CNP VARIANTS, THEIR CONJUGATES AND COMPOSITIONS

Abstract

Provided herein are pharmaceutical compositions comprising variants and prodrugs of C-type natriuretic peptide (CNP) disclosed herein. Also provided herein are methods of using the disclosed compositions in treating bone-related disorders or skeletal dysplasia, as well as pharmaceutical kits comprising the disclosed compositions.

Description

CNP VARIANTS, CONJUGATES AND FORMULATIONS THEREOFCROSS REFERENCE TO RELATED APPLICATIONS

[0001] Priority is claimed to U.S. Provisional Application Nos. 63 / 597,246 (filed on November 8, 2023); 63 / 623,961 (filed on January 23, 2024); 63 / 651,801 (filed on March 24, 2024); 63 / 660,347 (filed on June 14, 2024); 63 / 695,491 (filed on September 17, 2024); and 63 / 711,678 (filed on October 24, 2024), each of which is incorporated herein by reference in entirety.INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

[0002] The Sequence Listing, which is a part of the present disclosure, is submitted concurrently with the specification as a xml file. The name of the xml file containing the Sequence Listing is “70213_Seqlisting.xml", which was created on November 7, 2024 and is 98,392 bytes in size. The subject matter of the Sequence Listing is incorporated herein in its entirety by reference.FIELD OF THE DISCLOSURE

[0003] The present disclosure, relates, in general, to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants and methods of use. The CNP variants are useful as therapeutic agents for the treatment of diseases responsive to CNP, including but not limited to bone-related disorders, such as skeletal dysplasias (e.g., achondroplasia).BACKGROUND

[0004] 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)) is a small, single chain peptide in a family of peptides (ANP, BNP, CNP) having a 17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and have important roles in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the generation of cyclic- guanosine monophosphate (cGMP) (J. Hypertens., 10: 1111-1 114 (1992)). CNP is expressed widely, including in the central nervous system, reproductive tract, bone and endothelium of blood vessels (Hypertension, 49: 419-426 (2007)).

[0005] In humans, CNP is initially produced from the natriuretic peptide precursor C (NPPC) gene as a single chain 126-amino acid pre-pro polypeptide (Biochem. Biophys. Res. Commun., 168: 863-870 (1990)). Removal of the signal peptide yields pro-CNP,and further cleavage by the endoprotease furin generates an active 53-amino acid peptide (CNP-53), which is secreted and cleaved again by an unknown enzyme to produce the mature 22-amino acid peptide (CNP- 22) (Wu, J. Biol. Chem. 278: 25847- 852 (2003)). CNP-53 and CNP-22 differ in their distribution, with CNP-53 predominating 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 similarly to NPR-B.

[0006] Downstream signaling mediated by cGMP generation influences a diverse array of biological processes that include endochondral ossification. For example, knockout of either CNP or NPR-B in mouse models results in animals having a dwarfed phenotype with shorter long bones and vertebrae. Mutations in human NPR-B that block proper CNP signaling have been identified and result in dwarfism (Olney, etai, J. Clin.Endocrinol. Metab. 91(4): 1229-1232 (2006); Bartels, et ai, Am. J. Hum. Genet. 75: 27- 34 (2004)). In contrast, mice engineered to produce elevated levels of CNP display elongated long bones and vertebrae.

[0007] Therapeutic use of CNP (CNP22) has been limited by its short plasma half-life, which has been shown to be 2.6 minutes in vivo in humans (J Clin. Endocrinol. Metab., 78: 1428-35 (1994)). A CNP variant having a longer in vivo serum half-life and exhibiting similar or improved activity to that of wild-type CNP is important for a sustainable therapeutic strategy.SUMMARY

[0008] The present disclosure relates to novel variants of C-type natriuretic peptide (CNP) having increased circulating half-life and stability in aqueous media, pharmaceutical compositions comprising such CNP variants, and methods of using such CNP variants to treat disorders responsive to CNP, including but not limited to bone- related disorders such as achondroplasia.

[0009] In various embodiments, the disclosure provides a variant of C-type natriuretic peptide (CNP) selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1);PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 6); andPGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC(SEQ ID NO: 5).

[0010] In various embodiments, the disclosure provides a variant of C-type natriuretic peptide (CNP) selected from the group consisting of PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7).

[0011] In various embodiments, the variant peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or an NH2 group at the C-terminus.

[0012] In various embodiments, the variant peptide comprises a conjugate moiety. In various embodiments, the conjugate moiety is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugate moiety is on a lysine residue. In various embodiments, the conjugate moiety comprises one or more acid moieties. In various embodiments, the acid moiety is a hydrophobic acid.

[0013] In various embodiments, the conjugate moiety comprises one or more acid moieties linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu). In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6- dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu) or OEG (8-amino-3,6- dioxaoctanoic acid). In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu) linked to one or two or more OEG (8- amino-3,6-dioxaoctanoic acid). In various embodiments, the acid moiety is a fatty acid. Exemplary fatty acids include short chain, medium chain, or long chain fatty acids, or a dicarboxylic fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. Contemplated are C-6 to C-20 fatty acids, including but not limited to, C-6, C-8, C-10, C-12, C-14, C-16, C-18 or C-20 fatty acids, saturated or unsaturated. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids of the same.

[0014] In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-yGlu-C18DA. In various embodiments, the acid moiety and the hydrophilic spacer have the structure:, whereinrepresents the point of attachment to a CNP variant. In various embodiments, represents the point of attachment to ahydrolysable linker, wherein the hydrolysable linker is attached to a CNP variant. In various embodiments, the hydrolysable linker is capable of releasing intact CNP variant.

[0015] In various embodiments, the CNP variant has the structure:

[0016] In various embodiments, the International Union of Pure and Applied Chemistry (IUPAC) name of the CNP variant is(4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2- ((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl- L- glutaminyl- L-glutamyl- L-histidyl)pyrrolidine-2-carboxamido)-4-amino-4- oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4- hydroxyphenyl)propanamido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4- oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)acetamido)-4- methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl- 28-(( S)-sec-butyl)-34-(carboxymethyl)-40-(( S)-33,51-dicarboxy-8-(2-hydroxyethyl)- 6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazahenpentacontyl)-31- (3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)- 6,9,12,15,18,21 ,24,27,30,33,36,39,42,45,48,51 -hexadecaoxo-1 ,2-d ith ia- 5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4- carboxylic acid.

[0017] In various embodiments, the chemical formula of the CNP variant is C217H363N61O65S3

[0018] In various embodiments, the exact mass of the CNP variant is 4959.61.

[0019] In various embodiments, the molecular weight of the CNP variant is 4962.83.

[0020] In various embodiments, the CNP variant with the conjugate moiety is a component of modified release composition. In various embodiments, the modified release composition is an extended release composition. In various embodiments, the CNP variant comprising a conjugate moiety and hydrolysable linker is capable of releasing the CNP variant, wherein (i) less than about 20% of CNP variant is released by day 1; and (ii) about 90% of the CNP variant is released weekly, or about 90% of the CNP variant is released bi-weekly, or about 90% of the CNP variant is released monthly, at pH 7 to 7.6.

[0021] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of peptide is released by day 1, at pH 7.0 to 7.6; and (ii) about 90% of peptide is released weekly, or about 90% of peptide is released bi-weekly, or about 90% of peptide is released monthly, at pH 7 to 7.6. It is further contemplated that (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% about 65%, about 70%, or about 75% of peptide is released by day 1 , at pH 7.0 to 7.6; and (ii) about 70%, about 80%, or about 90% of peptide is released weekly; or about 70%, about 80%, or about 90% of peptide is released bi-weekly; or about 70%,about 80%, or about 90% of peptide is released every three weeks; or about 70%, about 80%, or about 90% of peptide is released monthly, at p H 7 to 7.6; or alternatively ii) about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released weekly; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released bi-weekly; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released every three weeks; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released monthly, at pH 7 to 7.6.

[0022] In va ri o us embodiments, the variant has the structure:PGQEH PQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU-C18DA) LDRIGSMSGLGC (SEQ ID NO: 5), orAC-PGQEH PQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8) .

[0023] In various embodiments, the variant is selected from the group consisting of Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8); Ac- PGQEH PNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 9); Ac- PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10); Ac- PGQEH PNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 11); Ac- PGQEH PQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 12); Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2(SEQ ID NO: 13); and Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14).

[0024] In various embodiments, the variant comprises one or more linker groups. In various embodiments, the linker is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is on a lysine residue.

[0025] In various embodiments, the linker is a cleavable or lysable linker. In some instances of those embodiments, the linker is a hydrolysable linker.

[0026] In various embodiments, the CNP variant is attached to the conjugate moiety via the linker. In various embodiments, the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety. In various embodiments, the linker is aminoethoxy-2-ethoxy acetic acid (AEEA). In various embodiments, the linker is a bicin- type or peptoid linker, which refers to a linker having a similar cleavage mechanism as bicin (bis-2-hydroxyethylglycinamide), but cleaving instead via an asymmetric N-alkyl peptide, i.e., a peptoid. In various embodiments, the linker is an electronic linker basedon nonenzymatic p-elimination. In various embodiments, the electronic linker comprises an SO2moiety. Examples of linkers as illustrated in a CNP conjugate are set out in Figure 1. See also Santi, et.al., Proc Natl Acad Sci USA 109:621 1- 6216, 2012). In various embodiments, cleavage of the cleavable or lysable linker, particularly the hydrolysable linker, releases the CNP variant from the conjugate moiety. An exemplary embodiment where this can occur is depicted in Figure 9.

[0027] In various embodiments, the conjugate moiety is a synthetic polymeric group. In various embodiments, the variant comprises a synthetic polymeric group coupled to the variant through a hydrolysable linker. In various embodiments, the synthetic polymeric group comprises a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG). In various embodiments, the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a 6 to 20 atom chain length. For purposes of this conjugate, the synthetic polymeric group is not a peptide.

[0028] In various embodiments, the variant peptide is made synthetically.

[0029] In various embodiments, the variant peptide is stable for 10 days at about 37°C, pH 7.0 to 7.6. In various embodiments, the variant peptide is stable for at least 10 days at about 37° C, pH 7.0 to 7.4. In various embodiments, the variant peptide is stable for at least 10 days at about 37° C, pH 7.2 to 7.6.

[0030] In various embodiments, the variant peptides are stable to deamidation. In various embodiments, the variant peptides are stable to oxidation. In various embodiments, the variant peptides are stable to deamidation, and / or oxidation, or combinations thereof. In various embodiments, methionine is replaced by nor-leucine. In various embodiments, there is little to no detectable deamidation after 10 days.

[0031] In various embodiments, the variant peptide has a half-life of about 10 days at about 37° C, pH 7.0 to 7.6. In various embodiments, the variant peptide has a half-life of about 10 days at about 37° C, pH 7.0 to 7.4. In various embodiments, the variant peptide has a half-life of about 10 days at about 37° C, pH 7.2 to 7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37° C, pH 7.0 to 7.6. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37° C, pH 7.0 to 7.4. In various embodiments, the variant peptide has a half-life of at least 10 days at about 37° C, pH 7.2 to 7.6. In various embodiments, the half-lifeis at least about 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 2 1 days, 22 days, 23 days, 24 days, 25 days, 30 days or more.

[0032] In various embodiments, the variant peptide has an EC50 from 0.1 to 10 nM in a cGMP assay. In various embodiments, the variant peptide has an EC50 from 0.1 to 25 nM in a cGMP assay.

[0033] In various embodiments, greater than 45% of the variant peptide is detected after 10 days in aqueous media at physiological conditions, e.g., about 37° C, pH 7.0 to 7.6. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the variant peptide is detected after 10 days in aqueous media at physiological conditions, e.g., about 37° C, pH 7.0 to 7.6.

[0034] In various embodiments, greater than 45% of the variant peptide is detected after 10 days in aqueous media at 37° C, pH 7.4. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the variant peptide is detected after 10 days in aqueous media at 37° C, pH 7.4.

[0035] In various embodiments, greater than 45% of the variant peptide is detected after 10 days in plasma at physiological conditions, e.g., about 37° C, pH 7.0 to 7.6. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the variant peptide is detected after 10 days in plasma at physiological conditions, e.g., about 37° C, pH 7.0 to 7.6.

[0036] In various embodiments, greater than 45% of the variant peptide is detected after 10 days in plasma at 37° C, pH 7.4. In various embodiments, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% of the variant peptide is detected after 10 days in plasma at 37° C, pH 7.4.

[0037] In various embodiments, the variant peptide is conjugated to a lipid, fatty acid, hydrophilic spacer, or linker, or optionally combinations thereof. In various embodiments, the linker is a hydrophilic polymer moiety. In various embodiments, the hydrophilic polymer moiety is a synthetic hydrophilic polymer moiety.

[0038] In various embodiments, the variant peptide has a longer half-life compared to Pro- Gly-CNP37. In various embodiments, the variant peptide has a longer half-life compared to CNP-22. In various embodiments, the variant peptide has a longer half-life compared to Pro- Gly-CNP37 and / or CNP-22. In various embodiments, the variant peptide has a longer half-life compared to Pro-Gly-CNP37 and / or CNP-22 in vitro and / or in vivo. In various embodiments, the CNP prodrug composition exhibits lower Cmax andhigher AUC compared to free drug, e.g., an equivalent composition lacking an acid moiety, a spacer, and a hydrolysable linker.

[0039] The disclosure further provides a pharmaceutical composition comprising a CNP variant described herein, and a pharmaceutically acceptable excipient, carrier or diluent.

[0040] In various embodiments, the composition is a lyophilized formulation prepared from a formulation that comprises a citric acid / citrate buffer or an acetic acid / acetate buffer having a pH from about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a formulation that further comprises an isotonicity-adjusting agent or a bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a formulation that further comprises an antioxidant selected from the group consisting of methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied as a lyophilized powder for reconstitution from 0.8 mg to 10 mg. In various embodiments, the CNP variant composition is supplied as a 0.8-mg or 2-mg lyophilized, preservative-free powder for reconstitution.

[0041] In various embodiments, the composition is a lyophilized formulation prepared from a formulation that comprises a histidine buffer (including salts thereof, solvates thereof, and solvates of salts thereof) or an L-histidine buffer having a pH from about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a formulation that further comprises an isotonicity-adjusting agent or a bulking agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a formulation that further comprises an antioxidant selected from the group consisting of methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied as a lyophilized powder for reconstitution from 1 mg to 300 mg. For example, in some embodiments the CNP variant composition is supplied as a lyophilized powder in an amount of 10 to 290 mg (e.g., 25 to 250 mg, 50 to 200 mg, and 75 to 150 mg). In some embodiments, the CNP variant composition is supplied as a lyophilized powder in an amount of 10 to 50 mg (e.g., 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, and 50 mg). In various embodiments, the CNP variant composition is supplied as a 13 mg or 39 mg lyophilized, preservative-free powder for reconstitution. Also contemplated herein are formulations comprising (a) a CNPvariant peptide described herein and (b) one or more components selected from the group consisting of a buffering agent, an isotonicity agent, a stabilizer and an anti-adsorbent agent. In particularly preferred embodiments, buffering agents employed in the formulations may be L-histidine, histidine monohydrochloride monohydrate, or a combination of the two. In yet other preferred embodiments, isotonicity agents employed in the formulations of the present invention may be trehalose dihydrate, D-mannitol, or a combination of the two. In other preferred embodiments, the stabilizer employed in the formulations of the present invention is L-methionine. In yet other preferred embodiments, the anti-adsorbent agent employed in the formulations of the present invention is polysorbate 80. In various embodiments, the formulations of the present invention are lyophilized, in liquid form, or in liquid form that has been reconstituted from a previously lyophilized form. In certain embodiments, the formulations of the present invention are preservative-free and, optionally, may be contained within a type 1 untreated borosilicate glass vial. Optionally, the formulations of the present invention have a pH in the range of between about 5.0 and about 6.0 (e.g., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, and 6.0). In some embodiments, the disclosed formulations have a pH of about 5.5. In other embodiments, the formulations of the present invention comprise a CNP variant peptide as described herein, L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine and polysorbate 80. In certain preferred embodiments, the CNP variant peptide is present at a concentration between about 10.0 mg / ml and about 30.0 mg / ml, the L-histidine is present at a concentration between about 0.18 mg / ml and about 0.50 mg / ml, the histidine monohydrochloride monohydrate is present at a concentration between about 0.75 mg / ml and about 2.26 mg / ml, the trehalose dihydrate is present at a concentration between about 30 mg / ml and about 70 mg / ml, the D-mannitol is present at a concentration between about 10 mg / ml and about 20.0 mg / ml, the L-methionine is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml and the polysorbate 80 is present at a concentration between about 0.01 mg / ml and about 0.1 mg / ml. In other embodiments, a particularly preferred embodiments, the CNP variant is present at a concentration of about 10.0 mg / ml or 30 mg / ml, the L-histidine is present at a concentration of about 0.347 mg / ml, the histidine monohydrochloride monohydrate is present at a concentration of about 1.627 mg / ml, the trehalose dihydrate is present at a concentration of about 58.00 mg / ml, the D-mannitol is present at a concentration of about 15.0 mg / ml, the L-methionine is present at a concentration of about 0.73 mg / ml and the polysorbate 80 is present at a concentration of about 0.05 mg / ml. In other embodiments, the CNP variant peptide is present at a concentration between about 0.4 mg / ml and about 3.5 mg / ml, the citric acid monohydrate is present at a concentrationbetween about 0.15 mg / ml and about 0.40 mg / ml, the sodium citrate dihydrate is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml, the trehalose dihydrate is present at a concentration between about 30 mg / ml and about 70 mg / ml, the D-mannitol is present at a concentration between about 10 mg / ml and about 20.0 mg / ml, the L-methionine is present at a concentration between about 0.5 mg / ml and about 1.5 mg / ml and the polysorbate 80 is present at a concentration between about 0.01 mg / ml and about 0.1 mg / ml. In another embodiment, the CNP variant is present at a concentration of about 0.8 mg / ml, 2.0 mg / ml, or 5.0 mg / ml, the citric acid monohydrate is present at a concentration of about 0.28 mg / ml, the sodium citrate dihydrate is present at a concentration of about 1.08 mg / ml, the trehalose dihydrate is present at a concentration of about 58.01 mg / ml, the D- mannitol is present at a concentration of about 15.0 mg / ml, the L-methionine is present at a concentration of about 0.73 mg / ml and the polysorbate 80 is present at a concentration of about 0.05 mg / ml.

[0042] In various embodiments, the composition is an extended release composition.

[0043] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (BMN 111) (SEQ ID NO: 1).

[0044] Also provided is a method of treating a bone-related disorder or skeletal dysplasia in a subject in need thereof comprising administering to the subject a composition comprising a CNP variant as described herein.

[0045] In various embodiments, the bone-related disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia.

[0046] In various embodiments, the bone-related disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, spondyloepimetaphyseal dysplasia and osteoporosis.

[0047] In various embodiments the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other skeletal dysplasias.

[0048] In various embodiments, the bone-related disorder, skeletal dysplasia or short stature disorder results from an NPR2 mutation, SHOX mutation (Turner’s syndrome / Leri Weill), or PTPN1 1 mutations (Noonan’s syndrome).

[0049] In various embodiments, the bone-related disorder, skeletal dysplasia or short stature disorder results from an NPR2 mutation, SHOX mutation (Turner’s syndrome / Leri Weill), or PTPN1 1 mutations (Noonan’s syndrome), or insulin growth factor 1 receptor (IGF1R).

[0050] In various embodiments, the CNP variants are useful to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature which is also known as dominant inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is a result of a mutation in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0051] In various embodiments, the CNP variants are useful to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature which is also known as dominant inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is a result of a mutation incollagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, SHOX, NPR2, NPPC, or FGFR3.

[0052] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.

[0053] In various embodiments, the bone-related disorder, skeletal dysplasia or short stature disorder results from a RASopathy. In various embodiments, the RASopathy is Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1 , or LEOPARD syndrome.

[0054] In one embodiment, the RASopathy is hereditary gingival fibromatosis type 1.

[0055] In various embodiments, the CNP variants are useful to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature which is also known as dominant inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is a result of a mutation in collagen (COL2A1 , COL11 A1 , COL9A2, COL10), aggrecan (ACAN), indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R).

[0056] In various embodiments, the CNP variants are useful to treat growth plate disorders and short stature, including familial short stature, dominant familial short stature which is also known as dominant inherited short stature, or idiopathic short stature. In various embodiments, the short stature or growth plate disorder is a result of a mutation in collagen (COL2A1 , COL11 A1 , COL9A2, COL10), aggrecan (ACAN), indian hedgehog (IHH), PTPN11, S H OX, NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1R).

[0057] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.

[0058] In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and having at least one parent with a height SDS of less than -1.0, - 1.5, -2.0 or -2.5, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of less than -1.0, -1.5, -2.0, -2.25, -2.5, or -3.0, and having at least one parent with a height SDS of less than -1.0, - 1.5, -2.0, -2.25 or -2.5, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in a gene 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 combinations thereof. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. In various embodiments, the short stature is associated with one or more mutations in a gene associated with short stature, such as, collagen (COL2A1 , COL11 A1 , COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, S H OX , NPR2, NPPC, FGFR3, or insulin growth factor 1 receptor (IGF1 R), or combinations thereof. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.

[0059] In various embodiments, the short stature is a result of mutations in multiple genes as determined by 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 mutation 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. Polygenic risk scores (PRS) were calculated for height as described in Example 4. PRS 1 refers to the lowest height, PRS 5 the tallest height.

[0060] Also provided is a method of elongating a bone or increasing long bone growth in a subject in need thereof, comprising administering to the subject a composition as described herein, and wherein the administering elongates a bone or increases long bone growth.

[0061] In various embodiments, the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.

[0062] In various embodiments, the composition provides an extended release composition.

[0063] In various embodiments, the composition is administered once every 5 days, once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

[0064] In various embodiments, the administration increases the annualized growth velocity (AGV) in the subject at 12 months, optionally compared to baseline or to a normal control. In various embodiments, the AGV in the subject increases over 1 year or over 2 years, or more.

[0065] In various embodiments, the administration improves the height Z score at 12 months, optionally compared to baseline or to a normal control.

[0066] In various embodiments, the subject is greater than 3 years old. In various embodiments, the subject is between 3 and 17 years old. In various embodiments, the subject has open epiphyses.

[0067] In various embodiments, the composition is administered at a dose from about 5 μg / kg to 500 μg / kg or from about 15 μg / kg to 350 μg / kg . In various embodiments, the CNP Prodrug is administered at a dose from about 5 μg / kg to 500 μg / kg , from about 15 μg / kg to 350 μg / kg , from about 25 μg / kg to 300 μg / kg , from about 50 μg / kg to 250 μg / kg or from about 75 μg / kg to 200 μg / kg . In various embodiments, the CNP variant is administered at a dose of about 15 μg / kg , 20 μg / kg , 25 μg / kg , 30 μg / kg , 35 μg / kg , 40 μg / kg , 45 μg / kg , 50 μg / kg , 60 μg / kg , 70 μg / kg , 75 μg / kg , 80 μg / kg , 90 μg / kg , 100 μg / kg , 125 μg / kg , 150 μg / kg , 175 μg / kg , 200 μg / kg , 225 μg / kg , 250 μg / kg , 275 μg / kg , 300 μg / kg , 325 μg / kg , 350 μg / kg , 400 μg / kg , 450 μg / kg , or 500 μg / kg .

[0068] In various embodiments, the administration does not result in cardiovascular (CV) side effects. In various embodiments, the CV side effect is change in systemic blood pressure, mean arterial pressure, systolic and / or diastolic blood pressures, pulse pressure or heart rate. In various embodiments, subjects receiving a CNP Prodrug to treat skeletal dysplasia as described herein have reduced or none of the cardiovascular (CV) side effects such as changes in systemic blood pressures (mean arterial pressure, systolic and diastolic blood pressures, pulse pressure) and heart rate observed with administration of non-prodrug CNP variant.

[0069] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0070] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or an NH2group 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 hydrolysable linker.

[0071] In various embodiments, the disclosure provides a method of elongating a bone or increasing long bone growth in a subject in need thereof, comprising administering to the subject a composition comprising a CNP variant described herein, and wherein the administering elongates a bone or increases long bone growth.

[0072] In various embodiments, the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.

[0073] In various embodiments, the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

[0074] In various embodiments, the composition is an extended release composition.

[0075] Further contemplated is a method of treating a CNP-responsive condition or disorder, comprising administering a CNP variant or composition as described herein to a subject, and monitoring the level of at least one bone- or cartilage-associated biomarker in the subject, wherein an increase in the level of the at least one bone- or cartilage- associated biomarker indicates a therapeutic effect of the CNP peptide or variant on the subject or the condition or disorder.

[0076] Further contemplated is a method of overcoming cell growth arrest induced by a constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3) comprising contacting a cell expressing the constitutively active FGFR-3 with a CNP variant or a composition as described herein.

[0077] Further contemplated is a method of stimulating cGMP production in a cell expressing natriuretic peptide receptor B (NPR-B) comprising contacting the cell expressing NPR-B with a CNP variant or a composition as described herein.

[0078] In various embodiments, the method further comprises adjusting the amount or frequency of administration of the CNP peptide or variant as described herein, wherein i) the amount or frequency of administration of the CNP peptide or variant is increased if the level of the at least one bone- or cartilage-associated biomarker is below a target level; or ii) the amount or frequency of administration of the CNP peptide or variant is decreased if the level of the at least one bone- or cartilage-associated biomarker is above a target level.

[0079] In various embodiments, the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, Collagen Type I C- Telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides oftype I procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X , and alkaline phosphatase.

[0080] In various embodiments, the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, Collagen Type I C-Telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N- propeptide (PINP), cross-linked N-telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP- variant bioactivity. Cartilage- and bone-associated biomarkers can be measured in any appropriate biological sample, including but not limited to tissues, blood, serum, plasma, cerebrospinal fluid, synovial fluid and urine. In some embodiments, the biomarkers are measured in blood, plasma, serum or urine from subjects undergoing efficacy / pharmacodynamic in vivo studies and / or from the conditioned media of ex vivo studies.

[0081] In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0082] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on 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 an 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 hydrolysable linker.

[0083] Also provided is a method of making a CNP variant described herein comprising synthesizing the peptide on a solid-phase resin using Fmoc amino acids.

[0084] In various embodiments, the method comprises acetylating the peptides by reacting the resin with NMP / AC2O / DIEA (10:1:0.1, v / v / v).

[0085] In various embodiments, the method comprises conjugating the peptide to a conjugate moiety, optionally on a lysine residue. In various embodiments, the methodcomprises cleaving the protective amino group on lysine, reacting the peptide with 2x Fmoc-amino PEG(2) followed by amino acid, followed by conjugation of the lipid or fatty acid moiety.

[0086] It is understood that each feature or embodiment, or combination, described herein is a non-limiting, illustrative example of any of the aspects of the invention and, as such, is meant to be combinable with any other feature or embodiment, or combination, described herein. For example, where features are described with language such as “one embodiment”, “some embodiments”, “certain embodiments”, “further embodiment”, “specific exemplary embodiments”, and / or “another embodiment”, each of these types of embodiments is a non-limiting example of a feature that is intended to be combined with any other feature, or combination of features, described herein without having to list every possible combination.

[0087] Such features or combinations of features apply to any of the aspects of the invention. Where examples of values falling within ranges are disclosed, any of these examples are contemplated as possible endpoints of a range, any and all numeric values between such endpoints are contemplated, and any and all combinations of upper and lower endpoints are envisioned.BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 illustrates use of a peptoid or electronic linker in a CNP conjugate described herein.

[0089] Figure 2 shows the stability of CNP variants in human plasma over a period of 24 hrs.

[0090] Figure 3 shows the stability of CNP variants under different culture conditions.

[0091] Figure 43 shows the effects of a CNP variant (Pro-Gly-CNP) on cells carrying either NPR2 homozygous or heterozygous mutations, as measured by cGMP stimulation.

[0092] Figure 5 shows the nucleotide and predicted protein sequence of the first exon in NPR2 mutant clones transfected into RCS cells.

[0093] Figure 6 shows exemplary NPR2 mutations analyzed for response to CNP.

[0094] Figure 7 shows exemplary mutations associated with short stature in FGFR3,IGF1R. NPPC, NPR2 and SHOX.

[0095] Figures 8A-8F illustrate the combined effect of PRS and rare coding variants on Height. Fig. 8A. Effects on height as a quantitative trait, samples were divided in five groups based on their PRS, violin-plots with horizontal lines representing the 25%, 50% and 75% percentile of height. Samples were grouped by carrying status of missense, loss of function or None in any of the five core genes. Fig. 8B. Effect reflected on Odds ratios for “Idiopathic Short Stature” or ISS. Odds for ISS using PRS=3 as reference vs the other PRS groups. Fig. 8C. Odds for ISS using PRS = 1 non-carriers as reference vs having missense and / or loss of function variants in core genes. Fig. 8D. Odds for ISS using PRS=2 non-carriers as reference vs having missense and / or loss of function variants in core genes. Fig. 8E. Odds for ISS using PRS =3 non-carriers as reference vs having missense and / or loss of function variants in core genes. Fig . 8 F. Odds for ISS using PRS=4 non-carriers as reference vs having missense and / or loss of function variants in core genes.

[0096] Figure 9 illustrates the mechanism of linker cleavage in an embodiment of the disclosure.

[0097] Figure 10 shows the components of a CNP variant, including the CNP, lipid, and linker.

[0098] Figure 11 shows the structure and chemical name of a CNP variant disclosed herein.

[0099] Figure 12 shows a schematic of Phase 1 dosing study of CNP Prodrug in healthy volunteers.

[0100] Figure 13 shows visual appearance of CNP prodrug citrate-based formulations

[0101] Figure 14 shows CNP variant content when stored in different formulation buffers was analyzed over time, at a range of temperatures from -20 to 37° C.

[0102] Figure 15 shows free drug content release from different formulation buffers analyzed over time at a range of temperatures.

[0103] Figure 16 shows linker area of the drug conjugate when stored in different formulation buffers over time at a range of temperatures.

[0104] Figure 17 shows CNP conjugate content when stored in different formulation buffers was analyzed over time at a range of temperatures.

[0105] Figure 18 shows variant concentration as measured by UV when stored in different formulation buffers was analyzed over time.

[0106] Figure 19 shows variant content in formulations with different excipients (histidine, glycine and sorbitol) over time at pH 5.5 or 6.

[0107] Figure 20 shows results of a manufacturing feasibility study for CNP prodrug.

[0108] Figure 21 shows batch analysis of formulated CNP prodrug in histidine or acetate buffer with trehalose and mannitol at different ratios (4:1, or 1:4, respectively).

[0109] Figure 22 shows area under the curve after reverse phase chromatography.

[0110] Figure 23 shows in-use stability and free drug content assessed over time at a range of temperatures and pH 5.2 to 6.0.

[0111] Figure 24 shows visual comparison of CNP prodrug in different formulations.

[0112] Figure 25 shows naso-anal lengths in WT and Raf1+ / L613Vmice after 6 weeks of treatment.

[0113] Figure 26 shows naso-anal lengths in WT and Rit+ / - mice treated for 10 weeks. IP, intraperitoneal; MEKi, mitogen-activated protein kinase kinase inhibitor; N A, naso anal;QAD, every other day; QD, daily; SC, subcutaneous; Veh., vehicle; WT, wild type. # Significantly different from corresponding vehicle-treated group by un-paired t-test. * Significantly different from CNP-treated genotype-match group by un-paired t-test.

[0114] Figure 27 shows whole body pCT images of WT mice treated with vehicle (left) or CNP Prodrug (right). pCT, micro computed tomography; WT, wild-type.

[0115] Figure 28 shows plasma pharmacokinetics of CNP prodrug and released vosoritide in mice following a single SC dose.

[0116] Figure 29 shows mean (±SD) plasma vosoritide and CNP prodrug concentrationtime profiles after a single dose of CNP prodrug.

[0117] Figure 30A and 30B show mean (± SD) concentration vs. time curves for CNP prodrug (Fig. 30A) and vosoritide (Fig. 30B) in NHP plasma following IV or SC administration of CNP prodrug.

[0118] Figure 31 A and 31 B show mean (± SD) concentration vs. time curves for CNP prodrug (Fig. 31A) and vosoritide (Fig. 31B) in male and female NHP plasma following IV or SC administration of CNP prodrug.

[0119] Figure 32A shows mean (±SD) plasma vosoritide and CNP prodrug concentrationtime and dose normalized mean plasma vosoritide and CNP prodrug concentration-time profile. Figure 32B shows mean (±SD) plasma vosoritide and CNP prodrug concentration-time and dose normalized mean plasma vosoritide and CNP prodrug concentration-time profile.

[0120] Figure 33 shows mean (±sd) plasma vosoritide and CNP prodrug concentrationtime comparing Day 1 vs Day 22.

[0121] Figure 34 shows binding of CNP Prodrug to NPR-B and NPR-C compared to BMN111.DETAILED DESCRIPTION

[0122] The present disclosure relates to stable CNP variants useful in treating skeletal dysplasias and bone growth disorders.

[0123] As used in the specification and the appended claims, the indefinite articles “a” and “an” and the definite article “the” include plural as well as singular referents unless the context clearly dictates otherwise.

[0124] The term “about” or “approximately” means an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term “about” or “approximately” means within 1 , 2, 3, or 4 standard deviations. In certain embodiments, the term “about” or “approximately” means 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 term “about” or “approximately” precedes the first numerical value in a series of two or more numerical values, it is understood that the term “about” or “approximately” applies to each one of the numerical values in that series.

[0125] The term “C-type natriuretic peptide” or “CNP” refers to a small, single chain peptide having a 17-amino acid loop structure at the C-terminal end (GenBank Accession No. NP_077720, for the CNP precursor protein, NPPC) and variants thereof. The 17-mer CNP loop structure is also referred to as CNP 17, the CNP ring, or CNP cyclic domain. CNP includes the active 53-amino acid peptide (CNP-53) and the mature 22-amino acid peptide (CNP-22), and peptides of varying lengths between the two peptides.

[0126] In various embodiments, a “CNP variant” is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to the wild type NPPC over the same number of amino acid residues. It is further contemplated that a CNP variant peptide may comprise from about 1 to about 53, or 1 to 39, or 1 to 38, or 1 to 37, or 1 to 35, or 1 to 34, or 1 to 31, or 1 to 27, or 1 to 22, or 10 to 35, or about 15 to about 37 residuesof the NPPC polypeptide. In one embodiment, a CNP variant may comprise a sequence of 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 acids derived from the NPPC polypeptide.

[0127] The term “conjugate moiety” refers to a moiety that is conjugated to the variant peptide. Conjugate moieties include a lipid, fatty acid, hydrophilic spacer, synthetic polymer, linker, or optionally, combinations thereof.

[0128] The term “effective amount” refers to a dosage sufficient to produce a desired result on a health condition, pathology, or disease of a subject or for a diagnostic purpose. The desired result may comprise a subjective or objective improvement in the recipient of the dosage. "Therapeutically effective amount" refers to that amount of an agent effective to produce the intended beneficial effect on health. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation. It will be understood that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors, including the activity of the specific compound employed; the bioavailability, metabolic stability, rate of excretion and length of action of that compound; the mode and time of administration of the compound; the age, body weight, general health, sex, and diet of the patient; and the severity of the particular condition.

[0129] "Substantially pure" or "isolated" means an object species is the predominant species present (i.e., on a molar basis, more abundant than any other individual macromolecular species in the composition), and a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. In one embodiment, a substantially pure composition means that the species of interest comprises at least about 70%, 75%, 80%, 85%, 90%, 95%, 98% or more of the macromolecular species present in the composition on a molar or weight basis. The object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) if the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), stabilizers (e.g., BSA), and elemental ion species are not considered macromolecular species for purposes of this definition. In an embodiment, the compounds of the disclosure are substantially pure or isolated. In another embodiment, the compounds of the disclosure are substantially pure or isolated with respect to the macromolecular starting materials used in their production. In yet another embodiment, the pharmaceutical compositions of the disclosure comprise a substantially pure or isolated CNP variant admixed with one or more pharmaceuticallyacceptable excipients, carriers or diluents, and optionally with another biologically active agent.

[0130] "Treatment" refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. In certain embodiments, “treatment” refers to administration of a compound or composition to a subject for therapeutic, prophylactic or diagnostic purposes.

[0131] A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs of the disease, for the purpose of decreasing the risk of developing pathology. The compounds or compositions of the disclosure may be given as a prophylactic treatment to reduce the likelihood of developing a pathology or to minimize the severity of the pathology, if developed.

[0132] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of pathology for the purpose of diminishing or eliminating those signs or symptoms. The signs or symptoms may be biochemical, cellular, histological, functional or physical, subjective or objective. The compounds of the disclosure may also be given as a therapeutic treatment or for diagnosis.

[0133] “Diagnostic" means identifying the presence, extent and / or nature of a pathologic condition. Diagnostic methods differ in their specificity and selectivity. While a particular diagnostic method may not provide a definitive diagnosis of a condition, it suffices if the method provides a positive indication that aids in diagnosis.

[0134] “Bone- or cartilage-associated biomarker” or “bone- or cartilage-associated marker” refers to a growth factor, enzyme, protein, or other detectable biological substance or moiety whose level is increased or decreased in association with, e.g., cartilage turnover, cartilage formation, cartilage growth, bone resorption, bone formation, bone growth, or combinations thereof. Such biomarkers may be measured before, during and / or after administration of a CNP variant as described herein. Exemplary bone- or cartilage-associated biomarkers include, but are not limited to, CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, propeptides of collagen type I and fragments thereof, collagen type I and fragments thereof, osteocalcin, proliferating cell nuclear antigen (PCNA), aggrecan chondroitin sulfate, collagen X , alkaline propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), cross-linked C-telopeptide of type Icollagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP- variant bioactivity. Cartilage- and bone-associated biomarkers can be measured in any appropriate biological sample, including but not limited to tissues, blood, serum, plasma, cerebrospinal fluid, synovial fluid and urine. In some embodiments, the biomarkers are measured in blood, plasma, urine, or serum from subjects undergoing efficacy / pharmacodynamic in vivo studies and / or from the conditioned media of ex vivo studies.

[0135] "Pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in subject animal, including humans and mammals. A pharmaceutical composition comprises a therapeutically effective amount of CNP variant, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier or diluent. In an embodiment, a pharmaceutical composition encompasses a composition comprising the active ingredient(s), and the inert ingredient(s) that make up the carrier, as well as any product that results, directly or indirectly, from combination, complexation or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present disclosure encompass any composition made by admixing a compound of the disclosure and a pharmaceutically acceptable excipient, carrier or diluent.

[0136] "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions (e.g., an oil / water or water / oil emulsion). Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifying agents, wetting agents, lubricants, glidants, sweetening agents, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients and diluents are described in Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend upon 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).

[0137] A "pharmaceutically acceptable salt" is a salt that can be formulated 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.

[0138] By “pharmaceutically acceptable” or “pharmacologically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual without causing any undesirable biological effects or without interacting in a deleterious manner with any of the components of the composition in which it is contained or with any components present on or in the body of the individual.

[0139] “Physiological conditions” refer to conditions in the body of an animal (e.g., a human). Physiological conditions include, but are not limited to, body temperature and an aqueous environment of physiologic ionic strength, pH and enzymes. Physiological conditions also encompass conditions in the body of a particular subject which differ from the “normal” conditions present in the majority of subjects, e.g., which differ from the normal human body temperature of approximately 37 °C or differ from the normal human blood pH of approximately 7.4.

[0140] By “physiological pH” or a “pH in a physiological range” is meant a pH in the range of approximately 7.0 to 8.0 inclusive, more typically in the range of approximately 7.2 to 7.6 inclusive.

[0141] As used herein, the term “subject” encompasses 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 apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a particular age or gender. 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. In various embodiments, the subject is older than 3, older than 2, older than 1, or older than 6 months in age.C-type natriuretic peptide variants

[0142] 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)) is a small, single chain peptide in a family of peptides (ANP, BNP, CNP) having a 17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and have important roles in multiple biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the generation of cyclic- guanosine monophosphate (cGMP) (J. Hypertens., 10: 1111-1 114 (1992)). CNP is expressed more widely, including in the central nervous system,reproductive tract, bone and endothelium of blood vessels (Hypertension, 49: 419-426 (2007)).

[0143] Natural CNP gene and polypeptide have been previously described. U.S.Patent No. 5,352,770 discloses isolated and purified CNP-22 from porcine brain identical in sequence to human CNP and its use in treating cardiovascular indications. U.S.Patent No. 6,034,231 discloses the human gene and polypeptide of pre-proCNP (126 amino acids) and the human CNP-53 gene and polypeptide. The mature CNP is a 22- amino acid peptide (CNP-22). Certain CNP variants are disclosed in US Patent 8,198,242, incorporated by reference herein.

[0144] In various embodiments, CNP of the disclosure includes truncated CNP ranging from human CNP-17 (hCNP-17) to human CNP-53 (hCNP-53), and having wild-type amino acid sequences derived from hCNP-53. Such truncated CNP peptides include:

[0145] In various embodiments, the CNP variant peptides are modified CNP-37 or CNP-38 peptides, optionally having mutation(s) / substitution(s) 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:

[0146] In various embodiments, CNP variants of the disclosure include

[0147] In various embodiments, the CNP variant further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus, C-terminus or attached to an internal amino acid side group. In various embodiments, the acetyl group is on the N-terminus of the peptide.

[0148] In various embodiments, the peptide variant further comprises an OH or an NH2group at the C-terminus.

[0149] In various embodiments, the CNP variants are selected from the group consisting of:and

[0150] In various embodiments, the CNP variants are selected from the group consisting of:

[0151] In various embodiments, the CNP variants are selected from the group consisting of:and

[0152] In various embodiments, the CNP variant is selected from the group consisting of Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO:8). In various embodiments, the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1). In various embodiments, the CNP variant is PGQEH PNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1).

[0153] In additional embodiments, for any of the CNP variants described herein that have asparagine (Asn / N) residue(s) and / or glutamine (Gln / Q) residue(s), whether they have a wild- type sequence or a non-natural amino acid sequence, any Asn residue(s) and / or any Gin residue(s) can independently be substituted with any other natural or unnatural amino acids, including conservative substitutions such as Asn to Gin. Such substitution(s) are designed in part to minimize or avoid any potential deamidation of asparagine and / or glutamine.

[0154] In one embodiment, the CNP variants are cyclized via formation of a disulfide bond between Cys6and Cys22as designated in the wtCNP22 peptide. Cys6can be a cysteine analog such as, e.g., homocysteine or penicillamine. In a further embodiment, the CNP variants can be cyclized by a covalent bond formed head-to- tail, side chain-to-side chain, side chain-to-head, or side chain-to-tail. In an embodiment, the covalent bond is formed between an amino acid at or toward the N- terminus and an amino acid at or toward the C-terminus of the peptide (referred to as “terminal” amino acids in this context). In another embodiment, the covalent bond is formed between the side chains of the two terminal amino acids. In yet another embodiment, the covalent bond is formed between the side chain of one terminal amino acid and the terminal group of the other terminal amino acid, or between the terminal groups of the two terminal amino acids.

[0155] Head-to-tail cyclization of the terminal amine to the terminal carboxyl group can be carried out using a number of methods, e.g., using p-nitrophenyl ester, 2,4,5- trichlorophenyl ester, pentafluorophenyl ester, the azide method, the mixed anhydride method, HATU, a carbodimide (e.g., DIG, EDC or DCC) with a catalyst such as HOBt, HONSu or HOAt, or on- resin cyclization.

[0156] In addition, the cyclic structure can be formed via a bridging group involving the side chains of amino acid residues of the CNP variant and / or the terminal amino acid residues. A bridging group is a chemical moiety that allows cyclization of two portions of the peptide. No n limiting examples of bridging groups include amides, thioethers, thioesters, disulfides, ureas, carbamates, sulfonamides, and the like. A variety of methods are known in the art for incorporation of units having such bridging groups. For example, a lactam bridge (i.e., a cyclic amide) can be formed between the N-terminal amino group or an amino group on a side chain and the C-terminal carboxylic acid or a carboxyl group on a side chain, e.g., the side chain of lysine or ornithine and the side chain of glutamic acid or aspartic acid. A thioester can be formed between the C-terminal carboxyl group or a carboxyl group on a side chain and the thiol group on the side chain of cysteine or a cysteine analog.

[0157] Alternatively, a cross link can be formed by incorporating a lanthionine (thiodialanine) residue to link alanine residues that are covalently bonded together by a thioether bond. In another method, a cross-linking agent, such as a dicarboxylic acid (e.g., suberic acid (octanedioic acid)), can link the functional groups of amino acid side chains, such as free amino, hydroxyl, and thiol groups.

[0158] Enzyme-catalyzed cyclization can also be used. For example, it has been reported that the thioesterase domain of tyrocidine synthetase can be used to cyclize a thioester precursor, a subtilisin mutant can be utilized to cyclize peptide glycolate phenylalanylamide esters, and the antibody ligase 16G3 can be employed to cyclize a p- nitrophenylester. For a review of peptide cyclization, see Davies, J. Peptide Sci., 9: 471- 501 (2003), incorporated herein by reference in its entirety.

[0159] In certain embodiments, the final product has a purity of at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99%.Peptide Conjugates

[0160] Peptide therapeutics are attractive biological therapeutic agents, but are often disadvantaged by low stability and short half-life in solution (Tang et al., Eur J Pharm Sci. 102:63-70, 2017). Attempts to improve efficacy of peptide therapeutics, by enhancing stability and / or increasing the half-life, include attempts to encapsulate hydrophilic peptides into biodegradable particles such as liposomes or polymer particles. However, this has been difficult due to the cationic nature of these peptides and their ability to electrostatically interact with liposomes of negatively charged polymers (Griesser et al., Int J Pharmaceutics 520:267-274, 2017). Generation of peptide conjugates has been one means used to enable better encapsulation of hydrophilic polymers into microparticles or liposomes (Lu et al., Mol. Pharmaceutics 15:216-225, 2018).

[0161] Peptides can be a string of amino acids from 5 to 100 amino acids. The peptide can have positively charged amino acids, negatively charged amino acids, or a mixture of both, such that the peptide is capable of interacting with charged moieties, e.g., a cation, anion or a combination thereof having charged species opposite to those in the peptide.

[0162] It is contemplated that the peptide is complexed to a moiety, e.g., a conjugate moiety, that confers increased stability or half-life. In various embodiments, the conjugate moiety is complexed via a non-covalent bond or is attached by a covalent bond. The moiety may be non- covalently attached with the peptide via electrostatic interactions. Alternatively, the moiety may be covalently associated to the peptide via one or more linker moieties. Linkers can be cleavable and non-cleavable linkers. Cleavable linkers may be cleaved via enzymes, nucleophilic / basic reagents, reducing agents, photo-irradiation, electrophilic / acidic reagents, organometallic and metal reagents, or oxidizing reagents. Linkers may also be self-immolative linkers. Linkers may also be traceless linkers. Exemplary linkers include, but are not limited to, N-succinimidyl-3- (2-pyridyldithiol) propionate (SPDP), iminothiolane (IT),bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6- di isocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4- dinitrobenzene), beta alanine, 4-aminobutyric acid (GABA), 2-aminoethoxy acid (AEA), aminoethoxy-2-ethoxy acetic acid (AEEA), 5 aminovaleric acid (AVA), 6-aminocaproic acid (Abx), a vicinal diol cleavable linker, Trimethyl Lock Lactonization, p-alkoxyphenyl carbamate, bicin, peptoid or bicin-type linkers, and electronic linkers as described herein.

[0163] In various embodiments, the linker is attached to a residue of the CNP variant within the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is attached to a lysine residue. In various embodiments, the linker is attached to a lysine residue in the CNP cyclic domain.

[0164] In various embodiments, the CNP variant is attached to the conjugate moiety via the linker. In various embodiments, the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety.

[0165] In various embodiments, the linker is a hydrolysable linker.

[0166] In various embodiments the linker is a peptoid or electronic linker. In various embodiments the linker is a peptoid linker. In various embodiments the linker is an electronic linker. In various embodiments, the linker comprises an SO2 moiety.Exemplary linkers are illustrated in Figure 1 and below. It is further contemplated that linkers in Figure 1 are modified by substitution on the R groups. For example, bicin-type linkers include the structures as set out below:

[0167] In various embodiments, the moiety conjugated to the peptide is a synthetic polymer such as polyethylene glycol, a linker, a lipid moiety or fatty acid, or a combination thereof. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a spacer and a linker. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a polyethylene glycol spacer or a polyethylene glycol derivative spacer, and a linker. In various embodiments, the CNP variant is conjugated with a fatty acid, an amino acid, a spacer, and a linker, wherein the spacer comprises a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, wherein the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or carbonyl groups.

[0168] In various embodiments, the CNP variant is conjugated with a fatty acid. It is hypothesized that the lipid technology increases the serum half-life of the CNP variant allowing for less frequent injections and / or improved oral delivery. In various embodiments, the fatty acid is a short chain, medium chain, long chain fatty acid, or a dicarboxylic fatty acid. In various embodiments, the fatty acid is saturated or unsaturated. 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 decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids of the same. In various embodiments, the fatty acid is conjugated to a lysine residue.

[0169] In various embodiments, it is contemplated that the CNP variants described herein comprise a conjugate moiety as described herein. It is contemplated that the conjugate moiety is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugate moiety is on a lysine residue. In various embodiments, the conjugate moiety comprises one or more acid moieties. In various embodiments, the acid moiety is a fatty acid.

[0170] In various embodiments, the conjugate moiety comprises an acid moiety linked to a hydrophilic spacer. In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain or any amino acid, or a combination of both, wherein the carbon atoms of the alkyl chain can be replaced by one or more of O, NH, N(C-1 to C-6 alkyl), or carbonyl groups. In various embodiments, the hydrophilic spacer is any amino acid. In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu). In various embodiments, the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain. Invarious embodiments, the hydrophilic spacer is a substituted C-6, C-8, C-10, C-12, C- 14, C-16, C-18 or C-20 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 to C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-18 alkyl chain. In various embodiments, the hydrophilic spacer is a substituted C-9 alkyl chain. In various embodiments, the hydrophilic spacer is one or more OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is one or two OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is OEG (8-amino-3,6-dioxaoctanoic acid). In various embodiments, the spacer is OEG (8-amino-3,6-dioxaoctanoic acid) or yGlu. In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu) linked to one or more OEG (8-amino-3,6-dioxaoctanoic acid) groups. In various embodiments, the hydrophilic spacer is gamma glutamic acid (yGlu) linked to one or two OEG (8-amino-3,6- dioxaoctanoic acid) groups (diEG). In various embodiments, the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-yGlu-d8DA.

[0171] In various embodiments, the CNP variant has the structure:. In various embodiments, the CNPvariant has the structure(SEQ ID NO: 1), or). In various embodiments, the CNP variant comprises Asn to Glu variantsof the above peptides.

[0172] In various embodiments, the disclosure contemplates use of hydrophilic or water soluble polymers (e.g., oxygenated alkyl chains, wherein the carbon atoms can be replaced with one or more oxygen atoms, such as polyethylene glycol (PEG) or polyethylene oxide (PEO) and the like). In various embodiments, the water soluble polymers can vary in type (e.g., homopolymer or copolymer; random, alternating or block copolymer; linear or branched; monodispersed or polydispersed), linkage (e.g., hydrolysable or stable linkage such as, e.g., amide, imine, aminal, alkylene, or ester bond), conjugation site (e.g., at the N-terminus, internal, and / or C-terminus), and length (e.g., from about 0.2, 0.4 or 0.6 kDa to about 2 , 5 , 10, 25, 50 or 100 kDa). Thehydrophilic or water-soluble polymer can be conjugated to the CNP variant by means of N-hydroxy succinimide (NHS)- or aldehyde-based chemistry or other chemistry, as is known in the art. In various embodiments, negatively charged PEG-CNP variants can be designed for reduced renal clearance, including but not limited to use of carboxylated, sulfated and phosphorylated compounds (Caliceti, Adv. Drug Deliv. Rev., 55: 1261-77 (2003); Perlman, J . Clin. Endo. Metab., 88: 3227-35 (2003); Pitkin, Antimicrob. Ag. Chemo., 29: 440-444 (1986); Vehaskari, Kidney Int’l, 22: 127-135 (1982)). In one embodiment, the PEG (or PEO) moiety contains carboxyl group(s), sulfate group(s), and / or phosphate group(s).

[0173] In another embodiment, the hydrophilic polymer (e.g., PEG or PEO) moieties conjugated to the N-terminus, C-terminus and / or internal site(s) of CNP variants described herein contain one or more functional groups that are positively charged under physiological conditions. Such moieties are designed, inter alia, to improve distribution of such conjugated CNP variants to cartilage tissues. In one embodiment, PEG moieties contain one or more primary, secondary or tertiary amino groups, quaternary ammonium groups, and / or other amine- containing (e.g., urea) groups.Methods of Making

[0174] Contemplated herein is also a method of making a composition comprising a CNP variant and optionally a conjugate moiety as described herein.

[0175] In various embodiments, the CNP variant is made synthetically using standard protein synthesis chemistry. For example, peptides are synthesized step-wise using a solid-phase resin and standard Fmoc chemistry. Peptides are cleaved from the resin using tri-fluroacetic acid (TFA) and purified by reverse phase high-performance liquid chromatography (RP-HPLC).

[0176] [In various embodiments, the method further comprises, acetylating the peptides by reacting the resin with NMP / Ac20 / DIEA, optionally at 10:1:0.1, v / v / v.

[0177] Further provided is a method wherein the peptide is conjugated to a conjugate moiety, optionally on a lysine residue. The step comprising cleaving the protective amino group on the lysine, reacting the peptide with 2x Fmoc-amino PEG(2) followed by amino acid, followed by conjugation of the lipid or fatty acid moiety. In various embodiments, the conjugate moiety comprises one or more lipids or fatty acids and a hydrophobic spacer.

[0178] The method further provides a step of cleaving the peptide from the resin by contacting with tri-fluoroacetic acid, and a step of purifying the peptide by reverse phase- HPLC.

[0179] In certain embodiments, the CNP variants described herein are produced by a recombinant process that comprises culturing in a medium a host cell comprising a first polynucleotide encoding a CNP variant polypeptide, optionally linked to a second polynucleotide encoding a cleavable peptide or protein under conditions that result in expression of a fusion polypeptide encoded by the polynucleotides. In some embodiments, the host cell is transformed with an expression vector comprising the polynucleotide encoding the CNP variant polypeptide, optionally linked to the polynucleotide encoding the cleavable peptide or protein. In certain embodiments, the fusion polypeptide is expressed as a soluble protein or as an inclusion body. The expressed fusion polypeptide can be isolated from the host cell or culture medium, and the isolated fusion polypeptide can be contacted with a cleaving agent to release the CNP variant.

[0180] Methods of making CNP variant peptides, including use of host cells, expression vectors, cleavable peptides, and culture parameters, are disclosed in U.S. Patent 8,198,242, hereby incorporated by reference.Methods of Use

[0181] Achondroplasia is a result of an autosomal dominant mutation in the gene for fibroblast growth factor receptor 3 (FGFR-3), which causes an abnormality of cartilage formation. FGFR-3 normally has a negative regulatory effect on chondrocyte growth, and hence bone growth. In achondroplasia, the mutated form of FGFR-3 is constitutively active, which leads to severely shortened bones. In humans activating mutations of FGFR-3 are the primary cause of genetic dwarfism. Mice having activated FGFR-3 serve as a model of achondroplasia, the most common form of the skeletal dysplasias, and overexpression of CNP rescues these animals from dwarfism. Accordingly, functional variants of CNP are potential therapeutics for treatment of the various skeletal dysplasias

[0182] By stimulating matrix production, proliferation and differentiation of chondrocytes and increasing long bone growth, the CNP variants of the disclosure are useful for treating mammals, including humans, suffering from a bone-related disorder, such as a skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, short stature, dwarfism,osteochondrodysplasias, thanatophoric dysplasia, osteogenesis congenita, achondrogenesis, chondrodysplasia congenita, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis congenita, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia congenita, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenital, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia. Short stature, growth plate disorder, bone-related disorder or skeletal dysplasias contemplated herein include disorders related to NPR2 mutation, SHOX mutation (Turner’s syndrome / Leri Weill), and PTPN1 1 mutations (Noonan’s syndrome).

[0183] By stimulating matrix production, proliferation and differentiation of chondrocytes and increasing long bone growth, the CNP variants of the disclosure are useful for treating mammals, including humans, suffering from a bone-related disorder, such as a skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis congenita, achondrogenesis, chondrodysplasia congenita, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis congenita, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia congenita, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenital, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia. Short stature, growth plate disorder, bone-related disorder or skeletal dysplasias contemplated herein include disorders related to NPR2 mutation, SHOX mutation (Turner’s syndrome / Leri Weill), PTPN1 1 mutations (Noonan’s syndrome) and IGF1R mutation.

[0184] Further provided herein, the CNP variants of the disclosure are useful for treating mammals, including humans, suffering from a bone-related disorder, such as a skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletaldysplasias include achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis congenita, achondrogenesis, chondrodysplasia congenita, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis congenita, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia congenit, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenital, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia and osteoporosis.

[0185] Additional short stature and growth plate disorders contemplated by the methods include disorders related to mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3.

[0186] Additional short stature and growth plate disorders contemplated by the methods include disorders related to mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0187] Also provided herein is treatment of short stature and growth plate disorders including disorders related to mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), S H OX, PTPN11, NPR2, NPPC, FGFR3, or IGF1R.

[0188] Further, the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other skeletal dysplasias.

[0189] Growth plate disorders include disorders that result in short stature or abnormal bone growth and that may be the result of a genetic mutation in a gene involved in bone growth, including collagen (COL2A1. COL1 1A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders include disorders that result in short stature or abnormal bone growth and that may be the result of a genetic mutation in a gene involved in bone growth, including collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN1 1, NPR2, NPPC, FGFR3, or IGFIR. In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy. In various embodiments, the growthplate disorder or short stature is associated with one or more mutations in the SHOX gene. In various embodiments, a subject with a growth plate disorder is heterozygous for a mutation in a growth plate gene. In various embodiments, the mutation is a loss-of- function mutation. 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 which is also known as dominant inherited short stature, or idiopathic short stature. See, e.g., Plachy et al., J Clin Endocrinol Metab 104: 4273- 4281, 2019.

[0190] Mutations in ACAN can give rise to familial osteochondritis dissecans and short stature and eventually osteoarthritis, characterized by areas of bone damage (or lesions) caused by the detachment of cartilage and sometimes bone from the end of the bone at a joint. It has been suggested that the disorganized cartilage network in growing bones impairs their growth, leading to short stature. A mutation associated with ACAN and short stature includes Val2303Met. See Stattin et al., Am J Hum Genet 86(2): 126-37, 2010. It is contemplated that patients with a mutation in ACAN resulting in short stature would benefit from treatment with CNP as administration may be able to increase height in these patients by the known interaction of CNP with FGFR3.

[0191] The natriuretic peptide system, including receptor NPR2, has been shown to be involved in regulation of endochondral bone growth (Vasques et al., Horm Res Pediat 82:222- 229, 2014). Studies have shown that homozygous or compound heterozygous loss-of-function mutations in NPR2 cause acromesomelic dysplasia type Maroteaux (AMDM), which is a skeletal dysplasia having extremely short stature (Vasquez et al., 2014, supra). There are reports implicating heterozygous loss-of-function (such as dominant negative) NPR2 mutations as a cause of short stature, whereas gain-of- function NPR2 heterozygous mutations have been found to be responsible for tall stature (Vasquez et al., 2014, supra). In view of CNP’s interaction with NPR2 to stimulate cGMP generation, increasing cGMP levels is desirable in these conditions and would have therapeutic benefit in the management of the complications from these diseases and conditions.

[0192] Heterozygous mutations of NPR2 are believed to result in idiopathic short stature and other forms of short stature. Mutations in the NPR2 gene are set out below and described in Amano et al., J Clin Endocrinol Metab 99:713-718, 2014, Hisado-Oliva et al., J Clin Endocrinol Metab 100 1133-1 142, 2015 and Vasques et al., J Clin Endocrinol Metab 98: E1636- 1644, 2013, hereby incorporated by reference. It iscontemplated that a subject having short stature to be treated with a CNP variant as described herein has a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and has at least one parent with a height SDS of less than -1.0, -1.5, -2.0 or -2.5, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between - 2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. However, because de novo mutations in NPR2 can result in short stature as defined by a height SDS of less than - 1.5, -2.0, -2.5, or -3.0, treatment of individuals who are heterozygous carriers of a deleterious mutation in NPR2 with neither parent having short stature is also contemplated. Further contemplated is treatment of individuals who are heterozygous for deleterious mutations in other growth plate genes with CNP to improve stature and / or enhance bone growth.

[0193] Exemplary NPR2 mutations in patients that may be treated with a CNP variant include:

[0194] NPPC’s role in skeletal growth is well documented (Hisado-Oliva et al., Genetics Medicine 20:91-97, 2018). The NPPC knock out mouse showed severe disproportionate form of dwarfism including shortening of limbs and endochondral ossification (Hisado-Oliva et al., 2018, supra). Human genome wide studies have shown a link between NPPC and height (Hisado-Oliva et al., 2018, supra). Although CNP haploinsufficiency has been believed to be a cause of short stature in humans, a recent study identified heterozygous mutations in families with short stature and hands (Hisado- Oliva et al., 2018, supra). These studies observed significant reduction in cGMP production as measured in heterozygous state (Hisado-Oliva et al., 2018, supra). Mutations in NPPC include a 355G>T missense mutation causing a Gly1 19Cys change and a 349C>G missense mutation causing a Argl 17Gly change. A CNP variant rescuing CGMP production may provide therapeutic benefit in the management of a disorder in patients having heterozygous loss-of-function NPPC mutations.

[0195] Isolated SHOX deficiency is one of the more prevalent monogenic causes of short stature (birth prevalence approximately 10 per 100,000 [Marchini et al., Endocr Rev. 37: 417—448, 2016]) (Genoni 2018). The SHOX gene, which is located on both the X and Y chromosomes, encodes for a transcription factor expressed throughout the growth plate with functions including influencing the natriuretic peptide receptor-type B (NPR-B) and fibroblast growth factor receptor 3 (FGFR3) pathways. The SHOX gene has been shown to be a repressor of FGFR3 transcription, and SHOX deficiency leads to increased FGFR3 signaling (Marchini, supra). Several pathogenic variants in SHOX are known to give rise to short stature. Individuals with pathogenic variants causing SHOX deficiency present with growth failure in the first year of life, height that often remains below -2.00 SDs compared to CDCpopulation norms, and no development of a pubertal growth spurt accentuating final statural impairment (Binder 2018; Fukami 2016; Jorge 2010).

[0196] Turner syndrome is a rare chromosomal disorder (birth prevalence 32 per 100,000 females [16 per 100,000 total population] [Martin-Giacalone 2023]) causing short stature and other phenotypic features in girls. It is caused by a structurally abnormal, or a partial or complete absence of 1 X chromosome which can in turn result in a loss of one copy of the SHOX gene (located on the X-chromosome). The absence of 1 copy of the SHOX gene results in disordered growth and skeletal anomalies (Gravholt 2019). Girls with Turner syndrome exhibit growth rate slowing after their first 3 years of life, with adult height deficits further impacted by an absent pubertal growth spurt. Individuals have final heights significantly shorter than average stature women, with a mean height deficit of 20 cm from that predicted by mid-parental height (Karlberg 1991; Rongen-Westerlaken 1997). Given the association of SHOX with FGFR3 and bone growth, it is contemplated that a subject having a homozygous or heterozygous SHOX mutation would benefit from treatment with CNP variants as described herein.

[0197] Leri-Weill dyschondrosteosis (LWD) is a rare genetic disorder characterized by shortening of the forearms and lower legs, abnormal misalignment of the wrist (Madelung deformity of the wrist), and associated short stature. LWD is caused by a heterozygous mutation in the short stature homeobox-containing (SHOX) gene or its regulatory elements located on the pseudoautosomal region 1 (PAR1) of the sex chromosomes. (See the Rare Disease Database and Carmona et al., Hum Mol Genet 20:1547-1559, 201 1). The disorder Langer mesomelic dysplasia arises when there are two SHOX mutations, and may result from a mutation on each chromosome, either a homozygous or compound heterozygous mutations. A subset of SHOX mutations give rise to idiopathic short stature. Turner syndrome results due to a deletion on the X chromosome that can include the SHOX gene. SHOX has been identified as involved in the regulation of FGFR3 transcription and contributes to 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 to support that SHOX has direct interactions with CNP / NPR2 as well (Marchini, supra). Given the association of SHOX with FGFR3 and bone growth, it is contemplated that a subject having a homozygous or heterozygous SHOX mutation would benefit from treatment with CNP variants as described herein.

[0198] RASopathies are a group of rare genetic conditions caused by mutations in genes of the Ras / mitogen-activated protein kinase (MAPK) pathway. RASopathies are a group of disorders characterized by increased signaling through RAS / MAPK pathway.This pathway leads to downstream activation of the RAF / M EK / ERK pathway. Short stature is a characteristic feature of certain RASopathies. For example, CNP signaling inhibits RAF and leads to decreased MEK and ERK activation.

[0199] Treatment of RASopathies are contemplated herein. RASopathies associated with short stature include Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, and LEOPARD syndrome. Hereditary gingival fibromatosis type 1 is also a RASopathy contemplated herein. RASopathy patients (including Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, LEOPARD syndrome, 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).

[0200] 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 called faciocutaneoskeletal (FCS) syndrome is caused by activating mutations in the H-Ras gene. Hereditary gingival fibromatosis type I (HGF) is caused by dominant mutations in the SOS1 gene (Son of Sevenless homolog 1), which encodes a guanine nucleotide exchange factor (SOS) that acts on the Ras subfamily of small GTPases. Neurofibromatosis type I (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 mutations in one of several genes, including PTPN1 1, which encodes SHP2, and SOS1, as well as K-Ras and Raf-1.

[0201] CNP has been demonstrated to be an effective therapy in RASopathy models. Ono etal. generated mice deficient in Nf1 in type 1 1 collagen producing cells (Ono et aL, Hum. Mol. Genet. 2013;22(15):3048-62). These mice demonstrated constitutive ERK1 / 2 activation, and decreased chondrocyte proliferation, and maturation. Daily injections of CNP in these mice led to decreased ERK phosphorylation and corrected the short stature. A mouse model of Cardiofaciocutaneous syndrome using a Braf mutation (p.Q241 R) (Inoue et al. Hum. Mol. Genet. 2019;28(1):74-83). exhibited decreased body length and reduced growth plate width with smaller proliferative and hypertrophic zones compared to wild type, and CNP administration led to increases in body length in these animals.

[0202] Mutations in multiple genes can cause Noonan syndrome, which is characterized by short stature, heart defects, bleeding problems, and skeletalmalformations. Mutations in the PTPNI 1 gene cause about half of all cases of Noonan’s syndrome. SOS1 gene mutations cause an additional 10 to 15 percent, and RAF1 and RIT 1 genes each account for about 5 percent of cases. Mutations in other genes each account for a small number of cases. The cause of Noonan syndrome in 15 to 20 percent of people with this disorder is unknown.

[0203] Noonan syndrome (birth prevalence 40 per 100,000 [NORD 2019]) is the most common RASopathy, a clinically defined group of disorders caused by a germline mutation in one of the genes encoding components of the RAS-MAPK pathway, typically resulting in increased signaling through this pathway. The RAS-MAPK pathway leads to downstream activation of RAF / MEK / extracellular-signal-regulated kinase (ERK). C-type natriuretic peptide (CNP) signaling intersects with this pathway by inhibiting RAF, leading to decreased MEK and ERK activation. Noonan syndrome primarily results from gain of function (GoF) mutations in genes encoding components of the RAS-MAPK pathway. Noonan syndrome is characterized by short stature in 50-70% of those affected (Bhambhani 2014), with typical facial features and cardiac defects in more than 80% of patients (Noonan 2005), and multisystem involvement in older children (Allanson 2021; Breilyn 2019). Growth failure occurs in the first year of life and the height of the child often remains below -2.00 SDs until puberty, when the growth is further affected due to an attenuated pubertal growth spurt (Carcavilla 2020). Approximately 50% of adults with Noonan syndrome have significantly reduced height (Noonan 2003).

[0204] The PTPN11, SOS1, RAF1, and RIT1 genes all encode for proteins that are important in the RAS / MAPK cell signaling pathway, which is needed for cell division and growth (proliferation), differentiation, and cell migration. Many of the mutations in the genes associated with Noonan syndrome cause the resulting protein to be turned on (active) and this prolonged activation alters normal RAS / MAPK signaling, which disrupts the regulation of cell growth and division, leading to the characteristic features of Noonan syndrome. See, e.g., Chen et al., Proc Natl Acad Sci U S A. 111(31 ):1 1473-8, 2014, Romano et al., Pediatrics. 126(4): 746-59, 2010, and Milosavljevic et al., Am J Med Genet 170(7): 1874-80, 2016. It is contemplated that a subject having mutations that activate the MAPK pathway would benefit from treatment with CNP variants as described herein to improve bone growth and short stature. It is also contemplated that a subject having mutations that activate the MAPK pathway would benefit from treatment with CNP variants as described herein to improve other comorbidities associated with an overactive MAPK pathway in other cells throughout the body where the NPR2 receptor is expressed on its surface.

[0205] Mutations in the PTPN11 gene, which encodes the non-receptor protein tyrosine phosphatase SHP-2, lead to disorders characterized by short stature such as Noonan’s Syndrome (Musente et al., Eur J Hum Genet 11 :201-206 (2003). Musente (supra) identifies numerous mutations in the PTPN1 1 gene that lead to short stature. Gain of function mutations lead to overactive signaling through SHP2 and inhibit Growth Hormone-induced IGF-1 release, thereby contributing to a decrease in bone growth (Rocca Serra-Nedelec, PNAS 109:4257- 4262, 2012). It is contemplated that a subject having a homozygous or heterozygous PTPN1 1 mutation would benefit from treatment with CNP variants as described herein to improve bone growth and short stature.

[0206] Mutations in the Indian hedgehog (IHH) gene, which is related to regulation of endochondral ossification, have also been associated with short stature syndromes (Vasques et al., J Clin Endocrinol Metab. 103:604-614, 2018). Many IHH mutations identified segregate with short stature in a dominant inheritance pattern. Given the association of IHH with bone growth and ossification, it is contemplated that subjects having a homozygous or heterozygous IHH mutation will benefit from treatment with a CNP variant as described herein.

[0207] Mutations in FGFR3, including N540K and K650N, lead to short stature and hypochondroplasia.

[0208] Insulin-like growth factor 1 receptor (IGF1R) is a heterotetrameric (a202) transmembrane glycoprotein with an intrinsic kinase activity. IGF1 R has been shown to have a role in prenatal and postnatal growth. Heterozygous mutations in IGF1R have been identified in Small for gestational age children (SGA) and individuals with familial short stature (Kawashima et al., Endocrine J. 59:179-185, 2012). Mutations in IGF1 R associated with short stature include R108Q / K1 15N, R59T, R709Q, G1050K, R481Q, V599E, and G1125A (Kawashima, supra).

[0209] Height is a highly heritable trait that can be influenced by the combined effect of hundreds or thousands of genes (Wood et al, 2014, Nature Genetics, 46:1 173-1 189. Short stature in an individual can be the result of the combined effect of these genes, without a single gene being the primary contributor. It is contemplated that such individuals with short stature defined by a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, can be beneficially treated with a CNP variant given the ability of CNP to increase the length of normal animals, for example, enhance bone growth and length of bones.

[0210] In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and having at least one parent with a height SDS of less than -1.0, -1.5, -2.0 or -2.5, optionally wherein the second parent has height within the normal range. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5. In various embodiments, the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less. In various embodiments, the short stature is associated with one or more mutations in a gene associated with short stature, such as, collagen (COL2A1, COL11A1, COL9A2, COL10), aggrecan (ACAN), Indian hedgehog (IHH), PTPN11, NPR2, NPPC, FGFR3, SHOX, or insulin growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, mutations in denticleless E3 Ubiquitin Protein Ligase Homolog (DTL), and pregnancy-associated plasma protein A2 (PAPPA2), or combinations thereof are also associated with short stature.

[0211] In various embodiments, the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.

[0212] In various embodiments, the short stature is a result of mutations in multiple genes as determined by polygenic risk score (PRS). Polygenic risk scores (PRS) were calculated for height using the largest published GWAS meta-analysis for height that did not include any samples from the UK Biobank project as described in Example 4. The cohort was divided into five PRS quintiles (PRS 1 being the lowest height, PRS 5 the tallest height). 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 mutation in one or more of FGFR3, IGF1 R, NPPC, NPR2 and SHOX, and a low PRS. In various embodiments, the subject has one or more mutation in denticleless E3 Ubiquitin Protein Ligase Homolog (DTL), or pregnancy-associated plasma protein A2 (PAPPA2). In various embodiments, the PRS is 1 or 2. In various embodiments, the PRS is 1. In various embodiments, the PRS is 2.

[0213] In addition, the CNP variants are useful for treating other bone-related conditions and disorders, such as rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also called vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets], and osteomalacia [including tumor-induced osteomalacia (also called oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)].

[0214] The CNP variants of the disclosure can also be used to treat osteoarthritis. Osteoarthritis is a degenerative disease of the articular cartilage and occurs frequently in the elderly. Osteoarthritis involves destruction of the cartilage and proliferative change in the bone and cartilage resulting from degeneration of articular components, with the change resulting in a secondary arthritis (e.g., synovitis). The extracellular matrix proteins, which are the functional entity of the cartilage, are reduced, and the number of chondrocytes decreases in osteoarthritis (Arth. Rheum. 46(8): 1986-1996 (2002)). By promoting the matrix production, growth and differentiation of chondrocytes, the CNP compositions are useful for countering the undesired effects of FGF-2 and increasing matrix synthesis in subjects suffering from arthritis, including osteoarthritis, thereby treating arthritis, including osteoarthritis.

[0215] In certain embodiments, the CNP variants and compositions and formulations comprising the same of the present disclosure are useful for improving one or more of the symptom(s) or physiological consequences of a skeletal dysplasia, wherein the improvement may be increased absolute growth, increased growth velocity, increased qualitative computed tomography (QCT) bone mineral density, improvement in growth plate morphology, increased long bone growth, improvement in spinal morphology, improved elbow joint range of motion and / or decreased sleep apnea. Additional symptoms that can be improved by CNP therapy include bone tissue mineral density (TMD), bone mineral density (BMD), bone strength, metacarpal length with more cortical area, or combinations thereof. In this regard, it is noted that the terms "improved", "improvement", "increase", "decrease" and grammatical equivalents thereof are all relative terms that when used in relation to a symptom or physiological consequence of a disease state, refer to the state of the symptom or physiological consequence of the disease after treatment with a CNP variant (or composition or formulation comprising the same) of the present invention as compared to the same symptom or physiological consequence of the disease before treatment with a CNP variant (or composition or formulation comprising the same) of the present invention (i.e., as compared to "baseline"). As described above, a "baseline" state can be determinedeither through measurement of the state in the subject prior to treatment (which can subsequently be compared to the state in the same subject after treatment), or through measurement of that state in a population of subjects suffering from the same affliction that share the same or similar characteristics (e.g., age, sex and / or disease state or progression).

[0216] Also provided is a method of overcoming cell growth arrest induced by a constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3) comprising contacting a cell expressing the constitutively active FGFR-3 with a CNP variant or a composition as described herein.

[0217] Further provided is a method of stimulating cGMP production in a cell expressing natriuretic peptide receptor B (NPR-B) comprising contacting the cell expressing NPR-B with a CNP variant or a composition as described herein.

[0218] In another aspect, provided herein is a method for increasing facial volume, facial sinus volume, and foramen magnum area in a subject (e.g., 6 months old or less) having a bone-related disorder, skeletal dysplasia or short stature comprising administering CNP variants as described herein. Also provided is a method of decreasing the incidence of sudden infant death, sleep disordered breathing, and necessity for neurosurgical decompression of the foramen magnum in a subject (e.g., 6 months old or less) having a bone-related disorder, skeletal dysplasia or short stature comprising administering CNP variants as described herein.

[0219] In various embodiments, the increase in facial volume, facial sinus volume, and foramen magnum area are measured by magnetic resonance imaging (MRI). In various embodiments, the change in facial volume, facial sinus volume, and foramen magnum area are compared to baseline levels, healthy control subjects or untreated control subjects.

[0220] In yet another embodiment, the disclosure provides CNP variants that in vitro or in vivo stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP level produced under the same concentration of wtCNP22 (e.g., 1 uM). In a still further embodiment, the CNP variants of the disclosure in vitro or in vivo stimulate the production of at least about 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140% or 150% of the cGMP level produced under the same concentration of wtCNP22 (e.g., 1 uM).

[0221] It is contemplated that any of the CNP variants described herein are useful in the methods.

[0222] In various embodiments, the CNP variant isPGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1).

[0223] In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is on the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or an NH2group 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 hydrolysable linker.

[0224] Efficacy of treatment is measured by various parameters. In various embodiments, efficacy is assessed as the change in annualized growth velocity from the baseline period to the intervention period. Efficacy will also be assessed as the change in height SDS from baseline to end of treatment as measured using the CDC growth curves, and growth velocity SDS will be based on the Bone Mineral Density in Childhood Study (Kelly et al. , J. Clin. Endocrinol. Metab. 2014;99(6):2104-21 12).

[0225] QoLISSY, the Quality of Life in Short Stature Youth, is assessed as directed (Quality of Life in Short Stature Youth - The QoLISSY Questionnaire User’s Manual. Lengerich: Pabst Science Publishers; 2013). The QoLISSY questionnaire is a diseasespecific clinical outcome assessment designed for short stature youth, which has both child self-report and parent-report versions and consists of questions across 8 domains, physical, social, emotional, coping, treatment, belief, future, and effects on parents. Domain total scores are then linearly transformed to standard scores on a scale of 0 to 100: min score = 0 (worst QoL), max score = 100 (best QoL).

[0226] In various embodiments, subjects receiving a CNP Prodrug to treat skeletal dysplasia as described herein have reduced or none of the cardiovascular (CV) side effects such as changes in systemic blood pressures (mean arterial pressure, systolic and diastolic blood pressures, pulse pressure) and heart rate observed with administration of non prodrug CNP variant.Pharmaceutical Compositions

[0227] The disclosure provides pharmaceutical compositions, including modified release compositions, comprising a CNP variant described herein, and one or more pharmaceutically acceptable excipients, carriers and / or diluents. In certain embodiments,the compositions further comprise one or more other biologically active agents (e.g., inhibitors of proteases, receptor tyrosine kinases, and / or the clearance receptor NPR-C).

[0228] The disclosure provides for modified release compositions comprising a conjugate moiety as described herein. Modified-release compositions include those that deliver a drug with a delay after its administration (delayed-release dosage) or for a prolonged period of time (extended-release dosage). Various embodiments of a CNP peptide conjugate provided herein include modified-release compositions, such as extended release, sustained or controlled release, and delayed release. The term “extended release composition” refers to a composition formulated in a manner in order to make the active ingredient / drug available over an extended period of time following administration (US Pharmacopeia). Extended-release dosage include sustained-release (SR) or controlled-release (CR) forms in which. 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 release of the drug substance for some period of time after initial administration.

[0229] In various embodiments, the modified release composition is an extended release composition.

[0230] In various embodiments, for the extended release composition, (i) less than about 20% of peptide is released by day 1 ; and (ii) about 90% of peptide is released weekly, or about 90% of peptide is released bi-weekly, or about 90% of peptide is released monthly, at pH 7 to 7.6.

[0231] In various embodiments, less than about 20% of peptide is released by day 1 at pH 7 to 7.6. In various embodiments, less than about 10% of peptide is released by day 1 at pH 7 to 7.6. It is further contemplated that (i) less than about 30%, or about 40%, or about 50% of peptide is released by day 1, at pH 7.0 to 7.6; and (ii) about 90% of peptide is released weekly, or about 90% of peptide is released bi-weekly, or about 90% of peptide is released monthly, at pH 7 to 7.6. It is further contemplated that (I) less than about 30%, or about 40%, or about 50%, or about 60% of peptide is released by day 1, at pH 7.0 to 7.6; and (ii) about 70%, about 80%, or about 90% of peptide is released weekly; or about 70%, about 80%, or about 90% of peptide is released biweekly; or about 70%, about 80%, or about 90% of peptide is released every three weeks; or about 70%, about 80%, or about 90% of peptide is released monthly, at pH 7to 7.6. In various embodiments, about 90% of peptide is released weekly, at pH 7 to 7.6. In various embodiments, about 90% of peptide is released biweekly, at pH 7 to 7.6. In various embodiments, about 90% of peptide is released monthly at pH 7 to 7.6. It is further contemplated that the release can be at a pH between pH 7.0 to 7.6, between pH 7.1 to 7.5, between p H 7.2 to 7.4, between pH 7.2 to 7.6, or between p H 7.0 to 7.4.

[0232] In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of peptide is released by day 1 , at pH 7.0 to 7.6; and (ii) about 90% of peptide is released weekly, or about 90% of peptide is released bi-weekly, or about 90% of peptide is released monthly, at pH 7 to 7.6. It is further contemplated that (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60% about 65%, about 70%, or about 75% of peptide is released by day 1, at pH 7.0 to 7.6; and (ii) about 70%, about 80%, or about 90% of peptide is released weekly; or about 70%, about 80%, or about 90% of peptide is released bi-weekly; or about 70%, about 80%, or about 90% of peptide is released every three weeks; or about 70%, about 80%, or about 90% of peptide is released monthly, at pH 7 to 7.6; or alternatively ii) about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released weekly; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released bi-weekly; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released every three weeks; or about 70%, about 75%, about 80%, about 85%, or about 90% of peptide is released monthly, at pH 7 to 7.6.

[0233] In various embodiments, the composition comprises an excipient, diluent or carrier. 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.

[0234] Non-limiting examples of excipients, carriers and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on. The compositions can contain liquids (e.g., water, ethanol); diluents of various buffer content (e.g., Tris-HCI, phosphate, acetate buffers, citrate buffers), pH and ionic strength; detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80); anti-oxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosol, benzyl alcohol, m-cresol); and bulking substances (e.g., lactose, mannitol, sucrose). The use of excipients, diluents and carriers in the formulation of pharmaceuticalcompositions is known in the art; see, e.g., Remington’s Pharmaceutical Sciences, 18^ Edition, pages 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0235] For example, carriers include without limitation 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(s). Non-limiting examples of carriers include phosphate buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). A carrier can be a solvent or dispersing medium containing, e.g., ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), a vegetable oil, and mixtures thereof. In some embodiments, the compositions are liquid formulations. In various embodiments, the CNP variant is reconstituted from a lyophilized powder. In certain embodiments, the formulations comprise a CNP variant in a concentration range from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 0.5 to 5 mg / ml, or from about 0.5 to 3 mg / ml, or from about 1 mg / ml to about 10 mg / ml. In various embodiments, the CNP variant is in 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 formulation comprises a CNP variant concentration of no less than about 10 mg / ml, no less than about 5 mg / ml, no less than about 1 mg / ml, no less than about 0.5 mg / ml, or no less than about 0.1 mg / ml. In various embodiments, the formulation comprises a CNP variant concentration of no more than about 300 mg / ml, no more than no more than about 250 mg / ml, no more than about 200 mg / ml, no more than about 150 mg / ml, no more than about 100 mg / ml, no more than about 50 mg / ml, or no more than about 40 mg / ml. In certain embodiments, the formulations comprise a CNP variant in a concentration range from about 1 mg / ml to about 300 mg / ml, or from about 5 mg / ml to about 300 mg / ml, or from about 10 mg / ml to about 300 mg / ml, or from about 1 mg / ml to about 150 mg / ml, or from about 1 mg / ml to about 75 mg / ml, or from about 1 to 75 mg / ml, or from about 5 mg / ml to about 50 mg / ml. In various embodiments, the CNP variant is in a concentration of 10 mg / ml to 30 mg / ml. In various embodiments, the CNP variant is at a concentration of 10 mg / ml. In various embodiments, the CNP variant is at a concentration of 30 mg / ml. In other embodiments, the formulations comprise a CNP variant in a concentration range from about 0.1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 0.5 to 5 mg / ml,or from about 0.5 to 3 mg / ml, or from about 1 mg / ml to about 10 mg / ml. In various embodiments, the CNP variant is in 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 at a concentration of 10 mg / ml. Exemplary concentrations of the CNP variant in the formulation is 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / ml, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 51 mg / ml, 52 mg / ml, 53 mg / ml, 54 mg / ml, 55 mg / ml, 56 mg / ml, 57 mg / ml, 58 mg / ml, 59 mg / ml, 60 mg / ml, 61 mg / ml, 62 mg / ml, 63 mg / ml, 64 mg / ml, 65 mg / ml, 66 mg / ml, 67 mg / ml, 68 mg / ml, 69 mg / ml, 70 mg / ml, 71 mg / ml, 72 mg / ml, 73 mg / ml, 74 mg / ml, 75 mg / ml, 76 mg / ml, 77 mg / ml, 78 mg / ml, 79 mg / ml, 80 mg / ml, 81 mg / ml, 82 mg / ml, 83 mg / ml, 84 mg / ml, 85 mg / ml, 86 mg / ml, 87 mg / ml, 88 mg / ml, 89 mg / ml, 90 mg / ml, 91 mg / ml, 92 mg / ml, 93 mg / ml, 94 mg / ml, 95 mg / ml, 96 mg / ml, 97 mg / ml, 98 mg / ml, 99 mg / ml, 100 mg / ml, 101 mg / ml, 102 mg / ml, 103 mg / ml, 104 mg / ml, 105 mg / ml, 106 mg / ml, 107 mg / ml, 108 mg / ml, 109 mg / ml, 110 mg / ml, 111 mg / ml, 112 mg / ml, 113 mg / ml, 114 mg / ml, 115 mg / ml, 116 mg / ml, 117 mg / ml, 118 mg / ml, 119 mg / ml, 120 mg / ml, 121 mg / ml, 122 mg / ml, 123 mg / ml, 124 mg / ml, 125 mg / ml, 126 mg / ml, 127 mg / ml, 128 mg / ml, 129 mg / ml, 130 mg / ml, 131 mg / ml, 132 mg / ml, 133 mg / ml, 134 mg / ml, 135 mg / ml, 136 mg / ml, 137 mg / ml, 138 mg / ml, 139 mg / ml, 140 mg / ml, 141 mg / ml, 142 mg / ml, 143 mg / ml, 144 mg / ml, 145 mg / ml, 146 mg / ml, 147 mg / ml, 148 mg / ml, 149 mg / ml, 150 mg / ml, 151 mg / ml, 152 mg / ml, 153 mg / ml, 154 mg / ml, 155 mg / ml, 156 mg / ml, 157 mg / ml, 158 mg / ml, 159 mg / ml, 160 mg / ml, 161 mg / ml, 162 mg / ml, 163 mg / ml, 164 mg / ml, 165 mg / ml, 166 mg / ml, 167 mg / ml, 168 mg / ml, 169 mg / ml, 170 mg / ml, 171 mg / ml, 172 mg / ml, 173 mg / ml, 174 mg / ml, 175 mg / ml, 176 mg / ml, 177 mg / ml, 178 mg / ml, 179 mg / ml, 180 mg / ml, 181 mg / ml, 182 mg / ml, 183 mg / ml, 184 mg / ml, 185 mg / ml, 186 mg / ml, 187 mg / ml, 188 mg / ml, 189 mg / ml, 190 mg / ml, 191 mg / ml, 192 mg / ml, 193 mg / ml, 194 mg / ml, 195 mg / ml, 196 mg / ml, 197 mg / ml, 198 mg / ml, 199 mg / ml, 200 mg / ml, 201 mg / ml, 202 mg / ml, 203 mg / ml, 204 mg / ml, 205 mg / ml, 206 mg / ml, 207 mg / ml, 208 mg / ml, 209 mg / ml, 210 mg / ml, 211 mg / ml, 212 mg / ml, 213 mg / ml, 214 mg / ml, 215 mg / ml, 216 mg / ml, 217 mg / ml, 218 mg / ml, 219 mg / ml, 220 mg / ml, 221 mg / ml, 222 mg / ml, 223 mg / ml, 224 mg / ml, 225 mg / ml, 226 mg / ml, 227 mg / ml, 228 mg / ml, 229 mg / ml, 230 mg / ml, 231 mg / ml, 232 mg / ml, 233 mg / ml, 234 mg / ml, 235 mg / ml, 236 mg / ml, 237 mg / ml, 238 mg / ml, 239 mg / ml, 240 mg / ml, 241mg / ml, 242 mg / ml, 243 mg / ml, 244 mg / ml, 245 mg / ml, 246 mg / ml, 247 mg / ml, 248 mg / ml, 249 mg / ml, 250 mg / ml, 251 mg / ml, 252 mg / ml, 253 mg / ml, 254 mg / ml, 255 mg / ml, 256 mg / ml, 257 mg / ml, 258 mg / ml, 259 mg / ml, 260 mg / ml, 261 mg / ml, 262 mg / ml, 263 mg / ml, 264 mg / ml, 265 mg / ml, 266 mg / ml, 267 mg / ml, 268 mg / ml, 269 mg / ml, 270 mg / ml, 271 mg / ml, 272 mg / ml, 273 mg / ml, 274 mg / ml, 275 mg / ml, 276 mg / ml, 277 mg / ml, 278 mg / ml, 279 mg / ml, 280 mg / ml, 281 mg / ml, 282 mg / ml, 283 mg / ml, 284 mg / ml, 285 mg / ml, 286 mg / ml, 287 mg / ml, 288 mg / ml, 289 mg / ml, 290 mg / ml, 291 mg / ml, 292 mg / ml, 293 mg / ml, 294 mg / ml, 295 mg / ml, 296 mg / ml, 297 mg / ml, 298 mg / ml, 299 mg / ml, or 300 mg / ml.

[0236] In further embodiments, the compositions comprise a buffer solution or buffering agent to maintain the pH of a CNP-containing solution or suspension within a desired range. No n limiting examples of buffer solutions include phosphate buffered saline, Tris buffered saline, and Hanks buffered saline. Buffering agents include without limitation sodium acetate, sodium phosphate, and sodium citrate. Mixtures of buffering agents can also be used. In certain embodiments, the buffering agent is histidine / L-histidine or histidine monohydrochloride monohydrate. In other embodiments, the buffering agent is acetic acid / acetate or citric acid / citrate. The amount of buffering agent suitable in a composition depends in part on the particular buffer used and the desired p H of the solution or suspension. In some embodiments, the buffering agent has a concentration of about 10 mM ± 5 mM. In certain embodiments, the p H of a composition is from about p H 3 to about pH 9, or from about p H 3 to about p H 7.5, or from about p H 3.5 to about pH 7, or from about p H 3.5 to about pH 6.5, or from about pH 4 to about p H 6, or from about pH 4 to about p H 5, or is at about pH 5.0 ± 1.0. In various embodiments, the pH is about 5.0 to about 6.0 (e.g., 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 p H is 5.5.

[0237] In other embodiments, the compositions contain an isotonicity-adjusting agent to render the solution or suspension isotonic and more compatible for administration. Non-limiting examples of isotonicity agents include NaCI, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol. In certain embodiments, the isotonicity agent is NaCI. In certain embodiments, NaCI is in a concentration of 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.

[0238] In yet other embodiments, the compositions comprise a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is in a concentration of about 0.4% ± 0.2%, or about 1% ± 0.5%, or about 1.5% ± 0.5%, or about 2.0% ± 0.5%.

[0239] In still other embodiments, the compositions contain an anti-adsorbent (e.g., to mitigate adsorption of a CNP variant to glass or plastic). Anti-adsorbents include without limitation benzyl alcohol, Polysorbate 20, and Polysorbate 80. In certain embodiments, the anti adsorbent is in a concentration from about 0.001% to about 0.5%, or from about 0.01% to about 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about 1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, or from about 1% to about 2%.

[0240] In additional embodiments, the compositions comprise a stabilizer. Nonlimiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine ((L- methionine), and ascorbic acid and salts thereof. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is in a concentration from about 0.1% to about 1%. In other embodiments, when the stabilizer is methionine, the stabilizer is in a concentration from about 0.01% to about 0.5%, or from about 0.01% to about 0.2%. In still other embodiments, when the stabilizer is glycerin, the stabilizer is in a concentration from about 5% to about 100% (neat).

[0241] In further embodiments, the compositions contain an antioxidant. Exemplary antioxidants include without limitation methionine and ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP is from about 0.1:1 to about 15:1 , or from about 1:1 to about 15:1 , or from about 0.5:1 to about 10:1, or from about 1:1 to about 10:1 or from about 3:1 to about 10:1.

[0242] Pharmaceutically acceptable salts can be used in the compositions, including without limitation mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), salts of organic acids (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and salts of amines (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A thorough discussion of pharmaceutically acceptable salts is found in Remingtons Pharmaceutical Sciences, 18thEdition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0243] The pharmaceutical compositions can be administered in various forms, such as tablets, capsules, granules, powders, solutions, suspensions, emulsions, ointments, and transdermal patches. The dosage forms of the compositions can be tailored to the desired mode of administration of the compositions. For oral administration, the compositions can take the form of, e.g., a tablet or capsule (including softgel capsule),or can be, e.g., an aqueous or nonaqueous solution, suspension or syrup. Tablets and capsules for oral administration can include 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 lubricating agents (e.g., magnesium stearate, sodium stearyl fumarate). If desired, flavoring, coloring and / or sweetening agents can be added to the solid and liquid formulations.

[0244] Other optional ingredients for oral formulations include without limitation preservatives, suspending agents, and thickening agents. Oral formulations can also have an enteric coating to protect the CNP variant from the acidic environment of the stomach. Methods of preparing solid and liquid dosage forms are known, or will be apparent, to those skilled in this art (see, e.g., Remington’s Pharmaceutical Sciences, referenced above).

[0245] Formulations for parenteral administration can be prepared, e.g., as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in a liquid medium prior to injection, 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 solution, Ringer's solution, isotonic sodium chloride solution), dispersing agents, wetting agents, emulsifying agents, suspending agents, and the like. In addition, sterile fixed oils, fatty esters, polyols and / or other inactive ingredients can be used. As further examples, formulations for parenteral administration include aqueous sterile injectable solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can contain suspending agents and thickening agents.

[0246] Compositions comprising a CNP variant can also be lyophilized formulations. In certain embodiments, the lyophilized formulations comprise a buffer and bulking agent, and optionally an antioxidant. Exemplary buffers include without limitation acetate buffers, citrate buffers, and histidine buffers. Exemplary bulking agents include without limitation mannitol (e.g., D-mannitol), sucrose, dextran, lactose, trehalose (e.g., trehalose dihydrate), and povidone (PVP K24). In certain embodiments, mannitol is in an amount from about 3% to about 10%, or from about 4% to about 8%, or from about 4% to about 6%. In certain embodiments, sucrose is in an amount from about 6% to about 20%, or from about 6% to about 15%, or from about 8% to about 12%. Exemplary anti-oxidants include, but are not limited to, methionine and ascorbic acid. In various embodiments, thelyophilized formulation comprises no less than about 10 mg of the CNP variant, no less than about 5 mg of the CNP variant, no less than about 3 mg of the CNP variant, no less than about 1 mg of the CNP variant, no less than about 0.5 mg of the CNP variant, or no less than about 0.1 mg. In various embodiments, the lyophilized formulation comprises no more than about 300 mg of the CNP variant, no more than about 250 mg of the CNP variant, no more than about 200 mg of the CNP variant, no more than about 150 mg of the CNP variant, no more than about 100 mg of the CNP variant, no more than about 50 mg of the CNP variant, or no more than about 40 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises about 1 mg to about 300 mg of the CNP variant, or from about 5 mg to about 300 mg of the CNP variant, or from about 10 mg to about 300 mg of the CNP variant, or from about 1 mg to about 150 mg of the CNP variant, or from about 5 mg to about 150 mg of the CNP variant, or from about 5 to 150 mg of the CNP variant, or from about 5 to 75 mg of the CNP variant, or from about 10 mg to about 50 mg of the CNP variant. The lyophilized formulation comprises 13 mg to 39 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 13 mg of theCNP variant. In various embodiments, the lyophilized formulation comprises 39 mg of theCNP variant. In other embodiments, the lyophilized formulation comprises about 0.1 mg to about 20 mg of the CNP variant, or from about 0.4 mg to about 20 mg of the CNP variant, or from about 1 mg to about 20 mg of the CNP variant, or from about 0.1 mg to about 10 mg of the CNP variant, or from about 0.1 mg to about 10 mg of the CNP variant, or from about 0.1 to 5 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 0.4 mg to 3.5 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 0.4 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 0.56 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 1.2 mg of the CNP variant. In various embodiments, the lyophilized formulation comprises 3.5 mg of the CNP variant. Exemplary concentrations of the CNP variant in the formulation is 0.1 mg, 0.11 mg, 0.12 mg, 0.13 mg, 0.14 mg, 0.15 mg, 0.16 mg, 0.17 mg, 0.18 mg, 0.19 mg, 0.2 mg, 0.21 mg, 0.22 mg, 0.23 mg,0.24 mg, 0.25 mg, 0.26 mg, 0.27 mg, 0.28 mg, 0.29 mg, 0.3 mg, 0.31 mg, 0.32 mg, 0.33 mg,0.34 mg, 0.35 mg, 0.36 mg, 0.37 mg, 0.38 mg, 0.39 mg, 0.4 mg, 0.41 mg, 0.42 mg, 0.43 mg,0.44 mg, 0.45 mg, 0.46 mg, 0.47 mg, 0.48 mg, 0.49 mg, 0.5 mg, 0.51 mg, 0.52 mg, 0.53 mg,0.54 mg, 0.55 mg, 0.56 mg, 0.57 mg, 0.58 mg, 0.59 mg, 0.6 mg, 0.61 mg, 0.62 mg, 0.63 mg,0.64 mg, 0.65 mg, 0.66 mg, 0.67 mg, 0.68 mg, 0.69 mg, 0.7 mg, 0.71 mg, 0.72 mg, 0.73 mg,0.74 mg, 0.75 mg, 0.76 mg, 0.77 mg, 0.78 mg, 0.79 mg, 0.8 mg, 0.81 mg, 0.82 mg, 0.83 mg,0.84 mg, 0.85 mg, 0.86 mg, 0.87 mg, 0.88 mg, 0.89 mg, 0.9 mg, 0.91 mg, 0.92 mg, 0.93 mg,0.94 mg, 0.95 mg, 0.96 mg, 0.97 mg, 0.98 mg, 0.99 mg, 1 mg, 1.5 mg, 2 mg, 2.5 mg, 3 mg,3.5 mg, 4 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, 10 mg, 10.5 mg, 11 mg, 11.5 mg, 12 mg, 12.5 mg, 13 mg, 13.5 mg, 14 mg, 14.5 mg, 15 mg, 15.5 mg, 16 mg, 16.5 mg, 17 mg, 17.5 mg, 18 mg, 18.5 mg, 19 mg, 19.5 mg, 20 mg,20.5 mg, 21 mg, 21.5 mg, 22 mg, 22.5 mg, 23 mg, 23.5 mg, 24 mg, 24.5 mg, 25 mg, 25.5 mg, 26 mg, 26.5 mg, 27 mg, 27.5 mg, 28 mg, 28.5 mg, 29 mg, 29.5 mg, 30 mg, 30.5 mg, 31 mg, 31.5 mg, 32 mg, 32.5 mg, 33 mg, 33.5 mg, 34 mg, 34.5 mg, 35 mg, 35.5 mg, 36 mg,36.5 mg, 37 mg, 37.5 mg, 38 mg, 38.5 mg, 39 mg, 39.5 mg, 40 mg, 40.5 mg, 41 mg, 41.5 mg, 42 mg, 42.5 mg, 43 mg, 43.5 mg, 44 mg, 44.5 mg, 45 mg, 45.5 mg, 46 mg, 46.5 mg, 47 mg, 47.5 mg, 48 mg, 48.5 mg, 49 mg, 49.5 mg, 50 mg, 50.5 mg, 51 mg, 51.5 mg, 52 mg,52.5 mg, 53 mg, 53.5 mg, 54 mg, 54.5 mg, 55 mg, 55.5 mg, 56 mg, 56.5 mg, 57 mg, 57.5 mg, 58 mg, 58.5 mg, 59 mg, 59.5 mg, 60 mg, 60.5 mg, 61 mg, 61.5 mg, 62 mg, 62.5 mg, 63 mg, 63.5 mg, 64 mg, 64.5 mg, 65 mg, 65.5 mg, 66 mg, 66.5 mg, 67 mg, 67.5 mg, 68 mg,68.5 mg, 69 mg, 69.5 mg, 70 mg, 70.5 mg, 71 mg, 71.5 mg, 72 mg, 72.5 mg, 73 mg, 73.5 mg, 74 mg, 74.5 mg, 75 mg, 75.5 mg, 76 mg, 76.5 mg, 77 mg, 77.5 mg, 78 mg, 78.5 mg, 79 mg, 79.5 mg, 80 mg, 80.5 mg, 81 mg, 81.5 mg, 82 mg, 82.5 mg, 83 mg, 83.5 mg, 84 mg,84.5 mg, 85 mg, 85.5 mg, 86 mg, 86.5 mg, 87 mg, 87.5 mg, 88 mg, 88.5 mg, 89 mg, 89.5 mg, 90 mg, 90.5 mg, 91 mg, 91.5 mg, 92 mg, 92.5 mg, 93 mg, 93.5 mg, 94 mg, 94.5 mg, 95 mg, 95.5 mg, 96 mg, 96.5 mg, 97 mg, 97.5 mg, 98 mg, 98.5 mg, 99 mg, 99.5 mg, 100 mg,100.5 mg, 101 mg, 101.5 mg, 102 mg, 102.5 mg, 103 mg, 103.5 mg, 104 mg, 104.5 mg, 105 mg, 105.5 mg, 106 mg, 106.5 mg, 107 mg, 107.5 mg, 108 mg, 108.5 mg, 109 mg, 109.5 mg, 110 mg, 110.5 mg, 111 mg, 111.5 mg, 112 mg, 112.5 mg, 113 mg, 113.5 mg, 114 mg, 114.5 mg, 115 mg, 115.5 mg, 116 mg, 116.5 mg, 117 mg, 117.5 mg, 118 mg, 118.5 mg, 119 mg,119.5 mg, 120 mg, 120.5 mg, 121 mg, 121.5 mg, 122 mg, 122.5 mg, 123 mg, 123.5 mg, 124 mg, 124.5 mg, 125 mg, 125.5 mg, 126 mg, 126.5 mg, 127 mg, 127.5 mg, 128 mg, 128.5 mg, 129 mg, 129.5 mg, 130 mg, 130.5 mg, 131 mg, 131.5 mg, 132 mg, 132.5 mg, 133 mg, 133.5 mg, 134 mg, 134.5 mg, 135 mg, 135.5 mg, 136 mg, 136.5 mg, 137 mg, 137.5 mg, 138 mg,138.5 mg, 139 mg, 139.5 mg, 140 mg, 140.5 mg, 141 mg, 141.5 mg, 142 mg, 142.5 mg, 143 mg, 143.5 mg, 144 mg, 144.5 mg, 145 mg, 145.5 mg, 146 mg, 146.5 mg, 147 mg, 147.5 mg, 148 mg, 148.5 mg, 149 mg, 149.5 mg, 150 mg, 150.5 mg, 151 mg, 151.5 mg, 152 mg, 152.5 mg, 153 mg, 153.5 mg, 154 mg, 154.5 mg, 155 mg, 155.5 mg, 156 mg, 156.5 mg, 157 mg,157.5 mg, 158 mg, 158.5 mg, 159 mg, 159.5 mg, 160 mg, 160.5 mg, 161 mg, 161.5 mg, 162 mg, 162.5 mg, 163 mg, 163.5 mg, 164 mg, 164.5 mg, 165 mg, 165.5 mg, 166 mg, 166.5 mg, 167 mg, 167.5 mg, 168 mg, 168.5 mg, 169 mg, 169.5 mg, 170 mg, 170.5 mg, 171 mg, 171.5 mg, 172 mg, 172.5 mg, 173 mg, 173.5 mg, 174 mg, 174.5 mg, 175 mg, 175.5 mg, 176 mg,176.5 mg, 177 mg, 177.5 mg, 178 mg, 178.5 mg, 179 mg, 179.5 mg, 180 mg, 180.5 mg, 181 mg, 181.5 mg, 182 mg, 182.5 mg, 183 mg, 183.5 mg, 184 mg, 184.5 mg, 185 mg, 185.5 mg, 186 mg, 186.5 mg, 187 mg, 187.5 mg, 188 mg, 188.5 mg, 189 mg, 189.5 mg, 190 mg, 190.5 mg, 191 mg, 191.5 mg, 192 mg, 192.5 mg, 193 mg, 193.5 mg, 194 mg, 194.5 mg, 195 mg,195.5 mg, 196 mg, 196.5 mg, 197 mg, 197.5 mg, 198 mg, 198.5 mg, 199 mg, 199.5 mg, 200 mg, 200.5 mg, 201 mg, 201.5 mg, 202 mg, 202.5 mg, 203 mg, 203.5 mg, 204 mg, 204.5 mg, 205 mg, 205.5 mg, 206 mg, 206.5 mg, 207 mg, 207.5 mg, 208 mg, 208.5 mg, 209 mg, 209.5 mg, 210 mg, 210.5 mg, 211 mg, 211.5 mg, 212 mg, 212.5 mg, 213 mg, 213.5 mg, 214 mg,214.5 mg, 215 mg, 215.5 mg, 216 mg, 216.5 mg, 217 mg, 217.5 mg, 218 mg, 218.5 mg, 219 mg, 219.5 mg, 220 mg, 220.5 mg, 221 mg, 221.5 mg, 222 mg, 222.5 mg, 223 mg, 223.5 mg, 224 mg, 224.5 mg, 225 mg, 225.5 mg, 226 mg, 226.5 mg, 227 mg, 227.5 mg, 228 mg, 228.5 mg, 229 mg, 229.5 mg, 230 mg, 230.5 mg, 231 mg, 231.5 mg, 232 mg, 232.5 mg, 233 mg,233.5 mg, 234 mg, 234.5 mg, 235 mg, 235.5 mg, 236 mg, 236.5 mg, 237 mg, 237.5 mg, 238 mg, 238.5 mg, 239 mg, 239.5 mg, 240 mg, 240.5 mg, 241 mg, 241.5 mg, 242 mg, 242.5 mg, 243 mg, 243.5 mg, 244 mg, 244.5 mg, 245 mg, 245.5 mg, 246 mg, 246.5 mg, 247 mg, 247.5 mg, 248 mg, 248.5 mg, 249 mg, 249.5 mg, 250 mg, 250.5 mg, 251 mg, 251.5 mg, 252 mg,252.5 mg, 253 mg, 253.5 mg, 254 mg, 254.5 mg, 255 mg, 255.5 mg, 256 mg, 256.5 mg, 257 mg, 257.5 mg, 258 mg, 258.5 mg, 259 mg, 259.5 mg, 260 mg, 260.5 mg, 261 mg, 261.5 mg, 262 mg, 262.5 mg, 263 mg, 263.5 mg, 264 mg, 264.5 mg, 265 mg, 265.5 mg, 266 mg, 266.5 mg, 267 mg, 267.5 mg, 268 mg, 268.5 mg, 269 mg, 269.5 mg, 270 mg, 270.5 mg, 271 mg,271.5 mg, 272 mg, 272.5 mg, 273 mg, 273.5 mg, 274 mg, 274.5 mg, 275 mg, 275.5 mg, 276 mg, 276.5 mg, 277 mg, 277.5 mg, 278 mg, 278.5 mg, 279 mg, 279.5 mg, 280 mg, 280.5 mg, 281 mg, 281.5 mg, 282 mg, 282.5 mg, 283 mg, 283.5 mg, 284 mg, 284.5 mg, 285 mg, 285.5 mg, 286 mg, 286.5 mg, 287 mg, 287.5 mg, 288 mg, 288.5 mg, 289 mg, 289.5 mg, 290 mg,290.5 mg, 291 mg, 291.5 mg, 292 mg, 292.5 mg, 293 mg, 293.5 mg, 294 mg, 294.5 mg, 295 mg, 295.5 mg, 296 mg, 296.5 mg, 297 mg, 297.5 mg, 298 mg, 298.5 mg, 299 mg, 299.5 mg, or 300 mg.

[0247] In various embodiments, the formulation comprises L-histidine, histidine monohydrochloride monohydrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI). In various embodiments, the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).

[0248] The disclosure also provides kits containing, e.g., bottles, vials, ampoules, tubes, cartridges and / or syringes that comprise a liquid (e.g., sterile injectable)formulation or a solid (e.g., lyophilized) formulation. The kits can also contain pharmaceutically acceptable vehicles or carriers (e.g., solvents, solutions and / or buffers) for reconstituting a solid (e.g., lyophilized) formulation into a solution or suspension for administration (e.g., by injection), including without limitation reconstituting a lyophilized formulation in a syringe for injection or for diluting concentrate to a lower concentration. Furthermore, extemporaneous injection solutions and suspensions can be prepared from, e.g., sterile powder, granules, or tablets comprising a CNP- containing composition. The kits can also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and / or needles.

[0249] As a non-limiting example, a kit can include syringes having a single chamber or dual chambers. For single-chamber syringes, the single chamber can contain a liquid CNP formulation ready for injection, or a solid (e.g., lyophilized) CNP formulation or a liquid formulation of a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) that can be reconstituted into a solution or suspension for injection. For dual-chamber syringes, one chamber can contain a pharmaceutically acceptable vehicle or carrier (e.g., solvent system, solution or buffer), and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or a liquid formulation of a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) which can be reconstituted into a solution or suspension, using the vehicle or carrier from the first chamber, for injection.

[0250] As a further example, a kit can include one or more pen injector or autoinjector devices, and dual-chamber cartridges. One chamber of a cartridge can contain a pharmaceutically acceptable vehicle or carrier (e.g., solvent system, solution or buffer), and the other chamber can contain a solid (e.g., lyophilized) CNP formulation or a liquid formulation of a CNP variant in a relatively small amount of a suitable solvent system (e.g., glycerin) which can be reconstituted into a solution or suspension, using the vehicle or carrier from the first chamber, for injection. A cartridge can comprise an amount of the CNP variant that is sufficient for dosing over a desired time period (e.g., 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, etc.). The pen injector or autoinjector can be adjusted to administer a desired amount of the CNP formulation from a cartridge.Administration and Dosing

[0251] The CNP variants, or pharmaceutical compositions or formulations comprising them, can be administered to subjects in various ways such as, e.g., subcutaneously, intraarticularly, intraperitoneally, intramuscularly, intradermally or orally. In oneembodiment, the CNP variant composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.

[0252] The CNP variants or compositions thereof can also be administered by implantation of a depot at the target site of action (e.g., an abnormal or degenerated joint or cartilage area).

[0253] Alternatively, the CNP variant can be administered sublingually under the tongue (e.g., sublingual tablet) by transdermal delivery (e.g., by means of a patch on the skin) or orally in the form of microspheres, microcapsules, liposomes (uncharged or charged (e.g., cationic)), polymeric microparticles (e.g., polyamides, polylactide, polyglycolide, poly(lactide-glycolide)), microemulsions, and the like.

[0254] The CNP variant compositions described herein can be administered to patients in need thereof at therapeutically effective doses to treat, ameliorate or prevent bone-related disorders (e.g., skeletal dysplasias, including achondroplasia). The CNP variant compositions described herein can be administered to patients in need thereof at therapeutically effective doses to treat, ameliorate or prevent bone-related disorders or short stature disorders (e.g., skeletal dysplasias, including achondroplasia, hypochondroplasia, or idiopathic short stature). The safety and therapeutic efficacy of the CNP variant can be determined by standard pharmacological procedures in cell cultures or experimental animals, such as, for example, by determining the LD50(the dose lethal to 50% of the population) and the ED50(the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED 50. Active agents exhibiting a large therapeutic index are normally preferred.

[0255] In certain embodiments, the CNP variant compositions described herein are administered at a dose in the range from about 3, 4, 5, 6, 7, 8, 9 or 10 nmol / kg to about 300 nmol / kg, or from about 20 nmol / kg to about 200 nmol / kg. In some embodiments, the CNP compositions are administered at a dose 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 other dose deemed appropriate by the treating physician. In other embodiments, the CNP variant compositions are administered at a dose of about 3, 4, 5, 6, 7, 8, 9 , 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950 or 1000 μg / kg , or about 0.5, 0.8, 1.0, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg / kg, or other dose deemed appropriate by the treating physician. In various embodiments, the CNP Prodrug is administered at a dose from about 5 μg / kg to 500 μg / kg , from about 15 μg / kg to 350 μg / kg , from about 25 μg / kg to 300 μg / kg , from about 50 μg / kg to 250 μg / kg or from about 75 μg / kg to 200 μg / kg . In various embodiments, the CNP variant is administered at a dose of about 15 μg / kg , 20 μg / kg , 25 μg / kg , 30 μg / kg , 35 μg / kg , 40 μg / kg , 45 μg / kg , 50 μg / kg , 60 μg / kg , 70 μg / kg , 75 μg / kg , 80 μg / kg , 90 μg / kg , 100 μg / kg , 125 μg / kg , 150 μg / kg , 175 μg / kg , 200 μg / kg , 225 μg / kg , 250 μg / kg , 275 μg / kg , 300 μg / kg , 325 μg / kg , 350 μg / kg , 400 μg / kg , 450 μg / kg , or 500 μg / kg . The doses of CNP or CNP variant described herein can be administered according to the dosing frequency / frequency of administration described herein, including without limitation daily, 2 or 3 times per week, weekly, every 2 weeks, every 3 weeks, monthly, etc. In various embodiments, the CNP or CNP variant is administered daily subcutaneously. In various embodiments, the CNP or CNP variant is administered weekly subcutaneously. In various embodiments, the CNP variant is administered at a dose of 2.5 μg / kg / day to 60 μg / kg / day, 10μg / kg / day to 45 μg / kg / day, or 15μg / 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.

[0256] In various embodiments, the pharmaceutical composition is a sustained release composition. In various embodiments, the pharmaceutical composition comprising the CNP prodrug has a longer half-life, improved Cmax and improved AUC compared to a non- sustained release composition comprising the same active CNP compound, i.e., free drug.

[0257] The frequency of dosing / administration of a CNP variant for a particular subject may vary depending upon various factors, including the disorder being treated and the condition and response of the subject to the therapy. The CNP variant can be administered in a single dose or in multiple doses per dosing. In certain embodiments, the CNP variant composition is administered, in a single dose or in multiple doses, once daily, once weekly, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months, or as deemed appropriate by the treating physician. In various embodiments, the CNP variant is administered for 3 months, 6 months, 12 months or more.

[0258] In some embodiments, a CNP variant composition is administered so as to allow for periods of growth (e.g., chondrogenesis), followed by a recovery period (e.g., osteogenesis). For example, the CNP composition may be administered subcutaneouslyor by another mode daily or multiple times per week for a period of time, followed by a period of no treatment, then the cycle is repeated. In some embodiments, the initial period of treatment (e.g., administration of the CNP variant composition daily or multiple times per week) is for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related embodiment, the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks. In certain embodiments, the dosing regimen of the CNP variant compositions is daily for 3 days followed by 3 days off; or daily or multiple times per week for 1 week followed by 3 days or 1 week off; or daily or multiple times per week for 2 weeks followed by 1 or 2 weeks off; or daily or multiple times per week for 3 weeks followed by 1 , 2 or 3 weeks off; or daily or multiple times per week for 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks followed by 1, 2, 3 or 4 weeks off.Biomarkers

[0259] For treatment of bone-related disorders, indicators of growth can be measured, such as long bone growth measurements in utero and neonatal and measurements of bone growth biomarkers such as CNP, cGMP, Collagen II, Collagen X, osteocalcin, and Proliferating Cell Nuclear Antigen (PCNA).

[0260] One CNP signaling marker is cGMP (guanosine 3’, 5’ cyclic monophosphate). The level of this intracellular signaling molecule increases after CNP binds to and activates its cognate receptor NPR-B. Elevated levels of cGMP can be measured from cell culture extracts (in vitro) after CNP exposure, conditioned media from bone ex-plant studies (ex vivo) after CNP exposure, and in the plasma (in vivo) within minutes of CNP administration subcutaneously, intravenously, or via other routes of administration known in the art.

[0261] Cartilage and bone-specific analytes (or cartilage- and bone-associated markers) can also be measured to assess CNP efficacy. For example, fragments of cleaved collagen type II are a cartilage-specific marker for cartilage turnover. Type II collagen is the major organic constituent of cartilage and fragments of type II collagen (cleaved collagen) are released into circulation, and subsequently secreted into the urine, following cartilage turnover. Cartilage turnover precedes new bone formation.

[0262] A bone-specific biomarker for bone formation which can be measured is N- terminal propeptides of type I procollagen (PINP). The synthesis of type I collagen is an important step in bone formation, as type I collagen is the major organic component inbone matrix. During collagen synthesis, propeptides are released from the procollagen molecule and can be detected in serum. In addition, fragments of collagen type I can be measured as a marker for bone resorption.

[0263] Other potential biomarkers for cartilage and bone formation and growth include aggrecan chondroitin sulfate (cartilage-specific marker for cartilage turnover), propeptides of type II collagen (cartilage-specific marker for cartilage formation), collagen type I C- telopeptide (CTx), alkaline phosphatase (bone-specific) and osteocalcin (bone-specific marker for bone formation). Biomarkers also include proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, and alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), cross-linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP- variant bioactivity. Cartilage- and bone-associated biomarkers can be measured, e.g., in serum from efficacy / pharmacodynamic in vivo studies and from the conditioned media of ex vivo studies, using commercially available kits.

[0264] In one embodiment, the level of at least one bone- or cartilage-associated biomarker is assayed or measured in a subject that has been administered a CNP variant or composition described herein in order to monitor the effects of the CNP composition on bone and cartilage formation and growth in vivo. For example, an increase in the level of at least one bone- or cartilage-associated biomarker may indicate that administration of a CNP variant or composition has a positive effect on bone growth and is a useful treatment for skeletal dysplasias and other bone- or cartilage-related diseases or disorders associated with decreased CNP activity.

[0265] Exemplary bone- or cartilage-associated biomarkers include, but are not limited to, CNP (e.g., endogenous levels of CNP), cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, collagen type I C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, collagen X, aggrecan chondroitin sulfate, and alkaline phosphatase

[0266] In various embodiments, the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II andfragments thereof, collagen type II and fragments thereof, Collagen Type I C-Telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, and alkaline phosphatase, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N- propeptide (PINP), cross-linked C-telopeptide of type I collagen (CTx), cross-linked N- telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, e.g., from PAXgene® RNA, and CNP-variant bioactivity. NTproCNP is an amino-terminal propeptide (NTproCNP) of CNP that is released from cells at an equimolar ratio with CNP. The biologically active forms of CNP are found in plasma in low concentrations due to the quick clearance rate of the peptide. NTproCNP is not cleared via the same mechanism and it is found in the circulation at 20- to 50-fold higher concentration (Olney et al., Clin Endocrinol (Oxf). 2012, 77:416—422).

[0267] Collagen type X biomarker (CXM) is a degradation fragment of collagen type X, comprising intact trimeric noncollagenous 1 (NCI) domain of type X collagen. CXM is released by active growth plates and decreases in samples as subjects age. CXM levels have been correlated with growth velocity in children (Coghlan et al., Sci Transl Med 2017, 9(419):eaan4669).

[0268] Bone-specific alkaline phosphatase (BSAP or BAP) is a bone growth biomarker produced by osteoblasts and osteoclasts in growth plates and mineralized bone. Changes in BSAP may reflect growth plate activity, bone growth, and / or bone remodeling activity.

[0269] A bone-specific biomarker for bone formation which can be measured is N-terminal propeptides of type I procollagen (PINP). The synthesis of type I collagen is an important step in bone formation, as type I collagen is the major organic component in bone matrix.During collagen synthesis, propeptides are released from the procollagen molecule and can be detected in serum. In addition, fragments of collagen type I can be measured as a marker for bone resorption.

[0270] Other potential biomarkers for cartilage and bone formation and growth include aggrecan chondroitin sulfate (cartilage-specific marker for cartilage turnover), propeptides of type II collagen (cartilage-specific marker for cartilage formation), collagen type I C- telopeptide (CTx), alkaline phosphatase (bone-specific) and osteocalcin (bone-specific marker for bone formation). Cartilage- and bone-associated biomarkers can be measured, e.g., in serum from efficacy / phamnacodynamic in vivo studies and from the conditioned media of ex vivo studies, using commercially available kits.

[0271] In various embodiments, biomarkers are measured by obtaining a biological sample from a subject who will be administered, is being administered or has been administered a CNP variant. Biomarkers can be measured using techniques known in the art, including, but not limited to, Western Blot, enzyme linked immunosorbant assay (ELISA), and enzymatic activity assay. The biological sample can be blood, serum, urine, or other biological fluids.Formulations

[0272] In additional embodiments, the disclosure contemplates use of pharmaceutical compositions and formulations comprising a CNP variant peptide, and one or more pharmaceutically acceptable excipients, carriers and / or diluents. In certain embodiments, the compositions further comprise one or more other biologically active agents (e.g., inhibitors of proteases, receptor tyrosine kinases, and / or the clearance receptor NPR-C).

[0273] Some of the possible formulations are disclosed in the examples section. In any formulation, the concentration of the CNP Variant in units of mg / mL is typically 0.5 or greater, 1 or greater, 5 or greater, 10 or greater, 15 or greater, 20 or greater, 25 or greater, or even 30 or greater. In any formulation, the concentration of the CNP Variant in units of mg / mL is typically 40 or less, 35 or less, 30 or less, 25 or less, 20 or less, 15 or less, 10 or less, or even 5 or less.

[0274] Non-limiting examples of excipients, carriers and diluents include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on. The compositions can contain liquids (e.g., water, ethanol); diluents of various buffer content (e.g., Tris-HCI, phosphate, acetate buffers, citrate buffers), pH and ionic strength; detergents and solubilizing agents (e.g., Polysorbate 20, Polysorbate 80 (sometimes called PS 80)); anti-oxidants (e.g., methionine, ascorbic acid, sodium metabisulfite); preservatives (e.g., Thimerosol, benzyl alcohol, m-cresol); and bulking substances (e.g., lactose, mannitol, sucrose). The use of excipients, diluents and carriers in the formulation of pharmaceutical compositions is known in the art; see, e.g., Remington's Pharmaceutical Sciences, 18^ Edition, pages 1435- 1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0275] For example, carriers include without limitation 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(s). Non-limiting examples of carriersinclude phosphate buffered saline, physiological saline, water, and emulsions (e.g., oil / water emulsions). A carrier can be a solvent or dispersing medium containing, e.g., ethanol, a polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), a vegetable oil, and mixtures thereof.

[0276] In some embodiments, the compositions are liquid formulations. In certain embodiments, the formulations comprise a CNP variant peptide in a concentration range from about 0 .1 mg / ml to about 20 mg / ml, or from about 0.5 mg / ml to about 20 mg / ml, or from about 1 mg / ml to about 20 mg / ml, or from about 0.1 mg / ml to about 10 mg / ml, or from about 0.5 mg / ml to about 10 mg / ml, or from about 1 mg / ml to about 10 mg / ml, or from about 2 mg / ml to about 10 mg / ml, or about 2 mg / ml or about 10 mg / ml. In other embodiments, the formulation may be a lyophilized formulation or may be a liquid formulation that was previously reconstituted from a lyophilized formulation.

[0277] In further embodiments, the compositions comprise a buffer solution or buffering agent to maintain the pH of a CNP-containing solution or suspension within a desired range. Non-limiting examples of buffer solutions include phosphate buffered saline, Tris buffered saline, and Hank's buffered saline. Buffering agents include without limitation sodium acetate, sodium phosphate, citric acid monohydrate and sodium citrate dihydrate.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 in a composition depends in part on the particular buffer used and the desired pH of the solution or suspension. For example, acetate is a more efficient pH buffer at pH 5 than pH 6, so less acetate may be used in a solution at pH 5 than at pH 6. In some embodiments, the buffering agent has a concentration of about 5-15 mM (e.g., 10 mM + 5 mM). In certain embodiments, the pH of a composition is from about pH 3 to about pH 7.5, or from about pH 3.5 to about pH 7, or from about pH 3.5 to about pH 6.5, or from about pH 4 to about pH 6, or from about pH 4 to about pH 5, or is at about pH 5.0 + 1.0, or is at about pH 5.5 + 1.0.

[0278] In other embodiments, the compositions contain an isotonicity agent to render the solution or suspension isotonic and more compatible for injection. Non-limiting examples of isotonicity agents include NaCI, trehalose, mannitol, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol. In certain embodiments, the isotonicity agent is trehalose or mannitol, which can be employed individually or in combination. In certain embodiments, trehalose or mannitol is in a concentration of 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 60mM + 20 mM. The ratio of trehalose to mannitol can be about 4:1, such as about 3:1 to about 5:1.

[0279] In various embodiments, the compositions may comprise a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is in a concentration of about 0.4% + 0.2%, or about 1% + 0.5%, or about 1.5% + 0.5%, or about 2.0% + 0.5%. In certain embodiments of the invention, the composition or formulation does not contain a preservative.

[0280] In various embodiments, the compositions contain an anti-adsorbent agent (e.g., to mitigate adsorption of a CNP variant to glass or plastic). Anti-adsorbent agents include without limitation benzyl alcohol, polysorbate 20, and polysorbate 80. In certain embodiments, the anti-adsorbent is in a concentration from about 0.001% to about 0.5%, or from about 0.01% to about 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about 1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, or from about 1 % to about 2%.

[0281] In various embodiments, the compositions comprise a stabilizer. Non-limiting examples of stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and salts thereof. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid or a salt thereof, the stabilizer is in a concentration from about 0.1% to about 1%.

[0282] In various embodiments, the compositions contain an antioxidant. An exemplary anti-oxidant is without limitation ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP variant peptide is from about 0.1:1 to about 15:1, or from about 1:1 to about 15:1, or from about 0.5:1 to about 10:1, or from about 1:1 to about 10:1 or from about 3:1 to about 10:1.

[0283] Pharmaceutically acceptable salts can be used in the compositions, including without limitation mineral acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), salts of organic acids (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and salts of amines (e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A thorough discussion of pharmaceutically acceptable salts is found in Remington's Pharmaceutical Sciences, 18thEdition, Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0284] Formulations for parenteral administration can be prepared, e.g., as liquid solutions or suspensions, as solid forms suitable for solubilization or suspension in a liquidmedium prior to injection, 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 solution, Ringer's solution, isotonic sodium chloride solution), dispersing agents, wetting agents, emulsifying agents, suspending agents, and the like. In addition, sterile fixed oils, fatty esters, polyols and / or other inactive ingredients can be used. As further examples, formulations for parenteral administration include aqueous sterile injectable solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and nonaqueous sterile suspensions, which can contain suspending agents and thickening agents.

[0285] Exemplary CNP peptide-containing formulations are described in U.S. Patents 8,198,242 and 8,598,121. Use of CNP formulations having a pH in the range from about 4 to about 6 is contemplated.

[0286] In various embodiments, CNP variant peptides can be formulated in pharmaceutical carriers for administration to subjects affected by skeletal dysplasia. In some embodiments, liquid formulations of CNP variant peptides are formulated according to any combinations of the ingredients and their amounts or concentrations are described below:

[0287] Compositions comprising a CNP variant peptide can also be lyophilized formulations. In certain embodiments, the lyophilized formulations comprise a buffer and bulking agent, and optionally an antioxidant. Exemplary buffers include without limitation acetate buffers and citrate buffers. Exemplary bulking agents include without limitation mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, mannitol and / or trehalose is in an amount from about 3% to about 10%, or from about 4% to about 8%, or from about 4% to about 6%. In certain embodiments, sucrose is in an amount from about 6% to about 20%, or from about 6% to about 15%, or from about 8% to about 12%.

[0288] In various embodiments, lyophilized formulations of CNP variant peptides are prepared from formulations formulated according to any combinations of the ingredients and their amounts or concentrations described below:

[0289] In various embodiments, a formulation comprising a CNP variant peptide has a pH of about 3-7, or about 3-6, or about 3.5-6.5, or about 4-6, or about 4-5, or about 4.5-5.5. In some embodiments, for pH 4-5.5 a suitable buffering agent is acetic acid / acetate (e.g.,sodium acetate), and for pH 5.5-6 a suitable buffering agent is citric acid / citrate. Citric acid / citrate (e.g., sodium citrate) is also a suitable buffering agent in the range of pH 3-6 or pH 4-6. In certain embodiments, the buffering agent has a concentration in the formulation of about 2-50nM, or about 2-40 nM, or about 2-30 nM, or about 5-30 nM, or about 2-20 nM, or about 5-20 nM, or about 5-15 nM.

[0290] Also to minimize or avoid deamidation of a CNP variant peptide, water can be removed from the formulation by lyophilization. In some embodiments, lyophilized formulations contain any combinations of the following components: buffer: sodium acetate and acetic acid, or sodium citrate and citric acid; isotonicity / bulking agent: mannitol (e.g., 3- 10%, 2-8% or 4-6%); sucrose (e.g., 6-20%, 5-15% or 8-12%); antioxidants: methionine and / or ascorbic acid with molal ratio of each antioxidant to CNP variant peptide from about 0.1 :1 to about 1:1, or from about 0.5:1 to about 5:1, or from about 1:1 to about 15:1, or from about 1:1 to about 10:1, or from about 3:1 to about 10:1.

[0291] Deamidation can also be minimized or avoided by storing a CNP composition (e.g., a liquid formulation or a lyophilized formulation) at lower temperature, such as at about 5 °C, 0 °C, -10 °C, -20 °C, -30 °C, -40 °C, -50 °C, -60 °C, -70 °C, -80 °C, -90 °C, or -100 °C.

[0292] To minimize or avoid oxidation of oxidizable residues (e.g., methionine) in a CNP variant peptide, the variant can be formulated with one or more antioxidants. Exemplary antioxidants include, but are not limited to, methionine, ascorbic acid, and thioglycerol. Oxidation of, e.g., methionine residues can also be minimized or prevented by purging oxygen from a liquid medium (if a liquid formulation) with nitrogen or argon, and / or by purging oxygen from a container or packaging with nitrogen or argon.

[0293] In some embodiments, to minimize or prevent adsorption (e.g., adsorption of a CNP variant peptide to plastic or glass), polysorbate 20, polysorbate 80 or benzyl alcohol, or a combination thereof, is added to a CNP formulation. In certain embodiments, each of the anti-adsorbent(s) is in a concentration from about 0.001% to about 0.5%, or from about 0.01% to about 0.5%, or from about 0.1% to about 1%, or from about 0.5% to about 1%, or from about 0.5% to about 1.5%, or from about 0.5% to about 2%, or from about 1 % to about 2%. Exemplary range(s) of anti-adsorbent(s) in the formulation include without limitation from about 0.001% to about 0.5% of Polysorbate 20, from about 0.001% to about 0.5% of Polysorbate 80, and / or from about 0.5% to about 1.5% of benzyl alcohol.

[0294] The disclosure also provides kits containing, e.g., bottles, vials, ampoules, tubes, cartridges and / or syringes that comprise a liquid (e.g., sterile injectable) formulation or a solid (e.g., lyophilized) formulation. The kits can also contain pharmaceutically acceptable vehicles or carriers (e.g., solvents, solutions and / or buffers) for reconstituting a solid (e.g., lyophilized) formulation into a solution or suspension for administration (e.g., by injection), including without limitation reconstituting a lyophilized formulation in a syringe for injection or for diluting concentrate to a lower concentration. Furthermore, extemporaneous injection solutions and suspensions can be prepared from, e.g., sterile powder, granules, or tablets comprising a CNP-containing composition. The kits can also include dispensing devices, such as aerosol or injection dispensing devices, pen injectors, autoinjectors, needleless injectors, syringes, and / or needles.

[0295] Additional aspects and details of the disclosure will be apparent from the following examples, which are intended to be illustrative rather than limiting.EMBODIMENTS1. A pharmaceutical composition comprising a variant of C-type natriuretic peptide (CNP) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), a pharmaceutically acceptable excipient, and a carrier or a diluent, wherein the CNP variant comprises an acid moiety, a spacer, a hydrolysable linker, and is characterized by a structure as shown in Figure 11.2. A pharmaceutical composition comprising (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2- ((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl- L- glutaminyl- L-glutamyl- L-histidyl)pyrrolidine-2-carboxamido)-4-amino-4- oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4- hydroxyphenyl)propanamido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4- oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)acetamido)-4- methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl- 28-(( S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)- 6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazahenpentacontyl)-31- (3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)- 6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecaoxo-1,2-dithia- 5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4- carboxylic acid, a pharmaceutically acceptable excipient, and a carrier or a diluent.3. The pharmaceutical composition of embodiment 1 or 2, wherein the pharmaceutical composition is lyophilized.4. The pharmaceutical composition of any one of embodiments 1-3, wherein the carrier or diluent comprises a buffer.5. The pharmaceutical composition of embodiment 4, wherein the buffer comprises a buffer selected from citrate, acetate, phosphate, TRIS, and a combination thereof.6. The pharmaceutical composition of embodiment 5, wherein the buffer further comprises histidine, a salt thereof, a solvate thereof, or a solvate of a salt thereof.7. The pharmaceutical composition of any one of embodiments 4-6, wherein the buffer is present in a concentration of 5-15 mM.8. The pharmaceutical composition of any one of embodiments 4-7, having a pH of 3-9.9. The pharmaceutical composition of embodiment 8, having a pH of 4-6.10. The pharmaceutical composition of embodiment 9, having a pH of 5-6.11. The pharmaceutical composition of embodiment 10, having a pH of 5.2 or 5.5.12. The pharmaceutical composition of any one of embodiments 1-11, wherein the pharmaceutically acceptable excipient is selected from a bulking agent, a tonicity agent, an antioxidant, a surfactant, a solubilizing agent, a stabilizing agent, and a combination thereof.13. The pharmaceutical composition of embodiment 12, wherein the bulking agent is selected from mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24), and a combination thereof.14. The pharmaceutical composition of embodiment 12 or 13, wherein the bulking agent comprises trehalose or a solvate thereof, mannitol, or a combination thereof.15. The pharmaceutical composition of embodiment 14, wherein the bulking agent comprises trehalose and mannitol in a weight ratio of 3:1 to 1 :1.16. The pharmaceutical composition of embodiment 15, wherein the weight ratio of trehalose to mannitol is 3.9:1.17. The pharmaceutical composition of any one of embodiments 13-16, wherein trehalose is present in an amount of 3-6 wt% of the composition.18. The pharmaceutical composition of embodiment 17, wherein trehalose is present in an amount of 3.5-5.8 wt% of the composition.19. The pharmaceutical composition of embodiment 18, wherein trehalose is present in an amount of 3.8-4.8 wt% of the composition.20. The pharmaceutical composition of any one of embodiments 12-19, comprising a tonicity agent selected from sodium chloride, dextrose, glucose, glycerin, sorbitol, xylitol, ethanol, and a combination thereof.21. The pharmaceutical composition of any one of embodiments 12-20, comprising an antioxidant selected from methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol, and combinations thereof.22. The pharmaceutical composition of embodiment 21 , wherein the antioxidant is methionine.23. The pharmaceutical composition of any one of embodiments 1-22, comprising a stabilizing agent or surfactant selected from glycine, sorbitol, polysorbate, and a combination thereof.24. The pharmaceutical composition of embodiment 23, comprising polysorbate.25. The pharmaceutical composition of any one of embodiments 1-24, wherein the pharmaceutical composition comprises L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.26. The pharmaceutical composition of any one of embodiments 1-24, wherein the pharmaceutical composition comprises citrate buffer, trehalose dihydrate, D-mannitol, L- methionine, and polysorbate.27. A pharmaceutical composition comprising (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2- ((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl- L- glutaminyl- L-glutamyl- L-histidyl)pyrrolidine-2-carboxamido)-4-amino-4- oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4- hydroxyphenyl)propanamido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4- oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)acetamido)-4- methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl- 28-(( S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)- 6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazahenpentacontyl)-31- (3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)- 6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4- carboxylic acid, a buffer comprising histidine or a salt thereof, and one or more pharmaceutically acceptable excipients.28. The pharmaceutical composition of embodiment 27, wherein the buffer comprises L-histidine hydrochloride monohydrate.29. The pharmaceutical composition of embodiment 27 or 28, comprising a pharmaceutically acceptable excipient selected from a bulking agent, a stabilizer, an antiadsorbent, a diluent, and a combination thereof.30. The pharmaceutical composition of any one of embodiments 27-29, wherein the pharmaceutically acceptable excipient comprises a bulking agent comprising trehalose or a solvate thereof.31. The pharmaceutical composition of embodiment 30, wherein the bulking agent comprises trehalose dihydrate.32. The pharmaceutical composition of any one of embodiments 27-31 , wherein pharmaceutically acceptable excipient comprises a bulking agent comprising mannitol.33. The pharmaceutical composition of embodiment 32, wherein the mannitol is D-mannitol.34. The pharmaceutical composition of any one of embodiments 27-33, wherein the pharmaceutically acceptable excipient comprises a stabilizer comprising methionine.35. The pharmaceutical composition of embodiment 34, wherein the methionine is L-methionine.36. The pharmaceutical composition of any one of embodiments 27-35, wherein the pharmaceutically acceptable excipient comprises an anti-adsorbent comprising polysorbate.37. The pharmaceutical composition of embodiment 36, wherein the polysorbate is polysorbate 80.38. The pharmaceutical composition of any one of embodiments 27-37, wherein the pharmaceutical composition is substantially free of a citrate buffer.39. A pharmaceutical composition comprising a C-type natriuretic peptide (CNP) variant that is (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)- 2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L- prolylglycyl- L-glutaminyl- L-glutamyl- L-histidyl)pyrrolidine-2-carboxamido)-4-amino-4- oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4-hydroxyphenyl)propanamido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4- oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)acetamido)-4- methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl- 28-(( S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)- 6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,29,34-hexaazahenpentacontyl)-31- (3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)ethyl)- 6,9,12,15,18,21 ,24,27,30,33,36,39,42,45,48,51 -hexadecaoxo-1 ,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4- carboxylic acid, a histidine buffer, trehalose dihydrate, D-mannitol, L-methionine, polysorbate 80, and water.40. The pharmaceutical composition of embodiment 39, wherein the CNP variant is present at a concentration of 10 mg / mL.41. The pharmaceutical composition of embodiment 39, wherein the CNP variant is present at a concentration of 30 mg / mL.42. The pharmaceutical composition of any one of embodiments 39-41 , wherein the histidine buffer comprises L-histidine and histidine monohydrochloride monohydrate.43. The pharmaceutical composition of embodiment 42, wherein L-histidine is present at a concentration of 0.35 mg / mL.44. The pharmaceutical composition of embodiment 42, wherein L-histidine is present at a concentration of 2.2 mM.45. The pharmaceutical composition of any one of embodiments 42-44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 1.6 mg / mL.46. The pharmaceutical composition of any one of embodiments 42-44, wherein L-histidine monohydrochloride monohydrate is present at a concentration of 7.8 mM.47. The pharmaceutical composition of any one of embodiments 39-46, wherein the trehalose dihydrate is present at a concentration of 58 mg / mL.48. The pharmaceutical composition of any one of embodiments 39-46, wherein the trehalose dihydrate is present at a concentration of about 127 mM to about 153 mM.49. The pharmaceutical composition of embodiment 48, wherein the trehalose dihydrate is present at a concentration of 127 mM.50. The pharmaceutical composition of embodiment 48, wherein the trehalose dihydrate is present at a concentration of 153.3 mM.51. The pharmaceutical composition of any one of embodiments 39-50, wherein the D-mannitol is present at a concentration of 15 mg / mL.52. The pharmaceutical composition of any one of embodiments 39-50, wherein the D-mannitol is present at a concentration of about 68 mM to about 82 mM.53. The pharmaceutical composition of embodiment 52, wherein the D-mannitol is present at a concentration of 68.1 mM.54. The pharmaceutical composition of embodiment 52, wherein the D-mannitol is present at a concentration of 82.3 mM.55. The pharmaceutical composition of any one of embodiments 39-54, wherein the L-methionine is present at a concentration of 0.7 mg / mL.56. The pharmaceutical composition of any one of embodiments 39-54, wherein the L-methionine is present at a concentration of 4.9 mM.57. The pharmaceutical composition of any one of embodiments 39-56, wherein the polysorbate 80 is present at a concentration of 0.05 mg / mL.58. The pharmaceutical composition of any one of embodiments 39-56, wherein the polysorbate 80 is present at a concentration of 0.005% (v / v).59. The pharmaceutical composition of any one of embodiments 1-58, wherein the composition exhibits lower Cmax and higher AUC compared to free drug, e.g., an equivalent composition lacking an acid moiety, a spacer, and a hydrolysable linker.60. A pharmaceutical kit comprising the pharmaceutical composition of any one of embodiments 1-58.61. A pharmaceutical kit comprising the pharmaceutical composition of any one of embodiments 39-58, wherein the CNP variant is present in amount of 13 mg.62. A pharmaceutical kit comprising the pharmaceutical composition of any one of embodiments 39-58, wherein the CNP variant is present in amount of 39 mg.63. A method of treating a bone-related disorder or skeletal dysplasia in a subject in need thereof comprising administering to the subject a composition according to any one of embodiments 1-59.64. The method of embodiment 63, wherein the bone-related disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, campomelic dysplasia, congenital lethalhypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short-rib polydactyly syndromes, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia, and spondyloepimetaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner’s syndrome / Leri Weill), PTPN11 mutations (Noonan’s syndrome), insulin growth factor 1 receptor (IGF1R) mutation, idiopathic short stature and osteoporosis.65. A method of elongating a bone or increasing long bone growth in a subject in need thereof, comprising administering to the subject a composition according to any one of embodiments 1 to 59, and wherein the administering elongates a bone or increases long bone growth.66. The method of any one of embodiments 63-64, wherein the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.67. The method of any one of embodiments 63-66 wherein the composition provides an extended release composition.68. The method of any one of embodiments 63-66, wherein the composition is administered, once every 5 days, once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months or once every six months.69. A method of treating a CNP-responsive condition or disorder, comprising -administering to a subject a composition according to any one of embodiments 1 to 58, and-monitoring the level of at least one bone- or cartilage-associated biomarker in the subject, wherein an increase in the level of the at least one bone- or cartilage- associated biomarker indicates a therapeutic effect of the CNP variant on the subject or the condition or disorder.70. The method of embodiment 69, further comprising adjusting the amount or frequency of administration of the composition, whereini) the amount or frequency of administration of the composition is increased if the level of the at least one bone- or cartilage-associated biomarker is below a target level; or ii) the amount or frequency of administration of the composition is decreased if the level of the at least one bone- or cartilage-associated biomarker is above a target level.71. The method of embodiment 69 or 70, wherein the at least one bone- or cartilage-associated biomarker is selected from the group consisting of CNP, cGMP, propeptides of collagen type II and fragments thereof, collagen type II and fragments thereof, collagen type I C-telopeptide (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen (PINP) and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, alkaline phosphatase, proliferating cell nuclear antigen (PCNA), propeptides of type I procollagen and fragments thereof, collagen type I and fragments thereof, aggrecan chondroitin sulfate, collagen X, CXM (noncollagenous 1 (NC1) domain of type X collagen), NTproCNP, N-terminal collagen type I pro-peptide, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / procollagen type I N-propeptide (PINP), cross-linked C-telopeptide of type I collagen (CTx), cross-linked N-telopeptide of type I collagen (NTx) tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomics readouts, and CNP-variant bioactivity.72. The method of any one of embodiments to 63-70, wherein the administration increases the annualized growth velocity (AGV) in the subject at 12 months, optionally compared to baseline or to a normal control.73. The method of embodiment 72, wherein the AGV in the subject increases over 1 year or over 2 years, or more.74. The method of any one of embodiment 63-72, wherein the administration improves the height Z score at 12 months, optionally compared to baseline or to a normal control.75. The method of any one of embodiments to 63-73, wherein the subject is greater than 3 years old.76. The method of any one of embodiments to 63-74, wherein the subject is between 3 and 17 years old.77. The method of any one of embodiments 63-75, wherein the subject has open epiphyses.78. The method of any one of embodiments 63-77, wherein the composition is administered at a dose from about 5 μg / kg to 500 μg / kg or from about 15 μg / kg to 350 μg / kg .79. The method of any one of embodiments 63-77, wherein the administration does not result in cardiovascular (CV) side effects.80. The method of embodiment 79, wherein the CV side effect is change in systemic blood pressure, mean arterial pressure, systolic and / or diastolic blood pressures, pulse pressure or heart rate.EXAMPLES

[0296] In these examples and the related schematics, tables, graphs, charts, and the like, “Free Drug” is sometimes used to refer to CNP that is not bound or conjugated to a linker and / or polymer.Example 1 : Synthesis of CNP Variants

[0297] CNP variant peptides were synthesized on solid-phase using a resin that would leave a C-terminal COOH on a Symphony / Prelude (Protein Technologies Inc., USA), Voyager (CEM GmbH, Germany), or Syroll (MultiSyntech, Germany) synthesizer.

[0298] All Fmoc-amino acids were purchased from Biosolve (Netherlands) or Bachem GmbH (Germany) with side-chain functional groups protected with N-f-Boc (KW), O-f-Bu (DESTY), N- Trt (HNQ), S-Trt (C), or N-Pbf (R) groups. A 5-fold excess of HBTU / HOBt / amino acid / DIPEA (1:1:1 :2) in NMP with a 20 minute activation time using double couplings was employed for every amino acid coupling step.

[0299] Acetylation (Ac) of the peptide was performed by reacting the resin with NMP / AC2O / DIEA (10:1:0.1, v / v / v) for 30 min at room temperature.

[0300] For conjugation of a moiety, the protective amino group on lysine was cleaved to create a reactive group. Standard Fmoc synthesis was used to react 2x Fmoc-amino PEG(2) followed by glutamic acid, followed by C18-diacid.

[0301] The completed peptide was cleaved from the resin by reaction with a TFA (40 mL / mmol resin) for 2 hours at room temperature. Crude peptide was filtered, precipitated with ice-cold Et2O followed by lyophilization and ultimately purification by preparative reverse phase-high-performance liquid chromatography (RP-HPLC). Final products and purity were confirmed by mass spectrometry.

[0302] CNP variants were generated based on the sequence of Pro-Gly CNP-37 (PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1)), optionallyhaving changes in different amino acid residues, and / or acetylation at the N-terminus, and / or other modifications, and include (amino acid changes underlined):

[0303] Fully reduced peptides were dissolved in 0.1 M Tris-buffer (pH 8.0) with or without guanidine HCI, containing 1 mM cysteine (SS-form) and 8 mM cysteine (SH-form) in a final concentration of 0.1 mg / ml and stirred at room temperature. Disuflide formation was monitored by HPLC-analysis until no further peak changes were observed. Mixtures were loaded on preparative RP-HPLC for purification.Example 2: Characterization of Synthesized CNP Variants

[0304] CNP variants synthesized as in Example 1 were analyzed by mass spectrometry and UV spectroscopy to determine purity and stability after 10 days.

[0305] Stability was measured using RP-HPLC analysis of purity over time TO to T10. Briefly, the CNP variants were diluted 1:5 in buffer: 5mM citrate, 6% sucrose, 1.5% mannitol, 0.7mg / ml methionine and 0.005% tween80, pH 5.6 An injection volume of 10 μL was injected onto a Phenomenex Aeris XB-C18 RP column (2 pm, 100A, 2.1 x 250 mm; p / n 00G- 4505-AN) HPLC conditions were: Mobile Phase A: H2O / 0.05% TFA, pH 3 w / NH4OH (~3mM final), Mobile Phase B: 70% CH3CN in H2O / 0.05% TFA, pH 3 w / NH4OH (~3mM final) and a flow rate of 0.25mL / min. Measurement was carried out with a column temperature of 55° C and UV: 214nm (bw 4nm); ref 360nm (bw 20nm), UV: 280nm (bw 4nm); ref 360nm (bw 20nm). CNP variants were held 10 days in PBS, pH 7.4, at 37° C and stability measurements obtained.

[0306] Results of stability measurements are shown in Table 1 below as % variant detected at T0 (0 days) or T10 (10 days).

[0307] CNP variants were tested for activity by a cGMP stimulation assay using a CatchPoint Cyclic-GMP Fluorescent Assay Bulk kit (Molecular Devices, R8075). Briefly, NIH3T3 cells (ATCC, CRL-1658) and HEK293 cells were seeded at 60,000 cells / well in a 96-well plate (96-well black imaging plates, Grenier, #655090). Culture media was as follows: NIH3T3 culture media: DMEM high glucose, pyruvate (Thermo, 11995-073) + 10% FBS + 1x Pen Strep (abbrev P / S, Thermo, cat# 15140122). NIH3T3 was the control system for the cGMP assay HEK293 culture media: EMEM + 10% FBS + 1x P / S + 1x GMAX. Serum free NIH3T3 media: DMEM + 1x P / S for treatment of cells with IBMX (CAS 28822-58-4); Serum free NIH3T3 media with BSA: DMEM + 1x P / S + 0.5 mg / mL BSA (Thermo, A9418- 100G) for treatment of cells with CNP.

[0308] Cells were incubated for 24 hours at 37° C, 5% CO2. For cells to be treated with CNP variants, plates were pre-treated with IBMX (Enzo life sciences, 89161-340, 1g) 15 minutes prior to use. IBMX is a potent, non-specific inhibitor of phosphodiesterases. An 800 mM stock solution of IBMX is diluted in IBMX dilution media (serum-free media (DMEM + 1x PBS mixed 1:1 with 1x PBS) to a 0.75 mM working stock.

[0309] CNP variants were prepared as follows: Dilute 10 mg / ml CNP solution 1:1000 in CNP dilution media (DMEM + 1x P / S + 0.5 mg / mL BSA). This solution is further diluted to obtain a CNP starting solution plated at 100 nM CNP / well. This 100 nM CNP solution is further serially diluted 1:5 for six dilutions to obtain a lower end concentration of 0.0064 nM CNP / well. This provides a 7-point dose curve for analysis.

[0310] For cell treatment, cells were removed from the incubator, growth media was removed from cells and cells treated with IBMX. 80 μL 0.75 mM IBMX was added to each well and cells returned to 37° C incubator for 15 minutes. After 15 minutes CNP (40 μL / well) was added to each test well and cells returned to 37° C incubator for 15 minutes. The platewas mixed by gentle tapping. The plate was imaged on a Solentim cell metric to visualize cells and determine if there is any cell lifting and then placed back into 37° C incubator.

[0311] The reaction was stopped and cells lysed by adding 40 μL lysis buffer (from cGMP kit). Plate was placed on a shaker for 5 minutes to complete lysis. The cell lysate was used in the cGMP assay.

[0312] The cGMP assay was carried out using a cGMP calibrator, rabbit anti-cGMP antibody and HRP-cGMP prepared as according to manufacturer’s protocol. 40 μL of calibrator was added to wells of an anti-cGMP antibody coated plate, and 40 μL of lysate to be analyzed added to the appropriate wells. 40 μL of reconstituted rabbit anti-cGMP antibody was added to all wells and plates placed on shaker five minutes for mixing. 40 μL of reconstituted HRP-cGMP was added to each well and incubated for 2 hours at room temperature. Plates were manually aspirated and washed 4x with 300 μL wash buffer. 100 μL of stoplight red substrate was added to each well, the plate covered and left at room temperature for at least 10 mins, protected from light. The plate was read for fluorescence intensity on a Spectramax M, or similar instrument, at excitation 530 nm and emission at 590 nm.

[0313] Table 1 shows that the CNP variants stimulate cGMP production, suggesting that the stable variants described herein are useful as therapeutics to treat bone-related disorders.Example 2A: Stability of CNP Variants or Conjugates in Plasma

[0314] Stability of different CNP variants or CNP conjugates were tested for stability in human plasma over a 24 hour period. Briefly, a C18 fatty acid was fused to gamma glutamic acid using 2 OEG spacers (see e.g., Lau et al., J. Med. Chem 58:7370-7380, 2015). For the chromatography, Waters UPLC H-Class connected with BEH C18 1.7 um, 2.1 x 150 mm;Mobile Phase A: 1% DMSO 0.1% Formic Acid in Water; Mobile Phase B: 1% DMSO 0.1% Formic Acid in CAN. Mass Spectrometry was carried out using an AB Sciex QTRAP. Each CNP variant was prepared at 200nM in Human Plasma Li Heparin and smaples Incubated at 37° C 5% CO2. The reaction was quenched at 0, 1, 2, 4, 8, 24 hours with 0.5M Sodium Citrate pH 4. Plasma proteins were precipitated with 0.2% Formic Acid in MeOH and samples prepared using WCX 96-well uElution plate and analyzed by LC-MS / MS 6500.

[0315] Variants CNP-R refers to CNP37 variants in which the K residues not in the ring portion are changed to R residues, and CNP Q / R refers to CNP37 variants in which N residues are changed to Q and K residues not in the ring portion are changed to R.

[0316] Figure 1 illustrates CNP conjugates having different linker structures. Figure 2 shows that lipidated conjugate has improved stability in plasma compared to PEGylated or non-conjugated peptides in plasma.

[0317] Stability of different conjugates was also analyzed at varying conditions. Briefly, the same protocol as above was used at 37° C (control), 37° C+ 0.5M NaCI, 37°C + protease inhibitors or 4° C. Figure 3 shows that PG-CNP37 and variants are prone to proteolysis in human plasma and PG-CNP37 shows greater stability compared to other variants.Example 3: Heterozygous NPR2 Mutations Are Responsive to CNP Treatment

[0318] To determine the effects of CNP on subjects with short stature resulting from NPR2 mutations, cellular models of NPR2 mutations were developed. Exemplary NPR2 mutations analyzed are set out in Figure 6. Rat chondrosarcoma (RCS) cells having either a knockout of, or heterozygous loss of function mutations in, the NPR2 gene were made by RNP transfection into RCS cells using 125ng NPR2 variants or wild-type NPR2 plasmid DNA transfected into RCS or HEK293 cells. Single cell clones were seeded and genotyped by Sanger sequencing. Cell models are able to reproduce published cGMP phenotypes of the different mutations.

[0319] The NPR2 clones were created by creating insertions and deletions in the first exon of NPR2 in RCS cells. The sequence of the first exon in NPR2 was confirmed by next- generation sequencing and is set out in Figure 5. NPR2 mutant cells were tested for activity in response to CNP administration by a cGMP stimulation assay using a CatchPoint Cyclic- GMP Fluorescent Assay after treatment with 6nM of Pro-Gly CNP37. RCS (rat chondrosarcoma) cells were seeded at 40,000 cells / well in RCS culture media: DMEM + 10% FBS + 1x Pen Strep. Figure 4 shows that adding exogenous Pro-Gly-CNP37 variant rescues cGMP readout in a NPR2 + / - rat chondrosarcoma cell model.

[0320] Previous activation data reports cGMP EC50 in the range of 40 to 360nM for activation of PRKG2 (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 the heterozygous NPR2 knockout cells, a CNP dose >0.163nM is able to achieve an intracellular concentration exceeding the EC50 range for PRKG2 activation cGMP (Figure 4). Whereas, in wild-type cells a CNP dose of 0.040nM is able to achieve the same cGMP concentration. These results demonstrate that CNP supplementation can achieve the cGMP levels necessary for PRKG2 activation and growth in cells with loss-of-fu notion mutations of NPR2.

[0321] These results also suggest that administration of CNP variants is useful to restore bone growth in subjects with short stature that have reduced activity of NPR2. It is further contemplated that treatment with CNP variants will be beneficial in subjects having mutations in other growth plate genes in which cGMP signaling may be impaired.Example 4: Identification of Mutations Associated With Short Stature

[0322] It is hypothesized that genes showing clear evidence of genetically-driven bidirectional effects are more likely to represent therapeutic targets that can be effectively modulated in a broad patient population. To identify genes that are core regulators of growth, the intersections of five gene lists were analyzed including, the list of genes from genomewide association study (GWAS). Core growth regulators would be the most likely to contain rare coding mutations with bidirectional effects (i.e. short stature or skeletal dysplasia AND tall stature or overgrowth).

[0323] Databases queried include: GWAS 2,067 non-repeating closest genes for each of the 3,290 independent genetic variants reported by a large GWAS meta-analysis of height using -700,000 individuals were extracted; HGMD The “allmut” table from HGMD version v2019_2 was queried looking for all pathogenic variants labelled as “DM” having either “short stature” and “tall stature or overgrowth” in the same genes; OMIM The list of OMIM genes related to growth disorders was previously described and was created using the keywords: short stature, overgrowth, skeletal dysplasia, brachydactyly.

[0324] First, the Human Gene Mutation Database (HGMD version v2019_2) was queried for genes associated with short or tall stature (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 stature or overgrowth genes.

[0325] Secondly, a manually curated list of 258 OMIM genes was used (248 short, 20 tall) which was created using the keywords: short stature, overgrowth, skeletal dysplasia, brachydactyly (Wood et al., Nat Genet 46:1173-86, 2014). Third, the intersection of these lists was compared with the list of genes from GWAS. At the intersection of these list there were three genes known to be associated with height (IGF1R, NPPC, NPR2) and two additional genes were identified (FGFR3, SHOX).

[0326] Additional analysis led to generation of a new group of five Core genes that showed significantly decreased height (β=-0.20, 95%CI [-0.26 to -0.14], p=4.04x10-11) and significantly increased risk for Idiopathic Short Stature (ISS) (OR=2.75, 95%CI [1.92 - 3.96].Each of the core five genes (FGFR3, IGF1R, NPPC, NPR2 and SHOX) were associated with height when considered individually, and also were associated with short stature when taken in combination with other mutations. Exemplary mutations in FGFR3, IGF1R, NPPC, NPR2 and SHOX are set out in Figure 7.

[0327] Combined LoF (loss of function) and Missense variants in NPR2 and IGF1R were also associated with increased risk for ISS (OR=3.31, P= 0.001, OR= 2.85, P=0.002, respectively). Entire gene deletions and / or mutations causing loss of protein function in SHOX, IGF1R, NPPC, NPR2 have been reported in familial short stature with various degrees of severity. Mutations in DTL, and pregnancy-associated plasma protein A2 (PAPPA2), or combinations thereof also contribute to skeletal dysplasia.

[0328] Analysis shows that carriers of variants in any of the five core genes are at approximately a 3-fold increased risk for ISS and account for 6.7% of the total ISS population. Furthermore, a dose-dependent rescue of NPR2 signaling in a cell model of NPR2 haploinsufficiency after adding exogenous CNP was demonstrated.

[0329] According to the omnigenic model (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, then their effects should be modulated by multiple weaker common genetic variants driving regulatory networks. To indirectly test this hypothesis, polygenic risk scores (PRS) were calculated for height using the largest published GWAS meta-analysis for height that did not include any samples from the UK Biobank project. The cohort was divided into five equally-sized (n=6,824) PRS quintiles (PRS 1 being the lowest height, PRS 5 the tallest height). There was a dose-dependent relationship between increasing PRS score and mean height (β=0.30 per each PRS quintile increase) (Fig. 8A). Carriers of LoF variants in the five core genes were consistently shorter than non-carriers across the five different PRS backgrounds. See Figure 8. 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.

[0330] The risk for ISS across PRS groups was calculated using PRS=3 as a reference. The lowest PRS group was associated with increased risk for ISS and the highest PRS group with a decreased risk (OR=5.43, P=8.58x10-34; OR=0.22, P=4.49x10-7 for PRS 1 and PRS 5 respectively). The effect of rare coding variants of the five core genes was evaluated for ISS stratified by PRS group. Carriers of any of the five core genes were at increased risk for 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 Figs. 8C-F). Consistent directions of effect were observed for carriers of each individual core gene on ISS risk stratified by PRS (Figs. 8C-F).

[0331] Further, additive effects of PRS, mostly coming from multiple common genetic variation with individual small effects, predicted 20.1% of the variance in height in the dataset. These additive effects of PRS appeared to have similar magnitude on carriers of rare coding variation of core genes as well as in non-carriers. This observation indicates that PRS may be a strong contributor in the differences in penetrance of rare pathogenic variants (especially in models of haploinsufficiency such as the ones described here). Supporting this idea, it was observed that two of eight NPR2 variant carriers with low NPR2 activity had a low-normal height. This data suggests that most ISS individuals possessing mutations in NPR2 may also have a polygenic background that made them more susceptible to the pathogenic effect of losing NPR2 activity.

[0332] These results support the idea that CNP-based treatments could be effective in NPR2 haploinsufficient patient populations. Further, results showing a significant bi- directional (LoF and GoF) correlation of cGMP levels and height in NPR2 carriers of the general population suggest that targeting this receptor with CNP analogs could be an effective therapy for all ISS individuals. Methods of identifying a variant gene associated with short stature that is a GoF or LoF variant using a reporter construct / barcode assay is described in PCT / US 24 / 32942, incorporated by reference herein.Example 5: CNP Formulations

[0333] Different buffering agents for CNP variant formulations were evaluated for particulates. Particularly, lyophilized CNP formulations reconstituted with water were analyzed by visual and UV concentration measurements to identify particulates. As shown in Figure 13, visual appearance shows that visible particles are formed in citrate-based formulations. Histidine buffer exhibited the best appearance out of all the different buffers including citrate.

[0334] As shown below, the UV analysis highlights the drop in the concentration after precipitation.Example 6

[0335] Variant content when stored in different formulation buffers was analyzed over time, up to 13 months, at a range of temperatures from -20 to 37° C. Results are shown in Figure 14.Example 7

[0336] Free drug content release from different formulation buffers was analyzed over time, up to 13 months, at a range of temperatures from -20 to 37° C. Results are shown in Figure 15.Example 8

[0337] Linker area of the drug conjugate when stored in different formulation buffers was analyzed over time, up to 13 months, at a range of temperatures from -20 to 37° C. Results are shown in Figure 16.Example 9

[0338] CNP conjugate content when stored in different formulation buffers was analyzed over time, up to 13 months, at a range of temperatures from -20 to 37° C. Results are shown in Figure 17.Example 10

[0339] Variant concentration as measured by UV when stored in different formulation buffers was analyzed overtime, up to 3 months. Results are shown in Figure 18.Example 11

[0340] Variant content in formulations with different excipients (histidine, glycine and sorbitol) was analyzed over time, up to 13 months, pH 5.5 or 6 was assessed. Results are shown in Figure 19. No precipitation was observed, and excipients did not reduce drug cleavage. There was no significant difference in the histidine formulation at pH 5.5 and 6.Example 12

[0341] A manufacturing feasibility study was carried out for CNP prodrug. Results are shown in Figure 20. Free drug cleavage was observed up to 0.5% at 5° C and 2.5% up to 25° C. There was no significant difference between pH 5.5 and 6.Example 13

[0342] Batches of formulated CNP prodrug were analyzed for product degradation over 1 month in histidine or acetate buffer with trehalose and mannitol at different ratios (4:1, or 1:4, respectively). Results are shown in Figure 21. Free drug increases as a function ofincreasing time, temperature and pH. Dextran containing formulations at pH 4 had lower free drug cleavage that is likely due to the lower pH.Example 14

[0343] Different formulations of CNP prodrug were tested with buffers, bulking agents and other excipients.Tonicity: concentration 5 mM Osmo- 5, 10, 20 mg / mLExample 15

[0344] As described above, additional analysis of buffering agents for precipitations was conducted at different time points and different temperatures. The evaluated formulations included 1mg / ml of CNP variant in either 5 mM citrate pH 5.5 (58 g / L trehalose, 15 g / L Mannitol, 0.73 g / L L-methionine, 0.05 g / L PS80) or 10 mM citrate pH 5.5 (58 g / L trehalose, 15 g / L Mannitol, 0.73 g / L L-methionine, 0.05 g / L PS80). As shown below, precipitates were identified in formulations with citrate. Precipitates were not identified in formulations with histidine.Example 16

[0345] Additional CNP prodrug formulations were generated.Formulation Buffers10 mM Histidine FB pH 5.5 with prodrugExample 17

[0346] Area under the curve of drug product after reverse phase chromatography was assessed. Results are shown in Figure 22.Example 18

[0347] Drug product stability was assessed under the following conditions:Full testing: appearance, osmolality, pH, UV concentration, RP-Perchlorate UPLCExample 19

[0348] In use stability and free drug content was assessed over time, up to 13 months, at a range of temperatures from 5 to 25° C and pH 5.2 to 6.0. Results are shown in Figure 23.Example 20

[0349] Buffering formulations for CNP prodrug are described.Buffer range: 5-15 mN concentrationTrehalose : mannitol ratio of about 4:1 (e.g, 3:1 to 5:5)

[0350] Antioxidant in the buffer is not necessary, but an antioxidant is added in some case. When used, L-methionine is particularly employed as an antioxidant.

[0351] PS 80 (polysorbate 80) is the preferred surfactant, because it minimizes loss of the CNP Variant because of the CNP Variant’s absorption on the materials (e.g., vessel in which the formulation is prepared).Example 21 - Visual comparison of different amounts of trehalose

[0352] Visual comparison of CNP prodrug in different formulations is shown in Figure 24. Trehalose at 3.5-5.5%, such as 3.8-4.8% trehalose, may be acceptable. 4.8% trehalose was most preferred, because of favorable properties by visual inspection (among other reasons).Example 22 (Prophetic) - Analytical techniques to analyze binding in rat plasma and / or and linker cleavage and release of CNP from the conjugate.Preparation of materials to be used in the analysis

[0353] For the purposes of this example, CNP means a CNP Variant; it is particularly envisaged that the analysis will be undertaken with the CNP variant shown in Figure 10, although it is also envisaged that other CNP variants as described herein could be analyzed by the same or analogous methods.0.1 M MethionineWeigh 60 mg methionine. Add 4.0 mL water in a 5 mL tube. Mix well, tightly capped and store on wet-ice and discard after use.0.5 M Sodium Citrate (pH 4.0)Weigh 36.8 g sodium citrate dihydrate. Dissolve in approximately 220mL of water. Adjust pH to 4.0 using 1 .0 M citric acid while stirring. Transfer the solution to a 250-mL volumetric flask and keep adding water until the meniscus of the solution reaches the graduation line. Put on the volumetric flask stopper and invert the flask to thoroughly mix the solution.Protease inhibitor cocktail1) Dissolve protease inhibitor cocktail (Sigma P2714) in 10 mL Milli-Q water to get inhibitor solutions A.2) Dissolve 250 mg 3-isobutyl-1 -methylxanthine (phosphodiesterase inhibitor) (Sigmal5879) in 11.25 mL methanol to get inhibitor solution B.3) Combine 0.83 mL of inhibitor solution B with 9.17 mL of inhibitor solution A to get 10.0 mL protease inhibitor cocktail. Keep the remaining inhibitor solution B at -20°C for up to 2 months.Treated rat plasma K2EDTA matrix1 ) Chill rat K2EDTA whole blood on ice and aliquot 10 mL into a 15-mL tube.2) Add 2.0 mL of 0.5 M sodium citrate (pH 4.0) and 60 CL of protease inhibitor cocktail into 10 mL ice-chilled rat K2EDTA whole blood.3) Gently mix and then centrifuge the 15-mL tube at 1 ,500 xg for approximately 15 minutes at room temperature.4) Take the supernatant as the treated rat plasma K2EDTA matrix.CNP Stock Solution

[0354] 10 mg of dry or lyophilized CNP is reconstituted in 1mL of formulation buffer to provide 10 mg / mL stock solution. The concentration is then corrected to approximately 2.3 mM based on absorbance readings at 275 nm.CNPox Stock Solution

[0355] 10 mg CNP111ox was reconstituted in 1 mL of formulation buffer to get 10 mg / mL Lip-BC-CNPox stock solution. After A275 nm reading, concentration is corrected to 8.64 mg / mL (2.10 mM).Lip-BC-CNP Stock Solution

[0356] 10 mg Lipid-BC-CNP (MW: 4962.11) was reconstituted in 1 mL of formulation buffer to get 10 mg / mL Lip-BC-CNP stock solution. After A275 nm reading, concentration is corrected to 6.88 mg / mL (1.38 mM).Lip-BC-CNPox Stock Solution

[0357] 10 mg Lipid-BC-CNPox was reconstituted in 1 mL of formulation buffer to get 10 mg / mL Lip-BC-CNPox stock solution. After A275 nm reading, concentration is corrected to 6.75 mg / mL (1.36 mM).Mix ISTD Stock Solution

[0358] Prepare all ISWS on ice. Add 10 μL of 5 pM CNP (old) and 10 μL of 5 pM CNPox (old) into 5 mL of 0.1% FA, 20% ACN in water. Vortex thoroughly prior to aliquoting. Keep on ice till use. Add 10 mL of 5 mM Lip-BC-CNPA(new) and 10 mL of 5 mM Lip-BC-CNPoxA(new) into 5 mL of formulation buffer. Vortex it thoroughly prior to aliquoting. Keep on ice till use.Standard Solutions

[0359] Prepare standard working solutions as follows using 0.1% FA, 20% ACN in water as the diluent:

[0360] Prepare CNP and CNPox standards using treated rat plasma K2EDTA in 1.5-mL protein low-binding tubes;

[0361] Prepare Lip-BC-CNP and Lip-BC-CNPox standards using treated rat plasma K2EDTA in 1.5-mL protein low-binding tubes;QC Solutions

[0362] Prepare Free Peptides CNP QCs as follows using citrate and inhibitors treated rat plasma K2EDTA as the diluent:

[0363] Prepare Lip-BC-CNP and Lip-BC-CNPox QCs as follows using citrate and inhibitors treated rat plasma K2EDTA as the diluent:LC-MS ANALYTICAL CONDITIONSChromatographic ConditionsMass Spectrometer ParametersExample 22

[0364] Buffering Formulation for CNP Variant are described.Example 23 - CNP Prodrug Drug Substance and Drug ProductThe following abbreviations are used herein:Drug Substance (DS)

[0365] CNP Prodrug is comprised of CNP, a pH responsive self-immolative linker, spacer, and C-18 fatty acid albumin binding domain (attached to the Lys27side chain).

[0366] The sequence of the CNP Prodrug is as follows: Pro1-Gly-Gln-Glu-His5-Pro-Asn- Ala-Arg-Lys10-Tyr-Lys-Gly-Ala-Asn15-Lys-Lys-Gly-Leu-Ser20-Lys-Gly-Cys-Phe-Gly25-Leu- Lys27(X)-Leu-Asp-Arg30-lle-Gly-Ser-Met-Ser35-Gly-Leu-Gly-Cys-OH(X) = OH-1,18-octadecandioyl-Glu(NH-PEG2-carboxy)2-NH-CH2CH2-Gly(N-CH2CH2OH)-OH

[0367] The drug substance is isolated as a hydrochloride salt. The empirical molecular formula is C217H363N61O65S3(as free base). The average molecular mass is 4962.77 m.u.Physico-chemical Properties

[0368] The available relevant physico-chemical properties of the DS are provided in Table .Table 3: Physico-chemical Properties of CNP ProdrugManufacture

[0369] The synthesis of the CNP Prodrug DS applies SPPS technology, utilizing Fmoc amino acids derivatives as building blocks to assemble the peptide onto a resin (solid support) and Fmoc protected building blocks to assemble the side chain (including the pH responsive self-immolative linker, spacer, and C-18 fatty acid albumin binding domain) out of Lys27. Post-synthesis, the peptide is cleaved from the resin, cyclized, purified and isolated, as a lyophilized powder. A description of the synthetic process is provided in Table 4 and 5.

[0370] The release of all starting materials is done in accordance with ICH Q7. These procedures are part of the standard quality program that ensures the high quality of the peptide and batch-to-batch consistency.Table 4: Manufacturing Flowchart for CNP Prodrug Drug SubstanceStep 2: SPPS2-CTC, 2-chlorotrityl chloride; SPPS, Solid-Phase Peptide Synthesis; TFA, trifluoroacetic acid.Table 5: Description of Manufacturing ProcessCharacterization

[0371] The structure of CNP Prodrug has been elucidated by various analytical techniques (Table ).Table 6: Structure Elucidation of CNP ProdrugBiological Activity

[0372] CNP Prodrug is inactive as a prodrug of CNP. Hydrolysis of the linker forms the active parent, CNP, and inactive metabolites consisting of the lipid and hydrolyzed linker. Pharmacodynamics of CNP Prodrug is being evaluated in mice and NHPs to demonstrate the dose response effect of CNP is retained when active CNP is released continuously from its albumin bound inactive prodrug form into the circulation compared to daily pulsatile delivery of CNP. Completed studies demonstrated the expected dose responsive endochondral skeletal growth effect of CNP is retained with CNP Prodrug treatment, as shown by increases in cellu larity and height of the hypertrophic zone of the growth plate and / or increased length of long bones.CNP Prodrug Drug Substance Preliminary Specification and Batch Analysis Results

[0373] The controls on residual solvents and elemental impurities will be based on ICH Q3C and ICH Q3D. Obligatory tests will be based on ICH Q6A. For the clinical batch analysis, a reference material was prepared and characterized to be used for reversed phase ion-pair high performance liquid chromatography mass spectrometry purity and impurity testing. Preliminary batch release data are provided herein.CNP Prodrug Drug Substance Stability Protocol

[0374] The stability of non-GMP (nonclinical DS batches) and GMP (clinical DS batches) are obtained as per ICH Q1 A conditions. The purity method used in analysis of stability samples will be confirmed as stability indicating by a forced degradation study performed as per ICH Q1 A. The stability data will be evaluated as per ICH Q1 E for setting of a retest date at the long-term storage condition which is -20 ± 5°C. Additional stability data will beobtained at intermediate condition of 5 ± 3°C and accelerated condition of 25 ± 2°C / 60 ± 5% relative humidity.

[0375] The stability protocol for CNP Prodrug DS is provided in Tables 7-9.Stability Data Summary

[0376] Up to 9 months representative stability data are currently available for non-GMP batch. The results show the DS is stable at both -20 ± 5°C and 5 ± 3 °C for 9 months.Stability studies at 25 ± 2 °C / 60% ± 5% RH conditions show elevated impurities and an increase in moisture content over 6 months. However, based on these results it is expected that the DS will be stable for at least 12 months.Table 7: CNP Prodrug Drug Substance Stability Protocol - Long Term (-20 ± 5°C)Table 8: Drug Substance Stability Protocol for Accelerated Storage Condition (5± 3 °C)Table 9: Drug Substance Stability Protocol for Accelerated Storage Condition (25± 2 °C / 60% ± 5% RH)CNP Prodrug Drug ProductCNP Prodrug CompoundingTable 10-1 : Drug Substance CharacteristicsTable 10-2: Drug Product Inactive Ingredients1 % (w / v) Polysorbate 80Preparation for 1% (w / v) Polysorbate 80 is described below in Table 11.Table 11: 1% (w / v) Polysorbate 80 Preparation InstructionsTable 12: CNP Prodrug Formulation Buffer Preparation InstructionsDrug Substance Target Calculation

[0377] Based on the required DP concentration and volume, the DS target amount to weigh is determined using the following equation:Drug Substance Target CalculationNote:- CNP Variant HCI Salt, Purity Factor P = 81.3%CNP equivalence Factor B = 0.827Figure 4.1.2 is an example determining the mass of DS in milligrams needed to prepare 100 mL of DP Solution at a 0.500 mg / mL concentration.Drug Substance Target CalculationDrug Product Solution Preparation InstructionsProcedure:1. Determine DS target amount using the calculation in Figure 4.1.1.2. Weigh the DS amount determined in Step 1. Record weight.3. Calculate the FB volume needed to achieve the required DP concentration using the actual DS amount weighted in Step 2 and the following equation:4. Add 98% to 99% of the calculated CNP Prodrug FB volume from Step 3 to the previously weighed DS in Step 2 while withholding 1-2% of the FB volume to account for pH adjustments in next steps.Note: The FB volume to be withheld is based on scale; the larger the scale the more volume is withheld.5. Mix until the DS is completely dissolved, and a homogeneous solution is achieved.6. Test and record the initial pH.7. Adjust the pH to 5.5 ± 0.1 using the 1 N sodium hydroxide. Record the volume used.8. Q.s.ad to final volume with CNP Prodrug FB, then mix gently until a homogenous solution is achieved.9. Verify that the pH is 5.5 ± 0.1. Record the final pH. Appearance: clear colorless liquidStorage requirements / stability: In-use: 25 °C, no more than 6 hours;Short-term: 2-8 °C, no more than 3 days; and Long term: -60 °C or less, no more than 1 month.Description and Composition of the Drug Product

[0378] CNP Prodrug DP is supplied as lyophilized, preservative-free, white to yellow powder for reconstitution with sterile water for injection (WFI). The reconstituted solution is colorless to yellow. The drug product contains 15.7 mg / vial or 47.2 mg / vial of CNP prodrug substance which is equivalent to 13 mg / vial or 39 mg / vial of CNP, respectively. The reconstituted solution contains 10 mg / mL or 30 mg / mL of CNP equivalent with a target pH of 5.5. Sterile WFI will also be supplied for reconstitution. The clinical DP will be supplied in sterile, Type I, single-dose, borosilicate glass vials with coated stopper and flip-off aluminum cap. The formulation buffer contains 10 mM histidine buffer, 58.00 mg / mL trehalose dihydrate, 15.00 mg / mL D-mannitol, 0.73 mg / mL L-methionine and 0.05 mg / mL polysorbate 80. It will be used as placebo for clinical study.Table 13: Composition of CNP Prodrug Drug ProductJP: Japanese Pharmacopoeia; Ph. Eur.: European Pharmacopoeia; q.s., as much as needed; USP: United States Pharmacopeia. a Nominal concentration per mL after reconstitution Each vial is filled to a target of 1.3 mi- prior to lyophiliziation. b Amount of CNP Equivalent (Molar ratio of CNP to CNP prodrug is 0.827) c Water removed during lyophilization

[0379] The container closure system for the CNP Prodrug DP is compliant with USP<660> and USP <381 > (Table 14).Table 14: CNP Prodrug Drug Product Container Closure Sysl ternERFE, ethylene tetrafluoroethylene; JP, Japanese Pharmacopoeia; Ph. Eur., European Pharmacopeia; USP, United States Pharmacopeia.Pharmaceutical Development

[0380] The DP being proposed for use in clinical studies is representative to the DP used in the animal toxicology studies that support the established no observed adverse effect level (NOAEL) as described herein. A description of chemistry and manufacturingdifferences between the DP proposed for clinical use and the DP used in the animal toxicology studies to establish the NOAEL is provided below.Table 15: Summary of Drug Product Samples for Nonclinical Studies and Clinical

[0381] The formulation development began with a series screening studies using different buffering species and pH levels ranging from pH 3 to pH 8. Based on the outcome of these studies, histidine buffer pH 5.5 was selected. The pH ranging studies indicated impurities are generated in liquid phase and a lyophilization process was selected as stabilization strategy. A series of studies was conducted with varying bulking agents and their concentrations to develop an isotonic and lyophilizable formulation for CNP Prodrug. Trehalose and mannitol with above mentioned amount and ratio (Table ) provided pharmaceutically elegant cake structure and isotonic formulation. L-methionine is added to CNP Prodrug formulation to derisk the potential for oxidation while polysorbate-80 is included to minimize adsorption of CNP Prodrug to product contacting material.

[0382] A preliminary in-use study was conducted, and no risk was detected. Compatibility studies with the manufacturing process components were conducted and no risk was observed. Compatibility studies for the administration components and container closure system are planned and will be available for IND submission.

[0383] Additionally, process development studies including mixing, filterability, and lyophilization cycle development & optimization were conducted to support the manufacturing of CNP Prodrug DP. Using the outcome of these studies, a DP development batch and engineering batch were manufactured to support GMP manufacturing. Both the development batch and engineering batch are on stability. Detailed data on these batches and stability data will be shared during IND submission.Manufacture

[0384] The manufacturing process is shown in the schematic below and Table 16. and a brief narrative overview is provided which includes controls that represent a combination of manufacturing process parameters, in-process controls / tests, and quality attributes.CNP Prodrug Drug Product Manufacturing Process Flow DiagramDP, drug product; DS, drug substance; GMP, Good Manufacturing Practices.Table 16: Narrative Description of CNP Prodrug Drug Product Manufacturing Process

[0385] The drug product manufacturing process is controlled and monitored carefully to ensure consistency of product composition and quality of the drug product to ensure product safety and efficacy.Drug Product Specification

[0386] The proposed DP specifications are provided in Table 17:

[0387] Table 23: Summary of Drug SubstanceRH, relative humidity.Table 24: CNP Prodrug Drug Substance Batch Analysis

[0388] Table

[0388] 18 depicts the specifications are based on the available knowledge of the product. All impurities > 0.10% in area by purity method RP-UPLC are monitored on release and stability.Drug Product Stability

[0389] Stability studies are ongoing on a non-GMP engineering batch and a GMP batch will be placed on stability once manufactured. The stability studies will be performed using principles from ICH Q1A and stability data will be evaluated using principles per ICH Q1E. The stability protocols for the CNP Prodrug DP are provided in Table 18 and Table . It is intended that the stability data obtained from the engineering batch will be used to support the stability of the clinical DP through the duration of the clinical study. The DP in-process tests provide adequate control of the process for both engineering and clinical batches.There are no manufacturing process differences and container closure differences between engineering and GMP batches.Table 19: CNP Prodrug Drug Product Stability Protocol - Long Term (5 ± 3°C)Table 20: Drug Product Stability Protocol for Accelerated Storage Condition (25 ± 2 °C / 60 ± 5%RH)relative retention time; SCX-HPLC, strong cation exchange high performance liquid chromatography; SEC-HPLC; size exclusion high performance liquid chromatography; TBD, to be determined; USP, United States Pharmacopeia; UV, ultraviolet. a Tests applies to GMP Batches only. b Container closure integrity at the TO is confirmed by a passing sterility result at release.Manufacturing Process

[0390] This example demonstrates a process for preparing a process for manufacturing CNP Prodrug, including linear chain and side chain assembly by solid phase synthesis (SPPS), TFA cleavage / deprotection, in-solution cyclization by h, reverse phase chromatography (RPC) purification and salt exchange, is described below:Manual solid phase synthesis (SPPS)

[0391] A manual SPPS of CNP Prodrug was performed on chlorotrityl chloride (CTC) resin. The table below described the coupling time, amino acid equivalent, Fmoc deprotection time, and AA residues acetylation that was needed during chain expansion.Table 21. SPPS of CNP ProdrugGeneral coupling protocol:• AA:DIC:Oxymapure=1:1.1:1 and AA was dissolved in DMF at 0.2-0.3 M concentration• 0.1 M Oxymapure was added to the Fmoc removal steps to minimize Cys racemization during base treatment.• Preactivation for 0.5-1 hour for AA coupling solution except for Arg, Cys, His, and Met.• Fmoc-Lys(Alloc)-OH was used at A-11 for side chain deprotection prior to side chain assembly.• Alloc removal used Pd(PPh3)4 / PPh3 / DMBA (0.1eq:0.5eq:5eq in DMF)• DEPBT was used for His coupling to suppress racemization (His:DEPBT:DIPEA = 2:2.2:3-3.5, pH~9)• Single AA coupling strategy was employed for the side chain assembly.• N2 blanketing was applied throughout the SPPS to minimize Cys and Met oxidation.1. Cleavage and Cyclization

[0392] For the cleavage, the following conditions were used: TFA: DTT : H2O : TIPS : NH4I = 87:7.5:2.5:2.5:0.5, 3 hours at room temperature under nitrogen atmosphere. Reverse precipitation was then performed by adding the cleavage mixture (cooled to 1-15°C) to 4V of 25 % n-Heptane / MTBE (pre-cooled to 4°C) over 15 min.

[0393] The bulk crude was then dissolved in HOAc+20% ACN at 30 mg / mL for 30 minutes. 2% I2 in ACN was added to start oxidation till orange color persisted then quenched by 1 % ascorbic acid / H2O after completion.2. Purification

[0394] In addition to a first reverse purification on C18 media (A: 0.1 M TEAP pH 2.3 in H2O, B: 0.1M TEAP pH 2.3 in ACN, 25-45% B in 100 min), a second purification / salt exchange was implemented in order to convert the TFA salt to an HCI salt media (A: 0.02% HCI in H2O, B: 0.02% HCI in ACN, 10-15% B in 3 min, 15-50% B in 75 min).3. Lyophilization

[0395] The final combined pool is filtered through 0.2 pm filter and lyophilized. Typical yield: 6 to 7.5%Procedure for manufactureDrug Substance - Additional Information

[0396] The synthesis of the CNP Prodrug peptide applies Solid-Phase Peptide Synthesis (SPPS) technology, applying A / -a-9-fluorenylmethoxycarbonyl (Fmoc) amino acids derivatives as building blocks to assemble the peptide onto a resin (solid support) and Fmoc protected building block to assemble the side chain out of Lys 27. Post-synthesis, CNP Prodrug drug substance (DS) is cleaved from the resin, cyclized, purified and isolated, as a lyophilized powder.

[0397] The specifications for release and stability testing will conform to the general principles of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q6A for new DS.

[0398] The acceptance criterion for purity of clinical batches is proposed to set to 95% area, with total impurities (55%).

[0399] Relevant ICH guidelines and United States Pharmacopeia (USP)ZEuropean Pharmacopoeia (Ph. Eur.) monographs were consulted for setting acceptance criteria for residual solvents (ie, ICH Q3C), elemental impurities (ie, ICH Q3D, and other elements were included for monitoring purposes only), microbiological quality (ICH Q4B), and bacterial endotoxins (ICH Q4B).

[0400] The endotoxin limit for CNP Prodrug DS based on the USP <85> and Ph. Eur. chapter 5.1.10 for parenteral products for intravenous (IV) administration. The endotoxin level for the CNP Prodrug drug product (DP) will also be controlled based on the clinical dosage and administration route to ensure product safety. The acceptance criteria for bioburden follow USP <61 >.

[0401] In conclusion, the selected test parameters and release acceptance criteria are considered appropriate to ensure the correct identity, suitable chemical / microbial purity, and reliable assay value of the CNP Prodrug DS at this stage of clinical development.Drug Product - Additional Information

[0402] Test parameters and acceptance criteria for release and shelf-life of the CNP Prodrug DP were selected to confirm the identity, purity, quality, and to ensure the product’s safety. The acceptance criteria have been developed and justified based on limited manufacturing experience and the general principles of ICH Q6A. The relative retention time and % area of all impurities at > 0.10% will be reported and monitored on release and stability testing. The tests for pH, sterility, endotoxins, osmolality, container content, particulate matter, and container closure integrity are conducted according to ICH Q6A guidelines for parenteral products, and their acceptance criteria are established based on all applicable guidance documents, relevant USP chapters for sterile products, and current manufacturing understanding.Table 23: Summary of Drug Substance BatchesRH, relative humidity.Table 24: CNP Prodrug Drug Substance Batch AnalysisCNP, C-type natriuretic peptide; GC, gas chromatography; GMP, Good Manufacturing Practices; ICP-MS, inductively coupled plasma mass spectrometry; KF, Karl Fischer; LAL, Limulus Amebocyte Lysate; MS, mass spectrometry; ND, not detected; Ph. Eur., European Pharmacopeia; RP-HPLC, reversed phase high performance liquid chromatography; RP- UPLC, reversed phase ultra performance liquid chromatography; RRT, relative retention time; USP, United States Pharmacopeia; w / w, weight / weight. a Total impurities includes impurities >0.05% by area. b Total impurities includes impurities >0.10% by area per the DS release acceptance criteria for clinical material.Table 25: CNP Prodrug Drug Product Preliminary SpecificationCNP, C-type natriuretic peptide; HPLC-CAD, high performance liquid chromatography with charged aerosol detection; Ph. Eur., European Pharmacopeia; RP-UPLC, reversed phase ultra performance liquid chromatography; RRT, relative retention time; SCX-HPLC, strong cation exchange high performance liquid chromatography; SEC-HPLC; size exclusion high performance liquid chromatography; USP, United States Pharmacopeia; UV, ultraviolet; TBD, to be determined.1Lyophilized DP is reconstituted in water prior to testing.Table 26: Overview of CNP Prodrug Non-clinical StudiesGLP, Good Laboratory Practice;NHP, non-human primate; NPR, natriurel ic peptide receptor;PK, pharmacokinetics; SD, Sprague-Dawley; WT, wild-type.Metabolites

[0403] CNP Prodrug is comprised of CNP bound via the Lys27side chain to a pH responsive self-immolative linker, a spacer, and C-18 fatty acid albumin binding domain. Upon hydrolysis of the linker, active CNP is formed along with 2 major pharmacologically inactive metabolite isoforms consisting of the albumin-binding C-18 fatty acid and hydrolyzed linker, referred to as Metabolites A and B, respectively. The structure of both inactive metabolite isoforms is shown below.Structure of Metabolite AStructure of Metabolite B

[0404] Both Metabolites A and B are expected to be formed collectively in vivo in NHPs at a similar proportion to CNP Prodrug as in humans as the rate of CNP Prodrug hydrolysis is constant at physiologic pH. Therefore, the toxicity of each metabolite isoform will be evaluated as part of general toxicity studies of CNP Prodrug. Metabolite A is unstable and rapidly forms Metabolite B by hydrolysis during synthesis and purification; therefore, the major metabolite isoform likely to be detected in plasma of both humans and nonclinical species is Metabolite B. Both metabolite isoforms will be evaluated using 2 in silico approaches.

[0405] As genetic toxicology studies of CNP Prodrug are not planned, Metabolite B will be further evaluated in an in vitro bacterial reverse mutation assay and an in vitro micronucleus assay. Metabolite A is too unstable to produce, as it spontaneously forms Metabolite B, so will not be evaluated in genetic toxicology studies.

[0406] Additional metabolite identification and characterization of metabolite toxicity and absorption, distribution, metabolism, and excretion properties will be conducted, as appropriate, in accordance with FDA Guidance on Safety Testing of Drug Metabolites (March 2020).

[0407] Inhibition of CYP enzymes (e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 / 5 enzymes) in human liver microsomes by CNP Prodrug or Metabolite B were evaluated in vitro. Neither CNP Prodrug nor Metabolite B inhibited the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 / 5 in vitro at concentrations up to 20 pM. For each tested CYP, no direct, time-dependent (30-minute pre-incubation), or metabolism dependent (30-minute pre-incubation with NADPH) inhibition of activity was observed for either CNP Prodrug or Metabolite B. Evaluation of inhibition ofCYP enzymes (e.g., CYP1A2, CYP2D6 and CYP3A4 enzymes) in human liver microsomes) in cultured human hepatocytes by CNP Prodrug or Metabolite B are ongoing.

[0408] In silico screening assessment of Metabolites A and B were conducted by means of Quantitative Structure activity Relationship (QSAR) evaluation The in silico analysis did not identify any hazards relevant to administration of CNP Prodrug. Neither Metabolite A nor B is likely to be mutagenic based on QSAR analysis.

[0409] Evaluations of in vitro transporter substrate assessment of CNP Prodrug and lipid- linker in different cell lines are ongoing.Phase 1 Study

[0410] BioMarin plans to initiate a comprehensive clinical development program, including a Phase 1 study in healthy adult volunteers followed by Phase 2 dose-range finding and a pivotal Phase 3, to support approval of CNP Prodrug in pediatric patients with ACH.Example 24: Pharmacodynamics of CNP Prodrug

[0411] CNP Prodrug is a pharmacologically inactive prodrug of CNP, that does not bind to NPR-B (internal data). Hydrolysis of the linker forms the active parent, CNP, and inactive metabolite isoforms consisting of the lipid and hydrolyzed linker. Therefore, the pharmacodynamics of CNP Prodrug relies on the pharmacologic activity of released CNP. CNP has an established mechanism of action and has been shown to increase endochondral bone growth in both WT and animal models of FGFR3-mediated chondrodysplasia is approved for treatment of ACH in children with open epiphyses.Therefore, pharmacology studies of CNP Prodrug were designed to confirm CNP released from CNP Prodrug retains the desired pharmacologic effect and to establish the sustained CNP exposure needed to achieve endochondral bone growth.

[0412] Pharmacodynamics of CNP Prodrug is being evaluated in mice and NHPs to demonstrate the dose response effect of CNP is retained when active CNP is released continuously from its albumin bound inactive prodrug form into the circulation compared to daily pulsatile delivery of CNP. Completed studies demonstrated the expected dose responsive endochondral skeletal growth effect of CNP is retained with CNP Prodrug treatment, as shown by increases in cellularity and height of the hypertrophic zone of the growth plate and / or increased length of long bones.

[0413] Growth effects of CNP Prodrug in young WT mice were compared to CNP in a study, entitled, “Five Weeks Growth Study of CNP Prodrug Compared to CNP in C57BL / 6J mice.” A total of 80, 3-week-old male C57BL / 6J mice (Jackson Laboratories) wererandomized into 7 treatment groups. Animals were 6.3 to 7.2 g at study initiation. Animals were treated for 5 weeks with CNP by daily SC injection at 0 (vehicle), 40, 150, or 500 μg / kg / day or with CNP Prodrug by SC injection once every other day (QAD) at 0 (vehicle), 80, 300, or 1000 μg / kg / dose. All doses were based on CNP content. The design of the study is shown in Table 24-1.Table 24-1 : Design of the Pharmacodynamics Study of CNP Prodrug in Young Wild- Type MiceCNP, C-type natriuretic peptide; M, male; NA, not applicable; QAD, once every other day; QD, once daily;. aThe dose volume for each animal was 5 μL / gram bodyweight. bVehicle, 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80. cCNP, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1 .5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80. dCNP Prodrug, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.

[0414] Animals were observed daily for general health. Body weights were collected prior to each dose (daily for Groups A, C, D, and E and QAD for Groups B, F, G, and H). Overall growth was assessed by weekly naso-anal and tail lengths, beginning prior to treatment using a ruler while the mouse was anesthetized with inhaled isoflurane. The femur, tibia, humerus, ulna, and vertebral column (superior cervical to inferior sacral vertebrae) were measured weekly from micro computed tomography (pCT) images performed during inhaled isoflurane anesthesia. The pCT images were also reviewed by the study veterinarian for evidence of abnormalities. After 5 weeks of treatment, animals were euthanized and necropsied.

[0415] There were no effects on body weight gains when differences at Baseline were accounted for. There were no changes in gait or ambulation observed or macroscopic abnormalities at necropsy. Dose-responsive significant increases in naso-anal length were observed in all CNP-treated mice compared to the corresponding vehicle-treated mice. Asshown in Table 25, significant increases in naso-anal length were observed only at 1000 μg / kg QAD for CNP Prodrug-treated mice. Tail lengths were only significantly increased in CNP-treated mice at doses of 150 and 500 μg / kg / day compared to concurrent controls. No effects on tail length were observed in CNP Prodrug-treated mice.Table 24-2: Naso-anal and Tail Length Growth in CNP and CNP Prodrug-Treated Young WT MiceANOVA, analysis of variance; QAD, every other day dosing; QD, daily dosing; SD, standard deviation. Bolded values Indicate significant difference (p <0.05, by one-way ANOVA with Tukey’s post hoc test for multiple comparisons from treatment control for % change from Baseline (Groups C, D, and E compared to Group A and Groups F, G, and H compared to Group B).

[0416] Estimated Cmaxand AUC for Prodrug and CNP are set out in Table 24-2.1.Table 24-2.1 : Estimated Cmaxand AUC for Prodrug and CNPAUC(O-T) presented where in T = 120 min for animals treated with CNP and 48 hours for animals treated with CNP prodrug, representing the typical range of detectable vosoritide in plasma of mice following daily vosoritide treatment and interval between doses, respectively. cEstimated exposures are extrapolated from WT male mice administered 600 μg / kg CNP.“Estimated exposures are extrapolated from WT mice administered 627 or 1891 μg / kg CNP prodrug (based on CNP content).

[0417] The level of cGMP, a biomarker of CNP binding to the NPR-B receptor, was measured in plasma collected at 0.5, 4, and 24 hours post-dose. While in CNP-treated mice levels of cGMP in plasma were elevated at 0.5 hours post-dose at all doses, levels were similar to vehicle-treated mice by 4 hours post-dose. In contrast, for CNP Prodrug, at the 1000 μg / kg QAD dose level, there was a sustained release of cGMP observed at all time points. When compared to cGMP in CNP-treated mice, levels were approximately a quarter of the peak observed with CNP treatment. There was no detectable cGMP in the plasma of mice treated with 80 or 300 μg / kg QAD CNP Prodrug.

[0418] While significant dose-responsive growth of the femur, tibia, and vertebral column was observed for CNP-treated mice, no significant growth was observed in CNP Prodrug- treated mice. This study demonstrated treatment with 1000 μg / kg QAD CNP Prodrug is pharmacologically active in WT mice, with an effect on naso-anal length and detectable plasma cGMP. Nonetheless, it was concluded that QAD dosing of CNP Prodrug does not allow sufficiently consistent CNP exposure in mice to produce a similar growth effect as CNP given daily.Growth Effects of CNP Prodrug in WT Mice Dosed Daily

[0419] Increased frequency (daily) dosing of CNP Prodrug was evaluated in a study, entitled, ‘A 5 Week Study of CNP and CNP Prodrug by Subcutaneous Administration in Juvenile Male Mice.”

[0420] A total of 40 male C57BL / 6NCrl mice (Charles River Laboratories) were randomized into 4 treatment groups on postnatal day (PND) 21. Animals were treated by daily SC injection of vehicle, 500 μg / kg / day CNP, or CNP Prodrug at 500 or 1600 μg / kg / day. All doses were based on CNP content. Dosing was initiated for all animals on PND 22 and treatment was intended to occur for 5 weeks (to PND 57); however, due to test article effects, treatment was terminated early, after up to 18 days of treatment. The design of the study is shown in Table 24-3.Table 24-3: Design of the Daily-Dosing Pharmacodynamics Study of CNP Prodrug inYoung Wild-Type MiceCNP, C-type natriuretic peptide;1each group consisted of 10 male mice;aThe dose volume for each animal was 5 mL / kg bodyweight.bVehicle, 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) polysorbate 80.cCNP, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.dCNP Prodrug, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.

[0421] Animals were observed for mortality, clinical observations, body weights. Digital radiographs were taken on PNDs 21 (Pretest), 29 (Study Day 8), and 36 (Study Day 15), while animals were under general anesthesia. In addition, radiographs were taken on PND 41 (Study Day 20) on animals exhibiting suspected fractures, including the remaining controls. At scheduled necropsy, the right femur, right humerus, and right ulna were trimmed of tissues and a caliper was used to measure length and width.

[0422] There were several animals that were euthanized early in the 500 μg / kg / day CNP- treated group (Group 2) and the 1600 μg / kg / day CNP Prodrug-treated group (Group 4). One Group 4 animal was euthanized on Study Day 4 due to poor / deteriorating condition. Due to the short duration treatment, this death was considered to be potentially related to poor adaption to weaning and was not likely related to treatment with CNP Prodrug. One Group 4 animal was euthanized on Study Day 8 due to slight limited use of the right forelimb that was the result of a suspected cage-related injury and is of uncertain relationship to treatment with CNP Prodrug, given the short duration of treatment. On Study Day 15-16, 4 / 10 Group 2 animals and 5 / 8 remaining Group 4 animals were euthanized due to suspected fractures and / or clinical signs of slight to moderate firm swollen bilateral hindlimbs and abnormal gait. As a result of these early deaths, the study was terminated on Study Day 20 and all surviving animals were euthanized and necropsied.

[0423] During the dosing period, there were bilateral CNP-related radiographic findings in males at 500 μg / kg / day at the distal tibia and calcaneus. On Study Day 8, all animals presented radiographic increased physis thickness and radiographic mixed reaction of the distal tibia. On Study Day 15, radiographic increased physis thickness and radiographic mixed reaction of the distal tibia were still generally observed in association with increased physis thickness of the calcaneus and suspected or confirmed fracture of the distal tibia in some animals. In addition, there were occasional epiphyseal fractures of the calcaneus. On Study Day 20, all remaining males presented radiographic increased physis thickness and radiographic mixed reaction of the distal tibia and suspected or confirmed fracture at the distal tibia. Most males presented epiphyseal fractures of the calcaneus and / or increased physis thickness of the calcaneus. Radiographic findings are summarized in Table 24.4.

[0424] During the dosing period, there were bilateral CNP Prodrug-related radiographic findings in males at > 500 μg / kg / day at the distal tibia and calcaneus, generally similar to those noted for CNP. On Study Day 8, all males at 1600 μg / kg / day presented radiographic increased physis thickness and radiographic mixed reaction of the distal tibiae. On Study Day 15, radiographic increased physis thickness and radiographic mixed reaction of the distal tibia were generally observed in animals treated with 500 or 1600 μg / kg / day CNP Prodrug or 500 μg / kg / day CNP. These findings were associated with an increased physis thickness of the calcaneus and suspected or confirmed fracture of the distal tibia for some animals treated with 500 μg / kg CNP or 1600 μg / kg / day CNP Prodrug. In addition, one male treated with 500 μg / kg / day CNP Prodrug presented an epiphyseal fracture of the calcaneus. On Study Day 20, prior to necropsy, only those animals with clinically suspected fractures (3 / 6500 μg / kg CNP-treated animals and 1 / 6500 μg / kg CNP Prodrug-treated animals) as well as 2 representative control animals had radiographs performed. As on Study Days 8 and 15, radiographic increased physis thickness and radiographic mixed reaction, i.e., regions of both increased and decreased density of bone tissue, of the distal tibia and calcaneus were observed. Epiphyseal fracture of the calcaneus was observed in 2 of the CNP-treated animals and the 500 μg / kg / day CNP Prodrug-treated animal. Radiographic findings are summarized in Table 24-4.Table 24-4: Test Article Related Radiographic Findings on Study Days 8, 15, and 20Lt, left; Rt, right.aRadiographs were performed only on surviving animals with suspected fractures due to clinical observations of hindlimb swelling and / or altered gait, along with two control animals for comparison purposes.

[0425] Radiographic bone measurements were conducted on Study Days 8 and 15. Despite the observed physis thickness increase observed in radiographs presence of fractures, there was no treatment-related effect on the length of the femur, tibia, ulna, humerus, or lumbar spine in mice treated with 500 μg / kg / day CNP Prodrug or CNP compared to controls at either time point. Animals given 1600 μg / kg / day had significantly higher greater femur (+11%) and lumbar spine (+12%) lengths at Study Day 15 compared with controls. No other CNP Prodrug-related effects on the length of the tibia, ulna, or humerus were noted. Interpretation of terminal ex vivo measurements was impacted by the reduced number of surviving animals in the 500 ug / kg / day CNP and 1600 ug / kg / day CNP Prodrug-treated groups. At the end of the dosing period, males administered CNP Prodrug at 500 μg / kg / day presented statistically significant longer femur (+7%), humerus (+4%), and ulna length (+7%), compared with controls; however, there was high inter-animal variability, suggesting the findings may be incidental. No treatment effect was detected in histomorphometry, but value of this end point was impacted by the number of early deaths and reduced number of animals surviving to Study Day 20.

[0426] Microscopic findings for animals euthanized on or after Study Day 15 are detailed in Table 24.5. Mildly to severely increased thickness of the physis in the distal tibia wasnoted at 500 μg / kg / day CNP and 500 or 1600 μg / kg / day CNP Prodrug, correlating to joint enlargement grossly. Increased thickness of the physis progressed to minimal to marked degeneration / necrosis of the physis in most animals and physeal fracture in a few animals, mostly in the 1600 μg / kg / day CNP Prodrug group. Physeal fractures correlated to joint enlargement observed clinically. Increased thickness physis of the distal tibia was accompanied by 2 secondary changes at the same doses. Minimally to mildly increased bone in the metaphysis, which was considered an elongation of the primary and secondary spongiosa, and minimal to mild cortical resorption, which was considered an elongation of the “cut-back” zone.

[0427] In the proximal tibia, the most important change was minimally decreased thickness of the physis, which was noted at 500 μg / kg / day CNP or CNP Prodrug, with a greater incidence in CNP Prodrug-treated animals. The change correlated to decreased physeal parameters measured by histomorphometry. Minimally increased physis thickness was noted in a few animals at > 500 μg / kg / day CNP Prodrug, and the incidence of the change was dose-related. The change was accompanied by minimally increased bone in the metaphysis in one 1600 μg / kg / day CNP Prodrug-treated animal, which was considered a secondary change. Minimal chondrocyte vacuolation was noted at 500 μg / kg / day CNP. It was characterized by vacuolation of chondrocytes in the hypertrophic zone.

[0428] In the calcaneus, changes were similar to the ones described in the distal tibia and consisted of mildly to moderately increased thickness of the physis, minimal to moderate physeal degeneration / necrosis, fracture, and mild to moderate cortical resorption, all noted in the dorsal aspect of the bone. Fracture correlated to joint enlargement noted grossly. Table 24-5: Test Article Related Microscopic Findings in Wild-Type Micea Numbers in parentheses represented the number of animals with the finding. b The incidence of vacuolation reflected the total incidence of the change irrespective of the location. c Number of animals examined for this tissue represents the number of animals in which findings were noted in the calcaneus in the section submitted for tibio-tarsal joint examination.

[0429] In conclusion, the increase in dose interval from QAD to daily SC dosing of CNP Prodrug resulted in an increase in plasma levels of CNP that led to adverse effects on the tibia and calcaneus in juvenile WT mice at doses of 500 or 1600 μg / kg / day. Similar effects were seen with a high daily dose of CNP (500 μg / kg / day). These changes reflect exaggerated pharmacologic effects that have been previously observed with CNP in rodents, occasionally in the absence of no or modest effects on bone lengths. The absence of a significant effect on growth in this study despite these effects should be considered in the context of the short treatment period and detectability of subtle growth effects. This fracture effect was not observed radiologically via pCT, as discussed below, in which WT mice of adifferent strain were treated with 500 μg / kg / day CNP or 1600 μg / kg / day CNP Prodrug daily for 6 weeks.Example 25: Mouse Models of Noonan Syndrome

[0430] Noonan Syndrome (NS) is a genetically inherited disease affecting the MAPK signaling pathway in multiple cell and tissue types, with distinct disruptions in heart and bone morphology. Manifestations of NS pathobiology include reduced height and moderate alterations of the facial bones as well as hypertrophic cardiomyopathy (HCM). The genetic etiology of NS involves autosomal-dominant mutations in major signaling components of the RAS signaling cascade such as protein tyrosine phosphatase non-receptor type 11, RIT1, and rapidly accelerating fibrosarcoma 1 (RAF-1) (Saint-Laurent 2024 Eur JPediatr;183:1011-1019). Mutations in these signaling intermediates result in reduced negative regulation pathway needed for normal ERK / MAPK homeostasis, resulting in elevated MAPK activity. As in ACH, this increased MAPK signaling can be reduced by treatment with CNP or CNP, which antagonizes MAPK signaling at the level of RAF-1. Therefore, these models are useful to demonstrating the efficacy of CNP Prodrug compared to CNP in the context of increased MAPK signaling.

[0431] This study entitled, “Pilot Pharmacodynamic Study of CNP and CNP Prodrug in Juvenile Wild Type or Raf1 L613V Heterozygous Male Mice Dosed Daily via Subcutaneous Administration for 6 Weeks” was conducted to assess effects on growth in the presence of the Raf1 L613V activating mutation of CNP and CNP Prodrug. These effects were contrasted with a small molecule MAPK kinase inhibitor (MEKi), PD0235901, that has been previously evaluated in this model (Wu 2011, J Clin Invest 121:1009-1025).

[0432] Raf1+ / L613Vmice express one WT copy of the Raf1 gene, and one copy containing a knock-in missense mutation where the leucine at residue 613 is substituted for valine (L613V). This mutation results in excessive activation of the MAPK pathway. Mice heterozygous for this mutation begin to develop phenotypes like those seen in NS patients by 5 weeks of age, including enlarged hearts, mild flattening of craniofacial features and mild reduction in naso-anal length compared to WT siblings (Wu supra).

[0433] In this study, male WT or Raf1+ / L613Vmice of 50% 129S1 / SvilmJ and 50% C57BL / 6J background were bred through crossing of male Raf1+ / L613V129S1 / SvlmJ mice (custom made via clustered regularly interspaced short palindromic repeats [CRISPR] Cas9 technique; Jackson Laboratories, Sacramento, CA) to female WT C57BL6 / J mice (JAX stock #000664, Jackson Laboratories, Sacramento, CA). Animals were genotyped on PND 7 via a tail tip snip. A total of 25 WT and 38 Raf1+ / L613Vmales weighing 8-12 g were assigned to 8groups following weaning at 3 weeks of age (PND 21-23). The study design is shown in Table 25-1.Table 25-1 : Design of the Pharmacodynamics Study in WT and Raf1+IL6nvNoonan’sSyndrome MiceCNP, C-type natriuretic peptide; F, female; M, male; MEKi, mitogen-activated protein kinase kinase inhibitor; IP, intraperitoneal; SC, subcutaneous; WT, wild-type.® Doses based on CNP content in μg / kg, except for Groups G and H that were treated with PD0235901 .bVehicle, 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) polysorbate 80.cCNP, Bag 11 , in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.dCNP Prodrug, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.eVehicle, 5% dimethyl sulfoxide (DMSO) in sterile 1X phosphate buffered saline.fMEKi PD0235901 , IUPAC name: N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4- iodophenyl)amino]-benzamide, in 5% dimethyl sulfoxide (DMSO) in sterile 1X phosphate buffered saline. s During Study Week 4, one mouse was found to have been mis-sexed at study initiation and was found to be a female. The animal was housed separately for the remainder of the study.

[0434] Following randomization into study, animals were anesthetized and subjected to a baseline echocardiogram, body weight, and naso-anal length measurement using a standard metric ruler. At 3 weeks of age, Raf1+ / L613Vwere approximately 0.46 cm or 6.72% shorter than age-matched WT littermates and body weights were correspondingly lower. Cardiac hypertrophy was present in this model, but a HCM phenotype was not observed at Baseline and did not develop, with or without CNP or CNP Prodrug treatment.

[0435] Treatment was initiated at 4 weeks of age. Wild-type and Raf1+ / L613Vmice were dosed by daily SC injection with vehicle, 500 μg / kg / day CNP, or 1600 μg / kg / day CNP Prodrug. All doses were based on CNP content. Comparator groups of Raf1+ / L613Vmice were by daily intraperitoneal (IP) injection of vehicle or 5 mg / kg / day MEKi. All animals were dosed for 6 weeks.

[0436] Animals were examined daily for body condition, behavior and gait and were weighed daily prior to dosing. During Study Week 5, and naso-anal length was measured, and animals were subjected to an echocardiogram under anesthesia. Prior to euthanasia in Study Week 6, mice in Groups A-F received a full body pCT scan to assess femur and tibia length and skull morphology. Animals were euthanized on Study Days 49-52 (Study Week 6) and necropsied. The right femur and tibia were collected from all WT animals. Hearts and spleens were weighed.

[0437] There were no test article-related deaths. One animal in Group H (5 mg / kg / day MEKi) did not recover from anesthesia following the Study Week 5 echocardiogram procedure. After 2 weeks of treatment, animals treated with either CNP or CNP Prodrug began to develop visibly elongated skeletal features and minor peripheral bone outgrowths resulting in hunched posture and curled paws. Mice treated with MEKi rapidly gained weight and developed mild obesity by Study Week 5.

[0438] As shown in Figure 25, by 8 weeks of age, the reduced naso-anal length phenotype in Raf1+ / L613Vmice observed at 3 weeks of age was no longer present as the lengths of vehicle-treated Raf1+ / L613Vmice were not significantly different from vehicle-treated WT animals. Wild-type mice treated with 1600 μg / kg / day CNP Prodrug had an increase in naso-anal length of 21.2% and Raf1+ / L613Vmice naso-anal lengths increased 12.4% compared to vehicle-treated mice. Wild-type mice treated with 500 μg / kg / day CNP exhibited a 7.8% increase in naso-anal length and Raf1+ / L613Vmice lengths increased 7.4% compared to vehicle-treated mice. Both WT and Raf1+ / L613Vmice treated with CNP Prodrug had significantly longer naso-anal lengths than mice treated with 500 μg / kg / day CNP. Mitogen- activated protein kinase kinase inhibitor treatment was also associated with an increase in naso-anal length compared to vehicle control, however these gains were smaller than those observed with CNP or CNP Prodrug treatment.

[0439] There were no fractures detected by pCT at the end of the 6-week treatment period, in contrast to the effects observed in C57BL / 6NCrl mice. As with naso-anal length, by 9 weeks of age, there was no difference in femur or tibia length found by pCT in vehicle- treated Raf1+ / L613Vmice compared to vehicle-treated WT mice. Both 1600 μg / kg / day CNP Prodrug and 500 μg / kg / day CNP induced statistically significant growth in the long bones of WT mice (Table 25-2). There was also a statistical difference between the growth effects of CNP Prodrug and CNP in WT mice, where CNP Prodrug at 1600 μg / kg / day induced greater growth than 500 μg / kg / day CNP in both femur and tibia (p < 0.0001 by 2-way analysis of variance with Tukey’s multiple comparisons test).Table 25-2: Femur and Tibia Lengths in WT and Raf1+ / L613VMice Treated for 6 WeeksANOVA, analysis of variance; WT, wild-type.Bold values are significantly different from control by two-way ANOVA with Tukey's multiple comparison test.

[0440] Raf1+ / L613Vmice skulls exhibited mild enlargement of intercanthal distance, reduced skull width, and reduced skull length compared to their WT siblings at 9 weeks of age. These data compare with reported skull phenotypes seen in other published reports for this model (Wu 2011) and relate to the skull phenotype observed clinically in patients with NS and ACH. As shown in Table 25-3, treatment with 1600 μg / kg / day CNP Prodrug or 500 μg / kg / day CNP improved this phenotype, non-significantly reducing the mean intercanthal distance and significantly increasing skull width and length. As with the femur and tibia, CNP Prodrug effects were greater for skull length and width than for CNP. Only skull length was significantly increased in WT mice treated with 1600 μg / kg / day CNP Prodrug compared to vehicle treated WT mice.Table 25-3: Skull Morphology in WT and Raf1+ / L613VMice Treated for 6 WeeksANOVA, analysis of variance; WT, wild-type.Bold values are significantly different from genotype-matched control by two-way ANOVA with Tukey’s multiple comparison test.

[0441] There were no test article related effects on echocardiography parameters, confirming neither CNP nor CNP Prodrug impacted the function of either normal or hypertrophic hearts.-0442] In conclusion, daily doses of 500 μg / kg / day of CNP and 1600 μg / kg / day induced characteristic overgrowth in both WT (50% 129S1 / SvilmJ and 50% C57BL / 6J) and Raf1+ / L613Vmice on the same background. The skull phenotype of Raf1+ / L613Vmice was normalized with treatment with CNP and, to a greater degree, with CNP Prodrug. Animals did not present with fractures either clinically or by pCT imaging, despite longer treatment. These animals initiated treatment at 4 weeks of age rather than at 3 weeks and have a different strain background, which may have influenced these differences. Treatment with CNP or CNP Prodrug did not lead to adverse effects on the heart despite the hypertrophy phenotype.Effect of CNP Prodrug on Increased MAPK Signaling at the Level of RIT1

[0443] This study was conducted to assess effects on growth of CNP and CNP Prodrug in the presence of the activating M90I mutation of Rit1 (Rit+ / -mice). These effects were contrasted with a small molecule MEKi, PD0235901.

[0444] Mutations in RIT1 are less frequently associated with short stature in NS than mutations to RAF1 associated with NS (Yaoita 2016). The M90I mutation in RIT1 increases signaling along the ERK1 / 2 MAPK pathway, which may be ameliorated by treatment with CNP. Mice heterozygous for the M90I mutation (Rit1+I-) have been previously shown to have shorter body lengths at 4 weeks of age and increased spleen and heart to body weight ratios compared to WT littermates (Castel 2019).

[0445] In this study, 30 WT and 45 Rit1+I- 8-week-old male mice were randomized into 10 groups. Animals were dosed daily by SC injection with vehicle or CNP at 500 μg / kg / day or QAD with vehicle or CNP Prodrug at 3280 μg / kg / dose. Comparator groups of R / f+ / 'mice were treated by daily IP injection of vehicle or 5 mg / kg / day MEKi. All animals were dosed for 10 weeks. The design of the study is shown in Table 25-4.Table 25-4: Design of the Pharmacodynamics Study in WT and Rit1+ / ' Noonan’sSyndrome MiceCNP, C-type natriuretic peptide; DMSO, dimethyl sulfoxide; IP, intraperitoneal; M, male; MEKi, mitogen-activated protein kinase kinase inhibitor; QAD, every other day; QD, daily; SC, subcutaneous; WT, wild-type.aDoses based on CNP content in μg / kg, except for Groups G and H that were treated with PD0235901 .bVehicle, 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) polysorbate 80.cCNP, Bag 11 , in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.dCNP Prodrug, in 0.005 mol / L citrate buffer solution, pH 5.5 containing 5.8% (w / v) trehalose dihydrate, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) Polysorbate 80.3Vehicle, 5% DMSO in sterile 1X phosphate buffered saline.fMEKi PD0235901 , IUPAC name: N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4- iodophenyl)amino]-benzamide, in 5% DMSO in sterile 1X phosphate buffered saline.

[0446] Animals were weighed prior to each dose. Naso-anal lengths, tail lengths, radiographic measurements of long bones (tibia, femur, ulna, humerus) and vertebrae, and echocardiograms were collected at Baseline, prior to treatment initiation, after 6 weeks of treatment. Radiographic measurements were also collected at Study Week 10 prior to termination. In Study Week 10, animals were euthanized and necropsied. Terminal body weights, naso-anal lengths, and tail lengths were measured and the heart, lung, liver, and spleen were weighed.

[0447] During the 10-week treatment period, visibly noticeable skeletal growth occurred in CNP and CNP Prodrug treated WT and, to a lesser degree, Rit+I-mice. The study was originally planned for 14 weeks of dosing but was terminated at 10 weeks due to the skeletal growth phenotype observed in CNP and CNP Prodrug-treated animals and the absence of a HCM phenotype, making further dosing futile.

[0448] As with Raf1+ / L613Vmice, Rit+!-mice did not exhibit a HCM phenotype, however hearts were enlarged relative to body weight, which was not meaningfully affected by treatment with CNP Prodrug or CNP. There were no adverse test article effects on cardiac function as assessed by echocardiography.

[0449] Naso-anal lengths were significantly increased from treatment and genotype- matched vehicle-treated controls for both 500 μg / kg / day CNP and 3280 μg / kg / dose QAD CNP Prodrug (Figure 26). In WT animals, treatment with 3280 μg / kg / dose QAD CNP Prodrug resulted in significantly longer naso-anal lengths than animals treated with CNP.There was no difference in dose-response in Rit+I-mice. Treatment with MEKi also resulted in increased naso-anal length, albeit to a lesser degree than achieved with either CNP or CNP Prodrug treatment. A similar trend was observed with tail lengths.

[0450] Figure 27 shows representative images at Baseline, 6 weeks, and 10 weeks of treatment for a vehicle-treated and CNP Prodrug-treated WT mice. CNP Prodrug treatment resulted in noticeable increases in long bone lengths and growth of the spinal column is evident. Early data on this study shows a clear treatment related effect of CNP Prodrug on skeletal growth, as animals reached the characteristic overgrowth phenotype observed previously for CNP in mice.CNP Prodrug Pharmcacodynamics in NHPs

[0451] Male cynomolgus monkeys were treated with CNP Prodrug 1 month to evaluate pharmacologic activity, as assessed by radiographic and microscopic evaluation of bones and assessment of biomarkers of osteogenesis. Pharmacokinetics and CV safety pharmacology were also evaluated Animals were 2 years old at study initiation and weighed 2.7 to 3.3 kg. The study design is shown in Table 25-5.Table 25-5: Design o the 1 -Month Pharmacology Study in Cynomolgus MonkeysCNP, C-natriuretic peptide; M, male; USP, United States Pharmacopeia; w / v, weight / volume.1each group consisted of 4 male monkeysa1 mL / kg dose volume.b0.005 mol / L citrate buffer solution, pH 5.5, containing 5.25% (w / v) sucrose, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) polysorbate 80 prepared in sterile water for injection, USP.cCNP Prodrug formulated in 0.005 mol / L citrate buffer solution, pH 5.5, containing 5.25% (w / v) sucrose, 1.5% (w / v) mannitol, 0.727 mg / mL methionine, and 0.005% (w / v) polysorbate 80 prepared in sterile water for injection, USP.

[0452] Animals were given vehicle or CNP Prodrug at 30 or 150 μg / kg / dose (based on CNP content) by SC injection once weekly on Study Days 1, 8, 15, 22, and 29. Doses were delivered in the back in the scapular area, alternating between the left (Study Days 1 and 15) and right (Study Days 8 and 22) sides. The final dose on Day 29 was delivered to a naive site in the mid back.

[0453] Animals were monitored daily for mortality or evidence of gross toxicity and detailed clinical observations and body weights were performed weekly during the dosing period. Food consumption was monitored qualitatively. Clinical chemistry and hematology samples were collected weekly and on the day of necropsy. Biomarkers of chondrocyte hypertrophic differentiation (Collagen X and fragment of the NC1 domain of Collagen X [Pro- C10 HP]), and bone turnover / formation, ie, type II collagen degradation (type II collagen neoepitope [T2CM]), osteoblast activity (bone-specific alkaline phosphatase [BAP] and fragment of N-terminal type I collagen [Pro C1 ]), and type II collagen formation (fragment of N-terminal type IIB procollagen [Pro C2]) were measured in serum from fasted animals prior to treatment initiation, prior to dosing on Study Days 1, 8, 15, 22, and 29 and prior to necropsy on Study Day 31. Telopeptide of type I collagen (CTx-la), a biomarkers of type I collagen degradation, and telopeptide of type II collagen (CTx-ll), a biomarker of type II collagen degradation, and cGMP, a biomarker of CNP cellular activity, were measured in urine collected overnight from fasted animals prior to treatment initiation and one and five days after each dose on Study Days 2, 6, 9, 13, 16, 20, 23, 27, and 30. Atrial natriuretic peptide (ANP) levels in plasma were measured in PK samples collected after each dose to confirm sustained supraphysiologic levels of CNP did not interfere with ANP clearance via NPR-C.

[0454] Prior to initiation of dosing, on Study Day 21 , and prior to necropsy on Study Day 31 , digital radiographs of femora / tibiae / fibulae, spine, and radii / ulnae were performed on allanimals under anesthesia. Radiographs were evaluated qualitatively and the physeal closure was scored for the right proximal tibia / fibula and radius / ulna and for the right distal and proximal femora. Tibia and ulna length (right and left) were derived from the radiographs. Body length and tail length were also measured directly on anesthetized animals at the same time radiographs flexible measuring tape.

[0455] On Study Day 31, all animals were euthanized and underwent a full necropsy. The sternum, costochondral junction of the sixth rib, proximal left tibia, injection sites, and gross lesions were collected and examined microscopically. Histomorphometry was performed on the left proximal tibial physis for each animal to measure the thickness of the total epiphyseal growth plate and proliferative and hypertrophic zones.

[0456] All animals survived to scheduled necropsy. There were no test article-related clinical observations or effects on body weight / body weight gains, hematology, or clinical chemistry. ANP levels in plasma were below the limit of quantification (BLQ) at most timepoints for CNP Prodrug-treated animals, precluding definitive conclusions on trends; however, the result suggests continuous delivery of CNP via CNP Prodrug treatment did not result in competition for the shared clearance receptor, NPR-C.

[0457] Change from Baseline in normalized cGMP was increased in dosed groups (Groups 2 and 3) from the second dose onwards, on the second day from weekly dosing (Days 9, 16, 23, and 30). Change from Baseline in normalized cGMP was generally lower on the sixth day after weekly dosing (Study Days 13, 20 and 27) compared to the second day after weekly dosing (Study Days 9, 16, 23, and 30). Increases in normalized cGMP were higher in Group 3 (150 μg / kg / week) compared to Group 2 (30 μg / kg / week), suggesting that CNP pharmacological activity was sustained in a dose-dependent manner through 30 days of dosing.

[0458] There were no detectable treatment effects on Collagen X, Pro-C10 HP, T2CM, Pro-C1, or Pro C2 in serum. Changes from Baseline of BAP concentrations were similar in all 3 groups except on Study Day 29 and Day 31 (on the day of or 2 days after the 5th dose), when positive changes were observed in animals dosed with 150 μg / kg / week CNP Prodrug. Changes from Baseline in CTxia concentration normalized to urine creatinine was similar in all 3 groups and no observable trend was noted. CTx-ll concentrations in urine were BLQ for most timepoints in all groups.

[0459] There were no apparent treatment-related radiographic changes or exacerbation of pre-existing radiographic findings. There were no meaningful treatment related changes intibia, ulna, body, or tail lengths when compared to vehicle-treated animals, likely due to the short duration of the study.

[0460] At necropsy, there were no treatment related macroscopic findings and all gross lesions were attributed to surgical implantation of the telemetry device. There were no CNP Prodrug related findings at the injection sites. Minimal increased cellularity of the hypertrophic zone of the physis was observed in 150 μg / kg / week treated animals in the sternum (4 / 4), proximal tibia (2 / 4), and the sixth rib costochondral junction (1 / 4), demonstrating pharmacologic activity in the hypertrophic chondrocytes, as expected and previously observed with CNP treatment. A small increase in mean thickness of the hypertrophic zone of the proximal tibial physis was observed in animals administered 150 μg / kg / week compared to concurrent controls. Although this change was not statistically significant, it was considered CNP Prodrug-related and correlated microscopically to increased cellularity of the hypertrophic zone in 2 animals at this dose. There were no CNP Prodrug-related changes at 150 μg / kg / week for total epiphyseal growth plate thickness and proliferative zone thickness and there were no changes at 30 μg / kg / week for any of the measured parameters, when compared to concurrent control animals.

[0461] In conclusion, CNP Prodrug was well-tolerated in male NHPs when given weekly at 30 or 150 μg / kg / week for a total of 5 doses over 1 month. Pharmacologic activity (ie, binding to NPR-B) was evident by increases in normalized urinary cGMP levels in urine at both dose levels; however, effects on growth plate cellularity and hypertrophic zone thickness were only observed in animals given 150 μg / kg / week CNP Prodrug, suggesting the 30 μg / kg / week dose level was subtherapeutic. Effects on the growth plate did not translate to increases in bone, body, or tail length during the short duration of this study.

[0462] As with the primary and secondary pharmacodynamics, the active component of CNP Prodrug, CNP, also informs the safety pharmacology profiling of CNP Prodrug. Safety pharmacology studies of CNP Prodrug have focused on effects on CV endpoints, as the altered PK of CNP (ie, blunting of Cmax) mitigates the observed effects of CNP on heart rate and blood pressure. Safety pharmacology evaluations of CNP are briefly summarized below followed by a detailed discussion of the safety pharmacology studies of CNP Prodrug.Safety Pharmacology of CNP

[0463] No CNP-related respiratory or central nervous system (CNS) parameter changes were observed after a single SC administration of CNP at 30, 100 or 300 μg / kg to rats. The no-observed-effect level (NOEL) was 300 μg / kg in these studies.

[0464] In NHPs, as expected based on CNP mechanism of action in the vasculature, direct CNP administration induced vascular smooth muscle relaxation and subsequent dose- responsive decreases in blood pressure and compensatory increases in heart rate. Overall, little or no changes in blood pressure / heart rate were observed in the conscious NHPs given ≤ 10 μg / kg CNP. An approximately 25% increase in heart rate was observed in conscious NHPs given 28 μg / kg CNP. In the GLP CV safety pharmacology study, No overt CV-related clinical signs were observed in conscious NHPs given 50 μg / kg while a < 10% decrease in blood pressure and an approximately 37% increase in heart rate were noted. The presence of overt CV-related clinical signs was observed in some conscious NHPs given > 200 μg / kg CNP. These clinical signs consisted of reduced activity in 5 / 8 animals from 40 to 60 minutes post-dose on Day 1 in the pilot CV study and of idiosyncratic short and repeated bouts of sternal or lateral recumbency in 3 / 8 animals during the first hour post-dose in the Phase 2 of the main CV study. In the 28-day repeat-dose toxicity study no overt CV-related clinical signs were noted in the NHPs treated with up to 300 μg / kg CNP. The discrepancy between studies regarding the presence of overt CV-related observations was likely related to the method of administration (remote or manual) and to the amount of time technical staff were present in the room based on the size of the animal cohorts. Attenuation of the effects on blood pressure decreases, but not in the heart rate increases, was observed after daily repeat CNP administrations and was possibly due to NPR-B desensitization in the vasculature at high repeat doses delivered. A CV naive-like response was observed following at least a 2- day washout period. It is possible that, because NPR-B distribution in the vasculature is mostly restricted to the peripheral vasculature, the severity of the measurable and observable hemodynamic changes was overall limited. Based on the absence of measurable changes in heart rate and blood pressure and CV related clinical observation, the NOEL of subcutaneously administered CNP to conscious NHPs was 10 μg / kg in this study. No overt CV-related clinical observations were observed in the conscious monkey given a SC administration of 550 μg / kg CNP. Across all studies, no prolongation of QT interval was observed.

[0465] As CNP was not shown to have any effects on the CNS or pulmonary function, no CNS or respiratory safety pharmacology studies of CNP Prodrug are planned.

[0466] The CV effects of CNP were expected to be ameliorated by the blunting of CNP Cmaxwith continuous release from CNP Prodrug rather than daily pulsatile treatment. This was confirmed in 2 studies in cynomolgus monkeys, a non-GLP study and a GLP-compliant study.

[0467] As described above, male cynomolgus monkeys were dosed weekly by SC injection of vehicle or CNP Prodrug at 30 or 150 μg / kg / week (based on CNP content) for a total of 5 doses. Animals’ heart rate, systemic arterial pressures (systolic, diastolic, pulse pressure, and mean arterial pressure), ECGs parameters (PR, RR, QRS, QT intervals, and corrected QT interval [QTc]), and body temperature were monitored remotely via surgically implanted DSI PhysioTel® Digital M11 telemetry devices continuously for 24 hours prior to initiation of dosing, then for a 20-hour period after each dose, starting at 25 hours post-dose. A qualitative evaluation of the ECG was also conducted at 36 hours post-dose by a veterinary cardiologist. No effects on any CV parameters were observed. This study confirmed that repeated pharmacologically active doses of CNP Prodrug, as expected, did not result in the reduction in blood pressure and increased heart rate typically observed with CNP as a single SC injection.

[0468] A GLP-compliant study designed to evaluate toxicity, described further below, and pharmacologic effects of CNP Prodrug and formed CNP, on the CV system in male and female cynomolgus monkeys was carried out

[0469] In this study, male and female cynomolgus monkeys were dosed with vehicle or escalating doses of 150, 300, and 500 μg / kg / dose CNP Prodrug (based on CNP content) by SC injection, with each dose given two weeks apart (Study Days 1, 15, and 29) to avoid accumulation. The design of the study is shown in Table 25-6.Table 25-6: Design of the Ascending Dose Toxicity and CV Safety Pharmacology Study of CNP Prodrug in Cynomolgus MonkeysCNP, C-type natriuretic peptide; CV, cardiovascular; F, female; M, male. a Dose volume of 0.3 mL / kg. b CNP Prodrug Vehicle (10 mM Histidine pH 5.5, 48 mg / mL Trehalose Dihydrate, 12.4 mg / mL D-Mannitol, 0.73 mg / mL L-Methionine, 0.05 mg / mL Polysorbate 80 in Sterile Water for Injection) c CNP Prodrug, formulated in 10 mM Histidine pH 5.5, 48 mg / mL Trehalose Dihydrate, 12.4 mg / mL D-Mannitol, 0.73 mg / mL L-Methionine, 0.05 mg / mL Polysorbate 80 in Sterile Water for Injection.

[0470] Animals’ heart rate, systemic arterial pressures (systolic, diastolic, pulse pressure, and mean arterial pressure), ECGs parameters (PR, RR, QRS, QT intervals, and QTc), and body temperature were monitored remotely via surgically implanted DSI PhysioTel® DigitalM11 telemetry devices continuously for 24 hours prior to initiation of dosing and then up to 72 hours after each dose on Study Days 1, 15, and 29.

[0471] There were no CNP Prodrug-related changes in systemic blood pressures (mean arterial pressure, systolic and diastolic blood pressures, pulse pressure) and heart rate, as assessed up to 72 hours post-dose at 150, 300, and 500 pg / kg. An apparent trend for decreased group means heart rate measurements was noted in CNP Prodrug-treated females at 500 pg / kg (Dose 3), more pronounced during the dark cycles, relative to the controls, with the changes occasionally attaining statistical significance. However, the individual heart rate measurements of the CNP Prodrug-treated females at 500 μg / kg remained comparable to their own pre-dose and / or pre-study values. This trend was attributed in part to the additional individual variability resulting from the increased heart rate measurements of one control animal on this occasion.

[0472] Together, these studies confirm that by reducing the steep peaks of CNP exposure seen with daily delivery, CV effects of CNP can be eliminated while maintaining pharmacologic activity on endochondral bone growth.Example 26: Pharmaccokinetics of CNP Prodrug

[0473] Pharmacokinetics of CNP has been extensively evaluated. The characterization of the nonclinical PK of subcutaneously administered CNP Prodrug was conducted in small animals (mice and rats) and large animals (NHPs). These studies were conducted (or are ongoing) in conjunction with toxicity studies that included 3 single-dose studies and 2 repeat-dose studies. Additionally, studies are planned to characterize metabolic stability, plasma protein binding and drug-drug interaction potential of CNP Prodrug and its metabolite (lipid-linker; Metabolite B) released after hydrolysis.Pharmacokinetics in Mice

[0474] In a single-dose PK study in 10-week old male C57BL / 6J mice, single SC injections of 500 μg / kg or 1600 μg / kg of CNP Prodrug were administered. Plasma concentration from each mouse was measured at single time point and for each time point approximately 3 mice were used. Average plasma concentration at each time point was used for non-compartment analysis to determine PK parameters. After SC injection, CNP Prodrug was slowly absorbed in plasma with median Tmaxfor a 500 μg / kg dose of 8 hours and 4 hours for a 1600 pg / kg dose. CNP Prodrug had a mean t½ of approximately 10.5 hours for both dose groups. Dose normalized area under the plasma concentration-time curve (AUC) and Cmaxvalues were similar in both dose groups, suggesting plasma exposure of CNP Prodrug increased proportionally with dose over a range of 500 to 1600 μg / kg . CNPwas slowly released into plasma with a mean Tmaxof 4 hours for the 500 μg / kg dose and 12 hours for the 1600 μg / kg dose. Similar to CNP Prodrug, released CNP from CNP Prodrug had a longer mean t½ of 9.6 hours compared to a SC dose of CNP for the 500 μg / kg dose and 9.9 hours for the 1600 μg / kg dose. Dose normalized plasma concentration were similar in both groups. Dose normalized CNP AUC and Cmaxwere similar in both dose groups, suggesting plasma exposure of CNP increased proportionally with dose between doses of 500 to 1600 μg / kg . Percent metabolite ratio (released CNP / CNP Prodrug) at Cmaxof was 0.07% for the 500 μg / kg dose and 0.1 % for the 1600 μg / kg dose. Percent metabolite ratio of AUC of released CNP compared with CNP Prodrug was 0.06% for the 500 μg / kg dose and 0.08% for the1600 μg / kg dose.Pharmacokinetics in Rats

[0475] In a single-dose PK study in adult rats, CNP Prodrug was administered at 560 μg / kg IV and 150 μg / kg , 280 μg / kg , 560 μg / kg , or 1680 μg / kg SC. After an IV dose of CNP Prodrug, CNP was slowly released, reaching Tmaxat 4 hours. Mean t½ of released CNP was10.8 hours, which was similar to CNP Prodrug (8.9 hours). Released CNP metabolite ratio of area under the plasma concentration-time curve from time 0 to infinity (AUCo-.) was 0.58%. Released CNP mean t½ was similar in both males (10.7 hours) and females (10.9 hours). However, exposure (AUC and Cmax) of released CNP was slightly higher in males (60900 h*pM and 2800 pM, respectively) compared to females (45800 h*pM and 2380 pM, respectively). After a SC dose, CNP Prodrug was slowly absorbed in plasma with median Tmaxat 8 hours for all dose groups. The CNP Prodrug mean t½ ranged from 6.72 hours for the 150 μg / kg SC dose to 10.5 hours for the 1680 μg / kg SC dose. Dose proportional increase in CNP Prodrug exposure were observed with the mean dose normalized exposure (AUCo-- / dose) of CNP Prodrug being similar for all SC doses ranging from 150 μg / kg to 1680 μg / kg and dose normalized plasma concentration curves were virtually overlapping.Bioavailability of CNP Prodrug SC doses ranged from 17.67% to 21.71%. No consistent trend for bioavailability between sexes was observed. With the slow release of CNP, the median CNP Tmaxwas slightly delayed relative to CNP Prodrug (Tmaxapproximately 10-12 hours post-dose). The t½ for released CNP was similar to CNP Prodrug, ranging from 7.6 to12.8 hours. Released CNP mean exposure (AUC0-∞) and as Cmaxincreased with increasing dose of 150 μg / kg to 280 μg / kg and 560 μg / kg to 1680 μg / kg but was similar for dose 280 μg / kg and 560 μg / kg . Percent metabolite ratio of AUC0-twas higher for the 2 lower dose groups (0.461% for the 150 μg / kg dose and 0.494% for the 280 μg / kg dose) compared to the 2 higher dose groups (0.294% for the 560 μg / kg and 0.323% for the 1680 μg / kg dose). No consistent trend based on sex in exposure of CNP Prodrug and released CNP wasobserved. CNP Prodrug Cmaxand AUC increased proportionally with increase in dose from dose range 280 μg / kg to 1680 μg / kg . For released CNP, Cmaxand AUC increased proportionally from dose 150 μg / kg to 280 μg / kg and 560 μg / kg to 1680 μg / kg . Pharmacokinetics in NHPs

[0476] In a repeat-dose study in NHPs, CNP Prodrug was administered by weekly SC injection of 30 μg / kg / week or 150 μg / kg / week, based on CNP content, for 1 month. After SC administration, CNP Prodrug was slowly absorbed with a median Tmaxof 24 hours after the first dose on Study Day 1 and 16 hours after multiple weekly doses on Study Day 22 at 30 μg / kg QW and at 48 hours after the first dose on Study Day 1 and 28 hours after multiple weekly doses on Study Day 22 at the 150 μg / kg / week dose. Compared to rodents, CNP Prodrug had a longer mean t½ of 60 to 65.3 hours, which was consistent for both 30 μg / kg QW and 150 μg / kg QW dose levels. After 3 weeks of 30 μg / kg QW dosing, 2.09, 2.36, 3.37, and 3.15-fold accumulation of CNP Prodrug was observed in Cmax, AUCtlast, AUC0-∞and trough plasma concentration (Ctrough) at 168 hours, respectively. After 3 weeks of 150 μg / kg QW dosing, 1.24, 1.5, 1.42 and 1.67 -fold accumulation in Cmax, AUCtlast, AUC0-∞and Ctrough at 168 hours, respectively of CNP Prodrug was observed. The apparent clearance of drug, apparent volume of distribution based upon the terminal phase and t½ values were similar on Day 1 and Day 22 indicating that CNP Prodrug PK remains consistent after repeat dosing in NHPs.

[0477] Plasma concentrations of released CNP were not quantifiable for the majority of animals treated with 30 μg / kg QW on Study Days 1 and 22. Demonstrating a slow release of CNP, at 150 μg / kg QW, plasma concentrations of released CNP had a median Tmaxof 8 hours after the first dose and 48 hours. After 3 weekly doses of 150 μg / kg QW, 1 .88, 2.62, 4.05 and 2.92-fold accumulation of released CNP in Cmax, AUC0-t,AUC0-∞and Ctrough at 168 hours, respectively, was observed. Because of slow release rate and quick clearance of released CNP, t½ of CNP was similar to CNP Prodrug (65.3 hours on Study Day 1 and 63.8 hours on Study Day 22 at 150 μg / kg QW). Percent metabolite ratio of AUC0-twas could only be computed for 150 μg / kg QW group. After a single dose, the metabolite ratio of AUC0-twas 0.1 % and after three weekly doses, on Day 22, it was was 0.14%.

[0478] A single-dose PK study was conducted in cynomolgus monkeys in which male and female 2 -year-old cynomolgus monkeys were given a single SC injection of 101 μg / kg , 202 μg / kg , and 336 μg / kg CNP Prodrug. Sample analysis from this study is currently ongoing. Toxicokinetics are also being evaluated in the ongoing 26-week repeat-dose pharmacologyand toxicology study in NHPs wherein animals are receiving weekly SC injections of 75 μg / kg / week, 200 μg / kg / week, and 350 μg / kg / week CNP Prodrug.Single-dose Toxicity of CNP Prodrug in NHPs

[0479] A GLP-compliant study was designed to evaluate toxicity and pharmacologic effects of CNP Prodrug and formed CNP, on the CV system in male and female cynomolgus monkeys.

[0480] In this study, male and female cynomolgus monkeys were dosed with vehicle or escalating doses of 150, 300, and 500 μg / kg / dose CNP Prodrug (based on CNP content) by SC injection, with each dose given two weeks apart (Study Days 1, 15, and 29) to avoid accumulation. Each dose was delivered into a unique injection site on the back. Animals in this study were transferred from a prior study following an approximately 7-week washout between studies and, therefore, were not naive to CNP Prodrug treatment. As a result, although this study is referred to as a single-dose study, animals were not naive to CNP Prodrug treatment at any dose level. The design of the study is shown in Table 26-1.Table 26-1 : Design of the Ascending Dose Toxicity and CV Safety Pharmacology Study of CNP Prodrug in Cynomolgus MonkeysCNP, C-type natriuretic peptide; CV, cardiovascular; F, female; M, male. a Dose volume of 0.3 mL / kg. b CNP Prodrug Vehicle (10 mM Histidine pH 5.5, 48 mg / mL Trehalose Dihydrate, 12.4 mg / mL D-Mannitol, 0.73 mg / mL L-Methionine, 0.05 mg / mL Polysorbate 80 in Sterile Water for Injection) c CNP Prodrug, formulated in 10 mM Histidine pH 5.5, 48 mg / mL Trehalose Dihydrate, 12.4 mg / mL D-Mannitol, 0.73 mg / mL L-Methionine, 0.05 mg / mL Polysorbate 80 in Sterile Water for Injection

[0481] Animals were observed daily at cageside for mortality or gross toxicity and detailed physicals were performed at least weekly during the dosing phase as well as following the 72-hour telemetry monitoring period after each dose. Animals were weighed twice weekly, with one weight collected the day prior to each dose. Funduscopic and biomicroscopic ophthalmology examinations were performed prior to dose initiation and on Study Day 32 (3 days after the final dose). Hematology and clinical chemistry clinical pathology parameters were evaluated approximately 96 hours after each dose of CNP Prodrug. Toxicokinetic samples were collected prior to (0 hour) and 72, 84, and 96 hours after each dose. Ant-drug antibody samples were collected prior to study initiation and on Study Day 33, but only theBaseline sample will be measured due to expected drug interference in the sample on Study Day 33.

[0482] On Study Day 33, 4 days after animals received the 500 μg / kg dose, all animals underwent a detailed necropsy. The brain, epididymides, adrenal glands, pituitary glands, prostate, thyroid and parathyroid gland, kidneys, liver with gall bladder, ovaries, spleen, testes, and thymus were weighed (when present). A comprehensive list of tissues was collected from each animal for microscopic examination (Table 26-2). A coronal section of the proximal left tibia was collected for histomorphometry of the epiphyseal growth plate.Table 26-2: Tissues Collected for Microscopic Evaluation in CNP Prodrug-Treated Cynomolgus Monkeys

[0483] All animals survived to their scheduled necropsy. There were no test article-related effects on ophthalmology, body temperature, hematology, clinical chemistry, or organ weights. The only test article-related clinical observations were at or adjacent to the injection site, which consisted of slight soft or moderate firm swelling in the lumbar region of the back of 2 male animals 3-4 days after the 500 μg / kg dose (Study Days 32 and 33). Red skin discoloration in the lumbar area was also noted for one female and one male on Study Day 33 (4 days after the 500 μg / kg dose). At necropsy on Study Day 33, dark foci were noted in some animals at all injection sites and nodule and / or mass were observed in a few animals at the 300 and 500 μg / kg injection sites (Table 26-3).Table 26-3: Macroscopic Findings Adjacent to the Injection Sites in CNP Prodrug- Treated Cynomolgus Monkeys

[0484] The injection site (left scapular, Site 1) used for the 150 μg / kg CNP Prodrug dose was associated with mild granulomatous inflammation of the SC tissue in treated males. The injection sites used for the 300 μg / kg CNP Prodrug dose (right scapular; Site 2) and 500 μg / kg CNP Prodrug (mid-dorsal, Site 3) doses were associated with mild to marked granulomatous inflammation. The incidence and severity of these findings is summarized in Table 26-4. Granulomatous inflammation within the SC tissue in treated animals was characterized variably by a core of eosinophilic necrotic debris and / or empty cavities rimmed by numerous epithelioid macrophages and multinucleated giant cells with fewer neutrophils which in turn were rimmed by lymphocytes and fibrous connective tissue. This change correlated to the macroscopic observations of dark focus, nodule and / or mass. Moderate to...

Claims

1. A pharmaceutical composition comprising a variant of the C-type natriuretic peptide (CNP) PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1), a pharmaceutically acceptable excipient and a carrier or diluent, wherein the CNP variant comprises an acidic portion, a spacer, a hydrolyzable linker and is characterized by the structure shown in Fig.

11.

2. A pharmaceutical composition comprising (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl-L-glutaminyl-L-glutamyl-L-histidyl)pyrrolidine-2-carbo xamido)-4-amino-4-oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4-hydroxyphenyl)propane amido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4-oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)ac (etamido)-4-methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,2 9,34-hexaazagenepentacontyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)eth yl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4-carboxylic acid, a pharmaceutically acceptable excipient and a carrier or diluent.

3. A pharmaceutical composition according to claim 1 or 2, characterized in that the pharmaceutical composition is lyophilized.

4. A pharmaceutical composition according to one of paragraphs 1-3, characterized in that the carrier or diluent includes a buffer.

5. The pharmaceutical composition according to claim 4, characterized in that the buffer includes a buffer selected from citrate, acetate, phosphate, TRIS and a combination thereof.

6. The pharmaceutical composition according to claim 5, characterized in that the buffer also includes histidine, its salt, its solvate or solvate of its salt.

7. A pharmaceutical composition according to one of paragraphs 4-6, characterized in that the buffer is present at a concentration of 5-15 mM.

8. A pharmaceutical composition according to one of claims 4-7, having a pH level of 3-9.

9. A pharmaceutical composition according to claim 8, having a pH level of 4-6.

10. A pharmaceutical composition according to claim 9, having a pH level of 5-6.

11. The pharmaceutical composition according to claim 10, having a pH level of 5.2 or 5.

5.

12. A pharmaceutical composition according to one of claims 1-11, characterized in that the pharmaceutically acceptable excipient is selected from a bulking agent, a tonicity regulating agent, an antioxidant, a surfactant, a solubilizing agent, a stabilizer, and a combination thereof.

13. The pharmaceutical composition according to claim 12, characterized in that the bulking agent is selected from mannitol, sucrose, dextran, lactose, trehalose and povidone (PVP K24) and combinations thereof.

14. The pharmaceutical composition according to claim 12 or 13, characterized in that the bulking agent includes trehalose or its solvate, mannitol, or a combination thereof.

15. The pharmaceutical composition according to claim 14, characterized in that the bulking agent includes trehalose and mannitol in a weight ratio of 3:1 to 1:

1.

16. The pharmaceutical composition according to claim 15, characterized in that the mass ratio of trehalose to mannitol is 3.9:

1.

17. A pharmaceutical composition according to one of claims 13-16, characterized in that trehalose is present in an amount of 3-6% by weight of the composition.

18. The pharmaceutical composition according to claim 17, characterized in that trehalose is present in an amount of 3.5-5.8% by weight of the composition.

19. The pharmaceutical composition according to claim 18, characterized in that trehalose is present in an amount of 3.8-4.8% by weight of the composition.

20. A pharmaceutical composition according to one of claims 12-19, comprising a tonicity adjusting agent selected from sodium chloride, dextrose, glucose, glycerin, sorbitol, xylitol, ethanol and a combination thereof.

21. A pharmaceutical composition according to one of claims 12-20, comprising an antioxidant selected from methionine, ascorbic acid, salt forms of ascorbic acid, thioglycerol and combinations thereof.

22. The pharmaceutical composition according to claim 21, characterized in that the antioxidant is methionine.

23. A pharmaceutical composition according to one of claims 1-22, comprising a stabilizer or surfactant selected from glycine, sorbitol, polysorbate and a combination thereof.

24. A pharmaceutical composition according to claim 23, comprising polysorbate.

25. A pharmaceutical composition according to one of claims 1-24, characterized in that the pharmaceutical composition includes L-histidine, histidine monohydrochloride monohydrate, trehalose dihydrate, D-mannitol, L-methionine and polysorbate.

26. A pharmaceutical composition according to one of claims 1-24, characterized in that the pharmaceutical composition includes a citrate buffer, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate.

27. A pharmaceutical composition comprising (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl-L-glutaminyl-L-glutamyl-L-histidyl)pyrrolidine-2-carbo xamido)-4-amino-4-oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4-hydroxyphenyl)propane amido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4-oxobutanamido)-6-aminohexanamido)-6-aminohexanamido)ac (etamido)-4-methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl-28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentaoxo-14,17,23,26-tetraoxa-5,8,11,20,2 9,34-hexaazagenepentacontyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10,37,43-triisobutyl-19-(2-(methylthio)eth yl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4-carboxylic acid, a buffer comprising histidine or a salt thereof, and one or more pharmaceutically acceptable excipients.

28. The pharmaceutical composition according to claim 27, characterized in that the buffer includes L-histidine hydrochloride monohydrate.

29. A pharmaceutical composition according to claim 27 or 28, comprising a pharmaceutically acceptable excipient selected from a bulking agent, a stabilizer, an anti-adsorbent, a diluent, and a combination thereof.

30. A pharmaceutical composition according to one of paragraphs 27-29, characterized in that the pharmaceutically acceptable excipient includes a bulking agent that includes trehalose or a solvate thereof.

31. The pharmaceutical composition according to claim 30, characterized in that the bulking agent includes trehalose dihydrate.

32. A pharmaceutical composition according to one of paragraphs 27-31, characterized in that the pharmaceutically acceptable excipient includes a bulking agent that includes mannitol.

33. The pharmaceutical composition according to claim 32, characterized in that the mannitol is D-mannitol.

34. A pharmaceutical composition according to one of paragraphs 27-33, characterized in that the pharmaceutically acceptable excipient includes a stabilizer that includes methionine.

35. The pharmaceutical composition according to claim 34, characterized in that the methionine is L-methionine.

36. A pharmaceutical composition according to one of paragraphs 27-35, characterized in that the pharmaceutically acceptable excipient includes an anti-adsorbent including polysorbate.

37. The pharmaceutical composition according to claim 36, characterized in that the polysorbate is polysorbate 80.

38. A pharmaceutical composition according to one of paragraphs 27-37, characterized in that the pharmaceutical composition contains virtually no citrate buffer.

39. A pharmaceutical composition comprising a variant of C-type natriuretic peptide (CNP) which is (4R,10S,16S,19S,22S,28S,31S,34S,37S,40S,43S,49S,52R)-52-(2-((S)-2-((S)-2-((S)-2-(2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-2-((S)-1-(L-prolylglycyl-L-glutaminyl-L-glutamyl-L-histidyl)pyrrolide in-2-carboxamido)-4-amino-4-oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3-(4-hydro xyphenyl)propanamido)-6-aminohexanamido)acetamido)propanamido)-4-amino-4-oxobutanamido)-6-aminohexanamido)-6 -aminohexanamido)acetamido)-4-methylpentanamido)-3-hydroxypropanamido)-6-aminohexanamido)acetamido)-49-benzyl -28-((S)-sec-butyl)-34-(carboxymethyl)-40-((S)-33,51-dicarboxy-8-(2-hydroxyethyl)-6,12,21,30,35-pentaoxo-14,1 7,23,26-tetraoxa-5,8,11,20,29,34-hexaazagenpentacontyl)-31-(3-guanidinopropyl)-16,22-bis(hydroxymethyl)-10.37 ,43-triisobutyl-19-(2-(methylthio)ethyl)-6,9,12,15,18,21,24,27,30,33,36,39,42,45,48,51-hexadecaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32,35,38,41,44,47,50-hexadecaazacyclotripentacontane-4-carboxylic acid, histidine buffer, trehalose dihydrate, D-mannitol, L-methionine, polysorbate 80 and water., 40. The pharmaceutical composition according to claim 39, characterized in that the CNP variant is present at a concentration of 10 mg / ml.

41. The pharmaceutical composition according to claim 39, characterized in that the CNP variant is present at a concentration of 30 mg / ml.

42. A pharmaceutical composition according to one of paragraphs 39-41, characterized in that the histidine buffer includes L-histidine and histidine monohydrochloride monohydrate.

43. The pharmaceutical composition according to claim 42, characterized in that L-histidine is present at a concentration of 0.35 mg / ml.

44. The pharmaceutical composition according to claim 42, characterized in that L-histidine is present at a concentration of 2.2 mM.

45. A pharmaceutical composition according to one of claims 42-44, characterized in that L-histidine monohydrochloride monohydrate is present at a concentration of 1.6 mg / ml.

46. ​​A pharmaceutical composition according to one of claims 42-44, characterized in that L-histidine monohydrochloride monohydrate is present at a concentration of 7.8 mM.

47. A pharmaceutical composition according to one of paragraphs 39-46, characterized in that trehalose dihydrate is present at a concentration of 58 mg / ml.

48. The pharmaceutical composition according to one of claims 39-46, characterized in that trehalose dihydrate is present in a concentration of from about 127 mM to about 153 mM.

49. The pharmaceutical composition according to claim 48, characterized in that trehalose dihydrate is present at a concentration of 127 mM.

50. The pharmaceutical composition according to claim 48, characterized in that trehalose dihydrate is present at a concentration of 153.3 mM.

51. A pharmaceutical composition according to one of paragraphs 39-50, characterized in that D-mannitol is present at a concentration of 15 mg / ml.

52. The pharmaceutical composition according to one of claims 39-50, characterized in that D-mannitol is present in a concentration of from about 68 mM to about 82 mM.

53. The pharmaceutical composition according to claim 52, characterized in that D-mannitol is present at a concentration of 68.1 mM.

54. The pharmaceutical composition according to claim 52, characterized in that D-mannitol is present at a concentration of 82.3 mM.

55. A pharmaceutical composition according to one of paragraphs 39-54, characterized in that L-methionine is present at a concentration of 0.7 mg / ml.

56. A pharmaceutical composition according to one of paragraphs 39-54, characterized in that L-methionine is present at a concentration of 4.9 mM.

57. A pharmaceutical composition according to one of paragraphs 39-56, characterized in that polysorbate 80 is present at a concentration of 0.05 mg / ml.

58. A pharmaceutical composition according to one of paragraphs 39-56, characterized in that polysorbate 80 is present at a concentration of 0.005% (volume / volume).

59. A pharmaceutical composition according to one of claims 1-58, characterized in that the composition exhibits a lower Cmax value and a higher AUC value compared to the free drug.

60. A pharmaceutical kit comprising a pharmaceutical composition according to one of paragraphs 1-58.

61. A pharmaceutical kit comprising a pharmaceutical composition according to one of paragraphs 39-58, characterized in that the CNP variant is present in an amount of 13 mg.

62. A pharmaceutical kit comprising a pharmaceutical composition according to one of paragraphs 39-58, characterized in that the CNP variant is present in an amount of 39 mg.

63. A method for treating a bone disorder or skeletal dysplasia in a subject in need thereof, comprising administering to the subject a composition according to one of claims 1-59.

64. The method according to claim 63, characterized in that the bone disorder or skeletal dysplasia is selected from the group consisting of osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondroplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, epiphyseal punctate chondrodysplasia, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, perinatal lethal type of osteogenesis imperfecta, short rib-polydactyly syndromes, ankylosing type of epiphyseal punctate chondrodysplasia, Jansen's metaphyseal osteodysplasia, congenital spondyloepiphyseal dysplasia, atelosteogenesis, diastrophic dysplasia, congenital short femur, Langer's mesomelic dysplasia, Nievergelt's mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral dysostosis, Kniest dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia,micromelia, Morquio syndrome, Kniest syndrome, metatrophic dysplasia and spondyloepimetaphyseal dysplasia, NPR2 mutations, SHOX mutations (Turner / Léry-Weill syndrome), PTPN11 mutations (Noonan syndrome), insulin-like growth factor receptor 1 (IGF1R) mutations, osteoporosis, and idiopathic short stature.

65. A method for elongating bone or increasing the growth of long tubular bones in a subject in need thereof, comprising administering to the subject a composition according to one of claims 1-59, characterized in that the administration allows for elongation of bone or increases the growth of long tubular bones.

66. The method according to one of paragraphs 63-64, characterized in that the composition is administered subcutaneously, intradermally, intraarticularly, orally or intramuscularly.

67. The method according to one of paragraphs 63-66, characterized in that the composition provides an extended release composition.

68. The method according to one of paragraphs 63-66, characterized in that the composition is administered once every 5 days, once a week, once every two weeks, once every three weeks, once every 4 weeks, once every 6 weeks, once every two months, once every three months, or once every six months.

69. A method of treating a CNP-responsive condition or disorder, comprising - introducing into the subject a composition according to one of paragraphs 1-58 and - observing the level of at least one bone or cartilage-related biomarker in a subject, wherein an increase in the level of at least one bone or cartilage-related biomarker indicates a therapeutic effect of the CNP variant on the subject or condition or disorder.

70. The method of claim 69, further comprising adjusting the amount or frequency of administration of the composition, wherein i) the amount or frequency of administration of the composition is increased if the level of at least one bone or cartilage-related biomarker is less than the target level; or II) the amount or frequency of administration of the composition is reduced if the level of at least one bone or cartilage-related biomarker exceeds the target level.

71. The method according to claim 69 or 70, characterized in that at least one bone- or cartilage-related biomarker is selected from the group consisting of CNP, cGMP, propeptides of type II collagen and fragments thereof, type II collagen and fragments thereof, C-telopeptide of type I collagen (CTx), osteocalcin, proliferating cell nuclear antigen (PCNA), procollagen type I propeptides (PINP) and fragments thereof, type I collagen and fragments thereof, aggrecan-chondroitin sulfate, collagen X, alkaline phosphatase, proliferating cell nuclear antigen (PCNA), procollagen type I propeptides and fragments thereof, type I collagen and fragments thereof, aggrecan-chondroitin sulfate, collagen X, CXM (non-collagenous 1 (NC1) domain of type X collagen), and alkaline phosphatase, NTproCNP, N-terminal propeptide of type I collagen, bone-specific alkaline phosphatase, amino-terminal propeptide of type I collagen / N-propeptide of type I collagen (PINP), cross-linked C-telopeptide of type I collagen (CTx),cross-linked N-telopeptide of type I collagen (NTx), tartrate-resistant acid phosphatase 5b (TRAP-5b), transcriptomic indicators and bioactivity of the CNP variant.

72. The method according to one of claims 63-71, characterized in that the administration increases the annual growth velocity (AGV) in the subject over 12 months, optionally compared to the baseline or to a normal control subject.

73. The method according to claim 72, characterized in that the AGV of the subject increases over the course of 1 year or over the course of 2 years or more.

74. The method according to one of claims 63-73, characterized in that the administration improves the growth Z-score at 12 months, optionally compared to the baseline or to a normal control subject.

75. The method according to one of paragraphs 63-74, characterized in that the subject is over 3 years of age.

76. The method according to one of paragraphs 63-75, characterized in that the subject is between 3 and 17 years old.

77. The method according to one of paragraphs 63-76, characterized in that the subject has open epiphyses.

78. The method according to one of paragraphs 63-77, characterized in that the composition is administered in a dose of from about 5 mcg / kg to 500 mcg / kg or from about 15 mcg / kg to 350 mcg / kg.

79. The method according to one of paragraphs 63-77, characterized in that the administration does not lead to cardiovascular (CV) side effects.

80. The method according to claim 79, wherein the cardiovascular side effect is a change in systemic arterial pressure, mean arterial pressure, systolic and / or diastolic blood pressure, pulse pressure or heart rate.