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

[0001] Divisional Application

[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 To Related Applications

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

[0005] The present patent provides compositions and methods for sustained delivery of therapeutic drugs in the form of stable aqueous formulations containing gel-forming polypeptide compounds that can sustain the release of therapeutic drugs. BACKGROUND

[0006] In common therapeutic modalities, polypeptides are an intermediate form between “small molecule” drugs and large molecule biologic and nucleic acid-based therapeutics for injection. Therapeutic peptides and proteins are typically administered parenterally, such as by subcutaneous injection. One advantage of synthetic peptides is that these molecules can be incorporated with unnatural amino acids to enhance the stability of the peptide chain and reduce the proteolytic ability of the protein, while providing better production reproducibility.

[0007] When used as therapeutic agents, most peptides have disadvantages. For example, such drugs are highly susceptible to proteolysis and can be rapidly cleared by the kidneys, resulting in a short half-life, requiring multiple injections. Many therapies require continuous or repeated administration over a long period of time, causing discomfort and inconvenience to patients. Therefore, in order to administer polypeptide and protein-based therapeutic agents over a long period of time, sustained-release formulations or half-life extension techniques have been developed. The use of sustained-release formulations also enables the delivery of therapeutic agents to a specific tissue or organ, thereby minimizing systemic adverse reactions as much as possible.

[0008] Techniques using nanostructures that can slow down the release of drugs can improve the pharmacokinetic and pharmacodynamic characteristics of therapeutic agents after entering the human body or after local treatment. This technique can sustainably modulate the transduction of cell signals and reduce the peak-trough phenomenon of therapeutic drugs (e.g., small molecule drugs, peptides, hormones, proteins, nucleic acids, cells, or prodrugs). High biocompatibility of nanomedicine carriers can potentially bring substantial improvements to drug delivery, thereby improving 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, oil and solid particles can establish a barrier to limit the dispersion of soluble therapeutic agents.

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

[0010] High molecular weight peptides and hydrogels that are biocompatible are used in a wide variety of applications in the biotechnology and medical fields, particularly in the controlled delivery and release of pharmaceutical and therapeutic agents (see U.S. Patent Nos. 5,034,229, 5,057,318, and 5,110,596), in addition to their use as scaffolds in tissue engineering. Hydrogels can comprise a network of monomers that self-assemble into a nanostructure supported by hydrogen bonding, or a network of monomers that form by hydrophobic interactions and van der Waals forces. For example, high molecular weight microcapsules and matrices comprising polylactic acid have been used to deliver small molecule, peptide, and protein pharmaceuticals (e.g., Kent et al., U.S. Patent No. 4,675,189). Hydrogel-like polymers can comprise PLA (polylactic acid), PGA (polyglycolic acid), poly(lactide-co-glycolide) (PLGA), polyalkyl cyanoacrylate, poly-epsilon-caprolactone, poly N-isopropylacrylamide (NIPA), cellulose ether, hyaluronic acid, lecithin, polyacrylic acid, poly omega-caprolactone, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), and agarose, as well as copolymers obtained by combining or modifying these substances. The use of gel polymer delivery systems for the sustained delivery of pharmaceuticals has numerous advantages and is well known in the art.

[0011] Polymers such as PLGA are gradually destroyed after they enter the body. For example, these types of sustained release formulations have been used for the delivery of GnRH analogs, with dosing times of weeks or months. Such formulations can improve the accuracy of dosing and improve patient compliance with therapy. However, there are disadvantages to the use of hydrogel-like polymers. Many of the formulations contain a support material and a pharmaceutical composition, where the support material can be immunogenic. After administration, the support material can not degrade and thus will accumulate in the body. The manufacturing or production process for the formulation can be complex. Furthermore, the loading of the therapeutic agent into the carrier is typically only a small part of the process of preparing the therapeutic agent.

[0012] The most widely studied self-assembling peptide hydrogels are hydrogels synthesized from modified oligopeptides with repeating amino acid sequences. For example, the synthetic material can be a tetraphenyl ethylene end-capped dipeptide (e.g., tetraphenyl ethylene end-capped di-glycine (TPE-GG), Yeh et al.,

[2016] A novel nanostructured supramolecular hydrogel self-assembled from tetraphenyl ethylene end-capped dipeptides, Soft Matter 12:6347-51; fluorenylmethoxycarbonyl (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 leads to necrosis, Biomaterials Sci. 3:298-307; Frith et al.,

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

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

[2014] Formation of extracellular matrix in a self-assembling minimal bioactive hydrogel based on aromatic amphiphilic peptides, Tissue Eng Part A 5:2041731414531593; Eckes et al.,

[2014] No beta-sheet structure required: combinatorial experimental and computational studies on self-assembly and gelation of ester-containing analogs of Fmoc-dipeptide aqueous gelators, Langmuir. 30:5287-96; palmitoyl-V3A3E3 and palmitoyl-A3V3E3, Fu and Nguyen,

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

[0013] In addition, various hydrogel substitutes synthesized from complex molecules have also been reported. Self-assembling gels can be prepared from mixtures of the bifunctional peptide LHRH-MPGANLS with siRNA (Liu et al.

[2017] Targeted delivery of siRNA to cytoplasm of hepatocarcinoma cells by bifunctional carrier peptide. Drug Delivery and Translational Research 7: 147-155); mixtures of self-assembling peptides with heparin (Liu et al.

[2016] Cationic self-assembling peptide / heparin composite hydrogels slowly release hepatocyte growth factor to improve survival and function of beta cells by modulating inflammatory responses. International Journal of Nanomedicine 11: 4875-4890); mixtures of glycosylated nucleosides with lipids (Kaplan et al.

[2016] Self-assembling nanofibrous hydrogels for the mechanical responsive delivery of therapeutic anti-TNFa antibodies. 52: 5860-3); cysteine, dipeptide of cysteines and sterically hindered non-natural amino acid (penicillamine) incorporated into peptides by oxime ester-mediated chemical ligation (Rasale et al.

[2016] Control of peptide self-assembly by native chemical ligation / desulfurization strategies. Chemistry-An Asian Journal. 11: 926-35); fusion proteins composed of the immunostimulatory Mycobacterium tuberculosis heat shock protein 70 (MtbHSP70) and avidin (protein that binds biotin) (Leblanc

[2014] VaxCelerate II: Rapid development of a self-assembling vaccine for Lassa fever. Human Vaccines and Immunotherapeutics 10: 3022-38). However, the use of these artificially synthesized sequences for drug delivery can elicit immunogenic responses and cause cytotoxic effects, limiting the use of these nanostructures in the medical field. In addition, most of these molecules require complex production processes.

[0014] Studies on self-assembling hydrogels have shown that the three-dimensional nanofibrillar network of the gel nanostructure is assumed to have diversity. For example, the palmitoyl-V3A3E3 gel contains a continuous structure mainly with alkyl tail chains, while the palmitoyl-A3V3E3 gel is separated from the interconnected micelles. Likewise, studies on a group of hydrogels containing repetitive sequences named MAX1 analogs (e.g. VKVKVKVKV(D)PPTKVKVKVKV-NH2) have shown that, although all analogs can form a network of entangled fibrils of similar diameter, the rate of self-assembly and hydrogelation of the analogs and the mechanical toughness are significantly altered (Chen et al.

[2014] Modulation of gelation kinetics and mechanical rigidity of beta-hairpin peptide hydrogels by hydrophobic amino acid substitution. 6: 14360-8). These data indicate that the internal structural arrangement of the peptide gels and the correlation with the structural stability and mechanical behavior of the hydrogel nanostructure remain to be fully determined.

[0015] Gels formed using polypeptides would provide significant advantages for formulations, however, the necessary conditions for rationally designing a therapeutic peptide that self-assembles into a gel are not well understood. It is still extremely difficult to predict whether a peptide will self-assemble into a gel in an aqueous solution, and to design a peptide that will form a polymer or gel on its own at a critical aggregation concentration. Furthermore, the behavior of a peptide can vary in solutions with different pH, ionic strength, temperature, or polymer excipients. There is a need to improve the performance of sustained release agents and formulations and to find therapeutic peptides that can form gel polymers on their own in order to improve the delivery of a variety of therapeutic agents in the clinic. SUMMARY

[0016] The present invention relates to compositions and methods of use of gel-forming polypeptide therapeutic agents. Formulations of these gel-forming polypeptides have advantages because the polypeptides can be maintained at effective levels in the body for longer periods of time (i.e., extended residence time) compared to conventional formulations, thereby increasing the time between doses and reducing peak-trough phenomena and total dose of the active ingredient. Methods of formulating and manufacturing such gel-forming peptides are provided in some embodiments. In certain embodiments, peptides that are capable of forming gels on their own are identified, and these peptides can be formulated according to the methods described herein to administer the gel-forming peptides in an optimal delivery format and to achieve a therapeutic purpose. In other embodiments, peptides that are not capable of forming gels on their own or that form only small amounts of gel are modified by attaching a structural motif that enhances gel formation, to obtain or enhance the ability to form a gel, and can be formulated according to the methods described herein to be useful in therapy.

[0017] Compositions and therapeutic formulations are provided in some embodiments that deliver an effective dose of a therapeutic agent in a gel configuration upon administration. For example, a therapeutic formulation in the form of a solution or a liquid gel can release a therapeutic peptide, and the formulation can transform into a gel upon administration. The resulting gel nanostructure can act as a physical barrier to reduce the rate of diffusion or dispersion of the therapeutic agent, reduce proteolysis, and reduce renal clearance of the therapeutic agent, thereby extending the residence time of the agent in the body. In some embodiments, the therapeutic agent is a gel-forming polypeptide that can be optionally modified to enhance the ability to form a gel and to reduce the minimum concentration of the gel-forming polypeptide required to form a gel configuration in the body. In some embodiments, the therapeutic agent is provided in combination with a gel-forming polypeptide. 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 formulation is in the form of an aqueous solution with very low ionic strength. In other embodiments, the formulation is provided in two containers, and the dry peptide is mixed with an aqueous excipient, which can have very low ionic strength, just prior to administration.

[0018] In some embodiments, the gel-forming polypeptides include agonists, antagonists, or non-functional analogs of polypeptide hormones and their cell surface receptor ligands, such as GPCRs, tyrosine kinase receptors, and the like, as well as salts of therapeutic drugs that act on cell surface targets or enzymes and their biologically active or inactive analogs. The soluble gel-forming polypeptides also include peptides that form gel carriers and prodrugs that exist in gel nanostructures. The gel-forming polypeptides comprise a portion of the sequence of a natural polypeptide hormone or are analogs that contain the sequence of a natural hormone and can be used as carrier polymers. Upon formation of a gel, these peptides can immediately control the delivery of a therapeutic drug or an intragel substance at a rate suitable for therapy while avoiding an immunogenic response.

[0019] In some embodiments, the gel-forming polypeptides or polypeptide compositions can form self-assembled gels in aqueous solutions. The gel-forming polypeptides can have physiologically relevant biological or therapeutic activities or can simply serve a structural role. The formulations can include other biologically active molecules. In such formulations, the gel-forming polypeptides in the gel can release the optional other biologically active agents into the physiological environment over an extended period of time. The gel nanostructures can also serve as a physical barrier to reduce the solubility of the monomeric peptides and the enzymatic degradation of the gel-forming polypeptides.

[0020] In some embodiments, the gel-forming therapeutic drugs include, but are not limited to, peptide drugs that are characterized by or modified to be characterized by one or more gel structure-enhancing motifs that promote the formation of intermolecular chemical bonds. The enhancing motifs can be sequences that contain self-assembly enhancing motifs that promote the self-assembly of the polypeptides in solution. The gel-forming motifs can induce the self-assembly of the polypeptides into hydrogels. Optionally, the gel-forming enhancing motifs can be derived from circulating hormones secreted by the human body. The gel-forming enhancing motifs can be fused or otherwise coupled to therapeutic drugs that are cell surface receptors, biological function or enzyme modulators, or therapeutic drugs that are antigens. The coupling can be covalent or non-covalent. In some embodiments, the gel-forming polypeptides are modified to contain a signaling motif that can activate or antagonize a signaling pathway mediated by a target receptor or can activate or antagonize a biological process or enzyme reaction. In some embodiments, the therapeutic drugs are small molecules, peptidomimetics, biologies, nucleic acids, antigens, organelles, or cells.

[0021] In some embodiments, the gel enhancing motif comprises a fragment of a circulating peptide ligand secreted by a cell surface receptor (e.g., a GPCR), including but not limited to the peptides identified herein as capable of forming a gel naturally. In some embodiments, the gel enhancing motif is at least about 2 amino acids in length and no more than about 52 amino acids in length, up to 5 amino acids, 7 amino acids, 9 amino acids, 12 amino acids, 15 amino acids, 18 amino acids, 21 amino acids, 24 amino acids, 52 amino acids in length. 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 2-52 amino acids in length having at least 50%, 75%, 90%, or 100% sequence identity to Pal-KVQKLSAPVDPSSPHSY. In some embodiments, the gel enhancing motif comprises or consists of a fragment of 6 amino acids in length having at least 50%, 75%, 90%, or 100% sequence identity to Pal-SSPHSY. In some embodiments, the gel enhancing motif comprises or consists of a fragment of 3 amino acids in length, Pal-HSY or Pal-KSY, provided that the peptide naturally contains a Y or SY residue at the amino terminus can be modified by the addition of only the amino acids required to form the HSY sequence. In some embodiments, the gel enhancing motif comprises or consists of a fragment of 2 amino acids in length, Pal-HS, provided that the peptide naturally contains an H, Y or S residue at the amino terminus can be modified by the addition of only the amino acids required to form the Pal-HS sequence. Alternatively, one or more residues in the amino acid sequence can be substituted in order to generate the HSY or HS motif. Alternatively, a KSY motif can be used. In certain embodiments, a palmitate residue is coupled to the gel enhancing motif. In certain embodiments, the motif is attached to a peptide with a mini-PEG at the carboxy terminus or amino terminus, or to the side chain of an amino acid. In some embodiments, the peptide comprises a detectable label, such as FITC.

[0022] In some embodiments, the therapeutic agent and the gel enhancing motif are covalently linked to the self-assembling gel forming molecule. In some embodiments, the therapeutic agent is encapsulated in a self-assembling gel forming polypeptide selected from the sequences in SEQ ID NOS: 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140, and 201-275. In some embodiments, the gel forming therapeutic agent or combination of gel forming peptide and therapeutic agent is administered to a subject, and the gel forming polypeptide / therapeutic agent acts at the site of administration and stays for an extended period of time.

[0023] In some embodiments, the gel-forming polypeptide is a prodrug that is only therapeutically active after being separated from the gel nanostructure and the carrier encapsulating the therapeutic drug.

[0024] Gel-forming polypeptides include, but are not limited to, the naturally occurring and engineered gel-forming polypeptides listed in Tables 1, 2, and 3 herein. Such polypeptides include CGRP, ADM, ADM2, pramlintide, oxytocin, kisspeptin, plamotropin, thrombopoietin peptide analogs, romiplostim analogs, urocortin 3, substance P, GLP-1, GnRH analogs and GLP-2 receptor ligand analogs, bombesin receptor antagonists, gamma-MSH, and opioid receptor gel-forming ligands, and analogs of the foregoing; other polypeptides and analogs thereof that activate the kempferid + thymosin alpha 1 -mediated pathway, the thymosin beta 4-mediated pathway, and the like. Gel-forming polypeptides also include polypeptides containing a self-assembly enhancing motif coupled to a functional sequence, including but not limited to ADM, ADM2, GnRH, GnRH antagonists, vasopressin, oxytocin, apelin, neuromedin, kisspeptin, bombesin, neocortin, enkephalin, substance P, sarcosine antihypertensin, calcitonin, pramlintide (amylin analog), exenatide 4, GLP-1, tildraptide (GLP-2 analog), afanopeptin (melanotan I), melanotan II, gamma-MSH, ACTH1-24, setmelanotide, PYYYY3-36, urocortin 2, urocortin 3, parathyroid hormone, HOE140 (BKR2 antagonist), bradykinin receptor 1 antagonist (BKR1 antagonist), plamotropin, semorelin, atrial natriuretic peptide (ANP), thymosin alpha 1, thymosin beta 4, vasoactive intestinal peptide (VIP), TAT cell-penetrating enhancing peptide, kallikrein inhibitor, antibacterial peptide (e.g., Temporin A), 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, insulin, relaxin, PTHrP, bobmesin receptor antagonist, and analogs of the foregoing.

[0025] In one embodiment, a method of treating a patient with a gel-forming formulation is provided, characterized in that the formulation, when administered in a gel configuration, delivers an effective dose and concentration of a therapeutic drug. Prior to administration, the therapeutic formulation can exist in the form of a liquid solution or a liquid gel. For example, the route of administration of the therapeutic formulation can include intramuscular, subcutaneous, intradermal, or intraperitoneal injection, infusion, or intranasal, intrauterine, intraocular, topical, oral, or intrarectal, wherein the composition forms a gel upon interaction with the patient's bodily fluids. BRIEF DESCRIPTION OF DRAWINGS

[0026] A full and enabling disclosure of the present application is set forth in the specification taken in conjunction with the accompanying drawings, in which:

[0027] Figures 1A-1C Figure 1 shows a graph of peptide gel formation by a peptide of SEQ ID NO: 1.

[0028] Figure 2 Figure 4 shows sustained color change in bullfrogs treated with MSH gel (peptide of SEQ ID NO: 224) compared to animals treated with wild-type analog [SEQ ID NO: 29] (100 nmoles / kg body weight). DETAILED DESCRIPTION

[0029] The present application provides compositions and methods for formulating and using gel-forming polypeptides. Therapeutic drugs can be formulated, manufactured, and produced using low-cost processes when administered in the form of a gel. The formulations have low immunogenicity because the gel-forming polypeptides are derived from simple sequences of human-secreted peptide hormones. The formulations allow the therapeutic drugs to retain similar distribution volumes as their wild-type or native analogs. The compositions of the present application can be prepared by simply mixing the active ingredient with an aqueous solution, and thus, the compositions can significantly reduce the volume, cost, and production time of candidate therapeutic drugs compared to known sustained-release formulations.

[0030] Without being limited by theory, it has been discovered that certain secreted polypeptide hormones can self-assemble into gel nanostructures without the need for adding artificial synthetic polymers or other carrier matrices to control the release characteristics of the peptides. These peptides can contain gel formation-enhancing motifs and can automatically form gels upon interaction with aqueous solutions. Compared to known sustained-release formulations containing artificial synthetic polymers, peptide-based formulations with this ability can significantly enhance the release capabilities of therapeutic drugs while reducing cost and production time. Furthermore, the use of these gel-forming polypeptides eliminates the need for organic solvents when formulating sustained-release formulations.

[0031] GPCRs and other surface receptors have ubiquitous physiological roles and are major targets for drugs. Researchers have designed novel agonists and inhibitors for a variety of GPCRs and other cell surface receptors to achieve receptor signaling in patients. However, a major drawback of many polypeptide and non-peptide therapeutics is short half-life and short residence time in the body. Short half-life is often associated with proteolytic sensitivity and / or renal clearance. The use of self-assembling peptide-based gel formulations is an important technology to extend the half-life and residence time of drugs in the body. Unlike artificially synthesized polymeric molecules such as PEG, PLA, PGA, PLGA, or collagen used to encapsulate therapeutics, certain therapeutic polypeptides such as lantreotide can self-assemble into gel nanostructures in aqueous solution. Upon injection of the drug into the patient, the lantreotide gel in the gel depot gradually releases lantreotide monomers, which slowly release lantreotide in the systemic circulation. This can extend the time of somatostatin receptor modulation in the patient.

[0032] For peptides identified to form gels on their own, formulations of these molecules can extend the residence time of these molecules in the body. Peptides that do not naturally form gels can be engineered by coupling to a gel-enhancing sequence motif. The gel-enhancing motif can be coupled to any part of the therapeutic to promote the ability to form a gel, but the quality of the final molecule is not significantly changed. The gel-enhancing motif can promote the ability of small molecule drugs, peptides, proteins, or other biological molecules to form gels and extend the residence time of the molecules in the body.

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

[0034] Definitions

[0035] In practicing the present application, those of skill in the art can employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, peptide chemistry, and immunology. Such techniques are explained fully in the literature, such as, e.g., "Molecular Cloning: A Laboratory Manual" Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. MJ. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and CC. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991); and updated or revised versions thereof.

[0036] While specific peptides are exemplified herein, it will be apparent to those of skill in the art that many alternative peptides and methods, any one of which is equally applicable to the practice of the present application, will be apparent in light of the present application. The methods of the present application, and the assays to determine their efficacy in particular patients or applications, can be practiced according to the guidance herein using standard procedures of the art. Thus, in practicing the present application, those of skill in the art can employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology. Such techniques are explained fully in the literature, such as, e.g., "Molecular Cloning: A Laboratory Manual" Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (ed. MJ. Gait, 1984); "Animal Cell Culture" (ed. R.I. Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology" (ed. D.M. Weir and CC. Blackwell); "Gene Transfer Vectors for Mammalian Cells" (ed. J.M. Miller and M.P. Calos, 1987); "Current Protocols in Molecular Biology" (ed. F.M. Ausubel et al., 1987); "PCR: The Polymerase Chain Reaction" (ed. Mullis et al., 1994); "Current Protocols in Immunology" (ed. J.E. Coligan et al., 1991); and updated or revised versions thereof.

[0037] The terms "treatment," "treating," and the like, as used herein refer to obtaining a desired pharmacologic and / or physiologic effect. A prophylactic effect refers to completely or partially preventing a disease or symptom thereof and a therapeutic effect refers to either inhibiting the disease and / or adverse effect caused by the disease from developing or making the disease and / or adverse effect caused by the disease less severe. "Treatment" as used herein covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or symptom from occurring in an individual which can be predisposed to the disease or symptom but has not yet been diagnosed as having it (e.g., a disease that can be associated with or caused by the primary disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing the disease to regress.

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

[0039] The term "gel" refers to a three-dimensional network of a liquid, semi-solid, or solid that has a volume in the range of a liquid medium. A hydrogel is a network formed from hydrophilic polymer chains, sometimes in the form of a colloidal gel, with water as the dispersion medium. Hydrogels have a high water content that can exceed 50%, 60%, 70%, 80%, or can be about 85% to 99.9%. Hydrogels are characterized by a significant plateau in the elastic modulus that extends for at least a few seconds, and a viscous modulus that is significantly less than the elastic modulus in the plateau region. The elastic modulus of a hydrogel is typically in the range of 10-10 2 kPa. Because hydrogels are prone to fracture and collapse under their own weight, tensile and bending tests are not suitable for evaluating the elastic modulus. Methods known in the art can be used to determine the characteristics of the gels described herein. For example, see Kocen et al.,

[2017] Biomaterials 12(2), Viscoelastic properties of hydrogel composites for tissue engineering under mechanical loading; Jonker et al.,

[2012] Chem. Mater. 24(5), pp 759-773, Peptide and protein-based hydrogels.

[0040] "Transparent aqueous solution" refers to a solution consisting of a flowing aqueous solution in which preferably 95% ± 5% of the polypeptide solute is completely dissolved, presenting a relatively transparent solution appearance, and is free to move upon tilting of the container or for a very short time. Depending on the purity of the solute used, a trace of solute or particles can be visible in the transparent solution. However, these particles are not sufficient to produce a milky or turbid appearance. On the other hand, the gel-forming polypeptide presents a liquid gel or semi-solid gel appearance. To assess this, the most critical criterion is to observe whether the viscosity of the water is increased. The appearance of a liquid gel is manifested by a uniform increase in viscosity, and a liquid gel with a similar appearance is honey or glycerol. The appearance of a liquid gel can be transparent or opaque. A liquid gel is not suitable as a suspension, which is a heterogeneous mixture consisting of insoluble particles. A liquid gel can be the continuous phase, while a gel is a homogeneous single-phase solution with high viscosity, but actually presents a semi-solid appearance.

[0041] A semi-solid gel is a formulation consisting of a solvent and one or more solutes, in which the solutes are completely or partially dissolved, such that the formulation presents a transparent or homogeneous opaque appearance, and due to the high viscosity, the solution is difficult to move for a very short time or upon tilting of the container. A semi-solid gel composition is not suitable as a suspension, which is a heterogeneous mixture consisting of a large number of visible insoluble particles, while a solution is not homogeneous.

[0042] An insoluble precipitate generated in a suspension or solution is an aqueous formulation consisting of one or more solutes, in which the solutes are not dissolved or partially dissolved, such that the formulation cannot present a liquid homogeneous composition like a transparent or opaque solution, or a more viscous homogeneous composition like a gel.

[0043] The terms "semi-solid suspension" and "semi-solid therapeutic composition" are used interchangeably herein to refer to a viscous paste suspension of a therapeutic polypeptide in a liquid solvent. To maintain the homogeneity of the semi-solid, the semi-solid suspension according to the present application comprises soluble semi-solid particles of a gel-forming polypeptide, and up to 50% by weight of the pharmaceutically acceptable formulation. The gel-forming polypeptide can be used by the composition according to the present application, and upon administration to a patient, the salt of the polypeptide should gelate in the body fluid. Upon gel formation, the peptide is immediately delivered at a rate suitable for drug therapy.

[0044] A self-assembling gel-forming polypeptide is a polypeptide that forms a gel upon contact with an aqueous solution, including physiological fluids such as blood. The process of gel formation by self-assembling peptides is defined as the process by which peptides spontaneously assemble in solution through non-covalent interactions (Whitesides et al.; Science 1991, 254, 1312-1319). These gels create a nanostructure that can modify the rheological properties.

[0045] Peptide gels presented to date include two major classes: one class is mainly composed of alternating hydrophilic and hydrophobic amino acid sequences (amphiphilic peptides), and the other class is peptides with hydrophobic groups that form nanofibers (peptide amphiphiles) (Mata et al.

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

[2010] Proc Natl Acad Sci U S A 107, 3293; Huang et al.

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

[2011] Proc Natl Acad Sci U S A 108, 13438; Capito et al.

[2008] Science 319, 1812-1816). For example, peptide amphiphiles with alkyl tail chains at the ends can be derived from the peptide motifs in collagen. Typically, peptide amphiphiles form by hydrogen bonding between the beta- sheet structures of the amino acids and interactions between the hydrophobic tail chains. Another way is that peptides composed of alternating hydrophilic and hydrophobic amino acid sequences can self-assemble into gels with parallel or antiparallel beta-sheet structures. Thus, peptide gels can include extended cross-beta structures that form tubes and fibers, as well as alpha-helical structures that form triple-helical collagen-like structures, coiled-coil structures, or coiled-coil bundles.

[0046] Self-assembly of gels is typically achieved by a combination of hydrogen bonding and van der Waals interactions between monomeric molecules (Reches and Gazit; Current Nanoscience 2006, 2, 105-111). Upon entry into solution, the potential of self-assembling peptide monomers to form a gel 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] Nat Mater 9, 594-601). For polypeptides containing charged residues, a higher salt concentration can decrease 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 entrapped in polypeptide hydrogels, conjugated to anionic polymers or structures (e.g., heparin) via covalent bonds or electrostatic interactions. Disadvantages of these systems and related systems include lack of specificity of the growth factor after conjugation or the need to degrade the covalent bond to achieve the desired effect. Furthermore, because these peptides have artificial sequences with repeating motifs (e.g., gel-forming peptides with FKFEFKFE motifs and RADARADA motifs) or are derived from large proteins (e.g., fibronectin and laminin) or non-circulating proteins (e.g., amyloid), these peptides can elicit adverse immunogenic reactions after administration. As described herein, peptide gels made from native or modified secreted circulating polypeptide sequences are less likely to elicit immunogenic reactions and are safer. Because most polymeric carriers typically comprise 75-99% of the mass of the sustained release formulation, use of gel-forming therapeutic agents can increase the effective payload of therapeutic drugs by 20-50-fold 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's body such that the patient receives a dose of the therapeutic substance over an extended period of time. The proportion of the therapeutic drug in the composition that is released within a time window will be determined by the desired release rate.

[0049] Aqueous excipients. In some embodiments of the present application, the formulation of the gel-forming peptide delivers the drug using an aqueous excipient, where the excipient can have very low ionic concentrations. The total ionic concentration of such excipients can 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 than about 0.01 mM, less than about 0.001 mM, less than about 0.0001 mM, less than about 0.00001 mM. By way of example, salts present in the excipient can include Na+, K+, Cl", Mn++, Mg++, Ca++, PO4", and the like.

[0050] The term "carrier peptide" refers to a gel-forming peptide that forms a gel in aqueous solution or upon contact with a body fluid and can be used to encapsulate a therapeutic drug or to associate with a therapeutic drug to achieve sustained delivery.

[0051] The term "prodrug peptide" refers to a gel-forming peptide that is not capable of acting on a biological target in the gel nanostructure and only becomes active upon dissociation from the gel nanostructure.

[0052] A gel-enhancing motif is an amino acid sequence that enhances the ability of a small molecule, peptide, biological agent, antigen, nucleotide, or molecular therapeutic to form a gel nanostructure in an aqueous solution. The gel-enhancing motif can comprise (or consist of) a fragment of a circulating peptide ligand secreted by a cell surface receptor. In some embodiments, the gel-enhancing motif is at least about 2 amino acids in length and no more than about 52 amino acids in length, up to 5 amino acids, 7 amino acids, 9 amino acids, 12 amino acids, 15 amino acids, 18 amino acids, 21 amino acids, 24 amino acids, 52 amino acids in length. 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 2-52 amino acids in length having at least 50%, 75%, 90%, or 100% sequence identity to Pal-KVQKLSAPVDPSSPHSY. In some embodiments, the gel-enhancing motif comprises (or consists of) a fragment of 6 amino acids in length having at least 50%, 75%, 90%, or 100% sequence identity to Pal-SSPHSY. In some embodiments, the gel-enhancing motif comprises (or consists of) a fragment of 3 amino acids in length, Pal-HSY or Pal-KSY, with the proviso that a peptide naturally containing a Y or SY residue at the amino terminus can be modified by the addition of only the amino acids necessary to form the HSY sequence. In some embodiments, the gel-enhancing motif comprises (or consists of) a fragment of 2 amino acids in length, Pal-HS, with the proviso that a peptide naturally containing an H, Y, or S residue at the amino terminus can be modified by the addition of only the amino acids necessary to form the Pal-HS sequence. Alternatively, one or more residues in the amino acid sequence can be substituted in order to generate the HSY or HS motif. Alternatively, a KSY motif can be used. In certain embodiments, a palmitate residue is coupled to the gel-enhancing motif. In certain embodiments, the motif is attached to a mini-PEG peptide at the carboxy terminus or amino terminus, or to the side chain of an amino acid. In some embodiments, the peptide comprises a detectable label, such as FITC.

[0053] In addition to the gel-enhancing amino acid sequence, other substances can be coupled to the therapeutic polypeptide to enhance the ability of the gel to form, including but not limited to fatty acids, polyethylene glycol, and the like. The addition of a fatty acid can enhance the ability of the gel to form and facilitate subsequent release of the peptide from the nanostructure, as the chemical linkage is susceptible to cleavage by esterase-mediated fatty acid cleavage.

[0054] As used herein, the term "PEGylation" refers to the chemical modification of a polypeptide with one or more polyethylene glycol groups. The polypeptide can be directly coupled to the PEG via an amino, thiol, hydroxyl, or carboxyl group (i.e., no linker). In some embodiments, only one amino acid in the PEGylated polypeptide contains a PEG group. In other embodiments, two or more amino acids in the PEGylated polypeptide contain a PEG group.

[0055] In some embodiments, the PEG is attached to the polypeptide via a linker. A linker refers to any biocompatible linker, where "biocompatible" means that the compound or group is not toxic and can be used in vitro or in vivo without causing injury, discomfort, illness, or death. The PEG can be bonded to the linker via an ether linkage, an ester linkage, a thioester linkage, or an amide linkage. Suitable biocompatible linkers include, but are not limited to, ester groups, amide groups, imide groups, carbamate groups, carboxyl groups, hydroxyl groups, carbohydrates, succinimidyl groups (which can include succinimidyl succinate (Ss), succinimidyl propionate (SPA), succinimidyl butyrate (SBA), succinimidyl carboxymethyl ester (SCM), succinimidyl succinamide (SSA), or N-hydroxysuccinimide (NHS)), epoxy groups, oxycarbonylimidazole groups (including carbonyldiimidazole (CDI)), nitrophenyl groups (which can include nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), trysylate groups, aldehyde groups, isocyanate groups, vinyl sulfone groups, tyrosine groups, cysteine groups, histidine groups, or primary amines. If a properly folded, intact polypeptide protein is reacted with a PEG coupling reagent, the PEG groups will preferentially react with surface residues, rather than buried residues, providing a practical, low cost method for protein PEGylation and synthesis of PEGylated polypeptides in the present application.

[0056] Methods for attaching PEG to polypeptides are known in the art, and any known method can be used to generate the PEGylated polypeptides described herein according to the methods of the present application. For example, see Park et al., Anti-Cancer Res. 1 :373-376 (1981); Zaplipsky and Lee, in Polyethylene Glycol Chemistry: Biotechnical 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, the PEG is a monomethoxy PEG molecule that reacts with a primary amine group on the polypeptide. Methods for modifying polypeptides with monomethoxy PEG are known in the art and are referred to as reductive alkylation. See, e.g., Chamow et al. (1994) Bioconjugate Chem. 5:133-140.

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

[0059] In many embodiments, the PEG has at least one hydroxyl group, such as a terminal hydroxyl group, that is modified to produce a functional group that reacts with an amino group, such as the epsilon amino group of a lysine residue, the free amino group at the N-terminus of a polypeptide, or any other amino group, such as an asparagine, glutamine, arginine, or histidine, thereby facilitating covalent modification of the polypeptide with the PEG.

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

[0061] In other embodiments, the PEG is derivatized to contain a terminal thiocarboxylic acid group (-COSH) that selectively reacts with an amino group to produce an amide derivative. Because of the reactive nature of the thioacid, certain amino groups are selective over others. For example, -SH exhibits sufficient leaving ability to react with the N-terminal amino group under appropriate pH conditions, such that the epsilon-amino group of a lysine residue is protonated and remains non-nucleophilic. On the other hand, certain accessible lysine residues can be selectively reacted with under appropriate pH conditions.

[0062] In other embodiments, the PEG contains a reactive ester, such as a N-hydroxysuccinimidyl ester at the end of the PEG chain. Such N-hydroxysuccinimidyl ester-containing PEG molecules react with selected amino groups under specific pH conditions, such as neutral conditions at a pH of 6.5 to 7.5. For example, the N-terminal amino group can be selectively modified under neutral pH conditions. However, accessible -NH2 groups in lysines can also react if the reactivity of the reagent is sufficiently high.

[0063] In some embodiments, the PEG conjugated with the peptide has a linear structure. In other embodiments, the PEG conjugated with the peptide has a branched 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 commonly used PEGs 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 that stated molecular weight. For example, suitable PEGs for coupling with peptides have molecular weights of about 0.2 kDa to 5 kDa, about 5 kDa to 10 kDa, about 10 kDa to 15 kDa, about 15 kDa to 20 kDa, about 20 kDa to 25 kDa, about 25 kDa to 30 kDa, about 30 kDa to 40 kDa, about 40 kDa to 50 kDa, about 50 kDa to 60 kDa, about 60 kDa to 70 kDa, about 70 kDa to 80 kDa, about 80 kDa to 90 kDa, or about 90 kDa to 100 kDa.

[0065] Peptides can be coupled with fatty acids, including but not limited to coupling at the amino terminus, such as C3-C fatty acids with straight or branched chains. 100 Alkyl groups; preferably C4-C substituted with halogens, hydroxyl groups, alkoxy groups, amino groups, alkylamino groups, alkylamino groups, dialkylamino groups, sulfates, or phosphates. 30 Alkyl groups, and the fatty acids can be saturated, monounsaturated, or diunsaturated fatty acids, for example, in ratios of 18:0, 24:0, and 24:1. The target fatty acids include, but are not limited to, palmitic acid, stearic acid, arachidic acid, lauric acid, myristic acid, myristoleic acid, palmitoleic acid, hexadecenoic acid, oleic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid.

[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 that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of a wild-type (i.e., native) peptide analog. For example, the gel-forming polypeptide can comprise SEQ ID NOS: 1-15, 48-58, 61, 64, 106-114, 116-124, 126-131, 139-140, 201-275, and derivatives thereof.

[0067] As used herein, the term gel-forming polypeptide ligand can refer to any polypeptide analog that exhibits gel-forming ability at a concentration equal to or lower than the concentration required for the wild-type analog to form a gel in the same aqueous solution, or a wild-type and modified polypeptide determined to form a gel at 6, 11, 20, or 30% w / w. However, in one embodiment, the gel-forming polypeptide of the present application can be shorter or longer than the polypeptides of SEQ ID NOS: 1-15, 48-58, 61, 64, 101-140, and 201-275, for example, polypeptides that are truncated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more residues at the amino and / or carboxy terminus, substituted with non-natural amino acids, or fused to other sequences.

[0068] Compositions

[0069] Peptide gels are an intelligent delivery system comprising biodegradable and highly biocompatible polypeptides, and are approved by regulatory agencies for the delivery of drugs. Peptide gels have clear advantages over other colloidal carriers such as nanoemulsions, polymeric nanoparticles, liposomes, solid lipid nanoparticles, etc., the most important of which is the strong drug delivery capacity (i.e., enhanced drug loading capacity and increased drug bioavailability) and lack of immunogenicity. In addition, the formulation can reduce production and prescription-related problems. However, the mechanism of self-assembly of polypeptides into gel nanostructures is not yet clear, and small changes in amino acid composition and other modifiers can change the tendency of polypeptides to self-assemble into gel nanostructures.

[0070] The present patent describes gel-forming polypeptides and therapeutic compositions comprising at least one polypeptide with or without a gel-enhancing motif, wherein the enhancing motif is a secreted polypeptide hormone component that is capable of enhancing gel-forming ability. The compositions can further include a functional therapeutic component, or the therapeutic component can be provided by the gel-forming peptide. Included as formulations are stable aqueous solutions, gels and liquid gels, characterized by a content of gel-forming polypeptide compound of 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 formulations can further include an aqueous vehicle with very low ionic concentration. Also provided are methods of making stable aqueous formulations of gel-forming polypeptides, characterized by dissolving 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 a gel-forming polypeptide in an aqueous vehicle with very low ionic concentration. The formulations can be used in a subject having a condition that can be ameliorated by a therapeutic polypeptide gel, characterized by administering to the subject an effective amount of the stable aqueous formulation.

[0071] The compositions and methods described in the present patent provide new and improved self-assembling gel-forming polypeptides, characterized by gel-enhancing motifs, improved gel-forming polypeptide carriers, improved use of therapeutic drugs by forming gel nanostructures, other self-assembling nanostructures and methods of making and using the same. Gel-forming therapeutic formulations utilize non-covalent electrostatic interactions to control the solubility and nanostructure of therapeutic molecules or carrier polypeptides, which can or can not include a gel-forming enhancing motif.

[0072] The physicochemical properties (e.g., swelling behavior), mechanical properties (e.g., compressive modulus), degradation rate, and active agent release kinetics of the hydrogels of interest can be adjusted by varying the amount of the current polypeptide. For example, the percentage can vary from about 0.01% to 50% by weight, such as from about 0.02% to 45%, 0.03% to 40%, 0.04% to 35%, 0.05% to 30%.

[0073] As mentioned 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., 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 of the hydrogel. The swelling ratio may vary depending on the hydrogel structure. The "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 a hydrogel can vary depending on its composition. Compressive modulus refers to the ability of the target hydrogel to withstand axial thrust; it 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 can also vary depending on 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 embodiments, the structure of the target hydrogel is designed to degrade under physiological conditions (e.g., in vivo) over a predetermined time period, 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 a substantially zero-order degradation rate, a substantially first-order degradation rate, and including a substantially second-order degradation rate.

[0077] In one embodiment, the self-assembling gel-forming polypeptide contains one or more acyl groups that 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 selected from the group consisting of LHRH receptor, vasopressin receptor, oxytocin receptor, apelin receptor, neurotensin receptor, oxytocin receptor, bombesin receptor, delta opioid receptor, mu opioid receptor, kappa opioid receptor, substance P receptor, angiotensin II receptor, calcitonin receptor, amylin receptor, GLP-1 receptor, GLP-2 receptor, glucagon receptor, calcitonin gene-related peptide (CGRP) receptor, adrenomedullin 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, orexin receptor, glucagon receptor, PTHrP receptor, insulin receptor, relaxin receptor, natriuretic peptide receptor, 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 adrenomedullin, adrenomedullin 2, and CGRP. In some embodiments, the gel-enhancing motif is a sequence derived from human adrenomedullin and / or adrenomedullin 2.

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

[0081] Ea-(Fa)n-Ga (II)

[0082] wherein Ea is a polypeptide motif derived from a cell surface receptor ligand or a therapeutic drug that enhances gel formation; Fa is a PEG group or a linking sequence; n is an integer from 0 to 40; and Ga is a therapeutic drug or a polypeptide motif derived from a cell surface receptor ligand that enhances gel formation.

[0083] In some embodiments, the gelation-enhancing motif is coupled to a therapeutic drug 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 drugs, anti-malignant cell proliferation drugs, anti-mitotic drugs, angiogenic drugs, anti-angiogenic drugs, anti-psychotic drugs, central nervous system (CNS) drugs, anti-coagulants, and fibrinolytic drugs; including LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, apelin analogs, neurotensin analogs, kinetensin analogs, kinetensin 234 analogs, bombesin analogs, bombesin receptor antagonists, bradykinin analogs, bradykinin receptor antagonist 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 peptide (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, insulin analogs, relaxin analogs, atrial natriuretic peptide (ANP) analogs, brain natriuretic peptide (BNP) analogs, C-type natriuretic peptide (CNP) analogs, glatiramer (copaxone), thymosin alpha 1 analogs, thymosin beta 4 analogs, cell-penetrating peptides, TAT peptides, kallikrein inhibitors, compstatin, temporin YIGSR antimicrobial peptides, RGD peptides, VGVAPG peptides, EEMQRR peptides, and YRSRKYSSWY peptides; coagulation factors, cytotoxic therapeutic agents, microbial antigens, viral antigens, tumor antigens, neoantigens, and pharmaceutically and cosmetically acceptable salts of these compounds.

[0084] In some embodiments, the target of the therapeutic drug in the present invention is a cell surface receptor (e.g., an opioid receptor and a romidepsin receptor) or an enzyme (e.g., when using a kallikrein inhibitor or a compstatin). In some embodiments, the therapeutic target is a biological function mediator (e.g., the target of glatiramer peptide is less clear, and it is believed to act by modifying immune processes that are currently thought to be the pathogenesis of multiple sclerosis; for thymosin alpha 1 and thymosin beta 4, it is believed to enhance cellular immunity in humans and experimental animals). In some embodiments, the therapeutic target is a cell membrane barrier (e.g., for temporin A antibacterial peptide derivatives, it is a cell penetrating peptide, such as the TAT peptide cell penetrating sequence). In some embodiments, the therapeutic target is a skin matrix component and matrix enzyme (e.g., matrix-modifying proteins 1, 4, 7, 8, and acetyl hexapeptide-3 matrix-modifying peptide).

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

[0086] The CLR / RAMP receptor ligand of the gel-forming polypeptide comprises an adrenomedullin, an adrenomedullin 2, and a homolog, variant, chimera, or functional fragment of CGRP, such as the sequences of SEQ ID NOS: 1-3, 48-58, and 106-114, 116-124, 126-131, 139-140, and 274-275, including but not limited to the sequences of SEQ ID NOS: 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275. In another embodiment, the gel-forming polypeptide comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% identity to the sequences of SEQ ID NOS: 1-3, 48-58, 106-114, 116-124, 126-131, 139-140, and 274-275. The term CLR / RAMP receptor ligand as used in this patent can refer to any functional peptide analog capable of activating or inhibiting CLR / RAMP receptors (CLR / RAMP 1, 2, and 3). In one embodiment, the CLR / RAMP receptor ligand is an analog of adrenomedullin, adrenomedullin 2, and CGRP, or the sequences of SEQ ID NOS: 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 polypeptide in the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0087] In some embodiments, the ADM, CGRP, or IMD sequence comprises a 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,

[0088] R1 is a functional group comprising a structure of (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;

[0089] R2 is a C-terminal modification moiety comprising a {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'-oxobisethylamine-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;

[0090] B0 is 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);

[0091] B1 is 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;

[0092] B2 is selected from the group consisting of a null residue, arginine, lysine, glutamine, glutamic acid, aspartic acid, asparagine, and a non-proteinogenic amino acid;

[0093] B3 is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, glutamine, aspartic acid, and a non-protein amino acid;

[0094] B4 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, and a non-protein amino acid;

[0095] B5 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, tyrosine, and a non-protein amino acid;

[0096] B6 is selected from the group consisting of a null residue, alanine, leucine, isoleucine, valine, methionine, phenylalanine, histidine, arginine, lysine, and a non-protein amino acid;

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

[0098] B8 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, serine, threonine, and a non-protein amino acid;

[0099] 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;

[0100] 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;

[0101] 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;

[0102] 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;

[0103] 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;

[0104] 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;

[0105] 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;

[0106] B16 is selected from the group consisting of a null residue, asparagine, glutamine, valine, alanine, glycine, isoleucine, leucine, and a non-protein amino acid;

[0107] B17 is selected from the group consisting of a null residue, asparagine, glutamine, serine, threonine, tyrosine, and a non-protein amino acid;

[0108] B18 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, phenylalanine, tyrosine, and a non-protein amino acid;

[0109] 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;

[0110] 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;

[0111] 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;

[0112] 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;

[0113] 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;

[0114] B24 is selected from the group consisting of a null residue, alanine, glycine, proline, serine, threonine, tyrosine, and a non-protein amino acid;

[0115] B25 is selected from the group consisting of a null residue, valine, alanine, glycine, isoleucine, leucine, proline, serine, threonine, and a non-protein amino acid;

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

[0117] 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;

[0118] 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.

[0119] The GnRH receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a GnRH receptor agonist or antagonist, such as SEQ ID NO: 15, or can comprise (or consist of) SEQ ID NO: 15. In another embodiment, the gel-forming GnRH receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 15 or SEQ ID NO: 201, or can comprise (or consist of) SEQ ID NO: 15 or SEQ ID NO: 201. As used herein, the term GnRH receptor ligand can refer to any functional analog capable of activating or inhibiting a GnRH receptor (e.g., Buserelin, Deslorelin, Fertirelin, Goserelin, Leuprolide, Nafarelin, and Triptorelin). However, the GnRH receptor ligand within the gel-forming polypeptide of the present invention can be shorter (e.g., 6-9 or fewer amino acids in length) or longer (e.g., 11-40 or more amino acids in length) than the provided sequences.

[0120] The GnRH receptor antagonist component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a GnRH receptor agonist or antagonist, such as SEQ ID NO: 27, including but not limited to SEQ ID NO: 27. In another embodiment, the gel-forming GnRH receptor antagonist comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 202 or 203, or can comprise (or consist of) SEQ ID NO: 202 or 203. As used herein, the term GnRH receptor antagonist can refer to any functional analog capable of inhibiting a GnRH receptor (e.g., Abarelix, Cetrorelix, Degarelix, Ganirelix, and Ozarelix). In one embodiment, the GnRH receptor antagonist is an analog of a GnRH receptor antagonist or SEQ ID NO: 27. However, the GnRH receptor antagonist within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-40 or more amino acids in length) than the provided sequences.

[0121] The vasopressin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a vasopressin receptor agonist or antagonist, such as SEQ ID NO: 17, including but not limited to SEQ ID NO: 17. In another embodiment, the gel-forming vasopressin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 204, or can comprise (or consist of) SEQ ID NO: 204. As used herein, the term vasopressin receptor ligand can refer to any functional analog capable of activating or inhibiting a vasopressin receptor (e.g., desmopressin, lysine vasopressin, arginine vasopressin, d[Leu4, Lys8]-VP, (d(CH2)51, Tyr(Me)2, Arg8)-vasopressin, and neurophysin). In one embodiment, the vasopressin receptor ligand is an analog of a vasopressin receptor ligand or SEQ ID NO: 17. However, the vasopressin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[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 SEQ ID NO: 5, including but not limited to SEQ ID NO: 5. In another embodiment, the gel-forming oxytocin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 5 or 205, or can comprise (or consist of) SEQ ID NO: 5 or 205. As used herein, the term oxytocin receptor ligand can refer to any functional analog capable of activating or inhibiting an oxytocin receptor (e.g., desamino oxytocin, meplumine oxytocin, oxytocin, WAY-267,464, neurophysin, barusiban, atosiban, and carbetocin). In one embodiment, the oxytocin receptor ligand is an analog of an oxytocin receptor agonist, antagonist or SEQ ID NO: 5. However, the oxytocin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[0123] The apelin receptor (i.e., APJ receptor and Apela / ELABELA / Toddler) ligand component of the gel-forming polypeptide includes homologs, variants, analogs, chimeras, or functional fragments of an apelin receptor agonist or antagonist, such as SEQ ID NO: 18, including but not limited to SEQ ID NO: 18. In another embodiment, the gel-forming apelin receptor ligand includes an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 206 or 262, or can include (or consist of) SEQ ID NO: 206 or 262. As used herein, the term apelin receptor ligand can refer to any functional analog capable of activating or inhibiting an apelin receptor (e.g., apelin 36, apelin 17, apelin 13, and Apela / ELABELA / Toddler). In one embodiment, the apelin receptor ligand is an apelin receptor agonist, antagonist analog, or SEQ ID NO: 18. However, the apelin receptor ligand within the gel-forming polypeptide of the present invention can be shorter (e.g., 5-40 or more amino acids in length) or longer than the provided sequence length.

[0124] The neurotensin receptor (NTSR1 and NTSR2) ligand component of the gel-forming polypeptide includes homologs, variants, analogs, chimeras, or functional fragments of a neurotensin receptor agonist or antagonist, such as SEQ ID NO: 19, including but not limited to SEQ ID NO: 19. In another embodiment, the gel-forming neurotensin receptor ligand includes an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 207, or can include (or consist of) SEQ ID NO: 207. As used herein, the term neurotensin receptor ligand can refer to any functional analog capable of activating or inhibiting a neurotensin receptor (e.g., beta-lactotensin, JMV-449, neurotensin 13, neuromedin 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 analog or SEQ ID NO: 19. However, the neurotensin receptor ligand within the gel-forming polypeptide of the present invention can be shorter (e.g., 6-12 or fewer amino acids in length) or longer (e.g., 14-40 or more amino acids in length) than the provided sequence length.

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

[0126] The bombesin receptor (BB1, BB2, and BB3 receptor) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a bombesin receptor agonist or antagonist, such as SEQ ID NO: 20 and 61, including but not limited to SEQ ID NO: 20 and 61. In another embodiment, the gel-forming bombesin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 61, 209, or 255, or can comprise (or consist of) SEQ ID NO: 61, 209, or 255. As used herein, the term bombesin receptor ligand can refer to any functional analog capable of activating or inhibiting a bombesin receptor (e.g., bombesin, neuromedin B, BIM 187, BIM 189, [D-Phe12, Leu14]-bombesin, alytesin, BIM 23042, [D-Phe12, Leu14]-bombesin, and gastrin-releasing peptide). In one embodiment, the bombesin receptor ligand is an analog of a bombesin receptor agonist, antagonist, or SEQ ID NO: 20 and 61. However, the bombesin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[0127] The new dermorphin or opioid receptor (i.e., delta opioid receptor, kappa opioid receptor, mu opioid receptor, and nociceptin 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 SEQ ID NO: 21, including but not limited to SEQ ID NO: 21. In another embodiment, the gel-forming opioid receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 211, 212, 213, or 214, or can comprise (or consist of) SEQ ID NO: 211, 212, 213, or 214. As used herein, the term opioid receptor ligand can refer to any functional analog capable of activating or inhibiting an opioid receptor (e.g., buprenorphine, leucine enkephalin, methionine enkephalin, dermorphin, DADLE, DPDPE, 7-helix indan hydromorphone, and N-phenethyl-14-ethoxymethyl dihydromorphinone). In one embodiment, the opioid receptor ligand is an analog of an opioid receptor agonist, antagonist, or SEQ ID NO: 21. However, the opioid receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 4-40 or more amino acids in length).

[0128] The gel-forming polypeptide of the present invention comprises a homolog, variant, analog, chimera, or functional fragment of an opioid receptor agonist or antagonist, such as SEQ ID NO: 22, including but not limited to SEQ ID NO: 22. In another embodiment, the gel-forming opioid receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 211, 212, 213, or 214, or can comprise (or consist of) SEQ ID NO: 211, 212, 213, or 214. As used herein, the term opioid receptor ligand can refer to any functional analog capable of activating or inhibiting an opioid receptor (e.g., leucine enkephalin, methionine enkephalin, dermorphin, DADLE, DPDPE, 7-helix indan hydromorphone, and N-phenethyl-14-ethoxymethyl dihydromorphinone). In one embodiment, the opioid receptor ligand is an analog of an opioid receptor agonist, antagonist, or SEQ ID NO: 22. However, the opioid receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 4-40 or more amino acids in length).

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

[0130] The substance P receptor (neurokinin 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 SEQ ID NO: 23, including but not limited to SEQ ID NO: 23. In another embodiment, the gel-forming substance P receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 215, 253, and 254, or can comprise (or consist of) SEQ ID NO: 215, 253, and 254. As used herein, the term substance P receptor ligand can refer to any functional analog capable of activating or inhibiting a substance P receptor (e.g., substance P, GR-73632, Aprepitant, Casopitant, Ezlopitant, Fosaprepitant, Lanepitant, Maropitant, and Vestipitant). In one embodiment, the substance P receptor ligand is an analog of a substance P receptor agonist, antagonist, or SEQ ID NO: 23. However, the substance P receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[0131] The angiotensin II receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an angiotensin II receptor agonist or antagonist, such as SEQ ID NO: 24, including but not limited to SEQ ID NO: 24. In another embodiment, the gel-forming angiotensin II receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 216, or can comprise (or consist of) SEQ ID NO: 216. As used herein, the term angiotensin II receptor ligand can refer to any functional analog capable of activating or inhibiting an angiotensin II receptor (e.g., saralasin). In one embodiment, the angiotensin II receptor ligand is an analog of an angiotensin II receptor agonist, antagonist, or SEQ ID NO: 24. However, the angiotensin II receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[0132] The calcitonin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a calcitonin receptor agonist or antagonist, such as SEQ ID NO: 28, including but not limited to SEQ ID NO: 28. In another embodiment, the gel-forming calcitonin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 217, or can comprise (or consist of) SEQ ID NO: 217. As used herein, the term calcitonin receptor ligand can refer to any functional analog capable of activating or inhibiting a calcitonin receptor (e.g., Miacalcin analog). In one embodiment, the calcitonin receptor ligand is an analog of a calcitonin receptor agonist, antagonist, or SEQ ID NO: 28. However, the calcitonin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[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 SEQ ID NO: 4, including but not limited to SEQ ID NO: 4. In another embodiment, the gel-forming amylin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 4 or 218, or can comprise (or consist of) SEQ ID NO: 4 or 218. The term amylin receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting an amylin receptor (e.g., pramlintide). In one embodiment, the amylin receptor ligand is an analog of an amylin receptor agonist, antagonist, or SEQ ID NO: 4. However, the amylin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[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 SEQ ID NOs: 14 and 26, including but not limited to SEQ ID NOs: 14 and 26. In another embodiment, the gel-forming GLP-1 receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NOs: 14, 219, 220, 221, or 269-272, or can comprise (or consist of) SEQ ID NOs: 14, 219, 220, 221, or 269-272. The term GLP-1 receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting a GLP-1 receptor (e.g., GLP-1 7-37, exenatide, [Glu13]exenatide, [Met(O)14]-exenatide, [N-acetyl-His1]-exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, and taspoglutide). In one embodiment, the GLP-1 receptor ligand is an analog of a GLP-1 receptor agonist, antagonist, or SEQ ID NO: 14 or 26. However, the GLP-1 receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[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 SEQ ID NO: 12, including but not limited to SEQ ID NO: 12. In another embodiment, the gel-forming GLP-2 receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 12 or 222, or can comprise (or consist of) SEQ ID NO: 12 or 222. As used herein, the term GLP-2 receptor ligand can refer to any functional analog capable of activating or inhibiting a GLP-2 receptor (e.g., Teduglutide and GLP2). In one embodiment, the GLP-2 receptor ligand is an analog of a GLP-2 receptor agonist, antagonist, or SEQ ID NO: 12. However, the GLP-2 receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-40 or more amino acids in length).

[0136] The melanocortin receptor (i.e., MC1R-MC5R) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a melanocortin receptor agonist or antagonist, such as SEQ ID NO: 29, 33, 34, 62, and 64, including but not limited to SEQ ID NO: 29, 33, 34, 62, and 64. In another embodiment, the gel-forming melanocortin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273, or can comprise (or consist of) SEQ ID NO: 64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273. As used herein, the term melanocortin receptor ligand can refer to any functional analog capable of activating or inhibiting a melanocortin receptor (e.g., a-MSH, b-MSH, g-MSH, ACTH1-24, corticorin, afamelanotide, BMS-470,539, bimoclomol, melanotan II, modimelanotide, setmelanotide, PF-00446687, PL-6983, THIQ, PF-219,061, UK-414,495, agouti-related peptide, and agouti signaling peptide). In one embodiment, the melanocortin receptor ligand is an analog of a melanocortin receptor agonist, antagonist, or SEQ ID NO: 29, 33, 34, 62, or 64. However, the melanocortin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 4-40 or more amino acids in length).

[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 SEQ ID NO: 30, including but not limited to SEQ ID NO: 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% identical to the sequence of SEQ ID NO: 229 or 264, or can comprise (or consist of) SEQ ID NO: 229 or 264. As used in this patent, the term neuropeptide Y receptor ligand can refer to any functional analog capable of activating or inhibiting a neuropeptide Y receptor (e.g., neuropeptide Y fragment 13-36, peptide Yy, peptide YY 3-36 fragment, [Leu31, Pro34]-neuropeptide Y, neuropeptide Y, BVD-10, GR-231, 118, [cPP1-7, NPY19-23, Ala31, Aib32, Gln34]-hPancreoPeptide, BVD10, and PancreoPeptide). In one embodiment, the neuropeptide Y receptor ligand is an analog of a neuropeptide Y receptor agonist, antagonist, or SEQ ID NO: 30. However, the neuropeptide Y receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequences (e.g., 5-40 or more amino acids in length).

[0138] The corticotropin-releasing hormone receptor (CRHR1 and CRHR2) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a corticotropin-releasing hormone receptor agonist or antagonist, such as SEQ ID NOs: 10 and 31, including but not limited to SEQ ID NOs: 10 and 31. In another embodiment, the gel-forming corticotropin-releasing hormone receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 10, 230, 231, or 265, or can comprise (or consist of) SEQ ID NO: 10, 230, 231, or 265. As used herein, the term corticotropin-releasing hormone receptor ligand can refer to any functional analog capable of activating or inhibiting a corticotropin-releasing hormone receptor (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 corticotropin-releasing hormone receptor ligand is an analog of a corticotropin-releasing hormone receptor agonist, antagonist, CRH, urocortin 1, urocortin 2, urocortin 3, or SEQ ID NO: 10 or 31. However, the corticotropin-releasing hormone receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length) than the provided sequences.

[0139] The parathyroid hormone receptor (PTH1R and PTH2R) ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a parathyroid hormone receptor agonist or antagonist, such as SEQ ID NO: 25, including but not limited to SEQ ID NO: 25. In another embodiment, the gel-forming parathyroid hormone receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 232 and 258, or can comprise (or consist of) SEQ ID NO: 232 and 258. As used herein, the term parathyroid hormone receptor ligand can refer to any functional analog capable of activating or inhibiting a parathyroid hormone receptor (e.g., parathyroid hormone, parathyroid hormone related protein, DPCA J1951, teriparatide, and abaloparatide). In one embodiment, the parathyroid hormone receptor ligand is an analog of a parathyroid hormone receptor agonist, antagonist, or SEQ ID NO: 25. However, the parathyroid hormone receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length) than the provided sequences.

[0140] The bradykinin receptor (BDKRB1 and BDKRB2) ligand component of the gel- forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a bradykinin receptor agonist or antagonist, such as SEQ ID NOs: 32 and 65; Firazyr or icatibant, including but not limited to SEQ ID NOs: 32 and 65. In another embodiment, the gel-forming bradykinin receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NOs: 233, 234, or 261, or can comprise (or consist of) SEQ ID NOs: 233, 234, or 261. The term bradykinin receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting a bradykinin receptor (e.g., bradykinin 1-8, [Leu8]-bradykinin 1-8, Sar- [D-Phe8]-des-Arg9-bradykinin, KRPPGFS-DpNal-I, [Phe8Ψ(CH-NH)-Arg9]-bradykinin, MEN11270, R715, R 892, and Hoe 140 (icatibant)). In one embodiment, the bradykinin receptor ligand is an analog of a bradykinin receptor agonist, antagonist, or SEQ ID NO: 32. However, the bradykinin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequences (e.g., 5-40 or more amino acids in length).

[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 SEQ ID NO: 8, including but not limited to SEQ ID NO: 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% sequence identity to SEQ ID NO: 8 or 235, or can comprise (or consist of) SEQ ID NO: 8 or 235. The term ghrelin / growth hormone secretagogue receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting the ghrelin / growth hormone secretagogue receptor (e.g., Alarelin, Capromorelin, Esaxarin (Hexarelin), Ghrelin (Lenorelin), GHRP-6, Ibuprofen (Mk-677), Ipratropium, Macimorelin, Pramlintide (GHRP-2), Ramelteon, SM-130, 686, Tabimorelin, and Ulimorelin). In one embodiment, the ghrelin / growth hormone secretagogue receptor ligand is an analog of a ghrelin / growth hormone secretagogue receptor agonist, antagonist, or SEQ ID NO: 8. However, the ghrelin / growth hormone secretagogue receptor (GHSR) ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-50 or more amino acids in length).

[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 SEQ ID NO: 35, including but not limited to SEQ ID NO: 35. 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% sequence identity to SEQ ID NO: 35, 236, or 237, or can comprise (or consist of) SEQ ID NO: 35, 236, or 237. The term growth hormone releasing hormone receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting the growth hormone releasing hormone receptor (e.g., CJC-1295, Domoic Acid, GHRH (Growth Hormone-Releasing Factor), Liraglutide, Semorelin (GHRH1-29), and Tesamorelin). In one embodiment, the growth hormone releasing hormone receptor ligand is an analog of a growth hormone releasing hormone receptor agonist, antagonist, or SEQ ID NO: 35. However, the growth hormone releasing hormone receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length than the provided sequence (e.g., 5-50 or more amino acids in length).

[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 SEQ ID NO: 67, including but not limited to SEQ ID NO: 67. In another embodiment, the gel-forming VIP receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 266, or can comprise (or consist of) SEQ ID NO: 266. As used herein, the term VIP receptor ligand can refer to any functional analog capable of activating or inhibiting a VIP receptor. In one embodiment, the VIP receptor ligand is an analog of a VIP receptor agonist, antagonist, or SEQ ID NO: 67. However, the VIP receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length) than the provided sequence.

[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 SEQ ID NO: 41, including but not limited to SEQ ID NO: 41. In another embodiment, the gel-forming natriuretic peptide receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 238, or can comprise (or consist of) SEQ ID NO: 238. As used herein, the term natriuretic peptide receptor ligand can refer to any functional analog capable of activating or inhibiting a natriuretic peptide receptor (e.g., atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, and nesiritide). In one embodiment, the natriuretic peptide receptor ligand is an analog of a natriuretic peptide receptor agonist and antagonist, atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, or SEQ ID NO: 41. However, the natriuretic peptide receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length) than the provided sequence.

[0145] The thymosin alpha 1 -like ligand receptor ligand component of the gel- forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of thymosin alpha 1 and thymosin alpha 1 -like ligands, such as SEQ ID NO: 13, including but not limited to SEQ ID NO: 13. In another embodiment, the gel-forming thymosin alpha 1 -like ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 13, 239, 240, 267, or 268, or can comprise (or consist of) SEQ ID NO: 13, 239, 240, 267, or 268. As used herein, the term thymosin alpha 1 -like ligand can refer to any functional analog capable of activating or inhibiting a thymosin alpha 1 -mediated signaling pathway (e.g., thymosin alpha 1 ). In one embodiment, the thymosin alpha 1 and thymosin alpha 1 -like ligand is an analog of thymosin alpha 1 or SEQ ID NO: 13. However, the thymosin alpha 1 -like ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0146] The thymosin beta 4 ligand receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of thymosin beta 4 ligand, such as SEQ ID NO: 66, including but not limited to SEQ ID NO: 66. In another embodiment, the gel-forming thymosin beta 4-like ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the sequence of SEQ ID NO: 263, or can comprise (or consist of) SEQ ID NO: 263. As used herein, the term thymosin beta 4 can refer to any functional analog capable of activating or inhibiting a thymosin beta 4-mediated signaling pathway (e.g., thymosin beta 4). In one embodiment, the thymosin beta 4 and thymosin beta 4-like ligand is an analog of thymosin beta 4 or SEQ ID NO: 263. However, the thymosin beta 4-like ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0147] The cell-penetrating peptide component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a cell-penetrating peptide, such as SEQ ID NO: 36, including but not limited to SEQ ID NO: 36. In another embodiment, the gel-forming cell-penetrating peptide comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 241, or can comprise (or consist of) SEQ ID NO: 241. As used herein, the term cell-penetrating peptide can refer to any functional peptide analog that facilitates the transfer of a molecule from outside a cell to inside a cell (see, e.g., Kalafatovic D and Giralt E

[2017] Cell-penetrating peptides: design strategies beyond primary structure and amphipathicity. Molecules 22(11). In one embodiment, the cell-penetrating peptide is an analog of a cell-penetrating peptide or SEQ ID NO: 36. However, the cell-penetrating peptide within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0148] The kallikrein modulator component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a kallikrein modulator, such as SEQ ID NO: 37, including but not limited to SEQ ID NO: 37. In another embodiment, the gel-forming kallikrein modulator comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 242, or can comprise (or consist of) SEQ ID NO: 242. As used herein, the term kallikrein modulator can refer to any functional analog capable of activating or inhibiting kallikrein (e.g., ecallantide). In one embodiment, the kallikrein modulator is an analog of a kallikrein activator and inhibitor or SEQ ID NO: 37. However, the kallikrein modulator within the gel-forming polypeptide of the present invention can be shorter or longer in length (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 homolog, variant, analog, chimera, or functional fragment of a polypeptide antibiotic or antimicrobial peptide, such as SEQ ID NO: 68, including but not limited to SEQ ID NO: 68. In another embodiment, the gel-forming polypeptide antibiotic comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 243, or can comprise (or consist of) SEQ ID NO: 243. As used herein, the term polypeptide antibiotic can refer to any functional analog capable of inhibiting the growth or proliferation of a microbe, bacteria, fungus, virus, tumor, or other pathogen. In one embodiment, the polypeptide antibiotic is an analog of a polypeptide antibiotic (e.g., temporin A, gramicidin A, B, C, and D, defensin, esculentin 1-21, cecropin, andropin, moricin, ceratotoxin, melittin, magainin, dermaseptin, bombinin, brevinin-1, esculentin, bforin II, CAP18, LL37, abaecin, apidaecin, prophenin, indolicidin, actinomycin, bacitracin, colistin, polymyxin B, actinomycin-D, bacitracin, bosepyvir, dalbavancin, daptomycin, enfuvirtide, oritavancin, telavancin, telaprevir, teixobactin, and vancomycin) or SEQ ID NO: 68. However, the polypeptide antibiotic within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0150] The complement modulator component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a complement modulator, such as SEQ ID NO: 11, including but not limited to SEQ ID NO: 11. In another embodiment, the gel-forming complement modulator comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of SEQ ID NO: 11 or 244, or can comprise (or consist of) SEQ ID NO: 11 or 244. As used herein, the term complement modulator can refer to any functional analog capable of activating or inhibiting a complement factor or complement cascade (e.g., a complement 5a and a compstatin analog). In one embodiment, the complement modulator is an analog of a complement activator or inhibitor (e.g., compstatin, complement C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9) or SEQ ID NO: 11. However, the complement modulator within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0151] The C5aR receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of the C5aR receptor ligand, such as Serial No. 11, including but not limited to Serial No. 11. In another embodiment, the gel-forming C5aR receptor ligand comprises about 70%, 75%, 80%, 85%, 90%, or 95% of the amino acid sequence that is sequence-identical to Serial No. 11 or 244, or may comprise Serial No. 11 or 244 (or consist of that sequence). 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 Serial No. 11. However, the C5aR receptor ligand length within the gel-forming peptide of the present invention may be shorter or longer (e.g., 5-50 or more amino acids in length).

[0152] Every 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 of about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with Serial No. 245, or may comprise Serial No. 245 (or consist of that sequence). As used herein, the term clopason immunomodulator may refer to any functional analogue (e.g., copolymer 1, Cop-1, or clopason, Glatopa, and M356) consisting of four amino acids found in basic myelin proteins (i.e., glutamic acid, lysine, alanine, and tyrosine) that is capable of activating or inhibiting glatiramer acetate-mediated signaling pathways. In one embodiment, the immunomodulator is a glatiramer acetate analogue (a mixture of analogues consisting of four amino acids found in basic myelin proteins) or Serial No. 42. However, the immunomodulator within the gel-forming peptide in this 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 about 70%, 75%, 80%, 85%, 90%, or 95% amino acid sequence identity with Serial Numbers 246, 247, 248, 249, or 250, or may comprise Serial Numbers 246, 247, 248, 249, or 250 (or consist of such sequences). The term matrix-modified protein as used in this patent may refer to any functional analog capable of activating or inhibiting matrix enzyme-mediated signaling pathways. In one embodiment, the matrix-modifying 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 that act as signaling regulators of extracellular matrix components, carrier peptides, and neurotransmitter function regulators, 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-modifying protein 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).

[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 herein may refer to any functional analogue capable of activating or inhibiting the thrombopoietin receptor (e.g., (Romelastine). In one embodiment, the thrombopoietin receptor ligand is an analog of thrombopoietin (THPO), megakaryocyte growth and development factor (MGDF), or Serial No. 9. However, the thrombopoietin 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).

[0156] The insulin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of an insulin receptor ligand, such as SEQ ID NO: 59, including but not limited to SEQ ID NO: 59. In another embodiment, the gel-forming matrix-modifying protein comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 251, or can comprise (or consist of) SEQ ID NO: 251. As used herein, the term insulin receptor ligand can refer to any functional analog capable of activating or inhibiting an insulin receptor (e.g., insulin detemir, insulin lispro, insulin aspart, insulin glulisine, insulin degludec, and insulin glargine). In one embodiment, the insulin receptor ligand is an analog of insulin or SEQ ID NO: 59. However, the insulin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-50 or more amino acids in length for the B chain and A chain, respectively).

[0157] The relaxin receptor ligand component of the gel-forming polypeptide comprises a homolog, variant, analog, chimera, or functional fragment of a relaxin receptor ligand, such as SEQ ID NO: 60, including but not limited to SEQ ID NO: 60. In another embodiment, the gel-forming matrix-modifying protein comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 252, or can comprise (or consist of) SEQ ID NO: 252. As used herein, the term relaxin receptor ligand can refer to any functional analog capable of activating or inhibiting a relaxin receptor (i.e., LGR7 and LGR8). In one embodiment, the relaxin receptor ligand is an analog of relaxin 1, relaxin 2, relaxin 3, INSL3, INSL7, or SEQ ID NO: 60. However, the relaxin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-50 or more amino acids in length for the B chain and A chain, respectively).

[0158] A 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 SEQ ID NO: 63, including but not limited to SEQ ID NO: 63. In another embodiment, the gel-forming PTHrP receptor ligand comprises an amino acid sequence having about 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to SEQ ID NO: 258, or can comprise (or consist of) SEQ ID NO: 258. The term PTHrP receptor ligand as used in this patent can refer to any functional analog capable of activating or inhibiting a PTHrP receptor (e.g., parathyroid hormone, parathyroid hormone-related protein, teriparatide, and abaloparatide). In one embodiment, the PTHrP receptor ligand is a PTHrP receptor agonist, antagonist, analog of PTHrP 1-34, abaloparatide, or SEQ ID NO: 63. However, the PTHrP receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer in length (e.g., 5-50 or more amino acids in length).

[0159] A variety of known methods in the art can be employed to alter the sequence of a polypeptide, thereby making targeted changes to the sequence. Typically, the sequence of the polypeptide will be substantially similar to the sequences provided herein, i.e., will have greater than 70%, greater than 80%, greater than 90%, greater than 95% sequence identity to the provided sequences. Changes to the sequence can be made by way of substitution, insertion, or deletion. In determining the minimal sequence changes to make, systematic scan mutagenesis can be employed, in which alanine or other residues are introduced systematically. Conservative substitutions of amino acids typically include substitutions among: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine). Non-conservative substitutions can include any unusual combinations of amino acids.

[0160] Targeted modifications that do not alter the primary sequence include chemical derivatization of the polypeptide, e.g., methylation, acetylation, acylation, pegylation, or carboxylation. Other methods include modification of glycosylation, e.g., modification of the glycosylation pattern of the polypeptide during synthesis and processing or during further processing steps; e.g., exposure of the polypeptide to enzymes that affect glycosylation, e.g., mammalian glycosylation enzymes or deglycosylation enzymes.

[0161] In other embodiments, the peptide comprises an optical isomer, enantiomer, diastereomer, tautomer, cis-trans isomer, racemate, prodrug, or pharmaceutically acceptable salt of the peptide. The peptide can be modified with C-terminal amidation. Optionally, the modified peptide can be modified with acylation. Optionally, one or more acylations can be made, with one reaction group being preferred.

[0162] Peptide formulations also include stereoisomeric mixtures or individual pure isomers or mixtures of substantially pure isomers. For example, optionally, the compounds of the present application can have one or more asymmetric centers at the carbon atom containing any of the substituent groups. Thus, the compounds can exist as enantiomers or diastereomers or as mixtures of enantiomers or diastereomers. When the compounds of the present application contain a double bond, the compounds can exist as geometric isomers (cis or trans), and when the compounds of the present application contain an unsaturated bond, the compounds can exist as tautomers, and the compounds also include these isomers and mixtures thereof. In preparing the compounds of the present application, the starting compounds can be used in racemic mixtures, enantiomeric mixtures, or diastereomeric mixtures. When the compounds obtained are mixtures of diastereomers or enantiomers, they can be separated by conventional methods, such as chromatography or fractional crystallization. In addition, the compounds of the present application include within their scope solvates, hydrates, or polymorphs.

[0163] Also included in the present application are polypeptides modified based on general molecular biological techniques and synthetic chemistry to improve their resistance to proteolysis, or to optimize solubility properties or to enhance their suitability as therapeutic agents. For example, the stability of a peptide can be enhanced by cyclization of the peptide backbone through the addition of disulfide bridges or lactam bridges (see Friedler et al.

[2000] J. Biol. Chem. 275:23783-23789). Such analogs of the polypeptides contain residues other than naturally occurring L-amino acids, for example, D-amino acids or unnatural synthetic amino acids.

[0164] The peptides of interest can be prepared by in vitro synthetic routes using conventional methods known in the art. A variety of commercial synthetic instruments are currently available, for example, automated synthesizers provided by Applied Biosystems, Inc., Foster City, CA, Beckman, and the like. These synthetic instruments can be used to substitute unnatural amino acids for natural amino acids. The particular sequence and method of preparation will be determined by convenience, economy, practicality, and purity required.

[0165] If desired, various groups can be introduced into the peptide during synthesis or expression to allow attachment to other molecules or surfaces. Thus, cysteine can be used to make a thioether, histidine to bind metal ion complexes, carboxyl groups to form amides or esters, and amines to form amides, etc.

[0166] The polypeptides can also be isolated and purified according to conventional methods of recombinant synthesis. A lysate of the expression host can be prepared and purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification techniques. In most cases, the composition used will be at least 20% by weight of the target product, usually at least about 75%, and preferably at least about 95%, taking into account contaminants involved in the product preparation and purification process. For therapeutic purposes, it is usually at least 99% by weight of the target product. Generally, the percentages are calculated on the basis of total protein.

[0167] Compositions and uses of gel-forming polypeptides

[0168] The present invention relates to the preparation and use of self-assembling gel-forming polypeptides. In some embodiments, the polypeptides are derived from human secreted peptide hormones, human peptide analogs, and polypeptides having a gel formation enhancing motif. The gel-forming polypeptides can be natural or engineered gel-forming polypeptides for use in treating a patient, the method of treatment characterized by administering to an individual in need of the method of treatment a therapeutic composition comprising an effective amount of a gel-forming polypeptide, and the composition can be used alone or in combination with other therapeutic agents.

[0169] A series of peptide sequences are provided that comprise or mimic natural polypeptide therapeutics but have the ability to form gels. Compositions and improved methods for preparing self-assembling polypeptides are characterized by solubilizing such polypeptides at appropriate concentrations or coupling them to a gel formation enhancing motif. The highly biocompatible and biodegradable polypeptide gels can be used to deliver therapeutic agents. When used as carrier proteins, the therapeutic agents are encapsulated / bound within the gel-forming polypeptide carrier; alternatively, the polypeptide gels themselves can be used as therapeutic agents.

[0170] The following objectives are achieved by applying this technology: (1) to make therapeutic analogs with gel formation ability that the wild type or known analogs of the peptides themselves cannot form gels; (2) to form advanced non-immunogenic polypeptide gel nanostructures that encapsulate therapeutic agents for slow release by reducing solubility and providing a surface barrier; (3) to reduce the degradation ability or clearance rate of polypeptide class therapeutic agents in vivo; and (4) to add natural or wild type hormones / therapeutic agents with gel formation ability to the formulation and the formulation stays in the body for a longer period of time. The reversible phase transition of the non-covalently bound monomers in the polypeptide gel nanostructures enables sustained delivery of biologically active therapeutic agents over a longer period of time compared to injection of aqueous solutions or crystalline particle suspension formulations. Generally, the resulting liquid or semi-solid gels pose a very low risk of immunogenic reactions and are easily degraded.

[0171] In some embodiments, the present application also includes one or more bioactive agents adsorbed or absorbed within the hydrogel, which delivers the one or more bioactive agents to the site of administration, which can include implanting the hydrogel of interest, coating an implant with the hydrogel, orally administering the hydrogel, and the like. The amount of bioactive agent encapsulated will depend on the duration of delivery, the site of administration, and the condition being treated. In some embodiments, the amount of bioactive agent encapsulated into the hydrogel of interest is 0.0001 pg or more, for example, 0.001 pg or more, 0.01 pg or more, 0.1 pg or more, 1 pg or more, 10 pg or more, 25 pg or more, 50 pg or more, 100 pg or more, 500 pg or more, 1000 pg or more, 5000 pg or more, and including 10,000 pg or more. When the bioactive agent is encapsulated into the hydrogel in liquid form, the concentration of the bioactive agent can be 0.0001 pg / mL or more, for example, 0.001 pg / mL or more, 0.01 pg / mL or more, 0.1 pg / mL or more, 0.5 pg / mL or more, 1 pg / mL or more, 2 pg / mL or more, 5 pg / mL or more, 10 pg / mL or more, 25 pg / mL or more, 50 pg / mL or more, 100 pg / mL or more, 500 pg / mL or more, 1000 pg / mL or more, 5000 pg / mL or more, and including 10,000 pg / mL or more.

[0172] An effective dose of a therapeutic polypeptide administered to a human patient as a gel formulation can be about 1.0 pg / kg body weight, 2.5 pg / kg body weight, 5.0 pg / kg body weight, 10.0 pg / kg body weight, 25.0 pg / kg body weight, 50.0 pg / kg body weight, 75.0 pg / 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, 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 more, or any dosage range of these parameters.

[0173] An effective amount can be maintained for a sufficient period of time to achieve treatment of the condition. Depending on the structure of the particular hydrogel employed, there can be differences in the release of the one or more bioactive agents from the hydrogel matrix. For example, the hydrogels of the present application can release the drug slowly. By designing the gel, it is possible to have it release a therapeutic amount of the polypeptide for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more, such as 1 week, 2 weeks, 3 weeks, 4 weeks or more.

[0174] "Slow release" refers to the sustained delivery of the one or more bioactive agents from the hydrogel structure. The hydrogel remains in contact with the administration site throughout the delivery period, such as 1 day or more, 2 days or more, 5 days or more, 10 days or more, 15 days or more, 30 days or more, and including 100 days or more.

[0175] The present application also includes methods of treatment by applying one or more of the gels of the present application to the subject. In some embodiments, the methods include administering a hydrogel solution or suspension to the subject, and the contact time of the hydrogel with the subject is sufficient to achieve the purpose of treating the subject. As described above, the purpose hydrogel can be applied to any suitable administration site in need of treatment, including, without limitation, the skin, bone, heart, liver, kidney, bladder, trachea, lung, tumor tissue, oral cavity (e.g., buccal and sublingual), and nose, throat, ear, uterus, and bladder, among others.

[0176] In certain embodiments, the methods can include applying one or more hydrogels and the contact time of the hydrogel with the subject is sufficient to deliver a target dose of the bioactive agent. For example, the target dose of the bioactive agent can be a total exposure or a mean daily exposure. For example, the target dose of the bioactive agent delivered by the purpose method can be 0.01 mg / day or more, such as 0.04 mg / day or more, 0.5 mg / day or more, 1.0 mg / day or more, 2 mg / day or more, 5 mg / day or more, and including 10 mg / day over a 4 week dosing interval.

[0177] As discussed herein, a gelation-enhancing motif was identified that improved the way in which a gel-forming molecule could be prepared. Coupling the gelation-enhancing motif resulted in a very small structural change that had little effect on the distribution profile of the therapeutic agents discussed herein. The effect of the modification on the distribution volume of the therapeutic drug was minimal, which was advantageous because it allowed the pharmacokinetics of the therapeutic drug to be distributed in the body similar to the wild-type polypeptide analog. In most cases, this characteristic was preferred because other half-life extension techniques such as macromolecular fusion (e.g., fusion with IgG, albumin, or PEG) would significantly increase the molecular mass of the therapeutic agent and decrease its distribution volume, thereby preventing the therapeutic agent from reaching the intended target sites outside the systemic circulation.

[0178] The discovery that stable gel formulations could be obtained after dissolving selected polypeptide compounds in aqueous solutions at appropriate concentrations (i.e., below, at, or above 11% w / w) was unexpected. The selection of 11% w / w as the initial point of testing was because serum protein levels are approximately 9-10% w / w. When the critical aggregation concentration was reached, such polypeptides were able to self-assemble into liquid or semi-solid gel nanostructures. In particular, it was envisioned that liquid or semi-solid gels could be formed from native or modified polypeptides.

[0179] Based on these discoveries, it was possible to have a higher drug loading of therapeutic polypeptides and other therapeutic agents in sustained release formulations compared to conventional compositions and formulations. The advantages described above could be achieved by using appropriate formulations or engineering polypeptides to form gel nanostructures at a certain concentration at which the wild-type polypeptide analogs would remain partially dissolved or undissolved in aqueous solutions, as known to those skilled in the art. By encapsulating therapeutic drugs in higher-order gel nanostructures formed from self-assembling polypeptides, the gel formulations could extend the delivery time of the therapeutic drugs, thereby improving patient compliance, increasing drug delivery efficiency and efficacy, and reducing adverse reactions. At the same time, this approach could reduce non-specific interactions, prevent enzymatic 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 distributed deeply in tissues.

[0180] The phase transition process between aqueous solution and gel nanostructure of the non-covalently bound polypeptide gel was dependent on dose, time, and other environmental factors, based on which the advantages described above and others were achieved. Typically, gel nanostructures dissociate into monomeric molecules in aqueous solutions because molecules in the outer surface of the gel gradually diffuse into the surrounding solution.

[0181] In preferred embodiments, all percentages mentioned in the present invention are weight (w / w) percentages.

[0182] Examples

[0183] Amylin, a B-type GPCR ligand, naturally forms solid amyloid fibrils in vivo; the related peptide CGRP forms liquid gel nanofibrils at selected concentrations. Based on the above observations, we hypothesized that peptides of the amylin / CGRP / Adrenomedullin / Adrenomedullin 2 family have a propensity to form gel nanofibrils upon appropriate formulation or engineering. We found that selected adrenomedullin agonist / antagonist acylates naturally form gel nanofibrils in aqueous solution, and that engineering selected polypeptides with hydrophobic tails, heads or side chains promotes their gel formation in the presence of a gel formation enhancing motif, further supporting the above hypothesis. In addition, we explored the propensity of natural and artificially synthesized therapeutic peptides to form gels in aqueous solution. Assay of the ability of a compound to form a gel in water: To assay the solubility of a polypeptide at room temperature, 1, 2, 3, 4, 5, 20 or 100 mg of a selected peptide was weighed into a clear plastic or glass vial, and an equal volume of deionized water was added to the vial. The room temperature was controlled to be about 22-25 °C while this was done. Soluble peptide samples dissolved immediately to give clear solutions. The solutions were clear and had flow properties, indicating that the solubility of the peptide was greater than the corresponding concentration (w / w). Insoluble peptide samples retained a granular or pasty appearance after 20 minutes. To distinguish the gel formation state of the peptide solutions, in addition to visual inspection, we performed a test tube tap test and a test tube tilt test.

[0184] To determine the solubility of a polypeptide at room temperature, 1, 2, 3, 4, 5, 20 or 100 mg of a selected peptide was weighed into a clear plastic vial and an equal volume of deionized water was added to the vial. The room temperature was controlled at about 22-25 °C while this was done. The soluble polypeptide sample dissolved immediately in water and remained clear and no significant change in viscosity was observed with the naked eye after 20 minutes of mixing with water. This result was observed to indicate that the peptide was highly soluble and did not form a gel nanostructure. If there were still visible particles of the peptide in the solution after 20 minutes of mixing with water, the peptide was considered to be insoluble or to form a precipitate at the given concentration. In this case, the quality of the peptide was further judged by two other criteria: (1) mobility after tapping the test tube (test tube tapping test) and (2) flowability 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 main body of the solution changed position was recorded. In this test, the score for an aqueous solution (e.g., water) was 9 or 10, i.e., the main body of the liquid changed position each time the test tube was tapped. Similarly, the score for insoluble peptide or peptide precipitate in solution was the same, i.e., 9 or 10, and the position of the insoluble peptide particles or peptide precipitate changed visibly with the naked eye each time the test tube was tapped. In contrast, a gel-forming peptide dissolved immediately or gradually in the aqueous solution and exhibited high viscosity after 20 minutes of mixing with water. A peptide with moderate viscosity could form a liquid gel, and the conformation of the liquid gel slowly changed when the test tube was tapped with a finger or tilted. To distinguish between a liquid gel and a semi-solid gel, we used the test tube tapping test and the test tube tilting test to detect the mobility of the solution. The selected peptide solution exhibited very high viscosity and formed a semi-solid gel, which did not flow like a liquid when the test tube was tapped with a finger or tilted. In the test tube tapping test, if the gel scored 0-4 (i.e., the main body of the gel changed position visibly with the naked eye 0-4 times after 10 independent tapping tests), it was determined to be a semi-solid gel. If the main body of the gel was observed to change position visibly with the naked eye 5-9 times after 10 independent tapping tests, it was determined to be a liquid gel. The test tube tilting test was used to further confirm whether the liquid gel had high viscosity. In this test, the plastic tube containing the solution or gel was tilted 90° and the movement of the solution or gel mass was observed under light. If the liquid moved to the bottom of the tube in less than 15 seconds, the peptide was determined to be a liquid solution. If the liquid or gel took more than 15 seconds to flow to the bottom of the tilted tube, the resulting aqueous material was determined to be a liquid gel.

[0185] To investigate the biological activity of the conjugated 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 kappa opioid receptor (OPRK1). The agonist activity of the peptides on MC1R, MC4R, CRHR2, PTHR1, and OPRK1 cAMP was determined using MC1R cAMP assay kit, MC4R cAMP assay kit, CRHR2 cAMP assay kit, CRHR2 cAMP assay kit, PTHR1 cAMP assay kit, and OPRK1 cAMP assay kit provided by Discoverx Inc (Fremont, California). The antagonist activity of the peptides on bradykinin receptor 2 (BDKRB2) and GnRH receptor (GnRHR or LHRHR) was determined using BDKRB2 inhibitor protein assay kit and GnRHR calcium flux assay kit provided by Discoverx, respectively.

[0186] To analyze Gs-coupled cAMP production, cAMP Hunter cell lines were expanded using frozen stocks and cells were seeded in white 384-well plates in a total volume of 20 μL and incubated at 37 °C for a period of time prior to assay. The modulation of cAMP was determined using DiscoverX HitHunter cAMP XS+ kit. To determine agonist activity, cells were incubated with samples to induce a biological response. The medium was aspirated from the cells and replaced with 15 μL of reagent (HBSS / 10 mM HEPES at a ratio of 2:1 with cAMP XS+ Ab). Intermediary dilutions of sample stocks were made to create 4X samples using assay buffer, and 5 μL of 4x sample was added to the cells and incubated at 37 °C or room temperature for 30 or 60 minutes. The vehicle concentration was 1%. The activity of the compounds was analyzed using CBIS data analysis suite (ChemInnovation, CA). For Gs-coupled agonist assays, the percent activity was calculated using the following formula: percent activity = 100% x (average RLU of test - average RLU of vehicle control) / (average RLU of MAX control - average RLU of vehicle control).

[0187] For inhibition protein assays, PathHunter cell lines (Discoverx Inc.) were expanded from frozen stocks and cells were seeded in a total volume of 20 μΐ^ into white 384-well plates and incubated at 37 °C for a period of time prior to assay. To measure agonist activity, cells were incubated with samples to induce a biological response. Samples stocks were diluted 5X in assay buffer to generate 5X samples, 5 μΐ^ of 5X sample was added to cells and incubated at 37 °C or room temperature for 90 to 180 minutes. The vehicle concentration was 1%. To measure antagonist activity, cells were pre-incubated with antagonist and then challenged with agonist at EC80 concentration. Samples stocks were diluted 5X in assay buffer to generate 5X samples, 5 μΐ^ of 5X sample was added to cells and incubated at 37 °C or room temperature for 30 minutes. The vehicle concentration was 1%. Then, 5 μΐ^ of 6X EC80 agonist in assay buffer was added to cells and incubated at 37 °C or room temperature for 90 or 180 minutes.

[0188] A single addition of 12.5 μΐ^ or 15 μΐ^ (50% v / v) of PathHunter Detection Reagent mix was then incubated for 1 hour at room temperature to generate the assay signal. After signal generation, the microplate was read using a PerkinElmer Envision™ chemiluminescent signal detector. The activity of compounds was analyzed using CBIS Data Analysis Suite (ChemInnovation, CA). For agonist mode assays, the percent activity was calculated using the following formula: Percent Activity = 100% x (Average RLU of test - Average RLU of vehicle control) / (Average RLU of MAX control ligand - Average RLU of vehicle control). For antagonist activity assays, the percent inhibition was calculated using the following formula: Percent Inhibition = 100% x (1 - (Average RLU of test - Average RLU of vehicle control) / (Average RLU of EC80 control - Average RLU of vehicle control)).

[0189] To analyze calcium flux, cell lines were expanded using frozen stocks and cells were seeded in a total volume of 20 μL into the bottom of clear poly-D-lysine coated black 384 well plates and incubated at 37°C for a period of time prior to assay. Assays were performed in lx dye loading buffer containing lx dye, lx Additive A and 2.5 mM probenecid (in HBSS / 20 mM HEPES). Cells were loaded with dye prior to assay. The media 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 samples to induce a biological response. After dye loading, cells were removed from the incubator and 10 μL of buffer containing HBSS / 20 mM HEPES was added. To determine EC80 for antagonist assays, agonist dose curves were generated and vehicle was added to the buffer during curve generation. Cells were incubated at room temperature for 30 minutes in the dark and the plate was equilibrated to room temperature. Samples stocks were diluted 4X in assay buffer. Agonist activity of compounds was determined using the FLIPR Tetra (MDS) by monitoring calcium mobilization for 2 minutes. Then, 10 μL of 4X sample (in HBSS / 20 mM HEPES) was added to the cells 5 seconds prior to the start of the assay.

[0190] To determine antagonist activity, cells were pre-incubated with samples and then challenged with agonist at the EC80 concentration. Samples stocks were diluted 3X in assay buffer. After dye loading, cells were removed from the incubator and 10 μL of 3X sample was added. Cells were incubated at room temperature for 30 minutes in the dark and the plate was equilibrated to room temperature. The vehicle concentration was 1%. Antagonist activity of compounds was determined using the FLIPR Tetra (MDS) by monitoring calcium mobilization for 2 minutes. Then, 10 μL of EC80 agonist (in HBSS / 20 mM HEPES) was added to the cells 5 seconds prior to the start of the assay. Activity of compounds was analyzed using the CBIS data analysis suite (ChemInnovation, CA) and percent activity was calculated using the following formula: percent activity = 100% x (mean RFU of test - mean RFU of vehicle control) / (mean RFU of MAX control ligand - mean RFU of vehicle control). For antagonist assays, percent inhibition was calculated using the following formula: percent inhibition = 100% x (1 - (mean RFU of test - mean RFU of vehicle control) / (mean RFU of EC80 control - mean RFU of vehicle control)).

[0191] To analyze the release of selected molecules from gels prepared with gel forming peptides, we use semi-permeable membranes (such as centrifugal filtration device) to detect the release levels. In these experiments, FITC-labeled small molecules, peptides, or nucleic acids were dissolved in solutions containing low concentrations of gel-forming or non-gel-forming peptides and loaded onto the upper chamber of a centrifugal filtration device. The chromatographic 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 fluorometry. The centrifugal filtration device was centrifuged at 2000 or 3000 rpm for 20 minutes, and the content of FITC-labeled molecules in the lower chamber was determined by fluorometry.

[0192] As shown in Table 1, we dissolved (or attempted to dissolve) the polypeptides 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 have an effect or no effect on the ability of compositions of the present invention to form liquid, solid, or semi-solid gels.

[0193] The gel-forming ability of a variety of secreted and therapeutic polypeptides or analogs thereof was determined, as shown in Table 1. All polypeptides were prepared by solid-phase synthesis, and the ability of these polypeptides to form semi-solid or liquid gels was analyzed at room temperature. To systematically analyze the gel-forming ability, the test was first performed in an aqueous solution at a peptide concentration of 11% w / w. The peptides analyzed included functional modulators of cell surface receptors, enzymes, complement factors, antibacterial peptides, immunomodulators, therapeutic peptides, cell-penetrating peptides, antigens, matrikines, and CGRP analogs, adrenomedullin, and adrenomedullin 2 (or intermedin). When the solubility in the aqueous vehicle was 11% w / w, adrenomedullin 1-52, pramlintide (amylin analog), oxytocin, kisspeptin, kappa receptor agonist, plamotropin, thrombopoietin analog (i.e., romiplostim analog), urocortin 3, bombesin receptor antagonist, ADM 2-52 acylate, and calphostin (SEQ ID NOS: 2, 4-11, 61, and 274) all formed transparent or opaque semi-solid gels in aqueous solution. Likewise, adrenomedullin analog (i.e., ADE43), CGRP, teduglutide (GLP-2 analog), thymosin alpha 1, GLP-1, gamma-MSH, acylated thymosin beta 4 analog, and GnRH analog (SEQ ID NOS: 1, 3, 12-15, 64, and 263) all formed liquid gels at a concentration of 11% w / w. On the other hand, a variety of other functional polypeptides (SEQ ID NOS: 16-47) presented as transparent liquid solutions or formed insoluble precipitates after 20 minutes at the selected concentration. Polypeptides that formed transparent aqueous solutions or insoluble precipitates did not possess the ability to form gel nanostructures. Abbreviations used in the table include SS (semi-solid gel) and LG (liquid gel).

[0194] A semi-solid gel formed from the peptide of SEQ ID NO: 1 is shown in Figure 1. The semi-solid gel maintains its position when held upright (A), tilted 90° (B), or inverted (C).

[0195] Table 1. Identification of secreted polypeptides that form gels in aqueous solution by themselves

[0196]

[0197]

[0198]

[0199] Studies of adrenomedullin and adrenomedullin 2 analogs (SEQ ID NOS: 48-58 [Table 1], 274-275 [Table 3]) show that selected analogs form liquid gels at 11% w / w and that minor modifications to the sequence can dramatically change the gel forming ability of the polypeptide. The parent peptide, wild-type truncated adrenomedullin analog (SEQ ID NO: 1), forms a liquid gel at 11% w / w and substitution of selected residues with D-amino acids results in the formation of a semi-solid gel by sequence 48. On the other hand, palmitoylation of sequence 274 results in an analog that forms a semi-solid gel at 6% w / w, a concentration at which the wild-type peptide and other adrenomedullin analogs do not. Other modifications in peptides of SEQ ID NOS: 49-58 result in analogs that retain the ability to form liquid gels at 11% w / w. Although gel formation is influenced by many physical and chemical factors, gel forming peptides (e.g., adrenomedullin and adrenomedullin 2) can contain sequence motifs that promote gel formation and changes in residues or side chains can alter the ability of these peptides to form gels.

[0200] These data also show that gel forming peptides [e.g., adrenomedullin, pramlintide (amylin analog), oxytocin, kisspeptin, kappa opioid receptor agonists, plamotrel, romidepsin analogs, urocortin 3, campath, CGRP, teduglutide (GLP-2 analog), thymosin alpha 1, GLP-1, GnRH, bombesin receptor antagonists, gamma-MSH, thymosin beta 4, and adrenomedullin analogs (SEQ ID NOS: 1-15, 61, 64, 26, and 274)] can be delivered by self-assembling gel formulations, i.e., without the use of additional gel forming agents. However, these gel forming peptides can contain gel formation enhancing motifs that promote self-assembly gel formation.

[0201] Based on this finding, we hypothesized that these self-assembling gel-forming peptides can contain a gelation-enhancing motif. Generally, polypeptides cannot form gel nanostructures, and these motifs can enhance the ability of polypeptides to form gels. Then, we tested the ability of calcitonin / amylin / CGRP / Adrenomedullin / Adrenomedullin 2 family peptide analogs to form semi-solid or liquid gels at room temperature by visual inspection, test tube tapping test, and test tube tilting test. To systematically analyze the gelation ability, the test was performed at a concentration of 11% w / w. As shown in Table 2, some modified and chimeric adrenomedullin / adrenomedullin 2 analogs can form semi-solid or liquid gels at a concentration of 11% w / w, as with wild-type adrenomedullin (SEQ ID NOs: 106-114 and 116-123). In contrast, after selected modifications (e.g., truncation and substitution; SEQ ID NOs: 101-105 and 115), the resulting polypeptides can form aqueous solutions or are insoluble at the same concentration.

[0202] Among these peptides, we found that analogs as short as 3 amino acids (SEQ ID NOs: 120-121) can form semi-solid gels at a concentration of 11% w / w or less. Although a single amino acid change in the adrenomedullin / adrenomedullin 2 peptide can result in an analog that remains in an aqueous solution or is insoluble, or forms a liquid or semi-solid gel, these data suggest that ADM / ADM2-derived peptides have a tendency to form liquid or semi-solid gels. This observation also supports the hypothesis that a gelation-enhancing motif derived from a self-assembling gel-forming peptide can convert a non-gel-forming peptide into a self-assembling gel-forming peptide. Although many truncated adrenomedullin / adrenomedullin 2 analogs can form semi-solid or liquid gels (SEQ ID NOs: 116-124 and 126-131), polypeptides composed of sequences from the corresponding regions of CGRP or amylin (SEQ ID NOs: 132-134) can remain in an aqueous solution or produce insoluble precipitates at the same concentration. Nonetheless, modification of the CGRP sequence with fragments of the adrenomedullin sequence can result in analogs that can form aqueous solutions, liquid gels, or semi-solid gels (SEQ ID NOs: 139-140). As previously described, a gelation control composed of a repeating sequence (SEQ ID NOs: 141-142) can also form semi-solid gels in an aqueous solution when the solubility is 11% w / w.

[0203]

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

[0205] The short sequence motifs in the adrenomedullin family peptides can form gels at low concentrations. Based on this discovery, we tested the hypothesis that inclusion of this gel formation enhancing motif can enhance the ability of other polypeptides to form gels. We tested a series of chimeric analogs for their ability to form semi-solid or liquid gels. These analogs included the gel formation enhancing motif found in SEQ ID NOS: 109, 119, and 120. The gel formation enhancing motifs in these polypeptides ranged in length from 3 to 17 amino acids. These motifs included motifs appended to the N-terminus, C-terminus, or incorporated into the sequence by amino acid substitution, or motifs appended to the side chain of an amino acid. The motifs were linked to the functional polypeptide either directly or through a miniPEG.

[0206] As shown in Table 1, Kisspeptin, Kappa opioid receptor agonist, Semorelin, Camptothecin, Bombesin receptor antagonist, Gamma-MSH, and Thymosin alpha 1 peptide (SEQ ID NOS: 6, 7, 8, 11, 13, 61, and 64) can form self-assembled gels by themselves at 11% w / w or lower concentrations, and coupling or replacing these polypeptides with the gel formation enhancing motif (Table 3, SEQ ID NOS: 208, 214, 235, 244, 255, and 260) did not significantly alter the ability of these polypeptides to form gels at 11% w / w concentrations. Although the gel formation ability of these modified peptides was not significantly improved, these modified gel forming peptides have an albumin binding acyl group and gel formation ability, and thus can have a longer circulating half-life. Therefore, these peptides are superior analogs, and these analogs are expected to have better pharmacokinetic and pharmacodynamic profiles compared to the wild-type analogs. Also, we found that acylated analogs of ADM2-52 and Thymosin beta 4 can form gels at 11% w / w concentrations, and these acylated analogs are superior analogs with an albumin binding acyl group and the ability to form gels by themselves.

[0207] On the other hand, we demonstrated that GnRH analogs, GnRH antagonists, vasopressin, sennatin, apralin, sennatin receptor antagonists, neocorynebacterium, enkephalin, substance P, calcitonin, pramlinin (amylin 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), adrenomedullin, thymosin α1, kallikrein inhibitors, and temporin The gel-forming abilities of antimicrobial peptide A, immunomodulatory glatiramer (or cropason), matrix-modified peptide 1, matrix-modified peptide 4, matrix-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 concentration, wild-type peptides only form liquid gels, aqueous solutions, or produce 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 natriuretic peptide (ANP), thymosin α1, thymosin β4, adrenomedullin, kallikrein inhibitors, and temporin are all potentially harmful substances. Antimicrobial peptides such as campstatin, 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, as well as oxytocin, ipalin peptide, neurotensin, inosin, PYY3-36, TAT cell penetration enhancement peptide, pramorelin, or urocortin 3 exhibit the characteristics of self-assembling gel-forming peptides, which can be used to slowly release therapeutic drugs in the human body that target 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).

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] By visual inspection, test tube tapping, and test tube tilting, we found that GnRH analog (SEQ ID NO: 15; Table 3) formed a liquid gel in distilled water at a concentration of 11% w / w. In contrast, GnRH analogs coupled with the gelation-enhancing motif (SEQ ID NO: 201) formed a semi-solid gel at concentrations of 11, 6, or 3% w / w. Likewise, while GnRH antagonist (SEQ ID NO: 27) failed to form a gel, GnRH antagonists coupled with the gelation-enhancing motif (SEQ ID NO: 202 and 203) formed a semi-solid gel at concentrations of 11% and 6% w / w. Wild-type vasopressin (SEQ ID NO: 17) failed to form a gel at a concentration of 11% w / w, but vasopressin coupled with the gelation-enhancing motif (SEQ ID NO: 204) formed a semi-solid gel at concentrations of 11% and 6% w / w. Wild-type oxytocin (SEQ ID NO: 5) was able to form a semi-solid gel at a concentration of 11% w / w, but failed to form a liquid or semi-solid gel at a concentration of 6% w / w. On the other hand, oxytocin analog coupled with the gelation-enhancing motif (SEQ ID NO: 205) formed 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 apelin (SEQ ID NO: 18) failed to form a gel at concentrations of 20%, 11%, or 6% w / w. On the other hand, apelin analogs coupled with the gelation-enhancing motif (SEQ ID NO: 206 and 262) formed a liquid gel at concentrations of 20% or 11% w / w. Likewise, wild-type neuromedin S (SEQ ID NO: 19) failed to form a gel at concentrations of 20% or 11% w / w, but neuromedin S coupled with the gelation-enhancing motif (SEQ ID NO: 207) formed a liquid gel at concentrations of 20% and 11% w / w. Wild-type bombesin (SEQ ID NO: 20) failed to form a gel at concentrations of 11% or 6% w / w. On the other hand, bombesin or bombesin receptor antagonist coupled with the gelation-enhancing motif produced analogs (SEQ ID NO: 209 and 255) that formed a semi-solid or liquid gel at concentrations of 11%, 6%, or 3% w / w. Unlike apelin, neuromedin S, and bombesin, kisspeptin peptide (SEQ ID NO: 6) formed a semi-solid gel at concentrations of 11% or 6% w / w, and kisspeptin analog coupled with the gelation-enhancing motif (SEQ ID NO: 208) formed a gel at the same concentrations.

[0219] Neuromedin B and enkephalin (SEQ ID NOs: 21 and 22) were unable to form gels at concentrations of 11% or 6% w / w. On the other hand, neuromedin B analogs coupled to the gelation-enhancing motif (SEQ ID NOs: 210 and 211) were able to form semi-solid gels at concentrations of 11% w / w or liquid gels at concentrations of 6% w / w. Enkephalin analogs coupled to the gelation-enhancing motif (SEQ ID NOs: 212 and 213) were even able to form semi-solid gels at concentrations of 3% w / w. However, the kappa opioid receptor agonist (SEQ ID NO: 7) was able to form semi-solid gels at concentrations of 11%, 6%, or 3% w / w. The kappa opioid receptor agonist analog coupled to the gelation-enhancing motif (SEQ ID NO: 214) was able to form semi-solid gels at concentrations of 11% and 6% w / w and was able to form a liquid gel at a concentration of 3% w / w.

[0220] Wild-type substance P analogs (SEQ ID NOs: 23 and 69) were only able to form liquid gels at concentrations of 20% w / w or were unable to form liquid gels at all. When substance P was coupled to the gelation-enhancing motif, the resulting analogs (SEQ ID NOs: 215, 253, and 254) were able to form semi-solid gels at concentrations of 11% or 20% w / w. The angiotensin receptor antagonist saralasin (SEQ ID NO: 24) was unable to form gels at concentrations of 11% w / w. When saralasin was coupled to the gelation-enhancing motif, the resulting analog (SEQ ID NO: 216) was able to form liquid gels at concentrations of 20% or 11% w / w.

[0221] Wild-type calcitonin (SEQ ID NO: 28) was unable to form a gel at a concentration of 11% w / w. On the other hand, calcitonin coupled with a gelation-enhancing motif (SEQ ID NO: 217) was 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 analogue pramlintide (SEQ ID NO: 4) did not form a gel at a concentration of 6% w / w, but was able to form a semi-solid gel at a concentration of 11% w / w. When pramlintide was coupled with a gelation-enhancing motif, the resulting analogue (SEQ ID NO: 218) was able to form a semi-solid gel at a concentration of 11% or 6% w / w. Unlike exenatide 4 (SEQ ID NO: 26), which was unable to form a gel at a concentration of 11% w / w, an exenatide 4 analogue coupled with a gelation-enhancing motif (SEQ ID NO: 219) was 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. Wild-type GLP-1 (SEQ ID NO: 14) was only able to form a liquid gel at a concentration of 11%. On the other hand, GLP-1 analogues coupled with a gelation-enhancing motif (SEQ ID NO: 220, 269, and 270) and an analogue substituted with a gelation-enhancing motif (SEQ ID NO: 221) were able to form a semi-solid gel at a concentration of 20% or 11% w / w. The GLP-2 analogue tedaglutide (SEQ ID NO: 12) was able to form a liquid gel at a concentration of 11%. On the other hand, a GLP-2 analogue coupled with a gelation-enhancing motif (SEQ ID NO: 222) formed a semi-solid gel at a concentration of 11% w / w and a liquid gel at a concentration of 6% w / w.

[0222] Melanotan I analogs, Afamelanoid and Melanotan II (SEQ ID NOs: 29 and 62) were unable to form a gel at concentrations of 20% or 11% w / w. In contrast, Afamelanoid analogs coupled with a gelation-enhancing motif (SEQ ID NOs: 223, 224, 256, 257, and 273) were able to form a semi-solid or liquid gel at concentrations of 20%, 11%, or 6% w / w. The ACTH1-24 peptide (SEQ ID NO: 33) was unable to form a gel at concentrations of 20% or 11% w / w. In contrast, ACTH1-24 analogs coupled with a gelation-enhancing motif (SEQ ID NOs: 225 and 226) were able to form a semi-solid gel at concentrations of 20% or 11% w / w. Setmelanotide (SEQ ID NO: 34), an agonist of the melanocortin 4 receptor (MC4R), was unable to form a gel at concentrations of 30%, 20%, or 11% w / w. When setmelanotide was fused to the gelation-enhancing motif of Pal-SSPHSY, the resulting analog (SEQ ID NO: 227) was able to form a gel at a concentration of 30% w / w. On the other hand, a setmelanotide analog with the Pal-HSY gelation-enhancing motif (SEQ ID NO: 228) was able to form a semi-solid gel at concentrations of 11%, 6%, or 3% w / w. Furthermore, we found that γ-MSH (SEQ ID NO: 64) was able to form a liquid gel at a concentration of 11% and was soluble at a concentration of 6% w / w. A γ-MSH analog coupled with a gelation-enhancing motif (SEQ ID NO: 260) was able to form a liquid gel at a concentration of 6% w / w.

[0223] The peptide YY receptor agonist PYY3-36 (SEQ ID NO: 30) was unable to form a gel at concentrations of 20% or 11% w / w. When the peptide was coupled to the gelation- enhancing motif, the resulting analogs (SEQ ID NOs: 229 and 264) were able to form a semi-solid or liquid gel at concentrations of 20% or 11% w / w. The corticotropin-releasing hormone receptor 2 (CRHR2) agonist urocortin 2 (UCN2) (SEQ ID NO: 31) was unable to form a gel at concentrations of 20% or 11% w / w. In contrast, the urocortin 2 analogs coupled to the gelation-enhancing motif (SEQ ID NOs: 230 and 265) were able to form a semi-solid or liquid gel at concentrations of 20% w / w. Unlike UCN2, urocortin 3 (UCN3) (SEQ ID NO: 10) was able to form a semi-solid gel at concentrations of 11% and 6% and a liquid gel at a concentration of 3% w / w. The UCN3 analog coupled to the gelation-enhancing motif (SEQ ID NO: 231) was still able to form a gel at these concentrations. On the other hand, parathyroid hormone (SEQ ID NO: 25) was unable to form a gel at concentrations of 11% or 6% w / w. When the N-terminus of this peptide was replaced with the gelation-enhancing motif, the resulting analog (SEQ ID NO: 232) was able to form 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) (SEQ ID NO: 32) was unable to form a gel at concentrations of 30%, 20%, or 11% w / w. However, when this peptide was coupled to the gelation-enhancing motif of Pal-SSPHSY, the resulting analog (SEQ ID NO: 233) was able to form a semi-solid gel at a concentration of 30% w / w. When HOE140 was coupled to the gelation-enhancing motif of Pal-HSY, the resulting analog (SEQ ID NO: 234) was able to form a semi-solid gel at a concentration of 20% w / w. Another bradykinin receptor antagonist (SEQ ID NO: 65) was unable to form a gel at concentrations of 11% or 6% w / w, while the modified analog (SEQ ID NO: 261) was 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.

[0224] Pramorelin (SEQ ID NO: 8) forms a semi-solid gel at 11% or 6% w / w and a liquid gel at 3% w / w. Similar to wild-type pramorelin, a pramorelin analog conjugated to a gelation-enhancing motif (SEQ ID NO: 235) forms a semi-solid gel at the same concentrations. Unlike pramorelin, semorelin (SEQ ID NO: 35) does not form a gel at 20% or 11% w / w. When a semorelin analog is fused to a shorter gelation-enhancing motif (SEQ ID NO: 237), the analog forms a semi-solid gel at 20% concentration, but not when fused to a longer gelation-enhancing motif (SEQ ID NO: 236).

[0225] Atrial natriuretic peptide (ANP) (SEQ ID NO: 41) does not form a gel at 20% or 11% w / w. In contrast, an ANP analog conjugated to a gelation-enhancing motif (SEQ ID NO: 238) forms a semi-solid gel at 20% and 11% w / w. The immunomodulator thymosin alpha 1 (SEQ ID NO: 13) forms a semi-solid gel at 20% w / w and a liquid gel at 11% w / w. When the N-terminus of thymosin alpha 1 is replaced with a gelation-enhancing motif (Pal-HSY) or a shorter gelation motif, a semi-solid gel is formed at 11% w / w (SEQ ID NOs: 240, 267, and 268). In contrast, an analog with a long gelation-enhancing motif (Pal-SSPHSY) (SEQ ID NO: 239) does not form a gel at the same concentration. In addition, we found that a thymosin beta 4 analog (SEQ ID NO: 263) forms a liquid gel at 11% and 20% w / w.

[0226] Vasoactive intestinal peptide (VIP) (SEQ ID NO: 67) does not form a gel at 20 or 11% w / w. In contrast, a VIP analog conjugated to a gelation-enhancing motif (SEQ ID NO: 266) forms a semi-solid gel at 11% w / w. In addition, we found that an adrenomedullin analog (SEQ ID NO: 274) forms a semi-solid gel at 20%, 11%, or 6% w / w, which is superior to wild-type adrenomedullin (SEQ ID NO: 2) or a shorter acylated analog (SEQ ID NO: 1).

[0227] The cell penetrating peptide (CPP) TAT (SEQ ID NO: 36) was not able to form a gel at a concentration of 20% or 11% w / w. When fused to the gelation-enhancing motif (SEQ ID NO: 241), the peptide was able to form a liquid gel at a concentration of 11% w / w. Likewise, the kallikrein inhibitor analog (SEQ ID NO: 37) was not able to form a gel at a concentration of 11% w / w. When the kallikrein inhibitor was fused to the gelation-enhancing motif, the resulting analog (SEQ ID 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. The Temporin A peptide, an antibacterial peptide, was fused to the gelation-enhancing motif, and the resulting analog (SEQ ID NO: 243) was able to form a semi-solid gel at a concentration of 11% w / w. On the other hand, the complement system modulator, compstatin (SEQ ID NO: 11), was able to form a semi-solid gel at a concentration of 11% w / w, while its analog containing the gelation-enhancing motif (SEQ ID NO: 244) was able to form a semi-solid gel at the same concentration. The immunomodulator glatiramer (or copaxone) (SEQ ID NO: 42) was not able to form a gel at a concentration of 20% or 11% w / w. When the peptide was coupled to the gelation-enhancing motif, the resulting analog (SEQ ID NO: 245) was able to form a semi-solid gel at a concentration of 20% w / w.

[0228] Many of the matrikines (or matrix-modifying peptides) that can modulate skin matrix enzymes (SEQ ID NOs: 39 and 44-47) were not able to form a semi-solid or liquid gel at a concentration of 11% w / w. However, when these peptides were fused to the gelation-enhancing motif, they were able to form a semi-solid or liquid gel at a concentration of 11% w / w. For example, the fusion peptides of SEQ ID NOs: 246, 247, 249, and 250 were all able to form a semi-solid gel at a concentration of 11% w / w; while the fusion peptide of SEQ ID NO: 248 was able to form a liquid gel at a concentration of 11% w / w and a semi-solid gel at a concentration of 20% w / w.

[0229] In another experiment, we investigated the effect of coupling known gel-forming peptide motifs on the ability of substance P analogs to form a semi-solid or liquid gel. While the substance P analog containing the gelation-enhancing motif (SEQ ID NO: 23) was able to form a semi-solid gel 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 result in analogs (SEQ ID NOs: 284-286; Table 3) that were able to form a gel.

[0230] To determine if peptide modifications would affect the biological activity of the gel forming peptides, we investigated the receptor modulatory activity of select gel forming peptides. Investigation of receptor activation activity against MC1R demonstrated that both the conjugated gel forming aphantom peptide (melanotan I) analog and the ACTH1-24 analog (SEQ ID NOS: 224 and 226) stimulated MC1R with EC50concentrations in the sub-nanomolar and nanomolar range, respectively (Table 4). The positive control, melanotan II peptide, had an EC50of 0.56 nM. Analysis of receptor activation activity against MC4R demonstrated that the conjugated gel forming setmelanotide analog (SEQ ID NO: 228) stimulated MC4R with an EC50of 0.15 nM. In this assay, the melanotan II peptide had an EC50of 2 nM, indicating that the select modified peptides also had superior receptor activation activity. Similarly, CRHR2 activity analysis demonstrated that the gel forming urocortin 2 and urocortin 3 analogs (SEQ ID NOS: 230 and 231) potently stimulated CRHR2 with EC50of 0.5 nM and 5.7 nM, respectively. The positive control, sauvagine, had an EC50of 3.6 nM, indicating that the select modified urocortin peptides also had superior receptor activation activity. Investigation of PTHR1 receptor activity demonstrated that the gel forming PTH analog (SEQ ID NO: 232) had an EC50of 14.1 nM against PTHR1, while the positive control, PTH (1-34) peptide, had an EC50of 0.8 nM. On the other hand, the gel forming kappa opioid receptor agonist (SEQ ID NO: 214) had an EC50of 272 nM against OPRK1, while the positive control, dynorphin A, had an EC50of 0.5 nM.

[0231] In addition, investigation of bradykinin receptor BDKRB2 activity using the Discoverx BDKRB2 inhibitor kit demonstrated that the conjugated bradykinin receptor antagonist (SEQ ID NO: 234) inhibited bradykinin-stimulated BDKRB2 signaling with an IC50of 53.3 nM (Table 4). In comparison, the positive control antagonist, HOE140, had an IC50of 131.8 nM, indicating that the gel forming antagonist was more potent than the wild-type peptide, HOE140. Furthermore, analysis of GnRH receptor activity using the Discoverx GNRHR calcium flux kit demonstrated that the gel forming GnRHR antagonist analogs (SEQ ID NOS: 202 and 203) inhibited LHRH-stimulated GnRHR signaling with IC50s of less than 0.51 nM, while the positive control, cetrorelix, had an IC50of 0.08 nM. Overall, the data demonstrated that these novel cell surface receptor ligand analogs were able to form hydrogels and retain the ability to modulate receptor activity.

[0232] Table 4. List of bioactivity of artificially synthesized gel forming ligands

[0233] Table 4. List of bioactivity of artificially synthesized gel forming ligands

[0233]

[0234]

[0235] To comprehensively 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 represents the ability to form a semi-solid gel, and LG represents the ability to form a liquid gel. Furthermore, blank represents the lack of gel-forming ability at the selected concentration. The t-test results show that coupling gel-forming enhancement motifs to 57 peptides significantly enhances their gel-forming ability.

[0236] Table 5. Thermograms showing the gel-forming tendency of ligands with gel-forming enhancement motifs.

[0237]

[0238]

[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 under equilibrium conditions. 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, we used a semi-permeable membrane (e.g., Release tests were conducted using a centrifugal filtration device. In these experiments, FITC-labeled therapeutic agents were dissolved in solutions containing low concentrations of gel-forming peptides or non-gel-forming peptides. Centrifugation separated freely moving soluble molecules in the lower chamber from soluble molecules polymerized with the gel nanostructure in the upper chamber. The motility of the 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 (SEQ ID NO: 119; 1 mg / 10 mL) reduced molecular motility by 3–40-fold. This reduction was dose-dependent, with much smaller reductions observed in samples with a gel-forming peptide concentration of 1 mg / 100 mL. In contrast, when FITC-labeled compounds were mixed with non-gel-forming peptides (SEQ ID NO: 133) in solution, molecular motility was not significantly reduced compared to the control sample.

[0241]

[0242]

[0243] Using The interaction of the gel-forming peptides with FITC-labeled small molecules (e.g., naloxone and dexamethasone) was investigated using the centrifugal filtration device. The results showed that the FITC-labeled molecules were retained in the upper chamber of the filter after mixing the molecules with the gel-forming peptides (SEQ ID NOs: 109 and 119) (Table 7). In the sample containing the peptide of SEQ ID NO: 109, the naloxone and dexamethasone molecules that passed through the filter were reduced by 30% compared to the control sample. Also, less than 15% of the naloxone and dexamethasone molecules passed through the filter in the sample containing the peptide of SEQ ID NO: 109 compared to the control sample. These data clearly indicate that the FITC-labeled molecules can aggregate or be encapsulated by the gel nanostructures prepared from the gel-forming peptides, which prevents the passage of these molecules through the filter. These data also imply that the solution of the gel-forming peptides can be used for slow delivery of therapeutic drugs even in the absence of observed gel structures / conformations.

[0244] To further demonstrate the long-term biological effects of the gel-forming peptides in vivo, we investigated the effect of a gel-forming melanocyte-stimulating hormone (MSH) analog (SEQ ID NO: 224) on the color change of the skin of the bullfrog (Rana catesbeiana) (Fig. 6). Figure 2 The skin color of the bullfrog changed dramatically within 2 hours after intraperitoneal injection of either the wild-type analog (afamelanotide, SEQ ID NO: 29; 100 nmol / kg body weight) or the gel-forming analog (SEQ ID NO: 224; 100 nmol / kg body weight). The skin color changed from green to black. The control animals injected with saline still had green skin. Four or 14 days after injection, only the bullfrogs injected with the gel-forming MSH analog had dark skin due to the expansion of melanocytes in the skin, which demonstrated the long-term effect of the gel-forming analog.

[0245] Table 7. Interaction of gel-forming peptides with small molecule drugs

[0246]

[0247] All publications and patent applications cited in this specification are herein incorporated 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 application, the application has been described in terms of specific embodiments only. It will be appreciated that, in light of the instant disclosure, those skilled in the art will be able to devise many modifications and variations of the specific embodiments without departing from the scope of the present application. Furthermore, it should be understood that the structure of the proteins can be changed without affecting the kind or amount of biological effect, in view of the biological functional equivalency. All such modifications and variations are intended to be included within the scope of the appended claims.

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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