INCRETIN ANALOGS AND USES THEREOF

Abstract

Incretin analogs are provided that have activity at each of the glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1) and glucagon (GCG) 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 type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and obesity.

Claims

1. A compound comprising: TABLE-US-00017 YX.sub.2QGTX.sub.6TSDX.sub.10SIX.sub.13LDX.sub.16X.sub.17AQX.sub.20X.sub.21FIX.sub.24X.sub.25LLEGGPSSGEPP PX.sub.39, wherein X.sub.2 is Aib, X.sub.6 is αMeF(2F), X.sub.10 can be Y or 4Pal, X.sub.13 can be L or αMeL, X.sub.16 is Orn, 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 moiety, X.sub.20 can be 4Pal, Iva or αMeL, X.sub.21 can be A or Aib, X.sub.24 can be E or e, X.sub.25 can be Y or αMeY, and X.sub.39 can be E or S (SEQ ID NO:5), and wherein a carboxy-terminal (C-terminal) amino acid optionally is amidated; or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the amino acid with a functional group available for conjugation at position X.sub.17 is selected from the group consisting of C, D, E, K and Q

3. The compound of claim 1, wherein the amino acid with a functional group available for conjugation at position X.sub.17 is K.

4. The compound 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 moiety are conjugated by a linker between the amino acid and the fatty acid moiety.

5. The compound of claim 4, wherein the linker comprises one to four amino acids.

6. The compound of claim 5, wherein the amino acids are E or γE.

7. The compound of claim 4, wherein the linker further comprises the following structure:
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.

8. The compound of claim 4, wherein the linker further comprises one to four (2-[2-(2-amino-ethoxy)-ethoxy]-acetyl) moieties.

9. The compound of claim 1, wherein X17 is 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-(γE).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 20.

10. The compound of claim 9, wherein a is 1.

11. The compound of claim 9, wherein a is 2.

12. The compound of claim 9, wherein b is 1.

13. The compound of claim 9, wherein b is 2.

14. The compound of claim 9, wherein c is 20.

15. The compound of claim 1, wherein X.sub.10 is Y.

16. The compound of claim 1, wherein X.sub.10 is 4Pal.

17. The compound of claim 1, wherein X.sub.13 is L.

18. The compound of claim 1, wherein X.sub.13 is αMeL.

19. The compound of claim 1, wherein X.sub.20 is 4Pal.

20. The compound of claim 1, wherein X.sub.20 is Iva.

21. The compound of claim 1, wherein X.sub.20 is αMeL.

22. The compound of claim 1, wherein X.sub.21 is A.

23. The compound of claim 1, wherein X.sub.21 is Aib.

24. The compound of claim 1, wherein X.sub.24 is E.

25. The compound of claim 1, wherein X.sub.24 is e.

26. The compound of claim 1, wherein X.sub.25 is Y.

27. The compound of claim 1, wherein X.sub.25 is αMeY.

28. The compound of claim 1, wherein X.sub.39 is E.

29. The compound of claim 1, wherein X.sub.39 is S.

30. A compound having a formula selected from the group consisting of SEQ ID NOS:6-11, or a pharmaceutically acceptable salt thereof.

31. A method of treating a disease selected from the group consisting of type 2 diabetes mellitus, dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis and obesity, the method comprising the step of: administering to an individual in need thereof an effective amount of a compound of claim 1.

32. A method of treating type 2 diabetes mellitus, the method comprising the step of: administering to an individual in need thereof an effective amount of compound of claim 1.

33. A pharmaceutical composition comprising: compound of claim 1; and a pharmaceutically acceptable carrier, diluent, or excipient.

34-43. (canceled)

Description

EXAMPLES

[0081] The following non-limiting examples are offered for purposes of illustration, not limitation.

[0082] Polypeptide Synthesis

Example 1: Synthesis of Incretin Analog 1

[0083] Example 1 is a compound represented by the following description:

TABLE-US-00005 (SEQ ID NO: 9) YAibQGT-αMeF(2F)-TSDYSILLDOK(2-[2-(2-amino- ethoxy)-ethoxy]-acetyl-γE-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ- 4Pal-AFIEYLLEGGPSSGEPPPE-NH.sub.2.

[0084] Below is a depiction of the structure of Example 1 using the standard single letter amino acid codes except for residues Aib2, αMeF(2F)6, O16, K17, 4Pal20 and E39 where the structures of these amino acid residues have been expanded:

##STR00006##

[0085] The peptide backbone of Example 1 is synthesized using Fmoc/t-Bu chemistry on a Symphony X peptide synthesizer (Gyros Protein Technologies; Tucson, Ariz.).

[0086] The resin is 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 with the following exceptions. Fmoc-Lys(Mtt)-OH is used for the K at position 17, and Boc-Tyr(tBu)-OH) is used for the Y at position 1. Fmoc groups are removed prior to each coupling step (2×7 min) using 20% piperidine in DMF. All standard amino acid couplings are performed for 1 hr to a primary amine and for 3 hr to a secondary amine using an equal molar ratio of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM) and Oxyma (0.9 mM), at a nine-fold molar excess over the theoretical peptide loading. Exceptions are couplings to Cα-methylated amino acids, which are coupled for 3 hr. After completing synthesis of the peptide backbone, the resin is thoroughly washed with DCM for six times to remove residual DMF. The Mtt protecting group on the K at position 17 is selectively removed from the peptide resin using two treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (2×40-minute treatment).

[0087] Subsequent attachment of the fatty acid-linker moiety is accomplished by coupling 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid (Fmoc-AEEA-OH, ChemPep, Inc.), Fmoc-glutamic acid α-t-butyl ester (Fmoc-E-OtBu, Ark Pharm, Inc.) and mono-OtBu-eicosanoic acid (WuXi AppTec, Shanghai, China). A three-fold excess of reagents (AA: PyAOP: DIPEA=1:1:1 mol/mol) is used for each coupling for 1 hr.

[0088] After synthesis is complete, the peptide resin is washed with DCM and then thoroughly air-dried. The dry resin is treated with 10 mL of a cleavage cocktail (TFA:triisopropylsilane:water, 92.5:2.5:5 by volume) for 30 min at 40° C. The resin is filtered off, washed twice each with 2 mL of neat TFA, and the combined filtrates are treated with five-fold excess volume of 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 cold 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 >98.0%. Subsequent lyophilization of the final main product pool yields the lyophilized peptide TFA salt. The molecular weight is determined by LC-MS (obsd: M+3=1633.7; Calc M+3=1633.8).

Example 2: Synthesis of Incretin Analog 2

[0089] Example 2 is a compound represented by the following description:

TABLE-US-00006 (SEQ ID NO: 10) YAibQGT-αMeF(2F)-TSDYSI-αMeL-LDOK(2-[2-(2-amino- ethoxy)-ethoxy]-acetyl-γE-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ- Iva-AFIE-αMeY-LLEGGPSSGEPPPS-NH.sub.2 .

[0090] Below is a depiction of the structure of Example 2 using the standard single letter amino acid codes except for residues Aib2, αMeF(2F)6, αMeL13, O16, K17, Iva20, αMeY25 and S39 where the structures of these amino acid residues have been expanded:

##STR00007##

[0091] 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: Synthesis of Incretin Analog 3

[0092] Example 3 is a compound represented by the following description:

TABLE-US-00007 (SEQ ID NO: 11) YAibQGT-αMeF(2F)-TSD-4Pal-SI-αMeL-LDOK(2-[2-(2- amino-ethoxy)-ethoxy]-acetyl-γE-CO-(CH.sub.2).sub.18-CO.sub.2H) AQ-αMeL-Aib-FIe-αMeY-LLEGGPSSGEPPPS-NH.sub.2.

[0093] Below is a depiction of the structure of Example 3 using the standard single letter amino acid codes except for residues Aib2, αMeF(2F)6, 4Pal10, αMeL13, O16, K17, αMeL20, Aib21, e24, αMeY25 and S39 where the structures of these amino acid residues have been expanded:

##STR00008##

[0094] 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.

[0095] In Vitro Function

Example 4: Binding Affinity

[0096] Radioligand competition binding assays are used to determine the equilibrium dissociation constant for the Example compounds and comparators. Such assays use SPA methods and membranes prepared from transfected HEK293 cells overexpressing human GIP receptor (hGIPR; SEQ ID NO:14), human GLP-1 receptor (hGLP-1R; SEQ ID NO:15) or human GCG receptor (hGCGR; SEQ ID NO:16).

[0097] 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).

[0098] 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).

TABLE-US-00008 TABLE 1 K.sub.d Determined from Saturation Binding Analysis. K.sub.d, nM hGIPR hGLP-1R hGCGR 0.14 1.2 3.9

[0099] K.sub.i values of Example compounds and comparators are shown in Table 2.

TABLE-US-00009 TABLE 2 In Vitro K.sub.i of Example Compounds and Comparators for hGIPR, hGLP-1R and hGCGR. K.sub.i, nM (SEM, n) Compound hGIPR hGLP-1R hGCGR hGIP 0.186 (0.001,101) — — hGLP-1 — 0.844 (0.023, 93) — hGCG — — 3.00 (0.12, 76) Example 1 0.111 (0.021, 7) 4.53 (0.855, 7) 0.629 (0.0415, 7) Example 2 0.158 (0.029, 7) 2.54 (0.501, 7) 0.950 (0.350, 7) Example 3 0.114 (0.007, 5) 3.63 (0.386, 5) 1.73 (0.296, 5) 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 = l/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.

[0100] As seen in Table 2, the Example compounds have binding affinity at each of the hGIPR, hGLP-1R and hGCGR.

Example 5: Functional hGIPR, hGLP-1R and hGCGR Assays

[0101] Methods: Functional activity is determined using cAMP formation in HEK-293 clonal cell lines expressing hGIPR, hGLP-1R or hGCGR. hGIPR, hGLP-1R or hGCGR receptor-expressing cells are treated with a control polypeptide or one of Examples 1 to 3 (20 point concentration-response curve in DMSO, 2.75-fold Labcyte Echo direct dilution, 384 well plate Corning Cat #3570) in DMEM (Gibco Cat #31053) supplemented with 1× GlutαMAX™ (Gibco Cat #35050), 0.1% bovine casein (Sigma C4765-10ML), 250 μM IBMX (3-Isobutyl-1-methylxanthine, Acros Cat #228420010) and 20 mM HEPES (Gibco Cat #15630) in a 20 μL assay volume (final DMSO concentration was 0.5%). Experiments also are performed under identical assay conditions with the addition of 1.0% fatty acid free, globulin free human serum albumin (Sigma Cat #A3782).

[0102] After a 30-min incubation at 37° C., the resulting increase in intracellular cAMP is quantitatively determined using a 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 (10 μL) followed by the antibody anti-cAMP-Eu.sup.3+-Cryptate, also in cell lysis buffer (10 μL). The resulting competitive assay is incubated for at least 60 min at room temperature, and then is 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 are converted to nM cAMP per well using a cAMP standard curve. The amount of cAMP generated (nM) in each well is converted to a percent of the maximal response observed with human GIP(1-42)NH.sub.2, hGLP-1(7-36)NH.sub.2 or hGCG. A relative EC.sub.50 value and percent top (E.sub.max) are derived by non-linear regression analysis using the percent maximal response vs. the concentration of peptide added, fitted to a four-parameter logistic equation.

[0103] Results: Functional data for hGIP(1-42)NH.sub.2, hGLP-1(7-36)NH.sub.2, hGCG and the Example compounds are provided below in Table 3 (0.1% bovine casein) and Table 4 (0.1% bovine casein, 1.0% human serum albumin).

TABLE-US-00010 TABLE 3 Functional cAMP Potency (EC.sub.50) and Efficacy (E.sub.max) for Peptides Incubated at 37° C. (in the presence of 0.1% bovine casein). hGLP-1R .sup.a hGCGR .sup.a hGIPR .sup.a EC.sub.50, nM, E.sub.max, % ± EC.sub.50, nM, E.sub.max, % ± EC.sub.50, nM, E.sub.max, % ± Compound SEM (n) .sup.b SEM .sup.c SEM (n) .sup.b SEM .sup.c SEM (n) .sup.b SEM .sup.c hGLP-1 0.395 103 ± 2 — — — — (7-36)NH.sub.2 0.032 (62) hGCG — — 2.17 103 ± 2 — — 0.17 (63) hGIP — — — — 1.08   98 ± 2 (1-42)NH.sub.2  0.14 (60) Example 1 0.345 106 ± 4 1.20 101 ± 3 0.0513 102 ± 2 0.033 (10) 0.12 (10) 0.0099 (10) Example 2 0.383 103 ± 3 1.02 103 ± 2 0.0256 101 ± 2 0.089 (10) 0.19 (10) 0.0052 (10) Example 3 0.451 107 ± 2 2.49  99 ± 4 0.0306 103 ± 2 0.079 (11) 0.37 (10) 0.0061 (10) NOTE: .sup.a Expression density is determined using homologous competition binding of [.sup.125I]GLP-1(7-36)NH.sub.2 at hGLP-1R (112 fmol/mg protein), [.sup.125I]GCG at hGCGR (98 fmol/mg protein) and [.sup.125I]GIP(1-42) at hGIPR (124 fmol/mg protein), .sup.b EC.sub.50, nM = the Geometric Mean with the Standard Error of the Mean followed by the number of observations in parenthesis. .sup.c E.sub.max, % = the Arithmetic Mean ± the Standard Error of the Mean for the percent of maximal response to GLP-1(7-36)NH.sub.2 at hGLP-1R, GCG at hGCGR or GIP(1-42)NH.sub.2 at hGIPR. All values shown are to three (3) significant digits.

TABLE-US-00011 TABLE 4 Functional cAMP Potency (EC.sub.50) and Efficacy (E.sub.max) for Peptides Incubated at 37° C. (in the presence of 0.1% bovine casein and 1.0% human serum albumin). hGLP-1R .sup.a hGCGR .sup.a hGIPR .sup.a EC.sub.50, nM, E.sub.max, % ± EC.sub.50, nM, E.sub.max, % ± EC.sub.50, nM, E.sub.max, % ± Compound SEM (n) .sup.b SEM .sup.c SEM (n) .sup.b SEM .sup.c SEM (n) .sup.b SEM .sup.c hGLP-1    0.368 104 ± 3 — — — — (7-36)NH.sub.2 0.030 (45) hGCG — —    2.54 102 ± 3 — — 0.30 (45) hGIP — — — —  0.756 96 ± 2 (1-42)NH.sub.2 0.090 (45) Example 1 180 102 ± 4 899 103 ± 8 22.3  100 ± 3  17 (7) 118 (6)  5.4 (7) Example 2 119 116 ± 4 537  112 ± 10 2.91 99 ± 2 15 (7)  99 (8) 0.46 (7) Example 3 114 111 ± 5 480  97 ± 9 3.72 105 ± 5  12 (6) 178 (6) 0.83 (7) NOTE: .sup.a Expression density is determined using homologous competition binding of [.sup.125I]GLP-1(7-36)NH.sub.2 at hGLP-1R (112 fmol/mg protein), [.sup.125I]GCG at hGCGR (98 fmol/mg protein) and [.sup.125I]GIP(1-42) at hGIPR (124 fmol/mg protein). .sup.b EC.sub.50, nM = the Geometric Mean with the Standard Error of the Mean followed by the number of observations in parenthesis. .sup.c E.sub.max, % = the Arithmetic Mean ± the Standard Error of the Mean for the percent of maximal response to GLP-1(7-36)NH.sub.2 at hGLP-1R, GCG at hGCGR or GIP(1-42)NH.sub.2 at hGIPR. All values shown are to three (3) significant digits.

[0104] As seen in Table 3, Example compounds stimulate cAMP from hGLP-1R, hGCGR and hGIPR in the presence of 0.1% casein.

[0105] As seen in Table 4, Example compounds stimulate cAMP from hGLP-1R, hGCGR and hGIPR in the presence of 0.1% bovine casein and 1% human serum albumin.

[0106] In Vivo Function

Example 6: Pharmacokinetics in Male Diet-Induced Obese (DIO) Mice

[0107] Methods: Male DIO mice are administered a single SQ dose of 200 nmol/kg (0.98 mg/kg) of Example compounds in 40 mM Tris-HCL pH8 buffer with 0.02% PS80 at a volume of 10 mL/kg. Blood is collected at 1, 3, 6, 12, 24, 48 and 72 hr post-dose for pharmacokinetic characterization. Plasma concentration of Example compounds are determined by a qualified Liquid Chromatography Mass Spectrometry (LC/MS) method at Q Squared Solutions BioSciences LLC (Ithaca, N.Y.). The Example compounds and an internal standard are extracted from 100% mouse plasma using protein precipitation followed by solid phase extraction. The intact mass of the Example compounds, which includes peptide plus acyl chain, are detected by a Q-Exactive™ Orbitrap® mass spectrometer.

[0108] Results: Data for the Example compounds are provided below in Table 5.

TABLE-US-00012 TABLE 5 Mean Plasma Pharmacokinetic Parameters Following a Single 200 nmol/kg SQ Dose to Male DIO Mice. t½ T.sub.max C.sub.max AUC.sub.0-inf CL/F Compound (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 38 12 817 55550 3.6 Example 2 18 12 1239 42479 4.7 Example 3 13 12 1072 32800 6.1 NOTE: Abbreviations: t½ = half-life, T.sub.max = time to maximum concentration, C.sub.max = maximum observed plasma concentration, AUC.sub.0-inf = area under the curve from time 0 hours to infinity, CL/F = clearance/bioavailability. N = 3 animals/group/time point.

[0109] As seen in Table 5, the Example compounds demonstrate an extended pharmacokinetic profile in DIO mice.

Example 7: In Vivo Effect on Insulin Secretion in Male Wistar Rats

[0110] Methods: An intravenous glucose tolerance test (ivGTT) in Male Wistar rats is used to estimate insulinotropic potency of the Example compounds. A GLP-1R agonist, semaglutide, is used as a positive control. Rats with surgically implanted cannulas in the jugular vein and carotid artery (Envigo; Indianapolis, Ind.) 280-320 g are housed one per cage in polycarbonate cages with filter tops. Rats are maintained on a 12-hr light-dark cycle at 21° C. and receive 2014 Teklad Global diet (Envigo) and deionized water ad libitum. Rats are randomized by body weight and dosed 1.5 mL/kg SQ with Example compounds 16 hr prior to glucose administration and then fasted. Stock concentrations of 211 nM/mL of Example compounds are diluted in 40 mM Tris-HCl buffer pH 8.0 with 0.02% PS80 to desired dosing concentrations; doses tested are vehicle, 0.1, 0.3, 1, 3, 10 and 30 nM/kg. Semaglutide is used as positive control in connection with each run of Example compounds (10 nM/kg dose).

[0111] 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 min post intravenous administration of glucose. Plasma insulin is determined using an electrochemiluminescence assay (Meso Scale; Rockville, Md.). Insulin AUC is examined compared to the vehicle control with n=6 animals per group.

[0112] Statistical analysis is performed using JMP with a one-way ANOVA followed by Dunnett's comparison to the vehicle control.

[0113] Results: Data for the Example compounds are provided below in Table 6.

TABLE-US-00013 TABLE 6 Effect of Vehicle, Semaglutide (Sema at 10 nmol/kg) and Example Compounds on Insulin Secretion During ivGTT in Wistar Rats. AUC.sub.30 min of Insulin After Bolus IV Glucose Dose (nmol/kg) Compound Vehicle 0.1 0.3 1 3 10 30 Sema Example 1 27.8 ± 36.1 ± 45.5 ± 70.4 ± 115.7 ± 145.9 ± 134.5 ± 80.9 ± 4.8 5.0 8.4 8.4 6.0 14.9 16.6 8.2 Example 2 28.9 ± 69.0 ± 62.6 ± 72.6 ± 101.5 ± 128.8 ± 159.0 ± 76.4 ± 7.2 7.4 5.1 4.7 18.2 11.7 14.9 5.6 Example 3 37.3 ± 67.0 ± 77.6 ± 86.9 ± 97.2 ± 119.8 ± 174.8 ± 76.6 ± 4.1 10.2 6.7 9.1 7.5 10.2 22.1 7.1 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.

[0114] As seen in Table 6, the Example compounds dose-dependently increase insulin secretion.

Example 8: In Vivo Studies on Weight Loss, Metabolism and Body Composition in DIO C57/B16 Mice

[0115] Methods: To investigate the effects of the Example compounds on parameters such as weight loss, metabolism and body composition, the Example compounds are dosed to C57B16 DIO mice. These animals, although not diabetic, display insulin resistance and dyslipidemia, all characteristics of metabolic syndrome, after being placed on a high fat diet for 20 weeks.

[0116] Here, DIO male C57/B16 mice at 20 weeks old are maintained on a calorie-rich diet are used in the following studies. Animals are individually housed in a temperature-controlled (23° C. to 26° C.) facility with 12-hr light/dark cycle (lights on 2100) and free access to food (TD95217) and water. After a 3-week acclimation to the facility, which includes 1 week of vehicle acclimation dosing, 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 g to 51 g.

[0117] All groups contain 6 mice. Example compounds and semaglutide are dissolved in vehicle (40 mM Tris-HCl at pH 8.0 with 0.02% PS-80) and are administered by SQ injection (10 mL/kg) to ad libitum-fed DIO mice 30 min to 120 min prior to the onset of the dark cycle every day for 14 days (Days 1 to 14). Body weight and food intake are measured daily throughout the study, including 1 day after last dose (Day 15).

[0118] Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of molecule. On Days 0 and 15, total fat mass is measured by nuclear magnetic resonance (NMR) using an Echo Medical System (Houston, Tex.) instrument. On Day 15, metabolic parameters are measured using Accu-Chek® Aviva® Glucometers (Roche; Indianapolis, Ind.) or a Hitachi clinical blood analyzer (Roche, Indianapolis, Ind.). Plasma insulin is determined using an electrochemiluminescence assay (Meso Scale Discovery, Rockville, Md.).

[0119] Data are presented as mean±SEM of 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 as * with p<0.05.

[0120] Results: Data for the Example compounds are provided below in Tables 7 and 8.

TABLE-US-00014 TABLE 7 Body Weight Change After Treatment with Semaglutide and Example Compounds After 15 Days. Dose Δ Body Δ Body Fat Mass Compound (nmol/kg) Weight (g) Weight (%) (%) Sema 10 −9.23 ± 0.58* −21.98 ± 2.58* −15.14 ± 1.93* Example 1 0.15625 −1.20 ± 0.15  −4.31 ± 0.75 −2.44 ± 0.63 0.625 −4.37 ± 0.08* −11.40 ± 0.92*  −7.38 ± 1.05* 2.5 −10.42 ± 1.40*  −24.43 ± 3.84* −17.05 ± 2.47* 10 −18.97 ± 0.81*  −43.71 ± 2.32* −30.07 ± 1.69* 20 −21.30 ± 0.94*  −48.88 ± 2.00* −32.15 ± 1.93* Example 2 0.15625 −1.72 ± 0.04  −2.65 ± 0.55 −1.91 ± 0.84 0.625 −4.48 ± 0.26*  −8.90 ± 1.38*  −5.87 ± 0.94* 2.5 −8.62 ± 0.21* −17.90 ± 1.01* −11.96 ± 0.88* 10 −20.10 ± 0.54*  −43.44 ± 1.30* −28.53 ± 1.24* 20 −21.77 ± 0.50*  −46.79 ± 1.40* −28.80 ± 0.81* Example 3 0.15625 −1.95 ± 0.13  −4.60 ± 0.71 −3.34 ± 0.60 0.625 −5.18 ± 0.25* −11.91 ± 0.99*  −8.03 ± 0.84* 2.5 −11.65 ± 1.68*  −25.98 ± 3.85* −18.55 ± 2.41* 10 −18.43 ± 0.68*  −41.10 ± 1.51* −28.50 ± 1.55* 20 −19.92 ± 0.34*  −44.22 ± 1.01* −29.23 ± 1.29* NOTE: “Δ Body weight (g)” refers to difference between body weight at day 15 between test and vehicle groups. “Δ Body weight (%)” refers to percent decrease in body weight between days 1 and 15 in test groups. “Fat mass (%)” refers to difference between fat mass between days 0 and 15 in test groups. “Sema” means semaglutide. All data is from single representative study. Percent change in body weight and fat mass for animals receiving vehicle is recorded and is less than 2% in each study. The “Δ Body weight (g)”, “Δ Body weight (%)”, and “Δ Fat mass (%)” and are statistically significantly different (*, p < 0.05) than control for semaglutide and all Examples at all doses tested, except for Example peptides at the low dose, 0.15625 nmol/kg.

[0121] As seen in Table 7, the Example compounds dose-dependently reduce body weight and fat mass.

TABLE-US-00015 TABLE 8 Effect of Treatment with Example Compounds on Blood Glucose, Insulin, Total Cholesterol and Triglycerides After 15 Days. Dose Δ Glucose Δ Insulin Δ Cholesterol Δ Trigs Compound (nmol/kg) (mg/dL) (ng/mL) (mg/dL) (mg/dL) Sema 10 −18.33 ± 2.81  −4.15 ± 2.23  −73.33 ± 4.17*  16.00 ± 0.38 Example 1 0.15625 −18.58 ± 1.04  −4.48 ± 1.70  −14.33 ± 4.37 −27.83 ± 0.44 0.625 −24.42 ± 4.44*  −6.03 ± 0.94  −39.17 ± 0.08  15.33 ± 2.75 2.5 −14.67 ± 0.77 −10.13 ± 4.21 −118.17 ± 1.57*  −2.83 ± 4.13 10 −35.08 ± 1.47* −11.14 ± 4.41* −164.00 ± 1.85* −43.00 ± 0.50 20 −56.00 ± 2.12* −11.40 ± 4.55* −179.17 ± 7.73* −52.50 ± 3.95* Example 2 0.15625  −9.50 ± 5.21  14.64 ± 3.28  −22.00 ± 4.71  9.67 ± 7.80 0.625  −6.17 ± 7.12  −9.35 ± 5.61  −43.50 ± 3.47*  −3.50 ± 3.73 2.5 −13.50 ± 3.92 −14.01 ± 7.49 −150.33 ± 1.70*  −2.33 ± 3.07 10 −41.00 ± 4.18* −14.52 ± 7.39 −199.33 ± 7.02*  −9.00 ± 5.09 20 −46.08 ± 4.21* −14.73 ± 7.52 −198.00 ± 5.46* −15.83 ± 6.63 Example 3 0.15625 −26.97 ± 6.52*   0.48 ± 1.80  −14.50 ± 5.03  40.67 ± 1.75 0.625 −19.47 ± 6.65*  −4.49 ± 3.03  −37.83 ± 4.80*  40.33 ± 1.58 2.5 −33.47 ± 1.69*  −7.25 ± 3.60* −136.50 ± 5.42*  −7.17 ± 6.52 10 −48.72 ± 6.41*  −7.61 ± 3.80* −162.50 ± 4.09*  29.00 ± 21.71 20 −55.97 ± 6.74*  −7.69 ± 3.79* −186.83 ± 7.59* −16.50 ± 1.60 NOTE: All data refers to difference at day 15 between test and vehicle groups and is from a single representative study. “Sema” means semaglutide. *p < 0.05 compared to Vehicle group; one-way ANOVA, Dunnett's.

[0122] As seen in Table 8, the Example compounds also reduce blood glucose, insulin (as a sign of increasing insulin sensitivity), cholesterol and triglycerides.

TABLE-US-00016 SEQUENCE LISTING The following nucleic and/or amino acid sequences are referred to in the disclosure above and are provided below for reference. SEP ID NO: 1-Human GIP YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEO ID NO: 2-Human GLP-1.sub.7-36 amide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2 SEP ID NO: 3-Human glucagon HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEP ID NO: 4-Human OXM HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEP ID NO: 5-Incretin analog YX.sub.2QGTX.sub.6TSDX.sub.10SIX.sub.13LDX.sub.16X.sub.17AQX.sub.20X.sub.21FIX.sub.24X.sub.25LLEGGPSSGEPPPX.sub.39, where: X.sub.2 is Aib, X.sub.6 is αMeF(2F), X.sub.10 can be Y or 4Pal, X.sub.13 can be L or αMeL, X.sub.16 is Orn, X.sub.17 is any amino acid with a functional group available for conjugation, X.sub.20 can be 4Pal, Iva or αMeL, X.sub.21 can be A or Aib, X.sub.24 can be E or e, X.sub.25 can be Y or αMeY, and X.sub.39 can be E or S SEQ ID NO: 6-Incretin analog Y-Aib-QGT-αMeF(2F)-TSDYSILLDOKAQ-4Pal-AFIEYLLEGGPSSGEPPPE SEQ ID NO: 7-Incretin analog Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDOKAQ-Iva-AFIE-αMeY- LLEGGPSSGEPPPS SEQ ID NO: 8-Incretin analog Y-Aib-QGT-αMeF(2F)-TSD-4Pal-SI-αMeL-LDOKAQ-αMeL-Aib-FIe-αaMeY- LLEGGPSSGEPPPS SEQ ID NO: 9-Incretin analog Y-Aib-QGT-αMeF(2F)-TSDYSILLDOK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl-γE-CO- (CH.sub.2).sub.18-CO.sub.2H)AQ-4Pal-AFIEYLLEGGPSSGEPPPE-NH.sub.2 SEQ ID NO: 10-Incretin analog Y-Aib-QGT-αMeF(2F)-TSDYSI-αMeL-LDOK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl- γE-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ-Iva-AFIE-αMeY-LLEGGPSSGEPPPS-NH.sub.2 SEQ ID NO: 11 Incretin analog Y-Aib-QGT-αMeF(2F)-TSD-4Pal-SI-αMeL-LDOK(2-[2-(2-amino-ethoxy)-ethoxy]- acetyl-γE-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ-αMeL-Aib-FIe-αMeY-LLEGGPSSGEPPPS-NH.sub.2 SEQ ID NO: 12-Artificial sequence GPSSGEPPPE SEQ ID NO: 13-Artificial sequences GPSSGEPPPS SEQ ID NO: 14-human GIP receptor MTTSPILQLLLRLSLCGLLLQRAETGSKGQTAGELYQRWERYRRECQETLAAAEP PSGLACNGSFDMYVCWDYAAPNATARASCPWYLPWHHHVAAGFVLRQCGSDG QWGLWRDHTQCENPEKNEAFLDQRLILERLQVMYTVGYSLSLATLLLALLILSLF RRLHCTRNYIHINLFTSFMLRAAAILSRDRLLPRPGPYLGDQALALWNQALAACR TAQIVTQYCVGANYTWLLVEGVYLHSLLVLVGGSEEGHFRYYLLLGWGAPALF VIPWVIVRYLYENTQCWERNEVKAIWWIIRTPILMTILINFLIFIRILGILLSKLRTRQ MRCRDYRLRLARSTLTLVPLLGVHEVVFAPVTEEQARGALRFAKLGFEIFLSSFQ GFLVSVLYCFINKEVQSEIRRGWHHCRLRRSLGEEQRQLPERAFRALPSGSGPGE VPTSRGLSSGTLPGPGNEASRELESYC SEQ ID NO: 15-human GLP-1 receptor MAGAPGPLRLALLLLGMVGRAGPRPQGATVSLWETVQKWREYRRQCQRSLTED PPPATDLFCNRTFDEYACWPDGEPGSFVNVSCPWYLPWASSVPQGHVYRFCTAE GLWLQKDNSSLPWRDLSECEESKRGERSSPEEQLLFLYIIYTVGYALSFSALVIAS AILLGFRHLHCTRNYIHLNLFASFILRALSVFIKDAALKWMYSTAAQQHQWDGLL SYQDSLSCRLVFLLMQYCVAANYYWLLVEGVYLYTLLAFSVLSEQWIFRLYVSI GWGVPLLFVVPWGIVKYLYEDEGCWTRNSNMNYWLIIRLPILFAIGVNFLIFVRVI CIVVSKLKANLMCKTDIKCRLAKSTLTLIPLLGTHEVIFAFVMDEHARGTLRFIKL FTELSFTSFQGLMVAILYCFVNNEVQLEFRKSWERWRLEHLHIQRDSSMKPLKCP TSSLSSGATAGSSMYTATCQASCS SEQ ID NO: 16-human GCG receptor MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTL VCNRTFDKYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRG PRGQPWRDASQCQMDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAIL GGLSKLHCTRNAIHANLFASFVLKASSVLVIDGLLRTRYSQKIGDDLSVSTWLSD GAVAGCRVAAVFMQYGIVANYCWLLVEGLYLHNLLGLATLPERSFFSLYLGIG WGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFLAILINFFIFVRI VQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRS AKLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNH RASSSPGHGPPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPF