WO2016054056A1 - Methods of treating pxe with tnap inhibitors - Google Patents

Abstract

Disclosed herein are methods of treating a disorder or a symptom of the disorder, characterized by medial vascular calcification in a subject, by administering to the subject a therapeutically effective amount of an inhibitor of tissue-nonspecific alkaline phosphatase (TNAP), or a pharmaceutical composition comprising a therapeutically effective amount of a TNAP inhibitor.

Description

METHODS OF TREATING PXE WITH TNAP INHIBITORS

STATEMENT OF GOVERNMENT INTEREST

[0001] The invention was made with government support through the Intramural

Research Program of the National Human Genome Research Institute, National Institutes of Health, and an extramural RO 1 grant from the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health; the U.S. government has certain rights in this invention.

BACKGROUND

[0002] Vascular calcification (VC) results from the deposition of hydroxyapatite

(HA) as a consequence of disordered calcium phosphate regulation in the blood vessel. The mechanism of VC is not fully understood, but it is thought to involve a phenotypic change in the vascular smooth muscle cells in the wall with activation of certain bone- forming programs. Vascular calcifications (VCs) are of similar composition to bone minerals (Lanzer, et al. 2014 Eur Heart J35: 1515-1525) and are intima-based and/or media-based.

[0003] Vascular calcifications of the media (VCms) are, in principle, deposits of

HA with a high degree of crystallization. VCm represents a group of distinct pathological conditions of differing etiologies but a common final consequence (media calcification). M5nckeberg's medial sclerosis represents the most common variety of medial

calcifications; it is frequently associated with type II diabetes (T2D) and chronic kidney disease, yet, in some cases, none of these diseases, and no other known risk factors for VCs are present. Diseases and/or conditions associated with media calcifications include: atherosclerosis, diabetes mellitus II, chronic renal disease, aging, hyperparathyroidism, Vitamin D disorders, Vitamin K deficiency, Vitamin K-antagonist coagulants, osteoporosis, Kawasaki disease, ACDC (arterial calcification due to deficiency of CD73), GACI (generalized arterial calcification of infancy), IBGC (idiopathic basal ganglia calcification), PXE (pseudoxanthoma elasticum), rheumatoid arthritis, Singleton-Merten syndrome, β-thalassemia, and calciphylaxis (Lanzer, et al. 2014 Eur Heart J 35: 1515- 1525). While VCm are common to all of these conditions, the associated phenotypes may vary.

[0004] Today, VCm is recognized as a key predictor of cardiovascular morbidity and mortality requiring consequent prevention and, where possible, vigorous treatment. In all patients with documented VCm, regardless of potential cause and underlying pathogenesis, secondary prevention and optimum treatment of associated diseases, if any, is required, in particular atherosclerosis, T2D, and chronic kidney disease-mineral and bone disorder (CKD-MBD).

[0005] The aging process of arteries is characterized by a gradual increase in stiffness and calcification, potentially associated with numerous pathogenic principles, including generation of reactive oxygen species, systemic inflammation, endothelial dysfunction, and disturbances in phosphate metabolism. Yet to date, no proven therapeutic principles exist to slow the aging process of the arteries. In patients with VCm-type M5nckeberg's medial sclerosis (MMS) without known risk factors, no treatment is available at present.

[0006] There are several genetically defined vascular diseases associated with primary medial vascular calcification. One monogenetic autosomal recessive disease that seems most closely to resemble the classical description of MMS is arterial calcification due to deficiency of CD73 (A CDC), a rare vascular disease hallmarked by tortuous arteries with accompanied medial calcifications and medial hyperplasia. Generalized arterial calcification of infancy (GACI, also referred to as idiopathic infantile arterial calcification) is another rare vascular disease with systemic progressive VC that develop in utero and during the early postnatal period. Histologically, it is characterized by medial calcifications and neointimal formation that lead to vessel occlusion. Familial idiopathic basal ganglia calcification (IBGC, also known as Fahr's disease) is an additional rare disease with a wide spectrum of neuropathological symptoms that have recently been attributed to the medial calcification of small blood vessel that supply the area of the basal ganglia. Finally, pseudoxanthoma elasticum (PXE, also known as Gronblad-Strandberg syndrome) is a genetic disease that causes fragmentation and mineralization of elastic fibers in some tissues. PXE may be caused by autosomal recessive mutations in the ATP- binding cassette sub-family C member 6 (ABCC6) gene on the short arm of chromosome 16 ( 16p 13.1) (herein after "Abcc6-associated PXE") (Chassaing, et al. 2005 J Med Genet 42(12):881-92; Finger, et al. 2009 Surv Ophthalmol 54(2):272-85).

[0007] HA can be a significant risk factor in the pathogenesis of cardiovascular disease and has been associated with myocardial infarction and coronary death (Detrano, et al. 2000 Curr Probl Cardiol 25:374-402). The link between cardiovascular disease and bone formation has been verified in vivo (Speer, et al. 2002 J Exp Med 196: 1047-1055; Bucay, et al. 1998 Genes Dev 12: 1260-1268; Steitz, et al. 2002 Am J Pathol 161 :2035- 2046; Myers, et al. 2003 Arterioscler Thromb Vase Biol 23 : 1021-1028).

[0008] Alkaline phosphatases (APs) are dimeric enzymes present in most organisms (Millan, JL 2006 "Mammalian alkaline phosphatases. From biology to applications in medicine and biotechnology." Wiley- VCH Verlag GmbH & Co,

Weinheim, Germany pp. 1-322). They catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate (Pi) and alcohol. In humans, three of the four isozymes are tissue-specific, i.e., the intestinal (IAP), placental (PLAP), and germ cell (GCAP) APs, while the fourth AP is tissue-nonspecific (TNAP) and is expressed in bone, liver and kidney.

[0009] TNAP is centrally involved in mechanisms that control normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleosidetriphosphate pyrophosphohydrolase-1 (NPP1) and the

Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PPi), a potent inhibitor of hydroxyapatite formation at concentrations normally found in plasma.

BRIEF SUMMARY OF THE EMBODIMENTS

[0010] Recent studies suggest that one role for TNAP in bone tissue is to hydro lyze extracellular PPi (ePPi) to avoid accumulation of the mineralization inhibitor (Johnson, et al. 2000 Am JPhys Regulatory and Integrative Physiology 279: R1365-1377- 17; Hessle, et al. 2002 Proc Natl Acad Sci USA 99:9445-9449; Johnson, et al. 2003 J Bone Min Res 18:994-1004). Normalization of ePPi levels in NPP1 null and ANK-deficient mice improves their soft-tissue ossification abnormalities (Johnson, et al. 2000 Am JPhys Regulatory and Integrative Physiology 279: Rl 365- 1377, 16; Hessle, et al. 2002 Proc Natl Acad Sci USA 99:9445-9449).

[0011] TNAP may be a useful therapeutic target for the treatment of diseases such as ankylosis and osteoarthritis, as well as arterial calcification. Of additional note, when simulated with osteogenic media, TNAP is elevated to relatively high levels in the cells of patients with pseudoxanthoma elasticum (PXE), whereas control cells do not have increased levels of TNAP. [0012] Thus, in one aspect, the invention provides the use of tissue-nonspecific alkaline phosphatase (TNAP) inhibitors to treat vascular calcification, arterial

calcification, or other cardiovascular diseases.

[0013] In another aspect, the invention provides a method of treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject a therapeutically effective amount of a TNAP inhibitor or a pharmaceutical composition comprising a therapeutically effective amount of a TNAP inhibitor. In one embodiment of a method according to the invention, the plasma TNAP levels of the subject are reduced or inhibited.

[0014] In one embodiment of a method or use or composition according to the invention, the symptom is calcification of the arterial media.

[0015] In another embodiment of a method or use or composition according to the invention, the disorder is selected from, but not limited to, the group consisting of general arterial calcification of infancy (GACI), diabetes mellitus (I or II), chronic kidney disease, dialysis-related calcification, calciphylaxis, Monckeberg's sclerosis, Ehlers-Danlos syndrome, Kawasaki disease, pseudoxanthoma elasticum (PXE), heterotropic

calcification/ossification in amputees, tibial artery calcification, bone metastasis, bioprosthetic heart valve calcification, Paget's disease of bone (PDB), arterial calcification and distal joint calcification, arterial calcification due to deficiency of CD73 (ACDC), and Keutel syndrome.

[0016] In another embodiment of a method or use or composition according to the invention, the disorder is characterized by increased TNAP levels in the subject. In a further embodiment, the increased levels/overexpression are/is in the vascular media.

[0017] In another embodiment of a method or use or composition according to the invention, the disorder is a monogenic disease. In a further embodiment, the monogenic disease is selected from general arterial calcification of infancy (GACI), pseudoxanthoma elasticum (PXE), and ACDC.

[0018] In another embodiment of a method or use or composition according to the invention, the disorder is additionally characterized by one or more of reduced plasma levels of PPi, genetic mutations, or clinical diagnostic evidence of calcification on biopsy and/or imaging.

[0019] In another embodiment of a method or use or composition according to the invention, the disorder is pseudoxanthoma elasticum (PXE). In a further embodiment, PXE is associated with one or more mutations in the ATP-binding cassette sub-family C member 6 gene ("Abcc6-associated PXE"). ABCC6 is a protein that is encoded, in humans, by the ABCC6 gene. It is a member of the superfamily of ATP-binding cassette (ABC) transporters. In a further embodiment, the disorder is Enppl -associated GACI with PXE-like skin and eye findings. ENPP1, ectonucleotide

pyrophosphatase/phosphodiesterase family member 1, is an enzyme that in humans is encoded by the ENPP1 gene.

[0020] In another embodiment of a method or use or composition according to the invention, the administration is oral, parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), by inhalation, extracorporeal, or topical (including transdermal, ophthalmic, vaginal, rectal, intranasal). In a further embodiment, the administration is oral.

[0021] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is a compound according to Formula I:

Formula I

[0022] Wherein:

[0023] X¹ is =N- or =C(R²)-;

[0024] R¹ and R⁴ are independently selected from the group consisting of hydrogen, halogen, -CN, -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, haloalkyl, haloalkoxy, optionally substituted phenyl, and optionally substituted 5- or 6- membered heteroaryl;

[0025] R², R³, and R⁵ are independently selected from the group consisting of hydrogen, halogen, -CN, -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, haloalkyl, haloalkoxy, optionally substituted phenyl, and optionally substituted 5- or 6- membered heteroaryl; [0026] R⁷ and R⁸ are independently hydrogen, optionally substituted alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted phenyl, or R⁷ and R⁸ together with the nitrogen atom to which they are attached form an optionally substituted heterocycloamino;

[0027] R⁹ is selected from the group consisting of hydrogen, optionally substituted alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, and optionally substituted phenyl; and

[0028] A is selected from the group consisting of -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted phenyl, and optionally substituted 5- or 6-membered heteroaryl.

[0029] In a specific embodiment of a method or use or composition according to the invention, the TNAP inhibitor is a compound having the chemical structure:

5 -(5 -chloro-2-methoxy-benzenesulfonylamino)-nicotinamide, (SBI- 425) or a derivative thereof (for example, side group changes to increase efficacy, certain derivatives are described in WO 2013/126608).

[0030] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is a small molecule compound. In still a further embodiment, the small molecule compound is selected from, but not limited to, the group consisting of L-homoarginine, levamisole, theophylline, and lansoprazole.

[0031] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is a biaryl sulfanilamide, pyrazole, triazole, or a derivative thereof.

[0032] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is a bisphosphonate. In a further embodiment, the bisphosphonate is selected from, but not limited to, the group consisting of etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, and zoledronate. [0033] In a specific embodiment of a method or use or composition according to the invention, the TNAP inhibitor is etidronate.

[0034] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is short hairpin RNA. Antisense RNA is contemplated in an additional embodiment.

[0035] In another embodiment of a method or use or composition according to the invention, the TNAP inhibitor is administered with at least one additional form of therapy. In a further embodiment, the additional form of therapy is selected from, but not limited to, the group consisting of vitamin D sterols (calcitriol, alfacalcidol, doxercalciferol, maxacalcitol, paricalcitol), calcimimetics, vitamins, vitamin analogs, antibiotics, lanthanum carbonate, lipid-lowering agents, anti-hypertensives, anti-inflammatory agents (steroidal, non-steroidal), inhibitors of pro-inflammatory cytokine, adenosine agonists, adenosine receptor agonists, and cardiovascular agents. In still a further embodiment, the additional form of therapy is administered before, concurrently with, or after the composition.

[0036] In one aspect, the invention provides a use of a TNAP inhibitor in the manufacture of a medicament for the treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject (a pharmaceutical composition comprising) a TNAP inhibitor. In one embodiment of a use according to the invention, the

administration is oral.

[0037] In another aspect, the invention provides a TNAP inhibitor for use in the treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject (a pharmaceutical composition comprising) a TNAP inhibitor. In one embodiment of an inhibitor according to the invention, the administration is oral.

[0038] In yet another aspect, the invention provides a pharmaceutical composition comprising a TNAP inhibitor and a pharmaceutically acceptable carrier for the treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification.

[0039] In still another aspect, the invention provides a method of selectively treating

pseudoxanthoma elasticum (PXE) comprising: selecting a subject for treatment with a TNAP inhibitor on the basis of the subject having increased TNAP levels in fibroblasts derived from the same, when stimulated under osteogenic conditions; and, selectively administering at least one of SBI-425 and etidronate to the subject. In another embodiment, the subject is selected for treatment on the basis of having increased TNAP levels in skin cells (fibroblasts) derived from these patients when stimulated under osteogenic conditions, as described above, and/or reduced plasma levels of PPi and/or genetic mutation(s), and/or clinical diagnostic evidence of calcification on biopsy or imaging. In these embodiments, the administration may be oral.

[0040] Additional embodiments of the disclosed methods, uses, and compositions are set forth, at least in part, in the description that follows, can be understood from the description, or may be learned by practicing the disclosed methods, uses, and

compositions. The foregoing brief description and the following detailed description are exemplary and explanatory and are not restrictive of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1A demonstrates that primary fibroblasts from patients with GACI,

ACDC, and PXE calcify in vitro, whereas control fibroblasts do not. Calcification is identified by strong alizarin red staining. Briefly, primary dermal fibroblasts were derived from control and patient skin biopsies. These fibroblasts cultures were expanded and subjected to 21 days of osteogenic stimulation. Osteogenic media consists of beta- glycerophosphate, ascorbic acid, and dexamethasone in aMEM base media. To explore what might be causing the observed in vitro calcification, after 5 days of aMEM (alpha- Minimal Essential Media; control media) or osteogenic stimulation, the cells were lysed and TNAP was measured with an enzymatic assay. Figure IB is a box and whiskers plot showing elevated TNAP in PXE dermal fibroblasts. When stimulated with osteogenic media, fibroblasts derived from PXE patients had increased TNAP. The data shown include experimental duplicates at passage 3 (3 technical replicates/sample), biological replicates indicated on graph, statistical analysis: Student's t-test; alpha = 0.05. Figure 1C is a box and whiskers plot showing elevated ENPP1 in AB CCd^KO/KO fibroblasts. The data shown include 2 technical replicates/sample. Student's t-test; alpha = 0.05. Figure ID is a box and whiskers plot showing elevated ALPL (the gene encoding TNAP) and ENPP1 mRNA. Normalized to 18S. Data shown includes 3 technical replicates/sample;

biological replicates indicated on graph. Statistical analysis: One-way ANOVA with Tukey's HSD; alpha = 0.05. P-values shown are from pairwise comparisons. [0042] Figure 2 A is a schematic representation of the extracellular ATP metabolism pathway showing TNAP inhibitor interaction. Figure 2B provides photomicrographs showing that TNAP inhibition with SBI-425 prevented in vitro calcification in PXE cell lines.

[0043] Figure 3 provides preliminary data from an in vivo treatment trial testing the efficacy of TNAP inhibition on the PXE calcification phenotype. The box and whiskers plot shows the average area of calcification for the Abcc6 knockout mouse model having received vehicle, SBI-425, or etidronate. Above each box and whiskers plot are corresponding coronal views of the mouse muzzle, obtained via microCT (with arrows pointing at the calcification), indicating that both SBI-425 and etidronate decreased vibrissae calcification. Beneath each box and whiskers plot are images showing calcification (or lack thereof) employing Alizarin Red and vonKossa staining.

[0044] 6 week-old mice were treated q.d. with P.O. vehicle, 30mg/kg SBI-425, or

240mg/kg etidronate for 14 weeks. Calcification (Figure 4A) and plasma TNAP activity (Figures 4B ad 4C) were quantified. P.O. TNAP inhibition attenuated calcification in vivo (Figure 4A). Figure 4A shows a box and whiskers plot showing the average area of calcification for the control (wild-type or Abcc6 heterozygous littermates) vs. Abcc6 knockout mice having received vehicle, SBI-425, or etidronate. Above each box and whiskers plot are corresponding coronal views of the mouse muzzle, obtained via microCT, indicating that both SBI-425 and etidronate decreased vibrissae calcification. Mice were imaged on a SPECT-CT at 3x magnification (1024 slices/mouse). Images were reconstructed and analyzed with ImageJ software. Briefly, a Z-stack was created to encompass the entire region of vibrissae calcification (rostrally, from the tip of the nose to the zygomatic arch). The images included are representative. To quantify the

calcification, a threshold was manually determined to calculate the total area of ectopic calcification. Data are an average of analysis by 6 blinded-observers. Biological replicates indicated on slide. Figure 4B shows a box and whiskers plot depicting residual plasma TNAP activity for each of the genotype/treatment models of Figure 4A. Assay performed at pH 9.8. SBI-425 inhibited residual plasma TNAP activity (Figures 4B).

DETAILED DESCRIPTION

[0045] It is clear to the person of ordinary skill in the art that the disclosed methods, uses, and compositions are not limited to a particular methodology, protocol, or reagent described, as each of these may vary. Furthermore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention.

Definitions

[0046] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art. Publications cited herein and the material for which they are cited are hereby specifically incorporated by reference. No admission is made that any reference constitutes prior art.

[0047] As used in the specification and the appended claims, the singular forms

"a," "an," and "the" include plural referents, unless the context clearly dictates otherwise. Thus, for example, reference to "a compound," "a composition," or "agent," and like terms, may include mixtures of two or more such compounds, compositions, agents, and the like.

[0048] "Optional" or "optionally" means that the subsequently described agent, material, event, or circumstance may or may not occur or be present, and that the description includes instances where the agent, material, event, or circumstance is present or occurs and instances where it is not present or does not occur.

[0049] Ranges can be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as "about" that particular value in addition to the value itself, even if it is not explicitly stated herein.

[0050] Reference herein to any numerical range expressly includes each numerical value (including fractional numbers and whole numbers) encompassed by that range. For example, reference herein to a dosage range of 0.001 to 1 mg/kg includes all whole numbers of and fractional numbers between the two. In a further illustration, reference herein to a range of "less than x" (wherein x is a specific number) includes whole numbers x-1, x-2, x-3, x-4, x-5, x-6, etc., and fractional numbers x-0.1, x-0.2, x-0.3, x-0.4, x-0.5, x- 0.6, etc. In yet another illustration, reference herein to a range of from "x to y" (wherein x is a specific number, and y is a specific number) includes each whole number of x, x+1, x+2...to y-2, y-1, y, as well as each fractional number, such as x+0.1, x+0.2, x+0.3...to y- 0.2, y-0.1.

[0051] By the term "therapeutically effective amount", for example, of a TNAP inhibitor, as provided herein is meant such amount as is capable of performing the function of the inhibitor. The exact amount required will vary, depending on known variables, such as the inhibitor employed, the condition of the subject, and the parameters of the therapeutic regimen. Thus, it is neither necessarily possible nor required to specify an exact "pharmaceutically effective amount." Rather, the appropriate effective amount may be determined by one of ordinary skill in the art using routine experimentation.

[0052] As used herein, the terms "administering" and "administration" refer to any method of providing a TNAP inhibitor or a pharmaceutical composition comprising a TNAP inhibitor to a subject. Such methods are well known to those skilled in the art and include, without limitation, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration, intravaginal administration, ophthalmic administration, intraaural administration, intracerebral administration, rectal administration, and parenteral administration, including injectable such as intravenous administration, infusion, intra-arterial administration, intramuscular administration, and subcutaneous administration. Administration can be continuous or intermittent.

[0053] The terms "subject", "patient", and "individual", as used herein, interchangeably refer to a multicellular animal (including mammals (e.g., humans, non- Human primates, murines, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, ayes, etc.), avians (e.g., chicken), amphibians (e.g. Xenopus), reptiles, and insects (e.g. Drosophila). "Animal" includes guinea pig, hamster, ferret, chinchilla, mouse, and cotton rat.

[0054] As used herein, the term "prodrug," means an agent that is not necessarily biologically active when administered but, upon administration, can be converted to a bioactive agent through metabolism or some other mechanism. A prodrug can comprise any covalently bonded substance that can release the active parent drug or other formulas or compounds disclosed herein in vivo, when such pro-drug is administered to a subject.

[0055] As used herein, the term "comprise" and variations of the term, such as

"comprising" and "comprises," means "including but not limited to," and is not intended to exclude, for example, other agents, additives, components, integers, or steps. [0056] The term "including", as well as other forms, such as "includes" and

"included", as used herein, is not limiting.

[0057] An individual, i.e., a subject, referred to as "suffering from" a condition

(i.e., disorder) herein has been diagnosed with and/or displays one or more symptoms of the condition.

[0058] As used herein, the term "at risk" for a condition, refers to a subject (e.g., a human) that is predisposed to developing the condition and/or expressing one or more symptoms of the condition. Such subjects include those at risk for failing to elicit an immunogenic response to a vaccine against the disease. Furthermore, the term subject "at risk" includes subjects "suffering from" the condition, i.e., a subject that is experiencing the disorder, and vice versa. It is not intended that the present invention be limited to any particular signs or symptoms. Thus, it is intended that the present invention encompasses subjects that are experiencing any range of disorder, from sub-clinical condition to fullblown disorder, including wherein the subject exhibits at least one of the indicia (e.g., signs and symptoms) associated with the disorder.

[0059] The terms "treat," "treatment," or "treating", as used herein, refer to any method used to partially or completely alleviate, ameliorate, relieve, abate, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of a disorder or condition or one or more symptoms or features of the disorder or condition (e.g., medial vascular calcification). The terms "treat," and "prevent" and the like do not necessarily imply 100% or complete treatment or prevention. Rather, there are varying degrees of treatment or prevention of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the inventive methods, uses, compositions, etc. can provide any amount or any level of treatment or prevention of medial vascular calcification and/or any of the disorders described herein in a mammal. Also, for purposes herein, "prevention" can encompass delaying the onset of the disorder, or a symptom or condition thereof. Treatment may be administered to a subject who does not exhibit signs of a disorder. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disorder or condition for the purpose of decreasing the risk of developing pathology associated with the disorder or condition.

[0060] A small hairpin RNA (ribonucleic acid) or short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression (e.g., TNAP expression) via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. The promoter choice is essential to achieve robust shRNA expression.

[0061] Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery.

[0062] The term "synthetic" or "synthesized", as used herein, refers to an agent that does not occur naturally, i.e., is artificially produced by man.

[0063] As used herein, the term "small molecule" refers to a chemical agent which can include, but is not limited to, a peptide, a peptidomimetic, an amino acid, an amino acid analog, a polynucleotide, a polynucleotide analog, an aptamer, a nucleotide, a nucleotide analog, an organic or inorganic compound (e.g., including heterorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1 ,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0064] Treatment methods

[0065] TNAP inhibitors, including those described herein, can be used to treat medial vascular calcification and/or a disorder or condition characterized by medial vascular calcification. Thus, disclosed herein are methods for treating medial vascular calcification and/or disorders or conditions characterized by medial vascular calcification in a subject, comprising administering to the subject a TNAP inhibitor or a pharmaceutical composition comprising a TNAP inhibitor.

[0066] Treatment methods include methods of ameliorating, reversing, and/or preventing symptoms of medial vascular calcification and/or disorders or condition characterized by medial vascular calcification. Treatment methods comprise, in certain embodiments, methods for assessing calcification following (or during) treatment.

[0067] Vascular Calcification [0068] Vascular calcification refers to the formation, growth, or deposition of extracellular matrix hydroxyapatite (calcium phosphate) crystal deposits in blood vessels. Vascular calcification encompasses coronary, valvular, aortic, and other blood vessel calcification. The term includes atherosclerotic and medial wall calcification.

[0069] Vascular calcification increases the risk of cardiovascular morbidity and mortality (Giachelli, C. 2004 J Am Soc Nephrol 15: 2959-64; Raggi, et al. 2002 J Am Coll Cardiol 39: 695-701). Vascular calcification is an important and potentially serious complication of chronic renal failure. Two distinct patterns of vascular calcification have been identified (Proudfoot & Shanahan, 2001 C Herz 26: 245-51). The first, medial calcification, occurs in the media of the vessel in conjunction with a phenotypic transformation of smooth muscle cells into osteoblast-like cells, while the other, atherogenesis, is associated with lipid-laden macrophages and intimal hyperplasia. Medial wall calcification can develop in relatively young persons with chronic renal failure, and it is common in patients with diabetes mellitus, even in the absence of renal disease. The presence of calcium in the medial wall of arteries distinguishes this type of vascular calcification from that associated with atherosclerosis, which occurs in atheromatous plaques along the intimal layer of arteries. The extent of arterial calcification in patients with atherosclerosis generally corresponds to severity of disease.

[0070] Some patients with end-stage renal disease develop a severe form of occlusive arterial disease called calciphylaxis or calcific uremic arteriolopathy, which is characterized by extensive calcium deposition in small arteries. In patients with this disease, arterial calcification and vascular occlusion lead to tissue ischemia and necrosis.

[0071] Current therapies to normalize serum mineral levels or to decrease, inhibit, or prevent calcification of vascular tissues or implants are of limited efficacy and cause unacceptable side effects. Therefore, there exists a need for an effective method of treating vascular calcification.

[0072] Medial calcification, medial wall calcification, or Monckeberg's sclerosis refer to calcification characterized by the presence of calcium in the medial wall of arteries.

[0073] Assessing vascular calcification

[0074] Methods of detecting and measuring vascular calcification (including medial vascular calcification) are well known in the art. For example, methods of measuring calcification include direct methods of detecting and measuring extent of calcium-phosphorus depositions in blood vessels. Other direct methods of measuring vascular calcification comprise in vivo imaging methods, such as plain film

roentgenography, coronary arteriography, fluoroscopy, including digital subtraction fluoroscopy, cinefluorography, conventional, helical, and electron beam computed tomography, intravascular ultrasound (IVUS), magnetic resonance imaging (MRI), and transthoracic (surface) and transesophageal echocardiography. Fluoroscopy and electron- beam computed tomography (EBCT) are commonly used to detect calcification non- invasively. Coronary interventionalists use cinefluorography and intravascular ultrasound (IVUS) to evaluate calcification in specific lesions before angioplasty. Vascular calcification can be assessed ex vivo by the Van Kossa and Alizarin red methods. Other direct methods of measuring calcification include immuno fluorescent staining and densitometry.

[0075] Methods of assessing vascular calcification (including medial vascular calcification) also include methods of measuring determinants and/or risk factors of vascular calcification. Such factors include, but are not limited to, serum levels of phosphorus, calcium, and calcium-phosphorus product, parathyroid hormone (PTH), low- density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), triglycerides, and creatinine. Methods of measuring these factors are well known in the art.

[0076] Other methods of assessing vascular calcification (including medial vascular calcification) include assessing factors of bone formation. Such factors include bone formation markers such as bone-specific alkaline phosphatase (BSAP), osteocalcin (OC), carboxyterminal propeptide of type I collagen (PICP), and aminoterminal propeptide of type I collagen (PINP); serum bone resorption markers such as cross-linked C-telopeptide of type I collagen (ICTP), tartrate-resistant acid phosphatase, TRACP and TRAP5B, N-telopeptide of collagen cross-links (NTx), and C-telopeptide of collagen cross-links (CTx); and urine bone resorption markers, such as hydroxyproline, free and total pyridinolines (Pyd), free and total deoxypyridinolines (Dpd), N-telopeptide of collagen cross-links (NTx), and C-telopeptide of collagen crosslinks (CTx).

[0077] As mentioned above, in one aspect, the invention provides a method of treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject a TNAP inhibitor or a pharmaceutical composition comprising a TNAP inhibitor. In one embodiment, administration of the inhibitor (or composition comprising the same) retards/attenuates or reverses the formation, growth or deposition of extracellular matrix hydroxyapatite crystal deposits. In another embodiment, administration of the inhibitor (or composition comprising the same) prevents the formation, growth, or deposition of extracellular matrix hydroxyapatite crystal deposits.

[0078] In another embodiment, administration of a TNAP inhibitor (or composition comprising the same) can reduce serum PTH without causing aortic calcification. In another embodiment, administration of the inhibitor (or composition comprising the same) can reduce serum creatinine level or can prevent increase of serum creatinine level. In another embodiment, administration of the inhibitor (or composition comprising the same) can attenuates parathyroid (PT) hyperplasia.

[0079] Disorders

[0080] Diseases associated with medial vascular calcification include

atherosclerosis, hyperparathyroidism, vitamin D disorder, vitamin K deficiency, osteoporosis, general arterial calcification of infancy (GACI), diabetes mellitus (I or II), chronic kidney disease, dialysis-related calcification, calciphylaxis, Monckeberg's sclerosis, Ehlers-Danlos syndrome, Kawasaki disease, pseudoxanthoma elasticum (PXE), IBGC, rheumatoid arthritis, Singleton-Merten syndrome, β-thalassemia, heterotropic calcification/ossification in amputees, tibial artery calcification, bone metastasis, bioprosthetic heart valve calcification, Paget's disease of bone (PDB), arterial calcification and distal joint calcification, arterial calcification due to deficiency of CD37 (ACDC), and Keutel syndrome.

[0081] The uses, methods, and compositions contemplated herein treat a disorder and/or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject.

[0082] In one embodiment, at least one TNAP inhibitor or a composition comprising at least one TNAP inhibitor is used for the treatment of PXE or a symptom of PXE. PXE is a rare disorder that results in calcification of the elastic fibers, specifically in the skin, small vessels, and Bruch's membrane of the eye. PXE is classically caused by biallelic mutations in Abcc6, which codes for an ATP-dependent exporter.

[0083] In another embodiment, at least one TNAP inhibitor or a composition comprising at least one TNAP inhibitor is used for the treatment of tumoral calcinosis or a symptom of tumoral calcinosis. In yet another embodiment, at least one TNAP inhibitor or a composition comprising at least one TNAP inhibitor is used for the treatment of ectopic calcification or a symptom of ectopic calcification. In still another embodiment, at least one TNAP inhibitor or a composition comprising at least one TNAP inhibitor is used for the treatment of a disorder characterized by or associated with a decrease in the amount of circulating PPi, genetic mutation, or clinical diagnostic evidence of calcification on biopsy or imaging, or another symptom of a disorder characterized by or associated with a decrease in the amount of circulating PPi.

[0084] Treatment population

[0085] The subjects to be treated according to the inventive methods may be at risk for developing a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, or they may already have developed the disorder but not yet exhibit the symptoms, or they may already have the disorder and exhibit the symptoms. Thus, the treatment may be prophylactic or therapeutic or preventative.

[0086] Diagnosis of the PXE treatment population, including pre-symptomatic population, can, in certain embodiments, be molecular, for example, via genetic testing (ABCC6 (primarily) and/or ENPP1 mutations). PXE is typically inherited in an autosomal recessive manner.

[0087] In other embodiments, diagnosis of the treatment population can be based on any one of a number of symptoms, for example, yellow-white small raised areas (papules) in the skin folds (in flexure areas like the neck, armpit, etc.), angioid streaks in the retinae (sometimes associated with retinal hemorrhage), some degree of loss of vision (up to blindness), arterial calcification, atherosclerosis in the heart, mitral valve prolapse, fragile small blood vessels leading to abnormal bleeding in the bowel and/or uterus, impairment of circulation to the legs, claudication. In individuals with characteristic skin findings, diagnosis can be established by histologic findings on biopsy of lesional skin, specifically, calcified dystrophic elastic fibers.

[0088] The more serious ophthalmologic, cardiovascular, and gastrointestinal symptoms tend to manifest later in life. "Peau d'orange", rippling of the retina, may already occur in children. A skin biopsy will also allow diagnosis in children.

[0089] Some subjects having ABCC6 mutations develop GACI and/or ACDC.

Generalized calcification of infancy (GACI), a disorder affecting the circulatory system, becomes apparent before birth or within the first few months of life and is characterized by abnormal accumulation of the mineral calcium, i.e., calcification, in the walls of the arteries. This calcification is often accompanied by thickening of the intima, which leads to arterial stenosis and stiffness. Heart failure may develop in affected individuals, and symptoms including difficulty breathing, edema in the extremities, cyanosis (skin, lips), severe hypertension, and cardiomegaly. Subjects with GACI may also have calcification in other organs and tissues, particularly around the joints. In addition, they may have hearing loss or softening and weakening of the bones (rickets). Because GACI is also (in fact, more often) associated with mutations in the ENPP1 gene, genetic testing for the disease may focus on ENPP1 and/or ABCC6.

[0090] ACDC (arterial calcification due to deficiency of CD73) is characterized by calcium buildup in the arteries and joints (mostly below the waist). Patients with ACDC suffer from insufficient blood supply to the lower extremities, arteriomegaly, chronic pain, difficulty moving, and an increased risk of cardiovascular problems. Genetic testing for ACDC is to detect a mutation in the NT5E gene (related to the ENPP1 gene), which produces CD73.

[0091] TNAP inhibitors

[0092] Described herein are therapeutic agents that can inhibit tissue-nonspecific alkaline phosphatases (TNAPs). These TNAP inhibitors can be used to treat a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject.

[0093] TNAP inhibitors contemplated herein include, without limitation, SBI-425 or a derivative thereof; a small molecule compound (for example, L-homoarginine, levamisole, theophylline, lansoprazole); a biaryl sulfanilamide, pyrazole, triazole, or derivative thereof; a bisphosphonate (for example, etidronate, clodronate, tiludronate, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, zoledronate); a prodrug; short hairpin RNA, or antisense RNA. In effect, contemplated herein are any agents that can inhibit tissue-nonspecific alkaline phosphatases (TNAPs).

[0094] Prodrugs

[0095] A prodrug is an agent that is administered in an inactive (or less than fully active) form, and is then converted to its active form through a normal metabolic process, such as hydrolysis of an ester form of the drug. In other words, a prodrug is a precursor chemical compound of a drug. Instead of administering a drug, a prodrug may be used in order to improve how the agent is absorbed, distributed, metabolized, and/or excreted (ADME). Prodrugs are often designed to improve bioavailability, for example, if the drug itself is poorly absorbed from the gastrointestinal tract. A prodrug may also be used to improve how selectively the drug interacts with cells or processes that are not its intended target. This reduces adverse or unintended effects of a drug.

[0096] Prodrugs can be prepared using methods known in the art (for example, by modifying a functional group present in a compound or drug in such a way that the modifications can be cleaved, either in routine manipulation in vivo, to the parent compound). A review of metabolic reactions and enzyme reactions involved in the hydrolysis of drugs and prodrugs can be found in Wehner, V. (2004), Hydrolysis in Drug and Prodrug Metabolism. Chemistry, Biochemistry, and Enzymology By Bernard Testa and Joachim M. Mayer. Angew. Chem. Int. Ed., 43: 544-545, which is hereby

incorporated by reference.

[0097] Prodrugs can comprise any suitable functional group that can be chemically or metabolically cleaved by solvolysis or under physiological conditions to provide the biologically active compound. Suitable functional groups include, e.g., carboxylic esters, amides, and thioesters. Depending on the reactive functional group(s) of the biologically active compound, a corresponding functional group of a suitable linker precursor can be selected to provide, e.g., an ester linkage, thioester linkage, or amide linkage in the prodrug.

[0098] Dosage

[0099] A therapeutically effective amount of a TNAP inhibitor refers to a dose that is adequate to treat a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject. For example, calcification is attenuated in a subject as a result of administration of a therapeutically effective amount of a TNAP inhibitor or pharmaceutical composition comprising the same. Amounts effective for a therapeutic or prophylactic use will depend on, for example, the stage and severity of the disease or disorder being treated, the age, weight, and general state of health of the patient, and the judgment of the prescribing physician. The size of the dose will also be determined by the specific TNAP inhibitor selected, method of administration, timing and frequency of administration, possible adverse side-effects that might accompany the administration of the inhibitor, and the desired physiological effect.

[00100] It will be appreciated by one of skill in the art that various disorders or symptoms may require prolonged treatment involving multiple administrations, perhaps using the TNAP inhibitor in each or various rounds of administration. By way of a non- limiting example, the dose of the TNAP inhibitor for the inventive methods of treating and/or preventing a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, can be about 0.001 to about 1 mg/kg body weight of the subject being treated per day, for example, about 0.001 mg, 0.002 mg, 0.005 mg, 0.010 mg, 0.015 mg, 0.020 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.5 mg, 0.75 mg, or 1 mg/kg body weight per day. The dose of the TNAP inhibitor for methods of treating and/or preventing a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, can be about 1 to about 1000 mg/kg body weight of the subject being treated per day, for example, about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 500 mg, 750 mg, or 1000 mg/kg body weight per day. In one embodiment, the dose of the TNAP inhibitor for the inventive methods of treating and/or preventing a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, is about 5 to about 100 mg/kg/day. In other embodiments, the dose of TNAP inhibitor is in any range falling within about 5 and about 100 mg/kg/day, for example, about 5 to about 50 mg/kg/day, about 5 to about 30 mg/kg/day, and the like. That dosage can be provided in a single dosage form (once) or multiple (2, 3, etc.) dosage forms per day.

[00101] In another embodiment, the amount or dose of the TNAP inhibitor administered is sufficient to effect a therapeutic or prophylactic response in the subject over a reasonable time frame. For example, the dose of the TNAP inhibitor should be sufficient to decrease TNAP activity, increase pyrophosphate levels, and/or treat (for example, attenuate) medial vascular calcification in a period of from about 2 hours or longer, e.g., about 12 to about 24 or more hours, from the time of administration. In certain embodiments, the time period could be even longer. For example, the period to effect may be on the order of weeks to years. The dose will be determined by the efficacy of the TNAP inhibitor and the condition of the subject, as well as the body weight of the subject to be treated.

[00102] Combination therapy

[00103] In the context of the methods, uses, compositions, and the like described herein, at least one TNAP inhibitor can be administered alone or in combination with at least one other therapeutic agent or therapeutic regimen for treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject. Exemplary (but non-limiting) therapeutic agents include another TNAP inhibitor, vitamin D sterols (for example, calcitriol, alfacalcidol, doxercalciferol, maxacalcitol, paricalcitol), RENAGEL®, calcimimetics, vitamins and their analogs, antibiotics, lanthanum carbonate, lipid-lowering agents (for example, LIPITOR®), antihypertensives, anti-inflammatory agents (steroidal, non-steroidal), inhibitors of proinflammatory cytokines (for example, ENBREL®, KINERET®), adenosine agonists, adenosine receptor agonists, and cardiovascular agents. Therapeutic regimens can be surgical or non-surgical. One exemplary therapeutic regimen is dialysis.

[00104] In some embodiments, the additional therapeutic agent(s) and/or therapeutic regimen(s) can be administered before, concurrently with, or after

administration of the TNAP inhibitor.

[00105] The dosage regimen for treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification, with combination therapy as described herein is selected in accordance with a variety of factors, including the type, age, weight, sex, and medical condition of the patient, the severity of the disease, the route of administration, and the specific therapeutic agent(s) and/or therapeutic regimen(s) employed, and, thus, can vary widely.

[00106] By "in combination with," it is not intended to imply that the at least one

TNAP inhibitor and additional therapeutic agent or therapeutic regimen must be administered at the same time or formulated for delivery together, although these methods of delivery are within the scope of the invention. It will be appreciated that therapeutically active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.

[00107] In general, each agent (in this context, one of the "agents" is the at least one

TNAP inhibitor or a composition comprising at least one TNAP inhibitor) will be administered at a dose and on a time schedule determined for that agent. Additionally, the invention encompasses the delivery of the compositions in combination with agents that may improve their bioavailability, reduce or modify their metabolism, inhibit their excretion, or modify their distribution within the body.

[00108] The particular combination of agents to employ in a combination regimen will take into account compatibility of the desired agents and/or regimens and the desired therapeutic effect to be achieved. It will be appreciated that the therapies employed may achieve a desired effect for the same purpose (i.e., treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject), or they may achieve different effects (for example, treating different symptoms).

[00109] In certain embodiments, the agents utilized in combination are utilized at levels that do not exceed the levels at which they are utilized individually. In additional embodiments, the levels utilized in combination will be lower than those utilized individually.

[00110] Administration

[00111] The at least one TNAP inhibitor, pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent can be administered in any suitable manner. For example, administration may be effected orally (including, for example, by gavage), parenterally (e.g., intravenous, subcutaneous, intra-arterial, intramedullary, intrathecal, intraperitoneal, intragastric, intraventricular, or intramuscular injection), subcutaneous ly, by inhalation, extracorporeally, topically or transcutaneous ly (including transdermally, ophthalmically, vaginally, rectally, interdermally, intradermally, intranasally, buccally, enterally, vitreally, sublingually), by intratracheal instillation, bronchial instillation, or the like. In one embodiment, the administration is oral (per os (P.O.)). In another embodiment, the oral administration of the at least one TNAP inhibitor, pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent results in an improved bioavailability of the inhibitor, composition, and/or agent.

[00112] "Topical intranasal administration" indicates delivery into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the inhibitor or composition or agent. Administration by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism. Delivery can also be directly to any area of the respiratory system (e.g., lungs) via intubation.

[00113] In another embodiment, the at least one TNAP inhibitor, pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent can be administered in conjunction with balloons tipped catheters and/or stents. The stents, catheters, and/or balloons can be linked with the inhibitor or composition or agent or administered concurrently with the same. By "linking" or "linked" is meant any method of placing the inhibitor or composition or agent onto the stent, such as soaking, coating, infusing, or any known chemical methods. Also contemplated herein are time released methods of attaching the inhibitor or composition or agent to a balloon or stent.

[00114] If combination therapy is employed, the method of administration may be the same or different for the TNAP inhibitor or pharmaceutical composition comprising a TNAP inhibitor and the additional therapeutic agent or regimen.

[00115] Parenteral administration of the at least one TNAP inhibitor,

pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Slow or sustained release systems may, in an additional embodiment, maintain a constant dosage.

[00116] The manner of administration can be chosen based, for example, on whether local or systemic treatment is desired, the area to be treated, and the specific agent or composition or regimen to be administered.

[00117] The manner of administration can also affect the amount of the at least one

TNAP inhibitor, pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent to be administered to the subject, i.e., the dosage. The appropriate amount can be determined by one of ordinary skill in the art using routine experimentation, given the teachings herein. Thus, effective dosages and schedules for administration can be determined empirically, and making such

determinations is within the skill in the art. The dosage range(s) for administration are those large enough to produce the desired effect, i.e., treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject.

[00118] The dosage should not be so large as to cause adverse side effects, for example, unwanted cross-reactions, anaphylactic reactions, and the like. Thus, the dosage can be adjusted by the individual physician in the event of any counter indications.

Dosage can vary not only in amount per administration, but also in administration schedule (i.e., how often administration is carried out), for example, in one or more dose administrations daily, for one or several days, weekly, for one or several weeks, etc. Additional guidance can be found in the literature for appropriate dosages. For example, a typical daily dosage of a TNAP inhibitor used alone might range from about 1 μg/kg to up to 100 mg/kg of body weight or more per day, depending on the above- iterated factors.

[00119] In an additional embodiment, the at least one TNAP inhibitor,

pharmaceutical composition comprising at least one TNAP inhibitor, and/or optional additional therapeutic agent to be administered to the subject may be administered prophylactically to subjects who are at risk for (or who have been newly diagnosed with) medial vascular calcification.

[00120] Pharmaceutical compositions

[00121] Also described herein are compositions comprising at least one tissue- nonspecific alkaline phosphatase (TNAP) inhibitor. Such compositions comprise, in one embodiment, a therapeutically effective amount of at least one TNAP inhibitor and a pharmaceutically acceptable carrier, excipient, and/or diluent.

[00122] The at least one TNAP inhibitor can, in certain embodiments, be combined, conjugated, or coupled with or to carrier(s) within a composition, for example, to make administration easier or to improve delivery of the inhibitor. Exemplary carrier examples include, without limitation, small molecules, pharmaceutical drugs, fatty acids, detectable markers, conjugating tags, nanoparticles, and enzymes. Ideally, the carrier is

pharmaceutically acceptable, i.e., not biologically or otherwise undesirable. Thus, the carrier does not have a significant undesirable biological effect or interact in a

significantly deleterious fashion with the inhibitor and/or any other optional component of the pharmaceutical composition. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.

[00123] In certain embodiments, pharmaceutically acceptable carriers include solutions, such as, but not limited to, sterile water, saline, Ringer's solution, dextrose solution, and buffered solutions at physiological pH. In other embodiments, carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, for example, films, liposomes, and microparticles. The selection of carrier may reflect the route of administration and/or the specific TNAP inhibitor and/or the concentration of the composition being administered. [00124] Pharmaceutical compositions further include, in certain embodiments, thickeners, diluents, buffers, preservatives, surface active agents, and/or certain active ingredients (antimicrobial agents, anti-inflammatory agents, anesthetics, and the like).

[00125] Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (for example, olive oil), and injectable organic esters (for example, ethyl oleate). Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions (for example, saline and buffered media). Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and/or inert gases and the like.

[00126] Formulations for topical administration can include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, and the like are likewise contemplated.

[00127] For oral administration, the composition comprising at least one TNAP inhibitor may be presented as capsules, tablets, dissolvable membranes, powders, granules, or as a suspension. Such a composition may have conventional additives, such as lactose, mannitol, corn starch, or potato starch. The composition also may be presented with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch, or gelatins. Additionally, the composition may be presented with disintegrators, such as corn starch, potato starch, or sodium carboxymethylcellulose. The composition may be further presented with dibasic calcium phosphate anhydrous or sodium starch glycolate.

Thickeners, flavorings, diluents, emulsifiers, and/or dispersing aids may be desirable. Finally, the composition may be presented with lubricants, such as talc or magnesium stearate.

[00128] Compositions administered as a pharmaceutically acceptable acid- or base- addition salt can be formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and/or organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.

[00129] The composition components (at least one TNAP inhibitor and optional additional components) may be in solution and/or suspension (for example, incorporated into microparticles, liposomes, or cells). These can be targeted to a particular cell type via antibodies, receptors, or receptor ligands.

[00130] In certain embodiments, the carrier molecule can be covalently linked to the TNAP inhibitor. In other embodiments, the TNAP inhibitor is conjugated to a coating molecule such as bovine serum albumin (BSA). Protein crosslinkers that can be used to crosslink the carrier molecule to the TNAP inhibitor are known in the art.

[00131] Targeting molecules (for example, antibodies or fragments thereof, ligands for specific receptors, or other proteins specifically binding to the surface of the cells to be targeted) can, in certain embodiments, be attached to the compositions and/or carriers described herein.

[00132] Liposomes, structures comprising an outer lipid bi- or multi-layer membrane surrounding an internal aqueous space, can be used to package the at least one TNAP inhibitor for delivery to cells. Liposomes can be multilamellar or unilamellar, or they can be multivesicular. A comprehensive review of lipid vesicles and methods for their preparation are described in "Liposome Technology", ed. G. Gregoriadis, CRC Press Inc., Boca Raton, Fla., Vol. I, II & III (1984), incorporated herein by reference.

[00133] In vivo/Ex vivo

[00134] A pharmaceutical composition comprising at least one TNAP inhibitor can, in certain embodiments, be administered in a pharmaceutically acceptable carrier and delivered to a subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (for example, uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis, and the like).

[00135] If ex vivo methods are employed, cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art. The composition can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate-mediated gene delivery, electroporation, microinjection, or proteoliposomes. The transduced cells can then be infused (for example, in a

pharmaceutically acceptable carrier) or homotopically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.

Kits

[00136] Kits comprising at least one TNAP inhibitor or a pharmaceutical composition comprising at least one TNAP inhibitor according to the invention are provided in an additional embodiment. Kits can include one or more other elements including, but not limited to, instructions for use; other reagents, e.g., a diluent, devices or other materials for preparing the composition for administration; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject.

Instructions for use can include instructions for therapeutic application, including suggested dosages and/or modes of administration, e.g., in a human subject, as described herein.

[00137] In another embodiment, a kit according to the invention can further contain at least one additional reagent, such as a diagnostic or therapeutic agent, e.g., a diagnostic agent to monitor a response to the composition according to the invention in the subject, or an additional therapeutic agent as described herein (see, e.g., the section herein describing combination therapy).

[00138] Diagnostic evaluation

[00139] Diagnostic evaluation can be carried out before, during, and/or after treatment: before, for example, to assess the extent of disease, during and/or after to assess the efficacy of the treatment.

[00140] Medial vascular calcification of the MMS type is indicated by measuring the ABI (ankle-brachial index); MMS is diagnosed with an ABI >1.1. In patients with suggested MMS (ABI > 1.1), the toe-brachial index (TBI) has been additionally proposed for segmental blood pressure measurements.

[00141] Imaging studies employing conventional X-ray or ultrasound allow unequivocal definition of VCm. CT scan can be particularly illustrative. Fragmentation of the internal elastic lamina is considered pathognomonic for pseudoxanthoma elasticum, or PXE.

[00142] It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

[00143] Various embodiments of the disclosure could also include permutations of the various elements recited in the claims as if each dependent claim was a multiple dependent claim incorporating the limitations of each of the preceding dependent claims as well as the independent claims. Such permutations are expressly within the scope of this disclosure.

[00144] While the invention has been particularly shown and described with reference to a number of embodiments, it would be understood by those skilled in the art that changes in the form and details may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention and that the various embodiments disclosed herein are not intended to act as limitations on the scope of the claims. All references cited herein are incorporated in their entirety by reference.

EXAMPLES

[00145] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

Example 1

[00146] The current understanding of PXE disease pathogenesis is based on the fact that the ATP -binding cassette sub-family C member 6 (ABCC6) is heavily expressed in the liver. It is believed that ABCC6 transports an unknown ligand out of the liver and into the circulation. This ligand prevents calcification at target organs such as the eye and skin. When the ABCC6 transporter is defective, as in PXE, there is no circulating factor released from the liver, and, thus, calcification of the target organs ensues.

[00147] Described herein are studies that are inconsistent with the current dogma of

PXE disease pathogenesis and have revealed a novel strategy for the affected patient population.

[00148] Molecular analysis

[00149] Genomic DNA was isolated from leukocytes. All exon and intron-exon boundaries of ABCC6 and ENPP1 were amplified by polymerase chain reaction (PCR) using genomic DNA as a template. PCR was performed in final volume of ΙΟμΙ containing 50ng of genomic DNA, Ι μΜ of forward and reserve primers, and 5μ1 of HotStart Master Mix (Qiagen). The PCR products were purified using ExoSap-IT (USB) for 45min at 37C and sequenced in both directions using the same primers and the Big Dye terminator kit v3.1 (Applied Biosystems). Reactions were purified over G-50 Sephadex beads. Linear amplification products were separated in an automated capillary sequencer (ABI PRISM 3130x1 Genetic Analyzer, Applied Biosystems). Sequences were analyzed with Sequencher software 4.8 (Gene Code Corporation).

[00150] Cell culture

[00151] Primary dermal fibroblasts were cultured from a 3-mm forearm punch- biopsy specimen, obtained from each patient, and grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, ImM L-glutamine, and 1% penicillin- streptomycin. Cells were fed twice a week and split 1 :2 at confluence.

[00152] In vitro calcification assay

[00153] A modified protocol for in vitro calcification was used for fibroblasts obtained from patients and controls. Cultures were treated with 0.1 μΜ dexamethasone, 50μΜ ascorbic acid-2-phosphate, and lOmM B-glycerol phosphate in alpha minimal essential medium supplemented with 10% fetal bovine serum and 1% penicillin- streptomycin (termed, osteogenic media) for 21 days, with replenishment of the medium every 4 or 5 days. On day 21, cells were washed with phosphate-buffered saline and fixed in 10% formalin for lOmins. After washing with water, a solution of 2% alizarin red S, pH 4.2, was used to stain calcium phosphate crystals. SBI-425 was dissolved in 100% DMSO. The TNAP-specific inhibitor compound 10μΜ SBI-425 or DMSO-control was added to the culture every time the cells were fed.

[00154] When stimulated with osteogenic media for 21 days, primary dermal fibroblasts derived from patients with biallelic mutations in ABCC6 could calcify in vitro, while normal and obligate heterozygous (data not shown) cell lines did not (Figure 1A). Briefly, primary dermal fibroblasts were derived from control and PXE patient skin biopsies. Fibroblast cultures were expanded and plated in 6-well plates. Once the cells reached confluency, they were treated with osteogenic media (consisting of beta- glycerophosphate, ascorbic acid, and dexamethasone in an aMEM media base) for 21 days. After 21 days, cells were fixed and stained with Alizarin red. Since PXE cell lines can calcify in vitro, these data indicate that PXE disease pathogenesis is cell-autonomous and not dependent on the liver.

[00155] Alkaline Phosphatase and ENPP1 enzyme assays [00156] Cells seeded in 6-well plates were grown with or without osteogenic media, subjected to mild lysis, and scraped into eppendorf tubes and kept on ice. Cell suspensions were spun at 12,000xg for 5mins. Supernatants were incubated with para- nitrophenylphosphate (pNPP) or para-nitrophenol-trimethylolpropane (pNP-TMP) substrates to assay for alkaline phosphatase or ENPPl activity, respectively. Both assays relied on the production of p-nitrophenol (pNP) to quantify enzymatic activity, as determined by absorption at 405nm.

[00157] Expression studies

[00158] RNA was isolated from cultured fibroblasts with the use of Trizol

(Invitrogen) and the RNeasy kit (Qiagen). RNA concentration and purity were measured on a Nanodrop ND-1000 apparatus (Nanodrop Technologies). First strand cDNA was synthesized using the high capacity RNA-to-cDNA kit (Life Technologies). Quantitative RT-PCR was performed utilizing 1 μg cDNA, TaqMan gene expression master mix reagents and Assay-on-Demand (Life Technologies) for ABCC6, ENPPl, and the house keeping genes 18S on a QuantStudio7 (Life Technologies) to determine relative gene expression.

[00159] To further substantiate a cell-autonomous model for PXE, primary dermal fibroblasts derived from patients with biallelic ABCC6 mutations showed increased tissue non-specific alkaline phosphatase (TNAP) activity in vitro (Figure IB). Briefly, fibroblasts derived from control and PXE patients were stimulated with aMEM or osteogenic media. After 5 days, cells were lysed, and TNAP activity was measured with an enzymatic assay. Increased TNAP depletes pyrophosphate (PPi), the primary inhibitor of calcification. These data indicate that decreasing TNAP could be exploited as a therapeutic strategy in PXE patients.

[00160] Figures 1C and ID further demonstrate that fibroblasts derived from PXE patients show a cell-autonomous phenotype (increased ENPPl enzyme activity and increased ALPL and ENPPl mRNA, respectively). Per Figure 1C, fibroblasts from patients with biallelic mutations in ABCC6 have increased ENPPl, indicating that ENPPl is upregulated as a compensatory mechanism to produce more PPi. Figure ID shows that the changes in the patient cells are happening at the enzymatic level in addition to the gene expression level.

Example 2 [00161] In view of the findings indicating that PXE pathogenesis is cell- autonomous and caused by defects in extracellular ATP metabolism, the pathway was targeted for therapeutic intervention. Bisphosphonates, specifically etidronate, are analogs of PPi and can directly bind to mineral preventing calcium phosphate crystal growth. Bisphosphonates have also been shown to inhibit TNAP activity and are currently being used to treat GACI and ACDC patients, though the clinical efficacy has not been well established. SBI-425 is a direct TNAP inhibitor and has the chemical structure:

(5-(5-chloro-2-methoxy-benzenesulfonylamino)-nicotinamide)

and was also used herein (Figure 2A).

[00162] Increased TNAP leads to in vitro calcification in the PXE patient cell lines.

Inhibiting TNAP proved to be a potential therapeutic target in vitro (Figure 2B). Briefly, fibroblasts derived from control and PXE patients were stimulated with aMEM or osteogenic media for 21 days and treated with DMSO (vehicle control) or 10μΜ SBI-425 (a TNAP-inhibitor, chemical structure shown supra). After 21 days, cells were fixed and stained with Alizarin red. Photomicrographs were obtained from the culture wells.

Alizarin red stains calcification a bright red color. In vitro, the custom-synthesized TNAP inhibitor SBI-425 prevented calcification, further indicating that the calcification is TNAP-mediated and could be responsive to anti-TNAP therapy.

Example 3

[00163] The first sign of calcification in PXE and GACI mouse models is in the fibrous capsule surrounding their vibrissae or whiskers on their snout, as evidenced by positive alizarin red staining. Abcc6 targeted knockout mice have a mild phenotype and indolent clinical course, whereas Enppl targeted mice develop robust calcification earlier and throughout their vasculature, closely recapitulating the human phenotypes.

[00164] To explore the potential interaction between Abcc6 and Enppl in vivo, an

Abcc6 knockout (KO) mouse was bred to an ENU-mutated Enppl mouse. Both mouse lines are available commercially through Jackson Laboratories. A cross between Abcc6 and £>y?p 7 -targeted mice revealed strong evidence for genetic interaction. By 15 weeks of age, Abcc6 KO mice show mild calcification, as evidenced by microCT of the mouse muzzle (data not shown). Abcc6 KO mice with one mutated Enppl allele showed acceleration and worsening of the calcification phenotype. Abcc6 KO mice with two targeted Enppl alleles were indistinguishable from Enppl homozygotes, suggested that, according to the basic principles of classical synthetic lethality, ABCC6 acts

downstream of ENPP 1.

Example 4

[00165] Mice were given ad libitum access to food and water. All animal experiments were approved by the Johns Hopkins Animal Care and Use Committee.

[00166] Mouse chow was formulated with etidronate (240mg/kg/day) or SBI-425

(30mg/kg/day), assuming 20g mouse. For etidronate, Purina made food pellets after incorporating etidronate. For SBI-425, powdered feed was mixed with SBI-425 (after being crushed with a mortar and pestle).

[00167] All mice were imaged on a SPECT/CT small animal machine at 6 weeks (baseline analysis), 12 weeks (interim analysis; 6 weeks on drug or placebo), and 20 weeks (final analysis; 14 weeks on drug or placebo) of age. Images were reconstructed and analyzed with ImageJ. Briefly, a Z-stack was created to encompass the entire region of vibrissae calcification (rostrally, from the tip of the nose to the zygomatic arch). To quantify this calcification, a threshold was manually determined to calculate the total area of ectopic calcification. Data are an average of analysis by 6 blinded-observers.

[00168] The in vitro findings were translated in vivo with a TNAP inhibitor treatment trial in a PXE (knockout) mouse model— the afore-mentioned Abcc6 KO model. The PXE mouse model was treated with two different drugs: etidronate (an FDA- approved drug) and SBI-425 (a custom-synthesized TNAP inhibitor). Mice were treated with drug (10 mg/kg IP qd (quaque die, per day)) for six weeks starting at six weeks of age. In this preliminary experiment using a small sample size, both drugs significantly decreased calcification in the mouse model, as evidenced by microCT and histology (Figure 3).

Example 5

[00169] Mouse plasma Alkaline Phosphatase and Pyrophosphate analysis [00170] At the conclusion of the treatment trial, mice were sacrificed humanely. Blood was collected by cardiac puncture with a 21 -gauge needle and placed in either lithium-heparin- or EDTA -coated microcontainers for alkaline phosphatase or

pyrophosphate analysis, respectively. Blood was spun at 150xg for 15mins at 4°C, and supernatant was collected and stored at -80°C until assays were performed.

[00171] Residual alkaline phosphatase levels were assayed as previously described

(Sergienko, et al. 2013 Methods Mol Biol 1053: 103-113). Briefly, plasma samples were thawed on ice and spun at 2,400xg for 10 mins. The plasma samples were mixed with substrates in buffer in 3: 1 volume ratio to initiate the reaction in a 1536-well clear-bottom assay plate, with ImM MgCl₂, 50μΜ ZnCl₂, ImM pNPP, and lOOmM diethanolamine buffer at pH 9.8 or Tris buffer at pH 7.5. 10μΜ of SBI-425 was spiked in control treatment plasma samples to determine non-TNAP phosphatase activity levels. Samples were read kinetically for an hour on the PHERAstar FS plate reader at OD380, to detect amount of pNP produced. If longer incubation was required due to slow activity at pH 7.5, the plate was sealed and read once every hour for 5 more hours.

[00172] Calcium Quantification

[00173] Calcium was extracted from formalin-fixed muzzles. Briefly, half of the dissected muzzle was fixed in 10% formalin and frozen at -80°C. The muzzle was freeze- dried with a lyophilizer and weighed. The tissue was physically macerated before homogenizing with a bead homogenizer (MP) for 15 mins at 4°C. Tissue was then soaked overnight in 10% formic acid. Samples were spun at max speed for 5mins before ΙΟμΙ of the supernatant was used in a Calcium Quantification Assay, as per manufacturer's instructions.

[00174] In at least one study, systemic administration of SBI-425 to a PXE mouse model did not prevent vibrissae calcification (data not shown). 6 week-old mice were treated every day with vehicle or drug. After an 8-week treatment trial, there was no difference in vibrissae calcification, even though SBI-425 successfully targeted and decreased circulating plasma TNAP activity. However, when SBI-425 and etidronate were administered orally, TNAP inhibition attenuated PXE vibrissae calcification (Figure 4A). Control and PXE mice were treated for 14 weeks; 30mg/kg/day SBI-425 or 240mg/kg/day etidronate or control. Demonstration that TNAP inhibition prevents calcification in vivo establishes TNAP as a common distal target in certain calcification disorders (for example, GACI, ACDC, and PXE), providing an attractive treatment option. [00175] The clinical efficacy of etidronate has not been well-established in GACI and ACDC patient populations. The etidronate dose used in the mouse trial described herein was 12 times higher than the FDA-approved dose. Furthermore, it has been demonstrated that first-generation bisphosphonates can lead to severely decreased bone density, with many adult patients on etidronate complaining of bone pain and gastric upset. Thus, the bones from the mice in the instant cohort are analyzed to determine the extent of bone compromise. SBI-425 is not expected to have a negative effect on bone density. Because SBI-425 is more potent than etidronate, and has a presumed reduced side effect profile, it is an attractive drug for the patient populations described herein.

[00176] In addition, SBI-425 inhibited residual plasma TNAP levels, whereas etidronate did not (Figure 4B). Blood was collected by cardiac puncture, transferred into lithium heparin-coated microtainer tubes and centrifuged at 1500xg for 15 min at 4°C to prepare plasma that was then stored at -80°C until analysis. TNAP was measured as described above. Biological replicates indicated in Figure 4B. A combination therapy of etidronate and SBI-425 is still contemplated.

Thus, a cross between Abcc6 knockout (KO) mice and Enppl -targeted mice revealed strong evidence for genetic interaction. Abcc6 KO mice with one mutated Enppl allele showed acceleration and worsening of the calcification phenotype. In contrast, Abcc6 KO mice with two targeted Enppl alleles were indistinguishable from Enppl homozygotes, suggesting that ABCC6 acts downstream of ENPP l. The strong expression of ABCC6 in the liver led to the prevailing hypothesis that calcification in PXE reflects failed liver secretion of an endocrine inhibitor of calcification. Contrary to this, it is described herein that, when provoked under osteogenic conditions, PXE patient fibroblasts had increased tissue non-specific alkaline phosphatase (TNAP) activity and showed a strong tendency for calcification that was prevented by a TNAP inhibitor, indicating a cell-autonomous defect. Breakdown products of ATP closely regulate TNAP activity, suggesting that PXE, like GACI, is caused by defects in local extracellular ATP metabolism. Additionally, an orally administered TNAP inhibitor attenuated ectopic calcification in Abcc6 KO mice. Taken together, these data indicate that PXE is caused by a reversible, liver-independent mechanism that induces defects in local extracellular ATP metabolism downstream of ENPPl and provides a novel therapeutic target for PXE and GACI patients.

Claims

1. A method of treating a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject a pharmaceutical composition comprising a TNAP inhibitor.

2. The method of claim 1, wherein the symptom is calcification of the arterial media.

3. The method of claim 1 or 2, wherein the disorder is selected from the group

consisting of atherosclerosis, hyperparathyroidism, vitamin D disorder, vitamin K deficiency, osteoporosis, generalized arterial calcification of infancy (GACI), diabetes mellitus (I or II), chronic kidney disease, dialysis-related calcification, calciphylaxis, M5nckeberg's sclerosis, Ehlers-Danlos syndrome, Kawasaki disease, pseudoxanthoma elasticum (PXE), IBGC, rheumatoid arthritis, Singleton- Merten syndrome, β-thalassemia, heterotropic calcification/ossification in amputees, tibial artery calcification, bone metastasis, bioprosthetic heart valve calcification, Paget's disease of bone (PDB), arterial calcification and distal joint calcification, arterial calcification due to deficiency of CD37 (ACDC), and Keutel syndrome.

4. The method of any one of claims 1 to 3, wherein the disorder is a monogenic disease.

5. The method of claim 4, wherein the monogenic disease is selected from

generalized arterial calcification of infancy (GACI), pseudoxanthoma elasticum (PXE), and arterial calcification due to deficiency of CD37 (ACDC).

6. The method of claim 1, wherein the disorder is characterized by reduced plasma levels of PPi, or genetic mutation, or clinical diagnostic evidence of calcification on biopsy/imaging.

7. The method of claim 1, wherein the disorder is pseudoxanthoma elasticum (PXE).

8. The method of claim 7, wherein the PXE is

PXE.

9. The method of any one of claims 1 to 8, wherein the administration is oral,

parenteral (e.g., intravenous, subcutaneous, intraperitoneal, or intramuscular injection), by inhalation, extracorporeal, or topical (including transdermal, ophthalmic, vaginal, rectal, intranasal).

10. The method of claim 9, wherein the administration is oral.

11. The method of any one of claims 1 to 12, wherein the TNAP inhibitor or is administered in a dosage of about 5 to about 100 mg/kg/day.

12. The method of claim 11, wherein the TNAP inhibitor is administered in a dosage of about 10 to about 50 mg/kg/day.

13. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is a

compound according to Formula I:

Formula I

wherein:

X¹ is =N- or =C(R²)-;

R¹ and R⁴ are independently selected from the group consisting of hydrogen, halogen, -CN, -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, haloalkyl, haloalkoxy, optionally substituted phenyl, and optionally substituted 5- or 6- membered heteroaryl;

R², R³, and R⁵ are independently selected from the group consisting of hydrogen, halogen, -CN, -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, optionally substituted alkoxy, haloalkyl, haloalkoxy, optionally substituted phenyl, and optionally substituted 5- or 6- membered heteroaryl;

R⁷ and R⁸ are independently hydrogen, optionally substituted alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted

heterocycloalkyl, optionally substituted phenyl, or R⁷ and R⁸ together with the nitrogen atom to which they are attached form an optionally substituted heterocycloamino;

R⁹ is selected from the group consisting of hydrogen, optionally substituted alkyl, haloalkyl, optionally substituted cycloalkyl, optionally substituted heterocycloalkyl, and optionally substituted phenyl; and A is selected from the group consisting of -C(0)-N(R⁷)-R⁸, -C(0)-0-R⁹, optionally substituted phenyl, and optionally substituted 5- or 6-membered heteroaryl, or

a pharmaceutically-acceptable salt, or derivative thereof.

14. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is a

compound having the chemical structure:

(5-(5-chloro-2-methoxy-benzenesulfonylamino)-nicotinamide)

or a pharmaceutically-acceptable salt, or derivative thereof.

15. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is a small molecule compound.

16. The method of claim 15, wherein the small molecule compound is selected from the group consisting of L-homoarginine, levamisole, theophylline, and lansoprazole.

17. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is a biaryl sulfanilamide, pyrazole, triazole, or a derivative thereof.

18. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is a

bisphosphonate.

19. The method of claim 18, wherein the bisphosphonate is selected from the group consisting of etidronate and clodronate.

20. The method of any one of claims 1 to 12, wherein the TNAP inhibitor is short hairpin RNA.

21. The method of any one of claims 1 to 20, wherein the TNAP inhibitor is

administered with at least one additional form of therapy.

22. The method of any claim 21, wherein the additional form of therapy is selected from the group consisting of another TNAP inhibitor, vitamin D sterols (calcitriol, alfacalcidol, doxercalciferol, maxacalcitol, paricalcitol), calcimimetics,

RENAGEL®, vitamins, vitamin analogs, antibiotics, lanthanum carbonate, lipid- lowering agents, anti-hypertensives, anti-inflammatory agents (steroidal, nonsteroidal), inhibitors of pro-inflammatory cytokine, adenosine agonists, adenosine receptor agonists, and cardiovascular agents.

23. The method of claim 21 or 22, wherein the additional form of therapy is

administered before, concurrently with, or after the composition.

24. Use of a TNAP inhibitor in the manufacture of a medicament for the treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject a pharmaceutical composition comprising a TNAP inhibitor.

25. A TNAP inhibitor for use in the treatment of a disorder or a symptom of the

disorder, wherein the disorder is characterized by medial vascular calcification in a subject, comprising administering to the subject a pharmaceutical composition comprising a TNAP inhibitor.

26. A pharmaceutical composition comprising a TNAP inhibitor and a

pharmaceutically acceptable carrier for the treatment of a disorder or a symptom of the disorder, wherein the disorder is characterized by medial vascular calcification.

27. A method of selectively treating

pseudoxanthoma elasticum

(PXE) comprising:

i) selecting a subject for treatment with a TNAP inhibitor on the basis of at least one of the following: the subject having reduced plasma levels of PPi; a genetic mutation, or clinical diagnostic evidence of calcification on biopsy/imaging; and,

ii) selectively administering at least one of the TNAP inhibitor of claim 14 and etidronate to the subject.