LONG ACTING AMYLIN RECEPTOR AGONISTS AND USES THEREOF

Abstract

The present disclosure relates to the field of medicine. More particularly, the disclosure is in the field of treatment of diabetes, obesity and/or chronic weight management, dyslipidemia and/or NASH. The disclosure relates to compounds that agonize the amylin receptor 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. A compound comprising: TABLE-US-00028 (SEQ ID NO: 14) Xaa.sub.1-C-Xaa.sub.3-TATCAT-Xaa.sub.10-Xaa.sub.11-Xaa.sub.12-AE-Xaa.sub.15-LVRSS- Xaa.sub.21-Xaa.sub.22-FGP-Xaa.sub.26-LPPTEVGSNTY, Xaa.sub.1 is K or γE; Xaa.sub.3 is E, N, or G; Xaa.sub.10 is G or Q; Xaa.sub.11 is Orn or K; Xaa.sub.12 is L or αMeL; Xaa.sub.15 is αMeF or F; Xaa.sub.21 is N or H; Xaa.sub.22 is NMeD, NMeN, or N; and Xaa.sub.26 is I or K, or a pharmaceutically acceptable salt thereof.

2. The compound of claim 1, wherein the compound is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, 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, and SEQ ID NO:13, or a pharmaceutical salt thereof.

3. A compound consisting of SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.

4. A compound consisting of SEQ ID NO:4 or a pharmaceutically acceptable salt thereof.

5. A compound consisting of SEQ ID NO:6, or a pharmaceutically acceptable salt thereof.

6. A compound consisting of SEQ ID NO:8, or a pharmaceutically acceptable salt thereof.

7. A method of treating Type 2 diabetes in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

8. A method of treating obesity in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

9. A method of treating dyslipidemia in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

10. A method of treating NASH in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

11. A method of lowering food intake in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

12. A method of lowering body weight in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

13. A method of lowering blood glucose in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

14. A method of lowering triglycerides in a patient in need thereof, comprising administering to the patient an effective amount of a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof.

15. A pharmaceutical composition comprising a compound according to any of claims 1-6, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers, diluents, or excipients.

16. A method of treating a condition in a patient in need thereof, selected from the group consisting of: clinical or pre-clinical diabetes, obesity, NASH, and dyslipidemia, comprising administering to the patient an effective amount of the compound of claim 1 and an effective amount of an additional agent.

17. The method of treatment according to claim 16 wherein the additional agent is an incretin or incretin analog.

18. The method of treatment according to claim 16 wherein the incretin or incretin analog is SEQ ID NO:21.

Description

Example 1: Preparation and Purification of Compounds I and II

[0088] Compound I and Compound II are made according to the following steps. First, Compound I (SEQ ID NO:1) is synthesized

using Fluorenylmethyloxycarbonyl (Fmoc)/tert-Butyl (t-Bu) chemistry on a Symphony 12-channel multiplex peptide synthesizer (Protein Technologies, Inc. Tucson, Ariz.).

[0089] Polystyrene Rink Amide MBHA resin LL resin (Novabiochem, sub: 0.35 meq/g, 100-200 mesh, Cat #855045) is used for the synthesis at 0.13 mmol scale. Standard sidechain protecting groups are used. Boc-Glu-OtBu is used for position 1. Fmoc-Lys(Mtt)-OH is used for the lysine at position 26. 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 60° C. using an equal molar ratio of Fmoc amino acid (0.3M), diisopropylcarbodiimide (0.9M) and Oxyma (0.9M), at a 7.7-fold molar excess over the theoretical peptide loading. The amino acid couplings following αMeF at position 15 and NMeN at position 22 are performed for 3 h and 6 h respectively at 60° C. For Compound I (SEQ ID NO:1), the resin is treated with cleavage cocktail at this time (conditions described after the procedures for addition of the fatty acid-linker moiety to generate Compound II). Below is a schematic of Compound I (SEQ ID NO:1) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeN at position 22, and tyrosine at position 37, where the structures of these amino acid residues have been expanded:

##STR00018##

[0090] Then, the resin is thoroughly washed with DCM 6 times to remove residual DMF. The Mtt protecting group on the lysine at position 26 is selectively removed from the peptide resin using two treatments of 30% hexafluoroisopropanol (Oakwood Chemical) in DCM (2×40-minute treatment). Subsequent attachment of the fatty acid-linker moiety is accomplished by coupling 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 1-hour long coupling.

[0091] 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: DODT: TIS: H.sub.2O=89:3:3:5 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. To make the thioacetal bridge, the pellet after air drying is dissolved in 5 mL of 20 mM potassium phosphate buffer and 3 mL of ACN. Once completely dissolved, under good stirring, TCEP in H.sub.2O (30 μM, 5 eq), diiodomethane (8 eq) and triethylamine (10 eq) are added and left to stir for 5-30 mins. LCMS is used to monitor the reaction, which usually completes in 5 mins. After the reaction is complete, 1 mL of H.sub.2O w/0.1% TFA is added to the mixture as well as 6 mL of acetic acid. More ACN is added as needed if the solution is cloudy.

[0092] The crude peptide is purified by RP-HPLC on a Phenomenex PhenylHexyl column (5 μm, 100 A, 250×21.2 mm, Part Number:00G-4257-P0-AX) with a linear gradient using a 100% acetonitrile and 0.1% TFA/water buffer system. The purity of the peptide is assessed using analytical RP-HPLC using a Waters SymmetryShield RP18 column (3.5 um, 6×100 mm, Part 186000179) and pooling criteria is >95%. The main pool purity of Compound II (SEQ ID NO:2) is found to be >98.4%. Subsequent lyophilization of the final main product pool yields the lyophilized peptide TFA salt. The molecular weight of Compound I (SEQ ID NO:1) is determined by LC/MS (Found: [M+3H].sup.3+=1315.2; Calc. [M+3H].sup.3+=1315.5; Found MW (avg)=3942.6; Calc. MW (avg)=3943.4). The molecular weight of Compound II (SEQ ID NO: 2) is determined by LC/MS (Found: [M+3H].sup.3+=1509.5; Calc. [M+3H].sup.3+=1509.7; Found MW (avg)=4525.5; Calc. MW (avg)=4526.1). Below is a schematic of Compound II (SEQ ID NO:2) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeN at position 22, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00019##

[0093] 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 compounds described herein.

Example 2: Preparation and Purification of Compounds III and IV

[0094] Compounds III and IV are made according to the processes outlined in Example 1.

[0095] Below is a schematic of Compound III (SEQ ID NO:3) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (wherein the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00020##

The molecular weight of Compound III (SEQ ID NO:3) is determined by LC/MS (Found: [M+3H].sup.3+=1314.9; Calc. [M+3H].sup.3+=1315.8; Found MW (avg)=3941.7; Calc. MW (avg)=3944.4).

[0096] Below is a schematic of Compound IV (SEQ ID NO:4) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00021##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1509.7; Calc. [M+3H].sup.3+=1510.0; Found MW (avg)=4526.1; Calc. MW (avg)=4527.1).

Example 3: Preparation and Purification of Compound V and VI

[0097] Compounds V and VI are made according to the processes outlined in Example 1.

[0098] Below is a schematic of Compound V (SEQ ID NO:5) using the standard single letter amino acid codes with the exception of cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00022##

The molecular weight of Compound V (SEQ ID NO:5) is determined by LC/MS (Found: [M+3H].sup.3+=1314.9; Calc. [M+3H].sup.3+=1315.5; Found MW (avg)=3941.7; Calc. MW (avg)=3943.4).

[0099] Below is a schematic of Compound VI (SEQ ID NO:6) using the standard single letter amino acid codes with the exception of the lysine at position 1, the cysteines at positions 2 and 7, Orn at position 11, αMeF at position 15, NMeD at position 22, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00023##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1509.4; Calc. [M+3H].sup.3+=1509.7; Found MW (avg)=4525.2; Calc. MW (avg)=4526.1).

Example 4: Preparation and Purification of Compound VII and VIII

[0100] Compounds VII and VIII are made according to the processes outlined in Example 1.

[0101] Below is a schematic of Compound VII (SEQ ID NO:7) using the standard single letter amino acid codes with the exception of the cysteines at positions 2 and 7, Orn at position 11, αMeL at position 12, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00024##

The molecular weight of Compound VII (SEQ ID NO:7) is determined by LC/MS (Found: [M+3H].sup.3+=1317.9; Calc. [M+3H].sup.3+=1318.1; Found MW (avg)=3950.7; Calc. MW (avg)=3951.4).

[0102] Below is a schematic of Compound VIII (SEQ ID NO:8) using the standard single letter amino acid codes with the exception of the lysine at position 1, the cysteines at positions 2 and 7, Orn at position 11, αMeL at position 12, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00025##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1512.2; Calc. [M+3H].sup.3+=1512.4; Found MW (avg)=4533.6; Calc. MW (avg)=4534.2).

Example 5: Preparation and Purification of Compound IX

[0103] Compound IX is made according to the processes outlined in Example 1.

[0104] Below is a schematic of Compound IX (SEQ ID NO:9) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00026##

The molecular weight is determined by LC/MS (Found: [+H].sup.3+=1558.3; Calc. [M+3].sup.3+=1558.8; Found MW (avg)=4671.9; Calc. MW (avg)=4673.3).

Example 6: Preparation and Purification of Compound X

[0105] Compound X is made according to the processes outlined in Example 1.

[0106] Below is a schematic of Compound X (SEQ ID NO:10) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00027##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1558.5; Calc. [M+3H].sup.3+=1558.8; Found MW (avg)=4672.5; Calc. MW (avg)=4673.3).

Example 7: Preparation and Purification of Compound XI

[0107] Compound XI is made according to the processes outlined in Example 1.

[0108] Below is a schematic of Compound XI (SEQ ID NO:11) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00028##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1553.2; Calc. [M+3H].sup.3+=1553.4; Found MW (avg)=4656.6; Calc. MW (avg)=4657.2).

Example 8: Preparation and Purification of Compound XII

[0109] Compound XII is made according to the processes outlined in Example 1.

[0110] Below is a schematic of Compound XII (SEQ ID NO:12) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00029##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1577.4; Calc. [M+3H].sup.3+=1577.8; Found MW (avg)=4729.2; Calc. MW (avg)=4730.3).

Example 9: Preparation and Purification of Compound XIII

[0111] Compound XIII is made according to the processes outlined in Example 1.

[0112] Below is a schematic of Compound XIII (SEQ ID NO:13) using the standard single letter amino acid codes with the exception of the glutamic acid (γE) at position 1 (where the peptide bond is formed using its side chain carboxylic acid group at the gamma position rather than the typical alpha position), the cysteines at positions 2 and 7, Orn at position 11, lysine at position 26, and tyrosine at position 37 where the structures of these amino acid residues have been expanded:

##STR00030##

The molecular weight is determined by LC/MS (Found: [M+3H].sup.3+=1481.2; Calc. [M+3H].sup.3+=1481.3; Found MW (avg)=1440.6; Calc. MW (avg)=4441.0).

Disulfide Bond Bridge Preparation

[0113] To make the disulfide bond, the oxidation solution is prepared by adding 10 drops of Iodine solution (2% Iodine in AcOH) to 40 mL of 20% MeCN/20% AcOH/60% water in a flask. Separately, a peptide solution is made by dissolving the peptide pellet after air drying in 5 ml of AcOH. Under stirring, the peptide solution is added dropwise to the oxidation solution. Additional iodine solution (2% Iodine in AcOH) is added as needed to maintain a light yellow solution in the flask. The light brown/yellow color is maintained for 5 min after all the peptide solution is added. Extra iodine is neutralized by adding one drop of saturated ascorbic acid to the oxidation solution. The solution is filtered with a 0.45 μm filter and the peptide is ready for purification.

Example 10: In Vitro Functional Activity of Amylin Agonist Peptides

[0114] The AMY1 and CT receptors are GPCRs that are functionally coupled to Gas proteins. Stimulation of these receptors results in an increased production of intracellular cAMP, which can be detected using standard in vitro technologies. In vitro activity of peptides is measured by the amount of cAMP formed in human AMY1R and CTR overexpressed cells.

[0115] Human CT receptors are stably expressed in human urinary bladder cells (UM-UC-3, or UMUC3) under control of a pcDNA expression vector. The UMUC3 cell line is cultured in MEM 1X (Meditech Inc., 17-305-CV) supplemented with 10% FBS, 1% antibiotic/antimycotic solution, 1 mM sodium pyruvate, 1X MEM NEAA, 1X GlutaMAX-I. The plasmid of human CTa-pcDNA3.1Hygro(+)(T2616) DNA is transfected into UMUC3 cells using LipofectAMINE 2000 Transfection reagent (Invitrogen, 11668-019). After 20 days under selection, the mRNA levels from different clones are measured to confirm the expression of hCTR. To determine the function of over-expressed hCTR cells, the intracellular cAMP levels in response to salmon calcitonin are measured and compared to the expression of hCTR mRNA in each clone.

[0116] Human AMY1R stable cell lines are generated by further transfecting human RAMP1-pCMVpuroPB (T14213) into UMUC3 hCTR clone cells. After selection, the mRNA levels human RAMP1 from different clones are measured to confirm the expression of human AMY1R.

[0117] hAMY1R 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. The hCTR cells are cultured in the same medium lacking the puromycin. Cultured cells are grown to 70% confluency and then incubated overnight with fresh medium.

[0118] On the day of the assay, 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 (hCTR) or 1500 cells/well (hAMY1R) are dispensed into each well. The plates are incubated at room temperature for 1 hour.

[0119] 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 (pmol/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 selective activity at the amylin receptor versus the calcitonin receptor as shown in Table 1.

TABLE-US-00001 TABLE 1 Comparison of Functional Activity Data at Amylin and Calcitonin Receptors Compound hAMYIR cAMP EC50 hCTR cAMP EC50 Number (pM, ± SE) (pM, ± SE) I 133 ± 12 18000 ± 1080 II  75 ± 12 2340 ± 272 III 156 ± 27 16300 ± 3030 IV 107 ± 16 6140 ± 764 V 139 ± 17 17000 ± 2440 VI 107 ± 15 13200 ± 2230 VII 173 ± 34 11900 ± 1530 VIII 107 ± 20  9690 ± 2010 IX 132 ± 20 18900 ± 3830 X  80 ± 13 14000 ± 3320 XI  38 ± 7.8 10500 ± 2200 XII 113 ± 34 25900 ± 7620 XIII  78 ± 14  9490 ± 1540 XV  43 ± 4.4 2400 ± 402 XVI 393 ± 51 103 ± 20

Example 11: In Vitro Binding Affinity of Amylin Agonist Peptides to hAMY1 and hCT Receptors

[0120] The membranes from human AMY1R and CTR overexpressed cells (described in Example 10) were isolated via a standard method and used for the binding assays. The equilibrium dissociation constants (Kd) for the various receptor/radioligand interactions are determined from saturation binding analysis using the same reagents and buffers as described below for compound testing. The Kd values determined for the receptor preparations used in this study are as follows: hAMY1R, 0.067 nM; human calcitonin receptor (hCTR), 0.046 nM.

hAMY1R Binding Protocol

[0121] The receptor binding affinity (Ki) of rAMY, hCT, and hAMY agonist peptides are determined from a competitive radioligand binding filter assay. The assay buffer consists of (in mM) 50 HEPES, pH 7.1, 5 MgCl.sub.2, 5 KCl, 0.2% (w/v) bacitracin, 0.003% (w/v) saponin and is used to dilute radioligand and membrane preparation. Binding reactions are carried out in 0.1 mL total reaction volume in a polystyrene 96 deep-well assay block. Iodinated rat amylin (ViTrax custom synthesis; 2200 Ci/mmol; 125I-rAMY) is initially diluted to approximately 50 pM in assay buffer. Test compounds and non-specific binding (NSB—defined as 300 nM rAMY) are added to aliquots of radioactivity buffer. Briefly, test compounds are diluted to 200 nM initial concentration, serially diluted in 4-fold steps in 125I-rAMY, and then 0.05 ml of diluted test compound, NSB, or total binding (defined as neat 125I-rAMY) are transferred to the 96 well polystyrene assay block. The binding reaction is initiated by the addition of 0.05 mL of 200 μg/mL hAMY1R diluted in assay buffer to the radioactivity. Assay blocks are gently vortexed, sealed with parafilm, and incubated at RT for approximately 20 hours. Thirty minutes before the incubation is complete, filter mats (Perkin Elmer printed filter mats Cat #1450-421) are soaked in a solution consisting of assay buffer minus saponin but supplemented with 0.1% (w/v) fatty-acid free bovine serum albumin (FAF-BSA) and 0.5% (v/v) polyethyleneimine (PEI). At the end of the incubation, bound ligand is separated from free by addition of an ice cold quench buffer consisting of 100 mM NaCl, 50 mM TRIS-HCl pH 7.1 and immediately collected over the filter mat by vacuum filtration with a TomTec 96 well filter harvester using the following filter protocol (Dry time, 7; Cycle 3 repeat, 0; Wash Time, 1; Soak Time, 1; 1st Aspirate Time, 4; Wash/Aspirate Time, 7; 2nd Aspirate, 4; air pressure 2 PSI). Final assay concentration range for peptides tested in response curves is 100 nM-0.00038 nM.

hCTR Binding Protocol

[0122] The receptor binding affinity (Ki) of rAMY, hCT, and hAMY agonist peptides on hCTR membranes is determined from a competitive radioligand binding filter assay. The assay procedures are similar to that of hAMY1R binding assay, except 125I-hCT (ViTrax custom synthesis; 2200 Ci/mmol) is used as the hot ligand. Binding reactions are carried out in 0.2 mL total reaction volume with 14 pM 125I-hCT and 20 μg/ml hCTR membrane and incubated for 20 hours. Final assay concentration range for peptides tested in response curves is 2500 nM-0.00128 nM. The compounds of the present application show selective activity at the amylin receptor versus the calcitonin receptor as shown in Table 2.

TABLE-US-00002 TABLE 2 In vitro Binding (Ki) at Human Amylin and Calcitonin Receptors Compound hAMYIR, Ki hCTR, Ki Number (nM ± SE) (nM ± SE) II 0.017 ± 0.006 202 ± 45  IV 0.0296 ± 0.0099 665 ± 113 VI 0.0445 ± 0.0082 235 ± 45  VIII 0.0435 ± 0.0072 927 ± 120 XV 0.0196 ± 0.0020 0.256 ± 0.087 XVI 0.701 ± 0.148 0.104 ± 0.043

Example 12: In Vivo Effects on Food Intake and Body Weights in Normal Rats

[0123] Male Sprague Dawley rats from Envigo RMS (Indianapolis, Ind.) are maintained on a chow diet (2014; Teklad Global, Envigo RMS, Indianapolis, Ind.) and single housed in a temperature-controlled facility (72.0° F.; 22.2° C.) with a reversed 12:12-hour light cycle (10 AM OFF) and free access to food and water. At 10 weeks of age, non-fasted body weights and initial food weights are recorded, and animals are administered a single subcutaneous injection (SC) of vehicle or acylated peptide (1 mL/kg), followed by daily measurements of body weight and food intake for 4 days post dose. Area under the curve analysis (AUC) is calculated for both body weight and food intake versus vehicle.

TABLE-US-00003 TABLE 3 Changes in Body Weight and Food Intake for 4 Days in Sprague Dawley Rats Following a Single Dose of Long-Acting Amylin Agonist Peptide Cumulative Food Compound Dose A Body Weight Intake Inhibition Number (nmol/kg, SC) (%)* (%)** II 0.1 1.8 −4.1 0.3 2.4 −3.7 1 0.5 −18.6 3 −3.3 −37.9 10 −7.4 −57.3 30 −10.1 −69.8 100 −12.9 −77.9 300 −14.9 −84.5 1000 −17.2 −87.1 IV 0.1 1.4 −1.1 0.3 1.8 −2.0 1 0.8 −5.6 3 −2.0 −20.5 10 −5.2 −46.8 30 −9.2 −59.2 100 −11.9 −68.1 300 −14.2 −79.2 1000 −16.4 −82.9 VI 0.1 2.2 2.3 0.3 1.2 −0.4 1 1.2 −0.4 3 −0.9 −23.8 10 −3.6 −36.4 30 −8.3 −58.9 100 −12.7 −73.4 300 −14.1 −76.9 1000 −16.2 −84.6 VIII 0.1 2.0 1.3 0.3 1.6 3.2 1 −1.0 −17.5 3 −3.9 −37.7 10 −7.2 −58.5 30 −10.8 −67.0 100 −13.8 −75.2 300 −13.6 −74.9 1000 −18.9 −84.7 *Change in average body weights at 96 hours post dose compared to initial weights of naïve animals. **Cumulative food intake of peptide-treated animals at 96 hours post dose versus vehicle treated animals at 96 hours post dose.

Example 13: In Vivo Effects on Food Intake and Body Weights in Diet-Induced Obese Rats

[0124] Male Long Evan rats from Envigo RMS (Indianapolis, Ind.) at 14 weeks of age are put on a high fat diet (40% of kcal from fat, TD.95217; Envigo RMS, Indianapolis, Ind.) until 35-45 weeks old. Animals are individually housed in a temperature-controlled facility (75.0° F.; 23.9° C.) with a 12-hour reverse light/dark cycle (lights OFF at 10 AM) and free access to food and water. Body weights are recorded and body composition (fat mass) is determined using Quantitative Nuclear Magnetic Resonance Analysis (ECHO MRI, 3-1 Composition Analyzer; Echo Medical Systems, Houston, Tex.), followed by randomization into experimental groups (n=5). Rats are administered subcutaneous injections of vehicle or peptide (1 mL/kg) every three days (day 1, 4, 7, 10, and 13). Daily body weights are recorded and changes at end of treatment (day 14) are calculated as percentages versus pre-treatment weights (day 1). Body composition is determined on day 14, and changes in fat mass and fat-free mass (body weight—fat mass) are calculated from pre-treatment values as gram changes.

TABLE-US-00004 TABLE 4 Changes in Body Weight in a 2-Week Study in Diet-Induced Obese Rats with Amylin Agonist Peptides Compound Dose Δ Body Weight Δ Fat Mass Δ Fat-Free Mass Number (nmol/kg, SC) (%)* (g)* (g)* II 1 −7.1 −43.8 −7.5 10 −9.0 −59.6 −5.6 100 −10.7 −61.0 −15.4 IV 1 −4.5 −31.0 −0.2 10 −8.5 −53.5 −6.4 100 −8.9 −52.9 −8.6 VI 1 −3.2 −25.5 1.9 10 −8.8 −51.9 −9.1 100 −9.8 −54.7 −15.2 VIII 1 −6.0 −36.6 −4.7 10 −9.0 −54.3 −7.2 100 −11.5 −56.4 −26.9 *Change in average body weights or fat mass or fat-free mass at 2 weeks comparedto initial weights of naive animals.

Example 14: Pharmacokinetics in Male Sprague Dawley Rats

[0125] The pharmacokinetics of Compound II (SEQ ID NO:2), Compound IV (SEQ ID NO:4), Compound VI (SEQ ID NO:6), and Compound VIII (SEQ ID NO:8) are evaluated following a single subcutaneous administration of 30 nmol/kg to male Sprague Dawley rats. Blood samples are collected at 1, 3, 6, 12, 24, 48, 72, 96 hours post SC dose. The resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K.sub.3EDTA) concentrations are determined using a qualified LC/MS method that measured the intact mass of the peptides. Each peptide and an analog as an internal standard are extracted from plasma. A high resolution Thermo Q-Exactive is used for LC/MS detection. Mean pharmacokinetic parameters are shown in Table 5.

TABLE-US-00005 TABLE 5 Mean Pharmacokinetic Parameters of peptides Following a Single Subcutaneous Administration of 30 nmol/kg to Male Sprague Dawley Rats. Compound T.sub. l/2 T.sub.max C.sub.max/D AUCINF/D Cl/F Number (hr) (hr) (kg*nmol/L/nmol) (hr*kg*nmol/L/nmol) (mL/hr/kg) II 52.8 ± 6.6 24.0 ± 0.0 4.9 ± 0.9 473 ± 63.8 2.1 ± 0.3 IV  66.9 ± 27.6 20.0−6.9 5.5 ± 2.1 551 ± 27.1 1.8 ± 0.1 VI 54.0 ± 8.4 24.0−0.0 5.4 ± 0.5 506 ± 26.6 2.0 ± 0.1 VIII  36.4 ± 2.93 16.0−6.9 3.8 ± 0.4 270 ± 22.4 3.7 ± 0.3 Abbreviations: T.sub.½ = half-life, T.sub.max = time to maximal concentration, C.sub.max/D = maximal plasma concentration divided by dose, AUCINF/D = AUCinf divided by dose, CL/F = clearance/bioavailability. Notes: Data are the mean, where n = 3/group.

Example 15: Pharmacokinetics in Male Cynomolgus Monkeys

[0126] The pharmacokinetics of Compound II (SEQ ID NO:2), Compound IV (SEQ ID NO:4), Compound VI (SEQ ID NO:6), and Compound VIII (SEQ ID NO:8) are evaluated following a single subcutaneous administration of 20 nmol/kg to male cynomolgus monkeys. Blood samples are collected at 1, 3, 6, 12, 24, 48, 72, 120, 168, 240, 336, 408, 504 hours post SC dose. The resulting individual plasma concentrations are used to calculate pharmacokinetic parameters. Peptide plasma (K.sub.3EDTA) concentrations are determined using a qualified LC/MS method that measured the intact mass of the peptides. Each peptide and an analog as an internal standard are extracted from plasma. A high resolution Thermo Q-Exactive is used for LC/MS detection. Mean pharmacokinetic parameters are shown in Table 6.

TABLE-US-00006 TABLE 6 Mean Pharmacokinetic Parameters of peptides Following a Single Subcutaneous Administration of 20 nmol/kg to Cynomolgus Monkeys. Compound T.sub.½ T.sub.max C.sub.max/D AUCINF/D Cl/F Number (hr) (hr) (kg*nmol/L/nmol) (hr*kg*nmol/L/nmol) (mL/hr/kg) II 159 ± 7.7 64.0 ± 13.9 6.7 ± 1.0 1752 ± 313 0.58 ± 0.1  IV  177 ± 13.5 48.0 ± 24.0 6.2 ± 1.0 1607 ± 104 0.62 ± 0.04 VI 85.0 ± 5.8 40.0 ± 13.9 4.9 ± 0.7 794 ± 27 1.26 ± 0.04 VIII 62.0 ± 4.7 28.0−18.3 7.2 ± 0.3 942 ± 67 1.07 ± 0.1  Abbreviations: T.sub.½ = half-life, T.sub.max = time to maximal concentration, C.sub.max/D = maximal plasma concentration divided by dose, AUCINF/D = AUCinf divided by dose, CL/F = clearance/bioavailability. Notes: Data are the mean, where n = 3/group.

Example 16: Immunogenicity Risk Assessment

Dendritic Cell (DC) Internalization Assay

[0127] This assay assesses the ability of human DCs to internalize tested antibodies. CD14+ cells are cultured and differentiated into immature DCs with IL-4 and GM-CSF. Tested antibodies, isotype control, or a positive control are pre-incubated with the detection agent (Fab-QSY7-TAMRA) in a 1:1 ratio to form a complex and then added to the cultures. Cells are incubated for one day. Upon internalization and cleavage, a positive TAMRA signal is detected by flow cytometry, and a normalized internalization index is calculated using IgG1-EN isotype control and anti-CXCR antibody.

MAPPS Assay (MHC-Associated Peptide Proteomics)

[0128] MAPPS profiles the MHC-II presented peptides on human dendritic cells treated with tested molecules. CD14+ cells, isolated from the PBMCs of normal human donors, are cultured and differentiated into immature DCs by incubation with IL-4 and GM-CSF. On day 4, culture media is replaced with fresh media containing tested molecules. On day 5, LPS is added to transform the cells into mature DCs. On day 6, cells are lysed in RIPA buffer with protease inhibitors. Immunoprecipitation of MHC-II complexes are performed using biotinylated anti-MHC-II antibody coupled to streptavidin beads. The bound complex is eluted and filtered. The isolated MHC-II peptides are analyzed by a mass spectrometer. Peptide identifications are generated by an internal proteomics pipeline using search algorithms with no enzyme and a bovine/human database with test sequences appended to the database. A KNIME workflow is used to process the identification files from the samples. Peptides identified from the test articles are aligned against the parent sequence of the test molecule. The output is used to determine the percentage of donors displaying MHC-II peptides from regions of the test molecule. Compound II, Compound IV, Compound VI, Compound VIII, a compound representative of U.S. Pat. No. 9,023,789 (hereinafter “Compound ′789”), and Pramlintide are tested in the MAPPs assay. Compound II, Compound IV, Compound VI, Compound VIII show no peptides displayed on MHC-II complexes in the assay. Compound ′789 displays a peptide cluster spanning residues 8-23 on MHC-II complexes. Pramlintide shows two peptide clusters that in total span residues 1-34 that are displayed on MHC-II complexes.

In-Silico TCEM (T-Cell Exposed Motif) Analysis

[0129] This analysis assesses the likelihood that specific peptide clusters, identified by MAPPS, will activate CD4+ T cells. MAPPS-identified peptide sequences containing non-germline residues are inputted into an ImmunoEpitope Database (IEDB) Analysis Resource MHCII binding prediction page. The IEDB-recommended prediction method is selected. The prediction considers the 27 most frequent HLA-DR, -DP and -DQ alleles to cover a significant fraction of human population. Each input sequence, with a length equal or greater than 15 residues, is divided into overlapping 15-mers offset 1 amino acid to span the entire sequence. For each peptide, a percentile rank is generated by comparing the peptide's score against the scores of five million random 15 mers selected from SWISSPROT database. Amino acids located at the putative P-1, P2, P3, P5, P7, and P8 positions of the register generate the TCEM, and risk is defined on the basis of presence of non-germline residues at these positions. Non-germline residues and likelihood of the core binding to multiple alleles are reported in a graphic rendering and considered for immunogenicity risk assessment.

MS Serum Binding

[0130] This assay assesses off-target binding of a test candidate to human serum proteins. Tested antibodies are coated onto an Immulon 4 HBX microplate. Following blocking, human serum is added and incubated overnight. The plate is washed, and bound proteins are eluted, reduced, alkylated, and digested. The peptides are analyzed by a mass spectrometer. Peptide identifications are generated by an internal proteomics pipeline using search algorithms with tryptic enzyme specificity and a human database with the test molecule sequences appended. Ions are quantified by internal proteomics tools (Chrom-Alignment, Metaconsense and Quant) and analyzed in JMP using Oneway analysis/Each Pair, Student's t test platform. Analysis on log 2auc for ions using JMP: Fit Y by X per each ion/Compare Means/All pairs, Tukey HSD.

T Cell Proliferation Assay

[0131] This assay assesses the ability of tested antibodies or tested MAPPS peptides to activate CD4+ T cells by inducing cellular proliferation. CD8+ T cell depleted PBMC's are prepared and labeled with CFSE. Each sample is tested with media control, keyhole limpet haemocyanin (KLH; positive clinical benchmark control), tested antibodies, or tested MAPPS peptides. Cultures are incubated for 7 days. On day 7, samples are analyzed by flow cytometry.

Pre-Existing Reactivity (ACE AssayFormat)

[0132] This assay assesses the presence of pre-existing antibodies (ADA) against the tested molecules in treatment-naive normal human serum (NHS). Diluted NHS is incubated overnight on a Pierce Streptavidin plate coated with biotinylated tested molecules. On the following day, the captured binding proteins are acid eluted, hard-coated to a Mesoscale (MSD) plate, and detected with a combination of biotin-labeled molecule and ruthenium-labeled streptavidin. If anti-drug antibodies are present, they will bind the labeled drug and the resulting signal is referred to as Tier 1 signal (expressed as electrochemiluminescence). This signal is confirmed in Tier 2 by adding excess unlabeled tested molecule in the detection step, which results in the suppression of the Tier 1 signal. The presence of pre-existing anti-drug antibodies is expressed as magnitude of the 90.sup.th percentile of Tier 2 inhibition. The 90.sup.th percentile of Tier 2 inhibition, is a statistical tool to assess the magnitude of specificity of Tier 1 reactivity. This 90.sup.th percentile is used to rank molecules in terms of ADA risk.

TABLE-US-00007 TABLE 7 Immunogenicity Risk Assessment Summary Compound Compound Compound Compound Compound Assay Pramlintide ‘789 II IV VI VIII MAPPS Assay High Risk High Risk Low Risk Low Risk Low Risk Low Risk 10 donors with 10/10 5/10 donors no peptides no peptides no peptides no peptides diverse MHC donors displayed displayed displayed displayed displayed displayed peptides peptides T Cell High Risk Low Risk Low Risk Low Risk Low Risk Low Risk Proliferation (7/7 positive (1/7 (0/9 (0/9 (1/9 (1/9 Assay: protein donors) positive positive positive positive positive 7-10 donors with donors) donors) donors) donors) donors) diverse MHC Pre-existing N/A 58.0% 48.8% 33.2% 43.9% 51.4% Reactivity 90.sup.th percentile T2 inhibition >50 donors Abbreviations: ACE = acid capture elution; ADA = anti-drug antibodies; CDR = complementarity determining region; DC = dendritic cell; H1 = VH CDR1; H2 = VH CDR2; H3 = VH CDR3; L1 = VL CDR1; L2 = VL CDR2; MAPPs = MHC-associated peptide proteomics; MHC = major histocompatibility complex; MS = mass spectrometry; T2 = Tier 2; TCEM = T cell exposed motif; VH = variable heavy; VL = variable light; VHFR3 = variable heavy framework 3

Example 17: In Vivo Efficacy in Diet Induced Obese (DIO) Rats of Compound II in Combination with Other Incretin Compounds

[0133] 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 XVII), oxyntomodulin analog (Compound XVIII), and a triagonist of glucagon, GLP-1, and GIP (Compound XIX). 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.

[0134] 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.

[0135] Each group contains five rats. Vehicle and Compound II (1 nmol/kg) are dissolved in vehicle (20 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.

[0136] 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.).

[0137] 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 XVII, Compound XVIII or Compound XIX 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 −35.90 ± 3.95* 149.94 ± 10.88* (1 nmol/kg) Compound XVII −54.20 − 6.52* 150.04 ± 5.71*  (10 nmol/kg) Compound XVIII −50.24 − 6.36* 165.86 ± 7.45*  (10 nmol/kg) Compound XIX −43.28 − 2.54* 157.50 ± 10.63* (3 nmol/kg) Compound 11 + −80.68 − 6.44**  98.60 ± 16.46* Compound XVII Compound II + −59,96 ± 6.87** 137.78 ± 14.66* Compound XVIII Compound II + −63.22 − 10.11** 118.84 ± 11.99* Compound XIX *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; .sup.#p<0.05 compared to either Compound XVII, Compound XVIII or Compound XIX group; .sup.+p<0.05 compared to Compound II.

[0138] There is greater weight loss for the combination of Compound II with Compound XVII, Compound XVIII or Compound XIX than with Compound II alone.

Sequences

[0139]

TABLE-US-00009 SEQ ID NO: 1: Compound I γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN- FGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7

TABLE-US-00010 SEQ ID NO: 2: Compound II γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN- FGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula (γE).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00011 SEQ ID NO: 3: Compound III γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7

TABLE-US-00012 SEQ ID NO: 4: Compound IV γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula (γE).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00013 SEQ ID NO: 5: Compound V KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPILPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7

TABLE-US-00014 SEQ ID NO: 6 Compound VI KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPILPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 1 is attached to a fatty acid linker moiety according to the formula (γE).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00015 SEQ ID NO: 7: Compound VII KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7

TABLE-US-00016 SEQ ID NO: 8: Compound VIII KCETATCATG-Orn-αMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 1 is attached to a fatty acid linker moiety according to the formula (γE).sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00017 SEQ ID NO: 9: Compound IX γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula AEEA.sub.2-γE-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00018 SEQ ID NO: 10: Compound X γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula γE-AEEA.sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00019 SEQ ID NO: 11: Compound XI γE-CNTATCATGKLAEFLVRSSNNFGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula (γE).sub.2-AEEA-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00020 SEQ ID NO: 12: Compound XII γE-CNTATCATQ-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula AEEA.sub.2-γE-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00021 SEQ ID NO: 13: Compound XIII γE-CGTATCATG-Orn-LAEFLVRSSNNFGPKLPPTEVGSNTY-NH.sub.2
wherein there is a thioacetal bridge between the cysteines at positions 2 and 7 wherein the lysine at position 26 is attached to a fatty acid linker moiety according to the formula γE.sub.2-CO—(CH.sub.2).sub.18—CO.sub.2H

TABLE-US-00022 SEQ ID NO: 14: Compound XIV Xaa.sub.1-C-Xaa.sub.3-TATCAT-Xaa.sub.10-Xaa.sub.11-Xaa.sub.12-AE-Xaa.sub.15-LVRSS- Xaa.sub.21-Xaa.sub.22-FGP-Xaa.sub.26-LPPTEVGSNTY-NH.sub.2.
wherein

[0140] Xaa.sub.1 is K or γE

[0141] Xaa.sub.3 is E, N, or G

[0142] Xaa.sub.10 is G or Q;

[0143] Xaa.sub.11 is Orn or K;

[0144] Xaa.sub.12 is L or αMeL

[0145] Xaa.sub.15 is αMeF or F

[0146] Xaa.sub.21 is N or H

[0147] Xaa.sub.22 is NMeD, NMeN, or N

[0148] Xaa.sub.26 is I or K

TABLE-US-00023 SEQ ID NO: 15: Compound XV; Pramlintide KCNTATCATQRLANFLVHSSNNFGPILPPTNVGSNTY-NH.sub.2
Wherein there is a disulfide bridge between the cysteines at positions 2 and 7

TABLE-US-00024 SEQ ID NO: 16: Compounds XVI; hCT CGNLSTCMLGTYTQDFNKFHTFPQTAIGVGAP-NH.sub.2
Wherein there is a disulfide bridge between the cysteines at positions 1 and 7

TABLE-US-00025 SEQ ID NO: 17: hAMY KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY-Nth
Wherein there is a disulfide bridge between the cysteines at positions 2 and 7

TABLE-US-00026 SEQ ID NO: 18 Compound XVII H-Aib-EGTFTSDVSSYLEGQAAK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl).sub.2-(γGlu)-CO-(CH.sub.2).sub.16-CO.sub.2H)EFIAWLVRGRG SEQ ID NO:19 Compound XVIII 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 SEQ ID NO: 20 Compound XIX Y-Aib-QGTFTSDYSI-aMeL-LDKK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)-(γGlu)-CO-(CH.sub.2).sub.18-CO.sub.2H)AQ-Aib- AFIEYLLEGGPSSGAPPPS-NH.sub.2 SEQ ID NO: 21 YX.sub.1EGTFTSDYSIX2LDKIAQKAFVQWLIAGGPSSGAPPPS
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.

TABLE-US-00027 SEQ ID NO: 22: Compound I Backbone γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeN- FGPKLPPTEVGSNTY-NH.sub.2 SEQ ID NO: 23: Compound III Backbone γE-CNTATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPKLPPTEVGSNTY-NH.sub.2 SEQ ID NO: 24: Compound V Backbone KCETATCATG-Orn-LAE-αMeF-LVRSSN-NMeD- FGPILPPTEVGSNTY-NH.sub.2 SEQ ID NO: 25: Compound VII Backbone KCETATCATG-Orn-aMeL-AEFLVRSSHNFGPILPPTEVGSNTY-NH.sub.2