Hydrophobic peptide salts for extended release compositions

Abstract

This invention generally relates to hydrophobic salts of hydrophilic peptides, which form substances with low solubility in aqueous solutions and are capable of sustained or continuous release of peptide components upon administration to an individual. It also discloses hydrophobic salts of C-type natriuretic peptides and their uses.

Description

Technical Field

[0001] [Cross-reference to related applications] []

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 885,705, filed August 12, 2019; U.S. Provisional Patent Application No. 62 / 935,052, filed November 13, 2019; U.S. Provisional Patent Application No. 62 / 963,354, filed January 20, 2020; U.S. Provisional Patent Application No. 62 / 964,848, filed January 23, 2020; and U.S. Provisional Patent Application No. 63 / 038,652, filed June 12, 2020, all of which are incorporated herein by reference in their entirety. [] Incorporate materials submitted electronically by way of citation []

[0003] The sequence list, which is part of this invention, is submitted as a formal document along with the specification. The formal document containing the sequence list is named "54627_Seqlisting.txt", created on August 6, 2020, and is 54,454 bytes in size. The subject of the sequence list is incorporated herein by reference in its entirety. Technical Field

[0004] This invention relates generally to hydrophobic salts of hydrophilic peptides, which form substances with low solubility in aqueous solutions and can continuously release peptide components when administered to an individual. Prior Technology

[0005] Sustained delivery of therapeutic agents is desirable, for example, to reduce the number of doses or the amount of drug that an individual can accept to achieve therapeutic benefit. However, certain types of active ingredients in pharmaceuticals are difficult to formulate into sustained-release compositions (e.g., enteric-coated, dose-responsive capsules or tablets, or microspheres, such as liposomes or nanoparticles), which can deliver therapeutic agents at certain sites in the body or have certain slow-release properties that allow the drug to slowly escape from the particles over time. Summary of the Invention

[0006] This invention relates to compositions comprising electrostatically charged peptide salts having low solubility in solution, such that the salts form solids or semi-solids in aqueous media. It is shown herein that such salts dissolve more slowly in aqueous solutions than in the non-salt form of the peptides and can be used for extended-release therapeutic agents without the need for reformulation in a typical extended-release form.

[0007] This document provides compositions comprising hydrophobic salts of electrostatically charged peptides, wherein the salt comprises an electrostatically charged peptide misaligned with a hydrophobic relative ion. In various embodiments, the salt is a hydrophobic peptide salt.

[0008] In various embodiments, the peptide salt contains at least about 10% by weight of peptides. In various embodiments, the peptide salt contains at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more by weight of peptides. In various embodiments, the peptide salt contains at least about 5% by weight of active peptides. In various embodiments, the peptide salt contains at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or more by weight of active peptides. In various embodiments, the active peptide percentage relative to the total peptides present in the salt is at least about 50%, 60%, 70%, 80%, or more. In various embodiments, the hydrophobic peptide salt has slow dissolution and is not immediately soluble in 1× phosphate-buffered saline (PBS) at a concentration of 1 mg / mL.

[0009] In various embodiments, hydrophobic relative ions are misaligned with electrostatic peptides via non-covalent bonds.

[0010] In various embodiments, the hydrophobic peptide salt further comprises a multivalent cation misaligned with the peptide-hydrophobic relative ion complex. In various embodiments, the electrostatic peptide, the hydrophobic relative ion, and the multivalent cation are misaligned via non-covalent bonds. In various embodiments, the multivalent cation misaligned with the peptide-hydrophobic relative ion complex is a metal cation.

[0011] In various embodiments, the peptide salt containing the hydrophobic relative ion has a cLogP of about 0 to about 10, or the conjugate acid of the hydrophobic relative ion has a pKa of -2 to 5, or both. In various embodiments, the peptide salt containing the hydrophobic relative ion has a cLogP of about 2 to about 9, or the conjugate acid of the hydrophobic relative ion has a pKa of less than about 5, or both. In various embodiments, the peptide salt containing the hydrophobic relative ion has a cLogP of about 2 to about 9, and the conjugate acid of the hydrophobic relative ion has a pKa of less than about 5. In various embodiments, the peptide salt containing the hydrophobic relative ion has a cLogP of about 2 to about 9, and the conjugate acid of the hydrophobic relative ion has a pKa of about 0 to about 5.

[0012] In various embodiments, the hydrophobic relative ionic system is selected from the group consisting of: deprotonated fatty acids, deprotonated cholic acid, naphthates and their derivatives, nicotinic acid salts and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextran sulfates, alkylbenzene sulfates, ionic surfactants, and combinations thereof. In various embodiments, the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidyl, decanoate, 2-naphthalenesulfonate, 1-heptanesulfonate, 1-octylsulfonate monohydrate, 1-decylsulfonate, dodecyl sulfate, dextran sulfate, and dodecylbenzenesulfonate. In various embodiments, the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthate, docusate, or dodecyl sulfate. In various embodiments, the relative ion is selected from the group consisting of: oleate, dihydroxynaphthate, deoxycholate, decanoate, and docusate.

[0013] In various embodiments, the charge of the polyvalent cation is +2, +3, or +4, or higher. In various embodiments, the charge of the polyvalent cation is +2, +3, or +4. In some embodiments, the charge of the polyvalent cation is +2. In some embodiments, the charge of the polyvalent cation is +3. In some embodiments, the charge of the polyvalent cation is +4. In various embodiments, the cation is a metal cation. In various embodiments, the cation includes metals selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au). In various embodiments, the cation includes zinc or calcium. In various embodiments, the cation is Mg2+, Zn2+, or Ca2+. In various embodiments, the cation is Zn2+ or Ca2+. In various embodiments, the cation is Zn2+. In various embodiments, the cation is Ca2+.

[0014] In various embodiments, the peptide salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle. In various embodiments, the peptide salt is in solid form. In various embodiments, the peptide salt is in amorphous form. In various embodiments, the peptide salt is in gel form. In various embodiments, the peptide salt is suspended in a gel or attached to a gel.

[0015] In various embodiments, the electrostatic peptide is a C-type natriuretic peptide (CNP). In various embodiments, the CNP is a CNP variant. The CNP and CNP variants covered herein are described more fully in the embodiments. In various embodiments, the CNP is misaligned with a hydrophobic relative ion to form a hydrophobic CNP salt complex. In various embodiments, the hydrophobic CNP salt further comprises a multivalent cation misaligned with the CNP-hydrophobic relative ion complex. In various embodiments, the multivalent cation is a metal cation.

[0016] In various embodiments, the CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37; SEQ ID NO: 1); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4) and their salts. In various embodiments, the CNP salt suitable for forming the hydrophobic CNP salts described herein is a CNP-acetate.

[0017] In various embodiments, CNP is selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0018] In various embodiments, the CNP variant peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus.

[0019] In various embodiments, CNP variants are selected from the following groups: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH(SEQ ID NO: 8); Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH(SEQ ID NO: 10); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11); Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12); Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13); and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0020] In various embodiments, the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthyl salt, docusate, or dodecyl sulfate. In various embodiments, if a polyvalent cation is present, the polyvalent cation includes zinc or calcium. In various embodiments, if a polyvalent cation is present, the polyvalent cation includes magnesium, zinc, or calcium. In various embodiments, the cation is Mg²⁺, Zn²⁺, or Ca²⁺. In various embodiments, the polyvalent cation is Zn²⁺ or Ca²⁺. In various embodiments, the polyvalent cation is Zn²⁺. In various embodiments, the polyvalent cation is Ca²⁺.

[0021] In various embodiments, the composition further includes an excipient, diluent, or carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier. Sterile pharmaceutical compositions comprising the hydrophobic salt compositions described herein are also provided.

[0022] This invention further covers sustained-release compositions comprising the hydrophobic peptide salts described herein. In various embodiments, the sustained-release composition is a delayed-release composition, a sustained-release composition, or a delayed-release composition.

[0023] In various embodiments, the extended-release composition comprises a hydrophobic peptide salt, wherein the peptide salt solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle is resuspended in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer solution.

[0024] In various embodiments, the oil comprises triglycerides or fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. In various embodiments, the fatty acids are short-chain, medium-chain, or long-chain fatty acids. In various embodiments, when the fatty acids are in triglycerides, the fatty acids are saturated or unsaturated, and may be medium-chain or long-chain fatty acids.

[0025] In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is hexanoic acid, caprylic acid, decanoic acid, or dodecanoic acid.

[0026] In various embodiments, for the extended-release composition, at pH 7 to 7.6, (i) less than about 20% of the peptide is released on day 1; and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly.

[0027] In various embodiments, less than about 20% of the peptide is released on day 1 at pH 7 to 7.6. It is further anticipated that (i) less than about 30%, or about 40%, or about 50%, or about 60% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Further expectations include (i) the release of less than about 30%, or about 40%, or about 50%, or about 60% of the peptide on day 1 at pH 7.0 to 7.6; and (ii) the release of about 70%, about 80%, or about 90% of the peptide weekly at pH 7 to 7.6; or about 70%, about 80%, or about 90% of the peptide every two weeks; or about 70%, about 80%, or about 90% of the peptide every three weeks; or about 70%, about 80%, or about 90% of the peptide monthly.

[0028] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Further expectations include (i) the release of less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide on day 1 at pH 7.0 to 7.6; and (ii) the release of about 70%, about 80%, or about 90% of the peptide weekly at pH 7 to 7.6; or about 70%, about 80%, or about 90% of the peptide every two weeks; or about 70%, about 80%, or about 90% of the peptide every three weeks; or about 70%, about 80%, or about 90% of the peptide monthly; or (ii) at pH 7.0 to 7.6. At levels 7 to 7.6, approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released weekly; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every two weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every three weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released monthly.

[0029] In various embodiments, approximately 90% of the peptide is released weekly at pH 7 to 7.6. In various embodiments, approximately 90% of the peptide is released every two weeks at pH 7 to 7.6. In various embodiments, approximately 90% of the peptide is released monthly at pH 7 to 7.6. Further, this release is anticipated at pH 7.0 to 7.6, pH 7.1 to 7.5, pH 7.2 to 7.4, pH 7.2 to 7.6, or pH 7.0 to 7.4.

[0030] In various embodiments, the extended-release composition includes an excipient, a diluent, or a carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier. In various embodiments, a sterile pharmaceutical composition comprising the extended-release composition is provided.

[0031] This article also provides a method for manufacturing hydrophobic peptide salt compositions, such as hydrophobic CNP salts, as described herein. Ionic surfactants are good candidates for relative ions because the polar head groups are permanently charged, regardless of the misalignment pH. Adjusting the pH at which misalignment occurs will generate different amounts of charge on the peptide, and this allows control of the peptide:surfactant misalignment stoichiometry, and may control the size of the resulting precipitate. Metal cations can be used as bridging bonds for the anionic side chains of peptide amino acids to bind with the anionic hydrophobic relative ions. The order and rate of addition of cations and relative ions to the peptide are important for minimizing precipitation of metal cations with the anionic hydrophobic relative ions.

[0032] In various embodiments, the present invention covers a method for preparing a composition comprising an electrostatic peptide salt, comprising: a) contacting an electrostatic peptide in an aqueous solution with a hydrophobic relative ion in the solution; b) mixing the electrostatic peptide solution and the hydrophobic relative ion solution in a manner sufficient to cause the peptide and the relative ion to form a complex, wherein the formation of the peptide-relative ion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle, including the hydrophobic peptide salt. In various embodiments, the peptide salt is a hydrophobic CNP salt.

[0033] In various embodiments, the method may include, as appropriate, contacting the electrostatically charged peptide in solution with a polyvalent cation in aqueous solution prior to step (b) to form a peptide-cation complex. In various embodiments, the polyvalent cation is a metal cation.

[0034] In various embodiments, mixing is carried out by adding a hydrophobic relative ionic solution dropwise to an electrostatically charged peptide solution. In various embodiments, the solutions are mixed by vortexing after each drop of hydrophobic relative ionic solution is added, or by other mixing methods known in the art.

[0035] In various embodiments, the method further includes step (c) washing the peptide salt in a buffer or water. In various embodiments, the washing is performed in an aqueous solution, such as a buffer or water.

[0036] In various embodiments, the method further includes step (d) of obtaining the peptide salt by centrifugation to form a peptide salt centrifuge block. In various embodiments, if the salt is in gel form, it is obtained by centrifugation or by decanting the liquid phase followed by lyophilization or other drying methods.

[0037] In various embodiments, the method further includes step (e) removing water from the peptide salt centrifuge block. It is contemplated that techniques known in this art can be used to remove water or another aqueous solution from the centrifuge block by lyophilization or drying.

[0038] In various embodiments, the method further includes resuspending the centrifuged block in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer solution. In various embodiments, the oil comprises triglycerides or fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. In various embodiments, the fatty acids in the triglycerides are saturated or unsaturated, or a combination thereof.

[0039] The fatty acid may be the oil itself or in a triglyceride. In various embodiments, the fatty acid is a short-chain, medium-chain, or long-chain fatty acid. In various embodiments, when the fatty acid is in a triglyceride, the fatty acid may be saturated or unsaturated, and may be a medium-chain or long-chain fatty acid. In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is hexanoic acid, caprylic acid, decanoic acid, or dodecanoic acid.

[0040] In various embodiments, the synthetic method is intended to use a peptide:hydrophobic relative ion ratio of at least one mole equivalent of the total number of positively charged amino acids in the peptide. In various embodiments, the peptide:hydrophobic relative ion ratio used in the synthetic method is 1:1 to 1:20, or 1:1 to 1:50. The peptide:relative ion ratio used in the synthetic method may be 1:2 to 1:15, 1:2 to 1:10, 1:2 to 1:8, 1:3 to 1:10, or 1:4 to 1:10. In various embodiments, the peptide:relative ion ratio is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In various embodiments, the peptide:hydrophobic relative ion ratio used in the synthesis method is at least two molar equivalents of hydrophobic relative ions to the total number of positively charged amino acids in the peptide. In various embodiments, the peptide:hydrophobic relative ion ratio used in the synthesis method is at least three molar equivalents of hydrophobic relative ions to the total number of positively charged amino acids in the peptide.

[0041] In various embodiments, the synthesis method is intended to use a peptide:cation ratio of at least one mole equivalent of cations to the total number of negatively charged amino acids in the peptide. In various embodiments, the peptide:cation ratio used in the synthesis method is 1:1 to 1:10. The peptide:cation ratio used in the synthesis method may be 1:2 to 1:10, 1:3 to 1:10, 1:1 to 1:5, 1:2 to 1:5, or 1:2 to 1:8. In various embodiments, the peptide:cation ratio used in the synthesis method is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In various embodiments, the peptide:cation ratio used in the synthesis method is at least two mole equivalents of cations to the total number of negatively charged amino acids in the peptide. In various embodiments, the peptide:cation ratio used in the synthesis method is at least three mole equivalents of cations to the total number of negatively charged amino acids in the peptide. Further anticipates the combination of the peptide:cation and peptide:hydrophobic relative ion ratios described above.

[0042] An exemplary ratio is one relative ion per positive charge on the peptide. For polyvalent cations, an exemplary ratio is approximately one metal cation per negatively charged site in the peptide, and two polyvalent cations per negatively charged site, or a 2 × mol excess. For example, one or two polyvalent cations, such as Zn²⁺ or Ca²⁺, can be used in combination with 6-8 or more relative ions (if hydrophobic interactions are involved).

[0043] In various embodiments, the hydrophobic relative ionic system is via non-covalent bond misalignment.

[0044] In various embodiments, if the salt complex further comprises a multivalent cation complexed with a peptide-relative ion complex, the cation is complexed via bond misalignment. In various embodiments, the electrostatic peptide, hydrophobic relative ion, and cation are complexed via non-covalent bond misalignment.

[0045] In various embodiments, the CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0046] Also provided is a method for treating bone-related conditions or skeletal dysplasia in an individual in need, comprising administering to the individual a composition comprising a hydrophobic salt of a C-type natriuretic peptide (CNP), including the composition as described herein and a delayed-release composition.

[0047] In various embodiments, bone-related conditions or skeletal dysplasia are selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, chondrodysplasia, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, flexor dysplasia, congenital lethal hypophosphatase syndrome, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, decreased cartilage production, pedicle-type punctate chondrodysplasia, Jansen-type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, skeletal dysplasia, malformation dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia. dysplasia), Robinow syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone dysplasia, Kniest dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, small limbs, Morquio syndrome, Kniest syndrome, metaphyseal dysplasia and vertebral epiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner's syndrome / Leri Weill)), PTPN11 mutation (Noonan's syndrome) and idiopathic short stature.

[0048] In various embodiments, CNP variants are suitable as adjunctive or alternative growth hormones for the treatment of idiopathic short stature and other skeletal dysplasia.

[0049] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville) or PTPN11 mutations (Nunan syndrome).

[0050] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville) or PTPN11 mutations (Nunan syndrome) or insulin-like growth factor 1 receptor (IGF1R).

[0051] In various embodiments, CNP variants are suitable for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0052] In various embodiments, growth plate syndrome or short stature is associated with one or more mutations in genes related to Lasso disease.

[0053] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by Lasso disease. In various embodiments, Lasso disease is Noonan syndrome, Costello syndrome, cardiomyoplasty syndrome, neurofibromatosis type 1, or Leopard syndrome.

[0054] In one embodiment, Lasso disease is hereditary type 1 gingival fibromatosis.

[0055] In various embodiments, CNP variants are suitable for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R).

[0056] In various embodiments, short stature is associated with one or more mutations in genes related to Lasso disease.

[0057] In various embodiments, the CNP variant is suitable for treating individuals with short stature who have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one parent has a height SDS of less than -1.0, -1.5, -2.0, or -2.5, whereby the second parent's height is within the normal range. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -3.0. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -2.5. In various embodiments, short stature is associated with mutations in one or more genes related to short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, short stature is associated with mutations in one or more genes related to Lasso disease.

[0058] In various embodiments, short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). In various embodiments, the individual has an NPR2 mutation and low PRS. In various embodiments, the individual has an FGFR3 mutation and low PRS. In various embodiments, the individual has an NPR2 mutation and low PRS. In various embodiments, the individual has an IGF1R mutation and low PRS. In various embodiments, the individual has an NPPC mutation and low PRS. In various embodiments, the individual has a SHOX mutation and low PRS. In various embodiments, the individual has one or more mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX and low PRS. In various embodiments, PRS is 1 or 2. In various embodiments, PRS is 1. In various embodiments, PRS is 2.

[0059] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus. In various embodiments, the variant includes one or more linking groups as described herein. In various embodiments, the linking group is a hydrolyzable linking group. In various embodiments, the variant is a hydrophobic salt of an electrostatically charged CNP peptide, the salt comprising an electrostatically charged CNP peptide misaligned with a hydrophobic relative ion.

[0060] The present invention also covers a method for bone lengthening or increasing long bone growth in an individual in need, comprising administering to the individual a sustained-release composition comprising a salt of C-type natriuretic peptide (CNP), including the composition as described herein and the extended-release composition, wherein the administration results in bone lengthening or increasing long bone growth.

[0061] In various embodiments, CNP is a variant of CNP. In various embodiments, CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); And its pharmaceutical salts. In various embodiments, CNP is CNP-acetate.

[0062] In various embodiments, CNP is selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0063] In various embodiments, the variant CNP further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on a side group of an amino acid of the peptide. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus.

[0064] In various embodiments, the variants are selected from the group consisting of: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH(SEQ ID NO: 8); Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH(SEQ ID NO: 10); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11); Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12); Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13); and Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0065] In various embodiments, the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0066] In various embodiments, the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0067] In various embodiments, the composition is a delayed-release composition.

[0068] Hydrophobic salts of C-type natriuretic peptides (CNPs) comprising CNPs in complexes with hydrophobic opposing ions are also provided. In various embodiments, the hydrophobic CNP salt further comprises a cation complexed with both the CNP and the hydrophobic opposing ion. In various embodiments, the hydrophobic CNP salt is a purified salt. In various embodiments, the salt has a purity of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or greater.

[0069] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on a side group of an amino acid of the peptide. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus. In various embodiments, the variant includes one or more linking groups as described herein. In various embodiments, the linking group is a hydrolyzable linking group. In various embodiments, the peptide includes a hydrophobic salt of an electrostatic peptide, the salt comprising an electrostatic peptide misaligned with a hydrophobic relative ion. Simple Explanation of the Diagram

[0070] Figures 1A-1D show the solubility curves of different hydrophobic CNP salts in water at pH 6.5.

[0071] Figure 2 shows the solubility curves of different hydrophobic CNP salts in water at pH 6.5. The control is CNP-acetate.

[0072] Figure 3 shows the effect of the CNP variant (Pro-Gly-CNP37) on cells carrying NPR2 isotype-binding or heterotype-binding mutations, as measured by cGMP stimulation.

[0073] Figure 4 shows the nucleotide sequence of the first exon and the predicted protein sequence in the NPR2 mutant pure line transfected into RCS cells.

[0074] Figure 5 shows an illustrative NPR2 mutation for response analysis to CNP.

[0075] Figure 6 shows exemplary mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX.

[0076] Figures 7A-7F illustrate the combined effects of PRS and rare coding variants on height. Figure 7A. Effect on height as a quantitative trait, samples were divided into five groups based on their PRS, with horizontal lines representing violin plots of the 25th, 50th, and 75th percentiles of height. Samples were grouped into any of the five core genes based on missense, loss-of-function, or non-carrier status. Figure 7B. Effect reflected by odds ratios for "idiopathic short stature" or ISS. Odds ratio of ISS with PRS=3 as a reference relative to other PRS groups. Figure 7C. Odds ratio of ISS with PRS=1 as a reference relative to ISS with missense and / or loss-of-function variants in the core genes. Figure 7D. Odds ratio of ISS with PRS=1 non-carriers as a reference relative to ISS with missense and / or loss-of-function variants in the core genes. Figure 7E. Odds ratio of ISS with PRS=2 non-carriers as a reference relative to ISS with missense and / or loss-of-function variants in the core genes. Figure 7F. Odds of winning the ISS relative to the core gene with missense and / or loss-of-function variants, using PRS=3 noncarriers as a reference.

[0077] Figure 8A shows the release curves of CNP Zn-bis(hydroxynaphthyl) salt. The data shown is the average of three wells. Figure 8B shows the release curves of different CNP peptide salts. The data shown is the average of four wells.

[0078] Figures 9A and 9B illustrate the dissolution curves of CNP bis(hydroxynaphthyl) salt in the form of cumulative release curves (Figure 9A) or release percentages (Figure 9B). The data shown are the average values ​​of three wells for 1×PBS data and the average values ​​of two wells for 1×PBS + 0.05% PS80 data.

[0079] Figures 10A to 10C show the dissolution curves of docusate salts after freeze-drying and storage (Figure 10A), or freshly prepared and subsequently analyzed (Figures 10B-10C).

[0080] Figure 11 shows the release curve of CNP salt in vivo over 7 days. Implementation

[0081] This invention relates to hydrophilic peptide salts, which are solid, semi-solid gels, or other salt forms capable of extending the release of the active peptide component when placed in an aqueous solution. For example, it is shown here unexpectedly that hydrophilic C-type natriuretic peptides (CNPs) complex with charged, hydrophobic relative ions under aqueous conditions to form peptide salts with low solubility. This invention demonstrates that peptide-hydrophobic relative ion salt complexes themselves, including salts containing peptide-relative ion-cation complexes, can be used in sustained-release or extended-release compositions without encapsulating the peptide complexes in liposomes or microspheres / nanoparticles. Such compositions are suitable for extended-release applications, such as the treatment of skeletal dysplasia and osteophyte disorders as described herein.

[0082] As used in this specification and the accompanying claims, unless the context clearly indicates otherwise, the indefinite articles “a” and “an” and the definite article “the” include both plural and singular indicators.

[0083] The terms "about" or "approximately" mean the acceptable error of a particular value as determined by someone generally skilled in the art, depending in part on how the value is measured or determined. In some embodiments, the terms "about" or "approximately" mean within 1, 2, 3, or 4 standard deviations. In some embodiments, the terms "about" or "approximately" mean within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of a given value or range. When the terms "about" or "approximately" precede the first value in a series of two or more values, it should be understood that the terms "about" or "approximately" apply to each value in the series.

[0084] As used herein, the term "electrostatic peptide" refers to a peptide, such as an amino acid string comprising 5 to 100 charged amino acids. A peptide may have positively charged amino acids, negatively charged amino acids, or a mixture of both, such that the electrostatic peptide has an overall net charge and is capable of interacting with other charged parts, such as cations, anions, or relative ions having charged particles opposite to those in a peptide that can bind to a relative ion. Electrostatic peptides may have a net positive charge or a net negative charge. When a peptide has a net positive charge, it can interact with charged parts having one or more negative charges. When a peptide has a net negative charge, it can interact with charged parts having one or more positive charges.

[0085] As used herein, the term "hydrophobic relative ion" refers to a group of electrostatically charged portions that are inherently hydrophobic and capable of interacting with hydrophilic peptides. In various embodiments, the hydrophobic relative ion is selected based on its cLogP value, the pKa value of its conjugate acid, or both. In various embodiments, the hydrophobic relative ion has a cLogP of about 0 to about 10, or its conjugate acid has a pKa of -2 to 5, or both. In various embodiments, the hydrophobic relative ion has a net negative charge and can interact with electrostatically charged peptides having a net positive charge. In various embodiments, the hydrophobic relative ion includes deprotonated fatty acids, deprotonated cholic acids, naphthates and their derivatives, nicotinic acid salts and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextran sulfate, and alkylbenzene sulfates. In some embodiments, the hydrophobic relative ion is an amphoteric ion (e.g., phosphatidylethanolamine). In various embodiments, the hydrophobic relative ion includes, but is not limited to, palmitate, deoxycholate, oleate, dihydroxynaphthyl salt, nicotinic acid salt, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidyl salt, decanoate, 2-naphthalenesulfonate, 1-heptanesulfonate, 1-octylsulfonate monohydrate, 1-decylsulfonate, dodecyl sulfate, dextran sulfate, and dodecylbenzenesulfonate. In some embodiments, the hydrophobic relative ion has a net positive charge and can interact with electrostatically charged peptides having a net negative charge.

[0086] As used herein, the term "peptide salt" or "hydrophobic peptide salt" refers to a complex between an electrostatically charged peptide and a relative ion, such that the components are in the complex and form a salt. The peptide and the relative ion may be non-covalently misaligned. In various embodiments, the peptide-relative ion salt further comprises a polyvalent cation, such that the complex contains a misaligned peptide-cation-relative ion. A peptide salt or hydrophobic peptide salt refers to both peptide-relative ion complexes and peptide-cation-relative ion complexes.

[0087] In various embodiments, the peptide and cation are non-covalently misaligned. In various embodiments, the peptide, cation, and hydrophobic relative ions in the peptide salt are non-covalently misaligned.

[0088] The term "C-type natriuretic peptide" or "CNP" refers to a small single-chain peptide (GenBank accession number NP_077720, for the CNP precursor protein NPPC) with a 17-amino acid ring structure at its C-terminus and its variants. CNPs are initially generated from the natriuretic peptide precursor C (NPPC) gene as a single-chain 126-amino acid proto-peptide, which is cleaved to produce pro-CNP and an active 53-amino acid peptide (CNP-53), which is secreted and cleaved again by an unknown enzyme to produce a mature 22-amino acid peptide (CNP-22). "CNP salt" or "hydrophobic CNP salt" refers to a salt that includes a relative ion, such as a hydrophobic relative ion, and, where applicable, a polyvalent cation, and includes CNP as described herein.

[0089] In various embodiments, the "CNP variant" is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to the wild-type NPPC on the same number of amino acid residues. In various embodiments, the CNP variant peptide may comprise about 1 to about 53, or 1 to 38, or 1 to 37, or 1 to 35, or 1 to 34, or 1 to 33, or 1 to 32, or 1 to 31, or 1 to 27, or 1 to 22, or 10 to 35, or about 15 to about 37 NPPC polypeptide residues. In one embodiment, a CNP variant may include a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, or 53 amino acid sequences derived from an NPPC polypeptide.

[0090] This document provides sustained-release compositions comprising the hydrophobic peptide salts described herein. Sustained-release compositions include those that delay drug delivery (delayed-release dose) or deliver drug over a prolonged period of time (continuous-release dose). Various examples of peptide salts provided herein include sustained-release compositions, such as delayed-release, sustained-release, or controlled-release, and delayed-release. The term "delayed-release composition" refers to a composition formulated in a manner that facilitates the manufacture of an active ingredient / drug available for use over a prolonged period of time following administration (US Pharmacopeia). Delayed-release doses include sustained-release (SR) or controlled-release (CR) forms. Sustained-release maintains drug release over a sustained period of time, but not necessarily at a constant rate, while CR maintains drug release over a sustained period of time at a nearly constant rate (Pharmaceutics: Drug Delivery and Targeting, Yvonne Perrie, Thomas Rades, Pharmaceutical Press, 2009). Delayed-release compositions or products are modified to delay the release of a drug substance over a period of time following the initial administration.

[0091] The term "effective dose" refers to a dose sufficient to produce the desired outcome for an individual's health condition, lesion, or disease, or sufficient for diagnostic purposes. The desired outcome may include subjective or objective improvement in the recipient of the dose. "Therapeutic effective dose" refers to a dose of medicine that effectively produces the expected beneficial effect on health. The appropriate "effective" dose in any individual case can be determined by a person skilled in the art using routine laboratory methods. It should be understood that the specific dose level and frequency for any particular patient can vary and will depend on a variety of factors, including the activity of the specific compound used; bioavailability, metabolic stability, rate of secretion, and duration of action of the mixture; the mode and timing of compound administration; the patient's age, weight, general health condition, sex, and diet; and the severity of the specific condition.

[0092] "Treatment" refers to preventive, therapeutic, or diagnostic treatment. In some embodiments, "treatment" means the administration of a compound or composition to an individual for therapeutic, preventive, or diagnostic purposes.

[0093] "Preventive" treatment is the administration of medication to individuals who do not exhibit symptoms of disease or only exhibit early symptoms of disease, with the aim of reducing the pathological risk. The compounds or compositions of the present invention can be provided as preventive treatment to reduce the likelihood of disease or to minimize the severity of disease (if present).

[0094] "Therapeutic" treatment is the administration of treatment to an individual exhibiting pathological signs or symptoms for the purpose of alleviating or eliminating such signs or symptoms. Signs or symptoms can be biochemical, cellular, histological, functional, or physical, subjective or objective. The compounds of this invention may also be provided as a therapeutic treatment or for diagnosis.

[0095] "Pharmaceutical composition" or "formulation" means a composition suitable for medicinal use in an individual animal (including humans and mammals). A pharmaceutical composition includes a therapeutically effective amount of a hydrophobic peptide salt (e.g., a CNP salt), another bioactive agent as appropriate, and a pharmaceutically acceptable excipient, carrier, or diluent as appropriate. In one embodiment, a pharmaceutical composition encompasses compositions comprising an active ingredient and an inert component constituting a carrier, as well as any product directly or indirectly resulting from the combination, compounding, or aggregation of any two or more components, or from the dissociation of one or more components, or from other types of reactions or interactions of one or more components. Therefore, the pharmaceutical composition of this invention encompasses any composition obtained by blending the compounds of this invention with a pharmaceutically acceptable excipient, carrier, or diluent.

[0096] "Pharmaceutically acceptable carriers" refers to any of the standard pharmaceutical carriers, buffers, and analogues, such as phosphate-buffered saline solutions, 5% dextran solutions, and emulsions (e.g., oil / water or water / oil emulsions). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifiers, wetting agents, lubricants, slip agents, sweeteners, flavoring agents, and colorants. Suitable pharmaceutical carriers, excipients, and diluents are described in Remington's Pharmaceutical Sciences, 19th edition (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended mode of administration of the active agent. Typical modes of administration include enteric (e.g., oral) or non-enteric (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or local, percutaneous, or mucosal administration).

[0097] "Medically acceptable salts" are salts that can be formulated into compounds for medicinal use, including but not limited to metal salts (such as sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0098] "Medically acceptable" or "pharmacologically acceptable" means a substance that is not biologically or otherwise undesirable and can be administered to an individual without causing any undesirable biological effects or without interacting in a harmful manner with any component of the composition containing it or with any component present on or in the individual's body.

[0099] "Physiological conditions" refers to the conditions within an animal's body (such as a human). Physiological conditions include, but are not limited to, body temperature and an aqueous environment with physiological ionic strength, pH, and enzymes. Physiological conditions also encompass the conditions within a specific individual that differ from the "normal" conditions present in most individuals, such as a temperature different from the normal human body temperature of approximately 37°C or a blood pH different from the normal human blood pH of approximately 7.4.

[0100] As used herein, the term "individual" encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammal class: humans, non-human primates such as chimpanzees and other ape and monkey species; livestock such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and similar animals. This term does not indicate a specific age or sex. In various embodiments, the individual is a human being. In various embodiments, the individual is a child or adolescent. In various embodiments, the individual is an infant.

[0101] [Electrically charged peptides and peptide salts] []

[0102] Peptide therapeutics are attractive biotherapeutic agents, but are generally disadvantaged due to their low stability in solution and short half-life (Tang et al., *Eur J Pharm Sci.* 102:63-70, 2017). Attempts to improve the efficacy of peptide therapeutics have included encapsulating hydrophilic peptides into biodegradable particles, such as liposomes or polymer particles. However, this has been challenging due to the cationic nature of these peptides and their ability to electrostatically interact with negatively charged polymer liposomes (Griesser et al., *Int J Pharmaceutics* 520:267-274, 2017). The generation of hydrophobic ion pairs between hydrophilic peptides and their hydrophobic moieties has become a means of better encapsulating hydrophilic polymers into microparticles or liposomes (Lu et al., *Molecular Pharmaceutics* 15:216-225, 2018). Hydrophobic ion pairs are formed when charged residues in the peptide interact with oppositely charged ions in the hydrophobic moieties (Tang et al., cf.). In some cases, this can cause the hydrophobic ion pairs to precipitate from solution, making them easier to encapsulate into liposomes or polymeric nanoparticles (Griesser et al., cf.).

[0103] This paper has discovered that hydrophobic ionic complexes between hydrophilic CNP peptides and hydrophobic counterparts generate CNP peptide salts. The generation of hydrophobic ion pairs between hydrophilic peptides and hydrophobic counterparts can be enhanced by first contacting the hydrophilic peptide with a polyvalent cation (e.g., a metal cation) to strengthen the interaction between the peptide and the hydrophobic counterpart. For example, a polyvalent cation can kinase with the negatively charged functional groups of the hydrophilic peptide, thereby increasing the number of positive charges available for kinase with hydrophobic counterparts, such as hydrophobic anions. Thus, the polyvalent cation can bridge the negative charges of the hydrophilic peptide and the hydrophobic counterpart. Furthermore, this invention demonstrates that the peptide-hydrophobic counterpart salt complex itself can be used in sustained-release or extended-release compositions without encapsulating the peptide complex in liposomes or microspheres / nanoparticles.

[0104] A charged peptide may be a string of 5 to 100 amino acids comprising charged amino acids and having a total net charge. The peptide may have positively charged amino acids, negatively charged amino acids, or a mixture of both, such that the charged peptide can interact with other charged portions, such as cations, anions, or relative ions or combinations thereof having charged particles opposite to those in the peptide. In various embodiments, the charged peptide has a net positive charge. A charged peptide with a net positive charge may misalign with a negatively charged hydrophobic relative ion, such as a hydrophobic relative ion with a net negative charge. In various embodiments, the charged peptide has a net negative charge. A charged peptide with a net negative charge may misalign with a positively charged hydrophobic relative ion, such as a hydrophobic relative ion with a net positive charge. In various embodiments, the charged peptide has at least two amino acids having the same charge type (e.g., two positively charged amino acids or two negatively charged amino acids).

[0105] Hydrophilic peptides are peptides that have high solubility in aqueous solutions. Hydrophilic peptides as defined herein include peptides of 5 to 100 amino acids having a net charge of +3 to +15, or +4 to +15, or +3 to +12, or +4 to +12, or +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, or +15. In various embodiments, the hydrophilic peptide has a solubility in aqueous solution greater than 10 mg / mL, or a solubility greater than 5 mg / mL. In various embodiments, hydrophilic peptides also refer to peptides with high solubility in aqueous solutions, such as peptides having a cLogP of less than 1.

[0106] Hydrophobic relative ions

[0107] To generate the electrostatically charged peptide salts as described herein, the peptide is misaligned with a relative ion. In the case of a hydrophilic peptide, the relative ion is a hydrophobic relative ion. In various embodiments, the hydrophobic relative ion has a net negative charge and forms a salt with a hydrophilic peptide having a net positive charge.

[0108] It is expected that the relative ion will bind to the charged peptide via non-covalent bonding. The relative ion can also bind to the peptide non-covalently via electrostatic interactions.

[0109] When a hydrophobic relative ion is used, the hydrophobic relative ion exhibits a cLogP of about 0 to about 10, or its conjugate acid exhibits a pKa of about -2 to about 5, or both. In various embodiments, the hydrophobic relative ion has a cLogP of about 2 to about 9, about 3 to 8, about 4 to 7, or about 5 to 9. In various embodiments, the cLogP is about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In various embodiments, the conjugate acid of the hydrophobic relative ion has a pKa of about -1 to 4, 0 to 3, 0 to 5, 1 to 4, or 2 to 5. In various embodiments, the conjugate acid of the hydrophobic relative ion has a pKa of about -2, -1, 0, 1, 2, 3, 4, or 5. Further hydrophobic relative ions having any of these values ​​and combinations of ranges between these values ​​are contemplated. In various embodiments, the hydrophobic relative ion has a cLogP of about 2 to about 9, or its conjugate acid has a pKa of less than about 5, or both. In various embodiments, the hydrophobic relative ion has a cLogP of about 2 to about 9, and its conjugate acid has a pKa of less than about 5.

[0110] In various embodiments, the relative ion is an anion. In various embodiments, the relative ion is an amphoteric ion. In various embodiments, the relative ion is an anionic or amphoteric detergent. In various embodiments, the hydrophobic relative ion is selected from the group consisting of: deprotonated fatty acids, deprotonated cholic acid, naphthates and their derivatives, nicotinic acid salts and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextran sulfates, alkylbenzene sulfates, and ionic surfactants. In various embodiments, the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidic acid, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octylsulfonate monohydrate, sodium 1-decylsulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In various embodiments, the hydrophobic relative ion is oleate, dihydroxynaphthate, deoxycholate, decanoate, or docusate.

[0111] In various embodiments, at least one hydrophobic relative ion is misaligned with a hydrophilic peptide (when a multivalent cation is absent) or with a complex comprising a hydrophilic peptide and a multivalent cation (when a cation is present). In various embodiments, at least two hydrophobic relative ions are misaligned with a hydrophilic peptide (when a cation is absent) or with a complex comprising a hydrophilic peptide and a multivalent cation (when a cation is present). In various embodiments, at least three hydrophobic relative ions are misaligned with a hydrophilic peptide (when a cation is absent) or with a complex comprising a hydrophilic peptide and a multivalent cation (when a cation is present). In various embodiments, at least four hydrophobic relative ions are misaligned with a hydrophilic peptide (when a cation is absent) or with a complex comprising a hydrophilic peptide and a multivalent cation (when a cation is present). In various embodiments, the positive charge of each hydrophilic peptide is misaligned with the hydrophobic relative ion. For example, if the peptide has four positively charged amino acids, it may misalign with four hydrophobic relative ions. Similarly, if a peptide has four positively charged amino acids and is misaligned with two cations for a total of six positive charges, then the peptide can be misaligned with six hydrophobic ions. In various embodiments, not all positive charges of a hydrophilic peptide (when no cations are present) or a complex comprising a hydrophilic peptide and a polyvalent cation (when cations are present) are misaligned with hydrophobic ions. For example, if a peptide has four positively charged amino acids, it can be misaligned with three, two, or one hydrophobic ions. Similarly, if a peptide has four positively charged amino acids and is misaligned with two cations for a total of six positive charges, then the peptide can be misaligned with five, four, three, two, or one hydrophobic ions.

[0112] Multivalent cations

[0113] In various embodiments, the peptide salt further comprises a multivalent cation misaligned with a peptide-relative ionic complex. It is anticipated that the cation misaligns with the charged peptide via non-covalent bonding. The cation may also non-covalently bind to the peptide via electrostatic interactions.

[0114] The polyvalent cation is expected to have a charge of +2, +3, or +4 or higher. In one embodiment, the cation has a charge of +2. In another embodiment, the cation has a charge of +3. In yet another embodiment, the cation has a charge of +4. In various embodiments, the polyvalent cation is a metal cation. Metal cations include metals from Group II and Group III. Suitable polyvalent cations may include metals selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au). In various embodiments, the polyvalent cation includes magnesium, zinc, or calcium. In various embodiments, the polyvalent cation includes zinc or calcium. In one embodiment, the polyvalent cation includes zinc. In another embodiment, the polyvalent cation includes calcium. In various embodiments, the polyvalent cation is selected from the group consisting of Mg2+, Zn2+, and Ca2+. In various embodiments, the polyvalent cation is Zn2+ or Ca2+. In one embodiment, the polyvalent cation is Zn2+. In another embodiment, the polyvalent cation is Ca2+.

[0115] In various embodiments, at least one polyvalent cation is misaligned with the hydrophilic peptide. In various embodiments, at least two cations are misaligned with the hydrophilic peptide. In various embodiments, at least three cations are misaligned with the hydrophilic peptide. In various embodiments, each negatively charged part of the hydrophilic peptide is misaligned with a polyvalent cation. For example, if the peptide has four negatively charged amino acids, it may be misaligned with four, three, two, or one polyvalent cation.

[0116] peptide salts

[0117] In various embodiments, the peptide salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle. In various embodiments, the peptide salt is in the form of a solid, semi-solid, or gel. In various embodiments, the peptide salt is in the form of a solid or gel. In various embodiments, the peptide salt is in the form of a solid. In various embodiments, the peptide salt is in the form of an amorphous substance. In various embodiments, the peptide salt is in the form of a gel. In various embodiments, the peptide salt is suspended in or attached to a gel.

[0118] The peptide salts are expected to be in particle form, and particularly in solid particle form. In various embodiments, the particles are 1 to 10,000 micrometers (μm), 1 μm to 2000 μm, 2 μm to 1000 μm, 5 μm to 500 μm, 10 μm to 1000 μm, 50 μm to 500 μm, 100 μm to 800 μm, 200 to 600 μm, 300 μm to 500 μm, 100 μm to 300 μm, 50 μm to 100 μm, or 10 μm to 50 μm. In various embodiments, the particles are nanoparticles. In various embodiments, the nanoparticles are approximately 5 nm to 1000 nm, 8 nm to 900 nm, 10 nm to 800 nm, 20 nm to 600 nm, 50 nm to 500 nm, 50 to 400 nm, 20 to 300 nm, 300 to 800 nm, or 200 to 600 nm.

[0119] In some embodiments, the hydrophilic peptide of the peptide salt is a CNP or a CNP variant as described herein, and the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidic acid, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octylsulfonate monohydrate, sodium 1-decylsulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In some embodiments, the hydrophilic peptide salt is a CNP or a CNP variant as described herein, and the hydrophobic relative ion is selected from the group consisting of: oleate, dihydroxynaphthate, deoxycholate, and decanoate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-dihydroxynaphthylate, CNP-deoxycholate, and CNP-decanoate. In some embodiments, the hydrophilic peptide salt is a CNP or a CNP variant as described herein, and the hydrophobic relative ion is selected from the group consisting of oleate, dihydroxynaphthylate, deoxycholate, decanoate, and docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-dihydroxynaphthylate, CNP-deoxycholate, CNP-decanoate, and CNP-docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-dihydroxynaphthylate, and CNP-docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-deoxycholate, CNP-decanoate, and CNP-docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate and CNP-dihydroxynaphthylate. In various embodiments, the peptide salt is CNP-oleate. In various embodiments, the peptide salt is CNP-dihydroxynaphthylate. In various embodiments, the peptide salt is CNP-docusate.

[0120] In various embodiments, the hydrophilic peptide of the peptide salt is Pro-Gly CNP37 (PG-CNP37), and the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidic acid, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octylsulfonate monohydrate, sodium 1-decylsulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In some embodiments, the hydrophilic peptide salt is PG-CNP37 as described herein, and the hydrophobic relative ion is selected from the group consisting of: oleate, dihydroxynaphthate, deoxycholate, and decanoate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate, PG-CNP37-dihydroxynaphthylate, PG-CNP37-deoxycholate, and PG-CNP37-decanoate. In some embodiments, the hydrophilic peptide salt is PG-CNP37 as described herein, and the hydrophobic relative ion is selected from the group consisting of oleate, dihydroxynaphthylate, deoxycholate, decanoate, and docusate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate, PG-CNP37-dihydroxynaphthylate, PG-CNP37-deoxycholate, PG-CNP37-decanoate, and PG-CNP37-docusate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate, PG-CNP37-dihydroxynaphthylate, and PG-CNP37-docusate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-deoxycholate, PG-CNP37-decanoate, and PG-CNP37-docusate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate and PG-CNP37-dihydroxynaphthylate. In various embodiments, the peptide salt is PG-CNP37-oleate. In various embodiments, the peptide salt is PG-CNP37-dihydroxynaphthylate. In various embodiments, the peptide salt is PG-CNP37-docusate.

[0121] In various embodiments, the peptide-relative ionic salt further comprises a polyvalent cation. In some embodiments, the hydrophilic peptide of the peptide salt is a CNP or a CNP variant as described herein; the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthyl salt, nicotinic acid salt, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidic acid, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octylsulfonate. The hydrates include sodium 1-decylsulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate; and the polyvalent cations include metals selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au). In various embodiments, the hydrophilic peptide salt is a CNP as described herein, and the hydrophobic relative ion is selected from the group consisting of: oleate, dihydroxynaphthate, deoxycholate, and decanoate; and the polyvalent cation is Zn2+ or Ca2+. In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (oleate), CNP-Ca+2 (dihydroxynaphthyl salt), CNP-Ca+2 (deoxycholate), CNP-Ca+2 (decanoate), CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthyl salt), CNP-Zn+2 (deoxycholate), and CNP-Zn+2 (decanoate). In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic relative ion is selected from the group consisting of: oleate, dihydroxynaphthyl salt, deoxycholate, decanoate, and docusate; and the polyvalent cation is Zn2+ or Ca2+. In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic relative ion is selected from the group consisting of: oleate, dihydroxynaphthyl salt, and docusate; and the polyvalent cation is Zn2+ or Ca2+. In various embodiments, the hydrophilic peptide salt is a CNP as described herein, the hydrophobic relative ion is selected from the group consisting of deoxycholate, decanoate, and docusate; and the polyvalent cation is Zn2+ or Ca2+. In various embodiments, the hydrophilic peptide salt is a CNP as described herein, the hydrophobic relative ion is selected from the group consisting of oleate and dihydroxynaphthylate; and the polyvalent cation is Zn2+ or Ca2+. In various embodiments, the hydrophilic peptide salt is a CNP as described herein, the hydrophobic relative ion is oleate; and the polyvalent cation is Zn2+ or Ca2+.In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic relative ion is bis(hydroxynaphthate), and the polyvalent cation is Zn²⁺ or Ca²⁺.

[0122] In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (oleate), CNP-Ca+2 (dihydroxynaphthylate), CNP-Ca+2 (deoxycholate), CNP-Ca+2 (decanoate), CNP-Ca+2 (docusate), CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthylate), CNP-Zn+2 (deoxycholate), CNP-Zn+2 (decanoate), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (oleate), CNP-Ca+2 (dihydroxynaphthylate), CNP-Ca+2 (docusate), CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthylate), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (oleate), CNP-Ca+2 (dihydroxynaphthyl salt), and CNP-Ca+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (deoxycholate), CNP-Ca+2 (decanoate), and CNP-Ca+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (oleate) and CNP-Ca+2 (dihydroxynaphthyl salt). In various embodiments, the peptide salt is CNP-Ca+2 (oleate). In various embodiments, the peptide salt is CNP-Ca+2 (dihydroxynaphthyl salt). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthyl salt), CNP-Zn+2 (deoxycholate), CNP-Zn+2 (decanoate), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthyl salt), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Zn+2 (deoxycholate), CNP-Zn+2 (decanoate), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Zn+2 (oleate) and CNP-Zn+2 (dihydroxynaphthyl salt). In various embodiments, the peptide salt is CNP-Zn+2 (oleate). In various embodiments, the peptide salt is CNP-Zn+2 (dihydroxynaphthyl salt).

[0123] In various embodiments, the peptide salt is selected from the group consisting of: PG-CNP37-Ca+2 (oleate), PG-CNP37-Ca+2 (dihydroxynaphthyl salt), PG-CNP37-Ca+2 (deoxycholate), PG-CNP37-Ca+2 (decanoate), PG-CNP37-Ca+2 (docusate), PG-CNP37-Zn+2 (oleate), PG-CNP37-Zn+2 (dihydroxynaphthyl salt), PG-CNP37-Zn+2 (deoxycholate), PG-CNP37-Zn+2 (decanoate), and PG-CNP37-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: PG-CNP37-Ca+2 (oleate), PG-CNP37-Ca+2 (dihydroxynaphthyl salt), PG-CNP37-Ca+2 (docusate), PG-CNP37-Zn+2 (oleate), PG-CNP37-Zn+2 (dihydroxynaphthyl salt), and CNP-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: PG-CNP37-Ca+2 (oleate), PG-CNP37-Ca+2 (dihydroxynaphthyl salt), and PG-CNP37-Ca+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: CNP-Ca+2 (deoxycholate), PG-CNP37-Ca+2 (decanoate), and PG-CNP37-Ca+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-Ca+2 (oleate) and PG-CNP37-Ca+2 (dihydroxynaphthyl salt). In various embodiments, the peptide salt is PG-CNP37-Ca+2 (oleate). In various embodiments, the peptide salt is PG-CNP37-Ca+2 (dihydroxynaphthyl salt). In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-Zn+2 (oleate), PG-CNP37-Zn+2 (dihydroxynaphthyl salt), PG-CNP37-Zn+2 (deoxycholate), PG-CNP37-Zn+2 (decanoate), and PG-CNP37-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-Zn+2 (oleate), PG-CNP37-Zn+2 (dihydroxynaphthyl salt), and PG-CNP37-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of: PG-CNP37-Zn+2 (deoxycholate), PG-CNP37-Zn+2 (decanoate), and PG-CNP37-Zn+2 (docusate). In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-Zn+2 (oleate) and PG-CNP37-Zn+2 (dihydroxynaphthyl salt).In various embodiments, the peptide salt is PG-CNP37-Zn+2 (oleate). In various embodiments, the peptide salt is PG-CNP37-Zn+2 (bis(hydroxynaphthyl)ate).

[0124] Preparation method

[0125] This article also covers methods for preparing compositions comprising hydrophobic peptide salts as described herein.

[0126] In various embodiments, the present invention provides a method for preparing a composition comprising an electrostatic peptide salt, comprising: a) contacting an electrostatic peptide in an aqueous solution with a hydrophobic relative ion in the solution; b) mixing the electrostatic peptide solution and the hydrophobic relative ion solution in a manner sufficient to cause the peptide and the relative ion to form a complex, wherein the formation of the peptide-relative ion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle, including the peptide salt. In various embodiments, when the peptide-relative ion salt further comprises a polyvalent cation, the method comprises, prior to step (b), contacting the electrostatic peptide in the solution with a polyvalent cation in the aqueous solution to form a peptide-cation complex. The peptide-cation complex is then contacted with the hydrophobic relative ion to form a peptide-cation-relative ion complex.

[0127] In various embodiments, the present invention provides a method for preparing a composition comprising an electrostatic peptide salt, comprising: a) contacting an electrostatic peptide in solution with a multivalent cation in an aqueous solution to form a peptide-cation complex; b) contacting the peptide-cation complex in the aqueous solution with a hydrophobic relative ion in the solution; and c) mixing the peptide-cation complex solution and the hydrophobic relative ion solution in a manner sufficient to cause the peptide-cation and relative ion to form a complex, wherein the formation of the peptide-cation relative ion complex results in the formation of a solid, semi-solid, gel, crystal, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle, including the peptide salt.

[0128] In various embodiments, mixing is carried out by adding a hydrophobic relative ionic solution dropwise to the peptide solution. The solution is mixed by vortexing after each drop of hydrophobic relative ionic solution is added, or by other mixing methods known in the art.

[0129] In various embodiments, the method further includes step (c) or (d) washing the peptide salt in a buffer or water. In various embodiments, the washing is performed in an aqueous solution, such as a buffer or water.

[0130] In various embodiments, the method further includes step (d) or (e) obtaining the peptide salt by centrifugation to form a peptide salt centrifuge block. In various embodiments, if the salt is in gel form, it is obtained by centrifugation or by decanting the liquid phase followed by lyophilization or other drying methods.

[0131] In various embodiments, the method further includes step (e) or (f) removing water from the peptide salt centrifuge block. It is contemplated that techniques known in this art can be used to remove water or another aqueous solution from the centrifuge block by lyophilization or drying.

[0132] In various embodiments, the method further includes resuspending the centrifuged block in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer solution. In various embodiments, the oil comprises triglycerides or fatty acids. In various embodiments, the fatty acids are saturated or unsaturated. In various embodiments, the fatty acids in the triglycerides are saturated or unsaturated, or a combination thereof.

[0133] The fatty acid may be the oil itself or in the triglyceride. In various embodiments, the fatty acid is a short-chain, medium-chain, or long-chain fatty acid. In various embodiments, the fatty acid in the triglyceride is saturated or unsaturated and may be a medium-chain or long-chain fatty acid. In various embodiments, the fatty acid is a C-6 to C-20 fatty acid. In various embodiments, the fatty acid is a C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acid. In various embodiments, the fatty acid is hexanoic acid, caprylic acid, capric acid, or dodecanoic acid.

[0134] In various embodiments, the method encompasses the use of at least one mole equivalent of a hydrophobic relative ion, such as a hydrophobic anion, relative to: 1) the total number of charged amino acids if a cation is present; or 2) the total number of positive charges if a cation is absent. Therefore, in various embodiments, the method encompasses the use of at least one mole equivalent of a hydrophobic relative ion relative to the total number of positive charges in a hydrophilic peptide (when a cation is absent), or relative to the total number of positive charges in a complex comprising a hydrophilic peptide and a cation (when a polyvalent cation is present). This ratio is referred to herein as the peptide:hydrophobic relative ion ratio. In various embodiments, the peptide:hydrophobic relative ion ratio is at least one mole equivalent of a hydrophobic relative ion relative to the total number of positively charged amino acids in a peptide (when a cation is absent) or a complex comprising a peptide and a cation (when a cation is present). In various embodiments, the peptide:hydrophobic relative ion ratio is from 1:1 to 1:20, or from 1:1 to 1:50. The peptide:relative ion ratio can be 1:2 to 1:15, 1:2 to 1:10, 1:2 to 1:8, 1:3 to 1:10, or 1:4 to 1:10. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:1. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:2. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:3. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:4. In various embodiments, the peptide:hydrophobicity relative ion ratio is 1:5. In various embodiments, the peptide:hydrophobic relative ion ratio is 1:6. In various embodiments, the peptide:hydrophobic relative ion ratio is 1:7. In various embodiments, the peptide:hydrophobic relative ion ratio is 1:8. In various embodiments, the peptide:hydrophobic relative ion ratio is 1:9. In various embodiments, the peptide:hydrophobic relative ion ratio is 1:10. In various embodiments, the peptide:hydrophobic relative ion ratio is a hydrophobic relative ion ratio of at least two molar equivalents relative to the total number of positively charged amino acids in the peptide (when no cation is present) or a complex comprising peptide and cation (when cation is present). In various embodiments, the peptide:hydrophobic relative ion ratio is a hydrophobic relative ion ratio of at least three molar equivalents relative to the total number of positively charged amino acids in the peptide (when no cation is present) or a complex comprising peptide and cation (when cation is present). In various embodiments, the peptide:hydrophobic relative ion ratio is a hydrophobic relative ion of at least four molar equivalents relative to the total number of positively charged amino acids in the peptide (when no cation is present) or a complex comprising the peptide and a cation (when a cation is present).In some embodiments, the peptide:hydrophobic relative ion ratio is a hydrophobic relative ion with a total positive charge of less than one mole equivalent relative to the peptide (when cations are absent) or a complex comprising peptides and cations (when cations are present). In various embodiments, the peptide salt has a hydrophobic relative ion with a positive charge of less than one mole equivalent relative to the peptide (when cations are absent) or a complex comprising peptides and cations (when cations are present). In various embodiments, the peptide salt has a hydrophobic relative ion with a positive charge of less than two mole equivalents relative to the peptide (when cations are absent) or a complex comprising peptides and cations (when cations are present). In various embodiments, the peptide salt has a hydrophobic relative ion with a positive charge of less than three mole equivalents relative to the peptide (when cations are absent) or a complex comprising peptides and cations (when cations are present). In various embodiments, the method encompasses introducing a hydrophobic relative ion, for example, with a total positive charge of 0.9, 0.8, 0.7, 0.75, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.1 molar equivalents relative to the peptide (when no cation is present) or a complex comprising the peptide and a cation (when a cation is present).

[0135] In various embodiments, the method encompasses a cation comprising at least one mole equivalent relative to the total number of negatively charged amino acids in the peptide. This ratio is referred to herein as the peptide:cation ratio. In various embodiments, the peptide:cation ratio is 1:1 to 1:10. The peptide:cation ratio may be 1:2 to 1:10, 1:3 to 1:10, 1:1 to 1:5, 1:2 to 1:5, or 1:2 to 1:8. In various embodiments, the peptide:cation ratio is 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. In various embodiments, the peptide:cation ratio is 1:1. In various embodiments, the peptide:cation ratio is 1:2. In various embodiments, the peptide:cation ratio is 1:3. In various embodiments, the peptide:cation ratio is 1:4. In various embodiments, the peptide:cation ratio is 1:5. In various embodiments, the peptide:cation ratio is 1:6. In various embodiments, the peptide:cation ratio is 1:7. In various embodiments, the peptide:cation ratio is 1:8. In various embodiments, the peptide:cation ratio is 1:9. In various embodiments, the peptide:cation ratio is 1:10. In various embodiments, the method encompasses comprising a cation of less than one mole equivalent relative to the total number of negatively charged amino acids in the peptide. In various embodiments, the method encompasses introducing a cation of, for example, 0.9, 0.8, 0.7, 0.75, 0.6, 0.5, 0.4, 0.3, 0.25, 0.2, or 0.1 mole equivalent relative to the total number of negative charges on the peptide. [] [C] [Type natriuretic peptide] []

[0136] C-type natriuretic peptide (CNP) (Biochem.Biophys.Res. Commun., 168: 863-870 (1990) (Genbank accession number NP_077720, for the CNP precursor protein NPPC) (J. Hypertens., 10: 907-912 (1992)) is a small single-chain peptide in the peptide family (ANP, BNP, CNP) with a 17-amino acid ring structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and plays an important role in a variety of biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (Journal of Hypertension 10: 1111-1114 (1992)). CNP is more widely found in the central nervous system, reproductive tract, bones, and vascular endothelium (Hypertension, 49: 419-426 (2007)).

[0137] In humans, CNPs are initially produced from the natriuretic peptide precursor C (NPPC) gene as a single-chain 126-amino acid pro-propeptide (Biochemical and Biophysical Research Exchange, 168: 863-870 (1990)). Removal of the signal peptide produces pro-CNP, which is further cleaved by the endopeptide furin to produce an active 53-amino acid peptide (CNP-53), which is secreted and cleaved again by an unknown enzyme to produce a mature 22-amino acid peptide (CNP-22) (Wu, Journal of Biochemistry 278: 25847-852 (2003)). The difference between CNP-53 and CNP-22 lies in their distribution. CNP-53 is predominantly found in tissues, while CNP-22 is mainly found in plasma and cerebrospinal fluid (J. Alfonzo, *Receptor Signal Transduction Research*, 26: 269-297 (2006)). Both CNP-53 and CNP-22 bind to NPR-B in a similar manner.

[0138] In various embodiments, the CNPs of the present invention comprise truncated CNPs ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53) and having a wild-type amino acid sequence derived from hCNP-53. Such truncated CNP peptides comprise: DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-53)(SEQ ID NO: 56); LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-52)(SEQ ID NO: 15); RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-51)(SEQ ID NO: 16); VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-50)(SEQ ID NO: 17); DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-49)(SEQ ID NO: 18); TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-48)(SEQ ID NO: 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47)(SEQ ID NO: 20); SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-46)(SEQ ID NO: 21); RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-45)(SEQ ID NO: 22); AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-44)(SEQ ID NO: 23); AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-43)(SEQ ID NO: 24); WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-42)(SEQ ID NO: 25); ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-41)(SEQ ID NO: 26); RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-40)(SEQ ID NO: 27); LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-39)(SEQ ID NO: 28); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-36)(SEQ ID NO: 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-35)(SEQ ID NO: 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-33)(SEQ ID NO: 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-32)(SEQ ID NO: 32); RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-31)(SEQ ID NO: 33); KYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-30)(SEQ ID NO: 34); YKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-29)(SEQ ID NO: 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-28)(SEQ ID NO: 36); GANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-27)(SEQ ID NO: 37); ANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-26)(SEQ ID NO: 38); NKKGLSKGCFGLKLDRIGSMSGLGC(CNP-25)(SEQ ID NO: 39); KKGLSKGCFGLKLDRIGSMSGLGC(CNP-24)(SEQ ID NO: 40); KGLSKGCFGLKLDRIGSMSGLGC(CNP-23)(SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC(CNP-22)(SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC(CNP-21)(SEQ ID NO: 42); SKGCFGLKLDRIGSMSGLGC(CNP-20)(SEQ ID NO: 43); KGCFGLKLDRIGSMSGLGC(CNP-19)(SEQ ID NO: 44); GCFGLKLDRIGSMSGLGC(CNP-18)(SEQ ID NO: 45); and CFGLKLDRIGSMSGLGC(CNP-17) (SEQ ID NO: 67).

[0139] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide, which, where applicable, has a mutation / substitution at the furin cleavage site (underlined) and / or contains glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants include, but are not limited to: QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N);SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37;SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37;SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37(M32N);SEQ ID NO: 49]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37;SEQ ID NO: 50); and GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Gly-CNP-37: SEQ ID NO: 51) [G] QEHPNARKYKGAN [PK] GLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 52); [G] QEHPNARKYKGAN [QK] GLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 53); [G] QEHPNARKYKGAN [QQ] GLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 54); and [G] QEHPNARKYKGAN [KP] GLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 55).

[0140] In various embodiments, CNP is selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0141] In various embodiments, the CNP variant peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus.

[0142] In other embodiments, for any of the CNPs and CNP variants described herein having aspartic (Asn / N) residues and / or glutamic acid (Gln / Q) residues, regardless of whether they have wild-type or non-natural amino acid sequences, any Asn residue and / or any Gln residue may be independently substituted with any other natural or non-natural amino acid, including conserved substitutions such as Asn to Gln. Such substitutions are partly designed to minimize or avoid any potential deacetylation of aspartic and / or glutamic acid.

[0143] Additional CNP peptides and variant systems are disclosed in U.S. Patent 8,198,242, which is incorporated herein by reference.

[0144] In various embodiments, the electrostatic peptide is a C-type natriuretic peptide (CNP) or a CNP variant, and the hydrophilic peptide salt is a salt of CNP or a CNP variant. In various embodiments, the CNP is a CNP variant as described herein.

[0145] In various embodiments, the CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); And its pharmaceutical salts. In various embodiments, CNP is CNP-acetate.

[0146] In various embodiments, when the hydrophobic peptide salt is a hydrophobic CNP salt, the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthyl salt, docusate, or dodecyl sulfate. In various embodiments, if a polyvalent cation is present, the cation includes zinc, magnesium, or calcium. In various embodiments, if a cation is present, the cation includes zinc. In various embodiments, the cation includes calcium. In various embodiments, if a polyvalent cation is present, the cation is Zn²⁺, Mg²⁺, or Ca²⁺. In various embodiments, if a cation is present, the cation is Zn²⁺. In various embodiments, if a cation is present, the cation is Ca²⁺.

[0147] In various embodiments, the present invention provides a hydrophobic salt of a C-type natriuretic peptide, comprising a CNP peptide misaligned with a hydrophobic relative ion. In various embodiments, the salt further comprises a multivalent cation, optionally a metal cation. In various embodiments, the salt is a purified CNP salt.

[0148] Methods for purifying hydrophobic salts are known in the art and are covered herein. In various embodiments, the purity of the hydrophobic salt is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or greater.

[0149] [How to use] []

[0150] Achondroplasia results from an autosomal dominant mutation in the gene for fibroblast growth factor receptor 3 (FGFR-3), leading to abnormal cartilage formation. FGFR-3 typically negatively regulates chondrocyte growth and, consequently, bone growth. In achondroplasia, the mutated form of FGFR-3 is constitutively active, resulting in severe bone shortening. In humans, activating mutations in FGFR-3 are a major cause of inherited dwarfism. Mice with activated FGFR-3 serve as a model of achondroplasia (the most common form of skeletal dysplasia), and overexpression of CNP rescues these animals from dwarfism. Therefore, CNP and its functional variants represent potential therapeutic agents for various skeletal dysplasias. []

[0151] By stimulating the production, proliferation, and differentiation of chondrocyte matrix and increasing long bone growth, the CNP salt of this invention is applicable to the treatment of mammals, including humans, suffering from bone-related disorders (such as skeletal dysplasia). Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, congenital osteogenesis, chondrodysplasia, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, flexor dysplasia, congenital lethal hypophosphatase syndrome, perinatal lethal osteogenesis, short rib polydactyly syndrome, decreased cartilage production, limb root type punctate chondrodysplasia, Janssen type metaphyseal dysplasia, and congenital vertebral epiphyseal dysplasia. Osteogenesis imperfecta, malformations, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Roche syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, small limbs, Moquer syndrome, Knifell's syndrome, metaphyseal dysplasia, vertebral epiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lerreville), and PTPN11 mutation (Nunnan syndrome).

[0152] By stimulating the production, proliferation, and differentiation of chondrocyte matrix and increasing long bone growth, the CNP variant of this invention is applicable to the treatment of mammals, including humans, suffering from bone-related disorders (such as skeletal dysplasia). Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, decreased chondrogenesis, short stature, dwarfism, osteochondrodysplasia, lethal skeletal dysplasia, osteogenesis imperfecta, chondrodysplasia, congenital osteogenesis imperfecta, homozygous achondroplasia, congenital osteogenesis imperfecta, flexor dysplasia, congenital lethal hypophosphatase disease, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, decreased chondrogenesis, pedunculated chondrogenesis, and other bone-related disorders. This includes conditions such as Sen-type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, osteogenesis imperfecta, tortuous osteogenesis imperfecta, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Roche syndrome, Reinhardt syndrome, acromegaly, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage formation, Roberts syndrome, acromegaly and midlimb dysplasia, small limbs, Moquer syndrome, Knifell's syndrome, metaphyseal dysplasia, and vertebral metaphyseal dysplasia. The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasia covered in this article include conditions involving NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville), PTPN11 mutations (Nunnan syndrome), and IGF1R mutations.

[0153] The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasia covered in this article include conditions involving NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville), and PTPN11 mutations (Nunan syndrome).

[0154] The additional short stature and growth plate disorders covered by the method include those involving mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0155] The additional short stature and growth plate disorders covered by the method include those involving mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0156] In addition, CNP salts are suitable as an adjunct or replacement for growth hormone in the treatment of idiopathic short stature and other skeletal dysplasia.

[0157] Growth plate disorders include conditions that result in short stature or abnormal bone growth and may be the result of mutations in genes involved in bone growth, such genes including collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders include conditions that result in short stature or abnormal bone growth and may be the result of mutations in genes involved in bone growth, such genes including collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R. In various embodiments, growth plate disorders or short stature are associated with mutations in one or more genes related to Lasso disease. In various embodiments, individuals with growth plate disorders exhibit heterozygous binding to mutations in growth plate genes. In various embodiments, the mutations are loss-of-function mutations. In various embodiments, the mutations are gain-of-function mutations. Growth plate disorders include, but are not limited to, familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. See, for example, Plachy et al., *Journal of Clinical Endocrinology and Metabolism* 104: 4273-4281, 2019.

[0158] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, eventually leading to osteoarthritis, characterized by areas of bone damage (or lesions) caused by cartilage and sometimes bone detachment from the bone ends at joints. Disruption of the cartilage reticular formation during bone growth has been proposed to impair its growth, resulting in short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., *American Journal of Human Genetics* 86(2):126-37, 2010. Patients with ACAN mutations that cause short stature are expected to benefit from CNP therapy, as administration may increase their height through the known interaction between CNP and FGFR3.

[0159] The natriuretic peptide system (including the receptor NPR2) has been shown to participate in the regulation of endochondral bone growth (Vasquez et al., *Horm Res Pediat* 82:222-229, 2014). Studies have shown that loss-of-function mutations in NPR2, whether isochromocytosis or complex agenesis, cause acromegaly-maroteaux (AMDM), a dysplasia characterized by extremely short stature (Vasquez et al., 2014, cf.). Reports suggest that loss-of-function (e.g., dominant-negative) NPR2 mutations are a cause of short stature, while gain-of-function NPR2 agenesis mutations have been found to be a cause of tall stature (Vasquez et al., 2014, cf.). Given that CNPs interact with NPR2 to stimulate cGMP production, increasing cGMP levels is desirable in these cases and will have therapeutic benefits in managing complications arising from these diseases and conditions.

[0160] It is believed that atypical conjugation mutations in NPR2 result in idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are described below and in reference to: Amano et al., *Journal of Clinical Endocrinology and Metabolism* 99: E713-718, 2014; Hisado-Oliva et al., *Journal of Clinical Endocrinology and Metabolism* 100: E1133-1142, 2015; and Vasques et al., *Journal of Clinical Endocrinology and Metabolism* 98: E1636-1644, 2013, which are incorporated herein by reference. Individuals expected to have short stature for treatment with a CNP variant as described herein have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0 and have at least one parent with a height SDS of less than -1.0, -1.5, -2.0, or -2.5, where the second parent's height is within the normal range. In various embodiments, the CNP variant is applicable to treating individuals with short stature having a height SDS of -2.0 to -3.0. In various embodiments, the CNP variant is applicable to treating individuals with short stature having a height SDS of -2.0 to -2.5. However, since de novo mutations in NPR2 can cause short stature as defined by a height SDS of less than -1.5, -2.0, -2.5, or -3.0, treatment is also included for individuals who are allotype carriers of a detrimental mutation in NPR2 and whose parents do not have short stature. Further research anticipates using CNP treatment for individuals with heterozygous mutations in other growth plate genes to improve body size and / or enhance bone growth.

[0161] Indicative NPR2 mutations in patients treatable with CNP variants include: disease Nucleotides mutation amino acid changes short stature 1669C>T Misunderstanding Arg557Cys short stature 2794C>T Misunderstanding Arg932Cys short stature 2905G>C Misunderstanding Val969Leu short stature 3058C>T Misunderstanding Arg1020Trp short stature 2972A>G Misunderstanding Glu991Gly short stature 1262C>T Misunderstanding Thr421Met short stature 766G>T Misunderstanding Asp256Tyr short stature 1982C>A Misunderstanding Thr661Lys short stature 2449G>A Misunderstanding Glu817Lys short stature 1517G>A Misunderstanding Arg506His short stature 1802G>C Misunderstanding Arg601Pro short stature 1481T>G Misunderstanding Ile494Ser short stature 142G>T Misunderstanding Ala48Ser short stature 1167G>T Misunderstanding Glu389Asp short stature 1249C>G Misunderstanding Gln417Glu short stature 328C>T Misunderstanding Arg110Cys short stature 2455C>T Misunderstanding Arg819Cys short stature 788G>C Misunderstanding Arg263Pro short stature 226T>C Misunderstanding Ser76Pro short stature 2710A>T Unrighteous Lys904Term 9:35809194:C:G Leu1009Val 9:35802761:G:C Leu615Phe 9:35799645:C:T Pro301Ser 9:35792928:C:T Arg174Cys 9:35801728:C:G His508Asp 9:35792713:T:C Val102Ala 9:35793980:T:A Tyr250Ter 9:35807085:C:T Thr861Ile 9:35793906:A:G Ile226Val 9:35808558:G:A Arg921Gln 9:35802741:G:A Glu609Lys 9:35802594:G:A Arg601His 9:35808663:T:A Leu956Gln 9:35808545:G:C Gly917Arg

[0162] The role of NPPC in bone growth is well-documented (Hisado-Oliva et al., *Genetics Medicine* 20:91-97, 2018). NPPC knockout mice exhibit severe disproportionate dwarfism, including shortened limbs and endochondral ossification (Hisado-Oliva et al., 2018, cf.). Human genome-wide studies have demonstrated an association between NPPC and height (Hisado-Oliva et al., 2018, cf.). Although haploinous CNP deficiency is generally believed to be the cause of short stature in humans, recent studies have identified atypical conjugation mutations in families with short stature and short hands (Hisado-Oliva et al., 2018, cf.). These studies observed significantly reduced cGMP production as measured by atypical conjugation (Hisado-Oliva et al., 2018, cf.). Mutations in NPPC include the 355G>T missense mutation causing alterations in Gly119Cys and the 349C>G missense mutation causing alterations in Arg117Gly. Rescuing CNP variants produced by CGMP can provide therapeutic benefits in the management of patients with loss-of-binding NPPC mutations.

[0163] Lerley-Vell chondrodysplasia (LWD) is a rare genetic disorder characterized by shortening of the forearm and lower leg, abnormal wrist dislocation (Madelung deformity), and associated short stature. LWD is caused by heterozygous mutations in the short stature homeobox (SHOX) gene or its regulatory elements located in pseudoautosomal region 1 (PAR1) of the sex chromosome. (See Rare Disease Database and Carmona et al., Human Molecular Genetics 20:1547-1559, 2011). LWD occurs when two SHOX mutations are present and can result from mutations on different chromosomes (isozygous or compound heterozygous mutations). A subset of SHOX mutations causes idiopathic short stature. Turner syndrome can result from deletions on the X chromosome containing the SHOX gene. SHOX has been identified as involved in FGFR3 transcriptional regulation and contributes to bone growth control (Marchini et al., Endocrinology Review 37: 417-448, 2016). SHOX deficiency leads to increased FGFR3 signaling, and some evidence supports that SHOX also interacts directly with CNP / NPR2 (Marchini, above). Given the association between SHOX and FGFR3 and bone growth, individuals with isotype-matching or atypical-matching SHOX mutations are expected to benefit from treatment with CNP variants as described in this article.

[0164] LAS diseases are a group of rare genetic disorders caused by mutations in genes involved in the Ras / mitogen-activated protein kinase (MAPK) pathway. LAS diseases are characterized by increased signaling via the RAS / MAPK pathway. This pathway leads to downstream activation of the RAF / MEK / ERK pathway. Short stature is a typical feature of some LAS diseases. For example, CNP signaling inhibits RAF and reduces MEK and ERK activation.

[0165] This article covers the treatment of Lasso disease. Lasso diseases associated with short stature include Noonan syndrome, Costello syndrome, caroface dermatosis, neurofibromatosis type 1, and Leopard syndrome. Hereditary gingival fibromatosis type 1 is also covered in this article. Patients with Lasso disease (including Noonan syndrome, Costello syndrome, caroface dermatosis, neurofibromatosis type 1, Leopard syndrome, and hereditary gingival fibromatosis type 1) include those with an atypical conjugation variant of one or more of the following genes: BRAF, CBL, HRAS, KRAS, LZTR1, MAP2K1, MAP2K2, MRAS, NF1, NRAS, PPP1CB, PTPN11, RAF1, RRAS, RIT1, SHOC2, SOS1, or SOS2 (Tajan et al., Endocrinology Review 2018;39(5):676-700).

[0166] CFC is caused by mutations in several genes in the Ras / MAPK signaling pathway, including K-Ras, B-Raf, Mek1, and Mek2. Costello syndrome (also known as facial cutaneous-bone (FCS) syndrome) is caused by activating mutations in the H-Ras gene. Hereditary type I gingival fibromatosis (HGF) is caused by dominant mutations in the SOS1 gene (Son of Sevenless homolog 1), which encodes guanine nucleotide exchange factor (SOS) acting on the Ras subfamily of small GTPases. Neurofibromatosis type I (NF1) is caused by mutations in the neurofibromatosis protein 1 gene, which encodes a negative regulator of the Ras / MAPK signaling pathway. Noonan syndrome (NS) is caused by mutations in one of several genes, including PTPN11 (which encodes SHP2), SOS1, K-Ras, and Raf-1.

[0167] CNP has been shown to be an effective treatment in models of LASU disease. Ono et al. induced mice lacking Nf1 in type II collagen-producing cells (Ono et al., Human Molecular Genetics 2013;22(15):3048-62). These mice exhibited constitutive ERK1 / 2 activation, reduced chondrocyte proliferation, and maturation. Daily injection of CNP into these mice reduced ERK phosphorylation and corrected for short stature. A mouse model of heart-face skin syndrome using the Braf mutant (p.Q241R) (Inoue et al., Human Molecular Genetics 2019;28(1):74-83) showed reduced body length, reduced growth plate width, and smaller hyperplasia and hypertrophy areas compared to wild-type, and CNP administration increased body length in these animals.

[0168] Mutations in multiple genes can cause Noonan syndrome, characterized by short stature, heart defects, bleeding problems, and skeletal deformities. Mutations in the PTPN11 gene account for approximately half of all Noonan syndrome cases. Mutations in the SOS1 gene cause an additional 10 to 15% of cases, and mutations in the RAF1 and RIT1 genes each account for approximately 5%. Mutations in other genes each account for a minority of cases. The cause of Noonan syndrome is unknown in 15 to 20% of cases.

[0169] The PTPN11, SOS1, RAF1, and RIT1 genes all encode proteins essential for the RAS / MAPK cell signaling pathway, which is necessary for cell division and growth (proliferation), differentiation, and cell migration. Many mutations in genes associated with Noonan syndrome enable the activation of these proteins, and this prolonged activation alters normal RAS / MAPK signaling, disrupting cell growth and division regulation, thus leading to the characteristic features of Noonan syndrome. See, for example, Chen et al., Proceedings of the National Academy of Sciences of the United States of America 111(31):11473-8, 2014; Romano et al., Pediatrics 126(4):746-59, 2010; and Milosavljević et al., American Journal of Medical Genetics 170(7):1874-80, 2016. Individuals with mutations activating the MAPK pathway are expected to benefit from treatment with CNP variants as described herein to improve bone growth and short stature. Individuals with mutations activating the MAPK pathway are also expected to benefit from treatment with CNP variants as described herein to improve other comorbidities associated with over-activation of the MAPK pathway in other cells throughout the body, where the NPR2 receptor is expressed on its surface.

[0170] Mutations in the PTPN11 gene (which encodes the non-receptor protein tyrosine phosphatase SHP-2) lead to conditions characterized by short stature, such as Noonan syndrome (Musente et al., *Eur J Hum Genet* 11:201-206 (2003). Musente (above) identified numerous mutations in the PTPN11 gene that result in short stature. Gain-of-function mutations lead to reduced bone growth through excessive activation of SHP2 signaling and inhibition of growth hormone-induced IGF-1 release (Rocca Serra-Nédélec, *Proceedings of the National Academy of Sciences (PNAS)* 109:4257-4262, 2012). Individuals with isotype-binding or aisotype-binding PTPN11 mutations are expected to benefit from treatment with CNP variants as described herein to improve bone growth and short stature.

[0171] Mutations in the Indian hedgehog factor (IHH) gene (which is involved in the regulation of endochondral ossification) are also associated with short stature syndrome (Vasques et al., *Journal of Clinical Endocrinology and Metabolism* 103:604-614, 2018). Many identified IHH mutations are segregated from short stature by a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, individuals with isotype-matching or atypical-matching IHH mutations are expected to benefit from treatment with CNP variants as described in this article.

[0172] Mutations in FGFR3, including N540K and K650N, lead to short stature and reduced cartilage production.

[0173] Insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (α2β2) transmembrane glycoprotein with intrinsic kinase activity. IGF1R has been shown to play a role in prenatal and postnatal growth. IGF1R heterozygous mutations have been identified in small-for-gestational-age (SGA) infants and individuals with familial short stature (Kawashima et al., *Endocrine J* 59:179-185, 2012). IGF1R mutations associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, hereinafter).

[0174] Height is a highly heritable trait that can be influenced by the combined effects of hundreds or thousands of genes (Wood et al., *Nature Genetics*, 46:1173-1189, 2014). Individual cases of short stature may result from the combined effects of these genes, with no single gene being the primary contributor. Given that CNPs increase the length of normal animals, for example, by enhancing bone growth and length, individuals with short stature as defined by the SDS (Stature Spectrum Distance) of less than -1.0, -1.5, -2.0, -2.5, or -3.0 cm are expected to be beneficially treated with CNP variants.

[0175] In various embodiments, the CNP variant is suitable for treating individuals with short stature who have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one parent has a height SDS of less than -1.0, -1.5, -2.0, or -2.5, whereby the second parent's height is within the normal range. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -3.0. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -2.5. In various embodiments, short stature is associated with mutations in one or more genes related to short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R) or combinations thereof.

[0176] In various embodiments, short stature is associated with one or more mutations in genes related to Lasso disease.

[0177] In various embodiments, short stature is the result of mutations in multiple genes as determined by a polygenic risk score (PRS). In various embodiments, the individual has an NPR2 mutation and low PRS. In various embodiments, the individual has an FGFR3 mutation and low PRS. In various embodiments, the individual has an NPR2 mutation and low PRS. In various embodiments, the individual has an IGF1R mutation and low PRS. In various embodiments, the individual has an NPPC mutation and low PRS. In various embodiments, the individual has a SHOX mutation and low PRS. In various embodiments, the individual has one or more mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX and low PRS. In various embodiments, PRS is 1 or 2. In various embodiments, PRS is 1. In various embodiments, PRS is 2.

[0178] In addition, CNP salts are suitable for treating other bone-related conditions and diseases, such as rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also known as vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets], and osteomalacia [including tumor-induced osteomalacia (also known as carcinogenic osteomalacia or carcinogenic hypophosphatemic osteomalacia)].

[0179] The CNP salts of this invention can also be used to treat osteoarthritis. Osteoarthritis is a degenerative disease of articular cartilage and occurs frequently in the elderly. Osteoarthritis involves cartilage destruction and proliferative changes in bone and cartilage caused by degeneration of joint components, which lead to secondary arthritis (e.g., synovitis). In osteoarthritis, extracellular matrix proteins (which are the functional entities of cartilage) are reduced, and the number of chondrocytes is reduced (Arth. Rheumatoid Arthritis 46(8): 1986-1996 (2002)). By promoting the production, growth, and differentiation of chondrocyte matrix, the CNP composition is suitable for counteracting the undesirable effects of FGF-2 and increasing matrix synthesis in individuals with arthritis (including osteoarthritis), thereby treating arthritis, including osteoarthritis.

[0180] In some embodiments, the CNP salts of the present invention and compositions and formulations comprising them are suitable for improving one or more symptoms or physiological consequences of skeletal dysplasia, wherein such improvement may be an increase in absolute growth, an increase in growth rate, an increase in qualitative computed tomography (QCT) bone mineral density, an improvement in growth plate morphology, an increase in long bone growth, an improvement in spinal morphology, an improvement in elbow range of motion, and / or a reduction in sleep apnea. In this regard, it should be noted that the terms “improved,” “increased,” “reduced,” and their grammatical equivalents, when used relative to the symptoms or physiological consequences of a disease state, are relative terms, referring to the state of the symptoms or physiological consequences of the disease after treatment with the CNP salt peptides of the present invention (or compositions or formulations comprising them) compared to the same symptoms or physiological consequences of the disease before treatment with the CNP salts of the present invention (or compositions or formulations comprising them) (i.e., compared to a “baseline”). As mentioned above, the "baseline" status can be determined by measuring the individual's status before treatment (which can then be compared with the status of the same individual after treatment), or by measuring the status of a group of individuals suffering from the same ailment with the same or similar characteristics (e.g., age, sex, and / or disease status or progression).

[0181] In another embodiment, the present invention provides a salt of a CNP variant that, when stimulated in vitro or in vivo, produces at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP level produced at the same wt CNP22 concentration (e.g., 1 μM). In another embodiment, a hydrophobic salt comprising the CNP or a CNP variant of the present invention, when stimulated in vitro or in vivo, produces at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP level produced at the same wt CNP22 concentration (e.g., 1 μM).

[0182] It is expected that any of the CNP variants described herein will be applicable to the method.

[0183] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37 (SEQ ID NO: 1)). In various embodiments, the peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus. In various embodiments, the variant includes one or more linking groups as described herein. In various embodiments, the linking group is a hydrolyzable linking group. In various embodiments, the peptide includes an electrostatic peptide salt comprising an electrostatic peptide misaligned with a hydrophobic relative ion.

[0184] Treatment efficacy was measured using various parameters. In various embodiments, efficacy was assessed as the change in growth rate per year from baseline to intervention period. Efficacy was also assessed as the change in height SDS from baseline to the end of treatment, as measured using CDC growth curves, and the growth rate SDS was based on bone mineral density in pediatric studies (Kelly et al., Journal of Clinical Endocrinology and Metabolism 2014;99(6):2104-2112).

[0185] Assess the quality of life of short stature youth according to the QoLISSY questionnaire (Quality of Life in Short Stature Youth - The QoLISSY Questionnaire User's Manual. Lengerich: Pabst Science Publishers; 2013).

[0186] [Pharmaceutical Compositions] [] []

[0187] This invention provides pharmaceutical compositions comprising sustained-release compositions including peptide salts described herein, and one or more pharmaceutically acceptable excipients and / or diluents. In some embodiments, the compositions further comprise one or more other bioactive agents (e.g., proteases, receptor tyrosine kinases, and / or inhibitors that scavenge receptor NPR-C).

[0188] This invention provides sustained-release compositions comprising hydrophobic peptide salts as described herein. In various embodiments, the sustained-release composition is a delayed-release composition. In various embodiments, the delayed-release composition comprises a hydrophobic CNP salt. In various embodiments, the hydrophobic relative ion in the CNP salt is oleate, deoxycholate, decanoate, dihydroxynaphthyl salt, docusate, or dodecyl sulfate. In various embodiments, if a polyvalent cation is present, the cation comprises zinc or calcium. In various embodiments, if a cation is present, the cation is Zn²⁺ or Ca²⁺.

[0189] The precipitated peptide complex exhibits prolonged release characteristics under the pH conditions at which precipitation occurs. The precipitated peptide complex can also be further processed into a matrix that provides an additional barrier to sustained release, such as slowly degrading microspheres, hydrogels, and the like. The hydrophobic CNP salt is expected to be a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle, and to be resuspended in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer solution. In various embodiments, the particles are 1 to 10,000 micrometers (μm), 1 μm to 2000 μm, 2 μm to 1000 μm, 5 μm to 500 μm, 10 μm to 1000 μm, 50 μm to 500 μm, 100 μm to 800 μm, 200 to 600 μm, 300 μm to 500 μm, 100 μm to 300 μm, 50 μm to 100 μm, or 10 μm to 50 μm. In various embodiments, the particles are nanoparticles. In various embodiments, the nanoparticles are approximately 5 nanometers (nm) to 1000 nm, 8 nm to 900 nm, 10 nm to 800 nm, 20 nm to 600 nm, 50 nm to 500 nm, 50 to 400 nm, 20 to 300 nm, 300 to 800 nm, or 200 to 600 nm.

[0190] In various embodiments, the oil comprises triglycerides or fatty acids, which may be saturated or unsaturated. Triglycerides and fatty acids as described herein are also contemplated for use with hydrophobic CNP salt compositions. In various embodiments, the fatty acid is hexanoic acid, caprylic acid, decanoic acid, or dodecanoic acid. In various embodiments, the fatty acid is hexanoic acid, caprylic acid, decanoic acid, dodecanoic acid, or docusate.

[0191] In various embodiments, for the extended-release composition, at pH 7 to 7.6, (i) less than about 20% of the peptide is released on day 1; and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released every two weeks, or about 90% of the peptide is released monthly.

[0192] In various embodiments, less than about 20% of the peptide is released on day 1 at pH 7 to 7.6. It is further anticipated that (i) less than about 30%, or about 40%, or about 50% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Further, it is anticipated that (i) less than about 30%, or about 40%, or about 50%, or about 60% of the peptide will be released on day 1 at pH 7.0 to 7.6; and (ii) about 70%, about 80%, or about 90% of the peptide will be released weekly at pH 7 to 7.6; or about 70%, about 80%, or about 90% of the peptide will be released every two weeks; or about 70%, about 80%, or about 90% of the peptide will be released every three weeks; or about 70%, about 80%, or about 90% of the peptide will be released monthly. In various embodiments, about 90% of the peptide will be released weekly at pH 7 to 7.6. In various embodiments, about 90% of the peptide will be released every two weeks at pH 7 to 7.6. In various embodiments, about 90% of the peptide will be released monthly at pH 7 to 7.6. Further, it is expected that this release may occur at pH 7.0 to 7.6, pH 7.1 to 7.5, pH 7.2 to 7.4, pH 7.2 to 7.6, or pH 7.0 to 7.4.

[0193] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, or bi-weekly, or monthly at pH 7 to 7.6. Further expectations include (i) the release of less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide on day 1 at pH 7.0 to 7.6; and (ii) the release of about 70%, about 80%, or about 90% of the peptide weekly at pH 7 to 7.6; or about 70%, about 80%, or about 90% of the peptide every two weeks; or about 70%, about 80%, or about 90% of the peptide every three weeks; or about 70%, about 80%, or about 90% of the peptide monthly; or (ii) at pH 7.0 to 7.6. At levels 7 to 7.6, approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released weekly; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every two weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every three weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released monthly.

[0194] In various embodiments, the extended-release composition includes an excipient, a diluent, or a carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0195] Non-limiting examples of excipients, carriers, and diluents include mediators, liquids, buffers, isotensants, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, etc. The composition may contain liquids (e.g., water, ethanol); various buffer contents (e.g., Tris-HCl, phosphate, acetate buffers, citrate buffers), diluents for pH and ionic strength; detergents and solubilizers (e.g., polysorbate 20, polysorbate 80); antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosol, benzyl alcohol, m-cresol); and bulking agents (e.g., lactose, mannitol, sucrose). The use of excipients, diluents and carriers in the formulation of pharmaceutical compositions is known in this art; see, for example, Remington’s Pharmaceutical Science, 18th edition, pp. 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0196] For example, carriers include, but are not limited to, diluents, mediators, and adjuvants, as well as implant carriers, and inert, non-toxic solid or liquid fillers and encapsulating materials that do not react with the active ingredient. Non-limiting examples of carriers include phosphate-buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). Carriers may be solvents or dispersion media containing, for example, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), vegetable oils, and mixtures thereof.

[0197] In some embodiments, the composition is a liquid formulation. In some embodiments, the formulation comprises a hydrophobic CNP salt in a concentration range of about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 0.5 to 5 mg / ml, or about 0.5 to 3 mg / ml, or about 1 mg / ml to about 10 mg / ml. In various embodiments, the concentration of the CNP variant is from 0.8 mg / mL to 2 mg / mL. In various embodiments, the concentration of the CNP variant is 0.8 mg / mL. In various embodiments, the concentration of the CNP variant is 2.0 mg / mL. In various embodiments, the CNP variant is reconstituted from lyophilized powder.

[0198] In other embodiments, the composition comprises a buffer solution or buffer to maintain the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffer solutions include phosphate-buffered saline, Tris-buffered saline, and Hank's buffered saline. Buffers comprise, but are not limited to, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffers may also be used. In some embodiments, the buffer is acetate / acetate or citric acid / citrate. The suitable amount of buffer in the composition depends in part on the specific buffer used and the desired pH of the solution or suspension. In some embodiments, the concentration of the buffer is about 10 mM ± 5 mM. In some embodiments, the pH of the composition is about pH 3 to about pH 9, or about pH 3 to about pH 7.5, or about pH 3.5 to about pH 7, or about pH 3.5 to about pH 6.5, or about pH 4 to about pH 6, or about pH 4 to about pH 5, or about pH 5.0 ± 1.0. In various embodiments, the pH is about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. In various embodiments, the pH is 5.5.

[0199] In other embodiments, the composition contains an isotonic modifier to make the solution or suspension isotonic and more compatible with drug administration. Non-limiting examples of isotonic agents include NaCl, dextran, glucose, glycerol, sorbitol, xylitol, and ethanol. In some embodiments, the isotonic agent is NaCl. In some embodiments, the concentration of NaCl is about 160 ± 20 mM, or about 140 mM ± 20 mM, or about 120 ± 20 mM, or about 100 mM ± 20 mM, or about 80 mM ± 20 mM, or about 60 mM ± 20 mM.

[0200] In other embodiments, the composition includes a preservative. The preservative includes, but is not limited to, m-cresol and benzyl alcohol. In some embodiments, the concentration of the preservative is about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%.

[0201] In other embodiments, the composition contains an anti-adsorption agent (e.g., to reduce the adsorption of CNP salts onto glass or plastic). The anti-adsorption agent includes, but is not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In some embodiments, the concentration of the anti-adsorption agent is from about 0.001% to about 0.5%, or from about 0.01% to about 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about 1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, or from about 1% to about 2%.

[0202] In other embodiments, the composition includes a stabilizer. Non-limiting examples of stabilizers include glycerol, glycerol, thioglycerol, methionine, and ascorbic acid and its salts. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or its salts, the stabilizer concentration is from about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer concentration is from about 0.01% to about 0.5%, or from about 0.01% to about 0.2%. In other embodiments, when the stabilizer is glycerol, the stabilizer concentration is from about 5% to about 100% (pure).

[0203] In other embodiments, the composition contains an antioxidant. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In some embodiments, the molar ratio of the antioxidant to CNP is about 0.1:1 to about 15:1, or about 1:1 to about 15:1, or about 0.5:1 to about 10:1, or about 1:1 to about 10:1, or about 3:1 to about 10:1.

[0204] Pharmaceutically acceptable salts may be used in compositions, including but not limited to inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, methanesulfonate, toluenesulfonate), and amine salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A thorough discussion of pharmaceutically acceptable salts can be found in Remington’s Medical Sciences, 18th edition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0205] Pharmaceutical compositions can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage form of the composition can be adapted to the desired mode of administration. For oral administration, the composition may be in the form of, for example, tablets or capsules (including soft gel capsules), or may be, for example, aqueous or non-aqueous solutions, suspensions, or syrups. Tablets and capsules for oral administration may contain one or more commonly used excipients, diluents, and carriers, such as mannitol, lactose, glucose, sucrose, starch, corn starch, sodium saccharin, talc, cellulose, magnesium carbonate, and lubricants (e.g., magnesium stearate, sodium stearyl fumarate). Flavoring agents, coloring agents, and / or sweeteners may be added to solid and liquid formulations where necessary. Other ingredients present, as appropriate, for oral formulations include, but are not limited to, preservatives, suspending agents, and thickeners. Oral formulations may also have an enteric coating to protect CNP salts from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms are known or will be obvious to those skilled in the art (see, for example, Remington’s Medical Sciences cited above).

[0206] Compounds prepared for non-enteric administration may be, for example, in the form of liquid solutions or suspensions, in a solid form suitable for dissolving or suspending in a liquid medium prior to injection, or in the form of emulsions. For example, sterile injectable solutions and suspensions may be formulated using techniques known in this art using suitable diluents, carriers, solvents (e.g., buffered aqueous solutions, Ringer's solution, isotonic sodium chloride solution), dispersants, wetting agents, emulsifiers, suspending agents, and the like. Additionally, sterile fixed oils, fatty esters, polyols, and / or other inactive ingredients may be used. As other examples, compounds prepared for non-enteric administration include aqueous sterile injectable solutions that may contain antioxidants, buffers, antibacterial agents, and solutes that make the compound isotonic with the intended recipient's blood; and aqueous and non-aqueous sterile suspensions that may contain suspending agents and thickeners.

[0207] Compositions including hydrophobic CNP salts can also be lyophilized formulations. In some embodiments, the lyophilized formulation includes buffers and bulking agents, and antioxidants, if applicable. Exemplary buffers include, but are not limited to, acetate buffers and citrate buffers. Exemplary bulking agents include, but are not limited to, mannitol, sucrose, polydextrose, trehalose, and povidone (PVP K24). In some embodiments, the amount of mannitol is about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In some embodiments, the amount of sucrose is about 6% to about 20%, or about 6% to about 15%, or about 8% to about 12%. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid.

[0208] In various embodiments, the formulation includes citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and sterile water for injection (WFI) as appropriate.

[0209] The present invention also provides kits containing, for example, bottles, vials, ampoules, tubes, cartridges, and / or syringes comprising liquid (e.g., sterile injectable) formulations or solid (e.g., lyophilized) formulations. The kit may also contain pharmaceutically acceptable mediators or carriers (e.g., solvents, solutions, and / or buffers) for reconstituted solid (e.g., lyophilized) formulations into solutions or suspensions for administration (e.g., by injection), including, but not limited to, reconstituted lyophilized formulations in syringes for injection or diluting concentrates to lower concentrations. Furthermore, ready-to-use injectable solutions and suspensions may be prepared from, for example, sterile powders, granules, or tablets comprising compositions containing CNPs. The kit may also include dispensing devices (such as sprays or injection dispensing devices), pen dispensers, self-dispensing dispensers, needle-free dispensers, syringes, and / or needles.

[0210] As a non-limiting example, the kit may include a syringe having a single chamber or a dual chamber. For a single-chamber syringe, the single chamber may contain a liquid CNP formulation ready for injection, or a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP salt in a relatively small amount of a suitable solvent system (e.g., glycerol), which can be reconstituted into an injectable solution or suspension. For a dual-chamber syringe, one chamber may contain a pharmaceutically acceptable mediator or carrier (e.g., a solvent system, solution, or buffer), and the other chamber may contain a liquid formulation of a solid (e.g., lyophilized) CNP formulation or CNP salt in a relatively small amount of a suitable solvent system (e.g., glycerol), which can be reconstituted into an injectable solution or suspension using a mediator or carrier from the first chamber.

[0211] As another example, the kit may include one or more pen syringes or auto-injector devices and a dual-chamber cartridge. One chamber of the cartridge may contain a pharmaceutically acceptable medium or carrier (e.g., a solvent system, solution, or buffer), and the other chamber may contain a solid (e.g., lyophilized) CNP formulation or a liquid formulation of CNP salt in a relatively small amount of a suitable solvent system (e.g., glycerol), which can be reconstituted into an injectable solution or suspension using the medium or carrier from the first chamber. The cartridge may include an amount of CNP salt sufficient for administration over a desired period of time (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen syringe or auto-injector can be adjusted to deliver the desired amount of CNP formulation from the cartridge.

[0212] [Drug administration and delivery] []

[0213] Hydrophobic CNP salts or pharmaceutical compositions or formulations comprising them may be administered to an individual in various ways, such as subcutaneously, intra-articularly, intraperitoneally, intramuscularly, intradermally, or orally. In one embodiment, the CNP peptide salt composition may be administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0214] Hydrophobic CNP salts or salt compositions can also be administered by implanting a drug reservoir at the target site of action (e.g., abnormal or degenerated joints or cartilage regions). Alternatively, CNP salts can be administered sublingually (e.g., via a skin patch) or in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamide, polylactide, polyglycolic acid, poly(lactide-glycolic acid)), microemulsions, and similar forms orally.

[0215] The hydrophobic CNP salt compositions described herein can be administered in therapeutically effective doses to patients in need to treat, improve, or prevent bone-related conditions (such as skeletal dysplasia, including achondroplasia). The safety and therapeutic efficacy of CNP salts can be determined using standard pharmacological procedures in cell cultures or laboratory animals, such as by determining the LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is therapeutically effective in 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Active agents exhibiting a large therapeutic index are generally preferred. []

[0216] In some embodiments, the hydrophobic CNP salt composition described herein is administered at a dose ranging from about 5 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP salt composition is administered at a dose of about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 125, 130, 140, 150, 160, 170, 175, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 750, 1000, 1250, 1500, 1750 or 2000 nmol / kg or other doses deemed appropriate by the treating physician. In other embodiments, the CNP salt composition is in the form of about 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 µg / kg, or about 0.5, 0.8, 1.0, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 Administered at a dose of mg / kg, or at other doses deemed appropriate by the treating physician. The dosage of the hydrophobic CNP salts described herein may be administered according to the dosing / administration frequency described herein, including but not limited to daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc. In various embodiments, the CNP salts are administered subcutaneously daily. In various embodiments, the CNP salts are administered subcutaneously weekly. In various embodiments, the CNP variant system is administered at doses ranging from 2.5 μg / kg / day to 60 μg / kg / day, 10 μg / kg / day to 45 μg / kg / day, or 15 μg / kg / day to 30 μg / kg / day. In various embodiments, the CNP variant system is administered at a dose of 15 μg / kg / day. In various embodiments, the CNP variant system is administered at a dose of 30 μg / kg / day.

[0217] The frequency of administration / dosage of hydrophobic CNP salts for a specific individual can vary depending on various factors, including the condition being treated, the individual's condition, and their response to the therapy. Hydrophobic CNP salts can be administered as a single dose or multiple doses. In some embodiments, the hydrophobic CNP salt composition is administered as follows: once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months, or at a frequency deemed appropriate by the treating physician. In various embodiments, CNP variants are administered for 3 months, 6 months, 12 months, or longer.

[0218] In some embodiments, a hydrophobic CNP salt composition is administered to allow a growth phase (e.g., chondrogenesis), followed by a recovery phase (e.g., osteogenic formation). For example, the CNP salt composition may be administered subcutaneously or by another modality for a period of time, daily or weekly, followed by a treatment-free period, and then the cycle is repeated. In some embodiments, the initial treatment period (e.g., daily or weekly administration of the CNP salt composition) lasts for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In one related embodiment, the treatment-free period lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In some embodiments, the CNP salt composition is administered once daily for 3 days, followed by a 3-day break; or once daily or multiple times weekly for 1 week, followed by a 3-day or 1-week break; or once daily or multiple times weekly for 2 weeks, followed by a 1- or 2-week break; or once daily or multiple times weekly for 3 weeks, followed by a 1, 2, or 3-week break; or once daily or multiple times weekly for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, followed by a 1, 2, 3, or 4-week break.

[0219] [Biomarkers] []

[0220] To treat bone-related conditions, growth indicators can be measured, such as the measurement of long bone growth in utero and newborns, as well as bone growth biomarkers such as CNP, cGMP, collagen II, collagen X, osteocalcitonin, and proliferating cell nuclear antigen (PCNA).

[0221] One CNP signaling marker is cGMP (3',5'-cyclic guanosine monophosphate). The levels of this intracellular signaling molecule increase after CNP binds to and activates its homologous receptor NPR-B. Elevated cGMP levels can be measured in cell culture extracts following CNP exposure (in vitro), in conditioned media from bone explant studies following CNP exposure (ex vivo), and in plasma within minutes of CNP administration via subcutaneous, intravenous, or other routes of administration known in this technique (in vivo).

[0222] Cartilage and bone-specific analytes (or cartilage and bone-related markers) can also be measured to assess CNP efficacy. For example, fragments of cleaved type II collagen are cartilage-specific markers of cartilage renewal. Type II collagen is a major organic component of cartilage, and fragments of type II collagen (cleaved collagen) are released into circulation and subsequently secreted into urine after cartilage renewal. Cartilage renewal precedes new bone formation.

[0223] Measurable bone-specific biomarkers for bone formation include the N-terminal propeptide of type I procollagen (PINP). Synthesis of type I collagen is a crucial step in bone formation, as it is a major organic component of the bone matrix. During collagen synthesis, the propeptide is released from procollagen molecules and is detectable in serum. Furthermore, fragments of type I collagen can be measurably used as markers of bone resorption.

[0224] Other potential biomarkers for cartilage and bone formation and growth include chondroitin sulfate (a cartilage-specific marker for cartilage renewal), type II collagen precursor peptide (a cartilage-specific marker for cartilage formation), alkaline phosphatase (bone-specific), and osteocalcitonin (a bone-specific marker for bone formation). Cartilage and bone-related biomarkers can be measured in serum, for example, using commercially available kits in in vivo efficacy / pharmacodynamic studies, and in conditioned media used in in vitro studies.

[0225] In one embodiment, the levels of at least one bone or cartilage-related biomarker are analyzed or measured in individuals administered the CNP salt or sustained-release composition described herein to monitor the effect of the CNP composition on bone and cartilage formation and growth in vivo. For example, an increase in the level of at least one bone or cartilage-related biomarker may indicate a positive effect of administration of the CNP salt or sustained-release composition on bone growth and is suitable for the treatment of skeletal dysplasia and other bone or cartilage-related diseases or conditions associated with decreased CNP activity. Exemplary bone or cartilage-related biomarkers include, but are not limited to, CNP (e.g., endogenous CNP levels), cGMP, type II collagen and its fragment precursor peptide, type II collagen and its fragment, osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I procollagen (PINP) and its fragment precursor peptide, type I collagen and its fragment, chondroitin sulfate, and alkaline phosphatase.

[0226] In various embodiments, biomarkers are measured by obtaining biological samples from individuals to which, are being, or have been given CNP salts or a continuously releasing composition. Biomarkers can be measured using techniques known in this art, including, but not limited to, Western ink dot assays, enzyme-linked immunosorbent assays (ELISA), and enzyme activity assays. Biological samples can be blood, serum, urine, or other biological fluids.

[0227] Further features and details of the invention will become apparent from the following examples, which are intended to be illustrative rather than limiting. Example

[0228] Example 1: Generation of hydrophobic CNP salts

[0229] To determine whether misaligning the hydrophilic peptide CNP with a hydrophobic relative ion to stabilize its charge would effectively improve CNP formulations for therapeutic purposes, experiments were conducted to alter the electrostatic charge of CNP in combination with other ionic components.

[0230] To optimize the electrostatic misalignment between the CNP peptide and the hydrophobic relative ionic substance, and thus the controlled precipitation of the peptide complex, the solution environment should allow for the ionization of a large number of both substances. Adjusting the pH of both solutions to an intermediate value between 10 (the pI of CNP) and the acidic pKa of the relative ion (e.g., the pKa of the exemplary relative ion oleic acid is approximately 4-5) is important for the yield of the peptide complex precipitation.

[0231] To prepare hydrophobic CNP salts, including CNP variants, CNP-acetate was placed in water or a buffer solution. In the first experiment, a stock solution containing 20 mg / mL CNP was prepared in water. 100 mM Tris, pH 9.00, was also effective as a solvent. Disodium dihydroxynaphthyl acetate, oleic acid, or sodium docusate was added in excess in moles relative to the number of oppositely charged groups on CNP; in this example, a concentration of 12 moles excess of the ion relative to CNP was added to the buffer solution. CNP has 6 positively charged groups at pH 7, and this was twice the number of positively charged groups in excess. It was found that in buffer solutions (76 mM acetate buffer, pH 4.84 and 100 mM phosphate buffer, pH 6.61), the relative ions were poorly soluble and formed suspensions of what appeared to be insoluble or poorly soluble relative ions in the buffer solution. However, in water and in 100 mM Tris at pH 9.00, the relative ions of oleic acid, disodium dihydroxynaphthyl acetate, and sodium docusate were soluble.

[0232] When adding a metal cation to promote salt precipitation, the metal cation was dissolved in water and added to the CNP solution without being added to the relative ion solution. It was observed that if the metal cation was added directly to the relative ion solution, it induced precipitation in the relative ion solution. The metal cation solution (e.g., ZnCl2) was dissolved in water at a high concentration (>100 mg / mL) so that a small volume could be added to the CNP solution without significantly changing the concentration or pH. Various concentrations of metal cation could be added, but in the initial experiment, it was added in excess at 4 moles relative to the CNP concentration (CNP contains two negatively charged amino acids, and the addition resulted in 2 moles of zinc per mole of negatively charged amino acids). The relative ion solution was then added dropwise to the CNP solution with or without the metal cation, and the tube was vortexed for one second at the highest setting after each addition.

[0233] After adding sufficient solution to achieve the desired CNP to relative ion ratio, centrifuge the reaction tube at 10,000 x g for 5 minutes to allow salt precipitation into a pellet. After centrifugation, remove the supernatant and add an equal volume of water to resuspend the pellet. Centrifuge the tube at 7500 x g for 3–5 minutes, and salt precipitation should be observed. After centrifugation, wash the pellet with water again to remove the salt and resuspend it. Centrifuge the tube again and remove the supernatant. Transfer the contents to vials (e.g., 6R borosilicate glass vials), stopper, freeze, and lyophilize.

[0234] Next, the solubility of the subsequent salt powders in various buffer solutions was evaluated. Initially, approximately 1 mg of powder was weighed into a tube, and an appropriate volume of solvent was added to achieve a concentration of 1 mg / mL. The tube was then shaken overnight at approximately 37°C. Oleate was found to be soluble in 20% acetic acid. Dihydroxynaphthyl salt was soluble in dimethyl sulfoxide (DMSO).

[0235] These experiments demonstrate the possibility of precipitating highly water-soluble peptides into water-insoluble / low-solubility aggregates with sizes ranging from 5 nanometers to 1 millimeter in diameter.

[0236] Example 2 – Characterization of CNP Salts

[0237] Hydrophobic salts of CNP were prepared as in Example 1, and precipitation and solubility were tested. Table 1 describes the solid precipitation or formation caused by CNP misalignment with various hydrophobic relative ions. Table 1 [Relative Ions] [Reaction Solvent] [PPT] CNP-oleate H2 O yes CNP-dihydroxynaphthyl salt H2 O yes CNP-deoxycholate H2 O yes CNP-decanoate H2 O gel CNP-oleate Na-Phos yes CNP-bishydroxynaphthyl acid salt Na-Phos yes CNP-deoxycholate Na-Phos yes CNP-decanoate Na-Phos gel CNP-Ca+2 (oleate) H2 O yes CNP-Ca+2 (bishydroxynaphthyl salt) H2 O yes CNP-Ca+2 (deoxycholate) H2 O yes CNP-Ca+2 (decanoate) H2 O yes CNP-Zn+2 (oleate) H2 O yes CNP-Zn+2 (bishydroxynaphthyl salt) H2 O yes CNP-Zn+2 (deoxycholate) H2 O yes CNP-Zn+2 (decanoate) H2 O yes

[0238] Solubility studies were also conducted on the salt precipitate. 1 mg of CNP salt was resuspended in 50 mL of 1× PBS (pH 6.5) at 37°C, and the dissolution of the solid and the release of CNP into the solution were measured for 7 days. The buffer solution was not changed daily. Figures 1A-1D and 2 show that the hydrophobic salt dissolves more slowly than the CNP-acetate composition.

[0239] Example 3: Heterologous conjugation NPR2 mutation responds to CNP treatment

[0240] To determine the effect of CNP on individuals with short stature caused by NPR2 mutations, a cell model of NPR2 mutations was developed. Exemplary NPR2 mutations analyzed are illustrated in Figure 5. Rat chondrosarcoma (RCS) cell lines with NPR2 gene knockout or loss-of-binding mutations were prepared by transfecting RNP into RCS cells with 125 ng of the NPR2 variant, or by transfecting wild-type NPR2 plastid DNA into RCS or HEK293 cells. Single-cell pure lines were seeded and genotyped using Sanger sequencing. The cell model was able to reproduce the published cGMP phenotypes of different mutations.

[0241] NPR2 pure lines were generated by inducing insertions and deletions in the first exon of NPR2 in RCS cells. The sequence of the first exon of NPR2 was confirmed by next-generation sequencing and is shown in Figure 4. The activity of NPR2 mutant cells in response to CNP-induced stimulation was tested using CatchPoint Cyclic-GMP fluorescence analysis after treatment with 6 nM Pro-Gly CNP37. In summary, NIH3T3 cells (ATCC, CRL-1658) and NPR-transfected HEK293 cells were seeded at 60,000 cells per well in 96-well discs (96-well black imaging disc, Grenier, #655090). RCS (rat chondrosarcoma) cell lines were seeded at 40,000 cells per well. The culture media were as follows: NIH3T3 medium: DMEM high glucose, pyruvate (Thermo, 11995-073) + 10% FBS + 1× Pen Strep (abbrev P / S, Thermo, catalog number 15140122). NIH3T3 served as a control system for cGMP analysis of HEK293 medium: EMEM + 10% FBS + 1× P / S + 1× GMAX. RCS medium: DMEM + 10% FBS + 1× Pen Strep. Serum-free NIH3T3 medium: DMEM + 1× P / S, used for treating cells with IBMX (CAS 28822-58-4); serum-free NIH3T3 medium containing BSA: DMEM + 1× P / S + 0.5 mg / mL BSA (Thermo, A9418-100G), used for treating cells with CNP.

[0242] Cell lines were cultured at 37°C and 5% CO2 for 24 hours. For cells treated with the CNP variant, the trays were pretreated with IBMX (Enzo Life Sciences, 89161-340, 1 g) 15 minutes before use. IBMX is a potent, non-specific inhibitor of phosphodiesterase. An 800 mM stock solution of IBMX was diluted to a 0.75 mM working stock in IBMX dilution medium (serum-free medium (DMEM + 1× PBS and 1× PBS 1:1 mixture)).

[0243] For cell handling, cells were removed from the incubator, growth medium was removed from the cells, and cells were treated with IBMX. 80 µL of 0.75 mM IBMX was added to each well, and the cells were transferred back to the 37°C incubator for 15 minutes. After 15 minutes, CNP (40 µL / well) was added to each test well, and the cells were transferred back to the 37°C incubator for 15 minutes. The cells were mixed by tapping and imaged on a Solentim cell metric to visualize them and determine if any cells floated. The cells were then returned to the 37°C incubator.

[0244] Terminate the reaction and lyse the cells by adding 40 µL of lysis buffer (from the cGMP kit). Place the dish on a shaker for 5 minutes to complete the lysis. The cell lysate is used for cGMP analysis.

[0245] cGMP analysis was performed using cGMP calibrator, rabbit anti-cGMP antibody, and HRP-cGMP prepared according to the manufacturer's protocol. 40 µL of calibrator was added to the wells of a disk coated with anti-cGMP antibody, and 40 µL of the solution to be analyzed was added to the appropriate wells. 40 µL of reconstituted rabbit anti-cGMP antibody was added to the wells, and the disk was placed on a shaker for five minutes to mix. 40 µL of reconstituted HRP-cGMP was added to each well and incubated at room temperature for 2 hours. The disk was manually aspirated and washed 4× with 300 µL of washing buffer. 100 µL of red indicator light was added to each well of the disk, covered, and kept at room temperature for at least 10 minutes, protected from light. The fluorescence intensity of the disk was read using a Spectramax M or similar instrument with excitation at 530 nm and emission at 590 nm.

[0246] Figure 3 shows the cGMP readings in a rat chondrosarcoma cell model rescued by the addition of the exogenous Pro-Gly-CNP37 variant. Previous activation data reported cGMP EC50 in the range of 40 to 360 nM for PRKG2 activation (Campbell et al., ACS Chem Biol 12, 2388-2398, 2017; Vaandrager et al., J Biol Chem 272, 11816-23, 1997; Pohler et al., FEBS Lett 374, 419-25, 1995). In heterologous NPR2 knockout cells, a CNP dose >0.163 nM achieved intracellular concentrations exceeding the EC50 range for PRKG2 activation cGMP (Figure 1). In wild-type cells, a CNP dose of 0.040 nM achieved the same cGMP concentration. These results indicate that CNP supplementation can achieve the cGMP levels required for PRKG2 activation and growth in cells with NPR2 loss-of-function mutations.

[0247] These results also indicate that administration of CNP variants is suitable for restoring bone growth in short-statured individuals with reduced NPR2 activity. Further, treatment with CNP variants is expected to benefit individuals with mutations in other growth plate genes where cGMP signaling is impaired. Example 4: Identifying mutations associated with short stature

[0248] It is hypothesized that genes demonstrating clear evidence of a dual effect of gene-driven growth are more likely to represent therapeutic targets that can be effectively regulated in a broad patient population. To identify genes that are core regulators of growth, the intersection of five gene lists was analyzed, including gene lists from genome-wide association studies (GWAS). Core growth regulators will most likely contain rare coding mutations with dual effects (i.e., short stature or skeletal dysplasia and tall stature or hyperglycemia).

[0249] The databases queried include: GWAS, which extracted 2,067 non-duplicative recent genes from each of the 3,290 independent genetic variants reported by a large GWAS height metathesis analysis using approximately 700,000 individuals; HGMD, which queried the "allmut" table from HGMD version v2019_2 to find all pathogenic variants marked "DM" that have "short stature" and "tall stature or overgrowth" in the same gene; and OMIM, which was previously described as a list of OMIM genes involved in growth disorders and created using the following keywords: short stature, overgrowth, skeletal dysplasia, brachydactyly.

[0250] First, a search was conducted in the Human Genetic Mutation Database (HGMD version v2019_2) for genes associated with short stature or tall stature (Stenson et al., *Human Genetics* 136:665-677, 2017). The literature reported 47 genes labeled with at least one pathogenic variant causing "short stature." Only 20 genes were labeled as tall stature or overgrowth genes. Second, a manually curated list of 258 OMIM genes (248 for short stature and 20 for tall stature) was created using the keywords: short stature, overgrowth, skeletal dysplasia, brachydactyly (Wood et al., *Nature Genetics* 46:1173-86, 2014). Third, the intersection of these lists was compared with a gene list from GWAS. At the intersection of these lists, there are three known genes associated with height (IGF1R, NPPC, NPR2), and two additional genes (FGFR3, SHOX) are identified.

[0251] Additional analysis yielded a new set of five core genes that showed significantly reduced height (β = -0.20, 95% CI [-0.26 to -0.14], p = 4.04 × 10⁻¹¹) and significantly increased risk of idiopathic short stature (ISS) (OR = 2.75, 95% CI [1.92–3.96]). Each of the five core genes (FGFR3, IGF1R, NPPC, NPR2, and SHOX) was associated with height individually and with short stature when used in combination with other mutations. Exemplary mutations of FGFR3, IGF1R, NPPC, NPR2, and SHOX are presented in Figure 6.

[0252] Combination loss-of-function (LoF) and missense variants in NPR2 and IGF1R were also associated with an increased risk of ISS (OR=3.31, P=0.001, OR=2.85, P=0.002, respectively). Whole gene deletions and / or mutations in SHOX, IGF1R, NPPC, and NPR2 that cause loss of protein function have been reported in familial short stature of varying severity.

[0253] Analysis showed that carriers of variants of any of the five core genes had an approximately 3-fold increased risk of ISS, accounting for 6.7% of the total ISS population. Furthermore, it demonstrated dose-dependent rescue of NPR2 signaling in haplo-insufficient cell models after the addition of exogenous CNP.

[0254] According to whole-genome models (Liu et al., Cell 177:1022-1034 e6 (2019); Boyle et al., Cell 169:1177-1186 (2017)), if these genes are core human growth genes, their effects should be regulated by multiple weaker common genetic variants driving the regulatory network. To indirectly test this hypothesis, a polygenic risk score (PRS) for height was calculated using the largest published GWAS meta-analysis for height, which did not include any samples from the UK Biobank project. The population was divided into five equal-sized (n=6,824) PRS quintiles (PRS 1 for the shortest height and PRS 5 for the tallest height). A dose-dependent relationship was found between the increase in PRS score and mean height (β=0.30 for each PRS quintile) (Figure 7A). Across the five different PRS backgrounds, carriers of the LoF variant in the five core genes were consistently shorter than non-carriers. See Figure 7. Data indicate that the combined effect of PRS and rare protein variants is consistent with the additive model: polygenic effects regulate the height of carriers and non-carriers.

[0255] Using PRS=3 as a reference, the risk of ISS across PRS groups was calculated. The lowest PRS group was associated with an increased risk of ISS, while the highest PRS group was associated with a decreased risk (OR=5.43, P=8.58×10⁻³⁴ for PRS 1 and OR=0.22, P=4.49×10⁻⁷ for PRS 5). The effects of rare coding variants of the five core genes on ISS stratified by PRS groups were assessed. In the top three quintiles, carriers of any of the five core genes had an increased risk of ISS (OR=2.64, P=3.09×10⁻⁵; OR=2.17, P=0.04; OR=5.29, P=1.58×10⁻⁵; OR=2.72, P=0.09, Fig. 7C-F). For carriers of each individual core gene, a consistent direction of effect on ISS risk stratified by PRS was observed (Fig. 7C-F).

[0256] Furthermore, the 20.1% height difference was predicted primarily from the additive effects of PRS on several common genetic variants with individual small effects. These additive effects of PRS appear to have similar magnitudes for carriers and non-carriers of rare coding variants of the core gene. This observation suggests that PRS can be a significant contributor to penetrance differences in rare pathogenic variants (especially in haplo-insufficient models, such as those described in this paper). Supporting this idea, two out of eight carriers of NPR2 variants with low NPR2 activity were observed to have short-to-normal height. This data suggests that most individuals with NPR2 mutations in ISS may also have a polygenic background, making them more susceptible to the pathogenic effects of loss of NPR2 activity.

[0257] These results support the view that CNP-based treatment can be effective in patients with insufficient NPR2 haplogroups. Furthermore, the significant bidirectional (LoF and GoF) correlation between cGMP levels and height in NPR2 carriers in the general population suggests that targeting this receptor with CNP analogs could be an effective therapy for all individuals with ISS.

[0258] Example 5: Release curve of CNP salt in vivo

[0259] The release of hydrophobic CNP salts was also analyzed.

[0260] In short, Pro-Gly-CNP37 Zn dihydroxynaphthate and Pro-Gly-CNP37 Zn-oleate were freshly prepared and placed in 15 mL of culture medium in a Pion microDiss dissolution apparatus on the day of preparation, with the impeller rotating at 250 RPM and the temperature control setpoint at 37.4℃. Every 24 hours for 4 days, the contents of the containers were transferred to VWR polypropylene "Falcon" tubes and centrifuged rapidly at 4000×g for 30 minutes. RP-UPLC samples were obtained / frozen, and the solids were resuspended in fresh culture medium (15 mL). The salts were run on RP-UPLC to obtain signals, and their concentrations were determined by comparison with a calibration curve. The released salts were determined by comparing the released mass of Pro-Gly-CNP37 used in salt preparation with the initial mass.

[0261] Figures 8A and 8B show the cumulative release curves of CNP peptide salts over 7 days as a percentage of the total amount of CNP peptide salts.

[0262] Another release curve was run using the above protocol. Approximately 16.6 mg of Pro-Gly-CNP37-acetate (the starting material for salt preparation) or Pro-Gly-CNP37 Zn-dihydroxynaphthyl salt was placed in each well. The CNP salt was prepared, lyophilized, sealed, and stored at 4°C before use. For the CNP acetate control, container 1 was in 1× PBS, and container 5 was in 1× PBS + 0.05% PS80. Every 24 hours for 7 days, the contents of the containers were transferred to VWR polypropylene "Falcon" tubes, centrifuged rapidly at 4000×g for 30 min, and the UPLC samples were obtained / frozen, and the solids were resuspended in fresh culture medium (15 mL). The CNP salt was run on RP-UPLC to obtain a signal, and the concentration was obtained by comparison with the calibration curve. The cumulative release curves after 7 days are shown in Figures 9A and 9B.

[0263] Pro-Gly-CNP-37-docusate and Pro-Gly-CNP-37-Zn-docusate salts were freshly prepared or prepared and lyophilized, and sealed in glass vials for subsequent dissolution in Pion. Every 24 hours for 4 days, the contents were transferred to VWR polypropylene "Falcon" tubes and centrifuged rapidly at 4000×g for 30 minutes. Samples were then collected / frozen, and the solids were resuspended in fresh culture medium (15 mL). Salts were quantified by LC-MS, and the released salt was obtained by comparison with the initial salt placed in the dissolution vessel.

[0264] Figures 10A to 10C show the cumulative release curves of docusate salt over 4 days (Figure 10A) or 7 days (Figures 10B and 10C).

[0265] Example 6: Release curve of CNP salt in vivo

[0266] Next, the release curve of the sample salt CNP-dihydroxynaphthyl salt was analyzed by examining the in vivo release curve of rats within 7 days after subcutaneous injection of CNP salt.

[0267] Figure 11 shows the release curve of Pro-Gly-CNP37 from saline to plasma over 7 days. The saline release was observed during the initial burst within 24 hours, as well as some saline release over time.

[0268] Any single embodiment in this document may be supplemented with one or more elements from any one or more other embodiments in this document.

[0269] Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover all modifications that are within the spirit and scope of the invention as defined by the appended claims, the foregoing specification, the following numbered paragraphs, and / or the accompanying drawings.

[0270] Example of an implementation plan:

[0271] Paragraph 1. A composition comprising an electrostatic peptide salt, the salt comprising an electrostatic peptide misaligned with a hydrophobic relative ion.

[0272] Paragraph 2. The composition as described in paragraph 1, wherein the hydrophobic relative ionic system is via non-covalent bond misalignment.

[0273] Paragraph 3. The composition as described in paragraph 1, wherein the hydrophobic relative ionic system is misaligned with an electrostatic peptide via a cleavable linker.

[0274] Paragraph 4. A composition as described in any of paragraphs 1 to 3, wherein the salt further comprises a cation misaligned with a peptide-relative ion complex.

[0275] Paragraph 5. The composition as described in paragraph 4, wherein the electrostatic peptide, the hydrophobic relative ion, and the cation system are non-covalently interleaved.

[0276] Paragraph 6. As in paragraphs 4 or 5, wherein the cation has a charge of +2, +3 or +4.

[0277] Paragraph 7. A composition as described in any of paragraphs 4 through 6, wherein the cation is a metal cation.

[0278] Paragraph 8. A composition of any one of paragraphs 4 to 7, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au).

[0279] Paragraph 9. A composition of any one of paragraphs 1 to 8, wherein the hydrophobic relative ion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0280] Paragraph 10. The composition of any one of paragraphs 1 to 9, wherein the hydrophobic relative ion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0281] Paragraph 11. The composition of any one of paragraphs 1 to 10, wherein the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidic acid, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octylsulfonate monohydrate, sodium 1-decylsulfonate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate.

[0282] Paragraph 12. The composition of any one of paragraphs 1 to 11, wherein the peptide salt is in the form of a solid, a semi-solid, a gel, a crystal, an amorphous, a nanoparticle, a microparticle, an amorphous nanoparticle, an amorphous microparticle, a crystalline nanoparticle, or a crystalline microparticle.

[0283] Paragraph 13. The composition of any one of paragraphs 1 to 12, wherein the electrostatic peptide is a C-type natriuretic peptide (CNP).

[0284] Paragraph 14. The composition as described in paragraph 13, wherein CNP is a CNP variant.

[0285] Paragraph 15. As in paragraphs 13 or 14, wherein CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and its medicinal salts.

[0286] Paragraph 16. The composition as described in paragraph 15, wherein CNP is CNP-acetate.

[0287] Paragraph 17. A composition of any one of paragraphs 13 to 16, wherein the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthenate, docusate, or dodecyl sulfate.

[0288] Paragraph 18. The composition of any of paragraphs 13 to 17, wherein if a cation is present, the cation is zinc or calcium.

[0289] Paragraph 19. The composition of any one of paragraphs 1 to 18 further includes an excipient, a diluent, or a carrier.

[0290] Paragraph 20. The composition as described in paragraph 19, wherein the excipient, diluent or carrier is a pharmaceutically acceptable excipient, diluent or carrier.

[0291] Paragraph 21. A sterile pharmaceutical composition comprising any one of the compositions described in paragraphs 1 to 20.

[0292] Paragraph 22. A delayed-release composition comprising an electrostatic peptide salt, the salt comprising an electrostatic peptide misaligned with a hydrophobic relative ion.

[0293] Paragraph 23. The extended-release composition as described in paragraph 22, wherein the hydrophobic relative ionic system is via non-covalent bond misalignment.

[0294] Paragraph 24. The extended-release composition as described in paragraph 23, wherein the hydrophobic relative ionic system is misaligned with the electrostatic peptide via a cleavable linker.

[0295] Paragraph 25. The extended-release composition of any one of paragraphs 22 to 24, wherein the salt further comprises a cation misaligned with a peptide-relative ion complex.

[0296] Paragraph 26. The extended-release composition as described in paragraph 25, wherein the electrostatic peptide, the hydrophobic relative ion, and the cation system are non-covalently interleaved.

[0297] Paragraph 27. The extended-release composition as described in paragraph 26, wherein the cation has a charge of +2, +3 or +4.

[0298] Paragraph 28. The extended-release composition of any of paragraphs 25 to 27, wherein the cation is a metal cation.

[0299] Paragraph 29. The extended-release composition of any one of paragraphs 25 to 28, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu) and gold (Au).

[0300] Paragraph 30. The extended-release composition of any one of paragraphs 22 to 29, wherein the hydrophobic relative ion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0301] Paragraph 31. The extended-release composition of any one of paragraphs 22 to 30, wherein the hydrophobic relative ion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0302] Paragraph 32. The extended-release composition of any one of paragraphs 22 to 31, wherein the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthyl salt, nicotinic acid salt, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidyl salt, decanoate, 2-naphthalenesulfonate, 1-heptanesulfonate, 1-octylsulfonate monohydrate, 1-decylsulfonate, dodecyl sulfate, dextran sulfate, and dodecylbenzenesulfonate.

[0303] Paragraph 33. The extended-release composition of any of paragraphs 22 to 32, wherein the CNP salt is in the form of a solid, a semi-solid, a gel, a crystal, an amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle.

[0304] Paragraph 34. The extended-release composition of any of paragraphs 22 to 33, wherein the electrostatic peptide is a C-type natriuretic peptide (CNP).

[0305] Paragraph 35. The extended-release composition as described in paragraph 34, wherein CNP is a CNP variant.

[0306] Paragraph 36. As in paragraphs 34 or 35, wherein CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); Its medicinal salts.

[0307] Paragraph 37. The extended-release composition as described in paragraph 36, wherein CNP is CNP-acetate.

[0308] Paragraph 38. The extended-release composition of any of paragraphs 34 to 37, wherein the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthenate, docusate, or dodecyl sulfate.

[0309] Paragraph 39. The extended-release composition of any of paragraphs 34 to 38, wherein if a cation is present, the cation is zinc or calcium.

[0310] Paragraph 40. The extended-release composition of any of paragraphs 33 to 39, wherein the peptide salt solid, semi-solid, gel, crystal, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle or crystalline microparticle is resuspended in an aqueous solution or oil.

[0311] Paragraph 41. The extended-release composition as described in paragraph 40, wherein the aqueous solution is water, saline, or a buffer solution.

[0312] Paragraph 42. The extended-release composition as described in paragraph 40, wherein the oil comprises triglycerides or fatty acids.

[0313] Paragraph 43. The extended-release composition as described in paragraph 42, wherein the fatty acids are saturated or unsaturated.

[0314] Paragraph 44. Extended-release compositions as described in paragraphs 42 or 43, wherein the fatty acids are C-6 to C-20 fatty acids.

[0315] Paragraph 45. The extended-release composition of any of paragraphs 42 to 44, wherein the fatty acid is hexanoic acid, caprylic acid, decanoic acid or dodecanoic acid.

[0316] Paragraph 46. The extended-release composition of any one of paragraphs 22 to 45, wherein at pH 7 to 7.6, (i) less than 20% of the peptide is released on day 1; and ii) Approximately 90% of the peptides are released by day 7, or by day 14, or by day 31.

[0317] Paragraph 47. A prolonged-release composition as described in any of paragraphs 22 to 46, wherein less than 20% of the peptide is released on day 1 at pH 7 to 7.6.

[0318] Paragraph 48. A prolonged-release composition as described in any of paragraphs 22 to 47, wherein approximately 90% of the peptide is released by day 7 at pH 7 to 7.6.

[0319] Paragraph 49. The extended-release composition of any one of paragraphs 22 to 47, wherein approximately 90% of the peptide is released by day 30 at pH 7 to 7.6.

[0320] Paragraph 50. The extended-release composition of any of paragraphs 22 to 49 further includes an excipient, a diluent, or a carrier.

[0321] Paragraph 51. The extended-release composition as described in paragraph 50, wherein the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0322] Paragraph 52. A sterile pharmaceutical composition comprising a delayed-release composition as described in any one of paragraphs 22 to 51.

[0323] Paragraph 53. A method for preparing a composition comprising an electrostatically charged peptide salt, comprising: a) To bring electrostatically charged peptides in aqueous solution into contact with hydrophobic relative ions in the solution; b) Mixing an electrostatic peptide solution with a hydrophobic relative ion solution in a manner sufficient to cause the peptide and the relative ion to form a complex, wherein the formation of the peptide-relative ion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle.

[0324] Paragraph 54. The method of paragraph 53 may include, as appropriate, contacting the electrostatically charged peptide in solution with the polyvalent cation in the aqueous solution prior to step (b) to form a peptide-cation complex.

[0325] Paragraph 55. The method of paragraphs 53 or 54 further includes step (c) washing the peptide salt in a buffer or water.

[0326] Paragraph 56. The method of paragraph 55 further includes step (d) of obtaining peptide salt by forming a peptide salt centrifuge block by centrifugation.

[0327] Paragraph 57. The method of paragraph 56 further includes step (e) removing water from the peptide salt centrifuge block.

[0328] Paragraph 58. The method of paragraph 57 further includes resuspending the centrifuged block in an aqueous solution or oil.

[0329] Paragraph 59. The method of any one of paragraphs 53 to 58, wherein the peptide:hydrophobic relative ion ratio is 1:1 to 1:20.

[0330] Paragraph 60. The method of any one of paragraphs 54 to 59, wherein the peptide:cation ratio is 1:1 to 1:10.

[0331] Paragraph 61. The method of any of paragraphs 53 to 60, wherein the hydrophobic relative ionic system is via non-covalent bonding or misalignment.

[0332] Paragraph 62. The composition as described in paragraph 61, wherein the hydrophobic relative ionic system is misaligned with an electrostatic peptide via a cleavable linker.

[0333] Paragraph 63. The method of any of paragraphs 54 to 62, wherein if the salt further comprises a cation misaligned with a peptide-relative ionic complex, the cation is misaligned via a covalent or non-covalent bond or a mixture thereof.

[0334] Paragraph 64. As in paragraph 63, wherein the cation is bound to the peptide-hydrophobic relative ion complex via a non-covalent bond.

[0335] Paragraph 65. The composition as described in paragraphs 63 or 64, wherein the electrostatic peptide, the hydrophobic relative ion, and the cation are non-covalently interleaved.

[0336] Paragraph 66. The method of any of paragraphs 54 to 65, wherein the cation has a charge of +2, +3 or +4.

[0337] Paragraph 67. The method of any one of paragraphs 54 to 66, wherein the cation is a metal cation.

[0338] Paragraph 68. The method of any one of paragraphs 54 to 67, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), and gold (Au). Further encompassing the method of any one of paragraphs 54 to 67, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au).

[0339] Paragraph 69. The method of any one of paragraphs 46 to 58, wherein the hydrophobic relative ion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0340] Paragraph 70. The method of any of paragraphs 53 to 69, wherein the hydrophobic relative ion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0341] Paragraph 71. The method of any one of paragraphs 53 to 70, wherein the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthyl salt, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), and phosphatidic acid.

[0342] Paragraph 72. The method of any one of paragraphs 53 to 71, wherein the electrostatic peptide is a C-type natriuretic peptide (CNP).

[0343] Paragraph 73. As in paragraph 72, where CNP is a CNP variant.

[0344] Paragraph 74. As in paragraphs 72 or 73, where CNP is selected from the following groups: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); And its salt.

[0345] Paragraph 75. As in paragraph 74, where CNP is CNP-acetate.

[0346] Paragraph 76. The method of any one of paragraphs 72 to 75, wherein the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthenate, docusate, or dodecyl sulfate.

[0347] Paragraph 77. The method of any of paragraphs 72 to 76, wherein if a cation is present, the cation is zinc or calcium.

[0348] Paragraph 78. A method for treating bone-related conditions or skeletal dysplasia in an individual in need, comprising administering to the individual a composition comprising a hydrophobic salt of a C-type natriuretic peptide (CNP) as described in any of paragraphs 1 to 52.

[0349] Paragraph 79. As in Paragraph 78, bone-related disorders or skeletal dysplasia are selected from the following groups: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, chondrodysplasia, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, flexor dysplasia, congenital lethal hypophosphatase syndrome, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, decreased cartilage production, pedicle type punctate chondrodysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, and skeletal dysplasia. Complete, malformed dysplasia, congenital short femur, Langer type limb midbone dysplasia, Nivig type limb midbone dysplasia, Roche syndrome, Reinhardt syndrome, acrodysplasia, peripheral bone developmental disorders, Knifedipine dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, small limbs, Moquer syndrome, Knifedipine syndrome, metaphytic dysplasia and vertebral epiphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Lerreville), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation and idiopathic short stature.

[0350] Paragraph 80. A method for bone lengthening or increasing long bone growth in an individual in need, comprising administering to the individual a delayed-release composition comprising a salt of a C-type natriuretic peptide (CNP) as described in any one of paragraphs 1 to 52, wherein the administration causes bone lengthening or increasing long bone growth.

[0351] Paragraph 81. The method of any of paragraphs 78 to 80, wherein CNP is a CNP variant.

[0352] Paragraph 82. As in any of paragraphs 78 to 81, where CNP is selected from the following groups: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1);LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); And its salt.

[0353] Paragraph 83. As in paragraph 83, where CNP is CNP-acetate.

[0354] Paragraph 84. The method of any of paragraphs 78 to 83, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0355] Paragraph 85. The method of any one of paragraphs 78 to 84, wherein the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months.

[0356] Paragraph 86. The method of any one of paragraphs 78 to 85, wherein the composition is a delayed-release composition.

[0357] Paragraph 87. A salt of a C-type natriuretic peptide comprising a CNP peptide misaligned with a hydrophobic relative ion.

[0358] Paragraph 88. Salts as described in paragraph 87, wherein the hydrophobic relative ionic system is via non-covalent bond misalignment.

[0359] Paragraph 89. Salts as described in paragraphs 87 or 88, wherein the hydrophobic relative ion has a cLogP of about 2 to about 9, or a pKa of less than about 5, or both.

[0360] Paragraph 90. Salts of any of paragraphs 87 to 89, wherein the hydrophobic relative ion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0361] Paragraph 91. Salts of any of paragraphs 87 to 90, wherein the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), and phosphatidic acid.

[0362] Paragraph 92. A salt as described in any of paragraphs 87 to 91 further includes cations misaligned with peptides and relative ions.

[0363] Paragraph 93. Salts as described in paragraph 92, wherein the cations are bonded via non-covalent bonds.

[0364] Paragraph 94. Salts as described in paragraphs 92 or 93, wherein the cations have a charge of +2, +3, or +4.

[0365] Paragraph 95. A salt such as any of paragraphs 92 to 94, wherein the cation is a metal cation.

[0366] Paragraph 96. Salts as described in any of paragraphs 92 to 95, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), and gold (Au). Further encompassing salts as described in any of paragraphs 92 to 95, wherein the cation is selected from the group consisting of: beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba), zinc (Zn), cadmium (Cd), boron (B), aluminum (Al), gallium (Ga), indium (In), thallium (Tl), iron (Fe), manganese (Mn), cobalt (Co), nickel (Ni), titanium (Ti), vanadium (V), platinum (Pt), copper (Cu), and gold (Au).

[0367] Paragraph 97. Salts as described in any of paragraphs 87 to 96, wherein the CNP salt is in the form of a solid, a semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle.

[0368] Paragraph 98. Salts of any of paragraphs 87 to 97, where CNP is a CNP variant.

[0369] Paragraph 99. For any of the salts in paragraphs 87 and 98, CNP is selected from the following groups: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37;SEQ ID NO: 1); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4); and its medicinal salts.

[0370] Paragraph 100. As in paragraph 99, the salt, where CNP is CNP-acetate.

[0371] Paragraph 101. Salts of any of paragraphs 87 to 100, wherein the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthenate, docusate, or dodecyl sulfate.

[0372] Paragraph 102. For any of the salts in paragraphs 87 to 101, if a cation is present, then the cation is zinc or calcium.

[0373] Paragraph 103. Salts such as any of those in paragraphs 87 through 102 are purified.

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Claims

1. A composition comprising a hydrophobic salt of a C-type natriuretic peptide (CNP), the salt comprising the CNP misaligned with a hydrophobic counterion and a polyvalent cation misaligned with a peptide-counterion complex, wherein the CNP is a CNP variant selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1); DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53) (SEQ ID NO: 56); LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 15); RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 16); VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 17); TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 20); (CNP-46) (SEQ ID NO: 21); RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 22); AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 23); (SEQ ID NO: 24);WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 25); ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 26); RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 27); LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 28); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3); EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 32); RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 33); KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 34); YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 36); GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 37);ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 38); NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 39); KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 40); KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC (CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 42); SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 43); KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 44); GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC (CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N);SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37;SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37;SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N);SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37;SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 52);GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 55); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 7).; 2. The composition of claim 1, wherein the polyvalent cation is Zn²⁺ or Ca²⁺.

3. The composition of claim 1, wherein the hydrophobic relative ionic system is selected from the group consisting of: deprotonated fatty acids, deprotonated cholic acid, ionic surfactants, naphthates and their derivatives, nicotinic acid salts and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextran sulfates, alkylbenzene sulfates, and combinations thereof.

4. The composition of claim 3, wherein the hydrophobic relative ion is selected from the group consisting of: palmitate, deoxycholate, oleate, dihydroxynaphthate, nicotinic acid, dodecyl sulfate, docusate, myristate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidyl salt, decanoate, 2-naphthalenesulfonate, 1-heptanesulfonate, 1-octylsulfonate monohydrate, 1-decylsulfonate, dextran sulfate, and dodecylbenzenesulfonate.

5. The composition of any one of claims 1 to 3, wherein the CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4), and salts thereof.

6. The composition of any one of claims 1 to 3, wherein the hydrophobic relative ion is oleate, deoxycholate, decanoate, dihydroxynaphthenate, docusate, or dodecyl sulfate.

7. The composition of any one of claims 1 to 3 further comprises an excipient, a diluent or a carrier.

8. A delayed-release composition comprising a salt of a C-type natriuretic peptide (CNP), the salt comprising an electrostatically charged peptide bound to a hydrophobic relative ion and a polyvalent cation bound to a peptide-relative ion complex, wherein the CNP is a variant of the CNP selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1); DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53) (SEQ ID NO: 56); LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 15); RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 16); VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 17); TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 20); (CNP-46) (SEQ ID NO: 21); RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 22); AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 23); (SEQ ID NO: 24);WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 25); ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 26); RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 27); LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 28); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3); EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 32); RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 33); KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 34); YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 36); GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 37);ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 38); NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 39); KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 40); KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC (CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 42); SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 43); KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 44); GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC (CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 52);GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 55); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 7).; 9. The extended-release composition of claim 8, wherein the salt of the C-type natriuretic peptide is resuspended in an aqueous solution or oil, wherein the oil contains triglycerides or fatty acids.

10. The extended-release composition of claim 8 or 9, wherein at pH 7 to 7.6, (i) less than 20% of the peptide is released on day 1; and (ii) about 90% of the peptide is released by day 7, or about 90% of the peptide is released by day 14, or about 90% of the peptide is released by day 31.

11. A method for preparing a composition as claimed in any one of claims 1 to 7 or a extended-release composition as claimed in any one of claims 8 to 10, comprising a) contacting the CNP in an aqueous solution with a hydrophobic relative ion in the solution; b) mixing the CNP solution with the hydrophobic relative ion solution in a manner sufficient to cause the peptide and the relative ion to form a complex, wherein the formation of the peptide-relative ion complex results in the formation of a solid, semi-solid, gel, crystal, amorphous, nanoparticle or microparticle form containing the CNP salt.

12. Use of a composition as claimed in any one of claims 1 to 7 or a delayed-release composition as claimed in any one of claims 8 to 10, for the preparation of a medicament for treating bone-related disorders or skeletal dysplasia or for elongating or increasing the growth of long bones.

13. As requested in item 12, wherein the bone-related condition or skeletal dysplasia is selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, decreased cartilage production, short stature, dwarfism, osteochondrodysplasia, lethal dysplasia, osteogenesis imperfecta, achondrogenesis, punctate chondrodysplasia, campomelic dysplasia, congenital lethal hypophosphatase disease, perinatal lethal osteogenesis imperfecta, short rib polydactyly syndrome, decreased cartilage production, pedicle type punctate chondrodysplasia, Janssen type metaphyseal dysplasia, congenital hypophosphatidylchondrosis, etc. Congenital vertebral epiphyseal dysplasia, athelosteogenesis, malformation, congenital short femur, Langer type midlimb dysplasia, Nivig type midlimb dysplasia, Roche syndrome, Reinhardt syndrome, acromegaly, peripheral bone dysplasia, Knifell's dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, small limbs, Moquer syndrome, Knifell's syndrome, metaphyseal dysplasia and vertebral epiphyseal metaphysema, NPR2 mutation, SHOX mutation, PTPN11 mutation, insulin-like growth factor 1 receptor (IGF1R) mutation and idiopathic short stature.

14. The use as claimed in claim 12 or 13, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

15. A hydrophobic salt of a C-type natriuretic peptide (CNP) comprising a CNP misaligned with a hydrophobic relative ion and a polyvalent cation misaligned with a peptide-relative ion complex, wherein the CNP is a CNP variant selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1); DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53) (SEQ ID NO: 56); LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (SEQ ID NO: 15); RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (SEQ ID NO: 15). NO: 16); VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-50) (SEQ ID NO: 17); TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-48) (SEQ ID NO: 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-47) (SEQ ID NO: 20); (CNP-46) (SEQ ID NO: 21); RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-45) (SEQ ID NO: 22); AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-44) (SEQ ID NO: 23); (SEQ ID NO: 24);WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-42) (SEQ ID NO: 25); ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-41) (SEQ ID NO: 26); RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-40) (SEQ ID NO: 27); LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-39) (SEQ ID NO: 28); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3); EHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-36) (SEQ ID NO: 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-35) (SEQ ID NO: 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-33) (SEQ ID NO: 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-32) (SEQ ID NO: 32); RKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-31) (SEQ ID NO: 33); KYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-30) (SEQ ID NO: 34); YKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-29) (SEQ ID NO: 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-28) (SEQ ID NO: 36); GANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-27) (SEQ ID NO: 37);ANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-26) (SEQ ID NO: 38); NKKGLSKGCFGLKLDRIGSMSGLGC (CNP-25) (SEQ ID NO: 39); KKGLSKGCFGLKLDRIGSMSGLGC (CNP-24) (SEQ ID NO: 40); KGLSKGCFGLKLDRIGSMSGLGC (CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC (CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC (CNP-21) (SEQ ID NO: 42); SKGCFGLKLDRIGSMSGLGC (CNP-20) (SEQ ID NO: 43); KGCFGLKLDRIGSMSGLGC (CNP-19) (SEQ ID NO: 44); GCFGLKLDRIGSMSGLGC (CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC (CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N);SEQ ID NO: 46]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID NO: 49]; MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID NO: 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 52);GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 55), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC(SEQ ID NO: 7).; 16. The hydrophobic salt of claim 15, wherein the salt is selected from the group consisting of: CNP-oleate, CNP-dihydroxynaphthylate, CNP-deoxycholate, CNP-decanoate and CNP-docusate.

17. The hydrophobic salt of claim 15 or 16, wherein the salt is selected from the group consisting of: CNP-Ca+2 (oleate), CNP-Ca+2 (dihydroxynaphthylate), CNP-Ca+2 (deoxycholate), CNP-Ca+2 (decanoate), CNP-Ca+2 (docusate), CNP-Zn+2 (oleate), CNP-Zn+2 (dihydroxynaphthylate), CNP-Zn+2 (deoxycholate), CNP-Zn+2 (decanoate), and CNP-Zn+2 (docusate).

18. The hydrophobic salt of claim 15 or 16, wherein the CNP is selected from the group consisting of: PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID NO: 1); LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-38) (SEQ ID NO: 2); QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-37) (SEQ ID NO: 3); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-34) (SEQ ID NO: 4), and salts thereof.

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