Incretin analogs and uses thereof

Abstract

Incretin analogs are provided that have activity at each of the GIP, GLP-1 and glucagon receptors. The incretin analogs have structural features resulting in balanced activity and extended duration of action at each of these receptors. Methods also are provided for treating diseases such as diabetes mellitus, dyslipidemia, fatty liver disease, metabolic syndrome, non-alcoholic steatohepatitis and obesity.

Claims

1. An incretin analog comprising: TABLE-US-00030 YX.sub.2QGTFTSDYSIX.sub.13LDKX.sub.17AX.sub.19X.sub.20AFIEYLLX.sub.28X.sub.29GPSSX.sub.34APPPS, wherein: X.sub.2 is Aib, X.sub.13 is L or αMeL, X.sub.17 is any amino acid with a functional group available for conjugation selected from the group consisting of K, C, E and D, and the functional group is conjugated to a C.sub.16-C.sub.22 fatty acid, X.sub.19 is Q or A, X.sub.20 is Aib, αMeK, Q or H, X.sub.28 is E or A, X.sub.29 is G or Aib, X.sub.34 is G or Aib, (SEQ ID NO:5), and wherein the C-terminal amino acid is optionally amidated; or a pharmaceutically acceptable salt thereof.

2. The incretin analog of claim 1, wherein the amino acid with the functional group available for conjugation at position X.sub.17 is K.

3. The incretin analog of claim 1, wherein the amino acid with the functional group available for conjugation at position X.sub.17 and the C.sub.16-C.sub.22 fatty acid are conjugated by a linker between the amino acid and the fatty acid.

4. The incretin analog of claim 3, wherein the linker comprises one to four amino acids.

5. The incretin analog of claim 4, wherein the amino acids are Glu or γGlu.

6. The incretin analog of claim 3, wherein the linker further comprises a structure of: H—{NH—CH.sub.2—CH.sub.2[O—CH.sub.2—CH.sub.2].sub.m—O—(CH.sub.2).sub.p—CO}.sub.n—OH, wherein m is any integer from 1 to 12, n is any integer from 1 to 12, and p is 1 or 2.

7. The incretin analog of claim 3, wherein the linker further comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

8. The incretin analog of claim 1, wherein X.sub.17 is a K chemically modified through conjugation to an epsilon-amino group of a K side-chain with the following structure: (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.a-(γGlu).sub.b-CO—(CH.sub.2).sub.c—CO.sub.2H, wherein a is 0, 1 or 2; b is 1 or 2; and c is an integer between 16 to 18.

9. The incretin analog of claim 8, wherein a is 1.

10. The incretin analog of claim 8, wherein b is 1.

11. The incretin analog of claim 8, wherein c is 18.

12. The incretin analog of claim 1, wherein X.sub.13 is αMeL.

13. The incretin analog of claim 1, wherein X.sub.20 is Aib.

14. The incretin analog of claim 1, wherein X.sub.28 is E.

15. The incretin analog of claim 1, wherein X.sub.29 is G.

16. The incretin analog of claim 1, wherein X.sub.34 is G.

17. The incretin analog of claim 1, wherein X.sub.19 is Q.

18. The incretin analog of claim 8, wherein a is 1, b is 1, c is 18, X.sub.13 is αMeL, X.sub.19 is Q, X.sub.20 is Aib, X.sub.28 is E, X.sub.29 is G and X.sub.34 is G.

19. An incretin analog having a formula selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21 SEQ ID NO:23 and SEQ ID NO:25, or a pharmaceutically acceptable salt thereof.

20. A method of treating a disease selected from the group consisting of diabetes mellitus, obesity, fatty liver disease, non-alcoholic steatohepatitis, dyslipidemia and metabolic syndrome, the method comprising a step of: administering to an individual in need thereof an effective amount of an incretin analog of claim 1.

21. The method of claim 20, wherein the disease is obesity or type II diabetes mellitus.

22. A pharmaceutical composition comprising: an incretin analog of claim 1; and a pharmaceutically acceptable carrier, diluent or excipient.

23. The method of claim 20, wherein the disease is obesity.

24. The method of claim 20, wherein the disease is type II diabetes mellitus.

25. The incretin analog of claim 19, having a formula of SEQ ID NO: 17, or a pharmaceutically acceptable salt thereof.

26. An incretin analog having a formula ##STR00026## (SEQ ID NO:17) or a pharmaceutically acceptable salt thereof.

Description

EXAMPLE 1

(1) Example 1 is a compound represented by the following description:

(2) TABLE-US-00001 (SEQ ID NO: 7) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-2-amino- ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ- αMeK-AFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2.

(3) Below is a depiction of the structure of Example 1 (SEQ ID NO:7) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17, αMeK20 and Aib29, where the structures of these amino acid residues have been expanded:

(4) ##STR00006##

(5) The peptide backbone of Example 1 is synthesized using Fluorenyimethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-Channel Multiplex Peptide Synthesizer (Protein Technologies, Inc. Tucson, Ariz.).

(6) The resin consists of 1% DVB cross-linked polystyrene (Fmoc-Rink-MBHA Low Loading Resin, 100-200 mesh, EMD Millipore) at a substitution of 0.3-0.4 meq/g. Standard side-chain protecting groups are used. Fmoc-Lys(Mtt)-OH) is used for the lysine at position 17, and Boc-Tyr(tBu)-OH) is used for the tyrosine at position 1. Fmoc groups are removed prior to each coupling step (2×7 minutes) using 20% piperidine in DMF. All standard amino acid couplings are performed for 1 hour to a primary amine and 3 hour to a secondary amine, using an equal molar ratio of Fmoc amino acid (0.3M), diisopropylcarbodiimide (0.9M) and Oxyma (0.9M), at a 9-fold molar excess over the theoretical peptide loading. Exceptions are couplings to Cα-methylated amino acids, which are coupled for 3 hours. After completion of the synthesis of the peptide backbone, the resin is thoroughly washed with DCM for 6 times to remove residual DMF. The Mtt protecting group on the lysine at position 17 is selectively removed from the peptide resin using two treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (2×40-minute treatment).

(7) Subsequent attachment of the fatty acid-linker moiety is accomplished by coupling of 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid α-t-butyl ester (Fmoc-Glu-OtBu, Ark Pharm, Inc.), mono-OtBu-eicosanedioic acid (WuXi AppTec, Shanghai, China). 3-fold excess of reagents (AA:PyAOP:DIPEA=1:1:1 mol/mol) are used for each coupling that is 1-hour long.

(8) After the synthesis is complete, the peptide resin is washed with DCM, and then thoroughly air-dried. The dry resin is treated with 10 mL of cleavage cocktail (trifluoroacetic acid:water:triisopropylsilane, 95:2.5:2.5 v/v) for 2 hours at room temperature. The resin is filtered off, washed twice each with 2 mL of neat TFA, and the combined filtrates are treated with 5-fold cold diethyl ether (−20° C.) to precipitate the crude peptide. The peptide/ether suspension is then centrifuged at 3500 rpm for 2 min to form a solid pellet, the supernatant is decanted, and the solid pellet is triturated with ether two additional times and dried in vacuo. The crude peptide is solubilized in 20% acetonitrile/20% acetic acid/60% water and purified by RP-HPLC on a Luna 5 μm Phenyl-Hexyl Preparative Column (21×250 mm, Phenomenex) with linear gradients of 100% acetonitrile and 0.1% TFA/water buffer system (30-50% acetonitrile in 60 min). The purity of peptide is assessed using analytical RP-HPLC and pooling criteria is >95%. The main pool purity of Example 1 is found to be 98.8%. Subsequent lyophilization of the final main product pool yields the lyophilized peptide TFA salt. The molecular weight is determined by LC-MS (obsd: M+4H+/4=1226.8; Calc M+4H+/4=1226.9).

EXAMPLE 2

(9) Example 2 is a compound represented by the following description:

(10) TABLE-US-00002 (SEQ ID NO: 6) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- ethoxy)-ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H) AQHAFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2.

(11) Below is a depiction of the structure of Example 2 (SEQ ID NO:6) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17 and Aib29, where the structures of these amino acid residues have been expanded:

(12) ##STR00007##

(13) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 2.

EXAMPLE 3

(14) Example 3 is a compound represented by the following description:

(15) TABLE-US-00003 (SEQ ID NO: 8) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ- αMeK-AFIEYLLEGGPSSGAPPPS-NH.sub.2.

(16) Below is a depiction of the structure of Example 3 (SEQ ID NO:8) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17, and αMeK20, where the structures of these amino acid residues have been expanded:

(17) ##STR00008##

(18) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 3.

EXAMPLE 4

(19) Example 4 is a compound represented by the following description:

(20) TABLE-US-00004 (SEQ ID NO: 9) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib- AFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2.

(21) Below is a depiction of the structure of Example 4 (SEQ ID NO:9) using the standard single letter amino acid codes with the exception of residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(22) ##STR00009##

(23) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, conjugate the fatty acid-linker moiety, examine the purity and confirm the molecular weight of Example 4.

EXAMPLE 5

(24) Example 5 is a compound represented by the following description:

(25) TABLE-US-00005 (SEQ ID NO: 10) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H) AAQAFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2.

(26) Below is a depiction of the structure of Example 5 (SEQ ID NO:10) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17, and Aib29, where the structures of these amino acid residues have been expanded:

(27) ##STR00010##

(28) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, conjugate the fatty acid-linker moiety, examine the purity and confirm the molecular weight of Example 5.

EXAMPLE 6

(29) Example 6 is a compound represented by the following description:

(30) TABLE-US-00006 (SEQ ID NO: 11) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H) AAQAFIEYLLEGGPSSGAPPPS-NH.sub.2.

(31) Below is a depiction of the structure of Example 6 (SEQ ID NO:11) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, and K17, where the structures of these amino acid residues have been expanded:

(32) ##STR00011##

(33) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 6.

EXAMPLE 7

(34) Example 7 is a compound represented by the following description:

(35) TABLE-US-00007 (SEQ ID NO: 12) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino- ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H) AQHAFIEYLLEGGPSSGAPPPS-NH.sub.2.

(36) Below is a depiction of the structure of Example 7 (SEQ ID NO:12) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13 and K17, where the structures of these amino acid residues have been expanded:

(37) ##STR00012##

(38) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 7.

EXAMPLE 8

(39) Example 8 is a compound represented by the following description:

(40) TABLE-US-00008 (SEQ ID NO: 13) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-αMeK- AFIEYLLEGGPSSGAPPPS-NH.sub.2.

(41) Below is a depiction of the structure of Example 8 (SEQ ID NO:13) using the standard single letter amino acid codes with the exception of residues Aib2, K17 and αMeK20, where the structures of these amino acid residues have been expanded:

(42) ##STR00013##

(43) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 8.

EXAMPLE 9

(44) Example 9 is a compound represented by the following description:

(45) TABLE-US-00009 (SEQ ID NO: 14) Y-Aib-QGTFTSDYSI-αMeL-LDKK((γGlu).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H) AQHAFIEYLLEGGPSSGAPPPS-NH.sub.2.

(46) Below is a depiction of the structure of Example 9 (SEQ ID NO:14) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13 and K17, where the structures of these amino acid residues have been expanded:

(47) ##STR00014##

(48) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 9.

EXAMPLE 10

(49) Example 10 is a compound represented by the following description:

(50) TABLE-US-00010 (SEQ ID NO: 15) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQHAFIEYLLEG GPSSGAPPPS-NH.sub.2.

(51) Below is a depiction of the structure of Example 10 (SEQ ID NO:15) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13 and K17, where the structures of these amino acid residues have been expanded:

(52) ##STR00015##

(53) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 10.

EXAMPLE 11

(54) Example 11 is a compound represented by the following description:

(55) TABLE-US-00011 (SEQ ID NO: 16) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIE YLLEGGPSSGAPPPS-NH.sub.2.

(56) Below is a depiction of the structure of Example 11 (SEQ ID NO:16) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17 and Aib20, where the structures of these amino acid residues have been expanded:

(57) ##STR00016##

(58) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 11.

EXAMPLE 12

(59) Example 12 is a compound represented by the following description:

(60) TABLE-US-00012 (SEQ ID NO: 17) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEY LLEGGPSSGAPPPS-NH.sub.2.

(61) Below is a depiction of the structure of Example 12 (SEQ ID NO:17) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17 and Aib20, where the structures of these amino acid residues have been expanded:

(62) ##STR00017##

(63) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 12.

EXAMPLE 13

(64) Example 13 is a compound represented by the following description:

(65) TABLE-US-00013 (SEQ ID NO: 18) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)-AQ-Aib-AFIE YLLE-Aib-GPSSGAPPPS-NH.sub.2.

(66) Below is a depiction of the structure of Example 13 (SEQ ID NO:18) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(67) ##STR00018##

(68) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 13.

EXAMPLE 14

(69) Example 14 is a compound represented by the following description:

(70) TABLE-US-00014 (SEQ ID NO: 19) Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEY LLE-Aib-GPSS-Aib-APPPS-NH.sub.2.

(71) Below is a depiction of the structure of Example 14 (SEQ ID NO:19) using the standard single letter amino acid codes with the exception of residues Aib2, αMeL13, K17, Aib20, Aib29 and Aib34 where the structures of these amino acid residues have been expanded:

(72) ##STR00019##

(73) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 14.

EXAMPLE 15

(74) Example 15 is a compound represented by the following description:

(75) TABLE-US-00015 (SEQ ID NO: 20) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFI EYLLA-Aib-GPSSGAPPPS-NH.sub.2.

(76) Below is a depiction of the structure of Example 15 (SEQ ID NO:20) using the standard single letter amino acid codes with the exception of residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(77) ##STR00020##

(78) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 15.

EXAMPLE 16

(79) Example 16 is a compound represented by the following description:

(80) TABLE-US-00016 (SEQ ID NO: 21) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIE YLLEGGPSSGAPPPS-NH.sub.2.

(81) Below is a depiction of the structure of Example 16 (SEQ ID NO:21) using the standard single letter amino acid codes with the exception of residues Aib2, K17, and Aib20, where the structures of these amino acid residues have been expanded:

(82) ##STR00021##

(83) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 16.

EXAMPLE 17

(84) Example 17 is a compound represented by the following description:

(85) TABLE-US-00017 (SEQ ID NO: 22) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIE YLLE-Aib-GPSSGAPPPS-NH.sub.2.

(86) Below is a depiction of the structure of Example 17 (SEQ ID NO:22) using the standard single letter amino acid codes with the exception of residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(87) ##STR00022##

(88) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 17.

EXAMPLE 18

(89) Example 18 is a compound represented by the following description:

(90) TABLE-US-00018 (SEQ ID NO: 23) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIE YLLEGGPSSGAPPPS-NH.sub.2.

(91) Below is a depiction of the structure of Example 18 (SEQ ID NO:23) using the standard single letter amino acid codes with the exception of residues Aib2, K17, and Aib20, where the structures of these amino acid residues have been expanded:

(92) ##STR00023##

(93) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 18.

EXAMPLE 19

(94) Example 19 is a compound represented by the following description:

(95) TABLE-US-00019 (SEQ ID NO: 24) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIE YLLE-Aib-GPSSGAPPPS-NH.sub.2.

(96) Below is a depiction of the structure of Example 19 (SEQ ID NO:24) using the standard single letter amino acid codes with the exception of residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(97) ##STR00024##

(98) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 19.

EXAMPLE 20

(99) Example 20 is a compound represented by the following description:

(100) TABLE-US-00020 (SEQ ID NO: 25) Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEY LLE-Aib-GPSSGAPPPS-NH.sub.2.

(101) Below is a depiction of the structure of Example 20 (SEQ ID NO:25) using the standard single letter amino acid codes with the exception of residues Aib2, K17, Aib20 and Aib29, where the structures of these amino acid residues have been expanded:

(102) ##STR00025##

(103) Similar processes to those described above for Example 1 are used to synthesize the peptide backbone, to conjugate the fatty acid-linker moiety, to examine the purity, and to confirm the molecular weight of Example 20.

(104) In Vitro Function

(105) Binding Affinity:

(106) Radioligand competition binding assays are run to determine the equilibrium dissociation constant for exemplary compounds and comparator molecules. Such assay use scintillation proximity assay (SPA) methods and membranes prepared from transfected HEK293 cells overexpressing the human GIP receptor (GIPR), GLP-1 receptor (GLP-1R) or human glucagon receptor (GcgR).

(107) The assays are performed in the presence of bacitracin as a non-specific blocking agent to prevent acylated moieties of test analogs from binding to protein components used in standard assay buffers (e.g., albumin).

(108) Competition curves are plotted as the percent specific inhibition (y-axis) versus log concentration of compound (x-axis) and analyzed using a four parameter nonlinear regression fit with variable slope (ABase or Genedata). K.sub.i values are calculated according to the equation K.sub.i=IC.sub.50/(1+(D/K.sub.d)), where IC.sub.50 is the concentration of compound resulting in 50% inhibition of binding, D is the concentration of radioligand used in the assay, and K.sub.d is the equilibrium dissociation constant for the receptor and the radioligand, determined from saturation binding analysis (shown in Table 1 below).

(109) TABLE-US-00021 TABLE 1 Equilibrium Dissociation Constants (K.sub.d) Determined from Saturation Binding Analysis. K.sub.d, nM GLP-1R GcgR GIPR 1.2 3.9 0.14

(110) K.sub.i values of exemplary analogs and comparator molecules are shown in Table 2.

(111) TABLE-US-00022 TABLE 2 In Vitro Binding Affinity (K.sub.i) of Examples and Comparators for Human GIPR, GLP-1R and GcgR. K.sub.i, nM (SEM, n) Molecule GcgR GIPR GLP-1R hGcg 3.1 (0.5, 4) hGIP 0.12 (0.02, 4) hGLP-1 1.2 (0.2, 4) Example 1 8.41 (2.71, 5) 0.0469 (0.00558, 4/5) 2.64 (0.501, 5) Example 2 3.71 (1.49, 2) 0.0665 (0.0377, 2) 4.50 (0.735, 2) Example 3 12.0 (2.74, 4) 0.0446 (0.00838, 4) 6.06 (0.849, 4) Example 4 2.55 (0.411, 4) 0.0374 (0.0137, 4) 3.54 (0.503, 6) Example 5 0.422 (0.0887, 3) 0.179 (0.0466, 3) 36.7 (8.99, 3) Example 6 0.835 (0.369, 3) 0.249 (0.0369, 3) 36.7 (10.5, 3) Example 7 16.3 (2.37, 5) 0.110 (0.0206, 5) 21.4 (3.53, 5) Example 8 30.4 (38.9, 3) 0.0958 (0.0295, 3) 29.4 (29.8, 3) Example 9 8.27 (0.855, 4) 0.126 (0.0274, 4) 11.5 (1.85, 4) Example 10 7.37 (1.38, 4) 0.118 (0.0363, 4) 12.0 (2.75, 4) Example 11 10.8 (1.11, 4) 0.0890 (0.0369, 3/4) 9.97 (1.53, 4) Example 12 5.60 (0.796, 4) 0.0570 (0.00322, 4) 7.17 (1.68, 4) Example 13 1.91 (0.128, 3) 0.0452 (0.00297, 3) 6.43 (1.89, 5) Example 14 2.64 (0.231, 4) 0.0350 (0.00326, 4) 6.27 (1.12, 6) Example 15 4.56 (2.68, 2) 0.0972 (n = 1/2) 5.80 (1.80, 3) Example 16 11.5 (1.97, 5) 0.106 (0.0182, 5) 16.1 (2.30,6) Example 17 5.81 (0.875, 3) 0.0895 (0.0290, 3) 10.2 (1.74, 3) Example 18 5.71 (0.588, 4) 0.0835 (0.0128, 4) 8.06 (2.04, 4) Example 19 5.20 (0.572, 3) 0.0789 (0.0261, 3) 12.0 (2.07, 3) Example 20 3.76 (0.397, 3) 0.109 (0.0247, 3) 9.23 (2.14, 3) NOTE: A qualifier (>) indicates the data did not reach 50% inhibition relative to maximum binding, whereby the K.sub.i was calculated using the highest concentration tested in the assay. n = 1/x means that only one value out of the total number of replicates (x) is used to express the mean. SEM is only calculated when n = 2 or greater non-qualified results exist.

(112) As seen in Table 2, exemplary analogs have binding affinity at each of the GIP, GLP-1 and glucagon receptors.

(113) Functional Activity:

(114) Functional activity is determined in GIP-R-GLP-1R- and GcgR-expressing HEK-293 clonal cell lines. Each receptor over-expressing cell line is treated with peptide (20 point CRC, 2.75-fold Labcyte Echo direct dilution) in DMEM (Gibco Cat #31053) supplemented with 1× GlutaMAX™ (Gibco Cat #35050), 0.25% FBS (Fetal Bovine Serum, Gibco Cat #26400), 0.05% fraction V BSA (Bovine Serum Albumin, Gibco Cat #15260), 250 μM IBMX and 20 mM HEPES (Gibco Cat #15630) in a 20 μl assay volume.

(115) After a 60-minute incubation at room temperature, the resulting increase in intracellular cAMP is quantitatively determined using the CisBio cAMP Dynamic 2 HTRF Assay Kit (62AM4PEJ). Briefly, cAMP levels within the cell are detected by adding the cAMP-d2 conjugate in cell lysis buffer followed by the antibody anti-cAMP-Eu.sup.3+-Cryptate, also in cell lysis buffer. The resulting competitive assay is incubated for at least 60 minutes at room temperature and then detected using a PerkinElmer Envision® Instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Envision units (emission at 665 nm/620 nm*10,000) are inversely proportional to the amount of cAMP present and were converted to nM cAMP per well using a cAMP standard curve.

(116) The amount of cAMP generated (nM) in each well is converted to a percent of the maximal response observed with either human GLP-1(7-36)NH.sub.2, human Gcg, or human GIP(1-42)NH.sub.2. A relative EC.sub.50 value is derived by non-linear regression analysis using the percent maximal response vs. the concentration of peptide added, fitted to a four-parameter logistic equation.

(117) Data for exemplary analogs and hGIP(1-42)NH.sub.2, hGLP-1(7-36)NH.sub.2 and hGcg are shown in Table 3 below.

(118) TABLE-US-00023 TABLE 3 Functional cAMP Potency (EC.sub.50) for Exemplary Analogs and Comparators in the Presence of FBS and BSA. cAMP EC.sub.50, nM (SEM, n) GcgR GIPR GLP-1R hGcg 0.0125 (0.000280014, 112) gGIP amide 0.133 (0.0082, 60) hGLP-1 amide 0.0591 (0.00091, 113) Example 1 2.54 (0.199, n = 6) 0.914 (0.0915, 6) 7.49 (1.02, 6) Example 2 2.66 (0.175, 5) 2.19 (0.392, 5) 12.8 (2.50, 5) Example 3 8.03 (0.997, 6) 1.91 (0.205, 6) 12.9 (1.58, 6) Example 4 2.49 (0.371, 7) 1.55 (0.245, 7) 10.4 (1.80, 7) Example 5 1.47 (0.171, 6) 4.86 (0.682, 6) 22.3 (3.78, 6) Example 6 1.99 (0.201, 6) 7.41 (0.667, 6) 21.2 (2.02, 6) Example 7 14.2 (2.24, 6) 4.38 (0.750, 6) 15.5 (2.40, 6) Example 8 6.24 (0.673, 6) 2.23 (0.164, 6) 9.39 (0.959, 6) Example 9 6.32 (0.290, 4) 4.17 (0.695, 4) 9.76 (1.98, 4) Example 10 8.42 (1.17, 4) 4.30 (0.987, 4) 19.3 (1.52, 4) Example 11 11.9 (0.727, 6) 1.50 (0.124, 6) 10.3 (0.808, 6) Example 12 6.61 (0.512, 6) 2.24 (0.303, 6) 12.4 (1.41, 6) Example 13 3.61 (0.197, 8) 1.76 (0.126, 8) 12.2 (1.00, 8) Example 14 4.05 (0.255, 7) 1.55 (0.165, 7) 14.4 (1.71, 7) Example 15 5.92 (1.10, 4) 1.47 (0.264, 4) 10.7 (1.85, 4) Example 16 13.2 (1.93, 6) 4.37 (0.589, 6) 19.0 (2.39, 6) Example 17 8.05 (1.26, 3) 2.38 (0.212, 3) 18.4 (3.75, 3) Example 18 5.71 (0.256, 4) 5.89 (1.05, 4) 16.1 (2.61, 4) Example 19 8.45 (0.828, 3) 3.13 (0.179, 3) 24.4 (2.85, 3) Example 20 3.97 (0.284, 3) 3.70 (1.02, 3) 20.6 (5.10, 3) NOTE: EC.sub.50 determination of human GLP-1(7-36)NH.sub.2 at human GLP-1R, human Gcg at human GcgR, and human GIP(1-42)NH.sub.2 at human GIP-R: the peptide concentration ranges were 448 pM to 99.5 nM. EC.sub.50 determination of Examples at human GLP-1R, human GcgR, and human GIP-R: the peptide concentration ranges were 51.5 fM to 11.4 μM.

(119) As seen in Table 3, in the presence of FBS and BSA, exemplary analogs have agonist activities as determined by human GIP-R, GLP-1R, and GcgR cAMP cAMP assays, which are lower than the native ligands.

(120) An additional set of cAMP assays are conducted in HEK293 cells expressing the human GLP-1, GIP and glucagon receptors. Using homogeneous time resolved fluorescence methods, assays are conducted to determine the intrinsic potency of exemplary analogs and comparator molecules performed in the presence of casein (instead of serum albumin) as a nonspecific blocker, which does not interact with the fatty acid moieties of the analyzed molecules.

(121) Intracellular cAMP levels are determined by extrapolation using a standard curve. Dose response curves of compounds are plotted as the percentage of stimulation normalized to minimum (buffer only) and maximum (maximum concentration of each control ligand) values and analyzed using a four parameter non-linear regression fit with a variable slope (Genedata Screener 13). EC.sub.50 is the concentration of compound causing half-maximal simulation in a dose response curve.

(122) Data are provided below in Table 4.

(123) TABLE-US-00024 TABLE 4 Functional Activation of hGLP-1R, hGIPR, hGcgR in the Presence of 0.1% Casein. cAMP EC.sub.50, nM (SEM, n) GcGR GIPR GLP-1R hGcg 0.0119 (0.00356, 163) hGIP amide 0.154 (0.037, 118) gGLP-1 amide 0.063 (0.022, 197) Example 1 0.114 (0.0203, 5) 0.0523 (0.0112, 5) 0.153 (0.0132, 12) Example 2 0.0553 (0.00975, 4) 0.0474 (0.00485, 4) 0.207 (0.0213, 6) Example 3 0.152 (0.0147, 7) 0.0376 (0.00284, 7) 0.107 (0.0108, 7) Example 4 0.0674 (0.00532, 15) 0.0648 (0.00507, 14) 0.180 (0.0144, 17) Example 5 0.0226 (0.00304, 10) 0.0757 (0.0127, 5) 0.147 (0.0204, 7) Example 6 0.0282 (0.00409, 7) 0.274 (0.0377, 7) 0.142 (0.0127, 10) Example 7 0.180 (0.0190, 6) 0.0798 (0.0111, 6) 0.109 (0.0134, 5) Example 8 0.120 (0.0210, 5) 0.114 (0.0101, 4) 0.117 (0.0151, 7) Example 9 0.139 (0.0281, 5) 0.0522 (0.00816, 4) 0.0931 (0.00852, 8) Example 10 0.123 (0.00784, 15) 0.0928 (0.00721, 16) 0.143 (0.0103, 12) Example 11 0.205 (0.0175, 11) 0.0425 (0.00744, 12) 0.123 (0.0119, 13) Example 12 0.122 (0.00931, 15) 0.0529 (0.00394, 18) 0.162 (0.0100, 18) Example 13 0.0815 (0.00835, 12) 0.0391 (0.00315, 14) 0.125 (0.00961, 13) Example 14 0.0876 (0.00687, 17/18) 0.0356 (0.00242, 20) 0.146 (0.0108, 17) Example 15 0.131 (0.0141, 10) 0.0689 (0.00730, 9) 0.253 (0.0197, 9) Example 16 0.174 (0.00882, 22) 0.114 (0.0100, 20) 0.157 (0.0105, 20) Example 17 0.135 (0.00643, 12) 0.0439 (0.00457, 11) 0.153 (0.0135, 10) Example 18 0.0861 (0.00631, 16) 0.123 (0.00954, 13) 0.141 (0.00862, 13) Example 19 0.0874 (0.0317, 2) 0.0455 (0.00516, 2) 0.143 (0.0187, 3) Example 20 0.0641 (0.00369, 12) 0.0572 (0.00527, 11) 0.149 (0.00937, 11)

(124) As seen in Table 4, exemplary analogs stimulate cAMP from human GIP, GLP-1 and glucagon receptors in the presence of 0.1% casein.

(125) In Vivo Studies

(126) Pharmacokinetics in Male Sprague Dawley Rats:

(127) The pharmacokinetics of the exemplary analogs are evaluated following a single subcutaneous administration of 100 nM/kg to male Sprague Dawley rats. Blood samples are collected over 120 hours, and resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K.sub.3 EDTA) concentrations are determined using a qualified LC/MS method that measured the intact mass of the analog. Each peptide and an analog as an internal standard are extracted from 100% specie specified plasma using methanol with 0.1% formic acid. A Thermo Q-Exactive, High Resolution Instrument, and a Thermo Easy Spray PepMap are combined for LC/MS detection. Mean pharmacokinetic parameters are shown in Table 5.

(128) TABLE-US-00025 TABLE 5 Mean Pharmacokinetic Parameters of Peptides Following a Single Subcutaneous Administration of 100 nMol/kg to Male Sprague Dawley Rats. C.sub.max/D AUCINF_D_obs T.sub.1/2 T.sub.max (kg*nmol/ (hr*kg*nmol/ Cl/F (mL/ (hr) (hr) L/nmol) L/nmol) hr/Kg) Example 1 11.7 8 3.5 95.7 10.4 Example 3 19.2 16 2.5 146 6.9 Example 4 19.9 16 2.9 140 7.2 Example 10 23.4 24 3.1 203.5 4.9 Example 11 24.3 20 3.7 215.1 4.7 Example 12 26.5 24 3.7 197.1 5.1 Example 13 21.7 20 3.8 205.7 4.9 Example 16 29.1 20 3.8 274.9 3.6 Example 18 34.8 24 5.0 284.3 3.6 Abbreviations: T.sub.1/2 = half-life, T.sub.max = time to maximal concentration, C.sub.max = maximal plasma concentration, AUCINF_D_obs = AUCinf divided by dose, CL/F = clearance/bioavailability. NOTE: Data are the mean, where n = 3/group.

(129) As seen in Table 5, exemplary analogs demonstrate an extended pharmacokinetic profile.

(130) In Vivo Effect on Insulin Secretion in Male Wistar Rats:

(131) An intravenous glucose tolerance test (ivGTT) in rats (male Wistar) is used to estimate insulinotropic potency of the exemplary analogs. The GLP-1 RA semaglutide is used as a positive control. Rats with surgically implanted cannulas in the jugular vein and carotid artery (Envigo, Indianapolis, Ind.; 280-320 grams) are housed one per cage in polycarbonate cages with filter tops. Rats are maintained on a 12-hour light-dark cycle at 21° C. and receive 2014 Teklad Global Diet (Envigo, Indianapolis) and deionized water ad libitum. Rats are randomized by body weight and dosed 1.5 mL/kg subcutaneous (sc) with exemplary analogs 16 hours prior to glucose administration, and then fasted. Stock concentrations of 211 nM of the exemplary analogs are diluted in Tris buffer pH 8.0 to 6.667 nMol/mL, 2.0 nMol/mL, 0.667 nMol/mL, 0.2 nMol/mL; doses tested are vehicle, 1, 3 and 10 nMol/kg, and, in some cases 0.3 and 30 nMol/kg. Semaglutide is used as positive control, and its insulinotropic effects are measured both in its own test (vehicle and 1, 3, 10 and 30 nMol/kg doses) and in connection with each run of the exemplary analogs (10 nMol/kg dose).

(132) A time 0 blood sample is collected into EDTA tubes after which glucose is administered (0.5 mg/kg, 5 mL/kg). Blood samples are collected for glucose and insulin levels at time 2, 4, 6, 10, 20, and 30 minutes post intravenous administration of glucose. Plasma insulin is determined using an electrochemiluminescence assay (Meso Scale, Gaithersburg, Md.). Insulin area under the curve (AUC) is examined and compared to the vehicle control with n=6 animals per group.

(133) Statistical analysis is performed using JMP with a one-way ANOVA followed by Dunnett's comparison to the vehicle control. Data are provided in Table 6 below.

(134) TABLE-US-00026 TABLE 6 Effect of Vehicle, Semaglutide and Exemplary Analogs on Insulin Secretion During Intravenous Glucose Tolerance Test in Anesthetized Wistar Rats. AUC.sub.30min of Insulin after a Bolus IV Glucose Dose (nmol/kg) Semaglutide 0 1 3 10 (10 nmol/kg) Ex. 1 11.1 ± 1.3   60.1 ± 10.3* 53.8 ± 5.1* 71.5 ± 6.1*  51.86 +/− 6.7* Ex. 3 12.6 ± 2.1  34.4 ± 2.9* 47.5 ± 4.0* 51.6 ± 5.4*   45.6 +/− 6.6* Ex. 4 13.7 ± 2.3  27.4 ± 3.0  52.9 ± 3.0* 70.8 ± 8.4*   44.7 +/− 3.7* Ex. 11 11.3 ± 2.1  43.0 ± 6.8* 59.2 ± 6.3* 62.0 ± 4.6*   55.1 +/− 9.0* Ex. 12 9.3 ± 1.8 32.4 +/− 2.4* 44.8 ± 2.3* 53.6 ± 6.0*+  40.2 +/− 2.5* Ex. 13 8.9 ± 1.2 25.8 ± 2.7  47.3 ± 8.0* 70.6 ± 4.8*   38.4 +/− 6.0* Ex. 16 14.4 ± 2.1  18.9 ± 3.5  50.3 ± 4.2* 50.1 ± 4.2*   56.3 +/− 7.7* Ex. 18 12.3 ± 2.0  27.6 ± 4.6  44.8 ± 8.3* 57.3 ± 10.0*  48.4 +/− 6.6* NOTE: results are expressed as Mean ± Standard Error of Means (SEM) of 6 rats per group. The statistical test is one-way ANOVA followed by Dunnett′s *p < 0.05 compared to vehicle; +p < 0.05 compared to semaglutide.

(135) As seen in Table 6, the exemplary analogs show dose-dependent increases in insulin secretion.

(136) Studies in Diet-Induced Obese C57BL/6 Mice:

(137) The exemplary incretin analogs as described herein are proposed as a treatment not only for diabetes but also for metabolic syndrome, which is a collection of co-morbidities (dyslipidemia, obesity, hepatic steatosis, etc.) associated with insulin resistance and diabetes. To investigate the effects of the exemplary analogs on parameters such as weight loss, metabolism, body composition and hepatic steatosis, they were dosed to C57BL/6 diet-induced obese (DIO) mice. These animals, although not diabetic, display insulin resistance, dyslipidemia and hepatic steatosis, all characteristics of metabolic syndrome, after being placed on a high-fat diet for 18 weeks.

(138) Specifically, DIO male C57BL/6 mice 24 to 25 weeks old maintained on a calorie-rich diet are used in the following studies. Mice are individually housed in a temperature-controlled (24° C.) facility with 12-hour light/dark cycle (lights on 22:00) and free access to food (TD95217) and water. After a minimum of 2 weeks acclimation to the facility, the mice are randomized according to their body weight, so each experimental group of animals would have similar body weight. The body weights range from 40 to 51 g.

(139) All groups contain 5-6 mice. Vehicle, exemplary analogs dissolved in vehicle (40 mM Tris-HCl at pH 8.0), and semaglutide dissolved in vehicle are administered by subcutaneous (SC) injection (10 mL/kg) to ad libitum fed DIO mice 30 to 90 minutes prior to the onset of the dark cycle every 3 days for 15 days. SC injections are made on Day 1, 4, 7, 10 and 13. Body weight and food intake are measured daily throughout the study.

(140) Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of vehicle, analog or semaglutide. On Days 0 and 15, total fat mass is measured by nuclear magnetic resonance (NMR) using an Echo Medical System Instrument (Houston, Tex.). On Day 15, animals are sacrificed prior to dark photoperiod, and the livers are removed and frozen. Liver triglycerides, determined from homogenates of livers collected at sacrifice, and plasma cholesterol are measured on a Hitachi Modular P clinical analyzer.

(141) Data are presented as mean±SEM of 5-6 animals per group in Tables 7 and 8 below. Statistical analysis is performed using repeated measures ANOVA, followed by Dunnett's method for multiple comparisons. Significant differences are identified at p<0.05.

(142) TABLE-US-00027 TABLE 7 Body Weight Change After Treatment with Exemplary Analogs After Fifteen Days. Treatment Dose 3 nmol/kg 10 nmol/kg 30 nmol/kg Δ from Δ from Δ from vehicle (g) % change vehicle (g) % change vehicle (g) % change Ex. 1 −5.42 ± 0.54 −10.04 ± 1.14  −9.26 ± 0.36 −20.20 ± 1.51 −21.36 ± 1.08 −44.82 ± 1.54 Ex. 3 −10.14 ± 0.72 −21.18 ± 1.98 Ex. 4 −11.58 ± 0.85 −23.64 ± 1.70 −19.98 ± 1.63 −43.88 ± 3.71 Ex. 5 −12.30 ± 2.20 −25.40 ± 4.56 Ex. 6 −11.72 ± 1.78 −24.02 ± 3.31 Ex. 7 −5.26 ± 0.49 −12.22 ± 1.19  −8.38 ± 0.50 −19.65 ± 1.27 −16.26 ± 2.23 Ex. 10  −5.4 ± 0.49 −12.70 ± 1.17 −10.98 ± 0.76 −24.95 ± 1.99 −17.08 ± 1.43 −39.51 ± 3.84 Ex. 11 −7.22 ± 0.38 −16.58 ± 0.88 −11.82 ± 1.72 −26.83 ± 4.04 −16.48 ± 1.98 −37.35 ± 3.91 Ex. 12 −8.40 ± 0.77 −19.33 ± 1.66 −10.34 ± 0.69 −23.37 ± 1.40 −17.44 ± 1.37 −46.60 ± 3.78 Ex. 13 −4.92 ± 0.86 −10.14 ± 1.87 −11.02 ± 0.77 −23.80 ± 1.25 −21.12 ± 2.09 Ex. 14 −6.12 ± 0.80 −13.38 ± 1.80 −14.76 ± 1.06 −32.32 ± 2.49 −20.04 ± 2.40 −45.26 ± 4.79 Ex. 16 −7.44 ± 0.74 −16.78 ± 1.88 −12.24 ± 1.66 −28.08 ± 4.23 −16.70 ± 1.67 −37.42 ± 3.58 Ex. 18 −13.16 ± 0.82 −28.48 ± 2.05 −19.54 ± 2.37 −43.02 ± 4.59 Ex. 20 −4.18 ± 0.50  −8.98 ± 1.26 −10.76 ± 1.57 −24.42 ± 3.74 −23.00 ± 0.59 −52.14 ± 1.74 Sema  −2.62 ± 0.49 −10.31 ± 1.25 −15.49 ± 2.44 NOTE: “Δ from vehicle” refers to difference between body weight at day 15 between test and vehicle groups. “% change” refers to percent decrease in body weight between days 1 and 15 in test groups. “Sema” means semaglutide. Percent decrease in body weight for animals receiving vehicle is recorded, and is less than about 1% in each study. The Δ from vehicle and % change data are statistically significantly different (p <0.05) than control for all Examples at all doses tested.

(143) As seen in Table 7 above, the exemplary analogs show dose-dependent reductions in body weight.

(144) Data for metabolic parameters at the 3 nmol/kg dose are provided below in Table 8.

(145) TABLE-US-00028 TABLE 8 Effect of Treatment with Exemplary Analogs at 3 nmol/kg on Blood Glucose, Insulin, Cholesterol, Alanine Aminotransferase (ALT) and Liver Triglycerides After Fifteen Days of Treatment. Metabolic Parameters (Mean ± SEM) Plasma Liver Glucose Insulin Cholesterol Triglycerides Treatment (mg/dL) (pg/mL) (mg/dL) ALT (IU/L) (mg/g tissue) Vehicle 149.7 ± 3.09   8549 ± 1265   234.6 ± 3.42   141.8 ± 11.5    290.4 ± 13.3  Semaglutide 137.8 ± 7.34   4196 ± 1014*  218.0 ± 9.23   99.8 ± 14.84  226.4 ± 23.8  Example 1 107.4 ± 4.38*  3439 ± 936.4* 191.2 ± 7.55*  118.0 ± 20.73   150.6 ± 37.0* Example 7 109.3 ± 5.78*  2656 ± 949*   169.2 ± 11.79* 51.0 ± 7.78*   97.9 ± 18.2* Example 10 109.6 ± 4.81*  571.4 ± 158.4*  181.0 ± 6.20*  77.2 ± 9.35*   126.8 ± 17.0*  Example 11 114.7 ± 3.051* 1569 ± 318.3* 178.2 ± 7.95*  56.2 ± 4.51*   93.8 ± 21.8* Example 12 114.9 ± 2.12*  1691 ± 231.3* 169.0 ± 8.15*  58.6 ± 6.03*   98.2 ± 13.2* Example 13 119.7 ± 4.19*  2903 ± 737.4* 192.2 ± 11.44* 54.4 ± 6.82*   94.5 ± 22.0* Example 14 111.5 ± 1.77*  1971 ± 499.8* 164.8 ± 5.85*  31.4 ± 2.79*   63.1 ± 7.3*  Example 16 110.1 ± 3.61*  3227 ± 1070*  173.8 ± 9.32*  20.6 ± 4.43*   40.2 ± 13.2* Example 18 102.9 ± 5.37*  1958 ± 460.5* 190.0 ± 13.71* 66.0 ± 10.21*  109.5 ± 22.9*  NOTE: *p < 0.05 compared to Vehicle group; one-way ANOVA, Dunnett′s.

(146) In addition to substantial weight loss, and as seen in Table 8, the exemplary analogs reduce blood glucose, plasma insulin (as a sign of increasing insulin sensitivity) and plasma cholesterol, as well as improve liver health demonstrated by decrease of plasma ALT and liver triglycerides.

(147) TABLE-US-00029 Human glucagon SEQ ID NO: 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNT Human GLP-1 (7-36) amide SEQ ID NO: 2 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 Human OXM SEQ ID NO: 3 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA Human GIP SEQ ID NO: 4 YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ Incretin analog SEQ ID NO: 5 YX.sub.2QGTFTSDYSIX.sub.13LDKX.sub.17AX.sub.19X.sub.20AFIEYLLX.sub.28X.sub.29GPSSX.sub.34APPPS where: X.sub.2 is Aib, X.sub.13 is L or αMeL, X.sub.17 is any amino acid with a functional group available for conjugation, and the functional group is conjugated to a C.sub.16-C.sub.22 fatty acid, X.sub.19 is Q or A, X.sub.20 is Aib, αMeK, Q or H, X.sub.28 is E or A, X.sub.29 is G or Aib, and X.sub.34 is G or Aib Incretin analog SEQ ID NO: 6 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQHAFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 7 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-αMeK-AFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 8 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-αMeK-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 9 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 10 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AAQAFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 11 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AAQAFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 12 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQHAFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 13 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-αMeK-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 14 Y-Aib-QGTFTSDYSI-αMeL-LDKK((γGlu).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H)AQHAFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 15 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQHAFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 16 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 17 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 18 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 19 Y-Aib-QGTFTSDYSI-αMeL-LDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLE-Aib-GPSS-Aib-APPPS-NH.sub.2 Incretin analog SEQ ID NO: 20 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLA-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 21 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 22 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 23 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 24 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu).sub.2- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2 Incretin analog SEQ ID NO: 25 Y-Aib-QGTFTSDYSILLDKK((2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γGlu)- CO—(CH.sub.2).sub.18—CO.sub.2H)AQ-Aib-AFIEYLLE-Aib-GPSSGAPPPS-NH.sub.2 Artificial sequence SEQ ID NO: 26 GPSSGAPPPS Artificial sequence SEQ ID NO: 27 GPSS-Aib-APPPS