Treatment methods for tissue calcification

Abstract

To provide a method of treating NPP1 deficiency or NPP1-associated disease such as idiopathic infantile arterial calcification (IIAC), pseudoxanthoma elasticum, vascular calcification in chronic kidney disease (VCCKD), insulin resistance, hypophosphatemic rickets, myocardial ischemia, joint calcification, angioid streaks of retina, and ossification of the posterior longitudinal ligament of the spine.SOLUTION: Tissue calcification is treated by administering soluble NPP1 to produce transient increase in serum pyrophosphate levels.SELECTED DRAWING: None

Description

[Background technology]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 62 / 094,943, filed on 19 December 2014, and U.S. Provisional Application No. 62 / 249,781, filed on 2 November 2015. The entire teachings of the above applications are incorporated herein by reference.

[0002] Reference to electronically submitted sequence listings The entire contents of the sequence listing submitted electronically in the ASCII text file (name: 081245-0208_ascii.txt; size: 88,556 bytes; and creation date: December 15, 2015) filed with this application are incorporated herein by reference.

[0003] Vascular calcification can be characterized by the formation of very small dispersed crystals of hydroxyapatite (HA) and by large calcified deposits in vascular tissue, such as arteries. (Amann, K. Clin J Am Soc Nephrol 2008, 3, 1599-605). Extracellular pyrophosphate (PPi) is an important endogenous inhibitor of vascular calcification by inhibiting HA formation. (Lomashvili, KA et al., J Am Soc Nephrol 2004, 15, 1392-1401; Fleisch, H. et al., Nature 1966, 212, 901-903).

[0004] Ectonucleotide pyrophosphatase pyrophosphorylase (NPP1) is an ectoenzyme that cleaves ATP to produce extracellular pyrophosphate (PPi). Pyrophosphate is a potent inhibitor of hydroxyapatite formation and, under normal conditions, functions to inhibit vascular calcification.

[0005] NPP1 deficiency in humans leads to a decrease in circulating PPi levels and is associated with conditions such as arterial calcification and generalized arterial calcification in infants (GACI). (Rutsch, F. et al., Am J Pathol 2001, 158, 543-554). When fed a high-phosphate diet, NPP1-deficient mice (Enpp1) - / - ) also shows decreased PPi levels and exhibits a phenotype similar to that of NPP1-deficient humans (Harmey, D. et al., Am J Pathol 2004, 164, 1199-1209). Vascular calcification is also a well-recognized common complication in patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) and is associated with increased morbidity and mortality (Giachelli, CJ Am Soc Nephrol 2004, 15, 2959-64; Raggi, P. et al., J Am Coll Cardiol 2002, 39, 695-701).

[0006] Ectonucleotide pyrophosphatase / phosphodiesterase 1 (NPP1 / ENPP1 / PC-1) deficiency is a rare disorder caused by mutations in the type II transmembrane glycoprotein NPP1. NPP1 cleaves various substrates, including phosphodiester bonds and pyrophosphate bonds of nucleotides and nucleotide sugars. NPP1 deficiency has been associated with idiopathic infantile arterial calcification (IIAC), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine.

[0007] IIAC is a rare, autosomal recessive, almost certainly fatal disorder characterized by stenosis of the muscular arteries due to calcification of the internal elastic membrane and proliferation of intimal smooth muscle cells. More than 160 cases of IIAC have been reported worldwide. Symptoms of this disease almost always appear by early infancy, and the disease generally leads to death by 6 months of age due to complications of ischemic cardiomyopathy and other obstructive arteriopathies, including renal artery stenosis.

[0008] Despite the association of NPP1 protein deficiency with serious conditions such as IIAC, there are currently no available treatments for patients with this disease, as well as for other calcification disorders resulting from abnormal bone metabolism leading to high systemic loadings of calcium and phosphorus; low levels of circulating and locally produced inhibitors for phosphate producers; or impaired renal excretion.

[0009] Current treatment options for preventing vascular calcification have limited efficacy and undesirable and / or unacceptable side effects. For example, very large doses of exogenous PPi are required for efficacy, and other inhibitors of hydroxyapatite formation inhibit bone calcification, potentially leading to osteomalacia. In particular, direct administration of exogenous PPi has been found to prevent calcification in uremic animal models (O'Neil, WCet al., Kidney Int 2011, 79, 512-517; Riser, BLet al., Nephrol Dial Transp 2011, 26, 3349-3357). However, this approach requires high doses due to the short half-life of PPi, leading to hyperphysiological plasma levels of PPi and resulting in local irritation. Bisphosphonates, non-hydrolyzable analogs of PPi, have been used to treat vascular calcification, for example, in animal models. (Fleisch, H. et al., Europ J Clin Invest 1970, 1, 12-18; Price, PA et al., Arteriosclerosis Throm and Vas Bio 2001, 21, 817-824; Price, PA et al., Kidney Int 2006, 70, 1577-1583; Lomashvili, KA et al., Kidney Int 2009, 75, 617-625). However, bisphosphonates also inhibit bone formation. Bisphosphonates can delay calcification in GACI subjects, but they cannot prevent it (Rutsch, F. et al., Circ Cardiovasc Genet 2008, 1, 133-140), and can lead to osteomalacia, as in animals. (Otero, JE, et al., J Bone Miner Res 2013, 28, 419-430).

[0010] Braddock, D. et al., (WO2014 / 126965A2) discloses compositions and methods for treating pathological calcification and ossification by administering NPP1. Quinn, A. et al., (WO2012 / 125182A1) discloses NPP1 fusion proteins for treating conditions including GACI, arterial calcification, insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine.

[0011] Despite considerable research in this field, there remains a need for novel therapies that effectively inhibit vascular calcification, preferably without causing osteomalacia. Effective and safe medicines are also needed for the treatment of IIAC, vascular calcification in chronic kidney disease (VCCKD), pseudoxanthoma elasticum (PXE), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine. [Overview of the project]

[0012] The present invention relates to the use of isolated recombinant human soluble NPP1 lacking the N-terminal cytosolic domain and transmembrane domain, and its fusion proteins, for the treatment of NPP1 deficiency or other progressive disorders characterized by the accumulation of calcium and other mineral deposits.

[0013] Remarkably, the protein of the present invention can be used to restore blood NPP1 activity and restore normal levels of pyrophosphate in subjects who have a deficiency in NPP1 activity or who show accumulation of calcium deposits in the bones, joints, heart, blood vessels, eyes, and / or skin.

[0014] More specifically, the NPP1 protein and NPP1 fusion protein of the present invention can be used to treat a subject having a low level of pyrophosphate, including, but not limited to, idiopathic infantile arterial calcinosis (IIAC, also known as infantile systemic arterial calcification), vascular calcification in chronic kidney disease (VCCKD), pseudoxanthoma elasticum (PXE), insulin resistance, hypophosphatemic rickets, joint calcification, myocardial ischemia, and ossification of the posterior longitudinal ligament of the spine, and other diseases or disorders related thereto. Any progressive disorder characterized by the accumulation of deposits of calcium and other minerals in arteries and / or connective tissues is within the scope of the present invention.

[0015] In some embodiments, the present invention relates to a method for reducing tissue calcification, preferably vascular calcification, in a subject in need thereof. The method comprises administering to a subject having low plasma pyrophosphate (PPi) or high inorganic phosphate (Pi) a therapeutically effective amount of a composition comprising soluble ectonucleotide pyrophosphatase phosphodiesterase (NPP1) two or more times. Each administration contains an amount of soluble NPP1 sufficient to achieve a transient increase in plasma PPi in the subject. A transient increase in plasma PPi characterized by a peak PPi level that is at least about 40% of the normal plasma PPi level and a return to the baseline PPi level within about 48 hours after a single administration. The period between administrations is at least 2 days.

[0016] The transient increase in plasma PPi is maintained for at least about 4 hours, preferably at least about 6 hours, at least about 8 hours, at least about 10 hours or at least about 12 hours.

[0017] The tissue calcification can be vascular calcification, such as venous or arterial calcification, and the calcification can be intimal or medial.

[0018] Subjects requiring treatment may have NPP1 deficiency, chronic kidney disease (CKD), end-stage renal disease (ESRD), infantile systemic arterial calcification (GACI), cardiovascular disorders, type II true diabetes, atherosclerosis or pseudoxanthoma elasticum (PXE). If the subject has low plasma PPi, the pre-treatment level of plasma pyrophosphate (PPi) in the subject is at least about 40% lower than that of normal plasma PPi level, and the subject is human. If the subject has a high level of Pi, the pre-treatment level of Pi in the subject is typically at least about 110% of the normal plasma Pi level.

[0019] The amount of sNPP1 administered per dose can be about 1.0 mg / kg to about 5.0 mg / kg of NPP1 or about 1.0 mg / kg to about 10.0 mg / kg of NPP1. The period between administrations of NPP1 is at least 2 days and can be longer, for example, at least 3 days, at least 1 week, 2 weeks or 1 month. sNPP1 can be administered by any suitable method, such as intravenously, subcutaneously, or intraperitoneally.

[0020] In a preferred embodiment, an NPP1 fusion protein is administered. Preferred fusion proteins include an NPP1 component, an immunoglobulin Fc region and optionally a targeting moiety. A preferred targeting moiety is Asp 10 is. Particularly preferred NPP1 fusion proteins for administration according to the methods disclosed herein have the amino acid sequences of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0021] Other features and advantages of the present invention will become apparent from the following detailed description of the invention and the claims.

Brief Description of the Drawings

[0022] [Figure 1]Figure 1 shows the amino acid sequence of the wild-type NPP1 protein (SEQ ID NO: 1). The cytosolic and transmembrane regions are underlined. Sites capable of N-glycosylation are shown in bold. The bold amino acid motif "PSCAKE" (SEQ ID NO: 17) is the start of soluble NPP1, which includes a cysteine-rich region. [Figure 2] Figure 2 shows the amino acid sequence (SEQ ID NO: 2) of sNPP1, which includes the cysteine-rich region, the catalytic region, and the C-terminal region. [Figure 3] Figure 3 shows the amino acid sequence of the sNPP1-Fc fusion protein (SEQ ID NO: 3). [Figure 4] Figure 4 shows the amino acid sequence of sNPP1-Fc-D10 (SEQ ID NO: 4). The Fc sequence is underlined. The D10 (SEQ ID NO: 18) targeting region is shown in bold. [Figure 5] Figure 5 shows the serum pyrophosphate levels in wild-type mice after intravenous administration (1 hour after infusion) and subcutaneous administration (4 hours after infusion) of sNPP1-Fc or sNPP1-Fc-D10. [Figure 6] Figure 6 shows the prevention of aortic calcification in Enpp1(- / -) mice treated with sNPP1-Fc-D10. Enpp1(- / -) mice were subcutaneously treated with either the solvent or 6 mg / kg of sNPP1-Fc-D10 every other day for 21 days. Aortic calcium levels for males and females are shown. [Figure 7] Figure 7 shows serum PPi and enzyme activity levels in Enpp1(- / -) mice treated intravenously with 6 mg / kg sNPP1-Fc-D10. Plasma was collected at 0, 4, 24, 48, and 72 hours, and NPP1 activity (dashed line) and PPi levels (solid line) were analyzed. Wild-type PPi levels were measured at 2.18 μM (data not shown). The dashed line from top to bottom shows PPi levels for wild-type, heterozygous Enpp1(+ / -), and homozygous Enpp1(- / -)asj mice (Li et al., 2013). The sNPP1-Fc profile was similar to that of sNPP1-Fc-D10. [Figure 8]Figure 8 shows the increased survival rate of Enpp1asj homozygous male mice treated with 5 mg / kg of sNPP1-Fc compared to solvent-treated mice. Wild-type and Enpp1asj mice were placed on a high-phosphorus, low-magnesium diet from birth. From 14 days of age, the solvent or sNPP1-Fc (5 mg / kg) was administered subcutaneously every other day. Kaplan-Meier survival curves showed that more than 50% of asj mice died before 6 weeks, and all animals died by 9 weeks. In contrast, 50% of sNPP1-Fc-treated animals survived past 7 weeks and remained alive at 9 weeks. [Figure 9] Figures 9A and 9B show the increased weight gain rate of Enpp1asj male mice treated with 5 mg / kg of sNPP1-Fc (Figure 9B) compared to solvent-treated mice (Figure 9A). Wild-type and Enpp1asj mice were placed on a high-phosphorus, low-magnesium diet from birth. From 14 days of age, the solvent or sNPP1-Fc (5 mg / kg) was subcutaneously injected every other day. The weight gain rates of wild-type (solid line) and Enpp1asj (circles) mice were plotted from 2 weeks to 9 weeks of age. In the solvent group (upper panel), all Enpp1asj animals died at 9 weeks (white circles). In contrast, in the sNPP1-Fc treatment group, 5 Enpp1asj mice were alive at 9 weeks (black circles), and 5 had died (white circles). [Figure 10] Figures 10A-10C show photographs of wild-type mice (Figure 10A, top), solvent-treated Enpp1asj mice (Figure 10B, center), and sNPP1-Fc-treated (5 mg / kg) Enpp1asj mice (Figure 10C, bottom). [Figure 11] Figure 11 shows the levels of fibroblast growth factor 23 in solvent-treated wild-type, solvent-treated Enpp1asj / asj, and sNPP1-Fc-treated (5 mg / Kg) Enpp1as / asj mice. [Figure 12A] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12A shows the amino acid sequence of soluble NPP1 (SEQ ID NO: 5) containing amino acids 107-925 of SEQ ID NO: 1. [Figure 12B]Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12B shows the amino acid sequence of soluble NPP1 (SEQ ID NO: 6) containing amino acids 187-925 of SEQ ID NO: 1. [Figure 12C] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12C shows the amino acid sequence (SEQ ID NO: 7) of the Fc region of human IgG1, including the hinge region. [Figure 12D] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12D shows the amino acid sequence of human IgG1 Fc (SEQ ID NO: 8), including a partial hinge region. [Figure 12E] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12E shows the amino acid sequence (SEQ ID NO: 9) of the NPP1-Fc fusion protein. The NPP1 component contains SEQ ID NO: 5, and the Fc sequence contains a hinge region. [Figure 12F] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12F shows the amino acid sequence (SEQ ID NO: 10) of the NPP1-Fc fusion protein. Soluble NPP1 contains SEQ ID NO: 5, and the Fc sequence includes a partial hinge region. [Figure 12G] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12G shows the amino acid sequence of the NPP1-Fc fusion protein (SEQ ID NO: 11). Soluble NPP1 contains SEQ ID NO: 6, and the Fc sequence includes a hinge region. [Figure 12H] Figure 12 shows the amino acid sequences of the soluble NPP1 compound, fusion partner, and fusion protein. Figure 12H shows the amino acid sequence of the NPP1-Fc fusion protein (SEQ ID NO: 12). Soluble NPP1 contains SEQ ID NO: 6, and the Fc sequence includes a partial hinge region. [Figure 13]Figure 13 shows autoradiograms of thin-layer chromatograms demonstrating the activity of recombinant NPP1 in vitro and in vivo. Figure 13A shows 100 nM ATP incubated with 130 ug / ml sNPP1-Fc-D10 at 37°C for 1 hour. Figure 13 shows autoradiograms of thin-layer chromatograms demonstrating the activity of recombinant NPP1 in vitro and in vivo. Figure 13B shows 100 nM ATP incubated with plasma from wild-type mice (WT), Enpp1- / - mice, and Enpp1- / - mice 2 hours after IV infusion of recombinant NPP1 (6 mg / kg). Figure 13 shows autoradiograms of thin-layer chromatograms demonstrating the activity of recombinant NPP1 in vitro and in vivo. Figure 13C shows 100 nM ATP incubated in the aorta of wild-type mice (WT), Enpp1- / - mice, and recombinant NPP1 (6 mg / kg) IV infusion 2 hours later. Pi: orthophosphate; ATP: adenosine triphosphate; PPi: pyrophosphate. [Figure 14] Figures 14A and 14B are histograms showing the time course of plasma NPP1 activity (Figure 14A, top) and plasma pyrophosphate concentration (Figure 14B, bottom) in Enpp1(- / -) mice after subcutaneous injection of recombinant NPP1 (5 mg / kg). [Figure 15] Figure 15 is a scatter plot showing the relationship between plasma NPP1 activity and plasma pyrophosphate (PPi) in Enpp1(- / -) mice (circles) and wild-type mice (squares) at various time points after subcutaneous injection of recombinant NPP1 (5 mg / kg). [Figure 16] Figure 16 is a histogram showing the synthesis of pyrophosphates in human blood. Figure 16A shows heparinized blood or plasma obtained from the same blood sample. Figure 16B shows centrifuged blood cells suspended in HEPES-buffered saline, with or without the buffy coat (red blood cells). Figure 16C shows isolated leukocytes or platelets suspended in HEPES-buffered saline. Samples were incubated at 37°C for 2 hours with or without recombinant NPP1 (145 ug / ml). [Figure 17]Figure 17 is a histogram showing the effect of recombinant NPP1 on aortic calcification in Enpp1(- / -) mice. Recombinant NPP1 was subcutaneously injected every 48 hours (6 mg / kg) into mice fed a high-phosphate diet. Each bar represents one animal, with its age in weeks indicated below. M: male pair; F: female pair. The dashed line shows the mean calcium content of the aorta of wild-type littermates. [Figure 18] Figure 18 is a histogram showing the effect of recombinant NPP1 on aortic calcification in uric rats with renal failure. Uric rats fed a high-adenine diet were subcutaneously injected with either sNPP1-Fc-D10 or a control five times a week for 21 days (5 mg / kg). Each bar represents one animal approximately 4 months old. [Modes for carrying out the invention]

[0023] definition Unless otherwise defined, all scientific and technical terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0024] In this specification, when referring to measurable values ​​such as quantity or duration, the term "approximately" means to include variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, insofar as such variations are appropriate for carrying out the methods of this disclosure.

[0025] The term "abnormal PPi:Pi ratio" refers to the ratio of plasma PPi to serum Pi that is at least 10% or at least 20% higher or lower than the normal PPi:Pi ratio for that type of subject (e.g., human). Abnormal PPi:Pi ratios can occur due to lower-than-normal levels of plasma PPi or higher-than-normal levels of serum Pi. The PPi:Pi ratio is ([PPi] / [Pi]). * It is expressed as 1000, and the normal ratio for humans is approximately 1.75.

[0026] As used herein, the term “fragment” in relation to the NPP1 protein refers to a partial sequence of the full-length NPP1. A “fragment” of a protein or peptide can be at least about 20 amino acids long; for example, at least about 50 amino acids long; at least about 100 amino acids long; at least about 200 amino acids long; at least about 300 amino acids long; or at least about 400 amino acids long (and any integer value in between). The size of the fragment may range from 4 amino acid residues to a sequence that is only 1 amino acid less than the complete amino acid sequence. Therefore, a protein that “contains at least a portion of the amino acid sequence of SEQ ID NO: 1” includes the full-length NPP1 and its fragments.

[0027] As used herein, the term “elevated serum Pi” refers to a subject’s serum inorganic phosphate (Pi) level that is at least 110% of the normal Pi level in a subject of that type (e.g., human). Preferably, it is a subject’s serum Pi level that is at least about 120%, at least about 150%, at least about 200%, or at least about 300% of the normal Pi level in a subject of that type. The normal human Pi level has been reported to be 1.5 ± 0.5 mM (Rutsch, F. et al., Circ Cardiovasc Genet 1:133-140 (2008)).

[0028] "Isolated" or "purified" soluble NPP1 protein or its bioactive fragment or fusion protein is substantially free of cellular material or other contaminating proteins from the cell or tissue origin from which the NPP1 protein, bioactive fragment, or NPP1 fusion protein is derived, or, if chemically synthesized, substantially free of chemical precursors or other chemicals. The phrase "substantially free of cellular material" includes the preparation of NPP1 protein, bioactive fragment, or NPP1 fusion protein by isolating or recombining the protein from cellular components of cells that produce the protein. In one embodiment, the phrase "substantially free of cellular material" includes the preparation of an NPP1 protein, bioactive fragment, or NPP1 fusion protein that does not contain about 30% (dry weight) or more of non-NPP1 protein / fragment / fusion protein (also referred herein as "contamination protein"), more preferably about 20% or more of non-NPP1 protein / fragment / fusion protein, even more preferably about 10% or more of non-NPP1 protein / fragment / fusion protein, and most preferably about 5% or more of non-NPP1 protein / fragment / fusion protein. When recombinantly producing an NPP1 protein, fusion protein, or its bioactive fragment, it is preferable that the culture medium is also substantially free, i.e., the culture medium does not occupy about 20% or more, more preferably about 10% or more, and most preferably about 5% or more of the volume of the protein preparation.

[0029] As used herein, the term "low plasma PPi" refers to a plasma pyrophosphate (PPi) level in a subject that is 50% or less of the normal PPi level in that type of subject (e.g., human). Preferably, it is a plasma PPi level in a subject that is approximately 40%, 30%, 20%, or 10% or less of the normal PPi level in that type of subject. The normal PPi level in humans has been reported to be 2.63 ± 0.47 μM (O'Neill et al., Nephrol Dial Transplant 2010, 25, 187-191). Pyrophosphate can be quantified enzymatically using a suitable known method, such as the uridine diphosphoglucose (UDPG) method (Ryan, LMet al., Arthritis Rheum 1979, 22, 886-91).

[0030] Scope: Throughout this disclosure, various aspects of the present invention may be presented in range form. It should be understood that the range form is for convenience and conciseness only and should not be interpreted as an inflexible limitation of the scope of the present invention. Therefore, the range description should be considered to have all possible subranges specifically disclosed and the individual numbers within those ranges. For example, the range description 1 to 6 should be considered to have the specifically disclosed subranges, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as the individual numbers within this range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the range.

[0031] As used herein, the term "subject" includes both mammals and non-mammals. Examples of mammals include, but are not limited to, humans, chimpanzees, apes, monkeys, cattle, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds and fish.

[0032] As used herein, the term “therapeutic dose” means a non-toxic but sufficient amount of an active agent (e.g., sNPP1 protein) that results in improved treatment, cure, prevention, or remission of a disease, disorder, or side effect, or a reduced rate of disease or disorder progression, compared to a corresponding subject who has not received that amount. This term also includes amounts that are effective in enhancing normal physiological function.

[0033] The term “treatment” includes the application or administration of the NPP1 protein, fragments and fusion proteins of the present invention to a subject for the purpose of restoring, curing, alleviating, reducing, modifying, relieving, remitting, preventing, improving, or influencing a disease or disorder, or the application or administration of the NPP1 protein, fragments and fusion proteins of the present invention to a subject having an NPP1-related disease or disorder, or another disease or disorder associated with low levels of blood pyrophosphate, or another progressive disorder characterized by the accumulation of calcium and other mineral deposits (mineralization). The term “treatment” includes any sign of success in treating or relieving an injury, lesion or condition, including any objective or subjective parameters such as improvement; remission; reduction of symptoms or making the injury, lesion or condition more tolerable to the subject; slowing of the rate of degeneration or debilitation; reduction of debilitation at the end of degeneration; or improvement of the subject’s physical or mental health. Treatment may be therapeutic or preventive. Treatment or remission of symptoms may be based on objective or subjective parameters, including the results of a physical examination.

[0034] Treatment method This invention relates to the use of isolated recombinant human soluble NPP1 ("sNPP1") and its fusion proteins lacking the N-terminal region (i.e., lacking the cytosolic and transmembrane domains) for the treatment of NPP1-related diseases and disorders. Remarkably, the proteins of this invention can be used to increase NPP1 activity in vivo and to increase or restore normal levels of serum pyrophosphate (PPi) in subjects. The proteins of this invention can also be used to prevent the accumulation of calcium deposits in joints, kidneys, heart (e.g., aorta), arteries, blood vessels, or the posterior longitudinal ligament of the spine.

[0035] Subjects may be human patients with low levels of pyrophosphate, NPP1 deficiency (NPP1 deficiency), a disease or disorder associated with low levels of pyrophosphate, or a progressive disorder characterized by the accumulation of calcium and other mineral deposits in elastic fibers (mineralization). Mineralization can occur in the heart, arteries, blood vessels, kidneys, spinal ligaments, skin, eyes, and gastrointestinal tract.

[0036] More specifically, the NPP1 protein and NPP1 fusion protein of the present invention can be used to treat subjects with NPP1-related diseases or disorders, including, but not limited to, idiopathic infantile arterial calcification (IIAC), insulin resistance, hypophosphatemic rickets, and ossification of the posterior longitudinal ligament of the spine, or other diseases such as vascular calcification in chronic kidney disease (VCCKD), myocardial ischemia, articular calcification, retinal pigment streaks, and pseudoxanthoma elastica (PXE).

[0037] Soluble NPP1 protein, fragments, and their NPP1 fusion proteins can be used to treat a wide variety of pathological conditions in subjects. For example, the treatment of pathological conditions in mammals, such as human patients, that can be improved by reducing and / or removing one or more calcified structures and / or preventing the formation of calcified structures is within the scope of the present invention.

[0038] In one particularly useful embodiment, the condition being treated is systemic arterial calcification (also known as idiopathic infantile arterial calcification and infantile arterial medial calcification).

[0039] In other embodiments, conditions such as pseudoxanthoma elasticum, vascular calcification in chronic kidney disease, insulin resistance, hypophosphatemic rickets, or ossification of the posterior longitudinal ligament of the spine can also be treated using the method described herein.

[0040] Generally, the dosage of the fusion protein administered to a subject will vary depending on known factors such as age, the recipient's health status and weight, the type of concomitant therapy, and the frequency of treatment. Typically, the dosage of the active ingredient (i.e., the fusion protein) can be approximately 0.0001 to 50 mg per kg of body weight. The exact dosage, frequency of administration, and duration of treatment can be determined by a physician skilled in the field of therapeutic protein administration.

[0041] A preferred embodiment of the present invention relates to a method for treating NPP1-related disease or other calcification disorders, comprising the step of administering a therapeutically effective amount of isolated soluble NPP1 protein (sNPP1), a bioactive fragment, or an NPP1 fusion protein to a subject. As defined herein, the therapeutically effective amount (i.e., effective dose) of the protein is in the range of approximately 0.001 to 50 mg / kg body weight. Those skilled in the art will understand that certain factors may influence the dose required to effectively treat a subject, including, but not limited to, the severity of the disease, treatment history, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective amount of protein may consist of a single treatment, and preferably a series of treatments. It will also be understood that the effective dose of protein used in treatment may increase or decrease over the course of a particular treatment.

[0042] As defined herein, a therapeutically effective dose (i.e., effective dose) of a protein or polypeptide is in the range of about 0.001 to 50 mg / kg body weight, preferably about 0.01 to 25 mg / kg body weight, more preferably about 0.1 to 20 mg / kg body weight, and even more preferably about 1 to 10 mg / kg, 2 to 9 mg / kg, 3 to 8 mg / kg, 4 to 7 mg / kg, or 5 to 6 mg / kg body weight. Those skilled in the art will understand that certain factors may influence the dose required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, treatment history, the subject's overall health and / or age, and other pre-existing conditions. Furthermore, treatment of a subject with a therapeutically effective dose of protein, polypeptide, or antibody may consist of a single treatment, and preferably a series of treatments.

[0043] In preferred cases, the dosage ranges from approximately 0.1 to 20 mg / kg body weight, administered once a week, twice a week, once every 10 days, once every 12 days, once every 14 days, once every 17 days, once every 20 days, once every 25 days, or once every 30 days. It should also be understood that the effective dose of soluble sNPP1 protein, its bioactive fragment, or fusion protein used in treatment may increase or decrease with the course of a particular treatment.

[0044] This invention specifies a therapeutically effective dose of sNPP1, its bioactive fragment, or fusion protein to be administered to a patient for a period determined by a skilled practitioner in the medical field, ranging from once every 5 days to once every 30 days. In one embodiment, the period will be for the remainder of the patient's life. In one embodiment, the administration frequency is once every 5 days to once every 25 days. In another embodiment, the administration frequency is once every 5 days to once every 21 days. In yet another embodiment, the administration frequency is once every 7 days to once every 14 days. sNPP1, its bioactive fragment, or fusion protein can be administered once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, or once every 14 days. In some embodiments, sNPP1, its bioactive fragment, or fusion protein is administered approximately once a week. In other embodiments, sNPP1, its bioactive fragment, or fusion protein is administered approximately once every two weeks. In one embodiment, the administration frequency is approximately once every 30 days.

[0045] In one embodiment, the patient is under 2 years of age. In some embodiments, approximately 0.1 mg, approximately 0.2 mg, approximately 0.3 mg, approximately 0.4 mg, approximately 0.5 mg, approximately 1 mg, approximately 2 mg, approximately 3 mg, approximately 5 mg, approximately 10 mg, approximately 15 mg, approximately 20 mg, approximately 25 mg, approximately 30 mg, approximately 35 mg, approximately 40 mg, or approximately 45 mg of sNPP1, a bioactive fragment, or a fusion protein is administered to a patient with NPP1 deficiency or other calcification disease. In some embodiments, approximately 0.5 to approximately 30 mg, approximately 0.5 to approximately 20 mg, approximately 0.5 to approximately 10 mg, or approximately 0.5 to approximately 5 mg is administered to the patient.

[0046] In one embodiment, approximately 1 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 2 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 3 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 4 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 5 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 6 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 7 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In one embodiment, approximately 8 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In another embodiment, approximately 9 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week. In yet another embodiment, approximately 10 mg / kg of sNPP1, a bioactive fragment, or a fusion protein is administered to the patient once a week.

[0047] In some embodiments, the patient's pre-treatment blood PPi level is about 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% of the normal PPi level observed in a normal human individual. In one embodiment, the patient's pre-treatment PPi level is about 50% or less of the normal PPi level observed in a normal human individual. In one embodiment, the patient's pre-treatment PPi level is about 40% or less of the normal PPi level observed in a normal human individual. In some embodiments, the patient's pre-treatment PPi level is about 30% or less of the normal PPi level observed in a normal human individual. In some embodiments, the patient's pre-treatment PPi level is about 30% or less of the normal PPi level observed in a normal human individual. In some embodiments, the patient's pre-treatment PPi level is about 20% or less of the normal PPi level observed in a normal human individual. In some embodiments, the patient's PPi level before treatment is less than or equal to about 10% of the normal PPi level observed in a healthy human individual. In some embodiments, the patient's PPi level before treatment is less than or equal to about 5% of the normal PPi level observed in a healthy human individual. In some embodiments, the patient does not exhibit a measurable PPi before treatment.

[0048] sNPP1, bioactive fragments, or fusion proteins can be administered, for example, by subcutaneous injection, intramuscular injection, and intravenous (IV) infusion or administration.

[0049] In one embodiment, sNPP1, a bioactive fragment, or a fusion protein is administered intravenously by IV infusion using any convenient method. In one example, sNPP1, a bioactive fragment, or a fusion protein can be administered by intravenous infusion via a peripheral line. In another example, sNPP1, a bioactive fragment, or a fusion protein can be administered by intravenous infusion via a peripherally inserted central venous catheter.

[0050] In another embodiment, sNPP1, a bioactive fragment, or a fusion protein is administered intravenously by IV injection.

[0051] In another embodiment, sNPP1, a bioactive fragment, or a fusion protein may be administered by intraperitoneal injection.

[0052] In another embodiment, sNPP1, a bioactive fragment, or a fusion protein may be administered by subcutaneous injection.

[0053] In another embodiment, sNPP1, a bioactive fragment, or a fusion protein may be administered by intramuscular injection.

[0054] In yet another embodiment, sNPP1, a bioactive fragment, or a fusion protein is administered in a capsule that is acceptable as a therapeutic protein drug. For example, the capsule may be an enteric-coated gelatin capsule.

[0055] In one embodiment, the method comprises administering the soluble NPP1 protein or NPP1 fusion protein of the present invention alone or in combination with other active agents(s). In one embodiment, the method comprises administering the NPP1 protein or NPP1 fusion protein of the present invention as a treatment to compensate for reduced or abnormal NPP1 expression or activity in subjects with NPP1 deficiency or other related diseases or disorders.

[0056] In one embodiment, isolated sNPP1 proteins, fragments, and fusion proteins can be administered before, after, or simultaneously with an active agent, or co-administered with other known therapeutic agents. Co-administration of the isolated sNPP1 proteins, fragments, and fusion proteins of the present invention with other therapeutic agents may result in two active agents acting by different mechanisms, leading to increased therapeutic effects. Such co-administration can solve the problem of drug resistance development.

[0057] In certain embodiments, this disclosure relates to a method for reducing vascular calcification in subjects in need. This method is based on the surprising discovery that administering a soluble form of NPP1 to animals having low plasma PPi levels (inhibitors or tissue calcification) or high serum Pi levels can transiently increase plasma PPi in the animals, and that this transient increase in plasma PPi can inhibit vascular calcification in the animals. Because the increase in plasma PPi is transient, the treatment can be modified to inhibit undesirable or pathological tissue calcification, such as vascular calcification, without inhibiting bone calcification or inducing osteomalacia.

[0058] In general, this disclosure relates to a method for reducing tissue calcification (e.g., vascular calcification) in a subject by administering soluble NPP1 (sNPP1) to the subject two or more times. Each dose contains an amount of soluble NPP1 sufficient to achieve a transient increase in plasma PPi in the subject, preferably returning to baseline PPi levels within about 48 hours after the dose. The period between each dose is generally at least two days.

[0059] Subjects requiring treatment can be of any age and sex, and preferably have low plasma PPi or high serum Pi (e.g., an abnormal PPi:Pi ratio). Low plasma PPi may result from, for example, congenital NPP1 deficiency, mutations in the gene encoding NPP1 leading to decreased expression or enzyme activity of active NPP1 (associated with NPP1 deficiency and autosomal recessive hypophosphatemic rickets), and mutations in the gene encoding MRP6 leading to the absence or dysfunction of MRP6 protein (associated with pseudoxanthoma elastica). Low plasma PPi or high serum Pi are also frequently seen in patients with chronic kidney disease, end-stage renal disease / failure, diabetes mellitus, and other conditions. Therefore, subjects requiring treatment may have chronic kidney disease (CKD), end-stage renal disease (ESRD), generalized arterial calcification in infants (GACI), type II diabetes mellitus, autosomal recessive hypophosphatemic rickets, cardiovascular disorders, atherosclerosis, and / or pseudoxanthoma elastica (PXE). The subjects are generally humans, but any other suitable mammal or non-mammalian may be used.

[0060] Tissue calcification is a progressive process, and individuals born with a congenital NPP1 deficiency may not show tissue calcification for several years. By initiating treatment as early as possible, calcification may be reduced and / or minimized in such subjects. In subjects with low plasma PPi levels or high serum Pi levels not due to germ cell mutations (e.g., those with an abnormal plasma PPi:Pi ratio), treatment should be initiated as early as possible (i.e., immediately after diagnosis of a condition, such as chronic kidney disease (CKD) or end-stage renal disease (ESRD)). In some embodiments, subjects being treated may be between 1 month and 24 months of age, under 1 year, under 2 years, under 3 years, under 4 years, or under 5 years.

[0061] Each single dose of sNPP1 administered to a subject contains an amount of sNPP1 sufficient to achieve a transient increase in plasma PPi. Preferably, the transient increase is characterized by a peak PPi level that is at least about 40% of the normal plasma PPi level, at least about 50% of the normal plasma PPi level, at least about 60% of the normal plasma PPi level, at least about 70% of the normal plasma PPi level, at least about 80% of the normal plasma PPi level, about 40% to 100% of the normal plasma PPi level, about 50% to 100% of the normal plasma PPi level, about 60% to 100% of the normal plasma PPi level, about 70% to 100% of the normal plasma PPi level, about 80% to 100% of the normal plasma PPi level, or about 100% to 200% of the normal plasma PPi level.

[0062] Preferably, the transient increase in plasma PPi after sNPP1 administration is maintained for at least about 4 hours, at least about 6 hours, at least about 8 hours, at least about 10 hours, or at least about 12 hours. Furthermore, it is preferable that the transient increase in plasma PPi returns to the subject's baseline PPi level within about 48 hours after a single dose, within about 3 days after a single dose, or within about 4 days after a single dose.

[0063] The pre-treatment low plasma PPi of the subject is approximately 50% or less, preferably 40% or less, of the normal PPi level observed in a normal subject (e.g., a human). In some embodiments, the pre-treatment subject's PPi level is approximately 30% or less of the normal PPi level. In other embodiments, the pre-treatment subject's PPi level is approximately 20% or less of the normal PPi level. In some other embodiments, the pre-treatment subject's PPi level is approximately 10% or less of the normal level. In some embodiments, the subject may not have a measurable PPi before treatment.

[0064] The elevated serum Pi levels of subjects before treatment are approximately 110% or more, preferably 125% or more, of the normal Pi levels observed in normal subjects (e.g., humans). In some embodiments, the Pi levels of subjects before treatment are approximately 150% or more of the normal PPi levels. In other embodiments, the Pi levels of subjects before treatment are approximately 200% or more of the normal PPi levels. In some other embodiments, the Pi levels of subjects before treatment are approximately 300% or more of the normal levels. While we do not wish to be bound by any particular theory, it is thought that elevated plasma Pi levels can be compensated for by inducing a transient increase in serum PPi, thereby temporarily restoring a normal or near-normal PPi:Pi ratio, and thereby inhibiting tissue calcification promoted by higher-than-normal levels of serum Pi.

[0065] The amount of sNPP1 sufficient to achieve a transient increase in plasma PPi can be easily determined by a clinician with the usual skills, for example, by administering the dose expected to produce a transient increase in plasma PPi, determining whether a transient increase occurs, and then making appropriate adjustments to the dose. The amount administered will be influenced by several conventional factors, including the specific sNPP1 used, age, the subject's health status and weight, the subject's sensitivity to the drug, and other relevant factors. Typically, the amount of sNPP1 administered in each dose is approximately 0.001 to 50 mg per kg of body weight, with preferred doses being 1 mg / kg to 5 mg / kg, 1 mg / kg to 10 mg / kg, 1 mg / kg to 20 mg / kg, 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, or 20 mg / kg. The exact dosage, frequency of administration, and duration of treatment can be determined by a physician skilled in the field of therapeutic protein administration.

[0066] In some preferred embodiments, each dose contains approximately 1.0 mg to 5.0 mg of sNPP1 per kg of body weight, approximately 1.0 mg to 10.0 mg of sNPP1 per kg of body weight, or approximately 1.0 mg to 20.0 mg of sNPP1 per kg of body weight.

[0067] The interval between doses is selected to allow the subject's serum PPi levels to return to baseline levels and is at least 2 days (48 hours), but can be longer as desired or instructed. For example, the interval between doses can be 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 12 days, 2 weeks, 3 weeks, or approximately 1 month.

[0068] In general, it is desirable to initiate treatment according to the methods described herein as soon as possible after diagnosis of low plasma PPi, high serum Pi, or NPP1 deficiency. Subjects born with congenital NPP1 deficiency may not show tissue calcification for several years. Initiating treatment as early as possible may reduce and / or minimize calcification in such subjects. In subjects with low plasma PPi levels or high serum Pi not due to germline mutations, treatment should be initiated as soon as possible after diagnosis of a condition such as chronic kidney disease (CKD) or end-stage renal disease (ESRD).

[0069] This method provides an effective way to reduce tissue calcification (e.g., vascular calcification) in subjects with low plasma PPi or high serum Pi, including those with an abnormal ratio of PPi to Pi. The tissue calcification is preferably vascular calcification, preferably arterial calcification, but can also be venous calcification. The vascular calcification can be intima or medial. Subjects treated according to the method herein may have NPP1 deficiency, systemic arterial calcification (GACI), also known as idiopathic infantile arterial calcification and infantile arterial medial calcification. Subjects treated may also have cardiovascular disorders, such as coronary artery disease and / or atherosclerosis. Subjects treated may have chronic kidney disease (CKD) or end-stage renal disease (ESRD). Subjects treated may have diabetes mellitus (e.g., type II diabetes). Subjects treated may have pseudoxanthoma elasticum (PXE).

[0070] sNPP1 can be administered by any preferred method or route of administration, such as extraintestinal, oral, or inhalation. Extraintestinal administration, such as intravenous infusion or drip infusion, subcutaneous injection, intraperitoneal injection, or intramuscular injection, is preferred.

[0071] If necessary, sNPP1 may be administered with one or more concomitant therapeutic agents. For concomitant therapy, sNPP1 and one or more other therapeutic agents are administered in such a way that their individual pharmacological activities substantially overlap in the subject. Therefore, any concomitant therapeutic agent can be administered before, simultaneously with, or after the administration of sNPP1. Concomitant therapy may result in two active agents acting by different mechanisms that lead to an increased therapeutic effect.

[0072] In addition to causing a transient increase in serum PPi, it is thought that administering sNPP1 according to the method described herein may alter the levels of specific proteins in subjects. For example, although we do not wish to be bound by any particular theory, it is thought that administering sNPP1 according to the method described herein may decrease the levels of osteopontin, osteoprotegerin, and fibroblast growth factor 23 (FGF-23) in subjects. Therefore, in addition to plasma PPi and serum Pi levels, the levels of these proteins may also be used to monitor the treatment and adjust the dosage.

[0073] sNPP1 The present invention utilizes soluble NPP1 containing the biologically active NPP1 domain of NPP1 (i.e., an NPP1 component containing at least one extracellular catalytic domain of spontaneously occurring NPP1 for pyrophosphatase and / or phosphodiesterase activity). The soluble NPP1 protein of the present invention contains at least an NPP1 domain essential for exerting pyrophosphatase and / or phosphodiesterase activity.

[0074] In one embodiment, soluble NPP1, its fragments, and fusion proteins can form functional homodimers or monomers. In a preferred embodiment, the soluble NPP1 protein or its NPP1 fusion protein can be assayed for pyrophosphatase activity and the ability to increase pyrophosphate levels in vivo.

[0075] The preferred soluble NPP1 protein and NPP1 fusion protein of the present invention are enzymatically active in vivo (e.g., in humans). In one embodiment, the soluble protein comprises an amino acid sequence having at least 60, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity to the following sequences. JPEG0007856872000001.jpg58150 (Sequence ID 2)

[0076] Any desired enzymatically active form of soluble NPP1 can be used in the methods described herein. Enzymatically active sNPP1 can increase PPi levels in suitable enzyme assays and can be assayed for pyrophosphatase activity, phosphodiesterase activity, or pyrophosphatase and phosphodiesterase activity. Typically, sNPP1 contains at least the N-terminal cytosolic domain and the NPP1 component lacking the transmembrane domain of spontaneously occurring transmembrane NPP1. In a preferred embodiment, the NPP1 component contains the cysteine-rich region (amino acids 99-204 of SEQ ID NO: 1) and catalytic region (amino acids 205-591 of SEQ ID NO: 1) of spontaneously occurring human NPP1. Typically, the NPP1 component also contains the C-terminal region (amino acids 592-925 of SEQ ID NO: 1) and has the amino acid sequence of SEQ ID NO: 2. However, the C-terminal region can be cleaved as needed. Therefore, preferred NPP1 components include the cysteine-rich region and catalytic region of human NPP1 (amino acids 99-591 of SEQ ID NO: 1) or the cysteine-rich region, catalytic region, and C-terminal region of human NPP1 (SEQ ID NO: 2). Other preferred NPP1 components contain only a portion of the cysteine-rich domain and have the sequence of amino acids 107-925 of SEQ ID NO: 1 or amino acids 187-925 of SEQ ID NO: 1.

[0077] The cysteine-rich region of NPP1 (i.e., amino acids 99-204 of SEQ ID NO: 1) facilitates the dimerization of sNPP1. sNPP1 can exist as a monomeric form of a functional homodimer, including in fusion proteins.

[0078] The amino acid sequence of the NPP1 component can be a variant of the naturally occurring NPP1 sequence, provided that the NPP1 component is enzymatically active. The NPP1 variant is enzymatically active and has at least 80%, at least 85%, at least 90%, at least 95%, and more preferably at least 96%, amino acid sequence identity with the corresponding portion of human NPP1 (e.g., the length of the cysteine-rich region, catalytic region, C-terminal region, cysteine-rich region and catalytic region, and cysteine-rich region, catalytic region and C-terminal region). Preferred NPP1 variants have at least 90%, preferably at least 95%, more preferably at least 97% amino acid sequence identity to (i) the amino acid sequence of residues 205-591 of SEQ ID NO: 1, (ii) the amino acid sequence of residues 99-591 of SEQ ID NO: 1, (iii) the amino acid sequence of residues 99-925 of SEQ ID NO: 1, (iv) the amino acid sequence of residues 107-925 of SEQ ID NO: 1, or (v) the amino acid sequence of residues 187-925 of SEQ ID NO: 1. Preferred locations for amino acid mutations are known from NPP1 structural studies and analyses of disease-related mutations in NPP1. For example, the following amino acid substitutions occur in certain disease-associated mutations that reduce NPP1 enzyme activity, and mutations at these positions should be avoided: Ser216, Gly242, Pro250, Gly266, Pro305, Arg349, Tyr371, Arg456, Tyr471, His500, Ser504, Tyr513, Asp538, Tyr570, Lys579, Gly586, Tyr659, Glu668, Cys726, Arg774, His777, Asn792, Asp804, Arg821, Arg888, and Tyr901. (See, for example, Jansen, S. et al., Structure 20:1948-1959 (2012)).

[0079] In one embodiment, the soluble NPP1 protein can be a fusion protein formed by recombinant fusion or chemical bonding (e.g., covalent, ionic, hydrophobic, and van der Waals forces) with a fusion partner. In another embodiment, the fusion protein has at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4.

[0080] To determine the degree of agreement between two amino acid sequences, the sequences are aligned for optimal comparison (for example, gaps can be inserted in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, allowing non-homologous sequences to be ignored for comparison). In a preferred embodiment, the length of the reference sequence to be aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., the sNPP1 amino acid sequence of SEQ ID NO: 2; amino acids 107-925 of SEQ ID NO: 1 or amino acids 187-925 of SEQ ID NO: 1). Then, the amino acid residues or nucleotides at the corresponding amino acid positions are compared. If there is an amino acid residue or nucleotide at a certain position in the first sequence that is the same as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acids correspond to amino acid or nucleic acid "homology"). The agreement rate between two sequences is a function of the number of identical positions shared by those sequences, taking into account the number of gaps that need to be inserted for optimal alignment of the two sequences, and the length of each gap.

[0081] Sequence comparison and determination of the agreement rate between two sequences can be performed using mathematical algorithms. In a preferred embodiment, the agreement rate between two amino acid sequences is determined using the Needleman and Wunsch (J Mol Biol 1970, 48, 444-453) algorithm, which is incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either the Blosum62 matrix or the PAM250 matrix, as well as gap weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6. In another embodiment, the agreement rate between two amino acids is determined using the E. Meyers and W. Miller algorithm (CABIOS, 1989, 4, 11-17), which is incorporated into the ALIGN program (version 2.0 or 2.0U), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4.

[0082] sNPP1 consists of or is essentially composed of the NPP1 components described herein. Alternatively, sNPP1 may be in the form of a fusion protein containing the NPP1 components and one or more other polypeptides called fusion partners, optionally via a linker suitable in each case, or as a conjugate between the NPP1 components and another molecule (e.g., PEG). When sNPP1 is in the form of a fusion protein, each fusion partner is preferably located at the C-terminus of the NPP1 components. While we do not wish to be bound by any particular theory, fusion proteins containing NPP1 components with cysteine-rich and catalytic regions, as well as one or more fusion proteins located at the C-terminus of the NPP1 components, are considered preferable to NPP1 fusion proteins of other configurations because they can be expressed at sufficient levels and are sufficiently stable for use as therapeutic proteins.

[0083] Any suitable fusion partner can be included in the fusion protein. Several fusion partners are known in the art that can advantageously provide specific benefits, such as reduced aggregation and immunogenicity, increased solubility, improved expression and / or stability, and improved pharmacokinetic and / or pharmacodynamic performance. See, for example, Strohl, WRBioDrugs 29:215-239 (2015). For example, albumin, albumin fragments, or albumin variants (e.g., human serum albumin and its fragments or variants) can be incorporated into the fusion protein, and such fusion proteins are known to be readily purifiable, stable, and have an improved plasma half-life. Suitable albumins, albumin fragments, or albumin variants that can be used in sNPP1 fusion proteins are disclosed, for example, in WO2005 / 077042A2 and WO03 / 076567A2, the entire contents of which are incorporated herein by reference. Fusion to human transferrin is also known to improve half-life. See, for example, Kim BJ et al., J Pharmacol Expr Ther 334(3):682-692(2010); and WO2000 / 020746. Peptides that bind to albumin or transferrin, such as antibodies or antibody fragments, can also be used. See, for example, EP0486525B1, US6,267,964B1, WO04 / 001064A2, WO02 / 076489A1, WO01 / 45746, WO2006 / 004603, and WO2008 / 028977. Similarly, immunoglobulin Fc fusion proteins are known. For example, see Czajkowsky DM et al., EMBO Mol Med 4(10):1015-1028 (2012), U.S. Patent No. 7,902,151, and U.S. Patent No. 7,858,297, the entirety of which is incorporated herein by reference.The fusion protein may also contain a CTP sequence (see also Fares et al., Endocrinol 2010, 151, 4410-4417; Fares et al., Proc Natl Acad Sci 1992, 89, 4304-4308; and Furuhashi et al., Mol Endocrinol 1995, 9, 54-63). The fusion partner is preferably an immunoglobulin Fc (e.g., Fc or human IgG1). Fc may include the CH1, CH2, and CH3 of human IgG1, and optionally the human IgG1 hinge region (EPKSCDKTHTCPPCP (SEQ ID NO: 13)) or a portion of the human IgG1 hinge region (e.g., DKTHTCPPCP (SEQ ID NO: 14) or PKSCDKTHTCPPCP (SEQ ID NO: 15)). In some fusion proteins, Fc may include the CH2 and CH3 of human IgG1, or, if necessary, the Fc of human IgG2 or human IgG4.

[0084] Preferably, the sNPP1 fusion protein includes an NPP1 component and a peptide that increases the half-life of the fusion protein, most preferably an immunoglobulin Fc (e.g., Fc or human IgG1). As used herein, “protein that increases the half-life of the fusion protein” means a protein that, when fused to soluble NPP1 or a bioactive fragment, increases the half-life of the soluble NPP1 polypeptide or bioactive fragment compared to the half-life of the soluble NPP1 polypeptide alone or the NPP1 bioactive fragment alone.

[0085] In one embodiment, the half-life of the NPP1 fusion protein is increased by 50% compared to the half-life of the NPP1 polypeptide or bioactive fragment alone. In another embodiment, the half-life of the NPP1 fusion protein is increased by 60% compared to the half-life of the NPP1 polypeptide or bioactive fragment alone. In yet another embodiment, the half-life of the NPP1 fusion protein is increased by 70% compared to the half-life of the NPP1 polypeptide or bioactive fragment alone. In yet another embodiment, the half-life of the NPP1 fusion protein is increased by 80% compared to the half-life of the NPP1 polypeptide or bioactive fragment alone. In yet another embodiment, the half-life of the NPP1 fusion protein is increased by 90% compared to the half-life of the NPP1 polypeptide or bioactive fragment alone.

[0086] In another embodiment, the half-life of the NPP1 fusion protein is 2, 3, 4, 5, 6, 7, 8, 9, or 10 times longer than the half-life of the NPP1 polypeptide or bioactive fragment alone. Methods for determining the half-life of a protein or fusion protein are known in the art. For example, numerous methods for testing the half-life of a protein are disclosed in Zhou et al., Determining Protein Half-Lives, Methods in Molecular Biology 2004, 284, 67-77. If necessary, the fusion protein can be conjugated to a polymer or other suitable compound that extends the half-life, for example, polyethylene glycol (PEG) can be conjugated to the NPP1 fusion protein.

[0087] In one embodiment, the peptide that increases the half-life of the fusion protein is a CTP sequence (see also Fares et al., 2010, Endocrinol., 151(9):4410-4417; Fares et al., 1992, Proc. Natl. Acad. Sci, 89(10):4304-4308; and Furuhashi et al., 1995, Molec. Endocrinol., 9(1):54-63).

[0088] In another embodiment, the peptide that increases the half-life of the fusion protein is the Fc domain of Ig.

[0089] Fusion partners may also be selected to target the fusion protein to a desired clinically or biologically important site (e.g., a site of calcification). For example, a peptide with high affinity for bone is described in U.S. Patent No. 7,323,542, the entire teaching of which is incorporated herein by reference. Peptides capable of increasing protein targeting to calcification sites may contain a sequence of at least about four acidic amino acids, such as glutamic acid or aspartic acid. Typically, a peptide that targets a fusion protein to a calcification site will contain 4 to 20 consecutive acidic amino acids, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids selected from glutamic acid or aspartic acid. The peptide may consist of glutamic acid residues only, aspartic acid residues only, or a mixture of glutamic acid and aspartic acid residues. A particularly preferred portion for targeting to calcification sites is Asp 10 (Sequence number 18).

[0090] In one embodiment, the NPP1 fusion protein of the present invention comprises an NPP1 polypeptide and a moiety that increases protein targeting to calcification sites, such as a sequence of acidic amino acids, such as glutamic acid or aspartic acid.

[0091] Suitable peptide linkers for use in fusion proteins are known, typically incorporating flexible and long structures that do not interfere with the function of the NPP1 component or fusion partner. Peptide linker sequences may contain any combination of Gly, His, Asn, and Ser residues. Useful peptide linkers include, but are not limited to, polyGly, polyHis, polyAsn, or polySer. Other nearly neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Amino acid sequences that can be usefully used as linkers are disclosed in Maratea et al., Gene 1985, 40, 39-46; Murphy et al., Proc Natl Acad Sci USA 1986, 83, 8258-8262; and U.S. Patents 4,935,233 and 4,751,180. Other suitable linkers can be obtained from spontaneously occurring proteins, such as the hinge regions of immunoglobulins. The preferred synthetic linker is (Gly4Ser) n Here, n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 19). It is preferable that n is 3 or 4. For example, in some embodiments, the linker is (Gly4Ser)3 (SEQ ID NO: 16), and the fusion protein contains a linker with the amino acid sequence GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer (SEQ ID NO: 16). Typically, the linker is 1 to about 50 amino acid residues long, or 1 to about 25 amino acids long. Often, the linker is about 8 to about 20 amino acids long.

[0092] A preferred NPP1 fusion protein comprises an NPP1 component from the N-terminus to the C-terminus, optionally a linker, an immunoglobulin Fc region (e.g., optionally human IgG1 Fc including the hinge or a portion thereof), optionally a second linker, and optionally a targeting region. Therefore, if the Fc region and optionally a targeting region are present, they are located C-terminal to the NPP1 component. The NPP1 component preferably comprises the cysteine-rich region and catalytic domain of NPP1, lacks the N-terminal cytosolic domain and transmembrane domain, and optionally contains a C-terminal region.

[0093] A preferred fusion protein includes the NPP1 component, comprising the cysteine-rich domain, catalytic domain, and C-terminal region of human NPP1 from the N-terminus to the C-terminus; and an Fc region comprising the hinge of human immunoglobulin. The Fc region is preferably derived from human IgG1. In certain embodiments, the fusion protein has 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% sequence identity to SEQ ID NO: 3. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO: 3.

[0094] Another preferred fusion protein comprises an NPP1 component from the N-terminus to the C-terminus, including the cysteine-rich domain, catalytic domain, and C-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO: 16)); and an Fc region including a hinge of human immunoglobulin. The Fc region is preferably derived from human IgG1.

[0095] Another preferred fusion protein comprises, from the N-terminus to the C-terminus, an NPP1 component comprising the cysteine-rich domain, catalytic domain, and C-terminal region of human NPP1; an Fc region comprising the hinge or a part thereof of human immunoglobulin; and a moiety that targets the fusion protein to a site of calcification. The Fc region is preferably derived from human IgG1. The moiety that targets the fusion protein to a site of calcification is Asp 10 (SEQ ID NO: 18), preferably. The Fc region is derived from human IgG1, and the moiety that targets the fusion protein to a site of calcification is Asp 10 (SEQ ID NO: 18), more preferably. In certain embodiments, the fusion protein has 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% sequence identity to SEQ ID NO: 4. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO: 4.

[0096] Another preferred fusion protein comprises, from the N-terminus to the C-terminus, an NPP1 component comprising the cysteine-rich domain, catalytic domain, and C-terminal region of human NPP1; a linker (e.g., (Gly4Ser)3 (SEQ ID NO: 16)); an Fc region comprising the hinge or a part thereof of human immunoglobulin; and a moiety that targets the fusion protein to a site of calcification. The Fc region is preferably derived from human IgG1. The moiety that targets the fusion protein to a site of calcification is Asp 10 (SEQ ID NO: 18), preferably. The Fc region is derived from human IgG1, and the moiety that targets the fusion protein to a site of calcification is Asp 10 (SEQ ID NO: 18), more preferably.

[0097] Another preferred fusion protein includes an NPP1 component from the N-terminus to the C-terminus, comprising a portion of the cysteine-rich domain of human NPP1, a catalytic domain, and a C-terminal region; optionally a linker (e.g., (Gly4Ser)3 (SEQ ID NO: 16)); and an Fc region comprising the hinge or a portion of human immunoglobulin. The Fc region is preferably derived from human IgG1. In certain embodiments, the fusion protein has 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% sequence identity to SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. A preferred fusion protein of this type has the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

[0098] In a particularly preferred embodiment, the fusion protein of SEQ ID NO: 3 is administered according to the method described herein. In another preferred embodiment, the fusion protein of SEQ ID NO: 4 is administered according to the method described herein. In another preferred embodiment, the fusion protein of SEQ ID NO: 9 is administered according to the method described herein. In another preferred embodiment, the fusion protein of SEQ ID NO: 10 is administered according to the method described herein. In another preferred embodiment, the fusion protein of SEQ ID NO: 11 is administered according to the method described herein. In another preferred embodiment, the fusion protein of SEQ ID NO: 12 is administered according to the method described herein.

[0099] The fusion proteins of the present invention can be prepared using standard methods, including recombinant techniques or chemical bonding, known in the art. Techniques useful for isolating and characterizing the nucleic acids and proteins of the present invention are known to those skilled in the art, and standard molecular biology and biochemistry manuals can be consulted to select suitable procedures for use without excessive experimentation. For example, Sambrook et al., 1989, “Molecular Cloning: A Laboratory Manual”, 2nd See ed., Cold Spring Harbor, and its entire contents are incorporated herein by reference.

[0100] Isolated recombinant human sNPP1, its fragments, and fusion proteins can be produced in any useful protein expression system, but are not limited to, cell cultures (e.g., CHO cells, COS cells, HEK203), bacteria (e.g., Escherichia coli (E. coli)), and genetically modified animals, including mammals and birds (e.g., chickens, quail, ducks, and turkeys). For expression, the construct encoding sNPP1 includes a suitable signal sequence (e.g., human Ig heavy chain, NPP2, NPP4, NPP7, or, for example, derived from human serum albumin) within a frame having the sNPP1 sequence, and is responsively linked to a suitable expression regulatory element.

[0101] sNPP1, including its fusion protein and physiologically acceptable salt forms, is formulated into pharmaceutical compositions for administration, typically according to the methods described herein. The pharmaceutical compositions typically include pharmaceutically acceptable carriers or excipients. Compositions containing such carriers, including the complex molecule, are prepared by known conventional methods (e.g., Remington's Pharmaceutical Sciences, 14). thFormulated according to (see ed., Mack Publishing Co., Easton, PA), the entire teaching is incorporated herein by reference. The carrier may include a diluent. In one embodiment, the pharmaceutical carrier may be a liquid and the fusion protein may be in solution form. The pharmaceutical carrier may be a wax, fat, or alcohol. In another embodiment, the pharmaceutically acceptable carrier may be a solid in the form of a powder, lyophilized powder, or tablet. In one embodiment, the carrier may include liposomes or microcapsules. The pharmaceutical composition may be in the form of a sterile lyophilized powder for reconstitution with a diluent and injection. The diluent may be water for injection, bacteriostatic water for injection, or sterile saline. The lyophilized powder may be produced by lyophilizing a solution of the fusion protein to obtain a dry form of the protein. As is known in the art, lyophilized proteins are generally more stable and have a longer shelf life than protein solutions. [Examples]

[0102] The present invention is further illustrated by the following embodiments. These embodiments are for illustrative purposes only and are not intended to limit the present invention in any way, nor should they be interpreted as limiting it.

[0103] method animal: Six-week-old wild-type male C57Bl / 6J mice were used. The mean body weight of these mice ranged from 21 to 22 g. The mice were administered sNPP1-Fc [1.04 mg / ml] or sNPP1-Fc-D10 [1.03 mg / ml] at a concentration of 5 mg / kg by subcutaneous (SC) or intravenous (IV) injection. Table 1 TIFF0007856872000002.tif127166

[0104] Two different strains of mice lacking NPP1 were used. - / -The mouse model had previously been described in Lomashvili, KA et al., Kidney Int 2014, 85, 1351-1356. To accelerate arterial calcification, a 1.5% phosphate mixture (final phosphorus content: 2%) was added to the diet using a ratio of NaH2PO4 and Na2HPO4 at a neutral pH, as previously described (O'Neill, WC et al., Kidney Int 2011, 79, 512-517).

[0105] Chronic kidney disease (CKD) model: Wild-type Sprague Dolly rats were used in a CKD model study. Rats were fed a diet containing 0.25–0.75% adenine and high levels of phosphorus (0.75–0.9% phosphorus compared to 0.4% in a normal diet). Excess dietary adenine saturates the normal adenine phosphoribosyltransferase salvage pathway and is instead metabolized to 2,8-dihydroxyadenine, which, due to its low solubility, precipitates in the renal tubules and forms crystals. These crystals cause tubular damage, inflammation, obstruction, and renal fibrosis, leading to a phenotype consistent with human CKD. The resulting renal damage and failure lead to impaired phosphate excretion, resulting in abnormally high serum phosphate levels and mineral metabolism disorders, such as systemic calcification of soft tissues. High levels of phosphorus in the diet accelerate arterial calcification. Rats fed a high-adenine diet develop uremia, hyperphosphatemia, secondary hyperparathyroidism, renal osteodystrophy, and vascular calcification.

[0106] Plasma preparation: Blood was collected by cardiac puncture and immediately mixed (9:1 volume:volt blood vs. 110 mM citrate solution). Serum collection may result in excessive pyrophosphate (PPi) release from platelets, and EDTA inhibition of coagulation may interfere with the assay. The tubes containing citrated blood were nutated for several minutes, then centrifuged at 2,000 × g for 10–15 minutes. The top layer of plasma was collected (100–300 μl), and approximately 200 μl was added to a 10 kDa Centricon. These tubes were then centrifuged at 12,000 × g for 10 minutes to deproteinize the plasma. After centrifugation, the flow-through fluid was collected in a new tube. Plasma and deproteinized samples were frozen at -20°C until analysis.

[0107] Fluorescence PPi assay: This assay uses a fluorescence-generating PPi sensor that exhibits fluorescence intensity proportional to the PPi concentration. A 10 kDa filtered sample (4 μl) was added to 46 μl of assay buffer. The PPi sensor stock solution (200×) was diluted with assay buffer, and 50 μl of this solution was added to the sample. After incubation at room temperature for 20 minutes, fluorescence (Ex / Em = 316 / 456 nm) was read in a black, plain 96-well plate.

[0108] Assay: NPP1 activity was measured as previously described (Villa-Bellosta, R. et al., Am J Physiol Heart Circ Physiol 2011, 301, H61-H68). Briefly, plasma was added to 20 times the volume of physiological buffer containing 200 nM ATP and 1.5 uCi[32P]ATP / ml at 37°C for 10 minutes. The reaction products were then separated by thin-layer chromatography on polyethyleneimine cellulose, and the amount of PPi produced was measured by autoradiogram densitometry. Plasma PPi was measured as previously described using enzyme assays based on the conversion of PPi and UDP glucose to UTP and glucose-1-phosphate by UDP glucose pyrophosphorylase in freshly filtered plasma through a 30 kD cutoff filter (Lomashvili, KA et al., Kidney Int 2014, 85, 1351-1356). All used water was pretreated with hydroxyapatite to remove contaminating PPi. Aortic calcium was measured by calorimetry in HCl acid extracts of dried aorta, as previously described (Lomashvili, KA et al., Kidney Int 2014, 85, 1351-1356). Calcium content was normalized to dry weight, and after subtracting the calcium content of normal mouse aorta, the slight decrease in calcification was assessed.

[0109] Blood cell fractionation: To prepare leukocytes and platelets, freshly collected heparinized human blood was centrifuged at 250 g for 15 minutes at room temperature. Plasma was removed, and platelets were obtained by centrifuging at 2200 g for 12 minutes. The pellet from the first centrifugation was resuspended in normal saline to the original blood volume, and four times the volume of lysis buffer (155 mmol / L ammonium chloride; 10 mmol / L sodium bicarbonate; 0.1 mmol / L EDTA, pH 7.4) was added on ice for 5-10 minutes. This was repeated after centrifugation and removal of the supernatant, and purified leukocytes were obtained after the final centrifugation.

[0110] Statistical analysis: Continuous variables are expressed as mean ± standard error, and differences are determined by Student's t-test. Aortic calcium content was analyzed after logarithmic transformation.

[0111] Example I

[0112] Background: Experiments were conducted to determine whether PPi levels increased in wild-type mice administered a variant of sNPP1. For single intravenous infusion therapy, a 1-hour time point was selected, and for single subcutaneous infusion therapy, a 4-hour time point was selected. PPi levels were quantitatively measured by the abcam PPi fluorescence assay.

[0113] Results: Raw data from 1 minute of readings (total of 9 readings) were averaged and converted to a percentage relative to normal plasma (WT). Table 2 Table 2 TIFF0007856872000003.tif53169

[0114] As shown in Figure 5, intravenous or subcutaneous infusion of the sNPP1 protein variant (5 mg / kg) in wild-type mice resulted in increased PPi concentrations exceeding normal plasma levels. Figure 5 shows the blood pyrophosphate levels in wild-type mice after intravenous administration (1 hour post-infusion) and subcutaneous administration (4 hours post-infusion) of sNPP1-Fc or sNPP1-Fc-D10.

[0115] Example II Enpp1(- / -) knockout mice were subcutaneously treated with either a solvent or 6 mg / kg of sNPP1-Fc-D10 every other day for 21 days. Aortic calcium levels are shown for males and females. Figure 6 shows the effective prevention of aortic calcification in Enpp1(- / -) mice treated with sNPP1-Fc-D10.

[0116] Example III Enpp1(- / -) knockout mice were intravenously treated with 6 mg / kg of sNPP1-Fc-D10, and serum PPi and enzyme activity levels were measured. As shown in Figure 7, plasma was collected at 0, 4, 24, 48, and 72 hours, and NPP1 activity (dashed line) and PPi levels (solid line) were analyzed. Wild-type PPi levels were measured at 2.18 μM (data not shown). The dashed line from top to bottom shows the PPi levels of wild-type, heterozygous Enpp1(+ / -), and homozygous Enpp1(- / -) mice (Li et al., 2013). The sNPP1-Fc profile was similar to that of sNPP1-Fc-D10.

[0117] Example IV Wild type and Enpp1 asj Mice were placed on a high-phosphorus, low-magnesium diet from birth. From 14 days of age, they were subcutaneously administered either a solvent or sNPP1-Fc (5 mg / kg) every other day. Kaplan-Meier survival curves showed that more than 50% of ASJ mice died before 6 weeks, and all animals died by 9 weeks. In contrast, 50% of sNPP1-Fc-treated animals survived past 7 weeks and remained alive at 9 weeks. Figure 8 shows the results of Enpp1 treatment with 5 mg / kg of sNPP1-Fc compared to solvent-treated mice. asj This shows an increased survival rate in homozygous male mice.

[0118] Example V Wild type and Enpp1 asj Mice were placed on a high-phosphorus, low-magnesium diet from birth, and their growth rate was determined by subcutaneous treatment with a solvent or sNPP1-Fc (5 mg / kg) every other day from 14 days of age. As shown in Figures 9A and 9B, wild-type (solid line) and Enpp1 asj (Round) The weight gain rate of mice was plotted from 2 weeks to 9 weeks of age. Figures 9A and 9B show the comparison between Enpp1 treated with 5 mg / kg sNPP1-Fc and solvent-treated mice. asj This shows an increased rate of weight gain in male mice. In the solvent group (upper panel), all Enpp1 mice were found to have increased weight gain at 9 weeks. asjThe animals died (white circles). In contrast, in the sNPP1-Fc treatment group, 5 Enpp1 asj Some mice survived (black circles), while five died (white circles). Figures 10A-10C show the wild type (Figure 10A, top) and the solvent-treated Enpp1. asj (Figure 10B, center), sNPP1-Fc treatment (5mg / kg) Enpp1 asj (Figure 10C, bottom) A photograph of a mouse is shown.

[0119] Example VI Wild type and Enpp1 asj In male mice, FGF-23 (fibroblast growth factor 23), a biomarker for phosphate metabolism, was measured. This was done in wild-type and Enpp1 mice. asj Mice were fed a high-phosphorus, low-magnesium diet (TD.00442, Harlan) from birth. From 18 days of age, they were subcutaneously administered either a solvent or sNPP1-Fc-D10 (5 mg / kg) every other day. All serum was collected 24 hours after administration and analyzed using the mouse FGF-23 ELISA kit (Kainos Co., Ltd., Tokyo, Japan). Enpp1 + / + - Solvent (solid black line), Enpp1 asj / asj - Solvent (black dotted line), and Enpp1 asj / asj In -sNPP1-Fc-D10 (gray solid line) mice, FGF-23 levels were measured at baseline (day 0), before the start of treatment, and during the course of treatment.

[0120] During the progression of the disease (up to day 9 [27 days old]), Enpp1 asj / asj FGF-23 levels increased in mice. However, Enpp1 treated with 5 mg / kg of sNPP1-Fc-D10 was different. asj / asj The mice showed a decrease in FGF-23 levels by day 17 of treatment compared to the solvent-treated group. * The p-value is < 0.05 by one-way ANOVA or Student's t-test. Figure 11 shows the solvent treatment Enpp1. asj (center), sNPP1-Fc (5mg / Kg) treated Enpp1 asj (Below) Shows the levels of fibroblast growth factor in mice.

[0121] Example VII: In vitro and in vivo activity

[0122] As shown in Figure 13A, recombinant sNPP1-Fc-D10 completely hydrolyzed ATP to PPi in vitro, and there was no hydrolysis of PPi to orthophosphate.

[0123] The enzyme activity in plasma is shown in Figure 13B. Significant activity was observed in the plasma of wild-type mice, with slightly more than one-third of ATP being converted to PPi within 10 minutes, corresponding to an activity of 7.6 ± 1.0 nmol / h / ml. The remainder was converted to orthophosphate by nucleotide triphosphatase. Enpp1 - / - Mouse-derived plasma was essentially deficient in NPP1, and small amounts of PPi represented PPi contaminated with [32P]ATP. Two hours after intravenous infusion of NPP1 (5 mg / kg), activity increased significantly to 10.3 ± 0.3 nmol / h / ml, and plasma PPi increased from 0.07 ± 0.02 to 1.00 ± 0.14 μM, compared to a level of 2.39 ± 0.37 μM in comparison wild-type mice.

[0124] As shown in Figure 13C, wild type or Enpp1 - / - NPP1 activity was not detected in the aorta of any mouse species, nor did it increase after NPP1 infusion. Activity was also not detected in the liver after recombinant NPP1 administration.

[0125] Enpp1 - / - Figure 14 shows the time course of plasma NPP1 activity and PPi concentration after subcutaneous injection of 5 mg / kg into mice. NPP1 activity and PPi concentration peaked 12 hours after injection, reaching levels of 195% and 41%, respectively, compared to wild-type littermates. These levels rapidly decreased and were virtually undetectable after 24 hours.

[0126] As shown in Figure 15, subcutaneous injection of sNPP1-Fc-D10 (5 mg / kg) showed a correlation between plasma PPi levels and plasma NPP1 activity. The correlation between plasma PPi and plasma NPP1 suggested that PPi was generated in circulation. This was investigated by incubating fresh human blood with recombinant NPP1 and then measuring the PPi in the plasma. Human blood was used because only a limited amount of blood could be obtained from mice. The amount of NPP1 to add to the blood was calculated to obtain levels similar to those obtained after injection in mice.

[0127] Figure 16A shows that administration of recombinant NPP1 increased plasma PPi when added to whole blood for 2 hours, but not when added to plasma alone, suggesting a cellular requirement. To investigate the role of erythrocytes in relation to other cells, blood was centrifuged to remove plasma with or without the buffy coat. HEPES-buffered saline was then added to return to the original hematocrit levels. As shown in Figure 16B, synthesis occurred only when the buffy coat was left, suggesting a need for leukocytes or platelets rather than erythrocytes. As shown in Figure 16C, incubation of isolated leukocytes or platelets in HEPES-buffered saline showed either released or generated PPi in both cases, but synthesis in response to exogenous NPP1 occurred only in leukocytes.

[0128] Example VIII: Treatment Model

[0129] A. NPP1 deficiency As shown in Figure 17, aging Enpp1 - / -Mice were placed on a high-phosphate diet and subcutaneously treated every other day with either a solvent or sNPP1-Fc-D10 (6 mg / kg) to assess the effect of recombinant NPP1 on arterial calcification. Each treated mouse was paired with a mouse of the same sex and nearly the same age that received only the same amount of solvent. After 18 days, the mean aortic calcium content was 61 ± 30 nmol / mg in solvent-treated mice and 8.8 ± 1.0 nmol / mg in mice treated with recombinant NPP1 (p=0.016). The content in wild-type littermates was 6.3 ± 3.4 nmol / mg (n=16). Six out of eight control aortas (80 ± 37 nmol / mg) and only one treated aorta (15 nmol / mg) showed elevated content (2 standard deviations higher than wild-type littermates). In pairs where calcification was present in the control aorta, this represented a 91±2% reduction in calcification.

[0130] To determine if NPP1 accumulation occurred after multiple infusions over a long period, plasma NPP1 activity and PPi were measured at the time of sacrifice (24 hours after infusion), but neither was detectable. Another group of Enpp1 - / - In mice, aortic NPP1 activity was undetectable after three injections of recombinant NPP1 every other day.

[0131] B. Chronic kidney disease This example discloses the efficacy of sNPP1-Fc-D10 in the treatment of chronic kidney disease (CKD) in a uremic rat model. To determine the effect of recombinant NPP1 on arterial calcification in uremic rats with renal failure, uremic rats were fed a high-adenine diet and subcutaneously injected either a control or sNPP1-Fc-D10 (5 mg / kg) five times a week, as shown in Figure 18. Twenty-one days after treatment, the mean aortic calcium content was 25.7 ± 4.9 nmol / mg in control-treated rats and 7.0 ± 1.0 nmol / mg (p = 0.0068) in rats treated with recombinant NPP1. The normal aortic calcium content was 5 nmol / mg.

[0132] Examples VII and VIII demonstrate the activity of sNPP1 and the efficacy of using sNPP1 in models of ectonucleotide pyrophosphate pyrophosphorylase deficiency and chronic kidney disease. These examples show that a transient increase in PPi is sufficient for effective treatment of vascular calcification and NPP1 deficiency. Equal parts Although the present invention has been presented and described in particular with respect to exemplary embodiments, those skilled in the art will understand that various modifications of form and detail are possible without departing from the scope of the invention as encompassed in the appended claims.

Claims

1. A pharmaceutical composition for treating, as needed, a disease caused by a mutation in the gene encoding the MRP6 protein in a subject, wherein the pharmaceutical composition comprises isolated recombinant human sNPP1, a fragment or fusion protein containing the NPP1 domain essential for exhibiting its pyrophosphatase and / or phosphodiesterase activity, and the pharmaceutical composition is for reducing vascular calcification in the subject.

2. A pharmaceutical composition according to claim 1, characterized in that the subject is a human patient.

3. A pharmaceutical composition according to claim 1 or 2, characterized in that a fragment or fusion protein containing the isolated recombinant human sNPP1, the NPP1 domain essential for exhibiting its pyrophosphatase and / or phosphodiesterase activity is administered in an amount of about 0.10 to about 50 mg / kg.

4. A pharmaceutical composition according to claim 3, characterized in that the amount is about 0.5 mg / kg.

5. A pharmaceutical composition according to claim 3, characterized in that the amount is about 1 mg / kg.

6. A pharmaceutical composition according to claim 3, characterized in that the amount is about 5.0 mg / kg.

7. A pharmaceutical composition according to claim 3, characterized in that the amount is about 6.0 mg / kg.

8. A pharmaceutical composition according to claim 3, characterized in that the amount is about 10 mg / kg.

9. A pharmaceutical composition according to claim 3, characterized in that the amount is about 15 mg / kg.

10. A pharmaceutical composition according to claim 3, characterized in that the amount is about 20 mg / kg.

11. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for administration about once a week.

12. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is administered approximately once every two weeks.

13. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is administered approximately once a month.

14. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for intravenous, subcutaneous, intrathecal, or intraperitoneal administration.

15. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for intravenous administration.

16. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for subcutaneous administration.

17. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for administration and is sufficient to normalize the blood level of PPi in the subject.

18. A pharmaceutical composition according to any one of claims 1 to 10, characterized in that it is for administration and is sufficient to prevent calcification of the affected tissue in the subject.

19. A pharmaceutical composition according to any one of claims 1 to 18, characterized in that the pharmaceutical composition contains the fusion protein of the isolated recombinant human sNPP1.

20. A pharmaceutical composition according to claim 19, characterized in that the isolated recombinant human sNPP1 fusion protein includes the Fc region of an immunoglobulin.

21. A pharmaceutical composition according to claim 19 or 20, characterized in that the isolated recombinant human sNPP1 fusion protein contains a targeting moiety.

22. A pharmaceutical composition according to claim 21, characterized in that the targeted portion comprises at least eight consecutive aspartic acid or glutamic acid residues.

23. A pharmaceutical composition according to any one of claims 1 to 22, characterized in that the sNPP1 is SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.

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