Hydrophobic peptide salts for extended release compositions

Abstract

The present disclosure generally relates to hydrophobic salts of hydrophilic peptides that form low-solubility materials in aqueous solutions and are capable of extended or sustained release of peptide components when administered to a subject. Hydrophobic salts of C-type natriuretic peptides and uses thereof are also disclosed. The present disclosure relates to compositions comprising salts of electrostatically charged peptides that have low solubility in solution, such that the salts form solid or semi-solid forms in aqueous media. It is shown herein that such salts dissolve more slowly than the non-salt forms of the peptides in aqueous solutions and can be used in extended-release therapeutics without the need for reformulation in typical extended-release formats.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 885,705 filed on 12 August 2019, U.S. Provisional Patent Application No. 62 / 935,052 filed on 13 November 2019, U.S. Provisional Patent Application No. 62 / 963,354 filed on 20 January 2020, U.S. Provisional Patent Application No. 62 / 964,848 filed on 23 January 2020, and U.S. Provisional Patent Application No. 63 / 038,652 filed on 12 June 2020, all of which are incorporated herein by reference.

[0002] Integration by referencing electronically submitted documents The sequence listing, which is part of this disclosure, is submitted as a text file along with the specification. The name of the text file containing the sequence listing is "54627_Seqlisting.txt", which was created on August 6, 2020, and has a size of 54,454 bytes. The contents of the sequence listing are incorporated herein by reference.

[0003] This disclosure generally relates to hydrophobic salts of hydrophilic peptides that form a low-solubility material in aqueous solution and enable extended or sustained release of peptide components when administered to a target. [Background technology]

[0004] Sustained-release therapeutics are desirable, for example, to reduce the number of administrations or to reduce the amount of drug a subject may receive to achieve a therapeutic effect. However, certain types of active ingredients in drugs are difficult to incorporate into sustained-release compositions (e.g., enteric-coated dose-responsive capsules or tablets, or microspheres such as liposomes or nanoparticles) that have a certain delayed-release property that allows the therapeutic agent to be delivered in vivo to a specific site or allows the drug to slowly escape from the particles over time. [Overview of the project]

[0005] This disclosure relates to compositions comprising salts of electrostatically charged peptides that have low solubility in solution, such that the salts form a solid or semi-solid in an aqueous medium. Such salts dissolve more slowly than the unsalted form of the peptide in aqueous solution and can be used in extended-release therapeutics without the need for re-formulation in a typical extended-release form, as shown herein.

[0006] Provided herein are compositions comprising a hydrophobic salt of an electrostatically charged peptide, the salt comprising an electrostatically charged peptide complexed with a hydrophobic counterion. In various embodiments, the salt is a hydrophobic peptide salt.

[0007] In various embodiments, the weight percentage of peptide in the peptide salt is at least about 10% by weight of peptide in the peptide salt. In various embodiments, the amount of peptide in the peptide salt is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or more by weight. In various embodiments, the weight percentage of active peptide in the peptide salt is at least about 5% by weight. In various embodiments, the weight percentage of active peptide in the peptide salt is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or more. In various embodiments, the percentage of active peptide compared to the total peptide present in the salt is at least about 50%, 60%, 70%, or more than 80%. In various embodiments, the hydrophobic peptide salt dissolves slowly and does not dissolve immediately in 1 mg / mL of 1× phosphate-buffered saline (PBS).

[0008] In various embodiments, hydrophobic counterions form complexes with electrostatically charged peptides via non-covalent bonds.

[0009] In various embodiments, the hydrophobic peptide salt further comprises a polyvalent cation complexed with a peptide-hydrophobic counterion complex. In various embodiments, the electrostatically charged peptide, hydrophobic counterion, and polyvalent cation complex via non-covalent bonds. In various embodiments, the polyvalent cation complexed with the peptide-hydrophobic counterion complex is a metal cation.

[0010] In various embodiments, the peptide salt containing the hydrophobic counterion has a cLogP of about 0 to about 10, or the conjugate acid of the hydrophobic counterion has a pKa of -2 to 5, or both. In various embodiments, the peptide salt containing the hydrophobic counterion has a cLogP of about 2 to about 9, or the conjugate acid of the hydrophobic counterion has a pKa of less than about 5, or both. In various embodiments, the peptide salt containing the hydrophobic counterion has a cLogP of about 2 to about 9, and the conjugate acid of the hydrophobic counterion has a pKa of less than about 5. In various embodiments, the peptide salt containing the hydrophobic counterion has a cLogP of about 2 to about 9, and the conjugate acid of the hydrophobic counterion has a pKa of about 0 to about 5.

[0011] In various embodiments, the hydrophobic counterion is selected from the group consisting of deprotonated fatty acids, deprotonated bile acids, naphthoates and their derivatives, nicotinates and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextrans sulfates, alkylbenzene sulfates, ionic surfactants, and any combination thereof. In various embodiments, the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, decanate, 2-naphthalene sulfonate, 1-heptanesulfonate, 1-octanesulfonate monohydrate, 1-decanesulfonate, dodecyl sulfate, dextrans sulfate, and dodecylbenzenesulfonate. In various embodiments, the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. In various embodiments, the counterion is selected from the group consisting of oleate, pamoate, deoxycholate, decanoate, and docusate.

[0012] In various embodiments, the polyvalent cation has a charge of +2, +3, or +4 or higher. In various embodiments, the polyvalent cation has a charge of +2, +3, or +4. In a particular embodiment, the polyvalent cation has a charge of +2. In a particular embodiment, the polyvalent cation has a charge of +3. In a particular embodiment, the polyvalent cation has a charge of +4. In various embodiments, the cation is a metallic cation. In various embodiments, the cation includes a metal 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), barium (V), platinum (Pt), copper (Cu), and gold (Au). In various embodiments, the cation comprises zinc or calcium. In various embodiments, the cation comprises Mg 2+ Zn 2+ , or Ca 2+ In various embodiments, the cation is Zn 2+ or Ca 2+ In certain embodiments, the cation is Zn 2+ In certain embodiments, the cation is Ca 2+ That is the case.

[0013] In various embodiments, the peptide salt may be 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 or linked to a gel.

[0014] In various embodiments, the electrostatically charged peptide is a C-type natriuretic peptide (CNP). In various embodiments, the CNP is a CNP variant. The CNP and CNP variants contemplated herein will be described more fully in the detailed description. In various embodiments, the CNP complexes with a hydrophobic counterion to form a hydrophobic CNP salt complex. In various embodiments, the hydrophobic CNP salt further comprises a polyvalent cation that complexes with the CNP-hydrophobic counterion complex to form a CNP-cation-hydrophobic counterion salt complex. In various embodiments, the polyvalent cation is a metal cation.

[0015] In various embodiments, CNP is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), The selection is made from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and their salts. In various embodiments, the CNP salt useful for forming the hydrophobic CNP salts described herein is CNP acetate.

[0016] In various embodiments, CNP is PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), and The group is selected from PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 7).

[0017] In various embodiments, the CNP variant peptide further comprises 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 the amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus.

[0018] In various embodiments, the CNP variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (Sequence ID 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 9), Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10) Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 11), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 12), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13), and The selection is made from the group consisting of Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0019] In various embodiments, the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. In various embodiments, when a polyvalent cation is present, the polyvalent cation comprises zinc or calcium. In various embodiments, when a polyvalent cation is present, the polyvalent cation comprises magnesium, zinc, or calcium. In various embodiments, the cation is Mg 2+ , Zn 2+ , or Ca 2+ . In various embodiments, the polyvalent cation is Zn 2+ or Ca 2+ . In various embodiments, the polyvalent cation is Zn 2+ . In various embodiments, the polyvalent cation is Ca 2+ .

[0020] In various embodiments, the composition further comprises an excipient, diluent, or carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier. Also provided is a sterile pharmaceutical composition comprising the hydrophobic salt composition described herein.

[0021] Further contemplated by the present disclosure is a release-modulating composition comprising the hydrophobic peptide salt described herein. In various embodiments, the release-modulating composition is an extended-release composition, a sustained-release composition, or a delayed-release composition.

[0022] In various embodiments, the extended-release composition comprises a hydrophobic peptide salt, and the peptide salt solid, semi-solid, gel, crystalline, amorphous, nanoparticles, microparticles, amorphous nanoparticles, amorphous microparticles, crystalline nanoparticles, or crystalline microparticles are resuspended in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer.

[0023] In various embodiments, the oil contains 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, if the fatty acids are in triglycerides, the fatty acids are saturated or unsaturated and may be medium-chain or long-chain fatty acids.

[0024] 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 a hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid.

[0025] In various embodiments, the extended-release composition is such that, at pH 7-7.6, (i) less than 20% of the peptide is released by day 1, and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released every other week, or about 90% of the peptide is released monthly.

[0026] In various embodiments, less than 20% of the peptide is released by day 1 at pH 7–7.6. Furthermore, (i) less than 30%, 40%, 50%, or 60% of the peptide is released by day 1 at pH 7.0–7.6, and (ii) approximately 90% of the peptide is released weekly, bi-weekly, or monthly at pH 7–7.6. Furthermore, the intention is that (i) at pH 7.0-7.6, less than 30%, 40%, 50%, or 60% of the peptides are released by day 1; and (ii) at pH 7-7.6, approximately 70%, 80%, or 90% of the peptides are released weekly, or approximately 70%, 80%, or 90% of the peptides are released every two weeks, or approximately 70%, 80%, or 90% of the peptides are released every three weeks, or approximately 70%, 80%, or 90% of the peptides are released monthly.

[0027] In various embodiments, (i) at pH 7.0–7.6, about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the peptide is released by day 1, and (ii) at pH 7–7.6, about 90% of the peptide is released weekly, or about 90% of the peptide is released every other week, or about 90% of the peptide is released monthly. Furthermore, (i) at pH 7.0-7.6, approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the peptide is released by day 1, and (ii) at pH 7.0-7.6, approximately 70%, 80%, or 90% of the peptide is released weekly, or approximately 70%, 80%, or 90% of the peptide is released every two weeks, or approximately 70%, 80%, or 90% of the peptide is released every three weeks, or approximately 70% of the peptide ii) Approximately 80% or 90% of the peptides are released monthly, or alternatively, ii) at pH 7-7.6, 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.

[0028] In various embodiments, approximately 90% of the peptide is released weekly at pH 7–7.6. In various embodiments, approximately 90% of the peptide is released bi-weekly at pH 7–7.6. In various embodiments, approximately 90% of the peptide is released monthly at pH 7–7.6. Furthermore, it is intended that the release may be at pH 7.0–7.6, pH 7.1–7.5, pH 7.2–7.4, pH 7.2–7.6, or pH 7.0–7.4.

[0029] In various embodiments, the extended-release composition comprises an excipient, diluent, or 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.

[0030] Methods for preparing the hydrophobic peptide salt compositions described herein, such as hydrophobic CNP salts, are also provided herein. Ionic surfactants are good candidates for counterions because polar head groups remain permanently charged regardless of the complex formation pH. Adjusting the pH at which complex formation occurs imparts different amounts of charge to the peptide, thereby allowing for stoichiometric control of peptide:surfactant complex formation and potentially controlling the size of the resulting precipitate. Metal cations can be used as bridges for the anionic side chains of peptide amino acids to bond to anionic hydrophobic counterions. The order and rate of cation and counterion addition to the peptide are important to minimize precipitation of metal cations by anionic hydrophobic counterions.

[0031] In various embodiments, the Disclosure envisions a method for preparing a composition comprising a salt of an electrostatically charged peptide, comprising: a) contacting an electrostatically charged peptide in an aqueous solution with a hydrophobic counterion in a solution; and b) mixing the electrostatically charged peptide solution with a hydrophobic counterion solution in a manner sufficient for the peptide and counterion to form a complex, the formation of which the peptide-counterion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle morphology comprising the hydrophobic peptide salt. In various embodiments, the peptide salt is a hydrophobic CNP salt.

[0032] In various embodiments, the method optionally includes, prior to step (b), contacting an electrostatically charged peptide in solution with a polyvalent cation in an aqueous solution to form a peptide-cation complex. In various embodiments, the polyvalent cation is a metal cation.

[0033] In various embodiments, mixing is carried out by dropwise addition of a hydrophobic counterion solution to an electrostatically charged peptide solution. In various embodiments, the solutions are mixed by vortexing after the addition of each droplet of hydrophobic counterion solution, or by other mixing means known in the art.

[0034] In various embodiments, this method further comprises step (c) washing the peptide salt with a buffer or water. In various embodiments, the washing is carried out in an aqueous solution, for example, a buffer or water.

[0035] In various embodiments, the method further comprises step (d) obtaining the peptide salt by centrifugation to form a peptide salt pellet. In various embodiments, if the salt is in gel form, the salt is obtained by centrifugation or by decanting the liquid phase followed by freeze-drying or other drying method.

[0036] In various embodiments, the method further comprises step (e) removing water from the peptide salt pellet. It is thought that water or another aqueous solution can be removed from the pellet by freeze-drying or drying using techniques known in the art.

[0037] In various embodiments, this method further comprises resuspending the pellets in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline solution, or 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, unsaturated, or a combination thereof.

[0038] Fatty acids may be present in the oil itself or in triglycerides. In various embodiments, fatty acids are short-chain, medium-chain, or long-chain fatty acids. In various embodiments, if fatty acids are present in triglycerides, they may be saturated or unsaturated and may be medium-chain or long-chain fatty acids. In various embodiments, fatty acids are C-6 to C-20 fatty acids. In various embodiments, fatty acids are C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acids. In various embodiments, fatty acids are hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid.

[0039] In various embodiments, the synthesis method intends to use a peptide:hydrophobic counterion ratio of at least 1 molar equivalent of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide. In various embodiments, the peptide:hydrophobic counterion ratio used in the synthesis method is 1:1 to 1:20, or 1:1 to 1:50. The peptide:counterion ratio used in the synthesis 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:counterion 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-to-hydrophobic counterion ratio used in the synthesis method is at least 2 molar equivalents of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide. In various embodiments, the peptide-to-hydrophobic counterion ratio used in the synthesis method is at least 3 molar equivalents of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide.

[0040] In various embodiments, the synthesis method intends to use a peptide:cation ratio of at least 1 molar equivalent of cations relative 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 2 molar equivalents of cations relative 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 3 molar equivalents of cations relative to the total number of negatively charged amino acids in the peptide. Further combinations of the above peptide:cation and peptide:hydrophobic counterion ratios are being considered.

[0041] An exemplary ratio is one counterion for each positive charge of the peptide. For polyvalent cations, the exemplary ratio is approximately one metal cation per negatively charged site of the peptide, and two polyvalent cations per negatively charged site, or a 2x molar excess. For example, Zn 2+ or Ca 2+ One or two of these polyvalent cations can be used in combination with 6 to 8 counterions, or more if hydrophobic interactions are involved.

[0042] In various embodiments, hydrophobic counterions are complexed via non-covalent bonds.

[0043] In various embodiments, if the salt complex further contains a polyvalent cation complexed with a peptide-counterion complex, the cation is complexed via non-covalent bonds. In various embodiments, the electrostatically charged peptide, hydrophobic counterion, and cation are complexed via non-covalent bonds.

[0044] In various embodiments, CNP is The group is selected from PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37)(SEQ ID NO: 1).

[0045] Also provided is a method for treating bone-related disorders or skeletal dysplasia in subjects requiring treatment of bone-related disorders or skeletal dysplasia, comprising administering a composition comprising a hydrophobic salt of type C natriuretic peptide (CNP), including compositions such as those described and extended-release compositions.

[0046] In various embodiments, bone-related disorders or skeletal dysplasia include osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondrodysplasia, dwarfism, osteochondrodysplasia, fatal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, isozygosyngeal chondrodysplasia, chondrodysplasia punctata, dysplasia of the limbs, congenital fatal hypophosphatasia, perinatal fatal osteogenesis imperfecta, short-rib chondrodysplasia, hypochondrodysplasia, rhombophyseal dysplasia, Janssen type metaphyseal dysplasia, congenital spondyloepophyseal dysplasia, growth retardation osteogenesis imperfecta, and torsion. The group is selected from the following: dysplasia skeletalis, congenital short femur, Langer type midleg dysplasia, Niebergeld type midleg dysplasia, Robinnow syndrome, Reinhardt syndrome, achondroplasia, peripheral dysplasia, Niest dysplasia, fibrochondroplasia, Roberts syndrome, acromegaly and midlimmen ankylodysplasia, brachylimmus, Morquio syndrome, Niest syndrome, complex organic dysplasia, and vertebral epiphyseal metaphysical dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Reliweil), PTPN11 mutation (Noonan syndrome), and idiopathic short stature.

[0047] In various embodiments, CNP variants are useful as growth hormone adjuvants or substitutes for treating idiopathic short stature and other skeletal dysplasias.

[0048] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature disorders are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Reliweil), or PTPN11 mutations (Noonan syndrome).

[0049] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature disorders are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Reliweil), or PTPN11 mutations (Noonan syndrome), or insulin growth factor 1 receptor (IGF1R).

[0050] In various embodiments, CNP variants are useful in treating growth plate disorders and short stature, including familial short stature, dominant familial short stature, also known as dominant inherited 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), aggrecan (ACAN), Indian Hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0051] In various embodiments, growth plate disorders or short stature are associated with mutations in one or more genes related to RAS disease.

[0052] In various embodiments, bone-related disorders, skeletal dysplasia, or short stature disorders are caused by RAS disease. In various embodiments, RAS disease is Noonan syndrome, Costello syndrome, cardiac facial cutaneous syndrome, neurofibromatosis type 1, or Leopard syndrome.

[0053] In one embodiment, RAS disease is hereditary gingival fibromatosis type 1.

[0054] In various embodiments, CNP variants are useful in treating growth plate disorders and short stature, including familial short stature, dominant familial short stature, also known as dominant inherited 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), aggrecan (ACAN), Indian Hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R).

[0055] In various embodiments, short stature is associated with mutations in one or more genes related to RAS disease.

[0056] In various embodiments, the CNP variant is useful for treating subjects with short stature who have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0 and have at least one parent whose height SDS is less than -1.0, -1.5, -2.0, or -2.5, and optionally the height of the second parent is within the normal range; in various embodiments, the CNP variant is useful for treating subjects with short stature who have a height SDS of -2.0 to -3.0; and in various embodiments, the CNP variant is useful for treating subjects with short stature who have a height SDS of -2.0 to -2.5. In various embodiments, short stature is associated with mutations in one or more genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or a combination thereof. In various embodiments, short stature is associated with mutations in one or more genes associated with RAS disease.

[0057] In various embodiments, short stature is the result of mutations in multiple genes, such as those determined by a polygenic risk score (PRS). In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in FGFR3 and a low PRS. In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in IGF1R and a low PRS. In various embodiments, the subject has a mutation in NPPC and a low PRS. In various embodiments, the subject has a mutation in SHOX and a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX and a low PRS. In various embodiments, the PRS is 1 or 2. In various embodiments, the PRS is 1. In various embodiments, the PRS is 2.

[0058] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is at the N-terminus of the peptide. In various embodiments, the acetyl group is on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker. In various embodiments, the variant is a hydrophobic salt of an electrostatically charged CNP peptide, which comprises an electrostatically charged CNP peptide complexed with a hydrophobic counterion.

[0059] This disclosure also envisions a method for lengthening bones or increasing long bone growth in subjects requiring bone lengthening or increased long bone growth, the method comprising administering to a subject a sustained-release composition comprising a salt of type C natriuretic peptide (CNP), including the compositions and extended-release compositions described herein, the administration of which lengthens bones or increases long bone growth.

[0060] In various embodiments, CNP is a CNP variant. In various embodiments, CNP is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), The group is selected from PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and their pharmaceutically acceptable salts. In various embodiments, CNP is CNP acetate.

[0061] In various embodiments, CNP is PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), and The group is selected from PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 7).

[0062] In various embodiments, the CNP variant further comprises 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 at the side of an amino acid in the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus.

[0063] In various embodiments, the variant is Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (Sequence ID 8), Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2, (Sequence ID 9) Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10) Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 11), Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (Sequence ID 12), Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13), and The selection is made from the group consisting of Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0064] In various embodiments, the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0065] 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.

[0066] In various embodiments, the composition is an extended-release composition.

[0067] Hydrophobic salts of C-type natriuretic peptide (CNP) containing CNP complexed with a hydrophobic counterion are also provided. In various embodiments, the hydrophobic CNP salt further comprises CNP and a cation complexed with a hydrophobic counterion. In various embodiments, the hydrophobic CNP salt is a purified salt. In some cases, the salt has a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or higher.

[0068] In various embodiments, the hydrophobic salt comprises a hydrophobic counterion selected from the group consisting of oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. In various embodiments, the hydrophobic salt comprises Zn 2+ or Ca 2+ It contains the cation.

[0069] In various embodiments, the hydrophobic salt is selected from the group consisting of CNP-oleate, CNP-pamoate, CNP-deoxycholate, CNP-decanoate, and CNP-docusate. In various embodiments, the hydrophobic salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate), CNP-Ca +2 (Deoxycholate), CNP-Ca +2 (Decanoate) CNP-Ca +2 (Docusate), CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate), CNP-Zn +2 (Deoxycholate), CNP-Zn +2 (decanoate), and CNP-Zn +2 Selected from the group consisting of (docuset).

[0070] In various embodiments, the hydrophobic salt comprises CNP 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 hydrophobic salt comprises CNP which is a CNP acetate.

[0071] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is at the N-terminus of the peptide. In various embodiments, the acetyl group is at the side of an amino acid of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker. In various embodiments, the peptide comprises a hydrophobic salt of an electrostatically charged peptide, the salt comprising an electrostatically charged peptide complexed with a hydrophobic counterion.

[0072] This disclosure also provides compositions comprising hydrophobic peptide salts, such as hydrophobic CNP salts, as described herein, for use in the treatment of skeletal dysplasia or bone-related disorders as described herein. In certain embodiments, this disclosure provides the use of compositions comprising hydrophobic peptide salts, such as hydrophobic CNP salts, in the preparation of agents for the treatment of skeletal dysplasia or bone-related disorders as described herein. In various embodiments, the hydrophobic peptide salt is a hydrophobic CNP salt as described herein.

[0073] Each feature, embodiment, or combination described herein is a non-limiting, exemplary example of any aspect of the Invention and is therefore intended to be combinatable with any other feature, embodiment, or combination described herein. For example, where a feature is described using terms such as “one embodiment,” “several embodiments,” “a particular embodiment,” “further embodiments,” “specific exemplary embodiments,” and / or “another embodiment,” each of these types of embodiments is a non-limiting example of a feature intended to be combinatable with any other feature or combination of features described herein, without the need to enumerate all possible combinations. Such features or combinations of features apply to any aspect of the Invention. Where examples of values ​​within a range are disclosed, each of these examples is intended as a possible endpoint with respect to a range, and every possible numerical value between such endpoints is intended, and every possible combination of upper and lower endpoints is assumed.

[0074] The headings in this specification are for the convenience of the reader and are not intended to be limiting. Further aspects, embodiments, and variations of the present invention will become apparent from the detailed description and / or drawings and / or claims. [Brief explanation of the drawing]

[0075] [Figure 1A] Figures 1A-1D show the solubility profiles of various hydrophobic CNP salts in water at pH 6.5. [Figure 1B] Figures 1A-1D show the solubility profiles of various hydrophobic CNP salts in water at pH 6.5. [Figure 1C] Figures 1A-1D show the solubility profiles of various hydrophobic CNP salts in water at pH 6.5. [Figure 1D] Figures 1A-1D show the solubility profiles of various hydrophobic CNP salts in water at pH 6.5. [Figure 2]Figure 2 shows the solubility profiles of various hydrophobic CNP salts in water at pH 6.5. CNP acetate is used as a control. [Figure 3] Figure 3 shows the effect of the CNP variant (Pro-Gly-CNP37) on cells with NPR2 homozygous or heterozygous mutations, as measured by cGMP stimulation. [Figure 4-1] Figure 4 shows the nucleotides and predicted protein sequences of the first exon of the NPR2 mutant clone transfected into RCS cells. [Figure 4-2] Figure 4 shows the nucleotides and predicted protein sequences of the first exon of the NPR2 mutant clone transfected into RCS cells. [Figure 4-3] Figure 4 shows the nucleotides and predicted protein sequences of the first exon of the NPR2 mutant clone transfected into RCS cells. [Figure 5] Figure 5 shows exemplary NPR2 mutations analyzed for their response to CNP. [Figure 6-1] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-2] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-3] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-4] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-5] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-6] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-7] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-8] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-9] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figure 6-10] Figure 6 shows examples of mutations associated with short stature in FGFR3, IGF1R, NPPC, NPR2, and SHOX. [Figures 7A-7F] Figures 7A–7F show 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. The violin diagram shows horizontal lines representing the 25th, 50th, and 75th percentiles of height. Samples were grouped by having a missense, loss of function, or none status in any of the five core genes. Figure 7B: Effect reflected in the odds ratio for “Idiopathic Short Stature” or ISS. Odds for ISS using PRS=3 as reference versus other PRS groups. Figure 7C: Odds for ISS using PRS=1 as reference versus odds for having a missense and / or loss of function variant in the core gene. Figure 7D: Odds for ISS using PRS=1 non-carrier as reference versus odds for having a missense and / or loss of function variant in the core gene. Figure 7E: Odds for ISS using PRS=2 non-carrier as reference versus odds for having a missense and / or loss of function variant in the core gene. See Figure 7F for odds of ISS using a non-carrier with PRS=3 and odds of having a core gene missense and / or loss-of-function variant. [Figure 8A] Figure 8A shows the release profile of Zn CNP pamoate. The data shown are the average of three wells. [Figure 8B] Figure 8B shows the release profiles of various CNP peptide salts. The data shown are averages of four wells. [Figure 9A]Figures 9A and 9B show the solubility profile of CNP pamoate as a cumulative release profile (Figure 9A) or release percentage (Figure 9B). The data shown are the average of 3 wells for 1xPBS data and 2 wells for 1xPBS + 0.05% PS80 data. [Figure 9B] Figures 9A and 9B show the solubility profile of CNP pamoate as a cumulative release profile (Figure 9A) or release percentage (Figure 9B). The data shown are the average of 3 wells for 1xPBS data and 2 wells for 1xPBS + 0.05% PS80 data. [Figure 10A] Figures 10A–10C show the solubility profiles of docusate salts after freeze-drying and storage (Figure 10A), or after analysis of newly prepared salts (Figures 10B–10C). [Figure 10B] Figures 10A–10C show the solubility profiles of docusate salts after freeze-drying and storage (Figure 10A), or after analysis of newly prepared salts (Figures 10B–10C). [Figure 10C] Figures 10A–10C show the solubility profiles of docusate salts after freeze-drying and storage (Figure 10A), or after analysis of newly prepared salts (Figures 10B–10C). [Figure 11] Figure 11 shows the release profile of CNP salts over 7 days in vivo. [Modes for carrying out the invention]

[0076] This disclosure relates to salts of hydrophilic peptides that are in solid, semi-solid gel, or other salt forms capable of extended release of peptide active components when placed in aqueous solution. For example, this disclosure unexpectedly shows that complex formation between hydrophilic C-type natriuretic peptides (CNPs) and charged hydrophobic counterions produces low-solubility peptide salts under aqueous conditions. This disclosure shows that peptide-hydrophobic counterion salt complexes themselves, including salts containing peptide-counterion-cationic complexes, can be used in modified or extended-release compositions without the need to encapsulate the peptide complex in liposomes or microspheres / nanoparticles. Such compositions are useful, for example, in extended-release applications in the treatment of skeletal dysplasia and bone growth disorders, as described herein.

[0077] As used in the specification and the attached claims, the indefinite articles "a" and "an," as well as the definite article "the," include plural and singular referents unless the context explicitly indicates otherwise.

[0078] The terms “about” or “approximately” mean within an acceptable margin of error for a particular value as determined by those skilled in the art, which depends in part on how that value is measured or determined. In certain embodiments, the terms “about” or “approximately” mean within one, two, three, or four standard deviations. In certain 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. Whenever the terms “about” or “approximately” precede the first number in a set of two or more numbers, it is understood that the terms “about” or “approximately” apply to each of those numbers.

[0079] As used herein, the term “electrostatically charged peptide” refers to a peptide containing charged amino acids, for example, a series of amino acids ranging from 5 to 100 amino acids. A peptide may have positively charged amino acids, negatively charged amino acids, or a mixture of both, and as a result, an electrostatically charged peptide may have an overall net charge and be able to interact with other charged moieties, such as cations, anions, or counterions, which may have a charge opposite to that of the peptide to which a counterion may bind. An electrostatically charged peptide may have a net positive charge or a net negative charge. If a peptide has a net positive charge, it may interact with charged moieties that have one or more negative charges. If a peptide has a net negative charge, it may interact with charged moieties that have one or more positive charges.

[0080] As used herein, the term “hydrophobic counterion” refers to a group of electrostatically charged portions that are inherently hydrophobic and capable of interacting with hydrophilic peptides. In various embodiments, hydrophobic counterions are selected based on their cLogP value, the pKa value of their conjugate acid, or both. In various embodiments, hydrophobic counterions have a cLogP of about 0 to about 10, or a pKa of their conjugate acid of about -2 to about 5, or both. In various embodiments, hydrophobic counterions have a net negative charge and can interact with electrostatically charged peptides that have a net positive charge. In various embodiments, hydrophobic counterions include deprotonated fatty acids, deprotonated bile acids, naphthoates and their derivatives, nicotinates and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextrans sulfates, ionic surfactants, and alkylbenzene sulfates. In some embodiments, the hydrophobic counterion is a zwitterion (e.g., phosphatidylethanolamine). In various embodiments, hydrophobic counterions include, but are not limited to, palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, decanoate, 2-naphthalene sulfonate, 1-heptanesulfonate, 1-octanesulfonate monohydrate, 1-decanesulfonate, dodecyl sulfate, dextrans sulfate, and dodecylbenzenesulfonate. In some embodiments, the hydrophobic counterion has a net positive charge and can interact with electrostatically charged peptides that have a net negative charge.

[0081] As used herein, the terms “peptide salt” or “hydrophobic peptide salt” refer to a complex of an electrostatically charged peptide with a counterion, such as a hydrophobic counterion, where the portion forms a complex and a salt. The peptide and counterion may form a complex via non-covalent bonds. In various embodiments, the peptide counterion salt further contains a polyvalent cation such that the complex contains a peptide-cation-counterion within the complex. Peptide salt or hydrophobic peptide salt refers to both peptide-counterion complexes and peptide-cation-counterion complexes.

[0082] In various embodiments, peptides and cations form complexes non-covalently. In various embodiments, peptides, cations, and hydrophobic counterions in peptide salts form complexes via non-covalent bonds.

[0083] The terms “C-type natriuretic peptide” or “CNP” refer to a small single-chain peptide having a 17-amino acid loop structure at its C-terminus (CNP precursor protein, GenBank deposit number NP_077720 for NPPC), and its variants. CNP is initially produced from the natriuretic peptide precursor C (NPPC) gene as a single-chain 126-amino acid prepropolypeptide that is cleaved to produce proCNP, and an active 53-amino acid peptide (CNP-53). This is secreted by an unknown enzyme and cleaved again to produce a mature 22-amino acid peptide (CNP-22). “CNP salt” or “hydrophobic CNP salt” refers to the salts described herein that contain CNP or a CNP variant, each containing a counterion such as a hydrophobic counterion, and optionally further containing a polyvalent cation.

[0084] In various embodiments, the “CNP variant” is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to wild-type NPPC over the same number of amino acid residues. In various embodiments, the CNP variant peptide may contain about 1 to about 53, or 1 to about 38, or 1 to about 37, or 1 to about 35, or 1 to about 34, or 1 to 33, or 1 to 32, or 1 to 31, or 1 to 27, or 1 to 22, or 10 to about 35, or about 15 to about 37 residues of the NPPC polypeptide. In one embodiment, the CNP variant comprises a 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 sequence derived from an NPPC polypeptide.

[0085] Provided herein are release-modulated compositions comprising the hydrophobic peptide salts described herein. Release-modulated compositions include those that deliver drugs with a delay after administration (delayed-release dose) or over a longer period (extended-release dose). Various embodiments of the peptide salts provided herein include release-modulated compositions such as extended-release, sustained-release, or controlled-release, and delayed-release. The term "extended-release composition" refers to a composition formulated to make the active ingredient / drug available over a longer period after administration (United States Pharmacopeia). Extended-release doses include sustained-release (SR) or controlled-release (CR) forms. Sustained-release maintains drug release over a sustained period, but not necessarily at a constant rate, while CR maintains drug release over a sustained period 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 the active pharmaceutical ingredient for a certain period after the initial administration.

[0086] The term “effective dose” refers to the amount of medication sufficient to produce the desired outcome with respect to the health condition, illness, or disease of the subject, or for diagnostic purposes. The desired outcome may include subjective or objective improvement in the recipient of the dose. “Therapeutic effective dose” refers to the amount of medication that is effective in producing the intended beneficial effect on health. The appropriate “effective” dose in individual cases can be determined by those skilled in the art using routine experiments. It will be understood that specific dose levels and frequencies of administration for a particular patient may vary and depend on a variety of factors, including the activity of the particular compound used, its bioavailability, metabolic stability, excretion rate and duration of action, the mode and timing of the compound's administration, the patient’s age, weight, general health, sex, and diet, as well as the severity of the particular condition.

[0087] "Treatment" refers to preventive treatment, therapeutic treatment, or diagnostic treatment. In certain embodiments, "treatment" refers to the administration of a compound or composition to a subject for therapeutic, preventive, or diagnostic purposes.

[0088] "Prophylactic" treatment is a treatment administered to subjects who show no signs or symptoms of a disease, or only early signs of a disease, with the aim of reducing the risk of developing the disease. The compounds or compositions of this disclosure may be given as prophylactic treatment to reduce the likelihood of developing a disease, or to minimize the severity of a disease if it does develop.

[0089] "Therapeutic" treatment is a treatment administered to an object exhibiting signs or symptoms of a disease, with the aim of reducing or eliminating those signs or symptoms. Signs or symptoms may be biochemical, cellular, histological, functional, or physical, subjective, or objective. The compounds of this disclosure may also be administered as therapeutic treatment or for diagnostic purposes.

[0090] "Pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in target animals, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of a hydrophobic peptide salt, e.g., a CNP salt, optionally another bioactive agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In one embodiment, a pharmaceutical composition encompasses a composition comprising an active ingredient, an inactive component constituting a carrier, and any product arising directly or indirectly from the dissociation of any two or more components, or one or more components, or from a combination, complex formation, or aggregation of one or more components of other types of reactions or interactions. Accordingly, a pharmaceutical composition of the Disclosure encompasses any composition prepared by mixing a compound of the Disclosure with a pharmaceutically acceptable excipient, carrier, or diluent.

[0091] "Pharmacologically acceptable carriers" refer to any of the standard pharmaceutical carriers and buffers, such as phosphate-buffered saline, a 5% aqueous solution of dextrose, 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, flow enhancers, sweeteners, flavoring agents, and colorants. Preferred pharmaceutical carriers, excipients, and diluents are listed in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). The preferred pharmaceutical carrier depends on the intended mode of administration of the active agent. Typical modes of administration include enteral (e.g., oral) or parenteral (e.g., subcutaneous, intramuscular, intravenous, or intraperitoneal injection, or topical, transdermal, or transmucosal administration).

[0092] A "pharmaceutically acceptable salt" is a salt that can be incorporated into a compound for pharmaceutical use, including but not limited to metal salts (e.g., sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0093] "Pharmacologically acceptable" or "pharmacologically acceptable" means a material that is not biologically or otherwise undesirable, that is, a material that can be administered to an individual without causing an undesirable biological effect and without interacting in a harmful manner with any composition or component containing it or any component present on or in the body of the individual.

[0094] "Physiological state" refers to the internal state of an animal (e.g., a human). Physiological conditions include, but are not limited to, body temperature, and physiological ionic strength, pH, and the aqueous environment of enzymes. A physiological state also includes the physical state of a particular subject, which differs from the "normal" state present in most subjects, for example, from the normal human body temperature of approximately 37°C or from the normal human blood pH of approximately 7.4.

[0095] As used herein, the term “subject” encompasses both mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other ape and monkey species; domesticated animals such as cattle, horses, sheep, goats, and pigs; and domesticated animals such as rabbits, dogs, and cats. Laboratory animals include rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish. This term does not indicate a specific age or sex. In various embodiments, the subject is human. In various embodiments, the subject is a child or adolescent. In various embodiments, the subject is an infant.

[0096] Electrostatically charged peptides and peptide salts Peptide therapies are attractive biological agents, but their low stability and short half-lives in solution often present disadvantages (Tang et al., Eur J Pharm Sci. 102:63-70, 2017). Attempts to improve the potency of peptide therapies include attempts to encapsulate hydrophilic peptides in biodegradable particles such as liposomes or polymer particles. However, this is difficult due to the cationic nature of these peptides and their ability to electrostatically interact with liposomes in negatively charged polymers (Griesser et al., Int J Pharmaceutics 520:267-274, 2017). The generation of hydrophobic ion pairs between hydrophilic peptides and their hydrophobic moieties is one method used to enable better encapsulation of hydrophilic polymers into microparticles or liposomes (Lu et al., Mol. Pharmaceutics 15:216-225, 2018). Hydrophobic ion pairs are formed when a charged residue of a peptide interacts with an ion that is oppositely charged to the hydrophobic moiety (Tang et al., see above). In certain cases, this can lead to the precipitation of hydrophobic ion pairs from solution, facilitating encapsulation into liposomes or macromolecular nanoparticles (Griesser et al., see above).

[0097] In this specification, it has been discovered that hydrophobic ion complexes of hydrophilic CNP peptides and hydrophobic counterions generate CNP peptide salts. The generation of hydrophobic ion pairs between hydrophilic peptides and hydrophobic counterions can be enhanced by first contacting the hydrophilic peptide with a polyvalent cation (e.g., a metal cation) to enhance the interaction between the peptide and the hydrophobic counterion. For example, a polyvalent cation can complex with the negatively charged functional group of the hydrophilic peptide, increasing the number of positive charges on the hydrophilic peptide that can be used for complex formation with hydrophobic counterions such as hydrophobic anions. Thus, a polyvalent cation can link the negative charge of the hydrophilic peptide with the negative charge of the hydrophobic counterion. Furthermore, this disclosure shows that the peptide-hydrophobic counterion salt complex or the peptide-cation-hydrophobic counterion salt complex itself can be used in controlled-release or extended-release compositions without the need to encapsulate the peptide complex in liposomes or microspheres / nanoparticles.

[0098] Electrostatically charged peptides can be a sequence of 5 to 100 amino acids containing charged amino acids, with an overall net charge. Peptides can have positively charged amino acids, negatively charged amino acids, or a mixture of both, and as a result, electrostatically charged peptides can interact with other charged moieties, such as cations, anions, or counterions, or combinations thereof having species oppositely charged to the peptide. In various embodiments, electrostatically charged peptides have a net positive charge. Electrostatically charged peptides with a net positive charge can complex with negatively charged hydrophobic counterions, such as a negatively charged hydrophobic counterion. In various embodiments, electrostatically charged peptides have a net negative charge. Electrostatically charged peptides with a net negative charge can complex with positively charged hydrophobic counterions, such as a positively charged hydrophobic counterion. In various embodiments, electrostatically charged peptides have at least two amino acids having the same type of charge (e.g., two positively charged amino acids or two negatively charged amino acids).

[0099] A hydrophilic peptide is a peptide that has high solubility in aqueous solution. Hydrophilic peptides as intended 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, or any range of these values. In various embodiments, hydrophilic peptides have a solubility greater than 10 mg / mL or greater than 5 mg / mL in aqueous solution. In various embodiments, hydrophilic peptides also refer to peptides having high solubility in aqueous solution, for example, those having a cLogP of less than 1.

[0100] Hydrophobic counterions To produce a salt of an electrostatically charged peptide as described herein, the peptide forms a complex with a counterion. In the case of a hydrophilic peptide, the counterion is a hydrophobic counterion. In various embodiments, the hydrophobic counterion has a net negative charge and forms a salt with the hydrophilic peptide which has a net positive charge.

[0101] The counterion is thought to form a complex with the charged peptide via non-covalent bonding. The counterion may also be non-covalently bonded to the peptide via electrostatic interactions.

[0102] When using hydrophobic counterions, the hydrophobic counterion exhibits a cLogP of about 0 to about 10, its conjugate acid exhibits a pKa of about -2 to about 5, or both. In various embodiments, the hydrophobic counterion 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 can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In various embodiments, the conjugate acid of the hydrophobic counterion 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 counterion has a pKa of about -2, -1, 0, 1, 2, 3, 4, or 5. Further considerations include hydrophobic counterions having any combination of these values ​​and ranges. In various embodiments, the hydrophobic counterion has a cLogP of about 2 to about 9, or its conjugate acid has a pKa of less than about 5, or both.

[0103] In various embodiments, the counterion is an anion. In various embodiments, the counterion is a zwitterion. In various embodiments, the counterion is an anionic or zwitterionic detergent. In various embodiments, the hydrophobic counterion is selected from the group consisting of deprotonated fatty acids, deprotonated bile acids, naphthoates and their derivatives, nicotinates and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextrans sulfates, alkylbenzene sulfates, and ionic surfactants. In various embodiments, the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate monohydrate, sodium 1-decanesulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In various embodiments, the hydrophobic counterion is oleate, pamoate, deoxycholate, decanoate, or docusate.

[0104] In various embodiments, at least one hydrophobic counterion complexes with a complex containing a hydrophilic peptide (if no polyvalent cation is present) or a hydrophilic peptide and a polyvalent cation (if a cation is present). In various embodiments, at least two hydrophobic counterions complexes with a complex containing a hydrophilic peptide (if no cation is present) or a hydrophilic peptide and a polyvalent cation (if a cation is present). In various embodiments, at least three hydrophobic counterions complexes with a complex containing a hydrophilic peptide (if no cation is present) or a hydrophilic peptide and a polyvalent cation (if a cation is present). In various embodiments, at least four hydrophobic counterions complexes with a complex containing a hydrophilic peptide (if no cation is present) or a hydrophilic peptide and a polyvalent cation (if a cation is present). In various embodiments, each positive charge of a hydrophilic peptide complexes with a hydrophobic counterion. For example, if a peptide has four positively charged amino acids, it may complex with four hydrophobic counterions. Similarly, if a peptide has four positively charged amino acids and complexes with two cations, resulting in a total of six positive charges, the peptide can complex with six hydrophobic counterions. In various embodiments, not all positive charges of a complex containing a hydrophilic peptide (where no cations are present) or a hydrophilic peptide and a polyvalent cation (where cations are present) will complex with hydrophobic counterions. For example, if a peptide has four positively charged amino acids, it may complex with three hydrophobic counterions, two hydrophobic counterions, or one hydrophobic counterion. Similarly, if a peptide has four positively charged amino acids and complexes with two cations, resulting in a total of six positive charges, the peptide can complex with five hydrophobic counterions, four hydrophobic counterions, three hydrophobic counterions, two hydrophobic counterions, or one hydrophobic counterion.

[0105] Polyvalent cations In various embodiments, the peptide salt further comprises a polyvalent cation complexed with the peptide-counterion complex. The cation is thought to complex with the charged peptide via non-covalent bonds. The cation may also be non-covalently bonded to the peptide via electrostatic interactions.

[0106] Polyvalent cations are thought to have a charge of +2, +3, or +4 or higher. In embodiments, the cation has a charge of +2. In embodiments, the cation has a charge of +3. In embodiments, the cation has a charge of +4. In various embodiments, the polyvalent cation is a metallic cation. Metallic cations include cations of Group II and Group III metals. Preferred 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 comprises zinc or calcium. In various embodiments, the polyvalent cation comprises zinc. In various embodiments, the polyvalent cation comprises calcium. In various embodiments, the polyvalent cation comprises Mg 2+ Zn 2+ , and Ca 2+ Selected from the group consisting of the following. In various embodiments, the polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the polyvalent cation is Zn 2+ In various embodiments, the polyvalent cation is Ca 2+ That is the case.

[0107] In various embodiments, at least one polyvalent cation complexes with the hydrophilic peptide. In various embodiments, at least two cations complexes with the hydrophilic peptide. In various embodiments, at least three cations complexes with the hydrophilic peptide. In various embodiments, each negative charge of the hydrophilic peptide complexes with a polyvalent cation. For example, if the peptide has four negatively charged amino acids, it can complex with four polyvalent cations, three polyvalent cations, two polyvalent cations, or one polyvalent cation.

[0108] Peptide salts 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 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 linked to a gel.

[0109] Peptide salts are considered to be in particulate form, particularly solid particles. In various embodiments, the particles are 1 to 10,000 micrometers (µm), 1 µm to 2,000 µm, 2 µm to 1,000 µm, 5 µm to 500 µm, 10 µm to 1,000 µm, 50 µm to 500 µm, 100 µm to 800 µm, 200 µm 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 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 nm to 400 nm, 20 nm to 300 nm, 300 nm to 800 nm, 200 nm to 600 nm, 100 nm to 300 nm, or 50 nm to 200 nm.

[0110] In some embodiments, the hydrophilic peptide of the peptide salt is CNP or a CNP variant as described herein, and the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate monohydrate, sodium 1-decanesulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In some embodiments, the hydrophilic peptide salt is CNP or a CNP variant as described herein, and the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, and decanoate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-pamoate, CNP-deoxycholate, and CNP-decanoate. In some embodiments, the hydrophilic peptide salt is CNP or a CNP variant as described herein, and the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, decanoate, and docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-pamoate, CNP-deoxycholate, CNP-decanoate, and CNP-docusate. In various embodiments, the peptide salt is selected from the group consisting of CNP-oleate, CNP-pamoate, 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-pamoate. In various embodiments, the peptide salt is CNP-oleate. In various embodiments, the peptide salt is CNP-pamoate.In various embodiments, the peptide salt is CNP docusate.

[0111] In various embodiments, the hydrophilic peptide of the peptide salt is Pro-Gly CNP37 (PG-CNP37), and the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate monohydrate, sodium 1-decanesulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In some embodiments, the hydrophilic peptide salt is PG-CNP37 as described herein, and the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, and decanoate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate, PG-CNP37-pamoate, PG-CNP37-deoxycholate, and PG-CNP37-decanoate. In some embodiments, the hydrophilic peptide salt is PG-CNP37 as described herein, and the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, decanoate, and docusate. In various embodiments, the peptide salt is selected from the group consisting of PG-CNP37-oleate, PG-CNP37-pamoate, 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-pamoate, 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-pamoate.In various embodiments, the peptide salt is PG-CNP37-oleate. In various embodiments, the peptide salt is PG-CNP37-pamoate. In various embodiments, the peptide salt is PG-CNP37-docusate.

[0112] In various embodiments, the peptide counterion salt further comprises a polyvalent cation. In some embodiments, the hydrophilic peptide of the peptide salt is CNP or a CNP variant, as described herein. The hydrophobic counterions include palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, sodium decanoate, sodium 2-naphthalenesulfonate, sodium 1-heptanesulfonate, sodium 1-octanesulfonate monohydrate, sodium 1-decanesulfonate, and dodecyl sulfate. The polyvalent cation is selected from the group consisting of sodium and sodium dodecylbenzenesulfonate, and includes a metal 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 CNP as described herein, and the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, and decanoate. The polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the peptide salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate), CNP-Ca +2 (Deoxycholate), CNP-Ca +2(Decanoate), CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate), CNP-Zn +2 (Deoxycholate), and CNP-Zn +2 The hydrophilic peptide salt is selected from the group consisting of (decanoate). In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic counterion is selected from the group consisting of oleate, pamoate, deoxycholate, decanoate and docusate, and the polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic counterion is selected from the group consisting of oleate, pamoate, and docusate, and the polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic counterion is selected from the group consisting of deoxycholate, decanoate and docusate, and the polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the hydrophilic peptide salt is CNP as described herein, the hydrophobic counterion is selected from the group consisting of oleate and pamoate, and the polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the hydrophilic peptide salt is CNP, as described herein, and the hydrophobic counterion is oleic acid. The polyvalent cation is Zn 2+ or Ca 2+ In various embodiments, the hydrophilic peptide salt is CNP, as described herein, and the hydrophobic counterion is pamoic acid. The polyvalent cation is Zn 2+ or Ca 2+ That is the case.

[0113] In various embodiments, the peptide salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate), CNP-Ca+2 (Deoxycholate), CNP-Ca +2 (Decanoate) CNP-Ca +2 (Docusate), CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate), CNP-Zn +2 (Deoxycholate), CNP-Zn +2 (Decanoate) and CNP-Zn +2 Selected from the group consisting of (docusate). In various embodiments, the peptide salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate), CNP-Ca +2 (Docusate), CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate) and CNP-Zn +2 Selected from the group consisting of (docusate). In various embodiments, the peptide salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate) and CNP-Ca +2 Selected from the group consisting of (docusate). In various embodiments, the peptide salt is CNP-Ca +2 (Deoxycholate), CNP-Ca +2 (Decanoate) and CNP-Ca +2 Selected from the group consisting of (docusate). In various embodiments, the peptide salt is CNP-Ca +2 (Oleate) and CNP-Ca +2 The peptide salt is selected from the group consisting of (pamoate). In various embodiments, the peptide salt is CNP-Ca +2 (Oleate). In various embodiments, the peptide salt is CNP-Ca +2 (Pamoate). In various embodiments, the peptide salt is CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate), CNP-Zn +2 (Deoxycholate), CNP-Zn +2 (Decanoate) and CNP-Zn +2selected from the group consisting of (docetate). In various embodiments, the peptide salt is CNP-Zn +2 (oleate), CNP-Zn +2 (pamoate) and CNP-Zn +2 (docetate). In various embodiments, the peptide salt is CNP-Zn +2 (deoxycholate), CNP-Zn +2 (decanoate) and CNP-Zn +2 (docetate). In various embodiments, the peptide salt is CNP-Zn +2 (oleate) and CNP-Zn +2 (pamoate). In various embodiments, the peptide salt is CNP-Zn +2 (oleate). In various embodiments, the peptide salt is CNP-Zn +2 (pamoate).

[0114] In various embodiments, the peptide salt is PG-CNP37-Ca +2 (oleate), PG-CNP37-Ca +2 (pamoate), PG-CNP37-Ca +2 (deoxycholate), PG-CNP37-Ca +2 (decanoate), PG-CNP37-Ca +2 (docetate), PG-CNP37-Zn +2 (oleate), PG-CNP37-Zn +2 (pamoate), PG-CNP37-Zn +2 (deoxycholate), PG-CNP37-Zn +2 (decanoate), and PG-CNP37-Zn +2 (docetate). In various embodiments, the peptide salt is PG-CNP37-Ca +2 (oleate), PG-CNP37-Ca +2 (pamoate), PG-CNP37-Ca +2 (docetate), PG-CNP37-Zn +2(Oleate), PG-CNP37-Zn +2 (Pamoate) and CNP-Zn +2 The peptide salt is selected from the group consisting of (docusate). In various embodiments, the peptide salt is PG-CNP37-Ca +2 (Oleate), PG-CNP37-Ca +2 (Pamoate) and PG-CNP37-Ca +2 Selected from the group consisting of (docusate). In various embodiments, the peptide salt is CNP-Ca +2 (Deoxycholate), PG-CNP37-Ca +2 (Decanoate) and PG-CNP37-Ca +2 The peptide salt is selected from the group consisting of (docusate). In various embodiments, the peptide salt is PG-CNP37-Ca +2 (Oleate) and PG-CNP37-Ca +2 The peptide salt is selected from the group consisting of (pamoate). In various embodiments, the peptide salt is PG-CNP37-Ca +2 (Oleate). In various embodiments, the peptide salt is PG-CNP37-Ca +2 (Pamoate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Oleate), PG-CNP37-Zn +2 (Pamoate), PG-CNP37-Zn +2 (Deoxycholate), PG-CNP37-Zn +2 (Decanoate), and PG-CNP37-Zn +2 The peptide salt is selected from the group consisting of (docusate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Oleate), PG-CNP37-Zn +2 (Pamoate) and PG-CNP37-Zn +2 The peptide salt is selected from the group consisting of (docusate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Deoxycholate), PG-CNP37-Zn +2 (Decanoate), and PG-CNP37-Zn +2The peptide salt is selected from the group consisting of (docusate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Oleate) and PG-CNP37-Zn +2 The peptide salt is selected from the group consisting of (pamoate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Oleate). In various embodiments, the peptide salt is PG-CNP37-Zn +2 (Pamoeto)

[0115] Manufacturing method This specification also seeks to provide a method for preparing compositions containing hydrophobic peptide salts as described herein.

[0116] In various embodiments, the Disclosure provides a method for preparing a composition comprising a salt of an electrostatically charged peptide, comprising: a) contacting an electrostatically charged peptide in aqueous solution with a hydrophobic counterion in solution; and b) mixing the electrostatically charged peptide solution with a hydrophobic counterion solution in a manner sufficient for the peptide and counterion to form a complex, wherein the formation of the peptide-counterion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle form comprising the peptide salt. In various embodiments, if the peptide counterion salt further comprises a polyvalent cation, the method comprises, prior to step (b), contacting an electrostatically charged peptide in solution with a polyvalent cation in aqueous solution to form a peptide-cation complex. The peptide-cation complex is then contacted with a hydrophobic counterion to form a peptide-cation-counterion complex.

[0117] In various embodiments, the Disclosure provides a method for producing a composition containing a salt of an electrostatically charged peptide, comprising (a) contacting an electrostatically charged peptide in solution with a polyvalent cation in an aqueous solution to form a peptide-cation complex; (b) contacting the peptide-cation complex in an aqueous solution with a hydrophobic counterion in solution; and (c) mixing the peptide-cation complex solution with a hydrophobic counterion solution in a manner sufficient for the peptide-cation and counterion to form a complex, wherein the formation of the peptide-cation counterion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, fine particle, amorphous nanoparticle, amorphous fine particle, crystalline nanoparticle or crystalline fine particle form containing the peptide salt.

[0118] In various embodiments, mixing is carried out by dropwise adding a hydrophobic counterion solution to a peptide solution. The solutions are mixed by vortexing after the addition of each droplet of hydrophobic counterion solution, or by other mixing means known in the art.

[0119] In various embodiments, the method further comprises step (c) or (d) of washing the peptide salt in a buffer or water. In various embodiments, the washing is carried out in an aqueous solution, for example, a buffer or water.

[0120] In various embodiments, the method further comprises step (d) or (e) of obtaining the peptide salt by centrifugation to form a peptide salt pellet. In various embodiments, if the salt is in gel form, the salt is obtained by centrifugation or by decanting the liquid phase and subsequently freeze-drying or by other drying methods.

[0121] In various embodiments, the method further comprises step (e) or (f) of removing water from the peptide salt pellet. It is thought that water or another aqueous solution can be removed from the pellet by freeze-drying or drying using techniques known in the art.

[0122] In various embodiments, this method further comprises resuspending the pellets in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline solution, or 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, unsaturated, or a combination thereof.

[0123] Fatty acids may be present in the oil itself or in the triglycerides. In various embodiments, the fatty acids are short-chain, medium-chain, or long-chain fatty acids. In various embodiments, the fatty acids in the triglycerides may be saturated or unsaturated and may be medium-chain or long-chain fatty acids. In various embodiments, the fatty acids are C-6 to C-20 fatty acids. In various embodiments, the fatty acids are C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 fatty acids. In various embodiments, the fatty acids are hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid.

[0124] In various embodiments, the method intends to use at least 1 molar equivalent of a hydrophobic counterion, such as a hydrophobic anion, for 1) the total number of charged amino acids when cations are present, or 2) the total number of positive charges when cations are absent. Therefore, in various embodiments, the method intends to use at least 1 equivalent of a hydrophobic counterion for the total number of positive charges of a hydrophilic peptide when cations are absent, or for the total number of positive charges of a complex containing a cation when hydrophilic peptides and polyvalent cations are present. This ratio is referred to herein as the peptide:hydrophobic counterion ratio. In various embodiments, the peptide:hydrophobic counterion ratio is at least 1 molar equivalent of a hydrophobic counterion for the total number of positively charged amino acids in the peptide (when cations are absent) or the complex containing the peptide and cations (when cations are present). In various embodiments, the peptide:hydrophobic counterion ratio is 1:1 to 1:20, or 1:1 to 1:50. The peptide:counterion ratio can be 1:2-1:15, 1:2-1:10, 1:2-1:8, 1:3-1:10, or 1:4-1:10. In various embodiments, the peptide:hydrophobic counterion 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 counterion ratio is 1:1. In various embodiments, the peptide:hydrophobic counterion ratio is 1:2. In various embodiments, the peptide:hydrophobic counterion ratio is 1:3. In various embodiments, the peptide:hydrophobic counterion ratio is 1:4. In various embodiments, the peptide:hydrophobic counterion ratio is 1:5. In various embodiments, the peptide:hydrophobic counterion ratio is 1:6. In various embodiments, the peptide:hydrophobic counterion ratio is 1:7. In various embodiments, the peptide:hydrophobic counterion ratio is 1:8. In various embodiments, the peptide:hydrophobic counterion ratio is 1:9. In various embodiments, the peptide:hydrophobic counterion ratio is 1:10.In various embodiments, the peptide:hydrophobic counterion ratio is at least 2 molar equivalents of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide (when cations are absent) or the peptide and cation-containing complex (when cations are present). In various embodiments, the peptide:hydrophobic counterion ratio is at least 3 molar equivalents of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide (when cations are absent) or the peptide and cation-containing complex (when cations are present). In various embodiments, the peptide:hydrophobic counterion ratio is at least 4 molar equivalents of hydrophobic counterions relative to the total number of positively charged amino acids in the peptide (when cations are absent) or the peptide and cation-containing complex (when cations are present). In some embodiments, the peptide:hydrophobic counterion ratio is less than 1 molar equivalent of hydrophobic counterions relative to the total number of positive charges in the peptide (when cations are absent) or the peptide and cation-containing complex (when cations are present). In various embodiments, the peptide salt has 1 molar equivalent of hydrophobic counterions relative to the positive charge of the peptide (when cations are absent) or the peptide and cation-containing complex (when cations are present). In various embodiments, the peptide salt has 2 molar equivalents fewer hydrophobic counterions relative to the positive charge of the peptide (when no cation is present) or the complex containing the peptide and cation (when a cation is present). In various embodiments, the peptide salt has 3 molar equivalents fewer hydrophobic counterions relative to the positive charge of the peptide (when no cation is present) or the complex containing the peptide and cation (when a cation is present). In various embodiments, this method aims to introduce, 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 molar equivalents of hydrophobic counterions relative to the total positive charge of the peptide (when a cation is present) or the complex containing the peptide and cation (when a cation is present).

[0125] In various embodiments, this method intends to contain at least 1 molar equivalent of cations 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 can 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 intends to include less than 1 molar equivalent of cations relative to the total number of negatively charged amino acids in the peptide. In various embodiments, the method intends to introduce, 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 molar equivalents of cations relative to the total number of negative charges on the peptide.

[0126] C-type natriuretic peptide C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168:863-870 (1990) (GenBank Accession No. NP_077720, for the CNP precursor protein, NPPC) (J. Hypertens., 10:907-912 (1992)) has a 17-amino acid loop structure (Levin et al.) CNP is a small, single-chain peptide belonging to a family of peptides (ANP, BNP, CNP) that possesses properties and plays an important role in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic guanosine monophosphate (cGMP) (J. Hypertens., 10: 1111-1114 (1992)). CNP is more widely expressed in the central nervous system, reproductive tract, bone, and vascular endothelium (Hypertension, 49: 419-426 (2007)).

[0127] In humans, CNP is initially produced from the natriuretic peptide precursor C (NPPC) gene as a single-stranded 126-amino acid prepropeptide (Biochem. Biophys. Res. Commun., 168:863-870 (1990)). Removal of the signal peptide generates pro-CNP, and further cleavage by the endoprotease furin produces an active 53-amino acid peptide (CNP-53). This is then secreted and cleaved again by an unknown enzyme to produce a mature 22-amino acid peptide (CNP-22) (Wu, J. Biol. Chem. 278:25847-852 (2003)). CNP-53 and CNP-22 have different distributions; CNP-53 is dominant in tissues, while CNP-22 is mainly found in plasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct. Res., 26:269-297 (2006)). Both CNP-53 and CNP-22 bind to NPR-B in the same way.

[0128] In various embodiments, the CNPs of this disclosure include shortened CNPs having wild-type amino acid sequences derived from hCNP-53, ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53). Such shortened CNP peptides include: DLRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-53)(Sequence ID 56) LRVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-52) (Sequence ID 15), RVDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (CNP-51) (Sequence ID 16) VDTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-50)(Sequence ID 17:), DTKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-49)(Sequence ID 18) TKSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-48)(Sequence ID 19), KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47)(Sequence ID 20), SRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-46)(Sequence ID 21), RAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-45)(Sequence ID 22), AAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-44)(Sequence ID 23), AWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-43)(SEQ ID NO: 24), WARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-42)(Sequence ID 25), ARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-41)(Sequence ID 26), RLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-40)(SEQ ID NO: 27), LLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-39)(Sequence ID 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)(Sequence ID 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)(Sequence ID 37), ANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-26)(Sequence ID 38), NKKGLSKGCFGLKLDRIGSMSGLGC(CNP-25)(Sequence ID 39), KKGLSKGCFGLKLDRIGSMSGLGC(CNP-24)(Sequence ID 40), KGLSKGCFGLKLDRIGSMSGLGC(CNP-23)(SEQ ID NO: 41), GLSKGCFGLKLDRIGSMSGLGC(CNP-22)(Sequence ID 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)(Sequence ID 67).

[0129] In various embodiments, the CNP variant peptide is a modified CNP-37 or CNP-38 peptide, optionally having mutations / substitutions at the furin cleavage site (underlined) and / or containing glycine or proline-glycine at the N-terminus. Exemplary CNP-37 variants include, but are not limited to, the following: 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:Sequence ID 51) [ka] (Sequence ID 52) [ka] (Sequence ID 53) [ka] (Sequence ID 54), and [ka] (Sequence ID 55).

[0130] In various embodiments, CNP is PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 5), PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 1), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6), PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5), and The group is selected from PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 7).

[0131] In various embodiments, the CNP variant peptide further comprises 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 the amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus.

[0132] In additional embodiments, any of the CNPs and CNP variants described herein having an asparagine (Asn / N) residue and / or a glutamine (Gln / Q) residue may have a wild-type sequence or a non-natural amino acid sequence, or any Asn residue and / or any Gln residue may be independently substituted with any other natural or non-natural amino acid, including a conserved substitution such as Asn to Gln. Such substitutions are partially designed to minimize or avoid potential deamidation of asparagine and / or glutamine.

[0133] Additional CNP peptides and variants are disclosed in U.S. Patent No. 8,198,242, which is incorporated herein by reference.

[0134] In various embodiments, the electrostatically charged 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, CNP is a CNP variant as described herein.

[0135] In various embodiments, CNP is 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), The group consists of and their pharmaceutically acceptable salts. In various embodiments, CNP is CNP acetate.

[0136] In various embodiments, when the hydrophobic peptide salt is a hydrophobic CNP salt, the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. In various embodiments, when a polyvalent cation is present, the cation comprises zinc, magnesium, or calcium. In various embodiments, when a cation is present, the cation comprises zinc. In various embodiments, the cation comprises calcium. In various embodiments, when a polyvalent cation is present, the cation comprises Zn 2+ Mg 2+ , or Ca 2+ In various embodiments, when cations are present, the cations are Zn 2+ In various embodiments, if a cation is present, the cation is Ca 2+ That is the case.

[0137] In various embodiments, the disclosure provides hydrophobic salts of C-type natriuretic peptides, including CNP peptides complexed with hydrophobic counterions. In various embodiments, the salts further comprise polyvalent cations, optionally metal cations. In various embodiments, the salts are purified CNP salts.

[0138] Methods for purifying hydrophobic salts are known in the art and are intended herein. In some cases, the hydrophobic salts have a purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or higher.

[0139] How to use Achondroplasia is the result of an autosomal dominant mutation in the fibroblast growth factor receptor 3 (FGFR-3) gene, causing abnormalities in chondrogenesis. FGFR-3 normally has a negative regulatory effect on chondrocyte growth, and therefore bone growth. In achondroplasia, the mutant FGFR-3 is constitutively active, leading to significant bone shortening. In humans, activation of FGFR-3 mutations is the primary cause of hereditary dwarfism. Mice with activated FGFR-3 serve as a model for achondroplasia, the most common form of skeletal dysplasia, and overexpression of CNP rescues these animals from dwarfism. Therefore, CNP and its functional variants are potential therapeutic agents for the treatment of various skeletal dysplasias.

[0140] By stimulating chondrocyte matrix production, proliferation, and differentiation, and increasing long bone growth, the CNP salts of this disclosure are useful for treating mammals, including humans, that suffer from bone-related disorders such as skeletal dysplasia. Non-limited examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, hypochondrodysplasia, short stature, dwarfism, osteochondrodysplasia, fatal osteodysplasia, congenital osteogenesis imperfecta, achondroplasia, congenital chondrodysplasia, congenital chondrodysplasia, flexor limb dysplasia, congenital fatal hypophosphatasia, perinatal fatal type of osteogenesis imperfecta, short-rib polydactyly syndrome, hypochondrodysplasia, rhizome-type congenital chondrodysplasia, Janssen-type metaphyseal dysplasia, congenital vertebral metaphyseal dysplasia, atherosclerotic dysplasia, and dystoma. This includes Lofe's dysplasia, congenital short femur, Langer's metalegurodysplasia, Niebergeld's metalegurodysplasia, Robinnow syndrome, Reinhardt syndrome, acromesomelic dysplasia, peripheral dysplasia, Nieist dysplasia, fibrochondroplasia, Roberts syndrome, acromesomelic dysplasia, micromeria, Morquio syndrome, Nieist syndrome, complex organic dysplasia, vertebral epiphyseal metaphysical dysplasia, NPR2 mutations, SHOX mutations (Turner syndrome / Noonan syndrome), and PTPN11 mutations (Nonan syndrome).

[0141] By stimulating chondrocyte matrix production, proliferation, and differentiation, and increasing long bone growth, the CNP variants of this disclosure are useful for treating mammals, including humans, that suffer from bone-related disorders such as skeletal dysplasia. Non-limited examples of CNP-responsive bone-related disorders and skeletal dysplasia include achondroplasia, hypochondrodysplasia, dwarfism, osteochondrodysplasia, lethal dysplasia, congenital osteogenesis imperfecta, achondroplasia, congenital chondrodysplasia, congenital chondrodysplasia, flexor limb dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndrome, hypochondrodysplasia, rhizomelic type congenital chondrodysplasia, and Janssen type metaphysical dysplasia. This includes adult, congenital vertebral metaphyseal dysplasia, osteogenesis imperfecta, dystrophy, congenital short femur, Langer midleg dysplasia, Niebergeld metaphyseal dysplasia, Robinnow syndrome, Reinhardt syndrome, acrodystosis, peripheral dysplasia, Nieist dysplasia, fibrochondroplasia, Roberts syndrome, acromesomelic dysplasia, micromeria, Morquio syndrome, Nieist syndrome, metabolic dysplasia, and vertebral metaphyseal dysplasia. The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasias referred to herein include disorders associated with NPR2 mutations, SHOX mutations (Turner syndrome / Reliweil), PTPN11 mutations (Noonan syndrome), and IGF1R mutations.

[0142] The short stature, growth plate disorders, bone-related disorders, or skeletal dysplasiasm referred to herein include disorders associated with NPR2 mutations, SHOX mutations (Turner syndrome / Reliweil), and PTPN11 mutations (Noonan syndrome).

[0143] Additional short stature and growth plate disorders targeted by this method include disorders associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian Hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0144] Additional short stature and growth plate disorders targeted by this method include disorders associated with mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0145] Furthermore, CNP salts are useful as growth hormone adjuvants or substitutes for treating idiopathic short stature and other skeletal dysplasias described herein.

[0146] Growth plate disorders include disorders resulting in short stature or abnormal bone growth and may be the result of genetic mutations in genes involved in bone growth, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders include disorders resulting in short stature or abnormal bone growth and may be the result of genetic mutations in genes involved in bone growth, including collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (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 RAS disease. In various embodiments, subjects with growth plate disorders are heterozygous for mutations in growth plate genes. In various embodiments, the mutations are loss-of-function mutations. In various embodiments, the mutation is a gain-of-function mutation. Growth plate disorders include, but are not limited to, familial short stature, dominant familial short stature (also known as dominant inherited short stature), or idiopathic short stature. See, for example, Plachy et al., J Clin Endocrinol Metab 104:4273-4281, 2019.

[0147] Mutations in ACAN can cause familial osteochondritis dissecans and short stature, and ultimately osteoarthritis characterized by areas of bone damage (or lesions) caused by detachment of cartilage and sometimes bone from the ends of the joint bones. A disordered cartilage network in growing bones is suggested to impair their growth and lead to short stature. Mutations associated with ACAN and short stature include Val2303Met. See Stattin et al., Am J Hum Genet 86(2):126-37, 2010. Patients with ACAN mutations resulting in short stature are thought to benefit from treatment with CNP, as administration may increase the height of these patients due to known interactions between CNP and FGFR3.

[0148] The natriuretic peptide system, including the receptor NPR2, has been shown to be involved in regulating endochondral bone growth (Vasquez et al., Horm Res Pediat 82:222-229, 2014). Studies have shown that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause distal intermediate limb dysplasia type Maroto (AMDM), a skeletal dysplasia characterized by very short stature (Vasquez et al., 2014, see above). While some reports suggest heterozygous loss-of-function (dominant-negative, etc.) NPR2 mutations as a cause of short stature, gain-of-function heterozygous NPR2 mutations are known to cause tall stature (Vasquez et al., 2014, see above). Considering the interaction of CNP with NPR2 to stimulate cGMP production, increasing cGMP levels would be desirable in these conditions and would have therapeutic benefits in managing complications from these diseases and conditions.

[0149] Heterozygous mutations in NPR2 are thought to result in idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are described in Amano et al., J Clin Endocrinol Metab 99:E713-718, 2014, Hisado-Oliva et al., J Clin Endocrinol Metab 100:E1133-1142, 2015, and Vasques et al., J Clin Endocrinol Metab 98:E1636-1644, 2013, which are incorporated herein by reference. Subjects with short stature treated with the CNP variants described herein have a height SDS less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one parent has a height SDS less than -1.0, -1.5, -2.0, or -2.5, and optionally, the height of the second parent is within the normal range. In various embodiments, the CNP variants are useful for treating subjects with short stature having a height SDS between -2.0 and -3.0. In various embodiments, the CNP variants are useful for treating subjects with short stature having a height SDS between -2.0 and -2.5. However, since denovo mutations of NPR2 can result in short stature as defined by a height SDS less than -1.5, -2.0, -2.5, or -3.0, treatment of heterozygous carriers of harmful mutations of NPR2 in whom neither parent has short stature is also conceivable. Furthermore, the aim is to treat individuals heterozygous for harmful mutations in other growth plate genes with CNP in order to improve height and / or enhance bone growth.

[0150] Exemplary NPR2 mutations in patients who may be treated with CNP variants include: [Table 2] [Table 3]

[0151] The role of NPPC in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). NPPC knockout mice exhibited severe dwarfism with disproportionate morphological features, including limb shortening and endochondral ossification (Hisado-Oliva et al., 2018, see above). Studies of the entire human genome have shown a relationship between NPPC and height (Hisado-Oliva et al., 2018, see above). CNP haploinsufficiency is thought to be a cause of short stature in humans, and recent studies have identified heterozygous mutations in families with short stature and hand deformities (Hisado-Oliva et al., 2018, see above). These studies observed a significant decrease in cGMP production when measured in the heterozygous state (Hisado-Oliva et al., 2018, see above). NPPC mutations include the 355G>T missense mutation, which causes changes in Gly119Cys, and the 349C>G missense mutation, which causes changes in Arg117Gly. CNP variants that rescue CGMP production may offer therapeutic benefits in managing disability in patients with heterozygous loss-of-function NPPC mutations.

[0152] Leri-Weill chondrodysplasia (LWD) is a rare genetic disorder characterized by shortening of the forearms and lower limbs, abnormal wrist deformity (madelung deformity of the wrist), and associated short stature. LWD is caused by heterozygous mutations in the short stature homeobox-containing (SHOX) gene or its regulatory elements located in pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See Rare Disease Database and Carmona et al., Hum Mol Genet 20:1547-1559, 2011). Langer-type midleg dysplasia occurs when there are two SHOX mutations and can result from mutations on each chromosome, either homozygous or compound heterozygous. A subset of SHOX mutations causes idiopathic short stature. Turner syndrome is caused by a deletion on the X chromosome that may contain the SHOX gene. SHOX has been shown to be involved in the regulation of FGFR3 transcription and contribute to the control of bone growth (Marchini et al., Endocr Rev.37:417-448, 2016). SHOX deficiency leads to increased FGFR3 signaling, and there is some evidence supporting that SHOX also directly interacts with CNP / NPR2 (Marchini, above). Given the association between SHOX, FGFR3, and bone growth, subjects with homozygous or heterozygous SHOX mutations are likely to benefit from treatment with the CNP variants described herein.

[0153] RAS disease is a group of rare genetic conditions caused by mutations in the Ras / mitogen-activated protein kinase (MAPK) pathway. RAS disease is a group of disorders 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 characteristic feature of certain RAS diseases. For example, CNP signaling inhibits RAF and reduces the activation of MEK and ERK.

[0154] This specification focuses on the treatment of RAS diseases. RAS diseases associated with short stature include Noonan syndrome, Costello syndrome, cardiac facial cutaneous syndrome, neurofibromatosis type 1, and Leopard syndrome. Hereditary gingival fibromatosis type 1 is also a RAS disease that is the focus of this specification. Patients with RAS diseases (including Noonan syndrome, Costello syndrome, cardiac facial cutaneous syndrome, neurofibromatosis type 1, Leopard syndrome, and hereditary gingival fibromatosis type 1) include patients with heterozygous variants in 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. Endocr. Rev. 2018, 39(5):676-700).

[0155] 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 dermatoskeletal (FCS) syndrome, is caused by the activation of mutations in the H-Ras gene. Hereditary gingival fibromatosis type 1 (HGF) is caused by a dominant mutation in the SOS1 gene (Son of Sevenless homolog 1), which encodes a guanine nucleotide exchange factor (SOS) that acts on the Ras subfamily of low molecular weight GTPases. Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin 1 gene, which encodes a negative regulator of the Ras / MAPK signaling pathway. Noonan syndrome (NS) is caused by a mutation in one of several genes, including PTPN11 which encodes SHP2, SOS1, K-Ras, and Raf-1.

[0156] CNP has been demonstrated as an effective treatment in RAS disease models. Ono et al. generated mice lacking Nf1 in type II collagen-producing cells (Ono et al., Hum.Mol.Genet. 2013, 22(15):3048-62). These mice showed constitutive ERK1 / 2 activation and reduced chondrocyte proliferation and maturation. Daily injection of CNP into these mice reduced ERK phosphorylation and corrected short stature. A mouse model of cardiac facial skin syndrome using the Braf mutation (p.Q241R) (Inoue et al. Hum.Mol.Genet. 2019, 28(1):74-83) showed reduced body length and growth plate width compared to the wild type, with smaller proliferation and hypertrophic zones, and CNP administration increased the body length of these animals.

[0157] Mutations in multiple genes can cause Noonan syndrome, characterized by short stature, heart defects, bleeding problems, and skeletal malformations. Mutations in the PTPN11 gene cause about half of all cases of Noonan syndrome. Mutations in the SOS1 gene cause another 10–15%, and the RAF1 and RIT1 genes each account for about 5% of cases. Mutations in other genes each explain a small number of cases. The cause of Noonan syndrome in 15–20 percent of people with this disorder is unknown.

[0158] The PTPN11, SOS1, RAF1, and RIT1 genes all encode proteins crucial in the RAS / MAPK cell signaling pathway, which is essential for cell division and growth (proliferation), differentiation, and migration. Many mutations in the genes associated with Noonan syndrome activate the resulting proteins, and this prolonged activation alters normal RAS / MAPK signaling, disrupting the regulation of cell growth and division and leading to the characteristics of Noonan syndrome. See Chen et al., Proc Natl Acad Sci US A.111(31):11473-8,2014, Romano et al., Pediatrics.126(4):746-59,2010, and Milosavljevic et al., Am J Med Genet 170(7):1874-80,2016. Subjects with mutations that activate the MAPK pathway are thought to benefit from treatment with CNP variants, such as those described herein, to improve bone growth and short stature. Subjects with mutations that activate the MAPK pathway are also expected to benefit from treatment with the CNP variants described herein to improve other comorbidities associated with the hyperactive MAPK pathway in other cells throughout the body on which the NPR2 receptor is expressed on its surface.

[0159] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, cause disorders characterized by short stature, such as Noonan syndrome (Musente et al., Eur J Hum Genet 11:201-206 (2003). Musente (above) has identified numerous mutations in the PTPN11 gene that lead to short stature. Acquisition of functional mutations leads to hyperactive signaling via SHP2, inhibiting growth hormone-induced IGF-1 release and thereby contributing to reduced bone growth (Rocca Serra-Nedelec, PNAS 109:4257-4262, 2012). Subjects with homozygous or heterozygous PTPN11 mutations are expected to benefit from treatment with CNP variants, such as those described herein, to improve bone growth and short stature.

[0160] Mutations in the Indian hedgehog (IHH) gene, which is associated with the regulation of endochondral ossification, are also linked to short stature syndrome (Vasques et al., J Clin Endocrinol Metab. 103:604-614, 2018). Many of the IHH mutations identified segregate in short stature in a dominant inheritance pattern. Given the association between IHH and bone growth and ossification, subjects with homozygous or heterozygous IHH mutations are likely to benefit from treatment with the CNP variants described herein.

[0161] Mutations in FGFR3, including N540K and K650N, cause short stature and chondrodysplasia.

[0162] The 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. Heterozygous mutations in IGF1R have been identified in children with premature gestational age (SGA) and individuals with familial short stature (Kawashima et al., Endocrine J. 59: 179-185, 2012). Mutations in IGF1R associated with short stature include R108Q / K115N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, see above).

[0163] Height is a highly heritable trait and can be influenced by the combined effects of hundreds or thousands of genes (Wood et al., Nature Genetics, 46:1173-1189, 2014). Short stature in individuals may not be primarily caused by a single gene, but rather by the combined effects of these genes. Such individuals with short stature, defined by height SDS less than -1.0, -1.5, -2.0, -2.5, or -3.0, are thought to be beneficially treatable with CNP variants, given the ability of CNP to increase length in normal animals, for example, by improving bone growth and bone length.

[0164] In various embodiments, the CNP variant is useful for treating subjects with height SDS less than -1.0, -1.5, -2.0, -2.5, or -3.0, and subjects with height SDS less than -1.0, -1.5, -2.0, or -2.5 but with at least one parent, optionally, where the height of the second parent is within the normal range. In various embodiments, the CNP variant is useful for treating subjects with height SDS between -2.0 and -3.0. In various embodiments, the CNP variant is useful for treating subjects with height SDS between -2.0 and -2.5. In various embodiments, short stature is associated with mutations in one or more genes associated with short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R), or a combination thereof.

[0165] In various embodiments, short stature is associated with mutations in one or more genes related to RAS disease.

[0166] In various embodiments, short stature is the result of mutations in multiple genes, such as those determined by a polygenic risk score (PRS). In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in FGFR3 and a low PRS. In various embodiments, the subject has a mutation in NPR2 and a low PRS. In various embodiments, the subject has a mutation in IGF1R and a low PRS. In various embodiments, the subject has a mutation in NPPC and a low PRS. In various embodiments, the subject has a mutation in SHOX and a low PRS. In various embodiments, the subject has one or more mutations in one or more of FGFR3, IGF1R, NPPC, NPR2, and SHOX and a low PRS. In various embodiments, the PRS is 1 or 2. In various embodiments, the PRS is 1. In various embodiments, the PRS is 2.

[0167] Furthermore, CNP salts are useful in treating other bone-related conditions and disorders 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 oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)).

[0168] The CNP salts of this disclosure can also be used to treat osteoarthritis. Osteoarthritis is a degenerative disease of articular cartilage that frequently occurs in older adults. Osteoarthritis involves cartilage destruction and proliferative changes in bone and cartilage due to degeneration of the components of the joint, which lead to secondary arthritis (e.g., synovitis). In osteoarthritis, the extracellular matrix proteins, which are the functional entities of cartilage, decrease, and the number of chondrocytes decreases (Arth. Rheum. 46(8):1986-1996(2002)). By promoting matrix production, growth, and differentiation of chondrocytes, the CNP composition counteracts the undesirable effects of FGF-2 and increases matrix synthesis in subjects suffering from arthritis, including osteoarthritis, thereby proving useful in treating arthritis, including osteoarthritis.

[0169] In certain embodiments, CNP salts and compositions and formulations comprising the same as those of the present disclosure are useful in improving one or more symptoms or physiological outcomes of skeletal dysplasia, the improvements of which may be increased absolute growth, increased growth rate, increased qualitative computed tomography (QCT) bone mineral density, improved growth plate morphology, increased long bone growth, improved spinal morphology, improved elbow joint range of motion, and / or reduced sleep apnea. In this regard, it should be noted that the terms “improved,” “improved,” “increased,” “decreased,” and their grammatical equivalents are all relative terms used in relation to the symptoms or physiological outcomes of a medical condition, and refer to the state of the symptoms or physiological outcomes of the disease after treatment with the CNP salts (or compositions or formulations) of the present invention compared to the same symptoms or physiological outcomes of the disease before treatment with the CNP salts (or compositions or formulations) of the present invention (i.e., compared to “baseline”). As described above, the “baseline” state can be determined either by measuring the subject’s condition before treatment (which can then be compared to the same subject’s condition after treatment), or by measuring that condition in a group of subjects suffering from the same distress who share the same or similar characteristics (e.g., age, sex, and / or disease state or progression).

[0170] In yet another embodiment, the Disclosure provides salts of CNP variants that stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP level produced at the same concentration of wtCNP22 (e.g., 1 μM), either in vitro or in vivo. In yet another embodiment, hydrophobic salts comprising CNP or CNP variants of the Disclosure stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% of the cGMP level produced at the same concentration of wtCNP22 (e.g., 1 μM).

[0171] Any of the CNP variants described herein is considered useful in this method.

[0172] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37(SEQ ID NO: 1)). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is at the N-terminus of the peptide. In various embodiments, the acetyl group is on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker. In various embodiments, the peptide comprises a salt of an electrostatically charged peptide, the salt comprising an electrostatically charged peptide complexed with a hydrophobic counterion.

[0173] The effectiveness of the treatment is measured by various parameters. In various embodiments, effectiveness is assessed as the change in annual growth rate from the baseline period to the intervention period. Effectiveness is also assessed as the change in height SDS from baseline to the end of treatment, measured using CDC growth curves, where growth rate SDS is based on childhood bone mineral density studies (Kelly et al., J. Clin. Endocrinol. Metab. 2014, 99(6):2104-2112).

[0174] The Quality of Life (QoLISSY) scale for young people with short stature is assessed according to instructions (Quality of Life for Young People with Short Stature - QoLISSY Questionnaire User's Manual. Lengerich: Pabst Science Publishers, 2013).

[0175] Pharmaceutical composition This disclosure provides pharmaceutical compositions comprising release-modulated compositions comprising the peptide salts described herein, as well as one or more pharmaceutically acceptable excipients, carriers, and / or diluents. In certain embodiments, the composition further comprises one or more other biologically active agents (e.g., protease inhibitors, receptor tyrosine kinases, and / or clearance receptor NPR-C).

[0176] This disclosure provides release-modulating compositions comprising hydrophobic peptide salts as described herein. In various embodiments, the release-modulating composition is an extended release composition. In various embodiments, the extended release composition comprises a hydrophobic CNP salt. In various embodiments, the hydrophobic counterion in the CNP salt is oleate, deoxycholate, decanoate, pamoate, 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 Zn 2+ or Ca 2+ That is the case.

[0177] The precipitated peptide complex may exhibit extended release characteristics under the pH conditions at which precipitation occurs. The precipitated peptide complex can also be used for further treatment into a matrix that provides additional barriers for sustained release, such as microspheres and hydrogels that decompose slowly. The hydrophobic CNP salt is a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle or crystalline microparticle, and is considered to be resuspended in an aqueous solution or oil. In various embodiments, the aqueous solution is water, saline, or a buffer. In various embodiments, the particles are from 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.

[0178] In various embodiments, the oil contains triglycerides or fatty acids that can be saturated or unsaturated. The triglycerides and fatty acids described herein are also contemplated to be used with the hydrophobic CNP salt composition. In various embodiments, the fatty acid is hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid. In various embodiments, the fatty acid is hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid or docusate.

[0179] In various embodiments, for the extended release composition, at pH 7 - 7.6, (i) less than about 20% of the peptide is released by day 1, and (ii) about 90% of the peptide is released weekly, or about 90% of the peptide is released bi - weekly, or about 90% of the peptide is released monthly.

[0180] In various embodiments, at pH 7 to 7.6, less than about 20% of the peptide is released by the end of day 1. Further, (i) at pH 7.0 to 7.6, less than about 30%, or about 40%, or about 50% of the peptide is released by the end of day 1, and (ii) it is contemplated that at pH 7 to 7.6, about 90% of the peptide is released weekly, or about 90% of the peptide is released bi-weekly, or about 90% of the peptide is released monthly. Further, (i) at pH 7.0 to 7.6, less than about 30%, or about 40%, or about 50%, or about 60% of the peptide is released by the end of day 1, and (ii) at pH 7.0 to 7.6, about 70%, about 80%, or about 90% of the peptide is released weekly, or about 70%, about 80%, or about 90% of the peptide is released bi-weekly, or about 70%, about 80%, or about 90% of the peptide is released every three weeks, or about 70%, about 80%, or about 90% of the peptide is released monthly. In various embodiments, at pH 7 to 7.6, about 90% of the peptide is released weekly. In various embodiments, at pH 7 to 7.6, about 90% of the peptide is released bi-weekly. In various embodiments, at pH 7 to 7.6, about 90% of the peptide is released monthly. Further, it is contemplated that the release can be at a pH of 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.

[0181] In various embodiments, (i) at pH 7.0–7.6, approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the peptides are released by day 1, and (ii) at pH 7–7.6, approximately 90% of the peptides are released weekly, or approximately 90% of the peptides are released every other week, or approximately 90% of the peptides are released monthly. Furthermore, (i) at pH 7.0–7.6, approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of the peptides are released by day 1. (ii) At pH 7-7.6, approximately 70%, 80%, or 90% of the peptides are released weekly, or approximately 70%, 80%, or 90% of the peptides are released every two weeks, or approximately 70%, 80%, or 90% of the peptides are released every three weeks, or approximately 70%, 80%, or 90% of the peptides are released monthly. Alternatively, ii) at pH 7-7.6, it is intended that approximately 70%, 75%, 80%, 85%, or 90% of the peptides be released weekly, or approximately 70%, 75%, 80%, 85%, or 90% of the peptides be released every two weeks, or approximately 70%, 75%, 80%, 85%, or 90% of the peptides be released every three weeks, or approximately 70%, 75%, 80%, 85%, or 90% of the peptides be released monthly.

[0182] In various embodiments, the extended-release composition comprises an excipient, diluent, or carrier. In various embodiments, the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0183] Non-limiting examples of excipients, carriers, and diluents include vehicles, liquids, buffers, isotonic agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, and auxiliaries. Compositions may include liquids (e.g., water, ethanol). Diluents for various buffer contents (e.g., Tris-HCl, phosphates, acetate buffers, citrate buffers), pH and ionic strength, detergents and solubilizers (e.g., polysorbate 20, polysorbate 80), antioxidants (e.g., methionine, ascorbic acid, sodium metabisulfite), preservatives (e.g., thimerosal, benzyl alcohol, m-cresol), and fillers (e.g., lactose, mannitol, sucrose). The use of excipients, diluents, and carriers in the formulation of pharmaceutical compositions is known in the art, for example, in Remington's Pharmaceutical Sciences, 18, incorporated herein by reference. th See Edition, pages 1435–1712, Mack Publishing Co. (Easton, Pennsylvania (1990)).

[0184] For example, carriers include, but are not limited to, diluents, vehicles 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, saline, water, and emulsions (e.g., oil / water emulsions). Carriers may be solvents or dispersion media, for example, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oils, and mixtures thereof.

[0185] In some embodiments, the composition is a liquid formulation. In certain embodiments, the formulation contains 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 CNP variant is at a concentration of 0.8 mg / mL to 2 mg / mL. In various embodiments, the CNP variant is at a concentration of 0.8 mg / mL. In various embodiments, the CNP variant is at a concentration of 2.0 mg / mL. In various embodiments, the CNP variant is reconstituted from lyophilized powder.

[0186] In further embodiments, the composition includes a buffer or buffering agent for maintaining the pH of the CNP-containing solution or suspension within a desired range. Non-limiting examples of buffers include phosphate-buffered saline, Tris-buffered saline, and Hanks-buffered saline. Buffering agents include, but are not limited to, sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffering agents can also be used. In certain embodiments, the buffering agent is acetic acid / acetate or citric acid / citrate. The amount of buffering agent suitable for the composition depends in part on the specific buffer used and the desired pH of the solution or suspension. In some embodiments, the buffering agent has a concentration of about 10 mM ± 5 mM. In certain 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 approximately 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.

[0187] In other embodiments, the composition includes an isotonic modifier to make the solution or suspension isotonic and more suitable for administration. Non-limiting examples of isotonic agents include NaCl, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol. In certain embodiments, the isotonic agent is NaCl. In certain 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.

[0188] In yet other embodiments, the composition includes a preservative. The preservative includes, but is not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is present in concentrations of about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%.

[0189] In yet another embodiment, the composition includes an anti-adsorption agent (for example, to reduce the adsorption of CNP salts to glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In a particular embodiment, the anti-adsorption agent is present in concentrations of about 0.001% to about 0.5%, or about 0.01% to about 0.5%, or about 0.1% to about 1%, or about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%.

[0190] In additional embodiments, the composition includes a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid, as well as salts thereof. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is present in a concentration of about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is present in a concentration of about 0.01% to about 0.5%, or about 0.01% to about 0.2%. In yet another embodiment, when the stabilizer is glycerin, the stabilizer is present in a concentration of about 5% to about 100% (neat).

[0191] In further embodiments, the composition includes antioxidants. Exemplary antioxidants include, but are not limited to, methionine and ascorbic acid. In certain embodiments, the molar ratio of 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.

[0192] Pharmaceutically acceptable salts, including but not limited to inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and amine salts (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine), can be used in the composition. A complete discussion of pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 18. th It is available in Edition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0193] 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 a composition can be adjusted to suit the desired mode of administration. For oral administration, a composition may take the form of, for example, tablets or capsules (including softgel capsules), or may be, for example, aqueous or non-aqueous solutions, suspensions, or syrups. Tablets and capsules for oral administration may use 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). Flavorings, colorants, and / or sweeteners may be added to solid and liquid formulations as needed. Other optional components of oral formulations include, but are not limited to, preservatives, suspending agents, and thickeners. Oral formulations may also be enterically coated to protect CNP salts from the acidic environment of the stomach. Methods for preparing solid and liquid dosage forms will be known or obvious to those skilled in the art (see, for example, Remington's Pharmaceutical Sciences referenced above).

[0194] Parenteral formulations can be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for solubilization in liquid media before injection or for suspension, or as emulsions. For example, sterile injectable solutions and suspensions can be formulated according to techniques known in the art using suitable diluents, carriers, solvents (e.g., buffered aqueous solutions, Ringer's solution, isotonic sodium chloride solution), dispersants, wetting agents, emulsifiers, suspending agents, etc. Furthermore, sterile non-volatile oils, fatty acid esters, polyols, and / or other inert components can be used. Further examples of parenteral formulations include aqueous sterile injectable solutions and aqueous and non-aqueous sterile suspensions that may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient, and these may include suspending agents and thickeners.

[0195] Compositions containing hydrophobic CNP salts may also be lyophilized formulations. In certain embodiments, the lyophilized formulations include buffers and extenders, and optionally antioxidants. Exemplary buffers include, but are not limited to, acetate buffer and citrate buffer. Exemplary extenders include, but are not limited to, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, the amount of mannitol is about 3% to about 10%, or about 4% to about 8%, or about 4% to about 6%. In certain 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.

[0196] In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0197] The disclosure also provides kits comprising, for example, bottles, vials, ampoules, tubes, cartridges, and / or syringes containing liquid (e.g., sterile injectable) or solid (e.g., lyophilized) formulations. The kits also include, but are not limited to, lyophilized formulations in syringes for reconstituting solid (e.g., lyophilized) formulations into solutions or suspensions (e.g., by injection) for administration, including but not limited to lyophilized formulations in syringes for reconstituting lyophilized formulations in syringes for injection or diluting concentrates to lower concentrations. Furthermore, immediate injection solutions and suspensions can be prepared from, for example, sterile powders, granules, or tablets containing CNP-containing compositions. The kits also include dispensing devices such as aerosol or injection dispensing devices, pen injectors, auto injectors, needleless injectors, syringes, and / or needles.

[0198] As a non-limiting example, the kit may include syringes having a single chamber or a dual chamber. In the case of a single-chamber syringe, the single chamber may contain a liquid CNP formulation ready for injection, or a liquid formulation of a solid (lyophilized) CNP formulation or CNP salt in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or turbidity for injection. In the case of a dual-chamber syringe, for injection, one chamber may contain a pharmaceutically acceptable vehicle 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., glycerin) that can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber.

[0199] As a further example, the kit may include one or more pen-injector or auto-injector devices and a dual-chamber cartridge. For injection, one chamber of the cartridge may contain a pharmaceutically acceptable vehicle or carrier (e.g., a solvent system, solution, or buffer), and the other chamber may contain a relatively small amount of a solid (e.g., lyophilized) CNP formulation or liquid formulation of a CNP salt in a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension using the vehicle or carrier from the first chamber for injection. The cartridge may contain a sufficient amount of CNP salt to administer over a desired period (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen-injector or auto-injector may be configured to administer the desired amount of CNP formulation from the cartridge.

[0200] Dosage and administration Hydrophobic CNP salts, or pharmaceutical compositions or formulations containing them, can be administered to a subject by various methods, such as subcutaneously, intra-articularly, intraperitoneally, intramuscularly, intradermally, or orally. In one embodiment, the CNP peptide salt composition is administered once a day, once a week, 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.

[0201] The hydrophobic CNP salt or salt composition can also be administered by implanting a depot at the target site of action (e.g., an abnormal or degenerated joint or cartilage region). Alternatively, the CNP salt can be administered sublingually (e.g., as a sublingual tablet), by transdermal delivery (e.g., by a patch on the skin), or orally by microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamide, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, etc.

[0202] The hydrophobic CNP salt composition described herein can be administered to a patient who needs it at a therapeutically effective dose to treat, ameliorate, or prevent a bone-related disorder (e.g., skeletal dysplasia including achondrogenesis). The safety and therapeutic efficacy of the CNP salt can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (the dose lethal to 50% of the population) and ED 50 (the therapeutically effective dose in 50% of the population). The dose ratio between the toxic effect and the therapeutic effect is the therapeutic index, which can be expressed as the LD 50 / ED 50 ratio. Usually, an active agent showing a large therapeutic index is preferred.

[0203] In certain embodiments, the hydrophobic CNP salt compositions described herein are administered in doses ranging from about 3 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg, or from about 3 nmol / kg to 100 nmol / kg. In some embodiments, the CNP salt composition is administered in doses of about 3, 4, 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 any other dose deemed appropriate by the treating physician. In other embodiments, the CNP salt composition is approximately 5, 6, 7, 8, 9, 10, 15, 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, 75 It is administered in doses of 0, 800, 850, 900, 950 or 1000 μg / kg, or approximately 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 mg / kg, or other doses deemed appropriate by the treating physician. The doses of hydrophobic CNP salts described herein may be administered according to the dosing frequency / dosing frequency described herein, which includes, but is not limited to, daily, two or three times a week, weekly, every two weeks, every three weeks, monthly, etc. In various embodiments, the CNP salt is administered subcutaneously daily. In various embodiments, the CNP salt is administered subcutaneously weekly. In various embodiments, the CNP variant is administered at doses of 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 is administered at a dose of 15 μg / kg / day.In various embodiments, the CNP variant is administered at a dose of 30 μg / kg / day.

[0204] The frequency of administration of hydrophobic CNP salts to a particular subject may vary depending on various factors, including the disorder being treated and the subject's condition and response to treatment. Hydrophobic CNP salts can be administered as a single dose or multiple times per dose. In certain embodiments, hydrophobic CNP salt compositions are administered as a single dose or multiple times, 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 as the treating physician deems appropriate. In various embodiments, CNP variants are administered for three months, six months, twelve months, or longer.

[0205] In some embodiments, the hydrophobic CNP salt composition is administered to allow for a growth period (e.g., cartilage formation) and a subsequent recovery period (e.g., bone formation). For example, the CNP salt composition is administered subcutaneously or in another manner multiple times daily or weekly for a period, followed by a period of no treatment, after which the cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the CNP salt composition multiple times daily or weekly) is 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 relevant embodiments, the period of no treatment lasts 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the dosing regimen for the CNP salt composition is daily for 3 days followed by a 3-day break, or daily or multiple times per week for 1 week followed by a 3-day or 1-week break, or daily or multiple times per week for 2 weeks followed by a 1-week or 2-week break, or daily or multiple times per week for 3 weeks followed by a 1, 2, or 3-week break, or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks followed by a 1, 2, 3, or 4-week break.

[0206] biomarkers In the treatment of bone-related disorders, growth indicators can be measured, such as intrauterine and neonatal long bone growth measurements, and bone growth biomarkers such as CNP, cGMP, collagen II, collagen X, osteocalcin, and proliferating cell nuclear antigen (PCNA).

[0207] One CNP signaling marker is cGMP (guanosine 3',5' cyclic monophosphate). Levels of this intracellular signaling molecule increase after CNP binds to and is activated by its congener receptor, NPR-B. Elevated levels of cGMP can be measured from cell culture extracts after CNP exposure (in vitro), conditioned media from extraskeletal plant studies after CNP exposure (ex vivo), and plasma (in vitro) within minutes of CNP administration via subcutaneous, intravenous, or other known routes of administration in the art.

[0208] The efficacy of CNP can also be evaluated by measuring cartilage and bone-specific analytes (or markers related to cartilage and bone). For example, fragments of cleaved type II collagen are cartilage-specific markers of cartilage turnover. Type II collagen is the major organic component of cartilage, and fragments of type II collagen (cleaved collagen) are released into the circulatory system and secreted into the urine following cartilage turnover. Cartilage turnover precedes the formation of new bone.

[0209] A measurable bone-specific biomarker for bone formation is the N-terminal propeptide of type I procollagen (PINP). Since type I collagen is a major organic component of the bone matrix, its synthesis is a crucial step in bone formation. During collagen synthesis, the propeptide is released from the procollagen molecule and can be detected in serum. Furthermore, fragments of type I collagen can be measured as a marker of bone resorption.

[0210] Other potential biomarkers related to cartilage and bone formation and growth include aggrecan chondroitin sulfate (a cartilage-specific marker for cartilage turnover), type II collagen propeptide (a cartilage-specific marker for chondrogenesis), alkaline phosphatase (bone-specific), and osteocalcin (a bone-specific marker for bone formation). Biomarkers related to cartilage and bone can be measured, for example, from serum in efficacy / pharmacodynamic in vivo studies and from conditioned media in ex vivo studies using commercially available kits.

[0211] In one embodiment, to monitor the effect of the CNP composition on bone and cartilage formation and in vivo growth, the level of at least one bone or cartilage-related biomarker is assayed or measured in subjects administered with the CNP salt or sustained-release composition described herein. For example, an increase in the level of at least one bone or cartilage-related biomarker may indicate that administration of the CNP salt or sustained-release composition has a positive effect on bone growth and is a useful treatment for skeletal dysplasia and other bone or cartilage-related diseases or disorders associated with decreased CNP activity. Exemplary bone or cartilage-related biomarkers include, but are not limited to, CNP (e.g., endogenous levels of CNP), cGMP, collagen type II propeptides and their fragments, collagen type II and its fragments, osteocalcin, proliferating cell nuclear antigen (PCNA), type I procollagen (PINP) propeptides and their fragments, type I collagen and its fragments, aggrecan chondroitin sulfate, and alkaline phosphatase.

[0212] In various embodiments, the biomarker is measured by obtaining a biological sample from a subject to which CNP salt or sustained-release composition is administered, being administered, or has been administered. The biomarker can be measured using techniques known in the art, including but not limited to Western blotting, enzyme-linked immunosorbent assay (ELISA), and enzyme activity assays. The biological sample may be blood, serum, urine, or other body fluid.

[0213] Further aspects and details of this disclosure will be apparent from the following examples, which are intended to be illustrative rather than restrictive. [Examples]

[0214] Example 1: Formation of hydrophobic CNP salts To determine whether stabilizing the charge of hydrophilic peptide CNP by complexing it with hydrophobic counterions is effective in improving therapeutic CNP formulations, experiments were conducted to alter the static charge of CNP by combining it with other ionic components.

[0215] To optimize the electrostatic complex formation of CNP peptides and hydrophobic counterionic species and control peptide complex precipitation, it is necessary to allow large populations of both species to be ionized in the solution environment. Adjusting the pH of both solutions to an intermediate value between 10 (pI of CNP) and the acidic pKa of the counterion (e.g., the pKa of the oleic counterionic species is approximately 4-5) is important for peptide complex yield.

[0216] To generate hydrophobic CNP salts containing CNP variants, CNP acetate is placed in water or a buffer. In the initial experiment, a stock solution containing 20 mg / mL of CNP was prepared in water. 100 mM Tris, pH 9.00 is also a suitable solvent. Disodium pamoate, oleic acid, or sodium docusate was placed in the buffer at a molar excess of counterions compared to the number of oppositely charged groups on CNP, in this case a 12 molar excess relative to CNP. CNP has 6 positively charged groups at pH 7, which is twice the number of positively charged groups. In buffers (76 mM acetate buffer pH 4.84 and 100 mM phosphate buffer pH 6.61), the counterions did not readily dissolve, forming suspensions of insoluble or poorly soluble counterions in the buffer. However, in water and 100 mM Tris pH 9.00, oleic acid, disodium pamoate, and sodium docusate were soluble counterions.

[0217] When metal cations were added to promote salt precipitation, when the metal cations were dissolved in water and added to the CNP solution rather than the counterion solution, and when the metal cations were added directly to the counterions, it was observed that precipitation of the counterion solution was induced. To allow small amounts to be added to the CNP solution without significantly changing the concentration or pH, the metal cation solution (e.g., ZnCl2) was dissolved in water at a high concentration (>100 mg / mL). Although metal cations could be added at various concentrations, in the first experiment, they were added in a 4-mol excess relative to the CNP concentration (CNP has two negatively charged amino acids, and this was added so that there were 2 moles of zinc per mole of negatively charged amino acids). Next, the counterion solution was added dropwise to the CNP solution with or without the metal cation, and after each addition, the tube was vortexed for 1 second at the highest setting.

[0218] After adding sufficient solution to achieve the desired ratio of CNP to counterions, the reaction tube was rotated in a 10,000xg centrifuge for 5 minutes to pelletize the salt precipitate. After rotation, the supernatant was removed, and the pellet was resuspended in an equal volume of water. In this case, the tube was rotated at 7500xg for 3-5 minutes, and the pelletization of the salt was observed. After rotation, the salt pellet was washed again with water and resuspended. The tube was rotated again, and the supernatant was removed. Next, the contents were transferred to a vial (e.g., a 6R borosilicate glass vial), stoppered, frozen, and lyophilized.

[0219] Next, the solubility of the salt powder in various buffers was evaluated. First, approximately 1 mg of powder was weighed into a tube, and an appropriate amount of solvent was added to make a concentration of 1 mg / mL. The tube was left to agitate overnight at approximately 37°C. It was found that oleate was soluble in 20% acetic acid. Pamoate was soluble in dimethyl sulfoxide (DMSO).

[0220] These experiments demonstrate that it is possible to precipitate highly water-soluble peptides with a size ranging from 5 nanometers to 1 millimeter in diameter into water-insoluble / low-soluble aggregates.

[0221] Example 2 -- Characterization of CNP salts Hydrophobic salts of CNP were prepared as in Example 1 and tested for precipitation and solubility. Table 1 shows that CNP complexed with various hydrophobic counterions resulted in the formation of precipitates or solids. [Table 1]

[0222] Dissolution studies were also conducted using salt precipitates. 1 mg of CNP salt was resuspended in 50 mL of 1XPBS, pH 6.5, at 37°C, and the dissolution of the solid and the release of CNP into solution were measured for 7 days. The buffer was not changed daily. Figures 1A-1D and 2 show that the dissolution of the hydrophobic salt was slower than the dissolution of the CNP-acetate composition.

[0223] Example 3: Heterozygous NPR2 mutations respond to CNP treatment. To determine the effect of CNP on subjects with short stature due to NPR2 mutations, a cell model of NPR2 mutations was developed. Exemplary NPR2 mutations analyzed are shown in Figure 5. Rat chondrosarcoma (RCS) cells with NPR2 gene knockout or heterozygous loss-of-function mutations were generated by RNP transfection into RCS cells using 125 ng of NPR2 variant or wild-type NPR2 plasmid DNA transfected into RCS or HEK293 cells. Single-cell clones were seeded and genotyped by Sanger sequencing. The cell model can reproduce the published cGMP phenotypes of various mutations.

[0224] NPR2 clones were created by making 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. NPR2 mutant cells were treated with 6 nM Pro-Gly CNP37 and then tested for activity in response to CNP administration by a cGMP stimulation assay using a CatchPoint cyclic-GMP fluorescence assay. Briefly, NIH3T3 cells (ATCC, CRL-1658) and HEK293 cells transfected with NPR were seeded at 60,000 cells / well in 96-well plates (96-well black imaging plates, Grenier, #655090). RCS (rat chondrosarcoma) cells were seeded at 40,000 cells / well. The culture medium was as follows: NIH3T3 medium: DMEM high glucose, pyruvate (Thermo, 11995-073) + 10% FBS + 1x PenStrep (abbreviated as P / S, Thermo, catalog no. 15140122). NIH3T3 was the control system for the cGMP assay HEK293 medium: EMEM + 10% FBS + 1x P / S + 1x GMAX. RCS medium: DMEM + 10% FBS + 1x PenStrep. Serum-free NIH3T3 medium: DMEM + 1x P / S for treating cells with IBMX (CAS28822-58-4), serum-free NIH3T3 medium containing BSA: DMEM + 1x P / S + 0.5 mg / mL BSA (Thermo, A9418-100G) for treating cells with CNP.

[0225] Cells were incubated at 37°C and 5% CO2 for 24 hours. For cells treated with the CNP variant, plates were pre-treated with IBMX (Enzo Life Sciences, 89161-340, 1 g) 15 minutes before use. IBMX is a potent nonspecific 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 + 1xPBS mixed 1:1 with 1xPBS)).

[0226] For cell treatment, cells were removed from the incubator, the growth medium was removed from the cells, and the cells were treated with IBMX. 80 μL of 0.75 mM IBMX was added to each well, and the cells were returned to a 37°C incubator for 15 minutes. After 15 minutes, CNP (40 μL / well) was added to each test well, and the cells were returned to a 37°C incubator for another 15 minutes. The plates were gently tapped to mix, and the cells were visualized using Solentim Celmetric imaging to determine if the cells were floating, before returning the plates to a 37°C incubator.

[0227] The reaction was stopped, and 40 μL of lysis buffer (from the cGMP kit) was added to lyse the cells. The plate was placed in a shaker for 5 minutes to complete the lysis. The cell lysate was used in the cGMP assay.

[0228] The cGMP assay 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 wells coated and seeded with anti-cGMP antibody, and 40 μL of analyte lysate was added to the appropriate wells. 40 μL of reconstituted rabbit anti-cGMP antibody was added to all wells, and the plate was mixed by shaking for 5 minutes. 40 μL of reconstituted HRP-cGMP was added to each well and incubated at room temperature for 2 hours. The plate was manually aspirated and washed four times with 300 μL of wash buffer. 100 μL of stoplight red substrate was added to each well, the plate was covered, protected from light, and left at room temperature for at least 10 minutes. The plates were read for fluorescence intensity at excitation 530 nm and emission 590 nm using a Spectramax M or similar instrument.

[0229] Figure 3 shows that the addition of the exogenous Pro-Gly-CNP37 variant rescues cGMP reads in an NPR2+ / - rat chondrosarcoma cell model. Previous activation data have reported cGMP EC50s in the range of 40–360 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 heterozygous NPR2 knockout cells, intracellular concentrations exceeding the EC50 range of PRKG2-activated cGMP can be achieved with a CNP dose >0.163 nM (Figure 1). In contrast, the same cGMP concentration can be achieved in wild-type cells with a CNP dose of 0.040 nM. These results demonstrate that CNP supplementation can achieve the cGMP levels necessary for PRKG2 activation and proliferation in cells with loss-of-function mutations in NPR2.

[0230] These results also suggest that administration of CNP variants may be useful in restoring bone growth in short-statured subjects with reduced NPR2 activity. Furthermore, treatment with CNP variants may be beneficial in subjects with mutations in other growth plate genes that may impair cGMP signaling.

[0231] Example 4: Identification of mutations associated with short stature Genes showing clear evidence of genetically driven bidirectional effects are hypothesized to represent therapeutic targets that can be effectively regulated across a broad patient population. To identify core growth regulators, we analyzed the intersection of five gene lists, including a list of genes from genome-wide association studies (GWAS). Core growth regulators are most likely to include rare coding mutations with bidirectional effects (i.e., short stature or skeletal dysplasia and tall stature or overgrowth).

[0232] The databases queried included: GWAS: For each of the 3,290 independent genetic variants reported by a large GWAS meta-analysis of height using approximately 700,000 individuals, 2,067 non-repeating closest genes were extracted. HGMD: The "allmut" table of HGMD version v2019_2 was queried to find all pathogenic variants labeled "DM" that have either "short stature" or "tall stature or overgrowth" for the same gene. OMIM: A list of OMIM genes associated with growth disorders has been previously described and was compiled using the keywords short stature, overgrowth, skeletal dysplasia, and brachydactyly.

[0233] First, genes associated with short or tall stature were queried in the Human Genetic Variation Database (HGMD version v2019_2) (Stenson et al., Hum Genet 136:665-677, 2017). There were 47 genes annotated with at least one pathogenic variant reported in the literature to cause "short stature". Only 20 genes were annotated as tall or overgrowth genes. Next, a manually curated list of 258 OMIM genes (248 for short stature, 20 for tall stature) created using the keywords: short stature, overgrowth, skeletal dysplasia, brachydactyly was used (Wood et al., Nat Genet 46:1173-86, 2014). Third, the common parts of these lists were compared with the gene list from GWAS. The common threads in these lists include three genes known to be associated with height (IGF1R, NPPC, NPR2), and two additional genes (FGFR3, SHOX) were identified.

[0234] Further analysis generated a new group of five core genes associated with a significant decrease in height (β=-0.20, 95%CI[-0.26~-0.14], p=4.04x10⁻¹¹) and a significant increase in the 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 when examined individually and with short stature when combined with other mutations. Examples of mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX are shown in Figure 6.

[0235] Combinations of 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 causing loss of protein function in SHOX, IGF1R, NPPC, and NPR2 have been reported in familial short stature of varying degrees of severity.

[0236] The analysis shows that carriers of variants in any of the five core genes have approximately three times the risk of ISS and account for 6.7% of the total ISS population. Furthermore, dose-dependent relief of NPR2 signaling in a cellular model of NPR2 haploinsufficiency was demonstrated after the addition of exogenous CNPs.

[0237] According to omnogenic 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, more common gene variants that drive the regulatory network. To indirectly test this hypothesis, we calculated polygene risk scores (PRS) for height using the largest published GWAS meta-analysis on height, excluding samples from the UK Biobank Project. The cohort was divided into five PRS quintiles of the same size (n=6,824) (PRS1 for the lowest height, PRS5 for the highest height). There was a dose-dependent relationship between the increase in PRS score and mean height (β=0.30 for each increase in PRS quintile) (Figure 7A). Carriers of LoF variants in five core genes were consistently shorter than non-carriers across five different PRS backgrounds. See Figure 7. The data suggest that the combined effect of PRS and rare protein variants is consistent with an additive model: polygenic effects modulated height in both carriers and non-carriers.

[0238] The risk of ISS across PRS groups was calculated using PRS=3 as a reference. 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=8.58x10⁻³⁴, OR=0.22, P=4.49x10⁻⁷ for PRS1 and PRS5, respectively). The effects of rare coding variants in the five core genes were evaluated for ISS stratified by PRS group. Carriers of any of the five core genes had a higher risk of ISS in the first three quintiles (OR=2.64, P=3.09x10⁻⁵, OR=2.17, P=0.04, OR=5.29, P=1.58x10⁻⁵, OR=2.72, P=0.09, Figures 7C-F). A consistent direction of influence was observed for carriers of each individual core gene on ISS risk stratified by PRS (Figures 7C-F).

[0239] Furthermore, the additive effect of PRS, primarily derived from multiple common genetic variations with small individual effects, predicted 20.1% of the height variance in the dataset. These additive effects of PRS appeared to be of similar magnitude for carriers and non-carriers of rare coding variants in the core gene. This observation suggests that PRS may significantly contribute to differences in penetrance of rare pathogenic variants (particularly in haploinsufficiency models such as those described here). Supporting this idea, two of the eight NPR2 variant carriers with low NPR2 activity were observed to have short normal height. This data suggests that most ISS individuals with NPR2 mutations may have a polygenic background that makes them susceptible to pathogenicity by losing NPR2 activity.

[0240] These results support the idea that CNP-based therapy may be effective in the NPR2 haploinsufficiency patient population. Furthermore, the results showing a significant bidirectional (LoF and GoF) correlation between cGMP levels and height in NPR2 carriers in the general population suggest that targeting this receptor with CNP analogs may be an effective treatment for all ISS individuals.

[0241] Example 5: Release profile of CNP salts in vitro Release analysis of hydrophobic CNP salts was also performed. Briefly, fresh Pro-Gly-CNP37 Zn pamoate and Pro-Gly-CNP37 Zn-oleate were prepared in 15 mL of medium and placed in a PionmicroDiss dissolution apparatus with an impeller rotating at 250 RPM and a temperature-controlled setting of 37.4°C on the same day they were prepared. For 4 days, every 24 hours, the contents of the container were transferred to VWR polypropylene "Falcon" tubes, spun down at 4000xg for 30 minutes, RP-UPLC samples were collected / frozen, and the solids were resuspended in fresh medium (15 mL). The salts were signaled by RP-UPLC, and the concentration was obtained by comparison with a calibration curve. The percentage of released salt was obtained by comparing the released mass with the initial mass of Pro-Gly-CNP37 used to prepare the salt.

[0242] Figures 8A and 8B show the cumulative release profile of CNP peptide salts over 7 days, expressed as a percentage of the total amount of CNP peptide salts.

[0243] Additional release profiles were performed using the protocol described above. Approximately 16.6 mg of Pro-Gly-CNP37-acetate (starting material for salt preparation) or Pro-Gly-CNP37Zn-pamoate salt was placed in each well. CNP salts were prepared, lyophilized, sealed, and stored at 4°C before use. For the CNP acetate control, container 1 was in 1x PBS and container 5 was in 1x PBS + 0.05% PS80. For 7 days, every 24 hours, the contents of the containers were transferred to VWR polypropylene "Falcon" tubes, spun down at 4000xg for 30 minutes, UPLC samples were collected / frozen, and the solids were resuspended in fresh medium (15 mL). CNP salts were analyzed by RP-UPLC, and concentrations were obtained from comparison of signals with calibration curves. Cumulative release profiles for 7 days are shown in Figures 9A and 9B.

[0244] Pro-Gly-CNP-37-docusate and Pro-Gly-CNP-37-Zn-docusate salts were freshly generated or prepared, lyophilized, and sealed in glass vials before use in Pion for dissociation. For 4 days, every 24 hours, the contents of the container were transferred to VWR polypropylene "Falcon" tubes, spun down at 4000xg for 30 minutes, samples were collected / frozen, and the solids were resuspended in fresh medium (15 mL). The salts were analyzed and quantified by LC-MS and compared to the initial salts placed in the dissolution container to determine the percentage of salt released.

[0245] Figures 10A–10C show the cumulative release profiles of docusate salt over 4 days (Figure 10A) or 7 days (Figures 10B and 10C).

[0246] Example 6: In vivo release profile of CNP salts Next, the release profile of the sample salt, CNP-pamoate, was analyzed in vivo in rats over 7 days after subcutaneous injection of CNP salt.

[0247] Figure 11 shows the release profile of Pro-Gly-CNP37 from salt to plasma over 7 days. Salt release was observed as an initial burst over 24 hours, with some salt release observed over time.

[0248] Any single embodiment of this specification may be supplemented by one or more elements from any one or more other embodiments of this specification.

[0249] Therefore, it is understood that the present invention is not limited to the specific embodiments disclosed, but is intended to encompass all modifications that fall within the spirit and scope of the invention as defined by the appended claims, the above description, the following numbered sections, and / or the appended drawings.

[0250] Examples of embodiments: Section 1. A composition comprising a salt of an electrostatically charged peptide, wherein the electrostatically charged peptide is complexed with a hydrophobic counterion.

[0251] Section 2. The composition according to Section 1, wherein hydrophobic counterions form a complex via non-covalent bonds.

[0252] Section 3. The composition according to Section 1, wherein a hydrophobic counterion forms a complex with an electrostatically charged peptide via a cleavable linker.

[0253] Section 4. The composition according to any one of Sections 1 to 3, further comprising a cation formed by the salt complexing with a peptide-counterion complex.

[0254] Section 5. The composition according to Section 4, wherein an electrostatically charged peptide, a hydrophobic counterion, and a cation form a complex via non-covalent bonds.

[0255] Section 6. The composition according to Section 4 or 5, wherein the cation has a charge of +2, +3, or +4.

[0256] Section 7. A composition according to any one of Sections 4 to 6, wherein the cation is a metal cation.

[0257] Section 8. A composition according to any one of Sections 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). In an alternative embodiment, a composition according to any one of sections 4 to 7 is provided, 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), thallium (Ti), vanadium (V), and gold (Au).

[0258] Section 9. A composition according to any one of Sections 1 to 8, wherein the hydrophobic counterion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0259] Section 10. The composition according to any one of Sections 1 to 9, wherein the hydrophobic counterion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0260] Section 11. A composition according to any one of Sections 1 to 10, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, 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-octanesulfonate monohydrate, sodium 1-decanesulfonate, sodium dodecyl sulfate, and sodium dodecylbenzenesulfonate. In an alternative embodiment, a composition is provided according to any one of sections 1 to 10, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, 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-octanesulfonate monohydrate, sodium 1-decanesulfonate, sodium dodecyl sulfate, sodium dodecylbenzenesulfonate, and an ionic surfactant.

[0261] Section 12. The composition according to any one of Sections 1 to 11, wherein the peptide salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticles, microparticles, amorphous nanoparticles, amorphous microparticles, crystalline nanoparticles, or crystalline microparticles.

[0262] Section 13. The composition according to any one of Sections 1 to 12, wherein the electrostatically charged peptide is a C-type natriuretic peptide (CNP).

[0263] Section 14. The composition according to Section 13, wherein CNP is a CNP variant.

[0264] Section 15. CNP is, PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), A composition according to Section 13 or 14, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and pharmaceutically salts thereof.

[0265] Section 16. The composition according to Section 15, wherein CNP is CNP-acetate.

[0266] Section 17. The composition according to any one of Sections 13 to 16, wherein the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate.

[0267] Section 18. The composition according to any one of Sections 13 to 17, wherein, if a cation is present, the cation is zinc or calcium.

[0268] Section 19. A composition according to any one of Sections 1 to 18, further comprising an excipient, diluent, or carrier.

[0269] Section 20. The composition according to Section 19, wherein the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0270] Section 21. A sterile pharmaceutical composition comprising any one of the compositions described in Sections 1 to 20.

[0271] Section 22. Extended release composition comprising a salt of an electrostatically charged peptide, wherein the salt comprises an electrostatically charged peptide that forms a complex with a hydrophobic counterion.

[0272] Section 23. The extended release composition described in Section 22, wherein hydrophobic counterions form a complex via non-covalent bonds.

[0273] Section 24. The extended release composition according to Section 23, wherein a hydrophobic counterion forms a complex with an electrostatically charged peptide via a linker capable of cleaving it.

[0274] Section 25. The extended release composition according to any one of Sections 22 to 24, further comprising a cation formed by the complexing of the salt with a peptide-counterion complex.

[0275] Section 26. The extended release composition according to Section 25, wherein an electrostatically charged peptide, a hydrophobic counterion, and a cation form a complex via non-covalent bonds.

[0276] Section 27. The extended release composition according to Section 26, wherein the cation has a charge of +2, +3, or +4.

[0277] Section 28. An extended release composition according to any one of Sections 25 to 27, wherein the cation is a metal cation.

[0278] Section 29. An extended-release composition according to any one of Sections 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).

[0279] Section 30. An extended release composition according to any one of Sections 22 to 29, wherein the hydrophobic counterion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0280] Section 31. An extended release composition according to any one of Sections 22 to 30, wherein the hydrophobic counterion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0281] Section 32. An extended-release composition according to any one of Sections 22 to 31, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, decanoate, 2-naphthalene sulfonate, 1-heptanesulfonate, 1-octanesulfonate monohydrate, 1-decanesulfonate, dodecyl sulfate, dextrans sulfate, and dodecylbenzenesulfonate.

[0282] Section 33. An extended release composition according to any one of Sections 22 to 32, wherein the CNP salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticles, microparticles, amorphous nanoparticles, amorphous microparticles, crystalline nanoparticles, or crystalline microparticles.

[0283] Section 34. An extended-release composition according to any one of Sections 22 to 33, wherein the electrostatically charged peptide is a C-type natriuretic peptide (CNP).

[0284] Section 35. The extended release composition according to Section 34, wherein CNP is a CNP variant.

[0285] Section 36. CNP is, PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), A composition according to Section 34 or 35, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and pharmaceutically salts thereof.

[0286] Section 37. The extended release composition according to Section 36, wherein CNP is CNP-acetate.

[0287] Section 38. An extended-release composition according to any one of Sections 34 to 37, wherein the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate.

[0288] Section 39. The extended-release composition according to any one of Sections 34 to 38, wherein, if a cation is present, the cation is zinc or calcium.

[0289] Section 40. An extended release composition according to any one of Sections 33 to 39, wherein a peptide salt solid, semi-solid, gel, crystalline, amorphous, nanoparticles, microparticles, amorphous nanoparticles, amorphous microparticles, crystalline nanoparticles, or crystalline microparticles is resuspended in an aqueous solution or oil.

[0290] Section 41. The extended release composition according to Section 40, wherein the aqueous solution is water, saline solution, or buffer solution.

[0291] Section 42. The extended-release composition according to Section 40, wherein the oil comprises a triglyceride or a fatty acid.

[0292] Section 43. The extended-release composition according to Section 42, wherein the fatty acid is saturated or unsaturated.

[0293] Section 44. The extended-release composition according to Section 42 or 43, wherein the fatty acid is a C-6 to C-20 fatty acid.

[0294] Section 45. An extended-release composition according to any one of Sections 42 to 44, wherein the fatty acid is hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid.

[0295] Section 46. At pH 7-7.6, (i) less than 20% of the peptide is released by day 1. ii) The extended-release composition according to any one of sections 22 to 45, wherein approximately 90% of the peptide is released by day 7, or approximately 90% of the peptide is released by day 14, or approximately 90% of the peptide is released by day 31.

[0296] Section 47. Extended release composition according to any one of Sections 22-46, wherein less than 20% of the peptide is released by day 1 at pH 7-7.6.

[0297] Section 48. An extended-release composition according to any one of Sections 22 to 47, wherein approximately 90% of the peptide is released by day 7 at pH 7 to 7.6.

[0298] Section 49. An extended-release composition according to any one of Sections 22 to 47, wherein approximately 90% of the peptide is released by day 30 at pH 7 to 7.6.

[0299] Section 50. An extended release composition according to any one of Sections 22 to 49, further comprising an excipient, diluent, or carrier.

[0300] Section 51. The extended-release composition according to Section 50, wherein the excipient, excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier.

[0301] A sterile pharmaceutical composition comprising the extended-release composition described in any one of Sections 22 to 51.

[0302] Section 53. a) Contacting electrostatically charged peptides in an aqueous solution with hydrophobic counterions in the solution, b) A method for producing a composition containing a salt of an electrostatically charged peptide, comprising mixing an electrostatically charged peptide solution with a hydrophobic counterion solution in a manner sufficient for the peptide and counterion to form a complex, wherein the formation of the peptide-counterion complex results in the formation of a solid, semi-solid, gel, crystal, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle morphology containing the peptide salt.

[0303] Section 54. The method according to Section 53, optionally comprising, before step (b), contacting an electrostatically charged peptide in solution with a cation in aqueous solution to form a peptide-cation complex.

[0304] Section 55. The method according to Section 53 or 54, further comprising step (c) washing the peptide salt with buffer or water.

[0305] Section 56. The method according to Section 55, further comprising step (d) obtaining a peptide salt by centrifugation and forming a peptide salt pellet.

[0306] Section 57. The method according to Section 56, further comprising step (e) removing water from the peptide salt pellet.

[0307] Section 58. The method according to Section 57, further comprising resuspending the pellets in an aqueous solution or oil.

[0308] Section 59. Peptide: The method according to any one of Sections 53 to 58, wherein the hydrophobic counter-ion ratio is 1:1 to 1:20.

[0309] Section 60. The method according to any one of Sections 54-59, wherein the peptide:cation ratio is 1:1 to 1:10.

[0310] Section 61. The method according to any one of Sections 53 to 60, wherein a hydrophobic counterion forms a complex via a non-covalent bond.

[0311] Section 62. The composition according to Section 61, wherein a hydrophobic counterion forms a complex with an electrostatically charged peptide via a cleavable linker.

[0312] Section 63. The method according to any one of Sections 54-62, wherein the salt further comprises a cation that has complexed with a peptide-counterion complex, and the cation complexes via covalent, non-covalent, or a mixture thereof.

[0313] Section 64. The method according to Section 63, wherein a cation forms a complex with a peptide-hydrophobic counterion complex via a non-covalent bond.

[0314] Section 65. The composition according to Section 63 or 64, wherein an electrostatically charged peptide, a hydrophobic counterion, and a cation form a complex via non-covalent bonds.

[0315] Section 66. The method according to any one of Sections 54 to 65, wherein the cation has a charge of +2, +3, or +4.

[0316] Section 67. The method according to any one of Sections 54 to 66, wherein the cation is a metal cation.

[0317] Section 68. The method according to any one of Sections 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). Furthermore, the method described in any one of sections 54 to 67 is intended, 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).

[0318] Section 69. The method according to any one of Sections 46-58, wherein the hydrophobic counterion has a cLogP of about 0 to about 10, or a pKa of about -2 to about 5, or both.

[0319] Section 70. The method according to any one of Sections 53 to 69, wherein the hydrophobic counterion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0320] Section 71. The method according to any one of Sections 53 to 70, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), and phosphatidate.

[0321] Section 72. The method according to any one of Sections 53 to 71, wherein the electrostatically charged peptide is a C-type natriuretic peptide (CNP).

[0322] Section 73. The method described in Section 72, wherein CNP is a CNP variant.

[0323] Section 74. CNP, PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4), The method described in Section 72 or 73, selected from the group consisting of and salts thereof.

[0324] Section 75. The method described in Section 74, wherein CNP is CNP acetate.

[0325] Section 76. The method according to any one of Sections 72-75, wherein the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate.

[0326] Section 77. The method according to any one of Sections 72-76, wherein, if a cation is present, the cation is zinc or calcium.

[0327] Section 78. A method for treating bone-related disorders or skeletal dysplasia in subjects requiring treatment of bone-related disorders or skeletal dysplasia, comprising administering a composition containing a hydrophobic salt of a type C natriuretic peptide (CNP) as described in any one of Sections 1 to 52.

[0328] Section 79. Bone-related disorders or skeletal dysplasia include osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondrodysplasia, dwarfism, osteochondrodysplasia, fatal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, isozygosyngeal chondrodysplasia, chondrodysplasia punctata, dysplasia of the limbs, congenital fatal hypophosphatasia, perinatal fatal osteogenesis imperfecta, short-rib chondrodysplasia, hypochondrodysplasia, rhombophyseal dysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, growth-impaired osteogenesis imperfecta, torsional dysplasia, congenital short femur, and The method described in Section 78, selected from the group consisting of Ninger's type metalegal dysplasia, Niebergeld type metalegal dysplasia, Robinnow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysplasia, Nieist dysplasia, fibrochondroplasia, Roberts syndrome, distal intermediate limb dysplasia, brachylimmus, Morquio syndrome, Nieist syndrome, complex organic dysplasia, and vertebral epiphyseal metaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Reliweil), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature.

[0329] Section 80. A method for lengthening bones or increasing long bone growth in subjects requiring lengthening bones or increasing long bone growth, comprising administering to the subject an extended-release composition comprising a salt of type C natriuretic peptide (CNP) as described in any one of Sections 1 to 52, wherein the administration lengthens bones or increases long bone growth.

[0330] Section 81. The method described in any one of Sections 78-80, wherein CNP is a CNP variant.

[0331] Section 82. CNP, PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34)(SEQ ID NO: 4), The method described in any one of sections 78-81, selected from the group consisting of and their salts.

[0332] Section 83. The method described in Section 83, wherein CNP is CNP acetate.

[0333] Section 84. The method according to any one of Sections 78-83, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

[0334] Section 85. The method according to any one of Sections 78-84, wherein the composition is administered once daily, once a week, 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.

[0335] Section 86. The method according to any one of Sections 78 to 85, wherein the composition is an extended-release composition.

[0336] Section 87. Salts of C-type natriuretic peptides containing CNP peptides that form complexes with hydrophobic counterions.

[0337] Section 88. A salt described in Section 87, wherein hydrophobic counterions are complexed via non-covalent bonds.

[0338] Section 89. Salts described in Section 87 or 88, wherein the hydrophobic counterion has a cLogP of about 2 to about 9, or a pKa of less than about 5, or both.

[0339] Section 90. A salt described in any one of Sections 87-89, wherein the hydrophobic counterion has a cLogP of about 2 to about 9 and a pKa of less than about 5.

[0340] Section 91. A salt described in any one of Sections 87-90, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristic acid, palmitic acid, stearic acid, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), and phosphatidic acid.

[0341] Section 92. A salt according to any one of Sections 87-91, further comprising a peptide and a cation complexed with a counterion.

[0342] Section 93. A salt described in Section 92, wherein the cation forms a complex via a non-covalent bond.

[0343] Section 94. The salts described in Section 92 or 93, wherein the cation has a charge of +2, +3, or +4.

[0344] Section 95. A salt described in any one of Sections 92-94, wherein the cation is a metal cation.

[0345] Section 96. A salt described in any one of Sections 92-95, 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). Furthermore, the salts described in any one of sections 92-95 are intended, 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).

[0346] Section 97. A salt according to any one of Sections 87-96, wherein the CNP salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle or crystalline microparticle.

[0347] Section 98. A salt described in any one of Sections 87-97, wherein CNP is a CNP variant.

[0348] Section 99. CNP, PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), A salt selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and their pharmaceutically acceptable salts, as described in any one of sections 87 to 98.

[0349] Section 100. The salt described in Section 99, wherein CNP is CNP-acetate.

[0350] Section 101. A salt described in any one of Sections 87-100, wherein the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate.

[0351] Section 102. A salt described in any one of Sections 87-101, wherein, if a cation is present, the cation is zinc or calcium.

[0352] Section 103. Refined salt as described in any one of Sections 87-102.

[0353] Section 104. A composition comprising a hydrophobic salt as described in any one of Sections 1-52 or 87-103, for use in the treatment of bone-related disorders or skeletal dysplasia, or for lengthening bone or increasing the growth of long bones.

[0354] Section 105. Use of compositions comprising hydrophobic salts described in any one of Sections 1-52 or 87-103 in the preparation of agents for treating bone-related disorders or skeletal dysplasia, or for lengthening bones or increasing the growth of long bones.

[0355] Section 106. The composition or use described in Section 104 or 105, wherein the composition is an extended-release composition. In certain embodiments, for example, the following are provided: (Item 1) A composition comprising a hydrophobic salt of C-type natriuretic peptide (CNP), wherein the salt comprises CNP that has formed a complex with a hydrophobic counterion. (Item 2) The composition according to item 1, wherein the salt further comprises a polyvalent cation complexed with the peptide-counterion complex. (Item 3) The composition according to item 2, wherein the CNP, hydrophobic counterion, and cation form a complex via non-covalent bonds. (Item 4) The composition according to item 2 or 3, wherein the cation has a charge of +2, +3, or +4. (Item 5) The composition according to any one of items 2 to 4, wherein the cation is a metal cation. (Item 6) The composition according to any one of items 2 to 5, wherein the cation comprises a metal 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). (Item 7) The composition according to any one of items 2 to 6, wherein the cation comprises zinc or calcium. (Item 8) The composition according to any one of items 1 to 7, wherein the hydrophobic counterion has a cLogP of about 0 to about 10, or its conjugate acid has a pKa of about -2 to about 5, or both. (Item 9) The composition according to any one of items 1 to 8, wherein the hydrophobic counterion has a cLogP of about 2 to about 9 and its conjugate acid has a pKa of less than about 5. (Item 10) The composition according to any one of items 1 to 9, wherein the hydrophobic counterion is selected from the group consisting of deprotonated fatty acids, deprotonated bile acids, ionic surfactants, naphthoates and their derivatives, nicotinates and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextrans sulfates, alkylbenzene sulfates, and any combination thereof. (Item 11) The composition according to any one of items 1 to 10, wherein the hydrophobic counterion is selected from the group consisting of palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, decanoate, 2-naphthalene sulfonate, 1-heptanesulfonate, 1-octanesulfonate monohydrate, 1-decanesulfonate, dodecyl sulfate, dextrans sulfate, and dodecylbenzenesulfonate. (Item 12) The composition according to any one of items 1 to 11, wherein the peptide salt is in the form of a solid, semi-solid, gel, crystalline, amorphous, nanoparticles, microparticles, amorphous nanoparticles, amorphous microparticles, crystalline nanoparticles, or crystalline microparticles. (Item 13) The composition according to any one of items 1 to 12, wherein the CNP is a CNP variant. (Item 14) The aforementioned CNP PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), A composition according to any one of items 1 to 13, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and salts thereof. (Item 15) The composition according to item 14, wherein the CNP is CNP acetate. (Item 16) The composition according to any one of items 1 to 15, wherein the hydrophobic counterion is oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. (Item 17) Cation is Zn 2+ or Ca 2+ The composition described in any one of items 2 to 16. (Item 18) A composition according to any one of items 1 to 17, further comprising an excipient, diluent, or carrier. (Item 19) The composition according to item 18, wherein the excipient, diluent, or carrier is a pharmaceutically acceptable excipient, diluent, or carrier. (Item 20) A sterile pharmaceutical composition comprising any one of the compositions described in item 1 to 19. (Item 21) An extended-release composition comprising a salt of type C natriuretic peptide (CNP), wherein the salt comprises an electrostatically charged peptide that has formed a complex with a hydrophobic counterion. (Item 22) The extended release composition according to item 21, wherein the salt further comprises a cation complexed with the peptide-counterion complex. (Item 23) An extended-release composition according to item 21 or 22, wherein a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle or crystalline microparticle of a peptide salt is resuspended in an aqueous solution or oil. (Item 24) The extended-release composition according to item 23, wherein the oil comprises a triglyceride or a fatty acid, and optionally the fatty acid is saturated or unsaturated. (Item 25) The extended-release composition according to item 23 or 24, wherein the fatty acid is a C-6 to C-20 fatty acid. (Item 26) The extended-release composition according to any one of items 23 to 25, wherein the fatty acid is hexanoic acid, octanoic acid, decanoic acid, or dodecanoic acid. (Item 27) The extended release composition according to item 23, wherein the aqueous solution is water, physiological saline, or a buffer solution. (Item 28) At pH 7-7.6 (i) Less than 20% of the peptide is released by day 1, (ii) The extended-release composition according to any one of items 21 to 27, wherein approximately 90% of the peptide is released by day 7, or approximately 90% of the peptide is released by day 14, or approximately 90% of the peptide is released by day 31. (Item 29) An extended-release composition according to any one of items 21 to 28, further comprising a pharmaceutically acceptable excipient, diluent, or carrier. (Item 30) A method for preparing a composition containing a hydrophobic salt of C-type natriuretic peptide (CNP), a) Contacting CNP in an aqueous solution with hydrophobic counterions in the solution, b) A method comprising mixing the CNP solution with the hydrophobic counterion solution in a manner sufficient for the peptide and counterion to form a complex, wherein the formation of the peptide-counterion complex results in the formation of a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle, or crystalline microparticle form containing the CNP salt. (Item 31) The method of item 30, optionally comprising, prior to step (b), contacting the CNP in solution with a polyvalent cation in an aqueous solution to form a peptide-cation complex. (Item 32) The method according to item 30 or 31, further comprising step (c) washing the hydrophobic CNP salt with a buffer or water. (Item 33) The method according to item 32, further comprising step (d) obtaining the hydrophobic CNP salt by centrifugation to form a CNP salt pellet. (Item 34) The method according to item 33, further comprising step (e) removing water from the CNP salt pellet. (Item 35) The method according to item 34, further comprising resuspending the pellets in an aqueous solution or oil. (Item 36) The method according to any one of items 30 to 35, wherein the peptide:hydrophobic counterion ratio is 1:1 to 1:20. (Item 37) The method according to any one of items 31 to 36, wherein the peptide:cation ratio is 1:1 to 1:10. (Item 38) A method for treating a bone-related disorder or skeletal dysplasia in a subject requiring treatment for such disorder or skeletal dysplasia, comprising administering to the subject a composition comprising a hydrophobic CNP salt as described in any one of items 1 to 29 or 44 to 52. (Item 39) The aforementioned bone-related disorders or skeletal dysplasias include osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondrodysplasia, dwarfism, osteochondrodysplasia, fatal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, isozygosyngeal chondrodysplasia, chondrodysplasia punctata, dysplasia of the limbs, congenital fatal hypophosphatasia, perinatal fatal osteogenesis imperfecta, short-rib chondrodysplasia, hypochondrodysplasia, rhombophyseal dysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, growth-impaired osteogenesis imperfecta, torsional dysplasia, congenital short femur, and Langer syndrome. The method described in item 38, selected from the group consisting of type metalegial dysplasia, Niebergeld type metalegial dysplasia, Robinnow syndrome, Reinhardt syndrome, achondroplasia, peripheral dysplasia, Nieist dysplasia, fibrochondroplasia, Roberts syndrome, distal intermediate limb dysplasia, brachylimia, Morquio syndrome, Nieist syndrome, complex organic dysplasia, and vertebral epiphyseal metaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Reliweil), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature. (Item 40) A method for lengthening bones or increasing long bone growth in a subject that requires lengthening bones or increasing long bone growth, comprising administering to the subject a composition comprising a hydrophobic CNP salt as described in any one of items 1-29 or 44-52, wherein the administration lengthens bones or increases long bone growth. (Item 41) The method according to any one of items 38 to 40, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly. (Item 42) The method according to any one of items 38 to 41, wherein the composition is administered once a day, once a week, 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. (Item 43) The method according to any one of items 38 to 42, wherein the composition is an extended release composition. (Item 44) A hydrophobic salt of C-type natriuretic peptide (CNP), containing CNP that has formed a complex with a hydrophobic counterion. (Item 45) The hydrophobic salt according to item 44, further comprising the peptide and a cation complexed with a counterion. (Item 46) The hydrophobic salt according to item 44 or 45, wherein the hydrophobic counterion is selected from the group consisting of oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate. (Item 47) Cation is Zn 2+ or Ca 2+ A hydrophobic salt as described in any one of items 44-46. (Item 48) The hydrophobic salt according to any one of items 44 to 46, wherein the salt is selected from the group consisting of CNP-oleate, CNP-pamoate, CNP-deoxycholate, CNP-decanoate, and CNP-docusate. (Item 49) The aforementioned salt is CNP-Ca +2 (Oleate), CNP-Ca +2 (Pamoate), CNP-Ca +2 (Deoxycholate), CNP-Ca +2 (Decanoate), CNP-Ca +2 (Docusate), CNP-Zn +2 (Oleate), CNP-Zn +2 (Pamoate), CNP-Zn +2 (Deoxycholate), CNP-Zn +2 (decanoate), and CNP-Zn +2 A hydrophobic salt selected from the group consisting of (docusate), as described in any one of items 45-48. (Item 50) The aforementioned CNP PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-38)(SEQ ID NO: 2), QEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-37)(SEQ ID NO: 3), A hydrophobic salt as described in any one of items 44 to 49, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and salts thereof. (Item 51) A hydrophobic salt according to any one of items 44 to 50, wherein the CNP is CNP-acetate. (Item 52) A purified hydrophobic salt as described in any one of items 44-51. (Item 53) A composition comprising a hydrophobic CNP salt as described in any one of items 1-29 or 44-52, for use in the treatment of bone-related disorders or skeletal dysplasia, or for lengthening bone or increasing the growth of long bones. (Item 54) Use of compositions comprising hydrophobic CNP salts as described in any one of items 1-29 or 44-52 in the preparation of agents for treating bone-related disorders or skeletal dysplasia, or for lengthening bones or increasing the growth of long bones.

Claims

1. A composition comprising a hydrophobic salt of C-type natriuretic peptide (CNP), wherein the salt comprises the CNP complexed with a hydrophobic counterion and a polyvalent cation complexed with the peptide-counterion complex, wherein the CNP is DLRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-53)(Sequence ID 56); LRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-52) (Sequence ID 15); RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 16); VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence ID 17); DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 18); TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 20); SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 21); RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 22); AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 23); AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-43) (Sequence ID 24); WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-42) (Sequence ID 25); ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-41) (Sequence ID 26); RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-40) (Sequence ID 27); LLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-39) (Sequence ID 28); LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (Sequence ID 2); QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-37) (Sequence ID 3); EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-36) (Sequence ID 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-35) (Sequence ID 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (Sequence ID 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-33) (Sequence ID 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-32) (Sequence ID 32); RKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-31) (Sequence ID 33); KYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-30) (Sequence ID 34); YKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-29) (Sequence ID 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-28) (Sequence ID 36); GANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-27) (Sequence ID 37); ANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-26) (Sequence ID 38); NKKGLSKGCFGLKLDRIGSMSGLGC(CNP-25) (Sequence ID 39); KKGLSKGCFGLKLDRIGSMSGLGC(CNP-24) (Sequence ID 40); KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC(CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 42); SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 43); KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 44); GCFGLKLDRIGSMSGLGC(CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC(CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N); Sequence ID No. 49]; MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37; Sequence ID 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 55); PGQEHPNARRYRGANRRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6); PGQEHPQARRYRGAQRRRGLSRRGCFGLKLDRIGSMSGLGC (Sequence ID 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 7) A composition which is a CNP variant selected from the group consisting of the following.

2. The composition according to claim 1, wherein the CNP, hydrophobic counterion, and cation form a complex via non-covalent bonds.

3. The composition according to claim 1, wherein the cation is a metal cation.

4. The aforementioned hydrophobic counterion, i) Deprotonated fatty acids, deprotonated bile acids, ionic surfactants, naphthoates and their derivatives, nicotinates and their derivatives, alkyl sulfonates, dialkyl sulfosuccinates, phospholipids, alkyl sulfonates, aryl sulfonates, alkylbenzene sulfonates, alkyl sulfates, aryl sulfates, dextrans sulfates, alkylbenzene sulfates, and any combination thereof; and / or ii) Palmitate, deoxycholate, oleate, pamoate, nicotinate, dodecyl sulfate, docusate, myristate, palmitate, stearate, phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PL), phosphatidate, decanoate, 2-naphthalene sulfonate, 1-heptanesulfonate, 1-octanesulfonate monohydrate, 1-decanesulfonate, dodecyl sulfate, dextrans sulfate, and dodecylbenzenesulfonate A composition according to any one of claims 1 to 3, selected from the group consisting of the following.

5. The aforementioned CNP is PGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (Sequence ID 2), QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-37) (Sequence ID 3), A composition according to any one of claims 1 to 4, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and salts thereof.

6. The composition according to claim 5, wherein the CNP is CNP-acetate.

7. The cation is Zn ２＋ or Ca ２＋ The composition according to any one of claims 1 to 6.

8. The composition according to any one of claims 1 to 7, further comprising an excipient, a diluent, or a carrier.

9. where the salt is CNP-Ca ＋２ (oleate), CNP-Ca ＋２ (pamoate), CNP-Ca ＋２ (deoxycholate), CNP-Ca ＋２ (decanoate), CNP-Ca ＋２ (docusate), CNP-Zn ＋２ (oleate), CNP-Zn ＋２ (pamoate), CNP-Zn ＋２ (deoxycholate), CNP-Zn ＋２ (decanoate), and CNP-Zn ＋２ The composition according to any one of claims 1 to 7, selected from the group consisting of (docusate).

10. A sterile pharmaceutical composition comprising the composition described in any one of claims 1 to 9.

11. An extended-release composition comprising a salt of type C natriuretic peptide (CNP), wherein the salt comprises an electrostatically charged peptide complexed with a hydrophobic counterion and a polyvalent cation complexed with the peptide-counterion complex, and the CNP is DLRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-53)(Sequence ID 56); LRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-52) (Sequence ID 15); RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 16); VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence ID 17); DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 18); TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 20); SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 21); RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 22); AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 23); AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-43) (Sequence ID 24); WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-42) (Sequence ID 25); ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-41) (Sequence ID 26); RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-40) (Sequence ID 27); LLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-39) (Sequence ID 28); LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (Sequence ID 2); QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-37) (Sequence ID 3); EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-36) (Sequence ID 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-35) (Sequence ID 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (Sequence ID 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-33) (Sequence ID 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-32) (Sequence ID 32); RKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-31) (Sequence ID 33); KYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-30) (Sequence ID 34); YKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-29) (Sequence ID 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-28) (Sequence ID 36); GANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-27) (Sequence ID 37); ANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-26) (Sequence ID 38); NKKGLSKGCFGLKLDRIGSMSGLGC(CNP-25) (Sequence ID 39); KKGLSKGCFGLKLDRIGSMSGLGC(CNP-24) (Sequence ID 40); KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC(CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 42); SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 43); KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 44); GCFGLKLDRIGSMSGLGC(CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC(CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N); Sequence ID No. 49]; MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37; Sequence ID 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 55); PGQEHPNARRYRGANRRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6); PGQEHPQARRYRGAQRRRGLSRRGCFGLKLDRIGSMSGLGC (Sequence ID 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 7) An extended-release composition which is a CNP variant selected from the group consisting of the following.

12. The extended release composition according to claim 11, wherein a solid, semi-solid, gel, crystalline, amorphous, nanoparticle, microparticle, amorphous nanoparticle, amorphous microparticle, crystalline nanoparticle or crystalline microparticle of a peptide salt is resuspended in an aqueous solution or oil.

13. The extended release composition according to claim 12, wherein the oil contains a fatty acid, and the fatty acid is a C-6 to C-20 fatty acid.

14. At pH 7-7.6 (i) Less than 20% of the peptide is released by day 1, (ii) The extended release composition according to any one of claims 11 to 13, wherein approximately 90% of the peptide is released by day 7, or approximately 90% of the peptide is released by day 14, or approximately 90% of the peptide is released by day 31.

15. The aforementioned salt is CNP-Ca ＋２ (Oleate), CNP-Ca ＋２ (Pamoate), CNP-Ca ＋２ (Deoxycholate), CNP-Ca ＋２ (Decanoate), CNP-Ca ＋２ (Docusate), CNP-Zn ＋２ (Oleate), CNP-Zn ＋２ (Pamoate), CNP-Zn ＋２ (Deoxycholate), CNP-Zn ＋２ (decanoate), and CNP-Zn ＋２ An extended release composition according to any one of claims 11 to 13, selected from the group consisting of (docusate).

16. The extended-release composition according to any one of claims 11 to 15, further comprising a pharmaceutically acceptable excipient, diluent, or carrier.

17. A hydrophobic salt of C-type natriuretic peptide (CNP) complexed with a hydrophobic counterion and a polyvalent cation complexed with the peptide-counterion complex, wherein the CNP is DLRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-53)(Sequence ID 56); LRVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-52) (Sequence ID 15); RVDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-51) (Sequence ID 16); VDTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-50) (Sequence ID 17); DTKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-49) (Sequence ID 18); TKSRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-48) (Sequence ID 19); KSRAAWARLLQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-47) (Sequence ID 20); SRAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-46) (Sequence ID 21); RAAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-45) (Sequence ID 22); AAWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-44) (Sequence ID 23); AWARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-43) (Sequence ID 24); WARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-42) (Sequence ID 25); ARLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-41) (Sequence ID 26); RLLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-40) (Sequence ID 27); LLQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-39) (Sequence ID 28); LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (Sequence ID 2); QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-37) (Sequence ID 3); EHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-36) (Sequence ID 29); HPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-35) (Sequence ID 30); PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (Sequence ID 4); NARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-33) (Sequence ID 31); ARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-32) (Sequence ID 32); RKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-31) (Sequence ID 33); KYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-30) (Sequence ID 34); YKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-29) (Sequence ID 35); KGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-28) (Sequence ID 36); GANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-27) (Sequence ID 37); ANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-26) (Sequence ID 38); NKKGLSKGCFGLKLDRIGSMSGLGC(CNP-25) (Sequence ID 39); KKGLSKGCFGLKLDRIGSMSGLGC(CNP-24) (Sequence ID 40); KGLSKGCFGLKLDRIGSMSGLGC(CNP-23) (SEQ ID NO: 41); GLSKGCFGLKLDRIGSMSGLGC(CNP-22) (SEQ ID NO: 68); LSKGCFGLKLDRIGSMSGLGC(CNP-21) (Sequence ID 42); SKGCFGLKLDRIGSMSGLGC(CNP-20) (Sequence ID 43); KGCFGLKLDRIGSMSGLGC(CNP-19) (Sequence ID 44); GCFGLKLDRIGSMSGLGC(CNP-18) (SEQ ID NO: 45); CFGLKLDRIGSMSGLGC(CNP-17) (SEQ ID NO: 67); QEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[CNP-37(M32N); SEQ ID NO: 46]; MQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-CNP-37; SEQ ID NO: 47); PQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Pro-CNP-37; SEQ ID NO: 48); GQEHPNARKYKGANKGLSKGCFGLKLDRIGSNSGLGC[Gly-CNP-37(M32N); Sequence ID No. 49]; MGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(Met-Gly-CNP-37; Sequence ID 50); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37: SEQ ID NO: 51); GQEHPNARKYKGANPKGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 52); GQEHPNARKYKGANQKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 53); GQEHPNARKYKGANQQGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 54); GQEHPNARKYKGANKPGLSKGCFGLKLDRIGSMSGLGC (Sequence ID 55); PGQEHPNARRYRGANRRRGLSRGCFGLKLDRIGSMSGLGC (Sequence ID 6); PGQEHPQARRYRGAQRRRGLSRRGCFGLKLDRIGSMSGLGC (Sequence ID 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (Sequence No. 7) A hydrophobic salt that is a CNP variant selected from the group consisting of the following.

18. The hydrophobic salt according to claim 17, wherein the hydrophobic counterion is selected from the group consisting of oleate, deoxycholate, decanoate, pamoate, docusate, or dodecyl sulfate.

19. The cation is Zn ２＋ or Ca ２＋ The hydrophobic salt according to claim 17 or claim 18.

20. The hydrophobic salt according to any one of claims 17 to 19, wherein the salt is selected from the group consisting of CNP-oleate, CNP-pamoate, CNP-deoxycholate, CNP-decanoate, and CNP-docusate.

21. The aforementioned salt is CNP-Ca ＋２ (Oleate), CNP-Ca ＋２ (Pamoate), CNP-Ca ＋２ (Deoxycholate), CNP-Ca ＋２ (Decanoate), CNP-Ca ＋２ (Docusate), CNP-Zn ＋２ (Oleate), CNP-Zn ＋２ (Pamoate), CNP-Zn ＋２ (Deoxycholate), CNP-Zn ＋２ (decanoate), and CNP-Zn ＋２ A hydrophobic salt according to any one of claims 17 to 20, selected from the group consisting of (docusate).

22. The aforementioned CNP is PGQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37, Sequence ID 1) LQEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-38) (Sequence ID 2), QEHPNARKYKGANKGLSKGCFGLKLDRIGSMSGLGC(CNP-37) (Sequence ID 3), A hydrophobic salt according to any one of claims 17 to 21, selected from the group consisting of PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(CNP-34) (SEQ ID NO: 4) and salts thereof.

23. The hydrophobic salt according to any one of claims 17 to 22, wherein the CNP is CNP-acetate.

24. A pharmaceutical composition comprising a hydrophobic CNP salt according to any one of claims 17 to 22, for use in the treatment of bone-related disorders or skeletal dysplasia, or for lengthening bones or increasing the growth of long bones.

25. The aforementioned bone-related disorders or skeletal dysplasias include osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondrodysplasia, dwarfism, osteochondrodysplasia, fatal dysplasia, osteogenesis imperfecta, achondroplasia, chondrodysplasia punctata, isozygosyngeal chondrodysplasia, chondrodysplasia punctata, dysplasia of the limbs, congenital fatal hypophosphatasia, perinatal fatal osteogenesis imperfecta, short-rib chondrodroplasia, hypochondrodysplasia, rhombophyseal dysplasia, Janssen type metaphyseal dysplasia, congenital vertebral epiphyseal dysplasia, growth-impaired osteogenesis imperfecta, torsional dysplasia, congenital short femur, and Langer type metacalar. A pharmaceutical composition according to claim 24, selected from the group consisting of dysplasia, Niebergeld type midleg dysplasia, Robinnow syndrome, Reinhardt syndrome, achondroplasia, peripheral dysplasia, Nieist dysplasia, fibrochondroplasia, Roberts syndrome, distal intermediate limb dysplasia, brachylimia, Morquio syndrome, Nieist syndrome, complex organic dysplasia, and vertebral epiphyseal metaphysical dysplasia, NPR2 mutation, SHOX mutation (Turner syndrome / Reliweil), PTPN11 mutation (Noonan syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature.

26. The pharmaceutical composition according to claim 24 or claim 25, wherein the composition is an extended-release composition.

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