DUAL AMYLIN AND CALCITONIN RECEPTOR AGONISTS AND USES THEREOF

Abstract

The present disclosure related to the field of medicine. More particularly, the disclosure is in the field of treatment of diabetes, obesity, and/or dyslipidemia. The disclosure relates to compounds that agonize both the calcitonin and amylin receptors and can lower food intake, body weight, glucose and/or triglycerides, so can be used to treat diabetes, obesity and/or dyslipidemia. The present disclosure also includes pharmaceutical compositions containing such compounds and therapeutic uses of such compounds and compositions.

Claims

1-13. (canceled)

14. A method of treating a condition selected from the group consisting of diabetes, obesity, NASH, and dyslipidemia, in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a GLP-1 agonist.

15. The method of claim 14, wherein the GLP-1 agonist is selected from the group consisting of Compound III (SEQ ID NO:6), Compound VIII (SEQ ID NO:12), and Compound IX (SEQ ID NO:13).

16. The method of claim 14, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the GLP-1 agonist.

17. The method of claim 15, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the GLP-1 agonist.

18. A method of treating a condition selected from the group consisting of diabetes, obesity, NASH, and dyslipidemia, in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a dual GIP/GLP-1 agonist.

19. The method of claim 18, wherein the dual GIP/GLP-1 agonist is selected from the group consisting of Compound VI (SEQ ID NO:9) and Compound VII (SEQ ID NO: 10).

20. The method of claim 18, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the dual GIP/GLP-1 agonist.

21. The method of claim 19, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the dual GIP/GLP-1 agonist.

22. A method of treating a condition selected from the group consisting of diabetes, obesity, NASH, and dyslipidemia, in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of a tri-agonist of glucagon, GIP, and GLP-1.

23. The method of claim 22, wherein the tri-agonist of glucagon, GIP, and GLP-1 is Compound V (SEQ ID NO:8).

24. The method of claim 22, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the tri-agonist of glucagon, GIP, and GLP-1.

25. The method of claim 23, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the tri-agonist of glucagon, GIP, and GLP-1.

26. A method of treating a condition selected from the group consisting of diabetes, obesity, NASH, and dyslipidemia, in a patient in need thereof, comprising administering to the patient an effective amount of a compound comprising SEQ ID NO:1, or a pharmaceutically acceptable salt thereof, in combination with an effective amount of an analog of oxyntomodulin.

27. The method of claim 26, wherein the analog of oxyntomodulin is Compound IV (SEQ ID NO:7).

28. The method of claim 26, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the analog of oxyntomodulin.

29. The method of claim 27, wherein the compound comprising SEQ ID NO:1 is administered in separate, simultaneous, or sequential combination with the analog of oxyntomodulin.

Description

Example 1: Preparation and Purification of Compound I and Compound II

[0074] Compound I and Compound II are made according to the following steps. First, Compound I is synthesized using Fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.).

[0075] Polystyrene Rink Amide AM LL resin (Novabiochem, sub: 0.33 meq/g, 100-200 mesh, Cat #855045) is used for the synthesis at 0.13 mmol scale.

Standard sidechain protecting groups are used. Fmoc-Lys(Mtt)-OH) is used for the lysine at position 11. Fmoc groups are removed prior to each coupling step (2×7 minutes) using 20% piperidine in DMF. All amino acid couplings are performed for 30 minutes at 50° C. using an equal molar ratio of Fmoc amino acid (0.3 mM), diisopropylcarbodiimide (0.9 mM) and Oxyma (0.9 mM), at a 7.7-fold molar excess over the theoretical peptide loading. N-termini is acetylated with 5% acetic anhydride, 5% DIEA in DMF for 30 minutes. Below is a schematic of Compound I (SEQ ID NO:1)

##STR00005##

Then, the resin is thoroughly washed with DCM for 6 times to remove residual DMF The Mtt protecting group on the lysine at position 11 is selectively removed from the peptide resin using two treatments of 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (2×40-minute treatment).

[0076] Synthesis of Compound II is made according to the following steps. 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-eicosanoic acid (WuXi AppTec, Shanghai, China). 3-Fold excess of reagents (AA:PyAOP:DIEA=1:1:1 mol/mol) are used for each coupling that is 1-hour long.

[0077] 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 (TFA: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 4-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 Waters Xselect Peptide CSH C18 prep column 130A 5 um 19×150 mm PN 186007021) with a linear gradient using 100% acetonitrile and 0.1% TFA/water buffer system (20-40% acetonitrile in 60 min). The purity of peptide is assessed using analytical RP-HPLC and pooling criteria is >95%. The main pool purity of Compound I is found to be >99.0%. Subsequent lyophilization of the final main product pool yields the lyophilized peptide TFA salt. The molecular weight is determined by LC/MS. Average MW=3447.8 Da. Expected mass: (M+3H): 1150.3, found 1150.0.

[0078] The main pool purity of Compound II 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. Average MW=4191.8 Da. Expected mass: (M+3H): 1398.2, found 1398.2. Below is a schematic of Compound II (SEQ ID NO:2).

##STR00006##

[0079] Similar processes to those described above and known to those of skill in the art may be used to synthesize the peptide backbone, conjugate the fatty acid-linker moiety, examine the purity, and confirm the molecular weight of the inventive compound described herein.

Example 2: In Vitro Functional Activity

[0080] The AMY1 and CT receptors are GPCRs that are functionally coupled to Gαs proteins. Stimulation of these receptors results in an increased production of intracellular cAMP, which can be detected using standard in vitro technologies. Human AMY1 or CT receptors are stably expressed in human urinary bladder cells (UM-UC-3) cells under control of a pcDNA (CALCR) or pCMV piggybac (RAMP1) expression vector. AMY1 cells are cultured in MEM 1X (Corning) supplemented with 10% FBS, 1% antibiotic/antimycotic solution, 1 mM sodium pyruvate, 1X MEM NEAA, 1X GlutaMAX-I, 200 μg/mL hygromycin B, and 0.4 μg/mL puromycin. CT cells are cultured in the same medium except that it lacks the puromycin. Cultured cells are grown to 70% confluency, and then incubated overnight with fresh medium.

[0081] On the assay day, 10 μL of assay buffer (phenol red free MEM (Corning, cat #17-305-CV), 0.1% casein, 0.5 mM IBMX, 5 mM HEPES, pH 7.4) is dispensed into each well of white poly-D-lysine coated 384-well plates (Corning cat #354661). Peptides diluted in DMSO are added (200 nL/well) in a 1:3 dilution series using ECHO acoustic liquid handler (Beckman). Cultured cells are detached with TrypLE Express (Gibco), resuspended in assay buffer, and 10 μL containing 1200 cells/well (hCT) or 1500 cells/well (hAMY1) are dispensed into each well. The plates are incubated at room temperature for 1 hour.

[0082] The amount of intracellular cAMP is quantitated using HTRF technology (Homogeneous Time Resolved Fluorescence; Cisbio) as per vendor instructions. Briefly, 10 μL cAMP-d2 conjugate and 10 μL anti-cAMP-cryptate conjugate in lysis buffer are incubated with the treated cells at room temperature for 60 min. The HTRF signal is immediately detected using an Envision plate reader (Perkin-Elmer) to calculate the ratio of fluorescence at 665 to 620 nm. The raw data are converted to cAMP amount (pmole/well) using a cAMP standard curve generated for each experiment. Relative EC.sub.50 values are calculated from the top-bottom range of the concentration response curve defined using 1 nM salmon CT (Bachem) as the maximum and buffer alone as the minimum with a four-parameter logistic curve fitting program (Genedata Screener® v12.0.4). The compounds of the present application show activity at the amylin and calcitonin receptors as shown in Table 1.

TABLE-US-00001 TABLE 1 Activity of Compounds I and II at Amylin and Calcitonin Receptors. Compound hAMY1 cAMP EC50 (pM) hCT cAMP EC50 (pM) Compound I 49.3 ± 8.04 (n = 4)  31.7 ± 5.8 (n = 3) Compound II 49.4 ± 5.14 (n = 7) 40.9 ± 14.7 (n = 4)  Pramlintide  44.0 ± 4.79 (n = 22)  2320 ± 408 (n = 18) Human calcitonin  397 ± 63.5 (n = 7) 106 ± 20.5 (n = 6)

Example 3: In Vivo Efficacy in Normal Rat Models

[0083] Male Sprague Dawley (SD) rats from Envigo Laboratories weighing approximately 300 grams are used to evaluate the effects of Compound II on acute reductions in food intake and body weight loss in vivo during a 96-hour evaluation study. The rats are maintained under approved Animal Care and Use protocols for Lilly Research Laboratories and are housed individually on a 12 hour reverse light cycle from 10 p.m. to 10 a.m. The morning of the study, baseline animal body and food weights are measured and vehicle (20 mM Tris pH8, 50 mg/mL D-mannitol, 0.02% PS80) or Compound II (SEQ ID NO:2) alone in vehicle (20 mM Tris pH8, 50 mg/mL D-mannitol, 0.02% PS80) are subcutaneously administered at different doses.

[0084] Animal body and food weights are measured at 24-, 48-, 72- and 96-hours post-administration. The daily food intake and the percent decrease in body weight are calculated. The results are listed below in Tables 2 and 3. All data is indicated as the mean daily food intake and percent body weight decrease. As demonstrated by the results, Compound II has an effect to reduce food consumption and reduce body weight acutely and in a dose-dependent manner. Also, even very low doses show efficacy in lowering food intake and body weight.

TABLE-US-00002 TABLE 2 Effect of Compound II on Daily Food Intake in Sprague-Dawley Rats. Dose Daily Food Intake (grams) (nmol/kg) 0 h 24 h 48 h 72 h 96 h Control 0.00 18.58 20.42 19.88 20.12 0.1 0.00 16.12 19.65 19.93 21.60 0.3 0.00 15.55 18.65 20.47 21.32 1 0.00 13.72 16.92 19.77 21.03 3 0.00 5.32 10.22 14.88 18.78 10 0.00 2.77 0.87 9.62 15.45 30 0.00 3.75 0.30 5.67 14.85

TABLE-US-00003 TABLE 3 Effect of Compound II on % Change in Body Weight in Sprague-Dawley Rats. Dose % Change in Body Weight (nmol/kg) 0 h 24 h 48 h 72 h 96 h Control 0.00 −0.38 0.54 1.27 1.61 0.1 0.00 −0.89 −0.16 0.61 2.06 0.3 0.00 −0.91 −0.64 0.39 1.50 1 0.00 −1.82 −1.44 −0.38 0.57 3 0.00 −3.63 −4.68 −4.66 −3.26 10 0.00 −5.67 −10.02 −9.88 −8.95 30 0.00 −6.24 −11.60 −12.43 −11.74

Example 4: In Vivo Efficacy in Diet Induced Obese (DIO) Rats

[0085] This study is conducted to investigate the effect of Compound II for diabetes and/or obesity conditions in DIO rats. Diet-induced obese (DIO) male Long Evans (Envigo) rats 24 to 30 weeks old, maintained on a calorie rich diet since arrival at Lilly (TD95217; Teklad, Madison, Wis.), are used in the following studies. Animals are individually housed in a temperature-controlled (24° C.) facility with 12-hour light/dark cycle (lights on 2200) and free access to food (TD95217) and water.

[0086] The rats are randomized according to their body weight, so that each experimental group of animals would have similar body weight. The body weights range from 514 to 710 grams.

[0087] Each groups contains five rats. Vehicle and Compound II (0.1 and 100 nmol/kg) dissolved in vehicle (Tris pH8, (50 mg/mL D-mannitol)+0.02% ps80) are administered by subcutaneous (SC) injection (1 mL/kg) to ad libitum fed DIO rats 30 to 60 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. Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of molecule. Body composition was assessed by quantitative nuclear magnetic resonance (QMNR, EchoMRI LLC, Houston, Tex.) on Days −1 (one day prior to treatment) and 15.

[0088] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek glucometers (Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD, Rockville, Md.).

[0089] All data are presented as mean±SEM of 5 animals per group. Statistical analysis is performed using repeated measures ANOVA, followed by Dunnett's method for multiple comparisons. Significant differences are identified at p<0.05.

[0090] Compound II reduces body weight and food intake in male DIO rats in a dose-dependent manner. Reduced body weight is likely primarily due to reduction in fat mass. In addition to substantial weight loss, reduced serum glucose and reduced insulin are observed with treatment using Compound II in a dose-dependent manner as shown in Tables 4 and 5.

[0091] Body composition measurements are done on Day 0 and Day 14. The change from the initial measurement is presented as Measurement on Day 0-Measurement on Day 14. All data are presented as mean±SEM of 5 animals per group from Day 14. Statistical analysis is performed using repeated measures ANOVA, followed by Dunnett's method for multiple comparisons. Significant differences are identified at p<0.05.

TABLE-US-00004 TABLE 4 The effect of Compound II on body weight, cumulative food intake and fat mass in DIO rats on Day 15 Compound Body Weight Cumulative Food Fat Mass (dose) Change (g) Intake (g) Change (g) Vehicle  8.36 ± 3.69 236.38 ± 6.08  3.58 ± 1.0 (1 mL/kg, SC) Compound II  4.32 ± 1.66 235.58 ± 6.34   4.93 ± 2.96 (0.1 nmol/kg) Compound II −18.74 ± 3.31* 197.14 ± 14.35  −10.62 ± 2.80* (0.3 nmol/kg) Compound II −38.30 ± 5.67* 163.56 ± 13.40* −22.42 ± 4.74* (1 nmol/kg) Compound II −59.84 ± 5.32* 125.70 ± 12.36* −38.09 ± 3.64* (3 nmol/kg) Compound II −73.06 ± 4.27* 110.12 ± 11.24* −43.26 ± 0.81* (10 nmol/kg) Compound II −77.24 ± 3.41* 103.14 ± 13.53* −41.94 ± 6.05* (30 nmol/kg) Compound II −85.84 ± 3.78*  89.36 ± 10.45* −40.77 ± 3.46* (100 nmol/kg) *Significant differences are identified at p < 0.05

TABLE-US-00005 TABLE 5 The effect of Compound II treatment on fasting blood glucose, insulin, insulin resistance index (HOMA-IR = fasting glucose[mmol/L] × Fasting Insulin [μU/ml])/22.5) and glucose area under the curve for 60 minutes (AUC 60 min) during oral glucose tolerance test (OGTT) in DIO rats Fasting Fasting HOMA-IR Glucose (mg/dL) Compound Glucose Insulin [FI] [(FG × AUC (60 min) (dose) [FG](mmol/L) (μU/ml) FI)/22.5] during OGTT Vehicle 7.10 ± 0.40 39.08 ± 4.77  12.52 ± 1.95  14582.25 ± 1299.63 (1 ml/kg, SC) Compound II 6.94 ± 0.22 41.68 ± 5.13  12.98 ± 1.69  14638.50 ± 607.28  (0.1 nmol/kg) Compound II 6.50 ± 0.13 35.06 ± 4.89  10.22 ± 1.56  11367.75 ± 486.94* (0.3 nmol/kg) Compound II 6.94 ± 0.43 18.38 ± 3.53* 5.82 ± 1.31* 11367.75 ± 703.77* (1 nmol/kg) Compound II 6.78 ± 0.28 23.84 ± 2.15* 7.26 ± 0.93* 11189.25 ± 657.87* (3 nmol/kg) Compound II 7.04 ± 0.20 28.08 ± 2.51  8.72 ± 0.62  10959.75 ± 507.17* (10 nmol/kg) Compound II 6.02 ± 0.38 15.16 ± 3.63* 4.02 ± 0.91*  9936.00 ± 752.37* (30 nmol/kg) Compound II  5.86 ± 0.32* 11.92 ± 2.46* 3.20 ± 0.76* 10014.00 ± 538.58* (100 nmol/kg) *Significant differences are identified at p < 0.05

Example 5: Pharmacokinetic Behavior of Compound II

[0092] Plasma concentration of Compound II is determined by a qualified Liquid Chromatography Mass Spectrometry (LC/MS) method at Q Squared Solutions BioSciences LLC, Ithaca, N.Y. Compound II and an analog as an internal standard are extracted from 100% species-specific plasma using protein precipitation followed by solid phase extraction. The intact mass of Compound II which includes peptide plus acyl chain is detected by a Q-Exactive Orbitrap™ mass spectrometer.

[0093] In a monkey pharmacokinetics study, male and female Cynomolgus monkeys are administered a single subcutaneous dose of 20 nmol/kg (0.084 mg/kg) of Compound II in Tris-mannitol buffer (pH 8.0) at a volume of 0.2 mL/kg. Blood is collected predose and at 1, 3, 6, 12, 24, 48, 72, 96, 120, 144, 168, 240, 336, 408, 504 hours postdose for pharmacokinetic characterization. The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Individual and Mean Plasma Pharmacokinetic Parameters Following a Single 20 nmol/kg Subcutaneous Dose of Compound II to Male and Female Cynomolgus Monkeys Cmax AUC.sub.0-inf CL/F Animal T.sub.1/2 Tmax (nmol/ (hr*nmol/ (mL/hr/ Compound ID (hr) (hr) L) L) kg) Compound II P0001 185 12 198 50355 0.40 P0101 170 72 138 44678 0.45 Mean 178 42 168 47516 0.42 Abbreviations: T.sub.1/2 = 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.
In a rat pharmacokinetic study, male Sprague Dawley rats are administered a single subcutaneous dose of 20 nmol/kg (0.084 mg/kg) of Compound II in Tris-mannitol buffer (pH 8.0) at a volume of 0.2 mL/kg. Blood is collected at 1-, 3-, 6-, 12-, 24-, 48-, 72-, 96-, 120-, and 144-hours post-dose for pharmacokinetic characterization. The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Individual and Mean Plasma Pharmacokinetic Parameters Following a Single 20 nmol/kg Subcutaneous Dose of Compound II (SEQ ID NO: 2) to Male Sprague Dawley Rats Cmax AUC.sub.0-inf CL/F Animal T.sub.1/2 Tmax (nmol/ (hr*nmol/ (mL/hr/ Compound ID (hr) (hr) L) L) kg) Compound II R0001 47 48 88 8409 2.38 R0002 60 12 81 8702 2.30 R0003 67 12 99 8655 2.31 Mean 58 24 90 8588 2.33 SD 10 21 9 157 0.04 Abbreviations: T.sub.1/2 = 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.

[0094] The extended half-lives demonstrated by Compound II in Cynomolgus Monkeys show therapies with once weekly dose of Compound II is possible in patients.

Example 6: In Vivo Efficacy in Diet Induced Obese (DIO) Rats of Compound II in Combination with Other Incretin Compounds

[0095] This study is conducted to investigate the effect of Compound II for diabetes and/or obesity conditions in DIO rats when administered in combination with other incretin compounds, including a GLP-1 agonist (Compound III), oxyntomodulin analog (Compound IV), and a triagonist of glucagon, GLP-1, and GIP (Compound V). Diet-induced obese (DIO) male Long Evans (Envigo) rats, maintained on a calorie rich diet since arrival at Lilly (TD95217; Teklad, Madison, Wis.), are used in the following studies. Animals are individually housed in a temperature-controlled (24° C.) facility with 12-hour light/dark cycle (lights on 2200) and free access to food (TD95217) and water.

[0096] The rats are randomized according to their body weight, so that each experimental group of animals would have similar body weight. The body weights range from 529 to 823 grams.

[0097] Each group contains five rats. Vehicle and Compound II (1 nmol/kg) are dissolved in vehicle (40 mM Tris-HCl pH8+0.02% PS80) and are administered by subcutaneous (SC) injection (1 mL/kg) to ad libitum fed DIO rats 30 to 90 minutes prior to the onset of the dark cycle every 3 days for 14 days. SC injections are made on Days 1, 4, 7, 10 and 13. Body weight and food intake are measured daily throughout the study. Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of molecule.

[0098] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek glucometers (Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD, Rockville, Md.).

[0099] All data are presented as mean±SEM of 5 animals per group. Statistical analysis is performed using one-way ANOVA, followed by Tukey's multiple comparison test to compare treatment groups to vehicle group or each other. Significant differences are identified at p<0.05.

TABLE-US-00008 TABLE 8 The Effect of Compound II With and Without Combinations with Compound III, Compound IV, or Compound V on Body Weight and Cumulative Food Intake. Body Weight Cumulative Food Treatment* Change (g)** Intake (g)*** Vehicle −3.42 ± 6.10 212.08 ± 16.84 (10 mL/kg) Compound II −53.84 ± 2.85* 137.38 ± 4.28* (1 nmol/kg) Compound III −54.20 ± 6.52* 150.04 ± 5.71* (10 nmol/kg) Compound IV −50.24 ± 6.36* 165.86 ± 7.45* (10 nmol/kg) Compound V −43.28 ± 2.54*  157.50 ± 10.63* (3 nmol/kg) Compound II +  −85.64 ± 5.08*#+   92.66 ± 11.52*#+ Compound III Compound II +  −85.43 ± 8.66*#  113.58 ± 9.19*# Compound IV Compound II +    −89.70 ± 11.63*#+  97.16 ± 6.45*# Compound V *Treatments were subcutaneously administered every three days on Day 1, 4, 7, 10 and 13. **Body weight measurements were made daily. Body weight change is the difference from Day −1 to Day 14 represented as grams. ***Cumulative food intake was the total food consumed throughout 14-day treatment period. Statistical analysis was done by one-way ANOVA followed by Tukey's. *p < 0.05 compared to vehicle group; #p < 0.05 compared to either Compound III, Compound IV or Compound V group; +p < 0.05 compared to Compound II.

Example 7: An Analogue of Compound II in Combination with an Agonist of GIP-GLP

[0100] This study is conducted to investigate the effect of an analog of Compound II for diabetes and/or obesity conditions in DIO rats when administered with Compound VI (SEQ ID NO:9), a dual agonist of GIP and GLP-1. Diet-induced obese (DIO) male Long Evans (Envigo) rats, maintained on a calorie rich diet since arrival at Lilly (TD95217; Teklad, Madison, Wis.), are used in the following studies. Animals are individually housed in a temperature-controlled (24° C.) facility with 12 hour light/dark cycle (lights on 2200) and free access to food (TD95217) and water.

[0101] The rats are randomized according to their body weight, so that each experimental group of animals would have similar body weight. The body weights range from 549 to 683 grams.

[0102] Each group contains five rats. Vehicle and Compound II (1 nmol/kg) dissolved in vehicle (10 mM Tris-HCl pH7.5, (50 mg/mL D-mannitol)+0.02% PS80) are administered by subcutaneous (SC) injection (1 mL/kg) to ad libitum fed DIO rats 30 to 90 minutes prior to the onset of the dark cycle every 3 days for 14 days. SC injections are made on Days 1, 4, 7, 10 and 13. Body weight and food intake are measured daily throughout the study. Absolute changes in body weight are calculated by subtracting the body weight of the same animal prior to the first injection of molecule.

[0103] At the end of the study, blood is collected to measure blood glucose and plasma insulin. Blood glucose is measured by AccuChek glucometers (Roche, Indianapolis, Ind.). Insulin is measured by ELISA (MSD, Rockville, Md.).

[0104] All data are presented as mean±SEM of 5 animals per group. Statistical analysis is performed using one-way ANOVA, followed by Tukey's multiple comparison test to compare treatment groups to vehicle group or each other. Significant differences are identified at p<0.05.

TABLE-US-00009 Body Weight Cumulative Food Treatment* Change (g)** Intake (g)*** Vehicle  6.18 ± 1.37 231.58 ± 1032  (10 ml/kg) Analog of Compound II −24.82 ± 3.24* 190.74 ± 6.56  (0.1 nmol/kg) Compound VI −18.10 ± 6.67* 196.54 ± 16.98 (3 nmol/kg) Analog of Compound II +  −39.84 ± 2.20*#   174 ± 6.48* Compound VI *Treatments were subcutaneously administered every three days on Day 1, 4, 7, 10 and 13. **Body weight measurements were made daily. Body weight change is the difference from Day −1 to Day 16 represented as grams. ***Cumulative food intake was the total food consumed throughout 16-day treatment period. Statistical analysis was done by one-way ANOVA followed by Tukey's. *p < 0.05 compared to vehicle group; #p < 0.05 compared to GIP-706.
An analogue of Compound II in combination with Compound VI caused more weight loss than individual treatment which is likely mainly attributable to significant decrease in cumulative food intake induced by combination treatment.

Example 8: Immunogenicity Risk Assessment

MAPPs Assay (MHC-Associated Peptide Proteomics):

[0105] Primary human dendritic cells from ten normal human donors are prepared from buffy coats by isolation of CD14+ cells and differentiated into immature dendritic cells by incubation with 20 ng/mL IL-4 and 40 ng/mL GM-CSF in complete RPMI media containing 5% Serum Replacement (Thermo Fisher Scientific, cat #A2596101) for three days at 37° C. and 5% CO.sub.2 as described (Knierman et al., “The Human Leukocyte Antigen Class II Immunopeptidome of the SARS-CoV-2 Spike Glycoprotein”, Cell Reports, 33, 108454 (2020)). Three micromolar of test antibody is added to approximately 5×10.sup.6 cells on day 4 and fresh media containing 5 μg/mL of LPS to transform the cells into mature dendritic cells is exchanged after 5-hour incubation. The matured cells are lysed in 1 mL of RIPA buffer with protease inhibitors and DNAse the following day. The lysates are stored at −80° C. until sample analysis.

[0106] An automated liquid handling system is used to isolate the HLA-II molecules from thawed lysate using biotinylated anti-pan HLA class II antibody (clone Tu39). The bound receptor-peptide complex is eluted with 5% acetic acid, 0.1% TFA. The eluted MHC-II peptides are passed over a prewashed 10 k MWCO filter to remove high molecular weight proteins. The isolated MHC-II peptides are analyzed by nano LC/MS using a Thermo easy 1200 nLC-HPLC system with a Thermo LUMOS mass spectrometer. The separation used a 75 μm×7 cm YMC-ODS C18 column for 65-minute gradient with a 250 nL/min flow rate and 0.1% formic acid in water as A solvent and 80% acetonitrile with 0.1% formic acid as B solvent. Mass spectrometry is run in full scan mode with 240,000 resolution followed by a 3 second data dependent MS/MS cycle comprised of ion trap rapid scans with HCD and EThcD fragmentation.

[0107] Peptide identifications are generated by an internal proteomics pipeline (Higgs et al., “Label-free LC-MS method for the identification of biomarkers”, Methods in Molecular Biology, 428, 209-230 (2008)) using multiple search algorithms with no enzyme search parameter against a bovine/human database containing the test antibody sequences. A KNIME workflow is used to process the identification files for the samples. Peptides identified from the test articles are aligned against the parent sequence. A summary is created for all donors that annotates the percent of donors that display non-germline residues, the number of different regions that display peptides with non-germline residues and the depth of peptide display at each region with non-germline residues. Increases in the extent of display of non-germline peptides is associated with increased risk for immunogenicity. Results for Compound II are shown in Table 9 and suggest a low level of immunogenicity risk associated with Compound II.

TABLE-US-00010 TABLE 9 MAPPs Results Total # of non- Total # of non- Test % # of germline residues germline peptides Compound Donors clusters from all clusters from all clusters Compound II 0% 0 n/a* n/a* *n/a designates totals are not applicable because there were no donors displaying non-germline residues.

T Cell Proliferation Assay

[0108] This assay assesses the ability of test candidate to activate CD4+ T cells by inducing cellular proliferation as described (Walsh et al., “Post-hoc assessment of the immunogenicity of three antibodies reveals distinct immune stimulatory mechanisms”, mAbs, 12, 1764829 (2020)). Cryopreserved PBMCs were used from ten healthy donors and the CD8+ T cells were depleted from the PBMCs and labeled with 1 μM Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE). PBMCs were seeded at 4×10.sup.6 cells/ml/well in AIM-V media (Life Technologies, cat #12055-083) containing 5% CTS™ Immune Cell SR (Gibco, cat #A2596101) and tested in triplicate in 2.0 mL containing the different test articles, media control, keyhole limpet haemocyanin (KLH; positive control), lixisenatide (assay positive control), or Compound II. Cells were cultured and incubated for 7 days at 37° C. with 5% CO.sub.2. On day 7, samples were stained with the following cell surface markers: anti-CD3, anti-CD4, anti-CD14, anti-CD19, and DAPI for viability detection by flow cytometry using a BD LSRFortessa™, equipped with a High Throughput Sampler (HTS). Data was analyzed using FlowJo® Software (FlowJo, LLC, TreeStar) and a Cellular Division Index (CDI) was calculated. Briefly, the CDI for each test candidate's MAPPs-derived peptide cluster was calculated by dividing the percent of proliferating CFSE.sup.dimCD4+ T cells from peptide-stimulated wells by the percent of proliferating CFSE.sup.dimCD4+ T cells in the unstimulated wells. A CDI of ≥2.5 was considered to represent a positive response. A percent donor frequency across all donors was evaluated. All donors produced a positive T cell response against KLH (100%). The clinical immunogenic positive control lixisenatide induced a positive T cell response of 50% of the cohort in this study. This falls within the expected range for this assay (40-90% Positive Donor Frequency). Results for Compound II are shown in Table 10 and suggest a low level of immunogenicity risk for Compound II.

TABLE-US-00011 TABLE 10 The Frequency of CD4+ T cell Responses Median Median CDI CDI Molecule % Positive (Positive (All Range Number of Tested Donors Donors) donors) High Low donors KLH 100.0 36.2 36.2 171.4 4.5 8/8 Lixisenatide 50 3.0 2.5 5.7 1.2 4/8 Compound II 12.5 19.7 1.0 19.7 0.7 1/8

TABLE-US-00012 Sequences Compound 1 (SEQ ID NO: 1) Acetyl-ASHLSTAVLGKLS-Aib-ELHKLEDYPRTDVGAESP-NH.sub.2 Compound II (SEQ ID NO: 2) Acetyl-ASHLSTAVLGK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(γ-Glu)-CO-(CH.sub.2).sub.18- CO.sub.2H)LS-Aib-ELHKLEDYPRTDVGAESP-NH.sub.2 Pramlintide (SEQ ID NO: 3) KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH.sub.2 with a disulfide bond between Cys 2 and Cys 7 Human calcitonin (SEQ ID NO: 4) CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH.sub.2 with a disulfide bond between Cys 1 and Cys 7 Human amylin (SEQ ID NO: 5) KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH.sub.2 with a disulfide bond between Cys 2 and Cys 7 Compound III (SEQ ID NO: 6) H-Aib-EGTFTSDVSSYLEGQAAK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO-(CH.sub.2).sub.16-CO.sub.2H)EFIAWLVRGRG Compound IV (SEQ ID NO: 7) H-Aib-QGTFTSDYSKYLDEKKAK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu)- CO-(CH.sub.2).sub.18-CO.sub.2H)EFVEWLLEGGPSSG-NH.sub.2 Compound V (SEQ ID NO: 8) 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 Compound VI (SEQ ID NO: 9) Y-Aib-EGTFTSDYSI-Aib-LDKIAQK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl).sub.2-(γGlu).sub.2- CO-(CH.sub.2).sub.18-CO.sub.2H)A-(1Nal)-VQWLIAGGPSSGAPPPS-NH.sub.2 Compound VII (SEQ ID NO: 10) YX.sub.1EGTFTSDYSIX.sub.2LDKIAQKAFVQWLIAGGPSSGAPPPS wherein X.sub.1 is Aib; X.sub.2 is Aib; K at position 20 is chemically modified through conjugation to the epsilon-amino group of the K side-chain with (2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu).sub.1-CO-(CH.sub.2).sub.18-CO.sub.2H; and the C-terminal amino acid is amidated as a C-terminal primary amide. CAS Registry Number: 2023788-19-2 Lixisenatide (SEQ ID NO: 11) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK-NH.sub.2 CAS Registry Number: 320367-13-3 Compound VIII (SEQ ID NO: 12) HAEGTFTSDVSSYLEGQAAK(γGlu-CO-(CH.sub.2).sub.14-CH.sub.3)EFIAWLVRGRG CAS Registry Number: 204656-20-2 Compound IX (SEQ ID NO: 13) Dulaglutide is a human GLP-1 receptor agonist which comprises a dimer of a GLP-1 analog fused at its C-terminus via a peptide linker to the N-terminus of an analog of an Fc portion of an immunoglobulin, and is identified by CAS registry number 923950-08-7, which provides the following chemical name: 7-37-Glucagon-like peptide I [8-glycine, 22-glutamic acid, 36-glycine] (synthetic human) fusion protein with peptide (synthetic 16-amino acid linker) fusion protein with immunoglobulin G4 (synthetic human Fc fragment), dimer. Each monomer of dulaglutide has the amino acid sequence set forth in SEQ ID NO: 13: HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAESKYGPP CPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVD GVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSL SLG