Methods and compositions for the treatment, amelioration and / or prevention of diffuse idiopathic osteoarthritis (DISH)

Abstract

The present disclosure, in one aspect, provides specific doses of ENPP1 agonists for the in vivo treatment of diffuse idiopathic osteoarthritis (DISH), ankylosing spondylitis and / or spondyloarthritis.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 325,554, filed March 30, 2022, the entire contents of which are incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under DK121326-01, AR080416-01 and AG067347A1 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Field The field of the invention relates in one aspect to the treatment and / or amelioration of DISH, ankylosing spondylitis and / or spondyloarthritis by enzyme therapy.

[0004] Sequence Listing This XML file, entitled "047162-7377WO1(01806)Sequence Listing.xml", created on September 29, 2022, contains a size of 149K bytes and is incorporated by reference in its entirety herein. [Background technology]

[0005] background Ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) is a type 2 transmembrane protein whose extracellular activity hydrolyzes the phosphodiester bond of extracellular nucleotides such as adenosine triphosphate (ATP) to generate adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). Because PPi is the main physiological inhibitor of hydroxyapatite deposition, biallelic ENPP1 deficiency leads to ectopic calcification at an early age and high mortality in infants due to arterial calcification and luminal narrowing (Ziegler et al., Generalized arterial calcification of infancy. In: Adam MP, Ardinger HH, Pagon RA, et al., (eds). Gene Reviews 2014). Surviving patients, and ENPP1-deficient individuals who do not show neonatal calcification, may develop hypophosphatemic rickets later in life, mediated by fibroblast growth factor 23 (FGF23) (Ferreira et al., Prospective phenotyping of long-term survivors of generalized arterial calcification of infancy (GACI), Genet Med. 2020;23:396-407 (Non-Patent Document 2)).

[0006] WO2014126965 relates to the treatment of pathological calcification and pathological ossification in diseases involving ENPP1 deficiency. Three proteins - ENPP1, tissue non-specific alkaline phosphatase (TNAP), and progressive ankylosis protein or ankyrin (ANK) - play a role in regulating the extracellular balance of inorganic pyrophosphate (PPi) and inorganic phosphate (Pi). Inorganic pyrophosphate (PPi) is generated by cleavage of extracellular nucleotide triphosphate (NTP) by ENPP1 or by translocation of PPi from intracellular to extracellular space by Ank. TNAP degrades PPi to generate Pi.

[0007] "ENPP1 deficiency" is characterized by a reduced level of ENPP1 enzyme activity in the serum and / or plasma of a subject. ENPP1 deficiency is a rare genetic disorder caused by inactivating mutations in the ENPP1 gene that encodes the ENPP1 enzyme. As described elsewhere herein, ENPP1 is an integral transmembrane protein whose extracellular domain has pyrophosphatase and phosphodiesterase activity. Thus, ENPP1 converts extracellular ATP into inorganic pyrophosphate (PPi) and AMP.

[0008] Calcification in biological systems is a complex process in which calcium salts are maintained in higher concentrations in non-circulating matrices than in local circulating or other mobile fluids. Normal calcification primarily results in the accumulation of calcium and related inorganic salts in crystalline patterns of similar arrangement and chemical composition within specialized intercellular matrices, both of which may vary between species. Pathological calcification, on the other hand, ultimately results in the accumulation of calcium and related inorganic salts in these specialized matrices as well as other intercellular, extracellular and cellular materials that exceed the normal range in chemical composition, or diversity of patterns, thereby resulting in several disease states.

[0009] Diffuse idiopathic skeletal hyperostosis (DISH) is a bone disorder characterized by abnormal new bone formation (Resnick et al., Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations, Radiology. 1975; 115: 513-524 (Non-Patent Document 3)). New bone forms mostly where ligaments and tendons attach to bone (enthesis areas), but there is also generalized sclerosis of bone, and bone overgrowth (osteophytosis) (Pillai & Littlejohn, Metabolic Factors in Diffuse Idiopathic Skeletal Hyperostosis-A Review of Clinical Data. The Open Rheumatology Journal. 2014; 8: 116-128 (Non-Patent Document 4)). The respective roles of ENPP1, TNAP and ANK for DISH are not understood. Furthermore, it is not known whether deficiency in one or more of these enzymes is part of the DISH phenotype. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Ziegler et al.,Generalized arterial calcification of infancy.In:Adam MP,Ardinger HH,Pagon RA,et al.,(eds).Gene Reviews 2014 [Non-Patent Document 2] Ferreira et al., Prospective phenotyping of long-term survivors of generalized arterial calcification of infancy(GACI), Genet Med.2020;23:396-407 [Non-Patent Document 3] Resnick et al., Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations, Radiology.1975;115:513-524 [Non-Patent Document 4] Pillai&Littlejohn,Metabolic Factors in Diffuse Idiopathic Skeletal Hyperostosis-A Review of Clinical Data.The Open Rheumatology Journal.2014;8:116-128 Summary of the Invention

[0011] Quick Overview The present disclosure provides a method for treating, ameliorating and / or preventing diffuse idiopathic osteoarthritis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof. In certain embodiments, the method comprises administering a compound of formula (I) or a salt or solvate thereof: PROTEIN-Z-DOMAIN-XY (I) administering to the patient a therapeutically effective amount of In (I), PROTEIN comprises the catalytic domain of ENPP1, DOMAIN is absent or at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin protein (ALB) and fragments thereof; X and Z are, independently, absent or a polypeptide comprising 1 to 20 amino acids; Y is absent or is a negatively charged bone-targeting sequence, thereby treating, ameliorating and / or preventing DISH, ankylosing spondylitis and / or spondyloarthritis in the patient.

[0012] In certain embodiments, the compound lacks a negatively charged bone-targeting sequence.

[0013] In certain embodiments, Y is absent.

[0014] In certain embodiments, the compound comprises a negatively charged bone-targeting sequence.

[0015] In certain embodiments, the patient has ENPP1 haploinsufficiency.

[0016] In certain embodiments, the patient does not have ENPP1 haploinsufficiency.

[0017] In certain embodiments, the patient is not ENPP1 deficient.

[0018] In certain embodiments, the patient is ENPP1 deficient.

[0019] In certain embodiments, the patient is administered the compound by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary and topical.

[0020] In certain embodiments, the compound is administered to the patient intravenously or subcutaneously.

[0021] In certain embodiments, administering the compound to the patient increases or prevents a further decrease in the patient's extracellular pyrophosphate concentration.

[0022] In certain embodiments, administering the compound to the patient reduces or prevents further increase in one or more of Achilles tendon calcification, spinal calcification, hip calcification, and bilateral calcification in the patient.

[0023] In certain embodiments, the DOMAIN comprises albumin.

[0024] In certain embodiments, the DOMAIN comprises an IgG Fc domain.

[0025] In certain embodiments, the PROTEIN lacks the ENPP1 transmembrane domain.

[0026] In certain embodiments, the compound is administered to the patient as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier.

[0027] In certain embodiments, the patient is a mammal.

[0028] In certain embodiments, the mammal is a human.

[0029] In certain embodiments, the PROTEIN comprises amino acid residues 99 (PSCAKE...) to 925 (...QED) of SEQ ID NO:1.

[0030] In certain embodiments, PROTEIN comprises amino acid residues 1-833 of SEQ ID NO:3.

[0031] In certain embodiments, the PROTEIN comprises the amino acid sequence shown in SEQ ID NO:2.

[0032] In certain embodiments, the PROTEIN comprises the amino acid sequence set forth in SEQ ID NO:3 or 4 or 5.

[0033] In certain embodiments, the DOMAIN increases the circulating half-life of the compound compared to the circulating half-life of a compound lacking the DOMAIN.

[0034] In certain embodiments, the patient has also been diagnosed with a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets.

[0035] In certain embodiments, the patient has not been diagnosed with a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemic, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets.

[0036] explanation In certain embodiments, the disease or disorder contemplated herein is diffuse idiopathic osteoarthritis (DISH). In certain embodiments, the disease or disorder contemplated herein is ankylosing spondylitis. In certain embodiments, the disease or disorder contemplated herein is spondyloarthritis. In certain embodiments, the disease or disorder contemplated herein is diffuse idiopathic osteoarthritis (DISH) and / or ankylosing spondylitis. In certain embodiments, the disease or disorder contemplated herein is DISH and / or spondyloarthritis. In certain embodiments, the disease or disorder contemplated herein is ankylosing spondylitis and / or spondyloarthritis.

[0037] The present invention provides methods for treating, ameliorating, preventing further onset and / or progression of, and / or preventing diffuse idiopathic osteoarthritis (DISH), ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof. The present invention further provides methods for treating and / or ameliorating DISH, ankylosing spondylitis and / or spondyloarthritis in a patient in need thereof.

[0038] In one aspect, the present disclosure relates to treating, ameliorating, preventing further development and / or progression of, and / or preventing DISH, ankylosing spondylitis and / or spondyloarthritis by administering a therapeutically effective amount of an ENPP1 agonist to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis.

[0039] In one aspect, the disclosure relates to treating, ameliorating, preventing further onset and / or progression, and / or preventing ENPP1 deficiency in a subject by administering a therapeutically effective amount of an ENPP1 agonist to the subject.

[0040] In one aspect, the disclosure relates to treating, ameliorating, preventing further onset and / or progression, and / or preventing one or more symptoms of ENPP1 haploinsufficiency in a subject by administering to the subject a therapeutically effective amount of an ENPP1 agent.

[0041] Treatment of DISH, ankylosing spondylitis and / or spondyloarthritis may be at about 0.1 mg per kilogram, about 0.2 mg per kilogram of subject, about 0.3 mg per kilogram of subject, about 0.4 mg per kilogram of subject, about 0.5 mg per kilogram of subject, about 0.6 mg per kilogram of subject, about 0.7 mg per kilogram of subject, about 0.8 mg per kilogram of subject, about 0.9 mg per kilogram of subject, about 1.0 mg per kilogram of subject, about 1.1 mg per kilogram of subject, about 1.2 mg per kilogram of subject, about 1.3 mg per kilogram of subject, about 1.4 mg per kilogram of subject, about 1.5 mg per kilogram of subject, about 1.6 mg per kilogram of subject, about 1.7 mg per kilogram of subject, about 1.8 mg per kilogram of subject, about 1.9 mg per kilogram of subject, about 2.0 mg per kilogram of subject, about 2.1 mg per kilogram of subject, about 2.2 mg per kilogram of subject, about 2.3 mg per kilogram of subject, about 2.4 mg per kilogram of subject, about 2.5 mg per kilogram of subject, about 2.6 mg per kilogram of subject, about 2.7 mg per kilogram of subject, about 2.8 mg per kilogram of subject, about 2.9 mg per kilogram of subject, about 3.0 mg per kilogram of subject, about 3.1 mg per kilogram of subject, about 3.2 mg per kilogram of subject, about 3.3 mg per kilogram of subject, about 3.4 mg per kilogram of subject, about 3.5 mg per kilogram of subject, about 3.6 mg per kilogram of subject, about 3.7 mg per kilogram of subject, about 3.8 mg per kilogram of subject, about 3.9 mg per kilogram of subject, about 3.1 mg per kilogram of subject, about 3 The method includes administering an ENPP1 agonist at a dose of about 1.3 mg per kilogram, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject does not have an ENPP1 deficiency.

[0042] In one aspect, the disclosure provides a method of administering about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, about 1.1 ... The subject has an ENPP1 deficiency, wherein the subject is administered an ENPP1 agonist at a dose of about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease.

[0043] In one aspect, the disclosure provides a method of administering about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, or about 1.4 mg per kilogram of the subject to a subject having DISH, ankylosing spondylitis, and / or spondyloarthritis. 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject does not have ENPP1 haploinsufficiency.

[0044] In one aspect, the disclosure provides for the administration of about 0.1 mg per kilogram of a medicament to a subject having DISH, ankylosing spondylitis and / or spondyloarthritis, about 0.2 mg per kilogram of a subject, about 0.3 mg per kilogram of a subject, about 0.4 mg per kilogram of a subject, about 0.5 mg per kilogram of a subject, about 0.6 mg per kilogram of a subject, about 0.7 mg per kilogram of a subject, about 0.8 mg per kilogram of a subject, about 0.9 mg per kilogram of a subject, about 1.0 mg per kilogram of a subject, about 1.1 mg per kilogram of a subject, about 1.2 mg per kilogram of a subject, about 1.4 mg per kilogram of a subject, about 1.6 mg per kilogram of a subject, about 1.8 mg per kilogram of a subject, about 1.9 mg per kilogram of a subject, about 1.1 mg per kilogram of a subject, about 1.2 mg per kilogram of a subject, about 1.4 mg per kilogram of a subject, about 1.5 mg per kilogram of a subject, about 1.6 mg per kilogram of a subject, about 1.7 mg per kilogram of a subject, about 1.8 mg per kilogram of a subject, about 1.9 ... and administering an ENPP1 agonist at a dose of about 2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby treating, reducing and / or ameliorating one or more symptoms of DISH, ankylosing spondylitis and / or spondyloarthritis disease, wherein the subject has ENPP1 haploinsufficiency.

[0045] In one aspect, the disclosure provides a method of administering about 0.1 mg per kilogram of ENPP1 to a subject having DISH, ankylosing spondylitis, and / or spondyloarthritis, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 0.1 mg per kilogram of the subject ...1 mg per kilogram of the subject The present invention relates to administering the ENPP1 agonist at a dose of about 0.9 mg per kilogram, about 1.0 mg per kilogram of subject, about 1.1 mg per kilogram of subject, about 1.2 mg per kilogram of subject, about 1.3 mg per kilogram of subject, about 1.4 mg per kilogram of subject, about 1.5 mg per kilogram of subject, about 1.6 mg per kilogram of subject, about 1.7 mg per kilogram of subject, about 1.8 mg per kilogram of subject, about 1.9 mg per kilogram of subject, or about 2.0 mg per kilogram of subject.As used herein, the physiological level of ENPP1 protein and / or activity in plasma and / or tissue is the amount or concentration of the ENPP1 agonist that is sufficient to achieve and maintain a certain physiological level of PPi in human serum.In certain embodiments, the ENPP1 agonist is ENPP1 and / or an ENPP1 construct that includes ENPP1 activity.

[0046] In one aspect, the disclosure provides an administration of about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 0.1 mg per kilogram of the subject ... The present invention relates to administering an ENPP1 agonist at a dose of about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject. As used herein, a physiological level of ENPP1 protein and / or activity in plasma and / or tissue is an amount or concentration of an ENPP1 agonist sufficient to achieve and maintain a certain physiological level of PPi in human serum. In certain embodiments, the ENPP1 agonist is ENPP1 and / or an ENPP1 construct comprising ENPP1 activity.

[0047] In one aspect, the disclosure provides a method of increasing circulating pyrophosphate (PPi) in a subject having DISH, ankylosing spondylitis, and / or spondyloarthritis, comprising administering to the subject about 0.1 mg per kilogram, about 0.2 mg per kilogram, about 0.3 mg per kilogram, about 0.4 mg per kilogram, about 0.5 mg per kilogram, about 0.6 mg per kilogram, about 0.7 mg per kilogram, about 0.8 mg per kilogram, about 0.9 mg per kilogram, ...1 mg per kilogram, about 0.2 mg per kilogram, about 0.3 mg per kilogram, about 0.4 mg per kilogram, about 0.5 mg per kilogram, about 0.6 mg per kilogram, about 0.7 mg per kilogram, about 0.8 mg per kilogram, about 0.9 mg per kilogram, about 0.1 mg per kilogram, about 0.2 mg per kilogram, about 0.3 mg per kilogram, about 0.4 mg per kilogram, about 0.5 mg per kilogram, about 0.6 mg per kilogram, about 0.7 mg per kilogram, about 0 administering an ENPP1 agonist at a dose of about 1.0 mg, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby increasing circulating PPi in the subject.

[0048] In one aspect, the disclosure provides a method of increasing circulating pyrophosphate (PPi) in a subject having ENPP1 haploinsufficiency, comprising administering to the subject about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject , about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby increasing circulating PPi in the subject.

[0049] In one aspect, the disclosure provides a method of ameliorating one or more symptoms of ENPP1 haploinsufficiency in a subject, comprising administering to the subject about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 1.0 mg per kilogram of the subject, about 1.1 mg per kilogram of the subject, about 1.2 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, about 2.0 mg per kilogram of the subject, about 2.1 mg per kilogram of the subject, about 2.2 mg per kilogram of the subject, about 2.3 mg per kilogram of the subject, about 2.4 mg per kilogram of the subject, about 2.5 mg per kilogram of the subject, about 2.6 mg per kilogram of the subject, about 2.7 mg per kilogram of the subject, about 2.8 mg per kilogram of the subject, about 2.9 mg per kilogram of the subject, about 3.0 mg per kilogram of the subject, about 3.4 mg per kilogram of the subject, about 3.5 mg per kilogram of the subject, about 3.6 mg per kilogram of the subject, about 3.7 mg per kilogram of the subject, about 3.8 mg per kilogram of the subject, about 3.9 mg per kilogram of the subject, about 3.8 mg per kilogram of the subject, about 3.9 mg per The present invention relates to a method comprising administering an ENPP1 agonist at a dose of about 1.2 mg per program, about 1.3 mg per kilogram of the subject, about 1.4 mg per kilogram of the subject, about 1.5 mg per kilogram of the subject, about 1.6 mg per kilogram of the subject, about 1.7 mg per kilogram of the subject, about 1.8 mg per kilogram of the subject, about 1.9 mg per kilogram of the subject, or about 2.0 mg per kilogram of the subject, thereby ameliorating one or more symptoms of ENPP1 deficiency or one or more symptoms of ENPP1 haploinsufficiency in the subject.

[0050] In one aspect, the disclosure provides a method of preventing and / or reversing the progression of, or minimizing and / or reducing pathological calcifications and / or bone masses present in a subject having DISH, ankylosing spondylitis and / or spondyloarthritis, comprising administering to the subject about 0.1 mg per kilogram, about 0.2 mg per kilogram of the subject, about 0.3 mg per kilogram of the subject, about 0.4 mg per kilogram of the subject, about 0.5 mg per kilogram of the subject, about 0.6 mg per kilogram of the subject, about 0.7 mg per kilogram of the subject, about 0.8 mg per kilogram of the subject, about 0.9 mg per kilogram of the subject, about 10 mg per kilogram of the subject, about 15 mg per kilogram of the subject, about 16 mg per kilogram of the subject, about 17 mg per kilogram of the subject, about 18 mg per kilogram of the subject, about 19 mg per kilogram of the subject, about 20 mg per kilogram of the subject, about 21 mg per kilogram of the subject, about 22 mg per kilogram of the subject, about 23 mg per kilogram of the subject, about 24 mg per kilogram of the subject, about 25 mg per kilogram of the subject, about 26 mg per kilogram of the subject, about 27 mg per kilogram of the subject, about 28 mg per kilogram of the subject, about 29 mg per kilogram of the subject, about 30 mg per kilogram of the subject, about 31 mg per kilogram of the subject, about 32 mg per kilogram of the subject, about 33 mg per kilogram of the subject, about 34 mg per kilogram of the subject, about 35 mg per kilogram of the subject, about 36 mg per kilogram of the subject, about 37 mg per kilogram of the subject, about 38 mg per kilogram of the subject, about 39 mg per kilogram of the subject, about 39 mg per kilo administering an ENPP1 agonist at a dose of about 1.0 mg per kilogram of subject, about 1.1 mg per kilogram of subject, about 1.2 mg per kilogram of subject, about 1.3 mg per kilogram of subject, about 1.4 mg per kilogram of subject, about 1.5 mg per kilogram of subject, about 1.6 mg per kilogram of subject, about 1.7 mg per kilogram of subject, about 1.8 mg per kilogram of subject, about 1.9 mg per kilogram of subject, or about 2.0 mg per kilogram of subject, thereby preventing and / or reversing the progression of pathological calcification and / or ossified skeletal mass in the subject or minimizing and / or reducing pathological calcification and / or ossified skeletal mass in the subject.

[0051] In some embodiments of any of the aforementioned methods, the method includes administering to the patient a therapeutically effective amount of an ENPP1 agonist.

[0052] In some embodiments, the ENPP1 agonist is a compound of formula (I) or a salt or solvate thereof: W-PROTEIN-Z-DOMAIN-XY (I) and In (I), W is absent or contains a signal sequence that enables transport of the compound to the extracellular space, PROTEIN contains the catalytic domain of ENPP1, DOMAIN is absent or at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin protein (ALB) and biologically active fragments thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is absent or a "bone-targeting" sequence group; m is independently an integer ranging from 1 to 15; and n is independently an integer ranging from 1 to 10.

[0053] In some embodiments, W is absent. In some embodiments, W comprises a signal sequence that enables transport of the compound to the extracellular space.

[0054] In some embodiments, Y is a "bone-targeting" sequence selected from the group consisting of: TIFF2025510963000001.tif32130. [Brief description of the drawings]

[0055] [Figure 1-1] Figures 1A-1B show spinal CT and bone scintigraphy of patient 1. Figure 1A shows the presence of multiple compression fractures in the spine detected by spinal CT, shown as white arrows. Figure 1B shows bone scintigraphy that revealed multiple accumulations in the ribs. Figures 1C-1H show radiographs of the hip, knee and Achilles tendons of patient 1. Figure 1C: right hip joint, Figure 1D: left hip joint, Figure 1E: right knee joint, Figure 1F: left knee joint, Figure 1G: right Achilles tendon, Figure 1H: left Achilles tendon. There was no sign of heterotopic ossification. [Figure 1-2] Please refer to the description of Figure 1-1. [Diagram 2] 2A-C show ENPP1 mutation pedigrees corresponding to patients 1, 2, and 3, respectively. Arrows indicate the probands. [Diagram 3]Figures 3A-G show radiographs of the spine CT, hip, knee and Achilles tendon of patient 2. Figure 3A: Spine CT showed paravertebral ligament ossification (white arrowheads) and multiple compression fractures (white arrows). Figures 3B-C correspond to radiographs of the right hip (Figure 3B) and left hip (Figure 3C), respectively. The figures show that no signs of heterotopic ossification were observed. Figures 3D-E correspond to radiographs of the right knee (Figure 3D) and left knee (Figure 3E), respectively. The figures show that no signs of heterotopic ossification were observed. Figures 3F and 3G correspond to radiographs of the right Achilles tendon (Figure 3F) and left Achilles tendon (Figure 3G), respectively. Signs of tibial enthesopathy (shown as white arrowheads) were detected in the right Achilles tendon. [Figure 4] Figures 4A-I show the spine CT, hip, knee and Achilles tendon radiographs of patient 3. Figures 4A-C correspond to the spine CT, which showed multiple paravertebral ossifications (white arrowheads) in the cervical (Figure 4A), thoracic (Figure 4B) and lumbar (Figure 4C) spine. Figures 4D-E show prominent ossifications (white arrowheads) around the right hip (Figure 4D) and left hip (Figure 4E) as detected on the radiographs. Figures 4F-G show the right knee (Figure 4F) and left knee (Figure 4G) radiographs showing no signs of heterotopic ossification. Figures 4H-I show prominent enthesopathy (white arrowheads) detected on the radiographs of the right Achilles tendon (Figure 4H) and left Achilles tendon (Figure 4I). [Diagram 5] Figures 5A-5G show spine CT scans and x-rays of the hip, knee and Achilles tendons of the son of patient 1. (Figure 5A) Spine CT scan, (Figure 5B) Right hip, (Figure 5C) Left hip, (Figure 5D) Right knee, (Figure 5E) Left knee, (Figure 5F) Right Achilles tendon, (Figure 5G) Left Achilles tendon. There were no signs of heterotopic ossification. [Figure 6] Figures 6A-6G show spine CT scans and radiographs of the hip, knee, and Achilles tendons of one of the sons of patient 3. (Figure 6A) Spine CT scan, (Figure 6B) Right hip, (Figure 6C) Left hip, (Figure 6D) Right knee, (Figure 6E) Left knee, (Figure 6F) Right Achilles tendon, (Figure 6G) Left Achilles tendon. There was slight enthesopathy in the left Achilles tendon (white arrowhead). [Figure 7]Figures 7A-7G show spine CT scans and radiographs of the hip, knee and Achilles tendons of another son of Case 3. (Figure 7A) Spine CT scan, (Figure 7B) Right hip, (Figure 7C) Left hip, (Figure 7D) Right knee, (Figure 7E) Left knee, (Figure 7F) Right Achilles tendon, (Figure 7G) Left Achilles tendon. There was slight enthesopathy in the left Achilles tendon (white arrowhead). [Figure 8] Figure 8 shows Sanger sequencing of the compound heterozygosity confirmed by complementary DNA for the ENPP1 variants in patient 3. Clone type 1 contained the ENPP1 variant c.536A>G, ​​whereas another ENPP1 variant c.1352A>G was located in clone type 2, suggesting a compound heterozygosity. [Figure 9] Figure 9 shows a non-limiting schematic diagram of the ENPP1 structure and the location of this variant. ENPP1 consists of a cytoplasmic domain (CD), a transmembrane domain (TM), and an extracellular domain composed of two somatomedin domains (SMB1 and SMB2), a catalytic domain, and a nuclease-like domain. N179S and Y451C are located in SMB2 and the catalytic domain, respectively. [Figure 10] Figure 10 shows a comparison of the enzyme activity of WT, N179S and Y451C. Compared to WT ENPP1, the N179S and Y451C variants showed a 55% and 70% decrease in the rate of the enzyme reaction, respectively. Bars indicate median and quartile values. ****: p<0.0001. [Figure 11] Figure 11 shows the complete unprocessed amino acid sequence of wild-type ENPP1 precursor protein (SEQ ID NO:1). The cytosolic and transmembrane regions are underlined. Potential N-glycosylation sites are in bold. PSCAKE (residues 99-104; boxed) is the start of the soluble ENPP1 protein portion that includes SMB1 (residues 104-144) and SMB2 (residues 145-189). [Figure 12]Figure 12 shows plasma PPi responses in WT and Enpp1asj mice administered the indicated weekly subcutaneous doses of vehicle or ENPP1 (Figure 12). *p<0.05, **p<0.01; (ANOVA, Kruskal-Wallis test). [Figure 13] FIG. 13 shows paravertebral osteophyte and ankylosis responses in 17-week-old WT and 17-week-old Enpp1asj male mice administered vehicle or ENPP1 constructs #1118 and #2000 at the indicated weekly subcutaneous doses. Construct #1118 was administered weekly from week 3, and construct #2000 was administered weekly from week 5. MicroCT images demonstrate attenuation of paravertebral osteophyte and ankylosis preferentially in construct #2000, evident in both Enpp1asj male and Enpp1asj female mice administered at a weekly dose of 1 mg / Kg. [Figure 14] FIG. 14 shows paravertebral osteophyte and ankylosis responses in 17-week-old WT and 17-week-old Enpp1asj female mice administered vehicle or ENPP1 constructs #1118 and #2000 at the indicated weekly subcutaneous doses. Construct #1118 was administered weekly from week 3, and construct #2000 was administered weekly from week 5. MicroCT images demonstrate preferential attenuation of paravertebral osteophyte and ankylosis in construct #2000, evident in both Enpp1asj male and female mice administered a weekly dose of 1 mg / Kg. [Figure 15A]15A-B show auditory brainstem responses in 17-week-old WT and 17-week-old Enpp1asj mice administered vehicle or ENPP1 constructs #1118 and #2000 at the indicated weekly subcutaneous doses. Construct #1118 was administered once a week from week 3, and construct #2000 was administered once a week from week 5. Stimulation frequency measurements demonstrate prevention of hearing loss in the low frequency (8 kHz) range by ENPP1 construct #1118 at a weekly dose of 2 mg / Kg, and by ENPP1 construct #2000 at weekly doses of 0.5 and 1 mg / Kg. ENPP1 construct #2000 administered at a weekly dose of 1 mg / Kg also provides preferential hearing improvement in ENPP1-deficient animals in the high frequency range (32 kHz). [Figure 15B] Please see the legend to Figure 15A. [Figure 16] FIG. 16 shows intact FGF23 levels in WT and 17-week-old ENPP1asj mice with vehicle or ENPP1 constructs #1118 and #2000 at the indicated weekly subcutaneous doses. Construct #1118 was administered weekly from week 3, and construct #2000 was administered weekly from week 5. The data demonstrate that intact FGF23 is preferentially suppressed with ENPP1 construct #2000 when administered at 1 mg / Kg and 4 mg / Kg per week. Statistical significance was assessed by ANOVA Kruskal-Wallis test followed by Dunn's post-hoc analysis to assess differences from WT levels (one-way ANOVA). Statistical significance is represented by p-values ​​with the following annotation: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] definition The terms used herein generally have their ordinary meaning in the art in the context of this disclosure and in the specific context in which each term is used.Specific terms are explained below or elsewhere in this specification to provide additional guidance to practitioners in describing the compositions and methods of this disclosure and the methods of making and using them.The scope or meaning of any use of a term will be clear from the specific context in which it is used.

[0057] As used herein, the term "DISH" refers to diffuse idiopathic skeletal hyperostosis, also known as Forestier's disease, which is a bone disorder characterized by abnormal new bone formation (Resnick et al., Diffuse idiopathic skeletal hyperostosis (DISH): Forestier's disease with extraspinal manifestations, Radiology. 1975; 115: 513-524). DISH causes ligaments and tendons in the body to calcify (harden) and / or new bone growths to appear around the spine and in skeletal areas (ossification masses). These calcification areas can sometimes form osteophytes (abnormal new bone growths) that can cause pain, stiffness and reduced mobility. New bone is most often formed at the sites where ligaments and tendons attach to bone (enthesis areas), but the relationship between enthesis calcification and perivertebral ossification masses is not understood. There is also generalized sclerosis of the bones, and overgrowth of bone (osteophytosis) (Pillai & Littlejohn, Metabolic Factors in Diffuse Idiopathic Skeletal Hyperostosis-A Review of Clinical Data, The Open Rheumatology Journal. 2014;8:116-128). Another aspect of the disease is the formation of large, mobile osteophytes resulting from abnormal bone growth. These ossifications are mostly found in the axial skeleton, with the thoracic region of the axial skeleton being the primary location. Also, peripheral tendon attachments such as the peripatellar ligament, Achilles tendon insertion, plantar fascia, shoulder, olecranon and metacarpophalangeal joints may have calcifications. In some cases, calcifications are found in the hip, knee, ankle, foot, shoulder, hand and ribs (Helfgott, Diffuse idiopathic skeletal hyperostosis (DISH). UpToDate. June 7, 2017).Common features and / or symptoms of DISH include focal and diffuse calcification and ossification of the anterior longitudinal ligament, calcification of the paravertebral connective tissue and annulus fibrosus, degeneration of peripheral annulus fibrosus fibers, anterolateral extension of fibrous tissue, hypervascularity, chronic inflammatory cell infiltration, periosteal new bone formation anterior to the vertebral bodies, ossification of the metacarpophalangeal joints, pain in the thoracic, lumbar, and / or cervical regions, radiculopathy, polyarticular pain, monoarticular synovitis, and / or dysphagia.

[0058] DISH manifests as accumulation of calcium salts (calcification) in tendons and ligaments, and abnormal new bone growth (ossification), for unknown reasons (Mader et al., Diffuse idiopathic skeletal hyperostosis: clinical features and pathogenic mechanisms, Nat Rev Rheumatol. December 2013; 9(12): 741-50 and Nascimento et al. Diffuse idiopathic skeletal hyperostosis: A review, Surgical Neurology International. 2014; 5(Suppl 3): S122-S125). Common risk factors for developing DISH include, but are not limited to, large waist circumference, BMI / obesity, hyperinsulinemia, diabetes mellitus, hyperuricemia, dyslipidemia, hypertension, coronary artery disease, and gout. DISH can be asymptomatic, in which case diagnosis is usually made based on radiographic images.

[0059] DISH is an age-related progressive musculoskeletal disease. The prevalence of DISH was 25% of men and 15% of women over 50 years of age in two large Midwestern hospital populations. In addition, up to 6.3% of the Japanese population and 25% of North American degenerative cervical myelopathy patients develop progressive calcification in the posterior longitudinal ligament of the spine in a condition known as OPLL. Progressive calcium deposition at the tendon attachment leads to spinal stenosis and spinal cord compression from growing paravertebral masses. The resulting myelopathy is often severely painful and debilitating. There is no effective treatment to prevent the progressive ossification responsible for the symptomatic course, and the factors involved in initiating and promoting ectopic calcification are poorly understood, so treatment usually consists of conservative chronic pain management with NSAIDs. Although surgical decompression is used to provide short-term relief in acute cases, progression of the tendon attachment in OPLL occurs more rapidly after surgery (laminoplasty) than in conservatively managed patients (24% vs. 70%, respectively), discouraging surgical intervention in all but severely symptomatic cases.

[0060] The subject with DISH, as used herein, refers to the subject diagnosed with DISH.Diagnosis of DISH is based on radiological and / or clinical findings and is defined by Resnick and Niwayana.X-ray of the thoracic and lumbar spine is the only imaging diagnostic method that is most useful for diagnosing DISH.Computed tomography (CT) scan can be used to evaluate symptoms that may be caused by complications such as fractures or pressure effects on the trachea, esophagus and veins. This allows the entity to be differentiated into ankylosing spondylitis or OPLL (Artner et al., Diffuse idiopathic skeletal hyperostosis: current aspects of diagnostics and therapy, 2012, Orthopade. 2012 Nov;41(11):916-22; Olivieri et al., Diffuse idiopathic skeletal hyperostosis may give the typical postural abnormalities of advanced ankylosing spondylitis, Rheumatology 2007 Nov 1;46(11):1709-11). The presence of fluid calcification and ossification primarily along the anterolateral aspect (anterior longitudinal ligament) of at least four consecutive vertebrae (spanning three disc spaces) with preserved disc height indicates DISH. Spinal and extraspinal features visible on radiographs and CT scans of patients with DISH (Radswiki & Baba, Diffuse idiopathic skeletal hyperostosis. Reference paper, Radiopaedia.org) include: Non-limiting spinal features: Fluid ossification: Bright red fluid ossification along the anterior or right anterolateral aspects of at least four adjacent vertebrae -The disc space is usually well maintained Ankylosis is more prevalent in the thoracic spine than in the cervical or lumbar spine Frequently incomplete May have interdigitating areas of protruding disc material during fluid ossification There is no sacroiliac arthritis or facet joint ankylosis, but anterior sacroiliac bridging, posterior bridging, and tendon attachment bridging may be present. Non-limiting extraspinal features: Enthesopathy of the iliac crest, ischial tuberosity and greater trochanter Frequent osteophyte formation in the appendicular skeleton (olecranon, calcaneus, patellar ligament) "Whiskering" Enthesizing Spurs Non-limiting clinical features: ·pain Reduced range of motion Increased risk of spinal fractures in some patients.

[0061] In some cases, DISH becomes symptomatic and the main clinical features include one or more of the following: pain, stiffness and reduced mobility (range of motion), dysphagia (caused by osteophyte compression), esophageal obstruction, hoarseness, cervical myelopathy, atlantoaxial subluxation, spinal stenosis, ossification of the posterior longitudinal ligament, spinal cord injury, dyspnea, foreign body sensation, neurological symptoms due to spinal cord compression, hypercholesterolemia (leading to cardiovascular comorbidities), and / or peripheral joint involvement.

[0062] The "enthesis" is the site of attachment of a tendon or ligament to bone and is structured into four zones: a dense fibrous connective tissue zone populated by fibroblast-type cells (tenocytes) and composed of collagen types I and III and decorin; a non-calcified fibrocartilage zone populated by fibrochondrocytes and composed of collagen types I and II and aggrecan; a calcified fibrocartilage zone populated by hypertrophic chondrocytes and composed of collagen types II and X and aggrecan; and a bone populated by osteoblasts, osteocytes and osteoclasts and composed of collagen type I (Calejo et al., Enthesis tissue engineering: biological requirements meet at the interface, Tissue Eng Part B Rev. 2019;25(4):330-356). Thus, the enthesis represents a musculoskeletal structure that allows a smooth transition between two very different tissues, tendon or ligament (flexible soft tissue) and bone (stiff hard tissue) (Calejo et al.,Enthesis tissue engineering: biological requirements meet at the interface,Tissue Eng Part B Rev.2019;25(4):330-356). An abrupt transition at this interface would lead to interzonal stress concentrations and increased risk of failure. Conversely, a gradual transition in composition and structure over the enthesis would mitigate stress concentrations (Genin et al.,Functional grading of mineral and collagen in the attachment of tendon to bone,Biophys J.2009;97(4):976-985). There is a progressive decrease in collagen fiber alignment and increase in mineral content from tendon to bone, creating a gradient of tissue stiffness (Genin et al., Functional grading of mineral and collagen in the attachment of tendon to bone, Biophys J. 2009;97(4):976-985).A reduction in tendon-to-bone attachment mineralization reduces the strength of this structure (Deymier et al.,Micro-mechanical properties of the tendon-to-bone attachment,Acta Biomater.2017;1(56):25-35), but animal models with expansion of calcified fibrocartilage also show reduced attachment strength (Marinovich et al.,The role of bone sialoprotein in the tendon-bone insertion,Matrix Biol.2016;52-54:325-338). Therefore, mineralization must be appropriately regulated to achieve optimal mechanical properties of the tendon attachment.

[0063] As used herein, "ankylosing spondylitis" refers to a type of arthritis characterized by long-term inflammation of the joints of the spine, typically where the spine joins the pelvis. Affected areas may include other joints such as the shoulder or hip, and eye and bowel problems, as well as back pain, may occur. Joint mobility in affected areas generally worsens over time.

[0064] The cause of ankylosing spondylitis is unknown, but is thought to involve a combination of genetic and environmental factors. Many affected individuals have a specific human leukocyte antigen known as the HLA-B27 antigen. The underlying mechanism is thought to be autoimmune or autoinflammatory. Diagnosis is typically based on symptoms, with support from medical imaging and blood tests. Ankylosing spondylitis is a type of seronegative spondyloarthropathy, meaning that tests will not show the presence of rheumatoid factor (RF) antibodies. There is no known cure for ankylosing spondylitis. Treatment may include medications, exercise, physical therapy, and rarely surgery. Medications used include NSAIDs, steroids, DMARDs such as sulfasalazine, and biologic agents such as TNF inhibitors. Approximately 0.1% and 0.8% of the human population are affected, typically in young adults. Men and women are equally affected, but women are more likely than men to experience inflammation rather than fusion.

[0065] As used herein, "spondyloarthritis" or "SpA" is characterized by inflammation of the axial skeleton (sacroiliitis, spondylitis), peripheral joints and tendon attachments. Extraskeletal manifestations such as anterior uveitis, psoriasis and inflammatory bowel disease may occur. HLA-B27 is the major genetic risk factor. The global prevalence of the entire SpA group ranges from 0.1% to 1.9%, with variations between countries and ethnicities. Nonsteroidal anti-inflammatory drugs (commonly called NSAIDs) provide symptomatic relief for most patients by reducing pain and swelling. Other drugs, called biologics, including anti-TNF drugs (TNF blockers) and anti-IL-17 drugs (IL-17 blockers), are effective in patients who do not respond adequately to NSAIDs.

[0066] "ENPP1 deficiency" is characterized by reduced levels of ENPP1 enzyme activity in the serum and / or plasma of a subject. ENPP1 deficiency is a rare genetic disorder caused by inactivating mutations in the ENPP1 gene, which encodes the ENPP1 enzyme. ENPP1 is an integral transmembrane protein whose extracellular domain possesses pyrophosphatase and phosphodiesterase activities. ENPP1 converts extracellular ATP to inorganic pyrophosphate (PPi) and AMP.

[0067] "Enzymatically active" with respect to an ENPP1 polypeptide, or as used herein, "enzymatically active" with respect to an ENPP1 polypeptide, is defined as having ATP hydrolysis activity to AMP and PPi, and / or AP3A hydrolysis to ATP. ENPP1 readily hydrolyzes ATP to AMP and PPi. The steady-state Michaelis-Menten enzyme constant of ENPP1 is determined using ATP as a substrate. ENPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction is confirmed by using standards of ATP, AMP, and ADP. The ATP substrate degrades over time in the presence of ENPP1, and the enzyme product AMP accumulates. Various concentrations of ATP substrate are used to derive the initial velocity of ENPP1 in the presence of ATP, and the data are curve-fitted to derive the enzyme rate constant. At physiological pH, the rate constant of ENPP1 is K m = 2 μM, and k cat =3.4±0.4s -1 It is.

[0068] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animals include rats, mice, cats, dogs, humans, cows and horses. Due to release from platelets, it is necessary to measure PPi in plasma, not serum. There are several ways to measure PPi, one of which is by an enzyme assay that uses a modified version of uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63).

[0069] Typically, plasma PPi levels in healthy human subjects range from about 1 μM to about 3 μM, and in some cases from 1 to 2 μM. The normal level of ENPP1 in plasma refers to the amount of ENPP1 protein required to maintain normal levels of plasma pyrophosphate (PPi) in healthy subjects. The normal level of PPi in healthy humans corresponds to 2 to 3 μM. Subjects with ENPP1 deficiency exhibit low PPi levels ranging from at least 10% lower than normal levels, at least 20% lower than normal levels, at least 30% lower than normal levels, at least 40% lower than normal levels, at least 50% lower than normal levels, at least 60% lower than normal levels, at least 70% lower than normal levels, at least 80% lower than normal levels, and combinations thereof. In patients suffering from GACI, PPi levels are found to be less than 1 μM, and in some cases below detectable levels. In patients with PXE, PPi levels are below 0.5 μM (Arterioscler Thromb Vasc Biol. 2014 Sep;34(9):1985-9; Braddock et al., Nat Commun. 2015;6:10006).

[0070] As used herein, the term "pathological calcification" refers to the abnormal deposition of calcium salts in soft tissues, secretory and excretory tracts of the body, causing them to harden. There are two types of calcification: dystrophic calcification, which occurs in dying and dead tissues, and metastatic calcification, which is characterized by an increase in extracellular calcium levels (hypercalcemia) that exceeds the homeostatic capacity of cells and tissues. Calcification can involve cells, as well as extracellular matrix components, such as collagen in basement membranes and elastic fibers in arterial walls. Some examples of tissues that are prone to calcification include gastric mucosa - the inner epithelial lining of the stomach, kidneys and lungs, cornea, systemic arteries and pulmonary veins.

[0071] As used herein, the term "pathological ossification" refers to the pathological condition in which bone occurs in tissue that is not in bone system or in connective tissue that does not normally show osteogenic properties.Ossification is classified into three types according to the nature of affected tissue or organ: endochondral ossification is the ossification that occurs in cartilage and replaces cartilage; intramembranous ossification is the ossification of bone that occurs in connective tissue and replaces connective tissue; deformed ossification, the development of bone material in normally soft body structures; also called heterotopic ossification.

[0072] A "deficiency" of ENPP1 refers to a condition in which a subject has less than 5%-10% of the normal level of ENPP1 in plasma. Normal levels of ENPP1 in healthy human subjects are approximately 10-30 ng / ml (Am J Pathol. 2001 Feb;158(2):543-554).

[0073] "Ectopic calcification" refers to a condition characterized by the pathological deposition of calcium salts in tissue, or bone growth in soft tissue.

[0074] "Ectopic soft tissue calcification" refers to inappropriate biomineralization occurring in soft tissues, typically composed of calcium phosphate, hydroxyapatite, calcium oxalate, and octacalcium phosphate, resulting in loss of hardening of the soft tissue. "Arterial calcification" refers to ectopic calcification occurring in arteries and heart valves, resulting in hardening and / or narrowing of the arteries. Arterial calcification is correlated with atherosclerotic plaque burden and increased risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection after angioplasty.

[0075] "Venous calcification" refers to ectopic calcification that occurs in veins and reduces the elasticity of the veins, restricting blood flow, which in turn can lead to elevated blood pressure and coronary artery defects.

[0076] "Vascular calcification" refers to the pathological deposition of minerals within the vascular system. It has various forms, including intimal calcification and medial calcification, but can also be found in the valves of the heart. Vascular calcification is associated with atherosclerosis, diabetes, certain genetic conditions, and kidney disease, especially CKD. Patients with vascular calcification are at increased risk for adverse cardiovascular events. Vascular calcification affects a wide variety of patients. Idiopathic infantile arterial calcification is a rare form of vascular calcification in which the arteries of newborns become calcified.

[0077] "Cerebral calcification" (BC) refers to a non-specific neuropathology in which deposition of calcium and other minerals occurs within blood vessel walls and tissue parenchyma, leading to neuronal death and gliosis. Cerebral calcification is frequently associated with a variety of chronic and acute brain disorders, including Down's syndrome, Lewy body disease, Alzheimer's disease, Parkinson's disease, vascular dementia, brain tumors, and / or various endocrinological conditions.

[0078] Calcification of cardiac tissue refers to the accumulation of deposits of calcium (and possibly other minerals) in cardiac tissue, such as aortic and coronary artery tissue.

[0079] As used herein, "mineral bone disorder (MBD)" refers to a disorder characterized by abnormal hormone levels that cause imbalance of calcium and phosphorus levels in a person's blood. Mineral and bone disorders commonly occur in people with CKD and affect most people with kidney failure who are undergoing dialysis.

[0080] As used herein, the term "early onset osteoporosis" refers to the early stages of osteoporosis, which are generally characterized by back pain, stooped posture, and / or slow loss of bone mass. Common causes include a low calcium diet, smoking, and age-related hormonal changes.

[0081] "Osteopenia" is a bone condition characterized by decreased bone density, which leads to weakening of bones and increased risk of fracture. Osteomalacia is a bone disorder characterized by decreased mineralization of newly formed bone. Osteomalacia is caused by severe vitamin D deficiency (which may be nutritional or caused by genetic syndrome) and by conditions that cause extremely low blood phosphate levels. Both osteomalacia and osteopenia increase the risk of breaking bone. Symptoms of osteomalacia include bone pain and muscle weakness, bone tenderness, difficulty walking and muscle spasms.

[0082] As used herein, "age-related osteopenia" refers to a condition in which bone density is lower than normal. In general, osteopenia patients have a bone density T-score of -1.0 to -2.5. If left untreated, osteopenia progresses to osteoporosis, which makes bones brittle and highly susceptible to fracture.

[0083] As used herein, "ossification of the posterior longitudinal ligament (OPLL)" refers to a hyperostotic (excessive bone growth) condition that results in ectopic calcification of the posterior longitudinal ligament. The posterior longitudinal ligament connects and stabilizes the bones of the spine. The thickened or calcified ligament can compress the spinal cord, causing myelopathy. Symptoms of myelopathy include difficulty walking, and difficulty controlling the bowels and bladder. OPLL can also cause radiculopathy, or compression of the nerve roots. Symptoms of cervical radiculopathy include pain, tingling, or numbness in the neck, shoulder, arm, or hand. OPLL differs from DISH because ossification occurs only in the posterior longitudinal ligament, unlike DISH, in which ossification also occurs in the thoracic region and anterior longitudinal ligament.

[0084] The clinical symptoms and signs caused by OPLL are classified into (1) myelopathy, or spinal cord lesion with motor and sensory impairment of the upper and lower limbs, spasticity, and bladder dysfunction; (2) cervical radiculopathy with pain and sensory impairment of the upper limbs; and (3) axial discomfort with pain and stiffness around the neck. The most common symptoms in the early stages of OPLL include paresthesias and tingling sensations in the hands, and clumsiness. With the progression of neuropathy, lower limb symptoms such as gait disturbance may appear. OPLL is detected using lateral plain radiographs, and the diagnosis and morphological details of cervical OPLL have been clearly demonstrated by magnetic resonance imaging (MRI) and computed tomography (CT).

[0085] OPLL is common in Americans with cervical myelopathy and more broadly in Asian populations. Myelopathy and loss of mobility gradually worsen with age, and there are no effective means of preventing the progression of paravertebral ossifications responsible for myelopathy and stiffness. Current treatment focuses on symptom relief, and although surgery can be helpful in the short term, the subsequent rapid progression of enthesopathy and recurrence of symptoms often complicates this procedure.

[0086] Patients with rapidly progressive OPLL exhibit elevated circulating FGF23, a central finding of the rare disorders X-linked hypophosphatemia (XLH) and autosomal recessive hypophosphatemic rickets (ARHR), both of which exhibit DISH and similar enthesopathy to OPLL (Kawaguchi, Y., et al., Serum biomarkers in patients with ossification of the posterior longitudinal ligament (OPLL): Inflammation in OPLL. PLoS One, 2017.12(5):p.e0174881; Kawaguchi, Y., et al., Increase of the Serum FGF-23 in Ossification of the Posterior Longitudinal Ligament. Global Spine J, 2019.9(5):p.492-498). Although these findings indicate a causative role for FGF23 (or the resulting hypophosphatemia) in the pathogenesis of enthesopathy, the mechanisms by which elevated FGF23 and / or decreased phosphate may induce enthesopathy or vertebral ossification are unclear.

[0087] "Hereditary hypophosphatemic rickets" as used herein refers to a disorder associated with low levels of phosphate in the blood (hypophosphatemia). Phosphate is a mineral essential for the normal formation of bones and teeth. Most commonly, it is caused by a mutation in the PHEX gene. Other genes that may be involved in this condition include the CLCN5 gene, the DMP1 gene, the ENPP1 gene, the FGF23 gene, and the SLC34A3 gene. Other signs and symptoms of hereditary hypophosphatemic rickets may include premature fusion of the skull bones (craniosynostosis), and dental abnormalities. The disorder may also cause abnormal bone growth where ligaments and tendons attach to joints (enthesopathy). In adults, hypophosphatemia is characterized by softening of the bones, known as osteomalacia. Another rare type of disorder is known as hereditary hypophosphatemic rickets with hypercalciuria (HHRH), in which, in addition to hypophosphatemia, the condition is characterized by the excretion of high levels of calcium in the urine (hypercalciuria).

[0088] "X-linked Hypophosphatemia (XLH)" As used herein, the term X-linked hypophosphatemia (XLH), also called X-linked overt hypophosphatemic rickets or X-linked vitamin D-resistant rickets, is an X-linked overt form of rickets (or osteomalacia) that differs from most cases of rickets in that vitamin D supplementation does not cure it. It can cause short stature and bone deformities, including genu varum (bow legs). It is associated with a mutation in the PHEX gene sequence (Xp.22) and the subsequent inactivity of the PHEX protein.

[0089] As used herein, "autosomal recessive hypophosphatemic rickets type 2 (ARHR2)" refers to an inherited renal phosphate wasting disorder characterized by hypophosphatemia, rickets and / or osteomalacia, and growth retardation. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is caused by a homozygous loss-of-function mutation in the ENPP1 gene.

[0090] As used herein, "autosomal overt hypophosphatemic rickets (ADHR)" refers to a rare genetic disease in which excessive loss of phosphate in urine leads to poorly formed bones (rickets), bone pain, and dental abscesses. ADHR is caused by mutations in fibroblast growth factor 23 (FGF23). ADHR is characterized by impaired bone mineralization, rickets, and / or osteomalacia, suppressed levels of calcitriol (1,25-dihydroxyvitamin D3), renal phosphate wasting, and low serum phosphate. Mutations in FGF23 make the protein more stable and uncleavable by proteases, leading to enhanced biological activity of FGF23. Enhanced activity of FGF23 mutants reduces the expression of sodium phosphate cotransporters, NPT2a and NPT2c, on the apical surface of proximal tubule cells, leading to renal phosphate wasting.

[0091] "Hypophosphatemic rickets" (previously called vitamin D-resistant rickets) is a disorder in which bones become painfully soft and easily bent due to low levels of phosphate in the blood. Symptoms may include bowed legs and other bone deformities; bone pain; joint pain; poor bone growth; and short stature. In some affected infants, the spaces between the skull bones close too early, leading to craniosynostosis. Most patients exhibit abnormalities of calcium phosphate metabolism, abnormalities of dental enamel, delayed tooth eruption, and an elongated head (dolichocephaly).

[0092] As used herein, the term "ENPP1 haploinsufficiency" refers to a genetic condition in which one copy of the ENPP1 gene is inactivated or deleted, and the remaining functional copies of the ENPP1 gene are insufficient to produce the gene product required to maintain normal function.The result of ENPP1 haploinsufficiency may, but does not necessarily, manifest in the form of low PPi levels and pathological calcification.Diseases such as DISH, or early onset osteoporosis, may in certain embodiments be associated with and / or caused by ENPP1 haploinsufficiency.

[0093] For most genes, a single copy is sufficient to support normal growth and development of diploid organisms, but a small subset of genes known as haploinsufficient (HI) genes show extreme sensitivity to reduced gene dosage. Given the relatively high frequency of gene inactivating mutations over the lifespan of an organism, and the cell-to-cell variability of gene expression, haploinsufficiency is a significant barrier to organismal fitness. Haploinsufficiency in genetics represents a model of dominant gene action in diploid organisms, where a single copy of a wild-type allele at a locus heterozygously combined with a variant allele is insufficient to produce a wild-type phenotype. Haploinsufficiency can result from de novo or heritable loss-of-function mutations in the variant allele, resulting in little or no gene product.

[0094] For example, it has been found that the N179S mutation in ENPP1 leads to the loss of function of ENPP1 protein.The presence of a single copy of the mutant ENPP1 gene with N179S mutation reduces PPi production.The present invention discloses that N179S mutation is found in certain patients with DISH or early onset of osteoporosis, and in certain embodiments, can serve as a genetic marker for the presence or future onset and / or progression of DISH or osteoporosis.

[0095] Similarly, it has been found that the Y451C mutation in ENPP1 leads to the loss of function of ENPP1 protein.The presence of a single copy of the mutant ENPP1 gene with Y451C mutation reduces PPi production.The present invention discloses that Y451C mutation is found in certain patients with DISH or early onset of osteoporosis, and in certain embodiments, can serve as a genetic marker for the presence or future onset and / or progression of DISH or osteoporosis.

[0096] As used herein, "pre-treatment" means treatment prior to the initiation of a treatment method described herein.

[0097] The term "subject" as used herein refers to an individual, for example, a mammal, for example, a human, a non-human primate (e.g., chimpanzee and other ape and monkey species), livestock (e.g., birds, fish, cows, sheep, pigs, goats and horses), domestic mammals (e.g., dogs and cats), or laboratory animals (e.g., rodents such as mice, rats and guinea pigs).The term includes subjects of any age or sex.In another embodiment, the subject is a mammal, preferably a human.

[0098] A disease or disorder is "alleviated" if the severity of a symptom of the disease or disorder, the frequency with which such symptoms are experienced by a patient, or both, are reduced.

[0099] As used herein, the terms "alteration," "defect," "variation," or "mutation" refer to a mutation in a gene in a cell, e.g., missense and nonsense mutations, insertions, deletions, frameshifts, and premature termination, that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes.

[0100] A "disease" is a condition in the health of an animal where the animal is unable to maintain homeostasis and where, if the disease is not ameliorated, the animal's health continues to deteriorate.

[0101] An animal "disorder" is a health state in which the animal is able to maintain homeostasis, but in which the animal's health state is less favorable than it would be in the absence of the disorder. If left untreated, the disorder does not necessarily result in a further deterioration of the animal's health state.

[0102] As used herein, the term "ENPP" or "NPP" refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0103] As used herein, the term "ENPP1 protein" or "ENPP1 polypeptide" refers to the ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein that cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars, and pyrophosphate bonds of nucleotides and nucleotide sugars. The ENPP1 protein has a transmembrane domain and a soluble extracellular domain. The extracellular domain is further subdivided into a somatomedin B domain, a catalytic domain (residues 186-586 of SEQ ID NO:1), and a nuclease domain (residues 524-885 of SEQ ID NO:1). The sequence and structure of wild-type ENPP1 are described in detail in PCT Application Publication No. WO 2014 / 126965 to Braddock et al., which is incorporated herein by reference in its entirety.

[0104] Mammalian ENPP1 polypeptides, variants or variant fragments thereof have been previously disclosed in International PCT Application Publication Nos. WO / 2014 / 126965 to Braddock et al., WO / 2016 / 187408 to Braddock et al., WO / 2017 / 087936 to Braddock et al., and WO2018 / 027024 to Braddock et al., all of which are incorporated by reference in their entireties.

[0105] As used herein, the term "ENPP1 precursor protein" refers to ENPP1 having its signal peptide sequence at the ENPP1 N-terminus. During proteolysis, the signal sequence (indicated in certain non-limiting embodiments by W in the compound of formula (I)) is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful within the present invention include, but are not limited to, albumin signal sequence, azurocidin signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence and / or ENPP5 signal peptide sequence.

[0106] As used herein, the term "ENPP1-Fc construct" refers to ENPP1 recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0107] As used herein, the term "Fc" refers to the human IgG (immunoglobulin) Fc domain. Subtypes of IgG, such as IgG1, IgG2, IgG3, and IgG4, are contemplated for use as the Fc domain.

[0108] As used herein, "Fc region or Fc polypeptide" is the portion of an IgG molecule that correlates with the crystallizable fragment obtained by papain digestion of the IgG molecule. The Fc region comprises the C-terminal halves of the two heavy chains of an IgG molecule linked by disulfide bonds. The Fc region does not have antigen binding activity, but contains carbohydrate moieties and binding sites for complement and Fc receptors, including the FcRn receptor. The Fc fragment contains the entire second constant domain CH2 (residues 231-340 of human IgG1 according to the Kabat numbering system) and the third constant domain CH3 (residues 341-447). The term "IgG hinge-Fc region" or "hinge-Fc fragment" refers to the region of an IgG molecule that consists of the Fc region (residues 231-447) and the hinge region (residues 216-230) extending from the N-terminus of the Fc region. The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence than the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0109] As used herein, the term "fragment" as applied to a nucleic acid refers to a subsequence of a larger nucleic acid. A "fragment" of a nucleic acid can be at least about 15, at least about 50-100, at least about 100-500, at least about 500-1000, at least about 1000-1500 nucleotides, at least about 1500-2500, or at least about 2500 nucleotides (and any integer value in between). As used herein, the term "fragment" as applied to a protein or peptide refers to a subsequence of a larger protein or peptide and can be at least about 20, at least about 50, at least about 100, at least about 200, at least about 300, or at least about 400 amino acids in length (and any integer value in between).

[0110] "Isolated" means changed or removed from the natural state. For example, a nucleic acid or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or polypeptide that is partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form, or may exist in a non-native environment, such as, for example, a host cell.

[0111] As used herein, the terms "patient," "individual," or "subject" refer to a human.

[0112] As used herein, the term "pharmaceutical composition" or "composition" refers to a mixture of at least one compound useful within the present invention and a pharma- ceutical acceptable carrier.The pharmaceutical composition facilitates the administration of the compound to the patient.There are multiple techniques of administering the compound in the art, including, but not limited to, subcutaneous administration, intravenous administration, oral administration, aerosol administration, inhalation administration, rectal administration, vaginal administration, transdermal administration, intranasal administration, buccal administration, sublingual administration, parenteral administration, intrathecal administration, intragastric administration, ocular administration, pulmonary administration and topical administration.

[0113] As used herein, the term "pharmacologically acceptable" refers to a material, such as a carrier or diluent, e.g., phosphate buffered saline (PBS), that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, i.e., that may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0114] As used herein, the term "plasma pyrophosphate (PPi) level" refers to the amount of pyrophosphate present in the plasma of an animal. In certain embodiments, the animals include rats, mice, cats, dogs, humans, cows, and horses. Due to release from platelets, it is necessary to measure PPi in plasma rather than serum. There are several ways to measure PPi, one of which is by an enzyme assay using a modified version of uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63). Typically, normal PPi levels in healthy subjects range from about 1 μM to about 3 μM, and in some cases 1-2 μM. In some cases, a "low" level of PPi refers to a condition in which a subject has 2% to 5% or less of the normal level of plasma pyrophosphate (PPi) (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979)).

[0115] The subject with defective ENPP1 expression tends to show low PPi levels, ranging from at least 10% lower than normal levels, at least 20% lower than normal levels, at least 30% lower than normal levels, at least 40% lower than normal levels, at least 50% lower than normal levels, at least 60% lower than normal levels, at least 70% lower than normal levels, at least 80% lower than normal levels, and combinations thereof.In patients with GACI, PPi levels are found to be less than 1 μM, and in some cases are below detectable levels.In patients with PXE, PPi levels are less than 0.5 μM (Arterioscler Thromb Vasc Biol.2014 Sep;34(9):1985-9;Braddock et al.,Nat Commun.2015;6:10006).

[0116] As used herein, the term "polypeptide" refers to a polymer composed of amino acid residues linked via peptide bonds, related naturally occurring structural variants, and synthetic non-naturally occurring analogues thereof.

[0117] As used herein, the term "PPi" refers to pyrophosphate.

[0118] As used herein, the term "prevent" or "prevention" refers to the absence of onset of a disorder or disease if the disorder or disease has not occurred, or the absence of further onset of a disorder or disease if the disorder or disease has already developed. The ability to prevent some or all of the symptoms associated with a disorder or disease is also considered.

[0119] As used herein, "sample" or "biological sample" refers to a biological material isolated from a subject. A biological sample may contain any biological material suitable for detecting mRNA, polypeptides or other markers of a physiological or pathological process in a subject, and may include fluids, tissues, cellular and / or non-cellular materials obtained from an individual.

[0120] As used herein, "substantially purified" refers to being essentially free of other components. For example, a substantially purified polypeptide is a polypeptide that is separated from other components that are normally associated with it in its natural state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity, and 100% purity.

[0121] As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound useful within the present invention (alone or in combination with another pharmaceutical agent), to a patient, or the application or administration of a therapeutic agent to a tissue or cell line isolated from a patient having a disease or disorder and / or symptoms of a disease or disorder (e.g., for diagnostic or ex vivo use), with the aim of curing, relieving, mitigating, altering, curing, improving, improving, affecting, and / or preventing a disease or disorder and / or symptoms of a disease or disorder, and / or minimizing the progression of a disease or disorder and / or symptoms of a disease or disorder. Such treatments can be specifically tailored or modified based on knowledge gained from the field of pharmacogenomics.

[0122] As used herein, the terms "prevent", "preventing" and "prevention" refer to inhibiting the onset of disease in a subject or reducing the occurrence of disease in a subject. Prevention can be complete (e.g., complete absence of pathological cells in a subject) or partial. Prevention also refers to reducing susceptibility to a clinical condition. In certain embodiments, "preventing" includes preventing the onset of a disease or disorder.

[0123] As used herein, the term "wild type" refers to a gene or gene product isolated from a natural source. The wild type gene is the most frequently observed in the population, and therefore the "normal" or "wild type" form of the human ENPP1 gene is arbitrarily designated. In contrast, the term "functionally equivalent" refers to an ENPP1 gene or ENPP1 gene product that exhibits alterations in sequence and / or functional properties (i.e., changes in characteristics) when compared with a wild type gene or wild type gene product. Naturally occurring mutants can be isolated, and are identified by the fact that they have changes in characteristics (including changes in nucleic acid sequence) when compared with a wild type gene or wild type gene product.

[0124] The term "functionally equivalent variant" as used herein refers to a polypeptide that is substantially homologous to the sequence of ENPP1 (as defined above) and maintains the enzymatic and biological activity of ENPP1. Methods for determining whether a variant maintains the biological activity of natural ENPP1 are widely known to those skilled in the art and include any of the assays used in the experimental part of this application. In particular, the functionally equivalent variant of ENPP1 delivered by a viral vector is encompassed by the present invention.

[0125] A functionally equivalent variant of ENPP1 is a polypeptide that is substantially homologous to the native ENPP1. The term "substantially homologous" relates to a protein sequence when said protein sequence has a degree of identity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% respectively to the abovementioned ENPP1 sequences.

[0126] The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known to those skilled in the art.The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul et al., NCBI NLM NIH Bethesda, Md.20894, Altschul et al., J.Mol.Biol.215:403-410(1990)), but other similar algorithms can also be used.Percent sequence identity is determined using BLAST and BLAST 2.0 with the parameters described herein.The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information.

[0127] A "functionally equivalent variant" of ENPP1 can be obtained by substituting nucleotides in a polynucleotide that account for the codon preferences in the host cell used to produce ENPP1, respectively. Such "codon optimization" can be determined by a computer algorithm that incorporates a codon frequency table, such as "Human high.cod" for codon preferences, provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.

[0128] As used herein, "about" in reference to a measurable value, e.g., amount, duration, etc., is meant to encompass variations of ±10% or ±5%, in certain embodiments ±1-5%, in certain embodiments ±5%, in certain embodiments ±4%, in certain embodiments ±4%, in certain embodiments ±3%, in certain embodiments ±2%, and in certain embodiments ±1% from the specified value (0.2 mg / kg or 0.6 mg / kg or 1.8 mg / kg), where such variations are appropriate for carrying out the disclosed methods.

[0129] This disclosure provides representative examples of protein sequences. The described protein sequences can be converted into nucleic acid sequences by performing reverse translation and codon optimization. There are several tools available in the art that allow such conversion, such as Expasy (https: / / www.expasy.org / ) and Bioinformatics Server (www dot bioinformatics dot org).

[0130] Ranges: Throughout this disclosure, various aspects according to the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible subranges, as well as individual numerical values ​​within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0131] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the methods and compositions of the disclosure. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0132] Detailed Description 1. ENPP1 agonist The ENPP1 agonist is an ENPP1 polypeptide. The ENPP1 polypeptide disclosed herein includes, but is not limited to, the naturally occurring polypeptide of the ENPP1 family, as well as any variant thereof (including mutants, fragments, fusions and peptidomimetics) that retains biological activity, such as, but not limited to, the catalytic activity of ENPP1.

[0133] The ENPP1 agonist may be in the form of the formula shown below: W-PROTEIN-Z-DOMAIN-XY (I) It can be expressed as: In (I), W is absent or contains a signal sequence that enables transport of the compound to the extracellular space, PROTEIN contains the catalytic domain of ENPP1, DOMAIN is absent or at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin protein (ALB) and biologically active fragments thereof; X and Z are, independently, absent or a polypeptide containing 1 to 20 amino acids; Y is absent or TIFF2025510963000002.tif32130, m is independently an integer ranging from 1 to 15; n is independently an integer ranging from 1 to 10.

[0134] In some embodiments, W is absent. In some embodiments, W comprises a signal sequence that enables transport of the compound to the extracellular space.

[0135] In some embodiments, the ENPP1 agent comprises a bone targeting domain. In some embodiments, the bone targeting domain is a negatively charged sequence of amino acids. In some embodiments, the negatively charged bone targeting domain is polyaspartic acid.

[0136] The term "ENPP1" or "ENPP1 polypeptide" refers to ectonucleotide pyrophosphatase / phosphodiesterase 1 protein (NPP1 / ENPP1 / PC-1) and ENPP1-related proteins from any species. ENPP1 protein contains a type II transmembrane glycoprotein that forms homodimers. Each monomer of ENPP1 protein contains a short intracellular N-terminal domain involved in targeting to the plasma membrane, a transmembrane domain, and a large extracellular region that contains several domains. The large extracellular region contains the SMB1 and SMB2 domains that have been reported to be involved in ENPP1 dimerization (Gijsbers et al., Biochem. J. 371; 2003: 321-330). Specifically, the SMB domain contains eight cysteine ​​residues arranged in four disulfide bonds, respectively, and has been shown to mediate ENPP1 homodimerization through covalent cystine inter- and intramolecular bonds. The protein cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars, and pyrophosphate bonds of nucleotides and nucleotide sugars. The ENPP1 protein functions to hydrolyze nucleoside 5' triphosphatases to either of the corresponding monophosphates, and also hydrolyzes diadenosine polyphosphates. The ENPP1 protein plays a role in purinergic signaling, which is involved in regulating cardiovascular, neurological, immune, musculoskeletal, hormonal, and hematological functions. An exemplary amino acid sequence of human ENPP1 precursor protein (NCBI accession NP_006199) is shown in FIG. 11 (SEQ ID NO:1). Human ENPP1 precursor protein contains an endogenous ENPP1 signal peptide sequence at the ENPP1 N-terminus. The amino acid numbering for any ENPP1-related polypeptide described herein is based on the numbering of the human ENPP1 precursor protein sequence provided in FIG. 2, unless otherwise specifically indicated. In certain embodiments, the ENPP1 precursor protein further contains an endogenous signal peptide sequence or a heterologous signal peptide sequence.During proteolysis, the signal peptide sequence is cleaved from the ENPP1 precursor protein to provide a mature ENPP1 protein.See, for example, Jansen et al.J Cell Sci.2005;118(Pt 14):3081-9.Exemplary signal peptide sequences that can be used with the polypeptides disclosed herein include, but are not limited to, the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the ENPP7 signal peptide sequence, and / or the ENPP5 signal peptide sequence.A non-limiting processed (mature) extracellular ENPP1 polypeptide sequence is shown in SEQ ID NO:2.

[0137] It is generally known in the art that ENPP1 is well conserved among vertebrates, with large stretches of the extracellular domain being substantially conserved. For example, Figures 7A-7B show multiple sequence alignments of the human ENPP1 extracellular domain compared to various ENPP1 orthologs. ENPP1 binding to various nucleotide triphosphates (e.g., ATP, UTP, GTP, TTP and CTP), pNP-TMP, 3',5'-cAMP, and 2'-3'-cGAMP is also highly conserved (see, for example, Kato et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81 and Mackenzie et al. Bone. 2012; 51(5): 961-8). Thus, from these alignments, it is possible to predict important amino acid positions with the extracellular domain that are important for normal ENPP1 activity, and to predict amino acid positions that are likely to be tolerant to substitutions without significantly altering normal ENPP1 activity. Therefore, the enzymatically active human ENPP1 polypeptide useful according to the composition of the present disclosure may contain one or more amino acids at the corresponding positions from the sequence of another vertebrate ENPP1, or may contain residues similar to those of human or other vertebrate sequences.The substitution of one or more amino acids at the corresponding positions may include conservative mutations or substitutions that are unlikely to change the shape of the polypeptide chain or change normal ENPP1 activity.Examples of conservative mutations or substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, the substitution of glutamic acid for aspartic acid, or the substitution of glutamine for asparagine.For example, ENPP1 polypeptides include polypeptides derived from the sequence of any known ENPP1 polypeptide having a sequence at least about 80% identical to the sequence of an ENPP1 polypeptide, preferably at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity.

[0138] 2. Enzymatic activity of ENPP1 ENPP1 proteins have been characterized in the art for structural and biological characteristics. In certain embodiments, the soluble ENPP1 proteins disclosed herein comprise pyrophosphatase and / or phosphodiesterase activity. For example, in some embodiments, ENPP1 proteins bind nucleotide triphosphates (e.g., ATP, UTP, GTP, TTP, and CTP), pNP-TMP, 3',5'-cAMP, and 2'-3'-cGAMP and convert nucleotide triphosphates to inorganic pyrophosphate [see, e.g., Kato et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81; Li, et al., Nat Chem Biol. 2014; 10(12): 1043-8; Jansen et al., Structure. 2012; 20(11): 1948-59; and Onyedibe et al., Molecules. 2019; 24(22)].

[0139] An "enzymatically active" or "biologically active" ENPP1 polypeptide exhibits pyrophosphatase and / or phosphodiesterase activity (e.g., can bind and / or hydrolyze ATP to AMP and PPi, and / or bind and / or hydrolyze AP3a to ATP). For example, the pyrophosphatase / phosphodiesterase domain of the ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to generate inorganic pyrophosphate (PPi) and is generally soluble. This activity can be measured using a pNP-TMP assay as previously described (Saunders et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright et al., 2015, Nat Comm. 6:10006). In certain embodiments, a soluble ENPP1 polypeptide has a pyrophosphatase activity of about 3.4 (±0.4) s for the substrate ATP. -1 enzyme -1 More than k cat has a value k cat is determined by measuring the rate of hydrolysis of ATP for the polypeptide. In certain embodiments, the soluble ENPP1 polypeptide has a K of about 2 μM or less for the substrate ATP. M has a value of K M is determined by measuring the rate of ATP hydrolysis for the polypeptide. In addition to the teachings herein, these references provide ample guidance on how to generate soluble ENPP1 proteins that retain one or more biological activities (e.g., conversion of nucleotides to inorganic pyrophosphate).

[0140] 3. Soluble ENPP1 In one embodiment, the present disclosure relates to ENPP1 agents, such as, but not limited to, ENPP1 polypeptides. As described herein, the term soluble ENPP1 polypeptide includes any naturally occurring extracellular domain of ENPP1 protein, as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain biological activity (e.g., enzymatic activity). Examples of soluble ENPP1 polypeptides include, for example, ENPP1 extracellular domain (SEQ ID NO:2). In certain embodiments, soluble ENPP1 polypeptides further include a signal sequence in addition to the extracellular domain of ENPP1 polypeptide. Exemplary signal sequences include the native signal sequence of ENPP1 polypeptide, or a signal sequence from another protein, such as the hENPP7 signal sequence. Examples of variant soluble ENPP1 polypeptides are provided in International Patent Application Publication Nos. WO 2012 / 125182, WO 2014 / 126965, WO 2016 / 187408, WO 2018 / 027024, WO 2020206302 and WO 2020 / 047520, the contents of all of which are incorporated herein by reference in their entireties.

[0141] 4. ENPP1 fusion protein In some embodiments, the ENPP1 polypeptide is a fusion protein comprising an ENPP1 polypeptide domain and one or more heterologous protein moieties (i.e., polypeptide domains heterologous to ENPP1). An amino acid sequence is understood to be heterologous to ENPP1 if it is not uniquely found in the form of ENPP1 represented by SEQ ID NO:1. In some embodiments, the heterologous protein moiety comprises an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is an IgG1 immunoglobulin Fc domain. In certain embodiments, the soluble ENPP1 polypeptide is C-terminally fused to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3) and / or human immunoglobulin 4 (IgG4). In other embodiments, the soluble ENPP1 polypeptide is N-terminally fused to the Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3) and / or human immunoglobulin 4 (IgG4). In some embodiments, the presence of the Fc domain improves the half-life, solubility, reduces immunogenicity and increases the activity of the soluble ENPP1 polypeptide. In certain embodiments, a portion of a natural human IgG protein (IgG1, IgG2, IgG3 and IgG4) may be used for the Fc portion (e.g., ENPP1-Fc). For example, the present disclosure provides a fusion protein comprising ENPP1 fused to a polypeptide comprising a CH1 domain, CH2 domain or CH3 domain derived from a constant domain of an immunoglobulin, e.g., human IgG1, human IgG2, human IgG3 and / or human IgG4. The Fc fragment may include regions of a native IgG, such as the hinge region (residues 216-230 of human IgG1 according to the Rabat numbering system), the entire second constant domain CH2 (residues 231-340), and the third constant domain CH3 (residues 341-447).As used herein, the term "ENPP1-Fc construct" refers to a soluble form of ENPP1 (e.g., the extracellular domain of an ENPP1 polypeptide) recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugation) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0142] An example of an amino acid sequence that can be used for the Fc portion of human IgG1 (G1Fc) is SEQ ID NO:6 (Table 1). In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:6.

[0143] In some embodiments, the heterologous protein portion comprises one or more domains selected from the group consisting of polyhistidine, FLAG tag, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP) or human serum albumin. The fusion domain may be selected to confer desired properties. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For affinity purification, matrices associated with affinity chromatography are used, such as glutathione-conjugated resins, amylase-conjugated resins, and nickel- or cobalt-conjugated resins. Many such matrices are available in "kit" form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen), which are useful with (HIS6) fusion partners. As another example, the fusion domain may be selected to facilitate detection of the ENPP1 polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, such as factor Xa or thrombin, that allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be isolated from the fusion domain by subsequent chromatographic separation.

[0144] 5. Linker In some embodiments, the ENPP1 fusion protein further comprises a linker (Z) disposed between the ENPP1 polypeptide domain and one or more heterologous protein moieties (e.g., Fc immunoglobulin domains). In certain embodiments, the soluble ENPP1 polypeptide is directly or indirectly fused to the Fc domain. In some embodiments, the soluble ENPP1 fusion protein comprises a linker between the Fc domain and the ENPP1 polypeptide. In some embodiments, the linker can be an amino acid spacer comprising 1 to 200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine and serine.

[0145] In some embodiments, the linker comprises a polyglycine linker or a Gly-Ser linker. In some embodiments, the spacer can contain a GA (SEQ ID NO:21), GS (SEQ ID NO:22), GG (SEQ ID NO:23), GGA (SEQ ID NO:24), GGS (SEQ ID NO:25), GGG (SEQ ID NO:26), GGGA (SEQ ID NO:27), GGGS (SEQ ID NO:28), GGGG (SEQ ID NO:29), GGGGA (SEQ ID NO:30), GGGGS (SEQ ID NO:31), GGGGG (SEQ ID NO:32), GGAG (SEQ ID NO:33), GGSG (SEQ ID NO:34), AGGG (SEQ ID NO:35), SGGGG (SEQ ID NO:36) or SGGG (SEQ ID NO:37) motif, e.g., a multiple or repeated motif. In some embodiments, the spacer can contain 2-12 amino acids including the motifs GA or GS, e.g., GA, GS, GAGA (SEQ ID NO:38), GSGS (SEQ ID NO:39), GAGAGA (SEQ ID NO:40), GSGSGS (SEQ ID NO:41), GAGAGAGA (SEQ ID NO:42), GSGSGSGS (SEQ ID NO:43), GAGAGAGAGA (SEQ ID NO:44), GSGSGSGSGS (SEQ ID NO:45), GAGAGAGAGAGA (SEQ ID NO:46), and GSGSGSGSGSGS (SEQ ID NO:47). In some embodiments, the spacer can contain 3 to 12 amino acids including the motifs GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO:48), GGSGGS (SEQ ID NO:49), GGAGGAGGA (SEQ ID NO:50), GGSGGSGGS (SEQ ID NO:51), GGAGGAGGAGGA (SEQ ID NO:52) and GGSGGSGGSGGS (SEQ ID NO:53).Further, in some embodiments, the spacer can contain 4-12 amino acids including the motifs GGAG (SEQ ID NO:54), GGSG (SEQ ID NO:55), e.g., GGAG (SEQ ID NO:56), GGSG (SEQ ID NO:57), GGAGGGAG (SEQ ID NO:58), GGSGGGSG (SEQ ID NO:59), GGAGGGAGGGAG (SEQ ID NO:60) and GGSGGGSGGGSG (SEQ ID NO:61). In some embodiments, the spacer can contain the motifs GGGGA (SEQ ID NO:62) or GGGGS (SEQ ID NO:63), e.g., GGGAGGGGAGGGGA (SEQ ID NO:64) and GGGSGGGGSGGGGGS (SEQ ID NO:65). In some embodiments of the invention, the amino acid spacer between the heterologous protein moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) and the soluble ENPP1 polypeptide can be GGG, GGGA (SEQ ID NO:27), GGGG (SEQ ID NO:29), GGGAG (SEQ ID NO:66), GGGAGG (SEQ ID NO:67), or GGGAGGG (SEQ ID NO:68).

[0146] In some embodiments, the spacer also includes amino acids other than glycine, alanine, and serine, e.g., TIFF2025510963000003.tif128150. In some embodiments, the spacer can contain a motif, e.g., multiple motifs or repeated motifs, of EAAAK (SEQ ID NO:85). In some embodiments, the spacer can contain a praline-rich sequence, e.g., (XP)n, where X can be any amino acid (e.g., A, K, or E) and n is 1-5, and a motif, e.g., multiple motifs or repeated motifs, of PAPAP (SEQ ID NO:86).

[0147] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0148] In some embodiments, the various elements of a fusion protein (e.g., an immunoglobulin Fc fusion protein) may be arranged in any manner consistent with the desired functionality. For example, a soluble ENPP1 polypeptide domain may be arranged C-terminal to a heterologous protein portion, or alternatively, a heterologous protein portion may be arranged C-terminal to a soluble ENPP1 polypeptide domain. The soluble ENPP1 polypeptide domain and the heterologous protein portion may be linked directly or indirectly within the fusion protein, and additional domains or amino acid sequences may be included C-terminal or N-terminal to either domain, or between the domains. A preferred fusion protein comprises an amino acid sequence set forth in any one of SEQ ID NOs:3-5.

[0149] In some embodiments, the soluble ENPP1 polypeptide of the present disclosure contains one or more heterologous moieties. Optionally, the soluble ENPP1 polypeptide contains one or more heterologous moieties selected from glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to lipid moieties, and amino acids conjugated to organic derivatization agents. In some embodiments, the soluble ENPP1 polypeptide disclosed herein is further modified. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the soluble ENPP1 polypeptide may contain non-amino acid elements such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the soluble ENPP1 polypeptide may be tested as described herein for other soluble ENPP1 polypeptides. When the polypeptide of the present disclosure is produced in a cell by cleaving the nascent form of the polypeptide, post-translational processing may also be important for the correct folding and / or function of the protein.Various cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) have specific cellular and characteristic mechanisms for such post-translational activity and can be selected to ensure the correct modification and processing of soluble ENPP1 polypeptide.

[0150] As used herein, the percent "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence after aligning the sequences and introducing gaps as necessary to achieve maximum percent sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. In some embodiments, alignment is performed using CLUSTAL OMEGA software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences to be compared.

[0151] 6. Solubility Determination In some embodiments, the activity of soluble ENPP1 polypeptide may be tested in cell-based assays or in vivo assays. For example, the effect of soluble ENPP1 polypeptide on the generation of inorganic pyrophosphate (PPi) can be measured. Specifically, the pyrophosphatase / phosphodiesterase domain of ENPP1 protein hydrolyzes extracellular nucleotide triphosphate to generate inorganic pyrophosphate (PPi), which is generally soluble. This activity can be measured using pNP-TMP assay and HPLC-based ATP hydrolysis assay as previously described (Saunders et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright et al., 2015, Nat Comm. 6:10006). The effect of soluble ENPP1 polypeptide on the expression of genes involved in ENPP1-related diseases, such as ARHR2 (e.g., transcription of fibroblast growth factor 23 in osteoblasts and osteoclasts) can be evaluated. This may optionally be done in the presence of one or more nucleotide triphosphates or other ENPP1 substrates, and the cells may be transfected to produce soluble ENPP1 polypeptides. Similarly, soluble ENPP1 polypeptides may be administered to mice or other animals, and the effect on ENPP1-associated disease may be assessed using art-recognized methods.

[0152] In some embodiments, the ENPP1 polypeptide used according to the methods described herein is an isolated polypeptide. As used herein, an isolated protein or polypeptide is one that is separated from the components of its natural environment. In some embodiments, the polypeptide of the present disclosure is purified to greater than 95%, greater than 96%, greater than 97%, greater than 98% or greater than 99% purity, for example, as determined by electrophoretic analysis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion-exchange HPLC or reverse-phase HPLC) analysis. Methods for assessing purity are well known in the art [see, for example, Flatman et al., 2007, J. Chromatogr. B 848:79-87]. In some embodiments, the soluble ENPP1 polypeptide used according to the methods described herein is a recombinant polypeptide.

[0153] 7. ENPP1 Production The ENPP1 polypeptide of the present disclosure can be produced by various techniques known in the art. For example, the polypeptide of the present disclosure can be synthesized using standard protein chemistry techniques, such as those described in Bodansky, Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptide of the present disclosure, including its fragment or variant, can be recombinantly produced using various expression systems well known in the art (e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus, yeast Pichia). The protein can be produced in adherent or suspension cells. In some embodiments, the fusion protein is expressed in CHO cells. To establish a stable cell line, the nucleic acid sequence encoding the ENPP1 construct is cloned into a suitable vector for large-scale protein production. In certain embodiments, the modified or unmodified polypeptides of the present disclosure can be produced by digestion of recombinantly produced full-length ENPP1 polypeptides, for example, by using proteases such as trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using commercially available software, for example, MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such polypeptides can be produced from recombinantly produced full-length ENPP1 polypeptides using chemical cleavage (e.g., cyanogen bromide, hydroxylamine, etc.).

[0154] 8. Expression Systems Many expression systems are known and can be used to produce ENPP1 fusion proteins, including bacteria (e.g., Escherichia coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells and insect cells. The desired protein can be produced in a conventional manner, for example, from a coding sequence inserted into the host chromosome or a free plasmid.

[0155] Yeast can be transformed with the coding sequence of the desired protein by any of the usual methods (e.g., electroporation). Methods for transforming yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194:182. Successfully transformed cells, i.e., cells containing the DNA construct of the present disclosure, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce ENPP1 polypeptide. Cells can be harvested and lysed, and their DNA content examined for the presence of DNA using methods such as those described by Southern, 1975, J. Mol. Biol, 98:503 and / or Berent et al., 1985, Biotech 3:208. Alternatively, antibodies can be used to detect the presence of protein in the supernatant.

[0156] Useful yeast plasmid vectors include pRS403-406, and pRS413-416, which are publicly available from Stratagene Cloning Systems, La Jolla, Calif., USA. Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2 and 1JRA3. Plasmids pRS413-416 are Yeast Centromere Plasmids (YCps).

[0157] Various methods have been developed to operably link DNA to vector via complementary cohesive ends.For example, complementary homopolymer tracts can be added to the DNA segment to be inserted into vector DNA.The vector and DNA segment are then linked by hydrogen bonds between complementary homopolymer tails to form recombinant DNA molecules.

[0158] Synthetic linkers containing one or more restriction sites provide an alternative method of joining DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3'-single-stranded ends by their 3'-5'-exonuclease activity and fill in recessed 3'-ends with their polymerization activity.

[0159] Thus, the combination of these activities produces blunt-ended DNA segments.The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.As a result, the products of this reaction are DNA segments with polymer linker sequences at their ends.These DNA segments can be cut with appropriate restriction enzymes and ligated into expression vectors that have been cut with enzymes that produce ends that are compatible with the ends of the DNA segments.

[0160] Single stably transfected cell clones are then established and screened for high expression clones of the desired ENPP1 fusion protein. Screening of single cell clones for ENPP1 protein expression can be accomplished in a high-throughput manner in 96-well plates using the synthetic enzyme substrate pNP-TMP as previously described (Albright et al., 2015, Nat. Commun. 6:10006). Once high expression clones are identified by screening, protein production can be achieved in shake flasks or bioreactors as previously described (Albright et al., 2015, Nat. Commun. 6:10006).

[0161] 9. ENPP1 Purification Purification of ENPP1 can be achieved using a combination of standard purification techniques known in the art. After purification, ENPP1-Fc is concentrated to 5-7 mg / ml and then diluted with Zn 2+ and Mg 2+ The antibody can be dialyzed into PBS supplemented with 0.1% MgCl (PBSplus) and frozen in aliquots of 200-500 pl at -80°C. The aliquots can be thawed immediately prior to use, and the specific activity of the solution adjusted to 31.25 au / ml (or approximately 0.7 mg / ml depending on the preparation) by dilution with PBSplus.

[0162] 10. Route and frequency of administration Polypeptide can be administered to subject acutely or chronically.In certain embodiments, the second dose of soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is administered to subject after a suitable time interval of about 2 days, about 4 days, about 1 week or about 1 month, or even less frequently, for example, once every few months, or even once a year or less.The frequency of administration is easily clear to those skilled in the art and depends on any number of factors, including, but not limited to, the type and severity of the disease being treated, and the type and age of patient.

[0163] The dose or frequency may be selected to maintain steady state levels of plasma PPi at a constant or steady state level and / or to achieve continuous plasma PPi levels that are either close to normal PPi levels (2-3 μM) or higher (30-50% higher) than normal PPi levels and do not revert to the low PPi levels the subject had prior to administration of the first dose of a construct disclosed herein.

[0164] Alternatively, the ENPP1 agonist may be administered at appropriate time intervals, either every 2 days, or every 4 days, once a week, or once a month, to achieve a constant level of enzymatic activity of ENPP1.

[0165] Alternatively, an ENPP1 agent according to the present disclosure is administered at appropriate time intervals, such as every two days, or every four days, or once a week, or once a month, depending on monitoring one or more symptoms of the disease or disorder of the subject.

[0166] Without wishing to be bound by theory, it is believed that maintaining the steady state concentration of plasma PPi at normal levels reduces and / or prevents the progression of pathological calcification in a subject.

[0167] In certain embodiments, the polypeptide is administered topically, locally, parenterally, or systemically to the subject, hi some embodiments, the polypeptide is administered subcutaneously.

[0168] As used herein, "parenteral administration" of a formulation includes any administration route characterized by physical damage to the tissue of a subject and administration of an ENPP1 agent through the tissue damage.Thus, parenteral administration includes, but is not limited to, administration of an ENPP1 agent by injection of a composition, application of a composition through a surgical incision, application of a composition through a tissue-penetrating non-surgical wound, etc.In particular, parenteral administration is intended to include, but is not limited to, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intrastemal injection, and kidney dialysis infusion techniques.

[0169] Dosage regimen can affect what constitutes an effective amount.For example, several divided doses and alternating doses can be administered in a given period (daily) or continuously, or doses can be continuously infused or bolus injections.Furthermore, the selection of the recited dose of the ENPP1 agonist can be indicated by the exigencies of therapeutic or prophylactic situation.

[0170] The administration of the compositions of the present disclosure (e.g., soluble ENPP1 polypeptides and fusion proteins thereof) to a patient, such as a mammal (i.e., human), can be performed using known procedures at dosages and for periods effective to treat the patient's disease or disorder. The effective amount of the ENPP1 agonist at the recited dosages required to achieve a therapeutic effect can vary according to factors such as the activity of the particular compound used; the time of administration; the rate of excretion of the compound; the duration of treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the medical field. The administration regimen can be adjusted to provide an optimal therapeutic response. The selected dosage is determined based on the biological activity of the therapeutic compound, which depends on the half-life and the area under the plasma time curve of the therapeutic compound.

[0171] 11. Prophylactic Administration Thus, given the disclosure herein, one of skill in the art will understand that preventing a disease or disorder in a subject encompasses administering an ENPP1 polypeptide to a subject as a prophylactic measure against the disease or disorder.

[0172] The relative amounts of active ingredients (e.g., soluble ENPP1 polypeptides and fusion proteins thereof), pharma- ceutically acceptable carriers, and any additional ingredients in the formulations disclosed herein will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. By way of example, the compositions may contain from about 0.1% to about 100% (w / w) active ingredients.

[0173] 12. ENPP1 Polypeptide Sequence (Table 1) Sequence TIFF2025510963000004.tif53168TIFF2025510963000005.tif229168TIFF2025510963000006.tif201168Regarding SEQ ID NO:118 and 119: X=Export sequence Y = bone targeting tag or stop codon (i.e., no amino acid indicated by *) Non-limiting examples of bone-targeting sequences include, without limitation, DDDDDDDD and / or EEEEEEEE. Non-bold, non-underlined font = linker region that can be of various lengths, etc. The amino acid composition of the linker can be variable. In certain non-limiting embodiments, the linker region can include residues such as histidine that prevent non-specific protease cleavage (i.e., HS or HG instead of RS or RG). Bold font = human IgG1 (optimized in BL-1118 with M883Y S885T T887E mutations highlighted in bold italics) Underlined font = extracellular domain of human ENPP1 (optimized in BL-1118 with I256T substitution highlighted in bold underline) TIFF2025510963000007.tif153138TIFF2025510963000008.tif255150 EXAMPLES

[0174] Experimental Example The present disclosure will be further described in detail with reference to the following experimental examples.These examples are provided for illustrative purposes only and are not intended to be limiting unless so specified.Therefore, the present disclosure should not be interpreted as being limited to the following examples in any way, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0175] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the disclosed compounds and practice the claimed methods. Thus, the following examples specifically point out preferred aspects of the disclosure, and are not to be construed as limiting in any way the remainder of the disclosure.

[0176] The materials and methods used in the following examples are now described.

[0177] Human subjects Patient data, including laboratory data, radiographs, computed tomography (CT) and bone mineral density (BMD), were collected retrospectively and skeletal and biochemical data of patients with ENPP1 mutations were reported.

[0178] Mutation analysis Mutation analysis (Table 2) of genes associated with hereditary hypophosphatemic rickets, osteogenesis imperfecta (OI), and osteoporosis-pseudoglioma syndrome (OPPG) was performed using next-generation sequencing with the MiSeq Sequencing System at the Kazusa DNA Research Institute, as previously reported (Fujiki et al., 2018). Direct sequencing of detected ENPP1 variants was performed in the family members of the probands, as previously reported (Fujiki et al., 2018) or by using the primers listed in Table 2. The pathogenicity of the detected variants was assessed by the in silico tools PolyPhen-2, SIFT, and Mutation Taster (Adzhubei et al., 2010; Kumar et al., 2009; Schwarz et al., 2010).

[0179] Table 2. Genes tested in this case series (A) and primers used to target sequences of variants in the ENPP1 gene (B). TIFF2025510963000009.tif89150OI, osteogenesis imperfecta; OPPG, osteoporosis-pseudoglioma syndrome

[0180] Complementary DNA cloning For patient 3, total RNA from peripheral blood mononuclear cells was used to synthesize ENPP1 cDNA using reverse transcription and polymerase chain reaction (RT-PCR). ENPP1 cDNA was ligated with pT7blue T-vector (Merck, Darmstadt, Germany) before transformation into Escherichia coli. The amplified vector was purified using the QIAprep Spin Miniprep kit (Qiagen, Redwood City, CA, USA) and subjected to sequencing in both alleles.

[0181] Measurement of intact FGF23 Intact FGF23 was measured by Determinar CL FGF23 (CL) according to the manufacturer's protocol (Minaris Medical, Tokyo, Japan). CL is a sandwich chemiluminescent enzyme immunoassay (CLEIA) that uses an anti-human FGF23 mouse monoclonal antibody. Using this method, the reference range for FGF23 is 16.1–49.3 pg / ml, and the cutoff value for FGF23-associated hypophosphatemia is 30 pg / ml (Ito et al., 2021; Kato et al., 2021).

[0182] Measurement of plasma PPi Plasma was collected from participants to measure plasma pyrophosphate (PPi) concentrations. After plasma isolation, samples were filtered by centrifugation through a 30 kDa membrane (PALL, Port Washington, NY, USA) to remove platelets and frozen at -80 °C within 1 h of blood collection for single use. Measurement of plasma [PPi] was performed using ATP sulfurylase as previously described (Jansen et al., 2013; Jansen et al., 2014) with minor modifications. Luminescence signals were read at room temperature by an EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA). Final plasma [PPi] was normalized by background subtraction [PPi] from appropriate controls. The reference range of [PPi] in healthy children and adolescents using the ATP sulfurylase method was recently reported to be 2,360 to 4,440 (Bernhard et al., 2022). These values ​​are similar to previous standard ranges previously reported in healthy adults ( O'Neill et al., 2010 ).

[0183] Enzyme kinetics assay of ENPP1 Human mutations were engineered into the hENPP1-Fc construct using Quikchange II-XL Site Directed Mutagenesis (Agilent Technologies, Santa Clara, CA, USA). After sequence verification, constructs were transfected into CHO-K1 cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA). 48 h after transfection, 10 μL of supernatant was mixed with 90 μL of assay buffer containing 250 mM Tris pH 8.0, 500 mM NaCl, 0.05% Triton X-100, and 1 mM thymidine 50-monophosphate p-nitrophenyl. The rate of p-nitrophenyl groups released from the chromogenic substrate was reported as change in OD405 nM / min for at least five replicates for each construct and normalized to %WT.

[0184] statistical analysis PPi levels were shown as mean ± standard deviation (SD). Comparison of the enzyme kinetics assay of ENPP1 between WT and mutants was analyzed by ANOVA. Significance was set at P < 0.05. Data analysis was performed by GraphPad Prism version 6.05 for Windows (GraphPad Software, San Diego, CA, USA).

[0185] Example 1: Identification of ENPP1 haploinsufficiency in patients Patient 1 was a 47-year-old man with a history of upper and lower extremity fractures. The patient was a social drinker and had a 20 pack-year smoking history. The patient had no history of ureterolithiasis or malabsorption syndromes, no history of osteoporosis-related treatment, and no family history of osteoporosis. At age 46, the patient presented with back pain diagnosed as a vertebral compression fracture, as well as pain in the knee, wrist, and ankle that began 1 month after the back pain. The patient was seen at a local hospital, and dual-energy x-ray absorptiometry (DEXA) of the lumbar and proximal femur showed T-scores of -3.8 and -2.6, respectively. A CT scan of the spine showed compression fractures at the 7th and 11th thoracic vertebrae (Figure 1A).

[0186] Bone scintigraphy showed multiple uptakes in the ribs, suggesting multiple fractures (Figure 1B). No signs of heterotopic ossification were detected in the hip and knee joints, Achilles tendon, or in the paravertebral ligaments (Figures 1C-H). The patient was diagnosed with early-onset osteoporosis, and laboratory tests were performed to confirm the absence of secondary osteoporosis and osteomalacia. One local hospital detected low normal serum phosphorus, along with slightly elevated serum alkaline phosphatase and bone alkaline phosphatase, while another hospital detected no abnormalities in serum calcium, phosphorus, and FGF23 (Supplementary Table 1).

[0187] Other endocrine function tests, such as thyroid-stimulating hormone, free testosterone, and cortisol (measured in the morning), were within normal ranges (Supplementary Table 1). Therefore, the patient was referred to the University of Tokyo Hospitals for further evaluation, and blood chemistries were reevaluated (Table 3). Laboratory data showed normal levels of serum albumin, normal corrected calcium (8.8 mg / dL), low normal serum phosphorus (2.7 mg / dL, reference range 2.7 to 4.6 mg / dL), normal FGF23 (28 pg / mL, reference range 16.1 to 49.3 pg / mL), and low 25-hydroxyvitamin D (7.2 ng / mL).

[0188] A diagnosis of mild phenotype of OI or OPPG was considered, and mutation analysis of genes related to OI or OPPG was performed without identifying a pathogenic variant (Table 2). Based on serum analytes measured at the first hospital (Supplementary Table 1) and the presence of low normal serum phosphorus at our hospital, a diagnosis of mild hypophosphatemic rickets was next considered, and mutation analysis of related genes was performed, which revealed a missense variant in ENPP1 (c.536A>G, ​​p.Asn179Ser[N179S]) (Table 3). Direct sequencing of the detected variant in ENPP1 was then performed on the patient's 18-year-old son, revealing an identical heterozygous ENPP1 variant (Figure 2A). The son showed low normal BMD - low Z scores in the hip (-1.6) and proximal femur (-0.8) - but no history of spontaneous fractures or heterotopic ossification in the spine, hip and knee joints, or Achilles tendon (Figures 5A-G and Table 3).

[0189] Patient 2 was a 77-year-old woman diagnosed with diffuse idiopathic osteoarthritis (DISH) who visited the University of Tokyo hospital after suffering from a vertebral compression fracture. At the time of evaluation, ossification of the paravertebral ligaments and multiple vertebral compression fractures were observed by CT (Fig. 3A). In addition, slight heterotopic ossification was detected in the Achilles tendon (Fig. 3F), but the hip and knee joints were intact (Fig. 3B, Fig. 3C, Fig. 3D, and Fig. 3E). The ossification detected by spinal CT was not evaluated histologically. The patient's biochemical profile showed low normal serum phosphorus (3.1 mg / dL, reference range 2.7–4.6 mg / dL) and high normal FGF23 (43.3 pg / mL, reference range 16.1–49.3 pg / mL). Due to the presence of heterotopic ossification in the paravertebral ligaments and Achilles tendon, a diagnosis of hereditary FGF23-related hypophosphatemia (specifically, ARHR or XLH) was considered. Testing for genes associated with hereditary hypophosphatemic rickets (Table 2) revealed the presence of a heterozygous missense variant in ENPP1 (c.1352A>G, p.Tyr451Cys[Y451C]) (Table 3).

[0190] Patient 3 was a 54-year-old woman who visited a local hospital complaining of hip, knee and back pain. The patient subsequently presented with ossification of the anterior / posterior longitudinal ligaments (OALL / OPLL), which was diagnosed as DISH, osteophytes around the bilateral hip joints, and enthesopathy of the bilateral Achilles tendons (Figure 4A-I). Laboratory data showed low normal serum phosphate (2.9 mg / dL) and high normal serum FGF23 (38.4 pg / mL, reference range 16.1-49.3 pg / mL). The biochemical profile combined with the findings of ectopic paravertebral ossification led to the conclusion of FGF23-associated hypophosphatemia, and genetic analysis (Table 2) revealed the same ENPP1 variants (c.536A>G, ​​and c.1352A>G) as in patients 1 and 2 (Table 3). Sanger sequencing was then performed on the patient's sons (aged 19 and 23 years) and revealed the heterozygous ENPP1 variant c.1352A>G in both sons (Figure 2C). Importantly, neither of these young adults showed calcific Achilles tendon enthesopathy without the presence of heterotopic ossification in the spine or hip (Figures 6A-G and 7A-G).

[0191] (Supplementary Table 1) Biochemical and hormonal data of case 1 before referral TIFF2025510963000010.tif99150 Abbreviations: RI, reference interval; eGFR, estimated glomerular filtration rate; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25-dihydroxyvitamin; ALP, alkaline phosphatase; BAP, bone alkaline phosphatase; TRACP-5b, tartrate-resistant acid phosphatase 5b; iPTH, intact parathyroid hormone; FGF23, fibroblast growth factor 23; TSH, thyroid-stimulating hormone.

[0192] (Table 3) Demographic, clinical and biochemical data in adults with monoallelic ENPP1 variants or compound heterozygous ENPP1 variants, c.536A>G or c.1352A>G. TIFF2025510963000011.tif210105TIFF2025510963000012.tif21065Abbreviations: RI, reference interval; M, male; F, female; SD, standard deviation; BMD, bone mineral density; NA, not applicable; NE, not investigated; PP i , pyrophosphate; eGFR, estimated glomerular filtration rate; 25(OH)D, 25-hydroxyvitamin D; 1,25(OH)2D, 1,25-dihydroxyvitamin; BAP, bone alkaline phosphatase; TRACP-5b, tartrate-resistant acid phosphatase 5b; iPTH, intact parathyroid hormone; FGF23, fibroblast growth factor 23; TPR, tubular phosphate reabsorption.

[0193] Example 2: Characterization of the effects of ENPP1 haploinsufficiency in patients Cloning of the complementary DNA for ENPP1 in patient 3 Complementary DNA of ENPP1 was cloned and sequenced to assess whether the two ENPP1 variants were located on the same or opposite alleles. Sequence data for each allele revealed that case 3 had compound heterozygosity for the ENPP1 variants (Figure 8).

[0194] In silico prediction of pathogenicity for detected ENPP1 variants The allele frequencies of N179S and Y451C detected in this case were reported to be 0.00010 and 0.00016, respectively, in the Genome Aggregation Database (GnomAD). However, in a whole-genome reference panel derived from 3552 general Japanese individuals constructed by the Tohoku Medical Megabank Organization (ToMMo), the allele frequencies of N179S and Y451C were reported to be 0.0071 and 0.0055, respectively (Table 4). Asn 179 is located in the somatomedin B domain 2 (SMB2), which is important for protein dimerization and stability, while Tyr 451 is located in the catalytic domain (Figure 9), and both Asn 179 and Tyr 451 are highly conserved across all species. The combination of in silico tools described in the methods labeled the N179S and Y451C mutations as pathogenic (Table 4).

[0195] Table 4. Allele frequencies and in silico predictions of ENPP1 variants, c.536A>G or c.1352A>G TIFF2025510963000013.tif27142

[0196] Measurement of plasma PPi Plasma PPi was measured in three probands (cases 1–3) and their family members. Patients with heterozygous ENPP1 variants showed plasma PPi levels of 1,000–2,000 nM (Table 3), whereas plasma PPi in patients with biallelic ENPP1 variants showed a similar concentration of 1,866 nM.

[0197] Enzyme kinetics assay of ENPP1 To further evaluate the impact of the mutations on ENPP1 catalytic activity, the enzymatic rates of all variants were evaluated in parallel in vitro assays compared to the WT ENPP1 isoform. The N179S and Y451C variants reduced the catalytic rate of ENPP1 by 55% and 70%, respectively, when compared to WT ENPP1 (Figure 10). Therefore, we classified the N179S and Y451C variants as "probably pathogenic" according to the ACMG guidelines (N179S:PM1+PP3+PS3, Y451C:PM1+PS3).

[0198] Selection results The results show the presence of clinical manifestations of two cases of monoallelic ENPP1 deficiency and a case of compound biallelic heterozygous ENPP1 deficiency in the primary subjects (patients 1–3), as well as three additional cases of ENPP1 haploinsufficiency in the children of these subjects.

[0199] Patient 1 showed early onset osteoporosis, whereas patient 2 showed spinal osteophytosis and a presumptive diagnosis of DISH and was further found to be osteoporotic (as evidenced by vertebral compression fractures, Figures 1A-1B and 3A-3G). Patient 3 had a compound heterozygous ENPP1 mutation composed of the individual mutations present in patients 1 and 2, such that the patient was ENPP1 homozygous deficient and showed prominent ossification in the paravertebral ligaments, as well as calcification around the hip joints and bilateral calcific enthesopathy of the Achilles tendons (Figures 4A-4I).

[0200] Moreover, all children carrying monoallelic ENPP1 variants inherited from the primary patient displayed musculoskeletal disease at a strikingly early age, including low bone mass (lumbar spine Z score -1.6) in the 18-year-old son of patient 1, and calcific enthesopathy of the Achilles tendon in the 19- and 23-year-old children of patient 3. The segregation of the genotype with the phenotype further supports the idea that ENPP1 deficiency plays a central role in the pathogenesis of the bone disorders described in this study.

[0201] Previously, evaluation of a family with homozygous ENPP1 mutations inducing GACI reported that haploinsufficient ENPP1 carriers were asymptomatic but had hypocalcemic and hypophosphatemic serum biochemistry, suggesting a possible role for ENPP1 in calcium and phosphate homeostasis (Kotwal et al., 2020). In contrast, Oheim et al. reported ENPP1 haploinsufficiency in an adult male with early-onset osteoporosis, a phenotype also present in ENPP1 homozygous deficient mice, termed ENPP1asj / asj (Oheim et al., 2020), supporting the idea that ENPP1 regulates bone mass in mammals. Indeed, the skeletal phenotype of patient 1 and his 18-year-old son supports the association of ENPP1 haploinsufficiency with early-onset osteoporosis.

[0202] This study extends the association of ENPP1 haploinsufficiency to patients with DISH. DISH is a systemic condition characterized by ossification of ligamentous and tendon attachments, especially around the thoracic spine. DISH patients occasionally experience pain and reduced range of motion, as well as an increased risk of spinal fractures (Mader et al., 2013). Although the pathogenesis is unknown, DISH is associated with older age (>50 years), male sex, obesity, hypertension, and diabetes mellitus (Kuperus et al., 2020)

[0203] In the case of patient 2, although the patient was over 50 years old, other risk factors for DISH were not present and instead the patient was found to be ENPP1 haploinsufficient. Considering that homozygous ENPP1 deficiency has been associated with spinal ligament ossification in human and mouse clinical and preclinical studies (Okawa et al., 1998; Nakamura et al., 1999; Saito et al., 2011; Hirao et al., 2016), impaired ENPP1 activity, which reduces plasma PPi, is a risk factor for the progressive paravertebral ossification and calcific enthesopathy present in this patient.

[0204] Both described ENPP1 variants (N179S and Y451C) are located in highly conserved sequences located in regions of ENPP1 that are important for dimerization and stability (Asn 179) and catalytic activity (Tyr 451). Furthermore, N179S and Y451C have been found to be deleterious by multiple in silico tools (Table 4), but have not been reported as pathogenic so far.

[0205] In addition, N179S and Y451C reduce enzyme activity by 55% and 70% compared to WT levels, respectively, which is similar to the residual enzyme activity present in other pathogenic variants in ENPP1 (Kotwal et al., 2020; Oheim et al., 2020; Rutsch et al., 2003; Stella et al., 2016; Thumbigere-Math et al., 2018). Collectively, these findings support the finding that ENPP1 variants N179S and / or Y451C are responsible for the skeletal phenotype and heterotopic ossification present in the three probands.

[0206] Although the mechanism by which ENPP1 regulates musculoskeletal calcification is still not fully understood, i.e., whether through catalytic or catalytic-independent protein signaling, both patients described in patients 1 and 2 exhibited low plasma [PPi] (1,646 nM and 1,748 nM, respectively, reference range 2,360–4,440 nM), consistent with a role for ENPP1 catalytic activity in the observed abnormal phenotype. Both ENPP1 deficiency and ABCC6 deficiency result in low plasma PPi levels (Lorenz-Depiereux et al., 2010; Levy-litan et al., 2010; Rutsch et al., 2001; Nitschke et al., 2012; Le Saux et al., 2000). Plasma PPi levels in ENPP1 haploinsufficient patients are intermediate between those in patients without ENPP1 deficiency and those with homozygous ENPP1 deficiency (Kotwal et al., 2020; Oheim et al., 2020). PPi levels in our patients also fell within the range of 1,000-2,000 nM, but unexpectedly, PPi concentrations in patient 3 with compound heterozygous ENPP1 deficiency were similar to those in haploinsufficient ENPP1 patients, suggesting the involvement of compensatory mechanisms (Kotwal et al., 2020).

[0207] In addition, secondary hyperparathyroidism was observed in patient 3, a finding that was also observed in other patients with homozygous ENPP1 deficiency (Kotwal et al., 2020, and Capelli et al., 2015), as well as in mouse models of ENPP1 deficiency. PTH is also elevated in other disorders induced by elevated FGF23, such as Hyp mice that develop secondary hyperparathyroidism due to impaired activation of vitamin D by the action of FGF23, and in humans with XLH and ARHR (Carpenter JBMR, 2011). A similar mechanism may be responsible for the secondary hyperparathyroidism in case 3, who was found to be vitamin D deficient, and other cases of homozygous ENPP1 deficiency (Kotwal et al., 2020). Because the patient described in patient 3 did not show osteomalacia, treatment with natural vitamin D instead of activated vitamin D may be suitable to improve secondary hyperparathyroidism and to prevent the worsening of secondary hyperparathyroidism.

[0208] Although the patients in this study showed normal intact FGF23, their levels were interpreted as elevated in relation to their low normal serum phosphorus levels, raising the possibility of a genetically induced phosphate wasting disorder. In this regard, the majority of patients described in the original report of ARHR2 have high normal or slightly elevated intact FGF23 in relation to low serum phosphorus. In Levy-Litan's description of three patients with rickets, two of the three had high normal intact FGF23 (50 pg and 47 pg, normal reference range 10 to 50 pg, (Levy-litan et al., 2010) and five of the seven ARHR2 patients reported by Lorenz-Depiereux had intact FGF23 at the upper limit of normal or slightly elevated upon repeated measurements. These ranges were similarly interpreted as high in relation to the low phosphate values ​​and the severe rickets phenotype displayed by the patients.

[0209] Without wishing to be limited by any theory, the present study demonstrated that ENPP1 haploinsufficiency induced a greater FGF23 elevation than homozygous ENPP1 deficiency, and the normal and high-normal levels of intact FGF23 associated with normal or low-normal serum phosphorus (2.7-3.1 mg / dL, reference range 2.7-4.6 mg / dL) raised suspicion of genetically induced hypophosphatemia and initiated genetic testing. This study shows that increased circulating FGF23 and hypophosphatemia occur in patients with calcific enthesopathy in rare metabolic disorders such as ARHR, XLH, and hypophosphatemic tumoral calcinosis (Okawa et al., 1998; Nakamura et al., 1999; Saito et al., 2011; Hirao et al., 2016; Maulding et al., 2021; Rutsch et al., 2001; Cheng et al., 2005; Albright et al., 2015), and in the general medical population of patients with rapidly progressive forms of OPLL (Albright et al., 2015; Ferreira et al., 2021). In contrast, calcific enthesopathy is not associated with FGF23-independent forms of hypophosphatemic rickets, such as SLC34A3 deficiency (Kotwal et al., 2020). Thus, FGF23 appears to be closely related to the development of enthesopathy.

[0210] Previous transcriptome analysis performed on whole bones of ENPP1-deficient mice reported that inactivating mutations in Enpp1 increase Fgf23 transcription and decrease Wnt10b and Wnt16 transcription (Maulding et al., 2021), suggesting that ENPP1 deficiency induces abnormal skeletal mineralization in part through impaired Wnt signaling. Patient 1 did not exhibit overt hypophosphatemic rickets with elevated FGF23 levels, and the bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP-5b) was in the normal range, suggesting the development of osteoporosis without high bone turnover, a finding consistent with an anabolic defect induced by defective Wnt signaling. Thus, the human observations are consistent with the osteoporosis mechanism suggested by transcriptome analysis on a mouse model of ENPP1 deficiency (Maulding et al., 2021).

[0211] Finally, it is important to note the difference in allele frequency of the variants described in this study. The frequency of the allele detected in patient 2 (Y451C) is approximately 30 times higher in Japanese (ToMMo: 0.0055) than in all other ethnicities (GnomAD: 0.00016). Given the observation that monoallelic ENPP1 Y451C mutations are associated with OPLL, this difference in variant frequency may explain the 10-40 times higher prevalence of OPLL in Japan (1.8-4.1%) than in the United States (0.12%) and Germany (0.1%) (Stapleton et al., 2011).

[0212] Example 3: Generation of ENPP1 fusion protein and treatment of DISH model mice ENPP1 generation One example of an ENPP1 fusion protein is ENPP1-Fc, although the ENPP1-Fc example can be applied to other ENNPP1 fusion proteins described herein, such as ENPP1-albumin.

[0213] ENPP1-Fc is a recombinant fusion protein containing the extracellular domain of human ENPP1 (soluble ENPP1) combined with the Fc fragment of IgG1 (rhENPP1-Fc). The recombinant extracellular domain of ENPP1-Fc contains its catalytic activity and is identical to the native ENPP1 enzyme. ENPP1-Fc is a recombinant human protein produced in CHO cells via an animal-derived component-free fed-batch cell culture process. The molecular weight of the ENPP1-Fc dimer is approximately 290 kDa. ENPP1-Fc is highly glycosylated and has a pI of approximately 6.0. Similar to endogenous ENPP1, the primary substrate of ENPP1-Fc is ATP, which is cleaved into AMP and PPi.

[0214] In a particular embodiment, the soluble ENPP1 protein was fused to a human Fc domain by a linker through a linker (comprising leucine, isoleucine and asparagine). The three ENPP1-Fc constructs purified from CHO cells are shown in Table 1 as SEQ ID NOs: 3, 4 and 5.

[0215] Purification of ENPP1-Fc could be achieved, for example, by a series of column chromatography steps including three or more of the following in any order: Protein A chromatography, Q Sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography. Purification could be completed using viral filtration and buffer exchange. After purification of the protein, the catalytic activity of the ENPP1-Fc protein could be evaluated using pNP-TMP as a chromogenic substrate.

[0216] Treatment of DISH model mice with ENPP1 agonists The best-established mouse model for spinal enthesopathy (DISH) is the Enpp1-deficient mouse, called ttw ("toe walking mouse"). ttw mice exhibit severe myelopathy and extensive paraspinal ligament calcification and osteophyte formation. To evaluate the relationship between PPi and the severity of enthesopathy, we used Enpp1 mice maintained on a normal chow diet at 23 weeks of age. asj Examining the Achilles tendons of mice.

[0217] Animals were administered either vehicle (PBS) or human ENPP1-Fc, which can normalize plasma [PPi], for one week after subcutaneous submilligram administration. Protocols for administration of ENPP1-Fc, radiographic analysis, and histological experiments are outlined in Ferreira et al., Musculoskeletal Comorbidities and Quality of Life in ENPP1-Deficient Adults and the Response of Enthesopathy to Enzyme Replacement Therapy in Murine Models. J Bone Miner Res, 2021). Optionally, experiments can be repeated using ENPP1 agents containing bone targeting domains (such as, but not limited to, ENPP1-Fc) for better delivery and even higher efficacy.

[0218] An ENPP1 agonist (such as, but not limited to, ENPP1-Fc) is administered to asj Mice were administered Enpp1 at 0.3 mg / kg / week from weeks 2 to 23. asj Plasma PPi was measured in mice and it was noted that PPi was significantly increased but not completely normalized compared to the WT pair (2235 nM vs. 1358 nM, respectively, Table 6).

[0219] (Table 6) 23-week-old Enpp1 WT Mice and treated and untreated Enpp1 asj / asj Plasma analytes in mice TIFF2025510963000014.tif54128

[0220] Achilles tendon enthesopathy was analyzed histologically and using custom MATLAB software to quantify red pixels in photomicrographs of alizarin-red stained sections. asj Mouse Achilles tendons are predicted to reveal substantial calcification throughout the entire length of the tendon, but Enpp1 treated with ENPP1-Fc asj It is predicted that tendon calcification will be inhibited in mice.

[0221] Without wishing to be bound by theory, enthesopathy may be dependent on plasma PPi, and by increasing plasma PPi with an ENPP1 agonist, e.g., an enzyme biologic such as ENPP1-Fc, enthesopathy may be prevented. Findings indicate that complete suppression of enthesopathy may be achievable upon dose escalation.

[0222] Effects of PPi on DISH model mice To evaluate the effects of plasma PPi on osteophyte formation, spinal fusion and ossification in a mouse model of DISH, Enpp1 asj Mice are administered vehicle or 1 mg / kg ENPP1-Fc from weeks 3 to 17 and their spines are analyzed by micro-CT. Figure 12: Mice administered ENPP1-Fc show high plasma PPi levels (~10 µM), several fold higher than WT levels.

[0223] The presence of elevated plasma PPi and reduced paravertebral ossification in a mouse model of DISH treated with an ENPP1 agonist such as ENPP1-Fc would indicate that such administration of ENPP1 to human subjects with DISH would also attenuate paravertebral osteophytes, ankylosis and spinal fusion.

[0224] Example 4: Treatment Protocol The ENPP1 agent (including but not limited to ENPP1, ENPP1-X, ENPP1-Fc, and / or ENPP1-Fc-X, where X is a bone-targeting tag described elsewhere herein) is administered at one of the following selected doses: 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2.0mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg , 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / k g, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1m g / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8.0mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5 mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9.0mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9 mg / kg, 10.0 mg / kg, and / or fractions or multiples thereof. Administration is subcutaneous (SC) at least once or twice every two months, at least once or twice a month, three times a month, and at least once or twice a week.

[0225] The first dose of the ENPP1 agonist may be administered on day 1. On day 8 and thereafter, the ENPP1 agonist is administered to the subject twice weekly at the selected dose of mg / kg of the ENPP1 agonist. The doses may be administered at approximately the same time on each administration day. Injection sites are alternated, with no site within 2 inches of any prior injection site within the prior two weeks.

[0226] The selected doses of the ENPP1 agonist are one of 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg or 1.8 mg / kg SC.Other selected doses of ENPP1 agonists by SC were 0.2mg / kg, 0.3mg / kg, 0.4mg / kg, 0.5mg / kg, 0.6mg / kg, 0.7mg / kg, 0.8mg / kg, 0.9mg / kg, 1.0mg / kg, 1.1mg / kg, 1.2mg / kg, 1.3mg / kg, 1.4mg / kg, 1.5mg / kg, 1.6mg / kg, 1.7mg / kg, 1.8mg / kg, 1.9mg / kg, 2.0mg / kg, 2.1mg / kg, 2.2mg / kg, 2.3mg / kg, 2.4mg / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 5.8mg / kg, 5.9mg / kg, 5.1mg / kg, 5.2mg / kg, mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8m g / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6.1mg / kg, 6.2mg / kg, 6.3mg / kg, 6.4mg / k g, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, The initial dose of the ENPP1 agonist may be administered on day 1. The initial dose of the ENPP1 agonist may be administered on day 1.After the first dose, the subject may be observed for 7 days to monitor safety and collect PK samples. On the 8th day and thereafter, the subject is administered the selected dose twice weekly. Administration of the ENPP1 agonist at the selected dose is continued as deemed appropriate by a medical professional.

[0227] The subject may be administered 8 doses of the ENPP1 agonist over a 29 day period, for example, 0.2 mg / kg, 0.6 mg / kg and 1.8 mg / kg doses provide 1.6 mg, 4.8 mg and 14.4 mg exposure per 29 days, respectively. Alternatively, the subject may be administered more or less than 8 doses, as deemed appropriate by a medical professional.

[0228] Similar to the endogenous ENPP1 enzyme, ENPP1 agonists cleave ATP to generate AMP and PPi, thereby increasing plasma PPi levels and AMP, which rapidly converts CD73 to adenosine. Replacement of the endogenous human enzyme is intended to correct the inherent deficiency and allow for improved health and mitigation of clinical complications associated with ENPP1 baseline patient, clinician and carer outcomes.

[0229] Example 5: Treatment of patients with ENPP1 haploinsufficiency ENPP1 agonists are administered to patients identified as having ENPP1 haploinsufficiency by subcutaneous injection on day 1 and twice weekly beginning on day 8 using doses selected as follows:

[0230] (Table 7) TIFF2025510963000015.tif23150

[0231] The ENPP1 agonist is administered at least twice weekly for a period determined by a medical professional at a selected dose of one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg SC. The patient's response to the enzyme replacement is appropriately monitored, for example, by tracking a reduction in one or more symptoms of ENPP1 deficiency, and / or by using the guidelines provided herein, as determined by a medical professional.

[0232] Example 6: Treatment of Patients Diagnosed with DISH DISH commonly involves calcification of tendons and ligaments around the spine. As the tendons and ligaments harden, some of these tissues may turn into bone. This usually occurs where the tissue connects with the bone. As a result, osteophytes, which are bony growths that develop along the edges of bones, develop. DISH commonly affects the upper back and neck, known as the thoracic and cervical spine. However, DISH can also affect the shoulders, elbows, hands, knees, hips, heels, and / or ankles.

[0233] In certain embodiments, a subject diagnosed with DISH is treated with an ENPP1 agonist administered at least twice weekly for a period determined by a medical professional at a selected dose of one of 0.2 mg / kg, 0.6 mg / kg or 1.8 mg / kg SC, IV and / or IP.

[0234] In certain embodiments, a subject diagnosed with DISH is receiving a stimulant therapy of 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, 1.0 mg / kg, 1.1 mg / kg, 1.2 mg / kg, 1.3 mg / kg, 1.4 mg / kg, 1.5 mg / kg, 1.6 mg / kg, 1.7 mg / kg, 1.8 mg / kg, 1.9 mg / kg, 2.0 mg / kg, 2.1 mg / kg, 2.2 mg / kg, 2.3 mg / kg, 2.4 mg / kg, 2.5 mg / kg, 2.6 mg / kg, 2.7 mg / kg, 2.8 mg / kg, 2.9 mg / kg, 3.0 mg / kg, 3.1 mg / kg, 3.2 mg / kg, 3.3 mg / kg, 3.4 mg / kg, 3.5 mg / kg, 3.6 mg / kg, 3.7 mg / kg, 3.8 mg / kg, 3.9 mg / kg, 4.0 mg / kg, 4.1 mg / kg, 4.2 mg / kg, 4.3 mg / kg, 4.4 mg / kg, 4.5 mg / kg, 4.6 mg / kg, 4.7 mg / kg, 4.8 mg / kg, 4.9 mg / kg, 5.0 mg / kg, 5.1 mg / kg, 5.2 mg / kg, 5.3 mg / kg, 5.4 mg / kg, 5.5 mg / kg, 5.6 mg / kg, 5.7 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 5.8 mg / kg, 5.9 mg / kg, 5.9 mg / kg, 5. / kg, 2.5mg / kg, 2.6mg / kg, 2.7mg / kg, 2.8mg / kg, 2.9mg / kg, 3.0mg / kg, 3.1mg / kg, 3.2mg / kg, 3.3mg / kg, 3.4mg / kg, 3.5mg / kg, 3.6mg / kg, 3.7mg / kg, 3.8mg / kg, 3.9mg / kg, 4.0mg / kg, 4.1mg / kg, 4.2mg / kg, 4.3mg / kg, 4.4mg / kg, 4.5mg / kg, 4.6mg / kg, 4.7mg / kg, 4.8mg / kg, 4.9mg / kg g, 5.0mg / kg, 5.1mg / kg, 5.2mg / kg, 5.3mg / kg, 5.4mg / kg, 5.5mg / kg, 5.6mg / kg, 5.7mg / kg, 5.8mg / kg, 5.9mg / kg, 6.0mg / kg, 6.1mg / kg, 6.2 mg / kg, 6.3mg / kg, 6.4mg / kg, 6.5mg / kg, 6.6mg / kg, 6.7mg / kg, 6.8mg / kg, 6.9mg / kg, 7.0mg / kg, 7.1mg / kg, 7.2mg / kg, 7.3mg / kg, 7.4mg / kg, 7.5mg / kg, 7.6mg / kg, 7.7mg / kg, 7.8mg / kg, 7.9mg / kg, 8.0mg / kg, 8.1mg / kg, 8.2mg / kg, 8.3mg / kg, 8.4mg / kg, 8.5mg / kg, 8.6mg / kg, 8.7mg / kg, 8.8mg / kg, 8.9mg / kg, 9.0mg / kg, 9.1mg / kg, 9.2mg / kg, 9.3mg / kg, 9.4mg / kg, 9.5mg / kg, 9.6mg / kg, 9.7mg / kg, 9.8mg / kg, 9.9mg / kg, 10.The patient is treated with an ENPP1 agonist at a selected dose of one of the following: 1. 0 mg / kg, and / or fractions or multiples thereof, SC, IV and / or IP, administered at least twice weekly for a period determined by a medical professional.

[0235] A DISH patient's response to enzyme replacement is suitably monitored, as determined by a medical professional, for example, by tracking a reduction in one or more symptoms of DISH using the guidelines provided herein.

[0236] Alternatively, subjects suspected to be at risk of DISH are treated with ENPP1 agonists by administering a selected dose of ENPP1 agonists.In certain embodiments, the common risk factors for developing DISH include, but are not limited to, large waist circumference, BMI / obesity, hyperinsulinemia, diabetes mellitus, hyperuricemia, dyslipidemia, hypertension, coronary artery disease and / or gout.DISH can be asymptomatic, and in that case, diagnosis is usually made based on X-ray images.In certain embodiments, DISH is associated with older age (over 50 years old), male, obesity, hypertension and / or diabetes mellitus.

[0237] Alternatively, subjects with radiographic evidence of DISH, lacking clinical symptoms of disease, may be treated with an ENPP1 agonist to prevent or minimize the known phenotypes associated with DISH: increased spinal fractures, reduced mobility, myelopathy and / or pain.

[0238] The following examples, in conjunction with the entire specification, provide guidance for determining treatment protocols and efficacy.

[0239] Example 7: Biomarkers associated with bone health In addition to low plasma PPi, ENPP1-deficient patients are biochemically characterized by low serum phosphate, high urinary phosphate, low renal TmP / GFR, normal calcium (Ca), low normal urinary Ca, normal 25-hydroxyvitamin D (25 OH D), low normal 1,25(OH)2D, high BAP, high intact FGF23, and normal and / or elevated PTH (IOF 2019).

[0240] Table 8 lists biomarkers that may be used in certain embodiments as additional determinants of bone health in a treated patient.

[0241] Table 8. Clinical intermediates and biomarkers TIFF2025510963000016.tif38150

[0242] Example 8: Efficacy of treatment with ENPP1 agonists Therapeutic efficacy can be assessed by measuring plasma PPi, as well as other plasma analytes, such as FGF23, Pi, FGF23, Pi, TmP / GFR, serum alkaline phosphatase (ALP), bone-specific ALP (BALP), carboxy-terminal cross-linked telopeptide of collagen type I (CTx), and / or procollagen type 1 N-terminal propeptide (P1NP). These analyte measurements can be used as PD markers associated with ENPP1 deficiency to determine the efficacy of ENPP1 agents. Changes in these analytes can be described as changes from baseline and in a time-dependent manner over the course of treatment. Dose linearity of PK and PD parameters can also be assessed.

[0243] Changes from baseline in plasma PPi levels, FGF23 levels, and urinary phosphorus excretion per creatinine clearance can be analyzed using paired difference t-tests.

[0244] Example 9: Drug concentration measurement In addition, blood samples may be obtained from the patient for measurement of ENPP1 agent concentrations in plasma and subsequent determination of PK parameters after the first dose (i.e., a single dose) and at / after multiple doses (i.e., steady state).

[0245] Example 10: Immunogenicity (anti-drug antibodies) If desired, anti-drug antibodies (ADA) may be used to measure immunogenicity to ENPP1 agents. A multi-layer approach may be used in immunogenicity testing. If ADA is detected in the initial screening, a confirmatory test may be performed to determine specificity. Samples may also be used to assess and further establish specificity confirmation (i.e., titer) and neutralizing antibody assays.

[0246] Example 11: Pharmacokinetic, Pharmacodynamic and Exploratory Biomarker Analysis Pharmacokinetic analysis may be performed on the PK population, and the PK parameters of the ENPP1 agonist may be summarized by descriptive statistical treatment. The dose linearity of the PK and PD parameters may also be evaluated. PK / PD analysis, immunogenicity analysis, and exploratory biomarker analysis may be determined.

[0247] Example 12: Additional Determinants of Efficacy Although restoring normal levels of PPi is the primary indicator of the effectiveness of treatment using an ENPP1 agonist, other physical measurements may be used, if desired, to help determine the effectiveness of treatment. These include one or more of the following:

[0248] 1. X-ray and imaging a. X-rays of skeletal severity. Standard x-rays may be obtained to detect the skeletal deformities of rickets. X-rays may be obtained, for example, at the wrists and knees. b. DEXA scan. A DEXA scan may be used to assess changes in bone density. c. Positron emission tomography. Computed tomography. Baseline Na18F-PET / HRpQCT (or HR-CT) can be a whole-body scan performed within one month of the first administration of the ENPP1 agonist to measure arterial and organ calcification, as well as skeletal abnormalities, at baseline and for subsequent intervention assessments. 18 F-PET measures bone turnover, and arterial microcalcification. High-resolution quantitative computed tomography (HRQCT) or HR-CT can determine bone microarchitecture in the non-dominant distal radius and tibia. Standard bone geometric parameters are calculated. d. Doppler echocardiogram. A baseline echocardiogram may be obtained within 3 days prior to the first administration of the ENPP1 agonist. Doppler echocardiogram may be used to measure cardiac and valvular cardiac function [LVEF, blood flow] calcification, and arteriosclerosis. e. Optical coherence tomography. Optical coherence tomography may be used to visualize neointimal proliferation. f. Peripheral Arterial Tonometry. Peripheral arterial tonometry (PAT) may be used to assess digital pulse wave amplitude (PWA) which corresponds to digital volumetric variations. g. Renal ultrasound. Renal ultrasound may be used to measure nephrocalcinosis, for example, within one week of starting an ENPP1 agonist. h. Bone histomorphology and bone biopsy. A bone biopsy may be performed as a baseline measurement. Tetracycline loading for 10 days prior to bone biopsy is preferred.

[0249] 2. Walking Ability Walking tests can be used as a submaximal exercise measure to measure functional capacity in ambulatory patients, combining cardiopulmonary, neuromuscular and musculoskeletal function. The 6 Minute Walk Test (6MWT) was originally developed by the American Thoracic Society (ATS 2002) for use in adults and is now commonly used in adult and pediatric populations (Mylius et al., 2016), as well as in children with neuromuscular diseases such as spinal muscular atrophy (Montes et al., 2018), Duchenne muscular dystrophy (McDonald et al., 2013) and infantile-onset Pompe disease (van der Meijden et al., 2018). The 2 Minute Walk Test (2MWT) is included in the NIH Toolbox and is increasingly used to measure the same characteristics.

[0250] The 6MWT and 2MWT may be administered to the patient before and during treatment at the discretion of the medical provider. If the subject is unable to complete at least the 2MWT at baseline, additional assessments during treatment may be at the discretion of the medical provider. Resting heart rate is obtained before and after the test. Distance walked during the first 2 minutes and the entire 6 minutes of the 6MWT may be recorded. Distance walked during the 2 and 6 minutes may be compared to age- and sex-matched normative data (percentage predicted).

[0251] 3. Dynamometric method At the discretion of the healthcare provider, strength may be assessed using dynamometry prior to and / or during treatment. Portable dynamometry is a direct measurement of strength commonly used in both children and adults. Muscle groups that may be assessed include shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension. Each muscle group may be measured twice on both sides. a. Grip Strength. At the provider's discretion, grip strength may be measured using a grip dynamometer prior to and / or during treatment. Equipment, and assessor instructions may be standardized across sites. Grip may be assessed bilaterally with one practice and one maximum force measurement for each hand, and results may be compared to age- and sex-matched normative data (if available). b. Range of Motion. Range of motion may be assessed using a goniometer, an instrument that tests joint angles and measures the degree of joint movement. The fixed arm of the goniometer is aligned with a designated bony landmark on the fixed body segment and the moving arm of the goniometer is aligned with a designated bony landmark on the moving limb. A fulcrum of the goniometer is designated for each motion measured using the motion axis and bony landmark. Range of motion may be assessed for one or more of the following: shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension.

[0252] 4. Hearing Test Moderate hearing loss is associated with ARHR2 (Brachet et al. 2014, Steichen-Gersdorf et al. 2015). Baseline hearing may be determined by one or more of the following: physical examination and otoscopy, immittance audiometry (commonly referred to as tympanic hearing testing), if available, standard pure tone audiometry (PTA) using frequencies up to 8 kHz (if there is a PTA threshold of >15 dB, subjects should also undergo bone conduction testing), and high frequency audiometry (HFA) using frequencies up to 16 kHz.

[0253] 5. Clinician Global Impression Scales The Clinical Global Impression of Patients (CGI-S) scale was developed for use in clinical trials supported by the American Mental Health Association to provide a brief, independent assessment of the clinician's view of the patient's overall functioning before and after the initiation of an investigational drug (Guy 1976). The CGI provides a clinician-determined overall summary measure that considers all available information, including information on the patient's medical history, psychosocial situation, symptoms, behavior, and the impact of symptoms on the patient's ability to function. The CGI-S may be administered before and / or during treatment, at the provider's discretion, and provides an overall assessment of change using a 7-point scale ranging from -3 (severe worsening) to +3 (marked improvement).

[0254] 6. Gross Motor Function Classification System-Expanded and Revised The Gross Motor Skills Classification System-Expanded and Revised (GMFCS-E&R) may be administered prior to and / or during treatment at the provider's discretion. The GMFCS-E&R classifies patient-initiated movements with an emphasis on mobility on a scale of 1 to 5.

[0255] 7. Patient-reported outcome measurement information system The Patient-Reported Outcomes Measures Information System (PROMIS) consists of various questionnaires developed by the National Institutes of Health (NIH) to assess physical, mental and social health from the patient's perspective (www dot healthmeasures dot net). These questionnaires have been used in clinical trials for people with chronic health conditions such as X-linked hypophosphatemia, arthritis, multiple sclerosis and neurofibromatosis. Each questionnaire contains 8–10 items that are rated by participants on a 5-point Likert scale ranging from 1 (never) to 5 (always). Scores are summed for each questionnaire, with higher scores indicating more domains (e.g., more fatigue, more physical function) being measured. Raw scores are converted to T-scores based on a mean of 50 and a standard deviation of 10, allowing comparison of the study sample to the general population. PROMIS scales may include Pain Interference (Short Form 8a), Pain Intensity (Version 3a), Physical Function-Upper Extremity (Custom Short Form), Physical Function-Mobility (Short Form 13a FACIT Fatigue), Fatigue (Short Form) and Cognitive Impact (Short Form 8a) and may be administered prior to and / or during treatment at the discretion of the healthcare provider. These assessments may be completed by the subject unassisted.

[0256] 8. Carer Global Impression Scales The Caregiver Global Impression of Status may be administered to the patient's carer prior to and / or during treatment, at the discretion of the healthcare provider. The Carer Global Impression of Change provides an overall rating of change using a 7-point scale ranging from -3 (severe deterioration) to +3 (marked improvement).

[0257] 9. Western Ontario and McMaster University Osteoarthritis Index The WOMAC is a patient-reported outcome used to assess activities of daily living, functional mobility, gait, general health, pain, and quality of life in patients with hip or knee pain (www dot sralab dot org). The assessment consists of 24 items and takes approximately 12 minutes to administer. The WOMAC may be administered before and / or during treatment at the discretion of the healthcare provider. The assessment may be completed by the subject unassisted.

[0258] Example 13: Comparison of soluble ENPP1-Fc (construct 1118, SEQ ID NO: 118) and bone-targeted ENPP1-Fc (construct 2000, SEQ ID NO: 119) on cervical spine and hearing phenotypes FIG. 13 shows the results of 17-week-old WT male mice and 17-week-old Enpp1 mice administered the indicated weekly subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Paravertebral osteophyte and ankylosis responses in male mice. Construct #1118 was administered weekly from week 3 and construct #2000 was administered weekly from week 5. MicroCT images show Enpp1 mice administered construct #2000. asj Preferentially demonstrating attenuation of paravertebral osteophytes and ankylosis in male mice.

[0259] FIG. 14 shows the results of 17-week-old WT female mice and 17-week-old Enpp1 mice administered the indicated weekly subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj Paravertebral osteophytes and ankylosis responses in female mice. Construct #1118 was administered weekly from week 3 and construct #2000 was administered weekly from week 5. MicroCT images show Enpp1 mice administered construct #2000. asj Preferentially demonstrating attenuation of paravertebral osteophytes and ankylosis in female mice.

[0260] FIG. 15A-B show 17-week-old WT and 17-week-old Enpp1 mice administered the indicated weekly subcutaneous doses of vehicle or ENPP1 constructs #1118 and #2000. asj1 shows auditory brainstem responses in mice. Construct #1118 was administered once a week from week 3, and construct #2000 was administered once a week from week 5. Measurements of stimulation frequency demonstrate prevention of hearing loss in the low frequency (8 kHz) range by ENPP1 construct #1118 at a weekly dose of 2 mg / Kg, and by ENPP1 construct #2000 at weekly doses of 0.5 and 1 mg / Kg. ENPP1 construct #2000 administered at a weekly dose of 1 mg / Kg also preferentially improves hearing in ENPP1-deficient animals in the high frequency range (32 kHz).

[0261] FIG. 16 shows the effect of ENPP1 constructs #1118 and #2000 on the expression of ENPP1 in WT and 17-week-old mice at weekly subcutaneous doses of vehicle or #1118 and #2000 as indicated. asj Intact FGF23 levels in mice are shown. Construct #1118 was administered weekly from week 3 and construct #2000 was administered weekly from week 5. Data demonstrate that ENPP1 construct #2000 preferentially suppresses intact FGF23 when administered at 1mg / Kg and 4mg / Kg per week. Statistical significance was assessed by ANOVA Kruskal-Wallis test followed by Dunn's post-hoc analysis to assess differences from WT (one-way ANOVA). Statistical significance is represented by p-values ​​with the following annotation: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0262] References TIFF2025510963000017.tif93150TIFF2025510963000018.tif224150TIFF2025510963000019.tif224149TIFF2025510963000020.tif224148TIFF2025510963000021.tif141150Additional References for Examples 3-12 TIFF2025510963000022.tif73146TIFF2025510963000023.tif210150

[0263] List of Aspects The following exemplary aspects are provided, the numbering of which should not be construed as designating an order of importance: Aspect 1: Methods of treating, ameliorating, preventing further development and / or progression of, and / or preventing diffuse idiopathic osteoarthritis (DISH), ankylosing spondylitis, and / or spondyloarthritis in a patient in need thereof, including: A compound of formula (I) or a salt or solvate thereof: PROTEIN-Z-DOMAIN-XY (I) administering to said patient a therapeutically effective amount of In (I), PROTEIN comprises the catalytic domain of ENPP1, DOMAIN is absent or at least one selected from the group consisting of human IgG Fc domain (Fc), human serum albumin protein (ALB) and fragments thereof; X and Z are, independently, absent or a polypeptide containing 1 to 20 amino acids; and Y is a negatively charged bone-targeting sequence, Thereby treating, ameliorating, preventing further development and / or progression of, and / or preventing DISH, ankylosing spondylitis, and / or spondyloarthritis in said patient. Aspect 2: The method of embodiment 1, wherein said patient has ENPP1 haploinsufficiency. Aspect 3: The method of embodiment 1, wherein said patient does not have ENPP1 haploinsufficiency. Aspect 4: The method of any of embodiments 1-3, wherein the patient is not ENPP1 deficient. Aspect 5: The method of any of aspects 1-3, wherein the patient is ENPP1 deficient. Aspect 6: The method of any of embodiments 1-5, wherein said patient is administered said compound by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical. Aspect 7: The method of any of embodiments 1-6, wherein said compound is administered to said patient intravenously or subcutaneously. Aspect 8: The method of any of embodiments 1-7, wherein administering said compound to said patient increases or prevents a further decrease in extracellular pyrophosphate concentration in said patient. Aspect 9: The method of any of embodiments 1-8, wherein administering said compound to said patient reduces or prevents further increase in one or more of Achilles tendon calcification, spinal calcification, hip calcification, and bilateral calcification in said patient. Aspect 10: The method of any of embodiments 1-9, wherein the DOMAIN comprises albumin. Aspect 11: The method of any of aspects 1-10, wherein the DOMAIN comprises an IgG Fc domain. Aspect 12: The method of any of aspects 1 to 11, wherein the PROTEIN lacks the ENPP1 transmembrane domain. Aspect 13: The method of any of embodiments 1-12, wherein said compound is administered to said patient as a pharmaceutical composition further comprising at least one pharma- ceutically acceptable carrier. Aspect 14: The method of any one of aspects 1 to 13, wherein the patient is a mammal. Aspect 15: The method of embodiment 14, wherein the mammal is a human. Aspect 16: 16. The method of any of aspects 1-15, wherein the PROTEIN comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO:1. Aspect 17: 17. The method of any of aspects 1 to 16, wherein the PROTEIN comprises amino acid residues 1 to 833 of SEQ ID NO:3. Aspect 18: The method of any one of aspects 1 to 16, wherein the PROTEIN comprises the amino acid sequence shown in SEQ ID NO:2. Aspect 19: The method of any one of aspects 1 to 16, wherein the PROTEIN comprises the amino acid sequence shown in SEQ ID NO: 3 or 4 or 5. Aspect 20: The method of any of embodiments 1-19, wherein said DOMAIN increases the circulating half-life of said compound compared to the circulating half-life of said compound lacking said DOMAIN. Aspect 21: 21. The method of any of embodiments 1-20, wherein said patient has also been diagnosed with a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemic, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets. Aspect 22: 21. The method of any of aspects 1-20, wherein said patient has not been diagnosed with a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemic, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets. Aspect 23: 23. The method of any of embodiments 1-22, wherein the patient has a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemic, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets. Aspect 22: 23. The method of any of aspects 1-22, wherein the patient does not have a disease or condition selected from the group consisting of early onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemic, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets.

[0264] INCORPORATION BY REFERENCE All publications and patents mentioned in this specification are herein incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0265] Other Aspects Although certain aspects of the subject matter have been described, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the appended claims. The full scope of the invention should be determined by reference to the claims, their full scope of equivalents, and the specification, as well as such variations.

Claims

1. A compound of formula (I) or a salt or solvate thereof: PROTEIN-Z-DOMAIN-XY (I) 1. A pharmaceutical composition for treating, ameliorating, preventing further development and / or progression of, and / or preventing diffuse idiopathic osteogenesis imperfecta (DISH) and / or spondyloarthritis in a patient in need thereof, comprising a therapeutically effective amount of In (I), the protein comprises the catalytic domain of ENPP1, DOMAIN is absent or at least one selected from the group consisting of a human IgG Fc domain (Fc), a human serum albumin protein (ALB), and a fragment thereof; X and Z are, independently, null or a polypeptide containing 1 to 20 amino acids; and Y is a negatively charged bone-targeting sequence; The pharmaceutical composition.

2. 2. The pharmaceutical composition of claim 1, wherein the patient has ENPP1 haploinsufficiency.

3. The pharmaceutical composition of claim 1, wherein the patient does not have ENPP1 haploinsufficiency.

4. The pharmaceutical composition of claim 1, wherein the patient is not ENPP1 deficient.

5. 2. The pharmaceutical composition of claim 1, wherein the patient is ENPP1 deficient.

6. 10. The pharmaceutical composition of claim 1, wherein the patient is administered the pharmaceutical composition by at least one route selected from the group consisting of oral, aerosol, inhalation, rectal, vaginal, transdermal, subcutaneous, intranasal, buccal, sublingual, parenteral, intrathecal, intragastric, ocular, pulmonary, and topical.

7. The pharmaceutical composition of claim 1, wherein the composition is administered intravenously or subcutaneously to the patient.

8. The pharmaceutical composition of claim 1, which increases or prevents further decline in extracellular pyrophosphate concentration in the patient.

9. The pharmaceutical composition of claim 1, which reduces or prevents further increase in one or more of Achilles tendon calcification, spinal calcification, hip calcification and bilateral calcification in the patient.

10. 10. The pharmaceutical composition of claim 1, wherein the domain comprises albumin.

11. 2. The pharmaceutical composition of claim 1, wherein the DOMAIN comprises an IgG Fc domain.

12. 2. The pharmaceutical composition of claim 1, wherein the protein lacks the ENPP1 transmembrane domain.

13. The pharmaceutical composition of claim 1, further comprising at least one pharmaceutically acceptable carrier.

14. 10. The pharmaceutical composition of claim 1, wherein the patient is a mammal.

15. 15. The pharmaceutical composition of claim 14, wherein the mammal is a human.

16. The pharmaceutical composition of any one of claims 1 to 15, wherein the protein comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO:

1.

17. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein said PROTEIN comprises amino acid residues 1 to 833 of SEQ ID NO:

3.

18. The pharmaceutical composition of any one of claims 1 to 15, wherein the PROTEIN comprises the amino acid sequence shown in SEQ ID NO:

2.

19. The pharmaceutical composition of any one of claims 1 to 15, wherein the PROTEIN comprises the amino acid sequence shown in SEQ ID NO: 3 or 4 or 5.

20. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein said DOMAIN increases the circulating half-life of said compound compared to the circulating half-life of said compound lacking said DOMAIN.

21. 16. The pharmaceutical composition of any one of claims 1-15, wherein said patient has also been diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets and hypophosphatemic rickets.

22. 16. The pharmaceutical composition of any one of claims 1 to 15, wherein said patient has not been diagnosed with a disease or condition selected from the group consisting of early-onset osteoporosis, osteopenia, age-related osteopenia, OPLL, hereditary hypophosphatemic rickets, X-linked hypophosphatemia, autosomal recessive hypophosphatemic rickets type 2, autosomal overt hypophosphatemic rickets, and hypophosphatemic rickets.

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