Cnp variants and conjugates thereof

Abstract

Generally speaking, this disclosure relates to stable variants of C-type natriuretic peptide (CNP) and their use in the treatment of bone-related conditions.

Description

[Technical Field] Cross-reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 901,093, filed September 16, 2019; U.S. Provisional Patent Application No. 62 / 935,050, filed November 13, 2019; U.S. Provisional Patent Application No. 62 / 963,350, filed January 20, 2020; U.S. Provisional Patent Application No. 62 / 964,852, filed January 23, 2020; and U.S. Provisional Patent Application No. 63 / 038,667, filed June 12, 2020, all of which are incorporated herein by reference in their entirety. Materials submitted electronically are also incorporated by reference. The sequence list, which is part of this disclosure, is submitted as part of the main document along with the specification. The main document containing the sequence list is named "54736_Seqlisting.txt", created on August 6, 2020, and is 54,429 bytes in size. The subject of the sequence list is incorporated herein by reference in its entirety. Generally, this disclosure relates to variants of C-type natriuretic peptide (CNP), pharmaceutical compositions including CNP variants, and methods of use. CNP variants can be used as therapeutic agents to treat diseases that respond to CNP, including (but not limited to) bone-related conditions such as skeletal dysplasia (e.g., achondroplasia). [Previous Technology] C-type natriuretic peptide (CNP) (Biochem.Biophys.Res. Commun., 168: 863-870 (1990) (Genbank accession number NP_077720, for the CNP precursor protein NPPC) (J. Hypertens., 10: 907-912 (1992)) is a small single-chain peptide in the peptide family (ANP, BNP, CNP) with a 17-amino acid ring structure (Levin et al., N. Engl. J. Med., 339: 863-870 (1998)) and plays an important role in many biological processes. CNP interacts with natriuretic peptide receptor-B (NPR-B, GC-B) to stimulate the production of cyclic-guanosine monophosphate (cGMP) (J. Hypertens., 10: 907-912 (1992)). 1111-1114 (1992). CNP is widespread, including manifestations in the central nervous system, reproductive tract, bone and vascular endothelium (Hypertension, 49: 419-426 (2007)). In humans, CNP is initially produced as a single-chain 126-amino-acid precursor peptide from the natriuretic peptide precursor C (NPPC) gene (Biochemistry and Biophysics Research Exchange, 168: 863-870 (1990)). Removal of the signal peptide produces pro-CNP, which is further cleaved by the endopeptide furin to produce an active 53-amino-acid peptide (CNP-53). This is secreted and cleaved again by an unknown enzyme to produce a mature 22-amino-acid peptide (CNP-22) (Wu, Journal of Biochemistry 278: 25847-852 (2003)). The difference between CNP-53 and CNP-22 lies in their distribution. CNP-53 is predominantly found in tissues, while CNP-22 is mainly found in plasma and cerebrospinal fluid (J. Alfonzo, *Receptor Signal Transduction Research*, 26: 269-297 (2006)). Both CNP-53 and CNP-22 bind to NPR-B in a similar manner. cGMP production-mediated downstream signaling influences many different biological processes, including endochondral ossification. For example, knockout of CNP or NPR-B genes in mouse models results in dwarf phenotypes with shorter long bones and vertebrae. Human NPR-B mutations that block normal CNP signaling have been identified and cause 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 exhibit elongated long bones and vertebrae. The therapeutic use of CNP (CNP22) is limited by its short plasma half-life, which has been shown to be 2.6 minutes in human vivo (Journal of Clinical Endocrinology and Metabolism, 78: 1428-35 (1994)). CNP variants with longer in vivo serum half-lives and exhibiting similar or modified activities to wild-type CNP are important for sustainable therapeutic strategies. [Summary of the Invention] This disclosure relates to novel variants of C-type natriuretic peptide (CNP) having increased circulating half-life and stability in aqueous media, pharmaceutical compositions including such CNP variants, and methods of using such CNP variants to treat conditions that respond to CNP, including (but not limited to) bone-related conditions such as achondroplasia. In various embodiments, this disclosure provides variants of C-type natriuretic peptide (CNP) selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); and PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5). In various embodiments, this disclosure provides variants of C-type natriuretic peptide (CNP) selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7). In various embodiments, the variant peptide further includes an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further includes an OH or NH2 group at the C-terminus. In various embodiments, the variant peptide includes a conjugated portion. In various embodiments, the conjugated portion is located on a residue of a CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the conjugated portion is located on a lysine residue. In various embodiments, the conjugated portion includes one or more acidic portions. In various embodiments, the acidic portions are hydrophobic acids. In various embodiments, the conjugated portion includes one or more acidic portions linked to hydrophilic spacer groups. In various embodiments, the hydrophilic spacer group is any amino acid. In various embodiments, the hydrophilic spacer group is γ-glutamic acid (γGlu). In various embodiments, the hydrophilic spacer group is OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the hydrophilic spacer group is γ-glutamic acid (γGlu) or OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the hydrophilic spacer group is γ-glutamic acid (γGlu) linked to one or more OEG (8-amino-3,6-dioxanoic acid). In various embodiments, the acidic portion is a fatty acid. Exemplary fatty acids include short-chain, medium-chain, or long-chain fatty acids or dicarboxylic fatty acids. In various embodiments, the fatty acid is saturated or unsaturated. Consider C-6 to C-20 fatty acids, including (but not limited to) C-6, C-8, C-10, C-12, C-14, C-16, C-18, or C-20 saturated or unsaturated fatty acids. In various embodiments, the fatty acid is decanoic acid, dodecanoic acid, myristic acid, palmitic acid, stearic acid, eicosanoic acid, or a diacid thereof. In various embodiments, the acidic moiety and the hydrophilic spacer group have the structure AEEA-AEEA-γGlu-C18DA. In various embodiments, the acidic moiety and the hydrophilic spacer group have the structure: [Figure], where "[Figure]" indicates the connection point with the CNP variant. In various embodiments, "[Figure]" indicates the connection point with a hydrolyzable linker, wherein the hydrolyzable linker is connected to the CNP variant. In various embodiments, the hydrolyzable linker is capable of releasing the complete CNP variant. In various embodiments, the CNP variant having the conjugated portion is a component of the sustained-release composition. In various embodiments, the sustained-release composition is a delayed-release composition. In various embodiments, the CNP variant including the conjugated portion and the hydrolyzable linker is capable of releasing the CNP variant, wherein at pH 7 to 7.6, (i) less than about 20% of the CNP variant is released on day 1; and (ii) about 90% of the CNP variant is released weekly, or about 90% of the CNP variant is released every two weeks, or about 90% of the CNP variant is released monthly. In various embodiments, (i) less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide is released on day 1 at pH 7.0 to 7.6; and (ii) about 90% of the peptide is released weekly, bi-weekly, or monthly at pH 7 to 7.6. Further consideration was given to (i) the release of less than about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the peptide on day 1 at pH 7.0 to 7.6; and (ii) the release of about 70%, about 80%, or about 90% of the peptide weekly at pH 7 to 7.6; or about 70%, about 80%, or about 90% of the peptide every two weeks; or about 70%, about 80%, or about 90% of the peptide every three weeks; or about 70%, about 80%, or about 90% of the peptide monthly; or alternatively ii) at pH 7.0 to 7.6. At levels 7 to 7.6, approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released weekly; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every two weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released every three weeks; or approximately 70%, 75%, 80%, 85%, or 90% of the peptides are released monthly. In various embodiments, the variant has the following structure: PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC (SEQ ID NO: 5) or Ac-PGQEHPQARRYRGAQRRGLSRGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 8). In various embodiments, the variant systems are selected from the group consisting of: Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 8); Ac-PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 9); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 10); Ac-PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 11); Ac-PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 12); Ac-PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-NH2 (SEQ ID NO: 13); and Ac- PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC-OH (SEQ ID NO: 14). In various embodiments, the variant includes one or more linker groups. In various embodiments, the linker is located on a residue of the CNP cyclic domain or at a site other than the CNP cyclic domain. In various embodiments, the linker is located on a lysine residue. In various embodiments, the connector is a hydrolyzable connector. In various embodiments, the CNP variant is linked to the conjugated moiety via a linker. In various embodiments, the linker is linked to the conjugated moiety via a hydrophilic spacer group of the conjugated moiety. In various embodiments, the linker is aminoethoxy-2-ethoxyacetic acid (AEEA). In various embodiments, the linker is a bicin-type linker or a peptide-like linker, which refers to a linker having a cleavage mechanism similar to bicin (bis-2-hydroxyethylglycinylamine), but alternatively via asymmetric N-alkyl peptide (i.e., peptide-like) cleavage. In various embodiments, the linker is an electron linker based on non-enzymatic β-elimination. In various embodiments, the electron linker includes an SO2 moiety. Examples of linkers as illustrated in the CNP conjugate are shown in Figure 1. See also Santi et al., Proceedings of the National Academy of Sciences of the United States of America (PNAS) 109:6211-6216, 2012. In various embodiments, the conjugated portion is a synthetic polymeric group. In various embodiments, the variant includes a synthetic polymeric group coupled to the variant via a hydrolyzable linker. In various embodiments, the synthetic polymeric group includes a hydrophilic polymeric portion. In various embodiments, the hydrophilic polymeric portion includes polyethylene glycol (PEG). In various embodiments, the hydrophilic polymeric portion includes polyethylene glycol (PEG) having a chain length of 6 to 20 atoms. In various embodiments, the mutant peptides were prepared synthetically. In various embodiments, the mutant peptide was stable for 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the mutant peptide was stable for at least 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the mutant peptide was stable for at least 10 days at about 37°C and pH 7.2 to 7.6. In various embodiments, the mutant peptide is stable against deacetylation. In various embodiments, the mutant peptide is stable against oxidation. In various embodiments, the mutant peptide is stable against deacetylation and / or oxidation, or a combination thereof. In various embodiments, methionine is replaced by oroleucine. In various embodiments, there is little or no detectable deacetylation after 10 days. In various embodiments, the mutant peptide has a half-life of about 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the mutant peptide has a half-life of about 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the mutant peptide has a half-life of about 10 days at about 37°C and pH 7.2 to 7.6. In various embodiments, the mutant peptide has a half-life of at least 10 days at about 37°C and pH 7.0 to 7.6. In various embodiments, the mutant peptide has a half-life of at least 10 days at about 37°C and pH 7.0 to 7.4. In various embodiments, the mutant peptide has a half-life of at least 10 days at about 37°C and pH 7.2 to 7.6. In various embodiments, the half-life is at least about 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 30 days or longer. In various embodiments, the variant peptide has an EC50 of 0.1 to 10 nM in cGMP analysis. In various embodiments, the variant peptide has an EC50 of 0.1 to 25 nM in cGMP analysis. In various embodiments, after 10 days in an aqueous medium at physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, greater than 45% of the variant peptide was detected. In various embodiments, after 10 days in an aqueous medium at physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptide was detected. In various embodiments, after 10 days in an aqueous medium at 37°C and pH 7.4, greater than 45% of the variant peptide was detected. In various embodiments, after 10 days in an aqueous medium at 37°C and pH 7.4, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptide was detected. In various embodiments, under physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, after 10 days in plasma, greater than 45% of the variant peptide was detected. In various embodiments, under physiological conditions, such as approximately 37°C and pH 7.0 to 7.6, after 10 days in plasma, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptide was detected. In various embodiments, after 10 days in plasma at 37°C and pH 7.4, greater than 45% of the variant peptide was detected. In various embodiments, after 10 days in plasma at 37°C and pH 7.4, greater than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the variant peptide was detected. In various embodiments, the variant peptide is conjugated with lipids, fatty acids, hydrophilic spacers, or linkers, or in combination thereof, as appropriate. In various embodiments, the linker is a hydrophilic polymer portion. In various embodiments, the hydrophilic polymer portion is a synthetic hydrophilic polymer portion. In various embodiments, the mutant peptide has a longer half-life compared to Pro-Gly-CNP37. In various embodiments, the mutant peptide has a longer half-life compared to CNP-22. In various embodiments, the mutant peptide has a longer half-life compared to Pro-Gly-CNP37 and / or CNP-22. In various embodiments, the mutant peptide has a longer half-life compared to Pro-Gly-CNP37 and / or CNP-22 in vitro and / or in vivo. This disclosure further provides a pharmaceutical composition comprising the CNP variant described herein and a pharmaceutically acceptable excipient, carrier, or diluent. In various embodiments, the composition is a lyophilized formulation prepared from a formulation comprising a citrate / citrate buffer or an acetate / acetate buffer with a pH of about 4 to about 6. In various embodiments, the lyophilized formulation is prepared from a formulation further comprising an isotonic modifier or a buildup agent selected from the group consisting of mannitol, sucrose, sorbitol, trehalose, polysorbate 80, and combinations thereof. In various embodiments, the lyophilized formulation is prepared from a formulation further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salts of ascorbic acid, thioglycerol, and combinations thereof. In various embodiments, the CNP variant composition is supplied in lyophilized powder form for reconstitution from 0.8 mg to 10 mg. In various embodiments, the CNP variant composition is supplied in 0.8 mg or 2 mg lyophilized preservative-free reconstitution powder form. In various embodiments, the composition is a delayed-release composition. In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC(Pro-Gly-CNP-37)(BMN111)(EQ ID NO: 1). Also provided is a method for treating bone-related conditions or skeletal dysplasia in individuals in need, comprising administering to an individual a composition comprising a CNP variant as described herein. In various embodiments, bone-related disorders or skeletal dysplasia are selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, lethal dysplasia, osteogenesis imperfecta, achondrogenesis, punctate chondrodysplasia, homozygous achondroplasia, punctate chondrodysplasia, camptomelic dysplasia, congenital lethal hypophosphatasia, and perinatal lethal type of osteogenesis. Imperfecta, short-rib polydactyly syndromes, decreased chondrogenesis, rhizomelic type of chondrodysplasia punctata, Jansen-type metaphyseal dysplasia, spondyloepiphyseal dysplasia congenita, skeletal dysplasia, malformation, congenital short femur, Langer-type mesomelic dysplasia, Nievergelt-type mesomelic dysplasia, Robinow syndrome, Reinhardt syndrome, acrodysostosis, peripheral bone developmental disorders, Kniest dysplasia. dysplasia, fibrocartilage hyperplasia, Roberts syndrome, acromegaly, limb dysplasia, Morquio syndrome, Kniest syndromeSyndrome, postnatal nutritional dysplasia, and spondyloepimetaphyseal dysplasia. In various embodiments, CNP variants are suitable as adjunctive or alternative growth hormones for the treatment of idiopathic short stature and other skeletal dysplasia. In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner's syndrome / Leri Weill) or PTPN11 mutations (Noonan's syndrome). In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by NPR2 mutations, SHOX mutations (Turner syndrome / Lerreville), or PTPN11 mutations (Nunan syndrome), or insulin growth factor 1 receptor (IGF1R). In various embodiments, CNP variants are suitable for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, or FGFR3. In various embodiments, growth plate disorders or short stature are associated with one or more mutations in genes related to RASopathy. In various embodiments, bone-related disorders, skeletal dysplasia, or short stature are caused by Lasso disease. In various embodiments, Lasso disease is Noonan syndrome, Costello syndrome, cardiomyoplasmosis, neurofibromatosis type 1, or Leopard syndrome. In one embodiment, Lasso disease is hereditary type 1 gingival fibromatosis. In various embodiments, CNP variants are suitable for treating growth plate disorders and short stature, including familial short stature, dominant familial short stature (also known as dominant hereditary short stature), or idiopathic short stature. In various embodiments, short stature or growth plate disorders are the result of mutations in collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R). In various embodiments, growth plate syndrome or short stature is associated with one or more mutations in genes related to Lasso disease. In various embodiments, the CNP variant is suitable for treating individuals with short stature who have a height SDS of less than -1.0, -1.5, -2.0, -2.5, or -3.0, and at least one parent has a height SDS of less than -1.0, -1.5, -2.0, or -2.5, whereby the second parent's height is within the normal range. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -3.0. In various embodiments, the CNP variant is suitable for treating individuals with a height SDS of -2.0 to -2.5. In various embodiments, short stature is associated with mutations in one or more genes related to short stature, such as collagen (COL2A1, COL11A1, COL9A2, COL10), agglutinin (ACAN), Indian hedgehog factor (IHH), PTPN11, NPR2, NPPC, FGFR3, or insulin-like growth factor 1 receptor (IGF1R), or combinations thereof. In various embodiments, growth plate syndrome or short stature is associated with mutations in one or more genes related to Lasso disease. In various embodiments, short stature is the result of mutations in multiple genes, as determined by a polygenic risk score (PRS). In various embodiments, an individual has a mutation in NPR2 and a low PRS. In various embodiments, an individual has a mutation in FGFR3 and a low PRS. In various embodiments, an individual has a mutation in NPR2 and a low PRS. In various embodiments, an individual has a mutation in IGF1R and a low PRS. In various embodiments, an individual has a mutation in NPPC and a low PRS. In various embodiments, an individual has a mutation in SHOX and a low PRS. In various embodiments, an individual has one or more mutations in FGFR3, IGF1R, NPPC, NPR2, and SHOX and a low PRS. In various embodiments, PRS is 1 or 2. In various embodiments, PRS is 1. In various embodiments, PRS is 2. The polygenic risk score (PRS) for height is calculated as described in Example 4. PRS 1 refers to the shortest height, and PRS 5 refers to the tallest height. In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker. In various embodiments, this disclosure provides a method for bone lengthening or increasing long bone growth in an individual in need, comprising administering to the individual a composition including a CNP variant described herein, wherein the administration causes bone lengthening or increasing long bone growth. In various embodiments, the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly. In various embodiments, the composition is administered once daily, once weekly, once every two weeks, once every three weeks, once every four weeks, once every six weeks, once every two months, once every three months, or once every six months. In various embodiments, the composition is a delayed-release composition. A method for treating CNP-responsive symptoms or conditions has been further considered, comprising administering to an individual a CNP variant or composition as described herein, and monitoring the levels of at least one bone-related or cartilage-related biomarker in the individual, wherein an increase in the level of at least one bone-related or cartilage-related biomarker indicates a therapeutic effect of the CNP peptide or variant on the individual or the symptom or condition. A method for overcoming cell growth inhibition induced by constitutively active mutant fibroblast growth factor receptor 3 (FGFR-3) was further considered, comprising contacting cells expressing constitutively active FGFR-3 with CNP variants or compositions as described herein. A method for stimulating cGMP production in cells expressing natriuretic natriuretic peptide receptor B (NPR-B) has been further considered, comprising contacting NPR-B cells with a CNP variant or composition as described herein. In various embodiments, the method further includes adjusting the dosage or frequency of CNP peptides or variants as described herein, wherein i) the dosage or frequency of CNP peptides or variants is increased if the content of at least one bone-related or cartilage-related biomarker is below a target content; or ii) the dosage or frequency of CNP peptides or variants is decreased if the content of at least one bone-related or cartilage-related biomarker is above a target content. In various embodiments, at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen and its fragment precursor peptide, type II collagen and its fragment, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I procollagen precursor peptide (PINP) and its fragment precursor peptide, type I collagen and its fragment, chondroitin sulfate, collagen X, and alkaline phosphatase. In various embodiments, the CNP variant is PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (Pro-Gly-CNP-37) (SEQ ID NO: 1). In various embodiments, the peptide further comprises an acetyl group. In various embodiments, the acetyl group is located at the N-terminus of the peptide. In various embodiments, the acetyl group is located on an amino acid side chain within the peptide sequence. In various embodiments, the peptide further comprises an OH or NH2 group at the C-terminus. In various embodiments, the variant comprises one or more linker groups as described herein. In various embodiments, the linker is a hydrolyzable linker. A method for preparing the CNP variant described herein is also provided, comprising synthesizing peptides on a solid-phase resin using Fmoc amino acids. In various embodiments, the method includes acetylation of the peptide by reacting a resin with NMP / Ac2O / DIEA (10:1:0.1, v / v / v). In various embodiments, the method includes conjugating the peptide with a conjugated portion present, as appropriate, on a lysine residue. In various embodiments, the method includes cleaving a protective amino group on the lysine, reacting the peptide with 2× Fmoc-aminoPEG(2), followed by reacting with an amino acid, and then conjugating the lipid or fatty acid portion.

Implementation Method

Claims

1. A variant of a C-type natriuretic peptide (CNP) selected from the group consisting of: PGQEHPQARRYRGAQRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 5); PGQEHPNARKYKGANKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 1); PGQEHPNARRYRGANRRGLSRGCFGLKLDRIGSMSGLGC (SEQ ID NO: 6); and PGQEHPQARKYKGAQKKGLSKGCFGLKLDRIGSMSGLGC (SEQ ID NO: 7); and a moiety comprising an AEEA-AEEA-γGlu-C18DA structure, wherein the AEEA-AEEA-γGlu-C18DA structure is misaligned with a lysine residue (K) of the CNP variant within the CNP cyclic domain.

2. A variant of claim 1, wherein the lysine residue is located at position 27 of the amino acid sequence of SEQ ID NO: 1 and 5-7.

3. A variant of claim 1 or 2, wherein the peptide is PGQEHPNARKYKGANKKGLSKGCFGLK(AEEA-AEEA-γGlu-C18DA)LDRIGSMSGLGC-OH (SEQ ID NO: 70).

4. A variant of claim 1 or 2, wherein the AEEA-AEEA-γGlu-C18DA structure is misaligned with the lysine residue via a linker.

5. A variant of claim 4, wherein the linker is a hydrolyzable linker.

6. A variant of claim 4, wherein the linker is a peptide linker.

7. A variant of claim 6, wherein the AEEA-AEEA-γGlu-C18DA structure and the peptide linker together comprise the structure: , wherein “ ” denotes the connection point with the hydrolyzable linker, and wherein the hydrolyzable linker is connected to the lysine residue within the cyclic domain of the CNP variant.

8. A variant of claim 4, wherein the linker is an electronic linker.

9. A variant of claim 8, wherein the AEEA-AEEA-γGlu-C18DA structure and the electronic linker together comprise the structure: , wherein “ ” represents the connection point with the hydrolyzable linker, and wherein the hydrolyzable linker is connected to the lysine residue within the cyclic domain of the CNP variant.

10. A variant of claim 1 or 2, wherein the peptide is prepared synthetically.

11. A pharmaceutical composition comprising a variant of the CNP as claimed in any one of claims 1 to 10, and a pharmaceutically acceptable excipient, carrier, or diluent.

12. The pharmaceutical composition of claim 11, which is a lyophilized formulation prepared from a compound containing an isotonic modifier or a building agent selected from the group consisting of mannitol, sucrose, sorbitol and combinations thereof; and / or wherein the lyophilized formulation is prepared from a compound further comprising an antioxidant selected from the group consisting of methionine, ascorbic acid, salts of ascorbic acid, thioglycerol and combinations thereof.

13. Use of a variant of any of claims 1 to 10 or a pharmaceutical composition of claims 11 or 12 for the preparation of a medicament for treating an individual’s bone-related conditions or skeletal dysplasia, or for bone lengthening or increasing the growth of long bones.

14. As requested in item 13, wherein the bone-related condition or skeletal dysplasia is selected from the group consisting of: osteoarthritis, hypophosphatemic rickets, achondroplasia, hypochondroplasia, short stature, dwarfism, osteochondrodysplasias, lethal dysplasia, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, campomelic dysplasia, congenital lethal hypophosphatasia, and perinatal lethal type of osteogenesis. Imperfecta, Short-rib Polydactyly Syndromes, Rhizomelic Type of Chondrodysplasia Punctata, Jansen-type Metaphyseal Dysplasia, Spondyloepiphyseal Dysplasia Congenita, Osteogenesis Imperfecta, Degenerative Dysplasia, Congenital Short Femur, Langer-type Mesomelic Dysplasia, Nievergelt-type Mesomelic Dysplasia, Robinow Syndrome, Reinhardt Syndrome, Acrodysostosis, Peripheral Bone Developmental Disorders, Kniest Dysplasia, Fibrochondrosis, Roberts Syndrome Syndrome, acromegaly, limb dysplasia, Morquio syndrome, Kniest syndromeSyndrome, spondyloepimetaphyseal dysplasia, NPR2 mutation, SHOX mutation (Turner's syndrome / Leri Weill), PTPN11 mutation (Noonan's syndrome), insulin growth factor 1 receptor (IGF1R) mutation, and idiopathic short stature.

15. As claimed in claim 13 or 14, wherein the composition is administered subcutaneously, intradermally, intra-articularly, orally, or intramuscularly.

16. As claimed in claim 13 or 14, wherein the composition is a delayed-release composition.

17. Use of a variant of any one of claims 1 to 10 or a pharmaceutical composition of claim 11 or 12 for the preparation of a medicament for treating CNP-responsive symptoms or conditions, wherein the treatment comprises administering to an individual a variant of any one of claims 1 to 10 or a pharmaceutical composition of claim 11 or 12, and monitoring the levels of at least one bone-related or cartilage-related biomarker in the individual, wherein an increase in the level of the at least one bone-related or cartilage-related biomarker indicates the therapeutic effect of the CNP variant on the individual or the symptom or condition.

18. As claimed in claim 17, wherein the treatment further comprises adjusting the dosage or frequency of the CNP variant, wherein i) the dosage or frequency of the CNP variant is increased if the content of at least one bone-related or cartilage-related biomarker is below a target content; or ii) the dosage or frequency of the CNP variant is decreased if the content of at least one bone-related or cartilage-related biomarker is above a target content.

19. As claimed in claims 17 or 18, wherein the at least one bone-related or cartilage-related biomarker is selected from the group consisting of: CNP, cGMP, type II collagen and its fragment precursor peptide, type II collagen and its fragment, type I collagen C-terminal peptide (CTx), osteocalcitonin, proliferating cell nuclear antigen (PCNA), type I procollagen precursor peptide (PINP) and its fragment precursor peptide, type I collagen and its fragment, chondroitin sulfate, collagen X, and alkaline phosphatase.

20. A method for preparing a variant of any one of claims 1 to 10, comprising synthesizing the peptide on a solid-phase resin using Fmoc amino acids.

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