Protein tyrosine-tyrosine analogs and methods of use thereof

Abstract

The present invention relates to protein tyrosine-tyrosine analogs and methods of use thereof. Disclosed are PYY analogs comprising modifications that increase half-life compared to native human PYY, and additional modifications that increase efficacy and selectivity to the NPY2 receptor. Also disclosed are pharmaceutical compositions comprising one or more PYY analogs described herein in a pharmaceutically acceptable carrier. Also disclosed are methods of making and using the PYY analogs, particularly for the treatment of obesity and obesity-related diseases and disorders, such as type II diabetes.

Description

This application is a divisional application of Chinese Patent Application No. 201980071360.1, "Protein Tyrosine-Tyrosine Analogs and Methods of Use Thereof", with a filing date of October 28, 2019. Technical Field

[0001] The present disclosure generally relates to biology and medicine, and more specifically, it relates to peptide tyrosine-tyrosine (PYY) analogs that can bind to neuropeptide Y (NPY) receptors such as the NPY2 receptor, compositions containing the same, and their therapeutic use in treating obesity and obesity-related diseases and conditions such as type II diabetes (T2DM). Background Art

[0002] PYY is a member of the pancreatic polypeptide (PP) family and is involved in regulating postprandial food intake and energy expenditure (see, Tatemoto (1982) Proc. Natl. Acad. Sci. 79:2514-2518). PYY is secreted by L cells of the gastrointestinal tract and has two major endogenous forms - PYY 1-36 (SEQ ID NO:1) and PYY 3-36 (SEQ ID NO:2). During fasting, PYY 1-36 is dominant compared to PYY 3-36 , while after feeding, PYY 3-36 is dominant compared to PYY 1-36 . Dipeptidyl peptidase-IV (DPP-IV) hydrolyzes the Pro 1-36 -Ile 2 bond of PYY 3 to produce PYY 3-36 , which is more selective for the NPY2 receptor than PYY 1-36 .

[0003] Plasma PYY 3-36 concentrations typically increase within 15 minutes of food intake, peak within 60 - 90 minutes, and remain elevated for up to 6 hours before returning to baseline (see Adrian et al. (1985) Gastroenterology 89:1070-1077; and De Silva & Bloom (2012) Gut Liver 6:10-20). In this way, it is believed that PYY 3-36 affects appetite via its direct central action and via its effect on bowel motility (i.e., its anorectic effect). Additionally, it is believed that PYY 3-36 mediates insulin sensitivity to thereby help lower blood glucose (i.e., its sensitizing effect).

[0004] Given the anorectic effect of PYY 3-36 of PYY3-36 has been studied as a potential therapeutic agent for weight regulation, particularly for the treatment of obesity and its associated diseases and conditions, including T2DM and cardiovascular disease (see, e.g., International Patent Application Publication No. 2002 / 47712; and Schwartz & Morton (2002) Nature 418:595-597).

[0005] Unfortunately, due to proteases and other clearance mechanisms, exogenously administered PYY 3-36 has a short half-life (e.g., about 10-15 minutes) (see, Lluis et al. (1989) Rev. Esp. Fisiol. 45:377-384; and Torang et al. (2016) Am. J. Physiol. Regul. Integr. Comp. Physiol. 310:R866-R874), which poses a challenge when using it as a therapeutic agent. Due to this short half-life, PYY 3-36 should be administered at least once a day to exert a therapeutic effect, which is inconvenient for individuals in need. Therefore, efforts have been made to increase the half-life of PYY 3-36 and / or increase its NPY2 receptor selectivity. For example, Rubinstein et al. described PYY analogs having a 9-fluorenylmethoxy-carbonyl (Fmoc) or 2-sulfo-9-fluorenyl-methoxycarbonyl (FMS) group to increase the half-life (see, International Patent Application Publication No. WO 2004 / 089279). In addition, DeCarr et al. described PYY analogs having an amino-terminal linked PEG moiety to increase the half-life (see, DeCarr et al. (2007) Bioorg. Med. Chem. Lett. 17:1916-1919; also see, Ortiz et al. (2007) J. Pharmacol. Exp. Ther. 323:692-700). In addition, Kofoed et al. described PYY analogs having an albumin-binding side chain, at least one modified group near the cleavage site of PYY (e.g., an N-methyl amino acid analog of the target amino acid residue), N-glycine, and / or arginine mimics to increase the half-life (see, International Patent Application Publication No. WO 2011 / 033068).

[0006] Despite the significant increase in the understanding of the role of PYY 3-36 in metabolism, there is still a need for additional PYY analogs, particularly PYY analogs with enhanced efficacy and selectivity at the NPY2 receptor. SUMMARY OF THE INVENTION

[0007] As shown above, additional PYY analogs are needed for therapeutic use. To address this need, the present disclosure first describes PYY analogs that include the following basic amino acid sequence (numbered relative to native human PYY 1-36 (SEQ ID NO:1)): 3 PKPEX 7 PX 9 X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY 36 (Formula I), wherein X 7 is any amino acid having a functional group available for conjugation and the functional group is conjugated to a C 16 -C 22 fatty acid, X 9 is E is G, X 10 is E is K, X 17 is L is W, X 18 is N is Q, X 22 is A is I, X 23 is E, D is S, and X 30 is E is W (SEQ ID NO:3), and wherein the carboxyl-terminal (C-terminal) amino acid is optionally amidated.

[0008] In some cases, the amino acid having a functional group available for conjugation at position X 7 can be C, D, E, K or Q. In a specific case, the amino acid having a functional group available for conjugation at position X 7 is K, and the amino acid sequence can be one of the following: 3 PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY 36 (SEQ ID NO:4), 3 PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY 36 (SEQ ID NO:5), 3 PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY 36 (SEQ ID NO:6), 3 PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY36 (SEQ ID NO:7), or 3 PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY 36 (SEQ ID NO:8).

[0009] In some cases, C 16 -C 22 fatty acids are conjugated to amino acids having functional groups available for conjugation via a linker. In certain cases, C 16 -C 22 fatty acids have a structure of -CO-(CH 2 ) a -CO 2 H, where a is an integer between 16 and 22. In specific cases, the fatty acid is C 18 diacid or C 20 diacid, such as palmitic acid, stearic acid, arachidic acid or eicosanoic acid, especially saturated C 18 diacid or C 20 diacid. Similarly, and in some cases, the linker can be one or more units of [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid (Ahx), glutamic acid (E), γ-glutamic acid (γE) or combinations thereof.

[0010] In specific cases, the PYY analog can be one of the following:

[0011] In some cases, the basic structure of the PYY analogs herein can further include the two amino-terminal (N-terminal) amino acids of native human PYY 1-36 (SEQID NO:1), which can subsequently be processed in vivo to PYY 3-36 analogs (i.e., the N-terminal "YP" residues of SEQ ID NO:1 can be cleaved in vivo from any of the PYY analogs).

[0012] In some cases, the PYY analog has a charge greater than -2, especially -3 or -4.

[0013] In some cases, the PYY analog has a greater binding affinity for the human NPY2 receptor than human PYY 3-36 (SEQ IDNO:2), such as about 2-fold to about 10-fold, especially about 2-fold to about 3-fold.

[0014] In some cases, the PYY analog has a half-life that is longer than that of human PYY 3-36 (SEQ ID NO:2), such as about 5 hours to about 24 hours longer, especially about 12 hours.

[0015] Second, a pharmaceutical composition is described that comprises at least one PYY analog of the present disclosure or a pharmaceutically acceptable salt thereof (e.g., trifluoroacetate, acetate, or hydrochloride) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition may further comprise a carrier, diluent, and / or excipient.

[0016] In addition, the pharmaceutical composition may comprise an additional therapeutic agent, such as, for example, other antidiabetic or anti-obesity agents, especially incretins. In some cases, the incretin may be glucagon (GCG) or a GCG analog. In other cases, the incretin may be glucagon-like peptide-1 (GLP-1), GLP-1(7-36) 酰胺 or a GLP-1 analog. In other cases, the incretin may be gastric inhibitory polypeptide (GIP) or a GIP analog. In other cases, the incretin may be a dual receptor agonist, such as oxyntomodulin (OXM) or an OXM analog, GLP-1 / GCG, or GIP / GLP-1. In other cases, the incretin may be an incretin analog having triple receptor activity (i.e., an incretin analog that is active at each of the GIP, GLP-1, and GCG receptors). In other cases, the additional therapeutic agent may be a DPP-IV inhibitor.

[0017] Third, a method of using a PYY analog of the present disclosure, especially using a PYY analog to treat obesity and obesity-related diseases and conditions (such as T2DM) is described. The method comprises at least the step of administering to an individual in need thereof an effective amount of a PYY analog or a pharmaceutically acceptable salt thereof as described herein.

[0018] In some cases, the PYY analog may be administered subcutaneously (SQ) to the individual. Similarly, and in some cases, the PYY analog may be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week), or monthly. In certain cases, the PYY analog may be administered every other day SQ, three times a week SQ, twice a week SQ, once a week SQ, every other week SQ, or once a month SQ. In a specific case, the PYY analog is administered once a week (QW) SQ.

[0019] Alternatively, the PYY analog can be administered orally to the individual. As above, the PYY analog can be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week), or monthly. In some cases, the PYY analog can be administered orally every other day, three times a week orally, twice a week orally, once a week orally, every other week orally, or once a month orally. In a specific case, the PYY analog is administered orally once a week.

[0020] The method can further include administering at least one PYY analog in combination with an effective amount of an additional therapeutic agent such as a DPP-IV inhibitor or an incretin (e.g., GCG or a GCG analog, GLP-1, GLP-1(7-36) 酰胺 or a GLP-1 analog, GIP or a GIP analog, OXM or an OXM analog, GIP / GLP-1, GLP-1 / GCG, or an incretin having triple receptor activity). The DPP-IV inhibitor or incretin can be administered simultaneously with, separately from, or sequentially to the PYY analog.

[0021] In some cases, the DPP-IV inhibitor or incretin can be administered at the same frequency as the PYY analog (i.e., every other day, twice a week, or even weekly). In other cases, the DPP-IV inhibitor or incretin is administered at a frequency different from that of the PYY analog. In other cases, the DPP-IV inhibitor or incretin is administered QW. In still other cases, the PYY analog is administered SQ, and the DPP-IV inhibitor or incretin can be administered orally.

[0022] In some cases, the individual is obese or overweight. In other cases, the individual is a person with diabetes (PwD), especially T2DM. In certain cases, the individual is obese with T2DM or overweight with T2DM.

[0023] The method can further include steps such as measuring or obtaining the weight and / or blood glucose and / or hemoglobin A1c (HbA1c) of the individual and comparing such obtained values with one or more baseline values or previously obtained values to evaluate the effectiveness of the treatment.

[0024] The method can also be combined with diet and exercise and / or can be combined with additional therapeutic agents other than those discussed above.

[0025] Fourth, the use of the PYY analogs herein in the treatment of obesity and obesity-related diseases and conditions (such as T2DM) is described, which optionally may be administered simultaneously, separately, or sequentially (i.e., in combination) with a DPP-IV inhibitor and / or an incretin (such as GCG or a GCG analog, GLP-1, GLP-1(7-36) 酰胺 or a GLP-1 analog, GIP or a GIP analog, OXM or an OXM analog, GIP / GLP-1, GLP-1 / GCG, or even an incretin having triple receptor activity).

[0026] Fifth, the use of the PYY analogs herein in the preparation of a medicament for the treatment of obesity and obesity-related diseases and conditions (such as T2DM) is described, wherein the medicament optionally further comprises a DPP-IV inhibitor and / or an incretin (such as GCG or a GCG analog, GLP-1, GLP-1(7-36) 酰胺 or a GLP-1 analog, GIP or a GIP analog, OXM or an OXM analog, GIP / GLP-1, GLP-1 / GCG, or even an incretin having triple receptor activity).

[0027] One advantage of the PYY analogs herein is that they can not only promote weight loss but also lower glucose. In this way, individuals (especially those susceptible or suffering from T2DM) can delay progression to exogenous insulin and can maintain target HbA1c goals. In addition, the PYY analogs herein can enhance glycemic control by improving insulin sensitization. The combined GIP / GLP-1 and PYY analogs can be used for glucose control (incretin + potential insulin sensitizer) and weight loss (synergistically). Specifically, the PYY analogs herein can cause up to about 12% weight loss when administered to individuals in need thereof and up to about 25% weight loss when administered to individuals in need thereof in combination with an additional therapeutic agent (such as an incretin).

[0028] Another advantage of the PYY analogs herein is that they can have a half-life of up to about 24 hours, thereby allowing once-weekly administration.

[0029] Another advantage of the PYY analogs herein is that they have increased physicochemical stability and compatibility when compared to native human PYY 3-36 (SEQ ID NO:2), and increased compatibility in formulations having an incretin when compared to native human PYY 3-36 (SEQ ID NO:2).

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar to or equivalent to those described herein can be used in the practice or testing of PYY analogs, pharmaceutical compositions, and methods, the preferred methods and materials are described herein.

[0031] Furthermore, the mention of an element by the indefinite article "a" or "an" does not exclude the possibility of there being more than one element, unless the context clearly requires there to be one and only one element. Thus, the indefinite article "a" or "an" generally means "at least one".

[0032] Definitions As used herein, "about" means within a statistically significant range of one or more values, such as, for example, specified concentrations, lengths, molecular weights, pH, sequence identities, lengths of time, temperatures, volumes, etc. Such numerical values or ranges can be within the order of magnitude of usually 20% within the given numerical value or range, more usually 10% within, and even more usually 5% within. The allowable deviation covered by "about" will depend on the particular system being studied and can be readily recognized by those skilled in the art.

[0033] As used herein, "amino acid" means a molecule that is characterized from a chemical perspective by the presence of one or more amine groups and one or more carboxylic acid groups and may contain other functional groups. As is known in the art, there is a set of twenty amino acids, which are designated as standard amino acids and are used as the building blocks for most peptides / proteins produced by any organism.

[0034] As used herein, "amino acid having a functional group available for conjugation" means any natural or unnatural amino acid having a functional group that can be conjugated to a fatty acid, for example, by means of a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azide, bromo, carboxyl, chloro, iodo, and thiol groups. In addition, examples of natural amino acids including such functional groups include C (thiol), D (carboxyl), E (carboxyl), K (amino), and Q (amide).

[0035] As used herein, "analog" means a compound, such as a synthetic peptide or polypeptide, that activates a target receptor and elicits at least one in vivo or in vitro effect elicited by the natural agonist for that receptor.

[0036] As used herein, "anorectic effect" means the ability of the PYY analogs herein to reduce appetite, result in lower food consumption, and ultimately lead to weight loss. The anorectic effect can also refer to the ability of the PYY analogs herein to increase intestinal motility.

[0037] As used herein, "C 16 -C22 "Fatty acid" means a carboxylic acid having from 16 to 22 carbon atoms. C suitable for use herein 16 -C 22 The fatty acid can be a saturated monoacid or a saturated diacid ("diacid" having a carboxyl group at each end).

[0038] As used herein, "AUC" means area under the curve.

[0039] As used herein, "effective amount" means an amount, concentration or dose of one or more of the PYY analogs herein or a pharmaceutically acceptable salt thereof that, upon administration in a single or multiple doses to an individual in need thereof, provides a desired effect (i.e., can produce a clinically measurable difference in the condition of the individual, such as for example a decrease in blood glucose, a decrease in HbA1c and / or a decrease in weight or body fat) in such individual being diagnosed or treated. A person skilled in the art can readily determine the effective amount by using known techniques and by observing results obtained in analogous situations. When determining the amount effective for an individual, many factors are considered, including but not limited to the species of mammal, its body size, age and general health, the particular disease or disorder involved, the degree or severity of the disease or disorder involved or severity, the response of the individual, the particular PYY analog administered, the mode of administration, the bioavailability characteristics of the formulation administered, the dosage regimen selected, the use of concomitant drug therapy, and other relevant circumstances.

[0040] As used herein, "half maximal effective concentration" or "EC 50 " means the concentration of a compound that results in 50% activation / stimulation of an assay endpoint such as a dose-response curve (e.g., cAMP).

[0041] As used herein, "in combination with" means administering at least one of the PYY analogs herein simultaneously, sequentially or in a single combination formulation with one or more additional therapeutic agents.

[0042] As used herein, "incretin analog" means a peptide or polypeptide that has structural similarity but multiple differences to each of GIP, GLP-1, GCG and OXM, especially native human GIP, GLP-1, GCG and OXM. Some incretin analogs also have affinity for and activity at two or even each of the GIP, GLP-1 and GCG receptors (i.e., agonist activity at two receptors such as OXM, GIP / GLP-1 or GLP-1 / GCG, or even agonist activity at all three receptors).

[0043] As used herein, "subject in need thereof" means a mammal, such as a human, having a condition, disease, disorder or symptom for which treatment or therapy is needed, including, for example, those recited herein. Specifically, the preferred subject to be treated is a human.

[0044] As used herein, "long-acting" means that the binding affinity and activity of the PYY analogs herein are longer lasting than those of native human PYY 1-36 (SEQ ID NO:1) and / or native human PYY 3-36 (SEQ ID NO:2), allowing for administration at a frequency of at least once daily or even three times weekly, twice weekly, once weekly or monthly. The time-action profile of the PYY analogs herein can be measured using known pharmacokinetic testing methods (such as those described in the following examples).

[0045] As used herein, "non-standard amino acid" means an amino acid that may occur naturally in cells but is not involved in peptide synthesis. Non-standard amino acids can be components of peptides and are often generated by modifying standard amino acids in a peptide (i.e., via post-translational modification). Non-standard amino acids can include D-amino acids, which have the opposite absolute chirality to the above-described standard amino acids.

[0046] As used herein, "obese" or "obesity" means a condition in which an individual has a body mass index (BMI) of > 30.0 kg / m 2 . See generally, "Overweight & Obesity" from the Centers for Disease Control and Prevention, available at cdc.gov / obesity / adult / defining.html; and "Definitions & Facts for Adult Overweight & Obesity" from the National Institutes of Health, available at iddk.nih.gov / health-information / weight-management / adult-overweight-obesity / definition-facts.

[0047] As used herein, "obesity-related disease or disorder" means any disease or disorder caused or exacerbated by obesity, including but not limited to angina, cardiovascular disease, cholecystitis, cholelithiasis, congestive heart failure, dyslipidemia, fatty liver disease, fertility complications, glucose intolerance, gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, osteoarthritis, polycystic ovary syndrome (PCOS), pregnancy complications, psychological disorders, sleep apnea and other respiratory problems, stress urinary incontinence, stroke, T2DM, uric acid kidney stones (kidney stones), and breast cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, prostate cancer, and rectal cancer.

[0048] As used herein, "overweight" means a condition in which an individual has a BMI of about 25.0 kg / m 2 to < 30 kg / m 2 . See, supra.

[0049] As used herein, "PYY" means peptide YY obtained from or derived from any species, such as a mammalian species, particularly a human. PYY includes native PYY (i.e., full-length) and its variants (i.e., additions, deletions, and / or substitutions of native PYY). Specific PYYs include but are not limited to native human PYY 1-36 (SEQ ID NO:1) and native human PYY 3-36 (SEQ ID NO:2).

[0050] As used herein, "one or more PYY analogs" means PYY-like peptides or polypeptides that elicit one or more of the actions of native PYY at one or more NPY receptors, such as the NPY2 receptor. In some cases, when compared to native PYY, particularly human PYY, such as native human PYY 1-36 (SEQ ID NO:1) and native human PYY 3-36 (SEQ ID NO:2), the PYY analogs herein can bind to NPY receptors, particularly the human NPY2 receptor, with higher or lower affinity but demonstrate a longer in vivo or in vitro half-life. In this way, the PYY analogs herein are synthetic compounds that act as NPY2 receptor agonists.

[0051] As used herein, "saturated" means that a fatty acid does not contain carbon-carbon double or triple bonds.

[0052] As used herein, "sensitizing effect" means the ability of the PYY analogs herein to increase the action of insulin and thereby help lower blood glucose.

[0053] As used herein, the term "treating" or "to treat" means attenuating, inhibiting, reversing, slowing down or stopping the progression or severity of an existing condition, disease, disorder or symptom.

[0054] Certain abbreviations are defined as follows: "ACR" means urinary albumin / creatinine ratio; "amu" means atomic mass unit; "tBoc" means tert-butoxycarbonyl; "cAMP" means cyclic adenosine monophosphate; "DMF" means dimethylformamide; "DMSO" means dimethyl sulfoxide; "EIA / RIA" means enzyme immunoassay / radioimmunoassay; "hr" means hour; "HTRF" means homogeneous time-resolved fluorescence; "IV" means intravenous; "kDa" means kilodalton; "LC-MS" means liquid chromatography-mass spectrometry; "MS" means mass spectrometry; "OtBu" means O-tert-butyl; "Pbf" means NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; "RP-HPLC" means reverse phase high performance liquid chromatography; "SQ" means subcutaneous; "SEM" means standard error of the mean; "TFA" means trifluoroacetic acid; and "Trt" means trityl.

[0055] PYY analog The PYY analogs herein have structural similarity to the native PYY peptide, but there are many structural differences. For example, when compared to native human PYY 1-36 (SEQ ID NO:1) and / or native human PYY 3-36 (SEQ ID NO:2), the PYY analogs described herein include modifications at one or more of positions 3, 7, 9, 10, 17, 18, 22, 23, 30 and 31 numbered relative to native human PYY 1-36 (SEQ ID NO:1). In certain cases, exemplary amino acid sequences of the PYY analogs herein include (specific changes relative to the corresponding residues of native human PYY (SEQ ID NO:1) are in bold): 3 PKPEX 7 PX 9 X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY 36 (SEQ ID NO:3), 3 PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY36 (SEQ ID NO:4), 3 PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY 36 (SEQ ID NO:5), 3 PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY 36 (SEQ ID NO:6), 3 PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY 36 (SEQ ID NO:7), and 3 PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY 36 (SEQ ID NO:8).

[0056] The PYY analogs herein result in sufficient activity at the human NPY2 receptor, but insufficient activity at the NPY1, NPY4, and NPY5 receptors. Similarly, the PYY analogs herein have beneficial properties related to their developability as therapeutic agents, including increased solubility in aqueous solution, increased chemical and physical formulation stability, extended pharmacokinetic profiles, and minimized immunogenic potential.

[0057] In some cases, the PYY analogs herein are C-terminally amino acylated to affect stability. In addition to the changes described herein, the analogs may include one or more additional amino acid modifications, provided, however, that the analogs remain capable of binding and activating the human NPY2 receptor.

[0058] The PYY analogs herein further include, for example, fatty acids (i.e., “acylation”) conjugated to natural or unnatural amino acids having a functional group available for conjugation via a linker. In some cases, the amino acids having a functional group available for conjugation may be C, D, E, K, and Q. In a specific case, the amino acid having a functional group available for conjugation is K, where conjugation is to the ε-amino of the K side chain.

[0059] Here, when compared to native human PYY 1-36 (SEQ ID NO:1), acylation of the PYY analog is at position 7. In this way, the fatty acid can act as an albumin binder to provide a longer-acting analog.

[0060] Regarding fatty acids, they can be conjugated to the functional groups of amino acids available for conjugation either directly or through a linker. The length and composition of the fatty acid affect the half-life of the PYY analog, the in vivo efficacy of the PYY analog, and the solubility and stability of the PYY analog. Conjugation to C 16 -C 22 The saturated mono- or di-carboxylic acids thus produced give rise to PYY analogs that exhibit desired half-life, desired in vivo efficacy, and desired solubility and stability characteristics.

[0061] Exemplary saturated C 16 -C 22 fatty acids used herein include but are not limited to hexadecanoic acid (i.e., palmitic acid, C 16 mono-acid), hexadecanedioic acid (C 16 di-acid), heptadecanoic acid (i.e., margaric acid, C 17 mono-acid), heptadecanedioic acid (C 17 di-acid), stearic acid (C 18 mono-acid), octadecanedioic acid (C 18 di-acid), nonadecylic acid (i.e., nonadecanoic acid, C 19 mono-acid), nonadecanedioic acid (C 19 di-acid), eicosanoic acid (i.e., arachidic acid, C 20 mono-acid), eicosanedioic acid (C 20 di-acid), heneicosanoic acid (i.e., heneicosylic acid, C 21 mono-acid), heneicosanedioic acid (C 21 di-acid), docosanoic acid (i.e., behenic acid, C 22 mono-acid), docosanedioic acid (C 22 di-acid), and their branched and substituted derivatives. In certain cases, C 16 -C 22 fatty acids can be saturated C 18 mono-acids, saturated C 18 di-acids, saturated C 19 mono-acids, saturated C 19 di-acids, saturated C 20 mono-acids, saturated C 20 di-acids, and their branched and substituted derivatives. In specific cases, C 16 -C 22 fatty acids can be palmitic acid or hexadecanoic acid, stearic acid or octadecanedioic acid, or arachidic acid or eicosanedioic acid.

[0062] To assist in conjugating fatty acids to natural or unnatural amino acids having functional groups available for conjugation, the PYY analogs herein can include linkers. In some cases, the linker can be at least one of AEEA, Ahx, E, or γE, and combinations thereof.

[0063] When the linker contains amino acids, it can have one to four E or γE amino acid residues. In some cases, the linker can contain one or two E and / or γE amino acid residues. For example, the linker can contain one or two E and / or γE amino acid residues. In other cases, the linker can contain one to four amino acid residues (such as, for example, E or γE amino acids) used in combination with AEEA or Ahx. Specifically, the linker can be a combination of E and γE amino acid residues with AEEA or Ahx. In still other cases, the linker can be a combination of one or two γE amino acid residues and one or two AEEA or Ahx. In a specific case, the linker can be a (AEEA)2·γE moiety, an Ahx·E·γE moiety, or an AEEA·γE moiety.

[0064] Exemplary linker-fatty acid moieties can contain (AEEA) 2 ·γE·C 20 diacid, Ahx·E·γE·C 18 diacid, or AEEA·γE·C 18 diacid. The structural features of these linker-fatty acid moieties result in analogs having improved half-lives compared to native human PYY 1-36 (SEQ ID NO:1) or native human PYY 3-36 (SEQ ID NO:2).

[0065] In summary, exemplary PYY analogs are:

[0066] Although PYY analogs are described as having thirty-four amino acids (such as the thirty-four amino acids of native human PYY 3-36 (SEQ ID NO:2)), it is contemplated that the PYY analogs herein can have an amino acid sequence based on native human PYY 1-36 (SEQ ID NO:1). That is, the PYY analogs can include the two N-terminal amino acids of native human PYY 1-36 (SEQ ID NO:1) (i.e., the "YP" residues at positions 1 and 2 of SEQ ID NO:1), which can then be cleaved in vivo when administered to an individual, as occurs upon endogenous release of native human PYY 1-36 (SEQ ID NO:1).

[0067] The half-life of the PYY analogs herein can be measured using techniques known in the art, including, for example, those described in the following examples. Similarly, the respective affinities of the PYY analogs herein for various NPY receptors (e.g., NPY2R, NPY5R) can be determined using techniques known in the art for measuring receptor binding levels, including, for example, those described in the following examples, and are typically expressed as inhibition constant (K i ) values. In addition, techniques known in the art, including, for example, the in vitro activity assays described below, can be used to measure the activity of the PYY analogs herein at each receptor and are typically expressed as EC 50 values.

[0068] Due to the above modifications, the half-life of the PYY analogs herein is longer than that of native human PYY 3-36 (SEQ ID NO:2). For example, the half-life of the PYY analog can be from about 5 hours to about 24 hours, from about 6 hours to about 23 hours, from about 7 hours to about 22 hours, from about 8 hours to about 21 hours, from about 9 hours to about 20 hours, from about 10 hours to about 19 hours, from about 11 hours to about 18 hours, from about 12 hours to about 17 hours, from about 13 hours to about 16 hours, or even from about 14 hours to about 15 hours. Alternatively, the half-life of the PPY analogs herein can be about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 23 hours or even about 24 hours, especially about 12 hours.

[0069] Similarly, the binding affinity of the PYY analogs herein for the NPY2 receptor is greater than that of native human PYY 3-36 (SEQ ID NO:2), such as from about 2-fold to about 10-fold. Alternatively, the binding affinity of the PYY analogs herein can be up to about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or even about 10-fold greater than that of native human PYY 3-36 (SEQ ID NO:2), especially from 2-fold to 3-fold greater.

[0070] Pharmaceutical composition The PYY analogs of the present invention can be formulated into pharmaceutical compositions, which can be administered by parenteral routes (such as intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal). Such pharmaceutical compositions and their preparation techniques are well known in the art. See, for example, Remington, “The Science and Practice of Pharmacy” (edited by D.B. Troy, 21st edition, Lippincott, Williams & Wilkins, 2006). In a specific case, the PYY analogs are administered SQ. However, alternatively, the PYY analogs can be formulated into forms for other pharmaceutically acceptable routes, such as, for example, tablets or other solids for oral administration; timed-release capsules; and any other forms currently in use, including creams, emulsions, inhalants, etc.

[0071] To improve their in vivo compatibility and effectiveness, the PYY analogs of the present invention can react with any of a number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and their commonly used preparation techniques are well known in the art (see, for example, Stahl et al., “Handbook of Pharmaceutical Salts: Properties, Selection and Use,” 2nd revised edition (Wiley-VCH, 2011)). Pharmaceutically acceptable salts used herein include sodium salts, trifluoroacetates, hydrochlorides and acetates.

[0072] The PYY analogs of the present invention can be administered by a physician or self-administered using an injection. It should be understood that those skilled in the art can easily determine the dosage size and injection volume. However, the injection volume can be ≤ about 2 mL or even ≤ about 1 mL, and the needle gauge can be ≥ about 27 G or even ≥ about 29 G.

[0073] The present disclosure also provides and thus encompasses novel intermediates and methods that can be used to synthesize the PYY analogs of the present invention or their pharmaceutically acceptable salts. The intermediates and PYY analogs herein can be prepared by various methods well known in the art. For example, methods using chemical synthesis are illustrated in the following examples. The specific synthesis steps of each route can be combined in different ways to prepare the PYY analogs described herein. Reagents and starting materials are readily available to those skilled in the art.

[0074] The PYY analogs of the present invention are generally effective within a wide dosage range. Exemplary dosages of the PYY analogs of the present invention or pharmaceutical compositions containing them can be in milligram (mg), microgram (μg), nanogram (ng) or picogram (pg) amounts per kilogram (kg) of the individual. In this way, the daily dosage can be from about 1 μg to about 100 mg.

[0075] Here, the effective amount of the PYY analog in the pharmaceutical composition can be a dose of about 0.25 mg to about 5.0 mg. However, those skilled in the art understand that in some cases, the effective amount (i.e., dose / dosage) may be lower than the lower limit of the foregoing range and be more than sufficient, while in other cases, the effective amount can be a larger dose and may be employed with acceptable side effects.

[0076] In addition to the PYY analog, the pharmaceutical composition can also contain additional therapeutic agents, especially other antidiabetic or anti-obesity drugs. In some cases, the additional therapeutic agent can be at least one of an incretin or a DPP-IV inhibitor. Exemplary incretins include but are not limited to GCG, GLP-1, GLP-1(7-36) 酰胺 , GIP, OXM, GCG analogs, GLP-1 analogs, GIP analogs, OXM analogs, GIP / GLP-1, GLP-1 / GCG, or even incretin analogs with triple receptor activity.

[0077] In this way, the pharmaceutical composition can contain an effective amount of the PYY analog of SEQ ID NO:9 and an incretin or a DPP-IV inhibitor, an effective amount of the PYY analog of SEQ ID NO:10 and an incretin or a DPP-IV inhibitor, an effective amount of the PYY analog of SEQ ID NO:11 and an incretin or a DPP-IV inhibitor, an effective amount of the PYY analog of SEQ ID NO:12 and an incretin or a DPP-IV inhibitor, or an effective amount of the PYY analog of SEQ ID NO:13 and an incretin or a DPP-IV inhibitor.

[0078] In those cases where the incretin is GLP-1 or a GLP-1 analog, it can be GLP-1 or a GLP-1 analog such as albiglutide, dulaglutide, liraglutide, semaglutide, or a combination thereof, especially dulaglutide.

[0079] Methods for preparing and using PYY analogs The PYY analogs of the present invention can be synthesized via any number of peptide synthesis methods known in the art using standard manual or automated solid-phase synthesis procedures. Automated peptide synthesizers are commercially available from, for example, Applied Biosystems (Foster City, CA) and Protein Technologies Inc. (Tucson, AZ). Reagents for solid-phase synthesis are readily available from commercial sources. The solid-phase synthesizer can be used according to the manufacturer's instructions to block interfering groups, protect amino acids during the reaction, couple, deprotect, and cap unreacted amino acids.

[0080] Typically, at room temperature, in an inert solvent (such as DMF, N-methylpyrrolidone, or dichloromethane), in the presence of a coupling agent (such as diisopropylcarbodiimide and 1-hydroxybenzotriazole), an N-α-carbamoyl-protected amino acid is coupled to the N-terminal amino acid on the growing peptide chain attached to the resin. The N-α-carbamoyl protecting group is removed from the resulting peptide resin using a reagent such as TFA or piperidine, and the coupling reaction is repeated with the next desired N-α-protected amino acid to add to the peptide chain. Suitable amine protecting groups are well known in the art and are described, for example, in Green & Wuts, “Protecting Groups in Organic Synthesis,” (John Wiley and Sons, 1991). The most commonly used examples include tBoc and Fmoc. After synthesis is complete, the peptide is cleaved from the solid-phase support using standard procedures under acidic conditions while deprotecting the side chains.

[0081] One of ordinary skill in the art will understand the synthesis of the peptide chains described herein with a C-terminal carboxamide. To synthesize a C-terminal amide peptide, a resin incorporating a Rink amide MBHA or Rink amide AM linker is typically used for Fmoc synthesis, while MBHA resin is typically used for tBoc synthesis.

[0082] The crude peptide is typically purified on a C8 or C18 column using RP-HPLC with a water-acetonitrile gradient in 0.05% to 0.1% TFA. The purity can be verified by analytical RP-HPLC. The identity of the peptide can be verified by MS. The peptide can be dissolved in aqueous buffer over a wide pH range.

[0083] One use of the PYY analogs of the present invention is for reducing blood glucose and / or body weight in an individual (particularly an individual who is overweight or obese and has T2DM). Administration of the PYY analogs as described herein can lead to glycemic control by improving insulin sensitization and weight loss. Thus, the PYY analogs of the present invention show the additional benefit of glucose-lowering potency and weight loss such that an individual can delay progression to insulin and maintain target HbA1c goals.

[0084] The method may include the steps described herein, and these may (but need not) be carried out in the order described. However, other orders are also conceivable. In addition, the single or multiple steps may be implemented in parallel and / or overlap in time and / or individually or in multiple repeated steps. In addition, the method may include additional unspecified steps.

[0085] Thus, such a method may include selecting individuals who are overweight and have T2DM or are predisposed to the disease. Alternatively, the method may include selecting individuals who are obese and have T2DM or are predisposed to the disease.

[0086] The method may also include administering to the individual an effective amount of at least one PYY analogue as described herein, which may be in the form of a pharmaceutical composition also as described herein. In some cases, the at least one PYY analogue / pharmaceutical composition may include additional therapeutic agents, such as an incretin or a DPP-IV inhibitor.

[0087] The concentration / dose / dosage of the at least one PYY analogue and optionally the incretin or DPP-IV inhibitor is discussed elsewhere herein.

[0088] Regarding the route of administration, the at least one PYY analogue or the pharmaceutical composition containing it may be administered according to known methods (such as, for example, orally; by injection (i.e., intraarterial, intravenous, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, intraportal or intralesional); by a sustained release system, or by an implant device). In certain cases, the at least one PYY analogue or the pharmaceutical composition containing it may be administered by bolus or continuously SQ.

[0089] Regarding the frequency of administration, the at least one PYY analogue or the pharmaceutical composition containing it may be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week) or monthly. In certain cases, the at least one PYY analogue or the pharmaceutical composition containing it is administered SQ every other day, three times a week SQ, twice a week SQ, once a week SQ, every other week SQ or monthly SQ. In a specific case, the at least one PYY analogue or the pharmaceutical composition containing it is administered SQ once a week (QW).

[0090] Alternatively, the at least one PYY analogue or a pharmaceutical composition comprising the same may be administered orally. As described above and with regard to the dosing frequency, the at least one PYY analogue or a pharmaceutical composition comprising the same may be administered daily, every other day, three times a week, twice a week, once a week (i.e., weekly), every two weeks (i.e., every other week) or monthly. In certain cases, the at least one PYY analogue or a pharmaceutical composition comprising the same is administered orally every other day, three times a week, twice a week, once a week or every other week. In a specific case, the PYY analogue is administered orally once a week.

[0091] In those cases where at least one PYY analogue or a pharmaceutical composition comprising the same is administered in combination with an effective amount of an incretin, the incretin may be GCG or a GCG analogue, GLP-1, GLP-1(7-36) 酰胺 or a GLP-1 analogue, GIP or a GIP analogue, OXM or an OXM analogue, GIP / GLP-1, GLP-1 / GCG, or even an incretin having triple receptor activity. The GCG, GCG analogue, GLP-1, GLP-1(7-36) 酰胺 , GLP-1 analogue, GIP, GIP analogue, OXM, OXM analogue, GIP / GLP-1, GLP-1 / GCG, or an incretin having triple receptor activity may be administered simultaneously with, separately from, or sequentially to the at least one PYY analogue or a pharmaceutical composition comprising the same.

[0092] Furthermore, the GCG, GCG analogue, GLP-1, GLP-1(7-36) 酰胺 , GLP-1 analogue, GIP, GIP analogue, OXM, OXM analogue, GIP / GLP-1, GLP-1 / GCG, or an incretin having triple receptor activity may be administered at the same frequency as the at least one PYY analogue or a pharmaceutical composition comprising the same (i.e., every other day, twice a week, or even weekly). Alternatively, the GCG, GCG analogue, GLP-1, GLP-1(7-36) 酰胺 , GLP-1 analogue, GIP, GIP analogue, OXM, OXM analogue, GIP / GLP-1, GLP-1 / GCG, or an incretin having triple receptor activity may be administered at a different frequency from the at least one PYY analogue or a pharmaceutical composition comprising the same. In other cases, the GCG, GCG analogue, GLP-1, GLP-1(7-36) 酰胺, GLP-1 analogs, GIP, GIP analogs, OXM, OXM analogs, GIP / GLP-1, GLP-1 / GCG, or an incretin with triple receptor activity is administered QW. In other cases, the PYY analog is administered SQ, and the GCG, GCG analogs, GLP-1, GLP-1(7-36) 酰胺 , GLP-1 analogs, GIP, GIP analogs, OXM, OXM analogs, GIP / GLP-1, GLP-1 / GCG, or an incretin with triple receptor activity can be administered orally.

[0093] It is further contemplated that the method can be combined with diet and exercise and / or can be combined with additional therapeutic agents other than those discussed above. Detailed Description The present invention also relates to the following embodiments: 1. A peptide tyrosine-tyrosine (PYY) analog comprising the following amino acid sequence: PKPEX 7 PX 9 X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY (Formula I), wherein X 7 is any amino acid having a functional group available for conjugation and the functional group is conjugated to a C 16 -C 22 fatty acid, wherein X 9 is E or G, wherein X 10 is E or K, wherein X 17 is L or W, wherein X 18 is N or Q, wherein X 22 is A or I, wherein X 23 is E, D or S, wherein X 30 is E or W (SEQ ID NO: 3), and wherein the C-terminal amino acid is optionally amidated. 2. The PYY analog of embodiment 1, wherein X 7 is selected from C, D, E, K and Q. 3. The PYY analog of embodiment 1, wherein X 7is K and is conjugated to C via the ε-amino group of the K side chain 16 -C 22 fatty acid. 4. The PYY analogue of embodiment 1, wherein the amino acid sequence is selected from: PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY (SEQ ID NO:4); PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY (SEQ ID NO:5); PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY (SEQ ID NO:6); PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY (SEQ ID NO:7); and PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY (SEQ ID NO:8). 5. The PYY analogue of any one of embodiments 1 to 4, wherein the C 16 -C 22 fatty acid is selected from hexadecanoic acid, hexadecanedioic acid, heptadecanoic acid, heptadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanoic acid, nonadecanedioic acid, eicosanoic acid, eicosanedioic acid, henicosanoic acid, henicosanedioic acid, docosanoic acid, docosanedioic acid and their branched and substituted derivatives. 6. The PYY analogue of embodiment 5, wherein the C 16 -C 22 fatty acid is C 18 -C 20 fatty acid. 7. The PYY analogue of embodiment 6, wherein the C 18 -C 20 fatty acid is a straight-chain fatty acid having the formula CO-(CH 2 ) x -CO 2 H, and wherein x is 18 or 20. 8. The PYY analogue of embodiment 7, wherein the C 18 -C 20 fatty acid is selected from palmitic acid, stearic acid, arachidic acid and eicosanoic acid. 9. The PYY analogue of any one of embodiments 1 to 8, wherein the C 16 -C 22 fatty acid is conjugated to an amino acid having a functional group available for conjugation via a linker. 10. The PYY analogue of embodiment 9, wherein the linker can be one or more units selected from [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid (Ahx), glutamic acid (E), γ-glutamic acid (γE), or combinations thereof. 11. A peptide tyrosine-tyrosine (PYY) analogue, comprising: 12. A peptide tyrosine-tyrosine (PYY) analogue, comprising: 13. A peptide tyrosine-tyrosine (PYY) analogue, comprising: 14. A peptide tyrosine-tyrosine (PYY) analogue, comprising: 15. A peptide tyrosine-tyrosine (PYY) analogue, comprising: 16. The PYY analogue of any one of embodiments 1 to 15, wherein the PYY analogue has a charge greater than -2. 17. The PYY analogue of any one of embodiments 1 to 16, wherein the PYY analogue has a greater binding affinity for the NPY2 receptor than PYY 3-36 (SEQ ID NO:2). 18. The PYY analogue of any one of embodiments 1 to 17, wherein the PYY analogue has a longer half-life than human PYY 3-36 (SEQ ID NO:2). 19. A pharmaceutical composition, comprising: at least one peptide tyrosine-tyrosine (PYY) analogue of any one of embodiments 1 to 18 or a salt thereof; and one or more pharmaceutically acceptable carriers, diluents, and excipients. 20. The pharmaceutical composition of embodiment 19, which further comprises an additional therapeutic agent. 21. The pharmaceutical composition of embodiment 20, wherein the additional therapeutic agent is selected from glucagon (GCG), GCG analogues, glucagon-like peptide-1 (GLP-1), GLP-1 7-36-酰胺 , GLP-1 analogues, gastric inhibitory polypeptide (GIP), GIP analogues, oxyntomodulin (OXM), OXM analogues, GIP / GLP-1, GLP-1 / GCG incretins, or an incretin analogue having triple receptor activity. 22. The pharmaceutical composition of embodiment 20, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor. 23. A method for treating obesity or an obesity-related disease or disorder, the method comprising the steps of: administering to an individual in need thereof an effective amount of a peptide tyrosine-tyrosine (PYY) analogue of any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof. 24. The method of embodiment 23, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered subcutaneously (SQ) to the individual. 25. The method of embodiment 23, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered orally to the individual. 26. The method of embodiment 24 or 25, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered daily, every other day, three times a week, twice a week, once a week or every two weeks. 27. The method of embodiment 23, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered once a week (QW) SQ. 28. The method of embodiment 23, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered orally once a week. 29. The method of any one of embodiments 23 to 28, further comprising administering an additional therapeutic agent. 30. The method of embodiment 29, wherein the additional therapeutic agent is selected from glucagon (GCG), GCG analogues, glucagon-like peptide-1 (GLP-1), GLP-1 7-36-酰胺 , GLP-1 analogues, gastric inhibitory polypeptide (GIP), GIP analogues, oxyntomodulin (OXM), OXM analogues, GIP / GLP-1, GLP-1 / GCG incretins, or an incretin analogue having triple receptor activity. 31. The method of embodiment 29, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor. 32. Use of a peptide tyrosine-tyrosine (PYY) analogue of any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the treatment of obesity. 33. Use of a peptide tyrosine-tyrosine (PYY) analogue of any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the treatment of an obesity-related disease or disorder. 34. Use of a peptide tyrosine-tyrosine (PYY) analogue of any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the treatment of type II diabetes. Use of a peptide tyrosine-tyrosine (PYY) analogue according to any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of obesity. 36. Use of a peptide tyrosine-tyrosine (PYY) analogue according to any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of obesity-related diseases or disorders. 37. Use of a peptide tyrosine-tyrosine (PYY) analogue according to any one of embodiments 1 to 18 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of type II diabetes. Examples

[0094] For purposes of illustration and not limitation, the following non-limiting examples are provided.

[0095] Example 1: PYY analogue 1.

[0096] A PYY analogue incorporating the inventive concept may have the following structure:

[0097] Here, the N-terminus is free and the C-terminal amino acid is amidated to a C-terminal primary amide. The K at position 7 is chemically modified by conjugating (Ahx-E-(γE)-CO-(CH 2 ) 16 -COOH to the ε-amino group of the side chain of K at position 7.

[0098] The PYY analogue according to SEQ ID NO:9 was generated by solid-phase peptide synthesis using the 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). At 25 °C, amino acid coupling was carried out for 3 h using 10 equivalents of amino acids, 0.9 M diisopropylcarbodiimide (DIC) and 0.9 M Oxyma (1:1:1 molar ratio) in DMF. Deprotection was carried out using a 25% piperidine solution in DMF.

[0099] After elongation of the peptide-resin as described above, the MTT protecting group present in Lys at position 7 was removed using 30% hexafluoroisopropanol (HFIP) in dichloromethane (DCM). The additional coupling / deprotection cycles for elongation of the side chain of Lys at position 7 using the Fmoc / t-Bu strategy involved Fmoc-6-aminohexanoic acid (Chem-Impex International catalog number 02490), Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OH)-OtBu (ChemPep catalog number 100703), and HOOC-(CH 2 ) 16 -COOtBu. In all couplings, 3 equivalents of the building block were used with PyBOP (3 equivalents) and DIEA (6 equivalents) in DMF at 25 °C for 3 h.

[0100] At 25 °C, cleavage from the resin and removal of side chain protecting groups were carried out simultaneously in a solution containing TFA:triisopropylsilane:1,2-ethanedithiol:methanol:benzyl mercaptan 80:5:5:5:5 (v / v) for 2 h, followed by precipitation with cold ether. The crude peptide was purified to >99% purity (15 - 20% purification yield) by RP-HPLC on a phenylhexyl column (Phenomenex, Luna; 5 μm, 100 Å), where the appropriate fractions were combined and lyophilized.

[0101] The purity of the PYY analog was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1659.2 (+ / -0.2); calculated: M+3H + / 3 = 1659.2; observed: M+4H + / 4 = 1244.6 (+ / -0.2); calculated: M+4H + / 4 = 1244.6; observed: M+5H + / 5 = 995.9 (+ / -0.2); calculated: M+5H + / 5 = 995.9).

[0102] Example 2: PYY analog 2.

[0103] A PPY analog incorporating the inventive concept can have the following structure:

[0104] As in Example 1, the N-terminus is free and the C-terminal amino acid is amidated to a C-terminal primary amide. However, in contrast, Lys at position 7 is modified with ([2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γE)-CO-(CH 2 )18 The -COOH is conjugated to the ε-amino group of the K side chain for chemical modification.

[0105] The PYY analogue according to SEQ ID NO:10 is generated by solid-phase peptide synthesis, similar to that described above in Example 1. Thus, after MTT cleavage with 3 equivalents of the building block in DMF at 25 °C for 3 h using PyBOP (3 equivalents) and DIEA (6 equivalents), FMOC-NHPEG 2 -CH 2 COOH and HOOC-(CH 2 ) 18 -COOtBu are attached to the side chain.

[0106] The purity of the PYY analogue is checked by analytical RP-HPLC and its identity is confirmed using LC / MS (observed: M+3H + / 3 = 1665.4 (+ / -0.2); calculated: M+3H + / 3 = 1665.5; observed: M+4H + / 4 = 1249.3 (+ / -0.2); calculated: M+4H + / 4 = 1249.4; observed: M+5H + / 5 = 999.7 (+ / -0.2); calculated: M+5H + / 5 = 999.7).

[0107] Example 3: PYY analogue 3.

[0108] A PYY analogue incorporating the inventive concept may have the following structure:

[0109] As in Example 1, the N-terminus is free and the C-terminal amino acid is amidated to a C-terminal primary amide. However, in contrast thereto, the K at position 7 is chemically modified by conjugating ([2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γE)-CO-(CH 2 ) 16 -COOH to the ε-amino group of the K side chain.

[0110] The PYY analogue according to SEQ ID NO:11 is generated by solid-phase peptide synthesis, similar to that described above in Example 1. Thus, after MTT cleavage with 3 equivalents of the building block in DMF at 25 °C for 3 h using PyBOP (3 equivalents) and DIEA (6 equivalents), FMOC-NHPEG 2 -CH 2 COOH and HOOC-(CH2 ) 16 -COOtBu is attached to the side chain.

[0111] The purity of the PYY analog was examined by analytical RP-HPLC and its identity was confirmed using LC / MS (observed: M+3H + / 3 = 1664.7 (+ / -0.2); calculated: M+3H + / 3 = 1664.9; observed: M+4H + / 4 = 1248.9 (+ / -0.2); calculated: M+4H + / 4 = 1248.9; observed: M+5H + / 5 = 999.3 (+ / -0.2); calculated: M+5H + / 5 = 999.3).

[0112] Example 4: PYY analog 4.

[0113] A PYY analog incorporated into the inventive concept may have the following structure:

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

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

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

[0117] Example 5: PYY analog 5.

[0118] A PYY analog incorporating the inventive concept may have the following structure:

[0119] As in Example 1, the N-terminus is free and the C-terminal amino acid is amidated to a C-terminal primary amide. However, in contrast, the K at position 7 is chemically modified by conjugating ([2-(2-amino-ethoxy)-ethoxy]-acetyl) 2 -(γE) 3 -CO-(CH 2 ) 18 -COOH to the ε-amino group of the K side chain.

[0120] The PYY analog according to SEQ ID NO:13 was generated by solid-phase peptide synthesis, similar to that described above in Example 1. Thus, after 3 h of MTT cleavage using 3 equivalents of the building block in DMF at 25 °C with PyBOP (3 equivalents) and DIEA (6 equivalents), FMOC-NHPEG 2 -CH 2 COOH and HOOC-(CH 2 ) 18 -COOtBu were attached to the side chain.

[0121] The purity of the PYY analog was examined by analytical RP-HPLC and its identity was confirmed using LC / MS (Observed: M + 3H + / 3 = 1732.2(+ / -0.2); Calculated: M + 3H + / 3 = 1732.3; Observed: M + 4H + / 4 = 1299.4(+ / -0.2); Calculated: M + 4H + / 4 = 1299.5; Observed: M + 5H + / 5 = 1039.7(+ / -0.2); Calculated: M + 5H + / 5 = 1039.8).

[0122] Example 6: In vitro activity of PYY analogs.

[0123] (1) In vitro binding to hNPY1, hNPY2, hNPY4 and hNPY5 receptors Objective: To evaluate the in vitro binding affinity (Ki) of the PYY analogs of Examples 1 to 5 to the following human (h) receptors in the absence of bovine serum albumin (BSA): hNPY1R, hNPY2R, hNPY4R, and hNPY5R. A competitive radioligand binding assay using the scintillation proximity assay (SPA) method was performed, and the competitive radioligand binding assay used membranes prepared from cell lines overexpressing various recombinant receptors and relevant 125 I]-labeled peptides. In each assay, the binding affinity to the relevant native peptides PYY 1-36 (SEQ ID NO:1), PYY 3-36 (SEQ ID NO:2), and pancreatic polypeptide 1-36 (PP 1-36 ; SEQ ID NO:14) was determined as a control.

[0124] Methods: The PYY analogs, native human PYY 1-36 and control PYY 3-36 were synthesized at Lilly Research Laboratories (Indianapolis, IN, USA) and characterized by LC / MS, NMR, and LC / UV analysis (99.5% purity). The peptide content was estimated to be 80% of the powder weight. The peptides were prepared as 10 mM stock solutions in 100% DMSO and kept frozen at -20 °C until just prior to testing in the assay.

[0125] For hNPY1R, transient overexpression was performed using CHO cells. Stable transfected cell lines for hNPY2R and hNPY4R were prepared by subcloning the receptor cDNA into the pcDNA3.1 expression plasmid and transfecting into human embryonic kidney (HEK) 293 cells, followed by selection with geneticin. The hNPY5R clone was performed at Multispan, Inc. (Hayward, CA).

[0126] To prepare crude cell membranes of hNPY1R, hNPY2R, mNPY2R, and hNPY4R, two different methods (described below) were utilized. hNPY5R membranes were purchased from Multipan, Inc. (#MCG1275).

[0127] Method 1 – For hNPY2R and hNPY4R membranes, the frozen cell pellet was lysed on ice in 10 mL of hypotonic homogenization buffer containing 50 mM Tris HCl (pH 7.5) and Roche Complete with EDTA (#1169749001) TMPer gram of wet cell paste of protease inhibitor. Disrupt the cell suspension 25 times using a glass Potter-Elvehjem homogenizer equipped with a pestle. Centrifuge the homogenate at 1100 x g for 10 minutes at 4 °C. Collect the supernatant and store on ice while resuspending the pellet in homogenization buffer and homogenizing again as described above. Centrifuge the homogenate at 1100 x g for 10 minutes. Combine the second supernatant with the first supernatant and centrifuge at 35000 x g for 1 hour at 4 °C. Resuspend the resulting membrane pellet in homogenization buffer containing protease inhibitor at approximately 1 to 3 mg / mL, quickly freeze in liquid nitrogen, and store as aliquots in an -80 °C freezer until use. Determine the protein concentration using a BCA protein assay kit (Pierce, #23225) with BSA as the standard.

[0128] Method 2 – For hNPY1R membranes, lyse the frozen cell pellet on ice in 5 mL of hypotonic homogenization buffer containing 25 mM Tris HCl (pH 7.5), 1 mM MgCl 2 , 25 units / mL DNase I (Invitrogen, #18047-019), and Roche Complete TM protease inhibitor per gram of wet cell paste. Disrupt the cell suspension 25 times using a glass Potter-Elvehjem homogenizer equipped with a pestle. Centrifuge the homogenate at 1800 x g for 15 minutes at 4 °C in a 50 mL conical tube. Collect the supernatant and store on ice while resuspending the pellet in homogenization buffer and homogenizing again as described above, except without using DNase I in the homogenization buffer. Centrifuge the homogenate at 1800 x g for 15 minutes. Combine the second supernatant with the first supernatant and centrifuge at 25000 x g for 30 minutes at 4 °C. Resuspend the resulting membrane pellet in homogenization buffer containing protease inhibitor at approximately 2 mg / mL, aliquot, and store in an -80 °C freezer until use. Determine the protein concentration using a BCA protein assay kit (Pierce, #23225) with BSA as the standard.

[0129] General binding assay method – Using the same reagents and buffers as described below for compound testing, determine the equilibrium dissociation constant (K d ) for various receptor / radio ligand interactions from saturation binding assays. The K dValues were as follows: hNPY2R, 0.0047 nM; hNPY1R, 0.07 nM; hNPY4R, 0.084 nM; and hNPY5R, 0.896 nM.

[0130] hNPY1R Receptor Binding Protocol – PYY analog peptides and PYY 125 I]-PYY 1-36 (#NEX341, 2200 Ci / mmol) were determined for receptor binding affinity (Ki) for hNPY1R from a competitive radioligand binding assay. This assay was performed using a SPA method with polyvinyltoluene (PVT) wheat germ agglutinin conjugated SPA beads (#RPNQ0001, Perkin Elmer). Assay buffer (25 mM HEPES (pH 7.5), 1 mM MgCl 1-36 2, 2.5 mM CaCl 2 2, and 0.2% w / v bacitracin (RPI, #32000)) was used to prepare the reagents. PYY analogs and PYY 2 were thawed and serially diluted 3-fold in 100% DMSO (10-point concentration-response curve) using a Tecan Evo liquid handler. The peptides were then serially diluted 20-fold into the assay buffer to reduce the DMSO level and peptide concentration and then added to the assay plates. Next, 5 μL of the serially diluted peptides or DMSO was transferred to a 384-well clear-bottom assay plate containing 45 μL of assay buffer or unlabeled PYY 1-36 control (non-specific binding or NSB, 10 nM final concentration). 1-36 Then, 50 μL of I]-PYY 125 (0.05 nM final concentration) and 50 μL of hNPY1R membrane (1.0 μg / well) were added. Finally, 50 μL of WGA SPA beads (50 μg / well) was added. The final DMSO concentration was 0.125%. The plates were sealed and mixed on a plate shaker (setting 6) for 1 minute and read on a PerkinElmer Trilux 1-36 scintillation counter after a 10-hour incubation / bead sedimentation time at room temperature. The final assay concentration range of the peptides tested in the response curve was: PYY analogs (2.5 μM to 0.13 nM) and PYY (10 nM to 0.5 pM). 1-36 (10 nM to 0.5 pM).

[0131] hNPY2 Receptor Binding Protocol – PYY analog peptides and PYY 1-36Receptor binding affinity (K i ) of hNPY2R. The final assay concentrations of the peptides tested in the response curves were in the range: PYY analogs (0.1 μM to 5 pM) and PYY 3-36 (10 nM to 0.5 pM).

[0132] hNPY4R receptor binding protocol – Determine the receptor binding affinity (K 1-36 ) of PYY analog peptides and PP i for hNPY4R from a competitive radioligand binding assay as described above for hNPY1R. The final assay concentrations of the peptides tested in the response curves were in the range: PYY analogs (1 μM to 10 pM) and PYY 1-36 (1 μM to 10 pM).

[0133] hNPY5R receptor binding protocol – Determine the receptor binding affinity (K 1-36 ) of PYY analog peptides and PYY i for hNPY5R from a competitive radioligand binding assay as described above for hNPY1R. The final assay concentrations of the peptides tested in the response curves were in the range: PYY analogs (1 μM to 10 pM) and PYY 1-36 (1 μM to 10 pM).

[0134] Data analysis of NPY receptor binding assays – Convert the raw counts per minute (CPM) data of the concentration curves of PYY analogs, PYY 1-36 , PYY 3-36 or PP 1-36 to percent specific inhibition as follows: Subtract the non-specific binding (NSB, determined in the presence of excess unlabeled PYY 1-36 , PYY 3-36 or PP 1-36 ) from each CPM value and divide by the total binding signal, also corrected by subtracting the non-specific binding, as shown in the following equation:

[0135] Analyze the data using a four-parameter (curve maximum, curve minimum, IC 50 , Hill slope) non-linear regression routine (Genedata Screener, version 13.0.5, Genedata AG, Basel, Switzerland). Calculate the affinity (K i ) from the relative IC 50 values based on the equation K d = IC 50 / (1 + D / K i), where D = the concentration of the radioligand in the experiment, IC 50 is the concentration that causes 50% inhibition of binding, and K d is the equilibrium binding affinity constant of the radioligand determined from the saturation binding assay (listed above). The qualifier (>) indicates that the data did not reach 50% inhibition compared to the maximum binding in the absence of the competitor. Therefore, K i was calculated using the highest concentration of the compound tested in the assay.

[0136] The reported K i values were calculated as the geometric mean, as follows: Geometric mean = 10 (Log10Ki值的算术平均值) .

[0137] The standard error of the mean (SEM) was calculated using the Δ method, as follows: where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0138] The selectivity of the peptide for hNPY2R (Y2) (compared to hNPY5R (Y5), hNPY4R (Y4), and / or hNPY1R (Y1)) was calculated by dividing the hNPY2R result (in nM).

[0139] Results: Table 1: In vitro binding to hNPY1R, hNPY2R, hNPY4R, and hNPY5R (K i ). * Known PYY analogues for comparison; see International Patent Application Publication No. WO 2016 / 198682, where Reference Example 1 corresponds to Compound 4 therein, Reference Example 2 corresponds to Compound 21 therein, and Reference Example 3 corresponds to Compound 32 therein. ND – Not detected NA – Not applicable.

[0140] As shown above, the PYY analogues of Examples 1 to 5 are highly selective for the hNPY2 receptor, and it was even confirmed that the binding affinity to the hNPY5, hNPY4, and hNPY1 receptors is reduced compared to that of native human PYY 3-36 (SEQ ID NO:2).

[0141] (2) In vitro cAMP activity on the human NPY2 receptor Objective: The in vitro functional activity of the PYY analogues of Examples 1 to 5 compared to native human PYY was determined by measuring the inhibition of forskolin-induced intracellular cAMP production in HEK 293 cells overexpressing recombinant human NPY2 receptor 3-36 compared to native human PYY

[0142] Method: As described above in the receptor binding assay, the PYY analogues and human PYY 3-36 (SEQ ID NO:2) were synthesized, characterized and stored

[0143] Receptor cloning – Stable transfected cells of hNPY2 receptor were prepared by subcloning the receptor cDNA into the pcDNA3.1 expression plasmid and transfecting it into HEK 293 cells, followed by selection with geneticin. Aliquots of the cells of passage 9 (1 x 10 7 cells / mL) were prepared and kept frozen in the vapor phase of a liquid nitrogen tank. These frozen aliquots were used at the time of assay. The cells maintained a viability of greater than 95% over several months

[0144] hNPY2R cAMP assay – Using HEK 293 cells overexpressing recombinant hNPY2R, the inhibition of forskolin-induced cAMP production by the PYY analogues or PYY 3-36 was measured. The frozen cell aliquots were thawed in a 37 °C water bath. The cells were transferred to a 50 mL tube with 10 mL of 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), 1xNEAA (from Life Tech 11140-050), 1 mM sodium pyruvate (from Life Tech 11360-070), 1x antibiotic-antimycotic (from Life Tech 15240-062)) and centrifuged at 1500 rpm for 5 minutes in a Beckman benchtop centrifuge. The supernatant was removed and the cell pellet was resuspended in 10 mL of cell culture medium, followed by passing through a 40 μm filter. An accurate count of the cell number and cell viability was determined using a Vi-Cell analyzer (Vi-Cell XR 2.03) from Beckman-Coulter. 8000 cells per well were seeded into a white 384-well assay plate (Corning, poly-D-lysine coated, white / opaque, catalog number 356661) using a Combi-Tip dispenser (Thermo Scientific). The plate was centrifuged at 1000 rpm for 1 second and incubated at 37 °C in 5% CO 2- Incubate in the incubator for 18 to 20 hours. Remove the medium from the assay plate by tapping on a paper towel. Add 10 μL of assay buffer [1X HBSS (Hyclone, #SH3026801), 20 mM HEPES (pH 7.5) (Hyclone #SH 30237.01), 0.1% w / v casein (CTL Scientific Supply Corp., #440203H), 500 μM IBMX (Sigma-Aldrich #I5876)] to the wells using a Combi-Tip dispenser, and then centrifuge at 1500 rpm for 10 seconds. Prepare a 2-fold dilution concentration-response curve (20 points) in 100% DMSO using acoustic dispensing technology (Labcyte Echo 550). Cells are treated with PYY analog or human PYY 3-36 for 45 minutes at 37°C (final DMSO concentration = 1%), and then stimulated with 1 μM forskolin (Sigma-Aldrich, #F6886) for 45 minutes at 37°C. Use CisBio cAMP-G i Dynamic Kit (#62AM9PEB) to quantify intracellular cAMP. Briefly, use the HTRF kit reagent to detect intracellular cAMP levels by adding the cAMP-d2 conjugate in cell lysis buffer (10 μL), followed by adding the antibody anti-cAMP-Eu 3+ - Cryptate in cell lysis buffer (10 μL) as well. The resulting competitive assay is incubated at room temperature for at least 60 minutes, and then read on a PerkinElmer Envision TM instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The final assay concentrations of the peptides tested in the response curve range from: PYY analog (0.1 μM to 0.2 pM) and human PYY 3-36 (10 nM to 0.02 pM). Prepare a standard curve of known cAMP concentrations (0.5 μM to 1 pM) in the assay buffer. Include wells without the addition of competitor or with an excess addition of human PYY 3-36 on each plate as the maximum response and inhibitor controls respectively.

[0145] Data analysis of the hNPY2 receptor cAMP assay – Use time-resolved fluorescence emission to calculate the fluorescence ratio (665 nM / 620 nm), which is inversely proportional to the amount of cAMP present. Convert the signals of PYY analog and human PYY 3-36 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 cAMP concentration (x-axis).

[0146] The amount of cAMP generated in each well (nM) was converted to a percentage of the maximal response observed with forskolin alone, as shown in the following equation: where the inhibitor control is the cAMP generated in the presence of an excess of added human PYY 3-36 present, the maximal response is the cAMP generated in the presence of forskolin alone, and the peptide is the cAMP generated in the presence of the test peptide.

[0147] The percent specific inhibition (y-axis) was plotted against the concentration of the competitor (x-axis) and analyzed using a four-parameter (top of curve, bottom of curve, IC 50 , Hill slope) non-linear regression convention (Genedata Screener, version 13.0.5, Genedata AG, Basel, Switzerland) as defined below:

[0148] The relative IC 50 value represents the concentration that causes 50% inhibition of forskolin-induced cAMP production.

[0149] The reported IC 50 value was calculated as the geometric mean as follows: Geometric mean = 10 (Log10IC50值的算术平均值) .

[0150] The standard error of the mean (SEM) was calculated using the Δ method as follows: where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0151] Results: Table 2: In vitro cAMP activity against the hNPY2 receptor * Known PYY analogs for comparison; see, International Patent Application Publication No. WO 2016 / 198682, where Reference Example 1 corresponds to Compound 4 therein, Reference Example 2 corresponds to Compound 21 therein, and Reference Example 3 corresponds to Compound 32 therein.

[0152] As shown above, the results from the cAMP assays confirmed the functional activity of the PYY analogs of Examples 1 to 5 against the hNPY2 receptor, where Example 2 showed the weakest potency, which was 12-fold less potent than human PYY 3-36 .

[0153] (3) In vitro GTPγS activity of the hNPR2 receptor Objective: Evaluate the receptor-mediated activation of G-protein by the PYY analogs of Examples 1 to 5. Use the non-hydrolyzable GTP analog GTPγ 35 S] to measure the receptor-mediated activation. Agonist-mediated stimulation of G-protein coupled receptors leads to the activation of membrane-associated Gαβγ-protein heterotrimeric complexes. This represents the first step in transducing extracellular signals to modify intracellular pathways. Here, GTPγ 35 S] functional assay was used to evaluate the potency of various PYY analogs at the hNPY2 receptor.

[0154] Method: As described above in the receptor binding assay, synthesize, characterize and store the PYY analogs, human PYY 1-36 (SEQ ID NO:1) and the control peptide PYY 3-36 (SEQ ID NO:2).

[0155] For each test peptide, use a Hamilton NIMBUS liquid handler to complete a concentration-response curve (CRC) of 1 / 3 log dilution (log concentration from -6.52 to -12.52) in assay buffer (20 mM HEPES (pH 7.4), 100 mM NaCl, 6 mM MgCl 2 2, 1 mM EDTA) supplemented with 0.2% bacitracin (U.S. Biologicals #11805) and 0.5% DMSO. The final assay concentrations of bacitracin and DMSO are 0.05% and 0.125% respectively. Prepare the hNPY2R membrane (Multispan #HTS066M) at a concentration of 7.5 μg / mL in assay buffer supplemented with 20 μM GDP (Sigma #G-7127) and 6 μg / mL saponin (Sigma #S-4521) and incubate the hNPY2R membrane at room temperature for 20 minutes before adding to the assay. Prepare GTPγS 35 S] (PerkinElmer #NEG030H) at a concentration of 0.6 nM in assay buffer. Prepare the WGASPA beads (PerkinElmer #RPNQ0001) at a concentration of 12 mg / mL in assay buffer. By first adding 100 μL to the hNPY2R membrane, then adding 50 μL of the CRC solution, and then adding 50 μL of GTPγS 35S] solution with a final volume of 200 μL. This assay was performed in a 96-well plate (Costar #3604). The plate was covered and placed on an orbital shaker (175 rpm, 45 minutes) at room temperature. Then, 25 μL of SPA beads were added, the plate was resealed and vortexed to mix, and then placed back on the orbital shaker at room temperature for 3 hours. Then the plate was centrifuged at 500 rpm for 5 minutes and counted for 1 minute per well on a PerkinElmer 2450 microplate counter. Basal binding (CPM) was determined in the absence of the PYY analog or human PYY 3-36 and used to calculate the percentage above basal value for each concentration of the peptide using the following equation: (PYY analog or human PYY 3-36 , CPM – basal CPM) / (basal CPM)*100. EC 50 (nM) values were determined by performing a non-linear regression analysis of the logarithm of the concentration and the percentage of the basal value in GraphPad Prism 7.0 (log(agonist) vs. response – variable slope (four parameters)) using the following equation: Y = Bottom + (Top - Bottom) / (1 + 10^((LogEC 50 -X)*Hill slope)). Using the column statistics function in GraphPad Prism 7.0, the geometric mean and standard error of the mean were calculated from the EC 50 (nM) values.

[0156] Results: Table 3: In vitro GTPγS activity against the hNPY2 receptor. * Known PYY analogs for comparison; see, International Patent Application Publication No. WO 2016 / 198682, where Reference Example 1 corresponds to Compound 4 therein, Reference Example 2 corresponds to Compound 21 therein, and Reference Example 3 corresponds to Compound 32 therein.

[0157] As shown above, the results from the GTPγ 35 S] functional assay confirmed the functional activity of the PYY analogs against the hNPY2 receptor, where Example 2 showed the weakest potency, with a potency 4-fold lower than that of human PYY 3-36 .

[0158] (4) Pharmacokinetics Objective: To study the pharmacokinetic properties of the PYY analogs.

[0159] Methods: LC / MS – Plasma concentrations of various PYY analogs were determined by LC / MS method. The method measures the whole compound; the time for peptide plus linkage is prolonged. For the determination, PYY analogs and internal standards were extracted from mouse, rat or monkey plasma (50 μL) using methanol with 0.1% formic acid. The samples were centrifuged and the supernatant was transferred to a Thermo Protein Precipitation Plate. The samples were loaded onto a Sep-Pak tC18 SPE μ elution plate conditioned with methanol and 0.1% formic acid in water. The SPE column was washed twice with 0.1% formic acid in water. Then the compounds were eluted using formic acid / water / acetonitrile (0.1:15:85), then dried and reconstituted, and then an aliquot (10 μL) was injected onto a Thermo Acclaim PepMap100 C18, 300 μm x 5 mm trap column and a Thermo Easy Spray PepMap C18, 75 μm x 15 cm column for LC / MS analysis. The column effluent was introduced into a Thermo Q-Exactive Plus mass spectrometer for detection and quantification.

[0160] Pharmacokinetics of PYY analogs in CD-1 mice – After a single subcutaneous administration of 200 nmol / kg, the plasma pharmacokinetics of PYY analogs were evaluated in male CD-1 mice. Blood samples were collected from 2 animals at each time point over 168 hours. Since non-continuous sampling was used to evaluate the kinetics of PYY analogs in mice, mean concentration versus time data were used to tabulate the pharmacokinetic parameters of PYY analogs after a single subcutaneous administration of 200 nmol / kg. Plasma concentrations of PYY analogs were measured for 120 hours after a single subcutaneous administration of 200 nmol / kg.

[0161] Pharmacokinetics of PYY analogs in SD rats – After a single subcutaneous administration of 50 nmol / kg, the plasma pharmacokinetics of PYY analogs were evaluated in male Sprague Dawley rats. Blood samples were collected from 2 animals at each time point over 168 hours. Since continuous sampling was used to evaluate the kinetics of PYY analogs in rats, individual animal concentration versus time data were used to tabulate the pharmacokinetic parameters of PYY analogs after a single subcutaneous administration of 50 nmol / kg. Plasma concentrations of PYY analogs were measured for 120 hours after a single subcutaneous administration of 50 nmol / kg.

[0162] Pharmacokinetics of PYY analogues in cynomolgus monkeys – After a single subcutaneous administration of 50 nmol / kg, the plasma pharmacokinetics of the PYY analogue were evaluated in male and female cynomolgus monkeys. Blood samples were collected over 504 hours. Since continuous sampling was used to evaluate the kinetics of the PYY analogue in monkeys, individual animal concentration versus time data were used to tabulate the pharmacokinetic parameters of the PYY analogue after a single subcutaneous administration of 50 nmol / kg. Plasma concentrations of the PYY analogue were measured over 504 hours after a single subcutaneous administration of 50 nmol / kg.

[0163] Results: Table 4: Mean pharmacokinetic parameters after a single subcutaneous administration to male CD-1 mice. Abbreviations: AUC inf = Area under the curve from 0 to infinity; CL / F = Clearance divided by bioavailability (F); Cmax = Maximum concentration; Tmax = Time to maximum concentration; T1 / 2 = Half-life.

[0164] Table 5: Mean pharmacokinetic parameters after a single subcutaneous administration to male Sprague-Dawley rats. Abbreviations: AUC inf = Area under the curve from 0 to infinity; CL / F = Clearance divided by bioavailability (F); C max = Maximum concentration; T max = Time to maximum concentration; T1 / 2 = Half-life.

[0165] Table 6: Mean pharmacokinetic parameters after a single subcutaneous administration to cynomolgus macaques. Abbreviations: AUC inf = Area under the curve from 0 to infinity; CL / F = Clearance divided by bioavailability (F); C max = Maximum concentration; T max = Time to maximum concentration; T1 / 2 = Half-life.

[0166] Results: These data confirm that the above compounds have a pharmacokinetic profile suitable for once-weekly administration.

[0167] (5) Solubility and stability Objective: To determine the soluble pH range and stability of the PYY analogue Methods: Visual solubility range evaluation – The lyophilized PYY analog powder was reconstituted in water at a concentration of 4 mg / mL and the pH was adjusted to pH 4 using citrate / phosphate buffer. The pH of the system was titrated upwards to pH 8 with 0.5 N NaCl and then titrated downwards to pH 4 with 0.5 N HCl.

[0168] Thermal stability assessment – PYY analog solutions in 10 mM or 20 mM sodium phosphate (pH 7.0) at a concentration of 1 mg / mL or 2 mg / mL were prepared and incubated at 4 °C and 40 °C for 4 weeks. Samples at the 4-week time point were analyzed by size exclusion chromatography (SEC) and RP-HPLC.

[0169] SEC method – Performed using a TOSOH TSKgel G2000SWxl, 7.8 mm ID x 30 cm, 5 µm column, where the mobile phase composition was 50 mM sodium phosphate, 300 mM NaCl with 20% acetonitrile (pH 7.0), over 30 minutes, flow rate 0.5 mL / min, λ – 214 nm.

[0170] RP method – Performed using a Cortecs C18, 2.7 µm, 4.6 x 50 mm column, with 20% - 45% acetonitrile / water with 0.085% TFA, over 10 minutes, flow rate 1 mL / min, λ - 214 nm.

[0171] Results: Table 7: Solubility and thermal stability of PYY analogs.

[0172] As shown above, all peptides were soluble at pH > 7.0. The stability evaluated by RP and SEC indicated that these peptides were relatively stable under severe heat stress.

[0173] Example 7: In vivo effects of PYY.

[0174] (1) In vivo effects on food intake and body weight in normal mice Purpose: To compare the effects of the PYY analogs of Examples 1 to 5 on reducing body weight and inhibiting food intake in normal mice after a single injection.

[0175] Method: Male C57Bl / 6 mice from Envigo RMS (Indianapolis, IN) were maintained on a chow diet (5008; LabDiet, St. Louis, MO) and individually 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 to 10 weeks of age, non-fasted body weight and initial food weight were recorded, and animals were administered a single subcutaneous injection of vehicle or peptide, followed by daily measurement of body weight and food intake for 3 days after dosing. The area under the curve (AUC) of body weight and food intake relative to vehicle was calculated. In each assay run, Example 4 at 30 nmol / kg was used as a benchmark for 100% potency for body weight and food intake.

[0176] Results: Table 8: Changes in body weight and food intake in C57 / Bl6 mice over 3 days after single administration of PYY analogs. * Known PYY analogs for comparison; see International Patent Application Publication No. WO 2016 / 198682, where Reference Example 1 corresponds to Compound 4 therein, Reference Example 2 corresponds to Compound 21 therein, and Reference Example 3 corresponds to Compound 32 therein. ** Example 4 at 30 nmol / kg (AUC) was set as 100% potency As shown above, the reduction in both body weight and food intake confirmed the in vivo potency of the PYY analogs, and the comparison of the doses required for full potency confirmed the increased potency.

[0177] (2) In vivo effects on food intake and body weight in diet-induced obese mice Objective: To study the effect of daily administration of the PYY analogs of Examples 1-5 (alone or in combination with a GLP-1 receptor agonist) over a two-week period on body weight reduction in diet-induced obese (DIO) mice.

[0178] Methods: Twenty-week-old DIO male C57Bl / 6 mice (Taconic) were maintained on a 60% fat diet (D12492; Research Diets, New Brunswick, NJ) upon arrival. The animals were individually housed in a temperature-controlled facility (74.0°F; 23.3°C) with a 12-hour light / dark cycle (lights on at 22:00) and free access to food and water. After a one-week acclimation period with daily vehicle administration, non-fasted body weights were measured and the animals were randomly assigned to experimental groups (n = 6) and received daily subcutaneous injections of vehicle, a GLP-1 receptor agonist (GLP-1RA; SEQ ID NO:15), a PYY analogue, or a combination of a PYY analogue plus GLP-1RA. After 2 weeks of dosing, non-fasted body weights were recorded and the change in mean body weight compared to vehicle was calculated. To determine the additive or synergistic effect of the combination of the PYY analogue and GLP-1RA, the potency above that of GLP-1RA alone (net effect) was calculated.

[0179] Results: Table 9: Body weight changes in a 2-week study in diet-induced obese mice with a PYY analogue alone or in combination with a GLP-1 receptor agonist. * Potency above GLP-1 receptor agonist (GLP-1RA).

[0180] As shown above, the weight loss with the PYY analogue alone and in combination with GLP-1RA confirmed the in vivo potency of the PYY analogue, where the magnitude of weight loss was compared.

[0181] (3) In vivo effects on body weight and glucose in diabetic and obese (db / db) mice Objective: To study the effect of daily administration of the PYY analogues of Examples 1 to 5 over a ten-day period on body weight and blood glucose levels in obese and diabetic mice (db / db).

[0182] Methods: Lepr db / db (db / db) male mice from Envigo RMS (Indianapolis, IN) were 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 to 9 weeks of age, using Accu- A blood glucose meter (Roche Diabetes Care, Inc., Indianapolis, IN) measures body weight and blood glucose levels, and then a vehicle or peptide is injected subcutaneously daily. After 10 days of administration, body weight and blood glucose levels are measured and the changes compared to vehicle treatment are calculated.

[0183] Results: Table 10: Effects on body weight and blood glucose in db / db mice treated for 10 days.

[0184] As shown above, the reduction of body weight and blood glucose levels with PYY analogs confirms the in vivo potency of the PYY analogs, where the magnitudes of body weight loss and glucose reduction were compared.

[0185] In summary, the PYY analogs herein show selectivity for NPY2R. They also show a dose-dependent reduction in body weight, as reflected in normal mice, diet-induced obese mice, and db / db mice, as well as a dose-dependent improvement in blood glucose in db / db mice, where the PYY analogs of Examples 1, 3, and 4 are the most effective, consistent with the in vitro profile.

Claims

1. A peptide tyrosine-tyrosine (PYY) analogue, which comprises the following amino acid sequence: PKPEX 7 PX 9 X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY (Formula I), wherein X 7 is any amino acid having a functional group available for conjugation and said functional group is conjugated to C 16 -C 22 fatty acid where X 9 is E or G, where X 10 is E or K, where X 17 is L or W, where X 18 is N or Q, where X 22 is A or I, where X 23 is E, D, or S, where X 30 is E or W (SEQ ID NO:3), and wherein the C-terminal amino acid is optionally amidated.

2. The PYY analogue of claim 1, wherein X 7 is selected from C, D, E, K, and Q.

3. The PYY analog of claim 1, wherein X 7 is K and is conjugated to C through the ε-amino group of the K side chain 16 -C 22 fatty acid.

4. The PYY analogue of claim 1, wherein the amino acid sequence is selected from:

5. A PYY analogue according to any one of claims 1 to 4, wherein said C 16 -C 22 fatty acids are selected from hexadecanoic acid, hexadecanedioic acid, heptadecanoic acid, heptadecanedioic acid, stearic acid, octadecanedioic acid, nonadecanoic acid, nonadecanedioic acid, eicosanoic acid, eicosanedioic acid, heneicosanoic acid, heneicosanedioic acid, docosanoic acid, docosanedioic acid and their branched and substituted derivatives.

6. The PYY analogue of claim 5, wherein said C 16 -C 22 fatty acid is a C 18 -C 20 fatty acid.

7. The PYY analogue of claim 6, wherein said C 18 -C 20 fatty acid is a straight-chain fatty acid having the formula CO-(CH 2 ) x -CO 2 H, and wherein x is 18 or 20.

8. The PYY analogue of claim 7, wherein said C 18 -C 20 fatty acid is selected from palmitic acid, stearic acid, arachidic acid, and eicosanoic acid.

9. A PYY analogue according to any one of claims 1 to 8, wherein said C 16 -C 22 fatty acid is conjugated to an amino acid having a functional group available for conjugation via a linker.

10. The PYY analogue of claim 9, wherein the linker can be one or more units selected from [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid (Ahx), glutamic acid (E), γ-glutamic acid (γE) or a combination thereof.

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