Protein tyrosine-tyrosine analogs and methods for using them

Abstract

To provide PYY analogs having improved potency and selectivity for the NPY2 receptor, pharmaceutical compositions including one or more of the PYY analogs in a pharmaceutically acceptable carrier, and methods of using the PYY analogs, especially for treating obesity and obesity-related diseases and disorders such as type II diabetes mellitus.SOLUTION: The present invention provides a peptide tyrosine-tyrosine (PYY) analog including a specific amino acid sequence.SELECTED DRAWING: None

Description

Technical Field

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

Background Art

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

[0003] Plasma PYY 3-36 concentration typically increases within 15 minutes of food intake and reaches 60-9 ​​​​​​​​​​It peaks within 0 minutes and remains elevated for up to 6 hours before returning to baseline. (Adrian et al. (1985) Gastroenterology 89: 1070-1077; and De Silva & Bloom (2012) Gut Liv (See er 6:10-20). Thus, PYY 3-36 This is its direct central effect Furthermore, it is thought to affect appetite through its effect on intestinal motility (i.e., appetite-suppressing effect). It is available. Furthermore, PYY 3-36 It mediates insulin sensitivity, thereby affecting blood glucose. It is thought to help lower the value (i.e., its sensitization effect).

[0004] PYY 3-36 In light of its appetite-suppressing effect, it is used for weight control, especially for obesity and other conditions. Potential treatment for diseases and disorders related to (including T2DM and cardiovascular disease) It has been investigated as a therapeutic agent (for example, International Patent Application Publication No. 2002 / 47712, Schwartz & Morton (2002) Nature 418:595-59 See 7).

[0005] Unfortunately, due to proteases and other clearance mechanisms, exogenously administered P YY 3-36 It has a short half-life (for example, about 10-15 minutes) (Lluis et al.) 1989) Rev. Esp. Fisiol. 45:377-384; and Torang et al.(2016)Am.J.Physiol.Regul.Integr.C (See omp.Physiol.310:R866-R874), this is used as a therapeutic agent. We will present challenges when using this product. Because of its short half-life, in order to exert its therapeutic effect, PYY3-36 It needs to be administered at least once a day, but this is for individuals who need it. It is inconvenient for the body. Therefore, PYY 3-36 To extend the half-life, and / or Efforts have been made to increase the selectivity of the NPY2 receptor. For example, Rubi Nstein et al. suggested that 9-fluorecarbonylcal may be used to extend the half-life. Bonyl (Fmoc) or 2-sulfo-9-fluorenyl-methoxycarbonyl (FMS) This document describes a PYY analog having the radical of (International Patent Application Publication No. 2004 / 0892). (See issue 79). Furthermore, DeCarr et al. have shown that to increase the half-life, A PYY analog with a mino-terminal PEG moiety is described (DeCarr et al.). l.(2007)Bioorg.Med.Chem.Lett.17:1916-191 See 9; also Ortiz et al. (2007) J. Pharmacol. Exp. See also Ther. 323:692-700). Furthermore, Kofoed et al. , albumin-binding side chain, at least one modified residue near the cleavage site of PYY (e.g., eye Target amino acid residues (N-methyl amino acid analogs), N-glycine, and / or half-life This invention describes a PYY analog having an arginine mimic for increasing (International Patent Application). (See Publication No. 2011 / 033068).

[0006] PYY in metabolism 3-36 Despite a significant increase in understanding of the role, further PYY analogs, particularly PYY analogs with improved efficacy and selectivity in the NPY2 receptor. It is still needed. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] As mentioned above, additional PYY analogs are needed for therapeutic purposes. [Means for solving the problem]

[0008] To address this need, this disclosure first describes (natural human PYY 1-36 (array Regarding the numbering of number 1), the following PYY analogues containing the following base-amino acid sequence are described: 3 PKPEX7PX9X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYL NX 30 LTRQRY 36 (Formula I), where X7 is any functional group that can be used for bonding. It is an amino acid, and its functional group is C 16 ~C 22 It is bonded to a fatty acid, and X9 is either E or G. , X 10 is E or K, X 17 is L or W, X 18 is N or Q, X 22 is A or I, and X 23 is E, D or S, X 30 is E or W (Sequence ID 3), where the carboxyl-terminal (C-terminal) amino acid may be amidated. stomach. In certain cases, the amino acids that have functional groups available for joining at position X7 are C, D, and E. It can be K or Q. In certain cases, a functional group available for bonding at position X7 The amino acid is K, and the amino acid sequence can be one of the following: 3 PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY36 (Distribution Column number 4), 3 PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY 36 (Distribution Column number 5), 3 PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY 36 (Distribution Column number 6); 3 PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY 36 (Distribution Column number 7), or 3 PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY 36 (Distribution Column number 8)

[0009] In some examples, C 16 ~C 22 Functional groups that can be used for bonding via linkers in fatty acids It is joined to an amino acid that has C. In certain cases, 16 ~C 22 Fatty acids are -CO-(C H2) a -It has the structure CO2H, where a This is an integer between 16 and 22. In certain examples... Fatty acids include C1 such as palmitic acid, stearic acid, arachidic acid, or eicosanoic acid. 8 diacid or C 20 Diacids, especially saturated carbon dioxide. 18 Diacid or C 20 It is a diacid. Similarly, in some cases Therefore, the linker is [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEE A) Aminohexanoic acid (Ahx), glutamic acid (E), gamma-glutamic acid (γE) or can be one or more units of a combination thereof.

[0010] In certain cases, the PYY analogues may be one of the following: [ka] [ka]

[0011] In some cases, the base structures of the PYY analogs described herein are those of natural human PYY. 1-36 ( It can further contain the two amino-terminal (N-terminal) amino acids of SEQ ID NO: 1), which means that After that, PYY in vivo 3-36 Can be processed into an analogue (i.e., sequence number) The N-terminal "YP" residue of 1 is cleaved in vivo by one of the PYY analogs. obtain).

[0012] In some cases, the charge of PYY analogs exceeds -2, and in particular can be -3 or -4.

[0013] In some cases, PYY analogs are found in the human NPY2 receptor, 3-36 The binding affinity is greater than that of (Sequence ID 2), for example, about 2 to about 10 times greater, especially about 2 It has twice to approximately three times greater binding affinity.

[0014] In some cases, PYY analogs are human PYY 3-36 Longer half-life than (Sequence ID 2) It has a long half-life, for example, about 5 hours to about 24 hours, and especially about 12 hours.

[0015] Secondly, at least one PYY analog or a pharmaceutically acceptable salt thereof as specified herein ( For example, a trifluoroacetate, acetate, or hydrochloride salt and a pharmaceutically acceptable carrier. A pharmaceutical composition is described. In some cases, the pharmaceutical composition includes a carrier, diluent and / or It may further contain excipients.

[0016] Furthermore, the pharmaceutical composition may contain, for example, other antidiabetic drugs or weight-loss agents, particularly incretins. Additional therapeutic agents such as may be included. In some cases, incretins may contain glucagon ( It may be GCG or a GCG analog. In other examples, incretins are glucagon-like compounds. Petit-1 (GLP-1), GLP-1 (7-36) アミド , or a GLP-1 analogue It is possible. In other examples, incretins are gastric suppressor peptides (GIPs) or GIP analogues. It is possible. In other examples, incretins are oxytomodulin (OXM) or OXM Related compounds, such as GLP-1 / GCG or GIP / GLP-1, are dual receptor agonists. It is possible. In other examples, incretins are incretin analogs that have triple receptor activity ( In other words, incretions that have activity at the respective receptors of GIP, GLP-1, and GCG. It may be an analogue of the compound. In other cases, additional therapeutic agents may be DPP-IV inhibitors.

[0017] Thirdly, in this specification, methods using PYY analogues, particularly for obesity and obesity-related This document describes how to use PYY analogues to treat diseases and disorders (such as T2DM). This method provides at least an effective amount of P as described herein to the individual that requires it. The process includes administering a YY analog or a pharmaceutically acceptable salt thereof.

[0018] In some cases, PYY analogues can be administered subcutaneously (SQ) to individuals. Similarly, In some cases, PYY analogues are administered daily, every other day, three times a week, twice a week, or once a week (i.e., every It can be administered weekly, every other week, or monthly. In certain cases... PYY analogs are administered every other day in SQ, three times a week in SQ, twice a week in SQ, and once a week in SQ. It can be administered every week with SQ, or once a month with SQ. In certain cases, P YY analogues are administered once a week via SQ (QW).

[0019] Alternatively, PYY analogs can be administered orally to individuals. As mentioned above, PYY analogs The frequency of visits is daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every week). It can be administered every other week or monthly. In certain cases, PYY analogues may be administered orally. Orally every other day, three times a week, twice a week, once a week, or every other week. Alternatively, it can be administered orally once a month. In certain cases, PYY analogs are administered orally once a week. It will be administered.

[0020] The method also involves an effective amount of additional therapeutic agents such as DPP-IV inhibitors or incretins. For example, GCG or GCG analogues, GLP-1, GLP-1(7-36) アミド if This includes GLP-1 analogs, GIP or GIP analogs, OXM or OXM analogs, G (IP / GLP-1, GLP-1 / GCG, or incretins with triple receptor activity) This may include administering at least one PYY analog in combination with DPP-I. V inhibitors or incretins may be administered simultaneously with, separately from, or sequentially with PYY analogs. It is possible.

[0021] In some cases, DPP-IV inhibitors or incretins are administered at the same frequency as PYY analogs. (That is, it can be administered every other day, twice a week, or once a week.) In other cases, D PP-IV inhibitors or incretins are administered at different frequencies than PYY analogs. In some cases, DPP-IV inhibitors or incretins are administered during the QW phase. In yet another case, PYY analogues are administered via SQ, and DPP-IV inhibitors or incretins are administered orally. It is possible.

[0022] In some cases, the individual is obese or overweight. In other cases, the individual has diabetes (Pw D), especially in people with T2DM. In certain cases, individuals are obese with T2DM or T2D Size M is too fat.

[0023] The method also includes the individual's body weight and / or blood glucose and / or hemoglobin A1c(H Measure or obtain bA1c) and obtain one or more baseline values ​​such obtained values. This may include steps such as evaluating the effectiveness of the treatment by comparing it with previously obtained values.

[0024] These methods can also be combined with diet and exercise, and / or It can be combined with additional therapeutic drugs other than those discussed above.

[0025] Fourth, regarding PYY analogs, obesity and obesity-related diseases such as T2DM and The use of this drug in the treatment of disorders is described herein, and this may optionally involve DPP-IV inhibition. Harmful agents and / or GCG or GCG analogs, GLP-1, GLP-1(7-36) アミド Alternatively, GLP-1 analogues, GIP or GIP analogues, OXM or OXM It has analogues, GIP / GLP-1, GLP-1 / GCG, or even triple receptor activity. Incretins such as incretins, simultaneously, separately or sequentially (i.e., in combination) (They may be administered together.)

[0026] Fifth, regarding PYY analogs, obesity and obesity-related diseases such as T2DM and The use of a drug for the manufacture of a drug for the treatment of a disorder is described herein, and in this case, the drug is Optionally, DPP-IV inhibitors and / or GCG or GCG analogs, GLP-1 GLP-1(7-36) アミド Alternatively, GLP-1 analogues, GIP, or GIP analogues. Body, OXM or OXM analogues, GIP / GLP-1, GLP-1 / GCG, or These may further include incretins such as incretins having triple receptor activity. [Effects of the Invention]

[0027] One advantage of the PYY analogues described herein is that they can promote weight loss. Not only can it do this, but it can also lower glucose levels. In this way, Individuals that are susceptible to or already suffering from T2DM may have exogenous insulin It can slow the progression to phosphorus and maintain the target HbA1c level. Furthermore, the PYY analogs described herein improve insulin sensitization, thereby controlling blood glucose levels. Control can be enhanced. When combined, GIP / GLP-1 and PYY analogues Glucose control (incretins + potential insulin sensitizers) and weight loss (synergistic effect) It can be used for both. In particular, the PYY analogues herein can be administered to individuals that require it. If used alone, it can cause a weight loss of up to approximately 12%, and for individuals that need it... When administered, it can lead to a weight loss of up to approximately 25% in conjunction with additional therapeutic agents such as incretins. It can be caused.

[0028] Another advantage of the PYY analogues described herein is that they have a half-life of up to approximately 24 hours. This allows for administration once a week.

[0029] Another advantage of PYY analogs in this specification is that they are similar to natural human PYY. 3-36 (Sequence 2) Compared to natural human PYY, it offers improved physicochemical stability and compatibility. 3-3 Compared to 6 (SEQ ID NO: 2), the suitability for formulations containing incretins was improved. That is the case. [Modes for carrying out the invention]

[0030] Unless otherwise defined, all technical and scientific terms used herein are: This disclosure has the same meaning as that generally understood by those skilled in the art in which this disclosure pertains. Any method and materials similar or equivalent to those described in the specification, PYY analogues, medical It can be used in the implementation or testing of pharmaceutical compositions and methods, but preferred methods and The materials are as described herein.

[0031] Furthermore, references to elements using the indefinite article "a" or "an" are in sentences. Unless explicitly required, pulses can have multiple elements. The possibility of this cannot be ruled out. Therefore, the indefinite article "a" or "an" is , usually means "at least one".

[0032] definition

[0033] As used herein, "approximately" refers to, for example, the stated concentration, length, molecular weight, pH. , the statistically meaningful values ​​of sequence identity, time frame, temperature, volume, etc. (there may be multiple values) It means within a certain range. Such a value or range is typically 20 of a given value or range. Within a range of size of %, more typically within 10%, and even more typically within 5% It is possible. The permissible variation encompassed by "approximately" depends on the specific system in the study, and This can be easily understood by the contractor.

[0034] As used herein, "amino acid" means, from a chemical standpoint, one or more amine groups This refers to a molecule characterized by the presence of one or more carboxylic acid groups and potentially containing other functional groups. As is known in the field, it is designated as a standard amino acid and is used by all living organisms. 20 amino acids that are used as building blocks for most peptides / proteins produced A set of no acids exists.

[0035] As used herein, "amino acids having functional groups available for joining" means, for example, Any natural or non-natural amino acid having a functional group that can be bonded to a fatty acid by a linker. It means 'no acid'. Examples of such functional groups include alkynyl groups, alkenyl groups, and amino acids. Examples include nitrate groups, azide groups, bromo groups, carboxyl groups, chloro groups, iodine groups, and thiol groups. Examples of natural amino acids containing such functional groups include, but are not limited to, these. As for C (thiol), D (carboxyl), E (carboxyl), K (amino) One example is Q (amide).

[0036] As used herein, “analog” means a substance that activates a target receptor and enhances the natural properties of that receptor. At least one in vivo or in vitro agonist-induced This refers to compounds such as synthetic peptides or polypeptides that induce an effect.

[0037] As used herein, "appetite-suppressing effect" means reducing appetite and consequently reducing food intake. This refers to the ability of the PYY analogues herein to reduce intake and ultimately lead to weight loss. The appetite-suppressing effect also refers to the ability of the PYY analogs herein to increase intestinal motility. In some cases, it may be the case.

[0038] When used herein, "C 16 ~C 22 "Fatty acids" are molecules with 16 to 22 carbon atoms. This means a carboxylic acid having C. 16 ~C 22 Fatty acids are saturating It can be a monoacid or a saturated diacid ("diacid" has a carboxyl group at each terminal).

[0039] As used herein, "AUC" means Area Under the Curve.

[0040] As used herein, “effective amount” refers to a single or multiple doses to an individual requiring it. A single dose will produce the desired effect in such an individual undergoing diagnosis or treatment (i.e., for example) For example, individual results may include a decrease in blood sugar, a decrease in HbA1c, and / or a decrease in weight or body fat. One or more of the following (which may produce clinically measurable differences in physical condition): This refers to the amount, concentration, or dose of PYY analogues or their pharmaceutically acceptable salts. The quantity is observed by using known methods and observing the results obtained under similar circumstances. Therefore, it can be easily determined by those skilled in the art. Determining the effective amount for an individual. In such cases, the species of mammal, its size, age, and overall health, as well as any specific diseases involved, should be considered. or the degree or severity of involvement of the disorder, disease or disability, the individual's response, the specific administration PYY analogues, mode of administration, bioavailability characteristics of administered preparations, and selected medications. This includes, but is not limited to, regimens, the use of concomitant medications, and other relevant circumstances. Numerous factors are considered.

[0041] When used in this specification, "median effective concentration" or "EC 50 " is a dose-response curve ( For example, it can lead to 50% activation / stimulation of assay endpoints such as cAMP. It refers to the concentration of the mixture.

[0042] As used herein, “in combination with” means at least one PY as defined herein. Y-related compounds, administered sequentially, simultaneously with, or with, one or more additional therapeutic agents. This means administering it as a single formulation combined with the other ingredients.

[0043] As used herein, "incretin analogues" refers to GIP, GLP-1, and GCG. and OXM, in particular, with natural human GIP, GLP-1, GCG and OXM respectively This refers to peptides or polypeptides that have structural similarities but also possess several differences from them. It tastes good. Some incretin analogs also affect GIP, GLP-1, and GCG receptors. Having affinity and activity in two or even each of them (i.e., OXM, Agonist activity at two receptors, such as GIP / GLP-1 or GLP-1 / GCG. , or even agonist activity at all three receptors.

[0044] As used herein, “the individual requiring it” means, for example, the individual listed herein. Having a condition, disease, disorder, or symptom that requires treatment or therapy, including those that are affected by This refers to mammals such as humans. In particular, humans are the preferred individuals to be treated.

[0045] As used herein, “long-acting” refers to the conjugating parent of a PYY analog. The compatibility and activity of natural human PYY 1-36 (Sequence ID 1) and / or natural human PYY 3-36 This means it lasts for a longer period than (Sequence ID 2), and at least 1 per day Administer once or even at a frequency of approximately three times a week, twice a week, once a week, or once a month. This makes it possible to do so. The time action profiles of PYY analogues in this specification are as follows: It can be measured using known pharmacokinetic testing methods, such as those used in the example.

[0046] As used herein, "non-standard amino acids" may occur naturally within cells, but are not necessarily the same as those that occur naturally within cells. This refers to amino acids that are not involved in peptide synthesis. Non-standard amino acids are components of peptides. There is a possibility, and often it is due to modification of standard amino acids within the peptide (i.e., (Produced through post-translational modification). Non-standard amino acids have the opposite properties to the standard amino acids mentioned above. It may contain D-amino acids that possess absolute chirality.

[0047] As used herein, "overweight" or "obese" means that an individual weighs 30.0 kg or more. / m 2 This refers to a state of having a body mass index (BMI) that is above average. This is available at cdc.gov / obesity / adult / defining.html Possible "Overweight & Obesity" by Centers for Disease Control and Prevention; and iddk.nih.gov / health-information / weight-m anagement / adult-overweight-obesity / defin Definitions&F See "Acts for Adult Overweight & Obesity".

[0048] As used herein, “obesity-related disease or disorder” includes angina pectoris, cardiovascular disease, Cholecystitis, gallstones, congestive heart failure, dyslipidemia, fatty liver disease, complications of infertility, impaired glucose tolerance Common conditions include gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, and osteoarthritis. Polycystic ovary syndrome (PCOS), pregnancy complications, mental disorders, sleep apnea, and others. Respiratory problems, stress urinary incontinence, stroke, T2DM, uric acid nephrolithiasis (kidney stones), This includes cancers of the breast, colon, endometrium, esophagus, gallbladder, kidney, prostate, and rectum, but this is not limited to cancers of the breast, colon, endometrium, esophagus, gallbladder, kidney, prostate, and rectum. This refers to any disease or disorder that is induced or aggravated by obesity, not limited to those mentioned above.

[0049] As used herein, "overweight" refers to an individual weighing 25.0 kg / m². 2 From 30 kg / m 2 This refers to a condition where the BMI is below a certain level. See ID.

[0050] As used herein, "PYY" means any mammalian species, particularly humans. PYY refers to the peptide YY obtained or derived therefrom. PYY stands for natural PYY (sand). (i.e., total length) and its variations (i.e., addition, deletion, and / or addition of natural PYY) Includes both (or substitution). Certain PYYs include natural human PYYs. 1-36 (Sequence 1) ) and natural human PYY 3-36 This includes, but is not limited to, (Sequence ID 2). .

[0051] As used herein, “PYY analog” (singular or plural) refers to the NPY2 receptor. PYY-like substances that induce one or more effects of native PYY in one or more NPY receptors, such as This refers to peptides or polypeptides. In some examples, the PYY analogs used herein are , binds to NPY receptors, particularly human NPY2 receptors, with higher or lower affinity. It is possible, but natural PYY, especially natural human PYY 1-36 (Sequence ID 1) and natural hyphae TopYY 3-36 When compared to human PYY (Sequence ID 2, etc.), in vivo and It can exhibit a longer half-life in vitro. Thus, according to this specification The PYY analogs are synthetic compounds that act as NPY2 receptor agonists.

[0052] As used herein, “saturated” means that the fatty acid has carbon-carbon double or triple bonds. It means that it does not include the combination.

[0053] As used herein, “sensitizing effect” means increasing the effect of insulin, and thereby This refers to the ability of the PYY analogues described herein to help lower blood sugar levels.

[0054] As used herein, “to treat, to heal” or “to heal, to treat” "In order to reduce, suppress, or mitigate the progression or severity of an existing condition, disease, disability, or symptom." It means to reverse, slow down, or stop.

[0055] Certain abbreviations are defined as follows: "ACR" stands for urinary albumin / urinary creatinine It refers to a ratio; "amu" refers to an atomic mass unit; "tBoc" refers to tert-butoxycarb "nil" refers to cyclic adenosine monophosphate; "cAMP" refers to cyclic adenosine monophosphate; "DMF" refers to dimethyl This refers to methylformamide; "DMSO" refers to dimethyl sulfoxide; "EIA / RI "A" refers to enzyme immunoassay / radioimmunoassay; "hr" refers to time; "HTRF" refers to homogenous time-resolved fluorescence; "IV" refers to intravenous fluorescence; "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 This refers to NG-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl. "RP-HPLC" refers to reverse-phase high-performance liquid chromatography; "SQ" refers to subcutaneous Refers to; "SEM" refers to the standard error of the mean; "TFA" refers to trifluoroacetic acid; "T "rt" refers to the tritil.

[0056] PYY related

[0057] The PYY analogues described herein are structurally similar to natural PYY peptides, but many There are structural differences. For example, natural human PYY 1-36 (Sequence ID 1) and / or Natural Human PYY 3-36 Compared to (Sequence ID 2), the PYY analogues described herein The body is made of natural human PYY 1-36Regarding the numbering of (SEQ ID NO:1), modifications at one or more of the 3rd, 7th, 9th, 10th, 17th, 18th, 22nd, 23rd, 30th and 31st positions are included. In certain examples, the exemplary amino acid sequences of PYY analogs herein include, among others: (specific changes relative to the corresponding residues of native human PYY (SEQ ID NO:1) are shown in bold): PKPEX7PX9X DASPEEX 3 X 10 RYYX 17 X 18 LRHYL 22 X 23 NX LTRQRPY 30 (SEQ ID NO:3) 3 PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY 36 (SEQ ID NO:4), (SEQ ID NO:5), 3 PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY 36 (SEQ ID NO:6), 3 PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY 36 (SEQ ID NO:7), and 3 PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY 36 (SEQ ID NO:8), and 3 PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY 36 (SEQ ID NO:9).

[0058] The PYY analogs herein exhibit sufficient activity at the human NPY2 receptor, but insufficient activity at the NPY 1, NPY4 and NPY5 receptors. Similarly, the PYY Related compounds improve solubility in aqueous solutions, enhance the stability of chemical and physical formulations, and contribute to pharmacokinetics. It as a therapeutic measure, including profile expansion and minimizing the potential for immunogenicity. It possesses beneficial attributes related to the potential for development.

[0059] In some cases, the PYY analogues herein may affect stability due to the C-terminal amine. It is amidated with an acid. In addition to the changes described herein, the analogs are hy Under the condition that it can bind to and activate the NPY2 receptor, It may include one or more additional amino acid modifications.

[0060] The PYY analogues described herein, for example, have functional groups available for joining via a linker. Fatty acids that are conjugated to natural or unnatural amino acids (i.e., "acylated") Includes. In some cases, amino acids with functional groups available for joining are C, D, E, K. It can be K. In certain cases, the amino acid that has a functional group available for conjugation is K. Here, the bonding is with respect to the ε-amino group of the K side chain.

[0061] Here, the acylation of PYY analogs is similar to that of natural human PYY. 1~36 Compare with (Sequence ID 1) In this case, it is in 7th place. In this way, fatty acids provide analogues that act for a longer period of time. It can act as an albumin-binding agent for that purpose.

[0062] Regarding fatty acids, they can be linked by either direct bonding or a linker, and are available for joining. It can be chemically bonded to the functional groups of amino acids. The length and composition of fatty acids are PYY Half-life of analogues, in vivo potency of PYY analogues, and dissolution of PYY analogues. It affects properties and stability. 16 ~C 22 Conjugation to saturated fatty acid monoacid or diacid is desired. Desired half-life, desirable efficacy in in vivo animal models, and desirable solubility. This yields PYY analogues exhibiting sexual and stability properties.

[0063] Exemplary saturated C for use in this specification 16 ~C 22 As for fatty acids, hexadecanoic acid (That is, palmitic acid, C 16 (C1), hexadecanoic acid (C1) 16 Diacid, heptadeca C acid (i.e., margaric acid, C 17 (C1 acid), heptadecanedioic acid) 17 Diacid), Ste C acid 18 (C monoacid), octadecane diacid (C) 18 Diacids), nonadecylic acid (i.e., Nonadecanoic acid, C 19 (C1 acid), nonadecanedioic acid) 19 Diacids), eicosanoic acid (i.e.) , arachidonic acid, C 20 (C2) eicosanoic acid (C2) (0 diacid), henicosanoic acid (i.e., henicosyl acid, C) 21 (monoacid), Hen'eikosa dioic acid (C 21 Diacids), docosanic acid (i.e., behenic acid, C 22 (monoacid), docosanedioic acid (C 22 Examples include, but are not limited to, diacides, and their branched and substituted derivatives. It is not done. In certain cases, C 16 ~C 22 Fatty acids are saturated C 18 monoacid, saturated C 18 Dioxide, saturated C 19 monoacid, saturated C 19 Diacid, saturated C 20 monoacid, saturated C 20Dioxides, and their Branched and substituted derivatives are possible. In certain examples, C 16 ~C 22 Fatty acids include palmitin. Acid or hexadeconic acid, stearic acid or Octadeconic acid, or arachidic acid or e It could be eicosanoic acid.

[0064] The process involves conjugating fatty acids to natural or unnatural amino acids that have functional groups available for conjugation. To assist this, the PYY analogues herein may include linkers. The linker is at least one of AEEA, Ahx, E, or γE, and also These combinations are possible.

[0065] If the linker contains amino acids, it can have 1 to 4 E or γE amino acid residues. In some cases, the linker can contain one or two E and / or γE amino acid residues. It can include a base. For example, a linker may have one or two E and / or γE It can contain any of the amino acid residues. In other examples, the linker is AEEA or 1 to 4 amino acid residues used in combination with Ahx (e.g., E or γE amino acids) It may contain acids, etc. Specifically, the linker contains E and γE amino acid residues and A It may be in combination with EEA or Ahx. In yet another example, the linker is one Alternatively, a combination of two γE amino acid residues and one or two AEEA or Ahx It is possible. In certain cases, the linker has the (AEEA)2·γE part and the Ahx·E·γE part. It may be a fraction or an AEA·γE portion.

[0066] An example of a linker fatty acid portion is 、 (AEEA)2·γE·C 20 Dioxide, Ahx· E·γE·C 18 Diacid, or AEEA·γE·C 18 Diacids are mentioned. Natural human pyropropyl alcohol (PY) Y 1-36 (Sequence ID 1) or natural human PYY 3-36 When compared with (Sequence ID 2) The structural characteristics of these linker fatty acid portions give rise to analogs with improved half-lives. ru.

[0067] In summary, the following are examples of PYY analogues: [ka] [ka]

[0068] PYY analogs are naturally occurring human PYY 3-36 34 A similar to (Sequence No. 2) Although it is stated that it contains mino acids, the PYY analogs described herein are natural human PYY 1- 36 It is intended that it may have an amino acid sequence based on (SEQ ID NO: 1). That is, PY Y-related organisms include naturally occurring human PYY 1-36 The two N-terminal amino acids of (Sequence No. 1) This can include the "YP" residues at positions 1 and 2 of sequence number 1, which then... Natural Human PYY 1-36 As happens when (Sequence ID 1) is released endogenously, When administered to the body, it can be cleaved in vivo.

[0069] The half-lives of the PYY analogs described herein include, for example, those described in the following examples: It can be measured using techniques known in the art. Similarly, various affinities of the PYY analogs herein for each of the human NPY receptors (e.g., NPY2R, NPY5R) can be measured using techniques known in the art, including, for example, those described in the following examples, and are generally represented as inhibition constant (K i) values. Further, the activity of the PYY analogs described herein at each of the receptors can also be measured using techniques known in the art, including, for example, the in vitro activity assays described below, and is generally represented as an EC 50 value. i As a result of the above modifications, the PYY analogs herein have a longer half-life than the half-life of native human PYY (SEQ ID NO: 2). For example, the PYY analog can have a half-life of 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 50 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 PYY analogs herein can have a half-life of about 5 hours, about 6 hours, about 7

[0070] <X hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 3-36 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, particularly about 12 hours.

[0071] ​​​​​​​​​Similarly, the PYY analogs of the present specification have, at the NPY2 receptor, a binding affinity greater than that of native human PYY 3- 36 (SEQ ID NO: 2), for example, a binding affinity of about 2-fold to about 10-fold Or, the PYY analogs of the present specification may have a binding affinity that 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 up to 10-fold greater than that of native human PYY 3-36 (SEQ ID NO: 2 ), especially a binding affinity that is 2-fold to 3-fold greater.

[0072] Pharmaceutical composition

[0073] The PYY analogs described herein can be formulated as a pharmaceutical composition for administration by a parenteral route (e.g., intravenous, intraperitoneal, intramuscular , subcutaneous, or transdermal). Such pharmaceutical compositions and the techniques for preparing them are well known in the art . For example, see Remington, "The Science and Practice of Pharmacy" (D.B. Troy ed., 21 Edition Lippincott, Williams & Wilkins, 2006 st ). In certain examples, the PYY analog is administered SQ. However, alternatively, the PYY analog can be formulated in a form for any other pharmaceutically acceptable route, such as, for example, tablets or other solids for oral administration; sustained release capsules ; and any other currently used forms, including creams, lotions, inhalants, etc . To improve their in vivo compatibility and effectiveness, the PYY analogs of the present specification

[0074] It reacts with any of several inorganic and organic acids / bases to form pharmaceutically acceptable acids / bases. It can form base addition salts. Pharmacologically acceptable salts and common methods for preparing them. The methodology is well known in the relevant technical field (for example, Stahl et al., "H andbook of Pharmaceutical Salts:Property es, Selection and Use, 2 nd Revised Edition (See Wiley-VCH, 2011). pharmaceutically acceptable for use herein. The salts used include sodium salts, trifluoroacetate salts, hydrochloride salts, and / or acetate salts. These are some examples.

[0075] The PYY analogues described herein are administered by a physician or self-administered by injection. It may be given. The gauge size and injection volume can be easily determined by those skilled in the art. It is understood. However, the injection volume is approximately 2 mL or less, and even approximately 1 mL or less, and needle g The size could be approximately 27GB or more, or even 29GB or more.

[0076] This disclosure also relates to the PYY analogs described herein, or their pharmaceutically acceptable salts. This specification provides, and therefore includes, novel intermediates and methods for synthesizing. Intermediates and PYY analogs were obtained using various methodologies well known in the art. It can be prepared by the following methods. For example, a method using chemical synthesis is illustrated in the following examples. To prepare the PYY analogues described herein, each of the described routes The specific synthesis steps can be combined in various ways. Reagents and starting materials are It is readily available to those skilled in the art.

[0077] The specific PYY analogs described herein are generally effective over a wide dosage range. Exemplary dosages of the PYY analogs or pharmaceutical compositions containing them herein are in milligrams (mg), micrograms (μg), nanograms (ng), or picograms (pg: picrogram) per kilogram (kg) of an individual. Thus, the daily dosage can be from about 1 μg to about 100 mg. Here, the effective amount of the PYY analog in the pharmaceutical composition can be in the dosage range 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., dosage / dosing amount) may be below the lower limit of the aforementioned range and yet be sufficient, while in other cases, the effective amount can be higher dosages and can be used with acceptable side effects.

[0078]

[0079] In addition to the PYY analogs, the pharmaceutical compositions can also contain additional therapeutic agents, particularly other anti-diabetic agents or weight loss agents. In some cases, the additional therapeutic agent can be at least one of an incretin or a DPP-IV inhibitor. Exemplary incretins include, but are not limited to, GCG, GLP-1, GLP-1(7-36), GIP, OXM, GCG analogs, GLP-1 analogs, GIP analogs, OXM analogs, GIP / GLP-1, GLP-1 / GCG, or incretin analogs having triple receptor activity. アミド

[0080] Thus, the pharmaceutical composition contains an effective amount of the PYY analog of SEQ ID NO: 9 and an incretin as well. Alternatively, DPP-IV inhibitors, effective doses of the PYY analog of Sequence ID No. 10, and incretins are also available. Alternatively, DPP-IV inhibitors, effective doses of the PYY analog of Sequence ID No. 11, and incretins are also available. Alternatively, DPP-IV inhibitors, effective doses of the PYY analog of Sequence ID No. 12, and incretins are also available. Alternatively, a DPP-IV inhibitor, or an effective amount of the PYY analog and incre of Sequence ID No. 13. This may include tin or DPP-IV inhibitors.

[0081] If the incretin is GLP-1 or a GLP-1 analog, then it is an albiglutin. G such as dulaglutide, lilaglutide, semaglutide, or combinations thereof. It may be LP-1 or a GLP-1 analog, particularly dulaglutide.

[0082] Preparation and use method for PYY analogues

[0083] The PYY analogues described herein are synthesized using standard manual or automated solid-phase synthesis procedures. It can be synthesized via any number of peptide synthesis methods known in the art. Automated peptide synthesizers are, for example, those used by Applied Biosystems (Calif). Foster City, Walnia and Protein Technologies I It is commercially available from nc. (Tucson, Arizona). Reagents for solid-phase synthesis are commercially available. It is readily available from [source]. Solid-phase synthesis apparatus blocks interfering groups, protects amino acids during reaction, Manufacturer's instructions regarding coupling, deprotection, and capping of unreacted amino acids. It can be used in accordance with the following.

[0084] Typically, amino acids protected with N-α-carbamoyl and growing peptides bound to the resin The N-terminal amino acid of the dot chain is an inert solvent such as DMF, N-methylpyrrolidone, or methylene chloride. In the medium, diisopropyl 1-carbodiimide and 1-hydroxybenzotriazole The coupling occurs at room temperature in the presence of coupling agents such as the synthesized peptide tree. Remove the N-α-carbamoyl protecting group from lipids using reagents such as TFA or piperidine. Then, a coupling reaction is performed using the N-α-protected amino acid that is needed to be added to the peptide chain. Repeat this process. Suitable amine protecting groups are well known in the art, for example, Gree n&Wuts, “Protecting Groups in Organic Syn The thesis, (John Wiley and Sons, 1991) They exist. The most commonly used examples are tBoc and Fmoc. Synthesis After completion, the peptide is deprotected of its side chains simultaneously using a standard processing method under acidic conditions. It is then cut from the solid support.

[0085] Those skilled in the art will understand that the peptide chains described herein are synthesized with a C-terminal carboxamide. This will be understood. In the synthesis of C-terminal amide peptides, typically Rink amide MBH A resin incorporating Rink amide AM linker is used in Fmoc synthesis. MBHA resin is commonly used in tBoc synthesis.

[0086] Typically, RP-HPLC is used with a C8 or C18 column, and the TF is 0.05%~0.1%. Purify the crude peptide using a water-acetonitrile gradient A. Purity is measured using analytical grade RP-HP. This can be confirmed by LC. Peptide identity can be confirmed by MS. Peptides can be used across a wide pH range. It can be solubilized in aqueous buffer solution within a certain range.

[0087] The use of one of the PYY analogs herein is for individuals, particularly those that are overweight or obese, and T2 This is intended to reduce blood glucose and / or body weight in individuals suffering from diabetes mellitus. As described in the detailed document, administering PYY analogs can lead to insulin sensitization and weight loss. By improving this, blood sugar control can be achieved. Thus, this statement The PYY analog in this book slows down the progression of insulin in individuals, targeting HbA1c levels. To maintain this, along with the further benefits of weight loss, the glucose-lowering effect show.

[0088] These methods may include the steps described herein, and these are not necessarily limited to those described herein. However, the steps can be performed in the order described. Other orders are also possible. Furthermore, Individual or multiple processes may occur in parallel and / or overlapping time, and / or This can be carried out individually or in a process that is repeated multiple times. Furthermore, these methods may be additional This may include unspecified steps.

[0089] Therefore, such methods do not include those who are overweight and have T2DM, or those with a predisposition to it. This may include selecting individuals with a causal relationship, or the method may involve obesity and T2DM. This may include selecting individuals that possess or have the same predisposition.

[0090] The method may also be in the form of a pharmaceutical composition as described herein. This may include administering an effective amount of at least one PYY analog to an individual as described. In some cases, at least one PYY analog / pharmaceutical composition may contain incretins. This may include additional therapeutic agents such as DPP-IV inhibitors.

[0091] At least one PYY analog and any incretin or DPP-IV inhibitor Concentration / dosage / dosage are discussed elsewhere in this specification.

[0092] Regarding the route of administration, at least one PYY analog or a pharmaceutical composition containing the same, for example, For example, orally; by injection (i.e., intra-arterial, intra-vein, intraperitoneal, intracerebral, intraventricular, intramuscular, intraocular, Intra-portal vein or intra-lesional; known methods such as sustained-release systems or implantable devices Therefore, it can be administered. In certain cases, at least one PYY analog or The pharmaceutical composition containing it can be administered by bolus injection or by sequential SQ administration. ru.

[0093] Regarding the frequency of administration, at least one PYY analog or a pharmaceutical composition containing the same is administered every Daily, every other day, three times a week, twice a week, once a week (i.e., every week), every other week (i.e., every week) It can be administered every other day or monthly. In certain cases, at least one PY Y analogue or pharmaceutical composition containing the same, every other day at SQ, three times a week at SQ, weekly at SQ It is administered twice a week, once a week, every other week, or monthly. Specific examples In this case, at least one PYY analog or a pharmaceutical composition containing the same is administered once a week via SQ It is administered (QW).

[0094] Alternatively, administer orally at least one PYY analog or a pharmaceutical composition containing the same. It is possible. As mentioned above, and also with respect to the frequency of administration, at least one PYY analog is possible. Or a pharmaceutical composition containing it may be taken daily, every other day, three times a week, twice a week, or once a week (i.e., It can be administered weekly, every other week, or monthly. Specific examples Therefore, at least one PYY analog or a pharmaceutical composition containing the same is administered orally every other day. It is administered orally three times a week, twice a week, once a week, or every other week. In certain cases, PYY analogues are administered orally once a week.

[0095] At least one PYY analog or a pharmaceutical composition containing the same contains an effective amount of incretin When administered in combination, incretins are GCG or GCG analogs. GLP-1, GLP-1(7)-36) アミド Or GLP-1 analogues, GIP or These are GIP analogs, OXM or OXM analogs, GIP / GLP-1, and GLP-1 / GC It may be G, or even an incretin with triple receptor activity. GCG, GCG derivatives limbus, GLP-1, GLP-1(7-36) アミド GLP-1 analogues, GIP, GIP Related compounds, OXM, OXM related compounds, GIP / GLP-1, GLP-1 / GCG, or IN Cretin triple receptor activity is a pharmaceutical composition containing at least one PYY analog. They can be administered simultaneously, separately, or sequentially.

[0096] Furthermore, GCG, GCG analogues, GLP-1, GLP-1(7-36) アミド GLP -1 analogues, GIP, GIP analogues, OXM, OXM analogues, GIP / GLP-1, GL Incretins having P-1 / GCG or triple receptor activity include at least one PY At the same frequency as Y analogues or pharmaceutical compositions containing them (i.e., every other day, twice a week, or Furthermore, it can be administered weekly. Alternatively, GCG, GCG analogues, GLP-1, GLP-1(7-36) アミド GLP-1 analogues, GIP, GIP analogues, OXM, O XM analogues, GIP / GLP-1, GLP-1 / GCG, or those with triple receptor activity Incretins are different from at least one PYY analog or a pharmaceutical composition containing the same. It can be administered at a frequency. Other examples include GCG, GCG analogues, GLP-1, and GLP. -1 (7-36) アミド GLP-1 analogues, GIP, GIP analogues, OXM, OXM derivatives Ink containing remnants, GIP / GLP-1, GLP-1 / GCG, or triple receptor activity. Retin is administered in QW. In other cases, PYY analogs are administered in SQ, GCG. GCG analogues, GLP-1, GLP-1(7-36) アミド , GLP-1 analogues, GI P, GIP analogues, OXM, OXM analogues, GIP / GLP-1, GLP-1 / GCG, Alternatively, incretins having triple receptor activity may be administered orally.

[0097] This method can be combined with diet and exercise, and / or discussed above. It is further intended that this drug can be combined with additional therapeutic agents other than those already mentioned. [Examples]

[0098] The following non-limiting embodiments are provided for illustrative purposes only, not limiting purposes.

[0099] Example 1: PYY analog 1.

[0100] A PYY analog incorporating the concept of the present invention may have the following structure. [ka]

[0101] Here, the N-terminus is free, and the C-terminal amino acid is an amide as a primary C-terminal amide. It is transformed. The 7th position K is (Ahx-E-(γE)-CO-(CH2) 16 -COOH It is chemically modified by bonding the K side chain at position 7 to the ε-amino group.

[0102] PYY analogs according to Sequence ID No. 9 are RAPP AM-Rink Amide resin (H4 0023 Polystyrene AM RAM (Rapp polymere GmbH) SymphonyX Automated Peptide Synthesizer (PTI Protein Techno Solid-phase peptide synthesis using the Fmoc / t-Bu strategy at logies Inc. This is how it is produced. Amino acid coupling is performed using 10 equivalents of amino acids and 0.9M diisopropyl alcohol. Lucarbodiimide (DIC) and 0.9M oxima (1:1:1 molar ratio) in DMF Use the solution and incubate at 25°C for 3 hours. Deprotection is performed using a 25% piperidine solution in DMF. cormorant.

[0103] After extending the peptide resin as described above, the MTT protecting group located at position 7 (K) is removed by dichloro Using 30% hexafluoroisopropanol (HFIP) in lomethane (DCM) Remove. Extend K at the 7th side chain using the Fmoc / t-Bu strategy. The aging / deprotection cycle includes Fmoc-6-aminohexanoic acid (Chem-Impex). International catalog number 02490), Fmoc-Glu (OtBu )-OH,Fmoc-Glu(OH)-OtBu(ChemPep Catalog #1007 03) and HOOC-(CH2) 16 -COOtBu is included. All coupling In this case, 3 equivalents of building blocks are used in the DMF, along with PyBOP (3 equivalents) and DIE. Use with A (6 equivalents) at 25°C for 3 hours.

[0104] Simultaneously cleavage from the resin and removal of side chain protecting groups, TFA:triisopropylsilane: 1,2-Ethanedithiol:Methanol:Thioanisole 80:5:5:5:5(v / v The process is carried out in a solution containing ) at 25°C for 2 hours, followed by precipitation with cold ether. The crude peptide is then prepared. R in a phenylhexyl column (Phenomenex, Luna; 5 μm, 100 A) The product is purified to a purity of over 99% (purification yield of 15-20%) by P-HPLC, and then divided into appropriate fractions. Pool and freeze-dry.

[0105] The purity of the PYY analog was analyzed by RP-HPLC, and its identity was confirmed using LC / MS. (Observation: M+3H) + / 3=1659.2(+ / -0.2);Calculation:M+3H + / 3= 1659.2; Observation: M+4H + / 4=1244.6(+ / -0.2);Calculation:M+4H + / 4=1244.6; Observation: M+5H + / 5=995.9(+ / -0.2);Calculation:M +5H + ( / 5 = 995.9).

[0106] Example 2: PYY analogue 2.

[0107] A PPY analog incorporating the concept of the present invention may have the following structure. [ka]

[0108] As in Example 1, the N-terminus is free, and the C-terminal amino acid is a C-terminal primary amide. It is then amidated. However, in contrast, the K at position 7 is ([2-(2-amino-e Toxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 18 -COOH It is chemically modified by bonding the K side chain to the ε-amino group.

[0109] The PYY analogues according to Sequence ID No. 10 are solid-phase particles similar to those described in Example 1 above. It is produced by butyl. Therefore, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) 18 -COOtBu is PyBOP (3 equivalents) in DMF and Using a 3-equivalent building block containing DIEA (6 equivalents), 3 hours at 25°C. It binds to the side chain after TT cleavage.

[0110] The purity of the PYY analog was analyzed by RP-HPLC, and its identity was confirmed using LC / MS. (Observation: M+3H) + / 3=1665.4(+ / -0.2);Calculation:M+3H + / 3= 1665.5; Observation: M+4H + / 4=1249.3(+ / -0.2);Calculation:M+4H + / 4=1249.4; Observation: M+5H + / 5=999.7(+ / -0.2);Calculation:M +5H + ( / 5 = 999.7).

[0111] Example 3: PYY analogue 3.

[0112] A PYY analog incorporating the concept of the present invention may have the following structure. [ka]

[0113] As in Example 1, the N-terminus is free, and the C-terminal amino acid is a C-terminal primary amide. It is then amidated. However, in contrast, the K at position 7 is ([2-(2-amino-e Toxy)-ethoxy]-acetyl)-(γE)-CO-(CH2) 16 -COOH It is chemically modified by bonding the K side chain to the ε-amino group.

[0114] The PYY analogue according to Sequence ID No. 11 is a solid-phase compound similar to that described in Example 1 above. It is produced by butyl. Therefore, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) 16 -COOtBu is PyBOP (3 equivalents) in DMF and Using a 3-equivalent building block containing DIEA (6 equivalents), 3 hours at 25°C. It binds to the side chain after TT cleavage.

[0115] The purity of the PYY analog was analyzed by RP-HPLC, and its identity was confirmed using LC / MS. (Observation: M+3H) + / 3=1664.7(+ / -0.2);Calculation:M+3H + / 3= 1664.9; Observation: M+4H + / 4=1248.9(+ / -0.2);Calculation:M+4H + / 4=1248.9; Observation: M+5H + / 5=999.3(+ / -0.2);Calculation:M +5H + ( / 5 = 999.3).

[0116] Example 4: PYY analogue 4.

[0117] A PYY analog incorporating the concept of the present invention may have the following structure. [ka]

[0118] As in Example 1, the N-terminus is free, and the C-terminal amino acid is a C-terminal primary amide. It is then amidated. However, in contrast, the K at position 7 is ([2-(2-amino-e Toxy)-ethoxy]-acetyl)2-(γE)-CO-(CH2) 16 -COOH It is chemically modified by bonding the K side chain to the ε-amino group.

[0119] The PYY analogues according to Sequence ID No. 12 are solid-phase particles similar to those described in Example 1 above. It is produced by butyl. Therefore, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) 16 -COOtBu is PyBOP (3 equivalents) in DMF and Using a 3-equivalent building block containing DIEA (6 equivalents), 3 hours at 25°C. It binds to the side chain after TT cleavage.

[0120] The purity of the PYY analog was analyzed by RP-HPLC, and its identity was confirmed using LC / MS. (Observation: M+3H) + / 3=1664.8(+ / -0.2);Calculation:M+3H + / 3= 1665.5; Observation: M+4H + / 4=1248.9(+ / -0.2);Calculation:M+4H + / 4=1249.4; Observation: M+5H + / 5=998.9(+ / -0.2);Calculation:M +5H + ( / 5 = 995.7).

[0121] Example 5: PYY analogue 5.

[0122] A PYY analog incorporating the concept of the present invention may have the following structure. [ka]

[0123] As in Example 1, the N-terminus is free, and the C-terminal amino acid is a C-terminal primary amide. It is then amidated. However, in contrast, the K at position 7 is ([2-(2-amino-e Toxy)-ethoxy]-acetyl)2-(γE)3-CO-(CH2) 18 -COOH It is chemically modified by bonding of the K side chain to the ε-amino group.

[0124] The PYY analogue according to Sequence ID No. 13 is a solid-phase compound similar to that described in Example 1 above. It is produced by butyl. Therefore, FMOC-NHPEG2-CH2COOH and HOOC-(CH2) 18 -COOtBu is PyBOP (3 equivalents) in DMF and Using a 3-equivalent building block containing DIEA (6 equivalents), 3 hours at 25°C. It binds to the side chain after TT cleavage.

[0125] The purity of the PYY analog was analyzed by RP-HPLC, and its identity was confirmed using LC / MS. (Observation: M+3H) + / 3=1732.2(+ / -0.2);Calculation:M+3H + / 3= 1732.3; Observation: M+4H + / 4=1299.4(+ / -0.2);Calculation:M+4H + / 4=1299.5; Observation: M+5H + / 5=1039.7(+ / -0.2);Calculation: M+5H+ ( / 5 = 1039.8).

[0126] Example 6: In vitro activity of PYY analogs.

[0127] (1) Inv to hNPY1, hNPY2, hNPY4, and hNPY5 receptors merging in URO

[0128] the purpose:

[0129] The following human (h) receptors: hNPY1R, hNPY2R, hNPY4R, and hNPY5 PYY analogs of Examples 1-5 in the absence of bovine serum albumin (BSA) relative to R In vitro binding affinity (K i ) evaluate competitive radioactive ligand binding with the membrane. Sei was prepared from cell lines overexpressing each recombinant receptor, and related [125 I] Signpost Peptide is used in each scintillation proximity assay (SPA) method. Related natural peptides D, PYY 1-36 (Sequence ID 1), PYY 3-36 (Sequence ID 2) and pancreatic polypeptide Chido 1-36 (PP 1-36 The binding affinity to (SEQ ID NO: 14) was used as a control for each up I'll decide on Sei.

[0130] method:

[0131] PYY analogue, natural, human PYY 1-36 and control PYY 3-36 Lilly Research Laboratories (Indianapolis, Indiana, USA) Synthesized using ), and analyzed by LC / MS, NMR, and LC / UV (purity 99.5%). The properties will be evaluated. The peptide content is estimated to be 80% of the powder mass. The peptide will be 100% DM Prepare a 10 mM stock solution in SO and store it frozen at -20°C until immediately before use in the assay. ru.

[0132] In the case of hNPY1R, transient overexpression is performed using CHO cells. Receptor cDNA This was subcloned into a pcDNA3.1 expression plasmid, and human fetal kidney (HEK) 29 After transfecting the cells, they are selected with Geneticin. Cell lines stably transfected for hNPY2R and hNPY4R Prepare the following. hNPY5R cloning is performed by Multispan, Inc. (California) It will be held in Hayward, Near.

[0133] Preparation of crude cell membranes for hNPY1R, hNPY2R, mNPY2R, and hNPY4R Therefore, two different methods (described below) are used. The hNPY5R membrane is Multip Purchase from an, Inc. (#MCG1275).

[0134] Method 1 - For hNPY2R and hNPY4R membranes, the frozen cell pellet was moistened 50 mM Tris HCl, pH 7.5, and EDTA per gram of cell paste. Roche complete (商標) Protease inhibitors (#11697490) Dissolve on ice in 10 mL of hypotonic homogenization buffer containing 01). Glass Potter-Elvehjem homogeneous mortar with flon(registered trademark) pestle. Disrupt the cell suspension with 25 strokes using an Izer. Homogenate at 4°C, 11 Centrifuge at 00 × g for 10 minutes. Collect the supernatant and store it on ice, while leveling the pellet. Resuspend in a saturating buffer and rehomogenize as described above. Homogenate at 1100 × g Centrifuge for 10 minutes. Combine the second supernatant with the first supernatant and centrifuge at 35000 × g, 4°C. Centrifuge for 1 hour. Add a protease inhibitor to the resulting membrane pellet at a concentration of approximately 1-3 mg / mL. Resuspend in the homogenizing buffer containing the solution, rapidly freeze in liquid nitrogen, and aliquot until ready to use. Store in a freezer at -80°C. Protein concentration is measured using BSA as the standard. The determination is made using a protein assay kit (Pierce, #23225).

[0135] Method 2 - For hNPY1R membranes, add 1 gram of moist cell paste to the frozen cell pellet. For example, 25 mM Tris HCl, pH 7.5, 1 mM MgCl2, 25 units / mL. Contains DNase I (Invitrogen, #18047-019) and EDTA Roche complete (商標) Protease inhibitor #118361700 Dissolve on ice in 5 mL of hypotonic homogenized buffer containing 01). Teflon (registered Using a glass Potter-Elvehjem homogenizer equipped with a pestle (trademark) Then, disrupt the cell suspension with 25 strokes. Add the homogenate to 50 mL of conical tube. Centrifuge in a microwave at 4°C and 1800×g for 15 minutes. Collect the supernatant and store on ice. Alternatively, the pellet is resuspended in homogenizing buffer and rehomogenized as described above, but DNa SE I is not used in homogenization buffer. Homogenate at 1800 x g for 15 minutes. Then, centrifuge. Combine the second supernatant with the first supernatant and centrifuge at 25000×g at 4°C for 30 minutes. The heart is separated. The resulting membrane pellet is homogenized with a protease inhibitor at approximately 2 mg / mL. Resuspend in buffer solution, divide equally, and store in a -80°C freezer until use. Protein concentration The degree is measured using BSA as the standard with the BCA protein assay kit (Pierce, #2322) Determine using 5).

[0136] General binding assay methods - equilibrium dissociation constants of various receptor / radioligand interactions (K d ) Using the same reagents and buffers as described below for compound testing, saturated compound Determined from the combined analysis. K was determined for the receptor preparations used in this study. d The values ​​are as follows: As stated above: hNPY2R, 0.0047nM; hNPY1R, 0.07nM; hNP Y4R, 0.084 nM; and hNPY5R, 0.896 nM.

[0137] hNPY1R receptor binding protocol - PYY analog peptides to hNPY1R PYY 1-36 Receptor binding affinity (K i ) to Perkin Elmer (Massachusetts Human recombinant

[125] obtained from Waltham, Setts. I ]-PYY 1-36 (#NE Determined by a competitive radioligand binding assay with X341 (2200 Ci / mmol). The assay uses polyvinyltoluene (PVT) wheat germ agglutinin-conjugated SPA beads (#R The assay will be performed using the SPA method with PNQ0001 (Perkin Elmer). Buffer (25mM HEPES, pH7.5, 1mM MgCl2, 2.5mM CaC l2 and 0.2% w / v bacitracin (RPI#32000) were used in the preparation of the reagent. PYY analogues and PYY 1-36 Unfreeze and use the Tecan Evo liquid handler. The mixture is then serially diluted three-fold in 100% DMSO (10-point concentration response curve). Dilute the peptide 20-fold in assay buffer and add it to the assay plate. Before doing so, reduce the DMSO level and peptide concentration. Then, add 5 μL of serially diluted peptide. Add tide or DMSO to 45 μL of assay buffer or unlabeled PYY 1-36 Contrast (non Corning® 363 (containing specific binding or NSB, final concentration 10 nM) 2. Transfer to a clear bottom assay plate. Next, add 50 μL of [ 125 I]-PYY 1-36 (most A final concentration of 0.05 nM and 50 μL of hNPY1R membrane (1.0 μg / well) are added. The last to be added is 50 μL of WGA SPA beads (50 μg / well). The final concentration of DMSO is 0.125%. Seal the plate and shake on a plate shaker. Mix for 1 minute (setting 6), incubate / stand at room temperature for 10 hours, then PerkinE lmer Trilux MicroBeta® Scintillation Counter Read the plate. The final assay concentration range of the peptide tested in the response curve is PYY Related compounds (2.5 μM to 0.13 nM) and PYY 1-36 (10nM~0.5pM) be.

[0138] hNPY2 receptor binding protocol - PYY analog peptides and PP 1-36 Receptor binding affinity (K i ) As described above for hNPY1R, Determined from a radioactive ligand binding assay. The final result of the tested peptide in the response curve. The assay concentration range is PYY analog (0.1 μM to 5 pM) and PYY 3-36 (10 The concentration is nM to 0.5pM.

[0139] As mentioned above, regarding the hNPY4R receptor binding protocol - hNPY1R, PYY analog peptides and PP for 4R 1-36 Receptor binding affinity (K i ) compete Determined from a radioactive ligand binding assay. The final result of the tested peptide in the response curve. The assay concentration range is for PYY analogs (2.5 μM to 0.13 nM) and PYY 1-36 (10 nM to 0.5 pM).

[0140] As mentioned above, regarding the hNPY5R receptor binding protocol - hNPY1R, PYY analog peptides and PYY 1-36 Receptor binding affinity (K i )of Determined from a competitive radioligand binding assay. The most effective response curve for the tested peptide is determined from the response curve. The final assay concentration range is PYY analog (1 μM to 10 pM) and PYY 1-36 ( (1 μM to 10 pM)

[0141] Data analysis of NPY receptor binding assays - PYY analogues, PYY 1-36 , PYY 3- 36 , or PP 1-36 The raw data of the concentration curve in counts per minute (CPM) is as follows: As shown in the formula, from the individual CPM values, nonspecific binding (NSB), and excess unlabeled P, respectively, YY 1-36 , PYY 3-36 , or PP 1-36 Subtracting the bond in the presence of, By dividing by the total binding signal (which is also corrected by subtracting nonspecific bindings), Convert to 1 / 20 specific inhibition.

number

[0142] 4 parameters (maximum curve value, minimum curve value, IC) 50 , Hill gradient) Nonlinear regression routine ( Genedata Screener, version 13.0.5, Genedata A The data will be analyzed using G (Basel, Switzerland). Affinity constant (K i ) to equality K i = I C 50 / (1+D / K d ) relative IC 50 Calculate from the values, in the formula D = in the experiment The concentration of the radioactive ligand, IC 50 This is the concentration that causes 50% inhibition of binding, K d teeth This is the equilibrium binding dissociation constant of the radioactive ligand, determined from saturated binding analysis (listed above). The modifier (>) represents a 50% increase in data compared to the maximum bond when no competing substance is present. This indicates that inhibition was not achieved, and therefore, K i The highest concentration of the compound tested by assay Calculate using degrees.

[0143] K i The reported values ​​are calculated as a geometric mean, as shown below.

number

[0144] The mean standard error (SEM) is calculated using the delta method as shown below;

number

[0145] hNPY2R(Y2) versus hNPY5R(Y5), hNPY4R(Y4), and / or The selectivity of the peptide for hNPY1R(Y1) was determined by the results for nM units of hNPY2R. Dividing is a difficult calculation.

[0146] result:

[0147] [Table 1]

[0148] As shown above, the PYY analogs of Examples 1-5 are very effective against the hNPY2 receptor. It is selective, and furthermore, natural human PYY 3-36 Compared to (SEQ ID NO: 2), hNPY 5. It shows decreased binding affinity to hNPY4 and hNPY1 receptors.

[0149] (2) In vitro cAMP activity for the human NPY2 receptor

[0150] the purpose:

[0151] Forskolin in HEK 293 cells overexpressing recombinant human NPY2 receptor By measuring the inhibition of inducible intracellular cAMP production, we can determine the natural human PYY 3-36 and The in vitro functional activity of the PYY analogs in Examples 1-5 will be determined by comparison.

[0152] method:

[0153] PYY analogues and human PYY 3-36 (SEQ ID NO: 2) is used in receptor binding assays. The data is then synthesized, characterized, and stored as described above.

[0154] Receptor cloning -An Statistically transfected cell lines hNPY2 The receptor cDNA is subcloned into a pcDNA3.1 expression plasmid, transfected into HEK 293 cells, and then selected with Geneticin to prepare the cells (1 × 10⁶ cells) after 9 passages. 7 Prepare aliquots of cells ( / mL) and freeze-store them in the gas phase of a liquid nitrogen tank. These frozen aliquots will be used in the assay. The cells will maintain a viability of over 95% for several months.

[0155] hNPY2R cAMP assay - PYY analogue or PYY 3-36 False Inhibition of choline-induced cAMP production is associated with HEK 29 overexpressing recombinant hNPY2R. Measurement is performed using 3 cells. Thaw frozen cell aliquots in a 37°C water bath. 10 mL of culture medium (Lif Tech 10082-147 containing 10% FBS) e Tech 11090-081 MEM cell culture medium, Life Tech 250 30-081 1 mM L-Glutamine, Life Tech 11140-050 ×NEAA, Life Tech 11360-070 1 mM sodium pyruvate, Life Tech 15240-062 contains 1 x 50 mL of antibiotic-antifungal agent. Transfer to a tube and centrifuge in a Beckman benchtop centrifuge at 1500 rpm for 5 minutes. Remove the supernatant, resuspend the cell pellet in 10 mL of cell culture medium, and then use a 40 μm strainer. Pass through the filter. The exact number of cells and cell viability can be measured using the Beckman-Coulte filter. Using Vi-Cell Analyzer (Vi-Cell XR 2.03) from Microsoft The decision will be made. Combi-Tip Dispenser (Thermo Scienti Using fic, 8000 cells per well were used in a white 384-well assay. Rate (Corning, poly-D-lysine coating, white / opaque, catalog number) Plate onto 356661). Centrifuge the plate at 1000 rpm for 1 second. Incubate at 37°C for 18-20 hours in an incubator with 5% CO2 control. Remove the culture medium from the assay plate by lightly flicking it on a paper towel. 10 μL of assay buffer [1X HBSS (Hyclone, #SH3026801)] , 20mM HEPES, pH7.5 (Hyclone#SH30237.01), 0. 1% w / v casein (CTL Scientific Supply Corp., #4 40203H), 500μM IBMX (Sigma-Aldrich #I5876) After adding ] to the wells, use the Combi-Tip Dispenser to dispense 150 Centrifuge at 0 rpm for 10 seconds. Obtain the concentration response curve (20 points) at a 2x dilution. -Stick dispensing technology (acoustic dispensing Using (Labcyte Echo 550 technology) 100% D Prepare with MSO. The cells are incubated at 37°C (final DMSO concentration = 1%) for 45 minutes in a PYY analog. or Human PYY 3-36 The mixture is then treated with 1 μM forskolin ( ) at 37°C for 45 minutes. Stimulate with Sigma-Aldrich (#F6886). CisBio cAMP-G i We quantified intracellular cAMP using the Dynamic kit (#62AM9PEB). In short, add the cAMP-d2 conjugate to the cell lysis buffer (10 μL). In addition, the antibody in cell lysis buffer (10 μL), anti-cAMP-Eu, is added in the same manner. 3+ - By adding cryptotate, the HTRF kit reagent can be used to detect intracellular cAM Detect the P level. The resulting competitive assay is then sterilized at room temperature for at least 60 minutes. Incubate, and then PerkinElmer Envision (商標) Equipment (3 Detection is performed using excitation at 20 nm and emission at 665 nm and 620 nm. The final assay concentration range of the peptide tested in the response curve was PYY analogues (0.1 μM~0. 2 pM) and human PYY 3-36 (10nM~0.02pM). Known cAMP Prepare standard curves for concentrations (0.5 μM to 1 pM) using assay buffer. If a competing substance is present... Wells that have not been treated, or human PYY 3-36 Each well to which the substance has been added Each plate contains both the maximum response and inhibitor controls.

[0156] Data analysis of the hNPY2 receptor cAMP assay using time-resolved fluorescence Then, calculate the fluorescence ratio (665nM / 620nm) which is inversely proportional to the amount of cAMP present. YY analogues and human PYY 3-36 The signal is measured in relative response units (665nm / 620 Emission at nm*10,000 (y-axis) vs. cAMP concentration (x-axis) plotted. Convert to nM cAMP per well using the AMP standard curve.

[0157] The amount of cAMP (nM) generated in each well is expressed by the following equation: Convert to the percentage of the maximum response observed with Skolin alone.

number

[0158] The specific inhibition rate (y-axis) is plotted against the concentration of the competing substance (x-axis), and is defined as follows: To that end, there are 4 parameters (upper part of the curve, lower part of the curve, IC). 50 (hill gradient) nonlinear regression route (Genedata Screener, version 13.0.5, Genedata) Analyze using AG (Basel, Switzerland):

number

[0159] Relative IC 50 The value inhibits forskolin-induced cAMP production by 50%. It represents concentration.

[0160] I C 50 The reported values ​​are calculated as a geometric mean, as shown below.

number

[0161] The mean standard error (SEM) is calculated using the delta method as shown below;

number

[0162] result:

[0163] [Table 2]

[0164] As described above, the results of the cAMP assay for the hNPY2 receptor are as follows: The functional activity of PYY analogs 5 was demonstrated, and Example 2 is human PYY 3-36 12 times lower This indicates the weakest possible effect.

[0165] (3) In vitro activity of GTPγS against the hNPR2 receptor

[0166] the purpose:

[0167] The receptor-mediated activation of G proteins by PYY analogs in Examples 1-5 will be evaluated. Consumption-mediated activation involves non-hydrolyzable GTP analogs, GTPγ[ 35 Measure using [S] This is possible. Stimulation of G protein-bound receptors via agonists can trigger membrane-bound Gαβ This leads to the activation of the γ protein heterotrimer complex, which transmits extracellular signals. This is the first step in modifying intracellular pathways. In this specification, GTPγ[ 35 S] Function upgrade We will use Sei to evaluate the efficacy of various PYY analogs at the hNPY2 receptor.

[0168] method:

[0169] PYY analogues, human PYY 1-36 (SEQ ID NO: 1) and control peptide PYY 3-36 (SEQ ID NO: 2) was synthesized and characterized as described above in a receptor binding assay. And it is saved.

[0170] For each test peptide, a 1 / 3 log dilution (logarithmic concentration -6.52 to -12.52) was performed. The concentration-response curve (CRC) of 0.2% bacitracin (USBiologicals) Assay buffer (20 mM HE) supplemented with #11805) and 0.5% DMSO. PES, pH7.4, 100mM NaCl, 6mM MgCl2, 1mM EDTA) This is completed using a Hamilton NIMBUS liquid handler. The final assay concentrations of syn and DMSO were 0.05% and 0.125%, respectively. NPY2R film (Multispan#HTS066M) with 20 μM GDP (Sigm a#G-7127) and 6 ug / mL saponin (Sigma#S-4521) were added. Prepare the assay buffer to a concentration of 7.5 ug / mL and test the hNPY2R membrane using the assay buffer. Incubate at room temperature for 20 minutes before adding. GTPγS[35 S](Perkin Prepare Elmer (#NEG030H) at a concentration of 0.6 nM in the assay buffer. GA SPA beads (PerkinElmer #RPNQ0001) are used in the assay buffer. Prepare a concentration of 12 mg / mL. For a final volume of 200 uL, first add 100 uL to h Add to the NPY2R membrane, then 50 μL of CRC solution, then GTPγS[ 35 S] solution 50 By adding uL, in a 96-well plate (Costar #3604), Perform the operation. Cover the plate and shake it in an orbital shaker (175 rpm) at room temperature for 45 minutes. Place it in the container. Next, add 25uL of SPA beads, reseal the plate, and vortex. After mixing, return to the orbital shaker at room temperature for 3 hours. Then, heat the plate at 500 rp. Centrifuged at m for 5 minutes and then measured in a PerkinElmer 2450 microplate counter. Count for 1 minute per well. Determine the basal binding (CPM) with PYY analogs or human P. YY 3-36 Determined in the absence of [someone], the following equation shows that the [number] of peptide concentrations exceeding the baseline value. - Used to calculate cents: (PYY analogues, or human PYY 3-36 , CP M-Basic CPM) / (Basic CPM)*100. E.C. 50 The (nM) value is the logarithm of the concentration and The percentage of the base value is calculated using the following equation for GraphPad Prism 7.0 Apply linear regression analysis (logarithmic (agonist) versus response - slope of the variable (4 parameters)). Determined by: Y = bottom + (top - bottom) / (1 + 10^((LogE C 50 -X)*hill gradient)). Geometric mean and standard error of the mean value in GraphPad P Using rism 7.0 column statistics functions for EC 50 Calculate from the (nM) value.

[0171] result:

[0172] [Table 3]

[0173] As mentioned above, GTPγ[ 35 The results of the functional assay showed that for the hNPY2 receptor The activity of the PYY analog was demonstrated, and Example 2 is human PYY 3-36 Four times less effective This shows the weakest effect of the force.

[0174] (4) Pharmacokinetics

[0175] the purpose:

[0176] We will investigate the pharmacokinetic properties of PYY analogs.

[0177] method:

[0178] LC / MS - The plasma concentrations of various PYY analogs are determined by LC / MS. The method measures the whole compound, the peptide, and the associated time extension. Using methanol containing 0.1% formic acid, the plasma of mice, rats, or monkeys (50 Extract PYY analogs and internal standards from the sample (μL). Centrifuge the sample and remove the supernatant from the thyroid gland. Transfer to an ermo Protein Precipitation Plate. Sample Sep-Pak tC18 SPE prepared with methanol and 0.1% formic acid in water. Load into a μElution plate. Dilute the SPE column twice with 0.1% formic acid in water. Wash. Next, use formic acid / water / acetronityl (0.1:15:85) to remove the compound. After elution, it is dried and reconstituted, and then an aliquot (10 μL) is heated in a thermometer. Acclaim PepMap100 C18, 300 μm × 5 mm capture column, and Thermo Easy Spray PepMap C18, 75 for LC / MS analysis The sample was injected into a μm × 15 cm column. The column effluent was used for detection and quantification using a Therm Direct the signal towards the Q-Exactive Plus mass spectrometer.

[0179] Pharmacokinetics of PYY analogs in CD-1 mice - Plasma pharmacokinetics of PYY analogs, 2 Evaluation will be performed in male CD-1 mice after a single subcutaneous administration of 00 nmol / kg. Blood samples will be taken. Collected from two animals at each point in time over a 168-hour period. Discontinuous sampling is used. To evaluate the dynamics of PYY analogs in mice, we used mean concentration versus time data. The pharmacokinetic parameters of PYY analogs after a single subcutaneous administration of 00 nmol / kg are shown in the table. Plasma concentrations of PYY analogs were measured up to 120 hours after a single subcutaneous administration of 200 nmol / kg. To release.

[0180] Pharmacokinetics of PYY analogs in SD rats - Plasma pharmacokinetics of PYY analogs, 50n Evaluation will be performed in male Sprague Dawley rats after a single subcutaneous administration of mol / kg. Blood samples will be collected from two animals at each point in time over a 168-hour period. To evaluate the dynamics of PYY analogs in rats using a pharmacokinetic, we compared the average concentration of individual animals with the time interval. Using the data from between, the pharmacokinetics of PYY analogs after a single subcutaneous administration of 50 nmol / kg were studied. The parameters are presented in a table. The plasma concentration of the PYY analog is measured by a single subcutaneous injection of 50 nmol / kg. Detection is possible up to 120 hours after administration.

[0181] Pharmacokinetics of PYY analogs in cynomolgus monkeys - Plasma pharmacokinetics of PYY analogs, 50 Evaluation will be performed in male and female cynomolgus monkeys after a single subcutaneous administration of nmol / kg. Blood sample Collect data over 504 hours. Use continuous sampling to collect PYY data from cynomolgus macaques. To evaluate the dynamics of related organisms, we used average concentration-time data for individual animals, and 50n The pharmacokinetic parameters of PYY analogs after a single subcutaneous administration at mol / kg are shown in the table. Plasma concentrations of analogues are detected up to 504 hours after a single subcutaneous administration of 50 nmol / kg.

[0182] result:

[0183] [Table 4]

[0184] [Table 5]

[0185] [Table 6]

[0186] result:

[0187] These data indicate that the above compounds have a pharmacokinetic profile suitable for once-weekly administration. This indicates that...

[0188] (5) Solubility and Stability

[0189] the purpose:

[0190] The soluble pH range and stability of PYY analogues are determined.

[0191] method:

[0192] Visual solubility range evaluation - Freeze-dried PYY analog powder in water at a concentration of 4 mg / mL Reconstitute the solution and adjust the pH to pH 4 with citrate / phosphate buffer. Set the system pH to 0.5N After adjusting the pH to 8 with NaCl, the solution was titrated to pH 4 with 0.5N HCl.

[0193] Thermal stability evaluation - 10 mM or 20 mM phosphate at concentrations of 1 mg / mL or 2 mg / mL A solution of PYY analogs in sodium, pH 7.0 was prepared and incubated at 4°C and 40°C for 4 weeks. Cure the samples. At the 4-week mark, perform size exclusion chromatography (SEC). ) and RP-HPLC are used for analysis.

[0194] SEC method - TOSOH TSKgelG2000SWxl, inner diameter 7.8mm x 30cm Using a 5 μm column, 50 mM sodium phosphate, 300 mM NaCl, pH 7 Using a mobile phase composition of 0.0, 20% acetonitrile, with a flow rate of 0.5 mL / min and λ-214n It took 30 minutes to get there in meters.

[0195] RP method - Cortecs C18, 2.7 um, 4.6 x 50 mm column, 0.08 Using 20%-45% acetonitrile / water containing 5% TFA, at a flow rate of 1 mL / min, λ- The process was performed at 214nm over a period of 10 minutes.

[0196] result:

[0197] [Table 7]

[0198] As shown above, all peptides dissolve above pH 7.0. RP and SEC Therefore, the stability being evaluated is that these peptides remain relatively stable under aggressive heat stress. This suggests that they are present.

[0199] Example 7: Effects of PYY in vivo.

[0200] (1) In vivo effects on food intake and body weight in normal mice

[0201] the purpose:

[0202] Examples 1-5 of PYY reduced the body weight of normal mice after a single injection and suppressed food intake. We will compare the effects of related organisms.

[0203] method:

[0204] Male C57Bl / from Envigo RMS (Indianapolis, Indiana) Six mice were fed solid feed (5008; LabDiet, St. Louis, Missouri). With a normal 12:12 light cycle, temperature-controlled environment where food and water are freely available. They were housed individually in a facility (74.0°F; 23.3℃). They were 9-10 weeks old and fasted. Record body weight and initial food weight, and administer a single subcutaneous injection of vehicle or peptide to the animals. The procedure is performed, and then body weight and food intake are measured daily for 3 days after administration. Area-of-Consumption (AUC) analysis is performed for both body weight and food intake relative to the vehicle. Example 4, with a concentration of 30 nmol / kg, was compared to body weight and food intake in each assay. It is used as a benchmark for 100% effectiveness.

[0205] result:

[0206] [Table 8]

[0207] As shown above, a decrease in both body weight and food intake is associated with PYY in vivo. The effectiveness of analogues and a comparison of the doses required for complete efficacy demonstrate an improvement in efficacy. ru.

[0208] (2) In vivo effects of diet-induced obesity on food intake and body weight in mice

[0209] the purpose:

[0210] In diet-induced obesity (DIO) mice, over a period of 2 weeks, either alone or GLP PYY analogs of Examples 1-5 that reduce body weight when combined with -1 receptor agonists The effects of daily administration will be investigated.

[0211] method:

[0212] 20-week-old male DIO C57Bl / 6 mice (Taconic) were 60% fat upon arrival. They are raised on a high-fat diet (D12492; Research Diets, New Jersey) (New Brunswick, California). 12-hour light / dark cycle (lights on at 22:00), They are kept in a temperature-controlled facility (74.0°F; 23.3°C) where they have free access to food and water. The animals are housed individually. After a one-week acclimatization period with daily vehicle administration, their non-fasting body weight is measured. The animals were measured and randomized into experimental groups based on body weight (n=6), and the vehicle and GLP-1 receptors were measured. Somatic agonist (GLP-1 RA; SEQ ID NO: 15), PYY analogue, or PYY analogue Administer a combination of GLP-1 RA and daily subcutaneous injections. Two weeks after administration, do not fast. Record the body weight at any given time and calculate the average change in body weight relative to the vehicle. Combine with GLP-1 RA. To determine the additive or synergistic effect of combined PYY analogs, GLP-1 RA The effect that exceeds the effect of a single factor (net effect) is calculated.

[0213] result:

[0214] [Table 9]

[0215] As shown above, PYY analogues can be used alone or in combination with GLP-1 RA. The weight loss observed when using this method demonstrates the effectiveness of PYY analogs in vivo, and here They are comparing the degree of weight loss.

[0216] (3) Body weight and glucose in diabetic and obese (db / db) mice In vivo effects

[0217] the purpose:

[0218] Body weight and blood glucose levels over 10 days in obese and diabetic mice (db / db) The effects of daily administration of PYY analogs in Examples 1-5, which reduce [a certain condition], will be investigated.

[0219] method:

[0220] Lepr from Envigo RMS (Indianapolis, Indiana) db / d b (db / db) Male mice were fed solid diet (5008; LabDiet, Missouri, Sen They are kept in a Truis environment with a normal 12:12 light cycle and have free access to food and water. Five animals are kept in each cage within a temperature-controlled facility (74.0°F; 23.3°C). It was accepted. At 8-9 weeks of age, the Accu-Check (registered trademark) blood glucose meter (Roche Di Abetes Care, Inc. (Indianapolis, Indiana) uses body care Measure weight and blood glucose levels, then administer the vehicle or peptide by subcutaneous injection daily. After day 0, measure weight and blood glucose levels and calculate the changes in response to vehicle treatment.

[0221] result:

[0222] [Table 10]

[0223] As shown above, the reduction in body weight and blood glucose levels by PYY analogs is observed in vivo. The effectiveness of PYY analogs is demonstrated here, and the magnitude of weight loss and glucose reduction is shown. They are comparing the differences.

[0224] In conclusion, the PYY analogs described herein exhibit selectivity for NPY2R. They also show dose-dependent reductions in body weight and dose-dependent improvements in blood glucose in db / db mice, as reflected in normal mice, diet-induced obese mice, and db / db mice, with the PYY analogs in Examples 1, 3, and 4 being the most effective, which are consistent with the in vitro profiles. array Sequence ID 1 - Human PYY 1-36 YPIKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY Sequence ID 2 - Human PYY 3-36 IKPEAPGEDASPEELNRYYASLRHYLNLVTRQRY Sequence ID 3-PYY analog PKPEX7PX9X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY Sequence ID 4-PYY analog PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY Sequence ID 5-PYY analog PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY Sequence ID 6-PYY analog PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY Sequence ID 7-PYY analog PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY Sequence ID 8-PYY analog PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY [Chemistry] [Chemistry] [Chemistry] [Chemistry] [Chemistry] Sequence ID 14-PP 1-36 APLEPVYPGDNATPEQMAQYAADLRRYINMLTRPRY Sequence ID 15-GLP-1 RA HGEGTFTSDVSSYLEEQAAKEFIAWLVKGRGGGGGSGGGGSGGGGSESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG The present invention may include the following embodiments. (1) Peptide tyrosine-tyrosine (PYY) analogs containing the following amino acid sequence: PKPEX 7 PX 9 X 10 DASPEEX 17 X 18 RYYX 22 X 23 LRHYLNX 30 LTRQRY(Formula I) (In the formula, X 7 is an amino acid having a functional group that can be used for joining, and the functional group is C 16 ~C 22 It is bonded to the fatty acid, X 9 is E or G, X 10 is E or K, X 17 is L or W, X 18 is N or Q, X 22 is A or I, X 23 is E, D, or S, X 30 is E or W (sequence code 3), The C-terminal amino acid may be amidated. (2) X7 The PYY analogue according to claim 1, wherein is selected from the group consisting of C, D, E, K, and Q. (3) X 7 However, K is C 16 ~C 22 The PYY analog according to claim 1, wherein the attachment to the fatty acid is via the epsilon-amino group of the K side chain. (4) The PYY analog according to claim 1, wherein the amino acid sequence is selected from the group consisting of the following: PKPEKPGEDASPEEWQRYYAELRHYLNWLTRQRY(Sequence ID 4); PKPEKPGEDASPEEWQRYYAELRHYLNELTRQRY(sequence number 5); PKPEKPEEDASPEEWQRYYIELRHYLNWLTRQRY(Sequence ID 6); PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY(sequence number 7); and PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY (Sequence ID 8). (5) Said C 16~C 22 A PYY analog according to any one of claims 1 to 4, wherein the fatty acid is selected from the group consisting of hexadecanoic acid, hexadecanedioic acid, heptadecanoic acid, heptadecanedioic acid, stearic acid, octadecanoic acid, nonadesylic acid, nonadecanedioic acid, eicosanoic acid, eicosanedioic acid, heneicosanoic acid, heneicosanedioic acid, docosanoic acid, docosanedioic acid, and their branched and substituted derivatives. (6) Said C 16 ~C 22 fatty acids are C 18 ~C 20 A PYY analog according to claim 5, which is a fatty acid. (7) Said C 18 ~C 20 Fatty acids are CO-(CH 2 ) x -CO 2 It is a straight-chain fatty acid containing H, where x The PYY analog according to claim 6, wherein is 18 or 20 (8) Said C 18 ~C 20 The PYY analog according to claim 7, wherein the fatty acid is selected from the group consisting of palmitic acid, stearic acid, arachidic acid, and eicosanoic acid. (9) Said C 16 ~C 22 A PYY analog according to any one of claims 1 to 8, wherein a fatty acid is bonded to an amino acid having a functional group that can be used for linker-mediated bonding. (10) The PYY analogue according to claim 9, wherein the linker may be one or more units selected from the group consisting of [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid (Ahx), glutamic acid (E), gamma-glutamic acid (γE), and combinations thereof. (11) Peptide tyrosine-tyrosine (PYY) analogs including the following:

change

change

change

change

change

Claims

1. Compounds containing an amino acid sequence selected from the following group: PKPEKPGEDASPEEEWQRYYAELRHYLNELTRQRY (Sequence ID 5); PKPEKPEEDASPEEEWQRYYIELRHYLNWLTRQRY (Sequence ID 6); PKPEKPGKDASPEEWNRYYADLRHYLNWLTRQRY (Sequence ID 7); and PKPEKPGEDASPEELQRYYASLRHYLNWLTRQRY (Sequence ID 8) (In the formula, each K, which is the fifth amino acid from the N-terminus, is C １６ ~C ２２ It is an amino acid bonded to a fatty acid, and the C １６ ~C ２２ A fatty acid is bonded to the amino acid via a linker, wherein the linker is one or more units selected from the group consisting of [2-(2-amino-ethoxy)-ethoxy)]-acetic acid (AEEA), aminohexanoic acid (Ahx), glutamic acid (E), gamma-glutamic acid (γE), and combinations thereof).

2. Said C １６ ~C ２２ The compound according to claim 1, wherein the bonding to the fatty acid is via the epsilon-amino group of the K side chain.

3. Said C １６ ~C ２２ The compound according to any one of claims 1 to 2, wherein the fatty acid is selected from the group consisting of hexadecanoic acid, hexadecanedioic acid, heptadecanoic acid, heptadecanedioic acid, stearic acid, octadecanediic acid, nonadesylic acid, nonadecanedioic acid, eicosanoic acid, eicosanedioic acid, heneicosanoic acid, heneicosanedioic acid, docosanic acid, and docosanedioic acid.

4. Said C １６ ~C ２２ The fatty acid is C １８ ~C ２０ The compound according to claim 3, wherein the fatty acid is a C

5. Said C １８ ~C ２０ Fatty acids are CO-(CH ２ ) ｘ -CO ２ It is a linear fatty acid containing H, where, ｘ The compound according to claim 4, wherein is 18 or 20.

6. Said C １８ ~C ２０ The compound according to claim 4, wherein the fatty acid is selected from the group consisting of stearic acid and arachidic acid.

7. Compounds including the following: 【Chemistry 2】

8. Compounds including the following: 【Transformation 3】

9. Compounds including the following: 【Chemistry 4】

10. Compounds including the following: 【Transformation 5】

11. The compound according to any one of claims 1 to 10, wherein the compound has a charge greater than -2.

12. The aforementioned compound, PYY ３－３６ A compound according to any one of claims 1 to 11, having a binding affinity for the NPY2 receptor greater than the binding affinity of (SEQ ID NO: 2).

13. The aforementioned compound is human PYY ３－３６ The compound according to any one of claims 1 to 12, having a half-life longer than that of (SEQ ID NO: 2).

14. At least one compound or salt thereof according to any one of claims 1 to 13, A pharmaceutical composition comprising one or more pharmaceutically acceptable carriers, diluents, and excipients.

15. The pharmaceutical composition according to claim 14, further comprising an additional therapeutic agent.

16. The aforementioned additional therapeutic agents include glucagon (GCG), GCG analogs, glucagon-like peptide-1 (GLP-1), and GLP-1 ７－３６－アミド The pharmaceutical composition according to claim 15, wherein the incretin is selected from the group consisting of GLP-1 analogues, gastric inhibitory peptide (GIP), GIP analogues, oxytomodulin (OXM), OXM analogues, GIP / GLP-1, GLP-1 / GCG, or incretin analogues having triple receptor activity.

17. The pharmaceutical composition according to claim 16, wherein the additional therapeutic agent is a dipeptidyl peptidase-IV (DPP-IV) inhibitor.

18. A compound according to any one of claims 1 to 13 or a pharmaceutically acceptable salt thereof, used for the treatment of obesity or obesity-related diseases or disorders.

19. The compound according to claim 18, wherein the compound or a pharmaceutically acceptable salt thereof is administered subcutaneously to an individual.

20. The compound according to claim 18, wherein the compound or a pharmaceutically acceptable salt thereof is administered orally to an individual.

21. The compound according to claim 19 or 20, wherein the compound or a pharmaceutically acceptable salt thereof is administered to an individual daily, every other day, three times a week, twice a week, once a week, or every other week.

Images