 0 + 0 Vehicle Exendin-4 daily 0 + 100 Vehicle Compound 33 0 + 100 Vehicle Liraglutide 0 + 100 Exendin-4 Exendin-4 100 + 100 Exendin-4 Vehicle 100 + 0 Liraglutide Liraglutide 100 + 100 Liraglutide Vehicle 100 + 0 Compound 33 Compound 33 100 + 100 Compound 33 Vehicle 100 + 0 Vehicle: PBS: Phosphate buffered saline Gibco (#70011, pH = 7.4).

(210) The GLP-1-Gastrin dual agonist Compound 33 lowered the area under the glucose curve (AUC) following an oral glucose challenge test and also the fasting blood glucose was decreased compared to vehicle control regardless if the treatment paradigm was prevention, treatment or holiday (FIGS. 9A-9C and 10A-10C). Also, plasma levels of insulin and C-peptide and blood levels of HbA1c were significantly lower in mice treated with Compound 33 compared to those treated with vehicle control (FIGS. 11A-11C). These data shows that Compound 33 improves glycemic control in diabetic db/db mice, and to a greater extent than does both Exendin-4 and Liraglutide. Notably, the effect of the compounds on glycemic control was sustained for several days after treatment was stopped, most pronounced in the mice treated with Compound 33.