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WO2025101863A1 - Variants de cnp, conjugués et formulations de ceux-ci - Google Patents

Variants de cnp, conjugués et formulations de ceux-ci Download PDF

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Publication number
WO2025101863A1
WO2025101863A1 PCT/US2024/055072 US2024055072W WO2025101863A1 WO 2025101863 A1 WO2025101863 A1 WO 2025101863A1 US 2024055072 W US2024055072 W US 2024055072W WO 2025101863 A1 WO2025101863 A1 WO 2025101863A1
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WIPO (PCT)
Prior art keywords
cnp
pharmaceutical composition
various embodiments
variant
concentration
Prior art date
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PCT/US2024/055072
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WO2025101863A9 (fr
Inventor
Golam JAKARIA
Tian Wei CHOU
Pauline BOURBON
Kidisti Araya
Colm Campbell
Nina V. MITCHELL
Olivier CHARMANT
Daniel J. Wendt
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Biomarin Pharmaceutical Inc
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Biomarin Pharmaceutical Inc
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Publication of WO2025101863A1 publication Critical patent/WO2025101863A1/fr
Publication of WO2025101863A9 publication Critical patent/WO2025101863A9/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/2242—Atrial natriuretic factor complex: Atriopeptins, atrial natriuretic protein [ANP]; Cardionatrin, Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Cardionatrin; Cardiodilatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin

Definitions

  • the present disclosure relates, in general, to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions comprising CNP variants and methods of use.
  • 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).
  • C-type natriuretic peptide (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)).
  • 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.
  • Downstream signaling mediated by cGMP generation influences a diverse array of biological processes that include endochondral ossification.
  • 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.
  • mice engineered to produce elevated levels of CNP display elongated long bones and vertebrae.
  • CNP22 CNP22
  • 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.
  • 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);
  • the variant peptide further comprises an acetyl group.
  • the acetyl group is on the N-terminus of the peptide.
  • the peptide further comprises an OH or an NH2 group at the C-terminus.
  • the variant peptide comprises a conjugate moiety.
  • the conjugate moiety is on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain.
  • the conjugate moiety is on a lysine residue.
  • the conjugate moiety comprises one or more acid moieties.
  • the acid moiety is a hydrophobic acid.
  • the hydrophilic spacer is gamma glutamic acid (yGlu) linked to one or two or more OEG (8- amino-3,6-dioxaoctanoic acid).
  • 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.
  • 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.
  • the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, arachidic acid, or diacids of the same.
  • the CNP variant has the structure:
  • the molecular weight of the CNP variant is 4962.83.
  • the CNP variant with the conjugate moiety is a component of modified release composition.
  • the modified release composition is an extended release composition.
  • 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.
  • (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.
  • the variant has the structure:
  • the variant comprises one or more linker groups.
  • 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.
  • the linker is a cleavable or lysable linker. In some instances of those embodiments, the linker is a hydrolysable linker.
  • the CNP variant is attached to the conjugate moiety via the linker.
  • the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety.
  • the linker is aminoethoxy-2-ethoxy acetic acid (AEEA).
  • 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.
  • the linker is an electronic linker based on nonenzymatic p-elimination.
  • the electronic linker comprises an SO 2 moiety. 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).
  • 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.
  • the conjugate moiety is a synthetic polymeric group.
  • the variant comprises a synthetic polymeric group coupled to the variant through a hydrolysable linker.
  • the synthetic polymeric group comprises a hydrophilic polymer moiety.
  • the hydrophilic polymer moiety comprises polyethylene glycol (PEG).
  • the hydrophilic polymer moiety comprises polyethylene glycol (PEG) having a 6 to 20 atom chain length.
  • the synthetic polymeric group is not a peptide.
  • the variant peptide is stable for 10 days at about 37°
  • 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.
  • 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.
  • 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.
  • 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-life is 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the variant peptide is conjugated to a lipid, fatty acid, hydrophilic spacer, or linker, or optionally combinations thereof.
  • the linker is a hydrophilic polymer moiety.
  • the hydrophilic polymer moiety is a synthetic hydrophilic polymer moiety.
  • 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 and higher AUC compared to free drug, e.g., an equivalent composition lacking an acid moiety, a spacer, and a hydrolysable linker.
  • the disclosure further provides a pharmaceutical composition
  • a pharmaceutical composition comprising a CNP variant described herein, and a pharmaceutically acceptable excipient, carrier or diluent.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CNP variant composition is supplied as a lyophilized powder for reconstitution from 1 mg to 300 mg.
  • 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).
  • 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).
  • the CNP variant composition is supplied as a 13 mg or 39 mg lyophilized, preservative-free powder for reconstitution.
  • formulations comprising (a) a CNP variant 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.
  • buffering agents employed in the formulations may be L-histidine, histidine monohydrochloride monohydrate, or a combination of the two.
  • isotonicity agents employed in the formulations of the present invention may be trehalose dihydrate, D-mannitol, or a combination of the two.
  • the stabilizer employed in the formulations of the present invention is L-methionine.
  • the anti-adsorbent agent employed in the formulations of the present invention is polysorbate 80.
  • the formulations of the present invention are lyophilized, in liquid form, or in liquid form that has been reconstituted from a previously lyophilized form.
  • the formulations of the present invention are preservative-free and, optionally, may be contained within a type 1 untreated borosilicate glass vial.
  • 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).
  • the disclosed formulations have a pH of about 5.5.
  • 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.
  • 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
  • the polysorbate 80 is present at a concentration between about 0.01 mg/ml and about 0.1 mg/ml.
  • 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
  • the polysorbate 80 is present at a concentration of about 0.05 mg/ml.
  • 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 concentration between 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
  • the polysorbate 80 is present at a concentration between about 0.01 mg/ml and about 0.1 mg/ml.
  • 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
  • the polysorbate 80 is present at a concentration of about 0.05 mg/ml.
  • the composition is an extended release composition.
  • 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 dysp
  • 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 dysp
  • the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other skeletal dysplasias.
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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, SHOX, NPR2, NPPC, or FGFR3.
  • the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.
  • the bone-related disorder, skeletal dysplasia or short stature disorder results from a RASopathy.
  • the RASopathy is Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1 , or LEOPARD syndrome.
  • the RASopathy is hereditary gingival fibromatosis type 1.
  • 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.
  • 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).
  • 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.
  • 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).
  • the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.
  • 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.
  • 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.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to - 3.0.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5.
  • 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.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5.
  • 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.
  • the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.
  • the short stature is a result of mutations in multiple genes as determined by polygenic risk score (PRS).
  • PRS polygenic risk score
  • the subject has a mutation in NPR2 and a low PRS.
  • the subject has a mutation in FGFR3 and a low PRS.
  • the subject has a mutation in NPR2 and a low PRS.
  • the subject has a mutation in IGF1R and a low PRS.
  • the subject has a mutation in NPPC and a low PRS.
  • the subject has a mutation in SHOX and a low PRS.
  • the subject has one or more mutation in one or more of FGFR3, IGF1R, NPPC, NPR2 and SHOX, and a low PRS.
  • the PRS is 1 or 2.
  • the PRS is 1.
  • 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.
  • the composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.
  • the composition provides an extended release composition.
  • 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.
  • the administration increases the annualized growth velocity (AGV) in the subject at 12 months, optionally compared to baseline or to a normal control.
  • the AGV in the subject increases over 1 year or over 2 years, or more.
  • the administration improves the height Z score at 12 months, optionally compared to baseline or to a normal control.
  • 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.
  • the composition is administered at a dose from about 5 ⁇ g/kg to 500 ⁇ g/kg or from about 15 ⁇ g/kg to 350 ⁇ g/kg .
  • the CNP Prodrug is administered at a dose from about 5 ⁇ g/kg to 500 ⁇ g/kg , from about 15 ⁇ g/kg to 350 ⁇ g/kg , from about 25 ⁇ g/kg to 300 ⁇ g/kg , from about 50 ⁇ g/kg to 250 ⁇ g/kg or from about 75 ⁇ g/kg to 200 ⁇ g/kg .
  • the CNP variant is administered at a dose of about 15 ⁇ g/kg , 20 ⁇ g/kg , 25 ⁇ g/kg , 30 ⁇ g/kg , 35 ⁇ g/kg , 40 ⁇ g/kg , 45 ⁇ g/kg , 50 ⁇ g/kg , 60 ⁇ g/kg , 70 ⁇ g/kg , 75 ⁇ g/kg , 80 ⁇ g/kg , 90 ⁇ g/kg , 100 ⁇ g/kg , 125 ⁇ g/kg , 150 ⁇ g/kg , 175 ⁇ g/kg , 200 ⁇ g/kg , 225 ⁇ g/kg , 250 ⁇ g/kg , 275 ⁇ g/kg , 300 ⁇ g/kg , 325 ⁇ g/kg , 350 ⁇ g/kg , 400 ⁇ g/kg , 450 ⁇ g/kg , or 500 ⁇ g/kg .
  • the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1).
  • 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 , and alkaline phosphatase.
  • CNP Collagen Type I C- Telopeptide
  • PCNA proliferating cell nuclear antigen
  • PINP propeptides of type I procollagen
  • aggrecan chondroitin sulfate collagen X
  • alkaline phosphatase alkaline phosphatase
  • 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 read
  • 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.
  • 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.
  • the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1).
  • the peptide further comprises an acetyl group.
  • the acetyl group is on the N-terminus of the peptide.
  • the acetyl group is on an amino acid side chain within the peptide sequence.
  • the peptide further comprises an OH or an NH2 group at the C-terminus.
  • the variant comprises one or more linker groups as described herein.
  • the linker is a hydrolysable linker.
  • the method comprises acetylating the peptides by reacting the resin with NMP/AC2O/DIEA (10:1:0.1, v/v/v).
  • 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.
  • 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.
  • Figure 2 shows the stability of CNP variants in human plasma over a period of 24 hrs.
  • Figure 3 shows the stability of CNP variants under different culture conditions.
  • 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.
  • Figure 5 shows the nucleotide and predicted protein sequence of the first exon in NPR2 mutant clones transfected into RCS cells.
  • Figure 6 shows exemplary NPR2 mutations analyzed for response to CNP.
  • Odds for ISS using PRS 1 non-carriers as reference vs having missense and/or loss of function variants in core genes.
  • Figure 10 shows the components of a CNP variant, including the CNP, lipid, and linker.
  • Figure 11 shows the structure and chemical name of a CNP variant disclosed herein.
  • Figure 12 shows a schematic of Phase 1 dosing study of CNP Prodrug in healthy volunteers.
  • Figure 13 shows visual appearance of CNP prodrug citrate-based formulations
  • 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.
  • Figure 15 shows free drug content release from different formulation buffers analyzed over time at a range of temperatures.
  • Figure 16 shows linker area of the drug conjugate when stored in different formulation buffers over time at a range of temperatures.
  • Figure 17 shows CNP conjugate content when stored in different formulation buffers was analyzed over time at a range of temperatures.
  • Figure 18 shows variant concentration as measured by UV when stored in different formulation buffers was analyzed over time.
  • Figure 19 shows variant content in formulations with different excipients (histidine, glycine and sorbitol) over time at pH 5.5 or 6.
  • Figure 20 shows results of a manufacturing feasibility study for CNP prodrug.
  • 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).
  • Figure 22 shows area under the curve after reverse phase chromatography.
  • 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.
  • Figure 24 shows visual comparison of CNP prodrug in different formulations.
  • Figure 25 shows naso-anal lengths in WT and Raf1 +/L613V mice after 6 weeks of treatment.
  • 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
  • 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.
  • Figure 28 shows plasma pharmacokinetics of CNP prodrug and released vosoritide in mice following a single SC dose.
  • Figure 29 shows mean ( ⁇ SD) plasma vosoritide and CNP prodrug concentrationtime profiles after a single dose of CNP prodrug.
  • 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.
  • 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.
  • 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.
  • Figure 33 shows mean ( ⁇ sd) plasma vosoritide and CNP prodrug concentrationtime comparing Day 1 vs Day 22.
  • Figure 34 shows binding of CNP Prodrug to NPR-B and NPR-C compared to BMN111.
  • the present disclosure relates to stable CNP variants useful in treating skeletal dysplasias and bone growth disorders.
  • 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.
  • C-type natriuretic peptide 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.
  • 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 residues of the NPPC polypeptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the compounds of the disclosure are substantially pure or isolated.
  • the compounds of the disclosure are substantially pure or isolated with respect to the macromolecular starting materials used in their production.
  • the pharmaceutical compositions of the disclosure comprise a substantially pure or isolated CNP variant admixed with one or more pharmaceutically acceptable excipients, carriers or diluents, and optionally with another biologically active agent.
  • Treatment refers to prophylactic treatment or therapeutic treatment or diagnostic treatment.
  • treatment refers to administration of a compound or composition to a subject for therapeutic, prophylactic or diagnostic purposes.
  • 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.
  • 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.
  • 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.
  • “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 I collagen (CTx), cross-linked N-telopeptide of type I collagen (
  • 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.
  • 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.
  • composition 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.
  • 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.
  • 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.
  • “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).
  • 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.
  • 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).
  • enteral e.g., oral
  • parenteral e.g., subcutaneous, intramuscular, intravenous or intraperitoneal injection; or topical, transdermal, or transmucosal administration.
  • 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.
  • metal salts e.g., sodium, potassium, magnesium, calcium, etc.
  • salts of ammonia or organic amines e.g., sodium, potassium, magnesium, calcium, etc.
  • pharmaceutically 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.
  • 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.
  • 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.
  • the term “subject” encompasses mammals and non-mammals.
  • 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.
  • non-mammals include, but are not limited to, birds, fish, and the like.
  • the term does not denote a particular age or gender.
  • the subject is human.
  • the subject is a child or adolescent.
  • the subject is an infant.
  • the subject is older than 3, older than 2, older than 1, or older than 6 months in age.
  • C-type natriuretic peptide (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)).
  • 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.
  • 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.
  • 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.
  • truncated CNP peptides include:
  • 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:
  • CNP variants of the disclosure include
  • the CNP variant further comprises an acetyl group.
  • the acetyl group is on the N-terminus, C-terminus or attached to an internal amino acid side group.
  • the acetyl group is on the N-terminus of the peptide.
  • the peptide variant further comprises an OH or an NH 2 group at the C-terminus.
  • the CNP variants are selected from the group consisting of: and
  • the CNP variants are selected from the group consisting of:
  • the CNP variants are selected from the group consisting of: and
  • the CNP variant is selected from the group consisting of Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO:8).
  • the CNP variant is Ac-PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1).
  • the CNP variant is PGQEH PNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-YGIU- C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 1).
  • 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.
  • the CNP variants are cyclized via formation of a disulfide bond between Cys 6 and Cys 22 as designated in the wtCNP22 peptide.
  • Cys 6 can be a cysteine analog such as, e.g., homocysteine or penicillamine.
  • 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.
  • 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).
  • 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.
  • 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.
  • p-nitrophenyl ester 2,4,5- trichlorophenyl ester, pentafluorophenyl ester
  • the azide method the mixed anhydride method
  • HATU e.g., a carbodimide (e.g., DIG, EDC or DCC) with a catalyst such as HOBt, HONSu or HOAt, or on- resin cyclization.
  • 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.
  • a lactam bridge i.e., a cyclic amide
  • a lactam bridge 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.
  • 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.
  • 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.
  • Enzyme-catalyzed cyclization can also be used.
  • 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
  • the antibody ligase 16G3 can be employed to cyclize a p- nitrophenylester.
  • 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 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.
  • 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).
  • 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.
  • 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.
  • 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-
  • 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.
  • the CNP variant is attached to the conjugate moiety via the linker.
  • the linker is attached to the conjugate moiety via the hydrophilic spacer of the conjugate moiety.
  • the linker is a hydrolysable linker.
  • 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.
  • 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.
  • bicin-type linkers include the structures as set out below: [0167]
  • 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.
  • the CNP variant is conjugated with a fatty acid, an amino acid, a spacer and a linker.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the conjugate moiety comprises an acid moiety linked to a hydrophilic spacer.
  • 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.
  • the hydrophilic spacer is any amino acid.
  • the hydrophilic spacer is gamma glutamic acid (yGlu).
  • the hydrophilic spacer is a substituted C-6 to C-20 alkyl chain.
  • 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.
  • 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.
  • yGlu gamma glutamic acid
  • the hydrophilic spacer is gamma glutamic acid (yGlu) linked to one or two OEG (8-amino-3,6- dioxaoctanoic acid) groups (diEG).
  • yGlu gamma glutamic acid
  • diEG 8-amino-3,6- dioxaoctanoic acid
  • the acid moiety and the hydrophilic spacer have the structure AEEA-AEEA-yGlu-d8DA.
  • the CNP variant has the structure: . In various embodiments, the CNP variant has the structure (SEQ ID NO: 1), or ). In various embodiments, the CNP variant comprises Asn to Glu variants of the above peptides.
  • 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).
  • 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).
  • 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).
  • 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
  • length e.g., from about 0.2, 0.4 or 0.6 k
  • the hydrophilic 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.
  • N-hydroxy succinimide NHS
  • 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.
  • the PEG (or PEO) moiety contains carboxyl group(s), sulfate group(s), and/or phosphate group(s).
  • 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.
  • PEG moieties contain one or more primary, secondary or tertiary amino groups, quaternary ammonium groups, and/or other amine- containing (e.g., urea) groups.
  • Contemplated herein is also a method of making a composition comprising a CNP variant and optionally a conjugate moiety as described herein.
  • the CNP variant is made synthetically using standard protein synthesis chemistry.
  • 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).
  • TFA tri-fluroacetic acid
  • RP-HPLC reverse phase high-performance liquid chromatography
  • the method further comprises, acetylating the peptides by reacting the resin with NMP/Ac 2 0/DIEA, optionally at 10:1:0.1, v/v/v.
  • 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.
  • the conjugate moiety comprises one or more lipids or fatty acids and a hydrophobic spacer.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the mutated form of FGFR-3 is constitutively active, which leads to severely shortened bones.
  • 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
  • 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, spondyloepiphyse
  • 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).
  • 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, spondyloepiphyse
  • 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.
  • the CNP variants of the disclosure are useful for treating mammals, including humans, suffering from a bone-related disorder, such as a skeletal dysplasia.
  • a bone-related disorder such as a skeletal dysplasia.
  • 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 congenit, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenital,
  • 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.
  • 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.
  • 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.
  • 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.
  • the CNP variants are useful as an adjunct or alternative to growth hormone for treating idiopathic short stature and other skeletal dysplasias.
  • 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.
  • 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.
  • 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 growth plate 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.
  • 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.
  • 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).
  • AMDM acromesomelic dysplasia type Maroteaux
  • NPR2 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.
  • 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.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between - 2.0 to -3.0.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5.
  • Exemplary NPR2 mutations in patients that may be treated with a CNP variant include:
  • NPPC neuropeptide
  • 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.
  • 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.
  • NPR-B natriuretic peptide receptor-type B
  • FGFR3 fibroblast growth factor receptor 3
  • 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.
  • LWD Leri-Weill dyschondrosteosis
  • SHOX short stature homeobox-containing gene or its regulatory elements located on the pseudoautosomal region 1 (PAR1) of the sex chromosomes.
  • 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.
  • 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.
  • 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 include Noonan syndrome, Costello syndrome, Cardiofaciocutaneous syndrome, Neurofibromatosis Type 1, LEOPARD syndrome, hereditary gingival fibromatosis type 1
  • RASopathy patients 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).
  • 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.
  • SOS guanine nucleotide exchange factor
  • Neurofibromatosis type I 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.
  • 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.
  • Noonan syndrome which is characterized by short stature, heart defects, bleeding problems, and skeletal malformations. 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.
  • 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.
  • GoF gain of function
  • 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).
  • 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.
  • 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.
  • IHH Indian hedgehog
  • IGF1R Insulin-like growth factor 1 receptor
  • a202 heterotetrameric 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).
  • 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.
  • 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.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -3.0.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of between -2.0 to -2.5.
  • the CNP variants are useful to treat a subject with short stature having a height SDS of -2.00 or less.
  • 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.
  • 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.
  • the growth plate disorder or short stature is associated with one or more mutations in a gene associated with a RASopathy.
  • 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).
  • PRS 1 being the lowest height, PRS 5 the tallest height.
  • the subject has a mutation in NPR2 and a low PRS.
  • the subject has a mutation in FGFR3 and a low PRS.
  • the subject has a mutation in NPR2 and a low PRS.
  • 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.
  • DTL denticleless E3 Ubiquitin Protein Ligase Homolog
  • PAPPA2 pregnancy-associated plasma protein A2
  • the PRS is 1 or 2. In various embodiments, the PRS is 1.
  • the PRS is 2.
  • 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)].
  • rickets including X-linked hypophosphatemic rickets (also called vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets)
  • osteomalacia including tumor-induced osteomalacia (also called oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)].
  • 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)).
  • 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.
  • 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.
  • 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”).
  • a “baseline” state can be determined either 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).
  • FGFR-3 constitutively active mutant fibroblast growth factor receptor 3
  • NPR-B natriuretic peptide receptor B
  • a method for increasing facial volume, facial sinus volume, and foramen magnum area in a subject comprising administering CNP variants as described herein.
  • 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 comprising administering CNP variants as described herein.
  • the increase in facial volume, facial sinus volume, and foramen magnum area are measured by magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the change in facial volume, facial sinus volume, and foramen magnum area are compared to baseline levels, healthy control subjects or untreated control subjects.
  • 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).
  • 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).
  • the CNP variant is any of the CNP variants described herein. It is contemplated that any of the CNP variants described herein are useful in the methods. [0222] In various embodiments, the CNP variant is
  • the peptide further comprises an acetyl group.
  • the acetyl group is on the N-terminus of the peptide.
  • the peptide further comprises an OH or an NH 2 group at the C-terminus.
  • the variant comprises one or more linker groups as described herein.
  • the linker is a hydrolysable linker.
  • 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).
  • 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).
  • 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.
  • CV cardiovascular
  • compositions including modified release compositions, comprising a CNP variant described herein, and one or more pharmaceutically acceptable excipients, carriers and/or diluents.
  • 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).
  • 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).
  • modified-release compositions such as extended release, sustained or controlled release, and delayed release.
  • 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.
  • the modified release composition is an extended release composition.
  • 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.
  • 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.
  • 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.
  • (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.
  • the composition comprises an excipient, diluent or carrier.
  • the extended release composition comprises an excipient, diluent or carrier.
  • the excipient, diluent or carrier is a pharmaceutically acceptable excipient, diluent or carrier.
  • excipients include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on.
  • 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).
  • buffer content e.g., Tris-HCI, phosphate, acetate buffers, citrate buffers
  • detergents and solubilizing agents e.g., Polysorbate 20, Polysorbate 80
  • anti-oxidants e.g., methionine, ascorbic acid, sodium metabisulfite
  • preservatives e.g
  • 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.
  • 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.
  • the compositions are liquid formulations.
  • the CNP variant is reconstituted from a lyophilized powder.
  • 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.
  • the CNP variant is in a concentration of 0.8 mg/ml to 2 mg/ml.
  • the CNP variant is at a concentration of 0.8 mg/ml.
  • 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.
  • 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.
  • 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.
  • 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.
  • the CNP variant is at a concentration of 30 mg/ml.
  • 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.
  • the CNP variant is in a concentration of 0.8 mg/ml to 2 mg/ml.
  • 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/m
  • the compositions comprise a buffer solution or buffering agent to maintain the pH of a CNP-containing solution or suspension within a desired range.
  • 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.
  • 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.
  • 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).
  • the p H is 5.5.
  • the compositions contain an isotonicity-adjusting agent to render the solution or suspension isotonic and more compatible for administration.
  • isotonicity agents include NaCI, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol.
  • the isotonicity agent is NaCI.
  • 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.
  • the compositions comprise a preservative.
  • Preservatives include, but are not limited to, m-cresol and benzyl alcohol.
  • 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%.
  • 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.
  • 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%.
  • the compositions comprise a stabilizer.
  • stabilizers include glycerin, glycerol, thioglycerol, methionine ((L- methionine), and ascorbic acid and salts thereof.
  • the stabilizer 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%.
  • the stabilizer 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%.
  • the stabilizer when the stabilizer is glycerin, the stabilizer is in a concentration from about 5% to about 100% (neat).
  • the compositions contain an antioxidant.
  • antioxidants include without limitation methionine and ascorbic acid.
  • 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.
  • compositions 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).
  • mineral acid salts e.g., hydrochloride, hydrobromide, phosphate, sulfate
  • organic acids e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate
  • salts of amines e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine.
  • 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.
  • 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.
  • excipients 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).
  • lubricating agents e.g., magnesium stearate, sodium stearyl fumarate.
  • flavoring, coloring and/or sweetening agents can be added to the solid and liquid formulations.
  • 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).
  • 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.
  • 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.
  • solvents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g
  • 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.
  • compositions comprising a CNP variant can also be lyophilized formulations.
  • 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).
  • 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%.
  • 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.
  • the lyophilized 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.
  • 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.
  • 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 the
  • the lyophilized formulation comprises 39 mg of the
  • 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.
  • the lyophilized formulation comprises 0.4 mg to 3.5 mg of the CNP variant.
  • the lyophilized formulation comprises 0.4 mg of the CNP variant.
  • the lyophilized formulation comprises 0.56 mg of the CNP variant.
  • 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,
  • 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,
  • the formulation comprises L-histidine, histidine monohydrochloride monohydrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).
  • the formulation comprises citric acid, sodium citrate, trehalose, mannitol, methionine, polysorbate 80, and optionally sterile water for injection (WFI).
  • 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.
  • 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.
  • a kit can include syringes having a single chamber or dual chambers.
  • 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.
  • a suitable solvent system e.g., glycerin
  • 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.
  • a pharmaceutically acceptable vehicle or carrier e.g., solvent system, solution or buffer
  • a suitable solvent system e.g., glycerin
  • 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 suitable solvent system e.g., glycerin
  • 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.
  • 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.
  • 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.
  • 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).
  • a depot at the target site of action (e.g., an abnormal or degenerated joint or cartilage area).
  • 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.
  • transdermal delivery e.g., by means of a patch on the skin
  • liposomes uncharged or charged (e.g., cationic)
  • polymeric microparticles e.g., polyamides, polylactide, polyglycolide, poly(lactide-glycolide)
  • 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 LD 50 (the dose lethal to 50% of the population) and the ED 50 (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.
  • 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.
  • 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.
  • 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 ⁇ g/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.
  • the CNP Prodrug is administered at a dose from about 5 ⁇ g/kg to 500 ⁇ g/kg , from about 15 ⁇ g/kg to 350 ⁇ g/kg , from about 25 ⁇ g/kg to 300 ⁇ g/kg , from about 50 ⁇ g/kg to 250 ⁇ g/kg or from about 75 ⁇ g/kg to 200 ⁇ g/kg .
  • the CNP variant is administered at a dose of about 15 ⁇ g/kg , 20 ⁇ g/kg , 25 ⁇ g/kg , 30 ⁇ g/kg , 35 ⁇ g/kg , 40 ⁇ g/kg , 45 ⁇ g/kg , 50 ⁇ g/kg , 60 ⁇ g/kg , 70 ⁇ g/kg , 75 ⁇ g/kg , 80 ⁇ g/kg , 90 ⁇ g/kg , 100 ⁇ g/kg , 125 ⁇ g/kg , 150 ⁇ g/kg , 175 ⁇ g/kg , 200 ⁇ g/kg , 225 ⁇ g/kg , 250 ⁇ g/kg , 275 ⁇ g/kg , 300 ⁇ g/kg , 325 ⁇ g/kg , 350 ⁇ g/kg , 400 ⁇ g/kg , 450 ⁇ g/kg , or 500 ⁇ g/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.
  • the CNP or CNP variant is administered daily subcutaneously.
  • the CNP or CNP variant is administered weekly subcutaneously.
  • the CNP variant is administered at a dose of 2.5 ⁇ g/kg/day to 60 ⁇ g/kg/day, 10 ⁇ g/kg /day to 45 ⁇ g/kg /day, or 15 ⁇ g/kg/day to 30 ⁇ g/kg/day.
  • the CNP variant is administered at a dose of 15 ⁇ g/kg/day.
  • the CNP variant is administered at a dose of 30 ⁇ g/kg/day.
  • the pharmaceutical composition is a sustained release composition.
  • 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.
  • 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.
  • 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.
  • the CNP variant is administered for 3 months, 6 months, 12 months or more.
  • 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).
  • the CNP composition may be administered subcutaneously or 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.
  • the initial period of treatment e.g., administration of the CNP variant composition daily or multiple times per week
  • the initial period of treatment 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.
  • the period of no treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks or 4 weeks.
  • 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.
  • 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).
  • CNP CNP
  • cGMP CGF
  • Collagen II Collagen II
  • Collagen X Collagen X
  • osteocalcin Proliferating Cell Nuclear Antigen
  • CNP signaling marker is cGMP (guanosine 3’, 5’ cyclic monophosphate).
  • 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.
  • Cartilage and bone-specific analytes can also be measured to assess CNP efficacy.
  • 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.
  • a bone-specific biomarker for bone formation which can be measured is N- terminal propeptides of type I procollagen (PINP).
  • PINP N- terminal propeptides of type I procollagen
  • 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.
  • propeptides are released from the procollagen molecule and can be detected in serum.
  • fragments of collagen type I can be measured as a marker for bone resorption.
  • 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/
  • 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.
  • 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.
  • 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
  • CNP e.g., endogenous levels of CNP
  • CNP collagen type II and fragments thereof
  • Cx collagen type I C-telopeptide
  • PCNA proliferating cell nuclear antigen
  • PINP propeptides of type I procollagen
  • collagen X aggrecan chondroitin sulfate
  • 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, 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
  • 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).
  • Collagen type X biomarker 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).
  • Bone-specific alkaline phosphatase 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.
  • a bone-specific biomarker for bone formation which can be measured is N-terminal propeptides of type I procollagen (PINP).
  • PINP N-terminal propeptides of type I procollagen
  • propeptides are released from the procollagen molecule and can be detected in serum.
  • fragments of collagen type I can be measured as a marker for bone resorption.
  • 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.
  • 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.
  • compositions and formulations comprising a CNP variant peptide, and one or more pharmaceutically acceptable excipients, carriers and/or diluents.
  • 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).
  • 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.
  • excipients include vehicles, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on.
  • 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).
  • buffer content e.g., Tris-HCI, phosphate, acetate buffers, citrate buffers
  • solubilizing agents e.g., Polysorbate 20, Polysorbate 80 (sometimes called PS 80)
  • anti-oxidants e.g., methionine, ascorbic acid, sodium metabisulfite
  • 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.
  • 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.
  • the compositions are liquid formulations.
  • 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.
  • the formulation may be a lyophilized formulation or may be a liquid formulation that was previously reconstituted from a lyophilized formulation.
  • compositions comprise a buffer solution or buffering agent to maintain the pH of a CNP-containing solution or suspension within a desired range.
  • 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.
  • 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.
  • the buffering agent has a concentration of about 5-15 mM (e.g., 10 mM + 5 mM).
  • 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.
  • the compositions contain an isotonicity agent to render the solution or suspension isotonic and more compatible for injection.
  • isotonicity agents include NaCI, trehalose, mannitol, dextrose, glucose, glycerin, sorbitol, xylitol, and ethanol.
  • the isotonicity agent is trehalose or mannitol, which can be employed individually or in combination.
  • 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 60 mM + 20 mM.
  • the ratio of trehalose to mannitol can be about 4:1, such as about 3:1 to about 5:1.
  • the compositions may comprise a preservative.
  • Preservatives include, but are not limited to, m-cresol and benzyl alcohol.
  • 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%.
  • the composition or formulation does not contain a preservative.
  • 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.
  • 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%.
  • the compositions comprise a stabilizer.
  • stabilizers include glycerin, glycerol, thioglycerol, methionine, and ascorbic acid and salts thereof.
  • the stabilizer 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%.
  • the compositions contain an antioxidant.
  • An exemplary anti-oxidant is without limitation ascorbic acid.
  • 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.
  • compositions 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).
  • mineral acid salts e.g., hydrochloride, hydrobromide, phosphate, sulfate
  • organic acids e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate
  • salts of amines e.g., isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine.
  • 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.
  • 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.
  • solvents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution
  • dispersing agents e.g
  • 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.
  • 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.
  • CNP variant peptides can be formulated in pharmaceutical carriers for administration to subjects affected by skeletal dysplasia.
  • liquid formulations of CNP variant peptides are formulated according to any combinations of the ingredients and their amounts or concentrations are described below:
  • compositions comprising a CNP variant peptide can also be lyophilized formulations.
  • 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).
  • 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%.
  • 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%.
  • 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:
  • 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.
  • a suitable buffering agent is acetic acid/acetate (e.g., sodium acetate)
  • a suitable buffering agent is citric acid/citrate.
  • Citric acid/citrate e.g., sodium citrate
  • 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.
  • 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.
  • 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
  • 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.
  • a CNP composition e.g., a liquid formulation or a lyophilized formulation
  • the variant can be formulated with one or more antioxidants.
  • 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.
  • polysorbate 20 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.
  • 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%.
  • a liquid e.g., sterile injectable
  • a solid e.g., lyophilized
  • 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.
  • pharmaceutically acceptable vehicles or carriers e.g., solvents, solutions and/or buffers
  • 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.
  • 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.
  • CNP C-type natriuretic peptide
  • 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-((S)-2-(2- ((S)-2-((S)-2-((S)-2-((S)-2-(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-aminohe
  • composition of embodiment 4, wherein the buffer comprises a buffer selected from citrate, acetate, phosphate, TRIS, and a combination thereof.
  • composition of embodiment 10 having a pH of 5.2 or 5.5.
  • composition of embodiment 12, wherein the bulking agent is selected from mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24), and a combination thereof.
  • composition of embodiment 12 or 13, wherein the bulking agent comprises trehalose or a solvate thereof, mannitol, or a combination thereof.
  • composition of embodiment 14, wherein the bulking agent comprises trehalose and mannitol in a weight ratio of 3:1 to 1 :1.
  • 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.
  • a tonicity agent selected from sodium chloride, dextrose, glucose, glycerin, sorbitol, xylitol, ethanol, and a combination thereof.
  • 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.
  • composition of any one of embodiments 1-22 comprising a stabilizing agent or surfactant selected from glycine, sorbitol, polysorbate, and a combination thereof.
  • 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.
  • 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-((S)-2-(2- ((S)-2-((S)-2-((S)-2-((S)-2-(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-amino
  • 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.
  • composition of embodiment 30, wherein the bulking agent comprises trehalose dihydrate.
  • composition of any one of embodiments 27-31 wherein pharmaceutically acceptable excipient comprises a bulking agent comprising mannitol.
  • 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)-1-(L- prolylglycyl- L-glutaminyl- L-glutamyl- L-histidyl)pyrrolidine-2-carboxamido)-4-amino-4- oxobutanamido)propanamido)-5-guanidinopentanamido)-6-aminohexanamido)-3
  • CNP C-type natriuretic
  • composition of any one of embodiments 39-41 , wherein the histidine buffer comprises L-histidine and histidine monohydrochloride monohydrate.
  • a pharmaceutical kit comprising the pharmaceutical composition of any one of embodiments 1-58.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • composition is administered subcutaneously, intradermally, intraarticularly, orally, or intramuscularly.
  • 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.
  • 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
  • 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 propeptid
  • Free Drug is sometimes used to refer to CNP that is not bound or conjugated to a linker and/or polymer.
  • 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.
  • Acetylation (Ac) of the peptide was performed by reacting the resin with NMP/AC 2 O/DIEA (10:1:0.1, v/v/v) for 30 min at room temperature.
  • CNP variants were generated based on the sequence of Pro-Gly CNP-37 (PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1)), optionally having changes in different amino acid residues, and/or acetylation at the N-terminus, and/or other modifications, and include (amino acid changes underlined):
  • CNP variants synthesized as in Example 1 were analyzed by mass spectrometry and UV spectroscopy to determine purity and stability after 10 days.
  • 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).
  • 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.
  • IBMX Enzo life sciences, 89161-340, 1g
  • 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.
  • 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.
  • CNP dilution media DMEM + 1x P/S + 0.5 mg/mL BSA
  • 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 ⁇ L of calibrator was added to wells of an anti-cGMP antibody coated plate, and 40 ⁇ L of lysate to be analyzed added to the appropriate wells. 40 ⁇ L of reconstituted rabbit anti-cGMP antibody was added to all wells and plates placed on shaker five minutes for mixing. 40 ⁇ L 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 ⁇ L wash buffer.
  • stoplight red substrate 100 ⁇ L 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.
  • 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
  • Variants CNP-R refers to CNP37 variants in which the K residues not in the ring portion are changed to R residues
  • 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.
  • 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.
  • 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.
  • RCS Rat chondrosarcoma
  • 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.
  • 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.
  • 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.
  • Trehalose : mannitol ratio of about 4:1 (e.g, 3:1 to 5:5)
  • Antioxidant in the buffer is not necessary, but an antioxidant is added in some case.
  • an antioxidant is added in some case.
  • L-methionine is particularly employed as an antioxidant.
  • PS 80 polysorbate 80
  • PS 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).
  • 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.
  • 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.
  • Lip-BC-CNP and Lip-BC-CNPox QCs as follows using citrate and inhibitors treated rat plasma K2EDTA as the diluent:
  • CNP Prodrug is comprised of CNP, a pH responsive self-immolative linker, spacer, and C-18 fatty acid albumin binding domain (attached to the Lys 27 side chain).
  • the sequence of the CNP Prodrug is as follows: Pro 1 -Gly-Gln-Glu-His 5 -Pro-Asn- Ala-Arg-Lys 10 -Tyr-Lys-Gly-Ala-Asn 15 -Lys-Lys-Gly-Leu-Ser 20 -Lys-Gly-Cys-Phe-Gly 25 -Leu- Lys 27 (X)-Leu-Asp-Arg 30 -lle-Gly-Ser-Met-Ser 35 -Gly-Leu-Gly-Cys-OH
  • the drug substance is isolated as a hydrochloride salt.
  • the empirical molecular formula is C 217 H 363 N 61 O 65 S 3 (as free base).
  • the average molecular mass is 4962.77 m.u.
  • 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 Lys 27 .
  • 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.
  • 2-CTC 2-chlorotrityl chloride
  • SPPS Solid-Phase Peptide Synthesis
  • TFA trifluoroacetic acid
  • 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.
  • 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 be obtained at intermediate condition of 5 ⁇ 3°C and accelerated condition of 25 ⁇ 2°C/60 ⁇ 5% relative humidity.
  • the DS target amount to weigh is determined using the following equation:
  • Figure 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.
  • Step 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:
  • the FB volume to be withheld is based on scale; the larger the scale the more volume is withheld.
  • Short-term 2-8 °C, no more than 3 days; and Long term: -60 °C or less, no more than 1 month.
  • 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 Product
  • JP 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
  • ERFE ethylene tetrafluoroethylene
  • JP Japanese Pharmacopoeia
  • Ph. Eur. European Pharmacopeia
  • USP United States Pharmacopeia.
  • 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.
  • NOAEL no observed adverse effect level
  • Table 15 Summary of Drug Product Samples for Nonclinical Studies and Clinical
  • DP drug product
  • DS drug substance
  • GMP Good Manufacturing Practices.
  • 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.
  • 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.
  • 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.
  • 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:
  • SPPS solid phase synthesis
  • RPC reverse phase chromatography
  • SPPS Manual solid phase synthesis
  • CTC chlorotrityl chloride
  • CNP Prodrug peptide 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.
  • SPPS Solid-Phase Peptide Synthesis
  • Fmoc A/-a-9-fluorenylmethoxycarbonyl
  • DS CNP Prodrug drug substance
  • 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.
  • 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.
  • CNP 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.
  • NLP non-human primate
  • NPR natriurel ic peptide receptor
  • PK pharmacokinetics
  • SD Sprague-Dawley
  • WT wild-type.
  • CNP Prodrug is comprised of CNP bound via the Lys 27 side chain to a pH responsive self-immolative linker, a spacer, and C-18 fatty acid albumin binding domain.
  • 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.
  • Metabolites A and B The structure of both inactive metabolite isoforms is shown below. Structure of Metabolite A
  • 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.
  • 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.
  • CYP enzymes e.g., CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 enzymes
  • 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.
  • 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.
  • 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.
  • CNP Prodrug 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.
  • CNP 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 ⁇ L/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.
  • cEstimated exposures are extrapolated from WT male mice administered 600 ⁇ g/kg CNP.
  • 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 ⁇ g/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 ⁇ g/kg QAD CNP Prodrug.
  • 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 ⁇ g/kg /day CNP, or CNP Prodrug at 500 or 1600 ⁇ g/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.
  • each group consisted of 10 male mice; a The dose volume for each animal was 5 mL/kg bodyweight.
  • b Vehicle 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.
  • c CNP 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.
  • 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 ⁇ g/kg /day at the distal tibia and calcaneus, generally similar to those noted for CNP.
  • Table 24-4 Test Article Related Radiographic Findings on Study Days 8, 15, and 20
  • 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 ⁇ g/kg/day CNP Prodrug or CNP compared to controls at either time point. Animals given 1600 ⁇ g/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.
  • mice 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 was noted at 500 ⁇ g/kg /day CNP and 500 or 1600 ⁇ g/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 ⁇ g/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.
  • Noonan Syndrome 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).
  • HCM hypertrophic cardiomyopathy
  • 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 J
  • Raf1 +/L613V mice 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).
  • CNP C-type natriuretic peptide
  • F female
  • M male
  • MEKi mitogen-activated protein kinase kinase inhibitor
  • IP intraperitoneal
  • SC subcutaneous
  • WT wild-type.
  • b Vehicle 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.
  • c CNP, 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.
  • d CNP 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.
  • e Vehicle 5% dimethyl sulfoxide (DMSO) in sterile 1X phosphate buffered saline.
  • MEKi 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.
  • DMSO dimethyl sulfoxide
  • 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.
  • Wild-type mice treated with 500 ⁇ g/kg /day CNP exhibited a 7.8% increase in naso-anal length and Raf1 +/L613V mice lengths increased 7.4% compared to vehicle-treated mice.
  • Both WT and Raf1 +/L613V mice treated with CNP Prodrug had significantly longer naso-anal lengths than mice treated with 500 ⁇ g/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.
  • Raf1 +/L613V mice 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 ⁇ g/kg /day CNP Prodrug or 500 ⁇ g/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 ⁇ g/kg /day CNP Prodrug compared to vehicle treated WT mice.
  • RIT1 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).
  • CNP 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.
  • b Vehicle 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.
  • c CNP, 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.
  • Naso-anal lengths were significantly increased from treatment and genotype- matched vehicle-treated controls for both 500 ⁇ g/kg/day CNP and 3280 ⁇ g/kg /dose QAD CNP Prodrug (Figure 26).
  • treatment with 3280 ⁇ g/kg /dose QAD CNP Prodrug resulted in significantly longer naso-anal lengths than animals treated with CNP.
  • 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.
  • 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 C-natriuretic peptide
  • M male
  • USP United States Pharmacopeia
  • w/v weight/volume. 1 each group consisted of 4 male monkeys a 1 mL/kg dose volume.
  • b 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.
  • c CNP 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.
  • 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.
  • 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 ]
  • type II collagen formation fragment of N-terminal type IIB procollagen [Pro C2]
  • 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.
  • CNP Prodrug was well-tolerated in male NHPs when given weekly at 30 or 150 ⁇ g/kg/week for a total of 5 doses over 1 month.
  • Pharmacologic activity ie, binding to NPR-B
  • results on growth plate cellularity and hypertrophic zone thickness were only observed in animals given 150 ⁇ g/kg /week CNP Prodrug, suggesting the 30 ⁇ g/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.
  • CNP C-type natriuretic peptide
  • CV cardiovascular
  • F female
  • M male
  • 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)
  • 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.
  • 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® Digital M11 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.
  • CNP Prodrug 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.
  • lipid-linker Metabolite B
  • CNP AUC and C max were similar in both dose groups, suggesting plasma exposure of CNP increased proportionally with dose between doses of 500 to 1600 ⁇ g/kg .
  • Percent metabolite ratio (released CNP/CNP Prodrug) at C max of was 0.07% for the 500 ⁇ g/kg dose and 0.1 % for the 1600 ⁇ g/kg dose.
  • Percent metabolite ratio of AUC of released CNP compared with CNP Prodrug was 0.06% for the 500 ⁇ g/kg dose and 0.08% for the1600 ⁇ g/kg dose.
  • CNP Prodrug was administered at 560 ⁇ g/kg IV and 150 ⁇ g/kg , 280 ⁇ g/kg , 560 ⁇ g/kg , or 1680 ⁇ g/kg SC. After an IV dose of CNP Prodrug, CNP was slowly released, reaching T max at 4 hours. Mean t1 ⁇ 2 of released CNP was
  • the CNP Prodrug mean t1 ⁇ 2 ranged from 6.72 hours for the 150 ⁇ g/kg SC dose to 10.5 hours for the 1680 ⁇ g/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 ⁇ g/kg to 1680 ⁇ g/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 T max was slightly delayed relative to CNP Prodrug (T max approximately 10-12 hours post-dose). The t1 ⁇ 2 for released CNP was similar to CNP Prodrug, ranging from 7.6 to
  • CNP Prodrug C max and AUC increased proportionally with increase in dose from dose range 280 ⁇ g/kg to 1680 ⁇ g/kg .
  • C max and AUC increased proportionally from dose 150 ⁇ g/kg to 280 ⁇ g/kg and 560 ⁇ g/kg to 1680 ⁇ g/kg .
  • CNP Prodrug was administered by weekly SC injection of 30 ⁇ g/kg /week or 150 ⁇ g/kg /week, based on CNP content, for 1 month. After SC administration, CNP Prodrug was slowly absorbed with a median T max of 24 hours after the first dose on Study Day 1 and 16 hours after multiple weekly doses on Study Day 22 at 30 ⁇ g/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 ⁇ g/kg /week dose.
  • CNP Prodrug Compared to rodents, CNP Prodrug had a longer mean t1 ⁇ 2 of 60 to 65.3 hours, which was consistent for both 30 ⁇ g/kg QW and 150 ⁇ g/kg QW dose levels. After 3 weeks of 30 ⁇ g/kg QW dosing, 2.09, 2.36, 3.37, and 3.15-fold accumulation of CNP Prodrug was observed in C max , AUC tlast , AUC 0- ⁇ and trough plasma concentration (Ctrough) at 168 hours, respectively.
  • Plasma concentrations of released CNP were not quantifiable for the majority of animals treated with 30 ⁇ g/kg QW on Study Days 1 and 22. Demonstrating a slow release of CNP, at 150 ⁇ g/kg QW, plasma concentrations of released CNP had a median T max of 8 hours after the first dose and 48 hours. After 3 weekly doses of 150 ⁇ g/kg QW, 1 .88, 2.62, 4.05 and 2.92-fold accumulation of released CNP in C max , AUC 0-t ,AUC 0- ⁇ and Ctrough at 168 hours, respectively, was observed.
  • t1 ⁇ 2 of CNP was similar to CNP Prodrug (65.3 hours on Study Day 1 and 63.8 hours on Study Day 22 at 150 ⁇ g/kg QW). Percent metabolite ratio of AUC 0-t was could only be computed for 150 ⁇ g/kg QW group. After a single dose, the metabolite ratio of AUC 0-t was 0.1 % and after three weekly doses, on Day 22, it was was 0.14%.
  • 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 ⁇ g/kg , 202 ⁇ g/kg , and 336 ⁇ g/kg CNP Prodrug. Sample analysis from this study is currently ongoing. Toxicokinetics are also being evaluated in the ongoing 26-week repeat-dose pharmacology and toxicology study in NHPs wherein animals are receiving weekly SC injections of 75 ⁇ g/kg /week, 200 ⁇ g/kg/week, and 350 ⁇ g/kg/week CNP Prodrug.
  • CNP C-type natriuretic peptide
  • CV cardiovascular
  • F female
  • M male
  • 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)
  • 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
  • the injection site (left scapular, Site 1) used for the 150 ⁇ g/kg CNP Prodrug dose was associated with mild granulomatous inflammation of the SC tissue in treated males.
  • the injection sites used for the 300 ⁇ g/kg CNP Prodrug dose (right scapular; Site 2) and 500 ⁇ g/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 marked severity at the localized injection site findings, corresponding to a dose of > 300 ⁇ g/kg CNP Prodrug were considered adverse under these study conditions.
  • the proliferative zone was not affected by CNP Prodrug treatment, as expected based on previous studies of CNP. These changes in the growth plate are likely mostly attributed to the 500 ⁇ g/kg dose of CNP Prodrug but may be a cumulative effect of the ascending doses of CNP Prodrug given in this study.
  • CNP Prodrug did not cause adverse systemic effects when given as a single ascending SC injections of 150, 300, or 500 ⁇ g/kg CNP Prodrug with a 14-day interval between each dose. Therefore, the NOAEL for systemic effects was identified as 500 ⁇ g/kg . Due to the severity of effects at injection sites 2 and 3, the localized NOAEL was determined to be 150 ⁇ g/kg . CNP has previously been shown to be mildly more irritating than vehicle at the injection site, so in this study, CNP Prodrug appears to be less tolerable at the SC injection site.
  • Toxicity was assessed by twice daily observations at cageside for mortality and gross toxicity, weekly detailed clinical observations, weekly body weights, daily qualitative food consumption, and clinical pathology (hematology and clinical chemistry) prior to each dose and prior to necropsy on Study Day 31. On Study Day 31, animals underwent a detailed necropsy. All 3 injection sites were collected and examined microscopically.
  • the 6-month duration was selected to allow for comparability to a similar previous study of CNP in juvenile NHPs.
  • a 10-month study of CNP was also conducted in NHPs, that study was conducted in older animals to assess impacts on sexual maturation and did not include extensive skeletal endpoints to evaluate pharmacologic effects.
  • the longer duration of the 10-month study in NHPs of CNP did not identify any unique effects related to CNP and the lower NOAEL reflected the older age and larger size of the animals that increased CNP plasma levels during the duration of the study rather than an impact of longer treatment.
  • animals Prior to study initiation, animals were implanted with DSI PhysioTel® Digital M11 telemetry implants, allowing for monitoring of blood pressure, ECG waveforms, and body temperature once pre-study and on Study Days 15, 57, 106, and 162 as well as during the recovery phase on Study Week 32.
  • the pre-study and recovery evaluations will be conducted for 24 hours, starting as close as possible to the expected time of monitoring during the dosing period.
  • animals During the dosing phase, animals will be monitored for 2 hours pre-dose and at least 24 hours post-dose.
  • Clinical pathology (hematology, coagulation, clinical chemistry, urinalysis, and urine chemistry) will be evaluated twice pre-study then on Study Days 4, 46, 81 , 123, 151 , and 184 of the dosing phase and during the recovery phase on Study Day 225.
  • Densitometry scans and assessments will be performed using DXA and peripheral quantitative computed tomography (pQCT). Dual energy X-ray absorptiometry scans of the whole body and right proximal femur will be conducted prior to study initiation, on Study Weeks 13/14 and 25/26 on all animals, and during the recovery phase during Study Week 32.
  • the right proximal tibia will be scanned by pQCT once prior to study initiation, on Study Weeks 13/14 and 25/26 on all animals and on Study Week 32 during the recovery phase.
  • Radiographs of bilateral femora, tibiae, radii and ulnae and the lumbar and thoracic spine will be conducted prior to study initiation, during Study Weeks 12/13 and 24/25 and during the recovery phase during Study Week 31. Radiographs will be evaluated qualitatively for abnormalities and for skeletal maturity. Tibia, femur, and ulna lengths (right and left) will be derived from the radiograph measurements. Toxicokinetic of CNP Prodrug and formed CNP will be evaluated on Study Days 1, 92, and 176 at 0, 8, 12, 24, 36, 48, 96, and 168 hours post-dose.
  • ANP Atrial natriuretic peptide
  • AUC 0-168 the area under the concentration time curve from time zero to 168 hours following dosing
  • AUC (0- ⁇ ) area under the concentration-time curve from time zero extrapolated to infinity
  • AUCtiast the area under the concentration versus time curve from the start of dose administration to the last observed quantifiable concentration
  • cGMP cyclic guanosine monophosphate
  • BAP Bone-specific Alkaline Phosphatase
  • BLQ Below the limit of quantitation
  • cGMP cyclic guanosine monophosphate
  • C max maximum observed concentration measured after dosing
  • CNP C-type natriuretic peptide (39 amino acid)
  • CTx-l Telopeptide of Type I collagen
  • CTx- II Telopeptide of Type II collagen
  • M male
  • NA Not applicable
  • NC Not calculated
  • Pro-C1 N terminal Pro-peptide of Type I collagen
  • CNP Prodrug is comprised of CNP bound via the Lys 27 side chain to a pH responsive self-immolative linker, a spacer, and C-18 fatty acid albumin binding domain.
  • 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.
  • Metabolite A is unstable and rapidly forms Metabolite B; therefore, the major metabolite isoform that is likely to be detected in plasma of both humans and nonclinical species is Metabolite B.
  • a hydrolysis assay of the CNP Prodrug and lipid linker in Phosphate buffered saline, pH 7.4 and 9.4 is conducted. Both Metabolites A and B are expected to be formed in vivo following hydrolysis of the bond to Lys 27 of active CNP. Formation would be based on the rate of vosoritide release from CNP Prodrug via hydrolysis of the linker at a constant rate (zero order kinetics) under physiological conditions
  • Metabolites A and B are expected to be formed 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, allowing the evaluation of toxicity of each to be incorporated into the general toxicity studies of CNP Prodrug, as recommended by FDA Guidance on Safety Testing of Drug Metabolites (March 2020).
  • An in silica screening assessment of Metabolites A and B will be conducted by means of evaluation by Derek (v. 6.3.0), Leadscope Model Applier (v. 2023-0.0.15), and Organisation for Economic Co-operation and Development (OECD) Quantitative Structure-activity Relationship (QSAR) Toolbox (v. 4.6).
  • OECD QSAR Toolbox will be utilized for the purposes of QSAR and read-across assessment of the metabolite isoforms.
  • Metabolite B will be evaluated by in vitro assays for mutagenicity and clastogenicity. Mutagenicity will be assessed in a non-GLP bacterial reverse mutation assay using S. typhimurium strains T98, TA100, TA1535, and TA1537 and E.coli strain WP2 uvrA using the plate incorporation method, with or without metabolic activation with rat liver post- mitochondrial fraction (S-9). Clastogenicity will be assessed in an in vitro micronucleus assay in human peripheral blood lymphocytes with or without metabolic activation with rat S- 9. Both assays will use internationally recognized and validated techniques in full accordance with the ICH Guideline S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use (June 2012).
  • CNP Prodrug treatment has the same primary pharmacodynamic effect as CNP: a dose-responsive increase in the hypertrophic zone of epiphyseal growth plates. As with CNP treatment, this effect on the growth plate leads to endochondral bone growth, as demonstrated by increased bone lengths and body length increases. As observed in the CNP nonclinical program, high doses, particularly in WT rodents, leads to exaggerated pharmacologic effects including overgrowth related DJD and/or fracture. These effects are currently being evaluated in a more translatable model to human patients, juvenile NHPs dosed weekly for 26 weeks.
  • Nonclinical PK studies have confirmed a long half-life in plasma in NHPs and a markedly shorter t1 ⁇ 2 in rodents, consistent with the expected effect on t1 ⁇ 2 with the albumin- binding lipid region of CNP Prodrug.
  • the low metabolite ratio and similar T max and t1 ⁇ 2 for released CNP to CNP Prodrug are consistent with the slow release of CNP from CNP Prodrug but an unchanged clearance mechanism for CNP.
  • a GLP ascending dose toxicity and CV safety pharmacology study of CNP Prodrug in NHPs has been conducted that identified a NOAEL for systemic toxicity of 500 ⁇ g/kg (based on CNP content). This study confirmed that with CNP Prodrug, the desired primary pharmacodynamics of CNP are retained; however, CNP related effects on hemodynamics (ie, reduce blood pressure and increased heart rate) are absent at doses up to 500 ⁇ g/kg CNP Prodrug in NHPs.
  • CNP Prodrug related changes at the injection site consisting of granulomatous inflammation at 150 ⁇ g/kg CNP Prodrug became adverse due to the presence of necrosis at single doses > 300 ⁇ g/kg CNP Prodrug in NHPs; however, the reproducibility of these findings is questionable given the absence of test article related injection site lesions in male NHPs dosed weekly with up to 150 ⁇ g/kg CNP Prodrug for 1 month and is being further investigated in the 6-month repeat-dose pharmacology and toxicology study in NHPs at doses up to 350 ⁇ g/kg QW prior to initiation of repeat-dose clinical studies.
  • the non-GLP study was designed to characterize the pharmacokinetic properties of CNP Prodrug and released vosoritide in adult wild type mice after a single subcutaneous (SC) dose of CNP Prodrug.
  • a total of 51 male C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME, USA) were 10 weeks old with body weights ranging from 20 to 30 grams at initiation of dosing. Animals were divided into 17 groups (3/group) where 8 groups were dosed 627 ⁇ g/kg (500 ⁇ g/kg target dose) CNP Prodrug (Groups A, C, E, G, J, L, N and Q), 8 groups were dosed 1891 ⁇ g/kg (1600 ⁇ g/kg target dose) CNP Prodrug (Groups B, D, F, H, K, M, P, and R), and 1 group (Group S) was untreated.
  • a terminal blood sample was collected via cardiac puncture from 3 mice/timepoint at 1 , 4, 8, 12, 24, 36, 48, or 60 hrs following subcutaneous administration of test article as specified by animal group. Samples were analyzed using UPLC-MS/MS analysis. LLOQ for vosoritide and CNP Prodrug was 50 and 2500 pM, respectively. For NCA analysis, the dose levels were converted from mass to molar units using the molecular weight (MW) of vosoritide (4102.78 g/mol). The design of the study is shown in Table 26.8. Table 26.8: Study Design of the Single Dose Pharmacokinetic Study of CNP Prodrug in
  • CNP C-natriuretic peptide
  • hr hour
  • M male
  • NA not applicable
  • PK pharmacokinetics
  • SC subcutaneous. 1 each group consisted of 3 male mice; a Doses are based on CNP content in CNP Prodrug.
  • FIG. 28 Shown in Figure 28 is plasma CNP Prodrug and released vosoritide concentrationtime profiles separated by Dose (A) and separated by analyte (B). Each plot shows a trendline of either CNP Prodrug or released vosoritide concentration in plasma and 95% confidence intervals (Cl) generated by the LOESS algorithm. Each data point represents the plasma concentration of CNP Prodrug and released vosoritide from a single mouse.
  • CNP Prodrug was slowly absorbed in plasma with median Tmax of 4-8 hrs following the 627 ⁇ g/kg dose and 4 hrs following the 1891 ⁇ g/kg dose.
  • CNP Prodrug showed a long mean t1 ⁇ 2 of approximately 10.5 hrs for both dose groups, likely due to albumin binding.
  • the dose-normalized area under the plasma concentration-time curve (AUC) and Cmax values were similar in both dose groups, suggesting that the plasma exposure of CNP Prodrug increased proportionally with dose over a range of 627 to 1891 ⁇ g/kg . This also suggests that the albumin binding was not saturated at the high dose of CNP Prodrug.
  • released vosoritide from CNP Prodrug had a longer mean t1 ⁇ 2 of 9.6 hrs for the 627 ⁇ g/kg dose and 9.9 hrs for the 1891 ⁇ g/kg dose compared to a t1 ⁇ 2 of 15 min following SC dose of vosoritide.
  • Dose-normalized plasma concentrations were similar in both groups.
  • Dose-normalized released vosoritide AUC and Cmax were similar in both dose groups, suggesting plasma exposure of vosoritide increased proportionally with dose between doses of 627 to 1891 ⁇ g/kg CNP Prodrug.
  • the vosoritide to CNP Prodrug ratio for Cmax was 0.07% for the 627 ⁇ g/kg dose and 0.1 % for the 1891 ⁇ g/kg dose.
  • the vosoritide to CNP Prodrug ratio for AUC was 0.06% for the 627 ⁇ g/kg dose and 0.08% for the 1891 ⁇ g/kg dose.
  • AUCtiast Area under the concentration-time curve from time zero to 60 hours by the linear trapezoidal method for increasing concentrations and log trapezoidal method; AUCtiast/Dose, The AUCtiast divided by pMol/kg of the dose administered; AUC (0- ⁇ ) , Area under the concentration-time curve from time zero to infinity following the last measurable concentration; AUC (0- ⁇ ) /Dose, The AUC 0- ⁇ divided by pMol/kg of the dose administered; AUC (0- ⁇ ) Ratio, Metabolite ratio of AUC (0- ⁇ ) of vosoritide to CNP Prodrug; C max , Maximum observed concentration; C max /Dose, The C max divided by pMol/kg of the dose administered; CL/F, Apparent clearance; C max Ratio, Metabolite ratio of C max of vosoritide to CNP Prodrug; CNP, C-type natriuretic peptide (39 amino acid); F, Bio
  • a total of 70 Sprague Dawley rats (35 males and 35 females; Charles River Labs International Inc., Springfield, NY, USA), were 9 weeks old with body weights ranging from 267 to 359 grams (males) and 193 and 237 grams (females) at study initiation and were divided into 7 groups (5 male and 5 female/group).
  • rats were administered a SC dose of 80 and 240 ⁇ g/kg of vosoritide, respectively.
  • rats were administered an IV dose of 560 ⁇ g/kg CNP Prodrug.
  • Groups 4 5, 6, and 7, rats were dosed SC at 150, 280, 560, and 1680 ⁇ g/kg CNP Prodrug, respectively.
  • plasma sampling for this study in rat was not terminal, which allows more timepoints to be assessed per animal, but less sample volume collected per timepoint.
  • the LLOQ of the assay for CNP Prodrug and CNP Prodrug (ox) was 250 pM. However, due to limited sample volume, a subset of samples was diluted resulting in an effective LLOQ 500 pM after 2-fold dilution. For PK analysis, non-oxidized and oxidized forms of CNP Prodrug and released vosoritide were combined at each time point.
  • CNP Prodrug T max was 8 hrs for all SC dose groups, indicating slow absorption into plasma following SC administration. Similar to the IV dose group, after SC dose, CNP Prodrug showed an extended mean t1 ⁇ 2 ranging from 6.72 to 10.5 hrs. Mean bioavailability of CNP Prodrug following SC administration was low, ranging from 17.67% to 21.71% across dose groups. No consistent effect of sex on bioavailability was observed.
  • T max was slightly delayed relative to CNP Prodrug (T max approximately 10-12 hrs).
  • the t1 ⁇ 2 for released vosoritide was similar to CNP Prodrug, ranging from 7.6- 12.8 hrs.
  • the long t1 ⁇ 2 for released vosoritide from CNP Prodrug compared to short t1 ⁇ 2 (0.113-0.203 hrs) groups administered SC vosoritide confirms that t1 ⁇ 2 for released vosoritide was determined by slow release rate of vosoritide instead of its clearance rate (flip-flop kinetics).
  • CNP Prodrug C max and AUC increased proportionally with dose from 280 ⁇ g/kg to 1680 ⁇ g/kg (Table 26.15).
  • mean C max and mean AUC increased proportionally with dose from 150 to 280 ⁇ g/kg and 560 to 1680 ⁇ g/kg but was similar for 280 and 560 ⁇ g/kg doses.
  • animals administered vosoritide C max increased with dose and a slightly higher but comparable increase in AUC was observed with increase in dose.
  • Percent metabolite ratio of released vosoritide AUC 0-t ranged from 0.294% to 0.494% and C max ranged from 0.312% to 0.725% after SC administration of CNP Prodrug.
  • Table 26.13 Summary Pharmacokinetic Parameters for CNP Prodrug after a Single IV or SC Dose in Rats [0531] Table 26.14: Summary of Dose-Normalized Exposures of Vosoritide and
  • the non-GLP study was designed to characterize the pharmacokinetic properties of CNP Prodrug and released vosoritide in NHPs after a single intravenous (IV) or SC dose of CNP Prodrug.
  • NHPs (12 male and 12 female; Charles River Laboratories, Laval, QC, Canada) were 22 to 36 months old with body weights ranging from 1.6 to 3.0 kg and were divided into 4 groups (3 males and 3 females per group).
  • NHPs were administered a single IV dose of 100.5 ⁇ g/kg CNP Prodrug and in Groups 2, 3, and 4, NHPs were administered a single SC dose of 102.3, 205.8, and 352.8 ⁇ g/kg CNP Prodrug, respectively.
  • These dose levels reflect ⁇ 70% of the intended nominal doses (which were 150, 150, 300, and 500 ⁇ g/kg , respectively) due to a formulation calculation error.
  • F:M AUC tlast ratios was 0.917 following the IV bolus administration. Following 102.3, 205.8, and 352.8 ⁇ g/kg SC administration, F:M C max ratios were 1.05, 1.59, and 1.73 and F:M AUC tlast ratios were 1.26, 1.66, and 2.13, respectively.

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Abstract

L'invention concerne des compositions pharmaceutiques comprenant des variants et des promédicaments de peptide natriurétique de type C (CNP) présentement divulgués. L'invention concerne également des méthodes d'utilisation des compositions présentement divulguées dans le traitement de troubles liés à l'os ou de la dysplasie squelettique, ainsi que des kits pharmaceutiques comprenant les compositions divulguées.
PCT/US2024/055072 2023-11-08 2024-11-08 Variants de cnp, conjugués et formulations de ceux-ci Pending WO2025101863A1 (fr)

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