Peptide tyrosine-tyrosine analogs and their uses

Abstract

The present invention relates to the pharmaceutical field, and specifically, to the following general formula (I): P-K-P-E-ψ-P-E-X 10 -D-X 12 -S-P-E-E-W-Q-R-Y-Y-X 22 -X 23 -L-R-H-Y-L-N-W-L-T-R-Q-R-Y-R1(I) A novel peptide tyrosine-tyrosine (PYY 3~36 ) analog or a salt or solvate thereof is provided, which contains the array structure as described above. The novel peptide YY analog (PYY 3~36 ) provided by the present invention has better pharmacological effects, longer action time, excellent bioavailability and safety.

Description

[Technical Field]

[0001] This invention relates to the field of biomedicine, and more specifically to novel peptide tyrosine-tyrosine analogs and their uses. [Background technology]

[0002] Peptide tyrosine-tyrosine (PYY) is a 36-amino acid enteric hormone primarily synthesized and secreted by intestinal L cells (Spreckley E, Murphy KG. Front Nutr. 2015;2:23). Two native forms of human PYY exist: PYY1-36 and PYY3-36. PYY3-36 is the major circulating form, produced by the N-terminal cleavage of PYY1-36 by dipeptidyl peptidase-4 (DPP-4). PYY3-36 exhibits Y2 selectivity 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 the NPY2 receptor and reduce appetite. Currently, PYY3-36 is being studied as a potential therapeutic agent for weight regulation, particularly for treating obesity and its complications. However, there remains a need for PYY3-36 analogs with improved NPY2 receptor selectivity and stimulating activity.

[0004] Due to protease and other clearance mechanisms, the half-life of exogenous PYY3-36 is approximately 10-15 minutes. Because of this short half-life, exogenous PYY3-36 needs to be administered at least once daily to exert its therapeutic effect. Therefore, it is necessary to extend the half-life of PYY3-36, for example, by adding fatty acid side chains to a PYY3-36 analog. [Prior art documents] [Non-patent literature]

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] Despite the continuous deepening of research on the role of PYY3-36 in metabolism, there is still a need for better PYY3-36 analogs, particularly those with better NPY2 receptor activity and selectivity, more potent in vivo efficacy, and longer half-life.

Means for Solving the Problems

[0007] The present invention aims to provide a novel peptide tyrosine-tyrosine (PYY b , 23 , 12 , , , c , a , 22 , , , 10 ,

[0008] ) analog or a salt or solvate thereof. More specifically, this novel peptide tyrosine-tyrosine (PYY 3~36 ) analog has better pharmacological effects, longer action time, excellent bioavailability and safety.

[0008] In one aspect, the present invention provides the following general formula (I): ​​​​​​​​​​​​​​​​​​(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 exemplary example, AEEAc-AEEAc-γGlu)), the carboxyl terminus of Y is linked to the ε-amino group of the side chain of Lys, and Z is -CO-(CH2) m - A Lys modified with the structure COOH, where 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 either S or E; R1 is either NH2 or OH; X 10 , X 12 , and X 22 (At least one of which is Aib) includes a sequence structure Novel peptide tyrosine-tyrosine (PYY 3~36 ) Provides analogues or salts or solvates thereof.

[0009] In one embodiment of the present invention, Y in general formula (II) is AEEAc-γGlu or AEEAc-AEEAc-γGlu, preferably AEEAc-γGlu.

[0010] In the present invention, as one embodiment, an analog is preferably 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) 16COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2 Selected from.

[0011] In the present invention, as one embodiment, an analog is more PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2 Selected from.

[0012] The analog described in the present invention is chemically modified at a specific site X7 of the peptide skeleton with a fatty acid side chain group, and at least one non-natural amino acid Aib is introduced at the specific site of the skeleton. The above modifications enable the peptide chain of the analog to have a more stable peptide alpha-helix structure and improved albumin-binding ability, thereby improving the stability of the peptide analog, enhancing the in vivo efficacy, and extending the duration of action of the peptide.

[0013] The present invention provides a pharmaceutical composition comprising an effective amount of any of the above analogues or their salts or solvates, and a pharmaceutically acceptable excipient, diluent, carrier, or pharmaceutical adjuvant.

[0014] In the present invention, one embodiment of the pharmaceutical composition is in the form of an injection or lyophilized powder, tablet, pill, lozenge, soft capsule, hard capsule, granule, powder, liquid, microneedle, suspension or syrup; another embodiment of the pharmaceutical composition is in the form of a microcapsule, microsphere, nanoparticle or liposome.

[0015] In the present invention, as one embodiment, the pharmaceutical composition is used for oral administration, inhalation administration, transdermal administration, or parenteral administration, and as one embodiment, parenteral administration is selected from intraperitoneal, intramuscular, intra-arterial, intravenous, subcutaneous, or intradermal injection.

[0016] In one embodiment of the present invention, the pharmaceutical composition is administered at a frequency of at least once a day or once a week.

[0017] In one embodiment, the use of an analog or a salt or solvate thereof described in any of the preceding embodiments is provided in the preparation of a drug for the treatment, prevention or reduction of overweight and obesity or obesity-related complications. In one embodiment, obesity-related disorders refer to, but are not limited to, any disease or disorder caused by or exacerbated by obesity, including, angina pectoris, cardiovascular disease, cholecystitis, cholelithiasis, congestive heart failure, heart failure with preserved ejection fraction, dyslipidemia, non-alcoholic steatohepatitis, fertility complication, impaired glucose tolerance, 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 mellitus, uric acid kidney stones, breast cancer, colon cancer, endometrial cancer, esophageal cancer, gallbladder cancer, kidney cancer, prostate cancer, or rectal cancer.

[0018] In one embodiment, the pharmaceutical composition may be used in combination with other drugs for treating the same or related disease and may include, but is not limited to, one or more of metformin, sulfonylurea, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulin, GLP-1, GCG, GIP, and FGF-21.

[0019] As used herein, the terms “overweight” or “obesity” refer to a condition in which an individual’s body mass index (BMI) exceeds a certain threshold. This threshold varies depending on race. For example, the U.S. Centers for Disease Control and Prevention’s “Overweight & Obesity” and the U.S. National Institutes of Health’s “Definitions & Facts for Adult Overweight & Obesity” may be referenced. For example, a BMI of 30 kg / m² for an American is considered to be 30 kg / m². 2 If the BMI is above 25.0 kg / m², it may be considered obese. 2 ~30kg / m 2 If so, that person may be considered overweight. According to the standards of the National Health Commission of the People's Republic of China, a Chinese person with a BMI of 24.0 kg / m² is considered overweight. 2 ~27.9 kg / m 2 If so, you are overweight, and your BMI is ≥28.0 kg / m². 2 Therefore, they are obese.

[0020] As used herein, the term "PYY" means peptide tyrosine-tyrosine derived from any species. PYY includes natural PYY (i.e., sequences 1-36, full length) and its variants (i.e., additions, deletions, and / or substitutions of natural PYY), including natural human PYY. 1~36 and natural human PYY 3~36 This includes, but is not limited to, those items.

[0021] The term "PYY" as used in this application 3~36 The "analog" is natural PYY 3~36 To produce stimulatory activity similar to that induced by PYY, one or more NPY receptors (e.g., NPY2 receptor) are induced. 3~36This refers to peptides or polypeptides of a certain type. In some cases, natural human PYY 3~36 In contrast to the PYY described herein 3~36 The analogs may bind to NPY receptors, such as the NPY2 receptor, with higher or lower affinity, while exhibiting longer in vivo or in vitro half-lives. Therefore, the PYY described herein 3~36 The analog is a synthetic analog that acts as an NPY2 receptor agonist.

[0022] The PYY receptor described herein 3~36 The "stimulatory activity" induced by the analog refers to the analog's ability to stimulate specific NPY receptor (e.g., NPY2 receptor) cells to produce cAMP. The cells used may be host cells that overexpress a specific NPY receptor, as constructed by those skilled in the art. The receptor's stimulatory activity is the EC of cAMP produced by the receptor cells stimulated by the analog. 50 It can be measured using the value EC. 50 The value refers to the drug concentration required to achieve half of the analog's maximum activity (50% activity) in a specific assay system.

[0023] As used herein, the term “treatment” means to reduce, reverse, slow down, or halt the progression or severity of an existing condition, disease, or symptom.

[0024] The specific meanings of the abbreviations used in this invention are as follows: MBHA: 4-toluenehydroamine DMF: N,N-dimethylformamide Aib: α-aminoisobutyric acid AEEAc:[2-(2-amino-ethoxy)-ethoxy]-acetyl cAMP: Cyclic adenosine monophosphate NPY2R: Neuropeptide Y receptor 2 NPY1R: Neuropeptide Y receptor 1 NPY5R: Neuropeptide Y receptor 5 HEK-293: Human fetal kidney cells 293 PBS: Phosphate-buffered saline Forskolin: HBSS: Hanks equilibrium salt solution HEPES: (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer IBMX:3-Isobutyl-1-methylxanthine EC 50 : Semi-effective concentration DPBS: Dulbecco's phosphate-buffered saline EDTA: Ethylenediaminetetraacetic acid FLIPR: Fluorescence Imaging Plate Reader LC-MS / MS: Liquid Chromatography Mass Spectrometry / Hyphenated Mass Spectrometry [Brief explanation of the drawing]

[0025] [Figure 1a] This figure shows the effects of compound 1, compound 2, compound 3, and reference example (Ref.) 1 on suppressing the accumulation of food intake after a single injection in normal mice. *P<0.05, **P<0.01, ***P<0.001 relative to vehicle (solvent); #P<0.05, ##P<0.01, ###P<0.001 relative to Ref.1-3 nmol / kg; ^P<0.05, ^^P<0.01, ^^^P<0.001 relative to Ref.1-10 nmol / kg; &P<0.05 relative to Ref.1-30 nmol / kg. [Figure 1b] This figure shows the effect of Compound 1, Compound 2, Compound 3, and Reference Example 1 on weight loss after a single injection in normal mice. *P<0.05, **P<0.01, ***P<0.001 relative to vehicle (solvent), and &P<0.05 relative to Ref.1-30 nmol / kg. [Figure 2] This figure shows the effect of combination therapy with compound 3 and the GLP-1 agonist semaglutide on weight loss in normal SD rats. *P<0.05, **P<0.01, ***P<0.001 relative to vehicle (solvent), and #P<0.05, ##P<0.01 relative to 20 nmol / kg of semaglutide. [Modes for carrying out the invention]

[0026] The present invention will be further described in detail below by the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. (Example 1)

[0027] Synthesis of analogs The analogues listed in the table below were synthesized by chemical synthesis.

[0028] [Table 1]

[0029] Note: Reference Example 1 was selected from Example 4 of U.S. Patent Application Publication No. 20200140514.

[0030] In sequence numbers 1 and 2, Y in X36 was amidated; In sequence numbers 3 through 5, the K of X7 passed through the fatty acid side chain group; AEEAc-γGlu-CO(CH2) 16 COOH modifies the K side chain, and Y in X36 is amidated; In Sequence ID No. 6, X7 is the fatty acid side chain group AEEAc-AEEAc-γGlu-CO(CH2) 16 The COOH group modified the K side chain, and the Y group in X36 was amidated.

[0031] analogue 1 PKPEK(AEEA-γGlu-CO-(CH2) 16 -COOH)PEED-Aib-SPEEWQRYY-Aib-SLRHYLNWLTRQRY-NH2 Analogue 1 was synthesized using the standard Fmoc chemical method.

[0032] 1) Resin preparation: A fixed amount of DMF was added to MBHA resin (0.28 mmol, 1.00 equivalent, substituted (Sub) 0.28 mmol / g), and the mixture was stirred under N2 at 20°C for 2 hours. The mixture was filtered to obtain the desired resin.

[0033] 2) Deprotection: 15.0 mL of DMF containing 20% ​​piperidine was added to the resin described above. The resin was stirred under N2 for 15 minutes, washed with DMF (15.0 mL x 5), filtered, and a deprotected resin was obtained.

[0034] 3) Coupling: Dissolve Fmoc-Tyr(tBu)-OH (3.00 equivalents), DIEA (6.00 equivalents), and HBTU (2.85 equivalents) in DMF (4.00 mL) to prepare a solution, add the resin obtained in step 2), and stir under N2 at 20°C for 20 minutes. Rinse the resin with DMF (15.0 mL x 5).

[0035] 4) Steps 2) and 3) were repeated, and the specified amounts of raw materials were added in the order listed in the table below. The following amino acids were then coupled to synthesize peptide chains.

[0036] [Table 2] JPEG2026512033000004.jpg152170

[0037] 5) A 3% N2H4·H2O / DMF solution was added to the above reaction system and reacted under N2 conditions twice for 30 minutes each time. The mixture was rinsed five times with DMF.

[0038] 6) Steps 2) and 3) were repeated, and the raw materials were added in the order listed in the table below. The peptide chains were modified with fatty acid side chains by coupling the following groups.

[0039] [Table 3]

[0040] Peptide chain cleavage and purification 1) After the final step of peptide chain synthesis was completed, the resin was washed with MeOH (25.0 mL x 3) and vacuum-dried. 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.

[0041] 2) The peptide chain was precipitated with an appropriate amount of cold isopropyl ether, centrifuged (at 3000 rpm for 2 minutes), and washed twice with isopropyl ether. The crude peptide was dried under vacuum for 2 hours.

[0042] 3) The crude peptide was purified using high-performance liquid chromatography (A: 0.075% TFA in H2O, B: ACN) to obtain analogue 1 as a white solid product (122.1 mg, 17.60 μmol, yield 6.29%, purity 97.73%). The obtained analogue was confirmed to be analogue 1 by LC-MS. The measured molecular weight was 1234.9 [M+4H]. 4+ The theoretical molecular weight was 4935.59.

[0043] All intermediates and analogues in the present invention are synthesized by the methods described above, and specific synthesis steps may employ different combinations of materials and methods to synthesize various corresponding analogues or salts thereof as described herein.

[0044] Analogue 2 PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYYAELRHYLNWLTRQRY-NH2 Analogue 2 was synthesized using the standard Fmoc chemical method, with the peptide chain synthesis, cleavage, and purification steps being the same as those described for Analogue 1.

[0045] The crude peptide was purified using high-performance liquid chromatography (A: 0.075% TFA in H2O, B: ACN) to obtain the white solid product PY031 (134 mg, 26.70 μmol, yield 6.67%, purity 95.99%). The obtained analog was identified as analog 2 by LC-MS. The measured molecular weight was 1227.4[M+4H]. 4+ The theoretical molecular weight was 4905.56.

[0046] analogue 3 PKPEK(AEEAc-γGlu-CO(CH2) 16 COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH2 Analogue 3 was synthesized using the standard Fmoc chemical method, with the peptide chain synthesis, cleavage, and purification steps being the same as those described for Analogue 1.

[0047] The crude peptide was purified using high-performance liquid chromatography (A: 0.075% TFA in H2O, B: ACN) to obtain analogue 3 as a white solid product (127.6 mg, 25.93 μmol, yield 6.48%, purity 95.55%). The obtained analogue was confirmed to be analogue 3 by LC-MS. The measured molecular weight was 1230.8[M+4H]. 4+ The theoretical molecular weight was 4919.59. (Example 2)

[0048] PYY 3~36 In vitro activity of analogues 1. cAMP assay to determine the in vitro functional activity of PYY analogs against the NPY2 receptor.

[0049] Objective: To measure the inhibition of forskolin-induced intracellular cAMP production in HEK293 cells overexpressing recombinant human NPY2 receptor, thereby determining the efficacy of PYY analogs from Example 1 against natural human PYY 3~36 Determination of in vitro functional activity compared to [another method].

[0050] 1) Preparation of analogues: Using Bravo, the test analogues were diluted four-fold to 10 concentration points in an analogue plate using 0.5 M Tris HCl to make them ready for use.

[0051] 2) Cell preparation: The cultured cells were collected in a 15 mL sterile centrifuge tube and centrifuged at 1000 rpm for 5 minutes, then the supernatant was discarded. 10 mL of 1 × PBS was added and gently mixed, then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded.

[0052] 3) Resuspend the cells in detection buffer (20 mM HEPES, 1 × HBSS, 0.1% casein, 0.5 mM IBMX), count them with a Vi cell counter, and measure 0.6 × 10⁻¹ using the detection buffer. 6 Diluted to / mL.

[0053] 4) Following the arrangement order of the Map analogs, 100 nL of the prepared analog was added to a 384-well cell reaction plate using Echo. After addition, the plate was centrifuged at 1000 rpm for 1 minute.

[0054] 5) Using a Multidrop Combi, 5 μL of 2 μM forskolin (prepared using detection buffer) was added to a 384-well cell reaction plate and centrifuged at 1000 rpm for 1 minute. 5 μL of detection buffer containing cells was added to the 384-well reaction plate to ensure a cell count of 3000 cells per well. The plates were sealed with clear sealing film and incubated in a room temperature incubator for 45 minutes.

[0055] 6) Using a Multidrop Combi, 10 μL of cAMP detection reagent solution (9.5 mL of 1 × cell lysis buffer, 1 × D2-cAMP solution, 1 × cAMP antibody) was added to the corresponding wells of the cell reaction plate. The plate was covered with a lid and incubated in the dark at room temperature for 1 hour.

[0056] 7) After a 1-hour incubation, the plates were placed in the EnVision microplate reader for measurement. The final value was the ratio of OD665nm to OD615nm.

[0057] 8) The cAMP value was calculated for each well: % activity = (signal value of sample well - mean signal of low-signal control well) / (mean signal of high-signal control well - mean signal of low-signal control well). Using the logarithm of the analog concentration on the x-axis and the cAMP activation rate on the y-axis, the "nonlinear regression (curve fitting) - log(antigen) vs. reaction variable gradient" model in GraphPad Prism 5.0 was used for fitting to the EC of the sample. 50 I calculated it.

[0058] result:

[0059] [Table 4]

[0060] 2. Determination of in vitro functional activity of PYY analogs against human NPY1 and Y5 receptors using calcium flux assay.

[0061] method: Day 1: Cell Plating 1) Culture medium, 1× DPBS, and 0.05% trypsin EDTA, preheated in a 37°C water bath for over 30 minutes, ready for use.

[0062] 2) The preheated culture medium, 1× DPBS, and 0.05% trypsin EDTA were removed, disinfected with 75% alcohol, and placed in a safety cabinet.

[0063] 3) The cultured cells were removed from the incubator, 1× DPBS was added, and the cells were incubated for a short time. The DPBS was then aspirated and removed, an appropriate amount of 0.05% trypsin-EDTA was added for cell digestion, and 10% FBS medium was added to terminate the digestion and disperse the cells.

[0064] 4) The dispersed cells were transferred to a 50 mL centrifuge tube using a pipette, centrifuged at 1000 rpm for 5 minutes, resuspended in culture medium to disperse, and then counted.

[0065] 5) Using culture medium, the cells were divided into 1 × 10⁶ cells. 6 The solution was diluted to cells / mL, and the cells were inoculated into 384-well polylysine-coated cell plates at a volume of 20 μL / well.

[0066] 6) The cells were incubated overnight in an incubator at 37°C with 5% CO2.

[0067] Day 2: FLIPR experiment Reagent preparation: 1) Preparation of 250 mM probenecid solution: Following the kit's instructions, 1 mL of FLIPR buffer solution was added to 77 mg of probenecid.

[0068] 2) Preparation of 2×(8μM)Fluo-4 Direct™ sample buffer: The required number of Fluo-4 Direct™ tubes were thawed beforehand, 10 mL of FLIPR buffer solution was added to each tube, and 0.2 mL of 250 mM probenecid solution was added. The mixture was then vortexed in the dark for more than 5 minutes.

[0069] Methods for detecting analogues: 1) Preparation of test analogues and positive reference agonists: Using Bravo, the test analogues were diluted fourfold to 10 concentrations, and 900 nL were transferred to analogue plates using Echo. Then, 30 μL of detection buffer (20 mM HEPES, 1 × HBSS, 0.1% casein) was added to each well. The initial concentrations of the test analogues and positive reference agonists were 1 μM.

[0070] 2) Remove the cell plate from the incubator (for cell Y1, the culture medium needs to be removed and 20 μL of detection buffer added, otherwise all will have a high signal; this is not necessary for Y5), add 20 μL of 2×Fluo-4 detection reagent per well, incubate in a 37°C incubator for 50 minutes, and then let stand at room temperature for 10 minutes.

[0071] 3) The cell plate, analog plate, and pipette tip were placed in the FLIPR instrument, the instrument was started, and 10 μL of the analog was transferred from the analog plate to the cell plate. In the FLIPR instrument, a calcium ion dye was excited under excitation wavelengths of 470 nm to 496 nm to produce emission wavelengths of 515 nm to 575 nm, and the fluorescence signal was read.

[0072] 4) The calcium ion activation rate was calculated for each well: %Activity = (Signal value of sample well - Mean signal of low-signal control well) / (Mean signal of high-signal control well - Mean signal of low-signal control well). Using the logarithm of the analog concentration on the x-axis and the activation rate of the calcium ion fluorescence signal on the y-axis, the "Nonlinear regression (curve fitting) - log(antigen) vs. reaction variable gradient" model in GraphPad Prism 5.0 was used for fitting to the EC of the sample. 50 I calculated it.

[0073] result:

[0074] [Table 5]

[0075] According to the data in Table 1 and Table 2, the PYY of the present invention 3~36 The analog exhibits high selectivity for NPY2R but low selectivity for NPY1R and NPY5R. (Example 3)

[0076] PYY 3~36In vivo efficacy testing of analogues 1. Effects on food intake and body weight in normal mice Objective: To compare the effects of single injection of analogues 1, 2, 3 and reference example 1 on weight loss and suppression of food intake in normal mice.

[0077] method: Six- to eight-week-old male C57BL / 6 mice were housed in a strictly controlled animal enclosure, maintaining a temperature of 20°C to 24°C and humidity of 30% to 70%. The photoperiod in the enclosure was 12 hours (starting at 7:00 am and ending at 7:00 pm). During the experiment, the animals were kept in single cages, and toys were provided in each cage. Throughout the experiment, the animals had free access to food (growth / reproductive feed for mice and rats) and water.

[0078] The animals' body weight when not fasting and their initial food intake before administration were recorded. The animals were then administered a single subcutaneous injection, and their daily body weight and food intake were recorded for three days after administration. Animal activity, water intake, diet, and changes in body weight were observed daily. Changes in body weight and food intake were calculated by comparing each group to their baseline levels (initial body weight and food intake before administration), and the body weight change was expressed as a percentage (baseline set to 100%).

[0079] Results: See Table 3, Figure 1a, and Figure 1b.

[0080] [Table 6]

[0081] The data in Figure 1a, Figure 1b, and Table 3 represent the PYY ratio of animal food intake and weight loss in this application. 3~36 This strongly supported the inhibitory effect of the analogues.

[0082] 2. PYY in normal SD rats 3~36 Pharmacological effects of combination therapy with an analog and the GLP-1 agonist semaglutide.

[0083] Objective: To study PYY in normal SD rats to reduce weight loss and suppress food intake. 3~36 Determination of the efficacy of combination therapy with an analog and the GLP-1 agonist semaglutide.

[0084] method: Six- to eight-week-old male SD rats were housed in an animal enclosure with strictly controlled environmental conditions. The temperature of the enclosure was maintained at 20-24°C, and the humidity at 30-70%. The photoperiod in the animal enclosure was 12 hours (starting at 7:00 am and ending at 7:00 pm). During the experiment, the animals were kept in single cages, and toys were provided in each cage. During the experiment, the animals had free access to food (growth / reproductive feed for mice and rats) and water.

[0085] Analogue 3 was used with 15 nmol / kg of analogue 3 and 20 nmol / kg of the GLP-1 agonist semaglutide (SEQ ID NO: 7) (Note: X26 in SEQ ID NO: 7 is the fatty acid side chain group AEEAc AEEAc-γGlu-CO(CH2) 16 The study involved subcutaneous administration of a combination of the two drugs (with the K side chain modified by COOH). The two drugs were administered separately once daily for a total of 8 days, and the animals' body weight changes were monitored daily during the administration period. The change in body weight compared to the baseline level (initial body weight before administration) for each group was calculated and expressed as a percentage (baseline being 100%).

[0086] Results: See Table 4 and Figure 2.

[0087] [Table 7]

[0088] The data in Table 4 and Figure 2 show the weight loss of animals treated with semaglutide in combination with "Analog 3" compared to the weight loss of animals treated with the GLP-1 agonist semaglutide alone, as per the PYY of this application. 3~36 This indicates that the in vivo effect of the analogues has been confirmed. (Example 4)

[0089] PYY 3~36 Pharmacokinetic experiments of analogues Objective: To investigate the pharmacokinetic properties of PYY analogs. method: Blood samples were collected from each animal at each time point after administration, and the blood samples were centrifuged at approximately 4°C and 3200g for 10 minutes. Plasma was collected separately, transferred to pre-labeled 96-well plates or polypropylene tubes, immediately frozen on dry ice, and stored at -60°C or below until LC-MS / MS analysis.

[0090] LC-MS / MS was employed to determine the concentrations of the measured analogs in blood samples. Data on changes in plasma concentration over time were plotted on a chart and analyzed using the non-partitioned method with the Phoenix WinNonlin 6.3 software program. Key PK parameters were calculated based on the administration route.

[0091] 1. Pharmacokinetics of PYY analogs in SD rats The plasma pharmacokinetics of the PYY analog were evaluated in male SD rats after a single subcutaneous administration of 0.492 mg / kg of analog 3. Blood samples were collected from three rats at post-administration time points of 0.5h, 1h, 2h, 4h, 8h, 12h, 16h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h, up to 168h. Because discontinuous sampling was used to evaluate the pharmacokinetics of the PYY analog in rats, the pharmacokinetic parameters of analog 3 after a single subcutaneous administration of 0.492 mg / kg were enumerated using concentration-time mean data.

[0092] 2. Pharmacokinetics of PYY analogs in cynomolgus monkeys The plasma pharmacokinetics of the PYY analog were evaluated in male cynomolgus monkeys after a single subcutaneous administration of 0.246 mg / kg of analog 3. Blood samples were collected from two cynomolgus monkeys at post-administration time points of 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 240h, 288h, 336h, 408h, and 504h, up to 504h. Because discontinuous sampling was used to evaluate the pharmacokinetics of the PYY analog in cynomolgus monkeys, the pharmacokinetic parameters of analog 3 after a single subcutaneous administration of 0.246 mg / kg were enumerated using concentration-time mean data.

[0093] result:

[0094] [Table 8]

[0095] The data in Table 5 confirm that the PYY analog of the present invention has a pharmacokinetic profile suitable for weekly administration.

Claims

1. The following general formula (I): P-K-P-E-ψ-P-E-X １０ -D-X １２ -S-P-E-E-W-Q-R-Y-Y-X ２２ -X ２３ -L-R-H-Y-L-N-W-L-T-R-Q-R-Y-R １ (I) (In the formula, ψ is the side chain of the following general formula (II): Y-Z(II) (where Y is (AEEAc or Glu) ａ -(AEEAc or Glu) ｂ -(AEEAc or Glu) ｃ (where a, b, and c are each independently 0 or 1, and a, b, and c are not all 0 at the same time), the carboxyl terminus of Y is linked to the ε-amino group of the side chain of Lys, and Z is -CO-(CH ２ ) ｍ -COOH, and m is an integer between 6 and 24), and Lys is modified with the structure of; X １０ is E or Aib; X １２ is A or Aib; X ２２ is A or Aib; X ２３ is S or E; R １ NH ２ or OH; X １０ , X １２ , and X ２２ (At least one of which is Aib) includes a sequence structure Peptide tyrosine-tyrosine (PYY ３～３６ ) Analogues or their salts or solvates.

2. The peptide tyrosine-tyrosine (PYY) peptide according to claim 1, characterized in that Y in general formula (II) is AEEAc-γGlu or AEEAc-AEEAc-γGlu, preferably AEEAc-γGlu. ３～３６ ) Analogues or their salts or solvates.

3. Analogues, PKPEK(AEEAc-γGlu-CO(CH ２ ) １６ COOH)PEED-Aib-SPEEWQRYY-Aib-SLRHYLNWLTRQRY-NH ２ 、 PKPEK(AEEAc-γGlu-CO(CH ２ ) １６ COOH) PE-Aib-DASPEEWQRYYAELRHYLNWLTRQRY-NH ２ ,or PKPEK(AEEAc-γGlu-CO(CH ２ ) １６ COOH)PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH ２ Peptide YY (PYY) according to claim 1, characterized in that it is selected from ３～３６ ) Analogues or their salts or solvates.

4. The analogue is PKPEK(AEEAc-γGlu-CO(CH ２ ) １６ COOH) PE-Aib-DASPEEWQRYY-Aib-ELRHYLNWLTRQRY-NH ２ Peptide YY (PYY) according to claim 1, characterized in that it is selected from ３～３６ ) Analogues or their salts or solvates.

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

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

7. The pharmaceutical composition according to claim 5, characterized in that it is in the form of microencapsulated formulations, microsphere formulations, nanoparticle formulations, or liposome formulations.

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

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

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

11. Use of an analog or salt or solvate thereof according to any one of claims 1 to 4, or a pharmaceutical composition according to any one of claims 5 to 10, for the preparation of drugs for the treatment, prevention or reduction of overweight and obesity or obesity complications.

12. The use according to claim 11, characterized in that obesity complications include angina pectoris, cardiovascular disease, cholecystitis, gallstones, congestive heart failure, heart failure with preserved ejection fraction, dyslipidemia, non-alcoholic steatohepatitis, reproductive complications, impaired glucose tolerance, gout, hypertension, hypothyroidism, hyperinsulinemia, insulin resistance, osteoarthritis, polycystic ovary syndrome, pregnancy complications, mental confusion, sleep apnea and other respiratory problems, stress urinary incontinence, stroke, type II diabetes mellitus, 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 may be used in combination with other drugs for treating the same or related disease, wherein the other drugs are selected from one or more of metformin, sulfonylurea, SGLT-1 / 2 inhibitors, DPP-4 inhibitors, insulin, GLP-1, GCG, GIP, and FGF-21.

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