A polypeptide tyrosine tyrosine analogue and a sustained-release preparation containing the same

Abstract

The present invention provides a polypeptide tyrosine tyrosine analog and a sustained-release preparation containing the same, namely, a PYY3-36 analog and an injectable sustained-release preparation of the PYY3-36 analog that can be used to treat obesity. The PYY3-36 analog provided by the present invention is encapsulated in a biocompatible, degradable material that is compatible with the material used, thereby stabilizing the PYY3-36 analog within the material. The resulting sustained-release microspheres, implants, and gels are then prepared to slowly release the drug in the body. This overcomes the short half-life and instability of the PYY3-36 drug, providing a novel treatment for obesity.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and more particularly to a polypeptide tyrosine tyrosine 3-36 (PYY3-36) analogue and a sustained-release preparation containing the same. Background Art

[0002] Obesity is a group of metabolic disorders characterized by excessive body fat accumulation, resulting in excess weight. With global economic development and the increasing availability of food, particularly high-calorie foods, the number of people suffering from obesity has increased significantly worldwide. According to the World Health Organization, the number of obese people worldwide in 2016 had tripled since 1975.

[0003] Obesity is primarily caused by an imbalance between excessive energy intake and insufficient energy expenditure. Excessive consumption of high-calorie foods high in sugar or fat, as well as a lack of physical activity due to work schedules and the convenience of modern transportation, can contribute to obesity. In rare cases, obesity is also linked to genetic factors, psychological disorders, and endocrine factors. Obesity has numerous negative impacts on human health and is closely linked to the development of numerous chronic and devastating diseases. Modern medical research has shown that obesity is directly linked to increased morbidity and mortality from related conditions, such as coronary heart disease, hypertension, and hyperlipidemia; venous or pulmonary embolism; stroke; congestive heart failure; type 2 diabetes; gout; degenerative arthritis; infertility; and menstrual disorders. Obesity also increases the incidence of serious cancers, including prostate, colon, pancreatic, liver, and endometrial cancers.

[0004] Currently, the main treatments for obesity include behavioral therapy, drug therapy, and surgical treatment: 1) Behavioral therapy, which mainly involves dietary control. By changing eating habits, food structure, and exercise, weight loss has been achieved. It has a significant effect on short-term weight loss, but it is difficult to maintain a long-term weight loss model. 2) Drug therapy. The therapeutic effect of existing drugs is limited, and due to the occurrence of side effects, the use of drugs is restricted. 3) Surgical treatment is currently the most effective treatment for obesity, but it is high-risk, has low patient compliance, high treatment costs, and can cause complications. Some patients need to undergo a second operation, so patients have a low degree of acceptance of surgical treatment. Due to the limitations of existing treatment methods, new drugs that can control weight have huge research and development prospects and market demand.

[0005] Peptide YY (PYY), a 36-amino acid human peptide, is secreted by specialized endocrine cells (L-cells) in the ileum and colon after a meal. Endogenous PYY is primarily found in two forms: PYY1-36 and PYY3-36. PYY3-36 is the predominant form of PYY in the bloodstream and is formed by hydrolysis of the two N-terminal amino acids of PYY1-36 by dipeptidyl peptidase 4 (DPP4). PYY has been shown to reduce appetite and contribute to weight loss in obese patients. Its mechanism of action is to inhibit gastrointestinal and pancreatic secretions, gastrointestinal motility, and increase colonic absorption of water and electrolytes by binding to the Y-receptor.

[0006] Of the two forms, PYY1-36 and PYY3-36, PYY3-36 is valued for its superior receptor selectivity. Studies have shown that PYY3-36 reduces appetite primarily by producing anorexia. While its precise mechanism of action remains uncertain, studies suggest that PYY3-36 may penetrate the blood-brain barrier via an unsaturated mechanism, interacting with AgRP and POMC neurons and Y2 receptors in the arcuate nucleus of the hypothalamus (Boggiano et al., 2005 Obesity Reviews 6:307-322). Studies have also shown that PYY3-36 can increase caloric expenditure and fat oxidation rates in obese and emaciated individuals. In addition to peripheral injection, administration by inhalation can also reduce daily caloric intake. Furthermore, recent studies have shown that the combined use of PYY3-36 and GLP-1 receptor agonists or GLP-1 analogs can induce a synergistic effect to further reduce caloric intake (Lepsen et al., 2016 Eur. J. Endocrinology 174:775-784).

[0007] As a potential drug for the treatment of obesity, PYY3-36 currently faces obstacles including its short half-life (only 15-30 minutes), a narrow therapeutic window, and the potential for side effects such as nausea and vomiting. Two main approaches are proposed: one is to create analogs of the molecule by modifying its backbone molecular structure, thereby improving or altering its chemical and physical properties in the body to prolong its half-life and reduce side effects. The other is to develop formulations that are compatible with PYY3-36 by integrating with pharmaceutical manufacturing processes and leveraging the peptide's unique characteristics to address its current shortcomings.

[0008] Currently, new sustained-release injection drug delivery systems are gradually replacing traditional dosage forms and are widely used in clinical medicine. Sustained-release injection drug delivery systems utilize biodegradable polymers that gradually degrade into products that are absorbed and metabolized by the human body, thereby addressing the toxicity and retention issues associated with excipient residues. In recent years, an increasing number of sustained-release injections have received regulatory approvals and entered the pharmaceutical market. Common controlled-release injections can last from one week to one year, or even longer. Dosage forms include liposomes, vesicles, suspensions, microspheres, emulsions, gels, and implants.

[0009] Therefore, there is still a need in the art to develop PYY analogs, especially PYY3-36 analogs, and in particular to develop sustained-release injection delivery systems for PYY3-36 analogs, so as to overcome the deficiencies of existing PYY3-36 preparations. Summary of the Invention

[0010] When formulating PYY3-36 into a sustained-release peptide pharmaceutical, excellent stability of PYY3-36 and excipients is required. Therefore, overcoming the compatibility of PYY3-36 with biodegradable polymers is a major challenge in its formulation. Therefore, the present invention aims to provide a novel PYY3-36 analog that, compared to PYY3-36, has several substituted amino acid residues, thereby improving its stability in the biodegradable polymers used in sustained-release formulations.

[0011] In response to the above technical problems, one object of the present invention is to provide a novel PYY3-36 analog having several amino acid substitutions at specific positions in the amino acid sequence compared to PYY3-36. Another object of the present invention is to provide pharmaceutical uses of the novel PYY3-36 analog. A further object of the present invention is to provide a sustained-release preparation prepared using the novel PYY3-36 analog.

[0012] The technical solutions of the present invention are as follows.

[0013] In one aspect, the present invention provides a polypeptide tyrosine tyrosine 3-36 (PYY3-36) analog or a pharmaceutically acceptable salt thereof, wherein the PYY3-36 analog comprises an amino acid sequence represented by the following general formula A, wherein the PYY3-36 analog has a total of 2, 3 or 4 amino acid substitutions at positions Xaa4, Xaa6, Xaa9, Xaa10 and Xaa11 of the amino acid sequence compared to PYY3-36:

[0014] Ile-Xaa4-Pro-Xaa6-Ala-Pro-Xaa9-Xaa10-Xaa11-Ala-Ser-Pro-Glu-Glu-Leu-Asn-Arg-Tyr-Try-Ala-Ser-Leu-Arg-His-Tyr-Leu-Asn-Leu-Val-Thr-Arg-Gln-Arg-Tyr-NH2

[0015] Formula A

[0016] in,

[0017] Xaa4 is Arg, Leu, Ile, 2-aminobutyric acid (Abu), homoarginine (hArg), Ala, 2-aminoisobutyric acid (Aib), D-Leu, D-Arg, or N,N-dimethylarginine (Arg(Me)2);

[0018] Xaa6 and Xaa10 are independently Glu, Gln, Asp, Asn, 2-aminoadipic acid (Aad), α-aminomalonic acid or its amide, α-aminopimelic acid or its amide, or α-aminosuberic acid (Asu) or its amide;

[0019] Xaa9 is Gly, Ala, Abu, Ser, Leu, Ile, Aib, β-Ala, D-Leu, or D-Ala;

[0020] Xaa11 is Asp, Ser, Thr, Glu, Aad, α-aminomalonic acid or its amide, α-aminopimelic acid or its amide, Asu or its amide, or Tyr or its halide. The Tyr halide is obtained by replacing the hydroxyl group on its benzene ring with a halogen element, such as chlorophenylalanine, fluorophenylalanine, bromophenylalanine, and iodophenylalanine.

[0021] The "PYY3-36" mentioned in the present invention refers to human PYY3-36, the molecular formula of which is C 180 H 279 N 53 O 54 The molar mass is 4049.48 g / mol. The amino acid sequence of PYY3-36 is shown in SEQ ID NO. 17.

[0022] The PYY3-36 analogues of the present invention having the above-mentioned general formula A are artificial sequences, which have amino acid substitutions (replacements) at amino acids 4, 6, 9, 10 or 11, respectively, compared to PYY3-36, wherein the numbering of the amino acid positions is based on PYY1-36.

[0023] The "pharmaceutically acceptable salts" of the present invention include salts formed between the PYY3-36 analogs and inorganic or organic acids, such as trifluoroacetate, acetate, hydrochloride, and the like.

[0024] According to a specific embodiment of the present invention, in the PYY3-36 analogue provided by the present invention:

[0025] Xaa4 is Arg, Leu, hArg, Aib or Arg(Me)2, preferably Arg or Leu;

[0026] Xaa6 is Glu, Gln or aminomalonic acid, preferably Glu;

[0027] Xaa9 is Gly, Ser, Ala, Aib, Abu or β-Ala, preferably Gly, Ala or Ser, more preferably Gly or Ser;

[0028] Xaa10 is Gln, Glu, Asp or Asu, preferably Asp or Gln;

[0029] Xaa11 is Asp, Ser, Thr, Tyr or 4-fluorophenylalanine, preferably Ser or Asp, more preferably Ser.

[0030] The PYY3-36 analogs provided herein can be synthesized using the Fmoc solid-phase synthesis method. Fmoc chemical synthesis involves using a polymer resin as a solid-phase reaction matrix. The amino termini of the amino acids are protected with Fmoc, and each amino acid is sequentially condensed with the corresponding coupling reagent in an organic solvent via coupling chemistry to complete the chemical synthesis of the peptide. Following synthesis, the resin is cleaved with a solvent, and the resulting peptide is purified using an HPLC C18 semi-preparative column with acetonitrile and aqueous TFA as the mobile phase.

[0031] According to a specific embodiment of the present invention, the PYY3-36 analogue having the general formula A of the present invention is:

[0032] PYY3-36 analog 1, the amino acid sequence is shown in SEQ ID NO. 1, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Gln (Q), and Xaa11 is Asp (D): I R P E AP SQD ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0033] PYY3-36 analog 2, the amino acid sequence is shown in SEQ ID NO. 2, wherein Xaa4 is Leu (L), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Gln (Q), and Xaa11 is Asp (D): I L P E AP SQD ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0034] PYY3-36 analog 3, the amino acid sequence is shown in SEQ ID NO. 3, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ala (A), Xaa10 is Glu (E), and Xaa11 is Asp (D): I R P E AP AED ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0035] PYY3-36 analog 4, the amino acid sequence is shown in SEQ ID NO. 4, wherein Xaa4 is Arg (R), Xaa6 is Gln (Q), Xaa9 is Ala (A), Xaa10 is Glu (E), and Xaa11 is Asp (D): I R P Q AP AED ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0036] PYY3-36 analog 5, the amino acid sequence is shown in SEQ ID NO. 5, wherein Xaa4 is Leu (L), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Asp (D), and Xaa11 is Ser (S): I L P E AP SDS ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0037] PYY3-36 analog 6, the amino acid sequence is shown in SEQ ID NO. 6, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ala (A), Xaa10 is Gln (Q), and Xaa11 is Ser (S): I R P E AP AQS ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0038] PYY3-36 analog 7, the amino acid sequence is shown in SEQ ID NO. 7, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ala (A), Xaa10 is Glu (E), and Xaa11 is Ser (S): I R P E AP AES ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0039] PYY3-36 analog 8, the amino acid sequence is shown in SEQ ID NO. 8, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Gly (G), Xaa10 is Gln (Q), and Xaa11 is Ser (S): I R P E AP GQS ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0040] PYY3-36 analog 9, the amino acid sequence is shown in SEQ ID NO. 9, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Gly (G), Xaa10 is Gln (Q), and Xaa11 is Asp (D): I R P E AP GQD ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0041] PYY3-36 analog 10, the amino acid sequence is shown in SEQ ID NO. 10, wherein Xaa4 is Leu (L), Xaa6 is Glu (E), Xaa9 is Gly (G), Xaa10 is Gln (Q), and Xaa11 is Asp (D): I L P E AP GQD ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0042] PYY3-36 analog 11, the amino acid sequence is shown in SEQ ID NO. 11, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Glu (E), and Xaa11 is Asp (D): I R P E AP SED ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0043] PYY3-36 analog 12, the amino acid sequence is shown in SEQ ID NO. 12, wherein Xaa4 is hArg, Xaa6 is aminomalonic acid, Xaa9 is Aib, Xaa10 is Glu (E), and Xaa11 is Asp (D): I- hH gP- aminomalonicacid -AP- Aib-ED ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0044] PYY3-36 analog 13, the amino acid sequence is shown in SEQ ID NO. 13, wherein Xaa4 is Aib, Xaa6 is Gln (Q), Xaa9 is Abu, Xaa10 is Glu (E), and Xaa11 is Thr (T): I- Aib -P Q AP- Abu- ET ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0045] PYY3-36 analog 14, the amino acid sequence is shown in SEQ ID NO. 14, wherein Xaa4 is dimethylarginine, Xaa6 is Glu (E), Xaa9 is β-Ala, Xaa10 is Asu, and Xaa11 is Thr (T): I- dimethylarginine -P E AP- β-Ala-Asu-T ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0046] PYY3-36 analog 15, the amino acid sequence is shown in SEQ ID NO. 15, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Glu (E), and Xaa11 is Tyr (Y): I R P E AP SEY ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0047] PYY3-36 analog 16, the amino acid sequence is shown in SEQ ID NO. 16, wherein Xaa4 is Arg (R), Xaa6 is Glu (E), Xaa9 is Ser (S), Xaa10 is Glu (E), and Xaa11 is 4-fluorophenylalanine: I R P E AP SE-fluorophenylalanine-ASPEELNRYYASLRHYLNLVTRQRY-NH2.

[0048] In another aspect, the present invention provides a pharmaceutical composition comprising the PYY3-36 analog or a pharmaceutically acceptable salt thereof. Optionally, the pharmaceutical composition may further comprise other pharmaceutical agents used in combination with the PYY3-36 analog or a pharmaceutically acceptable salt thereof, and / or may further comprise a pharmaceutically acceptable excipient.

[0049] In another aspect, the present invention provides the use of the PYY3-36 analog, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of a medicament for preventing, ameliorating, or treating obesity or obesity-related diseases. Alternatively, the present invention provides the use of the PYY3-36 analog, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition thereof, in the preparation of a product for reducing appetite, decreasing food intake, or inducing weight loss. The product may be a medicine or a healthcare product.

[0050] On the other hand, the present invention also provides a sustained-release preparation prepared from the PYY3-36 analogue or a pharmaceutically acceptable salt thereof. Therefore, the present invention provides a sustained-release preparation comprising the PYY3-36 analogue or a pharmaceutically acceptable salt thereof.

[0051] Preferably, the sustained-release preparation comprises the PYY3-36 analog or a pharmaceutically acceptable salt thereof and a biocompatible degradable material, such as an aliphatic polyester.

[0052] Preferably, the sustained-release preparation is a sustained-release microsphere, a sustained-release in-situ gel or a sustained-release implant, wherein the sustained-release in-situ gel is preferably a polymer precipitated gel or a thermosensitive hydrogel.

[0053] According to a specific embodiment of the present invention, the sustained-release preparation is sustained-release microspheres; further, it is sustained-release microspheres for injection, such as subcutaneous injection or intramuscular injection, and the microsphere particle size (D50) is preferably 5-100 μm.

[0054] Preferably, the sustained-release microspheres comprise the PYY3-36 analog or a pharmaceutically acceptable salt thereof and a biocompatible degradable material, wherein the PYY3-36 analog or a pharmaceutically acceptable salt thereof as an active drug is dispersed or embedded in the biocompatible degradable material.

[0055] More preferably, the sustained-release microspheres contain 0.2-10% (w / w) of a PYY3-36 analog or a pharmaceutically acceptable salt thereof, and 90-99.8% (w / w) of a biocompatible degradable material. The biocompatible degradable material is an aliphatic polyester. The aliphatic polyester is, for example, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polyglycolide-lactide copolymer (PLGA), polycaprolactone-glycolide copolymer (PCGA), polycaprolactone-lactide copolymer (PCLA), or sucrose acetate isobutyrate, or a mixture of two or more thereof.

[0056] Preferably, the biocompatible degradable material in the sustained-release microspheres is PLGA or PLA. Preferably, the LA:GA ratio in PLGA is 50-95:5-50, and the Mw is 10,000-120,000 Daltons, preferably 20,000-60,000 Daltons; and the Mw of PLA is 6,000-55,000 Daltons, preferably 15,000-55,000 Daltons.

[0057] For example, the present invention provides injectable sustained-release microspheres comprising a PYY3-36 analog or a salt thereof, which can be used for sustained release for one month, wherein the biocompatible degradable material is PLGA (e.g., LA:GA is 50:50; Mw 10,000-85,000 Daltons), PLGA (e.g., LA:GA is 55:45; Mw 15,000-55,000 Daltons), PLGA (e.g., LA:GA is 65:35; Mw 15,000-55,000 Daltons), or PLGA (e.g., LA:GA is 75:25; Mw 10,000-25,000 Daltons);

[0058] As another example, the present invention provides injectable sustained-release microspheres comprising a PYY3-36 analog or a salt thereof, which can be used for sustained release for three months. The biocompatible degradable material is PLGA (e.g., LA:GA is 75:25; Mw 50,000-120,000 Daltons), PLGA (e.g., LA:GA is 85:15; Mw 55,000-85,000 Daltons), PLGA (e.g., LA:GA is 95:05; Mw 30,000-85,000 Daltons), or PLA (e.g., LA:GA is 100:00; Mw 15,000-55,000 Daltons).

[0059] Preferably, the sustained-release microspheres provided by the present invention are prepared by a preparation method comprising the following steps:

[0060] (1) dissolving a PYY3-36 analog or a pharmaceutically acceptable salt thereof in water as the internal aqueous phase;

[0061] (2) dissolving a biocompatible degradable material in an organic solvent as the oil phase;

[0062] (3) dissolving a hydrophilic surfactant in water as the external aqueous phase;

[0063] (4) adding the inner aqueous phase in step (1) to the oil phase in step (2), and emulsifying the mixture by one or more methods selected from high shear, ultrasound, and high-pressure homogenization to obtain colostrum;

[0064] (5) adding the colostrum in step (4) to the external aqueous phase in step (3) and stirring uniformly to form a double emulsion;

[0065] (6) The emulsion prepared in step (5) is solidified into microspheres, and the suspension after solidification is filtered, collected, washed, and dried.

[0066] In the above method, the water is water for injection.

[0067] Preferably, in step (1), the mass concentration of the PYY3-36 analogue or a pharmaceutically acceptable salt thereof in the internal aqueous phase is 0.05-50%.

[0068] Preferably, in step (2), the organic solvent is one or more selected from dichloromethane, chloroform, ethyl acetate, methyl acetate, ether, acetone, dioxane, acetonitrile, and tetrahydrofuran, preferably dichloromethane. Preferably, the mass concentration of the biocompatible degradable material in the oil phase is 10-35%.

[0069] Preferably, in step (3), the hydrophilic surfactant is one or more selected from polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), sodium carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC), β-cyclodextrin, poloxamer 188, Pluronic F88, Pluronic F127, gelatin, glycine, lysine, histidine, arginine, aspartic acid, glutamic acid, Span, and Tween, preferably polyvinyl alcohol. Preferably, in step (3), the mass concentration of the hydrophilic surfactant in the external aqueous phase is 0.1-5%.

[0070] Preferably, in step (4), the inner water phase in step (1) and the oil phase in step (2) are mixed in a volume ratio of 1:5-1:50.

[0071] Preferably, in step (5), the colostrum in step (4) and the external aqueous phase in step (3) are mixed in a volume ratio of 1:10-1:100.

[0072] Preferably, in step (6), the microspheres are cured at 5-30°C.

[0073] According to another embodiment of the present invention, the sustained-release preparation is a sustained-release in-situ gel.

[0074] Preferably, the sustained-release in situ gel comprises the PYY3-36 analog or a pharmaceutically acceptable salt thereof, a biocompatible degradable material and a solvent.

[0075] More preferably, the sustained-release in situ gel comprises 0.05-5% (w / w) of the PYY3-36 analog or a pharmaceutically acceptable salt thereof, 15-87% (w / w) of a biocompatible degradable material, and 12-85% (w / w) of a solvent.

[0076] More specifically, the sustained-release in situ gel is a polymer precipitation gel, wherein the biocompatible degradable material is an aliphatic polyester. The aliphatic polyester is, for example, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polyglycolide-lactide copolymer (PLGA), polycaprolactone-glycolide copolymer (PCGA), polycaprolactone-lactide copolymer (PCLA), or sucrose acetate isobutyrate, or a mixture of two or more thereof. The solvent is an organic solvent, which can be one or more selected from methanol, ethanol, propanol, propylene glycol, ethyl acetate, ethyl lactate, dimethyl sulfoxide, and N-methyl-2-pyrrolidone.

[0077] Preferably, the polymer precipitation gel provided by the present invention is prepared by a preparation method comprising the following steps: dissolving a biocompatible degradable material in an organic solvent, and then blending with a PYY3-36 analog or a pharmaceutically acceptable salt thereof, for example, by stirring, vortexing or shaking at room temperature for 1-2 hours.

[0078] Alternatively, more specifically, the sustained-release in situ gel is a thermosensitive hydrogel, wherein the biocompatible degradable material is polyethylene glycol (PEG)-aliphatic copolyester or poloxamer. The polyethylene glycol-aliphatic copolyester is, for example, a binary or ternary block copolymer of PLGA-PEG-PLGA, PEG-PLGA, PEG-PLA, PEG-PCL, PEG-PGA, PEG-PCGA, PEG-PCLA, or a mixture of two or more thereof.

[0079] Preferably, the thermosensitive hydrogel provided by the present invention is prepared by a preparation method comprising the following steps: dissolving a biocompatible degradable material in water, and then blending with a PYY3-36 analog or a pharmaceutically acceptable salt thereof, for example, by stirring, vortexing or shaking at room temperature for 1-2 hours.

[0080] According to another embodiment of the present invention, the sustained-release preparation is a sustained-release implant. Preferably, the sustained-release preparation is an injectable sustained-release implant.

[0081] Preferably, the sustained-release implant comprises the PYY3-36 analogue or a pharmaceutically acceptable salt thereof and a biocompatible degradable material.

[0082] More preferably, the sustained-release implant comprises 5-35% (w / w) of a PYY3-36 analog or a pharmaceutically acceptable salt thereof, and 65-95% (w / w) of a biocompatible degradable material. The biocompatible degradable material is an aliphatic polyester. The aliphatic polyester is, for example, polylactide (PLA), polyglycolide (PGA), polycaprolactone (PCL), polyglycolide-lactide copolymer (PLGA), polycaprolactone-glycolide copolymer (PCGA), polycaprolactone-lactide copolymer (PCLA), or sucrose acetate isobutyrate, or a mixture of two or more thereof.

[0083] Preferably, the biocompatible degradable material in the sustained-release implant is PLGA, wherein preferably, the LA:GA ratio in PLGA is 50-75:25-50, and the Mw is 10,000 to 85,000 Daltons, preferably 10,000 to 20,000 Daltons.

[0084] For example, the present invention provides a PYY3-36 sustained-release implant, wherein the biocompatible degradable material is PLGA (e.g., LA:GA is 50:50; Mw 10,000-85,000 Daltons), PLGA (e.g., LA:GA is 55:45; Mw 15,000-55,000 Daltons), PLGA (e.g., LA:GA is 65:35; Mw 15,000-55,000 Daltons) or PLGA (e.g., LA:GA is 75:25; Mw 10,000-25,000 Daltons).

[0085] Preferably, the sustained-release implant provided by the present invention is prepared by a preparation method comprising the following steps:

[0086] (1) dissolving a biocompatible degradable material and a PYY3-36 analog or a pharmaceutically acceptable salt thereof in glacial acetic acid, and then freeze-drying to prepare a lyophilized powder;

[0087] (2) performing hot melt extrusion of the freeze-dried powder in step (1);

[0088] (3) Cutting the strips hot-melt extruded in step (2) into cylinders.

[0089] In the above method, preferably, in step (1), the mass ratio of the biocompatible degradable material, the PYY3-36 analogue or a pharmaceutically acceptable salt thereof, and the glacial acetic acid is: 12-19.96:0.04-8:80.

[0090] Preferably, in step (2), the hot melt extrusion is carried out at 85-100°C;

[0091] Preferably, in step (3), the strip is cut into cylinders with a diameter of 1.00-1.50 mm and a length of 5.0-15.0 mm.

[0092] Biodegradable polymers used in sustained-release formulations such as microspheres, gels, and implants are primarily aliphatic polyesters. When the inventors tested the stability of formulations made with PYY3-36 and these excipients, they found that the peptide drug began to show significant degradation starting on day 7, with the appearance and increasing levels of related substances. By day 17, approximately 20% of PYY3-36 had been degraded. It is speculated that the slightly higher-affinity functional groups in PYY3-36 may cause degradation of the excipients and the peptide drug itself, leading to the production of related substances. Therefore, the present invention, by modifying the amino acid residues at specific amino acid sites in PYY3-36, has produced a novel PYY3-36 analog. This effectively increases the compatibility of the peptide drug with biodegradable excipients, reduces the presence of related substances during preparation, and facilitates the preparation of the peptide drug into relevant dosage forms.

[0093] Based on this, the present invention also provides sustained-release formulations of the PYY3-36 analogs, including microspheres, implants, and in-situ gels. These sustained-release formulations overcome the short half-life of PYY3-36 or its analogs in oral tablets, capsules, conventional injections, and inhalants, resulting in an inability to stably release the drug within the effective therapeutic window. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:

[0095] Figure 1 Shown are the results of stability characterization of PYY3-36 formulations.

[0096] Figure 2 Shown are the results of stability characterization of PYY3-36 analog formulations.

[0097] Figure 3 The prepared PYY3-36 analog sustained-release microspheres are shown.

[0098] Figure 4 The 28-day in vitro cumulative release profile of the PYY3-36 analog sustained-release gel is shown.

[0099] Figure 5 The 7-day in vitro cumulative release profile of the PYY3-36 analog sustained-release gel is shown.

[0100] Figure 6 The 15-day in vitro cumulative release profile of the PYY3-36 analog sustained-release implant is shown.

[0101] Figure 7 The 28-day in vitro cumulative release profile of PYY3-36 analog sustained-release microspheres is shown.

[0102] Figure 8 The in vivo kinetic curve of PYY3-36 analog sustained-release microspheres in rats for 28 days is shown.

[0103] Figure 9 The in vivo kinetics of a PYY3-36 analog sustained-release implant in rats for 28 days are shown. DETAILED DESCRIPTION

[0104] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0105] The experimental methods in the following examples are conventional methods unless otherwise specified. The medicinal materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0106] PYY3-36: purchased from Kedao-Guangzhou Hewei Pharmaceutical Technology Co., Ltd., batch number 14180560, production number CD430696.

[0107] Example 1 Solid-phase synthesis of PYY3-36 and PYY3-36 analogs

[0108] PYY3-36 and PYY3-36 analog peptides were synthesized using a CSBio CS136S instrument according to the manufacturer's instructions. Characterization was performed by MALDI-MS. The results are shown in Table 1.

[0109] Table 1. PYY3-36 and PYY3-36 analogs

[0110] PYY3-36 analogs Amino acid sequence MW (calculated) MW (measured value) 1 SEQ ID NO.1 4106.6 4015.2 2 SEQ ID NO.2 4063.6 4062.2 3 SEQ ID NO.3 4091.6 4090.2 4 SEQ ID NO.4 4090.6 4091.2 5 SEQ ID NO.5 4022.5 4021.1 6 SEQ ID NO.6 4062.6 4061.3 7 SEQ ID NO.7 4063.6 4062.3 8 SEQ ID NO.8 4048.6 4047.2 9 SEQ ID NO.9 4076.6 4077.1 10 SEQ ID NO.10 4033.5 4032.2 11 SEQ ID NO.11 4107.6 4106.3 12 SEQ ID NO.12 4091.6 4090.2 13 SEQ ID NO.13 4019.5 4018.1 14 SEQ ID NO.14 4147.7 4146.2 15 SEQ ID NO.15 4155.6 4154.1 16 SEQ ID NO.16 4157.6 4156.2

[0111] Example 2 Pharmacological validation of PYY3-36 analogs

[0112] To evaluate the efficacy of PYY3-36 analogs, PYY3-36 and its analogs were administered to mice. Food intake and body weight were measured 24 hours after administration, and PYY3-36 and its analogs were compared in parallel as follows:

[0113] The experiment involved adult male C57BL / 6 mice (6-9 weeks old). Both blank and treatment groups were established, with 10 mice per group. Pre-dose weight was 20-30g. The mice were maintained at a temperature of 20-26°C, with a light cycle of approximately 12 hours. Food and water intake was unrestricted, except for fasting during experimental conditions.

[0114] The dosage of PYY3-36 and its analogs in the drug-treated group was 1 mg / kg, prepared by dissolving 0.2 mg / mL in 30 mM PBS buffer and adjusting the pH to 8.2. The blank control group was given PBS buffer.

[0115] Mice were fasted for 4 hours before dosing. Dosing was performed by intraperitoneal injection twice daily for seven days at 9:00 AM and 9:00 PM. Food intake and body weight were monitored for 24 hours before morning dosing. Changes in body weight and food intake for each treatment group compared to the control group are shown in Table 2.

[0116] Table 2. Effects of the PYY3-36 analogs of the present invention vs. PYY3-36 on food intake and body weight in mice

[0117]

[0118]

[0119] Note: Change in food intake (%): after 7 days, (average food intake of mice in the treatment group - average food intake of mice in the blank control group) / average food intake of mice in the blank control group × 100%; change in body weight (%): after 7 days, (average body weight of mice in the treatment group - average body weight of mice in the blank control group) / average body weight of mice in the blank control group × 100%

[0120] The results showed that after one week of administration, PYY3-36 and its analogs significantly reduced the mice's food intake, with no significant difference in their effects on food intake. However, there was no clear pattern in their effects on body weight. Furthermore, under the conditions of this experiment, there was no significant correlation between the effects of PYY3-36 and its analogs on food intake and body weight.

[0121] Example 3 Comparison of the stability of PYY3-36 sustained-release preparations and PYY3-36 analogue sustained-release preparations

[0122] PYY3-36 was used to prepare sustained-release microspheres (for the preparation method, see Example 5), sustained-release gels (for the preparation method, see Example 15) and sustained-release implants (for the preparation method, see Example 17).

[0123] The above-mentioned PYY3-36 preparation was placed in a vial and placed at 37°C. Appropriate amounts were taken at preset time points (0 days, 7 days, 15 days, and 30 days) to investigate the drug loading.

[0124] The determination method is high performance liquid chromatography. Weigh 50mg of sample, accurately add 2ml of acetonitrile, sonicate for 10 minutes to dissolve, then add 0.1% HAC solution to 10ml, sonicate for 3 minutes, and immediately centrifuge the suspension (speed 10000 rpm) for 10 minutes. The supernatant is used as the test solution. The chromatographic conditions include: octadecyl bonded silica gel as the chromatographic column packing (250mm*4.6mm*5μm, ), using an ultraviolet detector with a detection wavelength of 220 nm, 0.1% TFA water and 0.1% TFA acetonitrile as the mobile phase, and the gradient elution conditions are shown in Table 3. Injection volume: 10 uL.

[0125] Table 3. Gradient elution conditions

[0126] Time (min) Mobile phase A (%) Mobile phase B (%) 0 80 20 20 20 80 20.1 80 20 25 80 20

[0127] See the results Figure 1 .

[0128] Microsphere preparations were prepared using the method of Example 5 using the PYY3-36 analog.

[0129] The above-mentioned PYY3-36 analog preparation was placed in a vial and placed at 37°C. At the preset time points (0 days and 30 days), appropriate amounts were taken to examine the drug loading. The determination method was as described above for the PYY3-36 sustained-release preparation. The results are shown in Figure 2 .

[0130] The above stability results show that the compatibility stability of the PYY3-36 analogue of the present invention with excipients is significantly improved compared with PYY3-36 at 37° C. for 30 days.

[0131] Example 4 Preparation of sustained-release microspheres of PYY3-36 analogs

[0132] 1. Dissolve 0.1 g of PYY3-36 analog in 2 g of water for injection as the internal aqueous phase;

[0133] 2. Weigh 12.4 g of poly(glycolide-lactide) (PLGA 50 / 50, 40,000-50,000 Daltons) and add it to 49.6 g of dichloromethane as the oil phase;

[0134] 3. Prepare 2.5 L of an aqueous solution for injection containing 1.0% polyvinyl alcohol (PVA) as the external aqueous phase;

[0135] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0136] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0137] 6. The emulsion is solidified at 10°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0138] The particle size (D50) of the microspheres of the preparation was measured to be 40 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 0.7%, the encapsulation efficiency was 87.5%, and the yield was 82.1%.

[0139] Example 5 Preparation of sustained-release microspheres of PYY3-36 analogs

[0140] 1. Dissolve 0.2 g of PYY3-36 analog in 2 g of water for injection as the internal aqueous phase;

[0141] 2. Weigh 9.8 g of poly(lactide-co-glycolide) (PLGA 50 / 50, 20,000-35,000 Daltons) and add it to 39.7 g of dichloromethane as the oil phase;

[0142] 3. Prepare 2.2 L of an aqueous solution for injection containing 0.5% polyvinyl alcohol (PVA) as the external aqueous phase;

[0143] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0144] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0145] 6. The emulsion is solidified at 10°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0146] The particle size (D50) of the microspheres of the preparation was measured to be 38 μm; quantitative determination by HPLC showed that the drug loading of the microspheres was 1.8%, the encapsulation efficiency was 90%, and the yield was 80.3%. Figure 3 (Scanning electron microscope 2000x).

[0147] Example 6 Preparation of sustained-release microspheres of PYY3-36 analogs

[0148] 1. Dissolve 0.1 g of PYY3-36 analog in 2 g of water for injection as the internal aqueous phase;

[0149] 2. Weigh 9.5 g of poly (lactide-co-glycolide) (PLGA 75 / 25, 40,000-60,000 Daltons) and add it to 39.7 g of dichloromethane as the oil phase;

[0150] 3. Prepare 1 L of aqueous solution for injection containing 1.0% polyvinyl alcohol (PVA) as the external aqueous phase;

[0151] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0152] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0153] 6. The emulsion is solidified at 15°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0154] The particle size (D50) of the microspheres of the preparation was measured to be 35 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 0.9%, the encapsulation efficiency was 86.4%, and the yield was 82.4%.

[0155] Example 7 Preparation of sustained-release microspheres of PYY3-36 analogs

[0156] 1. Dissolve 0.8 g of PYY3-36 analog in 2 g of water for injection as the internal aqueous phase;

[0157] 2. Weigh 9.2 g of poly (lactide-co-glycolide) (PLGA 50 / 50, 50,000-60,000 Daltons) and add it to 37.3 g of dichloromethane as the oil phase;

[0158] 3. Prepare 2.3 L of an aqueous solution for injection containing 1.0% polyvinyl alcohol (PVA) as the external aqueous phase;

[0159] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0160] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0161] 6. The emulsion is solidified at 15°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0162] The particle size (D50) of the microspheres of the preparation was measured to be 38 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 6.9%, the encapsulation efficiency was 86.3%, and the yield was 85.4%.

[0163] Example 8 Preparation of sustained-release microspheres of PYY3-36 analogs

[0164] 1. Dissolve 0.2 g of PYY3-36 analog in 2 g of water for injection as the internal aqueous phase;

[0165] 2. Weigh 19.8 g of polylactic acid (PLA 100%, 6000-20000 Daltons) and add it to 79.2 g of dichloromethane as the oil phase;

[0166] 3. Prepare 3.5 L of an aqueous solution for injection containing 0.5% polyvinyl alcohol (PVA) as the external aqueous phase;

[0167] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0168] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0169] 6. The emulsion is solidified at 10°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0170] The particle size (D50) of the microspheres of the preparation was measured to be 42 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 0.8%, the encapsulation efficiency was 81%, and the yield was 80.3%.

[0171] Example 9Preparation of sustained-release microspheres of PYY3-36 analogs

[0172] 1. Dissolve 0.5 g of PYY3-36 analog in 4.5 g of water for injection as the internal aqueous phase;

[0173] 2. Weigh 9.5 g of poly(lactide-glycolide) copolymer (PLGA 85 / 15, 50,000-60,000 Daltons) and add it to 38.5 g of dichloromethane as the oil phase;

[0174] 3. Prepare 2.6 L of an aqueous solution for injection containing 0.5% polyvinyl alcohol (PVA) as the external aqueous phase;

[0175] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0176] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0177] 6. The emulsion is solidified at 20°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0178] The particle size (D50) of the microspheres of the preparation was measured to be 40 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 4.2%, the encapsulation efficiency was 84%, and the yield was 79.3%.

[0179] Example 10 Preparation of sustained-release microspheres of PYY3-36 analogs

[0180] 1. Dissolve 0.3 g of PYY3-36 analog in 1.7 g of water for injection as the internal aqueous phase;

[0181] 2. Weigh 14.7 g of poly(glycolide-lactide copolymer) (PLGA 95 / 05, 35,000-45,000 Daltons) and add it to 59.9 g of dichloromethane as the oil phase;

[0182] 3. Prepare 2.6 L of an aqueous solution for injection containing 1.5% polyvinyl alcohol (PVA) as the external aqueous phase;

[0183] 4. Add the oil phase from step 2 to the inner water phase from step 1, mix the inner water phase and the oil phase evenly, and high shear at 8000-20000 rpm to produce colostrum;

[0184] 5. The colostrum prepared in step 4 was uniformly injected into the external aqueous phase in step 3, and emulsified using a high shear mixer to form a double emulsion. The double emulsion was emulsified for 5 minutes.

[0185] 6. The emulsion is solidified at 20°C under magnetic stirring; after solidification, the suspension is filtered and collected using a filter to remove microspheres, and washed multiple times with distilled water. The microspheres are then collected, freeze-dried, and packaged into PYY3-36 analog sustained-release microsphere preparations of corresponding specifications.

[0186] The particle size (D50) of the microspheres of the preparation was measured to be 32 μm. Quantitative determination by HPLC showed that the drug loading of the microspheres was 1.6%, the encapsulation efficiency was 80%, and the yield was 80.2%.

[0187] Example 11 Preparation of sustained-release gel (polymer precipitated gel) formulation of PYY3-36 analogues

[0188] 1 g of PLGA (50 / 50, 15,000-20,000 Daltons) was dissolved in 1 ml of ethyl acetate, and then mixed with 1.2 mg of PYY3-36 analog at room temperature for 2 hours under stirring, and the mixture was packaged into PYY3-36 analog sustained-release injectable gel preparations of corresponding specifications.

[0189] Example 12 Preparation of sustained-release gel (polymer precipitated gel) formulation of PYY3-36 analogues

[0190] 1 g of PLA (20,000-30,000 Daltons) was dissolved in 1 ml of NMP, and then mixed with 2 mg of PYY3-36 analogues under stirring at room temperature for 2 hours. The mixture was then packaged into PYY3-36 analogue sustained-release injectable gel preparations of corresponding specifications.

[0191] Example 13 Preparation of sustained-release gel (polymer precipitated gel) formulation of PYY3-36 analogues

[0192] 1 g of PCL (10,000-15,000 Daltons) was dissolved in 1 ml of DMSO, and then mixed with 2.5 mg of PYY3-36 analogue under stirring at room temperature for 2 hours, and the mixture was packaged into PYY3-36 analogue sustained-release injectable gel preparations of corresponding specifications.

[0193] Example 14 Preparation of sustained-release gel (polymer precipitated gel) formulation of PYY3-36 analogues

[0194] 1 g of sucrose acetate isobutyrate was dissolved in 0.15 ml of ethanol and then mixed with 1.5 mg of PYY3-36 analog at room temperature for 2 hours under stirring, and the mixture was packaged into PYY3-36 analog sustained-release injectable gel preparations of corresponding specifications.

[0195] Example 15 Preparation of PYY3-36 analog sustained-release gel (thermosensitive hydrogel) preparation

[0196] 1 g of PLGA1000-PEG1500-PLGA1000 was dissolved in 5 ml of water, and then mixed with 4 mg of PYY3-36 analog at room temperature under stirring for 2 hours, and then packaged into PYY3-36 analog sustained-release injectable gel preparations of corresponding specifications.

[0197] Example 16 Preparation of PYY3-36 analog sustained-release gel (thermosensitive hydrogel) preparation

[0198] 1 g of poloxamer was dissolved in 5 ml of water, and then mixed with 4 mg of PYY3-36 analogue under stirring at room temperature for 2 hours, and the mixture was packaged into PYY3-36 analogue sustained-release injectable gel preparations of corresponding specifications.

[0199] Example 17 to Example 29 Preparation of PYY3-36 analog (analog 8) sustained-release implants

[0200] 1. Preparation of freeze-dried powder of raw materials and auxiliary materials: PLGA and PYY3-36 analogs are co-dissolved in glacial acetic acid, and then freeze-dried to prepare freeze-dried powder of raw materials and auxiliary materials;

[0201] 2. Hot melt extrusion: Set the hot melt extrusion temperature of the hot melt extruder (85-100°C). When the temperature reaches a stable level, add the freeze-dried powder into the feeder of the hot melt extruder for hot melt extrusion.

[0202] 3. Pulling and Cutting: Set the pulling speed of the puller and the length of the implant to cut the hot-melt extruded strip into cylinders of a certain length. The implant diameter range is 1.00mm to 1.50mm; the implant length range is 5.0mm to 15.0mm.

[0203] The sustained-release implants of other examples were prepared according to the above steps, and the release profiles of the obtained implants were tested with reference to the method of Example 33. See Table 4.

[0204] Table 4. Sustained-release implants and their release profiles

[0205]

[0206]

[0207] The implants extruded from Examples 19 and 29 exhibited a burst release phenomenon, presumably because the hot-melt extrusion temperature was relatively low and the raw materials and auxiliary materials were not fully melted, resulting in a burst release in the preparations. The implant extruded from Example 22 exhibited a burst release phenomenon, presumably because the PGA material had good hydrophilicity and the drugs on the surface and shallow layer of the implants caused a burst release in the preparations. The implant extruded from Example 27 exhibited a burst release phenomenon, presumably because the drug loading of the preparation was relatively high, resulting in a burst release in the preparations.

[0208] There is no burst release phenomenon in Examples 17, 19, 20, 21, 23, 24, 25, and 28, but the types of excipients selected in the prescriptions are different, and the periods of sustained and stable drug release are different.

[0209] Example 30 In vitro release of a PYY3-36 analog (analog 1) sustained-release gel formulation

[0210] Accurately weigh 30 mg of the gel preparation prepared in Example 11 and add it to a 15 mL centrifuge tube. Add 15 mL of preheated release medium, 0.05 M pH 7.4 phosphate buffer, and then place it in a 37°C incubator. At the corresponding time point, sample 1 mL and add 1 mL of the corresponding release medium.

[0211] The amount of PYY3-36 analogues in the release medium sample was detected by high performance liquid chromatography as described in Example 3, thereby obtaining the in vitro cumulative release of PYY3-36 analogues over time. Figure 4 .

[0212] Example 31 In vitro release of a PYY3-36 analog (analog 6) sustained-release gel formulation

[0213] 30 mg of the gel preparation prepared in Example 15 was accurately weighed and added to a 15 mL centrifuge tube. 15 mL of preheated release medium, 0.05 M pH 7.4 phosphate buffer, was added. The tube was then placed in a 37°C incubator. 1 mL of the sample was taken at the corresponding time point and 1 mL of the corresponding release medium was added.

[0214] The amount of PYY3-36 analogues in the sample was detected to obtain the in vitro cumulative release of PYY3-36 analogues over time. Figure 5 .

[0215] Example 32 In vitro release of a PYY3-36 analog (analog 8) sustained-release implant

[0216] Accurately weigh 10 mg of the implant prepared in Example 18 and add it to a 15 mL centrifuge tube. Add 15 mL of preheated release medium, 0.05 M pH 7.4 phosphate buffer, and then place it in a 37°C incubator. At the corresponding time point, sample 1 mL and add 1 mL of the corresponding release medium.

[0217] The amount of PYY3-36 analogues in the sample was detected to obtain the in vitro cumulative release of PYY3-36 analogues over time. Figure 6 .

[0218] Example 33 In vitro release of a PYY3-36 analog (analog 5) sustained-release microsphere formulation

[0219] Accurately weigh 10 mg of the microsphere preparation prepared in Example 5 and add it to a 15 mL centrifuge tube. Add 15 mL of preheated release medium 0.05 M pH 7.4 phosphate buffer, and then place it in a 37°C incubator. At the corresponding time point, sample 1 mL and add 1 mL of the corresponding release medium.

[0220] The amount of PYY3-36 analogues in the release medium sample was detected by high performance liquid chromatography as described in Example 3, thereby obtaining the in vitro cumulative release of PYY3-36 analogues over time. Figure 7 .

[0221] Example 34 In vitro release of a PYY3-36 analog (analog 5) sustained-release microsphere formulation

[0222] Accurately weigh 10 mg of the microsphere preparation prepared in Example 10 and add it to a 15 mL centrifuge tube. Add 15 mL of preheated release medium, 0.05 M pH 7.4 phosphate buffer, and then place it in a 37°C incubator. At the corresponding time point, sample 1 mL and add 1 mL of the corresponding release medium.

[0223] The HPLC method of Example 3 was used to determine the amount of PYY3-36 analog in the release medium sample, thereby obtaining the in vitro cumulative release of the PYY3-36 analog over time. The results showed that the release of the polypeptide analog lasted for nearly 3 months, with a slower release during the first 10 days and a gradual increase thereafter, with nearly 80% of the polypeptide ultimately released.

[0224] Example 35 Pharmacokinetics of a PYY3-36 analog (analog 5) sustained-release microsphere preparation

[0225] Male SD rats weighing 220-250 g were used as the research subjects. The PYY3-36 analog sustained-release microsphere formulation prepared according to Example 5 of the present invention was administered subcutaneously to the rats at a dose of 26 μg / kg. The formulation was administered intraperitoneally once, and 0.3 mL of blood was collected from the tail vein at a specific time after administration. The blood sample was placed in a centrifuge tube containing 30 μL of aprotinin, temporarily stored at room temperature, and centrifuged after blood coagulation to prepare a serum sample. The blood drug concentration (pg / mL) in the plasma sample at each time point was determined using an ELISA kit.

[0226] In vivo pharmacokinetic curves are shown in Figure 8 .

[0227] Example 36 Pharmacokinetics of a PYY3-36 Analog (Analog 8) Sustained-Release Implant

[0228] Male SD rats weighing 220-250 g were used as the study subjects. A sustained-release implant formulation of a PYY3-36 analog prepared according to Example 17 of the present invention was administered subcutaneously to the rats' abdomen at a dose of 26 μg / kg. The dose was administered intraperitoneally once, and 0.3 mL of blood was collected from the tail vein at a specific time after administration. The sample was placed in a centrifuge tube containing 30 μL of aprotinin, temporarily stored at room temperature, and centrifuged after blood coagulation to prepare serum samples. The plasma drug concentration (pg / mL) in the plasma samples at each time point was determined using an ELISA kit.

[0229] In vivo pharmacokinetic curves are shown in Figure 9 .

[0230] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the claims attached to the present invention. Sequence Listing <110> Livzon Pharmaceutical Group Co., Ltd. <120> A polypeptide tyrosine tyrosine analogue and a sustained-release preparation containing the same <130> LC19110075 <160> 17 <170> PatentIn version 3.3 <210> 1 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 1 Ile Arg Pro Glu Ala Pro Ser Gln Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 2 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 2 Ile Leu Pro Glu Ala Pro Ser Gln Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 3 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 3 Ile Arg Pro Glu Ala Pro Ala Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 4 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 4 Ile Arg Pro Gln Ala Pro Ala Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 5 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 5 Ile Leu Pro Glu Ala Pro Ser Asp Ser Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 6 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 6 Ile Arg Pro Glu Ala Pro Ala Gln Ser Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 7 <211> 34 <212> PRT <213> artificial <220> <223> Similar to PYY3-36 <400> 7 Ile Arg Pro Glu Ala Pro Ala Glu Ser Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 8 <211> 34 <212> PRT <213> artificial <220> <223> Similar to PYY3-36 <400> 8 Ile Arg Pro Glu Ala Pro Gly Gln Ser Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 9 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 9 Ile Arg Pro Glu Ala Pro Gly Gln Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 10 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 10 Ile Leu Pro Glu Ala Pro Gly Gln Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 11 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 11 Ile Arg Pro Glu Ala Pro Ser Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 12 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <220> <221> MISC_FEATURE <222> (2)..(2) <223> hArg <220> <221> MISC_FEATURE <222> (4)..(4) <223> aminomalonic acid <220> <221> MISC_FEATURE <222> (7)..(7) <223> Aib <400> 12 Ile Xaa Pro Xaa Ala Pro Xaa Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 13 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <220> <221> MISC_FEATURE <222> (2)..(2) <223> Aib <220> <221> MISC_FEATURE <222> (7)..(7) <223> Abu <400> 13 Ile Xaa Pro Gln Ala Pro Xaa Glu Thr Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 14 <211> 34 <212> PRT <213> artificial(人工) <220> <223> PYY3-36 analog <220> <221> MISC_FEATURE <222> (2)..(2) <223> dimethylarginine <220> <221> MISC_FEATURE <222> (7)..(7) <223> β-Ala <220> <221> MISC_FEATURE <222> (8)..(8) <223> Asu <400> 14 Ile Xaa Pro Glu Ala Pro Xaa Xaa Thr Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 15 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <400> 15 Ile Arg Pro Glu Ala Pro Ser Glu Tyr Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 16 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 analogs <220> <221> MISC_FEATURE <222> (9)..(9) <223> 4-Fluorophenylalanine <400> 16 Ile Arg Pro Glu Ala Pro Ser Glu Xaa Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr <210> 17 <211> 34 <212> PRT <213> artificial <220> <223> PYY3-36 <400> 17 Ile Lys Pro Glu Ala Pro Gly Glu Asp Ala Ser Pro Glu Glu Leu Asn 1 5 10 15 Arg Tyr Tyr Ala Ser Leu Arg His Tyr Leu Asn Leu Val Thr Arg Gln 20 25 30 Arg Tyr

Claims

1. A polypeptide tyrosine tyrosine 3-36 (PYY3-36) analogue or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of the PYY3-36 analogue is shown in SEQ ID NO.1 or SEQ ID NO.

5.

2. A pharmaceutical composition comprising the PYY3-36 analogue or a pharmaceutically acceptable salt thereof according to claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition further comprises other pharmaceutical agents used in combination with the PYY3-36 analog or a pharmaceutically acceptable salt thereof, and / or further comprises pharmaceutically acceptable excipients.

4. Use of the PYY3-36 analogue or a pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 2 or 3 in the preparation of a medicament for preventing, improving or treating obesity.

5. Use of the PYY3-36 analogue or pharmaceutically acceptable salt thereof according to claim 1 or the pharmaceutical composition according to claim 2 or 3 in the preparation of a product for inducing weight loss. A sustained-release preparation comprising the PYY3-36 analogue or a pharmaceutically acceptable salt thereof according to claim 1.

7. The sustained-release preparation according to claim 6, characterized in that The sustained-release preparation comprises the PYY3-36 analog or a pharmaceutically acceptable salt thereof and a biocompatible degradable material.

8. The sustained-release preparation according to claim 7, characterized in that The biocompatible degradable material is aliphatic polyester.

9. The sustained-release preparation according to claim 6 or 7, characterized in that The sustained-release preparation is a sustained-release microsphere, a sustained-release in-situ gel or a sustained-release implant.

10. The sustained-release preparation according to claim 9, characterized in that The sustained-release in-situ gel is a polymer precipitation gel or a thermosensitive hydrogel.

11. The sustained-release preparation according to claim 6 or 7, characterized in that The sustained-release microspheres contain 0.2-10% (w / w) of a PYY3-36 analog or a pharmaceutically acceptable salt thereof, and 90-99.8% (w / w) of a biocompatible degradable material; wherein the biocompatible degradable material is an aliphatic polyester.

12. The sustained-release preparation according to claim 11, characterized in that The sustained-release microspheres are prepared by a preparation method comprising the following steps: (1) dissolving a PYY3-36 analog or a pharmaceutically acceptable salt thereof in water as the internal aqueous phase; (2) dissolving a biocompatible degradable material in an organic solvent as the oil phase; (3) dissolving a hydrophilic surfactant in water as the external aqueous phase; (4) adding the inner aqueous phase in step (1) to the oil phase in step (2), and emulsifying the mixture by one or more methods selected from high shear, ultrasound, and high-pressure homogenization to obtain colostrum; (5) adding the colostrum in step (4) to the external aqueous phase in step (3) and stirring uniformly to form a double emulsion; (6) The emulsion prepared in step (5) is solidified into microspheres, and the suspension after solidification is filtered, collected, washed, and dried.

13. The sustained-release preparation according to claim 9, characterized in that The sustained-release in situ gel comprises the PYY3-36 analog or a pharmaceutically acceptable salt thereof, a biocompatible degradable material and a solvent.

14. The sustained-release preparation according to claim 13, characterized in that The sustained-release in situ gel comprises 0.05-5% (w / w) of a PYY3-36 analog or a pharmaceutically acceptable salt thereof, 15-87% (w / w) of a biocompatible degradable material, and 12-85% (w / w) of a solvent.

15. The sustained-release preparation according to claim 14, characterized in that The sustained-release in-situ gel is a polymer precipitation gel, wherein the biocompatible degradable material is an aliphatic polyester; and the solvent is an organic solvent.

16. The sustained-release preparation according to claim 15, characterized in that The organic solvent is one or more selected from methanol, ethanol, propanol, propylene glycol, ethyl acetate, ethyl lactate, dimethyl sulfoxide and N-methyl-2-pyrrolidone.

17. The sustained-release preparation according to claim 15 or 16, characterized in that The polymer precipitation gel is prepared by a preparation method comprising the following steps: dissolving a biocompatible degradable material in an organic solvent, and then blending the material with a PYY3-36 analogue or a pharmaceutically acceptable salt thereof.

18. The sustained-release preparation according to claim 13 or 14, characterized in that The sustained-release in-situ gel is a thermosensitive hydrogel, wherein the biocompatible degradable material is polyethylene glycol-aliphatic copolyester or poloxamer.

19. The sustained-release preparation according to claim 18, characterized in that The thermosensitive hydrogel is prepared by a preparation method comprising the following steps: dissolving a biocompatible degradable material in water, and then blending the material with a PYY3-36 analogue or a pharmaceutically acceptable salt thereof.

20. The sustained-release preparation according to claim 9, characterized in that The sustained-release implant comprises 5-35% (w / w) of a PYY3-36 analog or a pharmaceutically acceptable salt thereof and 65-95% (w / w) of a biocompatible degradable material, wherein the biocompatible degradable material is an aliphatic polyester.

21. The sustained-release preparation according to claim 20, characterized in that The sustained-release implant is prepared by a preparation method comprising the following steps: (1) dissolving a biocompatible degradable material and a PYY3-36 analog or a pharmaceutically acceptable salt thereof in glacial acetic acid, and then freeze-drying to prepare a lyophilized powder; (2) performing hot melt extrusion of the freeze-dried powder in step (1); (3) Cutting the strips hot-melt extruded in step (2) into cylinders.

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