Peptide tyrosine-tyrosine analog and use thereof

Abstract

The present invention relates to the field of medicines, and specifically provides a novel peptide tyrosine-tyrosine (PYY3-36) analog or a salt or solvate thereof, comprising the following sequence structure of general formula (I): P-K-P-E-ψ-P-E-X10-D-X12-S-P-E-E-W-Q-R-Y-Y-X22-X23-L-R-H-Y-L-N-W-L-T-R-Q-R-Y-R1 (I). The provided novel PYY3-36 analog has better pharmaceutical efficacy, longer duration of action, and excellent bioavailability and safety.

Description

Peptide tyrosine analogs and their applications Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to novel peptide-tyrosine analogs and applications thereof. Background Art

[0002] Peptide tyrosine yrosine (PYY) is a 36-amino acid gut hormone primarily synthesized and secreted by intestinal L cells (Spreckley E, Murphy KG. Front Nutr. 2015;2:23). Human PYY has two native forms: PYY1-36 and PYY3-36. PYY3-36 is the predominant circulating form, produced by dipeptidyl peptidase-4 (DPP-4) cleavage of the N-terminus of PYY1-36. PYY3-36 is Y2-selective and can inhibit NPY release by activating Y2 receptors on NPY-containing cells in the arcuate nucleus, thereby reducing appetite and promoting weight loss.

[0003] PYY3-36 can activate NPY2 receptors and reduce appetite. It has been studied as a potential therapeutic drug for weight regulation, especially for the treatment of obesity and obesity complications. However, there is still a need for PYY3-36 analogs that enhance NPY2 receptor selectivity and agonism.

[0004] Due to proteases and other clearance mechanisms, exogenous PYY3-36 has a half-life of approximately 10-15 minutes. Due to its short half-life, it requires at least daily administration to exert its therapeutic effect. Therefore, there is a need to increase the half-life of PYY3-36, for example by adding fatty acid side chains to increase the half-life of PYY3-36 analogs.

[0005] Despite the continuous deepening of research on the role of PYY3-36 in metabolism, there is still a need for better PYY3-36 analogs, especially PYY3-36 analogs with better NPY2 receptor activity and selectivity, stronger in vivo efficacy and longer half-life.

[0006] Summary of the Invention

[0007] The present invention provides a novel peptide tyrosine kinase (PYY 3-36 ) analogs or salts or solvates thereof, more specifically, this novel peptide tyrosine tyrosine (PYY 3-36 ) analogues have better efficacy, longer duration of action, excellent bioavailability and safety.

[0008] In one aspect, the present invention provides a novel peptide tyrosine tyrosine (PYY 3-36 ) analogs or salts or solvates thereof, comprising the following sequence structure of general formula (I):

[0009] PKPE-ψ-PEX 10 -DX 12 -SPEEWQRYYX 22 -X 23 -LRHYLNWLTRQRY-R1(I),

[0010] Wherein, ψ is a Lys with a side chain modified by a structure having the following general formula (II):

[0011] YZ(II), where Y is (AEEAc or Glu) a -(AEEAc or Glu) b -(AEEAc or Glu) c , wherein a, b, and c are each independently 0 or 1, and a, b, and c are not all 0 at the same time (as an example, it can be AEEAc-AEEAc-γGlu), the carboxyl end of Y is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m -COOH, m is an integer between 6 and 24;

[0012] X 10 is E or Aib;

[0013] X 12 A or Aib;

[0014] X 22 A or Aib;

[0015] X 23 is S or E;

[0016] R1 is NH2 or OH;

[0017] And X 10 、X 12 and X 22 At least one of them is Aib.

[0018] In the present invention, as one embodiment, Y in the general formula (II) is AEEAc-γGlu or AEEAc-AEEAc-γGlu, preferably AEEAc-γGlu.

[0019] In the present invention, as one embodiment, the analogue is preferably selected from:

[0020] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PEED-Aib-SPEEWQRYY-Aib-SLRHYLNWLTRQRY-NH2,

[0021] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYYAELRHYLNWLTRQRY-NH2, or

[0022] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2.

[0023] In the present invention, as one embodiment, the analogue is more preferably selected from:

[0024] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2.

[0025] The analogues of the present invention are chemically modified at a specific site X7 on the peptide backbone by a fatty acid side chain group, and at least one or more non-natural amino acids Aib are introduced at the specific site of the backbone. The above modifications enable the analogue peptide chain to have a more stable peptide α-helical structure and enhanced albumin binding ability, thereby achieving improved stability of the peptide analogue, enhanced in vivo efficacy and prolonged peptide action time.

[0026] The present invention provides a pharmaceutical composition comprising an effective amount of the analogue or a salt or solvate thereof according to any one of the aforementioned aspects, and a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

[0027] In the present invention, as one embodiment, the pharmaceutical composition is in the form of an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, microneedle, suspension or syrup; as one embodiment, the pharmaceutical composition is in the form of a microcapsule, microsphere, nanoparticle or liposome.

[0028] In the present invention, as one embodiment, the pharmaceutical composition is used for oral administration, inhalation administration, transdermal administration or parenteral administration; as one embodiment, the parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection.

[0029] In the present invention, as one embodiment, the pharmaceutical composition is administered at a frequency of at least once a day, or once a week.

[0030] In one aspect, the use of an analogue or a salt or solvate thereof as described in any of the foregoing aspects in the preparation of a medicament for treating, preventing or alleviating overweight and obesity or obesity-related diseases is provided. As one embodiment, the obesity-related diseases refer to any disease or condition caused or aggravated by obesity, including but not limited to angina pectoris, cardiovascular disease, cholecystitis, cholelithiasis, congestive heart failure, heart failure with preserved ejection fraction, dyslipidemia, non-alcoholic steatohepatitis, reproductive complications, glucose intolerance, gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, osteoarthritis, polycystic ovary syndrome, pregnancy complications, psychological disorders, sleep apnea and other respiratory problems, stress urinary incontinence, stroke, type II diabetes, uric acid kidney stones, breast cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, prostate cancer and rectal cancer.

[0031] In one aspect, the pharmaceutical composition can be used in combination with other drugs for treating the same or related diseases, including but not limited to one, two or more of metformin, sulfonylureas, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulins, GLP-1s, GCGs, GIPs, and FGF-21s.

[0032] "Overweight" or "obesity" as used herein refers to a condition where an individual's body mass index (BMI) exceeds a certain threshold. This threshold varies depending on race. For example, see the "Overweight & Obesity" guidelines of the Centers for Disease Control and Prevention and the "Definitions & Fcats for Adult Overweight & Obesity" of the National Institutes of Health. For Americans, a BMI > 30 kg / m 2 Obesity can be considered as a BMI of 25.0 kg / m 2 Up to 30kg / m 2 According to the standards of the National Health Commission of China, the BMI of Chinese people is 24.0kg / m 2 Up to 27.9kg / m 2 The condition is considered overweight, with a BMI ≥ 28.0 kg / m 2 The condition is obesity.

[0033] As used herein, "PYY" refers to peptide tyrosine tyrosine derived from any species. PYY includes native PYY (i.e., sequences 1-36, full length) and its variants (i.e., additions, deletions, and / or substitutions to native PYY). Specific PYYs include, but are not limited to, native human PYY. 1-36 and natural PYY 3-36 .

[0034] The "PYY" in the present invention3-36 "Analog" means that one or more NPY receptors (such as NPY2 receptors) produce a similar natural PYY 3-36 PYY agonist activity 3-36 In some cases, when compared to native human PYY 3-36 In comparison, the PYY in this article 3-36 Analogs may bind to NPY receptors, such as NPY2 receptors, with higher or lower affinity and demonstrate longer in vivo or in vitro half-lives. 3-36 The analogs are synthetic analogs that act as agonists of the NPY2 receptor.

[0035] The PYY in the present invention 3-36 The "agonistic activity" of an analog on the NPY receptor means that the analog can stimulate a specific NPY receptor (e.g., NPY2 receptor) cell to produce cAMP. The cells used can be host cells overexpressing a specific NPY receptor constructed by a person skilled in the art. The receptor agonistic activity can be measured by the EC of the analog to stimulate the receptor cell to produce cAMP. 50 The value is used as a measure. 50 The value refers to the drug concentration required to achieve half of the maximal activity (50% activity) of the analog in a specific assay system.

[0036] As used herein, the term "treat" or "treating" means to reduce, reverse, slow or stop the progression or severity of an existing condition, disease or symptom.

[0037] The abbreviations used in the present invention have the following specific meanings:

[0038] MBHA: 4-Toluene Hydrogenamine

[0039] DMF: N,N-dimethylformamide

[0040] Aib: α-amino isobutyric acid

[0041] AEEAc: [2-(2-amino-ethoxy)-ethoxy]-acetyl

[0042] cAMP: cyclic adenosine monophosphate

[0043] NPY2R: neuropeptide Y receptor 2

[0044] NPY1R: neuropeptide Y receptor 1

[0045] NPY5R: neuropeptide Y receptor 5

[0046] HEK-293: Human embryonic kidney cell line 293

[0047] PBS: Phosphate buffered saline

[0048] Forskolin: Forskolin

[0049] HBSS: Hank's balanced salt solution

[0050] HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer

[0051] IBMX: 3-isobutyl-1-methylxanthine

[0052] EC50: Half-maximal effect concentration

[0053] DPBS: Dulbecco's phosphate buffered saline

[0054] EDTA: Ethylenediaminetetraacetic acid

[0055] FLIPR: Fluorescence Imaging Detection System

[0056] LC-MS / MS: Liquid chromatography-mass spectrometry / mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1a. Cumulative food intake suppression effects of Compound 1, Compound 2, Compound 3, and Reference Example 1 in normal mice after a single injection. *P<0.05, **P<0.01, ***P<0.001 vs Vehicle, #P<0.05, ## P < 0.01, ### P<0.001vs Ref.1-3nmol / kg, ^P<0.05, ^^P<0.01, ^^^P<0.001vs Ref.1-10nmol / kg, & P < 0.05 vs Ref. 1-30 nmol / kg;

[0058] Figure 1b. Effects of Compound 1, Compound 2, Compound 3 and Reference Example 1 on weight loss in normal mice after a single injection. *P<0.05, **P<0.01, ***P<0.001 vs Vehicle, & P<0.05vs Ref.1-30nmol / kg.

[0059] Figure 2: Effects of combined treatment with compound 3 and the GLP-1 agonist semaglutide on body weight loss in normal SD rats. *P<0.05, **P<0.01, ***P<0.001 vs Vehicle, #P<0.05, ##P<0.01 vs Semaglutide_20 nmol / kg. DETAILED DESCRIPTION

[0060] The present invention is further described in detail by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0061] Example 1: Analog Synthesis

[0062] The analogs in the table below were all synthesized by chemical synthesis methods.

[0063] Table 1.1 PYY 3-36 List of analogs and molecular weight and purity

[0064] Note:

[0065] Reference Example 1 is selected from Example 4 of patent application US20200140514A1

[0066] Y of X36 in SEQ ID NO: 1 and SEQ ID NO: 2 is amidated;

[0067] In SEQ ID NO: 3 to SEQ ID NO: 5, K of X7 is through a fatty acid side chain group;

[0068] AEEAc-γGlu-CO(CH2) 16 The K side chain is modified by COOH, and the Y of X 36 is amidated;

[0069] In SEQ ID NO: 6, X7 is connected to the fatty acid side chain group AEEAc-AEEAc-γGlu-CO(CH2) 16 The side chain of K is modified with COOH, and the Y of X 36 is amidated.

[0070] Analog 1

[0071] PKPEK(AEEA-γGlu-CO-(CH2) 16 -COOH)PEED-Aib-SPEEWQRYY-Aib-SLRHYLNWLTRQRY-NH2

[0072] Analog 1 was synthesized using standard Fmoc chemistry.

[0073] 1) Resin preparation: MBHA resin (0.28 mmol, 1.00 eq, Sub 0.28 mmol / g) was added to a certain amount of DMF and stirred at 20°C under N2 for 2 hours. The mixture was then filtered to obtain the desired resin.

[0074] 2) Deprotection: Add 15.0 mL of DMF containing 20% ​​piperidine to the above resin, stir the resin under N2 for 15 minutes, wash with DMF (15.0 mL x 5), and filter to obtain the deprotected resin.

[0075] 3) Coupling: Dissolve Fmoc-Tyr(tBu)-OH (3.00 eq), DIEA (6.00 eq), and HBTU (2.85 eq) in DMF (4.00 mL) to prepare a solution. Add the resin obtained in step 2) and stir at 20°C under N2 for 20 minutes. Rinse the resin with DMF (15.0 mL x 5).

[0076] 4) Repeat steps 2)-3) and add a certain amount of raw materials in the order shown in the table below to couple the following amino acids to synthesize a peptide chain:

[0077] 5) Add 3% N2H4·H2O / DMF solution to the above reaction system, and react twice with N2 for 30 minutes each time. Rinse with DMF five times.

[0078] 6) Repeat steps 2)-3), add the raw materials in the order shown in the table below, couple the following groups, and modify the peptide chain with fatty acid side chains:

[0079] Peptide chain cleavage and purification

[0080] 1) After the final step of peptide chain synthesis, the resin was washed with MeOH (25.0 mL x 3) and dried under vacuum. The peptide-resin was then treated with lysis buffer (92.5% TFA / 2.5% 3-MPA / 2.5% TiS / 2.5% H2O) for 2 hours.

[0081] 2) Precipitate the peptide chain with an appropriate amount of cold isopropyl ether, centrifuge (3000 rpm for 2 minutes), wash twice with isopropyl ether, and dry the crude peptide under vacuum for 2 hours.

[0082] 3) The crude peptide was purified by high-performance liquid chromatography (HPLC) (A: 0.075% TFA in H₂O, B: ACN) to obtain analog 1 as a white solid (122.1 mg, 17.60 μmol, 6.29% yield, 97.73% purity). LCMS confirmed the identity of the obtained analog 1. The measured molecular weight was 1234.9 [M+4H]⁻¹⁺, and the theoretical molecular weight was 4935.59.

[0083] The intermediates and analogs in the present invention are synthesized according to the above methods. The specific synthesis steps can be combined with different materials and methods to synthesize a variety of corresponding analogs or salts thereof according to the present invention.

[0084] Analog 2

[0085] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYYAELRHYLNWLTRQRY-NH2

[0086] Analog 2 was synthesized using standard Fmoc chemistry, and the peptide chain synthesis, cleavage and purification procedures were the same as those described for Analog 1.

[0087] The crude peptide was purified by HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain a white solid product, PY031 (134 mg, 26.70 μmol, 6.67% yield, 95.99% purity). LCMS confirmed that the obtained analogue was analogue 2. The measured molecular weight was 1227.4 [M+4H] 4+ , the theoretical molecular weight is 4905.56.

[0088] Analog 3

[0089] PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2

[0090] Analog 3 was synthesized using standard Fmoc chemistry, and the peptide chain synthesis, cleavage and purification procedures were the same as those described for analog 1.

[0091] The crude peptide was purified by HPLC (A: 0.075% TFA in H2O, B: ACN) to obtain analog 3 as a white solid (127.6 mg, 25.93 μmol, 6.48% yield, 95.55% purity). LCMS confirmed the identity of the obtained analog 3. The measured molecular weight was 1230.8 [M+4H] 4+ , the theoretical molecular weight is 4919.59.

[0092] Example 2: PYY 3-36 In vitro activity of analogs

[0093] 1. cAMP assay to determine the in vitro functional activity of PYY analogs at the NPY2 receptor

[0094] Objective: To determine the effect of the PYY analogues of Example 1 on the activity of native human PYY by measuring the inhibition of forskolin-induced intracellular cAMP production in HEK 293 cells overexpressing recombinant human NPY2 receptor. 3-36 Compared with the in vitro functional activity.

[0095] 1) Analog preparation: The analog to be tested was diluted 4-fold with 0.5 M Tris-HCl in an analog plate by Bravo to 10 concentration points for use.

[0096] 2) Cell Preparation: Collect the cultured cells into a 15 mL sterile centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant. Add 10 mL of 1× PBS, mix gently, and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant.

[0097] 3) Resuspend the cells in assay buffer (20 mM HEPES, 1× HBSS, 0.1% casein, 0.5 mM IBMX), count the cells using a Vi-cell counter, and dilute the cells to 0.6×10 6 / mL.

[0098] 4) According to the order of the analogs in the Map, 100 nL of the prepared analogs were added to the 384-well cell reaction plate using Echo. After addition, centrifuge at 1000 rpm for 1 minute.

[0099] 5) Using a Multidrop Combi, add 5 μL of 2 μM Forskolin (prepared in assay buffer) to a 384-well cell reaction plate. Centrifuge at 1000 rpm for 1 minute. Add 5 μL of assay buffer containing cells to the 384-well plate, so that each well contains 3000 cells. Seal the plate with transparent sealing film and incubate in a room temperature incubator for 45 minutes.

[0100] 6) Using a Multidrop Combi, add 10 μL of cAMP detection reagent solution (9.5 mL of 1× cell lysis buffer, 1× D2-cAMP solution, and 1× cAMP-antibody) to the corresponding wells of the cell reaction plate. Cover the plate and incubate at room temperature in the dark for 1 hour.

[0101] 7) After 1 hour of incubation, place the plate in an EnVision microplate reader and read the final value as the ratio of OD at 665nm to OD at 615nm.

[0102] 8) Calculate the cAMP value for each well: % Activity = (sample well signal value - average low-signal control well signal value) / (average high-signal control well signal value - average low-signal control well signal value). The EC50 of each sample was calculated using the "Nonlinear regression (curve fit) - log (agonist) vs. response - Variable slope" model in GraphPad Prism 5.0, with the logarithmic value of the analog concentration as the abscissa and the cAMP activation rate as the ordinate.

[0103] result:

[0104] Table 1. In vitro cAMP activity of PYY analogs on hNPY2R receptor (mean values)

[0105] Note: Each analogue was tested at least 3 times independently

[0106] 2. Calcium flux method to determine the in vitro functional activity of PYY analogs at human NPY1 and Y5 receptors:

[0107] Day 1: Cell plating

[0108] 1) Preheat the culture medium, 1× DPBS, and 0.05% trypsin-EDTA in a 37°C water bath for at least 30 minutes and set aside.

[0109] 2) Remove the preheated culture medium, 1× DPBS, and 0.05% trypsin-EDTA, disinfect with 75% alcohol, and place in a biosafety cabinet.

[0110] 3) Remove the cultured cells from the incubator, add 1× DPBS, incubate for a while, then aspirate and add an appropriate amount of 0.05% trypsin-EDTA to digest the cells. Terminate the digestion with 10% FBS medium and disperse the cells.

[0111] 4) Use a pipette to draw the dispersed cells into a 50 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, resuspend in culture medium, disperse, and count.

[0112] 5) Dilute the cells to 1×10 6 cells / mL, and seeded cells at 20 μL / well in a 384-well poly-lysine-coated cell plate.

[0113] 6) Incubate in a 5% CO2, 37°C incubator overnight.

[0114] Day 2: FLIPR Experiment

[0115] Reagent preparation:

[0116] 1) Prepare 250 mM probenecid solution: According to the kit instructions, add 1 mL of FLIPR buffered saline to 77 mg of probenecid.

[0117] 2) Prepare 2× (8 μM) Fluo-4 Direct™ loading buffer: Thaw the number of Fluo-4 Direct™ tubes required for the experiment in advance, add 10 mL of FLIPR saline buffer to each tube, add 0.2 mL of 250 mM probenecid solution, and vortex for >5 minutes in the dark.

[0118] Analog detection method:

[0119] 1) Preparation of the test analogs and yang ginseng agonists: Dilute the test analogs 4-fold to 10 concentrations using Bravo. Transfer 900 nL to the analog plate using Echo. Add 30 μL of assay buffer (20 mM HEPES, 1× HBSS, 0.1% casein) to each well. The starting concentration of the test analogs and yang ginseng agonists was 1 μM.

[0120] 2) Remove the cell plate from the incubator (cell Y1 requires the removal of culture medium and the addition of 20 μL of detection buffer, otherwise the signal will be high; Y5 does not require this). Add 2x Fluo-4 detection reagent, 20 μL per well, and incubate in a 37°C incubator for 50 minutes, then let stand at room temperature for 10 minutes.

[0121] 3) Place the cell plate, analog plate, and pipette tip into the FLIPR instrument, start the instrument, and transfer 10 μL of analog from the analog plate to the cell plate. On the FLIPR instrument, excite the calcium dye to produce an emission wavelength of 515-575 nm under an excitation wavelength of 470-496 nm, and read the fluorescence signal.

[0122] 4) Calculate the calcium ion activation rate for each well: % Activity = (sample well signal value - average low-signal control well signal value) / (average high-signal control well signal value - average low-signal control well signal value). The logarithm of the analog concentration was plotted on the horizontal axis, and the activation rate of the calcium ion fluorescence signal was plotted on the vertical axis. The EC50 of the sample was calculated using the "Nonlinear regression (curve fit) - log (agonist) vs. response - variable slope" model in GraphPad Prism 5.0.

[0123] result:

[0124] Table 2. In vitro cAMP activity of PYY analogs at hNPY1R and Y5 receptors (mean values)

[0125] Note: Each analogue was tested at least three times independently, and ND means not detected.

[0126] As can be seen from the data in Tables 1 and 2, the PYY3-36 analogs of the present invention show high selectivity for NPY2R, but low selectivity for NPY1R and NPY5R.

[0127] Example 3: PYY 3-36 In vivo efficacy testing of analogs

[0128] 1. Effects on food intake and body weight of normal mice

[0129] Objective: To compare the effects of analogs 1, 2, 3 and reference example 1 on body weight loss and food intake suppression in normal mice after a single injection.

[0130] method:

[0131] Male C57BL / 6 mice, 6 to 8 weeks old, were housed in a strictly controlled vivarium with a temperature of 20–24°C and a humidity of 30–70%. The vivarium had a 12-hour light cycle (7:00 AM – 7:00 PM). During the experiment, animals were housed individually in cages and provided with toys. Animals had free access to food (mouse and mouse growth / breeding diet) and water.

[0132] Before dosing, non-fasted body weight and initial food weight were recorded. Following a single subcutaneous injection, body weight and food intake were recorded daily for 3 days. Animal activity, water and diet intake, and body weight changes were observed daily. Changes in body weight and food intake compared to the baseline levels (initial body weight and food intake before dosing) of each group were calculated, and body weight changes were expressed as percentages (baseline being 100%).

[0133] Results: See Table 3, Figure 1a, Figure 1b.

[0134] Table 3 Single subcutaneous injection of PYY 3-36 Body weight changes and cumulative food intake of C57BL / 6 mice after 3 days of treatment

[0135] The data in Figure 1a, Figure 1b and Table 3 strongly support the PYY 3-36 The analogs inhibit food intake and reduce body weight in animals.

[0136] 2. In normal SD rats, PYY 3-36 Pharmacodynamic effects of combination therapy with semaglutide and the GLP-1 agonist semaglutide.

[0137] Objective: To identify PYY 3-36 Effects of combined treatment with a GLP-1 agonist, semaglutide, and a GLP-1 analog on body weight loss and food intake suppression in normal SD rats.

[0138] method:

[0139] Male Sprague-Dawley rats, 6 to 8 weeks old, were housed in a strictly controlled animal enclosure with a temperature of 20 to 24°C and a humidity of 30 to 70%. The lighting cycle in the enclosure was 12 hours (7:00 AM to 7:00 PM). During the experiment, animals were housed in individual cages and provided with toys. Animals had free access to food (mouse and mouse growth / breeding diet) and water.

[0140] Analog 3 was tested at 15 nmol / kg of analog 3 and 20 nmol / kg of the GLP-1 agonist Semaglutide (SEQ ID NO: 7) (Note: X26 in SEQ ID NO: 7 is connected to the fatty acid side chain group AEEAc-AEEAc-γGlu-CO(CH2) 16 The combination of 1,2-dimethoprim-1 (1,2-dimethoprim-1) and 1,2-dimethoprim-1 (1,2-dimethoprim-1) (with COOH-modified K side chains) was administered subcutaneously once daily for 8 days. Animal body weight was monitored daily during dosing. Body weight change compared to baseline (initial weight before dosing) was calculated and expressed as a percentage (baseline = 100%).

[0141] Results: See Table 4 and Figure 2.

[0142] Table 4 Body weight changes in normal SD rats after combined treatment with analog 3 and GLP-1 agonist

[0143] The data in Table 4 and Figure 2 show that the weight loss of animals after administration of Semaglutide combined with "Analog 3" compared with the administration of GLP-1 agonist Semaglutide alone confirms the PYY 3-36 In vivo efficacy of analogs.

[0144] Example 4: PYY 3-36 Pharmacokinetic studies of analogues

[0145] Objective: To study the pharmacokinetic properties of PYY analogs.

[0146] method:

[0147] Blood samples were collected from each animal at each time point after dosing and centrifuged at 3200 g for 10 minutes at approximately 4°C. Plasma was collected separately, transferred to pre-labeled 96-well plates or polypropylene tubes, quickly frozen on dry ice, and stored at -60°C or lower until LC-MS / MS analysis.

[0148] LC-MS / MS was used to determine the concentration of the test analog in blood samples. Plasma concentration data over time were plotted and analyzed using a non-partitioned approach using Phoenix WinNonlin 6.3 software. PK parameters were calculated based on the route of administration.

[0149] 1. Pharmacokinetics of PYY analogs in SD rats

[0150] The plasma pharmacokinetics of the PYY analog were evaluated in male Sprague-Dawley rats following a single subcutaneous dose of 0.492 mg / kg of Analog 3. Blood samples were collected from three rats at each time point for 168 hours, 0.5, 1, 2, 4, 8, 12, 16, 24, 48, 72, 96, 120, 144, and 168 hours after dosing. Because non-continuous sampling was used to evaluate the kinetics of the PYY analog in rats, the pharmacokinetic parameters of Analog 3 following a single subcutaneous dose of 0.492 mg / kg are presented using mean concentration-time data.

[0151] 2. Pharmacokinetics of PYY analogs in cynomolgus monkeys

[0152] The plasma pharmacokinetics of the PYY analog were evaluated in male cynomolgus monkeys following a single subcutaneous dose of 0.246 mg / kg of Analog 3. Blood samples were collected from two cynomolgus monkeys at each time point, 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144, 168, 192, 240, 288, 336, 408, and 504 hours after dosing, for a total of 504 hours. Because non-continuous sampling was used to evaluate the kinetics of the PYY analog in cynomolgus monkeys, the pharmacokinetic parameters of Analog 3 following a single subcutaneous dose of 0.246 mg / kg are presented using mean concentration-time data.

[0153] result:

[0154] Table 5. Mean pharmacokinetic parameters of analog 3 in male rats and male cynomolgus monkeys after a single subcutaneous dose

[0155] Abbreviation: AUC inf = the area under the curve from 0 to infinity; C max = maximum concentration; T max = time at maximum concentration; T 1 / 2 = half-life.

[0156] The data in Table 5 demonstrate that the PYY analogs of the present invention have a pharmacokinetic profile suitable for once-weekly administration.

Claims

1. A novel peptide YY 3-36 ) analogs or salts or solvates thereof, which contain the following sequence structure of general formula (I): PKPE-ψ-PE-X10-D-X12-SPEEWQRYY-X22-X23-LRHYLNWLTRQRY-R1(I), in, ψ is a Lys with a side chain modified by a structure having the following general formula (II): YZ(II), where Y is (AEEAc or Glu) a -(AEEAc or Glu) b -(AEEAc or Glu) c , wherein a, b, and c are each independently 0 or 1, and a, b, and c are not 0 at the same time, the carboxyl end of Y is connected to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m -COOH, m is an integer between 6 and 24, X 10 is E or Aib; X 12 is A or Aib; X 22 is A or Aib; X 23 is S or E; R1 is NH2 or OH; And X 10 , X 12 and X 22 At least one of them is Aib.

2. The peptide YY according to claim 1 3-36 ) analog or a salt or solvate thereof, characterized in that In the general formula (II), Y is AEEAc-γGlu or AEEAc-AEEAc-γGlu, preferably AEEAc-γGlu.

3. The peptide YY according to claim 1 3-36 ) analog or a salt or solvate thereof, characterized in that The analogue is selected from: PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PEED-Aib-SPEEWQRYY-Aib-SLRHYLNWLTRQRY-NH2、 PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYYAELRHYLNWLTRQRY-NH2, or PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2。 4. The peptide YY according to claim 1 3-36 ) analog or a salt or solvate thereof, characterized in that The analogue is selected from PKPEK (AEEAc-γGlu-CO (CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2.

5. A pharmaceutical composition comprising an effective amount of the analogue or salt or solvate thereof according to any one of claims 1 to 4, and a pharmaceutically acceptable adjuvant, diluent, carrier or excipient.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of an injection, lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, solution, microneedle, suspension or syrup.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of microcapsules, microspheres, nanoparticles or liposomes.

8. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is used for oral administration, inhalation administration, transdermal administration or parenteral administration.

9. The pharmaceutical composition according to claim 8, characterized in that The parenteral administration is selected from intraperitoneal, intramuscular, intraarterial, intravenous, subcutaneous or intradermal injection administration.

10. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is administered at a frequency of at least once a day, or once a week.

11. Use of the analogue or salt or solvate thereof according to any one of claims 1 to 4 or the pharmaceutical composition according to any one of claims 5 to 10 in the preparation of a drug for treating, preventing or alleviating overweight and obesity or complications of obesity.

12. The use according to claim 11, characterized in that The complications of obesity include angina pectoris, cardiovascular disease, cholecystitis, cholelithiasis, congestive heart failure, heart failure with preserved ejection fraction, dyslipidemia, non-alcoholic steatohepatitis, fertility complications, glucose intolerance, gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, osteoarthritis, polycystic ovary syndrome, pregnancy complications, psychological disorders, sleep apnea and other respiratory problems, stress urinary incontinence stroke, type II diabetes, uric acid kidney stones, breast cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, prostate cancer or rectal cancer.

13. The use according to claim 11, characterized in that The pharmaceutical composition can be used in combination with other drugs for treating the same or related diseases, and the other drugs are selected from one, two or more of metformin, sulfonylureas, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulins, GLP-1s, GCGs, GIPs, and FGF-21s.