Gel-forming polypeptides

Abstract

A stable aqueous gel or semi-solid gel pharmaceutical composition comprising a water-soluble peptide having gel-forming ability, optionally in combination with appropriate excipients and therapeutic agents. Upon administration to an individual, the pharmaceutical composition forms a gel reservoir in which the gel nanostructure releases the peptide or encapsulated therapeutic drug for a longer period of time. The gel-forming compound can be formulated as an aqueous formulation, a suspension or a solid formulation, wherein the gel-forming polypeptide contained accounts for 0.01% to 99% of the total weight of the formulation. These formulations are useful for drug delivery and implantable drug depots, for long term delivery of therapeutic drugs, antigens or cells. Also provided are methods of preparing gel-forming compounds by gel formation enhancing motif modification compounds.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511547797.1 (22) Application Date 2019.03.14 (30) Priority Data 62 / 643,593 2018.03.15 US (62) Divisional Application Data 201980019009.8 2019.03.14 (71) Applicant: Edpesola Corporation Address: California, USA (72) Inventor: Xu Xiaoyu (74) Patent Agency: Beijing Hongquan Intellectual Property Agency Co., Ltd. 11363 Patent Attorneys: Xu Weiqun, Lin Chao (51) Int.Cl. A61K 9 / 06 (2006.01) A61K 45 / 06 (2006.01) A61K 47 / 60 (2017.01) A61K 47 / 42 (2017.01) A61K 38 / 23 (2006.01) A61K 38 / 22 (2006.01) A61K 38 / 095 (2019.01) (54) Invention Title: Gel-forming peptide (57) Abstract: A stable aqueous gel or semi-solid gel pharmaceutical composition comprising a water-soluble peptide with gel-forming ability, optionally combined with suitable excipients and therapeutic drugs. After individual administration, the pharmaceutical composition forms a gel reservoir, wherein the gel nanostructure releases the peptide or encapsulated therapeutic drug over a prolonged period of time. Gel-forming compounds can be formulated as aqueous formulations, suspensions, or solid formulations, wherein the gel-forming peptide comprises 0.01% to 99% of the total weight of the formulation. These formulations can be used for drug delivery and implantable drug reservoirs for long-term delivery of therapeutic drugs, antigens, or cells. A method for preparing gel-forming compounds by gel-forming enhanced motif-modified compounds is also provided. Claims (10 pages), Description (52 pages), Drawings (3 pages), CN 121421943 A 2026.01.30 CN 1 21 42 19 43 A 1. An aqueous pharmaceutical composition for the slow release of a therapeutic component, comprising: a) a self-assembling gel-forming polypeptide, having a concentration of at least about 0.01% (w / w) of the total weight of the composition; b) an aqueous excipient. 2. The pharmaceutical composition of claim 1, wherein the gel-forming polypeptide forms a non-covalently linked liquid gel or semi-solid gel in an aqueous solution at a concentration of about 11%-30% w / w or at a lower concentration. 3. The pharmaceutical composition of claim 1, wherein the aqueous excipient has a low ion concentration. 4. The pharmaceutical composition of any one of claims 1-3, further comprising an additive selected from buffers.Liquids, excipients, solvents, solubilizers, preservatives, stabilizers, surfactants, antioxidants, and mixtures thereof. 5. The pharmaceutical composition according to any one of claims 1-4, wherein the self-assembling gel-forming polypeptide is a G protein-coupled receptor (GPCR) ligand polypeptide or a peptide biological functional mediator. 6. The pharmaceutical composition according to claim 5, wherein the ligand is selected from analogs of amylin, CGRP, adrenomedullin (ADM), and adrenomedullin 2 (ADM 2 or IMD), and the analogs may be agonists, antagonists, chimeric analogs, or nonfunctional analogs. 7. The pharmaceutical composition according to any one of claims 1-4, wherein the self-assembling gel-forming peptide is an oxytocin analog, a kissing agonist, a κ opioid receptor agonist, pramorelin, a romilastine analog, urocortin 3, campstatin, GLP-1, GLP-2, thymosin α1, thymosin β4, γ-MSH, a scutellarin receptor antagonist, an opioid receptor ligand, or an analog of GnRH, said analog being an agonist, antagonist, chimeric analog, or nonfunctional analog. 8. The pharmaceutical composition according to any one of claims 1-7, wherein the concentration (by weight) of said gel-forming peptide is from 0.001% to 99% of the total weight of the composition, preferably from 0.1% to 30%. 9. The pharmaceutical composition according to any one of claims 1-8, wherein said gel-forming peptide comprises a sequence listed in any one of Tables 1, 2, or 3. 10. The pharmaceutical composition according to any one of claims 1-8, wherein the gel-forming polypeptide comprises a sequence selected from sequences of serial numbers 1-15, 61, 64, 263, 274 or the like. 11. The pharmaceutical composition according to any one of claims 1-4, wherein the gel-forming polypeptide is selected from ADM, CGRP, or IMD (ADM2) sequences containing the structure of formula I, as follows: R1-B0-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-R2, wherein R1 is a functional group containing the structure of formula (W')(X')n(Y')n(Z')n, wherein W' is a fatty acid, fatty acid diacid, fatty acid or cholesterol derivative or is empty; X' is a PEG group, glutamic acid, γ-glutamic acid, non-protein amino acid or is empty; Y' is a PEG group, glutamic acid, γ-glutamic acid, non-protein amino acid or is empty. -Glutamic acid, non-protein amino acid, or empty; Z' is protein amino acid, non-protein amino acid, or empty; R2 is a C-terminal modification fragment, including {NH2} amidation, {-CHO} peptide aldehyde, {-ol} alcohol peptide, {CMK} chloromethyl ketone, {FMK}Fluoromethyl ketone, {Cya} mercaptoethylamine, {pNA} p-nitroaniline, {-ONP} p-nitrophenol, {AMC} 7-amino-4-methylcoumarin, {AFC}, -OMe (C-terminus), -OEt (C-terminus), -OBzl (C-terminus), -OtBu (C-terminus), {-OSu} hydroxysuccinimide ester, -NHMe (C-terminus), -NHEt (C-terminus), -NH isopentylamino (C-terminus), NH(C2H)6 (C-terminus), -NHPh (C-terminus), {NHEt(O) EtNH-Fmoc} 2,2'-oxodiethylamine-Fmoc, {NHEt(EtNH-Myr)2}, -NH(OMe)Me (C-terminus), -TBzl (C-terminus), - B0 is selected from the group consisting of empty residues, any protein amino acid or non-protein amino acid, acylated histidine (acy-His), acylated arginine (acy-Arg), and acylated lysine (acy-Lys); B1 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, histidine, arginine, lysine, asparagine, glutamine, and non-protein amino acids; B2 is selected from the group consisting of empty residues, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids; B3 is selected from the group consisting of empty residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, glutamine, aspartic acid, and non-protein amino acids. B4 is the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids; B5 is the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, tyrosine, and non-protein amino acids; B6 is the group consisting of free residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, and non-protein amino acids; B7 is the group consisting of free residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, glutamine, asparagine, histidine, arginine, lysine, and non-protein amino acids; B8 is the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, serine, threonine, and non-protein amino acids; B9 is the group consisting of free residues, arginine, lysine, asparagine, glutamine, tryptophan, phenylalanine, serine, threonine, tyrosine, and non-protein amino acids.B10 consists of free residues, alanine, serine, threonine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids; B11 consists of free residues, tryptophan, phenylalanine, valine, alanine, glycine, isoleucine, leucine, proline, and non-protein amino acids; B12 consists of free residues, alanine, glycine, serine, threonine, proline, tyrosine, methionine, tryptophan, phenylalanine, and non-protein amino acids; B13 consists of free residues, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, methionine, leucine, phenylalanine, and non-protein amino acids; B14 consists of free residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and non-protein amino acids. B15 is selected from the group consisting of empty residues, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids; B16 is selected from the group consisting of empty residues, asparagine, glutamine, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids; B17 is selected from the group consisting of empty residues, asparagine, glutamine, serine, threonine, tyrosine, and non-protein amino acids; B18 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, phenylalanine, tyrosine, and non-protein amino acids; B19 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and non-protein amino acids; Claims 2 / 10 pages 3 CN 121421943 A B20 consists of free residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, proline, and non-protein amino acids; B21 consists of free residues, isoleucine, valine, serine, threonine, tyrosine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids; B22 consists of free residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, asparagine, glutamine, proline, and non-protein amino acids; B23 consists of free residues, serine, threonine, tyrosine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, and non-protein amino acids; B24 consists of free residues, alanine, glycine, proline, serine, threonine, tyrosine, and non-protein amino acids; B25 consists of free residues, valine, alanine, glycine, isoleucine, leucine, proline, serine, and threonine.The group consisting of non-protein amino acids; B26 consists of empty residues, histidine, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids; B27 consists of empty residues, valine, alanine, glycine, isoleucine, leucine, serine, threonine, tyrosine, and non-protein amino acids; B28 consists of empty residues, alanine, leucine, isoleucine, valine, phenylalanine, serine, threonine, tyrosine, and non-protein amino acids. 12. A functional self-assembling gel-forming polypeptide agonist or antagonist comprising a sequence of sequence numbers 1-15, 48-58, 61, 64, 263, 274, or an analogue thereof. 13. A gel-forming polypeptide comprising an amino acid sequence having at least 70% identity with an amino acid sequence selected from sequence numbers 1-15, 48-58, 61, 64, 263, and 274. 14. A gel-forming polypeptide comprising an amino acid sequence having at least 80% identity with an amino acid sequence selected from Serial Numbers 1-15, 48-58, 61, 64, 263, and 274. 15. A gel-forming polypeptide comprising a sequence selected from sequences of Serial Numbers 106-114, 116-124, 126-131, and 139-140, and analogues thereof. 16. A gel-forming polypeptide comprising an amino acid sequence having at least 70% identity with an amino acid sequence selected from Serial Numbers 106-114, 116-124, 126-131, and 139-140. 17. A gel-forming polypeptide comprising an amino acid sequence having at least 80% identity with an amino acid sequence selected from Serial Numbers 106-114, 116-124, 126-131, and 139-140. 18. A gel-forming polypeptide comprising a stereoisomer, derivative, analog, or peptide-like amino acid sequence selected from Serial Nos. 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140. 19. A method for preparing a self-assembled liquid gel or semi-solid gel, comprising: dissolving the self-assembled gel-forming polypeptide such that the concentration (by weight) of the resulting solution is at least about 0.01% (w / w) of the total weight of the composition; and dissolving it in an aqueous excipient. 20. The method of claim 19, wherein the polypeptide comprises any sequence from Serial Nos. 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140 or an analog thereof (or composed of the like). 21. A method for treating a subject suffering from a certain condition, which can be relieved by administering a gel-forming polypeptide preparation, said method comprising: (Claims 3 / 10, Page 4, CN 121421943)A. A formulation according to any one of claims 1-20, administered to the subject at an effective dose. 22. A pharmaceutical composition in liquid, semi-solid, or solid gel form, comprising one or more therapeutic agents; a water-soluble gel-forming polypeptide; and optionally excipients and / or therapeutic agents, wherein the composition forms a gel upon contact with the body when injected into a patient. 23. The pharmaceutical composition of claim 22, wherein the therapeutic agent is not covalently linked to the gel-forming polypeptide. 24. The pharmaceutical composition of claim 22 or 23, wherein the gel-forming polypeptide is a GPCR ligand-derived polypeptide or a peptide biological functional mediator. 25. The pharmaceutical composition of claim 24, wherein the gel-forming polypeptide comprises or consists of a gel-forming GPCR ligand or ligand fragment selected from serial numbers 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140 or the like. 26. The pharmaceutical composition according to any one of claims 22-25, wherein the therapeutic agent is selected from small molecule drugs, peptide drugs, macromolecular biological agents, antibodies, hormones, growth factors, antigens, nucleic acids, and nucleotides. 27. A method for treating a subject suffering from a condition that can be relieved by administration of a gel-forming polypeptide formulation, the method comprising: administering to the subject an effective dose of the formulation according to any one of claims 1-11, 12-21, or 22-26, wherein the therapeutic agent is released over a prolonged period of time. 28. The method according to claim 27, wherein: the therapeutic agent is released into systemic circulation, a local tissue or organ, or a surface, including the ocular surface, buccal surface, rectal surface, nasal surface, respiratory organ surface, gastrointestinal tract surface, urethral surface, uterine surface, or skin surface. 29. The method according to claim 27 or 28, wherein: the formulation is administered to the patient via parenteral, intramuscular, subcutaneous, intranasal, intrauterine, intraurethral, ​​intraocular, topical, oral, or intradermal route. 30. The method according to any one of claims 27-29, wherein the pharmaceutical preparation is used in combination with one or more other substances, said substances being selected from small molecules, polypeptides, proteins, enzymes, hormones, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, antibiotics, chemotherapeutic drugs, immunosuppressants, anti-inflammatory drugs, anti-tumor proliferative drugs, antimitotic drugs, angiogenic drugs, anti-angiogenic drugs, antipsychotic drugs, central nervous system (CNS) drugs, anticoagulants, and fibrinolytic drugs; said drugs include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophic agents.Nutritional factor-3 (Nt-3), neurotrophic factor-4 / 5 (Nt-4 / 5), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, myocardial nutrient-1, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor β1 (TGFβ1; TGFβ2, TGFβ3), activin, glial cell-derived neurotrophic factor (GDNF), mid-pregnancy factor, heparin-binding neurotrophic factor (HBNF), transforming growth factor α (TGFα), regulatory proteins (neuroregulatory proteins, ... ARIA), axonal ligand-1 (Al-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), transforming growth factor (TGF), interleukin (IL), colony-stimulating factors (CSF, MCF, GCSF, GMCSF), interferon (IFN), endothelial growth factor (VEGF, EGF), erythropoietin (EPO), angiopoietin (ANG), placental growth factor (PIGF), bone morphogenetic protein (BM). P), growth differentiation factor (GDF); antibodies, antigens, adenosine, adrenergic amines, acetylcholine, histamine derivatives, dopamine derivatives, glutamate derivatives, GABA derivatives, cannabinoid derivatives, prostaglandin derivatives, leukotrienes, thrombin analogs, lysophospholipid (LPA) derivatives, sphingosine 1-phosphate derivatives, LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, apralin analogs, neurotensin analogs, kissing agonist 234 analogs, frog claims 4 / 10 Page 5 CN 121421943 A Cortisol analogs, bradykinin analogs, bradykinin agonist analogs, opioid analogs, neocortigenes analogs, enkephalin analogs, substance P analogs, angiotensin II analogs, parathyroid hormone analogs, PTHrP analogs, GLP-1 analogs, GLP-2 analogs, glucagon analogs, GIP analogs, calcitonin analogs, amylin analogs, CGRP analogs, adrenomedullin analogs, adrenomedullin 2 analogs, neuropeptide Y (NPY) analogs, peptide YY (PYY) analogs, NPY Antagonist analogs, vasoactive intestinal polypeptide (VIP) analogs, urocortin analogs, urocortin 2 analogs, urocortin 3 analogs, bradykinin analogs, somatostatin analogs, endothelin analogs, adrenocorticotropic hormone (ACTH) analogs, melanin I analogs, melanin II analogs, melanocyte-stimulating hormone (MSH) analogs, melanocortin analogs, growth hormone-releasing hormone analogs, ghrelin analogs, HOE140 analogs, iticatide analogs, human growth hormoneAnalogs, insulin analogs, anti-inflammatory 1 analogs, thrombin activator analogs, neuromodulatory peptide U analogs, neuromodulatory peptide S analogs, heparin, interleukin-1 analogs, interleukin-2 analogs, factor V analogs, factor IX analogs, luteinizing hormone analogs, relaxin analogs, ghrelin analogs, follicle-stimulating hormone analogs, atrial natriuretic peptide (ANP). Analogs, brain natriuretic peptide (BNP) analogs, C-type natriuretic peptide (CNP) analogs, guanosine analogs, chemokine analogs, cytokine analogs, interferon analogs, erythropoietin analogs, thrombopoietin analogs, interleukin analogs, tumor necrosis factor (TNF) analogs; thrombopoietin peptide analogs, glatiramer (kiprasone), thymosin α1 analogs, thymosin β4 analogs, cell-penetrating peptides, TAT peptides, kallikrein inhibitors, phospholipase inhibitors, campstatin, temporin A antimicrobial peptide, anti-short bacillus peptide, BMP7-derived bone morphogenesis peptide-1, BMP7-derived bone morphogenesis peptide-2, PEDF (24-57), PEDF (58-101), PEDF (40-57), PEDF (44-77), PEDF (78-121), PEDF (98-114), PEDF-derived P14, PEDF-derived P17, PEDF-derived P18, PEDF-derived P23, PEDF-derived P34, PEDF-derived P44, FGF-derived FK18 peptide, Enfuviritide / Fuzeon peptide, Eptifibatide platelet aggregation inhibitor, YIGSR peptide, RGD peptide, VGVAPG peptide. EEMQRR peptides and YRSRKYSSWY peptides; toxins (such as botulinum toxin) and pharmaceutically acceptable salts of these compounds, or their analogues, fragments, or derivatives; salts of the following substances or analogues: ligands of adenosine receptors, adrenergic receptors, acetylcholine receptors, histamine receptors, dopamine receptors, calcium receptors, glutamate receptors, GABA receptors, cannabinoid receptors, prostaglandin receptors, leukotriene receptors, protease-activated receptors, lysophospholipid (LPA) receptors, sphingosine 1-phosphate receptors, LHRH receptors, vasopressin receptors, oxytocin receptors, apralin receptors, neurotensin receptors, kissing agonist receptors, and dermalin receptors. Opioid receptors, substance P receptors, angiotensin II receptors, parathyroid hormone receptors, GLP-1 receptors, GLP-2 receptors, glucagon receptors, calcitonin receptors, amylin receptors, calcitonin gene-related peptide (CGRP) receptors, adrenal medullary receptors, neuropeptide Y (NPY) receptors, peptide YY (PYY) receptors, vasoactive intestinal peptide (VIP) receptors, urocortin receptors, bradykinin receptors, somatostatin receptors, endothelin receptors, adrenocorticotropic hormone (ACTH) receptors, melanocyte-stimulating hormone (MSH) receptors, melanocyte-stimulating hormone receptors, and melanocyte-stimulating hormone receptors.Plasmin receptor, growth hormone-releasing hormone receptor, ghrelin receptor, insulin receptor, relaxin receptor, natriuretic peptide receptor, guanosine receptor, chemokine receptor, cytokine receptor, growth factor receptor, interferon receptor, erythropoietin receptor, growth hormone receptor, FSH receptor, LH receptor, TSH receptor, interleukin receptor, tumor necrosis factor (TNF) receptor, nerve growth factor receptor, platelet-derived growth factor (PDGF) receptor, colony-stimulating factor (CSF) receptor, bone morphogenetic protein (BMP) receptor, FGF receptor, growth differentiation factor receptor; glatiramer (kipasone) analogues, thymosin, campstatin, temporin A. YIGSR peptide, RGD peptide, VGVAPG peptide, YRSRKYSSWY peptide, and nucleotide derivatives, antibiotics, antibodies, enzyme inhibitors, enzymes, complement factors, urokinase, asparaginase, kallikrein, kallikrein inhibitors, coagulation factors, cytotoxic therapeutics, microbial antigens, viral antigens, tumor antigens, neoantigens, and cosmeceutical peptides and pharmaceutically acceptable salts of these compounds, or analogs, fragments, or derivatives thereof. 31. The composition according to any one of claims 1-11, 12-21, or 22-26, wherein the composition is a pharmaceutical composition, a cosmetic composition, or a dermal filler composition. Claims 5 / 10 pages 6 CN 121421943 A 32. A method of modifying a gel-forming polypeptide, the method comprising: coupling a therapeutic drug to a gel-forming enhancement motif; wherein the motif is an acylated or non-acylated amino acid sequence derived from a cell surface receptor secreted peptide ligand capable of self-assembling into a gel. 33. The method of claim 32, wherein the gel-enhancing motif is derived from any amino acid sequence of sequence numbers 1-15, 61, 64, 263, 274, 106-114, 116-124, 126-131, or 139-140. 34. The method of claim 32 or 33, wherein the gel-forming polypeptide comprises the structure of formula II, Ea-(Fa)n-Ga(II), where Ea is a gel-forming polypeptide motif derived from a cell surface receptor ligand or a therapeutic agent; Fa is a PEG group; n is an integer from 0 to 40; Ga is a therapeutic agent or a gel-forming polypeptide motif derived from a cell surface receptor ligand, and Ea, Fa, and Ga can be positioned and bound together via any side chain of an amino acid. 35. A modified gel-forming polypeptide with a structure of Formula II, Ea-(Fa)n-Ga(II), wherein: Ea is a gel-forming polypeptide with a structure of Formula I or containing a therapeutic drug; Fa is a PEG group; n is an integer from 0 to 40 or a covalent bond connecting the motif in Ea to the motif in Ga; Ga is a therapeutic drug or a gel-forming polypeptide containing a structure of Formula I.The polypeptide, Ea, Fa, and Ga, can be positioned and bound together via any side chain of the amino acids. 36. The gel-forming polypeptide according to claim 34 or 35, wherein the therapeutic agent is selected from small molecules, polypeptides, proteins, enzymes, hormones, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, antibiotics, chemotherapeutic agents, immunosuppressants, anti-inflammatory drugs, anti-tumor proliferative drugs, antimitotic drugs, angiogenic drugs, anti-angiogenic drugs, antipsychotic drugs, central nervous system (CNS) drugs, anticoagulants, or fibrinolytic agents. 37. The gel-forming polypeptide of claim 36, wherein the therapeutic agent is selected from: LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, apralin analogs, neurotensin analogs, kissing agonist analogs, kissing agonist 234 analogs, scutellarin analogs, bradykinin analogs, bradykinin agonist analogs, opioid analogs, neocortexin analogs, enkephalin analogs, substance P analogs, angiotensin II analogs, parathyroid hormone analogs, PTHrP analogs, GLP-1 analogs, GLP-2 analogs, glucagon analogs, GIP analogs, calcitonin analogs, amylin analogs, CGRP analogs, adrenomedullin analogs, adrenomedullin 2 analogs, neuropeptide Y (NPY) analogs, peptide YY (PYY) analogs, NPY antagonist analogs, and vasoactive intestinal polypeptides (VIP). Analogs, urocorticin analogs, urocorticin 2 analogs, urocorticin 3 analogs, bradykinin analogs, somatostatin analogs, endothelin analogs, adrenocorticotropic hormone (ACTH) analogs, melanin I analogs, melanin II analogs, melanocyte-stimulating hormone (MSH) analogs, melanocorticoid analogs, growth hormone-releasing hormone analogs, ghrelin analogs, HOE140 analogs, iticatide analogs, human growth hormone analogs, insulin analogs, anti-inflammatory 1 analogs, Thrombin activator analogs, neuromodulatory peptide U analogs, neuromodulatory peptide S analogs, heparin, interleukin-1 analogs, interleukin-2 analogs, factor V analogs, factor IX analogs, luteinizing hormone analogs, relaxin analogs, ghrelin analogs, follicle-stimulating hormone analogs, atrial natriuretic peptide (ANP) analogs, brain natriuretic peptide (BNP) analogs, C-type natriuretic peptide (CNP) analogs, guanosine analogs, chemokine analogs, cytokine analogs, interferon analogs, erythropoietin analogs, thrombopoietin analogs, interleukin analogs, tumor necrosis factor (TNF) analogs; thrombopoietin peptide analogs, glatiramer (kiprasone), thymosin α1 analogs, thymosin, cell-penetrating peptides, TAT peptides, kallikrein inhibitors, phospholipase inhibitors, campstatin, temporinA. Antimicrobial peptide, anti-short-barbituric peptide, BMP7-derived bone morphogenesis peptide-1, BMP7-derived bone morphogenesis peptide-2, PEDF (24-57), PEDF (58-101), PEDF (40-57), PEDF (44-77), PEDF (78-121), PEDF (98-114), PEDF-derived P14, PEDF-derived P17, PEDF-derived P18, PEDF-derived... (Claims 6 / 10, Page 7, CN 121421943 A) P23, PEDF-derived P34, PEDF-derived P44, FGF-derived FK18 peptide, Enfuviritide / Fuzeon peptide, epitubatide platelet aggregation inhibitor, YIGSR peptide, KTTKS peptide, RGD peptide, VGVAPG peptide, EEMQRR peptide, and YRSRKYSSWY peptide; toxins (such as botulinum toxin), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (Nt-3), neurotrophic factor-4 / 5 (Nt-4 / 5), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, myocardial nutrient-1, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor β1 (TGFβ1; TGFβ2, TGFβ3), activin, glial cell-derived neurotrophic factor (GDNF), mid-pregnancy factor, heparin-bound neurotrophic factor (HBNF), transforming growth factor α (TGFα), regulatory proteins (neuroregulatory proteins, ARIA), axonal ligand-1 (A1-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), transforming growth factor (TGF), interleukin (IL), colony-stimulating factors (CSF, MCF, ... GCSF, GMCSF), interferon (IFN), endothelial growth factor (VEGF, EGF), erythropoietin (EPO), angiopoietin (ANG), placental growth factor (PIGF), bone morphogenetic protein (BMP), growth differentiation factor (GDF); antibodies, antigens or their analogues, fragments or derivatives; salts of the following substances or analogues: ligands of adenosine receptors, adrenergic receptors, acetylcholine receptors, histamine receptors, dopamine receptors, calcium receptors, glutamate receptors, GABA receptors, cannabinoid receptors, prostaglandin receptors, leukotriene receptors, protease-activated receptors, lysophospholipid (LPA) receptors, sphingosine 1-phosphate receptors, LHRH receptors, vasopressin receptors, oxytocin receptors, apralin receptors, neurotensin receptors, kissing agonist receptors, serotonin receptors, opioid receptors, substance P receptors, angiotensin II receptors, parathyroid hormone receptors, GLP-1 receptors, GLP-2 receptors, and glucagon receptors.Glycosin receptor, calcitonin receptor, amylin receptor, calcitonin gene-related peptide (CGRP) receptor, adrenal medulla receptor, neuropeptide Y (NPY) receptor, peptide YY (PYY) receptor, vasoactive intestinal peptide (VIP) receptor, urocortin receptor, bradykinin receptor, somatostatin receptor, endothelin receptor, adrenocorticotropic hormone (ACTH) receptor, melanocyte-stimulating hormone (MSH) receptor, melanocortin receptor, growth hormone-releasing hormone receptor, ghrelin receptor, insulin receptor, relaxin receptor, natriuretic peptide receptor, Guanosine receptors, chemokine receptors, cytokine receptors, growth factor receptors, interferon receptors, erythropoietin receptors, growth hormone receptors, FSH receptors, LH receptors, TSH receptors, interleukin receptors, tumor necrosis factor (TNF) receptors, nerve growth factor receptors, platelet-derived growth factor (PDGF) receptors, colony-stimulating factor (CSF) receptors, bone morphogenetic protein (BMP) receptors, FGF receptors, growth differentiation factor receptors; glatiramer (kipasone) analogs, thymosin, campstatin, temporin A, YIGSR peptide, KTTKS peptide, RGD peptide, VGVAPG peptide, YRSRKYSSWY peptide, nucleoside derivatives, antibiotics, antibodies, enzyme inhibitors, enzymes, complement factors, urokinase, asparaginase, kallikrein, kallikrein inhibitors, coagulation factors, cytotoxic therapeutics, microbial antigens, viral antigens, tumor antigens, neoantigens, and pharmaceutically acceptable peptides and salts of these compounds, or analogs, fragments or derivatives thereof. 38. The gel-forming polypeptide according to any one of claims 34-37, wherein the gel-forming components Ea and Ga are selected from ligands of the following substances: adenosine receptor, adrenergic receptor, acetylcholine receptor, histamine receptor, dopamine receptor, calcium receptor, glutamate receptor, GABA receptor, cannabinoid receptor, prostaglandin receptor, leukotriene receptor, protease-activated receptor, lysophospholipid (LPA) receptor, sphingosine 1-phosphate receptor, LHRH receptor, vasopressin receptor, oxytocin receptor, apralin receptor, neurotensin receptor, kissing agonist receptor, scutellarin receptor, opioid receptor, substance P receptor, angiotensin II receptor, parathyroid hormone receptor, GLP-1 receptor, GLP- 2. Receptors, including glucagon receptor, calcitonin receptor, amylin receptor, calcitonin gene-related peptide (CGRP) receptor, adrenal medulla receptor, neuropeptide Y (NPY) receptor, peptide YY (PYY) receptor, vasoactive intestinal polypeptide (VIP) receptor, urocortin receptor, bradykinin receptor, somatostatin receptor, endothelin receptor, adrenocorticotropic hormone (ACTH) receptor, melanocyte-stimulating hormone (MSH) receptor, melanocortin receptor, growth hormone-releasing hormone receptor, ghrelin receptor, insulin receptor, relaxin receptor, natriuretic peptide receptor, guanosine receptor, and chemokine receptor.Cytokine receptors, growth factor receptors, interferon receptors, erythropoietin receptors, growth hormone receptors, FSH receptors, LH receptors, TSH receptors, interleukin receptors, tumor necrosis factor (TNF) receptors, nerve growth factor receptors, platelet-derived growth factor (PDGF) receptors, colony-stimulating factor (CSF) receptors, bone morphogenetic protein (BMP) receptors, growth differentiation factor receptors, and pharmaceutically acceptable salts of these compounds or their analogues, fragments, or derivatives. 39. The gel-forming polypeptide according to any one of claims 34-37, wherein the gel-forming components Ea and Ga are selected from sequences numbered 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140 or their pharmaceutically acceptable salts. 40. The gel-forming polypeptide according to any one of claims 34-37, wherein Ea and Ga comprise stereoisomers, derivatives, analogs, or peptide-like compounds of amino acid sequences selected from Serial Numbers 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140. 41. The gel-forming polypeptide according to any one of claims 34-37, wherein Ea or Ga is a gel-forming polypeptide whose amino acid sequence has at least 70% identity with an amino acid sequence selected from Serial Numbers 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140. 42. The gel-forming polypeptide according to any one of claims 34-37, wherein the Ea or Ga is a gel-forming polypeptide having an amino acid sequence that is at least 80% identical to an amino acid sequence selected from serial numbers 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140. 43. The gel-forming polypeptide according to any one of claims 34-37, wherein the gel-forming polypeptide comprises an amino acid sequence of serial numbers 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, or an analogue thereof. 44. The gel-forming polypeptide according to any one of claims 34-37, wherein the amino acid sequence comprising it is selected from serial numbers 201-275 or their analogues; wherein the functional component of the gel-forming polypeptide is a GnRH analogue or derivative, a GnRH antagonist, vasopressin, oxytocin, apralin, neurotensin, kissing agonist, taurine, or taurine receptor.Antagonists, neocortexin, enkephalin, κ receptor agonists, substance P, myoglobin, calcitonin, pramlinide (amylin analogue), exenatide 4, GLP-1, teduglutide (GLP-2 analogue), afanotide (melanotan I), melanotan II, γ-MSH, ACTH1-24, setmelanotide, PYY3-36, urocortin 2, urocortin 3, parathyroid hormone, VIP, bradykinin receptor 1 antagonists, HOE140 (BKR2 antagonists), sermorelin, atrial natriuretic peptide (ANP), thymosin α1, thymosin β4, adrenomedullin, adrenomedullin 2, TAT cell penetration enhancement peptide, kallikrein inhibitor, temporin A antimicrobial peptide. 45. Campstatin, glatiramer (or cropason), matrix-modified peptide 1, matrix-modified peptide 4, matrix-modified peptide 7, matrix-modified peptide 8, acetyl hexapeptide-3 matrix-modified peptide, and analogues thereof. 46. A gel-forming polypeptide according to any one of claims 34-44, wherein the relative activity of the polypeptide compared to a corresponding wild-type polypeptide ligand / enzyme / enzyme substrate / medium on at least one homologous receptor or cellular target is at least 0.01%. 47. A gel-forming polypeptide having at least 70% amino acid sequence identity with the amino acid sequence selected from Serial Numbers 201-275. 48. A pharmaceutical composition comprising a pharmaceutically acceptable excipient according to any one of claims 34-47 and a gel-forming polypeptide. 49. A pharmaceutical composition according to any one of claims 1-11, 22-26, or 48, wherein the active agent has therapeutic effects on cardiovascular, pulmonary, gastrointestinal, immune, tumor, skin, kidney, endocrine, ocular, musculoskeletal, or neuronal diseases. 50. A pharmaceutical composition according to any one of claims 1-11, 22-26, or 48-49, wherein the formulation is a liquid or liquid gel, and the administration route includes injection, infusion, or topical application. 51. A pharmaceutical composition according to any one of claims 1-11, 22-26, or 48-49, wherein the formulation slowly releases a polypeptide and / or a therapeutic agent in an individual. 52. A method for treating and / or preventing cardiovascular, pulmonary, gastrointestinal, immune, tumor, skin, kidney, endocrine, ocular, musculoskeletal, or neuronal diseases in an individual, or for treating and / or preventing conditions related to abnormal regulation of cellular processes, the method comprising: administering to the individual an effective dose of a pharmaceutical composition according to any one of claims 1-11, 22-26, or 48-49. 53. An apparatus for delivering a therapeutic drug to a subject, comprising any one of claims 12-18.54. A kit comprising a gel-forming polypeptide according to any one of claims 12-18 or a modified gel-forming polypeptide according to any one of claims 35-47, and optionally further comprising packaging and instructions. 55. The device according to claim 53 or the kit according to claim 54, further comprising at least one additional therapeutic agent. 56. A pharmaceutical composition comprising an aqueous solution or aqueous mixture, suspension, liquid gel or semi-solid gel pharmaceutical composition or solid gel pharmaceutical composition, said composition containing at least one gel-forming polypeptide compound with a water solubility greater than 0.01 mg / mL at room temperature, selected from serial numbers 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140 and 201-275 and their analogues and derivatives. 57. A method of inducing agonistic or antagonistic effects on the cell surface or intracellular receptor, enzyme, or biological process mediator in a desired object, comprising: administering to said object an effective dose of the pharmaceutical composition according to any one of claims 1-11, 22-26, or 48-49. 58. The method according to claim 57, wherein said receptor / enzyme / enzyme substrate / mediator is a GnRH receptor, vasopressin receptor, oxytocin receptor, apralin receptor, neurotensin receptor, kinase receptor, serotonin receptor, opioid receptor, substance P receptor, angiotensin receptor, ghrelin receptor, parathyroid hormone receptor, PTHrP receptor, GIP receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, calcitonin receptor, amylin receptor, CGRP receptor, adrenal medullary receptor, CGRP receptor, MSH receptor, melanocortin receptor, peptide Y receptor, peptide YY receptor, urocortin receptor, bradykinin receptor, etc. GHRH receptor, ACTH receptor, VIP receptor, thrombin receptor, somatostatin receptor, protease-activated receptor, natriuretic peptide receptor, insulin receptor, relaxin receptor, matrix proteins, cellular targets of matrix-modified metrikine peptides, thymosin, thymosin α1, thymosin β4, kallikrein inhibitors, thrombopoietin receptor-binding domains (or romistatin analogs), matrikine peptides, glatiramer (kiprasone), antibiotics and antimicrobial agents (e.g., temporin A), complement regulators (e.g., campstatin), molecules containing cell-penetrating peptides for intracellular delivery compounds (e.g., TAT peptides), microbial antigens, viral antigens, neoantigens, tumor antigens, or cytotoxic agents. 59. A method of inducing a sustained immune response in a patient or animal, comprising administering to said subject an effective dose of the pharmaceutical composition according to any one of claims 1-11, 22-26, or 48-49.60. A method for preparing a gel-forming compound for inducing a sustained immune response in a patient or animal, wherein the gel-forming compound is generated by conjugating a gel-forming enhancing motif to an antigen or neoantigen; said gel-forming enhancing motif is selected from amino acid sequences of serial numbers 1-15, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, and their analogues or derivatives. Claims 9 / 10 pages 10 CN 121421943 A 61. A method for delivering a gel-forming polypeptide according to any one of claims 12-18 or a modified gel-forming polypeptide according to any one of claims 35-47, comprising coating a gel formed by said polypeptide onto the surface of an implantable device or tissue. 62. A method of encapsulating a therapeutic agent, antigen, nanostructure, organelle, or cell with a gel-forming polypeptide of any one of claims 12-18 or a modified gel-forming polypeptide of any one of claims 35-47, comprising holding the therapeutic agent, antigen, nanostructure, organelle, or cell in a sealed space with a gel formed by said polypeptide. Claims 10 / 10 pages 11 CN 121421943 A Gel-forming polypeptide

[0001] Divisional Application Description

[0002] This patent application is a divisional application of Chinese Patent Application No. 2019800190098, filed on March 14, 2019, entitled “Gel-forming polypeptide”.

[0003] Cross-Reference

[0004] This patent application claims priority to U.S. Provisional Patent Application No. 62 / 643,593, filed on March 15, 2018, which is incorporated herein by reference in its entirety. Technical Field

[0005] This patent provides compositions and methods for sustained delivery of therapeutic drugs in the form of stable aqueous formulations containing gel-forming polypeptide compounds, providing sustained release of the therapeutic drug. Background Art

[0006] In common therapeutic modalities, polypeptides are an intermediate form between “small molecule” drugs and injectable macromolecular biological agents and nucleic acid therapeutic agents. Therapeutic peptides and proteins are typically administered parenterally, such as subcutaneously. One advantage of synthetic peptides is that these molecules can be incorporated into non-natural amino acids, improving peptide chain stability and reducing protein hydrolysis, while providing better production reproducibility.

[0007] Most peptides have disadvantages when used as therapeutic agents. For example, these drugs are highly sensitive to protein hydrolysis and are rapidly cleared by the kidneys, resulting in a short half-life and requiring multiple injections. Many therapies require continuous or repeated administration over a longer period, causing discomfort and inconvenience to patients. Therefore, in order to provide polypeptide and protein therapy over a longer period of time...Sustained-release formulations or half-life extension technologies have been developed for therapeutic agents. Sustained-release formulations also enable the delivery of therapeutic agents to specific tissues or organs, thereby minimizing systemic adverse reactions.

[0008] Technologies employing nanostructures capable of slowing drug release can improve the pharmacokinetic and pharmacodynamic characteristics of therapeutic agents after entry into the body or after local treatment. This technology can sustainably modulate cellular signal transduction and reduce peak-valley phenomena in therapeutic drugs (e.g., small molecule drugs, peptides, hormones, proteins, nucleic acids, cellular or prodrug formulations). Highly biocompatible nanomedicine carriers have the potential to substantially improve drug delivery, thereby enhancing efficacy and reducing systemic side effects. For example, oil suspensions and crystal particle suspensions have been widely used for sustained delivery of small molecule drugs. In these systems, the oil and solid particles can form a barrier that limits the dispersion of soluble therapeutic agents.

[0009] Gel polymers are now used as delivery carriers for small molecules and peptides, including gel-forming peptides such as lanreotide. At high concentrations, these gel-forming peptides form polymers and hydrogels, but in contrast, most peptides exist in aqueous solution or produce insoluble precipitates at high concentrations.

[0010] Biocompatible polymeric peptides and hydrogels, in addition to serving as supports in tissue engineering, have wide applications in biotechnology and medicine, particularly in the controlled delivery and release of drugs and therapeutics (see U.S. Patent Nos. 5,034,229, 5,057,318, and 5,110,596). Hydrogels may contain monomer network structures that form self-supporting nanostructures through hydrogen bonding or through hydrophobic interactions and van der Waals forces. For example, polymeric microcapsules containing polylactic acid and polymeric matrices have been used to deliver small molecule, peptide, and protein drugs (e.g., Kent et al., U.S. Patent No. 4,675,189). Hydrogel polymers may include PLA (polylactic acid), PGA (polyethanol), polylactide-glycolic acid copolymer (PLGA), polycyanoacrylate, poly(ε-caprolactone), poly(N-isopropylacrylamide) (NIPA), cellulose ether, hyaluronic acid, lecithin, polyacrylic acid, poly(ω-caprolactone), polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), and agarose, as well as copolymers obtained by combining or modifying these substances. Sustained drug delivery using gel polymer delivery systems has several advantages and is well known in the art.

[0011] Polymers (such as PLGA) are gradually destroyed after entering the body. For example, these types of sustained-release formulations have been used for the delivery of GnRH analogs over periods of weeks or months. Such formulations can improve dosage accuracy and improve patient well-being.Treatment adherence is a concern. However, the application of hydrogel polymers has drawbacks. Many formulations contain a support material and a pharmaceutical composition, wherein the support material may be immunogenic. After administration, the support material may not degrade and thus accumulate in the body. The manufacturing or production process of the formulation may be complex. Furthermore, loading the therapeutic agent into a carrier is usually only a small part of the therapeutic agent preparation process.

[0012] The most widely studied self-assembled peptide hydrogels are hydrogels synthesized from modified oligopeptides having repeating amino acid sequences. For example, the synthetic materials could be tetraphenylethylene-terminated dipeptides (e.g., tetraphenylethylene-terminated bisglycine peptide (TPE-GG), Yeh et al.,

[2016] A novel nanostructured supramolecular hydrogel of tetraphenylethylene-terminated dipeptide self-assembly, Soft Matter 12:6347-51); fluorene methoxycarbonyl (Fmoc) coupled diphenylalanine (Fmoc-FF), Fmoc-tyrosine or naphthalene dipeptide hydrogels, Truong et al.,

[2015] In vitro dissolution and degradation of Fmoc-diphenylalanine self-assembled gels at high concentrations leading to necrosis, Biomaterials Science 3:298-307; Frith et al.,

[2016] Self-assembly, structure formation and application of amphiphilic peptide small molecules, Philos Trans A Math Phys Eng Sci. 374(2072); Morris et al.,

[2015] Structural determinants in low molecular weight gelling agents, Soft Matter 11:1174-81; Zhou et al.,

[2014] Formation of extracellular matrix in minimally bioactive hydrogels based on the self-assembly of aromatic amphiphilic peptides, Journal of Tissue Engineering 5:2041731414531593; Eckes et al.,

[2014] No need for β-sheet structure: Experimental and computational study on the combinatorial method of self-assembly and gelation of Fmoc-dipeptide aqueous gelling agents containing ester analogs, Langmuir 30:5287-96; Palmitoyl-V3A3E3 and palmitoyl-A3V3E3, Fu and Nguyen,

[2015] Sequence-dependent structural stability of enantiomers self-assembling into columnar nanofibers, Biomacromolecules 16:2209-19).

[0013] In addition, various hydrogel substitutes synthesized from complex molecules have been reported. Self-assembled gels can be prepared from the following substances: a mixture of bifunctional peptide LHRH-MPGΔNLS and siRNA (Liu et al.,

[2017] siRNA targeted delivery to the cytoplasm of liver cancer cells via bifunctional carrier peptide, *Drug Delivery and Translational Research* 7:147-155); a mixture of self-assembled peptide and heparin (Liu et al.,

[2016] cationic self-assembled peptide / heparin composite hydrogel slowly releases hepatocyte growth factor through...Improving β-cell survival and function by modulating inflammatory responses (International Journal of Nanomedicine 11:4875-4890); Glycosylated nucleosides and lipid mixtures (Kaplan et al.,

[2016] Application of self-assembled nanofiber hydrogels in the delivery of mechanoresponsive therapeutic anti-TNF α antibodies, 52:5860-3); Incorporating cysteine, cysteine ​​dipeptides and sterically hindered non-natural amino acids (penicillamine) into peptides via oxygen-ester mediated chemical linkages (Rasale et al.,

[2016] Controlling peptide self-assembly via natural chemical linkage / desulfurization strategy, Chemistry-An Asian Journal. 11:926-35); Fusion protein composed of Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) with immunostimulatory effects and avidin (a protein that can bind biotin) (Leblanc

[2014] VaxCelerate II: Rapid development of self-assembled Lassa fever vaccines (Human Vaccines and Immunotherapy 10:3022-38). However, drug delivery using these synthetic sequences may trigger immunogenic reactions and cause cytotoxic effects, thus limiting the application of these nanostructures in the medical field. In addition, most of these molecules require complex manufacturing processes.

[0014] Studies on self-assembled hydrogels have shown that the three-dimensional nanofiber network of the gel nanostructure is assumed to be diverse. For example, palmitoyl-V3A3E3 gel contains a continuous structure dominated by alkyl tail chains, while palmitoyl-A3V3E3 gel is separated from the interconnected micelles of specification page 2 / 52, 13 CN 121421943 A. Similarly, studies on hydrogels containing repeating sequences of a group of analogues called MAX1 (e.g., VKVKVKVKV(D)PPTKVKVKVKV-NH2) have shown that although all analogues can form tangled fibrillary networks of similar diameter, the self-assembly and hydrogelation rates, as well as the mechanical toughness, of the analogues are significantly altered (Chen et al.,

[2014] Modulation of gel kinetics and mechanical stiffness of β-hairpin peptide hydrogels by hydrophobic amino acid substitution, 6:14360-8). These data suggest that the internal structural arrangement of peptide gels and its correlation with the structural stability and mechanical behavior of hydrogel nanostructures remain to be fully determined.

[0015] Using peptide-formed gels will provide significant advantages for formulations; however, the necessary conditions for the evidence-based design of therapeutic peptides that self-assemble into gels are not yet fully understood. Predicting whether a peptide will self-assemble into a gel in aqueous solution and designing a peptide that self-assembles into a polymer or gel at a critical aggregation concentration remains extremely difficult. Furthermore, the way peptides change may differ in solutions with different pH, ionic strength, temperature, or polymer excipients. In order to improve the critical aggregation concentration,The delivery efficacy of various therapeutic agents in bed urgently requires improvements in the performance of sustained-release agents and formulations, and the discovery of therapeutic peptides capable of forming gel polymers on their own.

[0016] This invention relates to compositions and methods of implementing gel-forming peptide therapeutic agents. Formulations of these gel-forming peptides offer certain advantages because, compared to conventional formulations, the peptides can maintain effective levels in vivo for a longer period (i.e., prolonged residence time), thereby increasing the dosing interval and reducing peak-and-trough phenomena of the active ingredient and the total dose. Some embodiments provide methods for formulating and manufacturing such gel-forming peptides. In some embodiments, peptides capable of naturally forming gels are identified, and these peptides can be formulated according to the methods described in this patent to deliver the gel-forming peptides in an optimal manner for therapeutic purposes. In other embodiments, peptides that cannot naturally form gels or only form small amounts of gel are modified by linking a structural motif with gel-forming enhancement properties to obtain or enhance gel-forming ability, and can be formulated by the methods described in this patent for therapeutic use.

[0017] Some embodiments provide compositions and therapeutic formulations in which a gel configuration is employed upon administration delivers an effective dose of the therapeutic drug. For example, a therapeutic formulation using a solution or liquid gel as a carrier releases a therapeutic peptide that transforms into a gel upon administration. The resulting gel nanostructure can act as a physical barrier, reducing the diffusion or dispersion rate of the therapeutic drug, decreasing the hydrolytic capacity of proteins, and reducing renal clearance of the therapeutic drug, thereby prolonging the duration of drug residence in the body. In some embodiments, the therapeutic drug is a gel-forming polypeptide that may be optionally modified to enhance gel-forming ability and reduce the minimum concentration of gel-forming polypeptide required to form a gel configuration in vivo. In some embodiments, the therapeutic drug and the gel-forming polypeptide are provided in combination. In some embodiments, the formulation does not contain a synthetic gel polymer carrier, such as silicon, chitosan, PLA, PGA, PLGA, etc. In some embodiments, the carrier of the formulation is an aqueous solution with extremely low ionic concentration. In other embodiments, the formulation is provided in two containers, with the dry peptide mixed with an aqueous excipient, which may have an extremely low ionic concentration, just before administration.

[0018] In some embodiments, gel-forming peptides include salts of peptide hormones and agonists, antagonists, or nonfunctional analogs of their cell surface receptor ligands, such as GPCRs, tyrosine kinase receptors, etc., and also include salts of therapeutic agents acting on cell surface targets or enzymes and their biologically active or inactive analogs. Soluble gel-forming peptides also include peptides that form a gel carrier and prodrugs present in the form of gel nanostructures. Gel-forming peptides contain a portion of the sequence of a natural peptide hormone or are analogs containing the sequence of a natural hormone, and can be used as carrier polymers. After gel formation, these peptides...It can immediately control the delivery of therapeutic drugs or substances within the gel at a rate suitable for treatment, while avoiding immunogenic reactions. Specification 3 / 52 pages 14 CN 121421943 A

[0019] In some embodiments, gel-forming peptides or peptide compositions can form self-assembled gels in aqueous solutions. Gel-forming peptides may have physiologically relevant biological or therapeutic activities, or may simply play a structural role. The formulation may contain other bioactive molecules. In such formulations, the gel-forming peptides in the gel can be released into the physiological environment over a longer period of time along with optional other bioactive agents. The gel nanostructure can also act as a physical barrier, reducing the solubility of monomeric peptides and the enzymatic degradation ability of gel-forming peptides.

[0020] In some embodiments, gel-forming therapeutic drugs include, but are not limited to, peptide drugs, characterized or modified in that they contain one or more gel structure reinforcing motifs that promote the formation of intermolecular chemical bonds. The reinforcing motif can be a sequence containing a self-assembly reinforcing motif that promotes the self-assembly of peptides in solution. The gel-forming motif can induce the peptide to self-assemble into a hydrogel. Optionally, the gel-forming enhancement motif may be derived from circulating hormones secreted by the human body. The gel-forming enhancement motif may be fused to or otherwise coupled to a therapeutic agent as a cell surface receptor, a regulator of biological function or enzyme, or as an antigen. The coupling may be covalent or non-covalent. In some embodiments, the gel-forming peptide is modified to include a signaling motif that can activate or antagonize a signaling pathway mediated by a target receptor, or activate or antagonize a biological process or enzymatic reaction. In some embodiments, the therapeutic agent is a small molecule, peptide, biologic, nucleic acid, antigen, organelle, or cell.

[0021] In some embodiments, the gel-enhancing motif comprises a circulating peptide ligand fragment secreted by a cell surface receptor (such as a GPCR), including but not limited to peptides identified herein as naturally gel-forming. In some embodiments, the length of the gel-enhancing motif is at least about 2 amino acids and no more than about 52 amino acids, with a maximum length of 5, 7, 9, 12, 15, 18, 21, 24, or 52 amino acids. In some embodiments, the gel-enhancing motif comprises, or is composed of, fragments of adrenomedullin, adrenomedullin 2, CGRP, or chimeric polypeptides derived therefrom. In some embodiments, the gel-enhancing motif comprises (or is composed of) a fragment of 2 to 52 amino acids in length, which has at least 50%, 75%, 90%, or 100% sequence identity with Pal-KVQKLSAPVDPSSPHSY. In some embodiments, the gel-enhancing motif comprises (or is composed of) a fragment of 6 amino acids in length.The gel-enhancing motif comprises a 3-amino acid fragment, Pal-HSY or Pal-KSY (or is composed of such fragments), provided that the peptide naturally containing a Y or SY residue at its amino terminus can be modified by adding only the amino acids required to form the HSY sequence. In some embodiments, the gel-enhancing motif comprises a 2-amino acid fragment, Pal-HS (or is composed of such fragments), provided that the peptide naturally containing an H, Y, or S residue at its amino terminus can be modified by adding only the amino acids required to form the Pal-HS sequence. Alternatively, one or more residues in the amino acid sequence may be substituted to generate the HSY or HS motif. Alternatively, the KSY motif may also be used. In some embodiments, palmitate residues are coupled to the gel-enhancing motif. In some embodiments, the motif is linked to a peptide with a mini-PEG at its carboxyl or amino terminus, or to a side chain of an amino acid. In some embodiments, the peptide contains a detectable marker, such as FITC.

[0022] In some embodiments, the therapeutic agent and the gel-enhancing motif are covalently linked to a self-assembled gel-forming molecule. In some embodiments, the therapeutic agent is encapsulated in a self-assembled gel-forming peptide selected from sequences numbered 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140, and 201-275. In some embodiments, a gel-forming therapeutic agent or a combination of a gel-forming peptide and a therapeutic agent is administered to a subject, the gel-forming peptide / therapeutic agent acting at the administration site and having a prolonged residence time.

[0023] In some embodiments, the gel-forming peptide is a prodrug that only acquires therapeutic activity upon separation from the gel nanostructure and the carrier encapsulating the therapeutic agent.

[0024] Gel-forming peptides include, but are not limited to, the natural and modified gel-forming peptides listed in Tables 1, 2, and 3 herein. These peptides include CGRP, ADM, ADM2, pramlineptide, oxytocin, kissing agonist, pramorelin, thrombopoietin peptide analogs, romilastine analogs, urocortin 3, substance P, GLP-1, GnRH analogs and GLP-2 receptor ligand analogs, scutellarin receptor antagonists, gel-forming ligands of γ-MSH and opioid receptors, and analogs of the above substances; other peptides and their analogs can activate the campstatin + thymosin α1-mediated pathway, the thymosin β4-mediated pathway, and similar pathways. Gel-forming peptides also include peptides containing self-assembly enhancement motifs coupled to functional sequences.These peptides include, but are not limited to, ADM, ADM2, GnRH, GnRH antagonists, vasopressin, oxytocin, apralin, neurotensin, kissing agonist, serotonin, neocorphin, enkephalin, substance P, myotropic hormone, calcitonin, pramlinide (amycin analog), exenatide 4, GLP-1, teduglutide (GLP-2 analog), afanotide (meranotine I), melanotine II, γ-MSH, ACTH1-24, setmelanotide, PYYYY3-36, urocortin 2, urocortin 3, parathyroid hormone, etc. HOE140 (BKR2 antagonist), bradykinin receptor 1 antagonist (BKR1 antagonist), pramorelin, sermorelin, atrial natriuretic peptide (ANP), thymosin α1, thymosin β4, vasoactive intestinal peptide (VIP), TAT cell penetration enhancing peptide, kallikrein inhibitor, antimicrobial peptide (such as Temporin A), glatiramer (or clopidogrel), matrix-modified peptide 1, matrix-modified peptide 4, matrix-modified peptide 7, matrix-modified peptide 8, acetyl hexapeptide-3 matrix-modified peptide, insulin, relaxin, PTHrP, bobmesin receptor antagonist, and analogues of the above substances.

[0025] In one embodiment, a method of treating a patient with a gel-forming formulation is provided, characterized in that: the formulation, when administered, delivers an effective dose and concentration of therapeutic drug. Prior to administration, the therapeutic formulation may be in the form of a liquid solution or a liquid gel. For example, the routes of administration of therapeutic formulations may include intramuscular, subcutaneous, intradermal or intraperitoneal injection, infusion or intranasal, intrauterine, intraocular, topical, oral or rectal administration, wherein the composition forms a gel upon interaction with the patient's bodily fluids. Brief Description of the Drawings

[0026] The most complete understanding of the invention can be obtained by reading the following detailed description in conjunction with the accompanying drawings. It should be emphasized that, by convention, the various features in the drawings are not drawn to scale. Rather, the dimensions of the various features have been arbitrarily enlarged or reduced for clarity. The drawings include the following illustrations.

[0027] Figures 1A-1C. Illustrations of peptide gels formed by sequence number: 1 peptide.

[0028] Figure 2. Bullfrogs treated with MSH gel (peptide sequence number: 224) exhibit persistent color changes compared to animals treated with a wild-type analog [sequence number: 29] (100 nmoles / kg body weight). Detailed Description

[0029] The present invention provides compositions and methods for formulating and using gel-forming peptides. When therapeutic drugs are administered in gel form, low-cost processes can be used for drug formulation, preparation, and production. Because gel-forming peptides are derived from simple sequences of peptide hormones secreted by the human body, the formulation exhibits low immunogenicity. The formulation allows the therapeutic drug to retain a distribution volume similar to its wild-type or natural analogue. The compositions of this invention can be obtained by simply mixing active ingredients.The composition is prepared by reacting the peptide with an aqueous solution, thus significantly reducing the volume, cost, and production time of the candidate therapeutic agent compared to agents containing known sustained-release formulations.

[0030] Without being limited by theory, it has been found that certain secretory peptide hormones can self-assemble into gel nanostructures without the addition of synthetic polymers or other carrier matrices to control the release characteristics of the peptide. These peptides may contain gel-forming enhancement motifs and can spontaneously form gels upon interaction with aqueous solutions. Peptide formulations with this capability can significantly enhance the release of therapeutic agents compared to known sustained-release formulations containing synthetic polymers, while reducing cost and production time. Furthermore, the application of these gel-forming peptides eliminates the need for organic solvents when formulating sustained-release formulations.

[0031] GPCRs and other surface receptors have universal physiological functions and are major drug targets. Researchers have designed novel agonists and inhibitors for various GPCRs and other cell surface receptors to achieve receptor signal transduction in patients. However, a major drawback of many peptide and non-peptide therapeutics is their short half-life and short residence time in vivo. A short half-life is often associated with sensitivity to proteolytic activity and / or renal clearance. The use of self-assembling peptide gel formulations is an important technique for prolonging the in vivo half-life and residence time of drugs. Unlike synthetic polymer molecules (such as PEG, PLA, PGA, PLGA, or collagen used to encapsulate therapeutics), certain therapeutic peptides (such as lanreotide) can self-assemble into gel nanostructures in aqueous solution. After the drug is injected into the patient, the lanreotide gel in the gel reservoir gradually releases lanreotide monomers, resulting in a slow release of lanreotide into the systemic circulation. This can prolong the regulation time of somatostatin receptors in the patient.

[0032] For peptides identified as self-gelling, their formulations can prolong the residence time of these molecules in vivo. Peptides that cannot naturally form gels can be modified by coupling with gel-enhancing motifs. Gel-enhancing motifs can be coupled to any part of the therapeutic agent to promote gel-forming ability, but the final molecular mass does not change significantly. Gel-enhancing motifs can promote the ability of small molecule drugs, peptides, proteins, or other biomolecules to form gels and prolong the residence time of molecules in vivo.

[0033] Another embodiment of the present invention includes a composition characterized by comprising a first self-assembled peptide incorporated with a first biological signal and a second self-assembled peptide incorporated with a second biological signal.

[0034] Definitions

[0035] In carrying out the present invention, those skilled in the art may employ conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, peptide chemistry, and immunology. These techniques are well explained in the literature.For example, *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Enzymatic Methods* (American Academic Press); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Techniques in Molecular Biology* (edited by F.M. Mosuubel et al., 1987); *PCR: Polymerase Chain Reaction* (edited by Mullis et al., 1994); *Current Techniques in Immunology* (edited by J.E. Coligan et al., 1991); and updated or revised versions of all of the above.

[0036] Although specific peptides have been exemplified herein, any of the many alternative peptides and methods that will be apparent to those skilled in the art when considering the invention are equally applicable to the practice of the invention. The methods of this invention, and the assays for determining their efficacy in a particular patient or application, can be performed using standard operating procedures in the art, as guided herein. Therefore, those skilled in the art can employ conventional techniques of molecular biology (including recombinant technologies), microbiology, cell biology, biochemistry, and immunology when carrying out this invention. These techniques are well explained in the literature, for example, *Molecular Cloning: A Laboratory Manual*, 2nd edition (Sambrook et al., 1989); *Oligonucleotide Synthesis* (edited by M.J. Gait, 1984); *Animal Cell Culture* (edited by R.R. Freshney, 1987); *Enzymological Methods* (American Academic Press); *Handbook of Experimental Immunology* (edited by D.M. Weir and C.C. Blackwell); *Gene Transfer Vectors for Mammalian Cells* (edited by J.M. Miller and M.P. Calos, 1987); *Current Techniques in Molecular Biology* (edited by F.M. Mosuubel et al., 1987); *PCR: Polymerase Chain Reaction* (edited by Mullis et al., 1994); *Current Techniques in Immunology* (edited by J.E. Coligan et al., 1991); and updated or revised versions of all the above-mentioned literature.

[0037] The term "therapeutic" as used in this patent refers to obtaining the desired pharmacological and / or physiological effects. Precautionary Information 6 / 52 pages 17 CN 121421943 A The function refers to complete or partial prevention of the disease or its symptoms; the therapeutic function refers to the effect on the disease and / or the symptoms caused by the disease.Adverse reactions result in partial or complete cure. The term "treatment" as used in this patent covers any treatment of mammalian, particularly human, diseases, including: (a) preventing a subject from developing a disease or disease symptoms, wherein the subject may be susceptible to the disease or symptoms but has not yet been diagnosed (e.g., a disease that may be related to or caused by a primary disease); (b) suppressing the disease, i.e., preventing its development; and (c) alleviating the disease, even if the disease subsides.

[0038] The terms "individual," "host," "subject," and "patient" are used interchangeably herein and refer to mammals, including but not limited to primates and humans.

[0039] The term "gel" refers to a three-dimensional network of liquid, semi-solid, or solid components with a volume in the liquid medium range. Hydrogels are networks formed by hydrophilic polymer chains, sometimes existing as colloidal gels, with water as the dispersion medium. Hydrogels are highly absorbent, containing water of 50%, 60%, 70%, 80%, or approximately 85% to 99.9%. Hydrogels are characterized by a distinct plateau in their elastic modulus that lasts for at least several seconds, while their viscous modulus is significantly smaller than that of the plateau region. The elastic modulus of hydrogels is typically in the range of 10–102 kPa. Because hydrogels are prone to fracture and collapse under their own weight, tensile and flexural tests are not suitable for assessing their elastic modulus. The characteristics of the gels described herein can be determined using methods known in the art. For example, see: Kocen et al.,

[2017] Biomedical Materials 12(2), Viscoelastic Properties of Hydrogel Composites for Tissue Engineering under Mechanical Loading; Jonker et al.,

[2012] Chemical Substances 24(5), pp. 759-773, Peptide and Protein Hydrogels.

[0040] "Transparent aqueous solution" refers to a solution consisting of a flowing aqueous solution, wherein preferably 95% ± 5% of the polypeptide solute is completely dissolved, exhibiting a relatively transparent solution appearance, and is free to move when the container is tilted or for a very short time. Depending on the purity of the solute used, trace amounts of solute or particles may be visible in the transparent solution. However, these particles are insufficient to produce an emulsion or turbid appearance. On the other hand, gel-forming polypeptides exhibit the appearance of a liquid gel or a semi-solid gel. The most critical criterion for assessing this is to observe whether the water viscosity increases. The appearance of a liquid gel is characterized by a uniform increase in viscosity; liquid gels with similar appearances include honey or glycerol. The appearance of a liquid gel can be transparent or opaque. Liquid gels are not suitable as suspensions, which are heterogeneous mixtures consisting of insoluble particles. A liquid gel can be a continuous phase, while a gel is a homogeneous single-phase solution with high viscosity, but actually exhibits a semi-solid appearance.

[0041] A semi-solid gel is a formulation consisting of a solvent and one or more solutes, wherein the solute is completely or partially dissolved.Dissolution results in a transparent or homogeneous opaque appearance of the formulation, and due to its high viscosity, the solution is difficult to move in a very short time or when the container is tilted. Semi-solid gel compositions are unsuitable as suspensions, which are heterogeneous mixtures composed of a large number of visible insoluble particles, while solutions are non-uniform.

[0042] Insoluble precipitates formed in suspensions or solutions are aqueous formulations composed of one or more solutes, wherein the solutes are insoluble or partially dissolved, making it impossible for the formulation to present a homogeneous liquid composition like a transparent or opaque solution, or a homogeneous composition with a higher viscosity like a gel.

[0043] The terms “semi-solid suspension” and “semi-solid therapeutic composition” are used interchangeably herein and refer to a viscous paste-like suspension containing a therapeutic polypeptide in a liquid solvent. To maintain the homogeneity of the semi-solid, the semi-solid suspensions of the present invention contain soluble semi-solid particles of gel-forming polypeptides and are pharmaceutically acceptable up to 50% by weight of the formulation. The gel-forming polypeptides can be used with the compositions of the present invention, and when administered to a patient, the salts of the polypeptides should gel in body fluids. Once the gel forms, the peptide can be delivered continuously at a rate suitable for drug therapy.

[0044] A self-assembling gel-forming peptide is a peptide that can form a gel upon contact with an aqueous solution, including physiological fluids such as blood. The process of self-assembling peptides forming a gel is defined as the spontaneous assembly of peptides in solution through non-covalent interactions (Whitesides et al.; Science 1991, 254, 1312-1319). These gels produce a nanostructure with altered rheological properties, as described in specification page 7 / 52, 18 CN 121421943 A.

[0045] Currently proposed peptide gels fall into two main categories: one is mainly composed of alternating hydrophilic and hydrophobic amino acid sequences (amphiphilic peptides), and the other is peptides with hydrophobic groups that form nanofibers (peptide amphiphiles) (Mata et al.,

[2010] Biomaterials 31, 6004; Shah et al.,

[2010] Proceedings of the National Academy of Sciences 107, 3293; Huang et al.,

[2010] Biomaterials 31, 9202; Webber et al.,

[2011] Proceedings of the National Academy of Sciences 108, 13438; Capito et al.,

[2008] Science 319, 1812-1816). For example, peptide amphiphiles with terminal alkyl tail chains can be derived from peptide motifs in collagen. Typically, peptide amphiphiles are formed through hydrogen bonds between the β-sheet structures of amino acids and interactions between hydrophobic tail chains. Another approach involves peptides composed of alternating sequences of hydrophilic and hydrophobic amino acids self-assembling into gels with parallel or antiparallel β-sheet structures. Therefore, peptide gels can include extended cross-beta knots forming tubular and fibrous structures.The structure, as well as the formation of triple helical collagen-like structures, helical barrel structures, or coiled helical bundles of α-helical structures.

[0046] The self-assembly of gels is usually achieved by a combination of hydrogen bonding between monomer molecules and van der Waals interactions (Reches and Gazit; Contemporary Nanoscience 2006, 2, 105-111). Upon entering solution, the potential for self-assembling peptide monomers to form gels increases rapidly until a critical concentration is reached. Solvent conditions (including pH, salt concentration, dielectric properties, and temperature) can alter the self-assembly process of almost all gel-forming peptides (Zhang et al.,

[2010] Nature Materials 9, 594-601). For peptides containing charged residues, higher salt concentrations can reduce the critical aggregation concentration (Ellis-Behnke et al.,

[2006] Nanomedicine: Nanotechnology, Biology and Medicine 2, 207-215). Similarly, pH can also affect the self-assembly properties of peptides (Aggeli et al.; Angewandte Chemie International Edition 2013.42, 5603-5606).

[0047] Growth factors have been physically encapsulated in peptide hydrogels and bound to anionic polymers or structures (such as heparin) via covalent bonds or electrostatic interactions. Disadvantages of these systems and related systems include the lack of specificity of the bound growth factors or the need to degrade the covalent bonds to achieve the desired effect. Furthermore, because these peptides have synthetic sequences containing repeating motifs (e.g., gel-forming peptides containing FKFEFKFE motifs and RADARAD motifs), or are derived from large proteins (such as fibronectin and laminin) or non-circulating proteins (such as amyloid), they may induce adverse immunogenic responses after administration. As described herein, peptide gels made from natural or modified secretory circulating peptide sequences are less likely to induce immunogenic responses and have better safety profiles. Since most polymer carriers typically account for 75-99% of the mass of sustained-release formulations, the use of gel-forming therapeutic agents can increase the effective loading of the therapeutic drug by 20-50 times or more.

[0048] The term "sustained-release" as used in this patent refers to the release of an active substance or carrier material in a patient, allowing the patient to receive a dose of the therapeutic substance over a longer period of time. The proportion of the therapeutic drug in the composition released within the time window will be determined by the expected release rate.

[0049] Aqueous excipients. In some embodiments of the invention, the formulation of the gel-forming peptide uses aqueous excipients to deliver the drug, wherein the excipients may have extremely low ion concentrations. The total ion concentration of such excipients may be less than about 50 mM, less than about 25 mM, less than about 15 mM, less than about 10 mM, less than about 5 mM, less than about 2.5 mM, less than about 1 mM, less than about 0.1 mM, less thanApproximately 0.01 mM, less than approximately 0.001 mM, less than approximately 0.0001 mM, less than approximately 0.00001 mM. For example, salts present in the excipient may include Na+, K+, Cl-, Mn++, Mg++, Ca++, PO4-, etc.

[0050] The term "carrier peptide" refers to a gel-forming peptide that forms a gel in aqueous solution or upon contact with bodily fluids and can be used to encapsulate therapeutic drugs or combination therapeutic drugs for sustained delivery.

[0051] The term "prodrug peptide" refers to a gel-forming peptide that cannot act on biological targets within a gel nanostructure and only becomes active upon separation from the gel nanostructure.

[0052] A gel-enhancing motif is an amino acid sequence that enhances the ability of small molecules, peptides, biologics, antigens, nucleotides, or molecular therapeutics to form gel nanostructures in aqueous solution as described on page 8 / 52 of the specification, 19 CN 121421943 A. The gel-enhancing motif may comprise (or consist of) a circulating peptide ligand fragment secreted by a cell surface receptor. In some embodiments, the gel-enhancing motif is at least about 2 amino acids long and no more than about 52 amino acids long, with a maximum length of 5, 7, 9, 12, 15, 18, 21, 24, or 52 amino acids. In some embodiments, the gel-enhancing motif comprises, or consists of, a fragment of adrenomedullin, adrenomedullin 2, CGRP, or a chimeric polypeptide derived therefrom. In some embodiments, the gel-enhancing motif comprises (or consists of) a fragment of 2 to 52 amino acids long, which has at least 50%, 75%, 90%, or 100% sequence identity with Pal-KVQKLSAPVDPSSPHSY. In some embodiments, the gel-enhancing motif comprises (or consists of) a 6-amino acid fragment having at least 50%, 75%, 90%, or 100% sequence identity with Pal-SSPHSY. In some embodiments, the gel-enhancing motif comprises (or consists of) a 3-amino acid fragment Pal-HSY or Pal-KSY, provided that the peptide naturally containing a Y or SY residue at its amino terminus can be modified by adding only the amino acids required to form the HSY sequence. In some embodiments, the gel-enhancing motif comprises (or consists of) a 2-amino acid fragment Pal-HS, provided that the peptide naturally containing an H, Y, or S residue at its amino terminus can be modified by adding only the amino acids required to form the Pal-HS sequence. Alternatively, one or more residues in the amino acid sequence may be substituted to generate the HSY or HS motif. Alternatively, the KSY motif may also be used. In some embodiments, a palmitate residue is coupled to the gel-enhancing motif. In some embodiments, the motif is coupled with a carboxyl group.The peptide is linked to a mini-PEG at the terminal or amino end, or to the side chain of an amino acid. In some embodiments, the peptide contains a detectable marker, such as FITC.

[0053] In addition to the amino acid sequence that has a gel-enhancing effect, other substances may be coupled to the therapeutic peptide to enhance the gel-forming ability, including but not limited to fatty acids, polyethylene glycol, etc. The addition of fatty acids can enhance the gel-forming ability and promote the subsequent separation of the peptide from the nanostructure, because the chemical bonds therein are easily broken by esterase-mediated fatty acid cleavage.

[0054] The term PEGylation used in this patent refers to the chemical modification of the peptide with one or more polyethylene glycol groups, i.e., PEGylation. The peptide may be directly coupled to PEG via an amino, thiol, hydroxyl, or carboxyl group (i.e., without a linker group). In some embodiments, only one amino acid in the PEGylated peptide contains a PEG group. In other embodiments, two or more amino acids in the PEGylated peptide contain PEG groups.

[0055] In some embodiments, PEG is bound to the peptide via a linker group. A linker group refers to any biocompatible linker group, where "biocompatible" means that the compound or group is non-toxic and can be used in vitro or in vivo without causing damage, discomfort, disease, or death. PEG can be bonded to linker groups via ether bonds, ester bonds, thioester bonds, or amide bonds. Suitable biocompatible linking groups include, but are not limited to, ester, amide, imide, carbamate, carboxyl, hydroxyl, carbohydrate, succinimide (which may include succinimide succinate (Ss), succinimide propionate (SPA), succinimide butyrate (SBA), succinimide carboxymethyl ester (SCM), succinimide succinamide (SSA) or N-hydroxysuccinimide (NHS)), epoxy, oxocarbonyl imidazole (including carbonyl diimidazole (CDI)), nitrophenyl (which may include nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), trysylate, aldehyde, isocyanate, vinyl sulfone, tyrosine, cysteine, histidine, or primary amine. If a properly folded, intact polypeptide protein is reacted with a PEG coupling agent, the PEG group will preferentially react with surface residues rather than masked residues, providing a practical, low-cost method for protein PEGylation and the synthesis of PEGylated polypeptides in this invention.

[0056] The method of linking PEG to a polypeptide is known in the art, and any known method can be used to generate the PEGylated polypeptides described in this invention. For example, see the following literature: Park et al., "Anticancer Manual 9 / 52 pages 20 CN 121421943 A"Research, 1:373-376 (1981); Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnology and Biomedical Applications, ed. J.M. Harris, Plenum Press, NY, Chapter 21 (1992); U.S. Patent No. 5,985,265; U.S. Patent No. 5,672,662 (Harris et al.) and WO 97 / 03106.

[0057] In many embodiments, PEG is a monomethoxy PEG molecule that reacts with a primary amine group on the polypeptide. A method known in the art for modifying polypeptides with monomethoxy PEG is reductive alkylation. For example, see: Chamow et al., (1994) Bioconjugate Chemistry 5:133-140.

[0058] Suitable polyethylene glycols for coupling with peptides are soluble in water at room temperature and have the general formula R(O-CH2-CH2)nO-R, where R is a protecting group such as hydrogen, alkyl, or alkanol, and where n is an integer from 1 to 1000. R is a protecting group, typically having 1 to 8 carbon atoms.

[0059] In many embodiments, PEG has at least one hydroxyl group, such as a terminal hydroxyl group, which, upon modification, can generate a functional group that reacts with an amino group, such as the ε-amino group of a lysine residue, the free amino group at the N-terminus of the peptide, or any other amino group, such as asparagine, glutamine, arginine, or histidine, thereby promoting covalent modification of the peptide by PEG.

[0060] In other embodiments, PEG undergoes derivatization, thereby reacting with a free carboxyl group in the peptide. Suitable PEG derivatives that react with a free carboxyl group at the carboxyl terminus of the peptide include, but are not limited to, PEG-amines and hydrazine derivatives of PEG (e.g., PEG-NH-NH2).

[0061] In other embodiments, PEG is derivatized to include a terminal thiocarboxylic acid group (-COSH), which selectively reacts with an amino group to generate an amide derivative. Due to the reactive nature of thioacids, some amino groups are selective for other amino groups. For example, -SH exhibits sufficient leaving ability to react with an N-terminal amino group under appropriate pH conditions, such that the ε-amino group in the lysine residue is protonated and remains non-nucleophilic. On the other hand, reaction under appropriate pH conditions may allow some accessible lysine residues to react selectively with it.

[0062] In other embodiments, PEG comprises a reactive ester, such as an N-hydroxysuccinimide ester at the end of the PEG chain. Such an N-hydroxysuccinimide-containing PEG molecule reacts with selected amino groups under specific pH conditions (e.g., neutral conditions at pH 6.5–7.5). For example, the N-terminal amino group may be selectively modified under neutral pH conditions. However, if the reagent is highly reactive, the accessible -NH2 group in the lysine may also react.

[0063] In some embodiments, the PEG coupled to the polypeptide has a straight-chain structure. In other embodiments, the PEG coupled to the polypeptide has a branched-chain structure. For example, branched PEG derivatives may include derivatives as described in U.S. Patent No. 5,643,575, star-PEG derivatives, and multi-arm PEG derivatives (e.g., derivatives described in Shearwater Polymers, Inc. catalogue “Polyethylene Glycol Derivatives 1997-1998”). Star PEG is described in the art, such as in U.S. Patent No. 6,046,305.

[0064] The molecular weight of PEGs commonly used is in the range of about 0.2 kDa to about 100 kDa. When describing PEG, the term “about” indicates that in the preparation of polyethylene glycol, some molecules will have a molecular weight greater than or less than said molecular weight. For example, suitable PEGs for coupling with peptides have molecular weights of approximately 0.2 kDa to 5 kDa, approximately 5 kDa to 10 kDa, approximately 10 kDa to 15 kDa, approximately 15 kDa to 20 kDa, approximately 20 kDa to 25 kDa, approximately 25 kDa to 30 kDa, approximately 30 kDa to 40 kDa, approximately 40 kDa to 50 kDa, approximately 50 kDa to 60 kDa, approximately 60 kDa to 70 kDa, approximately 70 kDa to 80 kDa, approximately 80 kDa to 90 kDa, or approximately 90 kDa to 100 kDa.

[0065] The peptide may be coupled with a fatty acid, including but not limited to coupling at the amino terminus, such as C3-C100 alkyl groups having a straight-chain or branched structure; preferably C4-C30 alkyl groups substituted with halogens, hydroxyl groups, alkoxy groups, amino groups, alkylamino groups, dialkylamino groups, sulfates, or phosphates, and the fatty acid may be a saturated fatty acid, a monounsaturated fatty acid, or a diunsaturated fatty acid, for example, in a ratio of 18:0, 24:0, and 24:1. The target fatty acid includes, but is not limited to, palmitic acid, stearic acid, arachidic acid, lauric acid, myristic acid, myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, linoleic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, etc.

[0066] In one embodiment, the gel-forming polypeptide comprises a homolog, variant, or functional fragment of a wild-type peptide analog, including a ligand of a GPCR. In another embodiment, the gel-forming polypeptide comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity to a wild-type (i.e., natural) peptide analog. For example, the sequence of the gel-forming polypeptide may include sequence numbers 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140, 201-275, and derivatives thereof.

[0067] The term gel-forming polypeptide ligand used in this patent may refer to any polypeptide analog that exhibits gel-forming ability at a concentration equal to or lower than that required for the wild-type analog to form a gel in the same aqueous solution, or wild-type and modified polypeptides that are identified as forming a gel at 6, 11, 20, or 30% w / w. However, in one embodiment, the length of the gel-forming polypeptide in this invention may be less than or greater than that of polypeptides in sequence numbers 1-15, 48-58, 61, 64, 101-140, and 201-275, for example, polypeptides truncated at the amino and / or carboxyl terminus by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues, polypeptides substituted with non-natural amino acids, or polypeptides fused with other sequences.

[0068] Composition

[0069] The peptide gel is a smart delivery system comprising biodegradable and highly biocompatible polypeptides, and is approved by regulatory agencies for use in drug delivery. Compared with other colloidal carriers such as nanoemulsions, polymer nanoparticles, liposomes, and solid lipid nanoparticles, peptide gels have significant advantages, the most important of which is their strong drug delivery capability (i.e., enhanced drug loading capacity and increased drug bioavailability) and lack of immunogenicity. Furthermore, this formulation can reduce problems related to manufacturing and applicable formulations. However, the mechanism by which peptides self-assemble into gel nanostructures is not yet clear, and small changes in amino acid composition and other modifications may alter the tendency of peptides to self-assemble into gel nanostructures.

[0070] This patent describes gel-forming peptides and therapeutic compositions comprising at least one peptide with or without a gel-enhancing motif, wherein the enhancing motif is a secretory peptide hormone component capable of enhancing gel-forming ability. The composition may further comprise a functional therapeutic component, or the therapeutic component may be provided by a gel-forming peptide. Provided as a formulation, the formulation comprises stable aqueous solutions, gels, and liquid gels, characterized in that the content of the gel-forming polypeptide compound contained is at least about 0.01%, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 30%, or more (w / w). The formulation may further comprise an aqueous excipient having a very low ionic concentration. A method for preparing a stable aqueous formulation of a gel-forming polypeptide is also provided, characterized in that at least about 0.01%, at least about 0.1%, at least about 1%, at least about 5%, at least about 10%, at least 12%, at least 15%, at least 18%, at least 20%, at least 30%, or more (w / w) of the gel-forming polypeptide is dissolved in an aqueous excipient having a very low ionic concentration. The formulation may be used on an individual suffering from a condition that can be relieved by a therapeutic polypeptide gel, characterized in that an effective amount of the stable aqueous formulation is administered to the subject.

[0071] The compositions and methods described in this patent provide novel and improved self-assembled gel-forming peptides, characterized by: gel-enhancing motifs, improved gel-forming peptide carriers, improved application of therapeutic drugs by forming gel nanostructures, other self-assembled nanostructures, and methods for their preparation and use. Gel-forming therapeutic formulations utilize non-covalent electrostatic interactions to control the solubility and nanostructure of therapeutic molecules or carrier peptides, said molecules or peptides may or may not contain gel-forming enhancing motifs.

[0072] The physicochemical properties (e.g., swelling properties), mechanical properties (e.g., compressive modulus), degradation rate, and surfactant release kinetics of the target hydrogel can be adjusted by varying the amount of the current peptide used. For example, the percentage variation by weight may range from approximately 0.01% to 50%, for example, from approximately 0.02% to 45%, 0.03% to 40%, 0.04% to 35%, and 0.05% to 30%.

[0073] As described above, the physicochemical and mechanical properties, as well as the surfactant release kinetics, of the target hydrogel can vary depending on the hydrogel structure. The target composition absorbs solvent (e.g., water) and swells under non-physiological conditions (e.g., in pure water) or physiological conditions (e.g., in contact with blood or plasma). The term “swelling” as used herein refers to isotropic (or anisotropic) swelling of the hydrogel structure, as solvent (e.g., water) molecules diffuse throughout the entire internal volume space of the hydrogel. The swelling ratio may vary depending on the structure of the hydrogel. “Swelling ratio” refers to the ratio of the weight of the hydrogel after solvent absorption to the dry weight of the hydrogel.

[0074] Similarly, the compressive modulus of the hydrogel can vary depending on the composition of the hydrogel. The compressive modulus refers to the ability of the target hydrogel to withstand axial thrust, which is the uniaxial compressive stress value reached when the material completely fails (e.g., crushed). In some embodiments, the compressive modulus of the target hydrogel may be in the range of 0.1 kPa to 35 kPa, for example, 0.2 kPa to 33 kPa, 0.3 kPa to 30 kPa, 0.4 kPa to 28 kPa, 0.5 kPa to 25 kPa, 0.6 kPa to 22 kPa, 0.7 kPa to 20 kPa, and including 1.0 kPa to 20 kPa.

[0075] The pore size of the hydrogel may also vary depending on the change in the hydrogel structure. In some embodiments, the pore size of the hydrogel ranges from 0.01 micrometers to 1000 micrometers, for example, 0.05 micrometers to 900 micrometers, 0.1 micrometers to 800 micrometers, 0.5 micrometers to 750 micrometers, 1.0 micrometers to 600 micrometers, 2.5 micrometers to 500 micrometers, 5.0 micrometers to 400 micrometers, and including 10.0 micrometers to 300 micrometers.

[0076] Under physiological conditions, the degradation rate of hydrogels can vary due to changes in structure and composition. In some embodimentsIn this embodiment, the structure of the target hydrogel is designed to degrade under physiological conditions (e.g., in vivo) for a predetermined period of time, such as 0.5 days or longer, 1 day or longer, 2 days or longer, 5 days or longer, 7 days or longer, 10 days or longer, 14 days or longer, 21 days or longer, 28 days or longer, 70 days or longer, and including 100 days or longer. In other embodiments, the hydrogel of interest degrades at a predetermined rate upon exposure to physiological conditions, such as substantially zero-order degradation rate, substantially first-order degradation rate, and including substantially second-order degradation rate.

[0077] In one embodiment, the self-assembling gel-forming polypeptide contains one or more acyl groups, which can improve gel-forming ability and increase sensitivity to esterase reactions.

[0078] In some embodiments, the gel-enhancing motif is a sequence derived from a cell surface receptor agonist or ligand, wherein the cell surface receptor is selected from LHRH receptor, angiotensin receptor, oxytocin receptor, apalamin receptor, neurotensin receptor, kissing agonist receptor, scutellarin receptor, delta-opioid receptor, μ-opioid receptor, κ-opioid receptor, substance P receptor, angiotensin II receptor, calcitonin receptor, amylin receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, and calcitonin gene-related peptide (CGRP) receptor. The receptors include: adrenal medulla receptor, melanocortin receptor, parathyroid hormone receptor, bradykinin receptor, neuropeptide Y (NPY) receptor, peptide YY (PYY) receptor, vasoactive intestinal peptide (VIP) receptor, urocortin receptor, somatostatin receptor, endothelin receptor, adrenocorticotropic hormone (ACTH) receptor, melanocyte-stimulating hormone (MSH) receptor, growth hormone-releasing hormone receptor, ghrelin receptor, glucagon receptor, PTHrP receptor, insulin receptor, relaxin receptor, natriuretic peptide receptor, and erythropoietin receptor.

[0079] In some embodiments, the gel-enhancing motif is a sequence derived from a human CLR / RAMP receptor agonist or ligand. In some embodiments, the gel-enhancing motif is a sequence derived from human adrenal medulla, adrenal medulla 2, and CGRP. In some embodiments, the gel-enhancing motif is a sequence derived from human adrenal medulla and / or adrenal medulla 2.

[0080] In some embodiments, the resulting structure has the following chemical formula:

[0081] Ea-(Fa)n-Ga(II) Specification 12 / 52 pages 23 CN 121421943 A

[0082] Wherein, Ea is a polypeptide motif or therapeutic agent derived from a cell surface receptor ligand that can enhance gel formation ability; Fa is a PEG group or linker sequence; n is an integer from 0 to 40; Ga is a therapeutic agent, or a polypeptide motif derived from a cell surface receptor ligand that can enhance gel formation ability.

[0083] In some embodiments, a gel-forming enhancement motif is coupled to a therapeutic agent selected from the group consisting of: small molecules, peptides, proteins, enzymes, hormones, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, antibiotics, chemotherapeutic agents, immunosuppressants, anti-inflammatory drugs, anti-malignant cell proliferation drugs, and antimitotic drugs. Angiogenic drugs, anti-angiogenic drugs, antipsychotic drugs, central nervous system (CNS) drugs, anticoagulants, and fibrinolytic drugs; said drugs include LHRHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, ipalin peptide analogs, neurotensin analogs, kissing agonist analogs, kissing agonist 234 analogs, scutellarin analogs, scutellarin receptor antagonists, bradykinin analogs, bradykinin receptor antagonist analogs, opioid analogs, neocorphin analogs, cerebroside analogs, substance P analogs, angiotensin II analogs, parathyroid hormone analogs, PTHrP analogs, GLP-1 analogs, GLP-2 analogs, glucagon analogs, GIP analogs, calcitonin analogs, amylin analogs, CGRP analogs, adrenomedullin analogs, adrenomedullin 2 analogs, neuropeptide Y (NPY) analogs, and peptide YY. (PYY) analogues, NPY antagonist analogues, vasoactive intestinal peptide (VIP) analogues, urocortin analogues, urocortin 2 analogues, urocortin 3 analogues, bradykinin analogues, somatostatin analogues, endothelin analogues, adrenocorticotropic hormone (ACTH) analogues, melanin I analogues, melanin II analogues, melanocyte-stimulating hormone (MSH) analogues, melanocortin analogues, growth hormone-releasing hormone analogues, ghrelin analogues, HOE140 analogues, insulin analogues, relaxin analogues, atrial natriuretic peptide (ANP) analogues, brain natriuretic peptide (BNP) analogues, C-type natriuretic peptide (CNP) analogues, glatiramer (kiprasone), thymosin α1 analogues, thymosin β4 analogues, cell-penetrating peptides, TAT peptides, kallikrein inhibitors, campstatin, temporin YIGSR antimicrobial peptide, RGD peptide, VGVAPG peptide, EEMQRR peptide, and YRSRKYSSWY peptide; coagulation factors, cytotoxic therapeutic agents, microbial antigens, viral antigens, tumor antigens, neoantigens, and cosmeceutical peptides and pharmaceutically acceptable salts of these compounds.

[0084] In some embodiments, the target of the therapeutic agent in this invention is a cell surface receptor (e.g., opioid receptors and romistase receptors) or an enzyme (e.g., when using a kallikrein inhibitor or campstatin). In some embodiments, the therapeutic target is a biological functional mediator (e.g., the target of glatirameride is less clear, but it is thought to modify currently considered multiple...).It plays a role in the immune process of the pathogenesis of sclerosis; for thymosin α1 and thymosin β4, it is believed to enhance cellular immunity in humans and experimental animals. In some embodiments, the therapeutic target is the cell membrane barrier (e.g., for temporin A antimicrobial peptide derivatives, it is a cell-penetrating peptide, such as the TAT peptide cell-penetrating sequence). In some embodiments, the therapeutic target is skin matrix components and matrix enzymes (e.g., matrix-modified proteins 1, 4, 7, 8 and acetyl hexapeptide-3 matrix-modified peptides).

[0085] The following provides non-limiting examples of peptides that can be applied to gel formation and to therapeutic gel-forming formulations.

[0086] The CLR / RAMP receptor ligands of gel-forming peptides include homologs, variants, chimeras, or functional fragments of adrenomedullin, adrenomedullin 2, and CGRP, such as sequences with sequence numbers 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275, including but not limited to sequences with sequence numbers 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275. In another embodiment, the gel-forming polypeptide comprises approximately 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity with sequences 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275. The term CLR / RAMP receptor ligand as used herein may refer to any functional peptide analog capable of activating or inhibiting CLR / RAMP receptors (CLR / RAMP1, 2, and 3). In one embodiment, the CLR / RAMP receptor ligand is an analogue of adrenomedullin, adrenomedullin 2, and CGRP, or sequences with sequence numbers 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275. However, the CLR / RAMP receptor ligand within the gel-forming peptide of the present invention may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0087] In some embodiments, the ADM, CGRP, or IMD sequence includes the structure of formula I: R1-B0-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-R2, where,

[0088] R1 is a functional group containing the structure of formula (W')(X')n(Y')n(Z')n, where W' is a fatty acid, fatty acid diacid, or fatty acid ester.Derivatives of fatty acids or cholesterol may be empty; X' is a PEG group, glutamic acid, γ-glutamic acid, non-protein amino acid or empty; Y' is a PEG group, glutamic acid, γ-glutamic acid, non-protein amino acid or empty; Z' is a protein amino acid, non-protein amino acid or empty;

[0089] R2 is a C-terminal modification fragment, including {NH2} amidation, {-CHO} peptide aldehyde, {-ol} alcohol peptide, {CMK} chloromethyl ketone, {FMK} fluoromethyl ketone, {Cya} mercaptoethylamine, {pNA} p-nitroaniline, {-ONP} p-nitrophenol, {AMC} 7-amino-4- Methylcoumarin, {AFC}, -OMe (C-terminal), -OEt (C-terminal), -OBzl (C-terminal), -OtBu (C-terminal), {-OSu} hydroxysuccinimide ester, -NHMe (C-terminal), -NHEt (C-terminal), -NH isopentylamino (C-terminal), NH(C2H)6 (C-terminal), -NHPh (C-terminal), {NHEt(O) EtNH-Fmoc}2,2'-oxodiethylamine-Fmoc, {NHEt(EtNH-Myr)2}, -NH(OMe)Me (C-terminal), -TBzl (C-terminal), -NHNH2 (C-terminal), -ED (C-terminal)-NH-CH2CH2-NH2 or -BD (C-terminal)-NH-CH2CH2CH2CH2-NH2NH2 group;

[0090] BO is selected from empty residues, any protein amino acid or non-protein amino acid, acylated histidine (acy-His). The group consisting of acylated arginine (acy-Arg) and acylated lysine (acy-Lys);

[0091] B1 is the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, histidine, arginine, lysine, asparagine, glutamine, and non-protein amino acids;

[0092] B2 is the group consisting of free residues, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids;

[0093] B3 is the group consisting of free residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, glutamine, aspartic acid, and non-protein amino acids;

[0094] B4 is the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids;

[0095] B5 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, tyrosine, and non-protein amino acids;

[0096] B6 is selected from the group consisting of empty residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, and non-protein amino acids;

[0097] B7 is selected from the group consisting of free residues, alanine, leucine, isoleucine, valine, methionine, phenylalanine, glutamine, asparagine, histidine, arginine, lysine, and non-protein amino acids;

[0098] B8 is selected from the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, serine, threonine, and non-protein amino acids;

[0099] B9 is selected from the group consisting of free residues, arginine, lysine, asparagine, glutamine, tryptophan, phenylalanine, serine, threonine, tyrosine, and non-protein amino acids;

[0100] B10 is selected from the group consisting of free residues, alanine, serine, threonine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids;

[0101] B11 is selected from the group consisting of free residues, tryptophan, phenylalanine, valine, alanine, glycine, isoleucine, leucine, proline, and non-protein amino acids; Specification 14 / 52 pages 25 CN 121421943 A

[0102] B12 is selected from the group consisting of empty residues, alanine, glycine, serine, threonine, proline, tyrosine, methionine, tryptophan, phenylalanine, and non-protein amino acids;

[0103] B13 is selected from the group consisting of empty residues, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, methionine, leucine, phenylalanine, and non-protein amino acids;

[0104] B14 is selected from the group consisting of empty residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and non-protein amino acids;

[0105] B15 is selected from the group consisting of empty residues, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids;

[0106] B16 is selected from the group consisting of free residues, asparagine, glutamine, valine, alanine, glycine, isoleucine, leucine, and non-protein amino acids;

[0107] B17 is selected from the group consisting of free residues, asparagine, glutamine, serine, threonine, tyrosine, and non-protein amino acids;

[0108] B18 is selected from the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, phenylalanine, tyrosine, and non-protein amino acids;

[0109] B19 is selected from the group consisting of free residues, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and non-protein amino acids;

[0110] B20 is selected from the group consisting of free residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, proline, and non-protein amino acids;

[0111] B21 is selected from the group consisting of empty residues, isoleucine, valine, serine, threonine, tyrosine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids;

[0112] B22 is selected from the group consisting of empty residues, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, asparagine, glutamine, proline, and non-protein amino acids;

[0113] B23 is selected from the group consisting of empty residues, serine, threonine, tyrosine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, and non-protein amino acids;

[0114] B24 is selected from the group consisting of empty residues, alanine, glycine, proline, serine, threonine, tyrosine, and non-protein amino acids;

[0115] B25 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, and non-protein amino acids;

[0116] B26 is selected from the group consisting of empty residues, histidine, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and non-protein amino acids;

[0117] B27 is selected from the group consisting of empty residues, valine, alanine, glycine, isoleucine, leucine, serine, threonine, tyrosine, and non-protein amino acids;

[0118] B28 is selected from the group consisting of empty residues, alanine, leucine, isoleucine, valine, phenylalanine, serine, threonine, tyrosine, and non-protein amino acids.

[0119] The GnRH receptor ligand component of the gel-forming polypeptide contains a homolog, variant, analog, chimera, or functional fragment of a GnRH receptor agonist or antagonist, such as sequence number 15, or may contain sequence number 15 (or consist of that sequence). In another embodiment, the gel-forming GnRH receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of sequence number 15 or 201, or may comprise or consist of sequence number 15 or 201. As used in this patent, the term GnRH receptor ligand may refer to any functional analogue capable of activating or inhibiting the GnRH receptor (e.g., buserreline, desserreline, futoreline, gosereline, leuproreline, nafareline, and triptoreline). However, the GnRH receptor ligand within the gel-forming polypeptide of this invention may be shorter (e.g., 6-9 or fewer amino acids) or longer (e.g., 11-40 or more amino acids).

[0120] The GnRH receptor antagonist component of the gel-forming peptide comprises a homolog, variant, analog, chimera, or functional fragment of a GnRH receptor agonist or antagonist, such as Serial No. 27, including but not limited to Serial No. 27. In another embodimentIn this invention, the gel-forming GnRH receptor antagonist comprises an amino acid sequence that has approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with sequence 202 or 203, or may comprise sequence 202 or 203 (or consist of that sequence). The term GnRH receptor antagonist as used herein may refer to any functional analogue capable of inhibiting GnRH receptors (e.g., abaric, cetrorex, degarelic, ganilex, and Ozarelix). In one embodiment, the GnRH receptor antagonist is an analogue of a GnRH receptor antagonist or sequence 27. However, the length of the GnRH receptor antagonist within the gel-forming polypeptide of this invention may be shorter or longer than the provided sequence length (e.g., 5–40 or more amino acids).

[0121] The angiotensin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an angiotensin receptor agonist or antagonist, such as Serial No. 17, including but not limited to Serial No. 17. In another embodiment, the gel-forming angiotensin receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to Serial No. 204, or may comprise Serial No. 204 (or consist of that sequence). The term angiotensin receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting angiotensin receptors (e.g., desaminoglycan, lysine vasopressin, arginine vasopressin, d[Leu4,Lys8]-VP, (d(CH2)51,Tyr(Me)2,Arg8)-vasopressin, and vasopressin). In one embodiment, the vasopressin receptor ligand is an analog of the vasopressin receptor ligand or Serial No. 17. However, the length of the vasopressin receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0122] The oxytocin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an oxytocin receptor agonist or antagonist, such as Serial No. 5, including but not limited to Serial No. 5. In another embodiment, the gel-forming oxytocin receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to Serial No. 5 or 205, or may comprise Serial No. 5 or 205 (or consist of that sequence). As used in this patent, the term oxytocin receptor ligand may refer to any functional analogue capable of activating or inhibiting oxytocin receptors (e.g., desmotocin, melotocin, oxytocin, WAY-267, 464, posterior oxytocin, baluciban, atociban, and carbetocin). In one embodiment, the oxytocin receptor ligand is an analogue or sequence of oxytocin receptor agonists or antagonists.Serial number 5. However, the oxytocin receptor ligand length within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids in length).

[0123] The apalaline peptide receptor (i.e., APJ receptor and Apela / ELABELA / Toddler) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an apalaline peptide receptor agonist or antagonist, such as Serial No. 18, including but not limited to Serial No. 18. In another embodiment, the gel-forming apalaline peptide receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to Serial No. 206 or 262, or may comprise Serial No. 206 or 262 (or consist of that sequence). As used in this patent, the term Apalaline peptide receptor ligand may refer to any functional analogue capable of activating or inhibiting the Apalaline peptide receptor (e.g., Apalaline peptide 36, Apalaline peptide 17, Apalaline peptide 13, and Apala / ELABELA / Toddler). In one embodiment, the Apalaline peptide receptor ligand is an analogue of an Apalaline peptide receptor agonist, antagonist, or Serial No. 18. However, the length of the Apalaline peptide receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids). Specification 16 / 52 pages 27 CN 121421943 A

[0124] The neurotensin receptor (NTSR1 and NTSR2) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a neurotensin receptor agonist or antagonist, such as Serial No. 19, including but not limited to Serial No. 19. In another embodiment, the gel-forming neurotensin receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of Serial No. 207, or may comprise Serial No. 207 (or consist of that sequence). As used herein, the term neurotensin receptor ligand may refer to any functional analogue capable of activating or inhibiting neurotensin receptors (e.g., β-lactotensin, JMV-449, neurotensin 13, neuroregulatory peptide N, Xenin 8, Kinetensin, PD-149,163, levocabastine, SR-48692, and SR-142,948). In one embodiment, the neurotensin receptor ligand is a neurotensin 13 analogue or Serial No. 19. However, the neurotensin receptor ligand length within the gel-forming polypeptide of the present invention may be shorter (e.g., 6-12 or fewer amino acids) or longer (e.g., 14-40 or more amino acids) than the provided sequence length.

[0125] The kissing agonist receptor (GPR54) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a kissing agonist receptor agonist or antagonist, such as Serial No. 6, including but not limited to Serial No. 6. In another embodiment, the gel-forming kissing agonist receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to Serial No. 6 or 208, or may comprise Serial No. 6 or 208 (or consist of that sequence). As used herein, the term kissing agonist receptor ligand may refer to any functional analogue capable of activating or inhibiting the kissing agonist receptor (e.g., kissing agonist 10, kissing agonist 13, kissing agonist 17, and kissing agonist 234 antagonists). In one embodiment, the kissing agonist receptor ligand is an analogue of a kissing agonist receptor agonist, an antagonist, or Serial No. 6. However, the length of the kissing agonist receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0126] The dermalin receptor (BB1, BB2, and BB3 receptor) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a dermalin receptor agonist or antagonist, such as sequence numbers 20 and 61, including but not limited to sequence numbers 20 and 61. In another embodiment, the gel-forming dermalin receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to sequence numbers 61, 209, or 255, or may comprise sequence numbers 61, 209, or 255 (or consist of such sequence). As used in this patent, the term "frogerin receptor ligand" may refer to any functional analogue capable of activating or inhibiting the frogerin receptor (e.g., frogerin, neuroregulatory peptide B, BIM187, BIM189, [D-Phe12,Leu14]-frogerin, alytesin, BIM23042, [D-Phe12,Leu14]-frogerin, and gastrin-releasing peptide). In one embodiment, the frogerin receptor ligand is an analogue of a frogerin receptor agonist, antagonist, or sequence numbers 20 and 61. However, the frogerin receptor ligand within the gel-forming polypeptide of this invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids in length).

[0127] The neocorticorhinone or opioid receptor (i.e., δ-opioid receptor, κ-opioid receptor, μ-opioid receptor, and pain-sensitive peptide receptor) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an opioid receptor agonist or antagonist, such as Serial No. 21, including but not limited to Serial No. 21. In another embodiment, the gel-forming opioid receptorThe ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of sequence numbers 211, 212, 213, or 214, or may comprise (or consist of) sequence numbers 211, 212, 213, or 214. As used in this patent, the term opioid receptor ligand may refer to any functional analogue capable of activating or inhibiting opioid receptors (e.g., buprenorphine, leucine enkephalin, methionine enkephalin, neocortisone, DADLE, DPDPE, 7-helical indole-1,4-ethoxymethyl dihydromorphone). In one embodiment, the opioid receptor ligand is an analogue of an opioid receptor agonist, antagonist, or sequence number 21. However, the length of the opioid receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 4-40 or more amino acids). Specification 17 / 52 pages 28 CN 121421943 A

[0128] The enkephalin or opioid receptor (i.e., δ opioid receptor, κ opioid receptor, μ opioid receptor and pain peptide receptor) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera or functional fragment of an opioid receptor agonist or antagonist, such as Serial No. 22, including but not limited to Serial No. 22. In another embodiment, the gel-forming opioid receptor ligand comprises an amino acid sequence that has approximately 70%, 75%, 80%, 85%, 90% or 95% sequence identity with Serial Nos. 211, 212, 213 or 214, or may comprise Serial Nos. 211, 212, 213 or 214 (or consist of the sequence). As used in this patent, the term opioid receptor ligand may refer to any functional analogue capable of activating or inhibiting opioid receptors (e.g., leucine enkephalin, methionine enkephalin, neoepeephalin, DADLE, DPDPE, 7-spiroindinomorphone, and N-phenylethyl-14-ethoxymethyldihydromorphone). In one embodiment, the opioid receptor ligand is an analogue of an opioid receptor agonist, antagonist, or Serial No. 22. However, the length of the opioid receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 4-40 or more amino acids).

[0129] The κ opioid receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a κ opioid receptor agonist or antagonist, such as Serial No. 7, including but not limited to Serial No. 7. In another embodiment, the gel-forming κ-opioid receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to sequence number 7 or 214, or may comprise sequence number 7 or 214 (or consist of that sequence). The term κ-opioid receptor ligand as used herein may refer to any functional analogue capable of activating or inhibiting κ-opioid receptors (e.g.,CR665, difelikefalin (CR845), and dynorphin). In one embodiment, the κ opioid receptor ligand is an analog of a κ opioid receptor agonist, antagonist, or sequence number 7 or 214. However, the length of the κ opioid receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 4-40 or more amino acids).

[0130] The substance P receptor (neurokine 1 receptor) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a substance P receptor agonist or antagonist, such as sequence number 23, including but not limited to sequence number 23. In another embodiment, the gel-forming substance P receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of sequences 215, 253, and 254, or may comprise sequences 215, 253, and 254 (or consist of such sequences). As used in this patent, the term "substance P receptor ligand" may refer to any functional analogue capable of activating or inhibiting the substance P receptor (e.g., substance P, GR-73632, aprepitant, cassopitant, ezlopitant, fosapirant, ranepitant, maropitant, and vertepitant). In one embodiment, the substance P receptor ligand is an analogue of a substance P receptor agonist, antagonist, or sequence number 23. However, the length of the substance P receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0131] The angiotensin II receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analogue, chimera, or functional fragment of an angiotensin II receptor agonist or antagonist, such as sequence number 24, including but not limited to sequence number 24. In another embodiment, the gel-forming angiotensin II receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of sequence number 216, or may comprise sequence number 216 (or consist of that sequence). The term angiotensin II receptor ligand as used herein may refer to any functional analogue capable of activating or inhibiting angiotensin II receptors (e.g., inosine vasopressin). In one embodiment, the angiotensin II receptor ligand is an analogue of an angiotensin II receptor agonist, antagonist, or sequence number 24. However, the length of the angiotensin II receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0132] The calcitonin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analogue, chimera, or functional fragment of a calcitonin receptor agonist or antagonist, such as sequence number 28, including but not limited to sequence number 28. In another embodiment...In embodiments, the gel-forming calcitonin receptor ligand comprises approximately 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence identical to sequence number 217, or may comprise sequence number 217 (or consist of that sequence). The term calcitonin receptor ligand as used herein may refer to any functional analogue capable of activating or inhibiting the calcitonin receptor (e.g., a miacalcin analogue). In one embodiment, the calcitonin receptor ligand is an analogue of a calcitonin receptor agonist, antagonist, or sequence number 28. However, the length of the calcitonin receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5–40 or more amino acids).

[0133] The amylin receptor (calcitonin receptor / RAMP 1, 2, and 3) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an amylin receptor agonist or antagonist, such as Serial No. 4, including but not limited to Serial No. 4. In another embodiment, the gel-forming amylin receptor ligand comprises an amino acid sequence of about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with Serial No. 4 or 218, or may comprise Serial No. 4 or 218 (or consist of that sequence). The term amylin receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting the amylin receptor (e.g., pramlintide). In one embodiment, the amylin receptor ligand is an analogue of an amylin receptor agonist or antagonist, or Serial No. 4. However, the length of the amylin receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0134] The GLP-1 receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a GLP-1 receptor agonist or antagonist, such as sequence numbers 14 and 26, including but not limited to sequence numbers 14 and 26. In another embodiment, the gel-forming GLP-1 receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to sequence numbers 14, 219, 220, 221, or 269-272, or may comprise sequence numbers 14, 219, 220, 221, or 269-272 (or consist of such sequences). The term GLP-1 receptor ligand used in this patent may refer to any functional analogue capable of activating or inhibiting the GLP-1 receptor (e.g., GLP-17-37, exenatide, [Glu13]exenatide, [Met(O)14]-exenatide, [N-acetyl-His1]-exenatide, liraglutide, lixisenatide, abirate, dulaglutide, solanine, etc.).(Marutide and Taslutide). In one embodiment, the GLP-1 receptor ligand is an analog of a GLP-1 receptor agonist, antagonist, or sequence number 14 or 26. However, the length of the GLP-1 receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0135] The GLP-2 receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a GLP-2 receptor agonist or antagonist, such as sequence number 12, including but not limited to sequence number 12. In another embodiment, the gel-forming GLP-2 receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of sequence number 12 or 222, or may comprise sequence number 12 or 222 (or consist of that sequence). As used in this patent, the term GLP-2 receptor ligand may refer to any functional analogue capable of activating or inhibiting the GLP-2 receptor (e.g., teduglutide and GLP2). In one embodiment, the GLP-2 receptor ligand is an analogue of a GLP-2 receptor agonist, antagonist, or sequence number 12. However, the length of the GLP-2 receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0136] The melanocortin receptor (i.e., MC1R-MC5R) ligand component of the gel-forming polypeptide comprises homologs, variants, analogues, chimeras, or functional fragments of melanocortin receptor agonists or antagonists, such as sequence numbers 29, 33, 34, 62, and 64, including but not limited to sequence numbers 29, 33, 34, 62, and 64. In another embodiment, the gel-forming melanocortin receptor ligand comprises an amino acid sequence having approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with sequence numbers 64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273, or may comprise sequence numbers 64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273 (or be composed of such sequences). The term "melanocortin receptor ligand" as used in this patent may refer to any functional analogue capable of activating or inhibiting melanocortin receptors (e.g., α-MSH, β-MSH, γ-MSH, ACTH1-24, corticotropin, afanotide, BMS-470,539, bumenotide, melanotan II, modimelanotide, setmelanotide, PF-00446687, PL-6983, THIQ, PF-219,061, UK-414,495, guinea pig-related peptide, and guinea pig-related peptide).(Guinea pig signal peptide). In one embodiment, the melanocortin receptor ligand is an analog of a melanocortin receptor agonist, antagonist, or sequence number 29, 33, 34, 62, or 64. However, the length of the melanocortin receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 4-40 or more amino acids).

[0137] The neuropeptide Y receptor (NPY1R, NPY2R, PPYR1, NPY5R) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a neuropeptide Y receptor agonist or antagonist, such as sequence number 30, including but not limited to sequence number 30. In another embodiment, the gel-forming neuropeptide Y receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of sequence number 229 or 264, or may comprise sequence number 229 or 264 (or consist of that sequence). As used in this patent, the term neuropeptide Y receptor ligand may refer to any functional analogue capable of activating or inhibiting the neuropeptide Y receptor (e.g., neuropeptide Y fragments 13-36, peptide Yy, peptide YY 3-36 fragments, [Leu31, Pro34]-neuropeptide Y, neuropeptide Y, BVD-10, GR-231, 118, [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-h pancreatic polypeptide, BVD10, and pancreatic polypeptide). In one embodiment, the neuropeptide Y receptor ligand is an analogue of a neuropeptide Y receptor agonist, antagonist, or sequence number 30. However, the length of the neuropeptide Y receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0138] The adrenocorticotropic hormone receptor (CRHR1 and CRHR2) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an adrenocorticotropic hormone receptor agonist or antagonist, such as sequence numbers 10 and 31, including but not limited to sequence numbers 10 and 31. In another embodiment, the gel-forming adrenocorticotropic hormone receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of sequence numbers 10, 230, 231, or 265, or may comprise sequence numbers 10, 230, 231, or 265 (or consist of such sequences). The term "adrenocorticotropic hormone receptor ligand" as used in this patent may refer to any functional analogue capable of activating or inhibiting adrenocorticotropic hormone receptors (CRH, urocortin 1, urocortin 2, urocortin 3, Stressin I, Antalarmin hydrochloride, Antisauvagine-30, LWH-234, CP-154, 526, NBI-27914, R-121).,919, Astressin-2B and Astressin-B). In one embodiment, the adrenocorticotropic hormone receptor ligand is an adrenocorticotropic hormone receptor agonist, antagonist, CRH, urocortin 1, urocortin 2, urocortin 3 analog or serial number 10 or 31. However, the adrenocorticotropic hormone receptor ligand length within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0139] The parathyroid hormone receptor (PTH1R and PTH2R) ligand component of the gel-forming polypeptide contains a homolog, variant, analog, chimera or functional fragment of a parathyroid hormone receptor agonist or antagonist, such as serial number 25, including but not limited to serial number 25. In another embodiment, the gel-forming parathyroid receptor ligand comprises, or may comprise, about 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence identity with sequences 232 and 258 (or may consist of sequences 232 and 258). As used herein, the term parathyroid receptor ligand may refer to any functional analogue capable of activating or inhibiting the parathyroid receptor (e.g., parathyroid hormone, parathyroid hormone-associated protein, DPCAJ1951, teriparatide, and abalotide). In one embodiment, the parathyroid receptor ligand is an analogue of a parathyroid receptor agonist, antagonist, or sequence 25. However, the length of the parathyroid receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5–50 or more amino acids).

[0140] The bradykinin receptor (BDKRB1 and BDKRB2) ligand components of the gel-forming peptides comprise homologs, variants, analogs, chimeras, or functional fragments of bradykinin receptor agonists or antagonists, such as sequence numbers 32 and 65; Firazyr or ateband, including but not limited to sequence numbers 32 and 65. In another embodiment, the gel-forming bradykinin receptor ligand comprises an amino acid sequence with approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to sequence numbers 233, 234, or 261, or may comprise sequence numbers 233, 234, or 261 (or consist of such sequences). As used in this patent, the term bradykinin receptor ligand may refer to any functional analogue capable of activating or inhibiting bradykinin receptors (e.g., bradykinin 1-8, [Leu8]-bradykinin 1-8, Sar-[D-Phe8]-des-Arg9-bradykinin, KRPPGFS-DβNal-I, [Phe8Ψ(CH-NH)-Arg9]-bradykinin, MEN11270, R715, R892, and Hoe140 (atebande)). In one embodiment, the bradykinin receptor ligand is bradykinin receptor ligand.The peptide receptor agonist, antagonist analogue, or sequence number 32. However, the bradykinin receptor ligand length within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids).

[0141] The ghrelin / growth hormone secretagogue receptor (GHSR) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a ghrelin / growth hormone secretagogue receptor (GHSR) agonist or antagonist, such as sequence number 8, including but not limited to sequence number 8. In another embodiment, the gel-forming ghrelin / growth hormone secretagogue receptor (GHSR) ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of sequence number 8 or 235, or may comprise sequence number 8 or 235 (or consist of that sequence). As used in this patent, the term ghrelin / growth hormone secretagogue receptor (GHSR) ligand may refer to any functional analogue capable of activating or inhibiting the ghrelin / growth hormone secretagogue receptor (e.g., alamolin, carmonorelin, esarorelin (hesararelin), ghrelin (lenorelin), GHRP-6, ibuprofen (Mk-677), iparorelin, masirelin, pramorelin (GHRP-2), remorelin, SM-130, 686, tamorelin, and ulimorelin). In one embodiment, the ghrelin / growth hormone secretagogue receptor ligand is an analogue of a ghrelin / growth hormone secretagogue receptor agonist, antagonist, or Serial No. 8. However, the length of the ghrelin / growth hormone secretagogue receptor (GHSR) ligand within the gel-forming polypeptide of this invention may be shorter or longer than the provided sequence length (e.g., 5-50 or more amino acids).

[0142] The growth hormone-releasing hormone receptor (GHRHR) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a growth hormone-releasing hormone receptor agonist or antagonist, such as Serial No. 35, including but not limited to Serial No. 35. In another embodiment, the gel-forming ghrelin / growth hormone secretagogue receptor (GHSR) ligand comprises about 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence identical to that of Serial No. 35, 236, or 237, or may comprise Serial No. 35, 236, or 237 (or consist of that sequence). The term growth hormone-releasing hormone receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting the growth hormone-releasing hormone receptor (e.g., CJC-1295, domorelin, GHRH (adrenergic troponin-releasing factor), rimoretarine, sermorelin (GHRH1-29), and temorelin). In one embodiment, the growth hormone-releasing hormone receptor ligand is a growth hormone-releasing hormone receptor agonist or antagonist.The length of the growth hormone-releasing hormone receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-50 or more amino acids).

[0143] The vasoactive intestinal peptide receptor (VIPR) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a vasoactive intestinal peptide receptor agonist or antagonist, such as Serial No. 67, including but not limited to Serial No. 67. In another embodiment, the gel-forming VIP receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of Serial No. 266, or may comprise Serial No. 266 (or consist of that sequence). The term VIP receptor ligand as used in this patent may refer to any functional analog capable of activating or inhibiting the VIP receptor. In one embodiment, the VIP receptor ligand is an analog of a VIP receptor agonist, antagonist, or Serial No. 67. However, the VIP receptor ligand length within the gel-forming polypeptide of the present invention may be shorter or longer than the provided sequence length (e.g., 5-50 or more amino acids).

[0144] The natriuretic peptide receptor (i.e., NPR1, NPR2, and NPR3) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a natriuretic peptide receptor agonist or antagonist, such as Serial No. 41, including but not limited to Serial No. 41. In another embodiment, the gel-forming natriuretic peptide receptor ligand comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of Serial No. 238, or may comprise Serial No. 238 (or consist of that sequence). As used in this patent, the term natriuretic peptide receptor ligand may refer to any functional analogue capable of activating or inhibiting natriuretic peptide receptors (e.g., atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, and nesiritide). In one embodiment, the natriuretic peptide receptor ligand is an analogue of natriuretic peptide receptor agonists and antagonists, atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, or Serial No. 41. However, the length of the natriuretic peptide receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5-50 or more amino acids).

[0145] The thymosin α1-like ligand receptor ligand component of the gel-forming polypeptide comprises thymosin α1 and homologs, variants, analogues, chimeras, or functional fragments of thymosin α1-like ligands, such as Serial No. 13, including but not limited to Serial No. 13. In another embodiment, the gel-forming thymosin α1-like ligand comprises a sequence corresponding to sequence numbers 13, 239, 240, 267, or 268.The amino acid sequence is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical, or may include sequence numbers 13, 239, 240, 267, or 268 (or consist of such sequences). The term thymosin α1-like ligand as used in this patent may refer to any functional analogue (e.g., thymosin α1) capable of activating or inhibiting thymosin α1-mediated signaling pathways. In one embodiment, thymosin α1 and the thymosin α1-like ligand are analogues of thymosin α1 or sequence number 13. However, the thymosin α1-like ligand within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5–50 or more amino acids in length).

[0146] The thymosin β4 ligand receptor ligand component of the gel-forming polypeptide comprises homologs, variants, analogues, chimeras, or functional fragments of the thymosin β4 ligand, such as sequence number 66, including but not limited to sequence number 66. In another embodiment, the gel-forming thymosin β4-like ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of Serial No. 263, or may comprise Serial No. 263 (or consist of the sequence thereof). The term thymosin β4 as used herein may refer to any functional analogue (e.g., thymosin β4) capable of activating or inhibiting thymosin β4-mediated signaling pathways. In one embodiment, thymosin β4 and the thymosin β4-like ligand are analogues of thymosin β4 or Serial No. 263. However, the thymosin β4-like ligand within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5–50 or more amino acids in length).

[0147] The cell-penetrating peptide component of the gel-forming polypeptide comprises homologs, variants, analogues, chimeras, or functional fragments of the cell-penetrating peptide, such as Serial No. 36, including but not limited to Serial No. 36. In another embodiment, the gel-forming cell-penetrating peptide comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of sequence number 241, or may comprise sequence number 241 (or consist of that sequence). The term cell-penetrating peptide used in this patent may refer to any functional peptide analog that facilitates the transfer of molecules from the extracellular space to the intracellular space (see examples in the following literature: Kalafatovic D and Giralt E

[2017] , Cell-penetrating peptides: Design strategies beyond primary structure and amphiphilicity, *Molecules* 22(11). In one embodiment, the cell-penetrating peptide is an analog of the cell-penetrating peptide or Serial No. 36. However, the cell-penetrating peptide within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5–50 or more amino acids in length).

[0148] The kallikrein regulatory component of the gel-forming polypeptide contains homologs, variants, analogs, chimeras, or functional fragments of the kallikrein regulatory factor, such as Serial No. 37, including but not limited to Serial No. 37. In another embodimentIn this invention, the gel-forming kallikrein regulator comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of Serial No. 242, or may comprise Serial No. 242 (or consist of the sequence thereof). The term kallikrein regulator as used herein may refer to any functional analogue capable of activating or inhibiting kallikrein (e.g., icarapotetide). In one embodiment, the kallikrein regulator is an analogue of kallikrein activators and inhibitors or Serial No. 37. However, the kallikrein regulator within the gel-forming polypeptide of this invention may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0149] The polypeptide antibiotic or antimicrobial peptide component of the gel-forming polypeptide comprises a polypeptide antibiotic or antimicrobial peptide source, variant, analog, chimera, or functional fragment of the same specification (pages 22 / 52, 33 CN 121421943 A), such as Serial No. 68, including but not limited to Serial No. 68. In another embodiment, the gel-forming peptide antibiotic comprises, or may comprise, about 70%, 75%, 80%, 85%, 90%, or 95% of an amino acid sequence identical to that of sequence number 243. The term peptide antibiotic as used in this patent may refer to any functional analogue capable of inhibiting the growth or proliferation of microorganisms, bacteria, fungi, viruses, tumors, or other pathogens. In one embodiment, the polypeptide antibiotic is an analogue of a polypeptide antibiotic (e.g., temporin A, bacitracin A, B, C and D, defensin, esculentin 1-21, silkworm antimicrobial peptide, andropin, silkworm antimicrobial peptide, ceratotoxin, bee venom peptide, Xenopus antimicrobial peptide, skin antimicrobial peptide, toad antimicrobial peptide, brevinin-1, esculentin, buforin II, CAP18, LL37, abaecin, apidaecin, prophenin, indolicidin, actinomycin, bacitracin, colistin, polymyxin B, actinomycin-D, bacitracin, bosavirin, dabavancin, daptomycin, enfuviride, orivoxetine, teicoplanin, terabixin, telapivoxetine, and vancomycin) or serial number 68. However, the polypeptide antibiotic within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0150] The complement regulator component of the gel-forming peptide comprises a homolog, variant, analog, chimeric, or functional fragment of a complement regulator, such as Serial No. 11, including but not limited to Serial No. 11. In another embodiment, the gel-forming complement regulator comprises about 70%, 75%, 80%, 85%, 90%, or 95% amino groups with sequence identity of Serial No. 11 or 244.The acid sequence may include or may contain sequence number 11 or 244 (or consist of that sequence). The term complement modulator as used in this patent may refer to any functional analogue capable of activating or inhibiting complement factors or complement cascades (e.g., complement 5a and campstatin analogues). In one embodiment, the complement modulator is an analogue of a complement activator or inhibitor (e.g., campstatin, complement C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9) or sequence number 11. However, the complement modulator within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5–50 or more amino acids in length).

[0151] The C5aR receptor ligand component of the gel-forming polypeptide contains homologs, variants, analogues, chimeras, or functional fragments of the C5aR receptor ligand, such as sequence number 11, including but not limited to sequence number 11. In another embodiment, the gel-forming C5aR receptor ligand comprises, or may comprise, about 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence identical to that of sequence number 11 or 244 (or may consist of sequence number 11 or 244). As used herein, the term C5aR receptor ligand may refer to any functional analogue capable of activating or inhibiting the C5aR receptor (e.g., complement 5a and campstatin analogues). In one embodiment, the C5aR receptor ligand is an analogue of a C5aR receptor activator or inhibitor (e.g., campstatin, complement C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9) or sequence number 11. However, the C5aR receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5–50 or more amino acids in length).

[0152] Each possibility

[0153] The gel-forming polypeptide's clopason immunomodulator component comprises a homolog, variant, analog, chimera, or functional fragment of an immunomodulator, such as Serial No. 42, including but not limited to Serial No. 42. In another embodiment, the gel-forming clopason immunomodulator comprises an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to Serial No. 245, or may comprise Serial No. 245 (or consist of that sequence). The term clopason immunomodulator as used herein may refer to any functional analogue (e.g., copolymer 1, Cop-1, or clopason, Glatopa, and M356) consisting of four amino acids (i.e., glutamic acid, lysine, alanine, and tyrosine) found in basic myelin proteins, and these analogues are capable of activating or inhibiting glatiramer acetate-mediated signaling pathways. In one embodiment, the immunomodulator is a glatiramer acetate analog (a mixture of analogs consisting of four amino acids found in basic myelin).(e.g., 5-60 or more amino acids in length). However, the immunomodulator within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., 5-60 or more amino acids in length).

[0154] The matrix-modified protein (or matrikine) component of the gel-forming polypeptide comprises homologs, variants, analogs, chimeras, or functional fragments of matrix-modified proteins, such as Serial Numbers 39 and 44-47, including but not limited to Serial Numbers 39 and 44-47. In another embodiment, the gel-forming matrix-modified protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of Serial Numbers 246, 247, 248, 249, or 250, or may comprise Serial Numbers 246, 247, 248, 249, or 250 (or consist of the sequence). As used in this patent, the term matrix-modified protein can refer to any functional analogue capable of activating or inhibiting matrix enzyme-mediated signaling pathways. In one embodiment, the matrix-modified protein is an analogue of the following substances (e.g., YIGSR, Pal-KTTKS, Pal-GHK, GERK, RGD, GQPR, VGVAPG, HFRW, YRSRKYSSWY, and hexapeptides): dermal extracellular matrix, collagen regulators, elastin regulators, keratinocyte / epidermal cell regulators, melanogen production regulators, structural peptides, carrier peptides, and neurotransmitter function regulators that act as signal regulators of extracellular matrix components, or sequence numbers 39 or 44-47 (see: Pai et al.,

[2016] Topical peptides for cosmeceuticals, Indian Journal of Dermatology, Venereology and Leprosy 83:9-18). However, the matrix-modified protein within the gel-forming polypeptide of this invention may be shorter or longer than the provided sequence length (e.g., 5-40 or more amino acids in length).

[0155] The thrombopoietin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of the thrombopoietin receptor ligand, such as Serial No. 9, including but not limited to Serial No. 9. In another embodiment, the gel-forming matrix-modified protein comprises an amino acid sequence of about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with Serial No. 9, or may comprise Serial No. 9 (or consist of that sequence). The term thrombopoietin receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting the thrombopoietin receptor (e.g., romistatin). In one embodiment, the thrombopoietin receptor ligand is an analogue of thrombopoietin (THPO), megakaryocyte growth and development factor (MGDF), or Serial No. 9. However, the gel in this invention...The thrombopoietin receptor ligand within the gel-forming polypeptide may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0156] The insulin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of the insulin receptor ligand, such as Serial No. 59, including but not limited to Serial No. 59. In another embodiment, the gel-forming matrix modified protein comprises an amino acid sequence that is approximately 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to Serial No. 251, or may comprise Serial No. 251 (or consist of that sequence). As used herein, the term insulin receptor ligand may refer to any functional analogue capable of activating or inhibiting the insulin receptor (e.g., insulin degludec, insulin lispro, insulin aspart, insulin liscous, insulin detemir, and insulin glargine). In one embodiment, the insulin receptor ligand is an analogue of insulin or Serial No. 59. However, the length of the insulin receptor ligand within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., the lengths of the B chain and A chain are 5-50 or more amino acids, respectively).

[0157] The relaxin receptor ligand component of the gel-forming polypeptide comprises homologs, variants, analogs, chimeras, or functional fragments of the relaxin receptor ligand, such as Serial No. 60, including but not limited to Serial No. 60. In another embodiment, the gel-forming matrix modified protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of Serial No. 252, or may comprise Serial No. 252 (or consist of that sequence). The term relaxin receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting the relaxin receptor (i.e., LGR7 and LGR8). In one embodiment, the relaxin receptor ligand is an analogue of relaxin 1, relaxin 2, relaxin 3, INSL3, INSL7, or Serial No. 60. However, the relaxin receptor ligand length within the gel-forming polypeptide of the present invention may be shorter or longer (e.g., the B chain and A chain lengths are 5-50 or more amino acids, respectively).

[0158] The parathyroid hormone-related peptide (PTHrP) receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a PTHrP receptor agonist or antagonist, such as Serial No. 63, including but not limited to Serial No. 63. In another embodiment, the gel-forming PTHrP receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% sequence identical to that of Serial No. 258, or may comprise Serial No. 258 (or consist of that sequence). The term PTHrP receptor ligand as used in this patent may refer to any functional analogue capable of activating or inhibiting the PTHrP receptor.(e.g., parathyroid hormone, parathyroid hormone-related protein, teriparatide, and abalotide). In one embodiment, the PTHrP receptor ligand is a PTHrP receptor agonist, antagonist, PTHrP 1-34, an analog of abalotide, or Serial No. 63. However, the PTHrP receptor ligand length within the gel-forming peptides of the present invention may be shorter or longer (e.g., 5-50 or more amino acids).

[0159] The sequence of the peptide can be altered using a variety of methods known in the art to produce targeted changes to the sequence. Typically, the sequence of the peptide is substantially similar to the sequence provided herein, i.e., the sequence identity with the provided sequence will be greater than 70%, greater than 80%, greater than 90%, or greater than 95%. The sequence alteration may be substitution, insertion, or deletion. Scanning mutagenesis with the systematic introduction of alanine or other residues can be used to identify key amino acids. Conservative substitutions of amino acids typically include the substitution of the following groups for each other: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine). Non-conservative substitutions may include any uncommon combination of amino acids.

[0160] Targeted modification methods that do not alter the primary sequence include chemical derivatization of the peptide, such as methylation, acetylation, acylation, polyethylene glycolation, or carboxylation. Other methods also include glycosylation modifications, such as modifying the glycosylation pattern of the peptide during its synthesis and processing or in further processing steps; for example, exposing the peptide to enzymes that can affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes.

[0161] In other embodiments, the peptide comprises an optical isomer, enantiomer, diastereomer, tautomer, cis-trans isomer, racemic mixture, prodrug, or pharmaceutically acceptable salt of the peptide. The peptide can be C-terminally amidated. Optionally, the modified peptide can be acylated. Optionally, one or more acylations can be performed, with one reactive group being preferred.

[0162] The peptide formulation also includes a mixture of stereoisomers or a mixture of individual pure isomers or substantially pure isomers. For example, optionally, the compounds of the present invention may have one or more asymmetric centers at the carbon atom containing any substituent. Therefore, the compound may exist in the form of enantiomers or diastereomers or mixtures thereof. When the compounds of the present invention contain a double bond, the compound may exist in the form of geometric isomers (cis compounds, trans compounds), and when the compounds of the present invention contain unsaturated bonds such as carbonyl groups, the compound may exist in the form of tautomers, and the compound may also include these isomers or mixtures thereof. When preparing the compounds of the present invention, starting compounds existing in the form of racemic mixtures, enantiomers or diastereomers may be used. When the obtained compound is diastereomerWhen isomers or enantiomers are present, they can be separated by conventional methods such as chromatography or fractional crystallization. Furthermore, the compounds in this invention include their intramolecular salts, hydrates, solvates, or polymorphs.

[0163] This invention also includes peptides modified with general molecular biology techniques and synthetic chemistry to improve their resistance to protein hydrolysis, optimize their solubility, or enhance their suitability as therapeutic agents. For example, the stability of the peptide can be enhanced by cyclizing the peptide backbone with cyclic disulfide bonds or lactam bonds (see Friedler et al.,

[2000] Journal of Biochemistry 275:23783-23789). Analogs of such peptides contain residues other than natural L-amino acids, such as D-amino acids or non-natural synthetic amino acids.

[0164] The target peptide can be prepared via in vitro synthesis using conventional methods known in the art. Various commercially available synthetic instruments are available, such as automated synthesizers provided by Applied Biosystems, Inc., Foster City, CA, Beckman, etc. These synthesizers can be used to replace natural amino acids with non-natural amino acids. The specific sequence and preparation method (page 25 / 52, CN 121421943 A) will be determined based on convenience, cost-effectiveness, and required purity.

[0165] If necessary, various groups can be introduced into the peptide during synthesis or expression to allow it to link with other molecules or surfaces. Therefore, cysteine ​​can be used to prepare thioethers, histidine can be used to link metal ion complexes, carboxyl groups can be used to form amides or esters, and amino groups can be used to form amides, etc.

[0166] The peptide can also be separated and purified according to conventional methods of recombinant synthesis. Lysates of the expressed host can be prepared and purified using HPLC, size exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, considering contaminants involved in the product preparation and purification methods, the weight of the composition used will be at least 20% of the target product weight, typically at least about 75%, preferably at least about 95%. For therapeutic purposes, it is typically at least 99% of the target product weight. Typically, the above percentages are calculated based on total protein.

[0167] Compositions and Uses of Gel-Forming Peptides

[0168] This invention relates to the preparation and use of self-assembled gel-forming peptides. In some embodiments, the peptides are derived from human-secreted peptide hormones, human peptide analogs, and peptides having gel-forming enhancement motifs. The gel-forming peptides may be natural or modified gel-forming peptides for treating patients, characterized in that a therapeutic composition comprising an effective dose of the gel-forming peptide is administered to an individual requiring the treatment, and the composition may be used alone or in combination with other therapeutic agents.

[0169] A series of peptide sequences are provided, which contain or mimic natural polypeptide therapeutic agents but have the ability to form gels. The compositions and improved methods for preparing self-assembled polypeptides are characterized by the ability to dissolve such polypeptides at suitable concentrations or to couple them with gel-forming enhancement motifs. Highly biocompatible and biodegradable polypeptide gels can be used to deliver therapeutic drugs. When used as a carrier protein, the therapeutic drug is encapsulated / bound within a gel-forming polypeptide carrier; alternatively, the polypeptide gel itself can also be used as a therapeutic drug.

[0170] By applying this technology, the following objectives can be achieved: (1) to prepare therapeutic analogs with gel-forming capabilities, where wild-type peptides or known analogs typically cannot form gels on their own; (2) to form high-level, non-immunogenic polypeptide gel nanostructures that encapsulate therapeutic drugs for slow release by reducing solubility and providing a surface barrier; (3) to reduce the degradation or clearance rate of polypeptide therapeutic drugs in vivo; and (4) to incorporate natural or wild-type hormones / therapeutic agents with gel-forming capabilities into formulations that have a longer residence time in vivo. Compared to injectable aqueous solutions or crystal particle suspensions, the reversible phase transition of non-covalently bound monomers in peptide gel nanostructures enables sustained delivery of bioactive therapeutic agents over a longer period. Typically, the resulting liquid or semi-solid gels pose a very low risk of immunogenicity and are readily degraded.

[0171] In some embodiments, the invention further includes one or more bioactive agents adsorbed or absorbed within the hydrogel, which delivers one or more bioactive agents to the administration site. Methods may include implanting the target hydrogel, coating the implant with the hydrogel, orally administering the hydrogel, etc. The amount of encapsulated bioactive agent will depend on the duration of delivery, the administration site, and the condition being treated. In some embodiments, the bioactive agent encapsulated in the target hydrogel contains 0.0001 μg or more, for example, 0.001 μg or more, 0.01 μg or more, 0.1 μg or more, 1 μg or more, 10 μg or more, 25 μg or more, 50 μg or more, 100 μg or more, 500 μg or more, 1000 μg or more, 5000 μg or more, and including 10,000 μg or more. When a bioactive agent is encapsulated in a hydrogel in liquid form, the concentration of the bioactive agent may be 0.0001 μg / mL or higher, for example, 0.001 μg / mL or higher, 0.01 μg / mL or higher, 0.1 μg / mL or higher, 0.5 μg / mL or higher, 1 μg / mL or higher, 2 μg / mL or higher, 5 μg / mL or higher, 10 μg / mL or higher, 25 μg / mL or higher, 50 μg / mL or higher, 100 μg / mL or higher, 500 μg / mL or higher, 1000 μg / mL or higher, 5000 μg / mL or higher, or even higher.Higher and including 10,000 μg / mL or higher. Instructions for Use 26 / 52 pages 37 CN 121421943 A

[0172] The effective dose of the therapeutic peptide administered to human patients as a gel formulation may be approximately 1.0 μg / kg body weight, 2.5 μg / kg body weight, 5.0 μg / kg body weight, 10.0 μg / kg body weight, 25.0 μg / kg body weight, 50.0 μg / kg body weight, 75.0 μg / kg body weight, 0.1 mg / kg body weight, 0.5 mg / kg body weight, 1.0 mg / kg body weight, 2.5 mg / kg body weight, 5.0 mg / kg body weight, 7.5 mg / kg body weight, 10.0 mg / kg body weight, and 25.0 mg / kg body weight. 50.0 mg / kg body weight, 75.0 mg / kg body weight, 1100 mg / kg body weight, 250 mg / kg body weight, 500 mg / kg body weight, 750 mg / kg body weight, 1 g / kg body weight, 2 g / kg body weight, 5 g / kg body weight, 10 g / kg body weight, 25 g / kg body weight to 50 g / kg body weight or higher, or any dose range of these parameters.

[0173] The effective dose can be maintained for a sufficiently long time to achieve treatment of the condition. Depending on the specific structure of the hydrogel used, the release of one or more bioactive agents in the hydrogel matrix may vary. For example, the hydrogel of the present invention can slowly release the drug. By designing the gel, it can be made to release a therapeutic dose of peptide for a release time of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or longer, such as 1 week, 2 weeks, 3 weeks, 4 weeks or longer.

[0174] "Sustained release" refers to the continuous delivery of one or more bioactive agents by a hydrogel structure. Throughout the delivery time, the hydrogel remains in contact with the administration site, for example, for 1 day or longer, 2 days or longer, 5 days or longer, 10 days or longer, 15 days or longer, 30 days or longer, and including 100 days or longer.

[0175] The invention also includes methods of treating a subject by applying one or more gels of the invention to the subject. In some embodiments, these methods include administering a hydrogel solution or suspension to the subject, and the contact time between the hydrogel and the subject is sufficient to achieve the purpose of treating the subject. As described above, the target hydrogel can be applied to any applicable administration site requiring treatment, including but not limited to skin, bone, heart, liver, kidney, bladder, trachea, lung, tumor tissue, oral cavity (e.g., cheek and sublingual), and nose, throat, ear, uterus, and bladder.

[0176] In some embodiments, these methods may include applying one or more hydrogels, and the contact time between the hydrogel and the subject is sufficient to deliver a target dose of the bioactive agent. For example, the target dose of the bioactive agent may be the total exposure.Or the average daily exposure. For example, the target dose of a bioactive agent delivered by the targeted method may be 0.01 mg / day or higher, for example, 0.04 mg / day or higher, 0.5 mg / day or higher over a 4-week dose interval, 1.0 mg / day or higher, 2 mg / day or higher, 5 mg / day or higher, and including 10 mg / day over a 4-week dose interval.

[0177] As discussed herein, a gel-forming enhancement motif has been identified, which improves the preparation of molecules capable of forming gels. Coupling the gel-forming enhancement motif results in extremely small structural changes that have minimal impact on the distribution characteristics of the therapeutic agents discussed herein. The modification method has minimal impact on the volume of distribution of the therapeutic agent, which is advantageous because it makes the pharmacokinetic distribution of the therapeutic agent in vivo similar to that of wild-type peptide analogs. In most cases, this property is preferred because other half-life extension techniques, such as macromolecular fusion (e.g., fusion with IgG, albumin, or PEG), will significantly increase the molecular weight of the therapeutic agent and reduce its distribution volume, thus preventing the therapeutic agent from reaching the intended target outside the systemic circulation.

[0178] The discovery that stable gel formulations can be obtained by dissolving selected peptide compounds in aqueous solution at appropriate concentrations (i.e., less than, equal to, or greater than 11% w / w) is unexpected. 11% w / w was chosen as the initial detection point because serum protein levels are approximately 9–10% w / w. When a critical aggregation concentration is reached, such peptides are capable of self-assembling into liquid gels or semi-solid gel nanostructures. Specifically, it is envisioned that liquid gels or semi-solid gels be formed from natural or modified peptides.

[0179] Based on these findings, therapeutic peptides and other therapeutic agents in sustained-release formulations can have higher drug loadings compared to conventional compositions and formulations. The aforementioned advantages can be obtained by using suitable formulations or modifying peptides to form gel nanostructures at a certain concentration, at which wild-type peptide analogs remain partially soluble or insoluble in aqueous solution, as is known to those skilled in the art. By encapsulating therapeutic drugs in more advanced gel nanostructures formed by self-assembled peptides, gel formulations can prolong the delivery time of therapeutic drugs, thereby improving patient compliance, increasing drug delivery efficiency and efficacy, and reducing adverse reactions. At the same time, this method can mitigate non-specific interactions, prevent enzyme degradation, and improve solubility. In addition, compared to therapeutic agents coupled or complexed with macromolecular carriers, gel-forming therapeutic agents are discrete molecules rather than complexes and can be deeply distributed in tissues.

[0180] The phase transition process between aqueous solution and gel nanostructures of non-covalently bonded peptide gels is dose-, time-, and other environmental factors, based on which the above and several other advantages are achieved. Typically, gel nanostructuresThe structure dissociates into monomer molecules in aqueous solution because molecules on the outer surface of the gel gradually diffuse into the surrounding solution.

[0181] In a preferred embodiment, all percentages mentioned in this invention are weight (w / w) percentages.

[0182] Example

[0183] Amylin (a type B GPCR ligand) naturally forms solid amyloid fibrous structures in vivo; the associated adrenal medulla peptide CGRP forms liquid gel nanostructures at selected concentrations. Based on the above observations, we hypothesize that peptides of the amylin / CGRP / adrenal medulla / adrenal medulla 2 family, after appropriate formulation or modification, have a tendency to form gel nanostructures. We found that selected adrenal medulla agonist / antagonist acylates naturally form gel nanostructures in aqueous solution, and that modification of selected peptides with hydrophobic tails, heads, or side chains promotes gel formation when gel-forming enhancing motifs are present, further supporting the above hypothesis. In addition, we explored the tendency of natural and synthetic therapeutic peptides to form gels in aqueous solutions. The ability of compounds to form gels in water was determined: To determine the solubility of peptides at room temperature, 1, 2, 3, 4, 5, 20, or 100 mg of selected peptides were weighed and placed in clear plastic or glass vials, and then an equal volume of deionized water was added. The room temperature was maintained at approximately 22–25°C during this procedure. Soluble peptide samples dissolved immediately, yielding a clear solution. The clear and fluid nature of the solution indicated that the peptide's solubility was greater than the corresponding concentration (w / w). After 20 minutes of reaction, insoluble peptide samples retained a particulate or pasty characteristic. To distinguish the gel-forming state of the peptide solution, in addition to visual inspection, we also performed a tube tapping test and a tube tilting test.

[0184] To determine the solubility of peptides at room temperature, 1, 2, 3, 4, 5, 20, or 100 mg of the selected peptide was weighed and placed in a transparent plastic vial, and then the same volume of deionized water was added to the vial. The room temperature was controlled at approximately 22–25 °C during this operation. Soluble peptide samples dissolved immediately in water, and after 20 minutes of mixing with water, the solution remained clear, with no significant change in viscosity observed by the naked eye. This result indicates that the peptide has high solubility and does not form gel nanostructures. If visible peptide particles remain in the solution after 20 minutes of mixing with water, it is considered that the peptide is insoluble at the given concentration or will precipitate. In this case, the quality of the peptide is further judged by two other criteria: (1) mobility after tapping the test tube (test tube tapping test) and (2) fluidity after tilting the test tube 90° (test tube tilting test). In the first test, the test tube was tapped 10 times with a finger, and the number of times the bulk position of the solution changed was recorded. In this test, the fluidity of the aqueous solution (e.g., water) was determined by the following criteria:A score of 9 or 10 indicates that the bulk of the liquid moves with each tap of the test tube. Similarly, insoluble peptides or peptide precipitates in the solution also receive a score of 9 or 10, and their positions change visibly with each tap. In contrast, gel-forming peptides dissolve immediately or gradually in aqueous solutions and exhibit high viscosity after 20 minutes of mixing with water. Peptides of moderate viscosity form liquid gels, whose conformation changes slowly when the test tube is tapped or tilted. To distinguish between liquid gels and semi-solid gels, we tested the mobility of the solution using a tapping and tilting test. The selected peptide solution exhibited extremely high viscosity and formed a semi-solid gel that did not flow like a liquid when the test tube was tapped or tilted. In the test tube tapping test, if the gel scores 0-4 points (i.e., in 10 independent tapping tests, the gel's main body position changes visibly 0-4 times after tapping), it is classified as a semi-solid gel. If in 10 independent tapping tests, the gel's main body position changes visibly 5-9 times, it is classified as a liquid gel. The high viscosity of the liquid gel is further confirmed by the tilting test (page 28 / 52, 39 CN 121421943 A) as described in the test tube instructions. In this test, the plastic tube containing the solution or gel is tilted 90°, and the movement of the solution or gel block is observed under light. If the liquid reaches the bottom of the tube in less than 15 seconds, the peptide is classified as a liquid solution. If the liquid or gel takes more than 15 seconds to flow to the bottom of the tilted tube, the resulting aqueous substance is classified as a liquid gel.

[0185] To investigate the bioactivity of the coupled peptides and mutant peptides, we performed in vitro receptor activation assays using cell cultures expressing recombinant melanocortin receptor 1 (MC1R), melanocortin receptor 4 (MC4R), CRH receptor 2 (CRHR2), PTH receptor 1 (PTHR1), or κ opioid receptor (OPRK1). The agonistic activity of the peptides against MC1R, MC4R, CRHR2, PTHR1, and OPRK1 cAMP was determined using the MC1R cAMP kit, MC4R cAMP kit, CRHR2 cAMP kit, CRHR2 cAMP kit, PTHR1 cAMP kit, and OPRK1 cAMP kit provided by Discoverx Inc. (Fremont, California). The antagonistic activity of the peptides against bradykinin receptor 2 (BDKRB2) and GnRH receptors (GnRHR or LHRHR) was determined using the BDKRB2 inhibitory protein kit and GnRHR calcium flux kit provided by Discoverx.

[0186] In order to analyze the generation of Gs-coupled cAMP, cAMP was amplified using a cryopreservation solution.Hunter cell lines were seeded at a total volume of 20 μL into white 384-well plates and incubated at 37°C for a period of time before assay. The regulatory effect of cAMP was determined using the DiscoverX HitHunter cAMP XS+ kit. To determine agonist activity, cells were co-incubated with the sample to induce a biological response. The culture medium was aspirated from the cells and replaced with 15 μL of reagent (HBSS / 10 mM HEPES to cAMP XS+Ab ratio of 2:1). The sample stock solution was intermediately diluted with assay buffer to generate 4X samples. 5 μL of the 4X sample was added to the cells and incubated at 37°C or room temperature for 30 or 60 minutes. The solvent concentration was 1%. The activity of the compounds was analyzed using the CBIS data analysis suite (ChemInnovation, CA). For the Gs-coupled agonist assay, the percentage of activity was calculated using the following formula: Percentage of activity = 100% × (mean RLU of test sample - mean RLU of solvent control) / (mean RLU of MAX control - mean RLU of solvent control).

[0187] For the inhibitory protein assay, the PathHunter cell line (Discoverx Inc.) was expanded using frozen stock solution, and the cells were seeded in white 384-well plates at a total volume of 20 μL and incubated at 37°C for a period of time before detection. To determine agonist activity, the cells were incubated with the sample to induce a biological response. The sample stock solution was diluted intermediately with detection buffer to generate a 5X sample, and 5 μL of the 5X sample was added to the cells and incubated at 37°C or room temperature for 90 to 180 minutes. The solvent concentration was 1%. To determine antagonist activity, the cells were pre-cultured with the antagonist and then an agonist challenge assay was performed at EC80 concentration. The sample stock solution was diluted intermediately with detection buffer to generate a 5X sample, and 5 μL of the 5X sample was added to the cells and incubated at 37°C or room temperature for 30 minutes. The solvent concentration was 1%. Then, 5 μL of 6X EC80 agonist containing detection buffer was added to the cells and incubated at 37°C or room temperature for 90 or 180 minutes.

[0188] A single addition of 12.5 μL or 15 μL (50% v / v) of the PathHunter Detection reagent mixture was followed by incubation at room temperature for 1 hour to generate an analytical signal. After signal generation, the microplate was read using a PerkinElmer Envision™ chemiluminescence signal detector. The activity of the compound was analyzed using the CBIS Data Analysis Suite (ChemInnovation, CA). For agonist mode assays, the percentage of activity was calculated using the following formula: Percentage of activity = 100% × (test sample)(mean RLU - mean RLU of solvent control) / (mean RLU of MAX control ligand - mean RLU of solvent control). For antagonistic activity assays, the inhibition percentage is calculated using the following formula: Inhibition percentage = 100% × (1 - (mean RLU of test sample - mean RLU of solvent control) / (mean RLU of EC80 control - mean RLU of solvent control)).

[0189] To analyze calcium flux, cell lines were expanded using frozen stock solution, and cells were seeded in 20 μL total volume into black 384-well plates with a clear bottom coated with poly-D-lysine and incubated at 37°C for a period of time before detection. The assay was performed in 1x dye loading buffer containing 1x dye, 1x additive A and 2.5 mM probenecid (dissolved in HBSS / 20 mM HEPES). Before the experiment, cells were loaded with dye. The culture medium was aspirated from the cells and replaced with 20 μL of dye loading buffer. Cells were incubated at 37 °C for 30 to 60 minutes. To determine agonist activity, cells were incubated with the sample to induce a biological response. After dye loading, cells were removed from the incubator and a buffer containing 10 μL HBSS / 20 mM HEPES was added. To determine the EC80 for the antagonist assay, an agonist dose curve was plotted, with the solvent added to the buffer during the plotting process. Cells were incubated at room temperature in the dark for 30 minutes to equilibrate the plate to room temperature. The sample stock solution was diluted with the detection buffer to generate a 4X sample. The agonist activity of the compound was determined using a FLIPR Tetra (MDS) assay, monitoring calcium mobilization for 2 minutes. Then, 10 μL of the 4X sample (dissolved in HBSS / 20 mM HEPES) was added to the cells 5 seconds before the start of the assay.

[0190] To determine the antagonist activity, cells were pre-cultured with the sample and then an agonist challenge assay was performed at the EC80 concentration. The sample stock solution was diluted with assay buffer to generate 3X samples. After dye loading, cells were removed from the incubator and 10 μL of 3X sample was added. Cells were incubated at room temperature in the dark for 30 minutes, and the plate temperature was equilibrated to room temperature. The solvent concentration was 1%. The antagonistic activity of the compound was determined using FLIPR Tetra (MDS), and calcium mobilization was monitored for 2 minutes. Then, 10 μL of EC80 agonist (dissolved in HBSS / 20 mM HEPES) was added to the cells 5 seconds before the start of the experiment. The activity of the compound was analyzed using the CBIS Data Analysis Suite (ChemInnovation, CA), and the percentage of activity was calculated using the following formula: Percentage of activity = 100% × (mean RFU of test sample - mean RFU of solvent control) / (mean RFU of MAX control ligand - ...The average RFU of the solvent reference. For antagonist tests, the inhibition percentage was calculated using the following formula: Inhibition percentage = 100% × (1 - (average RFU of the test sample - average RFU of the solvent reference) / (average RFU of the EC80 reference - average RFU of the solvent reference)).

[0191] To analyze the release of selected molecules from gels prepared from gel-forming peptides, we used a semi-permeable membrane (e.g., a centrifugal filter) to detect the release level. In these experiments, FITC-labeled small molecules, peptides, or nucleic acids were dissolved in a solution containing low concentrations of gel-forming or non-gel-forming peptides and loaded onto the upper chamber of a centrifugal filter through a 10 kDa or 20 kDa filter membrane. The column was centrifuged at 2000 or 3000 rpm for 20 minutes, and the content of FITC-labeled molecules in the lower chamber was determined by a fluorometer.

[0192] As shown in Table 1, we dissolved (or attempted to dissolve) peptides in water at room temperature to test the ability of various peptides to form stable gels. Peptides that formed liquid gels or semi-solid gels remained stable under the test conditions. These examples demonstrate that various modification methods may or may not affect the ability of the compositions of the present invention to form liquid, solid, or semi-solid gels.

[0193] The gel-forming abilities of a variety of secretory and therapeutic peptides or analogues were determined, as shown in Table 1. All peptides were prepared by solid-phase synthesis and their ability to form semi-solid or liquid gels was analyzed at room temperature. To systematically analyze gel-forming abilities, experiments were first conducted in aqueous solutions with a peptide concentration of 11% w / w. The peptides analyzed included functional regulators of cell surface receptors, enzymes, complement factors, antimicrobial peptides, immunomodulators, therapeutic peptides, cell-penetrating peptides, antigens, matrikine and CGRP analogues, adrenomedullin and adrenomedullin 2 (or pituitary mesothelin). When the concentration in aqueous excipients is 11% w / w, adrenomedullin 1-52, pramlintide (an amylin analog), oxytocin, kissing agonist, κ receptor agonist, pramorelin, thrombopoietin analog (i.e., romimistatin analog), urocortin 3, scutellarin receptor antagonist, ADM2-52 acylated, and campstatin (serial numbers: 2, 4-11, 61, and 274) all form transparent or opaque semi-solid gels in aqueous solution. Similarly, at a concentration of 11% w / w, adrenomedullin analog (i.e., ADE43), CGRP, teduglutide (a GLP-2 analog), thymosin α1, GLP-1, γ-MSH, acylated thymosin β4 analog, and GnRH analog (serial numbers: 1, 3, 12-15, 64, and 263) all form liquid gels. On the other hand, at selected concentrations, several other functional peptides (serial numbers: 16-47) either formed transparent liquid solutions or insoluble solutions after 20 minutes of reaction.Precipitates. Peptides that form transparent aqueous solutions or insoluble precipitates do not have the ability to form gel nanostructures. Abbreviations used in the table include SS (semi-solid gel) and LG (liquid gel). Specification 30 / 52 pages 41 CN 121421943 A

[0194] The semi-solid gel formed by the peptide with sequence number 1 is shown in Figure 1. The position of the semi-solid gel remains unchanged when it is held upright (A), tilted at 90° (B), or inverted (C).

[0195] Table 1 Identification of secretory peptides that self-gel in aqueous solution

[0196] Specification 31 / 52 pages 42 CN 121421943 A

[0197] Specification 32 / 52 pages 43 CN 121421943 A

[0198]

[0199] Studies on adrenomedullin and adrenomedullin 2 analogs (serial numbers: 48-58 [Table 1], 274-275 [Table 3]) have shown that at a concentration of 11% w / w, the selected analogs can form liquid gels, and minor modifications to the sequence can significantly alter the gel-forming ability of the peptides. At a concentration of 11% w / w, the parent peptide and wild-type truncated adrenomedullin analog (serial number: 1) form liquid gels, and after replacing selected residues with D-amino acids, sequence 48 forms a semi-solid gel. On the other hand, palmitoylation of sequence 274 allows the analog to form a semi-solid gel at a concentration of 6%, an advantage not found in wild-type peptides and other adrenomedullin analogs. Other modifications to the peptides in sequences 49-58 allow these analogs to retain the ability to form liquid gels at a concentration of 11% w / w. While gel-forming ability is influenced by numerous physical and chemical factors, gel-forming peptides (e.g., adrenomedullin and adrenomedullin 2) may contain sequence motifs that promote gel formation, and changes in residues or side chains can also alter the ability of these peptides to form gels.

[0200] These data also indicate that gel-forming peptides [e.g., adrenomedullin, pramlinide (an amylin analog), oxytocin, kissing agonist, κ opioid receptor agonists, pramorelin, romilastine analogs, urocortin 3, campstatin, CGRP, teduglutide (a GLP-2 analog), thymosin α1, GLP-1, GnRH, scutellarin receptor antagonists, γ-MSH, thymosin β4, and adrenomedullin analogs (serial numbers: 1-15, 61, 64, 26, and 274)] can be delivered via self-assembling gel formulations, i.e., without the use of additional gel-forming agents. However, these gel-forming peptides may contain gel-forming enhancement motifs that promote self-assembling gel formation.

[0201] Based on the finding that peptides of the CGRP / adrenomedullin family form liquid or semi-solid gels in aqueous solution, we hypothesized that these self-assembling gel-forming peptides may contain gel-forming enhancing motifs. Typically, peptides cannot form gel nanostructures, but these motifs can enhance their ability to form gels. We then tested the ability of calcitonin / amylin / CGRP / adrenomedullin / adrenomedullin 2 family peptide analogs to form semi-solid or liquid gels at room temperature using visual inspection, tube tapping, and tube tilting tests. For a systematic analysis of gel-forming ability, experiments were conducted at a concentration of 11% w / w. As shown in Table 2, some modified and chimeric adrenomedullin / adrenomedullin 2 analogs formed semi-solid or liquid gels at a concentration of 11% w / w, similar to wild-type adrenomedullin (serial numbers: 106-114 and 116-123). In contrast, after selective modifications (e.g., truncation and substitution; Serial numbers: 101-105 and 115), the resulting peptides either formed aqueous solutions or remained insoluble at the same concentration.

[0202] Among these peptides, we found that analogs as short as 3 amino acids (Serial numbers: 120-121) were able to form semi-solid gels at concentrations of 11% w / w or lower. Although changes to a single amino acid in the adrenomedullin / adrenomedullin 2 peptide resulted in analogs that remained aqueous solutions or remained insoluble, or formed liquids or semi-solid gels, these data suggest that ADM / ADM2-derived peptides have a tendency to form liquids or semi-solid gels. This observation also supports the hypothesis that gel-forming enhancement motifs derived from self-assembled gel-forming peptides can convert non-gel-forming peptides into self-assembled gel-forming peptides. While many truncated adremyelin / adremyelin 2 analogues can form semi-solid or liquid gels (Sequence Nos.: 116-124 and 126-131), polypeptides composed of sequences from corresponding regions of CGRP or amylin (Sequence Nos.: 132-134) can remain in aqueous solution or produce insoluble precipitates at the same concentration. Nevertheless, analogues capable of forming aqueous solutions, liquid gels, or semi-solid gels can be obtained by modifying CGRP sequences with adremyelin sequence fragments (Sequence Nos.: 139-140). As previously mentioned, gel-forming peptide reference standards composed of repeating sequences (Sequence Nos.: 141-142) also form semi-solid gels in aqueous solution at a solubility of 11% w / w. Specification page 34 / 52, page 45, CN 121421943 A

[0203] Specification page 35 / 52, page 46, CN 121421943 A

[0204] The abbreviations used in the table include SS (semi-solid gel), LG (liquid gel) and AS / IS (aqueous solution or insoluble gel).(Solution). Peptides that form transparent aqueous solutions or insoluble particles do not appear to have the ability to form gel nanostructures.

[0205] Based on the finding that short sequence motifs in adrenomedullin family peptides can form gels at low concentrations, we tested the hypothesis that the presence of such gel-forming enhancing motifs could enhance the ability of other peptides to form gels. We tested the ability of a series of chimeric analogs to form semi-solid or liquid gels. These analogs include gel-forming enhancing motifs found in serial numbers 109, 119, and 120. The length of the gel-forming enhancing motifs in these peptides ranges from 3 to 17 amino acids. These motifs include motifs attached to the N-terminus, C-terminus, or incorporated into the sequence through amino acid substitution, or motifs attached to the side chains of amino acids. The motifs are linked to the functional peptide directly or via miniPEG.

[0206] As shown in Table 1, kissing agonists, κ-opioid receptor agonists, sermorelin, campstatin, serotonin receptor antagonists, γ-MSH, and thymosin α1 peptides (serial numbers: 6, 7, 8, 11, 13, 61, and 64) can self-assemble into gels at concentrations of 11% w / w or lower, and their gel-forming ability is not significantly altered at 11% w / w when coupled to or replaced with gel-forming enhancement motifs (Table 3, serial numbers: 208, 214, 235, 244, 255, and 260). Although the gel-forming ability of these modified peptides is not significantly improved, these modified gel-forming peptides possess albumin-binding acyl groups and gel-forming ability, which would result in a longer circulating half-life. Therefore, these peptides belong to the category of dominant analogs, and these analogs are expected to have better pharmacokinetic and pharmacodynamic characteristics compared to wild-type analogs. Similarly, we found that acylated analogs of ADM2-52 and thymosin β4 can form gels at a concentration of 11% w / w, and these acylated analogs are dominant analogs with albumin-binding acyl groups and the ability to form gels themselves.

[0207] On the other hand, we have demonstrated that GnRH analogs, GnRH antagonists, vasopressin, sennatin, apralin, sennatin receptor antagonists, neocorynone, enkephalin, substance P, calcitonin, pramlinin (amycin analog), exenatide 4, GLP-1, teduglutide (GLP-2 analog), afanotide (melanotan I), melanotan II, ACTH1-24, setmelanotide, urocortin 2, parathyroid hormone, PYY3-36, VIP, HOE140 (bradykinin receptor 2 antagonist), bradykinin receptor 1 antagonist, sermorelin, atrial diuretic natriuretic peptide (ANP), adrenaline, thymosin α1, kallikrein inhibitors, temporin A antimicrobial peptide, immunomodulatory glatiramer (or clopidogrel), matrix-modified peptide 1, matrix-modified peptide 4, and matrixThe gel-forming abilities of modified peptide 7, matrix-modified peptide 8, and acetyl hexapeptide-3 matrix-modified peptides (serial numbers: 201-204, 209-213, 215, 217-228, 230, 232-234, 237-238, 242, 245-250, 253-254, 257, 261, 264, 266-270, 273-273-274) were significantly altered. Incorporation of gel-forming enhancing motifs into these peptides enabled them to form semi-solid gels at concentrations (i.e., 30%, 20%, 11%, or 6% w / w) where wild-type analogs could not form such gels. A phase transition was observed within 20 minutes of dissolution. At the selected concentrations, wild-type peptides formed only liquid gels, aqueous solutions, or insoluble precipitates.

[0208] Furthermore, we found that adding selected gel-forming enhancement motifs to oxytocin, apralin, neurotensin, myotropic antihypertensin, PYY3-36, melanocortin I, γ-MSH, urocortin 2, or TAT cell-penetration enhancing peptides partially improved the gel-forming ability of these peptides (serial numbers: 205-207, 216, 229, 241, 256, 260, 262, and 265). In these cases, the modified peptides formed liquid gels at the selected concentrations, while wild-type analogs (serial numbers: 5, 18, 19, 24, 29, 30, 31, 36, and 64) only formed aqueous solutions or were insoluble at these concentrations. However, at the selected test concentrations, these modified peptides failed to form semi-solid gels. Therefore, these data indicate that GnRH analogs, GnRH antagonists, vasopressin, serotonin, serotonin receptor antagonists, neocorynebacterium, enkephalin, substance P, calcitonin, pramlinide (amycin analog), exenatide 4, GLP-1, teduglutide (GLP-2 analog), afanotide (meranotine I), melanotine II, γ-MSH, ACTH1-24, setmelanotide, urocortin 2, parathyroid hormone, VIP, bradykinin receptor antagonists, HOE140, sermorelin, atrial diuretic natriuretic peptide (ANP), thymosin α1, thymosin β4, adrenomedullin, kallikrein inhibitors, and temporin are all potentially harmful substances. Antimicrobial peptides, including campstatin, glatiramer (or clopidogrel), matrix-modified peptide 1, matrix-modified peptide 4, matrix-modified peptide 7, matrix-modified peptide 8, acetyl hexapeptide-3, and oxytocin, ipalin, neurotensin-1, PYY3-36, TAT cell-penetration-enhancing peptide, pramorelin, or urocortin 3, exhibit characteristics of self-assembling gel-forming peptides and can be used for the slow release in the human body of therapeutic drugs targeting their receptors, enzyme substrates, cellular compartments, or biological mediators.

[0209] The abbreviations used in the table include SS (semi-solid gel), LG (liquid gel), and AS / IS (aqueous solution or insoluble). Instruction manual, pages 37 / 52, 48 CN 121421943 A

[0210] Instruction manual, pages 38 / 52, 49 CN 121421943 A

[0211] Instruction manual, pages 39 / 52, 50 CN 121421943 A

[0212] Instruction manual, pages 40 / 52, 51 CN 121421943 A

[0213] Instruction manual, pages 41 / 52, 52 CN 121421943 A

[0214] Instruction manual, pages 42 / 52, 53 CN 121421943 A

[0215] Instruction manual, pages 43 / 52, 54 CN 121421943 A

[0216]

[0217] Through visual inspection, test tube tapping test, and test tube tilting test, we found that when the concentration is 11% w / w, GnRH analogs (serial number: 15; Table 3) form liquid gels in distilled water. In contrast, a GnRH analog (serial number: 201) coupled with a gel formation enhancement motif (pages 44 / 52, CN 121421943 A) forms a semi-solid gel at concentrations of 11, 6, or 3% w / w. Similarly, while GnRH antagonists (serial number: 27) do not form gels, GnRH antagonists (serial numbers: 202 and 203) coupled with gel formation enhancement motifs form semi-solid gels at concentrations of 11% and 6% w / w. Wild-type vasopressin (serial number: 17) does not form a gel at a concentration of 11% w / w, but vasopressin (serial number: 204) coupled with a gel formation enhancement motif forms a semi-solid gel at concentrations of 11% and 6% w / w. Wild-type oxytocin (serial number: 5) can form a semi-solid gel at a concentration of 11% w / w, but cannot form a liquid or semi-solid gel at a concentration of 6% w / w. On the other hand, an oxytocin analog coupled with a gel-forming enhancement motif (serial number: 205) forms a semi-solid gel at a concentration of 11% w / w and a liquid gel at a concentration of 6% w / w.

[0218] Wild-type ipalin peptide (serial number: 18) cannot form a gel at concentrations of 20%, 11%, or 6% w / w. On the other hand, ipalin peptide analogs coupled with a gel-forming enhancement motif (serial numbers: 206 and 262) can form a liquid gel at concentrations of 20% or 11% w / w. Similarly, wild-type neurotensin (serial number: 19) failed to form a gel at concentrations of 20% or 11% w / w, but at concentrations of 20% and 11% w / w, neurotensin coupled with gel-forming enhancement motifs was observed.(Serial No.: 207) forms a liquid gel. Wild-type dinoflagrin (Serial No.: 20) cannot form a gel at concentrations of 11% or 6% w / w. On the other hand, analogues (Serial Nos.: 209 and 255) generated by coupling dinoflagrin or dinoflagrin receptor antagonists to gel-forming enhancement motifs can form semi-solid or liquid gels at concentrations of 11%, 6%, or 3% w / w. Unlike apalamin, neurotensin, and dinoflagrin, kissing agonist peptide (Serial No.: 6) can form a semi-solid gel at concentrations of 11% or 6% w / w, and kissing agonist analogue (Serial No.: 208) coupled with a gel-forming enhancement motif can form a gel at the same concentration.

[0219] Opioid receptor ligands such as neocortexin and enkephalin (Serial Nos.: 21 and 22) cannot form a gel at concentrations of 11% or 6% w / w. On the other hand, novel enkephalin analogs coupled with gel-forming enhancement motifs (serial numbers: 210 and 211) can form semi-solid gels at a concentration of 11% w / w, or liquid gels at a concentration of 6% w / w. Enkephalin analogs coupled with gel-forming enhancement motifs (serial numbers: 212 and 213) can even form semi-solid gels at a concentration of 3% w / w. However, κ-opioid receptor agonists (serial number: 7) can form semi-solid gels at concentrations of 11%, 6%, or 3% w / w. κ-opioid receptor agonist analogs coupled with gel-forming enhancement motifs (serial number: 214) can form semi-solid gels at concentrations of 11% and 6% w / w, and can form liquid gels at a concentration of 3% w / w.

[0220] Wild-type substance P analogs (serial numbers: 23 and 69) can only form liquid gels at a concentration of 20% w / w or cannot form liquid gels at all. When substance P is coupled with a gel-forming enhancement motif, the coupled analogs (serial numbers: 215, 253, and 254) are able to form semi-solid gels at concentrations of 11% or 20% w / w. At a concentration of 11% w / w, the angiotensin receptor antagonist inosine vasopressin (serial number: 24) cannot form a gel. When inosine vasopressin is coupled with a gel-forming enhancement motif, the resulting analog (serial number: 216) is able to form a liquid gel at concentrations of 20% or 11% w / w.

[0221] At a concentration of 11% w / w, wild-type calcitonin (serial number: 28) cannot form a gel. On the other hand, calcitonin coupled with a gel-forming enhancement motif (serial number: 217) is able to form a semi-solid gel at a concentration of 11% w / w and a liquid gel at a concentration of 6% w / w. The amylin peptide analog pralaminin (serial number: 4) does not form a gel at a concentration of 6% w / w, but it does form a semi-solid gel at a concentration of 11% w / w. Pralaminin forms an enhancing motif with the gel.Upon coupling, the resulting analog (serial number: 218) was able to form a semi-solid gel at a concentration of 11% or 6% w / w. Unlike exenatide 4 (serial number: 26), which could not form a gel at an 11% w / w concentration, the exenatide 4 analog (serial number: 219) coupled with a gel-forming enhancement motif could form a semi-solid gel at an 11% w / w concentration and a liquid gel at a 6% w / w concentration. Wild-type GLP-1 (serial number: 14) could only form a liquid gel at an 11% concentration. On the other hand, GLP-1 analogs coupled with a gel-forming enhancement motif (serial numbers: 220, 269, and 270) and an analog (serial number: 221) substituted with the gel-forming enhancement motif (see page 45 / 52 of the specification, CN 121421943 A) could form a semi-solid gel at a concentration of 20% or 11% w / w. The GLP-2 analog tiduglutide (serial number: 12) forms a liquid gel at a concentration of 11%. On the other hand, a GLP-2 analog coupled with a gel-forming enhancement motif (serial number: 222) forms a semi-solid gel at 11% w / w and a liquid gel at 6% w / w.

[0222] The melanotane I analog afanotide and melanotane II analogs (serial numbers: 29 and 62) fail to form gels at concentrations of 20% or 11% w / w. In contrast, afanotide analogs coupled with gel-forming enhancement motifs (serial numbers: 223, 224, 256, 257, and 273) are able to form semi-solid or liquid gels at concentrations of 20%, 11%, or 6% w / w. The ACTH1-24 peptide (serial number: 33) fails to form a gel at concentrations of 20% or 11% w / w. In contrast, ACTH1-24 analogs (serial numbers: 225 and 226) coupled with gel-forming enhancement motifs formed semi-solid gels at concentrations of 20% or 11% w / w. Setmelanotide (serial number: 34), an agonist of the melanocortin 4 receptor (MC4R), failed to form a gel at concentrations of 30%, 20%, or 11% w / w. When setmelanotide was fused with a gel-forming enhancement motif of Pal-SSPHSY, the resulting analog (serial number: 227) formed a gel at a concentration of 30% w / w. On the other hand, a setmelanotide analog with a Pal-HSY gel-forming enhancement motif (serial number: 228) formed a semi-solid gel at concentrations of 11%, 6%, or 3% w / w. Furthermore, we found that γ-MSH (serial number: 64) formed a liquid gel at a concentration of 11% and was soluble at a concentration of 6% w / w. A γ-MSH analogue (serial number: 260) coupled with a gel-forming enhancement motif was able to form a liquid gel at a concentration of 6% w / w.

[0223] At concentrations of 20% or 11% w / w, the peptide YY receptor agonist PYY3-36 (serial number: 30) failed to form a gel. When the peptide was coupled with a gel-forming enhancement motif, the resulting analogs (serial numbers: 229 and 264) were able to form semi-solid or liquid gels at concentrations of 20% or 11% w / w. At concentrations of 20% or 11% w / w, the adrenocorticotropic hormone receptor 2 (CRHR2) agonist urocortin 2 (UCN2) (serial number: 31) failed to form a gel. In contrast, urocortin 2 analogs coupled with a gel-forming enhancement motif (serial numbers: 230 and 265) were able to form semi-solid or liquid gels at a concentration of 20% w / w. Unlike UCN2, urocortin 3 (UCN3) (serial number: 10) formed semi-solid gels at concentrations of 11% and 6%, and liquid gels at a concentration of 3% w / w. The UCN3 analogue (Sequence No. 231) coupled with a gel-forming enhancement motif still formed gels at these concentrations. On the other hand, parathyroid hormone (Sequence No. 25) failed to form a gel at concentrations of 11% or 6% w / w. When the N-terminus of the peptide was replaced with a gel-forming enhancement motif, the resulting analogue (Sequence No. 232) formed a semi-solid gel at a concentration of 20% and a liquid gel at a concentration of 11% w / w. The bradykinin receptor ligand HOE140 (an antagonist of BKR2) (Sequence No. 32) failed to form a gel at concentrations of 30%, 20%, or 11% w / w. However, when the peptide was coupled with a gel-forming enhancement motif of Pal-SSPHSY, the resulting analogue (Sequence No. 233) formed a semi-solid gel at a concentration of 30% w / w. When HOE140 is coupled with a gel-forming enhancement motif of Pal-HSY, the resulting analog (serial number: 234) forms a semi-solid gel at a concentration of 20% w / w. Another bradykinin receptor antagonist (serial number: 65) fails to form a gel at concentrations of 11% or 6% w / w, while the modified analog (serial number: 261) forms a semi-solid gel at 11% w / w and a liquid gel at 6% w / w.

[0224] Pramoleline (serial number: 8) forms a semi-solid gel at concentrations of 11% or 6% w / w and a liquid gel at 3% w / w. Similar to wild-type pramoleline, the pramoleline analog (serial number: 235) coupled with a gel-forming enhancement motif forms a semi-solid gel at the same concentration. Unlike pramorelin, seromorelin (seromorelin 35) does not form a gel at concentrations of 20% or 11% w / w. However, seromorelin analogs can form a semi-solid at a concentration of 20% when fused with shorter gel-forming reinforcing motifs (seromorelin 237).A gel, but when fused with a longer gel-forming enhancement motif (serial number: 236), cannot form a semi-solid gel at this concentration.

[0225] Atrial natriuretic peptide (ANP) (serial number: 41) cannot form a gel at concentrations of 20% or 11% w / w. In contrast, an ANP analog (serial number: 238) coupled with a gel-forming enhancement motif can form a semi-solid gel at concentrations of 20% and 11% w / w. The immunomodulator thymosin α1 (serial number: 13) can form a semi-solid gel at a concentration of 20% w / w and a liquid gel at a concentration of 11% w / w. When the N-terminus of thymosin α1 is replaced by a gel-forming enhancement motif (Pal-HSY) or a shorter gel-forming motif, a semi-solid gel can be formed at a concentration of 11% w / w (serial numbers: 240, 267, and 268). On the other hand, analogs with a long gel-forming enhancement motif (Pal-SSPHSY) (serial number: 239) cannot form a gel at the same concentration. Furthermore, we found that a thymosin β4 analog (serial number: 263) can form a liquid gel at concentrations of 11% and 20% w / w.

[0226] Vasoactive intestinal peptide (VIP) (serial number: 67) cannot form a gel at concentrations of 20% or 11% w / w. In contrast, a VIP analog coupled with a gel-forming enhancement motif (serial number: 266) can form a semi-solid gel at a concentration of 11% w / w. Furthermore, we found that the adrenomedullin analog (Sequence No.: 274) could form a semi-solid gel at concentrations of 20%, 11%, or 6% w / w, which was superior to wild-type adrenomedullin (Sequence No.: 2) or the shorter acylated analog (Sequence No.: 1).

[0227] The cell penetration enhancing peptide (CPP) TAT (Sequence No.: 36) itself could not form a gel at concentrations of 20% or 11% w / w. When fused with a gel-forming enhancing motif (Sequence No.: 241), the peptide was able to form a liquid gel at a concentration of 11% w / w. Similarly, at a concentration of 11% w / w, the kallikrein inhibitor analog (Sequence No.: 37) could not form a gel. When the kallikrein inhibitor was fused with a gel-forming enhancing motif, the resulting analog (Sequence No.: 242) was able to form a semi-solid gel at a concentration of 11% and a liquid gel at a concentration of 6% w / w. Temporin A peptide is an antimicrobial peptide. When fused with a gel-forming enhancement motif, the resulting analogue (serial number: 243) forms a semi-solid gel at a concentration of 11% w / w. On the other hand, the complement system modulator campstatin (serial number: 11) forms a semi-solid gel at a concentration of 11% w / w.Solid gels, while analogs containing gel-forming enhancement motifs (Sequence No.: 244) can form semi-solid gels at the same concentration. The immunomodulator glatiramer (or clopidogrel) (Sequence No.: 42) fails to form a gel at concentrations of 20% or 11% w / w. When this peptide is coupled to a gel-forming enhancement motif, the resulting analog (Sequence No.: 245) is able to form a semi-solid gel at a concentration of 20% w / w.

[0228] Detection results for many matrix-modified peptides (or matrikine; Sequence Nos.: 39 and 44-47) that modulate skin matrix enzymes show that these peptides fail to form semi-solid or liquid gels at a concentration of 11% w / w. However, when these peptides are fused to gel-forming enhancement motifs, they can form semi-solid or liquid gels at a concentration of 11% w / w. For example, fusion peptides with serial numbers 246, 247, 249, and 250 can all form semi-solid gels at a concentration of 11% w / w; while fusion peptide with serial number 248 can form liquid gels at a concentration of 11% w / w and semi-solid gels at a concentration of 20% w / w.

[0229] In another experiment, we investigated the effect of coupling with known gel-forming peptide motifs on the ability of substance P analogs to form semi-solid or liquid gels. Although substance P analogs containing gel-forming enhancement motifs (serial number: 23) can form semi-solid gels at a concentration of 11% w / w, the addition of known gel-forming peptides containing repeating sequences (e.g., FKFEFKFE, QQRFEWEFEQQ, and VKVKVKVKV(D-Pro)PTKVKVKVKV) to substance P did not produce gel-forming analogs (serial numbers: 284-286; Table 3).

[0230] To determine whether peptide modification affects the bioactivity of gel-forming peptides, we investigated the receptor-modifying activity of selected gel-forming peptides. Studies on receptor activation activity against MC1R showed that both conjugated gel-forming afanotide (melanotide I) analogs and ACTH1-24 analogs (serial numbers: 224 and 226) stimulated MC1R, with EC50 concentrations ranging from sub-nanomolar to nanomolar levels (Table 4). The EC50 of the positive control melanotane II peptide was 0.56 nM. Analysis of receptor activation activity against MC4R showed that the conjugated gel-forming setmelanotide analog (serial number: 228) stimulated MC4R, with an EC50 of 0.15 nM. In this experiment, the EC50 of melanotane II peptide was 2 nM, indicating that the selected modified peptides also exhibited superior receptor activation activity. Similarly, CRHR2 activity analysis showed that gel-forming urocortin 2 and urocortin 3 were similar.The compounds (serial numbers: 230 and 231) potently stimulated CRHR2, with EC50 values ​​of 0.5 nM and 5.7 nM, respectively. The positive control, frog skin antihypertensive peptide, had an EC50 of 3.6 nM, indicating that the selected modified urocortin peptide also possessed superior receptor activation activity. Studies on PTHR1 receptor activity showed that the gel-forming PTH analogue (serial number: 232) had an EC50 of 14.1 nM for PTHR1, while the positive control, PTH(1-34) peptide, had an EC50 of 0.8 nM. On the other hand, the gel-forming κ-opioid receptor agonist (serial number: 214) had an EC50 of 272 nM for OPRK1, while the positive control, dynorphin A, had an EC50 of 0.5 nM.

[0231] Furthermore, a study of bradykinin receptor BDKRB2 activity using the Discoverx BDKRB2 Inhibitor Kit showed that the coupled bradykinin receptor antagonist (serial number: 234) inhibited the bradykinin-stimulated BDKRB2 signaling pathway with an IC50 of 53.3 nM (Table 4). In contrast, the positive control antagonist HOE140 had an IC50 of 131.8 nM, indicating that the gel-forming antagonist was more effective than the wild-type peptide HOE140. Additionally, a GnRH receptor activity analysis using the Discoverx GnRH Calcium Flux Kit showed that gel-forming GnRH antagonist analogs (serial numbers: 202 and 203) inhibited the LHRH-stimulated GnRH signaling pathway, with both having IC50 values ​​less than 0.51 nM, compared to an IC50 of 0.08 nM for the positive control cetrorex. Overall, the data indicate that these novel cell surface receptor ligand analogs are capable of forming hydrogels and retain the ability to modulate receptor activity.

[0232] Table 4 List of bioactivity of synthetically produced gel-forming ligands

[0233] Specification 48 / 52 pages 59 CN 121421943 A

[0234]

[0235] To fully understand the effect of gel-forming enhancement motifs on the gel-forming activity of various peptides, Table 5 lists the ability of each peptide to form semi-solid or liquid gels by comparing each pair of coupled and wild-type peptides. SS indicates the ability to form semi-solid gels, and LG indicates the ability to form liquid gels. In addition, blank represents the lack of gel-forming ability at the selected concentration. The results of the t-test showed that coupling gel-forming enhancement motifs to 57 peptides significantly enhanced the gel-forming ability of these peptides.

[0236] Table 5. Thermographs of ligands with gel-forming enhancement motifs to indicate gel formation tendency

[0237] Specification 49 / 52 pages 60 CN 121421943 A

[0238] Specification 50 / 52 pages 61 CN 121421943 A

[0239]

[0240] Because natural and modified gel-forming peptides form gel nanostructures through a continuous polymerization reaction involving electrostatic interactions between monomers, amphiphilic monomers can bind and separate in equilibrium. Therefore, gel-forming monomers can bind to each other even without observed gel nanostructures. To demonstrate that the self-assembled gel of this invention can be used as a carrier for other therapeutic agents, release assays were performed using semi-permeable membranes (e.g., centrifugal filters). In these experiments, FITC-labeled therapeutic agents were dissolved in solutions containing low concentrations of gel-forming or non-gel-forming peptides. Centrifugation separated freely moving soluble molecules in the lower chamber from soluble molecules polymerized with the gel nanostructures in the upper chamber. The mobility of FITC-labeled molecules was quantified using a fluorometer. As shown in Table 6, compared to samples without gel-forming peptides, mixing FITC-labeled morpholine molecules (nucleic acids), FITC-labeled GnRH molecules (peptides), and FITC-labeled dUTP molecules (nucleotides) with gel-forming peptides (serial number: 119; 1 mg / 10 ml) reduced molecular mobility by 3–40 times. This reduction was dose-dependent, and the reduction was much smaller in samples with a gel-forming peptide concentration of 1 mg / 100 mL. In contrast, when the FITC-labeled compound was mixed with a non-gel-forming peptide (Sequence No.: 133) in solution, the mobility of the molecules was not significantly reduced compared to the control sample.

[0241]

[0242] Specification 51 / 52 pages 62 CN 121421943 A

[0243] The interaction between gel-forming peptides and FITC-labeled small molecules (e.g., naloxone and dexamethasone) was studied using a centrifugal filtration apparatus, and the results showed that after these molecules were mixed with gel-forming peptides (Sequence Nos.: 109 and 119), the FITC-labeled molecules were retained in the upper chamber of the filter (Table 7). In the sample containing peptide 109, the number of naloxone and dexamethasone molecules passing through the filter was reduced by 30% compared to the control sample. Similarly, compared to the control sample, less than 15% of naloxone and dexamethasone molecules passed through the filter in the sample containing peptide 109. These data clearly demonstrate that FITC-labeled molecules can aggregate with or be encapsulated by gel nanostructures prepared from gel-forming peptides, which prevents these molecules from passing through the filter membrane. These data also imply that solutions of gel-forming peptides can be used for slow delivery of therapeutic drugs even without observing gel structure / conformation.

[0244] To further demonstrate the long-term biological effects of gel-forming peptides in vivo, we investigated the effect of a gel-forming melanocyte-stimulating hormone (MSH) analog (serial number: 224) on changes in skin color in bullfrogs (Figure 2). Intraperitoneal injectionFollowing injection of the wild-type analogue (alfano peptide, SEQ ID NO: 29; 100 nmol / kg body weight) or the gel-forming analogue (SEQ ID NO: 224, 100 nmol / kg body weight), the skin color of bullfrogs changed significantly. Within 2 hours, the skin color changed from green to black. The skin color of the control group animals remained green after injection of saline. Four or fourteen days after injection, only the bullfrogs injected with the gel-forming MSH analogue showed dark skin, which was due to the expansion of melanocytes in the skin. This result demonstrates that the gel-forming analogue has a long-term effect.

[0245] Table 7 Interactions between gel-forming peptides and small molecule drugs

[0246]

[0247] All publications and patent applications cited in this specification are incorporated herein by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0248] In order to include preferred embodiments of the invention, the invention has been described based on specific embodiments discovered or proposed by the inventors. Those skilled in the art will understand that, based on the disclosure of this invention, various modifications and alterations can be made to the specific embodiments listed without departing from the intended scope of this invention. Furthermore, for consideration of biological functional equivalence, the structure of the protein can be changed without affecting the type or amount of biological action. All such modifications are included within the scope of the appended claims. Specification 52 / 52 pages 63 CN 121421943 A Figure 1A Figure 1B Specification Drawings 1 / 3 pages 64 CN 121421943 A Figure 1C Specification Drawings 2 / 3 pages 65 CN 121421943 A Figure 2 Specification Drawings 3 / 3 pages 66 CN 121421943 A Abstract A stable aqueous gel or semi-solid gel pharmaceutical composition comprising a water-soluble peptide having gel-forming ability, optionally in combination with an appropriate excipient and a therapeutic agent. Following administration to an individual, the pharmaceutical composition forms a gel reservoir in which the gel nanostructure releases thepeptide or the encapsulated therapeutic agent(s) for a longer period of time. The gel-forming compounds can be formulated as aqueous formulations, suspensions or solid formulations, wherein the gel-forming polypeptide contained accounts for 0.01% to 99% of the total weight of the formulation. These formulations are useful in drug delivery and as implantable drug depot for long term delivery of therapeutic agents, antigens or cells. Also provided are methods of producing a gel-forming compound through the modification of a chemical compound with a gel-forming enhancing motif.

Claims

1. An aqueous pharmaceutical composition for slow release of a therapeutic component comprising: a) a self-assembling gel-forming polypeptide at a concentration of at least about 0.01% (w / w) of the total weight of the composition; b) an aqueous excipient.

2. The pharmaceutical composition of claim 1, wherein the gel-forming polypeptide forms a non-covalently linked liquid or semi-solid gel at a concentration of about 11-30% w / w in aqueous solution or at lower concentrations.

3. The pharmaceutical composition of claim 1, wherein the aqueous excipient is low in ionic concentration.

4. The pharmaceutical composition of any one of claims 1-3, further comprising an additive selected from the group consisting of buffers, excipients, solvents, solubilizers, preservatives, stabilizers, surfactants, antioxidants, and mixtures thereof.

5. The pharmaceutical composition of any one of claims 1-4, wherein the self- assembling gel-forming polypeptide is a G protein-coupled receptor (GPCR) ligand polypeptide or a peptide biological functional mediator.

6. The pharmaceutical composition of claim 5, wherein the ligand is selected from the group consisting of analogs of amylin, CGRP, adrenomedullin (ADM), and adrenomedullin 2 (ADM 2 or IMD), which can be agonists, antagonists, chimeric analogs, or non-functional analogs.

7. The pharmaceutical composition of any one of claims 1-4, wherein the self- assembling gel-forming polypeptide is an analog of oxytocin, kisspeptin, kappa opioid receptor agonist, pramlintide, a romiketin analog, urocortin 3, campath, GLP-1, GLP-2, thymosin alpha 1, thymosin beta 4, gamma-MSH, a bombesin receptor antagonist, an opioid receptor ligand, or an analog of GnRH, which can be agonists, antagonists, chimeric analogs, or non-functional analogs.

8. The pharmaceutical composition of any one of claims 1-7, wherein the concentration of the gel-forming polypeptide (by weight) is equivalent to 0.001% to 99% of the total weight of the composition, preferably 0.1% to 30%.

9. The pharmaceutical composition of any one of claims 1-8, wherein the gel-forming polypeptide comprises a sequence listed in any one of Tables 1, 2, or 3.

10. The pharmaceutical composition of any one of claims 1-8, wherein the gel-forming polypeptide comprises a sequence selected from the group consisting of SEQ ID NOS: 1-15, 61, 64, 263, 274, or an analog thereof.

11. The pharmaceutical composition of any one of claims 1-4, wherein the gel-forming polypeptide is selected from an ADM, CGRP, or IMD (ADM 2) sequence comprising the structure of Formula I: R1-B0-B1-B2-B3-B4-B5-B6-B7-B8-B9-B10-B11-B12-B13-B14-B15-B16-B17-B18-B19-B20-B21-B22-B23-B24-B25-B26-B27-B28-R2, wherein, ​ R1is a functional group comprising the structure of formula (W')(X')n(Y')n(Z')n, wherein W' is a fatty acid, a fatty diacid, a derivative of a fatty acid or cholesterol, or null; X' is a PEG group, glutamic acid, gamma-glutamic acid, a non-proteinogenic amino acid, or null; Y' is a PEG group, glutamic acid, gamma-glutamic acid, a non-proteinogenic amino acid, or null; Z' is a proteinogenic amino acid, a non-proteinogenic amino acid, or null; R2is a C-terminal modification moiety comprising {NH2} amidation, {-CHO} peptide aldehyde, {-ol} alcohol peptide, {CMK} chloromethyl ketone, {FMK} fluoromethyl ketone, {Cya} mercaptoethyl amination, {pNA} p-nitroaniline, {-ONP} p-nitrophenol, {AMC} 7-amino-4-methylcoumarin, {AFC}, -OMe (C-terminal), -OEt (C-terminal), -OBzl (C-terminal), -OtBu (C-terminal), {-OSu} hydroxysuccinimidyl ester, -NHMe (C-terminal), -NHEt (C-terminal), -NH isoamylamine (C-terminal), NH(C2H)6(C-terminal), -NHPh (C-terminal), {NHEt(O)EtNH-Fmoc} 2,2'-oxo bisethylamine-Fmoc, {NHEt(EtNH-Myr)2}, -NH(OMe)Me (C-terminal), -TBzl (C-terminal), -NHNH2(C-terminal), -ED (C-terminal) -NH-CH2CH2-NH2, or -BD (C-terminal) -NH-CH2CH2CH2CH2-NH2NH2 groups; B0is selected from the group consisting of a null residue, any proteinogenic or non-proteinogenic amino acid, acylated histidine (acy-His), acylated arginine (acy-Arg), acylated lysine (acy-Lys); B1is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, histidine, arginine, lysine, asparagine, glutamine, and a non-proteinogenic amino acid; B2is selected from the group consisting of a null residue, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and a non-proteinogenic amino acid; B3is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, glutamine, aspartic acid, and a non-proteinogenic amino acid; B4is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, and a non-proteinogenic amino acid; B5is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, tyrosine, and a non-proteinogenic amino acid; B6is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, and a non-proteinogenic amino acid; B7is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, methionine, phenylalanine, glutamine, asparagine, histidine, arginine, lysine, and a non-proteinogenic amino acid; B8 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, serine, threonine, and a non-protein amino acid; B9 is selected from the group consisting of a null residue, arginine, lysine, asparagine, glutamine, tryptophan, phenylalanine, serine, threonine, tyrosine, and a non-protein amino acid; B10 is selected from the group consisting of a null residue, alanine, serine, threonine, glutamine, glutamic acid, aspartic acid, asparagine, and a non-protein amino acid; B11 is selected from the group consisting of a null residue, tryptophan, phenylalanine, valine, alanine, glycine, isoleucine, leucine, proline, and a non-protein amino acid; B12 is selected from the group consisting of a null residue, alanine, glycine, serine, threonine, proline, tyrosine, methionine, tryptophan, phenylalanine, and a non-protein amino acid; B13 is selected from the group consisting of a null residue, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, methionine, leucine, phenylalanine, and a non-protein amino acid; B14 is selected from the group consisting of a null residue, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and a non-protein amino acid; B15 is selected from the group consisting of a null residue, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, valine, alanine, glycine, isoleucine, leucine, and a non-protein amino acid; B16 is selected from the group consisting of a null residue, asparagine, glutamine, valine, alanine, glycine, isoleucine, leucine, and a non-protein amino acid; B17 is selected from the group consisting of a null residue, asparagine, glutamine, serine, threonine, tyrosine, and a non-protein amino acid; B18 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, phenylalanine, tyrosine, and a non-protein amino acid; B19 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and a non-protein amino acid; B20 is selected from the group consisting of a null residue, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, proline, and a non-protein amino acid; B21 is selected from the group consisting of a null residue, isoleucine, valine, serine, threonine, tyrosine, glutamine, glutamic acid, aspartic acid, asparagine, and a non-protein amino acid; B22 is selected from the group consisting of a null residue, histidine, arginine, lysine, valine, alanine, glycine, isoleucine, leucine, asparagine, glutamine, proline, and a non-protein amino acid; B23 is selected from the group consisting of a null residue, serine, threonine, tyrosine, valine, alanine, glycine, isoleucine, leucine, methionine, phenylalanine, and a non-protein amino acid; B24 is selected from the group consisting of a null residue, alanine, glycine, proline, serine, threonine, tyrosine, and a non-protein amino acid; B25 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, and a non-proteinogenic amino acid; B26 is selected from the group consisting of a null residue, histidine, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and a non-proteinogenic amino acid; B27 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, serine, threonine, tyrosine, and a non-proteinogenic amino acid; B28 is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, phenylalanine, serine, threonine, tyrosine, and a non-proteinogenic amino acid.

12. A functional self-assembling gel forming polypeptide agonist or antagonist comprising a sequence selected from the group consisting of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, or an analog thereof.

13. A gel forming polypeptide comprising an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, and 274.

14. A gel forming polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, and 274.

15. A gel forming polypeptide comprising a sequence selected from the group consisting of SEQ ID NOS: 106-114, 116-124, 126-131, and 139-140, and analogs thereof.

16. A gel forming polypeptide comprising an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 106-114, 116-124, 126-131, and 139-140.

17. A gel forming polypeptide comprising an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOS: 106-114, 116-124, 126-131, and 139-140.

18. A gel forming polypeptide comprising a stereoisomer, derivative, analog, or peptidomimetic of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140.

19. A method for preparing a self-assembling liquid gel or semi-solid gel, comprising: dissolving a self-assembling gel forming polypeptide to a concentration of at least about 0.01% (w / w) of the total weight of the composition (by weight) in an aqueous vehicle.

20. The method of claim 19, wherein the polypeptide comprises (or consists of) a sequence selected from the group consisting of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, or an analog thereof.

21. A method for treating a subject having a condition that can be alleviated by administration of a gel forming polypeptide formulation, comprising: dissolving a self-assembling gel forming polypeptide to a concentration of at least about 0.01% (w / w) of the total weight of the composition (by weight) in an aqueous vehicle. administering to the subject an effective amount of a formulation according to any one of claims 1-20.

22. A pharmaceutical composition in the form of a liquid, semi-solid or solid gel comprising one or more therapeutic agents; a water-soluble gel-forming polypeptide, and optionally an excipient and / or a therapeutic agent, which composition forms a gel upon contact with the body when injected into a patient.

23. The pharmaceutical composition of claim 22, wherein the therapeutic agent is not covalently linked to the gel-forming polypeptide.

24. The pharmaceutical composition of claim 22 or 23, wherein the gel-forming polypeptide is a GPCR ligand-derived polypeptide or a peptide-based biological functional mediator.

25. The pharmaceutical composition of claim 24, wherein the gel-forming polypeptide comprises (or consists of) a gel-forming GPCR ligand or ligand fragment selected from the group consisting of SEQ ID NOs: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, or an analog thereof.

26. The pharmaceutical composition of any one of claims 22-25, wherein the therapeutic agent is selected from the group consisting of a small molecule drug, a peptide drug, a large molecule biologic, an antibody, a hormone, a growth factor, an antigen, a nucleic acid, and a nucleotide.

27. A method for treating a subject suffering from a condition that can be alleviated by administration of a gel-forming polypeptide formulation, the method comprising: administering to the subject an effective amount of a formulation according to any one of claims 1-11, 12-21, or 22-26, wherein the therapeutic agent is released over an extended period of time.

28. The method of claim 27, wherein: releasing the therapeutic agent into systemic circulation, into a local tissue or organ, or onto a surface, including an ocular surface, a buccal surface, a rectal surface, a nasal surface, a respiratory organ surface, a gastrointestinal tract surface, a urethral surface, a uterine surface, or a dermal surface.

29. The method of claim 27 or 28, wherein: administering the formulation to the patient parenterally, intramuscularly, subcutaneously, intranasally, intrauterinely, intraurethrally, intraocularly, topically, orally, or intradermally.

30. The method of any one of claims 27-29, wherein the pharmaceutical preparation is used in conjunction with one or more other substances selected from the group consisting of small molecules, polypeptides, proteins, enzymes, hormones, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, antibiotics, chemotherapeutics, immunosuppressants, anti-inflammatory agents, anti-malignant proliferation agents, anti-mitotic agents, angiogenic agents, anti-angiogenic agents, anti-psychotic agents, central nervous system (CNS) agents, anti-coagulants, and fibrinolytic agents; the agents including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (Nt-3), neurotrophin-4 / 5 (Nt-4 / 5), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, cardiotrophin-1, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor beta 1 (TGFβ1; TGFβ2, TGFβ3), activin, glial cell-derived neurotrophic factor (GDNF), pregnancy-associated plasma protein-A, heparin-binding neurotrophic factor (HBNF), transforming growth factor alpha (TGFα), modulin (neuromodulin, ARIA), axonal ligand-1 (Al-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), transforming growth factor (TGF), interleukin (IL), colony stimulating factor (CSF, MCF, GCSF, GMCSF), interferon (IFN), endothelial cell growth factor (VEGF, EGF), erythropoietin (EPO), angiogenin (ANG), placental growth factor (PIGF), bone morphogenic protein (BMP), growth differentiation factor (GDF).antibodies, antigens, adenosines, adrenergic amines, acetylcholine, histamine derivatives, dopamine derivatives, glutamic acid derivatives, GABA derivatives, cannabinoid derivatives, prostaglandin derivatives, leukotrienes, thrombin analogs, lysophospholipid (LPA) derivatives, sphingosine-1 -phosphate derivatives, LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, eledoisin analogs, neurotensin analogs, kisspeptin analogs, kisspeptin-234 analogs, bombesin analogs, bradykinin analogs, bradykinin agonist analogs, opioids analogs, neuropeptide FF analogs, enkephalin analogs, substance P analogs, angiotensin II analogs, parathyroid hormone analogs, PTHrP analogs, GLP-1 analogs, GLP-2 analogs, glucagon analogs, GIP analogs, calcitonin analogs, amylin analogs, CGRP analogs, adrenomedullin analogs, adrenomedullin 2 analogs, neuropeptide Y (NPY) analogs, peptide YY (PYY) analogs, NPY antagonist analogs, vasoactive intestinal polypeptide (VIP) analogs, urocortin analogs, urocortin 2 analogs, urocortin 3 analogs, bradykinin analogs, somatostatin analogs, endothelin analogs, adrenocorticotropic hormone (ACTH) analogs, melanotan I analogs, melanotan II analogs, melanocyte-stimulating hormone (MSH) analogs, melanocortin analogs, growth hormone-releasing hormone analogs, orexin analogs, HOE 140 analogs, eptaketa peptide analogs, human growth hormone analogs, insulin analogs, anti-inflammatory peptide 1 analogs, thrombin activatable analogs, neuromodin U analogs, neuromodin S analogs, heparin, interleukin-1 analogs, interleukin-2 analogs, factor V analogs, factor IX analogs, luteinizing hormone analogs, relaxin analogs, ghrelin analogs, follicle-stimulating hormone analogs, atrial natriuretic peptide (ANP) analogs, brain natriuretic peptide (BNP) analogs, C-type natriuretic peptide (CNP) analogs, guanylin analogs, chemokine analogs, cytokine analogs, interferon analogs, erythropoietin analogs, thrombopoietin analogs, interleukin analogs, tumor necrosis factor (TNF) analogs;Thrombopoietin peptide analogs, glatiramer (Cuproxone), thymosin alpha 1 analogs, thymosin beta 4 analogs, cell penetrating peptides, TAT peptides, kallikrein inhibitors, phospholipase inhibitors, krestin, temporin A antibacterial peptides, anti-gramicidin peptides, BMP7-derived bone formation peptide-1, BMP7-derived bone formation peptide-2, PEDF (24-57), PEDF (58-101), PEDF (40-57), PEDF (44-77), PEDF (78-121), PEDF (98-114), PEDF-derived P14, PEDF-derived P17, PEDF-derived P18, PEDF-derived P23, PEDF-derived P34, PEDF-derived P44, FGF-derived FK18 peptide, Enfuviritide / Fuzeon peptide, Eptifibatide platelet aggregation inhibitor, YIGSR peptide, RGD peptide, VGVAPG peptide, EEMQRR peptide, and YRSRKYSSWY peptide; toxins (such as botulinum toxin) and pharmaceutically acceptable salts of these compounds, or analogs, fragments or derivatives thereof; salts of the following substances or analogs: ligands for adenosine receptors, adrenergic receptors, acetylcholine receptors, histamine receptors, dopamine receptors, calcium receptors, glutamate receptors, GABA receptors, cannabinoid receptors, prostaglandin receptors, leukotriene receptors, protease-activated receptors, lysophospholipid (LPA) receptors, sphingosine-1 -phosphate receptors, LHRH receptors, vasopressin receptors, oxytocin receptors, apelin receptors, neurotensin receptors, kisspeptin receptors, bombesin receptors, opioid receptors, substance P receptors, angiotensin II receptors, parathyroid hormone receptors, GLP-1 receptors, GLP-2 receptors, glucagon receptors, calcitonin receptors, amylin receptors, calcitonin gene-related peptide (CGRP) receptors, adrenomedullin receptors, neuropeptide Y (NPY) receptors, peptide YY (PYY) receptors, vasoactive intestinal peptide (VIP) receptors, urocortin receptors, bradykinin receptors, somatostatin receptors, endothelin receptors, adrenocorticotropic hormone (ACTH) receptors, melanocyte-stimulating hormone (MSH) receptors, melanocortin receptors, growth hormone-releasing hormone receptors, ghrelin receptors, insulin receptors, relaxin receptors, natriuretic peptides receptors, guanylin receptors, chemokine receptors, cytokine receptors, growth factor receptors, interferon receptors, erythropoietin receptors, growth hormone receptors, FSH receptors, LH receptors, TSH receptors, interleukin receptors, tumor necrosis factor (TNF) receptors, nerve growth factor receptors, platelet-derived growth factor (PDGF) receptors, colony-stimulating factor (CSF) receptors, bone morphogenetic protein (BMP) receptors, FGF receptors, growth differentiation factor receptors;Glatiramer (Copaxone) analogues, thymosin, Krestin, temporin A, YIGSR peptide, RGD peptide, VGVAPG peptide, YRSRKYSSWY peptide, and nucleotide derivatives, antibiotics, antibodies, enzyme inhibitors, enzymes, complement factors, urokinase, asparaginase, kallikrein, kallikrein inhibitors, coagulation factors, cytotoxic therapeutic drugs, microbial antigens, viral antigens, tumor antigens, neoantigens, and cosmeceutical peptides and pharmaceutically acceptable salts of these compounds, or analogues, fragments or derivatives thereof.

31. The composition of any one of claims 1-11, 12-21, or 22-26, wherein the composition is a pharmaceutical composition, a cosmetic composition, or a dermal filler composition.

32. A method of engineering a gel-forming polypeptide, the method comprising: coupling a therapeutic agent to a gel-forming enhancing motif; wherein the motif is an acylated or unacylated amino acid sequence derived from a cell surface receptor secreted peptide ligand capable of self-assembly into a gel.

33. The method of claim 32, wherein the gel-enhancing motif is derived from any one of the amino acid sequences of SEQ ID NOs: 1-15, 61, 64, 263, 274, 106-114, 116-124, 126-131, or 139-140.

34. The method of claim 32 or 33, wherein the gel-forming polypeptide comprises the structure of Formula II, Ea-(Fa)n-Ga (II) wherein Ea is a gel-forming polypeptide motif derived from a cell surface receptor ligand or a therapeutic drug; Fa is a PEG moiety; n is an integer from 0 to 40; and Ga is a therapeutic drug or a gel-forming polypeptide motif derived from a cell surface receptor ligand, Ea, Fa, and Ga can be positioned and linked by any side chain of an amino acid.

35. An engineered gel-forming polypeptide having the structure of Formula II Ea-(Fa)n-Ga (II) wherein Ea is a gel-forming polypeptide motif having the structure of Formula I or a therapeutic drug; Fa is a PEG moiety; n is an integer from 0 to 40 or a covalent linkage between a motif in Ea and a motif in Ga; and Ga is a therapeutic drug or a gel-forming polypeptide comprising the structure of Formula I, Ea, Fa, and Ga can be positioned and linked by any side chain of an amino acid.

36. The gel-forming polypeptide of claim 34 or 35, wherein the therapeutic drug is selected from a small molecule, a polypeptide, a protein, an enzyme, a hormone, a polynucleotide, a nucleoprotein, a polysaccharide, a glycoprotein, a lipoprotein, a steroid, an analgesic, a local anesthetic, an antibiotic, a chemotherapeutic, an immunosuppressant, an anti-inflammatory, an anti-malignant proliferative, an anti-mitotic, an angiogenic, an anti-angiogenic, an antipsychotic, a central nervous system (CNS) drug, an anticoagulant, or a fibrinolytic drug. ​ 37. The gel-forming polypeptide of claim 36, wherein the therapeutic drug is selected from the group consisting of: LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, apelin analogs, neurotensin analogs, kisspeptin analogs, kisspeptin 234 analogs, bombesin analogs, bradykinin analogs, bradykinin agonist analogs, opioid analogs, neuropeptide FF analogs, enkephalin analogs, substance P analogs, angiotensin II analogs, parathyroid hormone analogs, PTHrP analogs, GLP-1 analogs, GLP-2 analogs, glucagon analogs, GIP analogs, calcitonin analogs, amylin analogs, CGRP analogs, adrenomedullin analogs, adrenomedullin 2 analogs, neuropeptide Y (NPY) analogs, peptide YY (PYY) analogs, NPY antagonist analogs, vasoactive intestinal polypeptide (VIP) analogs, urocortin analogs, urocortin 2 analogs, urocortin 3 analogs, bradykinin analogs, somatostatin analogs, endothelin analogs, adrenocorticotropic hormone (ACTH) analogs, melanotan I analogs, melanotan II analogs, melanocyte stimulating hormone (MSH) analogs, melanocortin analogs, growth hormone releasing hormone analogs, orexin analogs, HOE 140 analogs, eptaketa analogs, human growth hormone analogs, insulin analogs, anti-inflammatory 1 analogs, thrombin activatable analogs, neuromedial U analogs, neuromedial S analogs, heparin, interleukin-1 analogs, interleukin-2 analogs, factor V analogs, factor IX analogs, luteinizing hormone analogs, relaxin analogs, ghrelin analogs, follicle stimulating hormone analogs, atrial natriuretic peptide (ANP) analogs, brain natriuretic peptide (BNP) analogs, C-type natriuretic peptide (CNP) analogs, guanylin analogs, chemokine analogs, cytokine analogs, interferon analogs, erythropoietin analogs, thrombopoietin analogs, interleukin analogs, tumor necrosis factor (TNF) analogs.Thrombopoietin peptide analogues, glatiramer (Cuproxone), thymosin alpha 1 analogues, thymosin, cell penetrating peptides, TAT peptides, kallikrein inhibitors, phospholipase inhibitors, krestin, temporin A antibacterial peptides, anti-gramicidin peptides, BMP7-derived bone morphogenic peptide-1, BMP7-derived bone morphogenic peptide-2, PEDF (24-57), PEDF (58-101), PEDF (40-57), PEDF (44-77), PEDF (78-121), PEDF (98-114), PEDF-derived P14, PEDF-derived P17, PEDF-derived P18, PEDF-derived P23, PEDF-derived P34, PEDF-derived P44, FGF-derived FK18 peptide, Enfuviritide / Fuzeon peptide, Eptifibatide platelet aggregation inhibitor, YIGSR peptide, KTTKS peptide, RGD peptide, VGVAPG peptide, EEMQRR peptide, and YRSRKYSSWY peptide; toxins (e.g., botulinum toxin), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (Nt-3), neurotrophin-4 / 5 (Nt-4 / 5), ciliary neurotrophic factor (CNTF), leukemia inhibitory factor, cardiotrophin-1, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), transforming growth factor beta 1 (TGFβ1; TGFβ2, TGFβ3), activin, glial cell-derived neurotrophic factor (GDNF), midkine, heparin-binding neurotrophic factor (HBNF), transforming growth factor alpha (TGFα), modulin (neuromodulin, ARIA), axonal ligand-1 (Al-1), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), fibroblast growth factor (FGF), transforming growth factor (TGF), interleukin (IL), colony stimulating factor (CSF, MCF, GCSF, GMCSF), interferon (IFN), endothelial cell growth factor (VEGF, EGF), erythropoietin (EPO), angiogenin (ANG), placental growth factor (PIGF), bone morphogenic protein (BMP), growth differentiation factor (GDF); antibodies, antigens, or analogs, fragments, or derivatives thereof;salts of the following substances or analogs: ligands for adenosine receptors, adrenergic receptors, acetylcholine receptors, histamine receptors, dopamine receptors, calcium receptors, glutamate receptors, GABA receptors, cannabinoid receptors, prostaglandin receptors, leukotriene receptors, protease-activated receptors, lysophospholipid (LPA) receptors, sphingosine-1 -phosphate receptors, LHRH receptors, vasopressin receptors, oxytocin receptors, apelin receptors, neurotensin receptors, kisspeptin receptors, bombesin receptors, opioid receptors, substance P receptors, angiotensin II receptors, parathyroid hormone receptors, GLP-1 receptors, GLP-2 receptors, glucagon receptors, calcitonin receptors, amylin receptors, calcitonin gene-related peptide (CGRP) receptors, adrenomedullin receptors, neuropeptide Y (NPY) receptors, peptide YY (PYY) receptors, vasoactive intestinal peptide (VIP) receptors, urocortin receptors, bradykinin receptors, somatostatin receptors, endothelin receptors, adrenocorticotropic hormone (ACTH) receptors, melanocyte-stimulating hormone (MSH) receptors, melanocortin receptors, growth hormone-releasing hormone receptors, ghrelin receptors, insulin receptors, relaxin receptors, natriuretic peptides receptors, guanylin receptors, chemokine receptors, cytokine receptors, growth factor receptors, interferon receptors, erythropoietin receptors, growth hormone receptors, FSH receptors, LH receptors, TSH receptors, interleukin receptors, tumor necrosis factor (TNF) receptors, nerve growth factor receptors, platelet-derived growth factor (PDGF) receptors, colony-stimulating factor (CSF) receptors, bone morphogenetic protein (BMP) receptors, FGF receptors, growth differentiation factor receptors; glatiramer (copaxone) analogs, thymosin, compstatin, temporin A, YIGSR peptide, KTTKS peptide, RGD peptide, VGVAPG peptide, YRSRKYSSWY peptide, nucleoside derivatives, antibiotics, antibodies, enzyme inhibitors, enzymes, complement factors, urokinase, asparaginase, kallikrein, kallikrein inhibitors, coagulation factors, cytotoxic therapeutics, microbial antigens, viral antigens, tumor antigens, neoantigens, and cosmeceutical peptides and pharmaceutically acceptable salts of these compounds, or analogs, fragments, or derivatives thereof.

38. The gel-forming polypeptide of any one of claims 34-37, wherein the gel-forming components Ea and Ga are selected from ligands for adenosine receptors, adrenergic receptors, acetylcholine receptors, histamine receptors, dopamine receptors, calcium receptors, glutamate receptors, GABA receptors, cannabinoid receptors, prostanoid receptors, leukotriene receptors, protease-activated receptors, lysophospholipid (LPA) receptors, sphingosine-1 -phosphate receptors, LHRH receptors, vasopressin receptors, oxytocin receptors, apelin receptors, neurotensin receptors, kisspeptin receptors, bombesin receptors, opioid receptors, substance P receptors, angiotensin II receptors, parathyroid hormone receptors, GLP-1 receptors, GLP-2 receptors, glucagon receptors, calcitonin receptors, amylin receptors, calcitonin gene-related peptide (CGRP) receptors, adrenomedullin receptors, neuropeptide Y (NPY) receptors, peptide YY (PYY) receptors, vasoactive intestinal polypeptide (VIP) receptors, urocortin receptors, bradykinin receptors, somatostatin receptors, endothelin receptors, adrenocorticotropic hormone (ACTH) receptors, melanocyte-stimulating hormone (MSH) receptors, melanocortin receptors, growth hormone-releasing hormone receptors, ghrelin receptors, insulin receptors, relaxin receptors, natriuretic peptides receptors, guanylin receptors, chemokine receptors, cytokine receptors, growth factor receptors, interferon receptors, erythropoietin receptors, growth hormone receptors, FSH receptors, LH receptors, TSH receptors, interleukin receptors, tumor necrosis factor (TNF) receptors, nerve growth factor receptors, platelet-derived growth factor (PDGF) receptors, colony-stimulating factor (CSF) receptors, bone morphogenetic protein (BMP) receptors, growth differentiation factor receptors, and pharmaceutically acceptable salts or analogs, fragments, or derivatives thereof.

39. The gel-forming polypeptide of any one of claims 34-37, wherein the gel-forming components Ea and Ga are selected from the sequences of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, or a pharmaceutically acceptable salt thereof.

40. The gel-forming polypeptide of any one of claims 34-37, wherein the Ea and Ga comprise a stereoisomer, derivative, analog, or peptidomimetic of an amino acid sequence selected from the sequences of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140.

41. The gel-forming polypeptide of any one of claims 34-37, wherein the Ea or Ga is a gel-forming polypeptide having an amino acid sequence that is at least 70% identical to an amino acid sequence selected from the sequences of SEQ ID NOS: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140.

42. The gel-forming polypeptide of any one of claims 34-37, wherein the Ea or Ga is a gel-forming polypeptide having an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140.

43. The gel-forming polypeptide of any one of claims 34-37, wherein the gel-forming polypeptide comprises an amino acid sequence of SEQ ID NOs: 1-15, 48-58, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, or an analog thereof.

44. The gel-forming polypeptide of any one of claims 34-37, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-275, or an analog thereof; wherein the functional component of the gel-forming polypeptide is an analog or derivative of GnRH, a GnRH antagonist, vasopressin, oxytocin, apelin, neuromedin, kisspeptin, bombesin, bombesin receptor antagonist, neomorphin, enkephalin, kappa receptor agonist, substance P, relaxin, calcitonin, pramlintide (amylin analog), exenatide 4, GLP-1, teduglutide (GLP-2 analog), afenercept (melanotan I), melanotan II, gamma-MSH, ACTH1-24, setmelanotide, PYY3-36, urocortin 2, urocortin 3, parathyroid hormone, VIP, bradykinin receptor 1 antagonist, HOE140 (BKR2 antagonist), sermorelin, atrial natriuretic peptide (ANP), thymosin alpha 1, thymosin beta 4, adrenomedullin, adrenomedullin 2, TAT cell-penetrating peptide, kallikrein inhibitor, temporin A antimicrobial peptide, combretastatin, glatiramer (or copaxone), matrix modifying peptide 1, matrix modifying peptide 4, matrix modifying peptide 7, matrix modifying peptide 8, acetyl hexapeptide-3 matrix modifying peptide, and analogs thereof.

45. The gel-forming polypeptide of any one of claims 34-44, wherein the relative activity of the polypeptide is at least 0.01% compared to the corresponding wild-type polypeptide ligand / enzyme / enzyme substrate / mediator of at least one cognate receptor or cellular target.

46. A gel-forming polypeptide having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-275.

47. A gel-forming polypeptide having at least 80% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 201-275.

48. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and a gel-forming polypeptide of any one of claims 34-47.

49. A pharmaceutical composition according to any one of claims 1-11, 22-26 or 48, wherein the active agent has a therapeutic effect on cardiovascular, pulmonary, gastrointestinal, immune, oncological, dermal, renal, endocrine, ocular, musculoskeletal or neuronal disease.

50. A pharmaceutical composition according to any one of claims 1-11, 22-26 or 48-49, which is formulated as a liquid or liquid gel, and is administered by injection, infusion or topically.

51. A pharmaceutical composition according to any one of claims 1-11, 22-26 or 48-49, which is formulated to release a polypeptide and / or a therapeutic agent slowly in a subject.

52. A method of treating and / or preventing cardiovascular, pulmonary, gastrointestinal, immune, oncological, dermal, renal, endocrine, ocular, musculoskeletal or neuronal disease or treating and / or preventing a condition associated with abnormal regulation of a cellular process in a subject, the method comprising: administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1-11, 22-26 or 48-49.

53. A device for delivering a therapeutic agent to a subject, comprising a gel- forming polypeptide according to any one of claims 12-18 or an engineered gel-forming polypeptide according to any one of claims 35-47.

54. A kit comprising a gel-forming polypeptide according to any one of claims 12-18 or an engineered gel-forming polypeptide according to any one of claims 35-47, and optionally further comprising packaging and instructions.

55. The device of claim 53 or the kit of claim 54, further comprising at least one additional therapeutic agent.

56. A pharmaceutical composition comprising an aqueous solution or aqueous mixture, suspension, liquid gel or semi-solid gel pharmaceutical or solid gel pharmaceutical composition, the composition comprising at least one gel-forming polypeptide compound having an aqueous solubility greater than 0.01 mg / mL at room temperature, selected from the group consisting of SEQ ID NOs: 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140 and 201-275, and analogs and derivatives thereof.

57. A method of agonizing or antagonizing a cell surface or intracellular receptor, enzyme or biological process mediator in a subject in need thereof, the method comprising: administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1-11, 22-26 or 48-49.

58. The method of claim 57, wherein the receptor / enzyme / enzyme substrate / mediator is a GnRH receptor, a vasopressin receptor, an oxytocin receptor, an apelin receptor, a neuromedin receptor, a kisspeptin receptor, a bombesin receptor, an opioid receptor, a substance P receptor, an angiotensin receptor, a ghrelin receptor, a parathyroid hormone receptor, a PTHrP receptor, a GIP receptor, a GLP-1 receptor, a GLP-2 receptor, a glucagon receptor, a calcitonin receptor, an amylin receptor, a CGRP receptor, an adrenomedullin receptor, a CGRP receptor, a MSH receptor, a melanocortin receptor, a peptide Y receptor, a peptide YY receptor, a urocortin receptor, a bradykinin receptor, a GHRH receptor, an ACTH receptor, a VIP receptor, a thrombin receptor, a somatostatin receptor, a protease-activated receptor, a natriuretic peptide receptor, an insulin receptor, a relaxin receptor, a matrix protein, a cell target of metrikine matrix modifying peptides, a thymosin, a thymosin alpha 1, a thymosin beta 4, a kallikrein inhibitor, a thrombopoietin receptor binding domain (or a romiplostim analog), a matrikine peptide, a glatiramer (cuptagen), an antibiotic and an antibacterial drug (e.g., temporin A), a complement modulator (e.g., a compstatin), a cell penetrating peptide containing molecule for intracellular delivery of a compound (e.g., a TAT peptide), a microbial antigen, a viral antigen, a neoantigen, a tumor antigen, or a cytotoxic drug.

59. A method of eliciting a sustained immune response in a patient or animal comprising administering to the subject an effective dose of the pharmaceutical composition of any one of claims 1-11, 22-26, or 48-49.

60. A method of making a gel-forming compound for eliciting a sustained immune response in a patient or animal, wherein the method of making the gel-forming compound is coupling a gel formation enhancing motif to an antigen or a neoantigen; the gel formation enhancing motif is selected from the group consisting of the amino acid sequences in SEQ ID NOS: 1-15, 61, 64, 263, 274, 106-114, 116-124, 126-131, and 139-140, and analogs or derivatives thereof.

61. A method of delivering the gel-forming polypeptide of any one of claims 12-18 or the engineered gel-forming polypeptide of any one of claims 35-47, comprising coating a surface of an implantable device or a surface of a tissue with a gel formed by the polypeptide.

62. A method of encapsulating a therapeutic drug, an antigen, a nanostructure, an organelle, or a cell by the gel-forming polypeptide of any one of claims 12-18 or the engineered gel-forming polypeptide of any one of claims 35-47, comprising retaining the therapeutic drug, the antigen, the nanostructure, the organelle, or the cell within an enclosed space with a gel formed by the polypeptide.

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