Protein tyrosine-tyrosine analogs and methods of use thereof

Abstract

Disclosed are PYY analogs comprising modifications that increase half-life compared to native human PYY, and additional modifications that increase efficacy and selectivity for 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 treating obesity and obesity-related diseases and conditions (such as type II diabetes).

Description

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

[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. PYY is secreted by L cells in 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 Compared to PYY 3-36 dominant, and after eating, PYY 3-36 Compared to PYY 1-36 Dipeptidyl peptidase-IV (DPP-IV) hydrolyzes PYY 1-36 Pro 2 -Ile 3 Key to generate PYY 3-36 , which is more sensitive to NPY2 receptor than PYY 1-36 Be more selective.

[0003] Plasma PYY 3-36 Concentrations typically increase within 15 minutes of food ingestion, 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 PYY affects appetite both through its direct central effects and through its effects on intestinal motility (i.e., its anorectic effects). 3-36 Mediates insulin sensitivity to thereby help lower blood glucose (ie, its sensitizing effect).

[0004] Given PYY 3-36 Anorexic effect, PYY 3-36 It has been investigated as a potential therapeutic agent for weight regulation, particularly for the treatment of obesity and its related 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, exogenously administered PYY is not readily available due to proteases and other clearance mechanisms. 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 in using it as a therapeutic agent. Due to this short half-life, PYY 3-36 It must be administered at least once a day to exert a therapeutic effect, which is inconvenient for individuals in need thereof. Therefore, efforts have been made to increase PYY 3-36 The invention relates to a method for increasing the half-life of a PYY analogue by increasing the half-life of the PYY receptor and / or increasing its NPY2 receptor selectivity. For example, Rubinstein et al. described PYY analogues 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 analogues having an amino-terminal attached PEG moiety to increase the half-life (see, DeCarr et al. (2007)). Bioorg. Med. Chem. Lett. 17:1916-1919; see also, Ortiz et al. (2007) J. Pharmacol. Exp. Ther. 323:692-700). In addition, Kofoed et al. describe PYY analogs having an albumin binding side chain, at least one modified group close to the cleavage site of PYY (e.g., an N-methyl amino acid analog of the target amino acid residue), N-glycine and / or arginine mimetics to increase half-life (see, International Patent Application Publication No. WO 2011 / 033068).

[0006] Despite the PYY 3-36 The understanding of its role in metabolism has increased significantly, but there is still a need for additional PYY analogs, especially PYY analogs with enhanced efficacy and selectivity at the NPY2 receptor.

[0007] As indicated above, there is a need for additional PYY analogs for therapeutic use. To address this need, the present disclosure first describes PYY analogs comprising the following basic amino acid sequence (relative to native human PYY 1-36 (SEQ ID NO: 1) number):

[0008] (Formula I),

[0009] wherein X7 is any amino acid having a functional group available for conjugation and the functional group is conjugated to C 16 -C 22 Fatty acids, X9 is E is G, X 10 It's E, it's K, X 17 It's L, it's W, it's X 18 Yes N, Q, X 22 It's A, it's I, it's 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.

[0010] In some cases, the amino acid having a functional group available for conjugation at position X7 may be C, D, E, K or Q. In a specific case, the amino acid having a functional group available for conjugation at position X7 is K, and the amino acid sequence may be one of the following:

[0011]

[0012] or

[0013] .

[0014] In some cases, C 16 -C 22 The fatty acid is conjugated to an amino acid having a functional group available for conjugation via a linker. 16 -C 22 Fatty acids have -CO-(CH2) a -CO2H structure, where a is an integer between 16 and 22. In a specific case, the fatty acid is C 18 Diacid or C 20 Diacids such as palmitic acid, stearic acid, arachidic acid or eicosanoic acid, especially saturated C 18 Diacid or C 20 Likewise, and in some cases, the linker can be one or more units of [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminocaproic acid (Ahx), glutamic acid (E), γ-glutamic acid (γE), or a combination thereof.

[0015] In specific cases, the PYY analogue may be one of the following:

[0016]

[0017]

[0018]

[0019]

[0020]

[0021] In some cases, the base structure of the PYY analogs herein may further comprise native human PYY 1-36 The two amino terminal (N-terminal) amino acids of (SEQ ID NO: 1) can then be processed in vivo to PYY 3-36 analogs (ie, the N-terminal "YP" residue of SEQ ID NO: 1 can be cleaved in vivo from any of the PYY analogs).

[0022] In some cases, the PYY analogue has a charge greater than -2, in particular -3 or -4.

[0023] In some cases, the PYY analog has a higher binding affinity at the human NPY2 receptor than human PYY 3-36 The binding affinity of (SEQ ID NO: 2) is greater, such as about 2-fold to about 10-fold, in particular about 2-fold to about 3-fold.

[0024] In some instances, the PYY analog has a half-life that is longer than that of human PYY 3-36 The half-life of (SEQ ID NO: 2) is longer, such as about 5 hours to about 24 hours, especially about 12 hours.

[0025] Second, a pharmaceutical composition is described, which comprises at least one PYY analogue herein or a pharmaceutically acceptable salt thereof (eg, trifluoroacetate, acetate or hydrochloride) and a pharmaceutically acceptable carrier. In some cases, the pharmaceutical composition may further comprise a carrier, a diluent and / or an excipient.

[0026] In addition, the pharmaceutical composition may contain additional therapeutic agents, such as, for example, other antidiabetic or anti-obesity agents, in particular 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In other cases, the incretin may be a gastric inhibitory peptide (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 with triple receptor activity (i.e., an incretin analog with activity at each of the GIP, GLP-1 and GCG receptors). In other cases, the additional therapeutic agent may be a DPP-IV inhibitor.

[0027] Third, methods of using the PYY analogs herein, in particular using the PYY analogs to treat obesity and obesity-related diseases and conditions (such as T2DM) are described. The methods at least comprise the step of administering to an individual in need thereof an effective amount of a PYY analog as described herein or a pharmaceutically acceptable salt thereof.

[0028] In some cases, the PYY analogue can be administered subcutaneously (SQ) to the individual. Likewise, and in some cases, the PYY analogue 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 analogue can be administered SQ every other day, SQ three times a week, SQ twice a week, SQ once a week, SQ every other week, or SQ once a month. In specific cases, the PYY analogue is administered SQ once a week (QW).

[0029] Alternatively, the PYY analogue may be administered orally to the individual. As above, the PYY analogue may be administered daily, every other day, three times a week, twice a week, once a week (i.e., every week), every two weeks (i.e., every other week), or monthly. In some cases, the PYY analogue may be administered orally every other day, three times a week, twice a week, once a week, every other week, or once a month. In specific cases, the PYY analogue is administered orally once a week.

[0030] The method may also include administering at least one PYY analog 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 combination of GLP-1 analogs, GIP or GIP analogs, OXM or OXM analogs, GIP / GLP-1, GLP-1 / GCG or incretins with triple receptor activity). The DPP-IV inhibitor or incretin may be administered simultaneously, separately or sequentially with the PYY analog.

[0031] In some cases, the DPP-IV inhibitor or incretin may 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 different frequency than 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 may be administered orally.

[0032] In some cases, the individual is obese or overweight. In other cases, the individual is a person with diabetes (PwD), especially T2DM. In some cases, the individual is obese with T2DM or overweight with T2DM.

[0033] The method may also include steps such as measuring or obtaining the individual's weight and / or blood glucose and / or hemoglobin A1c (HbA1c) and comparing such obtained values ​​to one or more baseline values ​​or previously obtained values ​​to evaluate the effectiveness of the treatment.

[0034] The methods may also be combined with diet and exercise and / or may be combined with additional therapeutic agents other than those discussed above.

[0035] 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 can optionally be combined with DPP-IV inhibitors and / or incretins (such as GCG or GCG analogs, GLP-1, GLP-1 (7-36) 酰胺 or GLP-1 analogs, GIP or GIP analogs, OXM or OXM analogs, GIP / GLP-1, GLP-1 / GCG, or even incretins with triple receptor activity) are administered simultaneously, separately, or sequentially (ie, in combination).

[0036] Fifth, the use of the PYY analogs herein in the preparation of a medicament for treating 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 GLP-1 analogs, GIP or GIP analogs, OXM or OXM analogs, GIP / GLP-1, GLP-1 / GCG, or even incretins with triple receptor activity).

[0037] An advantage of the PYY analogs herein is that they can not only promote weight loss but also reduce glucose. In this way, individuals (particularly those susceptible to 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. Combined GIP / GLP-1 and PYY analogs can be used for glucose control (incretins + potential insulin sensitizers) and weight loss (synergy). Specifically, the PYY analogs herein can only cause up to about 12% weight loss when applied to individuals in need thereof and can cause up to about 25% weight loss when applied to individuals in need thereof in combination with additional therapeutic agents (such as incretins).

[0038] Another advantage of the PYY analogs herein is that they may have a half-life of up to about 24 hours, thereby allowing once-weekly administration.

[0039] Another advantage of the PYY analogs herein is that they have the ability to react with natural human PYY. 3-36 (SEQ ID NO: 2), and when compared to native human PYY 3-36 (SEQ ID NO: 2) has increased compatibility in formulations with incretins.

[0040] Unless defined otherwise, 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 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.

[0041] Furthermore, reference to an element by the indefinite article "a or an" does not exclude the possibility that more than one element is present, unless the context clearly requires that one and only one element is present. Thus, the indefinite article "a or an" generally means "at least one".

[0042] definition

[0043] As used herein, "about" means within a statistically significant range of one or more values, such as, for example, a specified concentration, length, molecular weight, pH, sequence identity, length of time, temperature, volume, etc. Such values ​​or ranges may be on the order of typically within 20%, more typically within 10%, and even more typically within 5% of a given value or range. The tolerance encompassed by "about" will depend on the particular system under study and may be readily recognized by those skilled in the art.

[0044] As used herein, "amino acid" means a molecule that is characterized from a chemical point of view by the presence of one or more amine groups and one or more carboxylic acid groups and may contain other functional groups. As known in the art, there is a set of twenty amino acids that are designated as standard amino acids and used as building blocks for most peptides / proteins produced by any organism.

[0045] As used herein, "amino acid having a functional group that can be used for conjugation" means any natural or non-natural amino acid having a functional group that can be conjugated to a fatty acid via, for example, a linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromine, carboxyl, chlorine, iodine, and thiol groups. In addition, examples of natural amino acids that include such functional groups include C (thiol), D (carboxyl), E (carboxyl), K (amino), and Q (amide).

[0046] 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 a natural agonist at that receptor.

[0047] As used herein, "anorexigenic effect" refers to the ability of the PYY analogs herein to reduce appetite, leading to lower food consumption and ultimately weight loss. Anorectic effect may also refer to the ability of the PYY analogs herein to increase intestinal motility.

[0048] As used herein, “C 16 -C 22 "Fatty acid" means a carboxylic acid having 16 to 22 carbon atoms. Suitable C 16 -C 22 Fatty acids can be saturated monoacids or saturated diacids ("diacids" have a carboxyl group at each terminus).

[0049] As used herein, "AUC" means area under the curve.

[0050] As used herein, "effective amount" means an amount, concentration or dosage of one or more PYY analogs herein or pharmaceutically acceptable salts thereof that provides the desired effect (i.e., can produce a clinically measurable difference in the individual's condition, such as, for example, a reduction in blood glucose, a reduction in HbA1c and / or a reduction in weight or body fat) in a diagnosed or treated individual following single or multiple dose administration to such an individual in need thereof. An effective amount is readily determined by one skilled in the art by using known techniques and by observing results obtained under similar circumstances. In determining an effective amount for an individual, a number of factors are considered, including, but not limited to, the species of mammal, its size, age and general health, the specific disease or condition involved, the extent or involvement or severity of the disease or condition, the response of the individual, the specific PYY analog administered, the mode of administration, the bioavailability characteristics of the administered formulation, the dosage regimen selected, the use of concomitant medications, and other relevant circumstances.

[0051] As used herein, "half maximal effective concentration" or "EC 50 ” refers to the concentration of compound that results in 50% activation / stimulation of an assay endpoint, such as a dose-response curve (e.g., cAMP).

[0052] As used herein, "in combination with" means administering at least one of the PYY analogues herein simultaneously, sequentially or in a single combined preparation with one or more additional therapeutic agents.

[0053] As used herein, "incretin mimetics" means peptides or polypeptides that have structural similarities to each of GIP, GLP-1, GCG and OXM, especially native human GIP, GLP-1, GCG and OXM, but have multiple differences. Some incretin mimetics also have affinity for two or even each of the GIP, GLP-1 and GCG receptors 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).

[0054] As used herein, "subject in need thereof" means a mammal, such as a human, having a condition, disease, disorder or symptom requiring treatment or therapy, including, for example, those listed herein. In particular, the preferred subject to be treated is a human.

[0055] As used herein, "long-acting" means that the PYY analogs herein have a binding affinity and activity that is greater than that of native human PYY. 1-36 (SEQ ID NO: 1) and / or native human PYY 3-36(SEQ ID NO: 2) for longer periods of time, allowing dosing at least once a day or even three times a week, twice a week, weekly or monthly. The temporal action profile of the PYY analogs herein can be measured using known pharmacokinetic test methods such as those described in the Examples below.

[0056] As used herein, "non-standard amino acids" means amino acids that may be naturally present in cells but are not involved in peptide synthesis. Non-standard amino acids may be components of peptides and are often generated by modifying standard amino acids in peptides (i.e., via post-translational modifications). Non-standard amino acids may include D-amino acids, which have an absolute chirality opposite to that of the standard amino acids described above.

[0057] As used herein, "obese" or "obese" means a person in which the body mass index (BMI) is >30.0 kg / m 2 See generally, "Overweight & Obesity" by the Centers for Disease Control and Prevention, available at cdc.gov / obesity / adult / defining.html; and "Definitions & Facts for Adult Overweight & Obesity" by the National Institutes of Health, available at iddk.nih.gov / health-information / weight-management / adult-overweight-obesity / definition-facts.

[0058] As used herein, "obesity-related disease or condition" means any disease or condition caused / aggravated 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 nephrolithiasis (kidney stones), and breast cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, prostate cancer, and rectal cancer.

[0059] As used herein, "overweight" means wherein the individual has a BMI of about 25.0 kg / m 2 To <30 kg / m 2 See, ibid.

[0060] As used herein, "PYY" means peptide YY obtained or derived from any species, such as a mammalian species, especially a human. PYY includes native PYY (i.e., full length) and variants thereof (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).

[0061] As used herein, "one or more PYY analogs" means a PYY-like peptide or polypeptide that elicits one or more effects of native PYY at one or more NPY receptors (such as NPY2 receptors). In some cases, when compared to native PYY, especially 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 may bind to NPY receptors, especially human NPY2 receptors, with higher or lower affinity, but demonstrate longer half-lives in vivo or in vitro. In this manner, the PYY analogs herein are synthetic compounds that act as NPY2 receptor agonists.

[0062] As used herein, "saturated" means that the fatty acids do not contain carbon-carbon double or triple bonds.

[0063] As used herein, "sensitizing effect" refers to the ability of the PYY analogs herein to increase the action of insulin and thereby help lower blood glucose.

[0064] As used herein, the terms "treating" or "to treat" means to lessen, inhibit, reverse, slow or stop the progression or severity of an existing condition, disease, disorder or symptom.

[0065] Certain abbreviations are defined as follows: “ACR” refers to urine albumin / urine creatinine ratio; “amu” refers to atomic mass unit; “tBoc” refers to tert-butyloxycarbonyl; “cAMP” refers to cyclic adenosine monophosphate; “DMF” refers to dimethylformamide; “DMSO” refers to dimethyl sulfoxide; “EIA / RIA” refers to enzyme immunoassay / radioimmunoassay; “hr” refers to hour; “HTRF” refers to homogeneous time-resolved fluorescence; “IV” refers to intravenous; “kDa” refers to kilodaltons; “LC-MS” refers to liquid chromatography-mass spectrometry; “MS” refers to mass spectrometry; “OtBu” refers to O-tert-butyl; “Pbf” refers to NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl; “RP-HPLC” refers to reversed-phase high performance liquid chromatography; “SQ” refers to subcutaneous; “SEM” refers to standard error of the mean; “TFA” refers to trifluoroacetic acid; and “Trt” refers to trityl.

[0066] PYY analogs

[0067] The PYY analogs herein have structural similarities to the natural PYY peptide, but there are many structural differences. For example, when compared to the natural human PYY 1-36 (SEQ ID NO: 1) and / or native human PYY 3-36 (SEQ ID NO: 2) when compared to PYY analogs described herein include analogs of PYY that are 1-36 (SEQ ID NO: 1). In some cases, exemplary amino acid sequences of PYY analogs herein include (specific changes relative to the corresponding residues of native human PYY (SEQ ID NO: 1) are in bold):

[0068]

[0069] and

[0070] .

[0071] The PYY analogs herein result in sufficient activity at the human NPY2 receptor, but insufficient activity at the NPY1, NPY4 and NPY5 receptors. Likewise, the PYY analogs herein possess beneficial attributes relevant to their developability as therapeutic treatments, including improved solubility in aqueous solutions, improved chemical and physical formulation stability, prolonged pharmacokinetic profiles and minimized immunogenic potential.

[0072] In some cases, the PYY analogs herein are amidated at the C-terminal amino acid 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 to and activating the human NPY2 receptor.

[0073] The PYY analogs herein further include fatty acids conjugated to natural or unnatural amino acids having a functional group available for conjugation (i.e., "acylated"), e.g., via a linker. In some cases, the amino acid 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, wherein the conjugation is to the ε-amino group of the K side chain.

[0074] Here, when compared with natural human PYY 1-36 (SEQ ID NO: 1), the acylation of the PYY analogue is at position 7. In this way, the fatty acid can act as an albumin binder to provide a longer acting analogue.

[0075] Regarding fatty acids, they can be conjugated to the functional groups of amino acids available for conjugation through direct bonds or through linker chemistry. The length and composition of the fatty acids 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 fatty mono- or di-acids thus produce PYY analogs that exhibit a desired half-life, desired in vivo efficacy, and desired solubility and stability characteristics.

[0076] Exemplary saturated C 16 -C 22 Fatty acids include, but are not limited to, hexadecanoic acid (i.e., palmitic acid, C 16 Monoacid), hexadecanedioic acid (C 16 diacid), heptadecanoic acid (i.e. margaric acid, C 17 monoacid), heptadecanedioic acid (C 17 Diacid), stearic acid (C 18 Monoacid), octadecane dioic acid (C 18 diacid), nonadecylic acid (i.e. nonadecylic acid, C 19 Monoacid), nonadecanedioic acid (C 19 Diacid), eicosanoic acid (i.e., arachidic acid, C 20 monoacid), eicosanedioic acid (C 20 diacid), heneicosylic acid (C 21 Monoacid), heneicosane dioic acid (C 21 dicarboxylic acid), behenic acid (behenic acid, C 22monoacid), docosanoic acid (C 22 Diacids) and their branched and substituted derivatives. In some cases, C 16 -C 22 Fatty acids can be saturated 18 Monoacid, saturated C 18 Diacid, saturated C 19 Monoacid, saturated C 19 Diacid, saturated C 20 Monoacid, saturated C 20 Diacids and their branched and substituted derivatives. In the specific case, C 16 -C 22 The fatty acid may be palmitic acid or hexadecanoic acid, stearic acid or octadecanedioic acid, or arachidic acid or eicosanoic acid.

[0077] To facilitate conjugation of fatty acids to natural or unnatural amino acids having functional groups available for conjugation, the PYY analogs herein may include a linker. In some cases, the linker may be at least one of AEEA, Ahx, E, or γE, and combinations thereof.

[0078] When the linker comprises amino acids, it may have one to four E or γE amino acid residues. In some cases, the linker may comprise one or two E and / or γE amino acid residues. For example, the linker may comprise one or two E and / or γE amino acid residues. In other cases, the linker may comprise 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 may be a combination of E and γE amino acid residues with AEEA or Ahx. In other cases, the linker may be a combination of one or two γE amino acid residues and one or two AEEA or Ahx. In specific cases, the linker may be a (AEEA)2•γE portion, an Ahx•E•γE portion, or an AEEA•γE portion.

[0079] An exemplary linker-fatty acid moiety may comprise (AEEA)2•γE•C 20 Diacid, Ahx•E•γE•C 18 Diacid or AEEA•γE•C 18 These structural features of the linker-fatty acid moiety lead to the 1-36 (SEQ ID NO: 1) or native human PYY 3-36 (SEQ ID NO: 2) compared to an analog with improved half-life.

[0080] In summary, exemplary PYY analogs are:

[0081]

[0082]

[0083]

[0084]

[0085]

[0086] Although PYY analogs are described as having thirty-four amino acids (like native human PYY 3-36 (34 amino acids of SEQ ID NO: 2), but it is contemplated that the PYY analogs herein may have amino acids based on natural human PYY 1-36 (SEQ ID NO: 1). That is, the PYY analogue may include natural 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 subsequently be cleaved in vivo upon administration to an individual, such as to endogenously release native human PYY 1-36 (SEQ ID NO: 1).

[0087] The half-life of the PYY analogs herein can be measured using techniques known in the art, including, for example, those described in the Examples below. Likewise, the respective affinities of the PYY analogs herein for various NPY receptors (e.g., NPY2R, NPY5R) can be measured using techniques known in the art for measuring receptor binding levels, including, for example, those described in the Examples below, and are generally expressed as inhibition constants (K i ) value. In addition, the activity of the PYY analogs herein at each receptor may also be measured using techniques known in the art, including, for example, the following in vitro activity assays, and is generally expressed as EC 50 value.

[0088] 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) has a longer half-life. For example, the half-life of the PYY analogue can be about 5 hours to about 24 hours, about 6 hours to about 23 hours, about 7 hours to about 22 hours, about 8 hours to about 21 hours, about 9 hours to about 20 hours, about 10 hours to about 19 hours, about 11 hours to about 18 hours, about 12 hours to about 17 hours, about 13 hours to about 16 hours, or even about 14 hours to about 15 hours. Alternatively, the half-life of the PPY analogue 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.

[0089] Similarly, the PYY analogs herein have a higher binding affinity at the NPY2 receptor than native human PYY 3-36 (SEQ ID NO: 2) with a greater binding affinity, such as about 2-fold to about 10-fold. Alternatively, the PYY analogs herein may have a binding affinity greater than that of native human PYY 3-36 The binding affinity of (SEQ ID NO:2) is 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, particularly 2-fold to 3-fold greater.

[0090] Pharmaceutical composition

[0091] The PYY analogs herein may be formulated as pharmaceutical compositions that can be administered by a parenteral route (e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, or transdermal). Such pharmaceutical compositions and techniques for their preparation are well known in the art. See, for example, Remington, "The Science and Practice of Pharmacy" (DB Troy, ed., 21st ed., Lippincott, Williams & Wilkins, 2006). In specific cases, the PYY analogs are administered SQ. Alternatively, however, the PYY analogs may be formulated in a form for other pharmaceutically acceptable routes, such as, for example, tablets or other solids for oral administration; timed-release capsules; and any other form currently used, including creams, emulsions, inhalants, and the like.

[0092] In order to improve its in vivo compatibility and effectiveness, the PYY analogs herein can be reacted with any of a number of inorganic and organic acids / bases to form pharmaceutically acceptable acid / base addition salts. Pharmaceutically acceptable salts and common preparation techniques thereof are well known in the art (see, e.g., 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.

[0093] The PYY analogs herein can be administered by a physician or self-administered using injection. It should be understood that the metering size and injection volume can be easily determined by one skilled in the art. However, the injection volume can be ≤ about 2 mL or even ≤ about 1 mL, and the needle meter can be ≥ about 27 G or even ≥ about 29 G.

[0094] The present disclosure also provides and therefore encompasses novel intermediates and methods that can be used to synthesize the PYY analogs herein or pharmaceutically acceptable salts thereof. 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 synthetic steps of each of the described pathways 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.

[0095] The PYY analogs herein are generally effective over a wide dosage range. Exemplary dosages of the PYY analogs herein or pharmaceutical compositions comprising the same may be in milligram (mg), microgram (μg), nanogram (ng) or picogram (pg) amounts per kilogram (kg) of subject. In this manner, a daily dose may be from about 1 μg to about 100 mg.

[0096] Here, the effective amount of the PYY analog in the pharmaceutical composition may 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 aforementioned range and more than sufficient, while in other cases, the effective amount may be a larger dose and may be adopted with acceptable side effects.

[0097] In addition to the PYY analog, the pharmaceutical composition may also include additional therapeutic agents, particularly other antidiabetic or anti-obesity agents. In some cases, the additional therapeutic agent may 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.

[0098] In this manner, the pharmaceutical composition may comprise an effective amount of a PYY analogue of SEQ ID NO:9 and an incretin or a DPP-IV inhibitor, an effective amount of a PYY analogue of SEQ ID NO:10 and an incretin or a DPP-IV inhibitor, an effective amount of a PYY analogue of SEQ ID NO:11 and an incretin or a DPP-IV inhibitor, an effective amount of a PYY analogue of SEQ ID NO:12 and an incretin or a DPP-IV inhibitor, or an effective amount of a PYY analogue of SEQ ID NO:13 and an incretin or a DPP-IV inhibitor.

[0099] In those cases where the incretin is GLP-1 or a GLP-1 analog, it may be GLP-1 or a GLP-1 analog such as albiglutide, dulaglutide, liraglutide, semaglutide or a combination thereof, in particular dulaglutide.

[0100] Methods of making and using PYY analogs

[0101] The PYY analogs herein 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. Solid phase synthesizers can be used to block interfering groups, protect amino acids during reactions, couple, deprotect and cap unreacted amino acids according to the manufacturer's instructions.

[0102] Typically, at room temperature, in an inert solvent (such as DMF, N-methylpyrrolidone or dichloromethane), in the presence of a coupling agent (such as diisopropyl-carbodiimide and 1-hydroxybenzotriazole), the N-α-carbamoyl protected amino acid and the N-terminal amino acid on the growing peptide chain attached to the resin are coupled. Reagents such as TFA or piperidine are used to remove the N-α-carbamoyl protecting group from the resulting peptide resin, and the coupling reaction is repeated with the next desired N-α-protected amino acid to be added to the peptide chain. Suitable amine protecting groups are well known in the art and are described in, for example, Green & Wuts, "Protecting Groups in Organic Synthesis," (John Wiley and Sons, 1991). The most commonly used examples include tBoc and Fmoc. After the synthesis is complete, standard treatment methods are used under acidic conditions to cut off the peptide from the solid support while deprotecting the side chain.

[0103] Those skilled in the art will appreciate the use of C-terminal carboxamides to synthesize the peptide chains described herein. For the synthesis of C-terminal amide peptides, resins incorporating Rink amide MBHA or Rink amide AM linkers are typically used for Fmoc synthesis, while MBHA resins are typically used for tBoc synthesis.

[0104] Crude peptides are typically purified using RP-HPLC on a C8 or C18 column using a water-acetonitrile gradient from 0.05% to 0.1% TFA. Purity can be verified by analytical RP-HPLC. The identity of the peptide can be verified by MS. Peptides can be dissolved in aqueous buffers over a wide pH range.

[0105] One use of the PYY analogs herein is for reducing blood glucose and / or body weight in individuals, particularly those who are overweight or obese and have 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 herein show glucose lowering efficacy with the additional benefit of weight loss, allowing individuals to delay progression to insulin and maintain target HbA1c goals.

[0106] The method may include the steps described herein, and these may (but not necessarily) be performed in the order described. However, other orders are also contemplated. In addition, single or multiple steps may be performed in parallel and / or overlap in time and / or individually or in multiple repeated steps. In addition, the method may include additional unspecified steps.

[0107] Thus, such methods may include selecting an individual who is overweight and has T2DM or is susceptible to the disease. Alternatively, the method may include selecting an individual who is obese and has T2DM or is susceptible to the disease.

[0108] 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 an additional therapeutic agent, such as an incretin or a DPP-IV inhibitor.

[0109] The concentration / dose / dosage of the at least one PYY analogue and optional incretin or DPP-IV inhibitor are discussed elsewhere herein.

[0110] Regarding the route of administration, the at least one PYY analogue or the pharmaceutical composition comprising the same can be administered according to known methods (such as, for example, orally; by injection (i.e., intraarterially, intravenously, intraperitoneally, intracerebrally, intraventricularly, intramuscularly, intraocularly, intraportally or intralesionally); by a sustained release system, or by an implant device). In some cases, the at least one PYY analogue or the pharmaceutical composition comprising the same can be administered SQ by bolus injection or continuously.

[0111] Regarding the frequency of administration, 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., every week), every two weeks (i.e., every other week), or monthly. In some cases, the at least one PYY analogue or a pharmaceutical composition comprising the same is administered SQ every other day, SQ three times a week, SQ twice a week, SQ once a week, SQ every other week, or SQ monthly. In a specific case, the at least one PYY analogue or a pharmaceutical composition comprising the same is administered SQ once a week (QW).

[0112] Alternatively, the at least one PYY analogue or a pharmaceutical composition comprising the same may be administered orally. As described above, and with respect to the frequency of administration, 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., every week), every two weeks (i.e., every other week), or monthly. In some 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 specific cases, the PYY analogue is administered orally once a week.

[0113] For 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 GLP-1 analogs, GIP or GIP analogs, OXM or OXM analogs, GIP / GLP-1, GLP-1 / GCG, or even incretins with triple receptor activity. 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 incretins with triple receptor activity can be administered simultaneously, separately or sequentially with at least one PYY analogue or a pharmaceutical composition comprising the same.

[0114] In addition, 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 incretins with triple receptor activity can be administered at the same frequency as at least one PYY analog or a pharmaceutical composition comprising the same (i.e., every other day, twice a week, or even weekly). Alternatively, 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 incretins with triple receptor activity may be administered at a different frequency than at least one PYY analog or a pharmaceutical composition comprising the same. In other cases, 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 incretins with triple receptor activity are administered QW. In other cases, the PYY analog is administered SQ and the GCG, GCG analog, GLP-1, GLP-1(7-36) 酰胺 , a GLP-1 analog, GIP, a GIP analog, OXM, an OXM analog, GIP / GLP-1, GLP-1 / GCG, or an incretin with triple receptor activity can be administered orally.

[0115] It is further contemplated that the methods may be combined with diet and exercise and / or may be combined with additional therapeutic agents other than those discussed above. Example

[0116] The following non-limiting examples are offered for purposes of illustration and not limitation.

[0117] Example 1: PYY analogue 1.

[0118] One PYY analogue incorporating the inventive concept may have the following structure:

[0119]

[0120] 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 synthesized by using (Ahx-E-(γE)-CO-(CH2) 16 -COOH was conjugated to the ε-amino group of the K side chain at position 7 for chemical modification.

[0121] 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). Amino acid coupling was performed in DMF using 10 equivalents of amino acid, 0.9 M diisopropylcarbodiimide (DIC) and 0.9 M Oxyma (1:1:1 molar ratio) at 25° C. for 3 h. Deprotection was performed using a 25% piperidine solution in DMF.

[0122] After elongation of the peptide-resin as described above, 30% hexafluoroisopropanol (HFIP) in dichloromethane (DCM) was used to remove the MTT protecting group present in K at position 7. Additional coupling / deprotection cycles using the Fmoc / t-Bu strategy to extend the side chain of K at position 7 involved Fmoc-6-aminohexanoic acid (Chem-Impex International Catalog No. 02490), Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OH)-OtBu (ChemPep Catalog No. 100703), and HOOC-(CH2) 16 -COOtBu. In all couplings, 3 equivalents of building blocks were used with PyBOP (3 equivalents) and DIEA (6 equivalents) in DMF at 25°C for 3 h.

[0123] Cleavage from the resin and removal of side chain protecting groups were performed simultaneously in a solution containing TFA:triisopropylsilane:1,2-ethanedithiol:methanol:thioanisole 80:5:5:5:5 (v / v) at 25° C. for 2 h, followed by precipitation with cold ether. The crude peptide was purified to >99% purity (15-20% purified yield) by RP-HPLC on a phenylhexyl column (Phenomenex, Luna; 5 μm, 100A), with the appropriate fractions combined and lyophilized.

[0124] The purity of the PYY analogues was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1659.2 (+ / -0.2); calculated value: M+3H + / 3 = 1659.2; Observed value: M+4H + / 4 = 1244.6 (+ / -0.2); calculated value: M+4H + / 4 = 1244.6; Observed value: M+5H + / 5 = 995.9 (+ / -0.2); calculated value: M+5H + / 5 =995.9).

[0125] Example 2: PYY analogue 2.

[0126] One PPY analogue incorporating the inventive concept may have the following structure:

[0127]

[0128] 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 cleaved by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 18 -COOH was conjugated to the ε-amino group of the K side chain for chemical modification.

[0129] The PYY analogue according to SEQ ID NO: 10 was generated from solid phase peptides similar to that described above in Example 1. Thus, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) were reacted with 3 equivalents of the building blocks after MTT cleavage with PyBOP (3 equivalents) and DIEA (6 equivalents) at 25°C in DMF for 3 h. 18 -COOtBu is attached to the side chain.

[0130] The purity of the PYY analogues was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1665.4 (+ / -0.2); calculated value: M+3H + / 3 = 1665.5; Observed value: M+4H + / 4 = 1249.3 (+ / -0.2); calculated value: M+4H + / 4 = 1249.4; Observed value: M+5H + / 5 = 999.7 (+ / -0.2); calculated value: M+5H + / 5 =999.7).

[0131] Example 3: PYY analogue 3.

[0132] One PYY analogue incorporating the inventive concept may have the following structure:

[0133]

[0134] 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 cleaved by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 16 -COOH was conjugated to the ε-amino group of the K side chain for chemical modification.

[0135] The PYY analogue according to SEQ ID NO: 11 was generated from solid phase peptides similar to that described above in Example 1. Thus, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) were reacted with 3 equivalents of the building blocks after MTT cleavage with PyBOP (3 equivalents) and DIEA (6 equivalents) in DMF at 25°C for 3 h. 16 -COOtBu is attached to the side chain.

[0136] The purity of the PYY analogues was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1664.7 (+ / -0.2); calculated value: M+3H + / 3 = 1664.9; Observed value: M+4H + / 4 = 1248.9 (+ / -0.2); calculated value: M+4H + / 4 = 1248.9; Observed value: M+5H + / 5 = 999.3 (+ / -0.2); calculated value: M+5H + / 5 =999.3).

[0137] Example 4: PYY analogue 4.

[0138] One PYY analogue incorporating the inventive concept may have the following structure:

[0139]

[0140] 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 cleaved by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 16 -COOH was conjugated to the ε-amino group of the K side chain for chemical modification.

[0141] The PYY analogue according to SEQ ID NO: 12 was generated from solid phase peptides similar to that described above in Example 1. Thus, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) were reacted with 3 equivalents of the building blocks after MTT cleavage with PyBOP (3 equivalents) and DIEA (6 equivalents) at 25°C in DMF for 3 h. 16 -COOtBu is attached to the side chain.

[0142] The purity of the PYY analogues was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1664.8 (+ / -0.2); calculated value: M+3H + / 3 = 1665.5; Observed value: M+4H + / 4 = 1248.9 (+ / -0.2); calculated value: M+4H + / 4 = 1249.4; Observed value: M+5H + / 5 = 998.9 (+ / -0.2); calculated value: M+5H + / 5 =995.7).

[0143] Example 5: PYY analogue 5.

[0144] One PYY analogue incorporating the inventive concept may have the following structure:

[0145]

[0146] 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 cleaved by using ([2-(2-amino-ethoxy)-ethoxy]-acetyl)2-(γE)3-CO-(CH2) 18 -COOH was conjugated to the ε-amino group of the K side chain for chemical modification.

[0147] The PYY analogue according to SEQ ID NO: 13 was generated from solid phase peptides similar to that described above in Example 1. Thus, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) were reacted with 3 equivalents of the building blocks after MTT cleavage with PyBOP (3 equivalents) and DIEA (6 equivalents) in DMF at 25°C for 3 h. 18 -COOtBu is attached to the side chain.

[0148] The purity of the PYY analogues was checked by analytical RP-HPLC and the identity was confirmed using LC / MS (observed: M+3H + / 3 = 1732.2 (+ / -0.2); calculated value: M+3H + / 3 = 1732.3; Observed value: M+4H + / 4 = 1299.4 (+ / -0.2); calculated value: M+4H + / 4 = 1299.5; Observed value: M+5H + / 5 = 1039.7 (+ / -0.2); calculated value: M+5H + / 5 =1039.8).

[0149] Example 6: In vitro activity of PYY analogues.

[0150] (1) In vitro binding to hNPY1, hNPY2, hNPY4, and hNPY5 receptors

[0151] Purpose:

[0152] The PYY analogs of Examples 1 to 5 were evaluated for their in vitro binding affinity (Ki) to the following human (h) receptors: hNPY1R, hNPY2R, hNPY4R and hNPY5R in the absence of bovine serum albumin (BSA). Competitive radioligand binding assays were used in the scintillation proximity assay (SPA) method using a 50 μg / mL cell suspension containing 10 μg / mL of recombinant 10 μg / mL cells overexpressing various recombinant receptors and the relevant [ 125 I] membranes prepared from cell lines labeled with peptides. In each assay, the expression of the relevant native peptide 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 used as a control.

[0153] method:

[0154] PYY analogs, natural human PYY 1-36 and control PYY3-36 Synthesized at Lilly Research Laboratories (Indianapolis, IN, USA) and characterized by LC / MS, NMR and LC / UV analysis (99.5% purity). Peptide content was estimated to be 80% of powder amount. The peptide was prepared as a 10 mM stock solution in 100% DMSO and kept frozen at -20°C until immediately prior to testing in the assay.

[0155] For hNPY1R, transient overexpression was performed using CHO cells. Stably 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. hNPY5R cloning was performed at Multispan, Inc. (Hayward, CA).

[0156] To prepare hNPY1R, hNPY2R, mNPY2R, and hNPY4R crude cell membranes, two different methods were utilized (described below). hNPY5R membranes were purchased from Multipan, Inc. (#MCG1275).

[0157] Method 1 - For hNPY2R and hNPY4R membranes, frozen cell pellets were lysed on ice in 10 mL of 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 was disrupted 25 times using a glass Potter-Elvehjem homogenizer equipped with a Teflon® pestle. The homogenate was centrifuged at 1100 x g for 10 minutes at 4°C. The supernatant was collected and stored on ice while the pellet was resuspended in homogenization buffer and homogenized again as described above. The homogenate was centrifuged at 1100 x g for 10 minutes. The second supernatant was combined with the first supernatant and centrifuged at 35000 x g for 1 hour at 4°C. The resulting membrane pellet was resuspended in homogenization buffer containing protease inhibitors at approximately 1 to 3 mg / mL, snap frozen in liquid nitrogen, and stored as aliquots in a -80°C freezer until use. Protein concentration was determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0158] Method 2 - For hNPY1R membranes, frozen cell pellets were lysed on ice in 5 mL hypotonic homogenization buffer containing 25 mM Tris HCl (pH 7.5), 1 mM MgCl2, 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 was disrupted 25 times using a glass Potter-Elvehjem homogenizer equipped with a Teflon® pestle. The homogenate was centrifuged at 1800 x g for 15 minutes at 4°C in a 50 mL conical tube. The supernatant was collected and stored on ice while the pellet was resuspended in homogenization buffer and homogenized again as described above, except that no DNase I was used in the homogenization buffer. The homogenate was centrifuged at 1800 x g for 15 minutes. The second supernatant was combined with the first supernatant and centrifuged at 25000 xg for 30 minutes at 4°C. The resulting membrane pellet was resuspended in homogenization buffer containing protease inhibitors at approximately 2 mg / mL, aliquoted, and stored in a -80°C freezer until use. Protein concentration was determined using a BCA protein assay kit (Pierce, #23225) with BSA as a standard.

[0159] General Binding Assay Methods - The equilibrium dissociation constants (K) for various receptor / radioligand interactions were determined from saturation binding analyses using the same reagents and buffers as described below for compound testing. d ). The K determined for the receptor preparation used in this study d The values ​​are as follows: hNPY2R, 0.0047 nM; hNPY1R, 0.07 nM; hNPY4R, 0.084 nM; and hNPY5R, 0.896 nM.

[0160] hNPY1R receptor binding protocol – Human recombinant [ 125 I]-PYY 1-36 (#NEX341, 2200 Ci / mmol) Determination of PYY analog peptide and PYY from competitive radioligand binding assays 1-36Receptor binding affinity (Ki) for hNPY1R. The assay was performed using the 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 CaCl2, and 0.2% w / v bacitracin (RPI, #32000)) was used for the preparation of reagents. PYY analogs and PYY 1-36 Thaw and serially dilute 3-fold in 100% DMSO (10-point concentration response curve) using a Tecan Evo liquid handler. Peptides were diluted 20-fold down into assay buffer to reduce DMSO levels and peptide concentrations before addition to the assay plate. Next, 5 µL of serially diluted peptide or DMSO was transferred to a plate containing 45 µL of assay buffer or unlabeled PYY. 1-36 A Corning® 3632 clear bottom assay plate containing a control (non-specific binding or NSB, 10 nM final concentration) was then added. 125 I]-PYY 1-36 (0.05 nM final concentration) and 50 µL hNPY1R membrane (1.0 µg / well). Finally, 50 µL WGA SPA beads (50 µg / well) were added. The final DMSO concentration was 0.125%. The plate was sealed and mixed on a plate shaker (setting 6) for 1 min and read on a PerkinElmer Trilux MicroBeta® scintillation counter after 10 h incubation / bead settling time at room temperature. The final assay concentration range of the peptides tested in the response curves was: PYY analogs (2.5 µM to 0.13 nM) and PYY 1-36 (10 nM to 0.5 pM).

[0161] hNPY2 receptor binding protocol - Determination of PYY analog peptides and PP from competitive radioligand binding assays as described above for hNPY1R 1-36 Receptor binding affinity (K) for hNPY2R i The final assay concentration range of the peptides tested in the response curves was: PYY analogs (0.1 µM to 5 pM) and PYY 3-36 (10 nM to 0.5 pM).

[0162] hNPY4R Receptor Binding Protocol - Determination of PYY analog peptides and PP from competitive radioligand binding assays as described above for hNPY1R 1-36 Receptor binding affinity (K) for hNPY4R iThe final assay concentration range of the peptides tested in the response curves was: PYY analogs (1 µM to 10 pM) and PYY 1-36 (1 µM to 10 pM).

[0163] hNPY5R receptor binding protocol - Determination of PYY analog peptides and PYY from competitive radioligand binding assays as described above for hNPY1R 1-36 Receptor binding affinity (K) for hNPY5R i The final assay concentration range of the peptides tested in the response curves was: PYY analogs (1 µM to 10 pM) and PYY 1-36 (1 µM to 10 pM).

[0164] Data analysis of NPY receptor binding assays – PYY analogs, PYY 1-36 ,PYY 3-36 or PP 1-36 The raw counts per minute (CPM) data of the concentration curves of 100 μg / mL were converted to percentage specific inhibition by subtracting nonspecific binding (NSB, respectively) from each CPM value in excess of unlabeled PYY. 1-36 ,PYY 3-36 or PP 1-36 The signal was corrected by subtracting nonspecific binding as shown in the following equation:

[0165] .

[0166] Using four parameters (curve maximum, curve minimum, IC 50 Data were analyzed using the nonlinear regression routine (GenedataScreener, version 13.0.5, Genedata AG, Basal, Switzerland). i =IC 50 / (1 + D / K d ) from the relative IC 50 The affinity (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 for the radioligand determined from the saturation binding analysis (listed above). The qualifier (>) indicates that the data did not reach 50% inhibition compared to maximal binding in the absence of competitor, therefore, the highest concentration of compound tested in the assay was used to calculate K i .

[0167] Reported Ki The values ​​are calculated as geometric means as follows:

[0168] .

[0169] The standard error of the mean (SEM) was calculated using the Δ method as follows:

[0170]

[0171] where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0172] The selectivity of the peptides for hNPY2R (Y2) over hNPY5R (Y5), hNPY4R (Y4) and / or hNPY1R (Y1) was calculated by dividing by the hNPY2R results (in nM).

[0173] result:

[0174] Table 1: In vitro binding to hNPY1R, hNPY2R, hNPY4R and hNPY5R (K i ).

[0175]

[0176] *Known PYY analogs for comparison; see International Patent Application Publication No. WO 2016 / 198682, wherein 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.

[0177] ND – Not Detected

[0178] NA – Not applicable.

[0179] As shown above, the PYY analogs of Examples 1 to 5 are highly selective for the hNPY2 receptor and even demonstrate similarity to native human PYY. 3-36 Compared with (SEQ ID NO: 2), the binding affinity to hNPY5, hNPY4 and hNPY1 receptors is reduced.

[0180] (2) In vitro cAMP activity on human NPY2 receptor

[0181] Purpose:

[0182] The PYY analogs of Examples 1 to 5 were determined to be comparable to native human PYY by measuring the inhibition of forskolin-induced intracellular cAMP production in HEK 293 cells overexpressing recombinant human NPY2 receptor. 3-36 Compared with the in vitro functional activity.

[0183] method:

[0184] PYY analogs and human PYY were synthesized, characterized and stored as described above in the receptor binding assay. 3-36 (SEQ ID NO:2).

[0185] Receptor Cloning - Stable transfected cells of the hNPY2 receptor were prepared by subcloning the receptor cDNA into the pcDNA3.1 expression plasmid and transfecting it into HEK293 cells followed by selection with geneticin. Aliquots of cells at passage 9 (1 x 10 7 Cells / mL) and kept frozen in the vapor phase of a liquid nitrogen tank. These frozen aliquots were used in the assays. The cells maintained greater than 95% viability over several months.

[0186] hNPY2R cAMP assay – using HEK 293 cells overexpressing recombinant hNPY2R to measure PYY analogs or PYY 3-36Inhibition of forskolin-induced cAMP production. Frozen cell aliquots were thawed in a 37°C water bath. Cells were transferred to 50 mL tubes 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), 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 tabletop centrifuge. The supernatant was removed and the cell pellet was resuspended in 10 mL of cell culture medium and then passed through a 40 µm filter. The accurate counting of cell number and cell viability was determined using the Vi-Cell analyzer (Vi-Cell XR 2.03) from Beckman-Coulter. 8000 cells per well were inoculated into a white 384-well assay plate (Corning, poly-D-lysine coated, white / opaque, catalog number (Cat. No.) 356661) using a Combi-Tip dispenser (Thermo Scientific). Plates were centrifuged for 1 second at 1000 rpm, and incubated at 37°C in a 5% CO2-controlled incubator for 18 to 20 hours. Culture medium was removed from the assay plate by patting on a paper towel. 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 SupplyCorp., #440203H), 500 µM IBMX (Sigma-Aldrich #I5876)] was added to the wells using a Combi-Tip dispenser, followed by centrifugation at 1500 rpm for 10 seconds. A concentration response curve (20 points) of 2-fold dilutions was prepared in 100% DMSO using acoustic dispensing technology (Labcyte Echo 550). Cells were incubated at 37°C with PYY analogs or human PYY 3-36 Treatment was performed for 45 min (final DMSO concentration = 1%), followed by stimulation with 1 µM forskolin (Sigma-Aldrich, #F6886) for 45 min at 37°C. iIntracellular cAMP was quantified using the kinetic kit (#62AM9PEB). Briefly, HTRF kit reagents were used to quantify intracellular cAMP by adding cAMP-d2 conjugate in cell lysis buffer (10 μL), followed by the addition of antibody anti-cAMP-Eu also in cell lysis buffer (10 μL). 3+ -Cryptate, to detect intracellular cAMP levels. The resulting competitive assay was incubated at room temperature for at least 60 minutes and then read on a PerkinElmer Envision™ instrument with excitation at 320 nm and emission at 665 nm and 620 nm. The final assay concentration range of the peptides tested in the response curve was: PYY analogs (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 assay buffer. Include wells on each plate with no competitor added or with excess human PYY added. 3-36 The wells with were used as maximum response and inhibitor control, respectively.

[0187] Data Analysis of hNPY2 Receptor cAMP Assay – Time-resolved fluorescence emission was used to calculate the fluorescence ratio (665 nM / 620 nm), which is inversely proportional to the amount of cAMP present. PYY analogs and human PYY were compared 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). 3-36 The signal was converted to nM cAMP per well.

[0188] 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:

[0189] ,

[0190] The inhibitor control was an excess of human PYY added 3-36 The maximum response is the cAMP produced in the presence of forskolin alone, and the maximum response is the cAMP produced in the presence of the test peptide.

[0191] The specific inhibition percentage (y-axis) was plotted against the concentration of competitor (x-axis) and the four parameters (top of curve, bottom of curve, IC 50 , Hill slope) nonlinear regression routine (Genedata Screener, version 13.0.5, Genedata AG, Basal, Switzerland):

[0192] .

[0193] Relative IC 50 Values ​​represent the concentrations causing 50% inhibition of forskolin-induced cAMP production.

[0194] Reported IC 50 The values ​​are calculated as geometric means as follows:

[0195] .

[0196] The standard error of the mean (SEM) was calculated using the Δ method as follows:

[0197]

[0198] where SD is the standard deviation, n is the number of independent runs, and ln(10) is the natural logarithm of 10.

[0199] result:

[0200] Table 2: In vitro cAMP activity on hNPY2 receptor

[0201]

[0202] *Known PYY analogs for comparison; see, International Patent Application Publication No. WO 2016 / 198682, wherein 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.

[0203] As shown above, the results from the cAMP assay confirmed the functional activity of the PYY analogs of Examples 1 to 5 on the hNPY2 receptor, with Example 2 showing the weakest potency, which was less potent than human PYY 3-36 12 times lower.

[0204] (3) In vitro GTPγS activity on hNPR2 receptor

[0205] Purpose:

[0206] Receptor-mediated activation of G-proteins was assessed by the PYY analogs of Examples 1 to 5. The non-hydrolyzable GTP analog GTPγ[ 35 S] measures receptor-mediated activation. Agonist-mediated stimulation of G-protein coupled receptors results in 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 assays were performed to evaluate the potency of various PYY analogs at the hNPY2 receptor.

[0207] method:

[0208] PYY analogs, human PYY 1-36 (SEQ ID NO: 1) and control peptide PYY 3-36 (SEQ ID NO:2).

[0209] For each test peptide, a concentration response curve (CRC) of 1 / 3 log dilution (log concentration -6.52 to -12.52) was completed using a Hamilton NIMBUS liquid handler in assay buffer (20 mM HEPES (pH 7.4), 100 mM NaCl, 6 mM MgCl2, 1 mM EDTA) supplemented with 0.2% bacitracin (USBiologicals #11805) and 0.5% DMSO. The final assay concentrations of bacitracin and DMSO were 0.05% and 0.125%, respectively. hNPY2R membranes (Multispan #HTS066M) were 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 incubated at room temperature for 20 minutes before addition to the assay. GTPγS[ 35 S] (PerkinElmer #NEG030H) was prepared at a concentration of 0.6 nM. WGA SPA beads (PerkinElmer #RPNQ0001) were prepared at a concentration of 12 mg / mL in assay buffer. The beads were prepared by first adding 100 uL to the hNPY2R membrane, followed by 50 uL of CRC solution, and then 50 uL of GTPγS[ 35 The assay was performed in a 96-well plate (Costar #3604) with 10% SPA beads and a final volume of 200 uL. The plate was covered and placed on an orbital shaker (175 rpm, 45 minutes) at room temperature. Then, 25 uL of SPA beads were added and the plate was resealed and vortexed to mix, then placed back on the orbital shaker for 3 hours at room temperature. The plate was then centrifuged at 500 rpm for 5 minutes and counted for 1 minute per well on a PerkinElmer 2450 microplate reader. 3-36 Basal binding (CPM) was determined in the absence of PYY and used to calculate the percentage above basal value for each concentration of peptide using the following equation: (PYY analog or human PYY 3-36 , CPM – Basic CPM) / (Basic CPM)*100. EC 50(nM) values ​​were determined by nonlinear regression analysis (log(agonist) vs. response - variable slope (four parameters)) of the logarithm of the concentration and the percentage of the basal value in GraphPad Prism 7.0 using the following equation: Y = bottom + (top - bottom) / (1 + 10^((LogEC 50 The column statistics function in GraphPad Prism 7.0 was used to calculate the slope of the EC 50 (nM) Values ​​were used to calculate the geometric mean and standard error of the mean.

[0210] result:

[0211] Table 3: In vitro GTPyS activity at the hNPY2 receptor.

[0212]

[0213] *Known PYY analogs for comparison; see, International Patent Application Publication No. WO 2016 / 198682, wherein 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.

[0214] As shown above, from GTPγ[ 35 The results of the functional assay confirmed the functional activity of PYY analogs on the hNPY2 receptor, among which Example 2 showed the weakest potency, with a potency lower than that of human PYY 3-36 4 times lower.

[0215] (4) Pharmacokinetics

[0216] Purpose:

[0217] To study the pharmacokinetic properties of PYY analogs.

[0218] method:

[0219] LC / MS – Plasma concentrations of various PYY analogs were determined by an LC / MS method. The method measures the bulk compound; peptides are linked for extended periods of time. For the assay, 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 ThermoProtein Precipitation Plate. The samples were loaded onto a Sep-Pak tC18 SPE µelution plate conditioned with methanol and 0.1% formic acid in water. The SPE column was washed twice with 0.1% formic acid in water. The compounds were then eluted using formic acid / water / acetonitrile (0.1:15:85), which was then dried and reconstituted before an aliquot (10 µL) was injected onto a ThermoAcclaim 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.

[0220] Pharmacokinetics of PYY Analogs in CD-1 Mice – The plasma pharmacokinetics of PYY analogs were evaluated in male CD-1 mice following a single subcutaneous dose of 200 nmol / kg. 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 present the pharmacokinetic parameters of PYY analogs following a single subcutaneous dose of 200 nmol / kg. Plasma concentrations of PYY analogs were monitored for 120 hours following a single subcutaneous administration of 200 nmol / kg.

[0221] Pharmacokinetics of PYY Analogs in SD Rats – The plasma pharmacokinetics of PYY analogs were evaluated in male Sprague Dawley rats following a single subcutaneous dose of 50 nmol / kg. Blood samples were collected from 2 animals at each time point over 168 hours. Since serial sampling was used to evaluate the kinetics of PYY analogs in rats, single animal concentration versus time data were used to present the pharmacokinetic parameters of PYY analogs following a single subcutaneous dose of 50 nmol / kg. Plasma concentrations of PYY analogs were monitored for 120 hours following a single subcutaneous administration of 50 nmol / kg.

[0222] Pharmacokinetics of PYY Analogs in Cynomolgus Monkeys – The plasma pharmacokinetics of PYY analogs were evaluated in male and female cynomolgus monkeys following a single subcutaneous dose of 50 nmol / kg. Blood samples were collected over 504 hours. Since serial sampling was used to evaluate the kinetics of PYY analogs in monkeys, individual animal concentration versus time data were used to present the pharmacokinetic parameters of PYY analogs following a single subcutaneous dose of 50 nmol / kg. Plasma concentrations of PYY analogs were measured for 504 hours following a single subcutaneous administration of 50 nmol / kg.

[0223] result:

[0224] Table 4: Mean pharmacokinetic parameters following a single subcutaneous administration to male CD-1 mice.

[0225]

[0226] Abbreviation: AUC inf = area under the curve from 0 to infinity; CL / F = clearance divided by bioavailability (F); Cmax = maximum concentration; Tmax = time at maximum concentration; T1 / 2 = half-life.

[0227] Table 5: Mean pharmacokinetic parameters following a single subcutaneous administration to male SD rats.

[0228]

[0229] Abbreviation: AUC inf = area under the curve from 0 to infinity; CL / F = clearance divided by bioavailability (F); C max = maximum concentration; T max = time at maximum concentration; T1 / 2 = half-life.

[0230] Table 6: Mean pharmacokinetic parameters following a single subcutaneous dose to cynomolgus monkeys.

[0231]

[0232] Abbreviation: AUC inf = area under the curve from 0 to infinity; CL / F = clearance divided by bioavailability (F); C max = maximum concentration; T max = time at maximum concentration; T1 / 2 = half-life.

[0233] result:

[0234] These data demonstrate that the above compounds have a pharmacokinetic profile suitable for once-weekly administration.

[0235] (5) Solubility and stability

[0236] Purpose:

[0237] Determining the pH range of solubility and stability of PYY analogs

[0238] method:

[0239] Visual Solubility Range Assessment - Lyophilized PYY analog powder was reconstituted in water at a concentration of 4 mg / mL and the pH was adjusted with citric acid / phosphate buffer to pH 4. The pH of the system was titrated up to pH 8 with 0.5 N NaCl and then titrated down to pH 4 with 0.5 N HCl.

[0240] Thermal Stability Assessment – ​​Solutions of PYY analogs in 10 mM or 20 mM sodium phosphate (pH 7.0) were prepared at a concentration of 1 mg / mL or 2 mg / mL and incubated for 4 weeks at 4°C and 40°C. Samples at the 4-week time point were analyzed by size exclusion chromatography (SEC) and RP-HPLC.

[0241] SEC Method – was performed using a TOSOH TSKgelG2000SWxl, 7.8 mm ID x 30 cm, 5 um column with a mobile phase composition of 50 mM sodium phosphate, 300 mM NaCl (pH 7.0) with 20% acetonitrile over 30 minutes at a flow rate of 0.5 mL / min, λ – 214 nm.

[0242] RP Method – Using a Cortecs C18, 2.7 um, 4.6 x 50 mm column, 20%-45% acetonitrile / water with 0.085% TFA over 10 minutes, flow rate 1 mL / min, λ-214 nm.

[0243] result:

[0244] Table 7: Solubility and thermal stability of PYY analogues.

[0245]

[0246] As shown above, all peptides were soluble at pH > 7.0. Stability as assessed by RP and SEC indicated that these peptides were relatively stable under severe thermal stress.

[0247] Example 7: In vivo effects of PYY.

[0248] (1) In vivo effects on food intake and body weight in normal mice

[0249] Purpose:

[0250] The PYY analogs of Examples 1 to 5 were compared for their effects of reducing body weight and inhibiting food intake in normal mice after a single injection.

[0251] method:

[0252] Male C57Bl / 6 mice from Envigo RMS (Indianapolis, IN) were maintained on a chowdiet (5008; LabDiet, St. Louis, MO) and housed individually in a temperature-controlled facility (74.0°F; 23.3°C) with a normal 12:12 hour lighting cycle and free access to food and water. At 9 to 10 weeks of age, unfasted body weight and initial food weight were recorded, and animals were given a single subcutaneous injection of vehicle or peptide, followed by daily measurements of body weight and food intake for 3 days post-dose. Body weight and food intake were calculated relative to the area under the curve analysis (AUC) of vehicle. In each assay run, 30 nmol / kg of Example 4 was used as a benchmark for 100% efficacy of body weight and food intake.

[0253] result:

[0254] Table 8: Changes in body weight and food intake in C57 / Bl6 mice over 3 days after a single administration of PYY analogs.

[0255]

[0256] *Known PYY analogs for comparison; see, International Patent Application Publication No. WO 2016 / 198682, wherein 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.

[0257] ** Example 4 of 30 nmol / kg (AUC) was set as 100% efficacy

[0258] As shown above, reductions in both body weight and food intake demonstrate the in vivo efficacy of the PYY analogues, with comparison of the doses required for full efficacy demonstrating an increase in efficacy.

[0259] (2) In vivo effects on food intake and body weight in diet-induced obese mice

[0260] Purpose:

[0261] The effects of daily administration of the PYY analogs of Examples 1-5, alone or in combination with a GLP-1 receptor agonist, on reducing body weight over a two week period were studied in diet-induced obese (DIO) mice.

[0262] method:

[0263] 20-week-old DIO male C57Bl / 6 mice (Taconic) were maintained on a 60% fat diet (D12492; Research Diets, New Brunswick, NJ) upon arrival. Animals were housed individually 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 of daily vehicle administration, non-fasting body weights were measured, and animals were randomized into experimental groups (n=6) based on body weight and administered daily subcutaneous injections of vehicle, GLP-1 receptor agonist (GLP-1 RA; SEQ ID NO: 15), PYY analogs, or a combination of PYY analogs plus GLP-1RAs. After 2 weeks of dosing, non-fasting body weights were recorded and the mean body weight change compared to vehicle was calculated. To determine the additive or synergistic effects of the combination of PYY analogs and GLP-1 RAs, the efficacy (net effect) was calculated to be higher than that of GLP-1 RA alone.

[0264] result:

[0265] Table 9: Body weight changes in a 2 week study with PYY analogues alone or in combination with a GLP-1 receptor agonist in diet-induced obese mice.

[0266]

[0267] *Higher potency than GLP-1 receptor agonists (GLP-1 RAs).

[0268] As shown above, the in vivo efficacy of PYY analogues was demonstrated by the reduction in body weight with PYY analogues alone and in combination with a GLP-1 RA, wherein the magnitude of the weight reduction was compared.

[0269] (3) In vivo effects on body weight and glucose in diabetic and obese (db / db) mice

[0270] Purpose:

[0271] The effects of daily administration of the PYY analogs of Examples 1 to 5 over a ten-day period on reducing body weight and blood glucose levels were studied in obese and diabetic mice (db / db).

[0272] method:

[0273] Lepr from Envigo RMS (Indianapolis, IN) db / db(db / db) male mice 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 lighting cycle and free access to food and water. At 8 to 9 weeks of age, body weight and blood glucose levels were measured using an Accu-Check® glucometer (Roche Diabetes Care, Inc., Indianapolis, IN) followed by daily subcutaneous injections of vehicle or peptide. After 10 days of dosing, body weight and blood glucose levels were measured and changes compared to vehicle treatment were calculated.

[0274] result:

[0275] Table 10: Effects on body weight and blood glucose in db / db mice treated for 10 days.

[0276]

[0277] As shown above, the in vivo efficacy of PYY analogs was demonstrated by reducing body weight and blood glucose levels with PYY analogs, wherein the magnitude of body weight reduction and glucose reduction was compared.

[0278] 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, and a dose-dependent improvement in blood glucose in db / db mice, with the PYY analogs of Examples 1, 3 and 4 being the most potent, consistent with the in vitro profiles.

[0279]

[0280]

[0281] Sequence Listing <110> Eli Lilly and Company <120> Protein tyrosine-tyrosine analogs and methods of use thereof <130> X21917 <160> 15 <170> PatentIn version 3.5 <210> 1 <211> 36 <212> PRT <213> Homo sapiens <400> 1 Tyr Pro Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu 1 5 10 15 Leu Asn Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr 20 25 30 Arg Gln Arg Tyr 35 <210> 2 <211> 34 <212> PRT <213> Homo sapiens <400> 2 Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 3 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MISC_FEATURE <222> (5) <223> Xaa at position 5 is any amino acid having a functional group available for conjugation and the functional group is conjugated to a C16-C22 fatty acid <220> <221> MISC_FEATURE <222> (7) <223> Xaa at position 7 is E or G <220> <221> MISC_FEATURE <222> (8) <223> Xaa at position 8 is E or K <220> <221> MISC_FEATURE <222> (15) <223> Xaa at position 15 is L or W <220> <221> MISC_FEATURE <222> (16) <223> Xaa at position 16 is N or Q <220> <221> MISC_FEATURE <222> (20)..(20) <223> Xaa at position 20 is A or I <220> <221> MISC_FEATURE <222> (21)..(21) <223> Xaa at position 21 is E, D, or S <220> <221> MISC_FEATURE <222> (28)..(28) <223> Xaa at position 28 is E or W <400> 3 Pro Lys Pro Glu Xaa Pro Xaa Xaa Asp Ala Ser Pro Glu Glu Xaa Xaa 1 5 10 15 Arg Tyr Tyr Xaa Xaa Leu Arg His Tyr Leu Asn Xaa Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 4 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 4 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ala Glu Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 5 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 5 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ala Glu Leu Arg His Tyr Leu Asn Glu Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 6 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 6 Pro Lys Pro Glu Lys Pro Glu Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ile Glu Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 7 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 7 Pro Lys Pro Glu Lys Pro Gly Lys Asp Ala Ser Pro Glu Glu Trp Asn 1 5 10 15 Arg Tyr Tyr Ala Asp Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 8 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <400> 8 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Gln 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 9 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (5) <223> The Lys at position 5 was chemically modified by conjugating the ε-amino group of the Lys side chain with Ahx-glutamic acid-γglutamic acid-CO-(CH2)16-CO2H <220> <221> MOD_RES <222> (34)..(34) <223> Amidation <400> 9 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ala Glu Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 10 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (5) <223> The Lys at position 5 was chemically modified by conjugating the ε-amino group of the Lys side chain with (AEEA)2-γ-glutamic acid-CO-(CH2)18-CO2H <220> <221> MOD_RES <222> (34)..(34) <223> Amidation <400> 10 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ala Glu Leu Arg His Tyr Leu Asn Glu Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 11 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (5) <223> The Lys at position 5 was chemically modified by conjugating the ε-amino group of the Lys side chain with AEEA-γ-glutamic acid-CO-(CH2)16-CO2H <220> <221> MOD_RES <222> (34)..(34) <223> Amidation <400> 11 Pro Lys Pro Glu Lys Pro Glu Glu Asp Ala Ser Pro Glu Glu Trp Gln 1 5 10 15 Arg Tyr Tyr Ile Glu Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 12 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (5) <223> The Lys at position 5 was chemically modified by conjugating the ε-amino group of the Lys side chain with (AEEA)2-γ-glutamic acid-CO-(CH2)16-CO2H <220> <221> MOD_RES <222> (34)..(34) <223> Amidation <400> 12 Pro Lys Pro Glu Lys Pro Gly Lys Asp Ala Ser Pro Glu Glu Trp Asn 1 5 10 15 Arg Tyr Tyr Ala Asp Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 13 <211> 34 <212> PRT <213> Artificial sequence <220> <223> Synthetic constructs <220> <221> MOD_RES <222> (5) <223> The Lys at position 5 was chemically modified by conjugating the ε-amino group of the Lys side chain with (AEEA)2-(γ-glutamic acid)3-CO-C18-CO2H <220> <221> MOD_RES <222> (34)..(34) <223> Amidation <400> 13 Pro Lys Pro Glu Lys Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Gln 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Trp Leu Thr Arg Gln 20 25 30 Arg Tyr <210> 14 <211> 36 <212> PRT <213> Homo sapiens <400> 14 Ala Pro Leu Glu Pro Val Tyr Pro Gly Asp Asn Ala Thr Pro Glu Gln 1 5 10 15 Met Ala Gln Tyr Ala Ala Asp Leu Arg Arg Tyr Ile Asn Met Leu Thr 20 25 30 Arg Pro Arg Tyr 35 <210> 15 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Construct <400> 15 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Glu 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly Gly 20 25 30 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser 35 40 45 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 50 55 60 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 65 70 75 80 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 85 90 95 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 100 105 110 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 115 120 125 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 130 135 140 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 145 150 155 160 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 165 170 175 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 180 185 190 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 195 200 205 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 210 215 220 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 225 230 235 240 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 245 250 255 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 260 265 270 Leu Gly

Claims

1. A peptide tyrosine-tyrosine (PYY) analogue comprising the following amino acid sequence: PKPEX7PX9X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY (Formula I), Where X7 is conjugated to C 16 -C 22 The fatty acid lysine, wherein the C 16 -C 22 The fatty acid is conjugated to lysine via a linker, wherein the linker may be one or more units selected from [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminocaproic acid (Ahx), glutamic acid (E), gamma glutamic acid (γE), and combinations thereof, Where X9 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 PYY analogue is (SEQ ID NO:9), or wherein the PYY analogue is (SEQ ID NO: 10), or wherein the PYY analogue is (SEQ ID NO: 11), or wherein the PYY analogue is (SEQ ID NO: 12), or wherein the PYY analogue is (SEQ ID NO: 13).

2. The PYY analogue of claim 1, wherein the PYY analogue has a charge greater than -2.

3. The PYY analogue of claim 1 or claim 2, wherein the PYY analogue has a greater binding affinity at the NPY2 receptor than PYY 3-36 (SEQ ID NO: 2).

4. The PYY analogue of claim 1 or claim 2, wherein the half-life of the PYY analogue is greater than that of human PYY 3-36 The half-life of (SEQ ID NO:2).

5. A pharmaceutical composition comprising: At least one peptide tyrosine-tyrosine (PYY) analogue or a salt thereof according to any one of claims 1 to 4; and One or more pharmaceutically acceptable carriers, diluents and excipients.

6. The pharmaceutical composition of claim 5, further comprising an additional therapeutic agent.

7. The pharmaceutical composition of claim 6, wherein the additional therapeutic agent is selected from the group consisting of glucagon (GCG), a GCG analog, glucagon-like peptide-1 (GLP-1), GLP-1 7-36-酰胺 , GLP-1 analogs, gastric inhibitory peptide (GIP), GIP analogs, oxyntomodulin (OXM), OXM analogs, incretins of GIP / GLP-1, GLP-1 / GCG, or incretin analogs with triple receptor activity.

8. The pharmaceutical composition of claim 6, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

9. Use of a peptide tyrosine-tyrosine (PYY) analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 in the preparation of a medicament for use in a method for treating obesity or an obesity-related disease or condition, wherein the method comprises the following steps: An effective amount of a peptide tyrosine-tyrosine (PYY) analogue according to any one of claims 1 to 4 or a pharmaceutically acceptable salt thereof is administered to an individual in need thereof.

10. The use according to claim 9, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered subcutaneously (SQ) to the individual.

11. The use according to claim 9, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered orally to the individual.

12. The use according to claim 10 or 11, 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.

13. The use of claim 9, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is administered once a week (QW) SQ.

14. The use according to claim 9, wherein the PYY analogue or a pharmaceutically acceptable salt thereof is orally administered once a week.

15. The use of any one of claims 9 to 11 and 13 to 14, further comprising administering an additional therapeutic agent.

16. The method of claim 15, wherein the additional therapeutic agent is selected from the group consisting of glucagon (GCG), a GCG analog, glucagon-like peptide-1 (GLP-1), GLP-1 7-36-酰胺 , GLP-1 analogs, gastric inhibitory peptide (GIP), GIP analogs, oxyntomodulin (OXM), OXM analogs, incretins of GIP / GLP-1, GLP-1 / GCG, or incretin analogs with triple receptor activity.

17. The use of claim 15, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

18. Use of a peptide tyrosine-tyrosine (PYY) analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of obesity.

19. Use of a peptide tyrosine-tyrosine (PYY) analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of an obesity-related disease or disorder.

20. Use of a peptide tyrosine-tyrosine (PYY) analogue or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of type II diabetes.

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