RU2794515C2 - Application of c-type natriuretic peptide variants for the treatment of skeletal dysplasia - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to pharmaceutical compositions containing CNP variant peptides for the treatment of skeletal dysplasias, one or more symptoms of skeletal dysplasias such as long bone growth or growth rate, and other disorders having skeletal dysplasia and/or a CNP-associated symptom or component.

EFFECT: obtaining a method of treatment of skeletal dysplasia.

45 cl, 1 tbl, 2 ex

Description

disclosure area

[0001] The disclosure generally relates to the use of C-type natriuretic peptide (CNP) variants for the treatment of skeletal dysplasia and to the peptide formulations of the CNP variants.

Disclosure background

[0002] The natriuretic peptide family consists of three structurally related peptides: atrial natriuretic peptide (ANP) (GenBank accession number NP_006163, for ANP precursor protein, NPPA), brain natriuretic peptide (BNP) (GenBank accession number NP_002512, for protein- BNP precursor, NPPB) and C-type natriuretic peptide (CNP) (Biochem. Biophys. Res. Commun., 168: 863-870 (1990) (GenBank accession number NP_077720, for CNP precursor protein, NPPC) (J. Hypertens ., 10: 907-912 (1992)).These small single chain peptides (ANP, BNP, CNP) have a 17-amino acid loop structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and play important roles in many biological processes.ANP and BNP bind to and activate natriuretic peptide receptor A (NPR-A), also called guanyl cyclase A (GC-A), leading to higher levels of intracellular cycling. guanosine monophosphate (cGMP) Similarly, CNP interacts with NPR-B (GC-B) to stimulate the formation of cGMP (J. Hypertens., 10: 1111-1114 (1992)). A third type receptor, NPR-C, binds each of the natriuretic peptides with high affinity and primarily functions to capture peptides from the extracellular compartment and direct the peptides to lysosomes where they are degraded (Science, 238: 675-678 (1987)) . ANP and BNP are primarily formed in cardiac muscle cells and are thought to play important roles in cardiovascular homeostasis (Science, 252: 120-123 (1991)). CNP is expressed more widely, including in the central nervous system, reproductive tract, bone, and endothelium of blood vessels (Hypertension, 49: 419-426 (2007)).

[0003] In humans, CNP is originally derived from the natriuretic peptide C (NPPC) precursor gene as a single chain 126-amino acid prepropolypeptide (Biochem. Biophys. Res. Commun., 168: 863-870 (1990)). Removal of the signal peptide yields pro-CNP, and further cleavage by the furin endoprotease creates an active 53-amino acid peptide (CNP-53), which is secreted and cleaved again to form 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 being predominant in tissues while CNP-22 is predominantly found in plasma and cerebrospinal fluid (J. Alfonzo, Recept. Signal. Transduct. Res., 26: 269-297 (2006)). The predominant form of CNP in cartilage is unknown. Both CNP-53 and CNP-22 bind similarly to NPR-B. In addition, both of them induce the formation of cGMP in a dose-dependent and similar manner (VT Yeung, Peptides, 17: 101-106 (1996)).

[0004] Natural CNP genes and polypeptides have been previously described. US Pat. No. 5,352,770 discloses isolated and purified CNP-22 sequence-identical to human CNP from porcine brain and its use in the treatment of cardiovascular indications. US Pat. No. 6,034,231 discloses a human proCNP gene and polypeptide (126 amino acids) and a human CNP-53 gene and polypeptide.

[0005] Clearance of CNP from the extracellular space occurs through the action of membrane-bound neutral endopeptidase (NEP), which rapidly degrades CNP (Biochem. J., 291 (Pt 1): 83-88 (1993)), and through NPR-C, which binds with CNP and directs it to lysosomes, where the destruction of CNP occurs. In a normal human, CNP has been shown to have an in vivo half-life of 2.6 minutes (J. Clin. Endocrinol. Metab., 78: 1428-35 (1994)). The low plasma concentration of CNP (J. Bone Moner. Res., 19 (Suppl. l) S20 (2004)) and its co-expression with NPR-B in many tissues suggests that CNP acts primarily through an autocrine/paracrine mechanism.

[0006] As discussed above, CNP binds to and activates the natriuretic peptide receptor B (NPR-B), also called guanylyl cyclase B (GC-B), leading to higher intracellular levels of cyclic guanosine monophosphate (cGMP). Downstream signaling mediated by cGMP formation affects a number of diverse biological processes that include endochondral ossification. Accordingly, increased or decreased levels of any of the components in this pathway may lead to aberrant bone or cartilage growth. For example, knocking out CNP or NPR-B in mouse models leads to animals that have a growth retardation phenotype with shorter long bones and vertebrae. Mutations in human NPR-B have been identified that block proper CNP signaling that lead to dwarfism (Olney, et al., J. Clin. Endocrinol. Metab. 91(4): 1229-1232 (2006); Bartels, et al. Am J Hum Genet 75: 27-34 (2004)). In contrast, mice engineered to produce elevated levels of CNP show elongated long bones and vertebrae.

[0007] Achondroplasia is the result of an autosomal dominant mutation in the fibroblast growth factor receptor 3 (FGFR-3) gene that causes abnormal cartilage formation. FGFR-3 generally has a negative regulatory effect on chondrocyte growth and thus bone growth. In achondroplasia, the FGFR-3 mutant is constitutively active, leading to severely shortened bones. It appears that both proliferation and differentiation of chondrocytes are impaired, leading to cartilage with a remarkably short growth plate (P. Krejci et al., J. Cell Sci. 118: 5089-5100 (2005)). Endochondral ossification is a process that controls the longitudinal growth of long bones. There are four zones of the growth plate - zone of rest, proliferation, hypertrophy and calcification. In the growth plate, NPR-B express proliferative cells while NPR-C express hypertrophic cells (Yamashite et al., J. Biochem. 127: 177-179 (2000)). During normal endochondral bone growth, chondrocytes form columns and proliferate in the zone of proliferation of the growth plate. These columns are disorganized in patients with achondroplasia. Additionally, the zone of hypertrophy is where cells become large and eventually undergo apoptosis (lysis), leading to osteocyte invasion and mineralization. Hypertrophic chondrocytes and the overall size of the zone in patients with achondroplasia are significantly smaller than in normal patients. CNP is an NPR-B agonist, a positive regulator of chondrocyte and bone growth. Downstream CNP/NPR-B signaling inhibits the FGFR-3 pathway at the level of mitogen-activated protein kinase (MAP K). Inhibition of MAP K promotes proliferation and differentiation of chondrocytes in areas of proliferation and hypertrophy in the growth plate, leading to bone growth.

[0008] In humans, FGFR-3 activating mutations are the first cause of genetic dwarfism. Mice with activated FGFR-3 serve as a model for achondroplasia, the most common form of skeletal dysplasia, and CNP overexpression saves these animals from dwarfism. Accordingly, CNPs and CNP functional variants are potential therapeutic agents for the treatment of various skeletal dysplasias.

[0009] The therapeutic use of CNP is currently limited by its short plasma half-life, which has been shown to be 2.6 minutes in vivo in humans (J Clin. Endocrinol. Metab., 78: 1428-35 (1994)). To increase CNP concentrations above innate levels (approximately 5 pM) normally found in human plasma, continuous infusion was required in all human and animal studies using systemically administered CNP. The half-life of CNP in human plasma is reduced by two mechanisms, which are degradation by neutral endopeptidase (NEP) and clearance by natriuretic peptide receptor C (NPR-C) (Growth Horm. & IGF Res., 16: S6-S14 (2006 )). A CNP variant that has a longer in vivo serum half-life and exhibits similar or improved activity relative to that of wild-type CNPs is important for a stable therapeutic strategy.

[0010] The biological activities of various CNP analogues and derivatives were evaluated. See, for example, US Pat. No. 7,276,481, PCT Publication No. WO 94/20534, which discloses a chimera of CNP-22 and the C-terminus of a 5-amino acid ANP, referred to as the peptide vasonathrin (VNP). US Pat. Nos. 8,198,242 and 8,598,121 disclose the use of CNP variants for the treatment of skeletal dysplasias such as achondroplasia. US Pat. Nos. 7,642,243 and 8,658,373 describe the use of CNP-22 or CNP-53 variants for the treatment of arthritis.

Brief disclosure

[0011] The present invention relates to the use of CNP variant peptides in order to treat skeletal dysplasias, to treat one or more symptoms associated with skeletal dysplasia, or to improve one or more consequences or physiological symptoms of skeletal dysplasia in a subject suffering from it, when the variant peptide CNPs are administered above a certain dose amount and/or at a certain dosage regimen, as described herein. It is disclosed herein that administration of CNP variant peptides can lead to improved growth rate in subjects with achondroplasia. In various embodiments, the CNP variant peptide is CNP-38 or Pro-Gly-CNP-37.

[0012] In various embodiments, the disclosure provides a method for treating skeletal dysplasia, which comprises administering a composition comprising a CNP variant peptide to a subject in need thereof in an amount of at least 7.5 μg/kg, wherein the administration treats skeletal dysplasia or improves at least one symptom or physiological consequence of skeletal dysplasia.

[0013] In one embodiment, the treatment is an improvement in one or more symptoms of skeletal dysplasia selected from the group consisting of increased absolute height, improved or increased growth rate, increased bone mineral density (BMD) QCT, improved growth plate morphology, increased long bone growth, improved spinal morphology, improved or increased elbow range of motion, and reduced sleep apnea.

[0014] In one embodiment, the skeletal dysplasia is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, campomelic dysplasia at birth, campomelic dysplasia at birth , osteogenesis imperfecta of the perinatal lethal type, syndromes of short ribs-polydactyly, hypochondroplasia, punctate chondrodysplasia of the rhizomelic type, Jansen's metaphyseal dysplasia, congenital spondyloepiphyseal dysplasia, atelosteogenesis, diastrophic dysplasia, congenital short femur, mesomelic dysplasia of Langer, mesomeliac dystaplasia syndrome Reinhardt, acrodysostosis, peripheral dysostosis, Knist dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Knist syndrome, metatrophic dysplasia and spondyloepimetaphyseal dysplasia. In a preferred embodiment, the skeletal dysplasia is achondroplasia.

[0015] A method of increasing long bone growth in a subject is also provided, which comprises administering a composition comprising a CNP variant peptide to a subject in need in an amount of at least 7.5 μg/kg, wherein the administration increases long bone growth. In a preferred embodiment, the subject has achondroplasia.

[0016] The disclosure also contemplates a method of increasing or increasing the growth rate in a subject, which comprises administering a composition comprising a CNP variant peptide to a subject in need thereof in an amount of at least 7.5 μg/kg, wherein the administration increases or increases the growth rate in subject. In a preferred embodiment, the subject has achondroplasia. In various embodiments, the increase or increase in growth rate is an increase in annual growth rate in the range of 25%-50% change from baseline in the subject. In one embodiment, the increase or increase in growth rate is an increase in annual growth rate of at least about 25%, more preferably at least about 40% change from baseline in the subject.

[0017] It is contemplated that an increase in growth rate can be assessed by measuring standing height, sitting height, weight, head circumference, shoulder length, forearm length, thigh length, calf length, hand length, and/or foot length.

[0018] In the various methods and compositions described herein, the CNP variant peptide may be selected from the group consisting of:

QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1];

MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2);

PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3);

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID No. 4];

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5);

MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37 or CNP-38: SEQ ID No. 7).

[0019] In various preferred embodiments, the CNP variant peptide is CNP-38 or Pro-Gly-CNP-37.

[0020] In the various methods described herein, the amount of CNP variant peptide administered is in the range of about 7.5 µg/kg to about 100 µg/kg, preferably about 7.5 µg/kg to about 80 µg/kg , more preferably from about 7.5 µg/kg to about 60 µg/kg. In various embodiments, the amount of CNP variant peptide administered is at least about 7.5 μg/kg, at least about 15 μg/kg, at least about 30 μg/kg, or at least about 60 μg/kg.

[0021] In the various methods described herein, the CNP variant peptide, or a composition or formulation containing it, is administered subcutaneously or parenterally, preferably subcutaneously. It is also contemplated that the CNP variant peptide is administered via other routes. Exemplary routes of administration include, but are not limited to, subcutaneous, intraarticular, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, intrathecal, intraperitoneal, intramuscular, intradermal, intrathecal, topical, transdermal, or transmucosal administration.

[0022] In the various methods described herein, a CNP variant peptide, or a composition or formulation containing it, is administered to a subject in a single treatment or multiple doses. Multiple doses may be administered once daily or in multiple doses over the course of treatment. In various embodiments, it is provided that the CNP variant peptide or composition or composition containing it is administered, in a single dose or in multiple doses, daily, every other day, every 3 days, 2 times a week, 3 times a week, weekly, once a 2 weeks, every 3 weeks, monthly, every 6 weeks, every 2 months, every 3 months, or as deemed appropriate by the attending physician. In particularly preferred embodiments, the CNP variant peptide, or a composition or formulation comprising it, is administered to a subject once daily for a period of at least one month, two months, three months, four months, five months, six months, seven months, eight months. , nine months, ten months, eleven months, twelve months or longer.

[0023] In certain embodiments of the methods described herein, the administration of a CNP variant peptide or a composition or formulation containing it is adjusted to allow periods of preventive or therapeutic treatment followed by a recovery period. For example, a CNP variant peptide or a composition or formulation containing it can be administered intra-articularly, subcutaneously, intravenously, or otherwise daily or several times a week for a period of time, followed by a period without treatment, then the cycle is repeated. In some embodiments, the initial treatment period (e.g., administration of the CNP variant peptide daily or several times a week) is 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related embodiment, the no-treatment period is 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the CNP variant peptide dosing regimen is daily for 3 days followed by 3 days of withdrawal; or daily or several times a week for 1 week, followed by 3 days or 1 week of withdrawal; or daily or several times a week for 2 weeks, followed by 1 or 2 weeks of withdrawal; or daily or several times a week for 3 weeks, followed by 1, 2, or 3 weeks of withdrawal; or daily or several times a week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, followed by 1, 2, 3, or 4 weeks of withdrawal.

[0024] In the various methods described herein, it is preferred that administration of a CNP variant peptide or a composition or formulation containing it does not cause or lead to an adverse event of magnitude two or higher in the subject. In other embodiments of the methods described herein, administration of a CNP variant peptide or a composition or composition containing it does not lead to a clinically significant change in blood hemoglobin concentration, blood platelet count, blood electrolyte concentration, blood urea nitrogen concentration, blood creatinine concentration, blood alkaline phosphatase concentration, blood alanine aminotransferase concentration and/or blood aspartate aminotransferase concentration in said subject.

[0025] In the various methods described herein, in preferred embodiments, administration of a CNP variant peptide, or a composition or formulation containing it, results in a change in (i) the ratio of upper body length to lower body length, (ii) the length ratio upper arm and forearm length or (iii) a thigh to calf length ratio between about -0.25 and about 0.25, about -0.20 and about 0.20, about -0.15 and about 0.15, about - 0.10 and about 0.10, or about -0.05 and about 0.05, compared to baseline (i.e., prior to administration of the CNP variant peptide or composition or formulation containing it).

[0026] In various embodiments, the disclosure provides for compositions or formulations comprising a CNP variant peptide, or the use of a composition or formulation comprising a CNP variant peptide in the methods described herein. In one embodiment, the composition or formulation further comprises a pharmaceutically acceptable excipient, carrier, or diluent. In certain embodiments, the composition is prepared from a formulation, liquid or lyophilized, that contains a citric acid/citrate buffer or an acetic acid/acetate buffer having a pH of about 4 to about 6. In various embodiments, the pH is about 5.5.

[0027] Also provided is a method of treatment as disclosed herein, which further comprises administering a second agent.

[0028] In various embodiments, CNP variant peptides used in the methods and compositions or compositions described herein can be attached to a hydrophobic acid and can be attached to one or more hydrophobic acids. Non-limiting examples of hydrophobic acids include linear or branched, saturated or unsaturated C ₅ -C ₁₂ carboxylic acids (eg, valeric acid, heptanoic acid, etc.) and natural fatty acids. Hydrophobic acids can be attached to the N-terminus, C-terminus and/or side chain of one or more amino acid residues. In one embodiment, hydrophobic acids are conjugated to the N-terminus.

[0029] In yet another embodiment, the CNP variant peptides used in the methods and compositions of the present invention are chimeric or fusion proteins that contain a CNP variant peptide and a cleavable peptide or protein or peptide tag. Exemplary cleavable proteins or peptides include, but are not limited to, histidine tags (eg, 6-His); TAF12: human transcription factor TAF12; KSI: ketosteroid isomerase; MBP: maltose-binding protein; β-Gal: β-galactosidase; GST: glutathione-S-transferase; Trx: thioredoxin; CBD: chitin binding domain; BMPM: BMP-2 mutation, SUMO, CAT, TrpE, staphylococcal protein A, streptococcal proteins, starch binding protein, endoglucanase A cellulose binding domain, Cex exoglucanase cellulose binding domain, biotin binding domain, recA, Flag, c-Myc, poly(His), poly (Arg), poly(Asp), poly(Gln), poly(Phe), poly(Cys), green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, biotin, avidin, streptavidin, antibody epitopes and their fragments.

[0030] In various embodiments described herein, the CNP variant peptide may be a monomer or a dimer. In a related embodiment, CNP variant dimeric peptide monomers can be attached N-terminus to N-terminus with or without a linker, N-terminus to C-terminus with or without a linker, or C-terminus to C-terminus with or without a linker.

[0031] In any of the embodiments described herein, CNP variant peptides may have substantially the same or improved biological activity as wild-type CNP-22. For example, CNP variant peptides may retain at least 50%, 60%, 70%, 80%, 90%, 95% or more wild-type CNP-22 activity, or may have higher activity than CNP-22, for example, in in relation to interaction with NPR-B (GC-B) to stimulate the formation of cGMP. Alternatively, or in addition, CNP variant peptides may retain at least 50%, 60%, 70%, 80%, 90%, 95% or more wild-type CNP-22 activity, or may have higher activity than CNP-22. , with respect to the regulation of endochondral bone growth and chondrocyte activity, including but not limited to chondrocyte proliferation, chondrocyte differentiation, inhibition of the mitogen-activated protein (MAP) kinase/MEK kinase (Raf-1) signal transduction pathway, and promotion of endochondral ossification. In any of the embodiments described herein, CNP variant peptides may comprise an amino acid sequence that is at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or more identical or homologous to amino acids. 6-22 or 1-22 from wild-type CNP-22.

[0032] In various embodiments, CNP variant peptides may optionally have conjugate(s) or extension(s), e.g., at the N- and/or C-terminus, in order to promote cartilage targeting, reduce renal clearance, and/ or increase resistance to NEP degradation. Such conjugate(s) or extension(s) may contain molecules or sequences formed or derived from, for example, poly Asp, poly Glu, cartilage targeting peptides, sialoprotein, PEG, carbohydrates, hydrophobic acids, (poly)peptides from NPPC or not CNP, or combinations thereof.

[0033] In addition, it is contemplated that CNP variant peptides can be conjugated to a hydrophobic polymeric or non-polymeric moiety, such as, for example, heptanoic acid, valeric acid, or fatty acids. The hydrophobic moiety can be conjugated to the side chain of an amino acid residue, including but not limited to lysine, serine, cysteine, or threonine, or can be attached to the N-terminus and/or C-terminus of the CNP variant.

[0034] In various embodiments, CNP variant peptides that can be used in the methods have a pI in the range of about 8 to about 10.5, or about 8.5 to about 10.

[0035] In various embodiments, the disclosure provides for the use of a pharmaceutical composition comprising a CNP variant peptide, optionally, another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier, or diluent. In various embodiments, the compositions are sterile pharmaceutical compositions suitable for parenteral injection. In some embodiments, the compositions comprise a substantially pure CNP variant peptide, such as at least about 90% or 95% pure. In some embodiments, the compositions contain less than about 5%, 4%, 3%, 2%, 1%, or 0.5% impurities such as other human proteins, porcine proteins, or CNP-53 or fragments thereof (other than the desired peptide CNP variant). In various embodiments, the sterile composition is administered to a subject to treat or prevent skeletal dysplasia or one or more of the symptoms or physiological consequences of skeletal dysplasia described herein.

[0036] CNP variant peptides that can be used herein favorably retain CNP activity and exhibit increased serum half-life. Retention of CNP activity can be shown, for example, as retention of the desired biological effect in vivo, or retention of at least about 50%, 60%, 70%, 80%, 90%, 95%, or at least about 100% of cGMP stimulatory activity in CNPs. -22, at the same concentration (eg, 1 μM CNP peptide or greater than ED ₈₀ ). In some embodiments, CNP variant peptides exhibit at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold or 40-fold increase in serum half-life compared to CNP-22.

[0037] In a related embodiment, the CNP variant peptides described herein have increased NEP resistance and exhibit an increased half-life compared to wild-type CNP-22. In one embodiment, the half-life of CNP variant peptides is increased by approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99 % or approximately 100% compared to wild-type CNP-22.

[0038] Also contemplated is the use of any of the above CNP variant peptides or a composition or formulation containing it described herein in the preparation of a medicament for the treatment of skeletal dysplasia and the symptoms or other physiological manifestations described herein associated with skeletal dysplasia. Also provided are syringes, e.g., disposable or pre-filled syringes, sterile sealed containers, e.g., vials, bottle, vial, and/or kits or packages containing any of the above CNP variant peptides or a composition or formulation containing it, optionally with appropriate instructions for use.

[0039] Also provided herein are compositions 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-adsorption agent. . In particularly preferred embodiments, the buffering agents used in the formulations may be citric acid monohydrate, sodium citrate dihydrate, or a combination of the two. In other preferred embodiments, the isotonicity adjusting agents used in the compositions of the present invention may be trehalose dihydrate, D-mannitol, or a combination of the two. In other preferred embodiments, the stabilizer used in the compositions of the present invention is L-methionine. In other preferred embodiments, the anti-adsorption agent used in the compositions of the present invention is polysorbate 80.

[0040] In various embodiments, the formulations of the present invention are lyophilized, liquid form, or liquid form that is reconstituted from a pre-lyophilized form. In certain embodiments, the compositions of the present invention do not contain preservatives and optionally can be contained in an untreated Type 1 borosilicate glass jar. Optionally, the compositions of the present invention have a pH in the range between about 5.0 and about 6.0, preferably about 5, 5.

[0041] In other embodiments, the formulations of the present invention comprise a CNP variant peptide at a concentration of at least about 2.0 mg/mL, 5.0 mg/mL, 10.0 mg/mL, or greater. In a particularly preferred embodiment, the formulation of the present invention contains a CNP variant peptide at a concentration of about 10.0 mg/mL. In particularly preferred embodiments, the CNP variant peptide of the formulation is CNP-38 or Pro-Gly-CNP-37.

[0042] In other embodiments, the formulations of the present invention comprise a CNP variant peptide as disclosed herein, citric acid monohydrate, sodium citrate dihydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate 80. In certain preferred embodiments, the peptide variant CNP is present at a concentration between about 2.0 mg/ml and about 10.0 mg/ml, citric acid monohydrate is present at a concentration between about 0.15 mg/ml and about 0.40 mg/ml, sodium citrate dihydrate is present in concentrations between about 0.5 mg/ml and about 1.5 mg/ml, trehalose dihydrate is present at a concentration between about 30 mg/ml and about 70 mg/ml, D-mannitol is present at a concentration between about 10 mg/ml and about 20.0 mg/ml, L-methionine is present at a concentration between about 0.5 mg/ml and about 1.5 mg/ml and Polysorbate 80 is present at a concentration between about 0.01 mg/ml and about 0.1 mg/ml ml. In a particularly preferred embodiment, the CNP variant is present at a concentration of approximately 10.0 mg/mL, citric acid monohydrate is present at a concentration of approximately 0.28 mg/mL, sodium citrate dihydrate is present at a concentration of approximately 1.08 mg/mL, trehalose dihydrate is present at concentration of approximately 58.01 mg/ml, D-mannitol is present at a concentration of approximately 15.0 mg/ml, L-methionine is present at a concentration of approximately 0.73 mg/ml, and polysorbate 80 is present at a concentration of approximately 0.05 mg/ml.

[0043] The CNP variant peptide-containing formulations described herein are effective and can be used in the various methods described herein.

[0044] It is understood that each feature or embodiment or combination described herein is a non-limiting, illustrative example of any of the aspects of the disclosure and is essentially intended to be combined with any other feature or embodiment or combination. described in the present description. For example, when features are described using phrases such as "one of the embodiments," "some embodiments," "an additional embodiment," "specific exemplary embodiments," and/or "another embodiment," each of these types of embodiments implementation is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features that are described in the present description, without the need to enumerate every possible combination. Such features or combinations of features are applicable to any of the aspects of the disclosure. When examples of values falling within ranges are disclosed, any of these examples are provided as possible limits of the range, any and all numeric values between such limits are provided, and any and all combinations of upper and lower limits are provided.

Detailed description of the disclosure

[0045] The present invention relates to methods of using CNP variant peptides to treat skeletal dysplasias, one or more of the symptoms or physiological consequences of skeletal dysplasias, and other disorders having skeletal dysplasia and/or a CNP-associated symptom or component. The present disclosure also relates to compositions and formulations comprising a CNP variant peptide and the use of such compositions or formulations for treating skeletal dysplasia or the symptoms or physiological consequences of skeletal dysplasia, increasing long bone growth in a subject in need thereof, or improving or increasing growth rate in a subject in need of it. this subject, for example, in a subject suffering from skeletal dysplasia.

A. Definitions

[0046] Unless otherwise indicated, the following terms used in this application, including the specification and claims, have the definitions given below.

[0047] As used in the description and the appended claims, the singular forms include the plural forms until the context clearly dictates otherwise.

[0048] The term "approximately" means an acceptable error for a particular value, as determined by a person skilled in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the term "about" means within 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.05% of the given value or range. Whenever the term "about" precedes the first numeric value in a sequence of two or more numeric values, it is understood that the term "about" applies to each one of the numeric values in that sequence.

[0049] Definitions of standard chemical terms can be found in reference books including Carey and Sundberg, Advanced Organic Chemistry, 3rd ed., Volumes A and B (Plenum Press, New York 1992). In the practice of the present disclosure, unless otherwise indicated, certain standard methods of synthetic organic chemistry, mass spectrometry, preparative and analytical chromatography, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art, may be used. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A.L. Lehninger, Biochemistry (Worth Publishers, Inc., 4th ed., 2004); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd ed., 1989); Methods In Enzymology (eds. S. Colowick and N. Kaplan, Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th ed. (Easton, Pennsylvania: Mack Publishing Company, 1990).

[0050] All publications, patents and patent applications cited herein, above or below, are hereby incorporated by reference in their entirety.

[0051] The following abbreviations are used for amino acids throughout the text:

Alanine: Ala (A) Arginine: Arg(R) Asparagine: Asn(N) Aspartic Acid: Asp(D) Cysteine: Cys(C) Glutamine: Gln(Q) Glutamic acid: Glu (E) Glycine: Gly (G) Histidine: His(H) Isoleucine: Ile(I) Leucine: Leu(L) Lysine: Lys(K) Methionine: Met(M) Phenylalanine: Phe(F) Proline: Pro (P) Serine: Ser(S) Threonine: Thr(T) Tryptophan: Trp(W) Tyrosine: Tyr(Y) Valine: Val(V)

[0052] The standard notation is used herein to describe polypeptide and peptide sequences: the left end of the polypeptide or peptide sequence corresponds to the amino end; the right end of the polypeptide sequence corresponds to the carboxy-terminus.

[0053] In one embodiment, the CNP variant peptides described herein that can be used in the methods are generated by recombinant means using a polynucleotide encoding a CNP variant peptide. CNP variant peptides expressed by such polynucleotides can be produced by methods comprising growing host cells in a culture medium under conditions suitable for expression of a CNP variant-encoding polynucleotide and isolating the expression product from the host cells or culture medium. The actual expression products may differ slightly from the encoded protein product, depending on any post-translational processing. Methods for producing CNP variant peptides of the present invention are disclosed in at least US Pat. No. 8,198,242, incorporated herein by reference.

[0054] The terms "identical" and percent "identity", in the context of two or more polynucleotide or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a particular percentage of nucleotides or amino acid residues that are the same, when comparison and alignment for maximum match, as measured using one of the following sequence comparison algorithms or by visual inspection.

[0055] The phrase "substantially homologous" or "substantially identical", in the context of two nucleic acids or polypeptides, generally refers to two or more sequences or subsequences that share at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 98% nucleotide or amino acid residue identity, when compared and aligned for maximum match, as measured using one of the following sequence comparison algorithms or by visual inspection. In certain embodiments, significant homology or identity exists in regions of sequences that are at least about 25, 50, 100, or 150 residues in length. In another embodiment, the sequences are substantially homologous or identical throughout the length of any one or both of the compared biopolymers.

[0056] When comparing sequences, usually one sequence functions as a reference sequence against which test sequences are compared. When using the sequence comparison algorithm, test and reference sequences are entered into the computer, coordinates of subsequences are set, if necessary, and program parameters of the sequence algorithm are set. The sequence comparison algorithm then calculates a percent sequence identity for the test sequence(s) relative to the reference sequence, based on the specified program parameters.

[0057] Optimal alignment of sequences for comparison can be performed, for example, using the local homology algorithm of Smith & Waterman, Adv. Appl. Math., 2: 482 (1981), using the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol., 48: 443 (1970), using the similarity search method of Pearson & Lipman, Proc. Natl. Acad. sci. USA, 85: 2444(1988), using computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI), or using visual viewing. One example of an efficient algorithm is PILEUP, which uses a simplification of the progressive alignment method by Feng & Doolittle, J. Mol. Evol., 35: 351-360 (1987) and which is similar to the method described by Higgins & Sharp, CABIOS, 5: 151-153 (1989). Another efficient algorithm for generating multiple sequence alignments is Clustal W (Thompson et al., Nucleic Acids Research, 22: 4673-4680 (1994)). An example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm (Altschul et al., J. Mol. Biol., 215: 403-410 (1990); Henikoff & Henikoff, Proc. Natl. Acad. Sci USA 89: 10915 (1989); Karlin & Altschul, Proc Natl Acad Sci USA 90: 5873-5787 (1993)). BLAST analysis software is publicly available through the National Center for Biotechnology Information.

[0058] "Wild type" (wt) is a term referring to the natural form, including sequence, of a polynucleotide, polypeptide, or protein in a particular species. The wild-type form is distinct from the mutant form of the polynucleotide, polypeptide or protein resulting from the genetic mutation(s).

[0059] In one embodiment, the first peptide, which is an "analogue" or "variant" or "derivative" of the second peptide, is a peptide that has sequence homology of at least about 50%, 60%, or 70%, but less 100% sequence homology, with the second peptide. Such analogs, variants, or derivatives may contain non-naturally occurring amino acid residues, including, but not limited to, homoarginine, ornithine, penicillamine, and norvaline, as well as naturally occurring amino acid residues.

[0060] The natriuretic peptide C (NPPC) precursor polypeptide is a 126 amino acid single chain prepropolypeptide that, when cleaved, ultimately leads to wild-type CNP-22 (wtCNP-22). Removal of the signal peptide from NPPC yields pro-CNP, and further cleavage with furin endoprotease produces the active 53-amino acid peptide (CNP-53), which is secreted and cleaved again to form the mature 22-amino acid peptide (CNP or CNP-22). In one embodiment, the "CNP variant peptide" is at least about 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% homologous to wild-type NPPC over the same the number of amino acid residues. It is further contemplated that a CNP variant peptide may comprise from about 1 to about 53, or from 1 to 38, or from 1 to 37, or from 1 to 35, or from 1 to 31, or from 1 to 27, or from 1 to 22, or 10 to 35, or about 15 to about 37 NPPC polypeptide residues. In one embodiment, the CNP variant may contain the 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.

[0061] The term "effective amount" means a dose sufficient to produce the desired result on the health, pathology or disease of the subject or for diagnostic purposes. The desired outcome may include subjective or objective improvement in the dose recipient. A "therapeutically effective amount" refers to that amount of an agent that is effective in producing the intended health benefit. An appropriate "effective" amount in any individual case can be determined by a person skilled in the art using standard experiments. It is understood that the specific dosage level and dosage frequency for any particular patient may vary and will depend on various factors, including the potency of the particular compound used; bioavailability, metabolic stability, excretion rate and duration of action of this compound; the method and time of administration of the compound; age, body weight, general health, sex and diet of the patient; and the severity of the particular condition.

[0062] "Treatment" refers to prophylactic treatment or therapeutic treatment or diagnostic treatment. In certain embodiments, "treatment" refers to administering a compound or composition to a subject for therapeutic, prophylactic, or diagnostic purposes.

[0063] A "prophylactic" treatment is a treatment administered to a subject who does not show signs of a disease, or only shows early signs of a disease, in order to reduce the risk of developing a pathology. The compounds or compositions of the disclosure may be given as a prophylactic treatment in order to reduce the likelihood of developing a pathology or to minimize the severity of the pathology, if it does.

[0064] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs or symptoms of a pathology in order to reduce or eliminate those signs or symptoms. 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.

[0065] "Pharmaceutical composition" or "formulation" refers to a composition suitable for pharmaceutical use in an animal subject, including humans and mammals. The pharmaceutical composition contains a therapeutically effective amount of a CNP variant peptide, optionally another biologically active agent, and optionally a pharmaceutically acceptable excipient, carrier or diluent. In one embodiment, a pharmaceutical composition encompasses a composition comprising the active ingredient(s) and the inert ingredient(s) that constitute the carrier, as well as any product that results, directly or indirectly, from the combination, complexation, or aggregation of any two or more ingredients or dissociation of one or more ingredients or other types of reactions or interactions of one or more ingredients. Accordingly, pharmaceutical compositions of the present disclosure encompass any composition obtained by mixing a compound of the disclosure and a pharmaceutically acceptable excipient, carrier, or diluent.

[0066] A "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, buffers, and the like, such as phosphate buffered saline, dextrose 5% aqueous solution, and emulsions (e.g., oil/water emulsion or water/oil emulsion) . Non-limiting examples of excipients include adjuvants, binders, fillers, diluents, disintegrants, emulsifying agents, wetting agents, lubricants, lubricants, sweeteners, flavoring agents, and coloring agents. Suitable pharmaceutical carriers, excipients and diluents are described in Remington's Pharmaceutical Sciences, 19th ed. (Mack Publishing Co., Easton, 1995). Preferred pharmaceutical carriers depend on the intended route of administration of the active agent. Typical routes of administration include enteral (eg, oral) or parenteral (eg, subcutaneous, intramuscular, intravenous, or intraperitoneal injection; or topical, transdermal, or transmucosal administration).

[0067] 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 ammonium or organic amine salts .

[0068] By "pharmaceutically acceptable" or "pharmacologically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual without causing any undesirable biological effects or without interacting in a deleterious manner with any from the components of the composition in which it is contained, or with any components present on or in the body of the individual.

[0069] "Physiological conditions" refers to conditions in the body of an animal (eg, human). Physiological conditions include, but are not limited to, body temperature and an aqueous environment of physiological ionic strength, pH, and enzymes. Physiological conditions also encompasses conditions in a particular subject's body that differ from the "normal" conditions present in most subjects, for example, that differ from the normal human body temperature of approximately 37°C or differ from the normal human blood pH of approximately 7.4.

[0070] As used herein, the term "subject" encompasses mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees and other great apes and marmosets; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice, guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. The term does not denote a specific age or gender.

[0071] The terms "polyethylene glycol", "PEG", "polyethylene oxide" and "PEO" are used interchangeably in the present description, unless otherwise indicated. The CNP variant conjugated through the amino group to the "PEOn" polymer associated with the number n has the general formula: CH ₃ - [-O-CH ₂ CH ₂ -] _n -C(=O)- NHR, where n is the number units of ethylene oxide and R denotes the rest of the peptide. The "PEOn" polymer may optionally have an alkylene group, (CH ₂ ) _m , where m is an integer from 1 to 5, between the carbonyl carbon and the ethylene oxide repeating units. Such a "PEOn" polymer (for example, PEO12 or PEO24) is monodispersed, i.e. it is one discrete polymer of a specific molecular weight. Similarly, a CNP variant conjugated via an amino group to a "PEGnK" polymer associated with an nK number is generally of the formula: CH ₃ -[-O-CH ₂ CH ₂ -] _p -C(=O)-NHR, where p is an integer greater than 1. The "PEGnK" polymer can also optionally have an alkylene group, (CH ₂ ) _m , where m is an integer from 1 to 5, between the carbonyl carbon and the ethylene oxide repeating units. However, such a "PEGnK" polymer (e.g., PEG1K, PEG2K, PEG5K, or PEG20K) is polydisperse, i.e., contains a mixture of polymers having a molecular weight distribution, where the number nK denotes the number average molecular weight (M _n ) of the polymer in kilodaltons. For example, "PEG2K" conjugated to a CNP variant refers to a PEG polydisperse polymer having a polymer number average molecular weight of approximately 2 kDa.

[0072] When a weight range of a polymer (eg, PEG) is given (e.g., in units of kDa), the range refers to the number average molecular weight range of the polymer, and not to the molecular weight range of multiple polymers in the polydisperse blend, unless explicitly stated otherwise.

B. CNP variant peptides

[0073] The use of CNP-22 as a therapeutic agent is limited by its short plasma half-life (J. Clin. Endocrinol. Metab., 78: 1428-35 (1994)). In human plasma, the concentration of CNP-22 is usually less than 5 pM. In humans, CNP-22 is degraded and cleared from circulation by NEP and NPR-C (Growth Hormone & IGF Res., 16: S6-S14). In all human and animal studies using systemically administered CNP-22, continuous infusion was used to increase the concentration of CNP-22 in subjects. A CNP peptide having a longer half-life and at least a similar level of functionality would be useful for a CNP-based therapeutic strategy. CNP variant peptides having improved properties are disclosed in WO 2009/067639 and WO 2010/135541 and US Pat.

[0074] In certain embodiments, the CNP variant peptides are derivatives of CNP-37 or CNP-38. CNP-37 variant peptides may contain the addition(s), deletion(s), and/or substitution(s) of an amino acid with naturally occurring or non-naturally occurring amino acid(s) or peptidomimetic(s) (e.g., isostere(s) at the peptide bond) in any one or more of the 37 provisions in CNP-37. Non-limiting examples of substitutions that can be made in CNP-37 based on CNP-22 numbering include K4R, G5S, G5R, G8S, K10R, G15S, S16Q, M17N, G19R, and combinations thereof.

[0075] In one embodiment, the CNP variant peptides are modified CNP-37 or CNP-38 peptides having mutation(s)/substitution(s) at the furin cleavage site, engineered to improve resistance to the furin protease in vivo, and/ or containing glycine or proline-glycine at the N-terminus, designed to improve plasma stability and prevent formation of pyroglutamine. Exemplary CNP-37 variants include, but are not limited to:

QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1];

MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2);

PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3);

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP-37 (M32N); SEQ ID No. 4];

PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5);

MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And

GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37 or CNP-38: SEQ ID No. 7).

C. Synthesis and purification of CNP variant peptides

[0076] In some embodiments, CNP variant peptides that can be used herein are obtained by recombinant expression using certain methods known in the art, in certain embodiments. See, for example, Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press (Cold Spring Harbor, N.Y. (1989)); DNA Cloning: A Practical Approach, Volumes I and II, eds. D. N. Glover, (1985); and Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0077] In certain embodiments, CNP variant peptides are produced by a recombinant process that includes culturing in a host cell medium containing a first polynucleotide encoding a CNP variant peptide linked to a second polynucleotide encoding a cleavable peptide or protein under conditions that lead to expression of a fusion polypeptide encoded by polynucleotides, wherein the fusion polypeptide comprises a CNP variant peptide directly connected to or indirectly connected to the cleavage peptide or protein via a linker. In some embodiments, the host cell is transformed with an expression vector containing a polynucleotide encoding a CNP variant peptide fused to a polynucleotide encoding a cleavable peptide or protein. In certain embodiments, the fusion polypeptide is expressed as a soluble protein or as an inclusion body. The expressed fusion polypeptide can be isolated from the host cell or culture medium, and the isolated fusion polypeptide can be contacted with a cleaving agent to release the CNP variant peptide.

[0078] Methods for obtaining peptides of CNP variants are described in US patents 8198242, 8377884 and 8598121, incorporated herein by reference.

[0079] The host cells used to produce CNP variant peptides can be bacteria, yeast, insect, non-mammalian vertebrate or mammalian cells. Bacterial cells include, without limitation, E. coli cell lines and strains. Non-limiting examples of E. coli cell lines and strains include cBL21, BL21(DE3), BL21(DE3)pLysS, BL21(DE3)pGro7, ArcticExpress(DE3), C41 [also called C41(DE3)], C43 [also called C43( DE3)], Origami B(DE3), Origami B(DE3)pLysS, KRX and Tuner(DE3). In one embodiment, CNP variant peptides and CNP fusion proteins are generated using BL21(DE3) cells. Mammalian cells include, but are not limited to, hamster, monkey, chimpanzee, dog, cat, bovine, pig, mouse, rat, rabbit, sheep, and human cells. Host cells can be immortalized cells (cell line) or non-immortalized (primary or secondary) cells and can be any of a wide range of cell types such as, but not limited to, fibroblasts, keratinocytes, epithelial cells (e.g. mammary epithelial cells, intestinal epithelial cells), ovarian cells (eg, Chinese hamster ovary or CHO cells), endothelial cells, glial cells, nerve cells, blood cells (eg, lymphocytes, bone marrow cells), chondrocytes, and other cells of bone origin and precursors of cells of these somatic types. Host cells containing the CNP variant DNA or RNA are cultured under conditions suitable for cell growth, DNA or RNA expression, and identification/selection of cells expressing the CNP variant peptide.

[0080] In certain embodiments, CNP variant peptides are produced by recombinant expression as fusion proteins comprising a CNP variant peptide and a cleavable transfer protein or cleavage tag (e.g., peptide tag), wherein the fusion protein comprises a CNP variant peptide directly fused to the cleavable carrier protein or label or indirectly connected to them through a linker. The use of a carrier protein or label facilitates, for example, the detection, isolation and/or purification of the fusion protein. Cleavable carrier proteins and tags include, but are not limited to, histidine (eg, 6-His) tags; TAF12 human transcription factor (TAF12), TAF12 fragments, TAF12 histone fold domain, TAF12 mutants and fragments thereof, TAF12(C/A), TAF12(D/E), TAF12(4D/4E), TAF12(6D/6E) , TAF12(10D/10E), TAF12(C/A & D/E), TAF12(C/A & 4D/4E), TAF12(C/A & 6D/6E), TAF12(C/A & 10D/10E ); ketosteroid isomerase (KSI); maltose binding protein (MBP); β-galactosidase (β-Gal); glutathione-S-transferase (GST); thioredoxin (Trx); chitin binding domain (CBD); BMP-2, BMP-2 mutants, BMP-2(C/A); SUMO and their mutants and fragments.

[0081] In other embodiments, the peptides of the CNP variants described herein are synthesized using a peptide synthesizer and purified according to methods known in the art, for example, according to the methods of Atherton and Sheppard, Solid Phase Peptide Synthesis: a Practical Approach , IRL Press (Oxford, England (1989)).

D. Methods of Use of CNP Variant Peptides, Pharmaceutical Compositions of CNP Variant Peptides and Routes of Administration

Ways to Use CNP Variants

Bone related disorders/skeletal dysplasias

[0082] Fibroblast growth factors (FGFs) play important roles in bone formation, and mutations in the FGF receptor genes (FGFRs 1, 2, and 3) give rise to various hereditary skeletal malformations (Curr. Biol., 5: 500-507 (1995) ). In particular, activating mutations in FGFR-3 are responsible for long bone disorders including achondroplasia, the most common form of human genetic dwarfism (Nature, 371: 252-254 (1994); Cell, 78: 335-342 (1994)), more moderately impaired hypochondroplasia (Ann. N.Y. Acad. Sci., 785: 182-187 (1996)), and more severe and neonatal lethal thanatophoric dysplasia (TD) types I and II (Hum. Mol. Genet., 5: 509-512 (1996); Nat. Genet., 9: 321-328 (1995)). Mouse models overexpressing FGF-2 and therefore activating FGFR-3 show shortened long bones and macrocephaly (Mol. Biol. Cell, 6: 1861-73 (1995)). According to this model, FGFR-3 deficient mice show marked skeletal overgrowth with wider growth plates (Nature Genet., 12: 390-397 (1996)).

[0083] By stimulating matrix formation, chondrocyte proliferation and differentiation, and increasing long bone growth, opening CNP variant peptides can be used to treat mammals, including humans, suffering from a bone-related disorder such as skeletal dysplasia. Non-limiting examples of CNP-responsive bone-related disorders and skeletal dysplasias include achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, thanatophoric dysplasia, osteogenesis congenita, achondrogenesis, chondrodysplasia congenita, homozygous achondroplasia, chondrodysplasia congenita, congenital hypochondroplasia, osteogenial dysplasia congenitata no perinatal lethal type, short ribe-polyidactylia syndromes, hypochondroPlasia, congenital chondrodispplasia of the risiselic type, metaphysar dysplasia of Jansen, congenital spondyloephysal dysplasia, atelostoogenesis, brilliant short femoral, mesulic dysplasia of the langir, mesulic dysphysia dysplasia. NDRM Reinhardt, acrodisostosis , peripheral dysostosis, Knist dysplasia, fibrochondrogenesis, Roberts syndrome, acromesomelic dysplasia, micromelia, Morquio syndrome, Knist syndrome, metatrophic dysplasia and spondyloepimetaphyseal dysplasia. In addition, CNP variants can be used as an adjunct or alternative to growth hormone for the treatment of idiopsic short stature and other skeletal dysplasias.

[0084] In addition, CNP variant peptides can be used to treat other bone-related conditions and disorders such as rickets, hypophosphatemic rickets [including X-linked hypophosphatemic rickets (also referred to as vitamin D-resistant rickets) and autosomal dominant hypophosphatemic rickets] and osteomalacia [including tumor-induced osteomalacia (also called oncogenic osteomalacia or oncogenic hypophosphatemic osteomalacia)].

[0085] In certain embodiments, CNP variant peptides and compositions and compositions containing them of the present invention can be used to improve one or more symptoms or physiological consequences of skeletal dysplasia, where the improvement can be increased absolute growth, increased growth rate, increased by quality computed tomography (QCT) bone mineral density, improved growth plate morphology, increased growth of long bones, improved spinal morphology, improved elbow range of motion, and/or reduced sleep apnea. In this regard, it should be noted that all terms "improved", "improvement", "increase", "decrease" and their grammatical equivalents are related terms which, when used in relation to a symptom or physiological consequence of a disease state, refer to a state of symptom or physiological outcome of a disease after treatment with a CNP variant peptide (or a composition or formulation containing it) of the present invention compared to the same symptom or physiological outcome of a disease prior to treatment with a CNP variant peptide (or a composition or formulation containing it) of the present invention ( i.e. compared to the “baseline”). As described above, a "baseline" state can be determined either by measuring a subject's condition before treatment (which can subsequently be compared to the same subject's condition after treatment) or by measuring that condition in a population of subjects with the same disease who have the same or similar characteristics (eg, age, sex and/or condition or disease progression).

Increasing or increasing the rate of growth

[0086] The compositions and compositions of the present invention can also be administered to increase or increase the growth rate in a subject suffering from skeletal dysplasia. In a preferred embodiment, the subject suffers from achondroplasia.

[0087] Growth rate measurements in subjects can be performed over time using standard methods well known in the art. In certain embodiments, measurements of parameters such as standing height, sitting height, mass, head circumference, shoulder length, forearm length, thigh length, calf length (knee to foot), hand length (wrist to toe) and/or foot length, can be performed for a precisely defined period of time in order to determine a particular growth rate, as measured by any particular parameter. Growth rate measurements over a well-defined period of time can be "annual" in which the growth rate calculated over a well-defined period of time is converted to an expected growth rate over a period of one year. Growth rate measurements in a subject prior to treatment with a CNP variant peptide of the present invention (i.e., "baseline" growth rate) can be compared with growth rate measurements during or after treatment with a CNP variant peptide of the present invention in order to determine the effect of treatment. , exerted on changes in the growth rate of the subject. A "baseline" of growth rate can also be determined in a population of subjects with the same general age, sex, and disease state as the individual being treated with the CNP variant peptide of the present invention.

[0088] Improvements in growth rate, as measured by assessing one or more of the parameters described above (e.g., standing height, etc.) induced or mediated by treatment of a subject in need thereof with a CNP variant peptide of the present invention (or a composition or composition containing it) can be quantified. As such, in certain embodiments, the annual increase or improvement in growth rate for any particular parameter in a subject treated with a CNP variant of the present invention (or a composition or formulation containing it) is at least about 10%. %, 20%, 25%, 30%, 35%, 40%, 45% or 50% or more of the base level. In certain embodiments, the methods described herein result in an annual increase in growth rate, as measured by standing height, of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45 %, or 50% or more of the baseline. In other embodiments, the methods described herein result in an annual increase in growth rate as measured by sitting height, weight, head circumference, upper arm length, forearm length, thigh length, calf length (knee to foot), hand length ( wrist to toe) or foot length of at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% or more of baseline. As disclosed herein, baseline comparison may be an annual growth rate as measured by a particular parameter before treating the same subject with a CNP variant peptide of the present invention, or may be a growth rate determined from a population of subjects with the same general age. , sex and disease state as the individual to be treated with the CNP variant peptide of the present invention.

[0089] Anthropometric data relating to the proportionality of certain body segments relative to others can also be obtained from subjects both before and after treatment with a CNP variant peptide in order to determine whether administration of a CNP variant peptide leads to changes in such proportionality. For example, ratios of upper body length (waist to top of head) and lower body length (waist to foot) can be calculated both before treatment with a CNP variant peptide (i.e., baseline) and after treatment with a CNP variant peptide in order to determine whether treatment with the CNP variant peptide has an undesirable effect on body proportion. Shoulder to forearm or thigh to calf length ratios (knee to foot) can also be calculated and compared to determine the effect of CNP variant peptide treatment on body proportion. In a preferred embodiment of the present invention, administration of a CNP variant peptide to a subject does not result in a significant change in body proportion, as measured by any particular ratio, from baseline. In this regard, in certain embodiments, treatment with a CNP variant peptide or a composition or formulation containing it of the present invention results in a change in any of the ratios described above of no more than 0.5, 0.25, 0.20, 0 .15, 0.10 or 0.05, preferably not more than 0.05.

[0090] In the various methods described herein, preferably, administration of the CNP variant peptide, or a composition or formulation containing it, does not lead to an undesirable event associated with an investigational drug of grade two or higher or grade three or higher in the subject being treated. . In this regard, methods for measuring and determining the extent of physiological events associated with treatment of a subject with a compound, as well as a list of adverse events of varying degrees, can be found at http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010- 06-14_QuickReference_8.5x11.pdf, which is incorporated herein by reference.

[0091] In addition, in the various methods described herein, it is preferred that administration of a CNP variant peptide or a composition or formulation containing it does not lead to a clinically significant change (increase or decrease from baseline) in one or more of the following physiological parameters; blood hemoglobin concentration, blood platelet count, blood electrolyte concentration, blood urea nitrogen concentration, blood creatinine concentration, blood alkaline phosphatase concentration, blood alanine aminotransferase concentration, or blood aspartate aminotransferase concentration. By "clinically significant change" is meant a change in any one or more of the above parameters that is observed to lead to a detectable/observable undesirable physiological change in the general health of the subject.

Pharmaceutical Compositions and Peptide Formulations of CNP Variants

[0092] In further embodiments, the disclosure provides for the use of pharmaceutical compositions and formulations comprising a CNP variant peptide and one or more pharmaceutically acceptable excipients, carriers, and/or diluents. In certain embodiments, the compositions further comprise one or more other biologically active agents (eg, inhibitors of proteases, receptor tyrosine kinases, and/or NPR-C clearance receptor).

[0093] Non-limiting examples of excipients, carriers, and diluents include carriers, liquids, buffers, isotonicity agents, additives, stabilizers, preservatives, solubilizers, surfactants, emulsifiers, wetting agents, adjuvants, and so on. The compositions may contain liquids (eg water, ethanol); diluents containing various buffers (eg Tris-HCl, phosphate, acetate buffers, citrate buffers), pH and ionic strength detergents and solubilizing agents (eg polysorbate 20, polysorbate 80); antioxidants (eg, methionine, ascorbic acid, sodium metabisulphite); preservatives (eg, Thimerosol, benzyl alcohol, m-cresol); and bulking agents (eg lactose, mannitol, sucrose). The use of excipients, diluents and carriers in pharmaceutical compositions is known in the art; see, for example, Remington's Pharmaceutical Sciences, 18th ed., pp. 1435-1712, Mack Publishing Co. (Easton, Pennsylvania (1990)), which is incorporated herein by reference in its entirety.

[0094] For example, carriers include, without limitation, diluents, carriers, and adjuvants, as well as implantable carriers, and inert, non-toxic solid or liquid excipients and encapsulating materials that do not react with the active ingredient(s). Non-limiting examples of carriers include phosphate buffered saline, saline, water, and emulsions (eg, oil/water emulsions). The carrier may be a solvent or dispersion medium containing, for example, ethanol, a polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), vegetable oil, and mixtures thereof.

[0095] In some embodiments, the compositions are liquid formulations. In certain embodiments, the formulations contain a CNP variant peptide in a concentration range of about 0.1 mg/ml to about 20 mg/ml, or about 0.5 mg/ml to about 20 mg/ml, or about 1 mg/ml up to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg /ml, or from about 2 mg/ml to about 10 mg/ml, or about 2 mg/ml or about 10 mg/ml. In other embodiments, the formulation may be a lyophilized formulation, or may be a liquid formulation that has been previously reconstituted from a lyophilized formulation.

[0096] In additional embodiments, the compositions contain a buffer solution or buffer agent in order to maintain the pH of the CNP-containing solution or suspension in the desired range. Non-limiting examples of buffer solutions include phosphate buffered saline, Tris buffered saline, and Hank's buffered saline. Buffer agents include, without limitation, sodium acetate, sodium phosphate, citric acid monohydrate, and sodium citrate dihydrate. Mixtures of buffer agents can also be used. In certain embodiments, the buffering agent is acetic acid/acetate or citric acid/citrate. The amount of buffering agent suitable for the composition depends in part on the particular buffer used and the desired pH of the solution or suspension. For example, acetate is a more effective pH buffer at pH 5 than at pH 6, so that less acetate can be used in solution at pH 5 than at pH 6. In some embodiments, the buffering agent has a concentration of approximately 10 mM±5 mM. In certain embodiments, the pH of the 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 to pH 6, or from about pH 4 to about pH 5, or about pH 5.0±1.0, or about pH 5.5±1.0.

[0097] In other embodiments, the compositions contain an isotonicity adjusting agent to render the solution or suspension isotonic and more compatible for injection. Non-limiting examples of isotonicity agents include NaCl, trehalose, mannitol, dextrose, glucose, glycerol, sorbitol, xylitol, and ethanol. In certain embodiments, the isotonicity adjusting agent is trehalose or mannitol, which can be used alone or in combination. In certain embodiments, the trehalose or mannitol is present at 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.

[0098] In various embodiments, the compositions may contain a preservative. Preservatives include, but are not limited to, m-cresol and benzyl alcohol. In certain embodiments, the preservative is present at a concentration of about 0.4%±0.2%, or about 1%±0.5%, or about 1.5%±0.5%, or about 2.0%±0. 5%. In certain embodiments, the composition or composition does not contain a preservative.

[0099] In various embodiments, the compositions contain an anti-adsorption agent (eg, to reduce the adsorption of the CNP variant to glass or plastic). Anti-adsorption agents include, but are not limited to, benzyl alcohol, polysorbate 20, and polysorbate 80. In certain embodiments, the anti-adsorption agent is present at a concentration of 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%.

[0100] In various embodiments, the compositions contain a stabilizer. Non-limiting examples of stabilizers include glycerol, glycerol, thioglycerol, methionine, and ascorbic acid and salts thereof. In some embodiments, when the stabilizer is thioglycerol or ascorbic acid, or a salt thereof, the stabilizer is present at a concentration of from about 0.1% to about 1%.

[0101] In various embodiments, the compositions contain an antioxidant. An exemplary antioxidant, without limitation, is ascorbic acid. In certain embodiments, the molar ratio of antioxidant to CNP variant peptide is from about 0.1:1 to about 15:1, or from about 1:1 to about 15:1, or from about 0.5:1 to about 10:1 , or from about 1:1 to about 10:1 or from about 3:1 to about 10:1.

[0102] Pharmaceutically acceptable salts can be used in the compositions, including, without limitation, inorganic acid salts (e.g., hydrochloride, hydrobromide, phosphate, sulfate), organic acid salts (e.g., acetate, propionate, malonate, benzoate, mesylate, tosylate), and amine salts (eg isopropylamine, trimethylamine, dicyclohexylamine, diethanolamine). A detailed discussion of pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Company, (Easton, Pennsylvania (1990)).

[0103] Formulations for parenteral administration can be prepared, for example, as liquid solutions or suspensions, as solid forms suitable for dissolution or suspension in a liquid medium prior to injection, or as emulsions. For example, sterile injectable solutions and suspensions can be formulated according to methods known in the art using suitable diluents, carriers, solvents (e.g., buffered aqueous solution, Ringer's solution, isotonic sodium chloride solution), dispersing agent, wetting agents, emulsifying agents. , suspending agents, and the like. In addition, sterile fatty oils, fatty acid esters, polyols, and/or other inactive ingredients can be used. As further examples, formulations for parenteral administration include aqueous sterile injectable solutions that may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickeners.

[0104] Exemplary CNP peptide-containing formulations are described in US Pat.

[0105] In various embodiments, CNP variant peptides can be formulated in pharmaceutical carriers for administration to subjects affected by skeletal dysplasia. In some embodiments, liquid formulations of CNP variant peptides are formulated according to any combination of ingredients, and their amounts or concentrations are described below:

Ingredient class Ingredient Concentration range Active Ingredient CNP variant peptide 10 mg/ml±9.9 mg/ml Buffer Acetic acid/acetate 10 mM±5 mM or
pH 5±1 Buffer Citric acid/citrate 10 mM±5 mM or
pH 5±1 Isotonic regulator NaCl 140 mM±20 mM Isotonic regulator Trehalose; mannitol 10%±5% preservative m-cresol 0.4%±0.1% or 0.2% Preservative / anti-adsorption agent benzyl alcohol 1.5%±0.5% Stabilizer Glycerol 5-100% (pure) Stabilizer Methionine 0.01-0.2% Stabilizer Ascorbic Acid/Ascorbate Salt 0.1-1% Stabilizer Thioglycerin 0.1-1% Anti-adsorption agent Polysorbate 20
Polysorbate 80
benzyl alcohol 0.001-0.5%
0.001-0.5%
0.5-1.5%

[0106] Compositions containing a CNP variant peptide may also be lyophilized formulations. In certain embodiments, the freeze-dried formulations contain a buffer and a bulking agent and optionally an antioxidant. Exemplary buffers include, but are not limited to, acetate buffers and citrate buffers. Exemplary bulking agents include, without limitation, mannitol, sucrose, dextran, lactose, trehalose, and povidone (PVP K24). In certain embodiments, the mannitol and/or trehalose is present in an amount of from about 3% to about 10%, or from about 4% to about 8%, or from about 4% to about 6%. In certain embodiments, sucrose is present in an amount of from about 6% to about 20%, or from about 6% to about 15%, or from about 8% to about 12%.

[0107] In various embodiments, freeze-dried formulations of CNP variant peptides are prepared from formulations formulated according to any combination of ingredients, and their amounts or concentrations are described below:

Ingredient class Ingredient Concentration range Active Ingredient CNP variant 10 mg/ml±9.9 mg/ml Buffer Acetic acid/acetate 10 mM±5 mM or
pH 5±1 Buffer Citric acid/citrate 10 mM±5 mM or
pH 5±1 Isotonicity corrector/volume builder Sorbitol 5%±3% Isotonicity corrector/volume builder Mannitol/trehalose 5%±3% Isotonicity corrector/volume builder sucrose 10%±5% preservative m-cresol 0.4%±0.2% Preservative / anti-adsorption agent benzyl alcohol 1.5%±0.5% Stabilizer Glycerin (glycerine) 5-100% (pure) Stabilizer Methionine 0.01-0.2% Stabilizer Ascorbic Acid/Ascorbate Salt 0.1-1% Stabilizer Thioglycerin 0.1-1% Anti-adsorption agent Polysorbate 20
Polysorbate 80
benzyl alcohol 0.001-0.5%
0.001-0.5%
0.5-1.5%

[0108] In various embodiments, the composition comprising the CNP variant peptide has a pH of about 3-7, or about 3-6, or about 3.5-6.5, or about 4-6, or about 4-5, or about 4.5-5.5. In some embodiments, for pH 4-5.5, acetic acid/acetate (eg, sodium acetate) is a suitable buffer, and citric acid/citrate is a suitable buffer for pH 5.5-6. Citric acid/citrate (eg sodium citrate) is also a suitable buffer in the pH 3-6 or pH 4-6 range. In certain embodiments, the buffering agent in the formulation has a concentration of about 2-50 mM, or about 2-40 mM, or about 2-30 mM, or about 5-30 mM, or about 2-20 mM, or about 5-20 mM , or approximately 5-15 mm.

[0109] Also, to minimize or avoid deamidation of the CNP variant peptide, lyophilization can remove water from the formulation. In some embodiments, the freeze-dried formulations contain any combination of the following: buffer: sodium acetate and acetic acid or sodium citrate and citric acid; isotonic/bulking agent: mannitol (eg 3-10%, 2-8% or 4-6%); sucrose (eg 6-20%, 5-15% or 8-12%); antioxidants: methionine and/or ascorbic acid with a molar 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.

[0110] Deamidation can also be minimized or avoided by storing the CNP composition (e.g., liquid formulation or lyophilized formulation) at a reduced temperature, such as 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.

[0111] In order to minimize or avoid oxidation of oxidizable residues (eg, methionine) in a CNP variant peptide, the variant can be formulated with one or more antioxidants. Exemplary antioxidants include, but are not limited to, methionine, ascorbic acid, and thioglycerol. Oxidation of, for example, methionine residues can also be minimized or prevented by blowing oxygen out of the liquid medium (in the case of a liquid formulation) with nitrogen or argon and/or by blowing oxygen out of the container or package with nitrogen or argon.

[0112] In some embodiments, polysorbate 20, polysorbate 80, or benzyl alcohol, or a combination thereof, is added to the CNP formulation to minimize or prevent adsorption (e.g., adsorption of the CNP variant peptide to plastic or glass). In certain embodiments, each of the anti-adsorption agents is present at a concentration of 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 about 0.5% to about 1%, or about 0.5% to about 1.5%, or about 0.5% to about 2%, or about 1% to about 2%. Exemplary range(s) of the anti-adsorption agent(s) in the formulation include, without limitation, from about 0.001% to about 0.5% of polysorbate 20, from about 0.001% to about 0.5% of polysorbate 80, and/or from about 0. 5% to approximately 1.5% benzyl alcohol.

[0113] The disclosure also provides kits containing, for example, bottles, vials, ampoules, tubes, cartridges, and/or syringes that contain a liquid (eg, sterile injectable) formulation or a solid (eg, lyophilized) formulation. The kits may also contain pharmaceutically acceptable carriers (e.g., diluents, solutions, and/or buffers) for reconstituting a solid (e.g., lyophilized) formulation into a solution or suspension for administration (e.g., by injection), including, but not limited to, reconstituting the lyophilized formulation in a syringe. for injection, or for diluting the concentrate to a lower concentration. In addition, extemporaneous injection solutions and suspensions can be obtained, for example, from sterile powder, granules or tablets containing a CNP-containing composition. The kits may also contain dispensing devices such as aerosol or injectable dispensers, pens, autoinjectors, needleless injectors, syringes and/or needles.

Doses and frequency of dosing

[0114] As used herein, the term "therapeutically effective amount" of an active agent (eg, CNP variant peptide) refers to an amount that provides a therapeutic effect in a patient. The amount may vary from one individual to another and may depend on a variety of factors, including the general physical condition of the patient. A therapeutically effective amount of a CNP variant peptide can be readily determined by one of ordinary skill in the art using commonly available materials and procedures. For example, the amount of CNP variant peptide used for therapy should give an acceptable growth rate based on growth charts for children aged 0 to 17 years with achondroplasia (214 women and 189 men) that list height for a specific age, head circumference and growth by segment (Horton WA et al., Standard growth curves for achondroplasia, J. Pediatr., 93: 435-8 (1978)). CDC charts can be used to estimate age-specific weight and age-specific weight or age-specific BMI. It is also possible to measure secondary outcomes for courses that are more chronic in nature.

[0115] The frequency of dosing for a particular individual may vary depending on various factors, including the disorder being treated and the individual's condition and response to therapy. In certain embodiments, a pharmaceutical composition comprising a CNP variant peptide is administered to a subject about once a day, once every two days, once every three days, or once a week, twice a week, three times a week, once every two weeks or monthly. In one embodiment, for the treatment of bone-related disorders (eg, skeletal dysplasias, including achondroplasia), a daily or weekly dose of the CNP variant peptide is administered to patients prior to and/or throughout adulthood.

[0116] In certain embodiments, the CNP variant peptides described herein are administered at a dose ranging from about 5 or 10 nmol/kg to about 300 nmol/kg, or from about 20 nmol/kg to about 200 nmol/kg. In some embodiments, CNP variant peptides are administered at a dose of about 5, 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 attending physician. In other embodiments, the CNP variant peptides are administered at a dose of about 5, 7.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170 , 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 mcg/kg or approximately 1, 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 such other dose as the attending physician deems appropriate. In various embodiments, the CNP variant peptide is administered at a dose of about 7.5 to 100 µg/kg, or about 15 to 75 µg/kg, or about 15 to 30 µg/kg, or about 2.5 µg/kg, 7 .5 µg/kg, 15.0 µg/kg, 30.0 µg/kg, or 60.0 µg/kg. Doses of the CNP variant peptides described herein may be administered according to the dosing frequency/frequency of administration described herein, including, but not limited to, daily, 2 or 3 times a week, weekly, every 2 weeks, every 3 weeks, monthly, etc. The administration of the CNP variant peptide can occur over an extended period of time, in some cases one month, three months, six months or more.

[0117] The frequency of dosing/administration of a CNP variant peptide for a particular subject may vary depending on various factors, including the disorder being treated and the condition and response of the subject to therapy. The CNP variant peptide can be administered in a single dose or in multiple doses per dosing. In certain embodiments, the CNP variant peptide is administered, in a single dose or in multiple doses, daily, every other day, every 3 days, 2 times a week, 3 times a week, weekly, every 2 weeks, every 3 weeks, monthly, every 6 weeks, every 2 months, every 3 months, or as deemed appropriate by the attending physician.

[0118] In some embodiments, a CNP variant peptide is administered to allow periods of growth (eg, chondrogenesis) followed by a recovery period (eg, osteogenesis). For example, the CNP variant peptide can be administered intravenously, subcutaneously, intra-articularly, or otherwise, daily or several times a week for a period of time, followed by a period without treatment, then the cycle is repeated. In some embodiments, the initial treatment period (e.g., administration of the CNP variant peptide daily or several times a week) lasts for 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks , 9 weeks, 10 weeks, 11 weeks or 12 weeks. In a related embodiment, the period without treatment lasts for 3 days, 1 week, 2 weeks, 3 weeks, or 4 weeks. In certain embodiments, the CNP variant peptide dosing regimen is daily for 3 days followed by 3 days of withdrawal; or daily or several times a week for 1 week, followed by 3 days or 1 week of withdrawal; or daily or several times a week for 2 weeks, followed by 1 or 2 weeks of withdrawal; or daily or several times a week for 3 weeks followed by 1, 2 or 3 weeks of withdrawal; or daily or several times a week for 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, followed by 1, 2, 3, or 4 weeks of withdrawal.

Methods of administration

[0119] CNP variant peptides or pharmaceutical compositions or formulations containing them can be administered to subjects in various ways, such as by injection subcutaneously, intraarticularly, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally or intrathecally. In one embodiment, CNP variant peptides are administered by a single subcutaneous, intra-articular, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection once a day, once every two days, once every three days, or once a week.

[0120] CNP variant peptides can also be administered by direct injection at or near the disease site. In addition, CNP variant peptides can be administered by depot implantation at the target site of action (eg, an area of abnormal or degenerated joint or cartilage). Alternatively, CNP variant peptides can be administered sublingually under the tongue (e.g., sublingual tablet) or by inhalation into the lungs (e.g., inhaler or aerosol spray), by nasal delivery (e.g., intranasal spray), by delivery to the eye (e.g., ocular drops) or via transdermal delivery (e.g. via a skin patch). CNP variant peptides can also be orally administered 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.

[0121] An additional mode of administration is mediated by an osmotic pump (e.g., Alzet pump) or a mini-pump (e.g., Alzet mini-osmotic pump) that allows for controlled, continuous and/or slow release delivery of a CNP variant peptide or pharmaceutical composition over a predetermined duration. period. An osmotic pump or minipump can be implanted subcutaneously, or near a target site (eg, long bones, epiphyses, etc.).

[0122] One of skill in the art will appreciate that CNP variant peptides or compositions thereof can also be administered in other ways. Determining the most efficient way to administer CNP variant peptides or compositions thereof is within the skill of one skilled in the art.

[0123] Additional aspects and details of the disclosure can be seen from the following examples, which are intended by way of illustration and not limitation.

E. Examples

Example 1 - Data from a six-month human clinical study for patients with achondroplasia

[0124] In order to determine the effect of CNP variant peptides on symptoms of skeletal dysplasia, such as achondroplasia, and on long bone growth and/or growth rate (as measured by the rate of increase in standing height of a patient after treatment compared to what was before treatment) in patients with achondroplasia, dose escalation studies were performed in children with achondroplasia (ACH) aged 5-14 years (inclusive) who have documented ACH, as documented on a clinical basis and confirmed by genetic testing.

[0125] Prior to administration of a formulation containing a CNP variant peptide as disclosed herein, serial measurements of height (in many different parameters) and ratio measurements of growth rates (at least in standing height of the patient) were collected at three-month intervals from study patients without treatment, to establish baseline levels of growth rate and body proportion ratio as described above. When baseline levels for growth rate and body proportions were obtained, a formulation containing a CNP variant peptide (particularly Pro-Gly-CNP-37; SEQ ID NO: 5) was then administered as a daily dose subcutaneously according to one of the following daily schedules. dosing: cohort 1 (n=8): daily dose of Pro-Gly-CNP-37 peptide 2.5 µg/kg, cohort 2 (n=8), daily dose of Pro-Gly-CNP-37 peptide 7.5 µg/kg kg; cohort 3 (n=10), daily dose of Pro-Gly-CNP-37 peptide 15.0 µg/kg. Subcutaneous administration was carried out daily for 6 months.

[0126] More specifically, the Pro-Gly-CNP-37 peptide was formulated in a sterile formulation without preservatives containing 2.0 mg/ml or 10 mg/ml of CNP peptide in combination with 0.28 mg/ml citric acid monohydrate, 1, 08 mg/ml sodium citrate dihydrate, 58.01 mg/ml trehalose dihydrate, 15 mg/ml D-mannitol, 0.73 mg/ml L-methionine, and 0.05 mg/ml polysorbate 80, pH 5.5. This liquid composition was reconstituted from the original lyophilized formulation containing the components described and supplied in a sterile disposable untreated type I borosilicate glass vial. This reconstituted liquid formulation was used in all of the human clinical treatment studies described herein.

[0127] During the course of administration of the CNP variant peptide, various assessments were performed, including body weight, measurement of bone length and bone growth, including the calculation of the ratio of shoulder length to forearm length, the ratio of thigh length to calf length, and the ratio of upper body length to length lower body. Growth rate measurements were also calculated by measuring the standing height of the patients, and the data were compared with baseline growth rate ratios previously obtained. Assessments were performed at approximately days 15, 22, 29, 43, 85, 127 and 183 after the first administration of the CNP variant peptide.

[0128] Research imaging assessment data included measurements and morphology of the spine, long bones, and limbs, as well as measures for growth plate, bone age, and bone mineral density. Tests include an anteroposterior (AP) x-ray of the pelvis to assess baseline for the hip; posteroanterior (PA) radiographs of the left hand and wrist for assessing bone age (Greulich, 1971, Stanford University Press.); (Tanner, 1975, Academic Press.), growth plates, hand length and cortical thickness; AP radiograph of the lower extremity for assessing growth plates, tibia length, cortical thickness and curvature, AP radiograph of the lumbar spine for assessing transverse interpedicular distance. Perform lateral radiographs of the lumbar spine to assess the angle of lumbothoracic lordosis, vertebral morphology, and other potential changes associated with spinal stenosis, QCT scans of the forearm and tibia to assess bone mineral density, growth plate morphology and bone length. This scan can be performed using a standard CT scanner, calibration phantom and specialized software using a predefined low dose protocol that avoids direct radiation to the head and torso.

[0129] Sleep Study: Untreated childhood sleep apnea is re-associated with poor functional and health outcomes, including negative effects on certain aspects of children's development, such as behavior and learning. Sleep apnea in children has been reported to be associated with cognitive impairments that include learning, memory, and visuospatial skills; speech, verbal fluency, school performance and performance. In addition, pediatric sleep disturbances are associated with growth abnormalities; changes in cardiovascular health, including systolic and diastolic blood pressure, autonomic regulation, cerebral oxygenation, and cerebral blood flow, indicate that obstructive sleep apnea syndrome may threaten long-term cardiovascular health; and systemic markers of inflammation (Marcus, 2012, Pediatrics). The sleep testing device is used to assess the presence and severity of sleep disturbance by measuring oxygen saturation, pulse rate, and airflow during overnight monitoring. Sleep apnea assessment includes, but is not limited to, determining the number of episodes of apnea and hypopnea per hour (apnea/hypopnea ratio, AHI).

[0130] Measures of elbow flexion-extension range of motion are measured using a goniometer.

[0131] Biological markers are assessed by change from baseline, and this includes, but is not limited to, assessment of cartilage renewal (CTX-II), chondrocyte and osteoblast activity (bone alkaline phosphatase), bone formation (P1NP), bone remodeling (osteocalcin); and CNP biological activity markers (cGMP, NT-proCNP and ANP), as well as additional biological markers. At scheduled time points, blood and urine samples are collected for biochemical markers of collagen and bone remodeling, as well as for markers of CNP activity.

[0132] Results : Change in growth rate resulting from treatment with CNP Pro-Gly-CNP-37 variant peptide was measured in cohort 1 (2.5 μg/kg/day), cohort 2 (7.5 μg/kg/day) and cohort 3 (15 µg/kg/day) for a total of 26 patients by measuring changes in standing height during treatment and comparing with baseline height ratios measured before treatment. The average change in growth rate for standing height in the study over 6 months is summarized in Table 1. As demonstrated in Table 1, no significant increase in growth rate was observed in patients with achondroplasia who were treated with Pro-Gly-CNP-37 2.5 μg /kg/day for 6 months (cohort 1). However, unlike patients in cohort 1, patients with achondroplasia who were treated with Pro-Gly-CNP-37 7.5 µg/kg/day for 6 months (cohort 2) showed a statistically significant increase (p-value =0.04) standing growth rate (compared to their pre-calculated baseline standing growth rate ratios). More specifically, patients in cohort 2 showed a mean increase in standing growth rate of 1.3 cm/yr, which represents a 45% increase in annual growth rate above baseline. Finally, patients with achondroplasia who were treated with Pro-Gly-CNP-37 15 µg/kg/day for 6 months (cohort 3) also showed a statistically significant increase (p-value=0.01) in growth rate by height. standing (compared to their pre-computed baseline growth rate ratio against standing height). More specifically, patients in cohort 3 showed a mean increase in growth rate for standing height of 2.0 cm/yr, which represents a 50% increase in annual growth rate above baseline. These data demonstrate that daily subcutaneous treatment of achondroplasia patients with at least 7.5 μg/kg of the CNP variant peptide for a period of 6 months or longer has a positive effect on bone growth, growth rate, and overall treatment of symptoms of achondroplasia.

Table 1

Effect analysis: 6-month annual growth rate growth rate Cohort 1
2.5 mcg/kg/daily
(n=8*) Cohort 2
7.5 mcg/kg/daily
(n=8) Cohort 3
15 mcg/kg/daily
(n=10) A basic level of
Average (cm/year) 3.8 2.9 4.0 After treatment
Average (cm/year) 3.4 4.2 6.1 Change from baseline
Average (cm/year)
95% Confidence Region (cm/yr)
p-value** -0.4
-1.8, 1.1
0.56 1.3
0.1, 2.5
0.04 2.0
0.6, 3.4
0.01 Percent increase from baseline
Based on average (%) NM 45 50

[0133] Body proportionality. Body proportionality ratios (upper body length and lower body length, upper arm length and forearm length and thigh length and calf length [knee to foot]) were calculated for all patients in all cohorts both before treatment (baseline) and during time course of treatment with the CNP variant peptide.

[0134] For Cohort 1, the mean baseline upper body length to lower body length ratio, the mean baseline upper arm to forearm length ratio, and the mean baseline thigh to calf length ratio were 2.09, 1.13, and 0, 67, respectively. After 6 months of daily subcutaneous treatment using 2.5 μg/kg of CNP variant peptide, the same body proportion ratios were determined to be 2.07, 1.11, and 0.67, respectively, giving mean changes in the same body proportion ratios. -0.02, -0.02 and 0.0, respectively.

[0135] For Cohort 2, the mean baseline upper body length to lower body length ratio, the mean baseline upper arm to forearm length ratio, and the mean baseline thigh to calf length ratio were 2.03, 1.14, and 0, 66, respectively. After 6 months of daily subcutaneous treatment with 7.5 μg/kg of CNP variant peptide, the same body proportion ratios were determined to be 2.03, 1.14, and 0.66, respectively, giving mean changes in the same body proportion ratios. 0.0, 0.0 and 0.0, respectively.

[0136] For Cohort 3, the mean baseline upper body length to lower body length ratio, the mean baseline upper arm to forearm length ratio, and the mean baseline thigh to calf length ratio were 1.91, 1.13, and 0, 69, respectively. After 6 months of daily subcutaneous treatment using 15 μg/kg of CNP variant peptide, the same body proportion ratios were determined to be 1.89, 1.08, and 0.69, respectively, giving mean changes in the same body proportion ratios of -0 .02, -0.05 and 0.0, respectively.

[0137] These data demonstrate that treatment of achondroplasia patients with the CNP variant peptide does not have significant adverse effects on overall body proportionality, as all measured changes in body proportionality ratios fell between -0.05 and 0.05, relative to baseline, after 6 months treatment with a CNP variant peptide.

[0138] Adverse events . All patients in each of the cohorts described above were regularly assessed for the presence of adverse events (as defined in http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5×11.pdf), in in particular due to treatment with a CNP variant peptide. None of the patients in any of the three cohorts studied had a grade two or higher adverse event that was defined as study drug related, demonstrating that treatment of achondroplasia patients with CNP variant peptides did not lead to serious physiological adverse events.

[0139] In addition, all patients in each cohort studied were regularly monitored for blood hemoglobin concentration, blood platelet count, blood electrolyte concentration, blood urea nitrogen concentration, blood creatinine concentration, blood alkaline phosphatase concentration, alanine aminotransferase concentration in the blood and the concentration of aspartate aminotransferase in the blood. The data obtained for each of the three cohorts studied showed that no clinically significant changes were observed in any of the safety parameters described above, which also confirms the basic safety of therapy for achondroplasia and associated symptoms with the CNP variant peptide.

Example 2 - Data from a twelve-month human achondroplasia clinical trial (cohort 3)

[0140] After a period of the first 6 months of treatment and evaluation in a clinical study, 10 patients with achondroplasia from cohort 3 described above in example 1 continued the clinical regimen of 15 μg/ml Pro-Gly-CNP-37, which was administered subcutaneously daily in for an additional 6 months (total 12 months). During the additional six-month treatment period, various assessments were performed, including body weight, measurement of bone length and bone growth, including calculation of the ratio of the length of the shoulder to the length of the forearm, the ratio of the length of the thigh to the length of the lower leg, and the ratio of the length of the upper body to the length of the lower body . Growth rate measurements were also calculated by measuring patients' standing height, and the data was compared to a baseline of growth rate ratios previously obtained.

[0141] Results : Change in growth rate following treatment for 12 months with CNP Pro-Gly-CNP-37 variant peptide was measured in cohort 3 (15 μg/kg/day) for a total of 10 patients by measuring changes in standing height during treatment and comparisons with baseline growth rate ratios measured before treatment. Data from this 12-month study indicated that patients with achondroplasia who were treated with 15 µg/kg/day of Pro-Gly-CNP-37 for 12 months (cohort 3) showed a statistically significant increase (p- value=0.017) standing growth rate (compared to their pre-calculated baseline growth rate to standing height ratio). More specifically, patients in cohort 3 showed a mean increase in standing growth rate of 1.9 cm/yr, resulting in an increase in annual growth rate of more than 47% above baseline. These data demonstrate that daily subcutaneous treatment of achondroplasia patients with at least 15 μg/kg of the CNP variant peptide for a period of 12 months or more has a positive effect on bone growth, growth rate, and overall treatment of symptoms of achondroplasia.

[0142] Body proportionality . Body proportionality ratios (upper body length and lower body length, upper arm length and forearm length and thigh length and calf length [knee to foot]) were calculated for 10 patients in cohort 3 both before treatment (baseline) and during time course of treatment with the CNP variant peptide.

[0143] For Cohort 3, the mean baseline upper body length to lower body length, the mean baseline upper arm to forearm length ratio, and the mean baseline thigh to calf length ratio were 1.91, 1.13, and 0, 69, respectively. After 6 months of daily subcutaneous treatment using 15 μg/kg of the CNP variant peptide, the same body proportion ratios were determined to be 1.88, 1.16, and 0.69, respectively, giving mean changes in the same body proportion ratios -0 .03, 0.03 and 0.0, respectively.

[0144] These data demonstrate that treatment of achondroplasia patients with the CNP variant peptide does not have a significant adverse effect on overall body proportionality, as all measured changes in body proportionality ratios fell between -0.05 and 0.05, relative to baseline, after 12 months treatment with a CNP variant peptide.

[0145] Adverse events . All patients in cohort 3 were regularly assessed for the presence of adverse events (as defined in http://evs.nci.nih.gov/ftp1/CTCAE/CTCAE_4.03_2010-06-14_QuickReference_8.5×11.pdf), in particular treatment with a CNP variant peptide during the course of treatment. No patient in cohort 3 had a grade two or higher adverse event that was defined as being related to study drug, demonstrating that treatment of achondroplasia patients with CNP variant peptides did not lead to serious physiological adverse events.

[0146] In addition, all patients in cohort 3 were regularly monitored for blood hemoglobin concentration, blood platelet count, blood electrolyte concentration, blood urea nitrogen concentration, blood creatinine concentration, blood alkaline phosphatase concentration, alanine aminotransferase concentration in blood and the concentration of aspartate aminotransferase in the blood. The data obtained in this study demonstrated that no clinically significant changes were observed in any of the safety parameters described above, which also confirms the basic safety of therapy for achondroplasia and associated symptoms with the CNP variant peptide.

[0147] Based on the human clinical data described above, daily subcutaneous doses of Pro-Gly-CNP-37 at 30 μg/kg/day and 60 μg/kg/day in human patients with achondroplasia should also be tested for effect and safety, as described above. Based on the data disclosed in the present description, it is fully reasonable to assume that such administration regimens will provide both efficacy and safety in the treatment of achondroplasia in human patients.

[0148] As expected, those skilled in the art will appreciate many modifications and variations of the disclosure as set forth in the embodiments and illustrative examples described herein. Therefore, the disclosure should be subject only to such restrictions as are set forth in the appended claims.

Claims (101)

1. Pharmaceutical composition for the treatment of skeletal dysplasia, which contains: (a) a CNP variant peptide selected from the group consisting of QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP 37 (M32N); SEQ ID No. 4]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37; SEQ ID No. 7); And (b) citric acid monohydrate, sodium citrate dihydrate, trehalose dihydrate, D-mannitol, L-methionine, and polysorbate 80, wherein the CNP peptide variant is in an amount of 0.1-2.0 mg/mL. 2. The pharmaceutical composition of claim 1, wherein the composition is lyophilized or in liquid form. 3. Pharmaceutical composition according to claim 1 or 2, which is reconstituted from a lyophilized composition. 4. Pharmaceutical composition according to any one of paragraphs. 1-3, where the composition contains 2.0 mg/ml of the indicated CNP peptide variant. 5. Pharmaceutical composition according to any one of paragraphs. 1-3, where the composition contains 0.5-2.0 mg/ml of the indicated CNP peptide variant. 6. A pharmaceutical composition according to claim 1, wherein the composition contains 0.8 mg/ml of said CNP peptide variant. 7. Pharmaceutical composition according to any one of paragraphs. 1-6, wherein said citric acid monohydrate is present at a concentration of 0.15 to 0.40 mg/ml, said sodium citrate dihydrate is present at a concentration of 0.5 to 1.5 mg/ml, said trehalose dihydrate is present at a concentration 30 to 70 mg/ml, said D-mannitol is present at a concentration of 10 to 20 mg/ml, said L-methionine is present at a concentration of 0.5 to 1.5 mg/ml, and said polysorbate 80 is present at a concentration of 0 .01 to 0.1 mg/ml. 8. The pharmaceutical composition according to claim 7, wherein said citric acid monohydrate is present at a concentration of 0.28 mg/ml, said sodium citrate dihydrate is present at a concentration of 1.08 mg/ml, said trehalose dihydrate is present at a concentration of 58.01 mg/ml ml, said D-mannitol is present at a concentration of 15 mg/ml, said L-methionine is present at a concentration of 0.73 mg/ml, and said polysorbate 80 is present at a concentration of 0.05 mg/ml. 9. Pharmaceutical composition according to any one of paragraphs. 1-8, where the composition does not contain preservatives. 10. Pharmaceutical composition according to any one of paragraphs. 1-9, where the composition is contained in a borosilicate glass vessel. 11. Pharmaceutical composition according to any one of paragraphs. 1-10, where the composition has a pH between 5.0 and 6.0. 12. Pharmaceutical composition according to any one of paragraphs. 1-11, where the composition has a pH of 5.5. 13. The use of a composition comprising a CNP variant peptide for the treatment of skeletal dysplasia in a subject, wherein the CNP variant peptide is administered to the subject in an amount of at least 7.5 μg/kg, selected from the group consisting of: QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP 37 (M32N); SEQ ID No. 4]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37 : SEQ ID No. 7), where the composition contains citric acid monohydrate, sodium citrate dihydrate, trehalose dihydrate, D-mannitol, L-methionine and polysorbate 80, and wherein the CNP peptide variant is present in an amount of 0.1-2.0 mg/mL. 14. Use according to claim 13, wherein the treatment leads to an improvement in one or more symptoms of skeletal dysplasia selected from the group consisting of increased absolute height, increased growth rate, increased bone mineral density (BMD) QCT, improved growth plate morphology, increased height long bones, improved spinal morphology, improved elbow range of motion, and reduced sleep apnea. 15. The use according to claim 13 or 14, wherein the skeletal dysplasia is selected from the group consisting of achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasia, thanatophoric dysplasia, osteogenesis imperfecta, achondrogenesis, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, campelic dysplasia congenital lethal hypophospatasia, imperfect osteogenesis of perinatal lethal type, short ribi-polyidactyli syndromes, hypochondroPlazia, dotted chondrodisplazia of a risiselic type, metaphysarium dysplasia, congenital spondyphysaria dysplasia, atelostoogenesis, diaboodic dysplant dyeine dysplasia, and maintenance of cormel nyvergelt dysplasia, syndrome Robinov, Reinhardt's syndrome, acrodysostosis, peripheral dysostosis, Knist's dysplasia, fibrochondrogenesis, Roberts' syndrome, acromesomelic dysplasia, micromelia, Morquio's syndrome, Knist's syndrome, metatrophic dysplasia and spondyloepimetaphyseal dysplasia. 16. Use according to claim 15, wherein the skeletal dysplasia is achondroplasia. 17. Application according to any one of paragraphs. 13-16 wherein said composition is administered once a day. 18. Application according to any one of paragraphs. 13-17 wherein said composition is administered subcutaneously. 19. Application according to any one of paragraphs. 13-18, where the composition contains 2.0 mg/ml of the indicated CNP peptide variant. 20. Application according to any one of paragraphs. 13-18, where the composition contains from 0.5 to 2.0 mg/ml of the indicated CNP peptide variant. 21. Application according to any one of paragraphs. 13-20, where (a) said composition containing said CNP peptide variant is administered to said subject in an amount of at least 15 μg/kg per day; (b) said composition containing said CNP variant peptide is administered to said subject in an amount of at least 30 μg/kg per day; or (c) said composition comprising said CNP variant peptide is administered to said subject in an amount of at least 60 μg/kg per day. 22. Application according to any one of paragraphs. 13-21, where the specified composition includes a composition according to any one of paragraphs. 1-12. 23. Application according to any one of paragraphs. 13-22, where said composition comprises the composition of claim 8. 24. Use of a composition comprising a CNP variant peptide to increase long bone growth in a subject, wherein the CNP variant peptide is administered to the subject in an amount of at least 7.5 μg/kg and is selected from the group consisting of: QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP 37 (M32N); SEQ ID No. 4]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37 : SEQ ID No. 7), and wherein the composition contains citric acid monohydrate, sodium citrate dihydrate, trehalose dihydrate, D-mannitol, L-methionine and polysorbate 80, and wherein the CNP peptide variant is in the amount of 0.1-2.0 mg/ml. 25. Use according to claim 24, wherein said subject suffers from achondroplasia. 26. Application according to paragraph 24 or 25, where (a) said composition is administered once a day; (b) said composition is administered once a day for a period of at least 6 months; and/or (c) said composition is administered subcutaneously. 27. Application according to any one of paragraphs. 24-26, where (a) said composition contains said CNP peptide variant for administration to said subject in an amount of at least 15 μg/kg per day; (b) said composition contains said CNP peptide variant for administration to said subject in an amount of at least 30 μg/kg per day; or (c) said composition contains said CNP peptide variant for administration to said subject in an amount of at least 60 μg/kg per day. 28. Application according to any one of paragraphs. 24-27, where the composition contains 2.0 mg/ml of the indicated CNP peptide variant. 29. Application according to any one of paragraphs. 24-27, where the composition contains from 0.5 to 2.0 mg/ml of the indicated CNP peptide variant. 30. Application according to any one of paragraphs. 24-28, while the specified composition contains the composition according to any one of paragraphs. 1-12. 31. Application according to any one of paragraphs. 24-29, where said composition contains the composition of claim 8. 32. Use of a composition comprising a CNP variant peptide to increase growth rate in a subject, wherein said CNP variant peptide is administered to the subject in an amount of at least 7.5 μg/kg and is selected from the group consisting of: QEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [CNP-37(M32N); SEQ ID No. 1]; MQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-CNP-37; SEQ ID No. 2); PQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-CNP-37; SEQ ID No. 3); GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSNSGLGC [Gly-CNP 37 (M32N); SEQ ID No. 4]; PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37; SEQ ID No. 5); MGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Met-Gly-CNP-37; SEQ ID No. 6); And GQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Gly-CNP-37 : SEQ ID No. 7), and where the composition contains citric acid monohydrate, sodium citrate dihydrate, trehalose dihydrate, D-mannitol, L-methionine and polysorbate 80, and where the CNP variant peptide is present in an amount of 0.1-2.0 mg/ml. 33. Use according to claim 32, wherein said subject suffers from achondroplasia. 34. Application according to claim 32 or 33, where the increase in growth rate is an increase in annual growth rate, as measured by standing growth, at least 25% above the baseline in the specified subject, or where the increase in growth rate is an increase in annual growth rate height, as measured by standing height, at least 40% above baseline in said subject. 35. Application according to any one of paragraphs. 32-34, where the increase in growth rate is measured by measuring standing height, sitting height, weight, head circumference, shoulder length, forearm length, thigh length, calf length, hand length, or foot length. 36. Application according to any one of paragraphs. 32-35, where (a) said composition is administered once a day; (b) said composition is administered once a day for a period of at least 6 months; (c) said composition is administered once a day for a period of at least 12 months; and/or (d) said composition is administered subcutaneously. 37. Application according to any one of paragraphs. 32-36, where the composition contains 2.0 mg/ml of the indicated CNP peptide variant. 38. Application according to any one of paragraphs. 33-36, where the composition contains from 0.5 to 2.0 mg/ml of the indicated CNP peptide variant. 39. Application according to any one of paragraphs. 32-38, where (a) said composition containing said CNP peptide variant is administered to said subject in an amount of at least 15 μg/kg per day; (b) said composition containing said CNP variant peptide is administered to said subject in an amount of at least 30 μg/kg per day; or (c) said composition comprising said CNP variant peptide is administered to said subject in an amount of at least 60 μg/kg per day. 40. Application according to any one of paragraphs. 32-39, where the specified composition includes the composition according to any one of paragraphs. 1-12. 41. Application according to any one of paragraphs. 32-39, where said composition comprises the composition of claim 8. 42. Application according to any one of paragraphs. 32-41, which does not lead to a clinically significant change in blood hemoglobin concentration, blood platelet count, blood electrolyte concentration, blood urea nitrogen concentration, blood creatinine concentration, blood alkaline phosphatase concentration, blood alanine aminotransferase concentration, or aspartate aminotransferase concentration in the blood of the specified subject. 43. Application according to any one of paragraphs. 32-42 which leads to (a) changing the ratio of the length of the upper body to the length of the lower body between -0.05 and 0.05; (b) measuring the ratio of the length of the shoulder and the length of the forearm between -0.05 and 0.05; and/or (c) a change in the ratio of femur length and tibia length between -0.05 and 0.05. 44. Application according to any one of paragraphs. 32-43, where said composition is administered parenterally. 45. Application according to any one of paragraphs. 32-44, wherein said composition is administered daily, 3 times a week, twice a week, once a week, or once every two weeks.