EP3764988A1 - Gel-forming polypeptides - Google Patents
Gel-forming polypeptidesInfo
- Publication number
- EP3764988A1 EP3764988A1 EP19768400.4A EP19768400A EP3764988A1 EP 3764988 A1 EP3764988 A1 EP 3764988A1 EP 19768400 A EP19768400 A EP 19768400A EP 3764988 A1 EP3764988 A1 EP 3764988A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analogs
- receptors
- gel
- peptide
- receptor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
- A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/585—Calcitonins
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/31—Fusion polypeptide fusions, other than Fc, for prolonged plasma life, e.g. albumin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
Definitions
- compositions and methods for sustained delivery of therapeutic agents provided as stable aqueous formulations of gel-forming polypeptide compounds that deliver sustained release of therapeutic agents.
- polypeptides represent a middle group between ‘small molecule’ drugs and larger injectable biologies and nucleic acid-based therapeutics.
- Therapeutic peptides and proteins are generally administered parenterally, e.g., by subcutaneous injection.
- An advantage of synthetic peptides is that they can incorporate non-natural amino acids to reduce instability and proteolytic cleavage liabilities as well as provide better manufacturing reproducibility.
- sustained-release formulation or half-life extension technologies have been developed to deliver polypeptide and protein therapeutics over a prolonged period of time.
- the use of sustained release formulations also allows the delivery of therapeutics to selected tissues or organs, in order to minimize systemic adverse effects.
- Techniques employing slow-release nano-structures can improve pharmacokinetic and pharmadynamic properties of therapeutics following delivery into the body, or following topical treatments.
- This technique can provide sustained regulation of cellular signaling and reduce the peak-to-trough effect of therapeutic agents (e.g. small molecule drugs, peptides, hormones, proteins, nucleic acids, cells, or pro-drugs).
- therapeutic agents e.g. small molecule drugs, peptides, hormones, proteins, nucleic acids, cells, or pro-drugs.
- Biocompatible nanoscale drug carriers have the potential to provide substantial improvement of drug delivery, and to improve efficacy and reduce systemic side effects.
- oil suspension and crystal particle suspension have been used extensively for sustained delivery of small molecule drugs. In these procedures, the oil and solid particle are used to create a barrier for the dispersion of soluble therapeutics.
- Polymeric gels have been used for the delivery of small molecules and polypeptides, including gel-forming peptides, such as lanreotide. These gel-forming peptides form polymers and aqueous gel at high concentrations, in contrast to the majority of polypeptides, which are either present as aqueous solution or becomes an insoluble precipitate at high concentrations.
- Biocompatible polymeric peptides and hydrogels have a wide variety of applications in biotechnology and medicine, especially in the controlled delivery and release of drugs and therapeutics, in addition to functioning as supports in tissue engineering (see, for example, (U.S. Patents Nos. 5,034,229; 5,057,318; and 5,110,596).
- Hydrogels can contain networks of monomers that interact to give self-supporting, hydrogen-bonded nanostructures, or that form through hydrophobic interactions and Van der Waals force.
- polymeric microcapsules and matrixes with polylactic polymers have been used for the delivery of small molecule, peptide, and protein drugs (e.g., Kent, et al., U.S. Patent No. 4,675,189).
- Hydrogel polymers may comprise PLA (poy-lactic acid), PGA (poly-glycolic acid), poly lactide-co-glycolides (PLGA), polyalkylcyanoacrylates, poly-e-caprolactones, poly-N-isopropyl acrylamide (NIPA), cellulose ether, hyaluronic acid, lecithin, polyacrylic acid, poly-s-caprolactone, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), and agarose as well as (co)polymer agents obtained by combination or modification of these.
- PLA poly-lactic acid
- PGA poly-glycolic acid
- PLGA poly lactide-co-glycolides
- NIPA polyalkylcyanoacrylates
- poly-e-caprolactones poly-N-isopropyl acrylamide
- NIPA poly-N-isopropyl acrylamide
- cellulose ether hyaluronic acid
- Polymers such as PLGA polymers, are progressively eroded when administered into the body. These types of sustained-release formulations have been used to deliver, for example, GnRH analogs over weeks or months. Such formulations have benefits including increased dosage accuracy and improved patient compliance. However there are disadtantages to the use of polymeric hydrogels. Many formulations contain a support material and a pharmaceutical composition, where the support material may have immunogenic properties. Supporting materials may not degrade after administration and will therefore accumulate in the body. Formulations may require complex fabrication or manufacturing procedures. Furthermore, the therapeutic cargo typically represents only a small portion of the therapeutic preparation.
- self-assembling peptide hydrogels are those made of modified oligopeptides with repeated amino acid sequences, such as those made of tetraphenylethylene-capped dipeptides (e.g., tetraphenylethylene-diglycine (TPE-GG), Yeh et al. 2016 A novel nanostructured supramolecular hydrogel self-assembled from tetraphenylethylene- capped dipeptides. Soft Matter.
- TPE-GG tetraphenylethylene-capped dipeptides
- self-assembled gel can be derived from mix of bifunctional peptide LHRH- MPGANLS and siRNA (Liu et al. 2017 Target-specific delivery of siRNA into hepatoma cells' cytoplasm by bifunctional carrier peptide. Drug Deliv Transl Res. 7:147-155), mix of self assembling peptide and heparin (Liu et al. 2016 Sustained release of hepatocyte growth factor by cationic self-assembling peptide/heparin hybrid hydrogel improves b-cell survival and function through modulating inflammatory response. Int J Nanomedicine.
- MAX1 analogs e.g., VKVKVKVKV(D)PPTKVKVKVKV-NH2
- VKVKVKVKV(D)PPTKVKVKVKV-NH2 a group of repeat-sequence-containing hydrogel named MAX1 analogs
- VKVKVKVKV(D)PPTKVKVKVKV-NH2 a study of a group of repeat-sequence-containing hydrogel named MAX1 analogs (e.g., VKVKVKVKV(D)PPTKVKVKVKV-NH2) indicated that the self-assembly and hydrogelation rates and mechanical stiffness of the analogs vary drastically despite that all analogs formed networks of entangled fibrils with the similar diameters (Chen et al. 2014 Tuning gelation kinetics and mechanical rigidity of b-hairpin peptide hydrogels via hydrophobic amino acid substitutions. 6:14360-8).
- compositions and methods are provided that relate to gel-forming polypeptide therapeutics.
- Formulations of these gel-forming polypeptides provide advantages, in that therapeutically effective levels of the polypeptide are maintained in vivo for extended periods of time (i.e. , increased resident time), thereby increasing treatment intervals and reducing the peak- to-trough effect and total dose of active ingredient, relative to conventional formulations.
- methods are provided for formulation and manufacture of such gel-forming peptides.
- peptides that naturally form gels are identified, and may be formulated for optimal delivery of the gel-forming peptide and for therapeutic use according to the methods described herein.
- peptides that do not naturally form gels, or that only weakly form gels are modified by joining to a gel-forming-enhancing motif to gain or increase the gel-forming capability, and may be formulated for therapeutic use according to the methods described herein.
- composition and therapeutic formulations comprising an effective dose of a therapeutic agent in a formulation that adopts a gel configuration on administration.
- the therapeutic formulation may, for example, provide the therapeutic peptide in a solution or liquid gel, which transitions to a gel upon administration.
- the resulting gel nanostructure can function as a physical barrier to reduce the diffusion or dispersion of the therapeutic agent, to reduce proteolytic degradation, and to reduce renal clearance of the therapeutic agent, thereby increasing the resident time.
- the therapeutic agent is a gel-forming polypeptide, which is optionally engineered to enhance gel formation, and reduce the minimal concentration of the gel-forming polypeptide required to provide a gel configuration in vivo.
- the therapeutic agent is provided in combination with a gel-forming polypeptide.
- the formulation is free of artificial polymeric gel carrier such as silicon, chitosan, PLA, PGA, PLGA, etc.
- the formulation is provided in a very low ionicity aqueous solution.
- the formulation is provided in a two part container, where a dry form of the peptide is mixed immediately prior to administration with an aqueous excipient, which may have very low ionicity.
- gel-forming polypeptides comprise salts of polypeptide hormones and their agonistic, antagonistic, or nonfunctional analogs of ligands for cell surface receptors, e.g. such as GPCRs, tyrosine kinase receptors, etc., and therapeutic agents that act on cell surface targets or enzymes, and salts of biologically active or inactive analogs thereof.
- Soluble, gel-forming polypeptides also include gel-forming carrier peptides and prodrugs in the form of a gel nanostructure.
- Gel-forming polypeptides which comprise a portion of a native polypeptide hormone sequence or analog of a native hormone sequence, can be used as carrier polymers and, once gelled, are capable of controlling the delivery of a therapeutic agent or agents within the gel at a rate suitable for therapeutic use, while avoiding the concern of immunogenic responses.
- a gel-forming polypeptide or combination of polypeptides forms a self-assembled gel in aqueous solution.
- the gel-forming polypeptide may have a physiologically relevant bioactivity or a therapeutic activity, or may provide a purely structural role.
- the formulation may comprise additional bioactive molecules.
- the gel-forming polypeptide in the gel can be released into the physiological environment over an extended period of time, along with the optional additional bioactive agent(s).
- the gel nanostructure also serves as a physical barrier that decreases the solubility of monomeric peptide and enzymatic degradation of the gel-forming polypeptide.
- a gel-forming therapeutic agent including without limitation a peptide agent, comprises or is modified to comprise one or more gel-enhancing motifs, which facilitate intermolecular bond formation.
- An enhancing motif may be a self-assembly-enhancing motif sequence, which promotes self-assembly of polypeptides in solution.
- the gel-formng motif induces self-assembly of the polypeptide into an aqueous gel.
- the gel-forming-enhancing motif is optionally derived from a secreted circulating human hormone.
- the gel-forming-enhancing motif may be fused or otherwise conjugated to a therapeutic agent that functions as a regulator of a cell surface receptor, a biological function, or an enzyme, or as an antigen.
- the conjugation may be covalent or non-covalent.
- a gel-forming polypeptide is modified to comprise a signaling motif that activates or antagonizes a target receptor-mediated signaling pathway, a biological process, or an enzyme reaction.
- the therapeutic agent is a small molecule, a peptidomimetic, a biological drug, a nucleic acid, an antigen, an organelle, or a cell.
- a gel-enhancing motif comprises a fragment of a secreted circulating peptide ligand of a cell surface receptor, e.g. a GPCR, including without limitation those peptides identified herein as naturally gel-forming.
- a gel-enhancing motif is at least about 2, and not more than about 52 amino acids in length, and may be up to 5, up to 7, up to 9, up to 12, up to 15, up to 18, up to 21 , up to 24, up to 52 amino acids in length.
- the gel-enhancing motif comprises or consists of a fragment of adrenomedullin, adrenomedullin 2, CGRP, or a chimeric polypeptide derived therefrom.
- the gel-enhancing motif comprises or consists of a 2-52 amino acid fragment with at least 50%, at least 75%, at least 90% or 100% sequence identity to Pal-KVQKLSAPVDPSSPHSY. In some embodiments, the gel-enhancing motif comprises or consists of a 6-amino-acid segment with at least 50%, at least 75%, at least 90% or 100% sequence identity with Pal-SSPHSY. In some embodiments, the gel-enhancing motif comprises or consists of the 3-amino-acid segment Pal- HSY or Pal-KSY, with the proviso that a peptide naturally containing a Y or SY residue at the amino terminus may be modified by the addition of just those amino acids required to make the HSY sequence.
- the gel-enhancing motif comprises or consists of the 2- amino-acid segment Pal-HS, with the proviso that a peptide naturally containing a H, Y, or S residue at the amino terminus may be modified by the addition of just those amino acids required to make the Pal-HS sequence.
- an amino acid sequence may be substituted at one or more residues to generate the HSY or HS motif.
- a KSY motif may alternatively be used.
- the gel-enhancing motif comprises a conjugated palmitate residue.
- the motif is linked to a peptide with mini-PEG at the carboxy or amino terminus, or to a side chain of an amino acid.
- the peptide comprises a detectable marker, e.g. FITC.
- a therapeutic agent and gel-enhancing motif are covalently linked as portions of a self-assembling gel-forming molecule.
- a therapeutic agent is encapsulated by the self-assembling gel-forming polypeptide selected from the list consisting of SEQ ID NOS: 1-15, 48-58, 61 , 64, 106-114, 116-124, 126-131 , 139-140, and 201-275.
- a gel-forming therapeutic; or combination of gel-forming peptide and a therapeutic is administered to a subject and the gel-forming polypeptide/therapeutic localizes in the delivery site and has an increased resident time.
- the gel-forming polypeptide is a prodrug that become an active therapeutic agent only after it is dissociated from the gel nanostructure, and carriers of an encapsulated therapeutic agent.
- Gel-forming polypeptides include, without limitation, the naturally gel-forming and engineered gel-forming polypeptides set forth in Tables 1 , 2 and 3 herein.
- Such polypeptides include CGRP, ADM, ADM2, Pramlintide, oxytocin, kisspeptin, Pralmorelin, thrombopoietin peptide analog, Romiplostim analog, urocortin 3, Substance P, GLP-1 , GnRH analog, and GLP- 2 receptor ligand analog, a bombesin receptor antagonist, gamma-MSH, and gel-forming ligands for opioid receptors, and their analogs thereof; etc.
- Gel-forming polypeptides also include polypeptides that contain a self-assembly-enhancing motif conjugated to a functional sequence, which include without limitation ADM, ADM2, GnRH, GnRH antagonist, vasopressin, oxytocin, apelin, neurotensin, kisspeptin, bombesin, deltorphin, enkephalin, substance P, saralasin, calcitonin, Pramlintide (amylin analog), exenatide 4, GLP-1 , Teduglutide (GLP-2 analog), afamelanotide (melanotan I), melanotan II, gamma-MSH, ACTH1-24, setmelanotide, PYY3-36, urocortin 2, urocortin 3, parat
- a method for treating a patient with a gel-forming formulation comprising administering an effective dose and concentration of a therapeutic agent in a formulation that adopts a gel configuration on administration.
- the therapeutic formulation may be a liquid solution or a liquid gel prior to administration.
- the therapeutic formulation may be administered by, for example, intramuscular, subcutaneous, intradermal, or intraperitoneal injection, infusion, or administered intranasally, intrauterinely, intraocularly, topically, orally, or intrarectally, wherein the composition forms a gel after interaction with the patient's bodily fluids.
- FIG. 1A-1C Graphic representation of a peptide gel formed by SEQ ID NO: 1 peptide.
- FIG. 2 Bull frogs treated with the MSH gel made of SEQ ID NO: 224 had lasting color change when compared to those treated with the wild-type analog SEQ ID NO: 29 (100 nmoles/kg body weight).
- compositions and methods are provided for the the formulation and use of gel-forming polypeptides.
- cost-effective processes can be used for formulation, preparation and manufacture.
- the immunogenicity of the formulation is low, as the gel-forming polypeptides are derived from simple secreted human peptide hormone sequences.
- the formulations allow the therapeutic agents to retain a volume of distribution similar to that of their wild-type or native counterparts.
- the composition according to the present invention may be prepared by simply mixing the active ingredient and an aqueous solution, thus dramatically reducing the volume, cost, and manufacturing time for these therapeutic candidates when compared to the preparation made with known sustained-release formulations.
- certain secreted polypeptide hormones can self-assemble to form gel nanostructures without the addition of an artificial polymer or other carrier matrix to control the peptide's release profile.
- These peptides may contain a gel-forming-enhancing motif, and automatically gel upon interaction with an aqueous solution.
- Peptide formulations that exploit this ability dramatically increase the loading capacity of a therapeutic agent, and at the same time reduce the cost and manufacturing time as compared to known sustained-release artificial polymer-based formulations.
- the use of these gel-forming polypeptides eliminates the need to use organic solvents in the formulation of a sustained release formulation.
- GPCRs and other surface receptors play a pervasive physiological role and are the leading target class for pharmaceuticals.
- researchers have designed novel agonists and inhibitors of a variety of GPCRs and other cell surface receptors to allow the regulation of receptor signaling in patients, however a major drawback of the usage of many polypeptide and nonpolypeptide therapeutics is their short half-life and resident time in vivo.
- the short half-life is commonly associated with a sensitivity to proteolysis and/or renal clearance.
- An important technique to lengthen in vivo half-life and resident time is the use of self-assembling peptide gel formulations.
- certain therapeutic polypeptides such as lanreotide can self-assemble into gel nanostructures in aqueous solution. After injecting into a patient, the lanreotide gel then gradually release the lanreotide monomer from the gel depot, allowing the sustained release of lanreotide in the circulation. This allows the prolonged modulation of somatostatin receptors in patients.
- Peptides that are identified to form gels by themselves can be formulated to extend the resident time of these molecules in vivo.
- Peptides that do not naturally form gels can be engineered by conjugation to gel-enhancing sequence motifs.
- the gel-enhancing motif can be conjugated to any part of a therapeutic to promote gel-forming capability without dramatically changing the mass of the resulting molecules.
- Gel-enhancing motifs can be used to promote gel forming capability of small molecule drugs, peptides, proteins, or other biomolecules and extend the resident time in vivo.
- compositions comprising first self assembling peptide incorporating a first biological signal and a second self-assembling peptide incorporating a second biological signal.
- the practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, peptide chemistry and immunology within the scope of those of skill in the art. Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook et al. , 1989);“Oligonucleotide Synthesis” (M.J. Gait, ed., 1984);“Animal Cell Culture” (R.l. Freshney, ed., 1987); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (D.M. Weir & C.C.
- the terms“treatment,”“treating,” and the like refer to obtaining a desired pharmacologic and/or physiologic effect.
- the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
- Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease or a symptom of a disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it (e.g., including diseases that may be associated with or caused by a primary disease; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
- the terms“individual,”“host,”“subject,” and“patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, primates and humans.
- hydrogel refers to a liquid, semi-solid, or solid three-dimensional network that spans the volume of a liquid medium.
- a hydrogel is a network of polymer chains that are hydrophilic, sometimes found as a colloidal gel in which water is the dispersion medium.
- Hydrogels are highly absorbent, and may comprise greater than 50% water, greater than 60%, greater than 70%, greater than 80%, and may be from about 85% to about 99.9% water. Hydrogels are characterized by an elastic modulus which exhibits a pronounced plateau extending to times at least of the order of seconds, and by a viscous modulus which is considerably smaller than the elastic modulus in the plateau region.
- the elastic modulus of hydrogels is generally within 10-10 2 kPa range.
- a“clear aquous solution” is a solution comprised of a flowing aqueous solution wherein preferably 95% ⁇ 5% of the polypeptide solute is completely dissolved so that the solution is relatively transparent and freely movable with a tilt of the container or a snap of the finger.
- a clear solution may have trace amounts of observable solute or particles depending on the purity of the solutes used. However, such particles are not sufficient to create a milky or cloudy appearance.
- the gel-forming polypeptides assume a liquid gel or semisolid gel appearance. The most critical criterium for this assessment is the increase in aqueous viscosity.
- a liquid gel appearance is that exhibit consistant increase in viscosity and to exhibit a liquid gel as exemplified by a honey or glycerine.
- the liquid gel could be clear or exhibit an opaque appearance.
- a liquid gel does not apply to a suspension which is a heterogeneous mixture composed of insoluble particles.
- a liquid gel could as a continuous phase, whereas the gel is a homogeneous, single-phase solution with high viscosity, but does reach a semisolid appearance.
- a semisolid gel is a formulation comprised of a solvent and one or more solutes wherein the solute may be completely or partially dissolved, so that the formulation assumes a transparent or consistent opaque appearance, and the high viscosity render the solution rather immovable upon the snap of a finger or a tilt of the container.
- the semisolid gel composition does not apply to a suspension which is a heterogeneous mixture composed of high levels of observable insoluble particles, whereas a solution is not homogeneous.
- An insoluble precipitates by a suspension or a solution is an aqueous formulation comprised of one or more solutes wherein the solute is not dissolved or partially dissolved, so that the formulation is not a consistent composition that could be as liquid as a clear or opaque solution or more viscous as a gel.
- a semisolid suspension according to the invention includes (1) a semisolid, soluble, gel-forming polypeptide particles and up to 50 percent, by weight, of a pharmaceutically acceptable formulation to provide the semisolid consistency.
- the salt form of the gel-forming polypeptides that can be used in the compositions of the invention should gel in bodily fluids when administered to a patient, and, once gelled, are capable of sustained delivery of the peptide at a rate suitable for a therapeutic use of the drug.
- a self-assembled gel-forming polypeptide is a polypeptide that forms a gel upon contact with an aqueous solution, including for example physiological fluids such as blood, etc.
- Self- assembly peptide gel formation has been defined as the spontaneous organization of peptides in solution via non-covalent interactions (Whitesides et al.; Science 1991 , 254, 1312-1319). These gels create a nanostructure that modify rheological properties.
- peptide gels belong to two major categories: one mainly consists of sequences of alternating hydrophobic and hydrophilic amino acids (an amphiphilic peptide), and the other is peptide with a hydrophobic group and forms nanofibers (a peptide amphiphile)(Mata et al., 2010 Biomaterials 31 , 6004; Shah et al., 2010 P Natl Acad Sci USA 107, 3293; Huang et al. 2010 Biomaterials 31 , 9202; Webber et al., 2011 P Natl Acad Sci USA 108, 13438. Capito, et. al.; 2008 Science 319, 1812-1816).
- peptide amphiphiles with alkyl tails on one terminus include amphiphiles derived from peptide motifs found in collagen.
- a peptide amphiphile is usually formed through hydrogen-bonding between beta-sheet forming amino acids and hydrophobic interactions of the tails.
- peptides consisting of alternating hydrophobic and hydrophilic amino acids can self-assemble into gels with parallel or anti-parallel beta sheets.
- peptide gels may include extended cross-beta structure that form tubes and fibers, and alpha-helices that form triple-helix collagen-like structures, helix barrrels, or coiled-coil bundles.
- Growth factors have been physically entrapped in polypeptide hydrogels, either covalently linked or bound electrostatically to anionic polymers or structures such as heparin.
- Drawbacks to these and related systems include non-specificity of bound growth factors or a requirement for degradation of covalent bonds to achieve the desired effect.
- these peptides may incur adverse immunogenic responses after administration due to artificial sequences with repeated motifs (e.g., the FKFEFKFE motif- and RADARADA motif-containing gel-forming peptides), or derivation from large proteins such as fibronectin and laminin or from noncirculating proteins such as amyloid protein.
- a peptide gel made of native or modified secreted circulating polypeptide sequences, as described herein, has a lower potential for immunogenic response and a better safety profile. Because most polymeric carrriers usually represent 75-99% of the mass in sustained release formulation, the use of a gel-forming therapeutics can increase the effective load of therapeutic drugs by more than 20-50 fold.
- sustained release means release of the active substance or a carrier material in a patient such that the patient receives a dose of the therapeutic substance over a prolonged period of time.
- the proportion of therapeutic agents in the composition that will be released in a time window will be determined by the release rate which it is desired to achieve.
- gel-forming peptides are formulated for delivery in an aqueous exceipient, where the excipient may be very low ionicity.
- excipients may be defined as less than about 50 mM total ion concentration, less than about 25 mM total ion concentration, less than about 15 mM total ion concentration, less than about 10 mM total ion concentration, less than about 5 mM total ion concentration, less than about 2.5 mM total ion concentration, less than about 1 mM total ion concentration, less than about 0.1 mM total ion concentration, less than about 0.01 mM total ion concentration, less than about 0.001 mM total ion concentration, less than about 0.0001 mM total ion concentration, less than about 0.00001 mM total ion concentration.
- the salts that are present may include, for example, Na + , K + , CI-, Mn ++ , Mg ++ , Ca ++ , P0 4 -, etc.
- carrier peptide refers to a gel-forming peptide that will form a gel in aqueous solution or upon contact with bodily fluids, and that can be used to encapsulate or associate with therapeutic agents for sustained delivery.
- prodrug peptide refers to a gel-forming peptide that cannot act on a biological target while in the gel nanostructure and only becomes active upon dissociation from the gel nanostructure.
- a gel-enhancing motif is an amino acid sequence that enhances the ability of a small molecule, a peptide, a biological, an antigen, a nucleotide, or a therapeutic molecule to form a gel nanostructure in aqueous solutions.
- a gel-enhancing motif may comprise or consist of a fragment of a secreted circulating peptide ligand of a cell surface receptor.
- a gel enhancing motif is at least about 2, and not more than about 52 amino acids in length, and may be up to 5, up to 7, up to 9, up to 12, up to 15, up to 18, up to 21 , up to 24, up to 52 amino acids in length.
- the gel-enhancing motif comprises or consists of a fragment of adrenomedullin, adrenomedullin 2, CGRP, or a chimeric polypeptide derived therefrom.
- the gel-enhancing motif comprises or consists of a 2-52 amino acid fragment with at least 50%, at least 75%, at least 90% or 100% sequence identity to Pal- KVQKLSAPVDPSSPHSY.
- the gel-enhancing motif comprises or consists of a 6-amino-acid segment with at least 50%, at least 75%, at least 90% or 100% sequence identity with Pal-SSPHSY.
- the gel-enhancing motif comprises or consists of the 3-amino-acid segment Pal-HSY or Pal-KSY, with the proviso that a peptide naturally containing a Y or SY residue at the amino terminus may be modified by the addition of just those amino acids required to make the HSY sequence.
- the gel-enhancing motif comprises or consists of the 2-amino-acid segment Pal-HS, with the proviso that a peptide naturally containing a H, Y, or S residue at the amino terminus may be modified by the addition of just those amino acids required to make the Pal-HS sequence.
- an amino acid sequence may be substituted at one or more residues to generate the HSY or HS motif.
- a KSY motif may alternatively be used.
- the gel-enhancing motif comprises a conjugated palmitate residue.
- the motif is linked to a peptide with mini- PEG at the carboxy or amino terminus, or to a side chain of an amino acid.
- the peptide comprises a detectable marker, e.g. FITC.
- additional agents can be conjugated to a therapeutic polypeptide to enhance gel formation, including without limitation fatty acids, pegylation, and the like.
- fatty acids can enhance the gel-forming capability, and enhance subsequent dissociation of the nanostructure due to the presence of chemical bonds that are susceptible to esterase-mediated cleavage of the fatty acid.
- PEGylated refers to polypeptides that are chemically modified with one or more polyethylene glycol moieties, i.e., PEGylated.
- the polypeptide may be coupled directly to PEG (i.e., without a linking group) through an amino group, a sulfhydryl group, a hydroxyl group, or a carboxyl group.
- the PEGylated polypeptide contains a PEG moiety on only one amino acid. In other embodiments, the PEGylated polypeptide contains a PEG moiety on two or more amino acids.
- PEG is attached to the polypeptide via a linking group.
- the linking group is any biocompatible linking group, where "biocompatible" indicates that the compound or group is non-toxic and may be utilized in vitro or in vivo without causing injury, sickness, disease, or death.
- PEG can be bonded to the linking group, for example, via an ether bond, an ester bond, a thiol bond or an amide bond.
- Suitable biocompatible linking groups include, but are not limited to, an ester group, an amide group, an imide group, a carbamate group, a carboxyl group, a hydroxyl group, a carbohydrate, a succinimide group (including, for example, succinimidyl succinate (SS), succinimidyl propionate (SPA), succinimidyl butanoate (SBA), succinimidyl carboxymethylate (SCM), succinimidyl succinamide (SSA) or N-hydroxy succinimide (NHS)), an epoxide group, an oxycarbonylimidazole group (including, for example, carbonyldimidazole (CDI)), a nitro phenyl group (including, for example, nitrophenyl carbonate (NPC) or trichlorophenyl carbonate (TPC)), a trysylate group, an aldehyde group, an isocyanate group, a vinylsulfone group, a
- the PEG groups will preferentially react with surface residues as opposed to buried residues, which provides practical, cost-efficent procedures for protein PEGylation and synthesis of the PEGylated polypeptides of the invention.
- Methods for attaching a PEG to a polypeptide are known in the art, and any known method can be used in accordance with the methods of the invention to produce a PEGylated polypeptide of the invention. See, for example, by Park et al, Anticancer Res., 1 :373-376 (1981); Zaplipsky and Lee, Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, J. M. Harris, ed., Plenum Press, NY, Chapter 21 (1992); U.S. Patent No. 5,985,265; U.S. Pat. No. 5,672,662 (Harris, et al.) and WO 97/03106.
- the PEG is a monomethoxy PEG molecule that reacts with primary amine groups on the polypeptide.
- Methods of modifying polypeptides with monomethoxy PEG via reductive alkylation are known in the art. See, e.g., Chamow et al. (1994) Bioconj. Chem. 5:133-140.
- Polyethylene glycol suitable for conjugation to a polypeptide is soluble in water at room temperature, and has the general formula R(0-CH 2 -CH 2 ) n O-R, where R is hydrogen or a protective group such as an alkyl or an alkanol group, and where n is an integer from 1 to 1000. Where R is a protective group, it generally has from 1 to 8 carbons.
- PEG has at least one hydroxyl group, e.g., a terminal hydroxyl group, which hydroxyl group is modified to generate a functional group that is reactive with an amino group, e.g., an epsilon amino group of a lysine residue, a free amino group at the N- terminus of a polypeptide, or any other amino group such as an amino group of asparagine, glutamine, arginine, or histidine, to facilitate covalent modification of a polypeptide with PEG.
- an amino group e.g., an epsilon amino group of a lysine residue, a free amino group at the N- terminus of a polypeptide, or any other amino group such as an amino group of asparagine, glutamine, arginine, or histidine, to facilitate covalent modification of a polypeptide with PEG.
- PEG is derivatized so that it is reactive with free carboxyl groups in the polypeptide.
- Suitable derivatives of PEG that are reactive with the free carboxyl group at the carboxyl-terminus of polypeptide include, but are not limited to PEG-amine, and hydrazine derivatives of PEG (e.g., PEG-NH-NH 2 ).
- PEG is derivatized such that it comprises a terminal thiocarboxylic acid group, -COSH, which selectively reacts with amino groups to generate amide derivatives.
- -SH a terminal thiocarboxylic acid group
- selectivity of certain amino groups over others is achieved.
- -SH exhibits sufficient leaving group ability in reaction with N-terminal amino group at appropriate pH conditions such that the e-amino groups in lysine residues are protonated and remain non-nucleophilic.
- reactions under suitable pH conditions may make some of the accessible lysine residues react with selectivity.
- the PEG comprises a reactive ester such as an N-hydroxy succinimidate at the end of the PEG chain.
- a reactive ester such as an N-hydroxy succinimidate at the end of the PEG chain.
- Such an N-hydroxysuccinimidate-containing PEG molecule reacts with select amino groups at particular pH conditions such as neutral 6.5-7.5.
- the N-terminal amino groups may be selectively modified under neutral pH conditions.
- accessible-NH 2 groups of lysine may also react.
- the PEG conjugated to the polypeptide polypeptide is linear. In other embodiments, the PEG conjugated to the polypeptide polypeptide is branched. Branched PEG derivatives such as those described in U.S. Pat. No. 5,643,575, "star-PEG's” and multi armed PEG'S such as those described in Shearwater Polymers, Inc. catalog "Polyethylene Glycol Derivatives 1997-1998.” Star PEGs are described in the art including, e.g., in U.S. Patent No. 6,046,305.
- PEG having a molecular weight in a range of from about 0.2 kDa to about 100 kDa is generally used, where the term "about,” in the context of PEG, indicates that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight.
- PEG suitable for conjugation to polypeptide has a molecular weight of from about 0.2 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 40 kDa, from about 40 kDa to about 50 kDa, from about 50 kDa to about 60 kDa, from about 60 kDa to about 70 kDa, from about 70 kDa to about 80 kDa, from about 80 kDa to about 90 kDa, or from about 90 kDa to about 100 kDa.
- Peptides may be conjugated to fatty acids, including without limitation conjugation at the amino terminus, for example where a linear or branched C3-C100 alkyl; preferably a C4-C30 alkyl optionally substituted with halo, hydroxy, alkoxy, amino, alkylamino, dialkylamino, sulfate, or phosphate, and which may by saturated, or mono- or di-unsaturated, e.g. 18:0, 24:0 and 24:1.
- Fatty acids of interest include, without limitation, palmitic acid; stearic acid; arachidic acid; lauric acid; myristic acid; myristoleic acid; palmitoleic acid; sapienic acid; oleic acid; linoleic acid; a- linolenic acid; arachidonic acid; eicosapentaenoic acid; erucic acid; docosahexaenoic acid; etc.
- the gel-forming polypeptide comprises a homolog, a variant, or a functional fragment of the wild-type counterpart peptide, including ligands for GPCR.
- the gel-forming polypeptide comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to a wild-type, i.e. naturally-occurring counterpart peptide.
- the gel-forming polypeptide may have the sequence set forth in, for example, SEQ ID NOS: 1-15, 48-58, 61 , 64, 106-114, 116-124, 126-131 , 139-140, 201-275, and derivatives thereof.
- gel-forming polypeptide ligand may refer to any polypeptide analogs which exhibit gel-forming capability at a concentration equal or lower than the concentration that allows the wild-type counterparts to form a gel in the sam aqueous solution, or wild-type and modified polypeptides that are identified to form gel at 6, 11 , 20, or 30% w/w.
- gel-forming polypeptides of the present invention can be shorter or longer than the SEQ ID NOS: 1-15, 48-58, 61 , 64, 101-140 and 201-275, e.g. truncated by 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or more residues at the amino and/or carboxy terminus, with substitution of nonnative amino acids or fused to an additional sequence.
- a peptide gel is a smart delivery system comprising biodegradable and biocompatible polypeptide(s), and is accepted by regulatory authorities for application in drug delivery.
- Peptide gels exhibit superior advantages over other colloidal carriers such as nanoemulsions, polymeric nanoparticles, liposomes, and solid lipid nanoparticles etc.
- One of the foremost qualifications is the high drug delivery capacity (i.e., enhanced drug loading capacity and increased drug bioavailability) and the lack of immunogenicity.
- this formulation reduces issues associated with production and suitable formulations.
- the mechanisms of self-assemble of polypeptides into gel nanostructure is not well understood, and minor changes in amino acid composition and other modifications may alter a polypeptide’s tendency to self-assemble into a gel nanostructure.
- compositions comprising at least one polypeptide with or without a gel-enhancing motif, wherein the enhancing motif is a component of a secreted polypeptide hormone that enhances gel formation.
- the composition may further include a functional therapeutic component., or the therapeutic component may be provided by the gel-forming peptide.
- formulations include stable aqueous solutions, gels and liquid gels, said formulations comprising 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 compound.
- the formulation may further comprise a very low ionicity aqueous excipient.
- Methods are also provided for preparing a stable aqueous formulation of a gel-forming polypeptide, comprising 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 or more (w/w) of a gel-forming polypeptide in a low ionicity aqueous excipient.
- the formulation may be administered to an individual suffering from a condition that may be alleviated by administration of a therapeutic polypeptide gel, said methods comprising administering to said subject an effective amount of the stable aqueous formulation.
- compositions and methods described herein provide novel and improved self assembling gel-forming polypeptides, comprising a gel-enhancing motif, improved gel-forming polypeptide carriers, improved usage of a therapeutic agent via the formation of a gel nanostructure, and other self-assembling nanostructures and methods of making and using same.
- the gel-forming therapeutic formulations utilize noncovalent electrostatics to control the solubility and nanostructure of therapeutic molecules or carrier polypeptides which may or may not contain a gel-forming-enhancing motif.
- Physicochemical properties e.g., swelling behavior
- mechanical properties e.g, compressive modulus
- degradation rates as well as active agent release kinetics of the subject hydrogels can be modulated by varying the amount of polypeptide present.
- the percentage can be varied between about 0.01% and about 50% by weight, and, such as from about 0.02% and about 45% by weight, such as from about 0.03% and 40% by weight, such as from 0.04% to 35% by weight and including from about 0.05% to 30% by weight.
- the physicochemical and mechanical properties as well as the active agent release kinetics of the subject hydrogels may vary depending on hydrogel structure.
- the subject compositions absorb solvent (e.g. water) and undergo swelling under nonphysiolgical condition (e.g., in pure water) or physiological conditions (e.g., in contact with blood or plasma).
- solvent e.g. water
- the term“swelling” as referred to herein is meant the isotropic (or anisotropic) expansion of the hydrogel structure as solvent (e.g., water) molecules diffuse throughout the internal volume of the hydrogel.
- the swelling ratio may vary.
- swelling ratio is meant the ratio of the hydrogel weight after absorption of solvent to the dry weight of the hydrogel.
- the compressive modulus of the hydrogels may vary depending on the composition of the hydrogel.
- compressive modulus is meant the capacity of the subject hydrogels to withstand axially directed pushing forces and is the value of uniaxial compressive stress reach when the material fails completely (e.g., crushed).
- the compressive modulus of the subject hydrogels could range from 0.1 kPa to 35 kPa, such as from 0.2 kPa to 33 kPa, such as from 0.3 kPa to 30 kPa, such as from 0.4 kPa to 28 kPa, such as form 0.5 kPa to 25 kPa, such as from 0.6 kPa to 22 kPa, such as from 0.7 kPa to 20 kPa and including a compressive modulus ranging from 1.0 kPa to 20 kPa.
- the pore sizes of the hydrogel may also vary depending on the structure of the hydrogel.
- the pore sizes of the hydrogel ranges from 0.01 microns to 1000 microns, such as 0.05 microns to 900 microns, such as 0.1 micron to 800 microns, such as 0.5 microns to 750 microns, such as 1.0 microns to 600 microns, such as 2.5 microns to 500 microns, such as 5.0 microns to 400 microns and including from 10.0 microns to 300 microns.
- the rate of degradation of hydrogels under physiological conditions may vary depending on the structure and composition.
- the subject hydrogels are structurally designed to degrade under physiological conditions (e.g., in vivo) over a predetermined duration, such as for example 0.5 days or longer, such as 1 day or longer, such as 2 days or longer, such as 5 days or longer, such as 7 days or longer, such as 10 days or longer, such as 14 days or longer, such as 21 days or longer, such as 28 days or longer, such as 70 days or longer and including 100 days or longer.
- hydrogels of interest are configured to degrade when exposed to physiological conditions at a predetermined rate, such as at a substantially zero-order degradation rate, such as at a substantially first order degradation rate and including at a substantially second-order degradation rate.
- the self-assembling gel-forming polypeptides comprise an or more acyl group to improve the gel-forming capability and to add susceptibility to esterase enzyme reaction.
- the gel-enhancing motif is a sequence derived from an agonist or ligand of a cell surface receptor selected from LHRH receptor, vasopressin receptors, oxytocin receptors, apelin receptor, neurotensin receptors, kisspeptin receptor, bombesin receptors, delta opioid receptor, mu opoid receptor, kappa opioid receptor, substance P receptors, angiotensin II receptors, calcitonin receptor, amylin receptors, GLP-1 receptor, GLP-2 receptor, glucagon receptor, calcitonin gene related peptide (CGRP) receptors, adrenomedullin receptors, melanocortin receptors, parathyroid hormone receptors, bradykinin receptors, neuropeptide Y (NPY) receptor, peptide YY (PYY) receptor, vasoactive intestinal polypeptide (VIP) receptor, urocortin receptors, somatostatin receptors, endothe
- the gel-enhancing motif is a sequence derived from an agonist or ligand of human CLR/RAMP receptors. 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.
- the resulting structure has the formula
- Ea is a gel-forming-enhancing cell-surface-receptor-ligand-derived polypeptide motif or a therapeutic agent
- Fa is a PEG group or a linker sequence
- n is an integral number from 0 to 40
- Ga is a therapeutic agent, or a gel-forming-enhancing cell-surface-receptor-ligand- derived polypeptide motif.
- a gel-forming-enhancing motif is conjugated to a therapeutic agent selected from the list consisting of small molecules, polypeptides, proteins, enzymes, hormones, polynucleotides, nucleoproteins, polysaccharides, glycoproteins, lipoproteins, steroids, analgesics, local anesthetics, antibiotics, chemotherapeutic, immunosuppressive agents, anti- inflammatory, antiproliferative, antimitotic, angiogenic, antiangiogenic, antipsychotic, central nervous system (CNS), anticoagulant, and fibrinolytic drugs; said drugs include LHRH analogs, LHRH antagonist analogs, vasopressin analogs, oxytocin analogs, apelin analogs, neurotensin analogs, kisspeptin analogs, kisspeptin 234 analogs, bombesin analogs, bombesin receptor antagonists, bradykinin analogs, bradykinin receptor atagonist analogs, opioid analogs, deltor
- the target of the therapeutic agent of the present invention is a cell surface receptor (e.g., opioid receptors and Romiplostim receptor) or enzyme (e.g., in the cases of kallikrein inhibitor or compstatin).
- the therapeutic target is a biological function mediator (e.g., in the case of Glatiramer peptide, which has a less well-defined target and is thought to act by modifying immune processes that are currently believed to be responsible for the pathogenesis of multiple sclerosis; in the case of thymosin alpha 1 and thymosin beta 4, they are believed to enhance cell-mediated immunity in humans as well as experimental animal).
- the therapeutic target is the cell membrane barrier (e.g., in the cases of antimicrobial temporin A derivative, a cell-penetrating peptide such as the TAT cell-penetraing sequence).
- the therapeutic target is the skin matrix components and matrix enzymes (e.g., matrix modifying proteins 1 , 4, 7, 8 and acetyl hexapeptide-3 matrix modifying peptide).
- matrix enzymes e.g., matrix modifying proteins 1 , 4, 7, 8 and acetyl hexapeptide-3 matrix modifying peptide.
- a CLR/RAMP receptor ligand of a gel-forming polypeptide comprises a homolog, a variant, a chimera, or a functional fragment of adrenomedullin, adrenomedullin 2, and CGRP, for example SEQ ID NOS:1-3, 48-58, and 106-114, 116-124, 126-131 , ,139-140, and 274-275 including without limitation the sequence of SEQ ID NOS: 1-3, 48-58, 106-114, 116-124, 126-131 ,, 139-140, and 274-275.
- the gel-forming polypeptide comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NOS: 1-3, 48-58, 106-114, 116-124, 126-131 , 139-140, and 274-275.
- CLR/RAMP receptor ligand may refer to any functional peptide analog which activates or inhibits a CLR/RAMP receptor (CLR/RAMP1 , 2 and 3).
- the CLR/RAMP receptor ligand is an analog of adrenomedullin, adrenomedullin 2, and CGRP, or SEQ ID NOS: 1-3, 48-58, 106-114, 116-124, 126-131 , 139-140, and 274-275.
- the CLR/RAMP 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).
- an 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- B 19- B20- B21 - B22- B23- B24- B25- B26- B27- B28- R2 , where:
- R1 a functional group comprising a structure of Formula (W’)(X’)n(Y’)n(Z’)n, wherein W is a fatty acid, a fatty diacid, a fatty acid or cholesterol derivative or empty;
- W is a fatty acid, a fatty diacid, a fatty acid or cholesterol derivative or empty;
- X’ is a PEG group, glutamic acid, g-glutamic acid, a non-proteinogenic amino acid, or empty;
- Y’ is a PEG group, glutamic acid, y-glutamic acid, a non-proteinogenic amino acid, or empty;
- Z’ is a proteinogenic amino acid, a non-proteinogenic amino acid, or empty;
- R2 is an C-terminal modification including an ⁇ NH2 ⁇ amidation, ⁇ -CHO ⁇ peptide aldehydes, ⁇ -ol ⁇ alcohol peptide, ⁇ CMK ⁇ chloromethylketone, ⁇ FMK ⁇ Fluoromethylketone, ⁇ Cya ⁇ Cysteamide, ⁇ pNA ⁇ p-nitroaniline, ⁇ -ONP ⁇ para-nitrophenol, ⁇ AMC ⁇ 7-Amino-4-methylcoumarin, ⁇ AFC ⁇ , -OMe (C-terminal), -OEt (C-terminal), -OBzl (C-terminal), -OtBu (C-terminal), ⁇ -OSu ⁇ hydroxysucinimide ester, -NHMe (C-terminal), NHEt (C-terminal), -NHisopen (C-terminal), NH(CH2)6 (C-terminal), - NHPh (C-terminal), ⁇ NHEt(0)EtNH-Fmoc ⁇ 2,2'-O
- B0 is selected from the group consisting of an empty residue, any proteinogenic amino acid or non-proteinogenic amino acid, acylated histidine (acy-His), acylated arginine (acy-Arg), acylated lysine (acy-Lys);
- B1 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, His, Arg, Lys, Asn, Gin and a non-proteinogenic amino acid;
- B2 is selected from the group consisting of an empty residue, Arg, Lys, Gin, Glu, Asp, Asn and a non-proteinogenic amino acid;
- B3 is selected from the group consisting of an empty residue, Ala, Leu, lie, Val, Met, Phe, His, Arg, Lys, Gin, Asp and a non-proteinogenic amino acid;
- B4 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu and a non-proteinogenic amino acid;
- B5 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Pro, Ser, Th, Tyr and a non-proteinogenic amino acid;
- B6 is selected from the group consisting of an empty residue, Ala, Leu, lie, Val, Met, Phe, His, Arg, Lys and a non-proteinogenic amino acid;
- B7 is selected from the group consisting of an empty residue, Ala, Leu, lie, Val, Met, Phe, Gin, Asn, His, Arg, Lys and a non-proteinogenic amino acid;
- B8 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Ser, Thr and a non-proteinogenic amino acid;
- B9 is selected from the group consisting of an empty residue, Arg, Lys, Asn, Gin, Trp, Phe, Ser, Thr, Tyr and a non-proteinogenic amino acid;
- B10 is selected from the group consisting of an empty residue, Ala, Ser, Thr, Gin, Glu, Asp, Asn and a non-proteinogenic amino acid;
- B11 is selected from the group consisting of an empty residue, Trp, Phe, Val, Ala, Gly, lie, Leu, Pro and a non-proteinogenic amino acid;
- B12 is selected from the group consisting of an empty residue, Ala, Gly, Ser, Thr, Pro, Tyr; Met, Trp, Phe and a non-proteinogenic amino acid;
- B13 is selected from the group consisting of an empty residue, Gin, Glu, Asp, and Asn, Val, Ala, Gly, lie, Leu, Met, Phe and a non-proteinogenic amino acid;
- B14 is selected from the group consisting of an empty residue, His, Arg, Lys, Val, Ala, Gly, lie, Leu, Met, Phe, Pro and a non-proteinogenic amino acid;
- B15 is selected from the group consisting of an empty residue, Arg, Lys, Gin, Glu, Asp, Asn, Val, Ala, Gly, lie, Leu and a non-proteinogenic amino acid;
- B16 is selected from the group consisting of an empty residue, Asn, Gin, Val, Ala, Gly, lie, Leu and a non-proteinogenic amino acid;
- B17 is selected from the group consisting of an empty residue, Asn, Gin, Ser, Thr, Tyr and a non-proteinogenic amino acid
- B18 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Phe, Tyr and a non-proteinogenic amino acid
- B19 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Met, Phe, Pro and a non-proteinogenic amino acid;
- B20 is selected from the group consisting of an empty residue, His, Arg, Lys, Val, Ala, Gly, lie, Leu, Pro and a non-proteinogenic amino acid;
- B21 is selected from the group consisting of an empty residue, lie, Val, Ser, Thr, Tyr, Gin, Glu, Asp, Asn and a non-proteinogenic amino acid;
- B22 is selected from the group consisting of an empty residue, His, Arg, Lys, Val, Ala, Gly, lie, Leu, Asn, Gin, Pro and a non-proteinogenic amino acid;
- B23 is selected from the group consisting of an empty residue, Ser, Thr, Tyr, Val, Ala, Gly, lie, Leu, Met, Phe and a non-proteinogenic amino acid;
- B24 is selected from the group consisting of an empty residue, Ala, Gly, Pro, Ser, Thr, Tyr and a non-proteinogenic amino acid;
- B25 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Pro, Ser, Thr and a non-proteinogenic amino acid;
- B26 is selected from the group consisting of an empty residue, His, Arg, Lys, Gin, Glu, Asp, Asn and a non-proteinogenic amino acid;
- B27 is selected from the group consisting of an empty residue, Val, Ala, Gly, lie, Leu, Ser, Thr, Tyr and a non-proteinogenic amino acid;
- B28 is selected from the group consisting of an empty residue, Ala, Leu, lie, Val, Phe, Ser, Thr, Tyr and a non-proteinogenic amino acid.
- a GnRH receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a GnRH receptor agonist or antagonist, for example SEQ ID NO: 15, or may comprise or consist of SEQ ID NO: 15.
- the gel-forming GnRH receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:15 or SEQ ID NO:201 , or may comprise or consist of SEQ ID NO:15 or SEQ ID NO:201.
- GnRH receptor ligand may refer to any functional analog (e.g., Buserelin, Deslorelin, Fertirelin, Goserelin, Leuprorelin, Nafarelin, and Triptorelin) which is capable of activating or inhibiting a GnRH receptor.
- a GnRH receptor ligand within the gel-forming polypeptide of the present invention can be shorter (e.g., 6-9 or less amino acids in length) or longer (e.g., 11-40 or more amino acids in length).
- a GnRH receptor antagonist component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a GnRH receptor antagonist, for example SEQ ID NO:27, including without limitation the sequence of SEQ ID NO:27.
- a gel-forming GnRH receptor antagonist comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 202 or 203, or may or may comprise or consist of SEQ ID NO:202 or 203.
- GnRH receptor antagonist may refer to any functional analog (e.g., Abarelix, Cetrorelix, Degarelix, Ganirelix, and Ozarelix) which inhibits a GnRH receptor.
- the GnRH receptor antagonist is an analog of a GnRH receptor antagonist or SEQ ID NO:27.
- 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 sequence.
- a vasopressin receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a vasopressin receptor agonist or antagonist, for example SEQ ID NO: 17, including without limitation the sequence of SEQ ID NO:17.
- the a gel-forming vasopressin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 204, or may comprise or consist of SEQ ID NO:204.
- vasopressin receptor ligand may refer to any functional analog (e.g., desmopressin, lypressin, argipressin, d[Leu4,Lys8]-VP, (d(CH2)51 ,Tyr(Me)2,Arg8)-vasopressin, and pitressin) which activates or inhibits a vasopressin receptor.
- the vasopressin receptor ligand is an analog of a vasopressin receptor ligand or SEQ ID NO: 17.
- the vasopressin receptor ligand 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.
- An oxytocin receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a oxytocin receptor agonist or antagonist, for example SEQ ID NO:5, including without limitation the sequence of SEQ ID NO:5.
- the a gel-forming oxytocin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 5 or 205, or may comprise or consist of SEQ ID NO:5 or SEQ ID NO:205.
- oxytocin receptor ligand may refer to any functional analog (e.g., demoxytocin, merotocin, oxytocin, WAY- 267,464, pitocin, barusiban, atosiban, and carbetocin) which activates or inhibits an oxytocin receptor.
- the oxytocin receptor ligand is an analog of an oxytocin receptor agonist, antagonist, or SEQ ID NO:5.
- the oxytocin receptor ligand 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 sequence.
- An apelin receptor (i.e., APJ receptor and Apela/ELABELA/Toddler) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an apelin receptor agonist or antagonist, for example SEQ I D NO: 18, including without limitation the sequence of SEQ ID NO:18.
- the gel-forming apelin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NOS: 206 and 262, or may comprise or consist of SEQ ID NOS: 206 and 262.
- apelin receptor ligand may refer to any functional analog (e.g., apelin 36), apelin 17, apelin 13, and Apela/ELABELA/Toddler) which activates or inhibits an apelin receptor.
- the apelin receptor ligand is an analog of an apelin receptor agonist, antagonist, or SEQ ID NO: 18.
- the apelin receptor ligand 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 sequence.
- a neurotensin receptor (NTSR1 and NTSR2) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a neurotensin receptor agonist or antagonist, for example SEQ ID NO: 19, including without limitation the sequence of SEQ ID NO:19.
- the gel-forming neurotensin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:207, or may comprise or consist of SEQ ID NO:207.
- neurotensin receptor ligand may refer to any functional ananlog (e.g, Beta- lactotensin, JMV-449, Neurotensin 13, Neuromedin N, Xenin 8, Kinetensin, PD-149,163, Levocabastine, SR-48692, and SR-142,948) which is capable of activating or inhibiting a neurotensin receptor.
- the neurotensin receptor ligand is a neurotensin 13 analog or SEQ ID NO: 19.
- a neurotensin receptor ligand within the gel-forming polypeptide of the present invention can be shorter (e.g., 6-12 or less amino acids in length) or longer (e.g., 14-40 or more amino acids in length) than theprovided sequence.
- a kisspeptin receptor (GPR54) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a kisspeptin receptor agonist or antagonist, for example SEQ ID NOS:6, including without limitation the sequence of SEQ ID NOS:6.
- the a gel-forming kisspeptin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NOS: 6 or 208 , or may comprise or consist of SEQ ID NO: 6 or 208.
- kisspeptin receptor ligand may refer to any functional analog (e.g., kisspeptin-10, kisspeptin-13, kisspeptin 17, and kisspeptin 234 antagonist) which activates or inhibits a kisspeptin receptor.
- the kisspeptin receptor ligand is an analog of a kisspeptin receptor agonist, antagonist, or SEQ ID NO:6.
- the kisspeptin receptor ligand 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 sequence.
- a bombesin receptor (BB1 , BB2 and BB3 receptors) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a bombesin receptor agonist or antagonist, for example SEQ ID NOS:20 and 61 , including without limitation the sequence of SEQ ID NOS:20 and 61.
- the a gel-forming bombesin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NOS: 61 , 209 or 255, or may comprise or consist of SEQ ID NO:61 , 209 or 255.
- bombesin receptor ligand may refer to any functional analog (e.g., bombesin, neuromedin B, BIM 187, BIM 189, [D-Phe12,Leu14]-bombesin, alytesin, BIM 23042, [D-Phe12,Leu14]-bombesin, and gastrin-releasing peptide) which activates or inhibits a bombesin receptor.
- the bombesin receptor ligand is an analog of a bombesin receptor agonist, antagonist, or SEQ ID NO:20 and 61.
- the bombesin receptor ligand 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 sequence.
- a deltorphin or an opioid receptor (i.e. , delta opioid receptor, kappa opioid receptor, mu opioid receptor and nociceptin receptor) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an opioid receptor agonist or antagonist, for example SEQ ID NO:21 , including without limitation the sequence of SEQ ID NO:21.
- the a gel-forming opioid receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 211 , 212, 213, or 214, or may comprise or consist of SEQ ID NO:211 , 212, 213, or 214.
- opioid receptor ligand may refer to any functional analog (e.g., buprenorphin, Leu- enkephalin, Met-enkephalin, deltorphins, DADLE, DPDPE, 7-spiroindanyloxymorphone, and N- phenethyl-14-ethoxymetopon) which activates or inhibits an opioid receptor.
- the opioid receptor ligand is an analog of an opioid receptor agonist, antagonist, or SEQ ID NO:21.
- the opioid receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 4-40 or more amino acids in length) than the provided sequence.
- An enkephalin or an opioid receptor (i.e., delta opioid receptor, kappa opioid receptor, mu opioid receptor and nociceptin receptor) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an opioid receptor agonist or antagonist, for example SEQ ID NO:22, including without limitation the sequence of SEQ ID NO:22.
- the a gel-forming opioid receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 211 , 212, 213, or 214, or may comprise or consist of SEQ ID NO:211 , 212, 213, or 214.
- opioid receptor ligand may refer to any functional analog (e.g., Leu-enkephalin, Met- enkephalin, deltorphins, DADLE, DPDPE, 7-spiroindanyloxymorphone, and N-phenethyl-14- ethoxymetopon) which activates or inhibits an opioid receptor.
- the opioid receptor ligand is an analog of an opioid receptor agonist, antagonist, or SEQ ID NO:22.
- the opioid receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 4-40 or more amino acids in length) than the provided sequence.
- a kappa opioid receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a kappa opioid receptor agonist or antagonist, for example SEQ ID NO:7, including without limitation the sequence of SEQ ID NO:7.
- the a gel-forming kappa opioid receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 7 or 214, or may comprise or consist of SEQ ID NO:7 or SEQ ID NO:214.
- kappa opioid receptor ligand may refer to any functional analog (e.g., CR665, difelikefalin (CR845), and dynorphins) which activates or inhibits a kappa opioid receptor.
- the kappa opioid receptor ligand is an analog of a kappa opioid receptor agonist, antagonist, or SEQ ID NO:7 or 214.
- the kappa opioid receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 4-40 or more amino acids in length) than the provided sequence.
- a substance P receptor (or neurokinin 1 receptor) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a substance P receptor agonist or antagonist, for example SEQ ID NO:23, including without limitation the sequence o f SEQ ID NO:23.
- the a gel-forming substance P receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:215, 253 and 254, or may comprise or consist of SEQ ID NO:215, 253, and 254.
- substance P receptor ligand may refer to any functional analog (e.g, substance P, GR-73632, Aprepitant, Casopitant, Ezlopitant, Fosaprepitant, Lanepitant, Maropitant, and Vestipitant) which activates or inhibits a substance P receptor.
- the substance P receptor ligand is an analog of a substance P receptor agonist, antagonist, or SEQ ID NO:23.
- the substance P receptor ligand 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 sequence.
- An angiotensin II receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an angiotensin II receptor agonist or antagonist, for example SEQ ID NO:24, including without limitation the sequence of SEQ ID NO:24.
- the a gel-forming angiotensin II receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:216, or may comprise or consist of SEQ ID NO:216.
- angiotensin II receptor ligand may refer to any functional analog (e.g., saralasin) which activates or inhibits an angiotensin II receptor.
- the angiotensin II receptor ligand is an analog of an angiotensin II receptor agonist, antagonist, or SEQ ID NO:24.
- the angiotensin II receptor ligand 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 sequence.
- a calcitonin receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a calcitonin receptor agonist or antagonist, for example SEQ ID NO:28, including without limitation the sequence of SEQ ID NO:28.
- the a gel-forming calcitonin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:217, or may comprise or consist of SEQ ID NO:217.
- calcitonin receptor ligand may refer to any functional analog (e.g., Miacalcin analogs) which activates or inhibits a calcitonin receptor.
- the calcitonin receptor ligand is an analog of a calcitonin receptor agonist, antagonist, or SEQ ID NO:28.
- the calcitonin receptor ligand 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 sequence.
- An amylin receptor (calcitonin receptor/RAM P1 , 2 and 3) ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an amylin receptor agonist or antagonist, for example SEQ ID NO:4, including without limitation the sequence of SEQ ID NO:4.
- the a gel-forming amylin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:4, or 218, or may comprise or consist of SEQ ID NO:4, or 218.
- amylin receptor ligand may refer to any functional analog which (e.g., Pramlintide) activates or inhibits an amylin receptor.
- the amylin receptor ligand is an analog of an amylin receptor agonist, antagonist, or SEQ ID NO:4.
- the amylin receptor ligand 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 sequence.
- a GLP-1 receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a GLP-1 receptor agonist or antagonist, for example SEQ ID NOS: 14 and 26, including without limitation the sequence of SEQ ID NOS: 14 and 26.
- the a gel-forming GLP-1 receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NOS: 14, 219, 220, -221 , or 269-272, or may comprise or consist of SEQ ID NO: 14, 219, 220, 221 , or 269- 272.
- GLP-1 receptor ligand may refer to any functional analog (e.g., GLP-1 7-37, exenatide, [Glu13] exenatide, [Met(0)14]-exenatide, [N-acetyl-His1]-exenatide, liraglutide, lixisenatide, albiglutide, dulaglutide, semaglutide, and taspoglutide) which activates or inhibits a GLP-1 receptor.
- the GLP-1 receptor ligand is an analog of a GLP- 1 receptor agonist, antagonist, or SEQ ID NO: 14 or 26.
- the GLP-1 receptor ligand 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 sequence.
- a GLP-2 receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a GLP-2 receptor agonist or antagonist, for example SEQ ID NO:12, including without limitation the sequence of SEQ ID NO:12.
- the a gel-forming GLP-2 receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 12 or 222, or may comprise or consist of SEQ ID NO:12 or 222.
- GLP-2 receptor ligand may refer to any functional analog (e.g., Tedglutide, and GLP2) which activates or inhibits a GLP-2 receptor.
- the GLP-2 receptor ligand is an analog of a GLP-2 receptor agonist, antagonist, or SEQ ID NO: 12.
- the GLP-2 receptor ligand 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 sequence.
- a melanocortin receptor (i.e. , MC1 R-MC5R) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a melanocortin receptor agonist or antagonist, for example SEQ ID NOS:29, 33, 34, 62, and 64 including without limitation the sequence of SEQ ID NOS: 29, 33, 34, 62, and 64.
- the gel-forming melanocortin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273, or may comprise or consist of SEQ ID NO:64, 223, 224, 225, 226, 227, 228, 256, 257, 259, 260, or 273.
- melanocortin receptor ligand may refer to any functional analog which activates or inhibits a melanocortin receptor (e.g., a-MSH, b-MSH, g-MSH, ACTH1-24, cosyntropin, afamelanotide, BMS-470,539, bremelanotide, Melanotan II, modimelanotide, setmelanotide, PF-00446687, PL-6983, THIQ, PF-219,061 , UK- 414,495, agouti-related peptide, and agouti signaling peptide).
- a melanocortin receptor e.g., a-MSH, b-MSH, g-MSH, ACTH1-24, cosyntropin, afamelanotide, BMS-470,539, bremelanotide, Melanotan II, modimelanotide, setmelanotide, PF-
- the melanocortin receptor ligand is an analog of a melanocortin receptor agonist, antagonist, or SEQ ID NO: 29, 33, 34, 62, or 64.
- the melanocortin receptor ligand within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 4-40 or more amino acids in length) than the provided sequence.
- a neuropeptide Y receptor (NPY1 R, NPY2R, PPYR1 , NPY5R) ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a neuropeptide Y receptor agonist or antagonist, for example SEQ ID NOS:30 including without limitation the sequence of SEQ ID NOS: 30.
- the gel forming neuropeptide Y receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:229 or 264, or may comprise or consist of SEQ ID NO:229, or 264.
- neuropeptide Y receptor ligand may refer to any functional analog (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]-hPancreatic polypeptide, BVD 10, and Pancreatic polypeptide) which activates or inhibits a neuropeptide Y receptor.
- any functional analog 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]-hPancre
- the neuropeptide Y receptor ligand is an analog of a neuropeptide Y receptor agonist, antagonist, or SEQ ID NO:30.
- the neuropeptide Y receptor ligand 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 sequence.
- a corticotropin receptor (CRHR1 and CRHR2) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a corticotropin receptor agonist or antagonist, for example SEQ ID NOS: 10 and 31 , including without limitation the sequence of SEQ ID NOS: 10 and 31.
- the gel forming corticotropin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 10, 230, 231 , or 265, or may comprise or consist of SEQ ID NO: 10, 230, 231 , or 265.
- corticotropin receptor ligand may refer to any functional analog (CRH, urocortin 1 , urocortin 2, urocortin3, Stressin I, Antalarmin hydrochloride, Antisauvagine-30, LWH-234, CP-154,526, NBI-27914, R-121 ,919, Astressin-2B, and Astressin-B) which activates or inhibits a corticotropin receptor.
- the corticotropin receptor ligand is an analog of a corticotropin receptor agonist, antagonist, CRH, urocortin 1 , urocortin 2, urocortin3, or SEQ ID NO: 10 or 31.
- the corticotropin 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).
- a parathyroid hormone receptor (PTH1 R and PTH2R) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a parathyroid hormone receptor agonist or antagonist, for example SEQ ID NOS:25 including without limitation the sequence of SEQ ID NO:25.
- the gel-forming parathyroid hormone receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:232, 258, or may comprise or consist of SEQ ID NO:232, 258.
- parathyroid hormone receptor ligand may refer to any functional analog (e.g., parathyroid hormone, parathyroid hormone-related protein, DPC AJ1951 , Teriparatide, and Abaloparatide) which activates or inhibits a parathyroid hormone receptor.
- the parathyroid hormone receptor ligand is an analog of a parathyroid hormone receptor agonist, antagonist, or SEQ ID NO:25.
- the parathyroid hormone 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) than the provided sequences.
- a bradykinin receptor (BDKRB1 and BDKRB2) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a bradykinin receptor agonist or antagonist, for example SEQ ID NOS:32 and 65; Firazyr or icatibant, including without limitation the sequence of SEQ ID NOS:32 and 65.
- the gel-forming bradykinin receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 233, 234, or 261 , or may comprise or consist of SEQ ID NO:233, 234, or 261.
- bradykinin receptor ligand may refer to any functional analog (e.g., bradykinin1-8, [Leu8]-bradykinin1-8, Sar-[D- Phe8]-des-Arg9-bradykinin, KRPPGFS-DpNal-l, [Phe8 ⁇ +>(CH-NH)-Arg9]-bradykinin, MEN 11270, R 715, R 892, and Hoe 140 (icatibant)) which activates or inhibits a bradykinin receptor.
- the bradykinin receptor ligand is an analog of a bradykinin receptor antagonist or SEQ ID NO:32.
- the bradykinin receptor ligand 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 sequence.
- a ghrelin/growth hormone secretagogue receptor (GHSR) ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a ghrelin/growth hormone secretagogue receptor (GHSR) agonist or antagonist, for example SEQ ID NO:8, including without limitation the sequence of SEQ ID NO:8.
- the gel-forming ghrelin/growth hormone secretagogue receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:8 or 235, or may comprise or consist of SEQ ID NO:8 or 235.
- ghrelin/growth hormone secretagogue receptor (GHSR) ligand may refer to any functional analog (e.g., Anamorelin, Capromorelin, Examorelin (hexarelin), ghrelin (lenomorelin), GHRP-6, Ibutamoren (MK-677), Ipamorelin, Macimorelin, Pralmorelin (GHRP-2), Relamorelin, SM-130,686, Tabimorelin, and Ulimorelin) which activates or inhibits a ghrelin/growth hormone secretagogue receptor.
- GHSR ghrelin/growth hormone secretagogue receptor
- the ghrelin/growth hormone secretagogue receptor ligand is an analog of a ghrelin/growth hormone secretagogue receptor agonist, antagonist, or SEQ ID NO:8.
- the ghrelin/growth hormone secretagogue receptor (GHSR) 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) than the provided sequences.
- a growth hormone-releasing hormone receptor (GHRHR) ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a growth hormone-releasing hormone receptor agonist or antagonist, for example SEQ ID NO:35, including without limitation the sequence of SEQ ID NO:35.
- the gel-forming ghrelin/growth hormone secretagogue receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:35, 236, or 237, or may comprise or consist of SEQ ID NO:35, 236 or 237.
- growth hormone releasing hormone receptor ligand may refer to any functional analog (e.g., CJC- 1295, Dumorelin, GHRH (somatorelin), Rismorelin, Sermorelin (GHRH 1-29), and Tesamorelin) which activates or inhibits a growth hormone-releasing hormone receptor.
- the growth hormone-releasing hormone receptor ligand is an analog of a growth hormone releasing hormone receptor agonist, antagonist, or SEQ ID NO:35.
- the growth hormone releasing hormone 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) than the provided sequences.
- a vasoactive intestinal peptide receptor (VI PR) ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a vasoactive intestinal peptide receptor agonist or antagonist, for example SEQ ID NO:67, including without limitation the sequence of SEQ ID NO:67.
- the gel-forming VIP receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:266, or may comprise or consist of SEQ ID NO:266.
- the term VIP receptor ligand, as used herein, may refer to any functional analog which activates or inhibits a VIP receptor.
- the VIP receptor ligand is an analog of a VIP receptor agonist, antagonist, or SEQ ID NO:67.
- the VIP 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) than the provided sequences.
- a natriuretic peptide receptor (i.e. , NPR1 , NPR2, and NPR3) ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a natriuretic peptide receptor agonist or antagonist, for example SEQ ID NOS:41 , including without limitation the sequence of SEQ ID NOS:41.
- the gel forming natriuretic peptide receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:238, or may comprise or consist of SEQ ID NO:238.
- natriuretic peptide receptor ligand may refer to any functional analog (e.g., atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, and nesiritide) which activates or inhibits a natriuretic peptide receptor.
- the natriuretic peptide receptor ligand is an analog of a natriuretic peptide receptor agonist, antagonist, atrial natriuretic peptide, brain natriuretic peptide, C-type natriuretic peptide, or SEQ ID NO:41.
- the natriuretic peptide 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).
- a thymosin a 1 - 1 i ke ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a thymosin a1 and thymosin a 1 - 1 i ke ligand, for example SEQ ID NO: 13, including without limitation the sequence of SEQ ID NO: 13.
- the gel-forming thymosin a1-like ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 13, 239, 240, 267, or 268, or may comprise or consist of SEQ ID NO: 13, 239, 240, 267, or 268.
- the term thymosin a1 -like ligand, as used herein, may refer to any functional analog (e.g., thymosin a1) which activates or inhibits the thymosin a1-mediated signaling pathway.
- the thymosin a1 and thymosin a 1 - 1 i ke ligand is an analog of thymosin a1 , or SEQ ID NO:13.
- the thymosin a1-like 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).
- a thymosin beta 4 ligand component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a thymosin beta 4 ligand, for example SEQ ID NO:66, including without limitation the sequence of SEQ ID NO:66.
- the gel-forming thymosin beta 4-like ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:263, or may comprise or consist of SEQ ID NO:263.
- thymosin beta 4 may refer to any functional analog (e.g., thymosin beta 4) which activates or inhibits the thymosin beta 4-mediated signaling pathway.
- the thymosin beta 4 and thymosin beta 4-like ligand is an analog of thymosin beta 4, or SEQ ID NO:263.
- the thymosin beta 4-like 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).
- a cell-penetrating peptide component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of cell-penetrating peptide, for example SEQ ID NO:36, including without limitation the sequence of SEQ ID NO:36.
- the gel-forming cell-penetrating peptide comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:241 , or may comprise or consist of SEQ ID NO:241.
- cell-penetrating peptide may refer to any functional peptide analog which promotes the transfer of a molecule from the extracellular space to the intracellular space (please see examples in Kalafatovic D and Giralt E 2017. Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity. Molecules. 22(11).
- the cell-penetrating peptide is an analog of a cell-penetrating peptide, or SEQ ID NO:36.
- the cell-penetrating peptide within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-50 or more amino acids in length).
- a kallikrein regulator component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of kallikrein regulator, for example SEQ ID NO:37, including without limitation the sequence of SEQ ID NO:37.
- the gel-forming kallikrein regulator comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:242, or may comprise or consist of SEQ ID NO:242.
- the term kallikrein regulator, as used herein, may refer to any functional analog which activates or inhibits a kallikrein enzyme (e.g., Ecallantide).
- the kallikrein regulator is an analog of a kallikrein activator, inhibitor, or SEQ ID NO:37.
- the kallikrein regulator within the gel forming polypeptide of the present invention can be shorter or longer (e.g., 5-50 or more amino acids in length).
- a polypeptide antibiotic or antimicrobial peptide component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a polypeptide antibiotics or antimicrobial peptide, for example SEQ ID NO:68, including without limitation the sequence of SEQ ID NO:68.
- the gel-forming polypeptide antibiotics comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:243, or may comprise or consist of SEQ ID NO:243.
- polypeptide antibiotics may refer to any functional analog which inhibits the growth or proliferation of a microorganism, bacteria, a fungus, a virus, a tumor, or another pathological agent.
- the polypeptide antibiotic is an analog of a polypeptide antibiotics (e.g., temporin A, Gramicidin A, B, C, and D peptides, defensins, esculentin 1-21 , cecropins, andropin, moricin, ceratotoxin, melittin, Magainin, dermaseptin, bombinin, brevinin-1 , esculentins, buforin II, CAP18, LL37, abaecin, apidaecins, prophenin, indolicidin, actinomycin, bacitracin, colistin, polymyxin B, actinomycin-D, Bacitracin, Boceprevir, Dalbavancin, Dapto
- a complement regulator component of a gel-forming polypeptide comprises a homolog, a chimera, a variant, an analog, a chimera, or a functional fragment of a complement regulator, for example SEQ ID NO: 11 , including without limitation the sequence of SEQ ID NO: 11.
- the gel-forming complement regulator comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:11 or 244, or may comprise or consist of SEQ ID NO:11 or 244.
- the term complement regulator, as used herein, may refer to any functional analog (e.g., complement component 5a and compstatin analogs) which activates or inhibits a complement factor or the complement cascade.
- the complement regulator is an analog of a complement activator or inhibitor (e.g., compstatin, complement component C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9), or SEQ ID NO:11.
- a complement activator or inhibitor e.g., compstatin, complement component C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9
- SEQ ID NO:11 SEQ ID NO:11
- the complement regulator within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-50 or more amino acids in length).
- a C5aR receptor ligand component of a gel-forming polypeptide comprises a homolog, a variant, a chimera, an analog, a chimera, or a functional fragment of a C5aR receptor ligand, for example SEQ ID NO:11 , including without limitation the sequence of SEQ ID NO:11.
- the gel-forming C5aR receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:11 or 244, or may comprise or consist of SEQ ID NO:11 or 244.
- C5aR receptor ligand may refer to any functional analog (e.g., complement component 5a and compstatin analogs) which activates or inhibits a C5aR receptor.
- the C5aR receptor ligand is an analog of a C5aR receptor activator or inhibitor (e.g., compstatin, complement component C5a, C2a, C4b, C3, C3a, C3b, C5b, C6, C7, C8, and C9), or SEQ ID NO:11.
- the C5aR 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).
- a Copaxone immunomodulator component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of an immunomodulator, for example SEQ ID NO:42, including without limitation the sequence of SEQ ID NO:42.
- the gel-forming Copaxone immunomodulator comprises an amino acid sequence that is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:245, or may comprise or consist of SEQ ID NO:245.
- Copaxone immunomodulator may refer to any functional analog which are composed of the four amino acids found in myelin basic protein, namely glutamic acid, lysine, alanine, and tyrosine and activates or inhibits the glatiramer acetate-mediated signaling pathway (e.g., Copolymer 1 , Cop-1 or Copaxone, Glatopa, and M356).
- the immunomodulator is an analog of glatiramer acetate (which is a mixture of analogs composed of the four amino acids found in myelin basic protein), or SEQ ID NO:42.
- the immunomodulator within the gel-forming polypeptide of the present invention can be shorter or longer (e.g., 5-60 or more amino acids in length).
- a matrix-modifying protein (or matrikine) component of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a matrix modifying protein, for example SEQ ID NOS:39, and 44-47, including without limitation the sequence of SEQ ID NOS:39, and 44-47.
- the gel-forming matrix modifying protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:246, 247, 248, 249, or 250, or may comprise or consist of SEQ ID NO:246, 247, 248, 249, or 250.
- matrix-modifying protein may refer to any functional analog which activates or inhibits a matrix enzyme-mediated signaling pathway.
- the matrix-modifying protein is an analog of a modulator of dermal extracellular matrix, collagen modulator, elastin modulator, keratinocytes/epidermal cell modulator, melanogenesis modulator, a structural peptide that act as signal modulators of the extracellular matrix component, as structural peptides, carrier peptides and neurotransmitter function modulators (e.g., YIGSR, Pal-KTTKS, Pal-GHK, GERK, RGD, GQPR, VGVAPG, HFRW, YRSRKYSSWY, and argireline peptides), or SEQ ID NO:39, or 44-47 (See Pai et al., 2016 Topical peptides as cosmeceuticals.
- the matrix-modifying protein 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 sequence.
- a thrombopoietin receptor ligand of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a thrombopoietin receptor ligand, for example SEQ ID NO:9 , including without limitation the sequence of SEQ ID NO:9.
- the gel-forming matrix-modifying protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:9, or may comprise or consist of SEQ ID NO:9.
- thrombopoietin receptor ligand may refer to any functional analog (e.g., Nplate ® (romiplostim)) which activates or inhibits a thrombopoietin receptor.
- the thrombopoietin receptor ligand is an analog of thrombopoietin (THPO), megakaryocyte growth and development factor (MGDF), or SEQ ID NO:9.
- THPO thrombopoietin
- MGDF megakaryocyte growth and development factor
- SEQ ID NO:9 SEQ ID NO:9.
- the thrombopoietin 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).
- An insulin receptor ligand of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a insulin receptor ligand, for example SEQ ID NO:59, including without limitation the sequence of SEQ ID NO:59.
- the gel-forming matrix-modifying protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO: 251 , or may comprise or consist of SEQ ID NO:251.
- insulin receptor ligand may refer to any functional analog (e.g., insulin degludec, Insulin lispro, Insulin aspart, Insulin glulisine, Insulin detemir, and Insulin glargine) which activates or inhibits an insulin receptor.
- the insulin receptor ligand is an analog of insulin, or SEQ ID NO:59.
- 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 B and A chain, respectively).
- a relaxin receptor ligand of a gel-forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a relaxin receptor ligand, for example SEQ ID NO:60, including without limitation the sequence of SEQ ID NO:60.
- the gel-forming matrix-modifying protein comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:252, or may comprise or consist of SEQ ID NO:252.
- the term relaxin receptor ligand, as used herein, may refer to any functional analog which activates or inhibits a relaxin receptor (i.e. , LGR7 and LGR8).
- the relaxin receptor ligand is an analog of relaxin 1 , relaxin 2, relaxin 3, INSL3, INSL7, or SEQ ID NO:60.
- 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 B and A chain, respectively).
- a parathyroid hormone related peptide (PTHrP) receptor ligand component of a gel forming polypeptide comprises a homolog, a variant, an analog, a chimera, or a functional fragment of a PTHrP receptor agonist or antagonist, for example SEQ ID NO: 63, including without limitation the sequence of SEQ ID NO: 63.
- the gel-forming PTHrP receptor ligand comprises an amino acid sequence that is about 70%, 75%, 80%, 85%, 90%, or 95% identical to SEQ ID NO:258, or may comprise or consist of SEQ ID NO:258.
- PTHrP receptor ligand may refer to any functional analog (e.g., parathyroid hormone, parathyroid hormone-related protein, Teriparatide, and Abaloparatide) which activates or inhibits a PTHrP receptor.
- the PTHrP receptor ligand is an analog of a PTHrP receptor agonist, antagonist, PTHrP 1-34, abaloparatide, or SEQ ID NO:63.
- the PTHrP 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).
- sequence of the polypeptide may be altered in various ways known in the art to generate targeted changes in sequence.
- the polypeptide will usually 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 with the provided sequence.
- the sequence changes may be substitutions, insertions or deletions. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids.
- Conservative amino acid substitutions typically include substitutions within the following groups: (glycine, alanine); (valine, isoleucine, leucine); (aspartic acid, glutamic acid); (asparagine, glutamine); (serine, threonine); (lysine, arginine); or (phenylalanine, tyrosine).
- Nonconservative substitutions could include any unusual amino acids.
- Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., methylation, acetylation, acylation, pegylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes.
- the peptide comprises an optical isomer, enantiomer, diastereomer, tautomer, cis-trans isomer, racemate, prodrug or pharmaceutically acceptable salt of a peptide.
- the peptides may be amidated at the C-termini.
- the modified peptides optionally contain an acylation modification.
- the number of acylation can be more than one, with one reactive group being preferable.
- Peptide formulations also include a mixture of stereoisomers, or each pure or substantially pure isomer.
- the present compound may optionally have one or more asymmetric centers at a carbon atom containing any one substituent. Therefore, the compound may exist in the form of enantiomer or diastereomer, or a mixture thereof.
- the present compound may exist in the form of geometric isomerism (cis- compound, trans-compound), and when the present compound contains an unsaturated bond such as carbonyl, then the present compound may exist in the form of a tautomer, and the present compound also includes these isomers or a mixture thereof.
- the starting compound in the form of a racemic mixture, enantiomer or diastereomer may be used in the processes for preparing the present compound.
- the present compound When the present compound is obtained in the form of a diastereomer or enantiomer, they can be separated by a conventional method such as chromatography or fractional crystallization.
- the present compound includes an intramolecular salt, hydrate, solvate or polymorphism thereof.
- polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
- the backbone of the peptide may be cyclized by adding cyclic disulfide bridge or lactam bridge to enhance stability (see Friedler et al. (2000) J. Biol. Chem. 275:23783-23789).
- Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D-amino acids or non-naturally occurring synthetic amino acids.
- the subject peptides may be prepared by in vitro synthesis, using conventional methods as known in the art.
- Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, CA, Beckman, etc.
- synthesizers By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids.
- the particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like.
- cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like.
- the polypeptides may also be isolated and purified in accordance with conventional methods of recombinant synthesis.
- a lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique.
- the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein.
- the present invention is directed to the generation and use of self-assembled gel-forming polypeptides.
- the polypeptides are derived from secreted human peptide hormones, human peptide analogs, and polypeptides with gel-forming-enhancing motifs.
- the gel-forming polypeptides may be used in a method of treating a patient with the native or engineered gel-forming polypeptide, the method comprising administering a therapeutic composition comprising an effective dose of a gel-forming polypeptide, alone or in combination with an additional therapeutic agent, to an individual in need thereof.
- compositions and improved methods for generating self-assembling polypeptides may comprise dissolving such polypeptides at suitable concentrations or conjugating them with a gel-forming-enhancing motif.
- Biocompatible and biodegradable polypeptide gels are useful for delivering a therapeutic agent, either as a carrier protein, wherein the therapeutic agent is encapsulated/associated within a gel-forming polypeptide carrier; or as a therapeutic agent itself.
- the reversable phase transition of noncovalently associated monomers in polypeptide gel nanostructure allows the sustained delivery of therapeutic agents in a bioactive form for an extended peirod of time when compared with an injection of an aqueous solution or a crystal particle suspension formulation.
- the resulting liquid or semisolid gels generally exhibit minimal immunogenic response and readily degradable.
- aspects of the invention further include one or more bioactive agents adsorbed or absorbed within the hydrogels and where the hydrogel is configured to deliver the one or more bioactive agent to a site of administration, such as by implanting the subject hydrogel, coating an implant with the hydrogel, ingesting the hydrogel.
- the amount of bioactive agent incorporated will depend on the duration of delivery, site of application as well as the condition being treated.
- the amount of bioactive agent incorporated into the subject hydrogels is 0.0001 mg or greater, such as 0.001 mg or greater, such as 0.01 mg or greater, such as 0.1 mV or greater, such as 1 mg or greater, such as 10 mg or greater, such as 25 mg or greater, such as 50 mg or greater, such as 100 mg or greater such as 500 mg or greater, such as 1000 mg or greater such as 5000 mg or greater and including 10,000 mg or greater.
- the concentration of bioactive agent may be 0.0001 mg/mL or greater, such as 0.001 mg/mL or greater, such as 0.01 mg/mL or greater, such as 0.1 mg/mL or greater, such as 0.5 mg/mL or greater, such as 1 mg/mL or greater, such as 2 mg/mL or greater, such as 5 mg/mL or greater, such as 10 mg/mL or greater, such as 25 mg/mL or greater, such as 50 mg/mL or greater, such as 100 mg/mL or greater such as 500 mg/mL or greater, such as 1000 mg/mL or greater such as 5000 mg/mL or greater and including 10,000 mg/mL or greater.
- the effective dose of a therapeutic polypeptide delivered as a gel formulation to a human patient may be from about 1.0 mg/kg weight, 2.5 mg/kg weight, 5.0 mg/kg weight, 10.0 mg/kg weight, 25.0 mg/kg weight, 50.0 mg/kg weight, 75.0 mg/kg weight, 0.1 mg/kg weight, 0.5 mg/kg weight, 1.0 mg/kg weight, 2.5 mg/kg weight, 5.0 mg/kg weight, 7.5 mg/kg weight, 10.0 mg/kg weight, 25.0 mg/kg weight, 50.0 mg/kg weight, 75.0 mg/kg weight, 1100 mg/kg weight, 250 mg/kg weight, 500 mg/kg weight, 750 mg/kg weight, 1 g/kg weight, 2 g/kg weight, 5 g/kg weight, 10 g/kg weight, 25 g/kg weight up to about 50 g/kg weight, or more, or any range of doses with these parameters.
- the effective dose may be maintained for a period of time sufficient to treat the condition.
- the release of the one or more bioactive agents from the hydrogel matrix may vary.
- hydrogels of the present invention may be configured to provide a sustained release.
- the gel may be designed to release a therapeutic dose of the polypeptide for 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or more day, e.g. 1 week, 2 weeks, 3 weeks, 4 weeks or more.
- sustained release is meant that the hydrogel is structured to provide for continuous delivery of one or more bioactive agents over the entire time hydrogel is maintained in contact with the site of administration, such as over the course of 1 day or longer, such as 2 days or longer, such as 5 days or longer, such as 10 days or longer, such as 15 days or longer, such as 30 days or longer and including 100 days or longer.
- aspects of the invention also include methods for treating a subject by applying one or more gel of the invention to the subject.
- methods include applying a hydrogel solution or suspension to the subject and maintaining the hydrogel in contact with the subject in a manner sufficient to treat the subject.
- hydrogels of interest may be applied to any suitable application site in need of treatment, including by not limited to the skin, bones, heart, liver, kidneys, bladder, trachea, lungs, tumor tissues, in the mouth such as buccally and sublingually and within the nose, throat, ears, uterus, and bladder etc.
- methods may include applying one or more hydrogels and maintaining the hydrogel in contact with the subject in a manner sufficient to deliver a target dosage of bioactive agent, such as for example as characterized by total bioactive agent exposure or by average daily bioactive agent exposure.
- a target dosage of bioactive agent delivered by subject methods may be 0.01 mg/day or greater, such as 0.04 mg/day or greater, such as 0.5 mg/day or greater over a 4 week dosage interval, such as 1.0 mg/day or greater, such as 2 mg/day or greater, such as 5 mg/day or greater and including 10 mg/day over a 4 week dosage interval.
- gel-forming-enhancing motifs are identified, which improve the generation of gellable molecules.
- the minimal structural change associated with conjugation of a gel- enhancing motif has minimal effects on the volume of distribution characteristics of the therapeutic agents discussed herein.
- a minimal effect on volume of distribution of said modification is beneficial, as it allows the therapeutic agent to distribute in vivo with pharmacokinetics similar to a wild-type counterpart polypeptide. This property is preferred in many situations because the use of other half-life extension techniques such as the fusion to a large molecule (e.g., fusion to an IgG, albumin, or PEG) would drastically increase the molecular mass and reduce the volume of distribution of the said therapeutics, thereby preventing them from reaching many intended targets outside of the general circulation.
- the gel-forming therapeutics represents discrete molecule, not a complex, and can have deepter distribution into tissues when compared to therapeutics that are conjugated or complexed with large carrier molecules.
- a gel nanostructure generally dissociates into monomeric molecules in an aqueous solution as the molecules in the peripheral surface of the gel gradually diffuse into the surrounding solution.
- all percentages mentioned in the present invention are weight/weight (w/w) percentages.
- amylin a type B GPCR ligand
- amylin a type B GPCR ligand
- the related adrenomedullin peptide CGRP forms liquid gel nanostructure at select concentrations
- the quality of peptides is further judged by two other criteria: (1) mobility following a tapping of the tube (tube tapping assay) and (2) mobility flowing tilting the tube by 90o (tilted tube assay).
- the tube tapping assay the tube is tapped with the finger for 10 times, and the number of times that the body of the solution changes position is recorded.
- An aqueous solution such as water will have a score of 9 or 10 in this assay; meaning that the body of the liqiud moves every time when one taps the tube.
- an insoluble or precipitated peptide in solution will exhibit the same score; that is a score of 9 or 10 and macroscopic change in positions of the insoluble peptide particles or precipitates can be observed with each tapping.
- the gel-forming peptides dissolve instantly or gradually in the aqueous solution and exhibit high viscosity at 20 min after mixing with water. Those with moderate viscosity form liquid gel that changes the conformation slowly when the container tube is tapped with a finger or tilted. To differentiate the liquid gels from semisolid gels, we tested the mobility of the solution based on the tube tapping assay and the tilted tube assay.
- Selected peptide solutions exhibited extremely high viscosity and formed semisolid gels that do not flow as a liquid when the container is tapped with a finger or tilted.
- the gel has a score of 0-4 (i.e., macroscopic change in the position of the body of gel after tapping was observed in 0-4 times out of 10 separate tapping tests), it is considered a semisolid gel. If macroscopic change in the position of the body of gel was observed 5-9 time out of 10 separate tapping tests, the gel is considered a liquid gel.
- the high viscosity of a liquid gel is further confirmed using the tilted tube assay.
- the plastic tube that contains the solution or gel was tilted 90o, and the movement of the solution or the gel mass is observed under the light, if the liquid body moves to the bottom of the tube in less than 15 second, the peptide is considered a liquid solution. If the liquid or gel body takes more than 15 seconds to flow to the bottom of the tilted tube, the resulting aqueous mass is considered a liquid gel.
- the agonistic activities toward MC1 R, MC4R, CRHR2, PTHR1 , and OPRK1 were assyed with an MC1 R cAMP assay, an MC4R cAMP assay, a CRHR2 cAMP assay, a PTHR1 cAMP assay and an OPRK1 cAMP assay from Discoverx Inc. (Fremont, California), respectively.
- the antagonistic activities toward bradykinin receptor 2 (BDKRB2) and GnRH receptor (GnRHR or LHRHR) were assayed with a BDKRB2 arrestin assay and a GnRHR calcium flux assay from Discoverx, respectively.
- cAMP Hunter cell lines were expanded from freezer stocks, and cells were seeded in a total volume of 20 pl_ into white walled, 384-well microplates and incubated at 37C for the appropriate time prior to testing.
- the cAMP modulation was determined using the DiscoverX HitHunter cAMP XS+ assay.
- agonistic activity determination cells were incubated with sample to induce response.
- Media was aspirated from cells and replaced with 15 mI_ 2:1 HBSS/10mM Hepes: cAMP XS+ Ab reagent.
- % Activity 100% x (mean RLU of test sample - mean RLU of vehicle control)/(mean RLU of MAX control - mean RLU of vehicle control).
- Path Hunter cell lines (Discoverx Inc.) were expanded from freezer stocks, and seeded in a total volume of 20 pL into white walled, 384-well microplates and incubated at 37C for the appropriate time prior to testing.
- agonist determination cells were incubated with sample to induce response.
- Intermediate dilution of sample stocks was performed to generate 5X sample in assay buffer, and 5 pL of 5X sample was added to cells and incubated at 37C or room temperature for 90 to 180 minutes. Vehicle concentration was 1 %.
- antagonistic activity determination cells were pre-incubated with antagonist followed by agonist challenge at the EC80 concentration.
- Assay signal was generated through a single addition of 12.5 or 15 pL (50% v/v) of PathHunter Detection reagent cocktail, followed by an one hour incubation at room temperature.
- the microplates were read following signal generation with a PerkinElmer EnvisionTM instrument for chemiluminescent signal detection.
- the compound activity was analyzed using a CBIS data analysis suite (Chemlnnovation, CA).
- % Activity 100% x (mean RLU of test sample - mean RLU of vehicle control)/(mean MAX control ligand - mean RLU of vehicle control).
- % Inhibition 100% x (1 - (mean RLU of test sample - mean RLU of vehicle control)/(mean RLU of EC80 control - mean RLU of vehicle control)).
- cell lines were expanded from freezer stocks and seeded in a total volume of 20 pL into black-walled, clear-bottom, Poly-D-lysine coated 384-well microplates and incubated at 37C for the appropriate time prior to testing. Assays were performed in 1 x Dye Loading Buffer consisting of 1x Dye, 1x Additive A and 2.5 mM Probenecid in HBSS/20 mM Hepes. Cells were loaded with dye prior to testing. Media was aspirated from cells and replaced with 20 pL Dye Loading Buffer. Cells were incubated for 30-60 minutes at 37C. For agonist determination, cells were incubated with sample to induce response.
- 1 x Dye Loading Buffer consisting of 1x Dye, 1x Additive A and 2.5 mM Probenecid in HBSS/20 mM Hepes. Cells were loaded with dye prior to testing. Media was aspirated from cells and replaced with 20 pL Dye Loading Buffer
- % Activity 100% x (mean RFU of test sample - mean RFU of vehicle control)/(mean MAX RFU control ligand - mean RFU of vehicle control).
- percentage inhibition 100% x (1 - (mean RFU of test sample - mean RFU of vehicle control)/(mean RFU of EC80 control - mean RFU of vehicle control)).
- the gel-forming capability of a variety of secreted and therapeutic polypeptides or their analogs was determined, shown in Table 1. All peptides were produced by solid phase synthesis, and their ability to form semisolid or liquid gel was analyzed at room temperature. To allow systematic analysis of the gel-forming ability, the assay was first conducted at a peptide concentration of 11% w/w in aqueous solution. The peptides analyzed included functional regulators of cell surface receptors, enzymes, complement factors, antimicrobial peptides, immunomodulators, therapeutic peptides, cell-penetrating peptide, antigens, matrikines, and analogs of CGRP, adrenomedullin and adenomedullin 2 (or intermedin).
- adrenomedullin 1-52 At 11% w/w in an aqueous excipient, adrenomedullin 1-52, Pramlintide (an amylin analog), oxytocin, kisspeptin, a kappa receptor agonist, Pralmorelin, a thrombopoietin analog (i.e., Romiplostim analog), urocortin 3, a bombesin receptor antagonist, an acylated ADM2-52, and compstatin (SEQ ID NOS: 2, ,4- 11 , 61 , and 274) consistently formed semisolid clear or opaque gel in aqueous solution.
- Pramlintide an amylin analog
- oxytocin kisspeptin
- a kappa receptor agonist i.e., Romiplostim analog
- urocortin 3 i.e., Romiplostim analog
- urocortin 3 a bombes
- an adrenomedullin analog i.e., ADE43
- CGRP CGRP
- Teduglutide a GLP-2 analog
- thymosin alpha- 1 GLP-1
- gamma-MSH thymosin alpha- 1
- GLP-1 gamma-MSH
- an acylated thymosin beta 4 analog and a GnRH analog
- SEQ ID NOS:1 , 3, 12-15, 64, and 263 consistently formed a liquid gel at 11% w/w.
- a variety of other functional polypeptides SEQ ID NOS: 16-47 either remain as clear liquid solution or form insoluble precipitates at the select concentration after 20 minutes of reaction.
- Polypeptides that remains as clear aquous solution or as insoluble precipitates lack the ability to form a gel nanostructure.
- Abbreviations used in the Table include SS for semisolid gel, LG for liquid gel.
- a graphic representation of semisolid gel formed by SEQ ID NO: 1 peptide is shown in Figure 1. The semisolid gel retains the position when the holding tube is positioned upright (A), tilted 90° (B) or inverted (C).
- palmitoylation modification in SEQ ID NO:274 allowed the analog to form semisolid gel at 6%, which is superior to the wild-type peptides and other adrenomedullin analogs.
- Other modifications as shown in the peptides of SEQ ID NOS:49-58 allowed these analogs to retain the ability to form liquid gel at 11 % w/w.
- gel-forming capability is regulated by many physical and chemical factors, gel-forming peptides, such as adrenomedullin and adrenomedullin 2 may contain sequence motifs that promote gel formation, and changes in residues or side chains can alter the gel-forming capability of these peptides.
- the gel-forming peptides such as adrenomedullin, Pramlintide (an amylin analog), oxytocin, kisspeptin, a kappa opioid receptor agonist, Pralmorelin, a Romiplostim analog, urocortin 3, compstatin, CGRP, Teduglutide (a GLP-2 analog), thymosin alpha 1 , GLP-1 , a GnRH, a bombesin receptor antagonist, gamma-MSH, thymosin beta 4, and an adrenomedullin analog (SEQ ID NOS:1-15, 61 , 64, 263, and 274) can be delivered using a self-assembling gel formulation, i.e. optionally in the absence of additional gel-forming agents.
- these gel-forming peptides could contain gel-forming-enhancing motifs that promote self-assemble gel formation.
- gel-forming-enhancing motifs derived from self- assemble gel-forming peptides may be used to render non-gel-forming peptides into self- assemble gel-forming peptides. While many truncated adrenomedullin/adrenomedullin 2 analogs form semisolid or liquid gels (SEQ ID NOS: 116-124 and 126-131), polypeptides composed of sequences from corresponding regions in CGRP or amylin (SEQ ID NOS: 132-134) remain as aqueous solution or insoluble precipetates at the same concentration.
- a gel- forming-enhancing motif in these peptides allows the formation of semisolid gel at a concentration (i.e., at 30%, 20%, 11%, or 6% w/w) that does not allow for semisolid gel formation by the wild- type counterpart.
- concentration i.e., at 30%, 20%, 11%, or 6% w/w
- the phase transition can be observed within 20 min after solubilization.
- the wild-type polypeptides only form liquid gel, aqueous solution, or stay as insoluble precipitates.
- the wild-type vasopressin (SEQ ID NO: 17) does not form gel at 11%, whereas the gel-forming-enhancing motif-conjugated vasopressin (SEQ ID NO:204) forms semisolid gel at 11 and 6% w/w.
- the wild type oxytocin (SEQ ID NO:5) is able to forms seimisolid gel at 11 %, but does not form liquid or semisolid gel at 6% w/w.
- the gel-forming-enhancing motif-conjugated oxytocin analog (SEQ ID NO:205) forms semisolid gel at 11% and liquid gel at 6% w/w.
- the wild-type apelin (SEQ I D NO: 18) does not form gel at 20, 11 , or 6% w/w.
- the gel-forming-enhancing motif conjugated apelin analog (SEQ ID NOS:206 and 262) forms liquid gel at 20 or 11% w/w.
- the wild-type neuroteinsn (SEQ ID NO: 19) does not form gel at 20 or 11% w/w
- the gel-forming-enhancing motif-conjugated neurotensin analog (SEQ ID NO:207) forms liquid gel at 20 and 11 % w/w.
- the wild type bombesin (SEQ ID NO:20) does not form gel at 11 or 6% w/w.
- the conjugation of a gel-forming- enhancing motif to bombesin or a bombesin receptor antagonist leads to analogs (SEQ ID NOS:209 and 255) that form semisolid or liquid gel at 11 , 6 or 3% w/w.
- analogs SEQ ID NOS:209 and 255 that form semisolid or liquid gel at 11 , 6 or 3% w/w.
- the kisspeptin peptide SEQ ID NO:6
- the gel-forming-enhancing motif-conjugated kisspeptin analog forms gel at the same concentrations.
- the ligands for opioid receptors including deltrophin and endephalin (SEQ ID NOS:21 and 22), do not form gels at 11 or 6% w/w.
- the gel-forming-enhancing motif conjugated deltorphin analogs (SEQ ID NOS:210 and 211) are able to form semisolid gel at 11 % and semisolid or liquid gel at 6% w/w.
- the the gel-forming-enhancing motif conjugated enkephalin analogs (SEQ ID NOS:212 and 213) can form semisolid gels at even 3% w/w. Nonetheless, a kappa opioid receptor agonist (SEQ ID NO:7) can form semisolid gel at 11 , 6 or 3% w/w.
- the corresponding gel-forming-enhancing motif conjugated analog (SEQ ID NO:214) retains the ability to form seimisolid gel at 11 and 6% w/w, and to form liquid gel at 3% w/w.
- the wild-type substance P analogs (SEQ ID NOS:23 and 69) only forms liquid gel at 20% w/w or not at all.
- the conjugated analog (SEQ ID NOS:215, 253, and 254) become able to form semisolid gel at 11 % or 20% w/w.
- the angiotensin receptor antagonist saralasin (SEQ ID NO:24) does not form gel at 11 % w/w.
- the gel-forming-enhancing motif is conjugated to saralasin causes the resulting analog (SEQ ID NO:216) to obtain the capability to form liquid gel at 20 or 11% w/w.
- the wild-type calcitonin (SEQ ID NO:28) does not form gel at 11 % w/w.
- the gel-forming-enhancing motif conjugated calcitonin (SEQ ID NO:217) is able to form semisolid gel at 11% and liquid gel at 6% w/w.
- the amylin peptide analog, Pramlinitide (SEQ ID N 0 : 4) , does not form gel at 6% but form semisolid gel at 11 %.
- the resulting analog (SEQ ID NO:218) is capable of forming semisolid gel at 11 or 6% w/w.
- the gel-forming-enhancing motif conjugated exenatide 4 analog forms semisolid gel at 11 % and liquid gel at 6% w/w.
- the wild-type GLP-1 (SEQ ID NO: 14) only forms liquid gel at 11 %.
- the gel-forming-enhancing motif conjugated GLP-1 analogs (SEQ ID NOS:220, 269, and 270) and gel-forming-enhancing motif-substituted analog (SEQ ID NO:221) form semisolid gels at 20 or 11 % w/w.
- the GLP-2 analog, Tedglutide (SEQ ID NO:12) forms liquid gel at 11 %.
- the gel-forming-enhancing motif conjugated GLP-2 analog forms semisolid gel at 11% and liquid gel at 6% w/w.
- the melanotan I analog afamelanotide and melanotan II analog do not form gel at 20 or 11% w/w.
- the gel-forming-enhancing motif conjugated afamelanotide analogs (SEQ ID NOS:223, 224, 256, 257, and 273) are able to form semisolid or liquid gel at 20, 11 , or 6% w/w.
- the ACTH1-24 peptide (SEQ ID NO:33) does not form gel at 20 or 11 % w/w.
- the gel-forming-enhancing motif conjugated ACTH1-24 analogs can form semisolid gel at 20 or 11% w/w.
- the setmelanotide peptide (SEQ ID NO:34), an agonist of the melanocortin 4 receptor (MC4R), does not form gel at 30, 20, or 11 % w/w.
- M4R melanocortin 4 receptor
- the resulting analog (SEQ ID NO:227) is able to form gel at 30% w/w.
- the setmelanotide analog (SEQ ID NO:228) with a Pal-HSY gel-forming-enhancing motif forms semisolid gel at 11 , 6, or 3% w/w.
- the gamma-MSH (SEQ ID NO: 64) forms liquid gel at 11 % and stay soluble at 6% w/w.
- the gel-forming-enhancing motif-conjugated gamma MSH analog (SEQ ID NO:260) was able to form liquid gel at 6% w/w.
- the peptide YY receptor agonist PYY3-36 (SEQ ID NO:30) does not form gel at 20 or 11 % w/w.
- the resulting analog (SEQ ID NOS:229 and 264) gains the ability to form semisolid or liquid gel at 20% or 11 % w/w.
- the corticotropin receptor 2 (CRHR2) agonist urocortin 2 (UCN2) peptide (SEQ ID NO:31) does not form gel at 20 or 11 % w/w.
- the gel-forming-enhancing motif conjugated urocortin 2 analogs (SEQ ID NOS:230 and 265), by contrast, gains the ability to form semisolid or liquid gel at 20% w/w.
- the urocortin 3 (UCN3) peptide (SEQ ID NO:10) forms semisolid gel at 11 and 6% and forms liquid gel at 3% w/w.
- the gel-forming-enhancing motif conjugated UCN3 analog (SEQ ID NO:231) retains the ability to form gel at these concentrations.
- the parathyroid hormone peptide (SEQ ID NO:25) does not form gel at 11 or 6% w/w.
- the resulting analog gains the ability to form semisolid gel at 20% and liquid gel at 11% w/w.
- the bradykinin receptor ligand HOE140 an antagonist of BKR2 (SEQ ID NO:32)
- the resulting analog gains the ability to form semisolid gel at 30% w/w.
- the resulting analog (SEQ ID NO:234) is able to form semisolid gel at 20% w/w.
- Another bradykinin receptor antagonist, SEQ ID NO 65 does not form a gel at 11 %or 6% w/w, whereas, the modified analog SEQ ID NO 261 forms semisolid gel at 11% and liquid gel at 6% w/w.
- the Pralmorelin peptide forms semisolid gel at 11 or 6% w/w and liquid gel at 3% w/w. Similar to the wild-type Pralmorelin peptide, the gel-forming-enhancing motif conjugated Pralmorelin analog (SEQ ID NO:235) forms semisolid gel at the same concentrations. Unlike Pralmorelin, the sermorelin peptide (SEQ ID NO:35) does not form gel at 20 or 11 % w/w.
- the sermorelin analog gains the ability to form semisolid gel at 20% when it is fused with a short gel-forming-enhancing motif (SEQ ID NO:237) but not when it is fused with a long gel-forming- enhancing motif (SEQ ID NO:236).
- the atrial natriuretic peptide (ANP)(SEQ ID NO:41) does not form gel at 20 or 11% w/w.
- the gel-forming-enhancing motif conjugated ANP analog (SEQ ID NO:238) gains the ability to form semisolid gel at 20 and 11% w/w.
- the immunoregulator thymosin alpha 1 (SEQ ID NO: 13) is capable of forming semisolid gel at 20% and liquid gel at 11% w/w.
- thymosin alpha 1 When the N- terminus of thymosin alpha 1 is substituted with a gel-forming-enhancing motif (Pal-HSY) or an even shorter gel-forming motif, it retains the ability to form semisolid gel at 11% w/w (SEQ ID NOS:240, 267 and 268).
- the analog (SEQ ID NO:239) with a long gel-forming- enhancing motif (Pal-SSPHSY) does not form gel at the same concentration.
- a thymosin beta 4 analog, SEQ ID NO 263, forms liquid gel at 11 % and 20% w/w.
- VIP vasoactive intestinal peptide
- SEQ ID NO:67 The vasoactive intestinal peptide (VIP) (SEQ ID NO:67) does not form gel at 20 or 11% w/w.
- the gel-forming-enhancing motif conjugated VIP analog (SEQ ID NO:266) gains the ability to form semisolid gel at 11 % w/w.
- the adrenomedullin analog SEQ ID NO 274 forms semisolid gel at 20, 11 , or 6% w/w, which is superior to the wild- type adrenomedullin SEQ ID NO:2 or a shorter acylated analog (SEQ ID NO:1).
- the cell-penetrating enhancing peptide (CPP) TAT by itself (SEQ ID NO:36) cannot form gel at 20 or 11 % w/w. It gains the ability to form liquid gel at 11% w/w when it is fused with a gel- forming-enhancing motif (SEQ ID NO:241).
- a kallikrein inhibitor analog (SEQ ID NO:37) does not form gel at 11 % w/w.
- the resulting analog gains the ability to form semisolid gel at 11 % and liquid gel at 6% w/w.
- the temporin A peptide which is an antimicrobial peptide, is fused with a gel-forming-enhancing motif, the resulting analog (SEQ ID NO:243) forms semisolid gel at 11% w/w.
- the complement system regulator compstatin (SEQ ID NO: 11) forms semisolid gel at 11 % w/w
- its gel-forming-enhancing motif-containing analog (SEQ ID NO:244) forms semisolid gel at the same concentration.
- the immunoregulator Glatiramer peptide (or Copaxone)(SEQ ID NO:42) does not form gel at 20 or 11% w/w.
- the resulting analog (SEQ ID NO:245) obtains the capability to form semisolid gel at 20% w/w.
- the positive control sauvagine peptide exhibits an EC50 of 3.6 nM, suggesting that select modified urocortin peptide also had a superior receptor-activation activity.
- Studies of PTHR1 receptor activities showed that the gel-forming PTH analog (SEQ ID NO:232) has an EC50 of 14.1 nM on PTHR1 whereas the positive control PTH(1-34) peptide has an EC50 of 0.8 nM.
- the gel-forming kappa opioid receptor agonist (SEQ ID NO:214) stimulated OPRK1 with an EC50 of 272 nM, and the positive control dynorphin A peptide has an EC50 of 0.5 nM.
- FITC-labled therapeutic agents were dissolved in solutions with a low concentration of gel-forming or nongel forming peptides, and centrifuged to separate the free-moving soluble molecules in the lower camber and those aggregrate with the gel nanostrucutres in the upper chamber.
- the movement of FITC-labled molecules was quantified by a fluorometer.
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