Methods and compositions for preventing or treating tissue calcification

Abstract

To provide methods and compositions for preventing or treating (e.g., slowing the progression of, arresting and / or reversing) tissue calcification in a subject in need of the prevention or treatment of the tissue calcification.SOLUTION: The invention provides methods of using menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) for preventing or treating tissue calcification in a subject with diabetes, chronic kidney disease, end-stage renal failure, or a subject undergoing hemodialysis and / or receiving anticoagulant therapy. The invention further provides methods and compositions for reducing one or more symptoms of chronic obstructive pulmonary disorder (COPD), including using menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), for preventing or treating the one or more symptoms of COPD.SELECTED DRAWING: None

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to and the benefit of U.S. Provisional Application No. 62 / 682,796, filed June 8, 2018, which is incorporated herein by reference in its entirety. FIELD OF THEINVENTION The present invention generally provides methods and compositions for preventing or treating (e.g., slowing, halting and / or reversing) tissue calcification in a subject in need thereof, and more specifically, the present invention relates to methods of using menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to prevent or treat (e.g., slowing, halting and / or reversing) tissue calcification in subjects with diabetes, chronic kidney disease (CKD), end stage renal disease, or subjects undergoing hemodialysis and / or anticoagulant therapy and / or statin therapy. The present invention also relates to methods and compositions for reducing one or more symptoms of chronic obstructive pulmonary disorder (COPD), including the use of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to prevent or treat (e.g., slowing, halting and / or reversing) one or more symptoms of COPD. [Background technology]

[0002] Under normal physiological conditions, plasma calcium and phosphate are present at concentrations approaching supersaturation levels and would be expected to precipitate in soft tissues (e.g., blood vessels) as crystalline hydroxyapatite. The finding that this process does not occur in healthy subjects suggested the existence of powerful chemical and biological means to block pathological calcification (Price, et al. (2002) "Discovery of a High Molecular Weight Complex of Calcium, Phosphate, Fetuin, and Matrix-Carboxyglutamic Acid Protein in the Serum of Etidronate-treated Rats," JOURNAL BIOL. CHEM. 277 (6): 3926-3934).

[0003] When the inhibition of calcification is disturbed, for example in subjects with diabetes and chronic kidney disease (CKD), pathological calcification of soft tissues (e.g., blood vessels) can occur. It is understood that diabetes can lead to CKD and end-stage renal disease (ESRD), which are characterized by uremia. Uremic toxins can promote the oxidation of vitamin K hydroquinone (KH2), which can, among other effects, disturb the cyclical regeneration of vitamin K (see FIG. 1). Also, certain treatments, including warfarin-based anticoagulation and statin therapy, can cause or contribute to dysregulation of vitamin K. Loss of functional vitamin K leads to the loss of a key regulator of mineralization, causing pathological calcification of tissues. In the case of arterial calcification, endothelial microvascular thrombosis is observed to occur, resulting in small vessel occlusion and death of the surrounding tissue.

[0004] Vitamin K is an essential enzymatic cofactor required for post-translational modification of vitamin K-dependent (VKD) proteins. Many VKD proteins are clinically relevant in CKD and ESRD patients, including, for example, central coagulation factors, such as factors II, VII, IX, and X, and extracellular matrix proteins, such as matrix Gla protein (MGP), activated protein C, and osteocalcin. Vitamin K is a group of fat-soluble vitamins that includes, among others, vitamin K1 (also known as phylloquinone), which is made by plants, and vitamin K2 (also known as menaquinone), which is made by bacteria in the gut microbiota. It is understood that the isoprenoid chain of vitamin K2 can contain 4 to 12 repeating isoprenoid units. For example, menaquinone-4 (or MK-4) contains 4 isoprenoid units, whereas menaquinone-7 (or MK-7) contains 7 isoprenoid units.

[0005] Regarding menaquinone-7 (MK-7), under normal conditions, MK-7 is reduced to menaquinol-7 (MKH2-7) (a form of vitamin K hydroquinone) by one or more NADPH-dependent reductase enzymes (e.g., quinone oxidoreductases). Only the reduced form of MK-7 (i.e., MKH2-7) functions as a cofactor for the enzyme gamma glutamate carboxylase (GGCX), which catalyzes the carboxylation of vitamin K-dependent proteins (see Figures 1 and 2). Enzymatic carboxylation of glutamic acid residues results in the oxidation of MKH2-7 to the 2,3-epoxide form (MK-7 2,3-epoxide). The final step of the vitamin K cycle requires the enzymatic reduction of vitamin MK-7 2,3-epoxide to MK-7 by vitamin K epoxide reductase complex subunit 1 (VKORC1, also referred to as VKOR). In some tissues, the paralog VKORC1L1 (VKORC1-Like-1) may also carry out this catalytic reaction.Warfarin is thought to block both the generation of the active forms of MKH2-7 and vitamin K2 and the regeneration of MK-2 from vitamin MK-7 2,3-epoxide, which may lead to the high incidence of calcification seen in patients receiving warfarin therapy.

[0006] Despite previous efforts, new clinical approaches are needed to prevent and / or reverse pathological calcification, particularly in subjects with diabetes, CKD, ESRD, and subjects receiving anticoagulant and / or statin therapy. Summary of the Invention

[0007] It has been discovered that menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) can be effectively used at high doses, e.g., at least 2 mg per day, to prevent, slow the progression of, halt and / or reverse tissue calcification in subjects, e.g., subjects with diabetes and / or chronic kidney disease.

[0008] In one aspect, the present invention provides a method for preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject with diabetes, chronic kidney disease or a combination thereof in need of preventing or treating tissue calcification, the method comprising administering at least 2 mg of substantially pure MK-7, MKH2-7 or a combination thereof per day to the subject, thereby preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification, wherein the MK-7, MKH2-7 or a combination thereof is administered in the form of a pharmaceutical composition. In certain embodiments, the subject is undergoing hemodialysis. In certain embodiments, the pharmaceutical composition comprises MK-7. In certain embodiments, the pharmaceutical composition comprises MKH2-7. In certain embodiments, the pharmaceutical composition comprises a combination of MK-7 and MKH2-7.

[0009] In another aspect, the present invention provides a method for preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject undergoing hemodialysis in need of preventing or treating tissue calcification, the method comprising administering at least 2 mg of substantially pure MK-7, MKH2-7 or a combination thereof per day to the subject, thereby preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification, wherein MK-7, MKH2-7 or a combination thereof is administered in the form of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises MK-7. In certain embodiments, the pharmaceutical composition comprises MKH2-7. In certain embodiments, the pharmaceutical composition comprises a combination of MK-7 and MKH2-7.

[0010] In certain embodiments of any of the above aspects, the subject has diabetes, for example type II diabetes.In certain embodiments, the subject has chronic kidney disease, for example stage 1, stage 2, stage 3, or end-stage renal disease (ESRD), for example stage 4 or stage 5.In certain embodiments, the subject is receiving non-warfarin-based anticoagulant therapy, for example oral anticoagulant therapy.

[0011] In another aspect, the present invention provides a method for preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject having stage 5 chronic kidney disease in need of preventing or treating tissue calcification and concurrently receiving oral non-warfarin-based anticoagulant therapy. The method comprises administering at least 2 mg per day of substantially pure MK-7, MKH2-7 or a combination thereof to the subject, thereby preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in the subject, wherein the MK-7, MKH2-7 or a combination thereof is administered in the form of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises MK-7. In certain embodiments, the pharmaceutical composition comprises MKH2-7. In certain embodiments, the pharmaceutical composition comprises a combination of MK-7 and MKH2-7. In certain embodiments, the subject is diabetic. In certain embodiments, the subject is undergoing hemodialysis.

[0012] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 to a subject increases the subject's serum T50 value (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the subject's serum T50 value prior to administration of MK-7 and / or MKH2-7, respectively.

[0013] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the subject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the ratio before administration of MK-7 and / or MKH2-7, respectively. In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 reduces the amount of uncarboxylated vitamin K-dependent protein in the subject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% or more) compared to the amount before administration of MK-7 and / or MKH2-7, respectively. In certain embodiments, the vitamin K-dependent protein is selected from matrix Gla protein (MGP), growth arrest specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, activated protein S, factor II, factor VII, factor IX, and factor X.

[0014] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 increases the plasma level of osteoprotegerin or fetuin-A (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the plasma concentration of osteoprotegerin or fetuin-A before administration of MK-7 and / or MKH2-7, respectively.

[0015] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 reduces the plasma level of D-dimer or high sensitivity C-reactive protein (hs-CRP) (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the plasma concentration of D-dimer or hs-CRP before administration of MK-7 and / or MKH2-7, respectively.

[0016] In certain embodiments of any of the above aspects, the subject has skin and / or vascular lesions, and administration of MK-7 and / or MKH2-7 reduces the size of the skin and / or vascular lesions compared to the size of the lesions before the start of the treatment regimen. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area of ​​the lesions by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%.

[0017] In yet another aspect, the invention provides a method of preventing or treating (e.g., slowing, halting and / or reversing the progression of) tissue calcification in a subject in need thereof, the method comprising administering at least 2 mg per day of substantially pure menaquinone-7 (MK-7), menaquinol-7 (MKH2-7), or a combination thereof, to a subject in need thereof, the method comprising: (i) increasing the serum T50 value of the subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the serum T50 value of the subject prior to administration of MK-7 and / or MKH2-7; and (ii) increases the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in the subject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the ratio before administration of MK-7 and / or MKH2-7; (iv) increases the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in the subject's plasma (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the ratio before administration of MK-7 and / or MKH2-7; or (v) increase the plasma level of osteoprotegerin or fetuin A (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or more) compared to the plasma concentration of osteoprotegerin or fetuin A prior to administration of MK-7 and / or MKH2-7; or - causing at least one of, or a combination of, a reduction in plasma levels of D-dimer or high sensitivity C-reactive protein (hs-CRP) (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma concentration of D-dimer or high sensitivity C-reactive protein (hs-CRP), thereby preventing or treating (e.g., slowing the progression of) tissue calcification in a subject;The present invention provides a method of treating or preventing osteoporosis, comprising administering to a subject a MK-7, MKH2-7, or combination thereof, to inhibit or reverse osteoporosis, wherein the MK-7, MKH2-7, or combination thereof is administered in the form of a pharmaceutical composition. In certain embodiments, the pharmaceutical composition comprises MK-7. In certain embodiments, the pharmaceutical composition comprises MKH2-7. In certain embodiments, the pharmaceutical composition comprises a combination of MK-7 and MKH2-7. The vitamin K-dependent protein can be selected from matrix Gla protein, growth arrest specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, activated protein S, factor II, factor VII, factor IX, and factor X.

[0018] In another aspect, the present invention provides a method for preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject in need thereof, the method comprising administering to the subject at least 2 mg per day of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) and a statin. In certain embodiments, the statin is selected from simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin. In certain embodiments, the MK-7 and / or MKH2-7 are administered in the same dosage form as the statin. In other embodiments, the MK-7 and / or MKH2-7 are administered in a separate dosage form from the statin. In certain embodiments, the administration of MK-7 and / or MKH2-7 prevents or reduces the vitamin K depletion effect of the statin.

[0019] In another aspect, the present invention provides a method for improving aortic compliance in a subject in need thereof, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day.

[0020] In another aspect, the present invention provides a method of preventing or treating (e.g., slowing, halting and / or reversing the progression of) peripheral vascular disease in a subject in need thereof, wherein the subject has ESRD or CKD, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day.

[0021] In another aspect, the present invention provides a method for preventing or treating (e.g., slowing, stopping and / or reversing) one or more symptoms of chronic obstructive pulmonary disease (COPD) in a subject who needs to prevent or treat one or more symptoms of chronic obstructive pulmonary disease (COPD), comprising administering at least 2 mg of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day to the subject, thereby preventing or treating (e.g., slowing, stopping or reversing) one or more symptoms of COPD, wherein MK7 and / or MKH2-7 are administered in a pharmaceutical composition.In certain embodiments, the one or more symptoms are selected from the group consisting of dyspnea, cough, mucus production, wheezing and elastinolysis.

[0022] In certain embodiments of any of the above aspects, the subject has diabetes, for example type II diabetes, or has been diagnosed with prediabetes.In certain embodiments, the subject has chronic kidney disease, for example stage 1, stage 2, stage 3, or end-stage renal disease (ESRD), for example stage 4 or stage 5.In certain embodiments, the subject is receiving non-warfarin-based anticoagulant therapy, for example oral anticoagulant therapy.

[0023] In certain embodiments, when non-warfarin-based anticoagulant therapy is used, anticoagulant therapy can include inhibitors of factor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban or fondaparinux) or factor IIa activity (e.g., dabigatran or argatroban).In certain embodiments, the subject has been previously exposed to warfarin-based anticoagulant therapy.

[0024] In certain embodiments, the subject is receiving a statin. For example, the statin can be selected from simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin, and fluvastatin.

[0025] In certain embodiments, the tissue calcification is soft tissue calcification. For example, the tissue calcification can be vascular or dermal calcification.

[0026] In certain embodiments of any of the above aspects, the method can include administering to the subject about 2 mg to about 1,000 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method can include administering to the subject about 5 mg to about 1,000 mg of MK-7 and / or MKH2-7 per day.

[0027] In certain embodiments of any of the above aspects, the method may include administering to the subject about 2 mg to about 750 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 5 mg to about 750 mg of MK-7 and / or MKH2-7 per day. In certain embodiments of any of the above aspects, the method may include administering to the subject about 2 mg to about 500 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 5 mg to about 500 mg of MK-7 and / or MKH2-7 per day. In certain embodiments of any of the above aspects, the method may include administering to the subject about 2 mg to about 250 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 5 mg to about 250 mg of MK-7 and / or MKH2-7 per day. In certain embodiments of any of the above aspects, the method may include administering to the subject about 2 mg to about 100 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 5 mg to about 100 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 10 mg to about 75 mg of MK-7 and / or MKH2-7 per day, for example, administering to the subject 10, 25, 50, or 75 mg of MK-7 and / or MKH2-7 per day.

[0028] In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. If the subject is undergoing hemodialysis, MK-7 and / or MKH2-7 can be administered to the subject for a period of time that includes at least the duration of the hemodialysis.

[0029] In certain embodiments, MK-7 and / or MKH2-7 are administered orally. MK-7 and / or MKH2-7 may be placed in a tablet, caplet, or capsule.

[0030] The above description describes several aspects and embodiments of the present invention. The patent application specifically contemplates all combinations and variations of the aspects and embodiments. These and other aspects and features of the present invention are set forth in the following detailed description and claims. [Brief description of the drawings]

[0031] The above and other objects, features and advantages of the present invention will become apparent from the following description of the preferred embodiment as illustrated in the accompanying drawings, in which like references identify common features in corresponding drawings.

[0032] [Figure 1] FIG. 1 is a schematic diagram showing the vitamin K cycle and the effects of uremia- and hemodialysis-induced vitamin K hydroquinone oxidation.

[0033] [Diagram 2] FIG. 2 is a schematic diagram showing the carboxylation of vitamin K-dependent proteins by gamma glutamate carboxylase (GGCX). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The present invention is based, in part, on the discovery that menaquinone-7 (MK-7) and / or the reduced form of MK-7, menaquinol-7 (MKH2-7), can be administered to a subject in need thereof to prevent or treat (e.g., slow, halt or reverse the progression of) tissue calcification in a subject, for example a subject with diabetes and / or chronic kidney disease.

[0035] Without wishing to be bound by theory, oxidative stress induced by retention of uremic toxins depletes functional vitamin K2 in vascular endothelium, leading to suboptimal concentrations of certain vitamin K-dependent proteins, including carboxylated MGP and activated protein C. The subsequent decrease in calcium-binding capacity of these and other vitamin-dependent proteins contributes to abnormal tissue calcification. High doses of substantially pure MK-7 and / or MKH2-7 can be used to increase carboxylation of vitamin K-dependent proteins, thereby preventing, slowing, stopping and / or reversing tissue calcification, where MK-7 and / or MKH2-7 are administered in the form of a pharmaceutical composition. MK-7 and / or MKH2-7 can be administered in a single dosage unit (e.g., one capsule) or multiple dosage units (e.g., multiple capsules), as long as the required amount of MK-7 and / or MKH2-7 is administered per day. In certain embodiments, the pharmaceutical composition comprises MK-7. In certain embodiments, the pharmaceutical composition comprises MKH2-7. In certain embodiments, the pharmaceutical composition comprises a combination of MK-7 and MKH2-7.

[0036] I. Menaquinone-7 (MK-7) and / or Menaquinol (MKH2-7) The compositions of the present invention include menaquinone-7 (MK-7), a form of vitamin K2. The IUPAC name for MK-7 is 2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-dione, and the chemical structure of MK-7 is shown in Formula I. [ka] (Formula I)

[0037] Also contemplated for use according to the methods herein is the reduced form of MK-7, menaquinol-7 (MKH2-7), which has an IUPAC name of 2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaenyl]-3-methylnaphthalene-1,4-diol, and the chemical structure of MKH2-7 is shown in Formula II. [ka] (Formula II)

[0038] Without wishing to be bound by theory, it is believed that supplemental MK-7 and / or MKH2-7 forms of vitamin K provide increased protection against non-specific oxidation of vitamin K intermediates caused by uremia and / or dialysis.

[0039] The long aliphatic chain of MK-7 is not synthesized by humans, but is synthesized in the colon by bacteria. Dietary sources of MK-7 include bacterially fermented foods such as natto (soybeans fermented with Bacillus subtilis), cheese, sauerkraut, buttermilk, pork, eel, plaice, and buckwheat bread. Additionally, MK-7 is available as a dietary supplement (e.g., Rejuvenation Therapeutics®; Bio-Tech TM Available as Vitamin K2 MK-7 from Pharmacal MK-7 Vitamin K2, Fayetteville AR; NOW® Foods MK-7 Vitamin K-2.

[0040] However, the present invention employs substantially pure MK-7 and / or MKH2-7 administered in the form of a pharmaceutical composition.

[0041] MK-7 can also be produced synthetically, for example, as described in Baj et al. (2016) "Convergent Synthesis of Menaquinone-7 (MK-7)," ORG. PROCESS RES. DEV. 20:1026-1033, WO 2010 / 034999 and WO 2010 / 035000 (also published as U.S. Patent Application No. 2011 / 0207967).

[0042] MKH2-7 can be synthesized from MK-7 by reducing MK-7 using conventional reduction reactions known in the art, for example, by reduction with zinc and acetic acid as described in Marchand et al. (1991) "Mild and Highly Selective Ultrasound-promoted Zinc / Acetic Acid Reduction of C=C Bonds in α,β-Unsaturated γ-Dicarbonyl Compounds," SYNTHESIS 1991(3):198-200. [ka]

[0043] In this method, MK-7 (e.g., 1 g, 1.54 mmol) is dissolved in glacial acetic acid (15 mL) and powdered zinc (e.g., 0.8 g, 12.3 mmol) is added. The resulting mixture can be sonicated for 0.5 hours or refluxed until the reaction is complete. The resulting mixture is filtered and the residue is washed with dichloromethane. The combined filtrates are concentrated in vacuo to obtain the pure reduced product. The procedure is carried out under argon. To stabilize MKH2-7 in its reduced form, it can be useful to mix it with one or more antioxidants, such as vitamin C, vitamin C esters (e.g., ascorbyl palmitate), and / or vitamin E, for example, in a capsule or softgel. Alternatively, prodrugs of MKH2-7 can be made, for example by esterifying one or both hydroxyl groups with various groups (e.g., acetate) to generate stable prodrugs that are metabolized in the subject to generate MKH2-7.

[0044] II. Dosage Form and Administration In certain embodiments, the composition useful according to the present invention comprises, consists essentially of, or consists of substantially pure MK-7 and / or MKH2-7.Substantially pure refers to a composition of active ingredients that comprises at least 95% by weight, at least 96% by weight, at least 97% by weight, at least 98% by weight, at least 99% by weight, or at least 99.5% by weight of MK-7 and / or MKH2-7.In certain embodiments, MK-7 and / or MKH2-7 are the only vitamins administered in the dosage form.

[0045] As used herein, the phrases "effective amount" and "therapeutically effective amount" refer to an amount of a compound (e.g., MK-7 and / or MKH2-7) sufficient to exert one or more beneficial or desired effects. An effective amount may be administered in one or more administrations, applications or dosages, and is not intended to be limited to a particular formulation or route of administration.

[0046] In another embodiment, the present invention relates to a method for the preparation of an effective amount of MK-7 and / or MKH2-7, for example, about 2 mg to about 1,000 mg, about 2 mg to about 750 mg, about 2 mg to about 500 mg, about 2 mg to about 250 mg, about 2 mg to about 200 mg, about 2 mg to about 150 mg, about 2 mg to about 100 mg, about 2 mg to about 50 mg, about 2 mg to about 25 mg, about 5 mg to about 1,000 mg, about 5 mg to about 750 mg, about 5 mg to about 500 mg, about 5 mg to about 250 mg, about 5 mg to about 200 mg, about 5 mg to about 150 mg, about 5 mg to about 100 mg, about 5 mg to about 50 mg, about 5 mg to about 25 mg, about 10 mg to about 1,000 mg, about 10 mg to about 750 mg, about 10 mg to about 500 mg, about 10 mg to about 250 10 mg to 200 mg, 10 mg to 150 mg, 10 mg to 100 mg, 10 mg to 50 mg, 10 mg to 25 mg, 15 mg to 1,000 mg, 15 mg to 750 mg, 15 mg to 500 mg, 15 mg to 250 mg, 15 mg to 200 mg, 15 mg~150 mg, 15 mg~100 mg, 15 mg~50 mg, 15 mg~25 mg, 25 mg~1,000 mg, 25 mg~750 mg, 25 mg~500 mg, 25 mg~250 mg, 25 mg~200 mg, 25 mg~150 mg, 25 mg~100 mg, about 25 mg to about 50 mg, about 50 mg ~ 1,000 mg, 50 mg ~ 750 mg, 10 mg ~ 500 mg, 50 mg ~ 250 mg, 50 mg ~ 200 mg, 50 mg ~ 150 mg, 50 mg ~ 100 mg, 75 mg ~ 1,000 mg, 75 mg ~ 750 mg, 75 mg ~ 500 mg, 75 mg ~ about 250 mg, about 75 mg - about 200 mg, about 75 mg - about 150 mg, about 75 mg - about 100 mg, about 100 mg - about 1,In certain embodiments, dosage forms, e.g., oral dosage forms, are provided that contain about 1,000 mg, about 100 mg to about 750 mg, about 100 mg to about 500 mg, about 100 mg to about 250 mg, about 100 mg to about 200 mg, or about 100 mg to about 150 mg. In certain embodiments, 2 mg, 5 mg, 10 mg, 25 mg, 75 mg, or 100 mg of substantially pure MK-7 and / or MKH2-7 are administered to a subject in a suitable dosage form, e.g., a tablet, caplet, or capsule (e.g., a liquid or gel capsule).

[0047] In certain embodiments, the method may include administering to the subject about 2 mg to about 100 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 2.5 mg to about 100 mg, about 3 mg to about 100 mg, about 4 mg to about 100 mg, or about 5 mg to about 100 mg of MK-7 and / or MKH2-7 per day. In certain embodiments, the method may include administering to the subject about 2 mg, 2.5 mg, about 3 mg, about 4 mg, about 5 mg, or about 7.5 mg of MK-7 and / or MKH2-7 per day. In other embodiments, the method may include administering to the subject about 10 mg to about 100 mg of MK-7 and / or MKH2-7 per day, for example, administering to the subject about 10, 25, 50, 75, or 100 mg of MK-7 and / or MKH2-7 per day.

[0048] It is understood that MK-7 and / or MKH2-7 may be administered in a single dosage unit (e.g., one capsule) or multiple (e.g., 2, 3, or 4) dosage units (e.g., multiple capsules). Compositions for use according to the invention may be formulated as one or more dosage units. Such dosage units may be administered once per day or multiple times per day (e.g., 1 to about 10 times, 1 to about 8 times, 1 to about 6 times, 1 to about 4 times, or 1 to 2 times), or as many times as necessary to elicit a therapeutic response. The dosage units may be packaged in a kit, e.g., a kit including one or more blister packages of about 1 to about 20 dosage units (e.g., capsules) per sheet, or a bottle including multiple dosage units (e.g., capsules).

[0049] In certain embodiments, pharmaceutical compositions comprising MK-7 and / or MKH2-7 can be formulated for administration in solid or liquid form, including drench (aqueous or non-aqueous solution or suspension), tablet (e.g. buccal, sublingual and / or targeted for systemic absorption), bolus, powder, granule, paste for application to tongue and / or topical cream. As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with one or more pharmaceutically acceptable carriers (inert or active) that make the composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers and additives, such as phosphate buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions) and various types of wetting agents. The composition can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see Martin, Remington's Pharmaceutical Sciences, 15th Ed., Mack Publ. Co., Easton, PA (1975).

[0050] In certain embodiments, the MK-7 and / or MKH2-7 compositions may be formulated in oils such as castor oil, sesame oil, medium chain triglyceride (MCT) oil, olive oil, soybean oil, or coffee bean oil. Additives suitable for use with the MK-7 and / or MKH2-7 compositions include antioxidants, bioavailability enhancers, solubility enhancers or solubilizers, stabilizers, and the like.

[0051] In certain embodiments, the MK-7 and / or MKH2-7 compositions comprise one or more antioxidants, such as one or more antioxidants selected from DL-α-tocopherol (vitamin E), ascorbic acid (vitamin C) or vitamin C esters, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), lecithin, citric acid, sesame oil, olive oil, soybean oil, and coffee bean oil.

[0052] In certain embodiments, the MK-7 and / or MKH2-7 compositions include any of the following: Tween® 20 (polyethylene glycol sorbitan monolaurate), Tween® 80 (polyethylene glycol sorbitan monooleate), Kolliphor® EL (polyoxyl castor oil, also known as Cremophor EL), Capmul® MCM (mono-diglycerides of medium chain fatty acids such as caprylic and capric acids), lecithin, Lauroglycol TM 90 (propylene glycol monolaurate (type II)), deoxycholic acid, Phosal® 50 PG (lecithin, sunflower mono-diglycerides, ascorbyl palmitate in propylene glycol), beeswax and emulsifiers selected from wax flakes.

[0053] In certain embodiments, the MK-7 and / or MKH2-7 compositions include any of the following: Maisine® CC (glyceryl monolinoleate), Gelucire® 44 / 14 (lauroyl polyoxyl-32 glyceride), Capmul® PG-8 NF (propylene glycol monoester of caprylic acid), Plurol® Oleique (polyglyceryl-6 dioleate), Lauroglycol TM 90 (Propylene Glycol Monolaurate (Type II), Labrasol® (Caprylocaproyl Polyoxyl-8 Glycerides), Captisol® (SBE-β-Cyclodextrin), Labrafil® M2125CS (Linoleoyl Polyoxyl-6 Glycerides) and Phosal® 50 PG (Lecithin, Sunflower Mono-Diglycerides, Ascorbyl Palmitate in Propylene Glycol).

[0054] In certain embodiments, the MK-7 and / or MKH2-7 compositions contain at least one additional ingredient selected from the group consisting of oleic acid, Kolliphor® EL (polyoxyl castor oil, also known as Cremophor EL), Vitamin E TPGS (D-α-tocopherol polyethylene glycol-1000 succinate), Maisine® CC (glyceryl monolinoleate), Gelucire® 44 / 14 (lauroyl polyoxyl-32 glyceride), Miglyol® 812N (ester of caprylic acid and glycerin derived from saturated coconut and palm kernel oils), Plurol® Oleique (polyglyceryl-6 dioleate), Lauroglycol TM 90 (Propylene Glycol Monolaurate (Type II), Labrasol® (Caprylocaproyl Polyoxyl-8 Glycerides), Kolliphor® EL (Polyoxyl Castor Oil), Captisol® (SBE-β-Cyclodextrin), Encapsin TM HPB (hydroxypropyl-β-cyclodextrin), Peceol TM(glycerol / glyceryl monooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride) and medium chain mono- and di-glycerides.

[0055] In certain embodiments, the MK-7 and / or MKH2-7 compositions include isopropyl myristate, Capmul® PG-8 NF (propylene glycol monoester of caprylic acid), Lauroglycol TM 90 (propylene glycol monolaurate (type II)), polyethylene glycol (PEG) and propylene glycol.

[0056] In certain embodiments, the MK-7 and / or MKH2-7 compositions comprise a stabilizer selected from the group consisting of oleic acid and DL-α-tocopherol.

[0057] In certain embodiments, the MK-7 and / or MKH2-7 compositions contain Sodium Oleate, Span 20 (sorbitan laurate), Span® 80 (sorbitan oleate), Vitamin E TPGS (D-α-tocopherol polyethylene glycol-1000 succinate), Lauroglycol TM 90 (propylene glycol monolaurate (type II)), Labrasol® (caprylocaproyl polyoxyl-8 glyceride), polyethylene glycol (PEG) and Captisol® (SBE-β-cyclodextrin).

[0058] Other suitable additives include one or more fatty acids selected from lauric acid, valeric acid, caproic acid, capric acid, caprylic acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, and arachidic acid and esters thereof.

[0059] In certain embodiments, the composition useful for carrying out the present invention can be delivered orally.The term "orally deliverable" or "oral administration" as used herein includes any form of delivery of the composition described herein to a subject, in which the composition is placed in the mouth of the subject, regardless of whether the composition is swallowed.Therefore, "oral administration" includes buccal and sublingual as well as esophageal administration.

[0060] In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least 2 weeks, for example, 2, 3, 4, 5 weeks or more. In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least 6 weeks. In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least 2 months, at least 3 months, at least 6 months, at least 1 year, at least 2 years, at least 3 years or indefinitely. In certain embodiments, MK-7 and / or MKH2-7 are administered at least as long as the subject is receiving anticoagulant therapy that is not warfarin or a drug that has a similar mechanism of action to warfarin (e.g., acenocoumarol and phenprocoumon), and / or statin therapy. If the subject is receiving hemodialysis, MK-7 and / or MKH2-7 can be administered to the subject for a period of time, including the period of hemodialysis.

[0061] In certain embodiments, MK-7 and / or MKH2-7 compositions are administered together with statins, such as simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin.MK-7 and / or MKH2-7 compositions can be formulated into a single dosage form with statins, such as dosage forms described herein.In other embodiments, MK-7 and / or MKH2-7 compositions can be formulated into a separate dosage form with statins.The formulation of statins, such as simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin, is well known in the art.

[0062] The terms "subject" and "patient" as used herein are used interchangeably and refer to an organism to be treated by the methods and compositions of the present invention. Such an organism is preferably a mammal (e.g., a human, a mouse, a rat, a guinea pig, a dog, a cat, a horse, a cow, a pig, or a non-human primate, such as a monkey, a chimpanzee, a baboon, and a rhesus monkey), more preferably a human.

[0063] III. Method The present disclosure relates, in part, to methods of preventing or treating, e.g., slowing, halting and / or reversing, tissue calcification in a subject in need thereof. In certain embodiments, the methods relate to preventing tissue calcification. In certain embodiments, the methods relate to slowing the progression of tissue calcification. In certain embodiments, the methods relate to halting tissue calcification. In certain embodiments, the methods relate to reversing tissue calcification.

[0064] In certain embodiments, the present disclosure relates to a method of improving aortic compliance in a subject in need thereof, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day. In certain embodiments, the present disclosure relates to a method of slowing, halting and / or reversing the progression of peripheral vascular disease in a subject in need thereof, the subject having ESRD or CKD, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day.

[0065] The term "treating" as used herein includes any effect that results in the improvement of a condition, disease, disorder, etc., or the improvement of symptoms thereof, such as, for example, alleviation, reduction, slowing, arrest, regulation, improvement, or elimination. Treating can be curing, improving, or at least partially improving a disorder. In certain embodiments, treating is curing a disease. The term "disorder" refers to the term disease, condition, or illness, and is used interchangeably, unless otherwise specified.

[0066] The method may include administering to a subject a menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) composition described herein (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50, 75 mg or more per day of substantially pure MK-7 and / or MKH2-7), thereby preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification. Tissue calcification refers to the accumulation of calcium salts in bodily tissues. In certain embodiments, the tissue calcification is soft tissue calcification. The term "soft tissue" includes any tissue that is not bone or tooth. In certain embodiments, the tissue calcification is calcification of blood vessels or skin.

[0067] 1. Diabetes, CKD, ESRD, hemodialysis In certain embodiments, the subject to be treated with the methods and / or compositions described herein has diabetes, for example type I or II diabetes, or has been diagnosed with prediabetes.Diabetes often leads to chronic kidney disease (CKD) because uncontrolled blood sugar and blood pressure damage the capillaries of the kidney.CKD often leads to the development of uremia (urea in the blood), which inhibits the cyclical regeneration of vitamin K and can cause tissue calcification.Therefore, in certain embodiments, the subject has CKD.

[0068] Patients with chronic kidney disease are classified into stages based on their glomerular filtration rate (GFR). Stage 1 patients have a GFR ≥ 90 mL / min / 1.73 m 2, stage 2 is 60-89 mL / min / 1.73 m 2 , stage 3 is 30-59 mL / min / 1.73 m 2 , stage 4 is 15-29 mL / min / 1.73 m 2 , stage 5 is <15 mL / min / 1.73 m 2 In certain embodiments, the subject has stage 3, stage 4 or stage 5 CKD. Patients with stage 4 or stage 5 CKD are considered to have end-stage renal disease (ESRD). Other types of renal disease, such as polycystic renal disease, can also cause ESRD. In certain embodiments, the subject has ESRD.

[0069] Patients with ESRD require hemodialysis. However, hemodialysis often results in the oxidation of many tissue proteins, and both CKD and ESRD patients often have a higher percentage of carbonyl proteins (15-fold) compared to normal controls. The percentage of carbonyl proteins can be even higher in patients undergoing hemodialysis, suggesting that hemodialysis contributes to the oxidative load.

[0070] As discussed above, it is believed that the oxidative load caused by hemodialysis leads to the oxidation of KH2 to MK-7.It is believed that the oxidation of KH2 by hemodialysis can block the ability of GGCX to function as a cofactor, and reduce the gamma-carboxylated form of vitamin K-dependent proteins.Therefore, the oxidative effect of hemodialysis may contribute to the high rate of soft tissue (e.g., vascular) calcification observed in CKD and ESRD populations.Therefore, in certain embodiments herein, the subject is undergoing hemodialysis.

[0071] 2. Anticoagulant therapy Vitamin K is necessary for the production of clotting factors. However, certain anticoagulants, such as certain oral anticoagulants, function as vitamin K antagonists to prevent blood clotting. Considering that tissue calcification can occur when vitamin K activity is unregulated, the method herein relates to preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject who is undergoing anticoagulant therapy and needs to prevent or treat tissue calcification, comprising administering to the subject menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) comprising compositions described herein, thereby preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification.

[0072] Exemplary anticoagulant therapy includes inhibitors of factor Xa activity or factor IIa activity. Inhibitors of factor Xa activity include oral therapy apixaban (e.g., Eliquis®, Bristol-Myers Squibb), rivaroxaban (e.g., Xarelto®, Janssen), betrixaban (e.g., Bevyxxa®, Portola Pharmaceuticals) and edoxaban (e.g., Savaysa®, Daiichi Sankyo), and subcutaneous therapy fondaparinux (e.g., Arixtra®, GlaxoSmithKline). Inhibitors of factor IIa (thrombin) activity include oral therapy dabigatran (e.g., Pradaxa®, Boehringer Ingelheim) and intravenous therapy argatroban (e.g., Pfizer).

[0073] In certain embodiments, the subject is simultaneously receiving non-warfarin-based anticoagulant therapy.Non-warfarin-based anticoagulant therapy can be based on anticoagulants that do not primarily act to block the conversion of vitamin K to vitamin K hydroquinone and / or the conversion of vitamin K 2,3-epoxide to vitamin K.Exemplary non-warfarin-based anticoagulants can act as inhibitors of factor Xa activity or inhibitors of factor IIa activity.Exemplary non-warfarin-based anticoagulants include apixaban, rivaroxaban, betrixaban, edoxaban, dabigatran, fondaparinux and argatroban.In certain embodiments, the subject has previously been exposed to warfarin-based anticoagulant therapy.

[0074] In certain embodiments, the method described herein relates to preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject in need thereof, where the subject has CKD or ESRD and is receiving oral non-warfarin-based anticoagulant therapy.The method can include administering at least 2 mg of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day to the subject, thereby preventing or treating (e.g., slowing, stopping or reversing) tissue calcification.The subject can also have diabetes, pre-diabetes, and / or undergo hemodialysis.

[0075] 3. Statin Therapy It has been observed that coronary artery calcification may increase with statin use (Saremi et al. (2012) "Progression of Vascular Calcification Is Increased With Statin Use in the Veterans Affairs Diabetes Trial (VADT)," DIABETES CARE 35:2390-2392). Lipophilic statins have been shown to inhibit the enzymatic activity of UbiA prenyltransferase domain-containing protein (UBIAD1), an enzyme involved in vitamin K synthesis (Nakagawa et al. (2010) "Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme," NATURE 468(7320):117-21). Furthermore, in vitro experiments seem to indicate that vitamin K synthesis is impaired in the presence of statins (Chen et al. (2017) "Does statins promote vascular calcification in chronic kidney disease?" EUR. J. CLIN. INVEST. 47(2): 137-148).

[0076] Without wishing to be bound by theory, it is believed that HMG coA reductase inhibitors directly inhibit MK-4 production in coronary vascular smooth muscle, which may explain why statins have not been shown to reduce cardiovascular mortality in patients with CKD or ESRD. One possible explanation for this observation is that CKD and ESRD patients are functionally deficient in vitamin K, and blocking endogenous vascular smooth muscle cell vitamin K (MK-4) production would only exacerbate calcification in the vascular media, thus mitigating the potential benefits of lipid reduction. Furthermore, the loss of vascular compliance associated with increased calcification may contribute to overall cardiovascular mortality.

[0077] Accordingly, provided are methods of preventing or treating (e.g., slowing, halting and / or reversing) tissue calcification in a subject in need thereof, wherein the subject is receiving a statin. The method may comprise administering to the subject a menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) composition described herein. In certain embodiments, the subject is receiving a statin, such as simvastatin (e.g., Zocor®, Merck & Co., Inc.), lovastatin (e.g., Mevacor®, Merck & Co., Inc.), atorvastatin (e.g., Lipitor®, Pfizer), pravastatin (e.g., Pravachol®, Bristol-Myers Squibb Co.), pitavastatin (e.g., Livalo®, Kowa Pharmaceuticals America), rosuvastatin (e.g., Crestor®, AstraZeneca), and fluvastatin (e.g., Lescol®, Novartis Pharmaceuticals).

[0078] Also provided are methods of preventing or treating (e.g., slowing, halting and / or reversing) tissue calcification in a subject in need thereof, the method comprising administering to the subject a menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) composition described herein and a statin, such as simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin. The menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) and the statin can be administered as separate dosage forms or in the same dosage form.

[0079] 4. Improve aortic compliance and halt and / or reverse peripheral vascular dysfunction In certain embodiments, the present disclosure relates to a method of halting and / or reversing peripheral vascular disease in a subject in need thereof, the subject having ESRD or CKD, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).In certain embodiments, the present disclosure relates to a method of improving aortic compliance in a subject in need thereof, the method comprising administering to the subject an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).

[0080] The term "vascular disorder" refers to any disease or disorder that affects blood vessels.This can include any inflammatory, metabolic, coagulative, embolic or degenerative disease, disorder or condition.In certain embodiments, the vascular disorder is vascular calcification, for example medial or intimal calcification.

[0081] One of the clinical consequences of vascular calcification is arteriosclerosis, which reduces vascular compliance (e.g., vascular elasticity), e.g., aortic compliance. Thus, according to the methods described herein, administration of an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) can improve aortic compliance. The reduction in aortic compliance can be assessed using aortic plethysmography (i.e., the ability of blood vessels to expand), see, for example, Inuma et al. (2012) HONG KONG JOURNAL OF NEPHROLOGY 14(2):48-53. The reduction in aortic compliance can also be assessed by measuring pulse wave velocity (PWV). PWV is the speed at which arterial pulses travel through the circulatory system and is used as a measure of arteriosclerosis. The higher the PWV, the higher the arteriosclerosis and therefore the lower the vascular compliance. Methods for measuring PWV are known in the art and are described, for example, in Pereira et al. (2015) "Novel Methods for Pulse Wave Velocity Measurement," J. MED. BIOL. ENG. 35:555-565. Briefly, PWV can be assessed by measuring regional PWV, such as carotid-femoral PWV or local PWV. Methods for measuring regional PWV are known in the art and include, for example, the use of a pulse transducer probe, a TY-360 pressure transducer, a Doppler unit synchronized with an electrocardiogram (ECG), a pulse transducer probe, photoplethysmography, Complior® (piezoelectric pressure transducer), SphygmoCor®, Arteriograph®, PulsePen® (tonometer and integrated ECG unit). Methods for measuring regional PWV are known in the art and include, for example, magnetic resonance imaging, ultrasound and angiography.

[0082] In certain embodiments, administration of MK-7 and / or MKH2-7 increases the aortic compliance of the blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the aortic compliance of the blood vessel by about 10% to 500%, about 50% to 200%, about 75% to 125%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the aortic compliance of the vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the vessel before administration of MK-7 and / or MKH2-7. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the aortic compliance of the vessel by about 10% to 500%, about 50% to 200%, about 75% to 125% compared to the vessel before administration of MK-7 and / or MKH2-7.

[0083] The coronary artery calcification score (CAC) can be used to estimate the degree of calcification in the thoracic arteries. A high CAC score indicates calcification, and treatment has the goal of halting or reversing the long-term increase in the CAC score, or slowing the rate of increase.

[0084] 5. Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease is a term that refers to progressive lung disease that makes breathing difficult. The two main forms of COPD are emphysema and chronic bronchitis. Symptoms of COPD include difficulty breathing, coughing, mucus production and wheezing. Elastin degradation (proteolysis of elastin) is also a key feature of COPD. It contributes to loss of flexibility in arteries and promotes calcification of the intima of blood vessels. It has also been shown to be a strong predictor of mortality in COPD patients (Rabinovich et al., (2016) "Circulating desmosine levels do not predict emphysema progression but are associated with cardiovascular risk and mortality in COPD," ERJ Express doi: 10.1183 / 13993003.01824-2015). MGP has been shown to inhibit the production of matrix metalloproteinases that promote elastin degradation. It has therefore been hypothesized that vitamin D is a key determinant of the rate of elastin degradation, and that low vitamin D levels result in low MGP activity that is insufficient to protect against elastin degradation (Piscaer et al., (2017) "Vitamin D deficiency: the linking pin between COPD and cardiovascular diseases?" RESP. RES. 18:189). Without wishing to be bound by theory, increased production of activated (carboxylated) MGP by administration of MK-7 and / or MKH2-7 inhibits the deleterious effects of elastin degradation in subjects with COPD, thereby preventing or slowing, halting or reversing the progression of one or more symptoms of COPD.

[0085] Reduction in symptoms of COPD, including dyspnea, coughing, mucus production and wheezing, can be measured by any means known in the art. A slowing in the rate of increase of one or more COP symptoms over time indicates a slowing in the progression of one or more symptoms of COPD, or a reduction in one or more symptoms, for example by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, indicates a reversal of one or more symptoms of COPD.

[0086] Elastin degradation can be measured by any method known in the art, including, for example, measuring blood isodesmosine levels in a subject. A slower rate of increase in isodesmosine levels over time indicates a slower progression of one or more symptoms of COPD, or a decrease in isodesmosine levels, for example by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, indicates a reversal of one or more symptoms of COPD.

[0087] IV. Biomarkers Useful for Determining Prevention, Slowing Progression, Halt, and / or Reversal of Tissue Calcification in a Subject In certain embodiments, biomarkers can be evaluated to determine whether the methods and / or compositions described herein are effective in preventing or treating (e.g., slowing, stopping and / or reversing) tissue calcification in a subject.These include, for example, measuring the T50 time of a subject and measuring the ratio of carboxylated vitamin K-dependent protein to non-carboxylated protein, as described in more detail below.

[0088] 1. Primary calciprotein particles (CPP) and serum T50 Under normal physiological conditions, pathological calcification is prevented, in part, by primary calciprotein particles (CPPs) (Price et al., supra). CPPs contain circulating calcium phosphate crystals complexed with two calcification-inhibitory proteins, fetuin A and matrix Gla protein (ibid.). Matrix Gla protein is a vitamin K-dependent protein, and the formation of CPPs depends on the gamma-carboxylated form of matrix Gla protein (ibid.). Preclinical studies suggest that the calciprotein system functions as an alternative means to prevent pathological calcification when humoral lines of defense such as pyrophosphate, magnesium, and albumin are overwhelmed. Binding of calcium phosphate crystals by primary CPPs occurs in a coordinated, time-dependent process.

[0089] The time required for 50% conversion of primary CPPs to secondary CPPs (T50) is an accurate and sensitive measure to determine the ability of plasma to sequester excess calcium phosphate crystals. Conversion from the primary to secondary form involves the formation of more elongated crystals. Elongation results in a more turbid solution, which can be detected by turbidimetry. Under conditions of high calcium phosphate loading, the time to reach 50% conversion is shortened because of the reduced reserves for calcium phosphate absorption. Thus, a short T50 time suggests a reduced capacity to absorb calcium phosphate crystals, while a long T50 time is consistent with a high capacity. A short T50 time is often associated with warfarin therapy or hemodialysis, and with a high uremic oxidative load, which is generally associated with an increased risk of myocardial infarction, heart failure, and all-cause mortality.

[0090] Without wishing to be bound by theory, it is believed that administration of MK-7 and / or MKH2-7 increases the T50 time and improves a subject's ability to prevent pathological calcification. Because subjects with CKD and ESRD exhibit reduced levels of carboxylated matrix Gla protein, which is vitamin K-dependent and essential for the formation of primary CPPs, administration of MK-7 and / or MKH2-7 according to the methods of the present disclosure can reduce the risk of pathological calcification and prevent the development of soft tissue (e.g., vascular) calcification.

[0091] Thus, in certain embodiments described herein, administration of MK-7 and / or MKH2-7 to a subject increases the subject's serum T50 value (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the subject's serum T50 value prior to administration of MK-7 and / or MKH2-7. Methods for measuring a subject's serum T50 level are known in the art (see, for example, Pasch et al. (2012) "Nanoparticle-based test measures overall propensity for calcification in serum." J. AM. SOC. NEPHROL. 23(10): 1744-52; Dahle et al., (2016) "Serum Calcification Propensity Is a Strong and Independent Determinant of Cardiac and All-Cause Mortality in Kidney Transplant Recipients." AM. J. TRANSPLANT 16(1): 204-12; and Smith et al. (2014) "Serum Calcification Propensity Predicts All-Cause Mortality in Predialysis CKD," J. AM. SOC. NEPHROL. 25(2):339-348).

[0092] In certain embodiments, the disclosure relates to a method of increasing serum T50 levels in a subject having a disorder described herein, the method comprising administering to the subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg per day of substantially pure MK-7 and / or MKH2-7), wherein administration of MK-7 and / or MKH2-7 to the subject increases the subject's serum T50 level (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the subject's serum T50 level prior to administration of MK-7 and / or MKH2-7.

[0093] 2. Carboxylation of Vitamin K-Dependent Proteins Despite dietary deficiencies, vitamin K levels may not be reduced in ESRD patients. For example, a study of 172 stable hemodialysis patients found that only 6% of patients had clinically significant deficiency of vitamin K (Holden et al. (2010) "Vitamins K and D Status in Stages 3-5 Chronic Kidney Disease," CLIN. J. AM. SOC. NEPHROL. 5:590-597). However, when patients were tested for levels of carboxylated osteocalcin, a vitamin K-dependent protein, approximately 60% of patients had reduced levels (ibid.). Similar results have been shown for other vitamin K-dependent proteins, such as PIVKA-II (ibid., and Pilkey et al. (2007) "Subclinical Vitamin K Deficiency in Hemodialysis Patients," AM. J. KIDNEY DIS. 49:432-439). These results are consistent with the hypothesis that in uremic patients, while total vitamin K levels are normal, production of the reduced form is blocked by the oxidative properties of urea (ibid.).

[0094] The observation that oxidative conditions can disrupt the vitamin K cycle suggests that the oxidative load generated during hemodialysis further contributes to the high rates of soft tissue (e.g., blood vessel) calcification observed within the ESRD population. Hemodialysis delivery is known to result in the oxidation of numerous tissue proteins, and both CKD and ESRD patients often have a higher rate (15-fold) of carbonyl proteins compared to normal controls.

[0095] Without wishing to be bound by theory, it is believed that the oxidative load caused by uremia and / or hemodialysis results in the oxidation of functional vitamin K hydroquinone (KH2) to non-functional native vitamin K. Oxidation of KH2 blocks the ability of GGCX to function as a cofactor and can reduce the gamma-carboxylated forms of vitamin K-dependent proteins. Thus, administration of MK-7 and / or MKH2-7 to subjects with diabetes, CKD and / or ESRD and / or subjects undergoing hemodialysis can increase the ratio of one or more carboxylated vitamin K-dependent proteins to non-carboxylated vitamin K-dependent proteins in these subjects, and can reduce the amount of one or more non-carboxylated vitamin K-dependent proteins in these subjects.

[0096] Thus, in certain embodiments, administration of MK-7 and / or MKH2-7 increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the plasma of the subject (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the ratio before administration of MK-7 and / or MKH2-7. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the plasma of the subject by about 10% to 500% or more, about 30% to about 500%, about 30% to about 200%, about 30% to about 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the subject's plasma by about 50% to about 500%, about 50% to about 200%, or about 50% to about 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the subject's plasma by about 70% to about 500%, about 70% to about 200%, or about 70% to about 100%.

[0097] In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the amount of non-carboxylated vitamin K-dependent protein in the plasma of the subject (e.g., by at least 5%, 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the amount before administration of MK-7 and / or MKH2-7. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the amount of non-carboxylated vitamin K-dependent protein in the plasma of the subject by about 5% to about 100%, about 5% to about 80%, about 5% to about 30%, about 20% to about 100%, or about 20% to about 100%.

[0098] In certain embodiments, the disclosure provides a method for increasing the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in plasma of a subject having a disorder described herein, the method comprising administering to a subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg per day). mg of substantially pure MK-7 and / or MKH2-7 to a subject, wherein administration of MK-7 and / or MKH2-7 to the subject increases the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in the plasma of the subject (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the ratio prior to administration of MK-7 and / or MKH2-7.

[0099] In certain embodiments, the disclosure relates to a method of reducing the amount of non-carboxylated Vitamin K-dependent protein in plasma of a subject having a disorder described herein, the method comprising administering to the subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg of substantially pure MK-7 and / or MKH2-7 per day), wherein administration of MK-7 and / or MKH2-7 to the subject reduces the amount of non-carboxylated Vitamin K-dependent protein in the subject's plasma (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the amount prior to administration of MK-7 and / or MKH2-7.

[0100] In certain embodiments, the vitamin K-dependent protein is selected from matrix Gla protein (MGP), growth arrest specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, or activated protein S. Carboxylated forms of MGP, Gas-6, PIVKA-II, osteocalcin, activated protein C and activated protein S can be measured using sandwich assays as known in the art (see, e.g., Nigwekar et al. (2017) "Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis," J. AM.SOC.NEPHROL. 28:1717-1722 (detection of MGP); Gla-type osteocalcin (GLa-OC) ELISA kit (Takara, Mountain View, CA); STA-Staclot Protein C (Stago, Asnieres sur Seine, France); and REAADS Protein S antibody (cat. no. K036-001, Diapharma Group, West Chester, OH)). An increase in the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in a subject following administration of MK-7 indicates successful rescue of the GGCX carboxylation system. Thus, effective MK-7 and / or MKH2-7 administration can increase the ratio of carboxylated to uncarboxylated forms of proteins in a subject.

[0101] PIVKA-II is an abnormal, non-carboxylated form of prothrombin, also known as des-gamma-carboxyprothrombin (DCP). Thus, elevated levels of PIVKA-II correspond to poorer vitamin K status. As a biomarker of vitamin K status, PIVKA-II has the advantage of being independent of renal function and lipid profile (E Elliot et al. (2014) "Assessment of potential biomarkers of subclinical vitamin K deficiency in patients with end-stage kidney disease," CAN. J. KIDNEY HEALTH DIS. 1:13). According to the methods described herein, the measurement of PIVKA-II in a subject indicates successful rescue of the GGCX carboxylation system. PIVKA-II plasma levels can be measured by any method known in the art, such as, for example, using ELISA (Diagnostica Stage, Parsippany, NJ) (see also, e.g., Dituri et al. (2012) "PIVKA-II plasma levels as markers of subclinical vitamin K deficiency in term infants," J. MATERNAL-FETAL & NEONATAL MEDICINE 25(9):1660-1663).

[0102] MGP is believed to be a potent inhibitor of calcification of the arterial wall and other soft tissues in non-human animals, and its activity depends on vitamin K-dependent carboxylation of gamma glutamate (Schurgers et al. (2007a) "Post-translational modifications regulate matrix Gla protein function: importance for inhibition of vascular smooth muscle cell calcification," J. THROMB. HAEMOST. 5:2503-2511; Westenfeld et al. (2012) "Effect of vitamin K2 supplementation on functional vitamin K deficiency in hemodialysis patients: a randomized trial," AM. J. KIDNEY DIS. 59(2):186-195; Schurgers et al. (2013) "Vitamin K-dependent carboxylation of matrix Gla-protein: a crucial switch to control ectopic mineralization," TRENDS MOL. MED. 2013;19(4):217-26; Marles et al. (2017) "US Pharmacopeial Convention safety evaluation of menaquinone-7, a form of vitamin K," NUTR. REV. 75(7):553-578). Uncarboxylated MGP, formed as a result of vitamin K deficiency, is associated with cardiovascular disease. Recent studies suggest poor vitamin K status in hemodialysis patients (Westenfeld et al. (2012), supra).Circulating inactive form of MGP (dp-ucMGP) has been shown to increase progressively in CKD patients (Schurgers et al. (2010) "The Circulating Inactive Form of Matrix Gla Protein Is a Surrogate Marker for Vascular Calcification in Chronic Kidney Disease: A Preliminary Report," CLIN J. AM. SOC. NEPHROL. 5(4):568-75), and to predict vitamin K status and correlate with vascular calcification in patients undergoing hemodialysis (Delanaye et al. (2014) "Dephosphorylated-uncarboxylated Matrix Gla protein concentration is predictive of vitamin K status and is correlated with vascular calcification in a cohort of hemodialysis patients," BMC NEPHROL. 15(1):145). Thus, plasma dp-ucMGP has been proposed as a surrogate marker of vascular calcification in CKD (Schurgers et al. (2010) supra). Additionally, lower levels of circulating desphospho-carboxylated MGP (dp-cMGP) have been proposed as a predictor of mortality in hemodialysis patients (Schlieper et al. (2011) "Circulating nonphosphorylated carboxylated matrix gla protein predicts survival in ESRD," J. AM. SOC. NEPHROL. 22(2):387-95).

[0103] Osteocalcin is a non-collagenous bone matrix protein synthesized by mature osteoblasts and involved in bone formation and regulation of bone mineralization (Elliott et al. 2014, supra; Marles et al. 2017, supra). The percentage of uncarboxylated osteocalcin is a sensitive marker of vitamin K status in bone, and subclinical vitamin K deficiency is defined by a greater than 20% increase in the percentage of uncarboxylated osteocalcin (Elliott et al. 2014, supra). Supplementation with MK-7 at doses of 100-200 μg / d for 4-12 weeks has been shown to significantly increase the ratio of carboxylated to noncarboxylated osteocalcin in a dose-dependent manner (Inaba et al. (2015), "Low-Dose Daily Intake of Vitamin K(2) (Menaquinone-7) Improves Osteocalcin γ-Carboxylation: A Double-Blind, Randomized Controlled Trials," J. NUTR. SCI. VITAMINOL 61(6):471-80).

[0104] Vitamin K-dependent gamma glutamic acid proteins are synthesized in the liver and help maintain normal blood clotting through a balance of both procoagulant factors (II, VII, IX, and X) and anticoagulant proteins (C and S) (Schurgers et al. (2007c) "Vitamin K-containing dietary supplements: comparison of synthetic vitamin K1 and natto-derived menaquinone-7," BLOOD 109(8):3279-83; Marles et al. 2017, supra). Protein C controls the clotting process by abolishing the procoagulant activity of factors V and VIII in the presence of the cofactor protein S (Marlar et al. (2017) "Assessment of Hereditary Thrombophilia: Performance of Protein C (PC) Testing," METHODS MOL. BIOL. 1646:145-151). Hypercoagulable states, e.g., deficiencies of protein C and / or protein S, have been reported in patients with calciphylaxis and have been proposed as factors that increase the likelihood of developing calciphylaxis (Wilmer et al. (2002) "Calciphylaxis: Emerging Concepts in Prevention, Diagnosis, and Treatment," SEMIN. DIAL. 15(3):172-86; Nigwekar et al. (2008) "Calciphylaxis from nonuremic causes: a systematic review," CLIN. J. AM. SOC. NEPHROL. 3(4):1139-43). In a randomized trial evaluating the effect of vitamin K2 supplementation on functional vitamin K deficiency in adult hemodialysis patients, patients with hemolysis (N=53) showed 4.5-fold higher dp-ucMGP and 8.4-fold higher noncarboxylated osteocalcin levels compared with healthy age-matched controls (N=50). PIVKA-II levels were assessed in 49 hemodialysis patients.Vitamin K2 supplementation induced dose- and time-dependent decreases in circulating dp-ucMGP, uncarboxylated osteocalcin, and PIVKA-II levels. Response rates in reducing dp-ucMGP levels were 77% and 93% in the groups receiving 135 and 360 μg menaquinone-7, respectively (Westenfeld et al. (2012) supra).

[0105] 3. Levels of other biomarkers of calcification In certain embodiments, administration of MK-7 and / or MKH2-7 increases the plasma level of osteoprotegerin or fetuin-A (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma concentration of osteoprotegerin or fetuin-A prior to administration of MK-7 and / or MKH2-7. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the plasma level of osteoprotegerin or fetuin A by about 10-50%, about 50-100%, about 100-200% or about 200-500% compared to the plasma concentration of osteoprotegerin or fetuin A before administration of MK-7 and / or MKH2-7. Osteoprotegerin and fetuin A are inhibitors of tissue calcification. Thus, an increase in the level of osteoprotegerin and / or fetuin A indicates that administration of MK-7 and / or MKH2-7 can prevent, slow, stop and / or reverse tissue calcification.

[0106] In certain embodiments, the disclosure provides a method of increasing plasma levels of osteoprotegerin or fetuin A in a subject having a disorder described herein, the method comprising administering at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg per day). mg of substantially pure MK-7 and / or MKH2-7 to a subject, wherein administration of MK-7 and / or MKH2-7 to the subject increases a plasma level of osteoprotegerin or fetuin-A (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma level of osteoprotegerin or fetuin-A prior to administration of MK-7 and / or MKH2-7.

[0107] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 reduces the plasma level of D-dimer (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma concentration of D-dimer before administration of MK-7 and / or MKH2-7. In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 reduces the plasma level of D-dimer by about 10-50%, about 50-100% or about 10-100% compared to the plasma concentration of D-dimer before administration of MK-7 and / or MKH2-7. D-dimer is indicative of the functional status of vitamin D metabolism. Protein C and protein S prevent the generation of thrombin and fibrin, and lack of functional vitamin K contributes to the loss of protein C and protein S, which can cause microthrombosis by fibrin degradation and the formation of D-dimers. Thus, the presence of D-dimers is an indicator of subclinical thrombosis. Thus, a reduction in plasma levels of D-dimers may indicate that administration of MK-7 and / or MKH2-7 can restore the GGCX carboxylation system, which may lead to preventing, slowing, halting, and / or reversing tissue calcification.

[0108] In certain embodiments, the disclosure relates to a method for reducing plasma levels of D-dimer in a subject having a disorder described herein, the method comprising administering to the subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg of substantially pure MK-7 and / or MKH2-7 per day), wherein administration of MK-7 and / or MKH2-7 to the subject reduces the plasma level of D-dimer (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma concentration of D-dimer prior to administration of MK-7 and / or MKH2-7.

[0109] In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 increases the plasma level of high sensitivity C-reactive protein (hs-CRP) (e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma concentration of high sensitivity C-reactive protein (hs-CRP) prior to administration of MK-7 and / or MKH2-7. In certain embodiments of any of the above aspects, administration of MK-7 and / or MKH2-7 increases the plasma level of high sensitivity C-reactive protein (hs-CRP) by about 10-50%, about 50-100%, or about 10-100%, compared to the plasma concentration of high sensitivity C-reactive protein (hs-CRP) before administration of MK-7 and / or MKH2-7. Hs-CRP is a biomarker of ongoing inflammation. The presence of inflammation enhances soft tissue (e.g., vascular) calcification. Thus, a reduction in the plasma level of hs-CRP indicates that administration of MK-7 and / or MKH2-7 can prevent, slow, halt, and / or reverse tissue calcification.

[0110] In certain embodiments, the disclosure provides a method for reducing plasma levels of high sensitivity C-reactive protein (hs-CRP) in a subject having a disorder described herein, the method comprising administering at least 2 mg / day (e.g., at least 2 mg per day, e.g., 10, 15, 25, 50 or 75 mg per day). mg of substantially pure MK-7 and / or MKH2-7 to a subject, wherein administration of MK-7 and / or MKH2-7 to the subject reduces a plasma level of high sensitivity C-reactive protein (hs-CRP) (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%) compared to the plasma level of hs-CRP prior to administration of the MK-7 and / or MKH2-7.

[0111] V. Lesions The methods and compositions described herein may be useful for slowing or halting the progression of, or halting and / or reversing, tissue lesions, such as skin lesions or vascular lesions.

[0112] 1. Skin lesions The final step of the vitamin K cycle requires the enzymatic reduction of vitamin K-7 2,3-epoxide back to MK-7 by vitamin K epoxide reductase complex subunit 1 (VKORC1, also called VKOR) (see Figure 1). In some tissues, the paralog VKORC1L1 (VKORC1-Like-1) can also carry out the reaction. Skin has the lowest expression of VKOR-C1, consistent with the clinical observation that vitamin K-dependent vascular calcification is more prevalent in skin. Low cutaneous expression of VKOR-C1 suggests that any condition or treatment that blocks vitamin K production, such as hemodialysis, predisposes the skin to pathological calcification.

[0113] Thus, the methods and compositions described herein relate in part to reducing the size of skin lesions.In certain embodiments, a subject has skin lesions, and administering MK-7 and / or MKH2-7 reduces the size of skin lesions.In certain embodiments, administering MK-7 and / or MKH2-7 reduces the total surface area of ​​skin lesions by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total volume of skin lesions by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (i.e., eliminates the skin lesions).

[0114] In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area or volume of skin lesions by about 30% to about 100%, about 30% to about 90%, about 30% to about 80%, about 30% to about 70%, or about 30% to about 60%. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area or volume of skin lesions by about 50% to about 100%, about 50% to about 90%, about 50% to about 80%, about 50% to about 70%, or about 50% to about 60%. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area or volume of skin lesions by about 70% to about 100%, about 70% to about 90%, or about 70% to about 80%.

[0115] The surface area or volume of one or more skin lesions can be measured by any method known in the art.When measuring surface area, the total surface area can be calculated if multiple lesions are present.The volume of lesion can be calculated from a three-dimensional model of the lesion that is constructed from digital images.

[0116] In another embodiment, the efficacy of administration of MK-7 and / or MKH2-7 is determined by von Kossa staining for calcium in skin biopsies. This well-established stain is a validated means of testing and quantifying the amount of interstitial calcium deposits in skin biopsies. In this approach, a skin biopsy is taken and the tissue is stained using the von Kossa method. Due to the binding of soft tissue and vascular deposits of phosphate, von Kossa staining can be used to determine whether the rate of soft tissue and vascular calcium phosphate deposition slows or reverses with administration of MK-7 and / or MKH2-7. Because calcium phosphate deposits are central to the pathogenesis of calciphylaxis, a decrease in von Kossa staining indicates healing of the condition resulting in calciphylaxis lesions. Kits for performing the von Kossa method are commercially available (see, e.g., abcam®, #ab150687).

[0117] In certain embodiments, administration of MK-7 and / or MKH2-7 reduces skin lesions (i.e., eliminates skin lesions) by approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 0% of the total surface area of ​​the lesions before treatment.

[0118] 2. Vascular lesions The methods and compositions described herein relate in part to vascular lesions.In certain embodiments, subject has vascular lesions, and administering MK-7 and / or MKH2-7 reduces vascular lesions.The reduction of lesions can be assessed using aortic plethysmography, which measures vascular compliance (i.e., the ability of blood vessels to expand) (see, for example, Inuma et al. (2012) HONG KONG JOURNAL OF NEPHROLOGY 14(2):48-53).

[0119] The reduction of lesions can also be assessed by measuring pulse wave velocity (PWV). PWV is the speed at which arterial pulses travel through the circulatory system and is used as a measure of arterial stiffness. The higher the PWV, the higher the arterial stiffness and therefore the lower the vascular compliance. Methods for measuring PWV are known in the art and are described, for example, in Pereira et al. (2015) supra. Briefly, PWV can be assessed by measuring regional PWV, such as carotid-femoral PWV, or local PWV. Ibid. Methods for measuring regional PWV are known in the art and include, for example, the use of pulse transducer probes, TY-360 pressure transducers, Doppler units synchronized with an electrocardiogram (ECG), pulse transducer probes, photoplethysmography, Complior® (piezoelectric pressure transducer), SphygmoCor®, Arteriograph®, PulsePen® (tonometer and integrated ECG unit). Id. Methods for measuring regional PWV are known in the art and include, for example, magnetic resonance imaging, ultrasound, and angiography.

[0120] In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of the blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the vascular compliance before treatment. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of the blood vessel by about 10-50%, about 50-10%, about 100-200% or about 200-500%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of the blood vessel by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% compared to the vascular compliance before treatment. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of the blood vessel by about 10-50%, about 50-100%, about 100-200% or about 200-500% compared to the vascular compliance before treatment.

[0121] VI. Kits for Medical Use Another aspect of the present invention provides kits for treating one or more of the disorders or conditions described herein. The kits may include: i) instructions for treating a medical disorder; and ii) one or more dosage units containing substantially pure MK-7 and / or MKH2-7.

[0122] Throughout this specification, when compositions are described as having, including, or comprising certain components, or when processes and methods are described as having, including, or comprising certain steps, it is additionally contemplated that there are compositions of the invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the invention that consist essentially of, or consist of, the recited process steps.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0124] Throughout this specification, when compositions and kits are described as having, including, or comprising particular components, or processes and methods are described as having, including, or comprising particular steps, it is contemplated that there are additionally compositions and kits of the invention that consist essentially of, or consist of, the recited components, and processes and methods according to the invention that consist essentially of, or consist of, the recited process steps.

[0125] In this application, when an element or component is referred to as being included in and / or selected from a listed list of elements or components, it is understood that the element or component can be any one of the listed list of elements or components, or the element or component can be selected from the group consisting of two or more of the listed list of elements or components.

[0126] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein may be combined in various ways without departing from the spirit and scope of the invention, whether expressly or implicitly described herein. For example, when a particular compound is mentioned, that compound may be used in various embodiments of the compositions and / or methods of the invention, unless otherwise understood from the context. In other words, within this application, the embodiments are described and depicted in a manner that allows a clear and concise application to be written and depicted, but it is intended and will be understood that the embodiments may be combined or separated in various ways without departing from the teachings and the invention. For example, it will be understood that all features described and depicted herein may be applicable to all aspects of the invention described and depicted herein.

[0127] The articles "a" and "an" are used in this disclosure, unless the context is inappropriate, to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0128] The term "and / or" means either "and" or "or," unless stated otherwise.

[0129] The phrase "at least one of" includes each of the listed objects following the phrase and combinations of two or more of the listed objects, unless otherwise understood from context and usage. The phrase "and / or" in the context of three or more listed objects should be understood to have the same meaning, unless otherwise understood from context.

[0130] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," "contains," or "containing," including their grammatical equivalents, generally should be understood as open-ended and open-ended, e.g., not excluding further, additional, unrecited elements or steps, unless expressly stated otherwise or understood otherwise from the context.

[0131] When the term "about" is used before a quantitative value, the invention also includes the specific quantitative value itself, unless expressly stated otherwise. As used herein, the term "about" refers to ±10% of the nominal value, unless otherwise stated or inferred.

[0132] When molecular weights, e.g., of polymers, are given rather than absolute values, it is to be understood that the molecular weight is an average molecular weight, unless otherwise stated or understood from the context.

[0133] As a general matter, compositions specifying percentages are by weight unless otherwise specified, and if a variable is not accompanied by a definition, the variable's preceding definition controls.

[0134] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable, and two or more steps or actions may be performed simultaneously.

[0135] Any and all examples herein, or the use of exemplary language herein, such as "such as" or "including," are intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0136] The present disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of the disclosure to the specific procedures described herein. It should be understood that the examples are provided to illustrate particular embodiments and are not intended to limit the scope of the disclosure thereby.

[0137] Example 1 - Administering MK-7 and / or MKH2-7 to reverse or slow the progression of tissue calcification in subjects with end stage renal disease (ESRD) This example describes administration of MK-7 and / or MKH2-7 to subjects with ESRD and / or undergoing stable hemodialysis. Administration of MK-7 and / or MKH2-7 is believed to result in changes in aortic compliance (by plethysmography), vascular calcification, and certain biomarker levels that are indicative of slowing, halting, or reversing the progression of tissue calcification.

[0138] Subjects with ESRD undergoing stable hemodialysis will receive MK-7 and / or MKH2-7 orally at 10 mg, 25 mg, or 50 mg once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. Dosage forms are 10 mg or 25 mg softgel capsules. Two 25 mg capsules will be administered once daily to the 50 mg dose cohort.

[0139] The coronary artery calcification score (CAC) is used to estimate the degree of calcification in thoracic arteries. A high CAC score indicates calcification, and treatment has the objective of halting or reversing or slowing the rate of increase in the CAC score over time. Aortic plethysmography is also used to measure arterial compliance, which decreases with increasing calcification. In addition, pulse wave velocity (PWV) is measured to assess arterial compliance. The above measurements are useful for estimating the usefulness of treatments intended to prevent, slow, stop or reverse vascular calcification. These measurements are used before and after treatment with MK-7 and / or MKH2-7 to assess the value of treatment.

[0140] Also, several biomarkers are evaluated to determine the effectiveness of MK-7 and / or MKH2-7 at three dose levels.Exemplary biomarkers include PIVKA-II; uncarboxylated and total matrix Gla protein (MGP); uncarboxylated, carboxylated and total osteocalcin protein; uncarboxylated, carboxylated and total protein C, osteoprotegerin, fetuin A and hs-CRP.Most conveniently, blood samples are taken during patient visits for hemodialysis to measure biomarkers.

[0141] It is believed that administration of MK-7 and / or MKH2-7 may result in (i) an increase in PIVKA-II, osteoprotegerin or fetuin A, indicative of a slowing, halting or reversal of the progression of tissue calcification; (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin and / or uncarboxylated protein C, indicative of a slowing, halting or reversal of the progression of tissue calcification; and / or (iii) a decrease in hs-CRP, indicative of a slowing, halting or reversal of the progression of tissue calcification and / or a decrease in inflammation.

[0142] Following daily administration of 10 mg, 25 mg or 50 mg of MK-7 and / or MKH2-7, at least one of PIVKA-II, undercarboxylated matrix Gla protein (MGP), uncarboxylated osteocalcin protein, uncarboxylated protein C, osteoprotegerin, fetuin A and hs-CRP is believed to indicate a slowing, halting or reversal of the progression of tissue mineralization.

[0143] Example 2 -Administration of MK-7 and / or MKH2-7 in subjects with stable end-stage renal disease (ESRD) undergoing hemodialysis This example describes the administration of MK-7 to a subject with ESRD undergoing hemodialysis and at risk of developing tissue calcification. The administration of MK-7 can result in changes in certain biomarker levels that indicate prevention of the development of tissue calcification, or the cessation or delay of the development of tissue calcification.

[0144] A number of subjects with stable ESRD but no calciphylaxis are enrolled in the study, and the subjects are orally administered a 10 mg capsule of MK-7 per day for 14 days. Levels of certain biomarkers, including non-carboxylated matrix Gla protein (MGP), non-carboxylated osteocalcin, osteoprotegerin, fetuin A, and hs-CRP, are assessed on days 1 and 15 (end of treatment) and changes between days 1 and 15 are calculated. Between days 1 and 15, the mean decrease in non-carboxylated MGP was -21.9% and the mean decrease in non-carboxylated osteocalcin was -55.5%. Subjects also unexpectedly showed a mean increase in osteoprotegerin and fetuin A, as well as a decrease in hs-CRP. Osteoprotegerin, fetuin A, and hs-CRP are not vitamin K-dependent proteins, and it is believed that changes in these proteins have not previously been demonstrated as a result of MK-7 intake. The mean increase in osteoprotegerin was 14.3%, the mean increase in fetuin-A was 23.1%, and the mean decrease in hs-CRP was -35.8%.

[0145] Based on studies, administration of MK-7 can result in a decrease in uncarboxylated MGP, uncarboxylated osteocalcin and hs-CRP and an increase in fetuin A and osteoprotegerin, which may indicate that administration of MK-7 prevents, or halts or slows the progression of tissue calcification in a subject.

[0146] Incorporation by Credit The entire disclosure of each of the patent and scientific documents referenced herein is incorporated by reference for all purposes.

[0147] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore to be considered in all respects as illustrative and not limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A medicament for treating tissue calcification in a pre-diabetic subject or a subject with diabetes, chronic kidney disease or a combination thereof in need of treating tissue calcification, the medicament comprising at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), wherein administration of at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to a subject increases plasma levels of osteoprotegerin or fetuin-A compared to plasma levels of osteoprotegerin or fetuin-A prior to administration of MK-7 and / or MKH2-7.

2. The method of claim 1, wherein the subject has diabetes.

3. The method of claim 2, wherein the subject has type II diabetes.

4. The method of claim 1, wherein the subject has been diagnosed with prediabetes.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the subject has chronic kidney disease.

6. The method according to any one of claims 1 to 4, wherein the subject has stage 4 or 5 chronic kidney disease / end stage renal disease.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the subject is undergoing hemodialysis.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the subject is undergoing non-warfarin-based anticoagulant therapy.

9. The pharmaceutical composition according to claim 8, wherein the anticoagulant therapy is oral anticoagulant therapy.

10. 10. The method of claim 8 or 9, wherein the anticoagulant therapy comprises an inhibitor of factor Xa activity or factor IIa activity.

11. The pharmaceutical described in claim 10, wherein the inhibitor of factor Xa activity is apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, elibaxaban or fondaparinux.

12. The pharmaceutical according to claim 10, wherein the inhibitor of factor IIa activity is dabigatran or argatroban.

13. 1. A medicament for treating tissue calcification in a subject undergoing hemodialysis in need of treating tissue calcification, the medicament comprising at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), wherein administration of at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to the subject increases plasma levels of osteoprotegerin or fetuin-A compared to plasma levels of osteoprotegerin or fetuin-A prior to administration of the medicament.

14. The method of claim 13, wherein the subject has diabetes.

15. The method according to claim 14, wherein the diabetes is type II diabetes.

16. The method of claim 13, wherein the subject has been diagnosed with prediabetes.

17. The pharmaceutical composition according to any one of claims 13 to 16, wherein the subject has chronic kidney disease.

18. The method of claim 17, wherein the subject has stage 4 or 5 chronic kidney disease / end stage renal disease.

19. The pharmaceutical composition according to any one of claims 13 to 18, wherein the subject is receiving non-warfarin-based anticoagulant therapy.

20. The pharmaceutical composition according to claim 19, wherein the anticoagulant therapy is oral anticoagulant therapy.

21. 21. The method of claim 20, wherein the anticoagulant therapy comprises an inhibitor of factor Xa activity or factor IIa activity.

22. The pharmaceutical described in claim 21, wherein the inhibitor of factor Xa activity is apixaban, rivaroxaban, betrixaban, edoxaban or fondaparinux.

23. The pharmaceutical described in claim 21, wherein the inhibitor of factor IIa activity is dabigatran or argatroban.

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