Methods and materials for treating skeletal disorders

Abstract

Methods and materials for treating skeletal disorders are provided herein. For example, methods and materials for using a dual guanylate cyclase A and B activator (e.g., a polypeptide having the sequence set forth in SEQ ID NO:1) as a therapeutic agent for the treatment of skeletal disorders (e.g., achondroplasia) are provided herein.

Description

[0001] Attorney Docket No. 07039-2324WO1

[0002] 2024-161

[0003] METHODS AND MATERIALS FOR TREATING SKELETAL DISORDERS

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS

[0005] This application claims priority from U.S. Provisional Application Serial No. 63 / 705.140, filed October 9, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0006] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0007] This invention was made with government support under HL132854, HL135790, and HL171847 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] SEQUENCE LISTING

[0009] This application contains a Sequence Listing that has been submitted electronically as an XML file named 07039-2324WOl_SL_ST26.xml. The XML file, created on August 19, 2025, is 15,491 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety'.

[0010] TECHNICAL FIELD

[0011] This document relates to methods and materials for using a dual guanylate cyclase A and B activator (e.g., MV81) as a therapeutic agent for the treatment of diseases responsive to particulate guanylyl cyclase receptors and cGMP activation. For example, this document relates to methods and materials for using MV81 to treat skeletal disorders (e.g., achondroplasia).

[0012] BACKGROUND

[0013] Skeletal disorders encompass a range of disorders affecting the bones, joints, and / or connective tissues. A number of factors can cause skeletal disorders, including injury', aging, nutrition, lifestyle factors, and / or genetics. Achondroplasia is a ty pe of skeletal disorder that affects the grow th of bones and cartilage. Currently, there is no cure for achondroplasia. Attorney Docket No. 07039-2324WO1

[0014] 2024-161

[0015] SUMMARY

[0016] This document is based, at least in part, on the identification of dual guanylate cyclase A and B activators that can promote bone growth, increase bone mass, and or increase bone strength. As demonstrated herein, a dual guanylate cyclase A and B activator (e.g., MV81) can induce a dose dependent increase in plasma cGMP levels in mice and rats. In addition, administration of MV81 to mice can increase tibia length and body length (e.g., naso-anal length, anal-tail length, and total length).

[0017] This document provides methods and materials for treating mammals having skeletal disorders. For example, this document provides methods and materials for using a dual guanylate cyclase A and B activator (e.g.. MV81) to treat mammals having achondroplasia, and / or other skeletal disorders, such as hypochondroplasia. thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, grow th hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), Lacrimo-Auriculo-Dento-Digital syndrome (LADD), Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans (SADDAN), osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome. In some cases, a dual guanylate cyclase A and B activator (e.g., MV81) can be used to treat mammals having short stature that may be related to FGFR3-related skeletal dysplasia and / or related / associated cardiorenal-metabolic comorbidities.

[0018] In a first aspect, this document provides a method for treating a skeletal disorder. The method can include or consist essentially of administering, to a mammal identified as having the skeletal disorder, a composition that contains a natriuretic peptide including the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two. three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NOT are maintained and are the only two cysteine residues of the natriuretic peptide. The mammal can be a human. The mammal can be a monkey, a dog, a cat, a pig. a horse, a cow. a sheep, a goat, a rabbit, a mouse, or a rat. The method can include administering the composition to the mammal in an amount Attorney Docket No. 07039-2324WO1 2024-161 effective to increase bone length, increase bone strength, increase bone density, and / or improve bone shape in the mammal. The method can include administering the composition to the mammal in an amount effective to reduce impaired bone growth in the mammal. The skeletal disorder can be achondroplasia. The skeletal disorder can be hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo-Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-ty pe mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3-related skeletal dysplasia. The composition can include a natriuretic peptide containing the amino acid sequence set forth in SEQ ID NO:1. The composition can include a natriuretic peptide containing the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide.

[0019] In another aspect, this document features the use of a composition containing a natriuretic peptide for treating a skeletal disorder in a mammal, wherein the natriuretic peptide includes the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NO: 1 are maintained and are the only tw o cysteine residues of the natriuretic peptide. The mammal can be a human. The mammal can be a monkey, a dog, a cat, a pig, a horse, a cow, a sheep, a goat, a rabbit, a mouse, or a rat. The composition can contain the natriuretic peptide in an amount effective to increase bone length, increase bone strength, increase bone density, and / or improve bone shape in the mammal. The composition can contain the natriuretic peptide in an amount effective to reduce impaired bone growth in the mammal. The skeletal disorder can be achondroplasia. The skeletal disorder can be hypochondroplasia, thanatophoric dysplasia type 1 and type 2. SHOX deficiency, Noonan syndrome. Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short Attorney Docket No. 07039-2324WO1 2024-161 stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo-Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3-related skeletal dysplasia. The composition can include a natriuretic peptide containing the amino acid sequence set forth in SEQ ID NO:1. The composition can include a natriuretic peptide containing the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six. seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0021] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0022] DESCRIPTION OF DRAWINGS

[0023] FIG. 1 includes a pair of graphs plotting plasma cGMP levels (left) and the change in urinary cGMP response (right) after an acute intravenous administration of MV81or vehicle in rats. **P<0.01, ****p<0.0001 versus baseline within group.

[0024] FIG. 2 is a graph showing the plasma cGMP response after chronic subcutaneous administration of MV81 (25. 50. or 100 pmol / kg / min) or vehicle (0 pmol / kg / min) for 28 days in normal wild-type mice. Attorney Docket No. 07039-2324WO1

[0025] 2024-161

[0026] FIG. 3 is a graph showing the effect on tibia length after chronic subcutaneous administration of MV81 (25. 50. or 100 pmol / kg / min) or vehicle (0 pmol / kg / min) for 28 days in normal wild-type mice.

[0027] FIG. 4 is a graph showing the correlation between plasma cGMP levels and tibia length after chronic subcutaneous administration of MV81or vehicle for 28 days in normal wild-type mice.

[0028] FIG. 5 is a graph plotting naso-anal length at baseline and after chronic subcutaneous administration of MV81 or vehicle for 28 days in normal wild-type mice.

[0029] FIG. 6 is a graph plotting the percent change in naso-anal length after chronic subcutaneous administration of MV81 or vehicle (0 pmol / kg / min) for 28 days in normal wild-type mice.

[0030] FIG. 7 is a graph showing correlation between plasma cGMP levels and the percent change in naso-anal length after chronic subcutaneous administration of MV81 or vehicle for 28 days in normal wild-ty pe mice.

[0031] FIG. 8 is a graph plotting anal-tail length at baseline and after chronic subcutaneous administration of MV81(25, 50, or 100 pmol / kg / min) or vehicle for 28 days in normal wild-ty pe mice.

[0032] FIG. 9 is a graph plotting the percent change in anal-tail length after chronic subcutaneous administration of MV81(25, 50, or 100 pmol / kg / min) or vehicle (0 pmol / kg / min) for 28 days in normal wild-type mice.

[0033] FIG. 10 is a graph showing the correlation between plasma cGMP levels and the percent change in anal -tail length after chronic subcutaneous administration of MV81 or vehicle for 28 days in normal wild-type mice.

[0034] FIG. 11 is a graph plotting the total length at baseline and after chronic subcutaneous administration of MV81 or vehicle for 28 days in normal wild-type mice.

[0035] FIG. 12 is a graph plotting the percent change in total length after chronic subcutaneous administration of MV81(25, 50, or 100 pmol / kg / min) or vehicle (0 pmol / kg / min) for 28 days in normal wild-type mice.

[0036] FIG. 13 is a graph showing the correlation between plasma cGMP levels and the percent change in total length after chronic subcutaneous administration of MV81 or vehicle for 28 days in normal wild-ty pe mice.

[0037] FIG. 14 is a graph showing the effect on tibia length after chronic subcutaneous administration of MV81 (50 pmol / kg / min) or vehicle for 3 months in R403Q mice. Attorney Docket No. 07039-2324WO1 2024-161

[0038] FIG. 15 is a graph plotting naso-anal length at baseline and at 1, 2, and 3 months after chronic subcutaneous administration of MV81(50 pmol / kg / min) or vehicle in R403Q mice. ****p<0.0001 versus baseline within the MV81 group.

[0039] FIG. 16 is a graph plotting anal -tail length at baseline and at 1, 2, and 3 months after chronic subcutaneous administration of MV81(50 pmol / kg / min) or vehicle for 3 months in R403Q mice. ****p<0.0001 versus baseline within MV81 group.

[0040] FIG. 17 is a graph plotting the change in total length a at baseline and at 1, 2, and 3 months after chronic subcutaneous administration of MV81 (50 pmol / kg / min) for 3 months in R403Q mice. **P<0.01 and ****p<0.0001 versus baseline within MV81 group.

[0041] FIG. 18 is a graph plotting tibia length in wild-type mice that underwent TAC for three weeks and were then treated with MV81 (50 pmol / kg / min) or vehicle for three weeks.

[0042] DETAILED DESCRIPTION

[0043] This document provides methods and materials for treating mammals having skeletal disorders, where the methods include administering to the mammals a dual guanylate cyclase A and B activator. As used herein, a “dual guanylate cyclase A and B activator” is a molecule that is capable of activating both guanylate cyclase A and guanylate cyclase B. In some cases, the dual guanylate cyclase A and B activator can be a natriuretic peptide. There are three endogenous natriuretic peptides in humans: atrial natriuretic peptide (ANP, encoded by the NPPA gene in humans), B-type natriuretic peptide (BNP, encoded by the NPPB gene in humans), and C-type natriuretic peptide (CNP, encoded by the NPPC gene in humans). There also are three natriuretic peptide receptors: guanylate cyclase A (GC-A, also referred to as NPR-A or NPR1), guanylate cyclase B (GC-B, also referred to as NPR-B or NPR2), and natriuretic peptide receptor C (NPR-C, also referred to as NPR3). ANP and BNP bind with high affinity to GC-A, whereas CNP binds with high affinity to GC-B. Both GC-A and GC-B contain intracellular domains that catalyze production of cyclic guanosine monophosphate (cGMP). The binding of ligands to GC-A or GC-B results in the production of cGMP, a key second messenger that participates in downstream signaling that leads to a variety of biological effects. Excess cGMP produced by GC-A and / or GC-B is egressed out of the cell, which can be measured in plasma. Thus, plasma cGMP is a biomarker of GC-A and / or GC-B target engagement. Attorney Docket No. 07039-2324WO1 2024-161

[0044] Non-limiting examples of skeletal disorders that can be treated using the methods described herein include achondroplasia, and / or other skeletal disorders such as hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, S ADD AN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities.

[0045] This document provides methods and materials for using a dual guanylate cyclase A and B activator to treat skeletal disorders. In some cases, a dual guanylate cyclase A and B activator can be a natriuretic peptide. For example, a natriuretic peptide such as MV81 (SEQ ID NO: 1) can be used to treat a skeletal condition as described herein. In some cases, MV81 can be used to treat mammals (e g., humans) having achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency. Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozy gous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities.

[0046] The methods and materials provided herein can be used to treat a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, Attorney Docket No. 07039-2324WO1 2024-161 idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets. CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia. SADDAN. osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities) in any appropriate mammal. Mammals that can be treated using the methods and materials provided herein include, without limitation, humans, monkeys, dogs, cats, pigs, horses, cows, sheep, goats, rabbits, mice, and rats. A mammal that can be treated using the methods and materials provided herein can be any appropriate age (e g., an infant, a toddler, a juvenile, or an adult).

[0047] The methods and materials provided herein can include using a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or having the amino acid sequence set forth in SEQ ID NO: 1 with one to ten (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide. Examples of such natriuretic peptides include, without limitation, those set forth in TABLE 1. Peptides having the sequences set forth in TABLE 1 can form ring structures due to disulfide bonds between the cysteine residues, which are underlined.

[0048] TABLE 1. Exemplary natriuretic peptides Attorney Docket No. 07039-2324WO1 2024-161

[0049] In some cases, an amino acid substitution can be a conservative amino acid substitution. For example, an amino acid residue of SEQ ID NO: 1 can be selected and replaced with an amino acid residue that does not differ significantly in its effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity7of the molecule at the target site, or (c) the bulk of the side chain. For example, naturally occurring residues can be divided into groups based on side-chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine); (2) neutral hydrophilic amino acids (cysteine, serine, and threonine); (3) acidic amino acids (aspartic acid and glutamic acid); (4) basic amino acids (asparagine, glutamine, histidine, lysine, and arginine); (5) amino acids that influence chain orientation (glycine and proline); and (6) aromatic amino acids (tryptophan, tyrosine, and phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Non-limiting examples of conservative substitutions include, without limitation, substitution of valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, leucine for Attorney Docket No. 07039-2324WO1

[0050] 2024-161 isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenyalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and / or leucine for valine. Further examples of conservative substitutions that can be made within SEQ ID NO:1 are set forth in TABLE 2.

[0051] TABLE 2. Examples of conservative amino acid substitutions

[0052] In some cases, an amino acid substitution can be a non-conservative amino acid substitution. For example, an amino acid residue of SEQ ID NO: 1 can be selected and replaced with an amino acid residue from a different class of amino acids as compared with the selected amino acid. For example, an acidic amino acid (aspartic acid or glutamic acid) present in SEQ ID NO:1 can be selected and replaced with a basic amino acid (asparagine, glutamine, histidine, lysine, or arginine). A polypeptide described herein (e.g.. a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, or having the amino acid sequence set forth in SEQ ID NO: 1 with one to ten amino acid additions, deletions, or substitutions, provided that the tw o cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide) can be obtained using any appropriate technique Attorney Docket No. 07039-2324WO1 2024-161 including, without limitation, solid phase polypeptide synthesis techniques such as those involving the use of an Applied BioSystems (Foster City, CA) Peptide Synthesizer or a Biosearch Inc. (San Rafael, CA) automatic peptide synthesizer. Disulfide bonds between cysteine residues can be introduced by mild oxidation of the linear polypeptides using KCN as described elsewhere (see, e.g., U.S. Patent No. 4,757,048). In some cases, a polypeptide described herein (e.g.. a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, or having the amino acid sequence set forth in SEQ ID NO: 1 with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NOT are maintained and are the only two cysteine residues of the natriuretic peptide) can be obtained recombinantly. For example, a polypeptide described herein (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1) can be obtained by expressing a recombinant nucleic acid encoding the polypeptide such as an expression vector encoding a polypeptide described herein within host cells. The resulting polypeptide then can be purified using, for example, affinity chromatographic techniques and HPLC. The extent of purification can be measured using any appropriate method including, without limitation, column chromatography, polyacrylamide gel electrophoresis, or high-performance liquid chromatography. In some cases, a polypeptide described herein (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NOT. or having the amino acid sequence set forth in SEQ ID NO: 1 with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide) can be designed or engineered to contain a tag sequence that allows the polypeptide to be purified (e.g., captured onto an affinity' matrix) and / or an enzy me or label sequence that allows the polypeptide to be detected. For example, a tag such as c- myc, hemagglutinin, polyhistidine, or FLAG™ tag (Kodak) can be used to aid polypeptide purification. Examples of enzyme and label sequences that can be used to aid polypeptide detection include, without limitation, alkaline phosphatase, GFP, RFP, luciferases, and Cas polypeptides. Such tags, enzymes, and labels can be inserted anywhere within the polypeptide, including at either the carboxyl terminus or the amino terminus. In some cases, the polypeptide described herein can have the structure and can be obtained as described elsewhere (see, WO 2021 / 183928).

[0053] Any appropriate method can be used to confirm that a polypeptide useful in the methods provided herein (e.g.. a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, or having the amino acid sequence set forth in SEQ ID NO: 1 with one to Attorney Docket No. 07039-2324WO1 2024-161 ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide) has one or more biological activities. For example, a polypeptide described herein can be confirmed as having a biological (e.g., promoting chondrocyte proliferation and differentiation) activity in vitro by, for example, measuring its effect on intracellular cGMP levels in chondrocytes. For example, a polypeptide described herein can be confirmed as having a biological (e.g., bone growth) activity in vivo by, for example, testing its effects on factors such as plasma cGMP generation, tibia length, and / or body length.

[0054] In some cases, a polypeptide described herein (e.g., a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1, or having the amino acid sequence set forth in SEQ ID NO: 1 with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of the natriuretic peptide) can be formulated as a pharmaceutical composition by admixture with one or more pharmaceutically acceptable non-toxic excipients or carriers. Pharmaceutical compositions can be prepared for parenteral administration, for example, in the form of liquid solutions or suspensions in aqueous physiological buffer solutions; for oral administration, for example, in the form of tablets or capsules; or for intranasal administration, for example in the form of powders, nasal drops, or aerosols. Compositions for other routes of administration can be prepared as desired using appropriate methods.

[0055] Formulations for parenteral administration can include as common excipients, sterile water, saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes, and combinations thereof. In some cases, biocompatible, biodegradable lactide polymer, lactide / glycolide copolymer, polyoxethylene-polyoxypropylene copolymers, or combinations thereof can be used as excipients for controlling the release of the polypeptide in vivo. Other suitable parenteral delivery’ systems that can be used include, without limitation, ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, liposomes, and combinations thereof. Formulations for inhalation administration can include excipients such as lactose. Inhalation formulations can be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate, deoxy cholate, or combinations thereof, or they can be oily solutions for administration in the form of nasal drops. If desired, a composition containing a polypeptide described herein (e.g., a polypeptide having the Attorney Docket No. 07039-2324WO1 2024-161 amino acid sequence set forth in SEQ ID NO: 1) can be formulated as a gel to be applied intranasally. Formulations for parenteral administration can include glycocholate for buccal administration.

[0056] For oral administration, tablets or capsules can be prepared using appropriate methods with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium laury l sulfate). Tablets can be coated using appropriate methods. Preparations for oral administration can be formulated to give controlled release of the polypeptide.

[0057] Nasal preparations can be presented in a liquid form or as a dry product. Nebulised aqueous suspensions or solutions can include carriers or excipients to adjust pH and / or tonicity.

[0058] This document also provides methods and materials for treating a mammal (e.g., a human) that has a skeletal disorder, is developing a skeletal disorder, and / or is likely to develop a skeletal disorder (e.g., a skeletal disorder such as achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets. CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, S ADD AN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities). For example, a mammal can be treated with one or more natriuretic peptides to increase bone length in the mammal, and / or to reduce a sign, symptom, or complication of a skeletal disorder described herein, and / or to counteract a sign, symptom, or complication of a skeletal disorder described herein. Signs, symptoms, and complications of skeletal disorders such as those described herein can include, without limitation, dwarfism, limb shortening, short fingers and toes, trident hands, varus Attorney Docket No. 07039-2324WO1 2024-161 or valgus deformities, spinal ky phosis or lordosis, central sleep apnea, obstructive sleep apnea, frequent ear infections, and / or hydrocephalus. Examples of natriuretic peptides that can be used alone or in combination to treat a mammal as described herein (e.g., to reduce or counteract a sign, symptom, or complication of a skeletal disorder described herein) include, without limitation, a polypeptide having the sequence set forth in SEQ ID NO: 1, and polypeptides having the sequence set forth in SEQ ID NO: 1 but with one to ten (e.g., one, two, three, four, five, six, seven, eight, nine, or ten) amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide.

[0059] In some cases, one or more additional therapeutic agents also can be administered to a mammal (e.g.. a human) in addition to one or more natriuretic peptides, in order to reduce a symptom of skeletal disorder described herein, and / or to reduce a symptom of a condition occurring in the mammal along with the skeletal disorder. For example, mammals (e.g.. adult humans) with achondroplasia also may have cardiovascular, renal, and / or metabolic comorbidities. In such cases, a mammal having a skeletal disorder as described herein along with a cardiovascular, renal, and / or metabolic can be treated with a natriuretic peptide described herein and one or more additional therapeutic agents such as, without limitation, an ACE inhibitor, an angiotensin II receptor blocker (e.g., a neprilysin inhibitor such as sacubitril-valsartan). an aldosterone antagonist, a diuretic (e.g.. furosemide, bumetanide. ethacrynic acid, torsemide. acetazolamide, dorzolamide, amiloride, spironolactone, eplerenone, triamterene, potassium canrenoate, bendroflumethiazide, or hydrochlorothiazide), a beta-blocker, and / or a statin. The one or more natriuretic peptides and the one or more additional therapeutic agents can be administered simultaneously (e.g.. in the same composition or in separate compositions that are administered at the same time), or sequentially.

[0060] Any appropriate route of administration can be used to administer a composition containing one or more natriuretic peptides described herein (or one or more natriuretic peptides described herein in combination with one or more additional therapeutic agents) to treat a mammal (e.g., a human) that has skeletal disorder, is developing skeletal disorder, and / or is likely to develop a skeletal disorder (e g., a skeletal disorder such as achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, Attorney Docket No. 07039-2324WO1 2024-161 osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities). For example, parenteral administration (e.g., by subcutaneous, intrathecal, intraventricular, intramuscular, or intraperitoneal injection, or by intravenous drip) can be used to administer a composition containing one or more natriuretic peptides described herein (or one or more natriuretic peptides described herein in combination with one or more additional therapeutic agents) to a mammal (e.g., a human). In some cases, the administration can be rapid (e.g., by injection) or can occur over a period of time (e.g., by slow infusion or administration of slow release formulations). In some cases, a composition containing one or more natriuretic peptides described herein (or one or more natriuretic peptides described herein in combination with one or more additional therapeutic agents) can be administered to a mammal (e.g., a human) via topical administration (e.g., transdermal, sublingual, ophthalmic, or intranasal), pulmonary administration (e.g., by inhalation or insufflation of powders or aerosols), or oral administration.

[0061] In some cases, a mammal having a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia ty pe 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets. CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities) can be instructed to self-administer one or more Attorney Docket No. 07039-2324WO1 2024-161 natriuretic peptides described herein (and, in some cases, one or more additional therapeutic agents).

[0062] A composition containing one or more natriuretic peptides, such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or containing one or more such natriuretic peptides in combination with one or more additional therapeutic agents, can be administered to a mammal (e.g., a human) in an appropriate amount, at an appropriate frequency, and for an appropriate duration effective to achieve a desired outcome (e.g., increased bone length, improved bone shape, increased density, and / or increased strength).

[0063] In some cases, a composition containing one or more natriuretic peptides, such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO:1. or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or containing one or more such natriuretic peptides in combination with one or more additional therapeutic agents, can be administered to a mammal identified as having a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia t pe 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, grow th hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson -Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur. Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal -metabolic comorbidities) to increase bone length, bone strength, or bone density by, for example, at least 5 percent (e.g., at least 10 percent, at least 15 percent, at least 20 percent, at least 25 percent, or at least 50 percent, at least 75 percent, or 100 percent), or to improve bone shape. Any Attorney Docket No. 07039-2324WO1 2024-161 appropriate method can be used to determine whether or not a mammal (e.g., a human) has experienced increased bone length (e.g.. X-ray, computerized tomography (CT) scan, magnetic resonance imaging (MRI), nuclear scan, dual-energy X-ray absorptiometry (DEXA), or anthropometric measurements). Any appropriate method can be used to determine whether or not a mammal (e.g., a human) has experienced increased bone strength (e.g., DEXA or CT scan). Any appropriate method can be used to determine whether or not a mammal (e.g., a human) has experienced increased bone densify (e.g., DEXA or CT scan). Any appropriate method can be used to determine whether or not a mammal (e.g., a human) has experienced improved bone shape (e.g., X-ray, CT scan, MRI, nuclear scan, DEXA, or anthropometric measurements).

[0064] In some cases, a composition containing one or more natriuretic peptides, such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO:1 , or a polypeptide having the sequence set forth in SEQ ID NO:1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or containing one or more such natriuretic peptides in combination with one or more additional therapeutic agents, can be administered to a mammal identified as having a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal -metabolic comorbidities) to reduce impaired bone growth by, for example, at least 5 percent (e.g., at least 10 percent, at least 25 percent, at least 50 percent, at least 75 percent, or 100 percent). Any appropriate method can be used to determine whether or not a mammal (e.g., a human) has experienced a reduction in impaired bone growth, including X-ray, CT scan, MRI, nuclear scan, DEXA, or anthropometric measurements. Attorney Docket No. 07039-2324WO1

[0065] 2024-161

[0066] An effective amount of a composition containing one or more natriuretic peptides, such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more such natriuretic peptides in combination with one or more additional therapeutic agents can be any amount that increases bone length, increases bone strength, increases bone density, improves bone shape, and / or reduces impaired bone growth in a mammal having a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-tj pe mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabohc comorbidities), without producing significant toxicity to the mammal. An effective amount can vary depending on the relative potency of the peptide and / or the route of administration, and can generally be estimated based on EC50 found to be effective in in vitro and in vivo animal models.

[0067] In some cases, an effective amount of a natriuretic peptide described herein (e.g., a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO:1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide) that is subcutaneously administered can be from about 0.001 pg / kg to about 500 pg / kg of body weight. For example, an effective amount of a subcutaneously administered natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1 can be from about 0.001 pg / kg to about 0.01 pg / kg, from about 0. 1 pg / kg to about 50 pg / kg, from about 0.01 pg / kg to about 0. 1 pg / kg, from about 0. 1 pg / kg to about 0.5 pg / kg, from about 0.5 Attorney Docket No. 07039-2324WO1 2024-161 pg / kg to about 1 ptg / kg, from about 1 pg / kg to about 10 .g / kg, from about 10 pg / kg to about 50 |ig / kg, from about 50 pg / kg to about 100 pg / kg. or from about 100 pg / kg to about 500 pg / kg.

[0068] In some embodiments, an effective amount of a natriuretic peptide described herein (e.g., a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide) that is subcutaneously administered can be from about 0.05 pg to about 10 mg. For example, an effective amount of a subcutaneously administered natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1 can be from about 0.05 pg to about 10 mg, from about 0.5 pg to about 1 mg, from about 0.5 pg to about 5 pg, from about 5 pg to about 10 pg, from about 10 pg to about 50 pg, from about 50 pg to about 100 pg, from about 100 pg to about 500 pg, from about 500 pg to about 1 mg, from about 1 mg to about 2.5 mg, from about 2.5 mg to about 5 mg, or from about 5 mg to about 10 mg.

[0069] In some embodiments, an effective amount of a natriuretic peptide described herein (e.g., a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide) that is orally administered can be from about 0.05 mg to about 1000 mg. For example, an effective amount of an orally administered natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1 can be from about 0.05 mg to about 750 mg, from about 0.5 mg to about 100 mg. from about 0.5 mg to about 1 mg, from about 1 mg to about 5 mg, from about 5 mg to about 10 mg, from about 10 mg to about 50 mg, from about 50 mg to about 100 mg, from about 100 mg to about 200 mg, from about 200 mg to about 500 mg, or from about 500 mg to about 1000 mg.

[0070] If a particular mammal fails to respond to a particular amount, then the amount can be increased by, for example, two-fold. After receiving this higher amount, the subject can be monitored for both responsiveness to the treatment and toxicity, and adjustments can be made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of Attorney Docket No. 07039-2324WO1 2024-161 multiple treatment agents, route of administration, and severity of the condition (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and ty pe 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, grow th hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozy gous NPR2 mutation, osteoarthritis, craniosynostosis (e.g.. Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities) may require an increase or decrease in the actual effective amount administered.

[0071] A composition containing one or more natriuretic peptides, such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the tw o cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more natriuretic peptides in combination with one or more additional therapeutic agents can be administered once or more than once, at any' appropriate frequency and for any appropriate duration. An effective frequency for administering a composition containing one or more natriuretic peptides such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more natriuretic peptides in combination with one or more additional therapeutic agents can be any frequency that increases bone length, increases bone strength, increases bone density', improves bone strength, and / or reduces impaired bone growth, without producing significant toxicity' to the mammal. For example, the frequency of administration can be once or more daily (e.g., once, twice, three times, or four rimes daily), biweekly, weekly, monthly, or even less frequently. The frequency of administration can remain constant or can be variable during the duration of treatment. In some cases, a course of treatment can include rest periods. For example, a Attorney Docket No. 07039-2324WO1 2024-161 composition containing one or more natriuretic peptides such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more natriuretic peptides in combination with one or more additional therapeutic agents can be administered over a two month period followed by a month rest period, and then repeated. As with the effective amount, various factors can influence the actual frequency of administration. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson- Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic ty pe of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities) may require an increase or decrease in administration frequency.

[0072] An effective duration for administering a composition containing one or more natriuretic peptides such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more natriuretic peptides in combination with one or more additional therapeutic agents can be any duration that increases bone length, increases bone strength, increases bone density, improves bone shape, and / or reduces impaired bone grow th, without producing significant toxicity to the mammal. In some cases, the effective duration can vary from weeks to months or even years. In general, an effective duration for administering a composition containing one or more natriuretic peptides such as a Attorney Docket No. 07039-2324WO1 2024-161 natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide, and / or one or more natriuretic peptides in combination with one or more additional therapeutic agents can range from about 1 month to about 10 years or more (e.g., from about 1 month to about 6 months, from about 6 months to about 12 months, from about 1 year to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, or more than 10 years). In some cases, an effective duration can be for as long as the individual mammal (e.g., a human) is alive. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome, Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome). LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities).

[0073] After administering a composition containing one or more natriuretic peptides such as a natriuretic peptide having the amino acid sequence set forth in SEQ ID NO: 1, or a polypeptide having the sequence set forth in SEQ ID NO: 1 but with one to ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues are maintained and are the only two cysteine residues of the natriuretic peptide to a mammal (e.g., a human) having a skeletal disorder (e.g., achondroplasia, hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, Attorney Docket No. 07039-2324WO1 2024-161 aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis (e.g., Muenke syndrome. Crouzon syndrome, Apert syndrome, Jackson-Weiss syndrome, Pfeiffer syndrome, or Crouzonodermoskeletal syndrome), LADD, Osteoglophonic dysplasia, SADDAN, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic ty pe of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, and / or short stature related to FGFR3-related skeletal dysplasia and / or related / associated cardio-renal-metabolic comorbidities), the mammal can be monitored to determine whether or not the treatment was effective against the skeletal disorder. For example, a mammal (e.g., a human) can be assessed after treatment to determine whether bone length has increased, or skeletal function has improved. Any appropriate method can be used to assess bone length and / or skeletal function. For example, imaging (X-ray, CT, MRI, or nuclear scan), biomarkers (ALP), and / or morphometric analysis can be used to monitor an effective response to a treatment described herein. In some cases, plasma or urine cGMP can be measured in order to guide dosing or as a marker of efficacy. For example, an increase in plasma or urine cGMP levels after administration of a natriuretic peptide described herein can indicate that the administered dose was appropriate and / or that the treatment was effective.

[0074] Exemplary Embodiments

[0075] Embodiment 1 is a method for treating a skeletal disorder, wherein said method comprises administering, to a mammal identified as having said skeletal disorder, a composition that comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two, three, four, five, six. seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

[0076] Embodiment 2 is the method of embodiment 1, wherein said mammal is a human. Embodiment 3 is the method of embodiment 1, wherein said mammal is a monkey, a dog, a cat, a pig, a horse, a cow-, a sheep, a goat, a rabbit, a mouse, or a rat.

[0077] Embodiment 4 is the method of any one of embodiments 1 to 3, w herein said method comprises administering said composition to said mammal in an amount effective to increase bone length, increase bone strength, increase bone density, and / or improve bone shape in said mammal. Attorney Docket No. 07039-2324WO1 2024-161

[0078] Embodiment 5 is the method of any one of embodiments 1 to 4, wherein said method comprises administering said composition to said mammal in an amount effective to reduce impaired bone growth in said mammal.

[0079] Embodiment 6 is the method of any one of embodiments 1 to 5, wherein said skeletal disorder is achondroplasia.

[0080] Embodiment 7 is the method of any one of embodiments 1 to 5. wherein said skeletal disorder is hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, grow th hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo-Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic ty pe of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3- related skeletal dysplasia.

[0081] Embodiment 8 is the method of any one of embodiments 1 to 7, w herein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ 1D NO: 1.

[0082] Embodiment 9 is the method of any one of embodiments 1 to 7, w herein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

[0083] Embodiment 10 is a use of a composition comprising a natriuretic peptide for treating a skeletal disorder in a mammal, wherein said natriuretic peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two. three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

[0084] Embodiment 11 is the use of embodiment 10, wherein said mammal is a human.

[0085] Embodiment 12 is the use of embodiment 10, wherein said mammal is a monkey, a dog, a cat, a pig, a horse, a cow, a sheep, a goat, a rabbit, a mouse, or a rat. Attorney Docket No. 07039-2324WO1 2024-161

[0086] Embodiment 13 is the use of any one of embodiments 10 to 12, wherein said composition comprises said natriuretic peptide in an amount effective to increase bone length, increase bone strength, increase bone density', and / or improve bone shape in said mammal.

[0087] Embodiment 14 is the use of any one of embodiments 10 to 13, wherein said composition comprises said natriuretic peptide in an amount effective to reduce impaired bone growth in said mammal.

[0088] Embodiment 15 is the use of any one of embodiments 10 to 14, wherein said skeletal disorder is achondroplasia.

[0089] Embodiment 16 is the use of any one of embodiments 10 to 14, wherein said skeletal disorder is hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozy gous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo-Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozy gous achondroplasia, rhizomelic ty pe of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3- related skeletal dysplasia.

[0090] Embodiment 17 is the use of any one of embodiments 10 to 1 , wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1.

[0091] Embodiment 18 is the use of any one of embodiments 10 to 16, wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

[0092] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims. Attorney Docket No. 07039-2324WO1

[0093] 2024-161

[0094] EXAMPLES

[0095] Example 1 : Effects of MV81 treatment on body and tibia length MA TERIALS AND METHODS

[0096] Acute Administration In Vivo Rat Experiments: A total of 13 Sprague Dawley Rats (male. 250-375 g) were randomly infused with one of the following: (i) vehicle (0.9% saline, n=5), (ii) MV81 (SEQ ID NO: 1) (100 pmol / kg / min, n=4), or (iii) MV81 (600 pmol / kg / min, n=4). Investigators conducting the in vivo study were not blinded to the treatments, but biochemical analysis was performed by a different investigator who was blinded to the treatments.

[0097] On the day of the experiment, the rats were anesthetized with isoflurane. Rats were kept on a heating pad at 37°C to maintain a normal body temperature for the entire study. Vascular and bladder cannulation procedures were conducted. Briefly, a poly ethylene-50 (PE-50) tube catheter was placed into a jugular vein for saline / peptide infusion and another PE-50 tube was placed into the carotid artery’ for blood sampling. Urine samples were passively collected from the bladder through a PE-90 tube catheter. A 15-minute equilibrium period was followed by a 30-minute pre-infusion period to collect baseline urine samples. The pre-infusion period was followed by a 45-minute continuous infusion of vehicle or MV81 (15-minute lead-in drug infusion, 30-minute clearance during drug infusion). Immediately after the end of the peptide infusion, another 30- minute washout clearance period was started before termination and sacrifice. Two additional urine samples were collected during the 30-minute infusion clearance and during the 30-minute washout clearance periods, respectively. To determine the levels of circulating cGMP at baseline, at the end of drug infusion, at the middle of the washout clearance period, and at the end of the washout clearance period, four blood samples were collected and placed in tubes containing EDTA on ice. After the blood samples were centrifuged at 2500 rpm at 4°C for 10 minutes, plasma was aliquoted. All plasma and urine samples were stored at -80°C until assayed.

[0098] For the biochemical analysis, plasma and urine cGMP levels were measured with a cGMP ELISA kit (Enzo Life Sciences, Farmingdale, NY).

[0099] Normal Wild-Type Mouse Studies: Male mice (4-5 months of age) on the 129Sv / Ev background were used to assess the dose response of chronic subcutaneous infusion of MV81 via osmotic minipump (Alzet) for 28 days according to the manufacturer’s instructions. For the dosing study, mice (n=3 per group) were randomly Attorney Docket No. 07039-2324WO1 2024-161 treated with vehicle (5% dextrose in water) or MV81 (25 pmol / kg / min, 50 pmol / kg / min, or 100 pmol / kg / min). Following 28 days of infusion, mice were euthanized and tibiae were harvested. Tibia length was measured with digital calipers. Plasma was extracted from blood collected in tubes with EDTA, and stored at -80°C. Levels of plasma cGMP were measured using a commercially available ELISA (Enzo). To measure the naso-anal length and tail length, mice were anesthetized with isoflurane and placed in a supine position, and the lengths were measured using a ruler. Total length was calculated as the sum of naso-anal and anal-tail length.

[0100] R4030 Mouse Model of Hypertrophic Cardiomyopathy: The R403Q mouse is an established model of hypertrophic cardiomyopathy. R403Q mice aged 9-17 weeks were randomly treated with vehicle (5% dextrose in water) or MV81 (50 pmol / kg / min) for 3 months (n=9-l 3 per group). Every 28 days, minipumps were changed and naso-anal length and anal-tail length were measured as described above. Plasma w as extracted from blood collected in tubes with EDTA. To measure naso-anal length and tail length, mice were anesthetized with isoflurane and placed in a supine position, and the lengths were measured using a ruler. Total length was calculated as the sum of naso-anal and anal-tail length. Tibiae were extracted and their lengths were measured with digital calipers. Plasma cGMP levels were measured using a commercially available ELISA (Enzo).

[0101] Transverse Aortic Constriction in Mice: C57 / B6 mice underwent transverse aortic constriction (TAC) using a 25G needle as a model for heart failure. Following three weeks of TAC, mice w ere randomized to vehicle or MV81 (50 pmol / kg / min) via continuous subcutaneous infusion using an osmotic minipump. After three w eeks of treatment with either MV81 or vehicle, mice were sacrificed, and tibia lengths were measured using digital calipers.

[0102] RESULTS

[0103] Plasma and Urinary cGMP in Normal Rats: Treating normal rats with MV81 increased plasma and urine cGMP levels in a dose dependent manner (FIG. 1). During the first 15 minutes of the washout period, compared to the vehicle, both 100 pmol / kg / min and 600 pmol / kg / min doses of MV81 resulted in a sustained elevation in plasma cGMP. Thirty minutes into the w ashout period, compared to the 100 pmol / kg / min dose of MV81 and vehicle, the 600 pmol / kg / min dose exhibited a sustained elevation in the plasma cGMP. Attorney Docket No. 07039-2324WO1 2024-161

[0104] Plasma cGMP Generation in Normal Wild-Type Mice: Chronic subcutaneous administration of MV81 for 28 days induced a dose-dependent increase in plasma cGMP levels in the normal wild-type mice (FIG. 2).

[0105] MV81 Increased Tibia Length: Chronic subcutaneous administration of MV81 for 28 days induced a dose-dependent increase in tibia length in normal wild-ty pe mice (FIG. 3). There was a significant correlation between the plasma cGMP levels and the tibia length following the chronic subcutaneous administration of vehicle or MV81 for 28 days in the normal wild-type mice (FIG. 4).

[0106] MV81 Increased Body Length and Tibia Length: Chronic subcutaneous administration of MV81 for 28 days induced a dose-dependent increase in naso-anal length, anal-tail length, and total length in the normal wild-type mice (FIGS. 5, 6, 8, 9, 10, and 12). Following chronic subcutaneous administration of MV81 for 28 days in the normal wild-type mice, a significant correlation was observed between the change in the naso-anal length and the plasma cGMP levels (FIG. 7), between the change in the anal- tail length and the plasma cGMP levels (FIG. 10), and between the change in the total length and the plasma cGMP levels (FIG. 13). In the R403Q mice, chronic subcutaneous administration of MV81 for 3 months increased the naso-anal length, the anal -tail length, and the total length (FIGS. 15, 16, and 17, respectively). In addition, chronic subcutaneous administration of MV81 for three months increased tibia length in the R403Q mice (FIG. 14). In wild-type mice with TAC. there was an increase in tibia length following treatment with MV81 for three weeks, compared to vehicle (FIG. 18).

[0107] OTHER EMBODIMENTS

[0108] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 07039-2324WO12024-161WHAT IS CLAIMED IS:

1. Use of a composition comprising a natriuretic peptide for treating a skeletal disorder in a mammal, wherein said natriuretic peptide comprises the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

2. The use of claim 1, wherein said mammal is a human.

3. The use of claim 1, wherein said mammal is a monkey, a dog, a cat, a pig, a horse, a cow, a sheep, a goat, a rabbit, a mouse, or a rat.

4. The use of claim 1, wherein said composition comprises said natriuretic peptide in an amount effective to increase bone length, increase bone strength, increase bone density, and / or improve bone shape in said mammal.

5. The use of claim 1, wherein said composition comprises said natriuretic peptide in an amount effective to reduce impaired bone growth in said mammal.

6. The use of claim 1, wherein said skeletal disorder is achondroplasia.

7. The use of claim 1, wherein said skeletal disorder is hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature, growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo- Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3- related skeletal dysplasia.Attorney Docket No. 07039-2324WO12024-1618. The use of claim 1, wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1.

9. The use of claim 1, wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

10. A method for treating a skeletal disorder, wherein said method comprises administering, to a mammal identified as having said skeletal disorder, a composition that comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with zero, one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues within SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

11. The method of claim 10, wherein said mammal is a human.

12. The method of claim 10, wherein said mammal is a monkey, a dog, a cat, a pig, a horse, a cow, a sheep, a goat, a rabbit, a mouse, or a rat.

13. The method of claim 10, wherein said method comprises administering said composition to said mammal in an amount effective to increase bone length, increase bone strength, increase bone density, and / or improve bone shape in said mammal.

14. The method of claim 10, wherein said method comprises administering said composition to said mammal in an amount effective to reduce impaired bone growth in said mammal.

15. The method of claim 10, wherein said skeletal disorder is achondroplasia.

16. The method of claim 10, wherein said skeletal disorder is hypochondroplasia, thanatophoric dysplasia type 1 and type 2, SHOX deficiency, Noonan syndrome, Costello syndrome, LEOPARD syndrome, idiopathic short stature, autosomal dominant short stature,Attorney Docket No. 07039-2324WO1 2024-161 growth hormone deficiency, hypophosphatemic rickets, CNP deficiency, aggrecan deficiency, heterozygous NPR2 mutation, osteoarthritis, craniosynostosis, Lacrimo-Auriculo- Dento-Digital syndrome, Osteoglophonic dysplasia, severe achondroplasia developmental delay acanthosis nigricans, osteogenesis imperfecta, achondrogenesis, chondrodysplasia punctata, homozygous achondroplasia, rhizomelic type of chondrodysplasia punctata, spondyloepiphyseal dysplasia congenita, congenital short femur, Langer-type mesomelic dysplasia, neurofibromatosis, and / or Legius syndrome, or short stature related to FGFR3- related skeletal dysplasia.

17. The method of claim 10, wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1.

18. The method of claim 10, wherein said composition comprises a natriuretic peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid additions, deletions, or substitutions, provided that the two cysteine residues in SEQ ID NO: 1 are maintained and are the only two cysteine residues of said natriuretic peptide.

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