ENPP1 for use in the treatment of calciphylaxis

Abstract

The present disclosure provides, among other things, provides an ENPP1 agent for treatment of calciphylaxis in a subject in need thereof.

Description

[0001] ENPP1 FOR USE IN THE TREATMENT OF CALCIPHYLAXIS

[0002] FIELD

[0003] The field of the invention relates to treatment of calciphylaxis found in chronic kidney disease and end stage renal disease patients.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically as a WIPO Standard ST.26 XML file via Patent Center, created on November 6, 2025, is entitled “70019_SeqListing.xml”, and is 159,504 bytes in size. The sequence listing is incorporated herein by reference in its entirety.

[0006] BACKGROUND ENPP1 polypeptides have been shown to be effective in treating certain diseases of ectopic tissue calcification. ENPPl-Fc has been shown to reduce generalized arterial calcifications in a mouse model for GACI (generalized arterial calcification of infants), which is a severe disease occurring in infants and involving extensive arterial calcification (Albright, et al., 2015, Nature Comm. 10006). Fusion proteins of ENPP1 also have been described to treat diseases of severe tissue calcification (see, e.g., PCT Application Publication Nos. WO 2014 / 126965 and WO 2016 / 187408), and a fusion protein of ENPP1 comprising a negatively charged bone-targeting domain has been described to treat GACI (PCT Application Publication Nos. WO 2011 / 113027 and WO 2012 / 125182).

[0007] Chronic kidney disease (CKD) impacts a large percentage of the global population. CKD Mineralization and Mineral Bone Disorder (CKD-MBD) is the broad term describing alterations in key circulating factors involved in mineralization, ectopic calcification, and bone abnormalities. Cardiovascular complications involving vascular calcification is one of the leading causes of death in this patient population. Plasma levels of pyrophosphate (PPi), a potent inhibitor of ectopic mineralization, are low in CKD and end-stage kidney disease patients. Currently no clinically approved treatments exist for treating CKD-MBD in which patients experience devastating effects on multiple systems of the body, leading to lifethreatening or debilitating complications.

[0008] Calciphylaxis is a rare, serious, life-threatening disease of vascular calcification characterized by occlusion of micro vessels in the subcutaneous adipose tissue and skin resulting in painful lesions due to ischemia. Once calciphylaxis has been diagnosed, the prognosis is generally poor with low survival. In a large study conducted in the United States of over 1,000 subjects with calciphylaxis, mortality rates were 27% at 6 months and 45% at 12 months after diagnosis. Currently, there is no approved treatment for calciphylaxis, resulting in a high unmet need to develop a treatment for this debilitating disease.

[0009] SUMMARY

[0010] In an aspect, the invention relates to a method of treating calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide to the subject once a week thereby treating calciphylaxis in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

[0011] In another aspect, the invention provides a method of treating calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby treating calciphylaxis in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

[0012] In another aspect, the invention provides a method of increasing the levels of serum or plasma pyrophosphate (Ppi) in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby elevating levels of PPi in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

[0013] In another aspect, the invention provides a method of treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby treating or reducing calcification in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

[0014] In another aspect, the invention provides a method of reducing pain in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby treating or reducing calcification in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

[0015] In another aspect, the invention provides a method of reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby elevating levels of PPi in said subject and reducing the number and / or wound size of the skin lesions, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide. For example, the skin lesions are painful lesions, lesions at injection sites, and / or skin lesions caused due to ischemia. In some of these methods, the lesion number and / or wound size is reduced due to reduction of calcification.

[0016] In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for the treatment of calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

[0017] In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for the treatment of calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week. In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for increasing the levels of serum or plasma pyrophosphate (PPi) in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

[0018] In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

[0019] In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for reducing pain in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

[0020] In another aspect, the invention provides for use of a dose of ENPP1 polypeptide for the preparation of a medicament for reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

[0021] In a further aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in treating calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0022] In another aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in treating calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0023] In another aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in increasing the levels of serum or plasma pyrophosphate (PPi) in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0024] In another aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0025] In another aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in reducing pain in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0026] In another aspect, the invention provides for a composition comprising a dose of ENPP1 polypeptide for use in reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

[0027] In some embodiments of any of the aforesaid methods, uses and compositions, administration of the ENNP1 polypeptide increases the serum or plasma level of a bone and / or mineral metabolism biomarker. For example, in some embodiment, of any of the aforesaid methods, uses and compositions the bone and / or mineral metabolism biomarker is iPTH, FGF23, BALP, or Phosphate.

[0028] In some embodiments of any of the aforesaid methods, uses and compositions, the subject is the age of at least 18 years but less than 75 years (>18 to <75 years). In some embodiments of any of the aforesaid methods, the subject is of an age greater than 65 but less than 75 years.

[0029] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has a loss of function mutation in one or more of ecto-5'-nucleotidase (NT5E), vitamin D receptor (VDR), and fibroblast growth factor 23 (FGF23) gene.

[0030] In some embodiments of any of the foresaid methods, uses and compositions, the ENPP1 polypeptide, medicament or composition is administered subcutaneously or is formulated for subcutaneous administration.

[0031] In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 polypeptide comprises a heterologous moiety, and wherein said heterologous moiety increases the circulating half-life of the ENPP1 agent relative to the circulating half-life of the ENPP1 agent lacking the heterologous moiety.

[0032] In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 polypeptide comprises a heterologous moiety and wherein said heterologous moiety comprises the Fc region of an immunoglobulin molecule.

[0033] In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 polypeptide comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 or residues 1 (FTAGLKPSCAKE) to 833 (QED) of SEQ ID NO:3.

[0034] In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 polypeptide comprises the amino acid sequence depicted in any one of SEQ ID NO: 3-5 and 116-128.

[0035] In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 polypeptide is a ENPP1 fusion polypeptide, said ENPP1 fusion polypeptide comprises ENPP1 component and a Fc domain, wherein said ENPP1 component comprises at least 95%, 96%, 97%, 98% or 99% of amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 and Fc domain comprises at least 95%, 96%, 97%, 98% or 99% of amino acid residues of SEQ ID NO: 6. In some embodiments of any of the aforesaid methods, uses and compositions, the ENPP1 component and said Fc domain are connected by means of a peptide linker, wherein said peptide linker has a length that is no less than two amino acids and no more than 36 amino acids.

[0036] In some embodiments of any of the aforesaid methods, uses and compositions, the subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein.

[0037] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has or is suspected of having one of generalized arterial calcification of infancy (GACI), autosomal recessive hypophosphatemic rickets type 2 (ARHR2), hypopyrophosphatemia and Pseudoxanthoma elasticum (PXE).

[0038] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has or is suspected of having one or more of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis juvenile dermatomyositis, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), autosomal hypophosphatemic rickets (ARHR2), osteoarthritis, calcification of atherosclerotic plaques, Chronic Kidney Disease (CKD), End Stage Renal Disease (ESRD), Pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

[0039] In some embodiments of any of the aforesaid methods, uses and compositions, the subject exhibits one or more of calcification of soft connective tissues, accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers of connective tissues, calcification in the eyes, calcification of cardiovascular system, calcification of skin, narrowing of the arteries, arteriosclerosis, ectopic calcification, narrowing of blood vessels in the lower extremities, claudication, abnormal pigmentation of cells of the retina, angioid streaks, abnormalities in the elastic membrane beneath the retina, choroidal neovascularization, visual impairment, vision loss, and blindness. In some embodiments of any of the aforesaid methods, uses and compositions, the subject has not undergone peritoneal dialysis or hemodiafiltration.

[0040] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has not been diagnosed with malignancy other than non-melanoma skin cancer or cervical carcinoma in the past year prior to said administration.

[0041] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has not been diagnosed with advanced liver disease such as liver cirrhosis.

[0042] In some embodiments of any of the aforesaid methods, uses and compositions, the subject has not had a myocardial infarction, stroke, or congestive heart failure requiring hospitalization within 6 months prior to said administration.

[0043] DESCRIPTION

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 shows the full, 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 soluble ENPP1 protein portion which includes SMB1 (residues 104-144) and SMB2 (residues 145-189).

[0046] Figure 2 illustrates certain domains of human ENPP1.

[0047] Figure 3 shows the amino acid sequence of a soluble ENPP1 polypeptide (SEQ ID NO: 2).

[0048] Figure 4A and Figure 4B show a multiple sequence alignment of various vertebrate soluble ENPP1 polypeptides and human soluble ENPP1 polypeptides (SEQ ID NOs: 1 and 7-10). The various soluble ENPP1 polypeptides correspond to the following species and represent regions of the specific NCBI accession number: Mouse (NCBI accession NP_001295256.1; SEQ ID NO:7), Cow (NCBI accession NP_001193141; SEQ ID NO: 8), Rabbit (NCBI accession NP_001162404.1; SEQ ID NO:9), Human (NCBI accession NP_006199.2; SEQ ID NO: 1), and Baboon (NCBI accession NP_001076211.2; SEQ ID NO: 10). Figure 5: Study Design Schematic Representation. Rats in the healthy group received normal chow for the duration of the study and did not receive calcitriol or vehicle. The diets for all rats, with or without adenine, were low in protein.

[0049] Figure 6: CKD model rats develop kidney damage. Healthy (blue), CKD vehicle treated group (red) and CKD ENPP1 treated (green) groups were analyzed starting at day -3 to day 60. Bodyweights are displayed in A, BUN in B, CRE in C, plasma phosphate in D, and plasma calcium in E. Ordinary one-way ANOVA used for statistical analysis # indicates significant result (p < 0.05) for healthy vs CKD vehicle, & indicates significant result (p < 0.05) for healthy vs CKD + ENPP1, % indicates significant result (p < 0.05) for CKD vehicle vs CKD + ENPP1.

[0050] Figure 7: Plasma levels of key biomarkers in CKD model. Plasma biomarkers including (A) terminal ENPP1 activity, (B) terminal iFGF23, (C) terminal sclerostin, and (D) PPi of healthy (blue), CKD vehicle treated group (red) and CKD group treated with ENPP1 (green). Brown-Forsythe and Welch ANOVA test used for FGF23 comparison. *** p = 0.0001 ** p=0.0065. Ordinary one-way ANOVA used for sclerostin comparison * p = 0.0109. Ordinary one-way ANOVA used for PPi comparison # indicates significant result (p < 0.05) for healthy vs CKD vehicle and & indicates significant result (p < 0.05) for healthy vs CKD + ENPP1.

[0051] Figure 8: ENPP1 prevents arterial calcification. A) Representative cross-sections of the iliac arteries and aorta, scale bars: 50 pm (iliac artery) or 100 pm (aorta). Arrows denote dark mineralization while arrowheads denote light mineralization. B) Quantification of light and dark mineral deposition. C) Quantification of total calcium from the iliac artery, descending aorta, and abdominal aorta. One-sided t-test. * p<0.05.

[0052] Figure 9: ENPP1 prevents renal calcification. A) Representative sagittal sections of kidney using Alizarin Red stain. Brown-black areas are adenine crystals (arrowheads), red staining indicates calcification and is denoted by arrows, and the star indicates distorted tubules. Scale bars: 2000 pm (left); 100 pm (right). B) Quantification of total calcium from kidneys. Onesided t-test. * p = 0.0499.

[0053] Figure 10: ENPP1 prevents osteomalacia. A) Representative images from Femurs from rats stained with Goldner’s Tri chrome stain. Dark green: mineralized bone; red: hypomineralized bone / collagen rich osteoid. Scale bars: 200 pm (left); and 100 pm (right). B) Quantification of osteoid parameters. Ordinary one-way ANOVA with Tukey’s post-test. **** p < 0.0001, * p = 0.0338.

[0054] Figure 11: Plasma chemistries. A) alkaline phosphatase, B) amylase C) albumin. Healthy control group is shown in blue, red is CKD vehicle treated group, and green is CKD group treated with ENPP1.

[0055] Figure 12: Plasma ENPP1 activity from three rats who do not show terminal activity. Activity levels are plotted for each animal at each of the interim timepoints. The dotted line shows background signal from the assay.

[0056] Figure 13: Tissue calcium data. (A) Vibrissae and (B) Spleen. Ordinary one-way ANOVA with Tukey’s post-test. ** p = 0.0011. **** p < 0.0001

[0057] Figure 14: Static bone parameters of femurs. Trabecular number (A), trabecular spacing (B), trabecular thickness (C), trabecular BV / TV (D), cortical thickness (E), and cortical BA / TA (F) were measured. Representative trabecular and cortical images are displayed in G. Ordinary one-way ANOVA with Tukey’s post-test. * p = 0.0456.

[0058] Figure 15: PPi Levels in Healthy Volunteers Compared to CKD Patients with and without Calciphylaxis.

[0059] Figure 16: Graphical correlation of plasma PPi and 6-month mortality among patients with calciphylaxis.

[0060] Figure 17: Schematic of study design of administration of ENPP1 polypeptide to treat subjects with calciphylaxis.

[0061] Figure 18: Schedule of Events (SOE) in SEAPORT 1 study.

[0062] Figure 19: Schedule of blood collection time points from subjects for PK / PD and genetic testing.

[0063] Figure 20: Schedule of follow up period post administration of ENPP1 polypeptide. Figure 21: SEAPORT 1 study patient demographics & disease history.

[0064] Figure 22: INZ-701 increases PPi in dialysis patients across the spectrum of PPi deficiency. Figure 23: Phosphate (Pi) decreased during INZ-701 Treatment.

[0065] Figure 24: FGF-23 decreased during INZ-701 Treatment.

[0066] Figure 25: PK during INZ-701 Treatment.

[0067] Figure 26: ENPP1 activity increased during INZ-701 Treatment.

[0068] Figure 27: Characteristics of calciphylaxis patients.

[0069] Figure 28: Median plasma PPi levels at enrollment and 6-week mortality among patients with calciphylaxis.

[0070] Figure 29: Correlation of PPi levels at enrollment and total number of calciphylaxis lesions.

[0071] Figure 30: Study schematic of SEAPORT 2 study.

[0072] Figure 31: Schedule of Events (SOE) in SEAPORT 2 study.

[0073] Figure 32: PK / PD Sampling Schedule and Fasting Requirements.

[0074] DEFINITIONS

[0075] The terms used in this specification generally have their ordinary meanings in the art, within the context of this disclosure and in the specific context where each term is used. Certain terms are discussed below or elsewhere in the specification, to provide additional guidance to the practitioner in describing the compositions and methods of the disclosure and how to make and use them. The scope or meaning of any use of a term will be apparent from the specific context in which the term is used.

[0076] An “ENPP1 Deficiency' is characterized by a reduced level of ENPP1 enzymatic activity in serum or plasma of a subject. ENPP1 Deficiency is a rare, genetic disorder caused by inactivating mutations in the AA77J / gene that encodes the ENPP1 enzyme. ENPP1 is an integral transmembrane protein whose extracellular domains carry pyrophosphatase and phosphodiesterase activities. ENPP1 converts extracellular ATP to inorganic pyrophosphate (PPi) and AMP. ENPP1 deficiency is an ultra-rare genetic disorder with an incidence of 1 in 64,000 pregnancies. ENPP1 Deficiency causes hypopyrophosphatemia and hypoadenosinemia which, in turn, leads to ectopic (especially arterial) calcification (described in literature as Generalized Arterial Calcification of Infancy [GACI]), skeletal dysfunction secondary to rickets and osteomalacia (described in literature as Autosomal Recessive Hypophosphatemic Rickets 2 [ARHR2]) and occlusive neo-intimal proliferation. Beyond symptomatic and palliative interventions, no targeted therapy exists for this disease. Thus, ENPP1 Deficiency has a high unmet medical need. Infants with ENPP1 Deficiency have high mortality in the first 0 to 6 months of life and children of ages 1-12 and adults with ENPP1 Deficiency experience ongoing risk for organ calcification and dysfunction, debilitating rickets that progresses to osteomalacia in adulthood with severe bone and joint pain, fatigue, muscle weakness, and repeated bone fractures, all symptoms that lead to poor quality of life and function. Hypopyrophosphatemia causes a reactive increase in fibroblast growth factor 23 (FGF23) leading to hyperphosphaturia (the ENPP1 Deficiency “biochemical axis”), an essential feature in the pathophysiology of ENPP1 Deficiency.

[0077] ENPP1 Deficiency is characterized biochemically by low plasma PPi levels and clinically characterized by vascular calcification in infants (GACI Type phenotype) and rickets (ARHR2 phenotype) post-infancy and intimal proliferation. GACI (generalized arterial calcification of infants) is a severe disease occurring in infants and involving extensive arterial calcification (Albright, etal., 2015, Nature Comm. 1 006).

[0078] Reduced ENPP1 enzymatic activity may occur via a reduction in the amount of ENPP1 enzyme present in serum or plasma of a subject relative to the amount of ENPP1 enzyme present in serum or plasma of a normal subject, and / or a reduction in the level of enzymatic activity of ENPP1 detected in serum or plasma of a subject relative to the level of enzymatic activity of ENPP1 detected in a normal subject, and / or via a defect in expression of ENPP1 gene at the transcriptional (RNA and / or mRNA) or translational levels, for example via a defect in the ENPP1 gene or a control element affecting ENPP1 gene expression, relative to normal expression the ENPP1 gene in a subject. ENPP1 enzymatic activity is defined below.

[0079] “ Enzymatically active” with respect to an ENPP1 polypeptide, or, as used herein, “enzymatic activity” with respect to an ENPP1 polypeptide, is defined as possessing ATP hydrolytic activity into AMP and PPi and / or AP3a hydrolysis to ATP. NPP1 readily hydrolyzes ATP into AMP and PPi. The steady-state Michaelis-Menten enzymatic constants of NPP1 are determined using ATP as a substrate. NPP1 can be demonstrated to cleave ATP by HPLC analysis of the enzymatic reaction, and the identity of the substrates and products of the reaction are confirmed by using ATP, AMP, and ADP standards. The ATP substrate degrades over time in the presence of NPP1, with the accumulation of the enzymatic product AMP. Using varying concentrations of ATP substrate, the initial rate velocities for NPP1 are derived in the presence of ATP, and the data is fit to a curve to derive the enzymatic rate constants. At physiologic pH, the kinetic rate constants of NPP1 are Km=144 pM and kcat=7.8 s-1.

[0080] As used herein the term “plasma pyrophosphate (PPi) levels” refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow and horse. It is necessary to measure PPi in the plasma rather than serum because of release from platelets. There are several ways to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase (Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63)' with modifications.

[0081] Typically, plasma PPi levels in healthy human subjects range from about 1pm to about 3 pM, in some cases between 1-2 pm. A normal level of ENPP1 in plasma refers to the amount of ENPP1 protein required to maintain a normal level of plasma pyrophosphate (PPi) in a healthy subject. A normal level of PPi in healthy humans corresponds to 2-3 pM. Subjects who have a deficiency of ENPP1 exhibit low PPi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. In patients afflicted with GACI, the PPi levels are found to be less than 1 pm and in some cases are below a detectable level. In patients afflicted with PXE, the PPi levels are below 0.5 pm. (Arterioscler Thromb Vase Biol. 2014 Sep; 34(9): 1985-9; Braddocket al., Nat Commun. 2015; 6: 10006.)

[0082] An “ABCC6 Deficiency” may be indicated in a number of ways, for example, by a reduced expression levels of ABCC6 enzyme in a subject. Subjects having ABCC6 deficiency can be identified by the presence of one or more physiological symptoms such as ocular calcification, skin calcification yellow papules, angioid streaks, visual impairment, calcification of soft connective tissues including the cardiovascular system, and / or the presence of ABCC6 mutations that affect the activity and expression of ABCC6 protein. Details on diagnosis and classification of ABCC6 mutations are known in art. (Expert Opin Orphan Drugs. 2014 Jun 1; 2(6): 567-577).

[0083] ABCC6 deficiency may result in PXE. Reduced ABCC6 enzymatic activity may occur via a reduction in the amount of ABCC6 enzyme present in a subject relative to the amount of ABCC6 enzyme present in a normal subject, and / or a reduction in the level of enzymatic activity of ABCC6 in a subject relative to the level of enzymatic activity of ABCC6 detected in a normal subject, and / or via a defect in expression of ABCC6 gene at the transcriptional (RNA and / or mRNA) or translational levels, for example via a defect in the ABCC6 gene or a control element affecting ABCC6 gene expression, relative to normal expression the ABCC6 gene in a subject.

[0084] A subject with ABCC6 deficiency can be identified by screening for ABCC6 gene mutations. Several methods for detecting gene mutations are known in art. For instance, detection of mutation in ABCC6 genes can performed by following the protocols described in J Med Genet. 2007 Oct; 44(10): 621-628. (Mutation detection in theABCC6 gene and genotypephenotype analysis in a large international case series affected by pseudoxanthoma elasticum, Ellen G Pfendner, et al)

[0085] “ABCC6 deficient patient” or “ABCC6 deficient subject” as used herein, refers to a patient having at least one pathogenic mutation in the ABCC6 gene that affects activity and / or expression of ABC66 protein.

[0086] “Pathological calcification”', as used herein, the term refers to the abnormal deposition of calcium salts in soft tissues, secretory and excretory passages of the body causing it to harden. There are two types, dystrophic calcification which occurs in dying and dead tissue and metastatic calcification which elevated extracellular levels of calcium (hypercalcemia), exceeding 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 prone to calcification include: Gastric mucosa - the inner epithelial lining of the stomach - Kidneys and lungs, Cornea, Systemic arteries and Pulmonary veins. “Pathological ossification”: as used herein, the term refers to a pathological condition in which bone arises in tissues not in the osseous system and in connective tissues usually not manifesting osteogenic properties. Ossification is classified into three types depending on the nature of the tissue or organ being affected, endochondral ossification is ossification that occurs in and replaces cartilage. Intramembranous ossification is ossification of bone that occurs in and replaces connective tissue. Metaplastic ossification the development of bony substance in normally soft body structures; called also heterotrophic ossification.

[0087] A “deficiency” of NPP1 refers to a condition in which the subject has less than or equal to 5%- 10% of normal levels of NPP1 in blood plasma. Normal levels of NPP1 in healthy human subjects is approximately between 10 to 30 ng / ml. (Am J Pathol. 2001 Feb; 158(2): 543-554.)

[0088] A “deficiency” of ABCC6 refers to a condition in which the subject has less than or equal to 5%-l 0% of normal levels of ABCC6 expression. ABCC6 protein is expressed in liver and levels of ABCC6 protein can be measured by means of liver biopsy.

[0089] A “low” level of PPi refers to a condition in which the subject has less than or equal to 2%-5% of normal levels of plasma pyrophosphate (PPi). Normal levels of Plasma PPi in healthy human subjects is approximately 1.8 to 2.6 pM. (Arthritis and Rheumatism, Vol. 22, No. 8 (August 1979))

[0090] A “defective ENPP1 protein” is an ENPP1 protein having reduced ENPP1 enzymatic activity, as described herein, which may be due to a loss of function mutation in ENPP1 or another protein that functions to reduce that activity or that functions so as to permit faster degradation of the ENPP1 protein. Such a mutation can be determined by comparing the ENPP1 wild type gene sequence to that of the mutant gene encoding a defective ENPP1 protein. Human subjects with defective ENPP1 protein tend to exhibit low PPi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. “Ectopic calcification ' refers to a condition characterized by a pathologic deposition of calcium salts in tissues or bone growth in soft tissues.

[0091] “Ectopic calcification of soft tissue" refers to inappropriate biomineralization, typically composed of calcium phosphate, hydroxyapatite, calcium oxalates and octacalcium phosphates occurring in soft tissues leading to loss of hardening of soft tissues. “Arterial calcification” refers to ectopic calcification that occurs in arteries and heart valves leading to hardening and or narrowing of arteries. Calcification in arteries is correlated with atherosclerotic plaque burden and increased risk of myocardial infarction, increased ischemic episodes in peripheral vascular disease, and increased risk of dissection following angioplasty.

[0092] “Venous calcification" refers to ectopic calcification that occurs in veins that reduces the elasticity of the veins and restricts blood flow which can then lead to increase in blood pressure and coronary defects.

[0093] “Vascular calcification" refers to the pathological deposition of mineral in the vascular system. It has a variety of 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 heredity conditions, and kidney disease, especially CKD. Patients with vascular calcification are at higher risk for adverse cardiovascular events. Vascular calcification affects a wide variety of patients. Idiopathic infantile arterial calcification is a rare form of vascular calcification where the arteries of neonates calcify.

[0094] As used herein, “parenteral afl&zzzzzAZratzozz” of a formulation includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the ENPP1 agent through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of an ENPP1 agent by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques. “Brain calcification '1(BC) refers to a nonspecific neuropathology wherein deposition of calcium and other mineral in blood vessel walls and tissue parenchyma occurs leading to neuronal death and gliosis. Brain calcification is” often associated with various chronic and acute brain disorders including Down’s syndrome, Lewy body disease, Alzheimer’s disease, Parkinson’s disease, vascular dementia, brain tumors, and various endocrinologic conditions.

[0095] Calcification of heart tissue refers to accumulation of deposits of calcium (possibly including other minerals) in tissues of the heart, such as aorta tissue and coronary tissue.

[0096] “Chronic kidney disease (CKD) ”, as used herein, the term refers to abnormalities of kidney structure or function that persist for more than three months with implications for health. Generally excretory, endocrine and metabolic functions decline together in most chronic kidney diseases. Cardiovascular disease is the most common cause of death in patients with chronic kidney disease (CKD) and vascular calcification is one of the strongest predictors of cardiovascular risk. With decreasing kidney function, the prevalence of vascular calcification increases, and calcification occurs years earlier in CKD patients than in the general population. Preventing, reducing, and / or reversing vascular calcification may result in increased survival in patients with CKD.

[0097] Clinical symptoms of chronic kidney diseases include itching, muscle cramps, nausea, lack of appetite, swelling of feet and ankles, sleeplessness, and labored breathing. Chronic kidney disease, if left untreated, tends to progress into end stage renal disease (ESRD). Common symptoms of ESRD include an inability to urinate, fatigue, malaise, weight loss, bone pain, changes in skin color, a frequent formation of bruises, and edema of outer extremities like fingers, toes, hands, and legs. Calciphylaxis or calcific uremic arteriolopathy (CUA) is a condition that causes calcium to build up inside the blood vessels of the fat and skin. A subpopulation of patients suffering from ESRD can also develop Calciphylaxis. Common symptoms of Calciphylaxis include large purple net-like patterns on skin, deep and painful lumps that ulcerate creating open sores with black-brown crust that fails to heal, skin lesions on the lower limbs or areas with higher fat content, such as thighs, breasts, buttocks, and abdomen. A person with calciphylaxis may have higher than normal levels of calcium (hypercalcemia) and phosphate (hyperphosphatemia) in the blood. They may also have symptoms of hyperparathyroidism. Hyperparathyroidism occurs when the parathyroid glands make excess parathyroid hormone (PTH). Reduced plasma pyrophosphate (PPi) levels are also present in vascular calcification associated with end stage renal disease (ESRD).

[0098] Vascular calcifications associated with ESRD contribute to poor outcomes by increasing pulse pressure, causing or exacerbating hypertension, and inducing or intensifying myocardial infarctions and strokes. Most patients with ESRD do not die of renal failure, but from the cardiovascular complications of ESRD, and it is important to note that many very young patients with ESRD on dialysis possess coronary artery calcifications. The histologic subtype of vascular calcification associated with CKD is known as Monckeburg’s sclerosis, which is a form of vessel hardening in which calcium deposits are found in the muscular layers of the medial vascular wall. This form of calcification is histologically distinct from intimal or neo-intimal vascular wall calcification commonly observed in atherosclerosis but identical to the vascular calcifications observed in human CKD patients, and in the rodent models of the disease described herein.

[0099] “CKD-MBD” as defined herein, refers to mineral bone disorder associated with chronic kidney disease. Chronic kidney disease (CKD) impacts 11-13% of the global population and ranks as one of the leading causes of mortality worldwide (Hill, et al., PLoS One. 2016 Jul 6;ll(7):e0158765; Kovesdy, etal., Kidney Int Suppl (2011). 2022 Apr; 12(1):7-11). CKD is divided into stages 1-5 based on the glomerular filtration rate, with stage 5 referred to as end stage kidney disease (ESKD) (Levey, et al., Ann Intern Med. 2003 Jul 15;139(2):137-47; Chen, etal., JAMA. 2019 Oct 1;322(13): 1294-1304). CKD patients, particularly those in the later stages of the disease, are prone to pathological and progressive calcification. CKD-Mineral Bone Disorder (CKD-MBD) is a syndrome covering three broad categories of abnormal mineralization seen in CKD / ESKD patients: 1) dysregulation of key circulating factors involved in mineralization such as calcium, phosphate, FGF-23, vitamin D, and parathyroid hormone (PTH), 2) bone abnormalities including alterations in mineralization and turnover and 3) ectopic calcification, particularly vascular calcification (Moe, etal., Kidney Int. 2006 Jun; 69(11): 1945-53).

[0100] Cardiovascular complications, directly or indirectly linked to vascular calcification, are the primary cause of death in CKD / ESKD patients (Thompson, etal., J Am Soc Nephrol. 2015 Oct;26(10):2504-ll). Vascular calcification seen in CKD exhibits a high degree of heterogeneity, occurring in both the tunica intima and tunica media of blood vessels, as well as within the aortic valves (Hutcheson, et al., Circ Res. 2023 Apr 14;132(8):993-1012). This calcification is widespread, as seen in the femoral, carotid, and coronary arteries (Sorensen, et al., MC Nephrol. 2020 Dec 9;21(1 ):534). The predominant form of vascular calcification in CKD manifests as medial calcification, contributing to increased vascular stiffness and elevating the risk of heart failure and myocardial infarction (Jablonski, et al., Hemodial Int. 2013 Oct; 17 Suppl 1(01): S17-21; Guerin, etal., Nephrol Dial Transplant. 2000 Jul; 15(7): 1014-21). Patients with medial calcification are at a higher risk of sudden cardiac death compared to those with intimal calcification, emphasizing the need for early detection and treatment (London, etal., Nephrol Dial Transplant. 2003 Sep;18(9): 1731-40). The rapid onset of vascular calcification, coupled with the lack of treatment options, highlights the urgent therapeutic need in this area (Kanbay, etal., Expert Rev Cardiovasc Ther. 2023 Feb;21(2):75-85).

[0101] Renal osteodystrophy refers to bone abnormalities that arise in CKD-MBD and can be classified into high or low bone turnover subtypes (Dalle Carbonare, et al., J Clin Med. 2021 Oct 8;10(19):4617). In low turnover bone disease, an increase in bone volume is observed (Modest, et al., R I Med J (2013). 2022 Oct 3; 105 (8): 22-27). There is considerable variability in the literature regarding the prevalence of the different types of renal osteodystrophy among CKD-MBD patients, with a recent report suggesting that newer treatment regimens are more likely to result in the low turnover bone disease (Dalle Carbonare, et al., J Clin Med. 2021 Oct 8;10(19):4617; Drileke, et al., Kidney Int. 2016 Feb;89(2):289-302). Calciphylaxis or Calcific Uremic Arteriolopathy (CUA), is a rare condition characterized by calcification in the arterioles and small arteries, resulting in non-healing necrotic skin lesions (Nigwekar, etal., N Engl J Med.

[0102] 2018 May 3;378(18): 1704-1714; Bajaj, et al., Mayo Clin Proc. 2018 Sep;93(9): 1202-1212). This disease is predominantly found among ESKD patients, where they often develop sepsis, leading to mortality rate exceeding 50% within one year. Currently, there are no current effective therapeutic or surgical options to prevent or treat calciphylaxis (Nigwekar, et al., Am J Kidney Dis. 2015 Jul;66(l): 133-46).

[0103] Numerous animal models exist that model CKD employing methods such as adenine induced kidney damage and surgical intervention involving nephrectomies (Shobeiri, etal., Am J Nephrol. 2010;31(6):471-81). Feeding rats an adenine enriched diet induces crystal deposition in the tubules leading to kidney damage. This results in a decline in kidney function that mirrors the progression of CKD and can also replicate the skeletal abnormalities described in patients (Diwan, el al., Nephrology (Carlton). 2018 Jan;23(l):5-ll).

[0104] Chronic kidney disease (CKD) affects around 850 million people globally and presents a major burden on the health care system (Jager, et al., Kidney Int. 2019 Nov;96(5): 1048-1050; Bello, etal., Lancet Glob Health. 2024 Mar;12(3):e382-e395). As the disease progresses and patients require dialysis the dysregulation in mineral metabolism becomes a major contributor to other co-morbidities such as vascular calcification and renal osteodystrophy which collectively are referred to as CKD-MBD, chronic kidney disease-mineral bone disorder. The major forms of renal osteodystrophy that have been described are osteitis fibrosa, adynamic bone disease, mixed uremic osteodystrophy, and osteomalacia (Sherrard, etal., Kidney Int. 1993 Feb;43(2):436-42). The dysregulation of mineral metabolism and bone disease are also driven by pathological changes in key biomarkers such as FGF-23, sclerostin and Dickkopf-related protein-1 (Dkk-1) the latter two of which inhibit WNT / p-catenin signaling (Nagy, et al., J Clin Med. 2022 Nov 30; 11(23).7130; Massy, etal., J Nephrol. 2017 Oct;30(5):629-634; Gutierrez, etal., JAmSoc Nephrol. 2005 Jul;16(7):2205-15).

[0105] ‘Mineral bone disorders (MBD) ”, as used herein, the term refers to a disorder characterized by abnormal hormone levels cause calcium and phosphorus levels in a person’s blood to be out of balance. Mineral and bone disorder commonly occurs in people with CKD and affects most people with kidney failure receiving dialysis.

[0106] Osteopenia is a bone condition characterized by decreased bone density, which leads to bone weakening and an increased risk of bone fracture. Osteomalacia is a bone disorder characterized by decreased mineralization of newly formed bone. Osteomalacia is caused by severe vitamin D deficiency (which can be nutritional or caused by a hereditary syndrome) and by conditions that cause very low blood phosphate levels. Both osteomalacia and osteopenia increase the risk of breaking a bone. Symptoms of osteomalacia include bone pain and muscle weakness, bone tenderness, difficulty walking, and muscle spasm. “Arterial calcification ” or “vascular calcification ” or “hardening of arteries ”, as used herein, the term refers to a process characterized by thickening and loss of elasticity of muscular arteries walls. The thickening and loss of elasticity occurs in two distinct sites, the intimal and medial layers of the vasculatures (medial vascular calcification). Intimal calcification is associated with atherosclerotic plaques and medial calcification is characterized by vascular stiffening and arteriosclerosis. This results in a reduction of arterial elasticity and an increased propensity for morbidity and mortality due to the impairment of the cardiovascular system’s hemodynamics.

[0107] “Age related osteopenia”, as used herein, refers to a condition in which bone mineral density is lower than normal. Generally, patients with osteopenia have a bone mineral density T-score of between -1.0 and -2.5. Osteopenia if left untreated progresses into Osteoporosis where bones become brittle and are extremely prone to fracture.

[0108] “Pseudoxanthoma elasticum (PXE) ”, as used herein, the term refers a progressive disorder that is characterized by the accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers. Elastic fibers are a component of connective tissue, which provides strength and flexibility to structures throughout the body. In PXE, mineralization can affect elastic fibers in the skin, eyes, and blood vessels, and less frequently in other areas such as the digestive tract. People with PXE may have yellowish bumps called papules on their necks, underarms, and other areas of skin that touch when a joint bends. Mineralization of the blood vessels that carry blood from the heart to the rest of the body (arteries) may cause other signs and symptoms of PXE. For example, people with this condition can develop narrowing of the arteries (arteriosclerosis) or a condition called claudication that is characterized by cramping and pain during exercise due to decreased blood flow to the arms and legs.

[0109] Pseudoxanthoma elasticum (PXE), also known as Gronblad-Strandberg syndrome, is a genetic disease that causes fragmentation and mineralization of elastic fibers in some tissues. The most common problems arise in the skin and eyes, and later in blood vessels in the form of premature atherosclerosis. PXE is caused by autosomal recessive mutations in the ABCC6 gene on the short arm of chromosome 16 (16pl3.1). In some cases, a portion of infants survive GACI and end up developing pseudoxanthoma elasticum (PXE) when they grow into adults. PXE is characterized by the accumulation of calcium and other minerals (mineralization) in elastic fibers, which are a component of connective tissue. Connective tissue provides strength and flexibility to structures throughout the body. Features characteristic of PXE that also occur in GACI include yellowish bumps called papules on the underarms and other areas of skin that touch when a joint bends (flexor areas); arterial stenosis, and abnormalities called angioid streaks affecting tissue at the back of the eye (retinal hemorrhage), which is detected during an eye examination.

[0110] “End stage renal disease ” (ESRD), as used herein, the term refers to an advanced stage of chronic kidney disease where kidneys of the patient are no longer functional. Common symptoms include fatigue associated with anemia (low blood iron), decreased appetite, nausea, vomiting, abnormal lab values including elevated potassium, abnormalities in hormones related to bone health, elevated phosphorus and / or decreased calcium, high blood pressure (hypertension), swelling in hands / legs / eyes / lower back (sacrum) and shortness of breath.

[0111] “Calcific uremic arteriolopathy (CUA)” or “calciphylaxis”, as used herein refers to a condition with high morbidity and mortality seen in patients with kidney disease, especially in those with end stage renal disease (ESRD). It is characterized by calcification of the small blood vessels located within the fatty tissue and deeper layers of the skin leading to blood clots, and the death of skin cells due to reduced blood flow caused by excessive calcification. Calciphylaxis is a rare, serious, life-threatening disease of vascular calcification characterized by occlusion of micro vessels in the subcutaneous adipose tissue and skin resulting in painful lesions due to ischemia.

[0112] "Hemodialysis ' (HD) as used herein refers to medical treatment that filters waste, excess fluids, and toxins from the blood when the kidneys are no longer able to do so. A dialyzer, or artificial kidney, filters the blood outside the body. A patient's blood is pumped through the dialyzer, which removes waste products and extra fluid. The blood is then returned to the body.

[0113] “Peritoneal dialysis" as used herein is a medical procedure used to treat kidney failure. It involves using the lining of the abdomen, called the peritoneum, as a natural filter to remove waste products and excess fluid from the blood. A cleansing fluid, known as dialysate, is introduced into the abdominal cavity through a catheter. The fluid absorbs waste products and is then drained away, carrying the waste out of the body. This process can be done at home and is an alternative to hemodialysis.

[0114] “Hemodiafiltration” as used herein is a renal replacement therapy that combines hemodialysis and hemofiltration. Hemodiafiltration (HDF) is a kidney replacement therapy that uses a combination of diffusion and convection to remove toxins from the blood. It uses a machine to filter blood through a dialyzer, removing waste products and excess fluids. The process involves both diffusion and convection, allowing for the removal of a wider range of toxins. It is often used for patients with severe kidney failure and provides more efficient clearance of middle and large molecular weight solutes compared to standard hemodialysis.

[0115] “Ischemia” or “ischaemia” is a restriction in blood supply to any tissue, muscle group, or organ of the body, causing a shortage of oxygen that is needed for cellular metabolism. Ischemia is generally caused by blockage or problems with blood vessels, with resultant damage to or dysfunction of tissue i.e. hypoxia and microvascular dysfunction. It also implies local hypoxia in a part of a body resulting from constriction (such as vasoconstriction, thrombosis, or embolism).

[0116] "Statins", as used herein, refers to a class of lipid-lowering medications that reduce illness and mortality in those who are at high risk of cardiovascular disease. They lower the level of cholesterol in the blood by reducing the production of cholesterol by the liver. Commonly known examples of statins include, but are not limited to, Atorvastatin (Lipitor), Lovastatin (Altoprev), Pitavastatin (Livalo, Zypitamag), Pravastatin (Pravachol), Rosuvastatin (Crestor, Ezallor), and Simvastatin (Zocor).

[0117] "Malignancy", as used herein, refers to the presence of cancerous cells that have the ability to spread to other sites in the body (metastasize) or to invade nearby (locally) and destroy tissues. Malignant cells tend to have fast, uncontrolled growth and do not die normally due to changes in their genetic makeup. There are several main types of malignancy. Carcinoma is a malignancy that begins in the skin or in tissues that line or cover internal organs. Sarcoma is a malignancy that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue. Leukemia is a malignancy that begins in blood-forming tissue, such as the bone marrow, and causes too many abnormal blood cells to be made. Lymphoma and multiple myeloma are malignancies that begin in the cells of the immune system. Central nervous system cancers are malignancies that begin in the tissues of the brain and spinal cord.

[0118] "Nonmelanoma Skin Cancer", as used herein, refer to any cancer that forms in the basal, squamous or Merkel cells of the skin. Melanoma is a cancer that develops in the skin's melanocytes.

[0119] "Sponsor of study" as used herein, refers to an individual, institution, company, or organization (for example, a contract research organization) that takes the responsibility to initiate, manage or finance the clinical trial but does not actually conduct the investigation.

[0120] “Hypophosphatemic rickets”, as used herein, refers to a disorder in which the bones become soft and bend easily, due to low levels of phosphate in the blood. Symptoms usually begin in early childhood and can range in severity from bowing of the legs, bone deformities, bone pain, joint pain, poor bone growth, and short stature.

[0121] “Hereditary Hypophosphatemic Rickets” as used herein refers to a disorder related to low levels of phosphate in the blood (hypophosphatemia). Phosphate is a mineral that is essential for the normal formation of bones and teeth. Most commonly, it is caused by a mutation in the PHEX gene. Other genes that can be responsible for the condition include the CLCN5, DMP1, ENPP1, FGF23, and SLC34A3 genes. Other signs and symptoms of hereditary hypophosphatemic rickets can 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 a softening of the bones known as osteomalacia. Another rare type of the disorder is known as hereditary hypophosphatemic rickets with hypercalciuria (HHRH) wherein in addition to hypophosphatemia, this condition is characterized by the excretion of high levels of calcium in the urine (hypercalciuria).

[0122] “X-linked hypophosphatemia (XLH) ”, as used herein, the term X-linked hypophosphatemia (XLH), also called X-linked dominant hypophosphatemic rickets, or X-linked Vitamin D-resistant rickets, is an X-linked dominant form of rickets (or osteomalacia) that differs from most cases of rickets in that vitamin D supplementation does not cure it. It can cause bone deformity including short stature and genu varum (bow leggedness). It is associated with a mutation in the PHEX gene sequence (Xp.22) and subsequent inactivity of the PHEX protein.

[0123] “Autosomal Recessive Hypophosphatemia Rickets type 2 (ARHR2) ”, as used herein, the term refers to a hereditary renal phosphate-wasting disorder characterized by hypophosphatemia, rickets and / or osteomalacia and slow growth. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is caused by homozygous loss-of-function mutation in the ENPP1 gene.

[0124] “Autosomal Dominant Hypophosphatemic Rickets (ADHR) ”, as used herein, refers to a rare hereditary disease in which excessive loss of phosphate in the urine leads to poorly formed bones (rickets), bone pain, and tooth abscesses. ADHR is caused by a mutation in the fibroblast growth factor 23 (FGF23). ADHR is characterized by impaired mineralization of bone, rickets and / or osteomalacia, suppressed levels of calcitriol (1, 25-dihydroxyvitamin D3), renal phosphate wasting, and low serum phosphate. Mutations in FGF23 render the protein more stable and uncleavable by proteases resulting in enhanced bioactivity of FGF23. The enhanced activity of FGF23 mutants reduce expression of sodium-phosphate co-transporters, NPT2a and NPT2c, on the apical surface of proximal renal tubule cells, resulting in renal phosphate wasting.

[0125] “Hypophosphatemic rickets” (previously called vitamin D-resistant rickets) is a disorder in which the bones become painfully soft and bend easily, due to low levels of phosphate in the blood. Symptoms may include bowing of the legs and other bone deformities; bone pain; joint pain; poor bone growth; and short stature. In some affected babies, the space between the skull bones closes too soon leading to craniosynostosis. Most patients display Abnormality of calcium-phosphate metabolism, Abnormality of dental enamel, Delayed eruption of teeth and long, narrow head (Dolichocephaly).

[0126] “Pre-treatment”, as used herein, means treatment prior to commencement of a treatment method described herein.

[0127] The term “subject”, as used herein, refers to a human individual or person. The term includes a subject of any age or sex. The term subject is interchangeable used herein with terms such as patient or participants of study and have the same meaning. The term “infant' as used herein, refers to a human in his or her first year of life.

[0128] Typically, infantile stage refers to the age from birth to under the age of 1 year. Under the age of 1 year is equal to under the age of 12 months. Thus, an infant as used herein refers to an age inclusive of the first hours and the first day of life up of the newborn to the age of 11 months but does not include 12 months of age.

[0129] The term “ children" or “ child' as used herein, refers to a human child and refers to a life stage of having an age between greater than or equal to one year but less than 13 years of age. (i.e. between the ages of 1-12).

[0130] The term "trabecular thickness" as used herein, refers to the average thickness of the trabeculae, which are the small, rod-like or plate-like structures found in spongy bone (also known as cancellous or trabecular bone). These structures form a porous network that provides structural support and helps distribute mechanical loads. Trabecular thickness is an important parameter in assessing bone quality and strength, often measured in studies related to osteoporosis and other bone-related conditions.

[0131] The term ''trabecular number" as used herein, refers to the number of trabeculae per unit length in a given volume of spongy bone. It is a measure used to assess the density and structural integrity of trabecular bone, often in the context of evaluating bone health and conditions like osteoporosis. Higher trabecular number indicates a denser and potentially stronger bone structure.

[0132] The term "trabecular spacing' or "trabecular separation" (Tb. Sp) as used herein, describes the average distance or space between the trabeculae (the small, rod- or plate-like structures) within cancellous (spongy) bone. It is a measure of how far apart these trabeculae are from each other and provides insight into the bone's microarchitecture. An increase in trabecular spaces can indicate a loss of bone density, which is commonly associated with conditions like osteoporosis. As the spaces between the trabeculae widen, the bone becomes weaker and more susceptible to fractures. This parameter is often assessed alongside trabecular thickness and other measures to evaluate bone quality, using imaging techniques such as micro-CT or MRI. The term “trabecular BV / TV” or bone volume to total volume ratio as used herein, is a measure used to assess the proportion of bone volume relative to the total volume of a given region of trabecular bone. It is an important parameter in evaluating bone density and strength, often used in studies of bone health and diseases like osteoporosis. A higher BV / TV ratio indicates a greater amount of bone within the total volume, suggesting stronger bone structure.

[0133] The term “cortical thickness ' as used herein, refers to the measurement of the thickness of the cortical bone, which is the dense and compact outer layer of bone. This parameter is important for assessing bone strength and structural integrity, as thicker cortical bone generally indicates stronger and more resilient bone. Cortical thickness is often evaluated in studies related to bone health, such as osteoporosis research.

[0134] The term “ cortical BA / TA" or bone area to total area ratio as used herein, is a measure used to assess the proportion of cortical bone area relative to the total cross-sectional area of a bone. It helps evaluate bone strength and structural integrity, with a higher ratio indicating a greater proportion of bone, suggesting stronger bone structure.

[0135] A disease or disorder is “alleviated' if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0136] As used herein the terms “alteration," “defect A “variation", or “mutation" refer to a mutation in a gene in a cell that affects the function, activity, expression (transcription or translation) or conformation of the polypeptide it encodes, including missense and nonsense mutations, insertions, deletions, frameshifts and premature terminations.

[0137] As used herein, 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, if necessary, to achieve the maximum percent sequence identity.

[0138] Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, 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 the CLUSTAL OMEGA software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0139] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0140] A “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0141] As used herein, the term "immune response" or "immune reaction" refers to the host's immune system to antigen in an invading (infecting) pathogenic organism, or to introduction or expression of foreign protein. The immune response is generally humoral and local; antibodies produced by B cells combine with antigen in an antigen-antibody complex to inactivate or neutralize antigen. Immune response is often observed when human proteins are injected into mouse model systems. Generally, the mouse model system is made immune tolerant by injecting immune suppressors prior to the introduction of a foreign antigen to ensure better viability.

[0142] As used herein, the term "immune suppression" is a deliberate reduction of the activation or efficacy of the host immune system using immune suppressant drugs to facilitate immune tolerance towards foreign antigens such as foreign proteins, organ transplants, bone marrow, and tissue transplantation. Non-limiting examples of immunosuppressant drugs include anti-CD4(GK1.5) antibody, Cyclophosphamide, Azathioprine (Imuran), Mycophenolate mofetil (Cellcept), Cyclosporine (Neoral, Sandimmune, Gengraf), Methotrexate (Rheumatrex), Leflunomide (Arava), Cyclophosphamide (Cytoxan) and Chlorambucil (Leukeran). As used herein, the term “ENPP” or “NPP” refers to ectonucleotide pyrophosphatase / phosphodiesterase.

[0143] As used herein, the term “ENPP1 protein” or “ENPP1 polypeptide” refers to ectonucleotide pyrophosphatase / phosphodiesterase-1 protein encoded by the ENPP1 gene. The encoded protein is a type II transmembrane glycoprotein and cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars and pyrophosphate bonds of nucleotides and nucleotide sugars. ENPP1 protein has a transmembrane domain and soluble extracellular domain. The extracellular domain is further subdivided into somatomedin B domain, catalytic domain (residues 186 to 586 of SEQ ID NO: 1), and the nuclease domain (residues 524 to 885 of SEQ ID NO: 1). The sequence and structure of wild-type ENPP1 is described in detail in PCT Application Publication No. WO 2014 / 126965 to Braddock, et al., which is incorporated herein in its entirety by reference.

[0144] As used herein, the term “INZ-70T refers to ENPP1-Fc fusion protein, wherein the ENPP1 polypeptide is connected to Fc domain by means of a peptide linker.

[0145] Mammal ENPP1 polypeptides, mutants, or mutant fragments thereof, have been previously disclosed in International PCT Application Publications No. WO / 2014 / 126965, WO / 2016 / 187408, WO / 2017 / 087936, W02018 / 027024, WO 2020 / 047520, WO 2020 / 206302 and WO 2023 / 191898, the contents of each of which are incorporated by reference in their entireties herein.

[0146] As used herein, the term “ENPP1 precursor protein" refers to ENPP1 with its signal peptide sequence at the ENPP1 N-terminus. Upon proteolysis, the signal sequence is cleaved from ENPP1 to provide the ENPP1 protein. Signal peptide sequences useful within the invention include, but are not limited to, Albumin signal sequence, Azuroci din signal sequence, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence.

[0147] As used herein, the term "ENPP1-Fc construct” refers to ENPP1 recombinantly fused and / or chemically conjugated (including both covalent and non-covalent conjugations) to an FcR binding domain of an IgG molecule (preferably, a human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0148] As used herein, the term “Fc” refers to a human IgG (immunoglobulin) Fc domain. Subtypes of IgG such as IgGl, IgG2, IgG3, and IgG4 are contemplated for use as Fc domains. As used herein, the “Fc region ” or “Fc polypeptide" is the portion of an IgG molecule that correlates to a crystallizable fragment obtained by papain digestion of an IgG molecule. The Fc region comprises the C-terminal half of the two heavy chains of an IgG molecule that are linked by disulfide bonds. It has no antigen binding activity but contains the carbohydrate moiety and the 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 IgGl, 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 a region of an IgG molecule consisting of the Fc region (residues 231 -447) and a 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 having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen binding site. The constant domain contains the CHI, CH2, and CH3 domains of the heavy chain and the CHL domain of the light chain.

[0149] 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, 50-100, 100-500, 500-1000, 1000-1500 nucleotides, 1500-2500, or 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, 50, 100, 200, 300 or 400 amino acids in length (and any integer value in between).

[0150] “Isolated" means altered or removed from the natural state. For example, a nucleic acid or a polypeptide naturally present in a living animal is not “isolated”, but the same nucleic acid or polypeptide partially or completely separated from the coexisting materials of its natural state is “isolated”. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0151] As used herein, the term “patient", “individual", or “subject" refers to a human.

[0152] As used herein, the term “pharmaceutical composition" or “composition" refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a patient. Multiple techniques of administering a compound exist in the art including, but not limited to, subcutaneous, intravenous, oral, aerosol, inhalational, rectal, vaginal, transdermal, intranasal, buccal, sublingual, parenteral, intrathecal, intragastrical, ophthalmic, pulmonary, and topical administration.

[0153] As used herein, the term “pharmaceutically acceptable'''' refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material 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; for example, phosphate-buffered saline (PBS).

[0154] As used herein, the term "pharmacodynamics ' (PD) refers to the study of the biochemical and physiological effects of therapeutic drugs on the body, as well as the mechanisms of their action. It involves the study of the interaction between drug molecules and target cells, tissues, or organs, and the subsequent effects, including therapeutic and adverse outcomes.

[0155] As used herein, the term "pharmacokinetics ' (PK) refers to the study of the movement of drugs within the body, focusing on the processes of absorption, distribution, metabolism, and excretion (ADME). It quantitatively analyzes how the body affects a drug over time, influencing its concentration in various tissues and fluids.

[0156] As used herein, the term “adverse event” (AE) refers to any untoward medical occurrence (e.g., sign, symptom, illness, disease, injury) in a study participant administered any amount of INZ-701 or other protocol-imposed intervention regardless of attribution. Treatment-emergent adverse events (TEAEs) are defined as any AE that newly appears, increases in frequency, or worsens in severity following initiation of INZ-701. A serious adverse reaction (SAR) is any AE for which there is a reasonable possibility that the drug caused the AE. The table A below shows Adverse Event Severity Grading scale.

[0157]

[0158] a Instrumental ADL refer to preparing meals, shopping for groceries or clothes, using the telephone, managing money, etc.

[0159] b Self-care ADL refer to bathing, dressing and undressing, feeding self, using the toilet, taking medications, and not bedridden. Source: CTCAE v5.0 (NCI 2017).

[0160] As used herein, the term “ Serious Adverse Event (SAE)” refers to an event that results in any of the following:

[0161] • Death

[0162] • A life-threatening AE; Note: Life-threatening refers to an event that places the study participant at immediate risk of death. This definition does not include a reaction that, had it occurred in a more severe form, might have caused death.

[0163] • A persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions.

[0164] • Inpatient hospitalization or prolongation of existing hospitalization. Examples of visits to a hospital facility that do not meet the seriousness criteria for hospitalization include:

[0165] - Outpatient surgery - ER visits that do not result in hospital admission

[0166] - Preplanned or elective procedures

[0167] - Protocol procedures

[0168] • A congenital anomaly or birth defect (in a child or fetus of a study participant exposed to INZ-701 prior to conception or during pregnancy).

[0169] • An important medical event that, based on appropriate medical judgment, may jeopardize the study participant and may require medical or surgical intervention to prevent one of the outcomes listed above (e.g., anaphylaxis).

[0170] The following table B provides the adverse event causality attribution guidance.

[0171]

[0172] As used herein, the term “plasma pyrophosphate (PPI) levels’" refers to the amount of pyrophosphate present in plasma of animals. In certain embodiments, animals include rat, mouse, cat, dog, human, cow, and horse. It is necessary to measure PPi in plasma rather than serum because of release from platelets. There are several ways to measure PPi, one of which is by enzymatic assay using uridine-diphosphoglucose (UDPG) pyrophosphorylase {Lust & Seegmiller, 1976, Clin. Chim. Acta 66:241-249; Cheung & Suhadolnik, 1977, Anal. Biochem. 83:61-63) with modifications. Typically, normal PPi levels in healthy subjects range from about 1pm to about 3 pM, in some cases between 1-2 pm. A subject who has defective ENPP1 expression tend to exhibit low PPi levels which range from at least 10% below normal levels, at least 20% below normal levels, at least 30% below normal levels, at least 40% below normal levels, at least 50% below normal levels, at least 60% below normal levels, at least 70% below normal levels, at least 80% below normal levels and combinations thereof. In patients afflicted with GACI, the PPi levels are found to be less than 1 pm and in some cases are below the level of detection. In patients afflicted with PXE, the PPi levels are below 0.5 pm. (Arterioscler Thromb Vase Biol. 2014 Sep;34(9): 1985-9; Braddocket al., Nat Commun. 2015; 6: 1 006.)

[0173] As used herein, the term “polypeptide” refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked via peptide bonds.

[0174] As used herein, the term “PPi” refers to pyrophosphate.

[0175] As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability of one to prevent some or all of the symptoms associated with the disorder or disease.

[0176] “Sample” or “biological sample” as used herein means a biological material isolated from a subject. The biological sample may contain any biological material suitable for detecting a mRNA, polypeptide or other marker of a physiologic or pathologic process in a subject, and may comprise fluid, tissue, cellular and / or non-cellular material obtained from the individual.

[0177] As used herein, “substantially purified’" refers to being essentially free of other components. For example, a substantially purified polypeptide is a polypeptide that has been separated from other components with which it is normally associated in its naturally occurring state. Non-limiting embodiments include 95% purity, 99% purity, 99.5% purity, 99.9% purity and 100% purity.

[0178] 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 invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder, a symptom of a disease or disorder or the potential to develop a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the potential to develop the disease or disorder. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

[0179] The terms “prevent”, “preventing”, and “prevention”, as used herein, refer to inhibiting the inception or decreasing the occurrence of a disease in a subject. Prevention may be complete (e.g., the total absence of pathological cells in a subject) or partial. Prevention also refers to a reduced susceptibility to a clinical condition.

[0180] As used herein, the term “wild-type” refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the human NPP1 genes. In contrast, the term “functionally equivalent” refers to a NPP1 gene or gene product that displays modifications in sequence and / or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. Naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics (including altered nucleic acid sequences) when compared to the wild-type gene or gene product.

[0181] The term “functional equivalent variant”, as used herein, relates to a polypeptide substantially homologous to the sequences of ENPP1 (defined above) and that preserves the enzymatic and biological activities of ENPP1. Methods for determining whether a variant preserves the biological activity of the native ENPP1 are widely known to the skilled person and include any of the assays used in the experimental part of said application. Particularly, functionally equivalent variants of ENPP1 delivered by viral vectors is encompassed by the present invention.

[0182] The functionally equivalent variants of ENPP1 are polypeptides substantially homologous to the native ENPP1. The expression “substantially homologous” relates to a protein sequence when said protein sequence has a degree of identity with respect to the ENPP1 sequences described above 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. The degree of identity between two polypeptides is determined using computer algorithms and methods that are widely known for the persons skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., etal., NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990)), though other similar algorithms can also be used. BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0183] “Functionally equivalent variants” of ENPP1 may be obtained by replacing nucleotides within the polynucleotide accounting for codon preference in the host cell that is to be used to produce the ENPP1 respectively. Such “codon optimization” can be determined via computer algorithms which incorporate codon frequency tables such as “Human high.cod” for codon preference as provided by the University of Wisconsin Package Version 9.0, Genetics Computer Group, Madison, Wis.

[0184] ‘About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, 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 (2.4 mg / kg), as such variations are appropriate to perform the disclosed methods.

[0185] The disclosure provides a representative example of protein sequences. The protein sequences described can be converted into nucleic acid sequences by performing revere translation and codon optimization. There are several tools available in art such as Expasy (https: / / www.expasy.org / ) and bioinformatics servers (http: / / www.bioinformatics.org) that enable such conversions.

[0186] Ranges: throughout this disclosure, various aspects according to the invention can 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 according to the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 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.

[0187] The use of the term "flat dose" or “flat” means a dose that is administered to a subject without regard for the body weight of the patient. The flat dose is therefore not provided as an mg / kg dose, but rather as an absolute amount of the agent (e.g., INZ-701). For example, a 50 kg person and a 100 kg person would receive the same dose (e g., 150 mg).

[0188] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0189] DETAILED DESCRIPTION

[0190] Calciphylaxis

[0191] Without being bound by theory, inventors postulate that the development of microvascular calcifications in patients with calciphylaxis is likely due to an active, cell-mediated process that depends upon the balance between the promoters and inhibitors of calcification. Among inhibitors of vascular calcification, inorganic pyrophosphate (PPi) stands out as a very potent inhibitor of hydroxyapatite crystal deposition (i.e., mineralization), and therefore low levels of PPi may lead to increased tissue mineralization. In addition, PPi is hydrolyzed by alkaline phosphatase (ALP) into inorganic phosphate (Pi), which promotes bone remodeling. A distorted PPi-Pi balance results in pathological tissue and vascular calcification as is observed in patients with CKD. These pathological consequences can have a devastating impact resulting in high morbidity and mortality in CKD and ESKD populations.

[0192] Calciphylaxis is a serious condition characterized by the calcification of micro blood vessels, which can lead to their occlusion and blockage. This blockage prevents adequate blood flow, resulting in the formation of painful lesions. These lesions occur because the surrounding tissue is deprived of oxygen and essential nutrients that are normally delivered through blood circulation. The lack of these vital elements can cause tissue ischemia, a condition where the tissue is starved of oxygen, leading to severe pain and potential tissue damage.

[0193] The underlying cause of this excessive calcification is believed to be low levels of plasma pyrophosphate, a natural inhibitor of calcification. To address this, the administration of ENPP1 polypeptide is proposed. ENPP1 Polypeptide cleaves extracellular adenosine triphosphate (ATP) to generate PPi and adenosine monophosphate (AMP), which is then metabolized to adenosine, thereby increasing plasma PPi levels and adenosine. ENPPl cleaves ENPP1 polypeptide is thus expected to increase the levels of pyrophosphate (PPi) in the bloodstream. By boosting PPi levels, the treatment aims to reverse or reduce the calcification within the micro blood vessels.

[0194] Improving blood flow through this mechanism can restore the delivery of nutrients and oxygen to the affected ischemic tissue. As a result, the tissue can begin to heal, reducing the size and severity of the lesions. This healing process is expected to alleviate the associated pain, significantly improving the patient's quality of life. Additionally, by addressing the root cause of calcification, this treatment approach may offer a more sustainable solution to managing calciphylaxis.

[0195] CKD-MBD

[0196] Without being bound by theory, inventors postulate that the various manifestations of CKD-MBD are interconnected, representing complex interactions among different tissues, signaling pathways, and hormones. Increased osteoid volume and vascular calcification, for instance, are intertwined, characterized by hypomineralization of bone and simultaneous ectopic mineralization of the vasculature that is sometimes referred to as paradoxical mineralization (Chen, etal., Arterioscler Thromb Vase Biol. 2020 May;40(5): 1078-1093). FGF23, through interactions with the FGF receptor 1 and klotho, mediates the downregulation of the phosphate transporters SLC34al and SLC34a3 which are expressed in the proximal tubules of the kidney. FGF23 also reduces the concentration of both 1,25 dihydroxy vitamin D and PTH.

[0197] As CKD progresses, klotho expression decreases. The loss of klotho, coupled with increasing nephrotic damage, impairs FGF23’s ability to regulate phosphate levels which leads to hyperphosphatemia. This is further exacerbated as 1,25 dihydroxy vitamin D levels decrease as the primary location for expression of Cyp27bl, the key enzyme required for active vitamin D synthesis, is in the kidney. The loss of active vitamin D results in secondary hyperparathyroidism leading to increased bone resorption and further hyperphosphatemia. FGF23 levels continue to elevate during the progression of CKD. Imbalances in the above hormones, vitamins, and minerals undoubtedly play a key role in both vascular calcification and skeletal abnormalities but recent work suggests that signaling pathways, most notably the WNT pathway, are also altered, suggesting that factors secreted from sites of vascular calcification can drive mineral bone disease.

[0198] The paradoxical mineralization seen in CKD-MBD patients is reminiscent of the rare mineralization disorder known as generalized arterial calcification of infancy (GACI), caused mainly by mutations in the ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) gene as well as a smaller subset of patients who have mutations in the ABCC6 gene, mutations in the later stages might cause the related disorder pseudoxanthoma elasticum (PXE). Similar to CKD-MBD, GACI presents with medial vascular calcification and develop osteomalacia in adulthood. Further supporting the connection between this family of disorders and CKD are that mutations in both ENPP1 and ABCC6 have been linked to increased vascular calcification in the context of CKD, CKD-MBD, GACI, and PXE patients exhibit low plasma pyrophosphate (PPi) levels, where PPi acts as a potent inhibitor of vascular calcification and is generated by the breakdown of ATP byENPPl.

[0199] PPi levels in dialysis and CKD patients were found to be significantly reduced and inversely correlated with vascular calcification. Additionally, calciphylaxis patients exhibited particularly low PPi levels, which correlated with mortality. AMP, and especially its breakdown product adenosine, serve as an important signaling molecules in the vasculature and is a potent inhibitor of intimal proliferation.

[0200] The underlying cellular and biochemical mechanisms in these diseases are related to CKD-MBD given the similar disease presentations including low PPi levels, medial vascular calcification, and osteomalacia. Therefore, the therapeutic effects of ENPP1 agent such as a recombinant human ENPP1-Fc fusion protein consisting of the catalytic domain of ENPP1 fused to the Fc of human IgGl, were evaluated in adenine-induced rat model of CKD-MBD.

[0201] Chronic Kidney Disease (CKD) and Mineral Bone Disorder associated with CKD (CKD-MBD) are thus related but distinct conditions. CKD is a progressive condition characterized by a gradual loss of kidney function over time. It involves the reduced ability of the kidneys to fdter waste, balance electrolytes, and maintain fluid balance. CKD-MBD is a systemic disorder of bone metabolism and mineral imbalance that occurs as a complication of CKD. It involves abnormalities in bone structure, calcium, phosphorus, parathyroid hormone (PTH), and vitamin D metabolism.

[0202] CKD can result from various conditions, such as diabetes, hypertension, glomerulonephritis, and polycystic kidney disease. It leads to reduced kidney function over time. However CKD-MBD develops as a direct consequence of CKD. Impaired kidney function disrupts the normal regulation of minerals and hormones, leading to bone and mineral abnormalities.

[0203] In CKD, the kidneys' ability to fdter blood is compromised, leading to the accumulation of waste products and fluid imbalances. It is assessed by the glomerular fdtration rate (GFR) and the presence of kidney damage markers (e.g., proteinuria). When kidneys lose function, they can't maintain normal levels of phosphorus, calcium, and vitamin D. This imbalance triggers increased PTH production (secondary hyperparathyroidism), leading to bone demineralization, abnormal bone turnover, and soft tissue calcification thereby causing CKD-MBD.

[0204] Symptoms of CKD may include fatigue, swelling (edema), nausea, loss of appetite, changes in urination, and high blood pressure. Early stages are often asymptomatic. However CKD-MBD maybe diagnosed through blood tests (to check calcium, phosphorus, PTH, and vitamin D levels), bone density scans, and sometimes bone biopsies. Imaging studies may reveal calcifications in blood vessels.

[0205] CKD impacts multiple body systems due to the accumulation of toxins and waste. It can lead to complications like anemia, cardiovascular disease, and fluid overload. However CKD- MBD specifically affects bone health, mineral metabolism, and the cardiovascular system due to calcifications. It is a major cause of morbidity and increased mortality in CKD patients.

[0206] CKD is a condition characterized by declining kidney function, while CKD-MBD is a specific complication of CKD that affects mineral and bone metabolism. Both conditions are interrelated, but CKD-MBD specifically deals with the consequences of altered mineral balance and bone health.

[0207] The experiments disclosed elsewhere herein show that treatment with ENPP1 agent such as ENPP1-Fc fusion protein effectively prevented calcification in the iliac arteries, aorta, and kidney of a rat model of CKD. Furthermore, the study demonstrated that this CKD rat model induces severe osteomalacia which is prevented upon treatment with ENPP1-Fc fusion protein.

[0208] 1. ENPP1 Agent

[0209] An ENPP1 agent is an ENPP1 polypeptide. ENPP1 polypeptides disclosed herein include naturally occurring polypeptides of the ENPP1 family as well as any variants thereof (including mutants, fragments, fusions, and peptidomimetic forms) that retain a biological activity. The terms “ENPP1” or “ENPP1 polypeptide” refers to ectonucleotide pyrophosphatase / phosphodiesterase 1 proteins (NPP1 / ENPP1 / PC-1) and ENPPl-related proteins, derived from any species. ENPP1 protein comprises a type II transmembrane glycoprotein that forms a homodimer. Each monomer of the ENPP1 protein comprises a short intracellular N-terminal domain involved in targeting to the plasma membrane, a transmembrane domain, and a large extracellular region comprising several domains. The large extracellular region comprises SMB1 and SMB2 domains, which have been reported to take part in ENPP1 dimerization (R. Gijsbers, H. etal., Biochem. J. 371; 2003: 321-330). Specifically, the SMB domains contain eight cysteine residues, each arranged in four disulphide bonds, and have 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. ENPP1 protein functions to hydrolyze nucleoside 5’ triphosphatase to either corresponding monophosphates and also hydrolyzes diadenosine polyphosphates. ENPP1 proteins play a role in purinergic signaling which is involved in the regulation of cardiovascular, neurological, immunological, musculoskeletal, hormonal, and hematological functions. An exemplary amino acid sequence of the human ENPP1 precursor protein (NCBI accession NP_006199) is shown in Figure 1 (SEQ ID NO: 1). The human ENPP1 precursor protein includes an endogenous ENPP1 signal peptide sequence at the ENPP1 N-terminus. Numbering of amino acids for all ENPP1-related polypeptides described herein is based on the numbering of the human ENPP1 precursor protein sequence provided in Figure 2 unless specifically designated otherwise. In certain embodiments, the ENPP1 precursor protein further comprises an endogenous or heterologous signal peptide sequence. Upon proteolysis, the signal peptide sequence is cleaved from the ENPP1 precursor protein to provide the mature ENPP1 protein. See, e.g, Jansen S, 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, ENPP1 signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and / or ENPP5 signal peptide sequence. The processed (mature) extracellular ENPP1 polypeptide sequence is shown in Figure 3 (SEQ ID NO: 2).

[0210] It is generally known in the art that ENPP1 is well-conserved among vertebrates, with large stretches of the extracellular domain substantially conserved. For example, Figure 4A and Figure 4B depict a multi -sequence alignment of a 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, e.g., Kato K. et al., Proc Natl Acad Sci USA. 2012;109(42): 16876-81 and Mackenzie NC, et al. Bone. 2012;51(5):961-8

[0211] Accordingly, from these alignments, it is possible to predict key amino acid positions with the extracellular domain that are important for normal ENPP1 activities as well as to predict amino acid positions that are likely to be tolerant to substitution without significantly altering normal ENPP1 activities. Therefore, an enzymatically active, human ENPP1 polypeptide useful in accordance with the presently disclosed compositions, may include one or more amino acids at corresponding positions from the sequence of another vertebrate ENPP1, or may include a residue that is similar to that in the human or other vertebrate sequences. Substitutions of one or more amino acids at corresponding positions may include conservative variations or substitutions that are not likely to change the shape of the polypeptide chain or alter normal ENPP1 activities. Examples of conservative variations, or substitutions, include the replacement 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, glutamic for aspartic acid, or 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, and preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identity.

[0212] 2. Enzymatic Activity of ENPP1

[0213] ENPP1 proteins have been characterized in the art in terms of structural and biological characteristics. In certain embodiments, soluble ENPP1 proteins disclosed herein comprise pyrophosphatase and / or phosphodiesterase activity. For instance, in some embodiments, the ENPP1 protein binds nucleotide triphosphates (e.g, ATP, UTP, GTP, TTP, and CTP), pNP-TMP, 3',5'-cAMP, and 2'-3'-cGAMP; and converts nucleotide triphosphates into inorganic pyrophosphate [see, e.g., Kato K. etal., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81; Li L, etal. Nat ChemBiol. 2014; 10(12): 1043-8; Jansen S, etal. Structure. 2012;20(ll):1948-59; and Onyedibe KI, et al. Molecules. 2019;24(22)\.

[0214] “Enzymatically active” or “Biologically active” ENPP1 polypeptides exhibit pyrophosphatase and / or phosphodiesterase activity (e.g., is capable of binding and / or hydrolyzing ATP into AMP and PPi and / or AP3a into ATP). For example, the pyrophosphatase / phosphodiesterase domain of an ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphates (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, the soluble ENPP1 polypeptide has a kcatvalue for the substrate ATP greater than or equal to about 3.4 (±0.4) s-1enzyme-1, wherein the kcat is determined by measuring the rate of hydrolysis of ATP for the polypeptide. In certain embodiments, the soluble ENPP1 polypeptide has a KM value for the substrate ATP less than or equal to about 2 pM, wherein the K is determined by measuring the rate of hydrolysis of ATP for the polypeptide. In addition to the teachings herein, these references provide ample guidance for how to generate soluble ENPP1 proteins that retain one or more biological activities (e.g., conversion of nucleotides into inorganic pyrophosphate).

[0215] 3. Soluble ENPP1

[0216] In one embodiment, the disclosure relates to ENPP1 polypeptides. As described herein, the term soluble ENPP1 polypeptide, includes any naturally occurring extracellular domain of an ENPP1 protein as well as any variants thereof (including mutants, fragments and peptidomimetic forms) that retain a biological activity (e.g.. enzymatically active). Examples of soluble ENPP1 polypeptides include, for example, an ENPP1 extracellular domain (SEQ ID NO: 2) as shown in Figure 3. In certain embodiments, the soluble ENPP1 polypeptides further comprise a signal sequence in addition to the extracellular domain of an ENPP1 polypeptide. Exemplary signal sequences include the native signal sequence of an ENPP1 polypeptide, or a signal sequence from another protein, such as a hENPP7 signal sequence or Azurocidin signal sequence.

[0217] 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, WO 2020 / 047520, WO 2020 / 206302, and WO 2023 / 191898, the contents of all of which are incorporated herein by reference in their entirety.

[0218] 4. ENPP1 Fusion Proteins

[0219] In some embodiments, the ENPP1 polypeptide is a fusion protein comprising an ENPP1 polypeptide domain and one or more heterologous protein portions (z.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 portion comprises an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is an Fc domain of an IgGl immunoglobulin. In certain embodiments, the soluble ENPP1 polypeptide is C-terminally fused to the Fc domain of human immunoglobulin 1 (IgGl), 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 (IgGl), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In some embodiments, the presence of an Fc domain improves half life, solubility, reduces immunogenicity, and increases the activity of the soluble ENPP1 polypeptide. In certain embodiments, portions of the native human IgG proteins (IgGl, IgG2, IgG3, and IgG4), may be used for the Fc portion (e.g., ENPPl-Fc). For instance, the present disclosure provides fusion proteins comprising ENPP1 fused to a polypeptide comprising a constant domain of an immunoglobulin, such as a CHI, CH2, or CH3 domain derived from human IgGl, IgG2, IgG3, and / or IgG4. The Fc fragment may comprise regions of the native IgG such as the hinge region (residues 216- 230 of human IgGl, according to the Rabat numbering system), the entire second constant domain CH2 (residues 231-340), and the third constant domain CH3 (residues 341- 447).

[0220] An example of an amino acid sequence that may be used for the Fc portion of human IgGl (GIFc) is SEQ ID NO: 6 (Table 1). In part, the disclosure provides polypeptides comprising, consisting essential of, or consisting of amino acid sequences with 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.

[0221] 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, an immunoglobulin heavy-chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. A fusion domain may be selected so as to confer a desired property. For example, some fusion domains are particularly useful for isolation of the fusion proteins by affinity chromatography. For the purpose of affinity purification, relevant matrices for affinity chromatography, such as glutathione-, amylase-, and nickel- or cobalt- conjugated resins are used. Many of such matrices are available in “kit” form, such as the Pharmacia GST purification system and the QIAexpress™ system (Qiagen) useful with (HI Se) fusion partners. As another example, a fusion domain may be selected so as to facilitate detection of the ENPP1 polypeptide. Examples of such detection domains include the various fluorescent proteins (e.g., GFP) as well as “epitope tags,” which are usually short peptide sequences for which a specific antibody is available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus haemagglutinin (HA), and c-myc tags. In some cases, the fusion domains have a protease cleavage site, such as for Factor Xa or thrombin, which allows the relevant protease to partially digest the fusion proteins and thereby liberate the recombinant proteins therefrom. The liberated proteins can then be isolated from the fusion domain by subsequent chromatographic separation.

[0222] 5. Linkers

[0223] In some embodiments, the ENPP1 fusion protein further comprises a linker positioned between the ENPP1 polypeptide domain and the one or more heterologous protein portions (e.g., an Fc immunoglobulin domain). 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, a linker can be an amino acid spacer including 1-200 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. In some embodiments, the linker comprises a polyglycine linker or a Gly-Ser linker. In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of 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). In some embodiments, a spacer can contain 2 to 12 amino acids including motifs of 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, a spacer can contain 3 to 12 amino acids including motifs of 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). In yet some embodiments, a spacer can contain 4 to 12 amino acids including motifs of 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, a spacer can contain motifs of GGGGA (SEQ ID NO: 62) or GGGGS (SEQ ID NO: 63), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 64) and GGGGSGGGGSGGGGS (SEQ ID NO: 65). In some embodiments of the invention, an amino acid spacer between a heterologous protein portion (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 a human serum albumin) and a soluble ENPP1 polypeptide may 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).

[0224] In some embodiments, a spacer can also contain amino acids other than glycine, alanine, and serine, e g., LIN (SEQ ID NO: 69), TGGGG (SEQ ID NO: 70), AAAL (SEQ ID NO: 71), AAAK (SEQ ID NO: 72), AAAR (SEQ ID NO: 73), EGKSSGSGSESKST (SEQ ID NO: 74), GSAGSAAGSGEF (SEQ ID NO: 75), AEAAAKEAAAKA (SEQ ID NO: 76), KESGSVSSEQLAQFRSLD (SEQ ID NO: 77), GENL YFQSGG (SEQ ID NO: 78), SACYCELS (SEQ ID NO: 79), RSIAT (SEQ ID NO: 80), RPACKIPNDLKQKVMNH (SEQ ID NO: 81), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG(SEQ ID NO: 82), AAANSSIDLISVPVDSR (SEQ ID NO: 83), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 84), NSS (SEQ ID NO: 87), ESS (SEQ ID NO: 88), RQQ (SEQ ID NO: 89), KR (SEQ ID NO: 90), (R)m; m=0-15 (SEQ ID NO: 91), DSSSEEKFLRRIGRFG (SEQ ID NO: 92), EEEEEEEPRGDT (SEQ ID NO: 93), APWHLSSQYSRT (SEQ ID NO: 94), STLPIPHEFSRE (SEQ ID NO: 95), VTKHLNQISQSY (SEQ ID NO: 96), (E)m; m=l-15 (SEQ ID NO: 97), RSGSGGS (SEQ ID NO: 98), (D)m; m=l-15 (SEQ ID NO: 99), LVIMSLGLGLGLGLRK (SEQ ID NO: 100), VIMSLGLGLGLGLRK (SEQ ID NO: 101), IMSLGLGLGLGLRK (SEQ ID NO: 102), MSLGLGLGLGLRK (SEQ ID NO: 103), SLGLGLGLGLRK (SEQ ID NO: 104), LGLGLGLGLRK (SEQ ID NO: 105), GLGLGLGLRK (SEQ ID NO: 106), LGLGLGLRK (SEQ ID NO: 107), GLGLGLRK (SEQ ID NO: 108), LGLGLRK (SEQ ID NO: 109), GLGLRK (SEQ ID NO: 110), LGLRK (SEQ ID NO: 111), GLRK (SEQ ID NO: 112), LRK (SEQ ID NO: 113), RK (SEQ ID NO: 114), or (K)m; m=l-15 (SEQ ID NO: 115). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of EAAAK (SEQ ID NO: 85). In some embodiments, a spacer can contain motifs, e.g., multiple or repeating motifs, of praline- rich sequences such as (XP)n, in which X may be any amino acid (e.g., A, K, or E) and n is from 1-5, and PAPAP (SEQ ID NO: 86).

[0225] 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.

[0226] In some embodiments, different elements of the fusion proteins (e.g., immunoglobulin Fc fusion proteins) may be arranged in any manner that is consistent with desired functionality. For example, a soluble ENPP1 polypeptide domain may be placed C-terminal to a heterologous protein portion, or alternatively, a heterologous protein portion may be placed C-terminal to a soluble ENPP1 polypeptide domain. The soluble ENPP1 polypeptide domain and the heterologous protein portion may be directly or indirectly linked in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between the domains. Preferred fusion proteins comprise the amino acid sequence set forth in any one of SEQ IDNOs: 3-5, and 116-128.

[0227] In some embodiments, soluble ENPP1 polypeptides of the present disclosure contain one or more heterologous moi eties. Optionally, a soluble ENPP1 polypeptide includes one or more heterologous moieties selected from: a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, an amino acid conjugated to a lipid moiety, and an amino acid conjugated to an organic derivatizing agent. In some embodiments, a 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 glycols, lipids, polysaccharide or monosaccharide, and phosphates. Effects of such non-amino acid elements on the functionality of a soluble ENPP1 polypeptide may be tested as described herein for other soluble ENPP1 polypeptides. When a polypeptide of the disclosure is produced in cells by cleaving a nascent form of the polypeptide, post-translational processing may also be important for correct folding and / or function of the protein. Different cells e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the soluble ENPP1 polypeptides.

[0228] 6. Determining Solubility

[0229] In some embodiments, the activity of soluble ENPP1 polypeptides may also be tested in a cell-based or in vivo assay. For example, the effect of a soluble ENPP1 polypeptide on the production of inorganic pyrophosphates (PPi) can be measured. Specifically, the pyrophosphatase / phosphodiesterase domain of an ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to produce inorganic pyrophosphates (PPi) and is generally soluble. This activity can be measured using a pNP-TMP assay as well as an HPLC-based ATP hydrolysis assay, as previously described Saunders, etal., 2008, Mol. Cancer Ther. 7(10): 3352-62; Albright, et al., 2015, Nat Comm. 6:10006). The effect of soluble ENPP1 polypeptides on the expression of genes involved in ENPP1 associated diseases such as ARHR2 {e.g., transcription of fibroblast growth factor 23 in osteoblasts and osteoclasts) can be assessed. This may, as needed, be performed in the presence of one or more nucleotide triphosphates or other ENPP1 substrates, and cells may be transfected so as to produce a soluble ENPP1 polypeptide. Likewise, a soluble ENPP1 polypeptide may be administered to a mouse or other animal and effects on ENPP1 associated diseases may be assessed using art-recognized methods.

[0230] In some embodiments, ENPP1 polypeptides to be used in accordance with the methods described herein are isolated polypeptides. As used herein, an isolated protein or polypeptide is one which has been separated from a component of its natural environment. In some embodiments, a polypeptide of the disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity as determined by, for example, electrophoretic e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic {e.g., ion exchange or reverse phase HPLC) analyses. Methods for assessment of purity are well known in the art [see, e.g., Flatman et al., (2007) J. Chromatogr. B 848:79-87], In some embodiments, soluble ENPP1 polypeptides to be used in accordance with the methods described herein are recombinant polypeptides. 7. ENPP1 Production

[0231] ENPP1 polypeptides of the disclosure can be produced by a variety of art-known techniques. For example, polypeptides of the disclosure can be synthesized using standard protein chemistry techniques such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant G. A. (ed), Synthetic Peptides: A User's Guide, W. H. Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396;

[0232] Milligen / Biosearch 9600). Alternatively, the polypeptides of the disclosure, including fragments or variants thereof, may be recombinantly produced using various expression systems [e.g., E. coli, Chinese Hamster Ovary (CHO) cells, COS cells, baculovirus, Yeast Pichia] as is well known in the art. The protein can be produced in either adherent or suspension cells. In some embodiments, the fusion protein is expressed in CHO cells. To establish stable cell lines the nucleic acid sequence encoding ENPP1 constructs are cloned into an appropriate vector for large scale protein production. In a further embodiment, the modified or unmodified polypeptides of the disclosure may be produced by digestion of recombinantly produced full-length ENPP1 polypeptides by using, for example, a protease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzyme (PACE). Computer analysis (using commercially available software, e.g., MacVector, Omega, PCGene, Molecular Simulation, Inc.) can be used to identify proteolytic cleavage sites. Alternatively, such polypeptides may be produced from recombinantly generated full-length ENPP1 polypeptides using chemical cleavage {e.g., cyanogen bromide, hydroxylamine, etc.).

[0233] 8. Expression Systems

[0234] Many expression systems are known and can be used for the production of ENPP1 fusion protein, including bacteria (for example E. coli and Bacillus subtilis), yeasts (for example Saccharomyces cerevisiae, Kluyveronmyces lactis and Pichia pastoris), filamentous fungi (for example Aspergillus), plant cells, animal cells and insect cells. The desired protein can be produced in conventional ways, for example from a coding sequence inserted in the host chromosome or on a free plasmid. The yeasts can be transformed with a coding sequence for the desired protein in any of the usual ways (e.g., electroporation). Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194: 182. Successfully transformed cells, i.e., cells that contain a 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 an ENPP1 polypeptide. Cells can be harvested and lysed, and their DNA content examined for the presence of the DNA using a method, such as that described by Southern, 1975, J. Mol. Biol, 98:503 and / or Berent, etal., 1985, Biotech 3:208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies.

[0235] Useful yeast plasmid vectors include pRS403 — 406 and pRS413 — 416 and are generally available from Stratagene Cloning Systems, La Jolla, CA, USA Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3. Plasmids pRS413 — 416 are Yeast Centromere plasmids (YCps).

[0236] A variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tract can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

[0237] Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which are enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5' -exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities.

[0238] The combination of these activities thus generates 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 is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. As a result, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments can be cleaved with an appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

[0239] Clones of single, stably transfected cells are then established and screened for high expressing clones of the desired ENPP1 fusion protein. Screening of the single cell clones for ENPP1 protein expression can be accomplished in a high-throughput manner in 96 well plates using the synthetic enzymatic substrate pNP-TMP as previously described {Albright, et al., 2015, Nat. Commun. 6:10006). Upon identification of high expressing clones through screening, protein production can be accomplished in shaking flasks or bioreactors are previously described in Albright, etal., 2015, Nat. Commun. 6:10006.

[0240] 9. ENPP1 Purification

[0241] Purification of ENPP1 can be accomplished using a combination of standard purification techniques known in the art. Following purification, ENPPl-Fc can be dialyzed into PBS supplemented with Zn2+ and Mg2+ (PBSplus) concentrated to between 5 and 7 mg / ml, and frozen at -80 °C in aliquots of 200-500 pl. Aliquots can be thawed immediately prior to use and the specific activity of the solution can be adjusted to 31.25 au / ml (or about 0.7 mg / ml depending on the preparation) by dilution in PBSplus.

[0242] 10. Route and Frequency of Administration

[0243] The polypeptide may be administered acutely or chronically to the subject. In certain embodiments, a second dosage of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is administered after a suitable time interval of about after two days, after four days, after a week, or after a month to the subject or even less frequently, such as once every several months or even once a year or less. The frequency of the dose is readily apparent to the skilled artisan and depends upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.

[0244] A dose amount or frequency may be selected so that the steady state level of plasma PPi is maintained at a constant or steady state level, and / or so as to achieve a continuous level of plasma PPi that is either close to the normal (2-3 pM) level or above (30-50% higher than) normal levels of PPi and does not return to the lower level of PPi that the subject had prior to the administration of first dosage of constructs disclosed herein.

[0245] Alternative, the ENPP1 agent may be administered at appropriate time intervals of either every 2 days, or every 4 days, every week or biweekly or once every three weeks or once every month so as to achieve a constant level of enzymatic activity of ENPP1.

[0246] Alternatively, an ENPP1 agent according to the disclosure is administered at an appropriate time interval of every 2 days, or every 4 days, or every week or biweekly or once every three weeks or once every month by monitoring one or more symptoms of a subject’s disease or disorder.

[0247] Without wishing to be bound by theory, it is believed that maintaining a steady state concentration of plasma PPi at normal levels reduces and / or prevents progression of pathological calcification of subjects.

[0248] In certain embodiments, the polypeptide is administered locally, regionally, parenterally or systemically to the subject. In some embodiments, the polypeptide is administered subcutaneously.

[0249] The regimen of administration may affect what constitutes an effective amount. For example, several divided dosages, as well as staggered dosages may be administered in a given time period (daily) or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, selection of a recited dose of an ENPP1 agent may be indicated by the exigencies of the therapeutic or prophylactic situation.

[0250] 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., a human), may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder in the patient. An effective amount of the recited dosages of an ENPP1 agent necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed; the time of administration; the rate of excretion of the compound; the duration of the treatment; other drugs, compounds or materials used in combination with the compound; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. A selected dosage is determined based on the biological activity of the therapeutic compound which in turn depends on the half-life and the area under the plasma time of the therapeutic compound curve.

[0251] 11. Prophylactic Administration

[0252] Armed with the disclosure herein, one skilled in the art would thus appreciate that the prevention of a disease or disorder in a subject encompasses administering to a subject an ENPP1 polypeptide as a preventative measure against the disease or disorder.

[0253] The relative amounts of the active ingredient (e.g., soluble ENPP1 polypeptides and fusion proteins thereof), the pharmaceutically acceptable carrier, and any additional ingredients in a formulation disclosed herein will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between about 0.1% and about 100% (w / w) active ingredient.

[0254] 12. Diseases Relating to Low PPi

[0255] In some embodiments, the disclosure contemplates methods of reducing or preventing progression of diseases caused by lower levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptides disclosed herein to increase the plasma PPi of the subjects to normal (2-3 pM) or above (30-50% higher than) normal levels and then to maintain the plasma PPi at a constant normal or above normal level thereafter. The method further comprises administering additional therapeutic effective amounts at intervals of two days, three days, one week or one month in order to maintain the Plasma PPi of the subject at a constant normal or above normal level to reduce or prevent the progression of pathological calcification or ossification. In certain embodiments, a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to raise pyrophosphate (PPi) levels in a subject having PPi level lower than normal level (which is around 2pM). In other embodiments, a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to reduce or prevent progression of pathological calcification or ossification in a subject having PPi levels lower than normal level. In some embodiments, a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to treat ENPP1 deficiency (e.g., GACI and ARHR2) manifested by a reduction of extracellular PPi concentration in a subject. In certain embodiments, the steady state level of plasma PPi achieved after administration of a first dosage of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is maintained for a time period of at least 2 days, at least 4 days, at least a week or at least a month.

[0256] In some embodiments, the disclosure contemplates methods of reducing or preventing progression of a disease caused by lower than normal levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 3-5, and 116-128.) to increase and / or sustain the plasma PPi of the subjects to a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level. In certain embodiments, the method further comprises further administration of the polypeptide disclosed herein every two days, three days, one week, or one month in order to maintain the plasma PPi levels at a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level, thus preventing the progression of pathological calcification or ossification.

[0257] 13. Treatment / Indications

[0258] The ENPP1 agent disclosed herein may be used in methods of treating, reversing, or preventing progression of diseases associated with an ENPP1 deficiency or an ABCC6 deficiency as disclosed herein.

[0259] The ENPP1 agent disclosed herein may be used in methods of treating, reversing, or preventing progression of CKD-MBD or mineral bone disorder associated with chronic kidney disease as disclosed herein. The ENPP1 agent disclosed herein may be used in methods of treating, reversing, or preventing progression of calciphylaxis associated with chronic kidney disease (CKD) or end stage renal disease (ESRD) as disclosed herein.

[0260] In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject.

[0261] In an aspect, the disclosure relates to administering to a subject having an ENPP1 deficiency or an ABCC6 deficiency such as CKD-MBD, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of Pi and / or PPi in the plasma of the subject. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0262] In an aspect, the disclosure relates to a method for preventing progression of or reducing vascular calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce vascular calcification in the subject.

[0263] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce pathological calcification in the subject.

[0264] In an aspect, the disclosure relates to a method for preventing the progression of or reducing tissue calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject.

[0265] In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject. In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of Pi and / or PPi in the plasma of the subject. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0266] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological ossification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject.

[0267] In an aspect, the disclosure relates to a method for ameliorating one or more symptoms of CKD-MBD in a subject, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby ameliorate one or more symptoms of CKD-MBD in the subject.

[0268] In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of Pi and / or PPi in the plasma of the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0269] In an aspect, the disclosure relates to a method for preventing progression of or reducing vascular calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce vascular calcification in the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency.

[0270] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce pathological calcification in the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency. In an aspect, the disclosure relates to a method for preventing the progression of or reducing tissue calcification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency.

[0271] In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency.

[0272] In an aspect, the disclosure relates to administering to a subject having CKD-MBD, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of Pi and / or PPi in the plasma of the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0273] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological ossification in a subject with CKD-MBD, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency.

[0274] In an aspect, the disclosure relates to a method for ameliorating one or more symptoms of CKD-MBD in a subject, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby ameliorate one or more symptoms of CKD-MBD in the subject and wherein said subject does not have or is not diagnosed with ENPP1 deficiency or ABCC6 deficiency.

[0275] In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. In an aspect, the disclosure relates to administering to a subject having an ENPP1 deficiency or an ABCC6 deficiency and having calciphylaxis, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of Pi and / or PPi in the plasma of the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0276] In an aspect, the disclosure relates to a method for preventing progression of or reducing vascular calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce vascular calcification in the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0277] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce pathological calcification in the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0278] In an aspect, the disclosure relates to a method for preventing the progression of or reducing tissue calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0279] In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of Pi and / or PPi in the plasma of the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 pM respectively.

[0280] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological ossification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0281] In an aspect, the disclosure relates to a method for ameliorating one or more symptoms of calciphylaxis in a subject, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby ameliorate one or more symptoms of calciphylaxis in the subject, wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0282] In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective dose in order to restore a physiological level of Pi and / or PPi in the plasma of the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 μM respectively.

[0283] In an aspect, the disclosure relates to a method for preventing progression of or reducing vascular calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce vascular calcification in the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0284] In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce pathological calcification in the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0285] In an aspect, the disclosure relates to a method for preventing the progression of or reducing tissue calcification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0286] In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of ENPP1 in the plasma or tissues of the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0287] In an aspect, the disclosure relates to administering to a subject having calciphylaxis, an ENPP1 agent at a therapeutically effective amount in order to restore a physiological level of Pi and / or PPi in the plasma of the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis. The physiological level of Pi and PPi in human serum (and in mammals generally) is 1-3 mM and 2-3 uM respectively. In an aspect, the disclosure relates to a method for preventing the progression of or reducing pathological ossification in a subject with calciphylaxis, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby prevent the progression of or reduce tissue calcification in the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0288] In an aspect, the disclosure relates to a method for ameliorating one or more symptoms of calciphylaxis in a subject, the method comprising: administering to the subject an ENPP1 agent at a therapeutically effective amount to thereby ameliorate one or more symptoms of calciphylaxis in the subject and wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein, and wherein the subject has chronic kidney disease (CKD) or end stage renal disease (ESRD) and is undergoing hemodialysis.

[0289] In some embodiments of any of the aforesaid methods, the subject is between the age of 18 but less than 70 years (>18 to <70 years).

[0290] In some embodiments of any of the aforesaid methods, the subject has a loss of function mutation in one or more of ecto-5 '-nucleotidase (NT5E), vitamin D receptor (VDR), and fibroblast growth factor 23 (FGF23) gene.

[0291] In some embodiments of any of the aforesaid methods, the subject is of age greater than 65 but less than 70 and the ENPP1 polypeptide is administered subcutaneously.

[0292] In some embodiments of any of the aforesaid methods, the ENPP1 polypeptide comprises a heterologous moiety, and wherein said heterologous moiety increases the circulating half-life of the ENPP1 agent relative to the circulating half-life of the ENPP1 agent lacking the heterologous moiety.

[0293] In some embodiments of any of the aforesaid methods, the ENPP1 polypeptide comprises a heterologous moiety and wherein said heterologous moiety comprises the Fc region of an immunoglobulin molecule. In some embodiments of any of the aforesaid methods, the ENPP1 polypeptide comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 or residues 1 (FTAGLKPSCAKE) to 833 (QED) of SEQ ID NO:3.

[0294] In some embodiments of any of the aforesaid methods, the ENPP1 polypeptide comprises the amino acid sequence depicted in anyone of SEQ ID NO: 3-5 and 116-128.

[0295] In some embodiments of any of the aforesaid methods, the subject has or is suspected of having one of generalized arterial calcification of infancy (GACI), autosomal recessive hypophosphatemic rickets type 2 (ARHR2), hypopyrophosphatemia and Pseudoxanthoma elasticum (PXE).

[0296] In some embodiments of any of the aforesaid methods, the subject has or is suspected of having one or more of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis, juvenile dermatomyositis, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), autosomal hypophosphatemic rickets (ARHR2), osteoarthritis, calcification of atherosclerotic plaques, Chronic Kidney Disease (CKD), End Stage Renal Disease (ESRD), Pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

[0297] In some embodiments of any of the aforesaid methods, the subject exhibits one or more of calcification of soft connective tissues, accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers of connective tissues, calcification in the eyes, calcification of cardiovascular system, calcification of skin, narrowing of the arteries, arteriosclerosis, ectopic calcification, narrowing of blood vessels in the lower extremities, claudication, abnormal pigmentation of cells of the retina, angioid streaks, abnormalities in the elastic membrane beneath the retina, choroidal neovascularization, visual impairment, vision loss, and blindness. In some embodiments of any of the aforesaid methods, the ENPP1 polypeptide is a ENPP1 fusion polypeptide, said ENPP1 fusion polypeptide comprises ENPP1 component and a Fc domain, wherein said ENPP1 component comprises at least 95% of amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 and Fc domain comprises at least 95% of amino acid residues of SEQ ID NO: 6.

[0298] In some embodiments of any of the aforesaid methods, the ENPP1 component and said Fc domain are connected by means of a peptide linker, wherein said peptide linker has a length that is no less than two amino acids and no more than 36 amino acids.

[0299] In some embodiments of any of the aforesaid methods, the subject has not undergone peritoneal dialysis or hemodiafiltration.

[0300] In some embodiments of any of the aforesaid methods, the subject has not been diagnosed with malignancy other than non-melanoma skin cancer or cervical carcinoma in the past year prior to said administration.

[0301] In some embodiments of any of the aforesaid methods, the subject has not been diagnosed with advanced liver disease such as liver cirrhosis.

[0302] In some embodiments of any of the aforesaid methods, the subject has not had a myocardial infarction, stroke, or congestive heart failure requiring hospitalization within 6 months prior to said administration.

[0303] An ENPP1 agent (such as, but not limited to, ENPP1, or ENPPl-Fc or ENPPl-Fc mutant thereof) is administered at one of the following selected doses: 0.1 mg / kg, 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, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7.0 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8.0 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9.0 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10.0 mg / kg, and / or fractions or multiples thereof. Administration is subcutaneous (SC) at least once or twice bimonthly, at least once or twice monthly, three times monthly, at least once or twice weekly.

[0304] The first dose of the ENPP1 agent may be administered on Day 1. On Days 8 and thereafter, the ENPP1 agent is administered to a subject at a selected dose of the ENPP1 agent mg / kg doses twice weekly. The dose may be administered at approximately the same time on each dosing day. The site of injection is alternated, with no site within 2 inches of any prior site of injection within the prior 2 weeks.

[0305] A selected dose of the ENPP1 agent is one of 0.1 mg / kg, 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. Another selected dose of the ENPP1 agent by SC is one 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, 6.0 mg / kg, 6.1 mg / kg, 6.2 mg / kg, 6.3 mg / kg, 6.4 mg / kg, 6.5 mg / kg, 6.6 mg / kg, 6.7 mg / kg, 6.8 mg / kg, 6.9 mg / kg, 7.0 mg / kg, 7.1 mg / kg, 7.2 mg / kg, 7.3 mg / kg, 7.4 mg / kg, 7.5 mg / kg, 7.6 mg / kg, 7.7 mg / kg, 7.8 mg / kg, 7.9 mg / kg, 8.0 mg / kg, 8.1 mg / kg, 8.2 mg / kg, 8.3 mg / kg, 8.4 mg / kg, 8.5 mg / kg, 8.6 mg / kg, 8.7 mg / kg, 8.8 mg / kg, 8.9 mg / kg, 9.0 mg / kg, 9.1 mg / kg, 9.2 mg / kg, 9.3 mg / kg, 9.4 mg / kg, 9.5 mg / kg, 9.6 mg / kg, 9.7 mg / kg, 9.8 mg / kg, 9.9 mg / kg, 10.0 mg / kg, and / or fractions or multiples thereof.

[0306] The first dose of the ENPP1 agent may be administered on Day 1. After the first dose, a subject may be observed for 7 days to monitor safety and to collect PK samples. On Days 8 and thereafter, a subject receives a selected dose twice weekly. Administration of the ENPP1 agent at a selected dose is continued as considered appropriate by the medical professional.

[0307] A subject may receive 8 doses of the ENPP1 agent over the course of a 30 day period of time, for example, resulting in an exposure of 1.6 mg, 4.8 mg, and 14.4 mg per 30 days, respectively, for dose amounts of 0.2 mg / kg, 0.6 mg / kg, and 1.8 mg / kg. Or a subject may receive more or less than 8 doses, as considered appropriate by a medical profession.

[0308] A subject may receive 4 doses of the ENPP1 agent over the course of a 30 day period of time, for example, resulting in an exposure of 0.8 mg, 2.4 mg, and 7.2 mg per 30 days, respectively, for dose amounts of 0.2 mg / kg, 0.6 mg / kg, and 1.8 mg / kg..

[0309] In some embodiments, the present disclosure relates to the use of an ENPP1 agent, such as a polypeptide having amino acid sequences as set forth in, e.g., SEQ ID NOs: 3-5 and 116- 128.

[0310] In certain embodiments, the pathological calcification is selected from the group consisting of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis, juvenile dermatomyositis,

[0311] In certain embodiments, the subject has or is suspected of having one of Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, autosomal hypophophatemic rickets (ARHR2), osteoarthritis, calcification of atherosclerotic plaques, Chronic Kidney Disease (CKD), End Stage Renal Disease (ESRD), Pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

[0312] In some embodiments, the disclosure contemplates methods of reducing or preventing progression of ectopic calcification of soft tissue in a subject having CKD-MBD or Calciphylaxis, including reducing, ameliorating, or preventing vascular calcification, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein.

[0313] In some embodiments, the ENPP1 deficiency is GACI. In some embodiments, the ENPP1 deficiency is ARHR2.

[0314] In certain embodiments, the polypeptide is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, wherein the ENPP1 precursor protein undergoes proteolytic processing to the polypeptide disclosed herein. In some embodiments, in the ENPP1 precursor protein the signal peptide sequence is conjugated to the ENPP1 polypeptide N-terminus. Upon proteolysis, the signal sequence is cleaved from the ENPP1 precursor protein to provide the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the group consisting of ENPP1 signal peptide sequence, Azurocidin signal peptide sequence, ENPP2 signal peptide sequence, ENPP7 signal peptide sequence, and ENPP5 signal peptide sequence.

[0315] It will be appreciated by one of skill in the art, when armed with the present disclosure including the methods detailed herein, that the disclosure is not limited to treatment of a disease or disorder once it is established. Particularly, the symptoms of the disease or disorder need not have manifested to the point of detriment to the subject; indeed, the disease or disorder need not be detected in a subject before treatment is administered. That is, significant pathology from disease or disorder does not have to occur before the present ENPP1 polypeptides may provide benefit.

[0316] In certain aspects, the disclosure relates to methods for preventing diseases and disorders in a subject, in that a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder from developing. Therefore, the disclosure relates to methods for preventing or delaying onset, or reducing progression or growth, of a disease or disorder in a subject, comprising administering an ENPP1 polypeptide to a subject prior to detection of the disease or disorder. In certain embodiments, the ENPP1 polypeptide is administered to a subject with a strong family history of the disease or disorder, thereby preventing or delaying onset or progression of the disease or disorder.

[0317] 14. ENPP1 Agent- Some non-limiting examples of ENPP1 Polypeptide Sequences Table 1: Sequences

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327] SEQ ID NO: 7 (Mouse NPP1- NCBI accession NP 001295256.1)

[0328] 1 MERDGDQAGH GPRHGSAGNG RELESPAAAS LLAPMDLGEE PLEKAERARP AKDPNTYKVL 61 SLVLSVCVLT TILGCIFGLK PSCAKEVKSC KGRCFERTFS NCRCDAACVS LGNCCLDFQE 121 TCVEPTHIWT CNKFRCGEKR LSRFVCSCAD DCKTHNDCCI NYSSVCQDKK SWVEETCESI 181 DTPECPAEFE SPPTLLFSLD GFRAEYLHTW GGLLPVISKL KNCGTYTKNM RPMYPTKTFP 241 NHYSIVTGLY PESHGIIDNK MYDPKMNASF SLKSKEKFNP LWYKGQPIWV TANHQEVKSG 301 TYFWPGSDVE IDGILPDIYK VYNGSVPFEE RILAVLEWLQ LPSHERPHFY TLYLEEPDSS 361 GHSHGPVSSE VIKALQKVDR LVGMLMDGLK DLGLDKCLNL ILISDHGMEQ GSCKKYVYLN 421 KYLGDVNNVK WYGPAARLR PTDVPETYYS FNYEALAKNL SCREPNQHFR PYLKPFLPKR 481 LHFAKSDRIE PLTFYLDPQW QLALNPSERK YCGSGFHGSD NLFSNMQALF IGYGPAFKHG 541 AEVDSFENIE VYNLMCDLLG LIPAPNNGSH GSLNHLLKKP IYNPSHPKEE GFLSQCPIKS 601 TSNDLGCTCD PWIVPIKDFE KQLNLTTEDV DDIYHMTVPY GRPRILLKQH RVCLLQQQQF 661 LTGYSLDLLM PLWASYTFLS NDQFSRDDFS NCLYQDLRIP LSPVHKCSYY KSNSKLSYGF 721 LTPPRLNRVS NHIYSEALLT SNIVPMYQSF QVIWHYLHDT LLQRYAHERN GINWSGPVF 781 DFDYDGRYDS LEILKQNSRV IRSQEILIPT HFFIVLTSCK QLSETPLECS ALESSAYILP 841 HRPDNIESCT HGKRESSWVE ELLTLHRARV TDVELITGLS FYQDRQESVS ELLRLKTHLP 901 IFSQED

[0329] SEQ ID NO: 8 (Cow NPP1-NCBI accession NP 001193141.1)

[0330] 1 MERDSCAGGG SRGGEGGRGP REGLAGNGRD PGPGRAAEAS GEPQAAASLL APMDLGEEPL 61 ERAARARPAK DPNTYKVLSL VLSVCVLTTI LGCIFGLKPS CAKEIKSCKG RCFERTFGNC 121 RCDAACVDLG NCCLDYQETC IEPERIWTCT KFRCGEKRLS RSLCSCSDDC KDKGDCCINH 181 GSVCRGEKSW AEEECDSIDE PQCPAGFETP PTLLFSLDGF RAEYLHTWGG LLPVISKLKT 241 CGTYTKNMRP VYPTKTFPNH YSIVTGLYPE SHGIIDNNIY DPQMNANFAL KNKEKFNPEW 301 YKGEPIWLTA KYQGLKTGTF FWPGSDVKIN GIFPDIYKIY NVSVPFEERI LAILKWLQLP 361 KDERPHFYTL YLEEPDSSGH SYGPVSSEVI RALQRVDNMV GMLMDGLKEL NLHRCLNLIL 421 ISDHGMEQGS CKKYVYLNKY LGDTKDYKW YGPAARLRPS DVPDKYYSFD YEGIAKNLSC 481 QEPNQHFKPY LKHFLPKRLH FAKNDRIERL TFYLDPQWQL ALNPSERKYC GGGFHGSDNT 541 FLNMQALFIG YGPGFKHSTE VDSFENIEVY NLMCDLLNLT PAPNNGTHGS LNHLLSNPVY 601 TPKHPKEVRP LVQCPFTRAP RESLDCSCDP SILPIVDFQT QLNLTMAEEK TIKRGALPYG 661 RPRVLQNSTV CLLYQHQFVS GYSRDILMPL WTSYTIGRND SFSTEDFSNC LYQDLRIPLS 721 PVHKCSFYKN NAKLSYGLLS PPQLHKGSSQ VYSEALLTTN IVPMYQSFQV IWHYLHGTLL 781 QRYAEERNGL NWSGPVFDS DYDGRYDSLE TLKQNSKIIR NLEVLIPTHF FLVLTSCKNT 841 SQTPLQCENL DAMAFILPHK TDNSESCAHG KHESLWVEEL LKLHTARITD VEHITGLSFY 901 QERKEPISDI LKLKTHLPTF NQED

[0331] SEQ ID NO: 9 (Rabbit NPP 1- NCBI accession NP 001162404.1)

[0332] 1 MERDGCAGGG SRGGEGGRAP REGPAGNSRD PGRSHAAEAP GNPQAAASLL APMDVGEEPL 61 EKAARARTAK DPNTYKVLSL VLSVCVLTTI LGCIFGLKPS CAKEVKSCKG RCFERTFGNC 121 RCDAACVELG NCCLDYQETC IEPEHIWTCN KFRCGEKRLT RSLCACSDDC KDQGDCCINY 181 SSVCQGEKSW VEEPCESINE PQCPAGFETP PTLLFSLDGF RAEYLHTWGG LLPVISKLKK 241 CGTYTKNMRP VYPTKTFPNH YSIVTGLYPE SHGIIDNKMY DPKMNASFSL KSKEKFNPEW 301 YKGEPIWVTA KYQGLKSGTF FWPGSDVEIN GIFPDIYKMY NGSVPFEERI LAVLQWLQLP 361 KDERPHFYTL YLEEPDSSGH SYGPVSSEVI KALQRVDNMV GMLMDGLKEL NLHRCLNLIL 421 VSDHGMEQGS CKKYIYLNKY LGDVKNIKVI YGPAARLRPS DVPDKYYSFN YEGIARNLSC 481 REPNQHFKPY LKHFLPKRLH FAKSDRIEPL TFYLDPQWQL ALNPSERKYC GSGFHGSDNI 541 FSNMQALFVG YGPGFKHGIE VDTFENIEVY NLMCDLLNLT PAPNNGTHGS LNHLLKNPVY 601 TPKHPKEVHP LIQCPFTRNP RDNLGCSCNP SILPIEDFQT QFNLTVAEEK NIKHETLPYG 661 RPRVLQKKNT ICLLSQHQFM SGYSQDILMP LWTSYTVDRN DSFSTEDFSN CLYQDFRISL 721 SPVHKCSFYK NNTKVSYGFL SPPQLNKNSR GIYSEALLTT NIVPMYQSFQ VIWRYFHDTL 781 LRKYAEERNG VNWSGPVFD FDYDGRYDSL EILRQKRRVI RNQEILIPTH FFIVLTSCKD 841 ASQTPLHCEN LDTLAFILPH RTDNSESCLH GKHESSWVEE LLMLHRARIT DVEHITGLSF 901 YQQRKEPVSD ILKLKTHLPT FSQED

[0333] SEQ ID NO: 10 (Baboon NPP1-NCBI accession NP 001076211.2)

[0334] 1 MERDGCAGGG SQGGGKGGRG PREGLAGNGR DPSHGQASEA PGDPQAAASL LAPMDLGEEP 61 LEKAAGARPA KDPNTYKVLS LVLSVCVLTT ILGCIFGLKP SCAKEVKSCK GRCFERTFGN 121 CRCDVACVDL GNCCLDYQET CIEPERIWTC NKFRCGEKRL SRSLCACSDD CKERGDCCIN 181 YSAVCQGEKS WVEETCENIN EPQCPEGFEM PPTLLFSLDG FRAEYLHTWG GLLPVISKLK 241 KCGTYAKNMR PVYPTKTFPN HYSIVTGLYP ESHGIIDNKM YDPKMNASFS LKSKEKFNPE 301 WYKGEPIWLT AKYQGLRSGT FFWPGSDVKI NGIFPDIYKI YNGSVPFEER ILAILKWLRL 361 PKDERPHFYT LYLEEPDSSG HSYGPVSSEV IKALQRVDNM VGMLMDGLKE LNLHQCLNLI 421 LISDHGMEQG SCKKYIYLNK YLGDTKNIKV IYGPAARLRP SDVPEKYYSF NYENIARNLS 481 CREPNQHFKP YLKHFLPKRL HFAKSDRIEP LTFYLDPQWQ LALSPSERKY CGSGFHGSDN 541 VFSNMQALFV GYGPGFQHGI EVDSFENIEV YNLMCDLLNL TPAPNNGTHG SLNHLLKNPI 601 YTPKHPKEVQ PSVQCPLAGS PRDSLGCSCN PSILPIVDFQ TQFNLTTAEE KNINRASLPY 661 GRPRLLQKKS SVCLLYQHQF VSGYSHDVLM PLWTSYTVNR NDSFSTEDFS NCLYQDLRIS 721 FSPIHNCSFY KNNAKLSYGF LSPPQLSKDS SQIYSEALLT SNIVPMYQSF QVIWRYFHDT 781 LLQRYAEERN SINWSGPVF DSDYDGRYDS SEALKRNRRV IRNQEILIPT HFFIVITSCK 841 NTSQTPLQCD NLDPLAFILP HRSDNSESCV HEKRESSWIE ELLMMHRARI MDVEHITGLS 901 FYQERKEPVS DILKLKTHLP TVSQED

[0335] Treatment Frequency

[0336] An ENPP1 agent is administered at least once or twice bimonthly, at least once or twice monthly, three times monthly, at least once or twice weekly.

[0337] In some embodiments, the ENPP1 agent is administered once a week. In some embodiments, the ENPP1 agent is administered twice a week. In some embodiments, the ENPP1 agent is administered once every two weeks. In some embodiments, the ENPP1 agent is administered once every three weeks. In some embodiments, the ENPP1 agent is administered once a month. In some embodiments, the ENPP1 agent is administered once every five weeks. In some embodiments, the ENPP1 agent is administered once every six weeks. In some embodiments, the ENPP1 agent is administered once every seven weeks.

[0338] In some embodiments, the ENPP1 agent is administered once every two months. In some embodiments, the ENPP1 agent is administered once every nine weeks. In some embodiments, the ENPP1 agent is administered once every ten weeks. In some embodiments, the ENPP1 agent is administered once every eleven weeks. In some embodiments, the ENPP1 agent is administered once every three months.

[0339] Treatment Protocols

[0340] The following protocols may be used as guidance for treating Calciphylaxis or CKD-MBD at the recited dosages of the ENPP1 agent, as determined to be appropriate by the medical practitioner. Treatment protocols set forth herein rely on the use of abbreviated terms, whose full names are set forth in the glossary table C below.

[0341]

[0342]

[0343]

[0344] EXEMPLIFICATION

[0345] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain embodiments and embodiments of the present invention and are not intended to limit the invention.

[0346] Example 1. Generation of ENPP1 fusion proteins

[0347] One example of an ENPP1 agent is ENPP1 fusion protein, preferably the ENPP1 fusion protein is ENPPl-Fc. However, the exemplification of ENPPl-Fc can be applied to other ENPP1 fusion proteins as set forth herein. ENPPl-Fc is a recombinant fusion protein that contains the extracellular domains of human ENPP1 (soluble ENPP1) coupled with an Fc fragment of IgGl (rhENPPl-Fc). The recombinant extracellular domains of ENPPl-Fc contain its catalytic activity and are identical to the native ENPP1 enzyme. ENPPl-Fc is a recombinant human protein produced in CHO cells via a fed batch cell culture process that is free of animal-derived components. The molecular weight of the ENPPl-Fc dimer is approximately 290 kDa; ENPPl-Fc is highly glycosylated and has a pl of approximately 6.0. Like endogenous ENPP1, the primary substrate for ENPPl-Fc is ATP, which is cleaved to AMP and PPi.

[0348] In a specific embodiment, soluble ENPP1 protein was fused to a human Fc domain with a linker via a linker (comprising a leucine, isoleucine, and asparagine). Four ENPP1-Fc constructs are shown in Table 1 as SEQ ID NOs: 3-5, and 116-128 as purified from CHO cells.

[0349] Purification of ENPPl-Fc could be achieved by a series of column chromatography steps, including, for example, 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. The purification could be completed with viral filtration and buffer exchange. Following purification of the protein, the catalytic activity of the ENPPl-Fc protein could be evaluated using pNP-TMP as a chromogenic substrate.

[0350] Example 2: Measurement of Plasma Inorganic Pyrophosphate

[0351] Low plasma PPi levels are a characteristic of ENPP1 Deficiency such as CKD-MBD and are used as an indicator of treatment efficacy. ENPPl-Fc specifically cleaves ATP to generate AMP and PPi. The therapeutic goal of ENPP1 ERT is to normalize extracellular PPi levels and correct clinical abnormalities associated with ENPP1 Deficiency.

[0352] PPi is measured by obtaining patient plasma samples. Determined PPi data may be used to adjust dose levels. PPi levels may also serve as the primary PD marker for PK / PD analysis.

[0353] The concentration of Pi and PPi in mammals is 1-3 mM and 2-3 pM respectively. Example 3: Biomarkers Associated with Bone Health

[0354] In addition to low plasma PPi, patients with CKD-MBD are characterized biochemically by low serum phosphate, high urine phosphate, low renal TmP / GFR, normal calcium (Ca), low-normal urine Ca, normal 25-hydroxy Vitamin D (25 OH D), low-normal 1,25(OH)2D, high BAP, high intact FGF23, and normal PTH (IOF 2019).

[0355] Biomarkers that may be used as additional determinants of bone health of a treated patient are set forth in Table 2.

[0356] Table 2: Clinical Intermediates and Biomarkers

[0357]

[0358] Example 4: Efficacy of Treatment with ENPPl-Fc

[0359] Treatment efficacy may be assessed by measuring plasma PPi as well as measuring other plasma analytes, such as FGF23, Pi, TmP / GFR, serum alkaline phosphatase (ALP), bone-specific ALP (BALP), amylase, albumin, alkaline phosphatase, carboxy terminal cross-linked telopeptide of type I collagen (CTx), and procollagen type 1 N-terminal propeptide (P1NP). These analyte measurements may be used as a PD markers associated with ENPP1 Deficiency to determine the efficacy for ENPPl-Fc. Changes in these analytes may be described as changes from baseline and in a time-dependent manner over the course of treatment. Dose linearity of PK and PD parameters also may be assessed.

[0360] Changes from baseline in plasma PPi levels, FGF23 levels and Urinary phosphorus excretion per creatinine clearance may be analyzed using a t test of paired differences to test the null hypothesis that the change from baseline of PPi levels is equal to zero. Example 5: Additional Determinators of Efficacy

[0361] Although restoring a normal level of PPi is the primary indicator of efficacy of treatment using NEPPl-Fc, other physical measurements also may be used, if desired to assist in determining treatment efficacy. These include one or more of the following.

[0362] X-Rays for Skeletal Severity. Standard X-rays may be obtained to detect rachitic skeletal deformities. Obtain X-rays may be obtained, for example, on the wrists and knees.

[0363] DEXA Scan. DEXA scans may be used to evaluate changes in bone density.

[0364] Positron Emission Tomography - Computed Tomography. Baseline Na18F- PET / HRpQCT (or HR-CT) may be a full body scan done within 1 month of first dose of ENPP1-Fc to measure calcification of arteries and organs and skeletal abnormalities at baseline and for future interventional assessments. The Na18F-PET measures bone turnover as well as microcalcification of the arteries. High-resolution quantitative computed tomography (HRQCT) or HR-CT can determine bone microstructure at the non-dominant distal radius and tibia.

[0365] Standard bone geometric parameters are calculated.

[0366] Doppler Echocardiogram. A baseline echocardiogram may be obtained within 3 days prior to a first dose of ENPPl-Fc. Doppler echo may be used to measure heart function [LVEF, blood flow] calcification of heart and valves, and arterial stiffness.

[0367] Optical Coherence Tomography. Optical coherence tomography may be used to visualize neointimal proliferation.

[0368] Peripheral Arterial Tonometry. Peripheral arterial tonometry (PAT) may be used to assess digital pulse wave amplitude (PWA), which corresponds to digital volume variation.

[0369] Renal Ultrasound. Renal ultrasound may be used, for example, within 1 week of starting ENPPl-Fc, to measure renal calcification.

[0370] Bone Histomorphology and Bone Biopsy. Bone biopsy may be performed as a baseline measurement. Tetracycline loading for 10 days prior to bone biopsy is preferred. Example 6: Biochemical Assays

[0371] Processed plasma was analyzed for comprehensive clinical chemistry on an Abaxis VS2 clinical chemistry analyzer for all interim and terminal time points at Explora Biolabs, Inc., San Diego, CA. Plasma sclerostin levels in plasma was assayed using the Mouse / Rat SOST ELISA kit (Cat. #MSST00, R+D Biosystems). (See Figure 7C and 11)

[0372] ENPP1 activity was measured with a colorimetric assay adapted from assays previously described (Jansen, etal., Structure. 2012 Nov 7;20(ll):1948-59). Briefly, samples were diluted as needed in rat plasma (Cat. #RAT00PLLHP2102 BIOIVT, USA) and 10 gL of these diluted samples were incubated at 37°C with 90 gL of assay buffer (1 M Tris pH 8.0, 50 mM NaCl, 20 gM CaC12, 20 gM ZnC12) containing 2 mM pNP-TMP (Cat. #T4510, MilliporeSigma USA). The production of p-nitrophenol was analyzed kinetically by measuring the absorbance at 405 nm using a SPECTRAmax Plus 384 plate reader (Molecular Devices). The activity was calculated as the change in absorbance over time (mOD / min). (See Figure 12)

[0373] Plasma PPi was measured by a luminescent assay, adapted from assays previously described (Jansen, etal., Arterioscler Thromb Vase Biol. 2014 Sep; 34(9): 1985-9; Khan, et al., Dis Model Meeh. 2018 Oct 8;ll(10):dmm035691). Briefly, blood was collected at noted timepoints and filtered by centrifugation for 20 minutes at 14,000 x g at 4°C using an ultra-0.5 ml 50 kDa MWCO filter (Cat. # UFC505096, Ami con, USA) to generate filtered plasma for use in the assay. ATP sulfurylase is used to convert PPi to ATP in the presence of adenosine 5'-phosphosulfate (APS) and ATP levels were then measured by luminescent assay to obtain total luminescent signals. A standard curve was created by spiking known concentrations of PPi (Cat. #sc251047, Santa Cruz into water. Filtered plasma samples were mixed with assay buffer (40 mM Hepes pH 7.4, 8 mM CaC12, 2 mM MgSO4) containing 16 gM adenosine 5 '-phosphosulfate (Cat. #A5508, Millipore Sigma), 0.1 U / mL ATP sulfurylase (Cat. #M0394L, NEB). Reactions were incubated at 37°C for 40 minutes and followed by 10 mins at 90°C to deactivate ATP sulfurylase. Resulting ATP was then quantified by mixing the reaction mixture in a 1: 1 ratio with BactiterGlo detection reagent (Cat. G8230, Promega, USA). To account for endogenous ATP present in the plasma, a blank reaction was run for each sample with heat-inactivated ATP sulfurylase. The luminescent signals from this reaction were subtracted from the total luminescent signals to calculate the plasma PPi levels. (See Figure 7D)

[0374] Tissue calcium was measured in snap frozen samples. Calcium was extracted from tissues with acid and calcium quantified with a colorimetric assay using the O-Cresolphthalein Complexone method as described previously (Li, et al., Prog Biomater. 2013 Nov 14;2(1):13). Briefly, tissues were decalcified in IN HC1 at room temperature overnight. The acid extract was centrifuged to remove any tissue debris and diluted, as needed, in additional IN HC1. A 5 pL aliquot of sample was incubated with 195 pL of calcium liquicolor reagent (Cat. #0150-250, Stanbio Lab, USA) for 10 minutes at room temperature. The absorbance of the sample was read at 550 nm using a Molecular Devices SPECTRAmax Plus 384 plate reader. A standard curve for calculation of calcium concentration was generated in PBS using the calcium reference standard provided in the kit. The tissue calcium content (nmol Ca / mg) was calculated by converting calcium concentration to mass based on the acid volume and normalized to tissue weight. (See Figure 11 and 13)

[0375] Example 7: Imaging Protocols

[0376] Bone Imaging

[0377] All pCT scanning was performed and evaluated using Scanco pCT35 (Scanco Medical, AG, Switzerland). An X-ray energy intensity of 55kV with a current of 114 pA and 300 ms integration time was used. Quantitative analyses were carried out using ILP software (Scanco Medical, AG). Midshaft (cortical) femora were scanned with 12 pm voxels (1024 x 1024 pixels) and 160 slices were used for evaluation of all three groups. Distal (trabecular) femur area was scanned with 12 pm voxels (1024 x 1024 pixels) and 300 slices starting from 250 pm away from the growth plate were used for evaluation.

[0378] Histomorphometry

[0379] Necropsied femurs were embedded in methylmethacrylate (MMA) and cut in 11 pm sections and placed onto glass slides for Goldner’s Tri chrome staining to evaluate osteoid measurements. Osteoid measurements were evaluated with OsteoMeasure (OsteoMetrics) using the recommended settings. Two slides per sample were measured at a 50 pm depth difference by two different personal and each reading is counted individually.

[0380] Histology

[0381] Necropsied and fixed kidneys, ascending aortae, iliac arteries, and heart tissues were embedded in paraffin, cut in 5 pm sections and placed onto glass slides for von Kossa (Cat.#S 1890 / S 1895 / S248 Poly Scientific), or Alizarin Red (Cat VB-3008, Vitro Vivo Biotech) staining as per the manufacturer recommendations. Kidneys were sectioned sagittally to obtain cortex and medullary structures. Aortas and arteries were cross sectioned to evaluate the medial layers. Hearts were transverse-sectioned to obtain the ventricle walls. Three levels were sectioned from each block. Samples were imaged at 20x with a Axioscan 7 scanning microscope (Zeiss) and stitched together with 10% overlap.

[0382] Image Analysis

[0383] Segmentation and visualization of artery images was performed using the APEER arivis Cloud network Al-based automated image analysis tool (Zeiss).

[0384] Aorta and artery image analysis:

[0385] Aortas images were sorted into two classes using the APEER annotation tools. The first class being the aorta or iliac artery, with or without calcification, and the second class consisted into background. After regions were obtained that adequately represented the aorta or artery, the model was downloaded for image analysis in Zen 3.5 (Zeiss). Within the analysis software the aorta or artery automatic segmentation was set with the APEER model, and two subclasses of calcification (light brown and dark brown to black), were set with global thresholding. Analysis was completed on two to three different whole tissue levels per animal and the mean percent area calcified was calculated.

[0386] Kidney image analysis.

[0387] Three models were produced using the annotation tools of APEER, the kidney was sorted into kidney and background, the calcification was segmented into calcification and background, and the adenine crystals were segmented into adenine crystals and background. After all regions were adequately represented, the models were downloaded for image analysis in Zen 3.8 (Zeiss). The kidney, calcification, and adenine crystals automatic segmentation were set with their respective APEER models and were completed with one whole tissue level per animal and the mean percent area calcified was calculated with the analysis software.

[0388] Example 8: Treatment of CKD-MBD in CKD rat model

[0389] CD®-IGS (Sprague-Dawley) purpose-bred and specific pathogen free, naive male rats, at 13-14 weeks-old were used for this study. Animals were acclimated at least 3 days prior to the start of the study. All animal diets were supplied by Envigo-Harlan Teklad (Inotiv).

[0390] Animals were fed Teklad Global 18% Protein Rodent Diet (Cat. #2018) prior to the start of the study and changed over to custom diets on day 0 of the study. To induce kidney damage and vascular calcification, a custom diet made of adenine, low protein, and calcitriol injections was used, as shown in Figure 5.

[0391] Rats assigned to the healthy control group (“Healthy”) received base diet with a low protein content (low protein diet), 2.5% (TD.200666) for the duration of the study. Rats assigned to the CKD groups received low protein diet + 0.75% Adenine (TD.200667) for the first week of the study followed by a change to low protein diet + 0.25% Adenine

[0392] (TD.200668, Calcitriol (Cat. #D1530 Millipore-Sigma, USA) administered subcutaneously (SC) every-other-day at 100 ng / kg beginning on day 7 for the remainder of the study, OX-38 (Cat. #BE0308, BioXCell) administered intraperitoneally (IP) at an initial 5 mg / kg daily loading dose from days 1 to 5 and then 1 mg / kg three times a week for the remainder of the study and either PBS vehicle (“CKD, vehicle”) or 10 mg / kg of ENPP1 agent such as INZ-701 (“CKD, INZ-701”), INZ-701 is ENPPl-Fc fusion polypeptide, every other day starting at day 2 and continuing for the remainder of the study. On day 59, 6 animals per group were placed into metabolic cages for the last 22-24 hours of the study while the remaining animals were euthanized by isoflurane overexposure. The rats in metabolic cages were euthanized on day 60. For simplicity animals euthanized on day 59 and 60 are combined for analyses herein.

[0393] All rats were given acidified water (pH 2.5-3,0) ad libitum and bedded with hardwood chips (Cat #7115, Sani-Chip, Envigo++++). Rats were housed in standard disposable caging until transferred to individual metabolic rat cages (Cat. #650-0100, Nalgene) for the last 22-24 hours of the study. When supplementation or hydration was given, all animals, regardless of group received the same treatment. Hydrogel was offered on the cage bottom following interim bleeds and for the final 3 weeks of the study. Nutri-Cal (Vetoquinol) was offered in 1 cm3 doses / cage, starting at week 2 once / week, and increasing in frequency to thrice / week for the final 4 weeks of the study. Animal body weights and clinical observations were gathered prior to study initiation and at least twice per week for the duration of the study. These studies were approved by the Institutional Animal Care and Use Committee at Explora Biolabs (Charles River Laboratories).

[0394] Body Weights and Plasma Chemistry

[0395] SPRD-rats fed the 0.75% adenine diet (CKD groups) experienced a precipitous drop in weight relative to the healthy control rats (Figure 6A). By day 7 they exhibited a statistically significant 20% reduction in weight compared to the healthy controls. Following a switch to a 0.25% adenine diet after 1 week, the rat weights generally stabilized for the duration of the experiment, with body weights remaining about 30% lower than those of the healthy group at the terminal timepoint.

[0396] INZ-701 treatment (CKD, INZ-701) had no impact on body weight as compared to vehicle treated animals (CKD, vehicle). Upon initiation of the 0.75% adenine diet, the BUN levels in rats rose roughly 6-folds. After the switch to 0.25% adenine diet the BUN levels dropped close to baseline level and then steadily rose throughout the duration of the study. At the terminal timepoint, BUN levels were roughly 5 times higher in the rats fed adenine diet compared to the rats on baseline diet. Animals dosed with INZ-701 showed slightly lower BUN levels, averaging roughly 15.6 mg / dL compared to the vehicle-dosed rats which averaged 20 mg / dL (Figure 6B).

[0397] After a week on 0.75% adenine diet, the plasma creatinine levels had doubled compared to the healthy control group. Plasma creatinine levels continued to rise over the course of the experiment ending at approximately 2.4 mg / dL in the CKD groups compared to 0.4 mg / dL in the healthy group (Figure 6C). Plasma phosphate levels in the adenine diet groups began to diverge from the healthy group starting at day 21 and slowly elevated throughout the study (Figure 6D). At the terminal timepoint, plasma phosphate in the CKD vehicle group averaged 14 mg / dL, while in the CKD INZ-701 dosed group averaged 11.4 mg / dL compared to 7 mg / dL in the healthy control group. Thus, plasma phosphate levels are slightly improved by INZ-701 dosing though the effect is not statistically significant. Plasma calcium levels were elevated in the CKD groups averaging roughly 11.8 mg / dL with no impact of INZ-701 dosing, compared to 10.6 mg / dL in the healthy group (Figure 6E).

[0398] In order to ensure that INZ-701 maintained drug exposure throughout the study, plasma ENPP1 activity was assessed at the terminal timepoint. All but three of the rats in the CKD-INZ-701 group displayed increased drug exposure compared to healthy or CDK, vehicle groups animals (Figure 7A). To determine when these three rats lost drug exposure, plasma ENPP1 activity was assessed (Figure 12) at weekly interim timepoints. Of the three rats that did not show exposure at the terminal timepoint, two lost exposure between days 36 and 42 while one lost exposure between days 49 and the terminal timepoint. As all of these animals had drug exposure for over half of the study the decision was made to retain the animals in the analysis.

[0399] FGF23 levels were significantly elevated in the CKD groups compared to the healthy control group. CKD-vehicle group rats showed average intact FGF23 plasma levels of around 81,000 pg / mL compared to less than 500 pg / mL in the healthy group. CKD-INZ-701 group lowered FGF23 levels to an average of 49,000 pg / mL, though the effect was not statistically significant due to the heterogeneity of the values (Figure 7B).

[0400] Sclerostin levels were assessed in the terminal plasma to determine if INZ-701 dosing impacted these values. The rats in the healthy group exhibited average plasma SOST levels of 207.4 pg / mL compared to 1309 pg / mL in the CKD-vehicle group. Dosing with CKD-INZ-701 results in a slight reduction of SOST levels, down to an average of 970.1 pg / mL, though the effect was not significant.

[0401] Plasma pyrophosphate was assessed at multiple timepoints throughout the study. Before the start of the study all rats showed equivalent PPi values and up to day 21 the PPi levels in all groups were statistically similar. However, at day 36 PPi levels were elevated in CKD groups compared to healthy. The PPi levels in the healthy group average 413 nM compared to 1817.5 and 2341 nM in the CKD vehicle and INZ-701 dosed groups, respectively. PPi levels remain elevated in both CKD groups at day 49 averaging 957.5 and 1336.7 nM in the CKD vehicle- and INZ-701-dosed groups respectively, compared to 292 nM in the healthy control group. By the terminal timepoint PPi levels were equivalent, averaging 855, 1028.8, and 1407 nM in the healthy, CKD-vehicle, and CKD-INZ-701 groups, respectively. It should also be noted that the CKD groups exhibited much higher heterogeneity in PPi levels compared to the healthy control animals over virtually all timepoints except baseline. Interestingly, the divergence of PPi levels in the CKD groups begins at the same point at which phosphate levels begin to increase in these animals suggesting a global disruption of these pathways.

[0402] Calcification

[0403] Iliac arteries from INZ-701 treated CKD rats show an average of 5.2 nmol of calcium / mg of tissue, less than half of the vehicle treated group which shows 13.3 nmol of calcium / mg of tissue. Histological examination of iliac arteries validated the trends observed with calcium quantification. In CKD vehicle rats, von Kossa staining revealed extensive circumferential calcification along the elastic fibers (figure 8A). Upon treatment with INZ-701, the level of calcium staining was noticeably lower and image analysis shows a reduction in both light calcification from an average calcified area of 5.5% in CKD animals to 1.5% in CKD animals treated with INZ-701, and dark calcification from an average calcified area of 4.4% in CKD animals to 0.5% in CKD animals treated with INZ-701 (figure 8B).

[0404] Calcium content in the descending thoracic aortas of vehicle treated rats averaged roughly 35 nmol / mg of tissue compared to only 4.6 nmol of calcium / mg of tissue. Much the same trend was observed in the abdominal aorta where mean calcium content was 50.9 and 19.1 nmol of calcium / mg of tissue in vehicle treated and INZ-701 treated rats respectively. Like the observations of the iliac arteries the calcium in the ascending aortas in vehicle treated rats appeared through multiple sections and was often circumferential compared to the more punctate patterns found in INZ-701 treated rats (figure 8C). Image analysis shows the ascending aortas from INZ-701 treated CKD rats have an average area of less than 1% that is heavily or lightly calcified, noted by the dark and light calcification staining, where the CKD rats show an average area of 3.2% and 2% calcified for heavy and light calcium deposition respectively (figure 8C).

[0405] In order to assess kidney calcification, the right kidney was removed from at the terminal timepoint and snap frozen for quantitative calcium analysis. Kidneys from CKD-vehicle treated rats showed an average of 8.6 nmol of calcium / mg of tissue compared to only 3.2 nmol of calcium / mg of tissue in INZ-701 -treated rats. Kidneys from CKD rats treated with either vehicle or INZ-701 displayed extensive adenine crystallization and distorted tubules. Image analysis of sections stained with Alizarin red revealed a trend toward reduced calcification levels with INZ-701 treatment, (figure 9A-B).

[0406] Histomorphometry

[0407] CKD model rats showed mineralization imbalances as measured by histomorphometry. The average osteoid area in CKD vehicle treated rats was increased to 0.29mm2compared to 0.001 mm2in the healthy group. The osteoid area in INZ-701 treated rats was statistically indistinguishable from the healthy group, averaging 0.05 mm2. Osteoid thickness went from an average of 5.84mm in the healthy rats to 40 mm in CKD vehicle treated rats. INZ-701 restored the osteoid thickness to 16.8 mm. Osteoid volume per bone volume went from 0.12% in the healthy control rats to 33.8% in the CKD vehicle group. INZ-701 again restored the OV / BV to an average of 7.4%. (figure 10A-B)

[0408] Bone

[0409] Bone parameters were assessed via pCT from six randomly selected animals from each group. Only one parameter showed a significant difference between groups, trabecular spacing. CKD vehicle treated animals had an average spacing of 0.486 mm while healthy group animals showed an average spacing of 0.779 mm. INZ-701 treated animals were not significantly different from either group with an average spacing of 0.527 mm. All other parameters assessed did not vary significantly between groups indicating that there were minimal structural bone abnormalities in this experiment, (figure 14 A-G) Conclusions

[0410] The CKD rat model described herein serves as an excellent proxy for CKD-MBD, recapitulating key aspects of the disorder including renal damage, hyperphosphatemia, medial vascular calcification, osteomalacia, and increased circulating levels of iFGF23 and sclerostin. Dosing with INZ-701 prevents calcification of the kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, and spleen. In addition to the quantitative decrease in calcification there is a histological change in the appearance of the calcification. CKD animals dosed with vehicle often show circumferential arterial calcification that extends through multiple histological sections and extensive von Kossa staining. Animals dosed with INZ-701 show punctate patterns of calcification that do not extend through multiple sections. Thus, dosing with INZ-701 results in both quantitative and qualitative improvement in ectopic calcification.

[0411] CKD rats dosed with INZ-701 show reduced average iFGF23 levels compared to vehicle treated CKD rats by the end of the study. FGF23 levels are elevated in CKD patients and rapid elevation is strongly associated with increased mortality Recent studies have shown that patients with slowly rising FGF23 levels were at a roughly 4.5-fold higher risk of death compared to patients with stable FGF23 levels and patients with rapidly rising FGF23 levels had about a 15-fold increase in mortality showing the critical predictive and biological role of FGF23 in CKD pathology (Isakova, etal., J Am Soc Nephrol. 2018 Feb;29(2):579-590). FGF23 is a central regulator of mineral metabolism with elevations resulting in secondary hypoparathyroidism and severe vitamin D deficiency which, coupled with hyperphosphatemia, result in the vascular mineralization and bone dysregulation characteristic of CKD-MBD. The ability of INZ-701 to reduce FGF23 suggests that INZ-701 may prove beneficial to patients with this disease. Consistent with the ability of INZ-701 to reduce FGF23 levels, treatment was able to significantly reduce plasma phosphate levels by the end of the study. Hyperphosphatemia is a key driving factor for vascular calcification so reduction of phosphate levels may suggest a partial mechanism by which INZ-701 prevents calcification in this model. This model induces osteomalacia as seen by the significant increase in osteoid area, thickness, and OV / BV in the vehicle treated CKD rats compared to healthy rats. INZ-701 normalizes the osteoid area and OV / BV to levels that are statistically indistinguishable from healthy rats. Osteoid thickness in INZ-701 treated rats is significantly lower than the vehicle treated CKD rats. This effect is not simply due to calcitriol dosing as both CKD vehicle and INZ-701 dosed rats received equivalent doses of calcitriol.

[0412] ENPP1 and ABCC6 deficient mouse models show low circulating PPi levels which are corrected with INZ-701 treatment leading to prevention of ectopic calcification and bone phenotypes. Unlike human CKD patients, these CKD model rats do not show low circulating PPi levels (O’Neill, et al., Nephrol Dial Transplant. 2010 Jan;25(1):187-91; Lomashvili, et al., J Am Soc Nephrol. 2005 Aug; 16(8): 2495-500). While dialysis does reduce circulating PPi levels by 30%, the PPi levels were no different between patients who were and were not receiving dialysis showing that low PPi levels are not simply an artifact of dialysis (0 Neill, et al., Nephrol Dial Transplant. 2010 Jan;25(1): 187-91) ■

[0413] Thus, in this CKD rat model vascular calcification develops despite normal levels of circulating PPi indicating that INZ-701 does not function by simply correcting circulating PPi concentrations. It is believed that INZ-701 works in one of three ways either by increasing PPi concentration in the local environment, increasing adenosine concentrations, or by manipulation of the WNT pathway. Circulating INZ-701 can utilize local ATP substrate not normally accessible either physically or temporally to endogenous ENPP1 enzyme which is membrane bound. Access to additional substrate could increase local PPi levels at the sites of vascular calcification preventing progression. ENPP1 hydrolyzes ATP to generate AMP in addition to PPi. AMP is further degraded to adenosine by the action of CD73. Adenosine generation provides another possible mechanism by which INZ-701 could function in this model. Adenosine plays a key role in osteoblast development with higher levels of adenosine resulting in commitment of mesenchymal stem cells to osteoblasts (Takedachi, etal., J Cell Physiol. 2012 Jun;227(6):2622-31). Perhaps additional adenosine generated by INZ-701 could explain the prevention of the bone phenotype shown here. The fact that INZ-701 was able to prevent both vascular calcification and osteomalacia suggests this second, PPi independent mechanism, is important as a previous report showed Ppi supplementation prevented vascular calcification but not osteomalacia (Opdebeeck 2021). ENPP1 deficient mice expressing a catalytically inactive version of ENPP1 displayed low Ppi levels and developed vascular calcification but showed largely normal bone parameters compared to vehicle dosed mice.

[0414] CKD patients show a broad disruption in the WNT pathway with a variety of studies noting an increase in either circulating levels or expression of WNT inhibitors. Preclinical models of CKD have noted increases in either expression or circulating concentration of Sfrpl, Dkkl, and Sost (Mace, et al., J Bone Miner Res. 2021 Mar;36(3):510-522; Mace, etal., JBMR Plus. 2022 Mar l;6(4):el0610; Carrillo-Lopez, etal., Kidney Int. 2016 Jul;90(l):77-89; Fang, etal., J Am Soc Nephrol. 2014 Aug;25(8): 1760-73). The increase in WNT inhibitors has also been extensively documented in CKD patients. Elevated SOST levels have been noted in numerous studies (Pelletier, etal., Clin J Am Soc Nephrol. 2013 May;8(5):819-23; Kanbay, et al., J Clin Endocrinol Metab. 2014 Oct;99(10): E1854-61; Sabbagh, etal., J Bone Miner Res. 2012 Aug;27(8): 1757-72; Malluche, etal., Clin J Am Soc Nephrol. 2014 Jul;9(7): 1254-62)'

[0415] Cells from ENPP1 deficient mice have been shown to have elevated expression of the WNT inhibitors Dkk3 and Sfrpl. Another possibility is that CKD-MBD subjects may have reduced expression of ENPP1 and such loss of ENPP1 can lead to an increase of WNT inhibitors then addition of excess of soluble ENPP1 in the form of INZ-701 may be able to reduce the levels of WNT inhibitors yielding a second possible mechanism by which INZ-701 could function in this model. The results show an increase in SOST levels in the CKD model rats, the levels of SOST trend downward with treatment of INZ-701. The correlation between upregulation of WNT inhibitors in both ENPP1 deficient mice and in CKD suggests that the ability of ENPP1 to regulate these WNT inhibitors provides a second hypothesis to explain the results described here, particularly the prevention of osteomalacia.

[0416] The results thus support that INZ-701 administration can both prevent vascular calcification and osteomalacia in a rat CKD model. These positive effects occur in the absence of low PPi levels suggesting that INZ-701 is working by generating PPi at a local level and / or by restoring WNT pathways.

[0417] A high-adenine fed animal model of CKD has been developed to evaluate INZ-701 to prevent vascular calcifications. Administration of 10 mg / kg of INZ-701 in uremic rats showed reduced total calcium content in the large arteries (between 61 and 86%) and organs (between 15 and 63%) as compared to the vehicle group. Histological examination of the arteries showed that uremic rats dosed with vehicle showed extensive, often circumferential, medial calcification that extended over multiple sections. In contrast, uremic rats dosed with INZ-701 had a reduction in calcification that was evident by von Kossa staining. As calciphylaxis represents a continuum of vascular calcification complications with pain and skin changes, it is expected that treatment with INZ-701 for human subject would be beneficial in treating or ameliorating calciphylaxis.

[0418] Example 9: Treatment Protocol for CKD-MBD patients

[0419] Human patient or subject suffering from CKD-MBD is treated by administering a subcutaneous injection containing approximately 0.1 mg / kg to 10 mg / kg of ENPP1 agent such as ENPPl-Fc, once or twice a week. Successful treatment of CKD-MBD is observed by monitoring one or more aforesaid parameters through periodic blood and urine tests as discussed for mouse models. The CKD-MBD patient does not have ENPP1 deficiency or ABCC6 deficiency and also does not have a defective ENPP1 protein.

[0420] Instead of histological analysis which requires staining of kidney slices or arterial tissues which is not feasible to perform in living patients, a medical professional can use noninvasive visualization techniques commonly known in art such as CT scan, ultrasound, or intravenous pyelography to visualize the presence of calcifications and the reduction of calcifications in response to delivery of ENPPl-Fc in patients suffering from CKD-MBD.

[0421] Intravenous pyelography is an X-ray exam that uses a contrast medium, which functions as a dye, to help visualize the urinary tract and detect the presence of renal calcifications. Computed tomography is a noninvasive imaging technique that uses X-ray technology to depict internal structures of the body such as the urinary tract. Renal calcifications are visible on CT scans. CT scans collect X- ray images from different angles around the body to generate detailed cross-sectional images as well as three-dimensional images of the body's internal structures and organs. CT scan can also be used in arteries to detect the presence and subsequent reduction of calcification following treatment. A computer analyzes the radiation transmitted through the body to reconstruct the images of the internal structures and organs.

[0422] A medical doctor having skills in visualizing soft tissue calcification, cardiac calcification, kidney calcification, and myocardial infarction can undertake the treatment of a subject afflicted with CKD-MBD by administering a therapeutically effective amount of ENPP1 agent such as ENPPl-Fc. The exact dosage can be determined by a medical professional.

[0423] The physician or a medical professional thus has the option to control the dosage based on the rate and extent of improvement of symptoms. Successful treatment is observed by a medical professional of skill in the art by observing one or more positive symptoms such as improved kidney function, and improved urine creatine levels (normal creatine levels in urine for men are 40 — 278 mg / dL and 29 — 226 mg / dL for women), and improved urine-urea levels (normal urea levels in the urine for adults are 26 — 43 g 24 h), normal serum-creatine levels (normal serum creatinine range is 0.6-1.1 mg / dL in women and 0.7—1.3 mg / dL in men), normal vitamin D levels (20ng / ml to 50 ng / rnL is considered adequate for healthy people). A level less than 12 ng / mL indicates vitamin D deficiency), normal blood urea nitrogen levels (BUN level for healthy adults is 7-20 mg / dL), weight gain, increase in serum PPi levels (at least about 4-5 pm), reduction in calcification (25%, or 50%, or 70%, or 90% or 100% reduction of calcification in arteries and / or kidneys) of arterial tissues and or reduction of calcification in kidney tubules visualized by noninvasive techniques such as CT or ultrasound scans.

[0424] Like the endogenous ENPP1 enzyme, the ENPP1 agent cleaves ATP to generate AMP and PPi, thereby increasing plasma PPi levels and into AMP which CD73 coverts rapidly to adenosine. Such ENPPl-Fc administration is expected to correct the mineral imbalance, regulate PPi levels and allow for improved health and mitigation of clinical complications associated with CKD-MBD.

[0425] Example 10: Treatment of subjects having CKD and ESRD who are undergoing hemodialysis.

[0426] In a large study conducted in the United States of over 1,000 subjects with calciphylaxis, mortality rates were 27% at 6 months and 45% at 12 months after diagnosis. Approximately 50% of patients are bedridden or wheelchair-bound, and more than 70% require hospitalization for severe ulcers. Morbidity is related to severe pain, non-healing wounds, recurrent hospitalizations, and adverse effects of current supportive care Nigwekar et al., A Nationally Representative Study of Calcific Uremic Arteriolopathy Risk Factors. J Am Soc Nephrol. 2016;27(ll):3421-3429).

[0427] The disorder typically affects patients with ESKD requiring HD and may also occur in patients with preserved kidney function and different stages of CKD. The CKD population has a high prevalence of abnormal extraosseous tissue and vascular calcifications (Itani et al., Calciphylaxis on bone scan: correlation between molecular and cross-sectional findings. Radiol Case Rep. 2016; 12(1): 175-1782016, Nigwekar etal., Calciphylaxis. N Eng J Med 2018; 378: 1704-1714).

[0428] Histopathology of the arterial lumens show micro vessels undergo progressive narrowing first by calcification within the medial layer of vessel walls (also known as medial calcification) and proliferation of endothelial cells and fibrosis underneath the intima (also known as subintimal fibroplasia). When thrombosis later develops in the vessel lumen, ischemic injuries develop. The development of microvascular calcifications in patients with calciphylaxis (and in patients with other vascular calcifications) is likely to be an active, cell-mediated process that depends on the balance between the promoters and inhibitors of calcification.

[0429] It is postulated that calciphylaxis occurs due to the dysregulation of molecular calcification inhibitors in the vessel wall. Serum samples from patients with calciphylaxis show impaired inhibition of calcium phosphate precipitation. Additionally, risk factors associated with onset of calciphylaxis support the dysregulation of calcium phosphate interaction, including higher serum phosphorous, higher intact parathyroid hormone (iPTH), and use of active vitamin D, cinacalcet, or warfarin. This dysregulation of calcium phosphate interaction leads to a predisposition to ectopic or vascular calcification.

[0430] The balance between PPi and Pi maintains inhibition of ectopic vascular and tissue mineralization and proper bone remodeling.

[0431] An Open-label Exploratory Study is performed to evaluate the Safety, Pharmacokinetics, and Pharmacodynamics ofENPPl polypeptide such as ENPPl-Fc (INZ-701) in subjects with Chronic Kidney disease (CKD) or End-Stage Kidney Disease (ESRD) Undergoing Hemodialysis (HD)

[0432] Approximately 15 subjects with >18 to <70 years of age with end-stage kidney disease (ESKD) undergoing hemodialysis (HD). This is a Phase 1 study to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of multiple doses of INZ-701 in study participants with HD-dependent ESKD.

[0433] An exploratory observational study in study participants with calciphylaxis and different stages of CKD revealed lower levels of PPi as compared with age, sex, race, and CKD stage-matched controls. Furthermore, in this pilot study, lower PPi levels were associated with a significantly increased 6-month mortality rate among study participants with calciphylaxis. In addition, a greater number of skin lesions was associated with lower PPi levels. Based on these initial observations it is hypothesized that increasing PPi in this population may have a positive effect on tissue mineralization and vascular calcifications and subsequently improve clinical outcomes in calciphylaxis.

[0434] INZ-701 is a recombinant human ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) fused to the Fc fragment of IgGl (rhENPPl-Fc), is a therapeutic protein currently in clinical development for the treatment ofENPPl and ATP -binding cassette sub-family C member 6 (ABCC6) Deficiencies. Like endogenous ENPP1, INZ-701 cleaves extracellular adenosine triphosphate (ATP) to generate PPi and adenosine monophosphate (AMP), which is then metabolized to adenosine, thereby increasing plasma PPi levels and adenosine. It is hypothesized that augmenting endogenous human enzyme with INZ-701 in patients with calciphylaxis will increase plasma PPi levels and, in turn, address the associated clinical manifestations, including skin lesions and pain.

[0435] Because CKD patients, and particularly calciphylaxis patients, experience very low

[0436] levels of PPi, the administration of INZ-701 is hypothesized to increase PPi levels within the normal range, thus potentially inhibiting abnormal calcifications. The purpose of this study is to determine if multiple doses of INZ-701, given once per week over 4 weeks are safe and increase PPi levels in HD-dependent ESKD or CKD study participants who have low PPi levels. In addition, the effect of hemodialysis (HD) on the pharmacokinetics (PK) and pharmacodynamics (PD) of INZ-701 and PPi levels are evaluated. Genetic testing is also performed to determine how variants in the participant’s ENPP1, ecto-5 '-nucleotidase

[0437] (NT5E), vitamin D receptor (VDR), and fibroblast growth factor-23 (FGF23) genetic make-up may impact PPi levels.

[0438] Objective & Endpoints

[0439]

[0440]

[0441] AE=adverse event; AESI=adverse event of special interest; BALP=bone specific alkaline phosphatase; CKD-MBD=chronic kidney disease-mineral bone disease; iFGF23=intact fibroblast growth factor-23; iPTH=intact parathyroid hormone;

[0442] PK=pharmacokinetic(s); PPi=inorganic pyrophosphate; SAE=serious adverse event; TEAE=treatment-emergent adverse event.

[0443] Study Design:

[0444] SEAPORT 1 (INZ701-401) is a Phase 1, open-label study to evaluate the safety, PK, and PD of multiple doses of INZ-701, given once per week over 4 weeks, in study participants with ESKD undergoing HD. Study participants aged >18 to <70 years with confirmed low levels of PPi may participate if all other eligibility criteria are met.

[0445] The study consists of an up to 30-day Screening Period, a 26-day Treatment and Assessment Period comprised of several in-clinic visits, an End of Study (EOS) Visit 30 days after the last dose of INZ-701, if necessary, a Safety Visit 60 days after the last dose of INZ-701, and a Follow-Up Period lasting up to 365 days after the last dose. The details of time period of administration are in study scheme shown in Figure 17. Blood collection period and follow up period are shown in Figures 19 and 20 respectively.

[0446] Screening Period

[0447] The Screening Period lasts up to 30 days, during which PPi is collected to determine eligibility. Screening procedures are conducted in a manner and order to minimize study participant burden. Study participants are enrolled into the study if they meet all eligibility criteria. If the PPi does not meet the eligibility criterion it may be retested within the 30-day screening window.

[0448] Treatment and Assessment Period

[0449] The Treatment and Assessment Period lasts 26 days; study participants have study visits coinciding with hemodialysis (HD) days: Baseline Visit on Day 1, INZ-701 administration on Days 3, 10, 17 and 24; and Safety Visits on Days 5 and 26. For all study participants, INZ-701 is administered as a subcutaneous (SC) dose on Days 3, 10, 17 and 24 following the pre-HD blood collection and prior to the start of HD. Hemodialysis characteristics are recorded at each visit. Study participants enrolled in this study are required to comply with the diet prescribed by their treating physician and / or the Investigator and complete a food diary beginning on Day 1 (48 hours before INZ-701 administration) and ending on Day 26. Blood samples are collected per the schedule of events (SOE). (See Fig. 18-19) Fasting is required as specified in the SOE.

[0450] End of Study Visit

[0451] All study participants complete an EOS Visit 30 days after the last dose of INZ-701 and if necessary, a Safety Visit 60 days after their last dose of INZ-701. Blood samples are collected as per schedule noted in Figure 19.

[0452] Follow-Up Period

[0453] The Follow-Up Period begins after the EOS Visit or Safety Visit. During the Follow-Up Period, safety, PPi, ENPP1 activity, anti-drug antibodies (ADA) assessments, and genetic testing is conducted. Participants are followed for >1 year if they have increasing ADA titers or have serious adverse events (SAEs) related to AD As as determined by the Sponsor. See Figure 20.

[0454] Safety Monitoring

[0455] Study participants are followed for safety assessments to the completion of hemodialysis (HD) or for a minimum of 3 hours following INZ-701 administration and includes monitoring and managing signs and symptoms of hypersensitivity reactions. Safety, including immunogenicity, are evaluated throughout the study. The Sponsor reviews individual and cumulative clinical safety data on an ongoing basis, including but not limited to SAEs suspected unexpected serious adverse reactions (SUSARs), and adverse events of special interest (AESIs).

[0456] Dose / Dose Modification

[0457] The dose of INZ-701 (ENPPl-Fc) is 1.8 mg / kg once weekly. INZ-701 is administered as a subcutaneous injection once weekly for 4 weeks on Days 3, 10, 17, and 24. The study participant’s post-HD body weight on Day 1 is used to calculate the dose to be administered. Estimated Duration of Study Participant Involvement

[0458] Study participant involvement is up to 419 days, consisting of a Screening Period of up to 30 days, a 26-day Treatment and Assessment Period, an EOS Visit 30 days after the last dose of INZ-701, and if necessary, a Safety Visit 60 days after the last dose of INZ-701 and a Follow-Up Period lasting up to 365 days after last dose. Participants (subjects) are followed for >1 year if they have increasing ADA titers or have SAEs related to AD As as determined by the Sponsor.

[0459] Eligibility Criteria

[0460] Inclusion

[0461] Individuals meeting the following inclusion criteria may participate:

[0462] 1. Study participants must provide written or electronic consent after the nature of the study has been explained, and prior to any research-related procedures, per International Council for Harmonisation (ICH) Good Clinical Practice (GCP).

[0463] 2. Have ESKD or CKD and are receiving HD treatment.

[0464] 3. Are compliant with receiving 3 treatments of HD per week with a functioning arteriovenous (AV) fistula, AV graft, or central venous catheter.

[0465] 4. PPi level <700 nmol / L at Screening.

[0466] 5. Must be willing and able to comply with the diet prescribed by their treating physician and / or the Investigator.

[0467] 6. Male or female aged >18 years to <70 years.

[0468] 7. Women of child-bearing potential (WOCBP) as defined in Clinical Trials Coordination Group (CTCG 2024) and provided in Appendix B, must have a negative serum pregnancy test at Screening and within 3 days of INZ-701 administration.

[0469] 8. WOCBP and partners of fertile males who are WOCBP must be using or agree to use one highly effective form of contraception (per CTCG 2024) and a barrier method from at least 1 month before the first dose of INZ-701 to 30 days after the last dose (greater than 5 half-lives of INZ-701). WOCBP and partners of fertile males who are WOCBP must also agree to not donate ova from the time of the first INZ-701 dose to 30 days after the last dose. 9. Males who are sexually active must agree to use condoms from the time of the first INZ-701 dose to 30 days after the last dose. Males must also agree to not donate sperm from the time of the first INZ-701 dose to 30 days after the last dose.

[0470] 10. In the opinion of the Investigator, study participants are able and willing to complete all study procedures per protocol.

[0471] Exclusion

[0472] Individuals meeting any of the following exclusion criteria may not participate:

[0473] 1. Study participants receiving other types of dialysis than HD (e.g., peritoneal dialysis, hemodi afiltration).

[0474] 2. Study participants who are hospitalized.

[0475] 3. In the opinion of the Investigator, presence of any clinically significant disease or laboratory abnormality that may impact study participation and / or confound interpretation of the study results.

[0476] 4. Malignancy within the last year, except non-melanoma skin cancers or cervical carcinoma in situ.

[0477] 5. Advanced liver disease manifesting as liver cirrhosis.

[0478] 6. Myocardial infarction, stroke, or congestive heart failure requiring hospitalization within the last 6 months.

[0479] 7. Known intolerance to INZ-701 or any of its excipients.

[0480] 8. Weight >125 kg.

[0481] 9. Concurrent participation in another interventional clinical study and / or receipt of any other investigational new drug within 5 half-lives of the last dose of the other investigational product or from 4 weeks prior to the first dose of INZ-701, whichever is longer, or use of an investigational device, through completion of participation in the study.

[0482] Study Procedures and Assessments

[0483] Assessments are performed as indicated in the SOE. (See Figure 18). Safety Assessments:

[0484] Assessments to determine the safety of INZ-701 include evaluation of adverse events (AEs), ECG, vital signs and physical examination, clinical safety laboratory evaluations, and immunogenicity data.

[0485] Pharmacodynamic Assessments:

[0486] PD assessments include evaluation of PPi levels.

[0487] Pharmacokinetic Assessments:

[0488] Blood samples (see Figure 19) are obtained from all study parti cipants(subjects) for measurement of INZ-701 (ENPPl-Fc) concentration in serum and subsequent determination of the following PK parameters:

[0489] • Time to maximum serum concentration (Tmax)

[0490] • Maximum serum concentration (C max)

[0491] • Area under the concentration-time curve over the dosing interval (AUCtau)

[0492] • Clearance after extravascular administration of drug (CL / F)

[0493] • ENPP 1 -F c activity.

[0494] CKD Metabolic Bone Disease Function Analysis

[0495] Assessments for CKD-MBD function include 25-hydroxy vitamin D, intact PTH, calcium, phosphate, intact fibroblast growth factor 23 (iFGF23), and bone specific alkaline phosphatase (BALP) analysis.

[0496] Physical Assessments

[0497] A physical examination includes an assessment of systemic organ involvement and inspection of:

[0498] • General appearance

[0499] • Skin

[0500] • Cardiovascular system

[0501] • Respiratory system

[0502] • HD access (AV fistula, AV graft, central venous catheter) Height and Weight are measured

[0503] Vital Signs

[0504] Vital signs assessed in this study include systolic blood pressure and diastolic blood pressure (mm Hg), heart rate (beats per minute), respiration rate (breaths per minute), and temperature (°C). Vital signs are measured after the study participant has been at rest >5 minutes.

[0505] • Blood pressure (systolic and diastolic) measurements are taken throughout the study while study participants are seated.

[0506] • Respiratory rate is measured over at least 15 seconds and adjusted per minute.

[0507] • Body temperature is measured using either oral or tympanic methods, but the method should be consistent throughout the study for a given study participant.

[0508] Immunogenicity

[0509] Anti-drug antibodies to INZ-701 are assessed by a central laboratory using a validated method. Samples are stored to enable future testing including determinations of whether the antibodies are directed against the Fc or ENPP1 enzyme portions of INZ-701. Samples may be used to assess and further establish assays for specificity confirmation (ie, titer) and neutralizing antibodies.

[0510] Plasma Inorganic Pyrophosphate

[0511] Change in plasma PPi concentration (ie, pretreatment versus on-treatment) is the primary endpoint and pharmacodynamic marker of INZ-701 activity in this study. PPi is measured throughout the study. See Figures 18-20.

[0512] Electrocardiogram

[0513] Standard 12-lead ECGs are collected at time points indicated in the SOE (Figure 18). Additional ECGs are performed per site specific safety monitoring procedures and / or if clinically indicated. Hemodialysis Characteristics

[0514] The following information related to HD is collected and recorded for each HD treatment on Days 1, 3, 5, 10, 17, 24, and 26:

[0515] • HD time: start and stop times

[0516] • Blood flow (QB)

[0517] • Dialysate flow (QD)

[0518] • Dialysis membrane and size

[0519] • Ca bath

[0520] • Ultrafiltration: calculated using pre- and post-HD weight

[0521] Hematology

[0522] Routine blood chemistry panels including liver function tests, a hematology panel including complete blood count with differential is conducted to assess eligibility and as a measure of safety throughout the study.

[0523] Genetic Testing

[0524] Approximately 10 mL blood sample for DNA isolation is collected during the Follow-Up Period (Figure 20) from parti cipants(subjects) who have consented to participate in the genetic analysis component of the study. Using the subject DNA, mutational analysis for ENPP1, NT5E, VDR and FGF23 genes is conducted. The effect of participants’ ENPP1, NT5E, VDR, and FGF23 genetic variants on the PPi is then determined.

[0525] plasma concentrations are measured.

[0526] Results

[0527] Evidence of low PPi in calciphylaxis is supported by an exploratory study designed to measure PPi in patients with calciphylaxis and to examine whether PPi levels are predictive of clinical outcomes. PPi was measured in patients with calciphylaxis at different stages of CKD and matched for age, sex, race, and CKD stage in patients without calciphylaxis. Results from this study revealed that PPi was significantly decreased in patients with calciphylaxis (both in those with and without HD-dependent CKD) as compared to matched CKD controls (Figure 15). Median PPi value (interquartile range [IQR]) was 248 nM (110 to 323) in cases as compared to controls; 661 nM (361 to 799) p< 0.0001. PPi levels in both CKD populations were significantly lower as compared to healthy volunteer PPi levels. Furthermore, lower PPi levels were associated with a significantly increased 6-month mortality rate among patients with calciphylaxis (Figure 16). In addition, a greater number of skin lesions was associated with lower PPi levels (data not shown).

[0528] Instead of histological analysis which requires staining of kidney slices or arterial tissues which is not feasible to perform in living patients, a medical professional can use noninvasive visualization techniques commonly known in art such as CT scan, ultrasound, or intravenous pyelography to visualize the presence of calcifications and the reduction of calcifications in response to delivery of ENPPl-Fc in patients suffering from Calciphylaxis.

[0529] Intravenous pyelography is an X-ray exam that uses a contrast medium, which functions as a dye, to help visualize the urinary tract and detect the presence of renal calcifications. Computed tomography is a noninvasive imaging technique that uses X-ray technology to depict internal structures of the body such as the urinary tract. Renal calcifications are visible on CT scans. CT scans collect X- ray images from different angles around the body to generate detailed cross-sectional images as well as three-dimensional images of the body's internal structures and organs. CT scan can also be used in arteries to detect the presence and subsequent reduction of calcification following treatment. A computer analyzes the radiation transmitted through the body to reconstruct the images of the internal structures and organs.

[0530] A medical doctor having skills in visualizing soft tissue calcification, cardiac calcification, kidney calcification, and myocardial infarction can undertake the treatment of a subject afflicted with Calciphylaxis by administering a therapeutically effective amount of ENPP1 agent such as ENPPl-Fc at 1.8 mg / kg dosage.

[0531] The physician or a medical professional thus has the option to control the dosage based on the rate and extent of improvement of symptoms. Successful treatment is observed by a medical professional of skill in the art by observing one or more positive symptoms such as improved kidney function, and improved urine creatine levels (normal creatine levels in urine for men are 40 — 278 mg / dL and 29 — 226 mg / dL for women), and improved urine-urea levels (normal urea levels in the urine for adults are 26 — 43 g / 24 h), normal serum-creatine levels (normal serum creatinine range is 0.6-1.1 mg / dL in women and 0. 7 — I. 3 mg / dL in men), normal vitamin D levels (20ng / ml to 50 ng / mL is considered adequate for healthy people). A level less than 12 ng / mL indicates vitamin D deficiency), normal blood urea nitrogen levels (BUN level for healthy adults is 7-20 mg / dL), weight gain, increase in serum PPi levels (at least about 4-5 pm), reduction in calcification (25%, or 50%, or 70%, or 90% or 100% reduction of calcification in arteries and / or kidneys) of arterial tissues and or reduction of calcification in kidney tubules visualized by noninvasive techniques such as CT or ultrasound scans.

[0532] Like the endogenous ENPP1 enzyme, the ENPP1 agent cleaves ATP to generate AMP and PPi, thereby increasing plasma PPi levels and into AMP which CD73 coverts rapidly to adenosine. Such ENPPl-Fc administration is expected to correct the mineral imbalance, regulate PPi levels and allow for improved health and mitigation of clinical complications associated with Calciphylaxis.

[0533] Of 21 ESKD patients screened, median PPi levels were 582 nM. Nine patients screened failed with PPi>700 nM and 1 passed screening but withdrew before Day 1. Eleven patients with ESKD and PPi <700 nM were treated with four weekly doses of subcutaneous INZ-701 at 1.8 mg / kg. One patient remains in follow-up for safety observation. Patient demographics and disease history is shown in Figure 21.

[0534] INZ-701 increased PPi in dialysis patients across the spectrum of PPi deficiency (Figure 22). Key mediators of mineral metabolism phosphate (Pi) & FGF-23 decreased during INZ-701 treatment (Figures 23-24). INZ-701 exposure was as expected for 1.8 mg / kg weekly based on treatment of non-hemodialysis patients. 1.2 mg / kg / week dose (1.5-fold lower than SEAPORT 1) tested in adults with ENPP1 Deficiency (INZ701-101 study) showed mean exposure of 3,715 ng / mL. Mean SEAPORT 1 exposure was 6,732 ng / mL, a 1.8-fold difference, confirming dose proportionality (Figure 25). Increase in mean ENPP1 activity mirrored INZ-701 exposure (Figure 26). These results showed that PPi levels in screened ESKD patients (n=21) were lower than what has been reported in healthy volunteers. INZ-701 demonstrated a favorable safety profile, with no drug-related TEAEs observed. Mean PPi levels increased into the normal range by week 3 and was associated with reductions in mineral metabolism biomarkers (phosphate and FGF-23). Drug exposure was proportional to the dose received. These findings suggest that the PPi-adenosine pathway is impacted in ESKD patients, and that INZ-701 may normalize PPi levels in those patients, supporting further clinical development in calciphylaxis.

[0535] Example 11: Correlation of low PPi with calciphylaxis and risk of mortality.

[0536] Patients aged > 18 with the diagnosis of calciphylaxis have been recruited into Partners calciphylaxis Biorepository and Patient Registry (NCT03032835), and the patient characteristics are shown in Figure 27. Clinical data and blood samples were collected at the time of enrollment. For hemodialysis patients, plasma PPi levels were collected prior to dialysis. Plasma PPi levels were measured using an ATP Sulfuryl ase / Luminescence-based method. Among 70 patients, median PPi levels were 568 nM [IQR: 253-1205] at enrollment. For every 100 nM lower in plasma PPi levels at enrollment, there was a 25% higher risk of 6-week mortality (p = 0.008). Among ESKD dependent dialysis patients, there was a 54% higher risk of 6-week mortality for every 100 nM lower in plasma PPi levels (p = 0.01). 28 calciphylaxis cases that survived at 6 weeks were included in a longitudinal study. Among the 28 patients, median PPi levels were 1155 nM [IQR: 835-1456] at the 6-week follow up, and 12-week mortality among patients in the longitudinal study was 7%. Decrease in plasma PPi levels over the 6-week period was associated with higher 12-week mortality (p = 0.04). These results showed that low plasma PPi levels predicted higher 6-week mortality in calciphylaxis patients (Figure 28) and were correlated with higher numbers of skin lesions (Figure 29), suggesting a higher severity of calciphylaxis is associated with a higher mortality risk among calciphylaxis patients.

[0537] Example 12: Treatment of subjects with calciphylaxis.

[0538] SEAPORT 2 is a Phase 2 single-arm, open-label study to evaluate the safety and pharmacodynamics of subcutaneous (SC) INZ-701 in study participants with hemodialysis (HD)-dependent end stage kidney disease (ESKD) who have active calciphylaxis. Key outcomes will include the effect of treatment with INZ-701 on plasma pyrophosphate levels and the safety and tolerability of multiple weekly SC dose administrations in this population that may be at risk of disease exacerbation from dermis trauma. Previous studies have established the safety profile and dosing regimens for INZ-701 in patients having ATP -binding cassette sub-family C member 6 (ABCC6) deficiency and ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) deficiency, and in stable ESKD patients on HD without calciphylaxis.

[0539] Data from a completed Phase 1 / 2 study in adults (>18 to <65 years) with ENPP1 Deficiency (Study INZ701-101) showed increases in circulating levels of PPi to within normal. In addition, steady state INZ-701 levels were observed after 4 doses and no safety signals were identified.

[0540] Furthermore, increases in PPi levels have been observed in a completed Phase 1 / 2 study in adults (>18 to <70 years) with ABCC6 Deficiency (Study INZ701-201). These study participants have a mutation in the ABCC6 gene, but do have endogenous ENPP1 enzyme, suggesting a similar effect is plausible in the CKD and ESKD populations. No safety signals with INZ-701 treatment were identified in this ABCC6-deficient population either.

[0541] SEAPORT 1 (INZ701-401), a Phase 1 open-label study evaluating the safety, pharmacokinetics (PK) and pharmacodynamics (PD) of INZ-701 in HD-dependent ESKD patients with baseline PPi levels <700 nmol / L, demonstrated a reliable increase in ENPP1 activity (without effect from HD) at a dose of 1.8 mg / kg given weekly by SC injection. The drug was well-tolerated and without significant impact on calcium and phosphorus levels, and an increase in PPi levels was observed during the administration period. This study indicates INZ-701 can be safely administered to HD-dependent patients and improves their abnormally low PPi levels. There were no skin-related adverse events (AEs).

[0542] The purpose of SEAPORT 2 (INZ701-402) is to determine if SC INZ-701, given once per week over 12 weeks, is safe and effective at increasing PPi in HD-dependent ESKD study participants with calciphylaxis. Two different doses will be used to test whether PPi can be increased to different maximums, and whether the dose of INZ-701 alters the risk of developing calciphylaxis lesions at INZ-701 injection sites.

[0543] Ill Objectives and Endpoints

[0544]

[0545]

[0546] Study Design

[0547] This study is a multi center, open-label, Phase 2 study to evaluate the safety and pharmacodynamics of INZ-701 in HD-dependent ESKD adults with calciphylaxis. The study

[0548] will consist of an up to 30-day Screening Period, a 7-day Pre-treatment Baseline Window, a 12-week Treatment Period followed by an 8-week Observation Period, during which outcomes will continue to be monitored. Study Schematic is illustrated in Figure 30.

[0549] Study Procedures and Assessments

[0550] Procedures and assessments are performed as indicated in the SOE. (See Figure 31). Screening Period

[0551] The Screening Period will last up to 30 days (Day -37 to Day -8). Day 1 will be coordinated amongst the participants, dialysis center, and Investigator site for agreement on the start of the Treatment Period.

[0552] Pre-treatment Baseline Window

[0553] The Pre-treatment Baseline Window is Day -7 to Day -1 and is intended to collect baseline PPi levels prior to drug administration.

[0554] Treatment Period

[0555] The Treatment Period begins on Day 1 and lasts for 12 weeks, during which participants receive weekly INZ-701 injections (plus standard of care). INZ-701 will be administered on Days 1, 8, 15, 22 etc. Administration of INZ-701 will occur in the dialysis setting (at an in-center HD during the participants scheduled dialysis treatment) and is required on administration days that coincide with blood sampling.

[0556] Observation Period

[0557] All participants will enter the 8-week Observation Period after INZ-701 is no longer being administered, for washout and ongoing monitoring of outcomes.

[0558] Standard of Care

[0559] Standard of care for calciphylaxis includes intravenous (IV) sodium thiosulfate (STS), which may be administered at the discretion of the treating physician. There is no constraint on the duration of STS administration. Standard of care may also include parathyroidectomy, medication optimization (including changes to SC concomitant medications), renal replacement therapy (RRT) modality change, or RRT dose change.

[0560] INZ-701 Dosage and Formulation

[0561] INZ-701 will be administered as a Low Dose (flat 100 mg or 1.2 mg / kg) or High Dose (flat 150 mg or 1.8 mg / kg) concentration once weekly by SC injection. See Dose / Dose Modification Section below for details. End of Study

[0562] The End of Study (EOS) is defined as the date of the last protocol-specified visit / assessment for the last study participant enrolled in the study.

[0563] Safety Monitoring

[0564] Safety, including immunogenicity, will be evaluated from the date of informed consent continuing throughout the study. The Sponsor will review individual and cumulative clinical safety data on an ongoing basis, including but not limited to serious adverse events (SAEs), suspected unexpected serious adverse reactions (SUSARs), and adverse events of special interest (AESIs).

[0565] Adverse events will be characterized in the below possible categories. Note: AEs may fall into more than one category.

[0566] • SAEs

[0567] • SUSARs

[0568] • AESIs

[0569] o Systemic hypersensitivity reactions, including anaphylaxis

[0570] o Seizures

[0571] • Injection Site Reactions

[0572] • Hemodi aly si s-Rel ated Events

[0573] Safety monitoring for the development of new calciphylaxis lesions at the site of recent INZ-701 administration will be continuous.

[0574] Early Treatment Stoppage / Discontinuation

[0575] All study participants who discontinue INZ-701 should continue weekly visual inspection of INZ-701 injection sites and complete an EOS Visit 30 days after their last dose of INZ-701.

[0576] Early Study Discontinuation

[0577] Study participants may choose to terminate their participation in the study at any time for any reason or they may be withdrawn by the Investigator. The Sponsor is to be notified within 24 hours of the Investigator's awareness of all early discontinuations. All study participants who discontinue the study should complete an EOS Visit as soon as possible. Dose / Dose Modification

[0578] INZ-701 will be administered as a SC injection once weekly for 12 weeks during the Treatment Period. The first 5 participants enrolled in the study will receive the Low Dose. The following 5 participants enrolled will receive the High Dose. The study participant’s current estimated dry weight (EDW) will be used to calculate the dose administered each week.

[0579] Low Dose:

[0580] Participant weight 50 kg-100kg - Flat 100 mg dose weekly

[0581] Participant weight <50 kg or >100 kg - 1.2 mg / kg dose weekly

[0582] High Dose:

[0583] Participant weight 50 kg-100 kg - Flat 150 mg dose weekly

[0584] Participant weight <50 kg or >100 kg - 1.8 mg / kg dose weekly

[0585] Estimated Duration of Study Participation

[0586] Study participation consists of a Screening Period of up to 30 days, Pre-Treatment Baseline plus Treatment Period of 13 weeks, and Observation Period of 8 weeks, for a maximum duration of approximately 25 weeks.

[0587] Eligibility Criteria

[0588] Inclusion: Individuals eligible to participate must meet all the following inclusion criteria:

[0589] 1. Study participants must provide written or electronic consent after the nature of the study has been explained, and prior to any research-related procedures, per International Council on for Harmonisation (ICH) Good Clinical Practice (GCP). 2. Study participants must have HD-dependent kidney disease. Participants who initiate HD in the hospital are candidates once they transition to outpatient HD.

[0590] 3. Are compliant with their dialysis schedule in the opinion of the treating physician. 4. Diagnosed with calciphylaxis with at least 1 skin lesion with ulceration. In the absence of an ulcerated lesion (presence of 1 or more nonulcerated skin lesions), the investigator may establish the diagnosis of calciphylaxis by skin biopsy and clinicopathological correlation, and / or diagnostic agreement of a multi-disciplinary team, typically consisting of 2 independent physicians such as a nephrologist and dermatologist, depending on site expertise.

[0591] a. Clinical diagnosis is defined as diagnosis without tissue confirmation. b. Clinical diagnosis is defined by the investigator, and might include history, physical examination, and noninvasive testing.

[0592] c. There is no limit on the length of time since the diagnosis of cal ciphyl axis. The data used for diagnosis for a participant with a calciphylaxis recurrence (after a remission or resolution) will be the date of the recurrence.

[0593] 5. Aged 18 years to 75 years.

[0594] 6. Women of child-bearing potential (WOCBP) as defined in Clinical Trials Facilitation and Coordination Group (CTFG 2020), must have a negative serum pregnancy test at Screening and within 3 days of INZ-701 administration.

[0595] 7. In the opinion of the Investigator, study participants are able and willing to complete all study procedures per protocol.

[0596] Exclusion: Individuals meeting any of the following exclusion criteria may not participate:

[0597] 1. Coexisting or competing non-cal ciphyl axis skin disorder such as vasculitis.

[0598] 2. Diagnostic skin biopsy specimen showing any acute non-calciphylaxis lesion.

[0599] 3. Has received intralesional STS dosing within 60 days prior to first dose (Day 1). 4. Participating in another interventional study within 30 days of first dose (Day 1).

[0600] Participation in a registry is not an exclusion.

[0601] 5. Known intolerance to INZ-701.

[0602] 6. Scheduled surgical parathyroidectomy during the study period.

[0603] Prohibited Concomitant Medications

[0604] Study participants may continue or discontinue medications as clinically indicated by their treating physicians and / or Investigator. IV Sodium thiosulphate may be used at the discretion of the investigator; intralesional STS is prohibited. Dialysis Prescription

[0605] There are no restrictions on the HD prescription during the study. Investigators may increase or decrease HD parameters. KT / V, HD hours per week, and other measures of HD intensity will be collected and analyzed.

[0606] Statistical Considerations

[0607] A full description of the statistical evaluations, general considerations, and procedures for handling missing data will be provided in the Statistical Analysis Plan (SAP).

[0608] Determination of Sample Size

[0609] The sample size for this study was determined by clinical and practical considerations rather than statistical considerations. Approximately 10 patients will be enrolled to sufficiently address the safety objectives of the study.

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[0653] INCORPORATION BY REFERENCE

[0654] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0655] OTHER EMBODIMENTS

[0656] While specific embodiments of the subject matter have been discussed, 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 claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

CLAIMS1. A method of treating calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide to the subject once a week thereby treating calciphylaxis in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

2. A method of treating calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby treating calciphylaxis in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

3. A method of increasing the levels of serum or plasma pyrophosphate (PPi) in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby elevating levels of PPi in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

4. A method of treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby treating or reducing calcification in said subject, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

5. A method of reducing pain in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby elevating levels of PPi in said subject and reducing pain, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

6. A method of reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, said method comprising administering a dose of ENPP1 polypeptide once a week to the subject thereby elevating levels of PPi in said subject andreducing the number and / or wound size of the skin lesions, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide.

7. The method of any one of claims 1-6, wherein administration of the ENNP1 polypeptide increases serum or plasma level of a bone and / or mineral metabolism biomarker.

8. The method of claim 7, wherein the bone and / or mineral metabolism biomarker is iPTH, FGF23, BALP, or Phosphate.

9. The method of any one of claims 1-8, wherein said subject is the age of at least 18 years but less than 75 years (> 18 to <75 years).

10. The method of any one of claims 1-9, wherein the subject has a loss of function mutation in one or more of ecto-5 '-nucleotidase (NT5E), vitamin D receptor (VDR), and Fibroblast growth factor 23(FGF23) gene.

11. The method of any one of claims 1-10, wherein the ENPP1 polypeptide is administered subcutaneously.

12. The method of any one of claims 1-11, wherein the ENPP1 polypeptide comprises a heterologous moiety, and wherein said heterologous moiety increases the circulating half-life of the ENPP1 agent relative to the circulating half-life of the ENPP1 agent lacking the heterologous moiety.

13. The method of any one of claims 1-11, wherein the ENPP1 polypeptide comprises a heterologous moiety and wherein said heterologous moiety comprises the Fc region of an immunoglobulin molecule.

14. The method of any one of claims 1-13, wherein the ENPP1 polypeptide comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO:1 or residues 1 (FTAGLKPSCAKE) to 833 (QED) of SEQ ID NO:3.

15. The method of any one of claims 1-13, wherein the ENPP1 polypeptide comprises the amino acid sequence depicted in anyone of SEQ ID NO: 3-5 and 116-128.

16. The method of any one of claims 1-11, wherein the ENPP1 polypeptide is a ENPP1 fusion polypeptide, said ENPP1 fusion polypeptide comprises ENPP1 component and an Fc domain, wherein said ENPP1 component comprises at least 95% of amino acid residues 99 (PSCAKE) to925 (QED) of SEQ ID NO: 1 and Fc domain comprises at least 95% of amino acid residues of SEQ ID NO: 6.

17. The method of claim 16, wherein said ENPP1 component and said Fc domain are connected by means of a peptide linker, wherein said peptide linker has a length that is no less than two amino acids and no more than 36 amino acids.

18. The method of any one of claims 1-17, wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein.

19. The method of any one of claims 1-17, wherein the subject has or is suspected of having one or more of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis, juvenile dermatomyositis, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), autosomal hypophosphatemic rickets (ARHR2), hypopyrophosphatemia, osteoarthritis, calcification of atherosclerotic plaques, End Stage Renal Disease (ESRD), pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

20. The method of any one of claims 1-17, wherein said subject exhibits one or more of calcification of soft connective tissues, accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers of connective tissues, calcification in the eyes, calcification of cardiovascular system, calcification of skin, narrowing of the arteries, arteriosclerosis, ectopic calcification, narrowing of blood vessels in the lower extremities, claudication, abnormal pigmentation of cells of the retina, angioid streaks, abnormalities in the elastic membrane beneath the retina, choroidal neovascularization, visual impairment, vision loss, and blindness.

21. The method of any one of claims 1-20, wherein the subject has not undergone peritoneal dialysis or hemodiafiltration.

22. The method of any one of claims 1-21, wherein the subject has not been diagnosed with malignancy other than non-melanoma skin cancer or cervical carcinoma in the past year prior to said administration.

23. The method of any one of claims 1 -22, wherein the subject has not been diagnosed with advanced liver disease such as liver cirrhosis.

24. The method of any one of claims 1-23, wherein the subject has not had a myocardial infarction, stroke, or congestive heart failure requiring hospitalization within 6 months prior to said administration.

25. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for the treatment of calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

26. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for the treatment of calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

27. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for increasing the levels of serum or plasma pyrophosphate (PPi) in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

28. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

29. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for reducing pain in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

30. Use of a dose of ENPP1 polypeptide for the preparation of a medicament for reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide and is formulated for administration once a week.

31. The use of any one of claims 25-30, wherein the medicament, after being administered, increased serum or plasma level of a bone and / or mineral metabolism biomarker.

32. The use of claim 31, wherein the bone and / or mineral metabolism biomarker is iPTH, FGF23, BALP, or Phosphate.

33. The use of any one of claims 25-32, wherein said subject is the age of at least 18 years but less than 75 years (> 18 to <75 years).

34. The use of any one of claims 25-33, wherein the subject has a loss of function mutation in one or more of ecto-5 '-nucleotidase (NT5E), vitamin D receptor (VDR), and Fibroblast growth factor 23(FGF23) gene.

35. The use of any one of claims 25-34, wherein the medicament is for subcutaneous administration.

36. The use of any one of claims 25-35, wherein the ENPP1 polypeptide comprises a heterologous moiety, and wherein said heterologous moiety increases the circulating half-life of the ENPP1 agent relative to the circulating half-life of the ENPP1 agent lacking the heterologous moiety.

37. The use of any one of claims 25-35, wherein the ENPP1 polypeptide comprises a heterologous moiety and wherein said heterologous moiety comprises the Fc region of an immunoglobulin molecule.

38. The use of any one of claims 25-37, wherein the ENPP1 polypeptide comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 or residues 1 (FTAGLKPSCAKE) to 833 (QED) of SEQ ID NO: 3.

39. The use of any one of claims 25-37, wherein the ENPP1 polypeptide comprises the amino acid sequence depicted in anyone of SEQ ID NO: 3-5 and 116-128.

40. The use of any one of claims 25-35, wherein the ENPP1 polypeptide is a ENPP1 fusion polypeptide, said ENPP1 fusion polypeptide comprises ENPP1 component and an Fc domain, wherein said ENPP1 component comprises at least 95% of amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 and Fc domain comprises at least 95% of amino acid residues of SEQ ID NO:

641. The use of claim 40, wherein said ENPP1 component and said Fc domain are connected by means of a peptide linker, wherein said peptide linker has a length that is no less than two amino acids and no more than 36 amino acids.

42. The use of any one of claims 25-41, wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein.

43. The use of any one of claims 25-41, wherein the subject has or is suspected of having one or more of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis, juvenile dermatomyositis, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), autosomal hypophosphatemic rickets (ARHR2), hypopyrophosphatemia, osteoarthritis, calcification of atherosclerotic plaques, End Stage Renal Disease (ESRD), pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

44. The use of any one of claims 25-41, wherein said subject exhibits one or more of calcification of soft connective tissues, accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers of connective tissues, calcification in the eyes, calcification of cardiovascular system, calcification of skin, narrowing of the arteries, arteriosclerosis, ectopic calcification, narrowing of blood vessels in the lower extremities, claudication, abnormal pigmentation of cells of the retina, angioid streaks, abnormalities in the elastic membrane beneath the retina, choroidal neovascularization, visual impairment, vision loss, and blindness.

45. The use of any one of claims 25-44, wherein the subject has not undergone peritoneal dialysis or hemodiafiltration.

46. The use of any one of claims 25-45, wherein the subject has not been diagnosed with malignancy other than non-melanoma skin cancer or cervical carcinoma in the past year prior to said administration.

47. The use of any one of claims 25-46, wherein the subject has not been diagnosed with advanced liver disease such as liver cirrhosis.

48. The use of any one of claims 25-47, wherein the subject has not had a myocardial infarction, stroke, or congestive heart failure requiring hospitalization within 6 months prior to said administration.

49. A composition comprising a dose of ENPP1 polypeptide for use in treating calciphylaxis in a subject having or diagnosed with chronic kidney disease (CKD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

50. A composition comprising a dose of ENPP1 polypeptide for use in treating calciphylaxis in a subject having or diagnosed with end stage renal disease (ESRD) and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

51. A composition comprising a dose of ENPP1 polypeptide for use in increasing the levels of serum or plasma pyrophosphate (PPi) in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

52. A composition comprising a dose of ENPP1 polypeptide for use in treating or reducing calcification in one or more of kidneys, ascending aorta, descending thoracic aorta, carotid artery, iliac artery, skin, micro vessels in the subcutaneous adipose tissue and spleen in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

53. A composition comprising a dose of ENPP1 polypeptide for use in reducing pain in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg,about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

54. A composition comprising a dose of ENPP1 polypeptide for use in reducing the number and / or wound size of skin lesions in a subject having calciphylaxis and undergoing hemodialysis, wherein the dose is flat 100 mg, flat 150 mg, about 1.2 mg / kg, or about 1.8 mg / kg of the ENPP1 polypeptide, and the composition is formulated for administration once a week.

55. The composition of any one of claims 49-54, wherein the composition, after being administered, increased serum or plasma level of a bone and / or mineral metabolism biomarker.

56. The use of claim 55, wherein the bone and / or mineral metabolism biomarker is iPTH, FGF23, BALP, or Phosphate.

57. The composition of any one of claims 49-56, wherein said subject is the age at least 18 years but less than 75 years (>18 to <75 years).

58. The composition of any one of claims 49-57, wherein the subject has a loss of function mutation in one or more of ecto-5 '-nucleotidase (NT5E), vitamin D receptor (VDR), and Fibroblast growth factor 23(FGF23) gene.

59. The composition of any one of claims 49-58, wherein the composition is for subcutaneous administration.

60. The composition of any one of claims 49-59, wherein the ENPP1 polypeptide comprises a heterologous moiety, and wherein said heterologous moiety increases the circulating half-life of the ENPP1 agent relative to the circulating half-life of the ENPP1 agent lacking the heterologous moiety.

61. The composition of any one of claims 49-59, wherein the ENPP1 polypeptide comprises a heterologous moiety and wherein said heterologous moiety comprises the Fc region of an immunoglobulin molecule.

62. The composition of any one of claims 49-61, wherein the ENPP1 polypeptide comprises amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 or residues 1 (FTAGLKPSCAKE) to 833 (QED) of SEQ ID NO:3.

63. The composition of any one of claims 49-61, wherein the ENPP1 polypeptide comprises the amino acid sequence depicted in anyone of SEQ ID NO: 3-5 and 116-128.

64. The composition of any one of claims 49-59, wherein the ENPP1 polypeptide is a ENPP1 fusion polypeptide, said ENPP1 fusion polypeptide comprises ENPP1 component and an Fc domain, wherein said ENPP1 component comprises at least 95% of amino acid residues 99 (PSCAKE) to 925 (QED) of SEQ ID NO: 1 and Fc domain comprises at least 95% of amino acid residues of SEQ ID NO: 6.

65. The composition of claim 64, wherein said ENPP1 component and said Fc domain are connected by means of a peptide linker, wherein said peptide linker has a length that is no less than two amino acids and no more than 36 amino acids.

66. The composition of any one of claims 49-65, wherein said subject is not ENPP1 deficient or is not ABCC6 deficient or does not have a defective ENPP1 protein.

67. The composition of any one of claims 49-65, wherein the subject has or is suspected of having one or more of kidney and bladder stones, dental pulp stones, gall stones, salivary gland stones, chronic calculous prostatitis, testicular microliths, calcification in hemodialysis patients, atherosclerosis, malacoplakia, scleroderma (systemic sclerosis), calcinosis cutis, calcific aortic stenosis, calcific tendonitis, synovitis and arthritis, diffuse interstitial skeletal hyperostosis, juvenile dermatomyositis, Generalized Arterial Calcification of Infancy (GACI), Ossification of the Posterior Longitudinal Ligament (OPLL), autosomal hypophosphatemic rickets (ARHR2), hypopyrophosphatemia, osteoarthritis, calcification of atherosclerotic plaques, End Stage Renal Disease (ESRD), pseudoxanthoma elasticum (PXE), ankylosing spondylitis, hardening of the arteries, calciphylaxis, and systemic lupus erythematosus.

68. The composition of any one of claims 49-65, wherein said subject exhibits one or more of calcification of soft connective tissues, accumulation of deposits of calcium and other minerals (mineralization) in elastic fibers of connective tissues, calcification in the eyes, calcification of cardiovascular system, calcification of skin, narrowing of the arteries, arteriosclerosis, ectopic calcification, narrowing of blood vessels in the lower extremities, claudication, abnormal pigmentation of cells of the retina, angioid streaks, abnormalities in the elastic membrane beneath the retina, choroidal neovascularization, visual impairment, vision loss, and blindness.

69. The composition of any one of claims 49-68, wherein the subject has not undergone peritoneal dialysis or hemodiafiltration.

70. The composition of any one of claims 49-69, wherein the subject has not been diagnosed with malignancy other than non-melanoma skin cancer or cervical carcinoma in the past year prior to said administration.

71. The composition of any one of claims 49-70, wherein the subject has not been diagnosed with advanced liver disease such as liver cirrhosis.

72. The composition of any one of claims 49-71, wherein the subject has not had a myocardial infarction, stroke, or congestive heart failure requiring hospitalization within 6 months prior to said administration.

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