PROTEIN TYROSINE-TYROSINE ANALOGS AND METHODS OF USING THE SAME

Abstract

PYY analogs are disclosed that include modifications that increase half-life when compared to native, human PYY, as well as additional modifications that increase potency and selectivity to the NPY2 receptor. Pharmaceutical compositions also are disclosed that include one or more of the PYY analogs described herein in a pharmaceutically acceptable carrier. Methods of making and using the PYY analogs also are disclosed, especially for treating obesity and obesity-related diseases and disorders such as type II diabetes mellitus.

Claims

1-28. (canceled)

29. A method of treating obesity or an obesity-related disease or disorder, the method comprising the step of: administering to an individual in need thereof an effective amount of a Peptide Tyrosine-Tyrosine (PYY) analog comprising a structure selected from the group consisting of: ##STR00009##

30. The method of claim 29, wherein the PYY analog or pharmaceutically acceptable salt thereof is subcutaneously (SQ) administered to the individual.

31. The method of claim 29, where the PYY analog or pharmaceutically acceptable salt thereof is orally administered to the individual.

32. The method of claim 29, wherein the PYY analog or pharmaceutically acceptable salt thereof is administered daily, every other day, three times a week, two times a week, one time a week, or biweekly.

33. The method of claim 29, wherein the PYY analog or pharmaceutically acceptable salt thereof is administered SQ one time a week (QW).

34. The method of claim 29, wherein the PYY analog or pharmaceutically acceptable salt thereof is administered orally one time a week.

35. The method of claim 29 further comprising administering an additional therapeutic agent.

36. The method of claim 35, wherein the additional therapeutic agent is an incretin selected from the group consisting of glucagon (GCG), a GCG analog, glucagon-like peptide-1 (GLP-1), GLP-1.sub.7-36-amide, a GLP-1 analog, gastric inhibitory peptide (GIP), a GIP analog, oxyntomodulin (OXM), an OXM analog, a GIP/GLP-1, a GLP-1/GCG, or an incretin analog having triple receptor activity.

37. The method of claim 35, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

38. The method of claim 29, wherein the obesity-related disease or disorder is type II diabetes mellitus.

39. A pharmaceutical composition comprising: at least one Peptide Tyrosine-Tyrosine (PYY) analog comprising a structure selected from the group consisting of: ##STR00010##

40. The pharmaceutical composition of claim 39, further comprising an additional therapeutic agent.

41. The pharmaceutical composition of claim 40, wherein the additional therapeutic agent is an incretin selected from the group consisting of glucagon (GCG), a GCG analog, glucagon-like peptide-1 (GLP-1), GLP-1.sub.7-36-amide, a GLP-1 analog, gastric inhibitory peptide (GIP), a GIP analog, oxyntomodulin (OXM), an OXM analog, a GIP/GLP-1, a GLP-1/GCG, or an incretin analog having triple receptor activity.

42. The pharmaceutical composition of claim 40, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

Description

EXAMPLES

[0098] The following non-limiting examples are offered for purposes of illustration, not limitation.

Example 1: PYY Analog 1

[0099] One PYY analog incorporating the inventive concept can have a structure of:

##STR00003##

[0100] Here, the N-terminus is free, and the C-terminal amino acid is amidated as a C-terminal primary amide. The K at position 7 is chemically modified through conjugation to the ε-amino group of the K side chain at position 7 with (Ahx-E-(γE)-CO—(CH.sub.2).sub.16—COOH.

[0101] The PYY analog according to SEQ ID NO:9 is generated by solid-phase peptide synthesis using Fmoc/t-Bu strategy on a SymphonyX Automated Peptide Synthesizer (PTI Protein Technologies Inc.) starting from RAPP AM-Rink Amide resin (H40023 Polystyrene AM RAM, Rapp polymere GmbH). Amino acid couplings are performed using 10 equivalents of amino acid, 0.9 M diisopropylcarbodiimide (DIC) and 0.9 M Oxyma (1:1:1 molar ratio) in DMF for 3 h at 25° C. Deprotections are carried out using 25% piperidine solutions in DMF.

[0102] After elongating the peptide-resin as described above, the MTT protecting group present in K at position 7 is removed using 30% Hexafluoroisopropanol (HFIP) in dichloromethane (DCM). Additional coupling/deprotection cycles using a Fmoc/t-Bu strategy to extend the K at position 7 side chain involve Fmoc-6-aminohexanoic acid (Chem-Impex International Catalog #02490), Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OH)-OtBu (ChemPep Catalog #100703) and HOOC—(CH.sub.2).sub.16—COOtBu. In all couplings, 3 equivalents of the building block are used with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

[0103] Concomitant cleavage from the resin and side chain protecting group removal are carried out in a solution containing TFA:triisopropylsilane:1,2-ethanedithiol:methanol:thioanisole 80:5:5:5:5 (v/v) for 2 h at 25° C. followed by precipitation with cold ether. Crude peptide is purified to >99% purity (15-20% purified yield) by RP-HPLC on a Phenyl Hexyl Column (Phenomenex, Luna; 5 100 A), where suitable fractions are pooled and lyophilized.

[0104] The purity of the PYY analog is examined by analytical RP-HPLC, and identity is confirmed using LC/MS (observed: M+3H.sup.+/3=1659.2 (+/−0.2); calculated: M+3H.sup.+/3=1659.2; observed: M+4H.sup.+/4=1244.6 (+/−0.2); calculated: M+4H.sup.+/4=1244.6; observed: M+5H.sup.+/5=995.9 (+/−0.2); calculated: M+5H.sup.+/5=995.9).

Example 2: PYY Analog 2

[0105] One PYY analog incorporating the inventive concept can have a structure of:

##STR00004##

[0106] As in Example 1, the N-terminus is free, and the C-terminal amino acid is amidated as a C-terminal primary amide. In contrast, however, the K at position 7 is chemically modified through conjugation to the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γE)-CO—(CH.sub.2).sub.18—COOH.

[0107] The PYY analog according to SEQ ID NO:10 is generated by solid-phase peptide, similar to that described above in Example 1. Thus, FMOC-NHPEG.sub.2-CH.sub.2COOH and HOOC—(CH.sub.2).sub.18—COOtBu is attached to the side chain after MTT cleavage using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

[0108] The purity of the PYY analog is examined by analytical RP-HPLC, and identity is confirmed using LC/MS (observed: M+3H.sup.+/3=1665.4 (+/−0.2); calculated: M+3H.sup.+/3=1665.5; observed: M+4H.sup.+/4=1249.3 (+/−0.2); calculated M+4H.sup.+/4=1249.4; observed: M+5H.sup.+/5=999.7 (+/−0.2); calculated: M+5H.sup.+/5=999.7).

Example 3: PYY Analog 3

[0109] One PYY analog incorporating the inventive concept can have a structure of:

##STR00005##

[0110] As in Example 1, the N-terminus is free, and the C-terminal amino acid is amidated as a C-terminal primary amide. In contrast, however, the K at position 7 is chemically modified through conjugation to the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl)-(γE)-CO—(CH.sub.2).sub.16—COOH.

[0111] The PYY analog according to SEQ ID NO:11 is generated by solid-phase peptide, similar to that as described above in Example 1. Thus, FMOC-NHPEG.sub.2-CH.sub.2COOH and HOOC—(CH.sub.2).sub.16—COOtBu are attached to the side chain after MTT cleavage using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

[0112] The purity of the PYY analog is examined by analytical RP-HPLC, and identity is confirmed using LC/MS (observed: M+3H.sup.+/3=1664.7 (+/−0.2); calculated: M+3H.sup.+/3=1664.9; observed: M+4H.sup.+/4=1248.9 (+/−0.2); calculated: M+4H.sup.+/4=1248.9; observed: M+5.sup.+/5=999.3 (+/−0.2); calculated: M+5H.sup.+/5=999.3).

Example 4: PYY Analog 4

[0113] One PYY analog incorporating the inventive concept can have a structure of:

##STR00006##

[0114] As in Example 1, the N-terminus is free, and the C-terminal amino acid is amidated as a C-terminal primary amide. In contrast, however, the K at position 7 is chemically modified through conjugation to the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γE)-CO—(CH.sub.2).sub.16—COOH.

[0115] The PYY analog according to SEQ ID NO:12 is generated by solid-phase peptide, similar to that as described above in Example 1. Thus, FMOC-NHPEG.sub.2-CH.sub.2COOH and HOOC—(CH.sub.2).sub.16—COOtBu are attached to the side chain after MTT cleavage using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

[0116] The purity of the PYY analog is examined by analytical RP-HPLC, and identity is confirmed using LC/MS (observed: M+3H.sup.+/3=1664.8 (+/−0.2); calculated: M+3H.sup.+/3=1665.5; observed: M+4H.sup.+/4=1248.9 (+/−0.2); calculated: M+4H.sup.+/4=1249.4; observed: M+5H.sup.+/5=998.9 (+/−0.2); calculated: M+5H.sup.+/5=995.7).

Example 5: PYY Analog 5

[0117] One PYY analog incorporating the inventive concept can have a structure of:

##STR00007##

[0118] As in Example 1, the N-terminus is free, and the C-terminal amino acid is amidated as a C-terminal primary amide. In contrast, however, the K at position 7 is chemically modified through conjugation to the ε-amino group of the K side chain with ([2-(2-amino-ethoxy)-ethoxy]-acetyl).sub.2-(γE).sub.3-CO—(CH.sub.2).sub.18—COOH.

[0119] The PYY analog according to SEQ ID NO:13 is generated by solid-phase peptide, similar to that as described above in Example 1. Thus, FMOC-NHPEG.sub.2-CH.sub.2COOH and HOOC—(CH.sub.2).sub.18—COOtBu are attached to the side chain after MTT cleavage using 3 equivalents of the building block with PyBOP (3 equiv) and DIEA (6 equiv) in DMF for 3 h at 25° C.

[0120] The purity of the PYY analog is examined by analytical RP-HPLC, and identity is confirmed using LC/MS (observed: M+3H.sup.+/3=1732.2 (+/−0.2); calculated: M+3H.sup.+/3=1732.3; observed: M+4H.sup.+/4=1299.4 (+/−0.2); calculated: M+4H.sup.+/4=1299.5; observed: M+5H.sup.+/5=1039.7 (+/−0.2); calculated: M+5H.sup.+/5=1039.8).

Example 6: In Vitro Activity of PYY Analogs

[0121] (1) In Vitro Binding to hNPY1, hNPY2, hNPY4, and hNPY5 Receptors

[0122] Purpose:

[0123] To assess the in vitro binding affinity (K.sub.i) of the PYY analogs of Examples 1 to 5 in the absence of bovine serum albumin (BSA) to the following human (h) receptors: hNPY1R, hNPY2R, hNPY4R and hNPY5R. Competitive radioligand binding assays with membranes prepared from cell lines overexpressing each of the recombinant receptors and the relevant [.sup.125I]-labeled peptides are used in a scintillation proximity assay (SPA) method. The binding affinity for the associated native peptides, PYY.sub.1-36 (SEQ ID NO:1), PYY.sub.3-36 (SEQ ID NO:2) and Pancreatic Polypeptide.sub.1-36 (PP.sub.1-36; SEQ ID NO:14), is determined in each assay as a control.

[0124] Methods:

[0125] PYY analogs, native, human PYY.sub.1-36 and control PYY.sub.3-36 are synthesized at Lilly Research Laboratories (Indianapolis, Ind., USA) and are characterized by LC/MS, NMR, and LC/UV analysis (99.5% purity). Peptide contents are estimated at 80% powder mass. The peptides are prepared as 10 mM stock solution in 100% DMSO and kept frozen at −20° C. until just prior to testing in the assays.

[0126] For hNPY1R, transient overexpression is performed using CHO cells. Stably transfected cell lines are prepared for hNPY2R and hNPY4R by subcloning receptor cDNA into pcDNA3.1 expression plasmid and transfecting into human embryonic kidney (HEK) 293 cells followed by selection with Geneticin. hNPY5R cloning is performed at Multispan, Inc. (Hayward, Calif.).

[0127] For the preparation of hNPY1R, hNPY2R, mNPY2R and hNPY4R crude cell membranes, two different methods (described below) are utilized. hNPY5R membranes are purchased from Multipan, Inc. (#MCG1275).

[0128] Method 1—For hNPY2R and hNPY4R membranes, frozen cell pellets are lysed on ice in 10 mL hypotonic homogenization buffer containing 50 mM Tris HCl, pH 7.5, and Roche Complete™ Protease Inhibitors with EDTA (#1169749001) per gram of wet cell paste. The cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes. The homogenate is centrifuged at 4° C. at 1100×g for 10 minutes. The supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer and rehomogenized as described above. The homogenate is centrifuged at 1100×g for 10 minutes. The second supernatant is combined with the first supernatant and centrifuged at 35000×g for 1 hour at 4° C. The resulting membrane pellet is resuspended in homogenization buffer containing protease inhibitors at approximately 1 to 3 mg/mL, quick frozen in liquid nitrogen, and stored as aliquots in a −80° C. freezer until use. Protein concentration is determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0129] Method 2—For hNPY1R membranes, frozen cell pellets are lysed on ice in 5 mL hypotonic homogenization Buffer containing 25 mM Tris HCl, pH 7.5, 1 mM MgCl.sub.2, 25 units/mL DNase I (Invitrogen, #18047-019) and Roche Complete™ Protease Inhibitors without EDTA (#11836170001) per gram of wet cell paste. The cell suspension is disrupted using a glass Potter-Elvehjem homogenizer fitted with a Teflon® pestle for 25 strokes. The homogenate is centrifuged at 4° C. at 1800×g for 15 minutes in a 50 mL conical tube. The supernatant is collected and stored on ice while the pellets are resuspended in homogenization buffer and rehomogenized as described above, except DNase I is not used in the homogenization buffer. The homogenate is centrifuged at 1800×g for 15 minutes. The second supernatant is combined with the first supernatant and centrifuged at 25000×g for 30 minutes at 4° C. The resulting membrane pellet is resuspended in homogenization buffer containing protease inhibitors at approximately 2 mg/mL, aliquoted, and stored in a −80° C. freezer until use. Protein concentration is determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0130] General binding assay methods—The equilibrium dissociation constants (K.sub.d) 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 K.sub.d values determined for the receptor preparations used in this study are as follows: hNPY2R, 0.0047 nM; hNPY1R, 0.07 nM; hNPY4R, 0.084 nM; and hNPY5R, 0.896 nM.

[0131] hNPY1R receptor binding protocol—The receptor binding affinity (K.sub.i) of PYY analog peptides and PYY.sub.1-36 for hNPY1R is determined from a competitive radioligand binding assay with human recombinant [.sup.125I]-PYY.sub.1-36 (#NEX341, 2200 Ci/mmol) obtained from Perkin Elmer (Waltham, Mass.). The assay is performed with a SPA method using polyvinyltoluene (PVT) wheat germ agglutinin-coupled SPA beads (#RPNQ0001, Perkin Elmer). Assay buffer (25 mM HEPES, pH 7.5, 1 mM MgCl2, 2.5 mM CaCl.sub.2), and 0.2% w/v Bacitracin (RPI, #32000)) is used for preparation of reagents. PYY analogs and PYY.sub.1-36 are thawed and 3-fold serially diluted in 100% DMSO (10 point concentration response curves) using a Tecan Evo liquid handler. A 20-fold step-down dilution of peptide into assay buffer is made to reduce the level of DMSO and peptide concentration prior to addition into the assay plate. Next, 5 μL serially diluted peptide or DMSO is transferred into a Corning® 3632 clear bottom assay plate containing 45 μL assay buffer or unlabeled PYY.sub.1-36 control (nonspecific binding or NSB, at 10 nM final concentration). Then, 50 μL [.sup.125I]-PYY.sub.1-36 (0.05 nM final concentration) and 50 μL hNPY1R membranes (1.0 μg/well) are added. The final addition is 50 μL of WGA SPA beads (50 μg/well). Final DMSO concentration are 0.125%. Plates are sealed and mixed on a plate shaker (setting 6) for 1 minute and read with a PerkinElmer Trilux MicroBeta® scintillation counter after 10 hours of incubation/bead settling time at room temperature. Final assay concentration ranges for peptides tested in response curves are: PYY analogs (2.5 μM to 0.13 nM) and PYY.sub.1-36 (10 nM to 0.5 pM).

[0132] hNPY2 receptor binding protocol—The receptor binding affinity (K.sub.i) of PYY analog peptides and PP.sub.1-36 for hNPY2R is determined from a competitive radioligand binding assay as described above for hNPY1R. Final assay concentration ranges for peptides tested in response curves are: PYY analogs (0.1 μM to 5 pM) and PYY.sub.3-36 (10 nM to 0.5 pM).

[0133] hNPY4R receptor binding protocol—The receptor binding affinity (K.sub.i) of PYY analog peptides and PP.sub.1-36 for hNPY4R is determined from a competitive radioligand binding assay as described above for hNPY1R. Final assay concentration ranges for peptides tested in response curves are: PYY analogs (2.5 μM to 0.13 nM) and PYY.sub.1-36 (10 nM to 0.5 pM).

[0134] hNPY5R receptor binding protocol—The receptor binding affinity (K.sub.i) of PYY analog peptides and PYY.sub.1-36 for hNPY5R is determined from a competitive radioligand binding assay as described above for hNPY1R. Final assay concentration ranges for peptides tested in response curves are: PYY analogs (1 μM to 10 pM) and PYY.sub.1-36 (1 μM to 10 pM).

[0135] Data analysis for NPY receptor binding assays—Raw count per minute (CPM) data for concentration curves of PYY analogs, PYY.sub.1-36, PYY.sub.3-36, or PP.sub.1-36 are converted to percent specific inhibition by subtracting nonspecific binding (NSB, binding in the presence of excess unlabeled PYY.sub.1-36, PYY.sub.3-36, or PP.sub.1-36, respectively) from the individual CPM values and dividing by the total binding signal, also corrected by subtracting nonspecific binding, as shown in the equation below:

[00001]$\%\mathrm{Specific}\mathrm{Inhibition}=100-\left[\frac{\mathrm{CPM}\mathrm{for}\mathrm{Analog}\mathrm{or}\mathrm{Control}-\mathrm{CPM}\mathrm{for}\mathrm{NSB}}{\mathrm{CPM}\mathrm{for}\mathrm{Total}\mathrm{Binding}-\mathrm{CPM}\mathrm{for}\mathrm{NSB}}\times 100\right].$

[0136] Data are analyzed using four-parameter (curve maximum, curve minimum, IC.sub.50, Hill slope) nonlinear regression routines (Genedata Screener, version 13.0.5, Genedata AG, Basal, Switzerland). The affinity (K.sub.i) is calculated from the relative IC.sub.50 value based upon the equation K.sub.i=IC.sub.50/(1+D/K.sub.d) where D=the concentration of radioligand in the experiment, IC.sub.50 is the concentration causing 50% inhibition of binding, and K.sub.d is the equilibrium binding affinity constant of the radioligand determined from saturation binding analysis (listed above). A qualifier (>) indicates that the data did not reach 50% inhibition, compared to maximum binding in the absence of competitor, whereby the K.sub.i is calculated using the highest concentration of the compound tested in the assay.

[0137] Reported values for K.sub.i are calculated as the geometric mean as shown below: Geometric Mean=10.sup.(Arithmetic Mean of Log 10 Ki Values).

[0138] Standard error of the mean (SEM) is calculated using the delta method as shown below:

[00002]$\begin{array}{cc}\mathrm{SEM}=\mathrm{Geometric}\mathrm{Mean}\times \frac{\mathrm{SD}\mathrm{of}\log \mathrm{transformed}\mathrm{data}}{\mathrm{Square}\mathrm{root}\mathrm{of}n}\times \ln \left(10\right),& \end{array}$

where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0139] Selectivity of peptides for hNPY2R (Y2) versus hNPY5R (Y5), hNPY4R (Y4), and/or hNPY1R (Y1) are calculated by dividing by the hNPY2R results in nM.

[0140] Results:

TABLE-US-00002 TABLE 1 In Vitro Binding (K.sub.i) to hNPY1R, hNPY2R, hNPY4R and hNPY5R. hNPY2R hNPY5R hNPY4R hNPY1R Fold Fold Fold Peptide (nM) (nM) (nM) (nM) (Y5/Y2) (Y4/Y2) (Y1/Y2) PP.sub.1-36 — — 0.07 — — — — PYY.sub.1-36 0.007 0.37 5 0.06 52 714 8.6 PYY.sub.3-36 0.008 3.6 27 7 450 3375 875 Example 1 0.011 59 626 256 5363 56909 23272 Example 2 0.070 >1000 >1840 >1460 >14285 >26286 >20857 Example 3 0.009 36.4 112 >1550 4044 12444 >193750 Example 4 0.005 ND 280 >148 NA 56000 >29600 Example 5 0.030 ND 560 >1530 NA 18666 >51000 Ref. 1* 0.016 189 — — 11812 — — Ref. 2* 0.021 112 — — 5333 — — Ref. 3* 0.013 469 — — 36076 — — *Known PYY analog for comparison; see, Intl. Patent Application Publication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4 therein Ref. 2 corresponds to Compound 21 therein, and Ref. 3 corres.00070011ponds to Compound 32 therein. ND—not detected NA—not applicable

[0141] As shown above, the PYY analogs of Examples 1-5 are highly selective for the hNPY2 receptor, even demonstrating reduced binding affinity to hNPY5, hNPY4 and hNPY1 receptors versus native, human PYY.sub.3-36 (SEQ ID NO:2).

[0142] (2) In Vitro cAMP Activity on the Human NPY2 Receptor

[0143] Purpose:

[0144] To determine the in vitro functional activity of the PYY analogs of Examples 1 to 5 compared to native, human PYY.sub.3-36 by measuring inhibition of forskolin-induced intracellular cAMP production in HEK 293 cells overexpressing the recombinant human NPY2 receptor.

[0145] Methods:

[0146] PYY analogs and human PYY.sub.3-36 (SEQ ID NO:2) are synthesized, characterized and stored as described above in the receptor binding assays.

[0147] Receptor cloning—A stably transfected cell line is prepared hNPY2 receptor by subcloning receptor cDNA into pcDNA3.1 expression plasmid and transfecting it into HEK 293 cells followed by selection with Geneticin. Aliquots of cells (1×10.sup.7 cells/mL) at passage 9 are made and kept frozen in the vapor phase of a liquid nitrogen tank. These frozen aliquots are used at the time of the assay. Cells maintain greater than 95% viability over several months.

[0148] hNPY2R cAMP assay—Inhibition of forskolin-induced cAMP production by PYY analogs or PYY.sub.3-36 is measured using HEK 293 cells overexpressing recombinant hNPY2R. Frozen aliquots of cells are thawed in a 37° C. water bath. Cells are transferred to a 50 mL tube with 10 mL of culture medium (MEM cell culture medium from Life Tech 11090-081 with 10% FBS from Life Tech 10082-147, 1 mM L-Glutamine from Life Tech 25030-081, 1×NEAA from Life Tech 11140-050, 1 mM sodium pyruvate from Life Tech 11360-070, 1× antibiotics-antimycotics from Life Tech 15240-062) and are centrifuged 5 minutes at 1500 rpm in a Beckman tabletop centrifuge. The supernatant is removed and the cell pellet is resuspended in 10 mL of cell culture medium followed by passage through a 40 μm strainer. An accurate count of cell number and cell viability is determined using a Vi-Cell Analyzer from Beckman-Coulter (Vi-Cell XR 2.03). 8000 cells per well are plated into a white 384 well assay plate (Corning, Poly-D-Lysine coated, white/opaque, cat #356661) using a Combi-Tip Dispenser (Thermo Scientific). Plates are centrifuged 1 second at 1000 rpm and incubated 18 to 20 hours at 37° C. in a 5% CO.sub.2-controlled incubator. Culture medium is removed from the assay plates by flicking gently on paper towels. 10 of assay buffer [1×HBSS (Hyclone, #SH3026801), 20 mM HEPES, pH 7.5 (Hyclone #SH30237.01), 0.1% w/v Casein (CTL Scientific Supply Corp., #440203H), 500 μM IBMX (Sigma-Aldrich #15876)] is added to the wells using the Combi-Tip Dispenser followed by centrifugation for 10 seconds at 1500 rpm. A concentration response curve (20 point) at 2-fold dilutions is prepared in 100% DMSO using acoustic dispensing technology (Labcyte Echo 550). The cells are treated with PYY analogs or human PYY.sub.3-36 for 45 minutes at 37° C. (final DMSO concentration=1%), then stimulated with 1 μM forskolin (Sigma-Aldrich, #F6886) for 45 minutes at 37° C. The intracellular cAMP is quantified using a CisBio cAMP-G.sub.i Dynamic Kit (#62AM9PEB). Briefly, cAMP levels within the cell are detected using the HTRF kit reagents by adding cAMP-d2 conjugate in cell lysis buffer (10 μL) followed by adding 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 minutes at room temperature, then read on a PerkinElmer Envision™ instrument with excitation at 320 nm and emission at 665 nm and 620 nm. Final assay concentration ranges for peptides tested in response curves are: PYY analogs (0.1 μM to 0.2 pM) and human PYY.sub.3-36 (10 nM to 0.02 pM). A standard curve of known cAMP concentrations (0.5 μM to 1 pM) is prepared in assay buffer. Wells in the absence of added competitor or with an excess of added human PYY.sub.3-36 are included on each plate as Maximum Response and Inhibitor Controls, respectively.

[0149] Data analysis for hNPY2 receptor cAMP assay—Time-resolved fluorescence emission is used to calculate a fluorescence ratio (665 nM/620 nm), which is inversely proportional to the amount of cAMP present. Signals for PYY analogs and human PYY.sub.3-36 are converted to nM cAMP per well using a cAMP standard curve plotted as relative response units (emission at 665 nm/620 nm*10,000, y-axis) versus concentration of cAMP (x-axis).

[0150] The amount of cAMP generated (nM) in each well is converted to a percent of the maximal response observed with forskolin only as shown in the equation below:

[00003]$\%\mathrm{Specific}\mathrm{Inhibition}=\frac{\mathrm{Peptide}-\mathrm{Inhibitor}\mathrm{Control}}{\mathrm{Maximum}\mathrm{Response}-\mathrm{Inhibitor}\mathrm{Control}},$

where Inhibitor Control is the cAMP produced in the presence of added excess human PYY.sub.3-36, Maximum Response is the cAMP produced in the presence of forkolin only, and Peptide is the cAMP produced in the presence of test peptide.

[0151] Percent specific inhibition (y-axis) is plotted against the concentration of competitor (x-axis) and analyzed using a four-parameter (curve top, curve bottom, IC.sub.50, Hill slope) nonlinear regression routine (Genedata Screener, version 13.0.5, Genedata AG, Basal, Switzerland) as defined below:

[00004]$\begin{array}{cc}y=\mathrm{bottom}+\frac{\mathrm{top}-\mathrm{bottom}}{1+{\left(\frac{x}{IC50}\right)}^{\mathrm{Hi1l}\mathrm{Slope}}}.& \end{array}$

[0152] The relative IC.sub.50 value represents the concentration causing 50% inhibition of forskolin-induced cAMP production.

[0153] Reported values for IC.sub.50 are calculated as the geometric mean as shown below: Geometric Mean=10.sup.(Arithmetic Mean of Log 10 IC50 Values).

[0154] Standard error of the mean (SEM) is calculated using the delta method as shown below:

[00005]$\begin{array}{cc}\mathrm{SEM}=\mathrm{Geometric}\mathrm{Mean}\times \frac{\mathrm{SD}\mathrm{of}\log \mathrm{transformed}\mathrm{data}}{\mathrm{Square}\mathrm{root}\mathrm{of}n}\times \ln \left(10\right),& \end{array}$

where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0155] Results:

TABLE-US-00003 TABLE 2 In Vitro cAMP Activity on the hNPY2 Receptor. hNPY2R EC.sub.50 Peptide (nM) PYY.sub.1-36 0.12 PYY.sub.3-36 0.15 Example 1 0.07 Example 2 1.23 Example 3 0.15 Example 4 0.06 Example 5 0.55 Ref. 1* 0.30 Ref. 2* 0.23 Ref. 3* 0.32 *Known PYY analog for comparison; see, Intl. Patent Application Publication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4 therein, Ref. 2 corresponds to Compound 21 therein, and Ref. 3 corresponds to Compound 32 therein.

[0156] As shown above, results from the cAMP assay demonstrate functional activity of the PYY analogs of Examples 1-5 on the hNPY2 receptor, with Example 2 showing the weakest potency at 12-fold lower potency versus human PYY.sub.3-36.

[0157] (3) In Vitro GTPγS Activity on the hNPR2 Receptor

[0158] Purpose:

[0159] To assess receptor-mediated activation of G-proteins by the PYY analogs of Examples 1 to 5. Receptor-mediated activation can be measured using the non-hydrolyzable GTP analog, GTPγ[.sup.35S]. Agonist-mediated stimulation of G-protein-coupled receptors results in the activation of membrane-associated Gαβγ-protein heterotrimeric complexes. This represents a first step in transducing extracellular signals to modify intracellular pathways. Herein, a GTPγ[.sup.35S] functional assay is used to assess the potency of various PYY analogs at the hNPY2 receptor.

[0160] Methods:

[0161] PYY analogs, human PYY.sub.1-36 (SEQ ID NO:1) and control peptide PYY.sub.3-36 (SEQ ID NO:2) are synthesized, characterized and stored as described above in the receptor binding assay.

[0162] For each test peptide, concentration response curves (CRC) with ⅓ log dilutions (logarithmic concentrations from −6.52 to −12.52) are completed with a Hamilton NIMBUS liquid handler in assay buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 6 mM MgCl.sub.2, 1 mM EDTA) supplemented with 0.2% Bacitracin (U.S. Biologicals #11805) and 0.5% DMSO. Final assay concentrations of Bacitracin and DMSO are 0.05% and 0.125%, respectively. hNPY2R membranes (Multispan #HTS066M) are prepared to a concentration of 7.5 ug/mL in assay buffer supplemented with 20 uM GDP (Sigma #G-7127) and 6 ug/mL Saponin (Sigma #S-4521) and hNPY2R membranes are incubated at room temperature for 20 minutes prior to addition to the assay. GTPγS[.sup.35S] (PerkinElmer #NEG030H) is prepared in assay buffer to a concentration of 0.6 nM. WGA SPA beads (PerkinElmer #RPNQ0001) are prepared at concentration of 12 mg/mL in assay buffer. The assay is performed in a 96-well plate (Costar #3604) by first adding 100 uL to the hNPY2R membranes, then 50 uL of CRC solution, then 50 uL of GTPγS[.sup.35S] solution for a final volume of 200 uL. The plate is covered and placed on an orbital shaker (175 rpm for 45 minutes) at room temperature. Then, 25 uL of SPA beads are added and plate is re-sealed and vortexed to mix, then placed back on orbital shaker for 3 hours at room temperature. The plate is then centrifuged for 5 minutes at 500 rpm and counted on a PerkinElmer 2450 Microplate counter for 1 minute per well. Basal binding (CPM) is determined in the absence of PYY analog or human PYY.sub.3-36 and is used to calculate a percent above basal value for each concentration of peptide with the following equation: (PYY analog, or human PYY.sub.3-36, CPM−Basal CPM)/(Basal CPM)*100. EC.sub.50 (nM) values is determined by subjecting the logarithm of concentrations and percent of basal values to non-linear regression analysis (log(agonist) vs. response—Variable slope (four parameters)) in GraphPad Prism 7.0 using the equation: Y=Bottom+(Top−Bottom)/(1+10{circumflex over ( )}((Log EC.sub.50−X)*Hill Slope)). Geometric mean and standard error of the mean are calculated from EC.sub.50 (nM) values using the column statistics function in GraphPad Prism 7.0.

[0163] Results:

TABLE-US-00004 TABLE 3 In Vitro GTPγS Activity on the hNPY2 Receptor. hNPY2R EC.sub.50 Peptide (nM) PYY.sub.1-36 0.42 PYY.sub.3-36 0.43 Example 1 0.08 Example 2 1.83 Example 3 0.06 Example 4 0.04 Example 5 0.16 Ref. 1* 0.22 Ref. 2* 0.13 Ref. 3* 0.11 *Known PYY analog for comparison; see, Intl. Patent Application Publication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4 therein, Ref. 2 corresponds to Compound 21 therein, and Ref. 3 corresponds to Compound 32 therein.

[0164] As shown above, results from the GTPγ[.sup.35S] functional assay demonstrate activity of the PYY analogs on the hNPY2 receptor, with Example 2 showing the weakest potency at 4-fold lower potency versus human PYY.sub.3-36.

[0165] (4) Pharmacokinetics

[0166] Purpose:

[0167] To investigate the pharmacokinetic properties of the PYY analogs.

[0168] Methods:

[0169] LC/MS—Plasma concentrations of various PYY analogs are determined by LC/MS methods. The methods measure the whole compound; peptide plus linked time extension. For the assay, PYY analogs and an internal standard are extracted from mouse, rat or monkey plasma (50 μL) using methanol with 0.1% formic acid. The samples are centrifuged and supernatant is transferred to a Thermo Protein Precipitation Plate. The samples are loaded on a Sep-Pak tC18 SPE μElution Plate that is conditioned with methanol and 0.1% formic acid in water. The SPE columns are washed twice with 0.1% formic acid in water. The compounds are then eluted using Formic Acid/Water/acetronitrile (0.1:15:85), which are then dried and reconstituted prior to injecting an aliquot (10 μL) onto a Thermo Acclaim PepMap100 C18, 300 μm×5 mm trap column and Thermo Easy Spray PepMap C18, 75 μm×15 cm column for LC/MS analysis. The column effluent is directed into a Thermo Q-Exactive Plus Mass Spectrometer for detection and quantitation.

[0170] Pharmacokinetics of PYY analogs in CD-1 mice—The plasma pharmacokinetics of the PYY analogs are evaluated in male CD-1 mice following a single subcutaneous dose of 200 nmol/kg. Blood samples are collected from 2 animals per time point over 168 hours. Since non-serial sampling is used to evaluate the kinetics of the PYY analogs in mice, the mean concentration versus time data are used to tabulate the pharmacokinetic parameters for the PYY analogs following a single subcutaneous dose of 200 nmol/kg. Plasma concentrations of PYY analogs are detected through 120 hours following a single subcutaneous administration of 200 nmol/kg.

[0171] Pharmacokinetics of PYY analogs in SD rats—The plasma pharmacokinetics of the PYY analogs are evaluated in male Sprague Dawley rats following a single subcutaneous dose of 50 nmol/kg. Blood samples are collected from 2 animals per time point over 168 hours. Since serial sampling was used to evaluate the kinetics of the PYY analogs in rats, individual animal concentration versus time data were used to tabulate the pharmacokinetic parameters for the PYY analogs following a single subcutaneous dose of 50 nmol/kg. Plasma concentrations of PYY analogs are detected through 120 hours following a single subcutaneous administration of 50 nmol/kg.

[0172] Pharmacokinetics of PYY analogs in cynomolgus monkeys—The plasma pharmacokinetics of the PYY analogs are evaluated in male and female cynomolgus monkeys following a single subcutaneous dose of 50 nmol/kg. Blood samples are collected over 504 hours. Since serial sampling is used to evaluate the kinetics of the PYY analogs in monkeys, individual animal concentration versus time data are used to tabulate the pharmacokinetic parameters for the PYY analogs following a single subcutaneous dose of 50 nmol/kg. Plasma concentrations of PYY analogs are detected through 504 hours following a single subcutaneous administration of 50 nmol/kg.

[0173] Results:

TABLE-US-00005 TABLE 4 Mean Pharmacokinetic Parameters Following a Single Subcutaneous Dose to Male CD-1 Mice. Dose T.sub.1/2 T.sub.max C.sub.max AUC.sub.inf CL/F Peptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 200 15 6 1140 29795 6.71 Example 2 200 27 12 1014 56198 3.56 Example 3 200 26 12 1360 52751 3.79 Example 4 200 15 12 916 31257 6.40 Abbreviations: AUC.sub.inf = area under the curve from 0 to infinity; CL/F = clearance divided by bioavailability (F); C.sub.max = maximum concentration; T.sub.max = time at maximal concentration; T.sub.1/2 = half-life.

TABLE-US-00006 TABLE 5 Mean Pharmacokinetic Parameters Following a Single Subcutaneous Dose to Male SD Rats. Dose T.sub.1/2 T.sub.max C.sub.max AUC.sub.inf CL/F Peptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 50 22 12 221 8621 5.83 Example 3 50 38 12 399 21094 2.39 Example 4 50 19 8 219 8289 6.41 Abbreviations: AUC.sub.inf = area under the curve from 0 to infinity; CL/F = clearance divided by bioavailability (F); C.sub.max = maximum concentration; T.sub.max = time at maximal concentration; T.sub.1/2 = half-life.

TABLE-US-00007 TABLE 6 Mean Pharmacokinetic Parameters Following a Single Subcutaneous Dose to Cynomolgus Monkeys. Dose T.sub.1/2 T.sub.max C.sub.max AUC.sub.inf CL/F Peptide (nmol/kg) (hr) (hr) (nmol/L) (hr*nmol/L) (mL/hr/kg) Example 1 50 101 9 500 64552 0.775 Example 2 50 131 18 583 123173 0.408 Example 3 50 148 9 599 105839 0.509 Abbreviations: AUC.sub.inf = area under the curve from 0 to infinity; CL/F = clearance divided by bioavailability (F); C.sub.max = maximum concentration; T.sub.max = time at maximal concentration; T.sub.1/2 = half-life.

[0174] Results:

[0175] These data demonstrate that the above compounds have a pharmacokinetic profile suitable for once weekly administration.

[0176] (5) Solubility & Stability

[0177] Purpose:

[0178] To determine the soluble pH ranges and stability of the PYY analogs

[0179] Methods:

[0180] Visual solubility range assessment—Lyophilized PYY analog powders are reconstituted in water at 4 mg/mL concentrations, and the pH is adjusted with citric acid/phosphate buffer to pH 4. pH of the system is titrated up with 0.5 N NaCl to pH 8 and then titrated down with 0.5 N HCl to pH 4.

[0181] Thermal stability evaluation—PYY analog solutions in 10 mM or 20 mM sodium phosphate, pH 7.0 at 1 mg/mL or 2 mg/mL concentration are prepared and incubated at 4° C. and 40° C. for 4 weeks. The samples at 4-week time point are analyzed by size exclusion chromatography (SEC) and RP-HPLC.

[0182] SEC method—Performed using a TOSOH TSKgelG2000SWxl, 7.8 mm ID×30 cm, 5 um column, with mobile phase composition of 50 mM sodium phosphate, 300 mM NaCl, pH 7.0 with 20% acetonitrile over 30 minutes with a flow rate of 0.5 mL/min, λ-214 nm.

[0183] RP method—Performed using a Cortecs C18, 2.7 um, 4.6×50 mm column, 20%-45% acetonitrile/water with 0.085% TFA over 10 minutes with a flow rate 1 mL/min, λ-214 nm.

[0184] Results:

TABLE-US-00008 TABLE 7 Solubility and Thermal Stability of the PYY Analogs. Thermal Stability (10 or 20 mM Phosphate, pH 7.0) Solubility RP % Main Peak SEC % Main Peak Peptide (pH range) 4° C. 40° C. 4° C. 40° C. Example 1 >5.7 95.8 92.9 98.9 95.7 Example 2 >5.5 99.7 99.3 99.9 99.6 Example 3 >6.1 99.5 97.1 95.2 92.4 Example 4 >6.8 96.8 97.5 99.6 98.7 Example 5 >5.8 96.8 95.2 99.7 97.5

[0185] As shown above, all the peptides are soluble at pH>7.0. The stability, as assessed by RP and SEC, suggests that these peptides are relative stable under the aggressive thermal stress.

Example 7: In Vivo Effects of PYY

[0186] (1) In Vivo Effects on Food Intake and Body Weights in Normal Mice

[0187] Purpose:

[0188] To compare the effect of the PYY analogs of Examples 1 to 5 to reduce body weight and suppress food intake in normal mice after a single injection.

[0189] Methods:

[0190] Male C57Bl/6 mice from Envigo RMS (Indianapolis, Ind.) are maintained on a chow diet (5008; LabDiet, St. Louis, Mo.) and single housed in a temperature-controlled facility (74.0° F.; 23.3° C.) with a normal 12:12-hour light cycle and free access to food and water. At 9-10 weeks of age, non-fasted body weights and initial food weights are recorded, and animals are administered a single subcutaneous injection of vehicle or peptide, followed by daily measurements of body weight and food intake for 3 days post dose. Area under the curve analysis (AUC) is calculated for both body weight and food intake versus vehicle. Example 4 at 30 nmol/kg is used as benchmark of 100% efficacy for body weight and food intake in each run of the assay.

[0191] Results:

TABLE-US-00009 TABLE 8 Changes in Body Weight and Food Intake for 3 Days in C57/B16 Mice Following a Single Dose of PYY Analog. Dose Δ Body Weight Δ Food Intake Peptide (nmol/kg, SC) (%)** (%)** Example 1 30 89 103 100 108 105 Example 2 100 71 71 300 117 78 Example 3 30 98 93 100 111 100 Example 4 3 22 12 10 41 48 30 100 100 Example 5 100 70 99 300 129 118 Ref. 1* 100 87 79 300 127 104 Ref. 2* 100 62 67 300 110 85 Ref. 3* 100 50 78 300 104 99 *Known PYY analog for comparison; see, Intl. Patent Application Publication No. WO 2016/198682, where Ref. 1 corresponds to Compound 4 therein, Ref. 2 corresponds to Compound 21 therein, and Ref. 3 corresponds to Compound 32 therein. **Example 4 at 30 nmol/kg (AUCs) set at 100% efficacy

[0192] As shown above, reductions in both body weight and food intake demonstrate efficacy of the PYY analogs in vivo, where comparisons on doses required for full efficacy demonstrate improvements in efficacy.

[0193] (2) In Vivo Effects on Food Intake and Body Weights in Diet-Induced Obese Mice

[0194] Purpose:

[0195] To investigate the effect of daily dosing of the PYY analogs of Examples 1-5 to reduce body weight, either alone or in combination with a GLP-1 receptor agonist, over a two-week period in diet-induced obese (DIO) mice.

[0196] Methods:

[0197] DIO male C57Bl/6 mice (Taconic) at 20 weeks of age are maintained on a 60% fat diet upon arrival (D12492; Research Diets, New Brunswick, N.J.). Animals are individually housed in a temperature-controlled facility (74.0° F.; 23.3° C.) with a 12-hour light/dark cycle (lights on 22:00) and free access to food and water. After a one-week acclimation period of daily vehicle dosing, non-fasted body weights are measured, and animals are randomized by body weight into experimental groups (n=6) and administered daily subcutaneous injections of vehicle, a GLP-1 receptor agonist (GLP-1 RA; SEQ ID NO:15), PYY analogs, or combinations of PYY analogs plus the GLP-1 RA. After 2 weeks of dosing, non-fasted body weights are recorded and changes in average body weight versus vehicle are calculated. To determine additive or synergistic effects of PYY analogs in combination with a GLP-1 RA, efficacy above that of the GLP-1 RA alone (net effect) is calculated.

[0198] Results:

TABLE-US-00010 TABLE 9 Changes in Body Weight in a 2-Week Study in Diet-Induced Obese Mice with PYY Analogs Alone or in Combination with a GLP-1 Receptor Agonist. Dose Δ Body Weight (%) Peptide (nmol/kg, SC) PYY Alone PYY + GLP − 1RA* Example 1 1 −4 −18 3 −6 −26 10 −15 −31 Example 2 3 ND −1 10 ND −6 30 2 −11 Example 3 1 −3 −16 3 −6 −28 10 −23 −30 Example 4 0.3 0 −3 1 −1 −12 3 −4 −17 10 −12 −21 Example 5 1 −2 −2 3 0 −11 10 −2 −18 30 −3 ND *Efficacy above that of a GLP-1 receptor agonist (GLP-1 RA)

[0199] As shown above, reductions in body weight with the PYY analogs, both alone and in combination with the GLP-1 RA, demonstrate efficacy of the PYY analogs in vivo, where comparisons are made on the magnitude of weight loss.

[0200] (3) In Vivo Effects on Body Weight and Glucose in Diabetic and Obese (Db/Db) Mice

[0201] Purpose:

[0202] To investigate the effect of daily dosing of the PYY analogs of Examples 1 to 5 to reduce body weight and blood glucose levels over a ten-day period in obese and diabetic mice (db/db).

[0203] Methods:

[0204] Lepr.sup.db/db (db/db) male mice from Envigo RMS (Indianapolis, Ind.) are maintained on a chow-style diet (5008; LabDiet, St. Louis, Mo.) and housed 5 animals per cage in a temperature-controlled facility (74.0° F.; 23.3° C.) with a normal 12:12-hour light cycle and free access to food and water. At 8-9 weeks of age, body weights and blood glucose levels using Accu-Check® Glucometers (Roche Diabetes Care, Inc., Indianapolis, Ind.) are measured, followed by daily subcutaneous injections of vehicle or peptide. After 10 days of dosing, body weights and blood glucose levels are measured and changes versus vehicle treatment are calculated.

[0205] Results:

TABLE-US-00011 TABLE 10 Effects on Body Weight and Blood Glucose in db/db Mice Treated for 10 Days. Dose Δ Body Weight Δ Glucose Peptide (nmol/kg, SC) (%) (%) Example 1 1 −1 7 3 −1 −10 10 −10 −56 30 −14 −59 Example 2 30 −5 −40 100 −9 −60 300 −17 −67 Example 3 1 0 0 3 −5 −7 10 −9 −50 30 −15 −53 Example 4 3 −2 −23 10 −12 −61 30 −17 −62 Example 5 10 −4 −24 30 −11 −59 100 −17 −66

[0206] As shown above, reductions in body weight and blood glucose levels with the PYY analogs demonstrate efficacy of the PYY analogs in vivo, where comparisons are made on the magnitude of weight loss and glucose lowering.

[0207] In conclusion, the PYY analogs herein show selectivity toward NPY2R. They also show dose-dependent reductions in body weight, as reflected in normal mice, diet-induced obese mice and db/db mice, as well as dose-dependent improvement in blood glucose in db/db mice, with the PYY analogs of Examples 1, 3 and 4 being the most efficacious, in line with the in vitro profile.

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