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 of related applications) This application claims priority and benefit under U.S. Provisional Application No. 62 / 682,796, filed on 8 June 2018, which, by attribution, is part of this Specified in whole. (Field of invention) The present invention generally provides methods and compositions for preventing or treating tissue calcification (e.g., slowing, stopping, and / or reversing its progression) in subjects who require prevention or treatment of tissue calcification, and more specifically, the present invention relates to the use of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) for preventing or treating tissue calcification (e.g., slowing, stopping, and / or reversing its progression) in subjects with diabetes mellitus, 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 disease (COPD), comprising the use of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to prevent or treat one or more symptoms of COPD (e.g., slowing, stopping, and / or reversing their progression). [Background technology]

[0002] Under normal physiological conditions, plasma calcium and phosphates are present at near-supersaturated concentrations, and as a result, precipitation in soft tissues (e.g., blood vessels) as crystalline hydroxyapatite can be expected. The finding that this process does not occur in healthy subjects suggests the existence of powerful chemical and biological mechanisms 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] For example, impaired inhibition of calcification in individuals with diabetes and chronic kidney disease (CKD) can lead to pathological calcification of soft tissues (e.g., blood vessels). Diabetes is understood to contribute to CKD and end-stage renal disease (ESRD), characterized by uremic toxicity. Uremic toxicity promotes the oxidation of vitamin K hydroquinone (KH2), which can impair the cyclical regeneration of vitamin K, among other effects (see Figure 1). Furthermore, certain treatments, including warfarin-based anticoagulation and statin therapy, can cause or contribute to abnormal regulation of vitamin K. Loss of functional vitamin K results in the loss of a key regulator of mineralization, leading to pathological calcification of tissues. In cases of arterial calcification, endothelial microangiogenic thrombosis has been observed, resulting in small vascular occlusions and the death of surrounding tissue.

[0004] Vitamin K is an essential enzyme cofactor required for the post-translational modification of vitamin K-dependent (VKD) proteins. Many VKD proteins are clinically relevant in patients with chronic kidney disease (CKD) and endoscopy-associated neuropathy (ESRD), including, for example, central coagulation factors such as factors II, VII, IX, and X, and intercellular matrix proteins such as matrix Gla protein (MGP), activated protein C, and osteocalcin. Vitamin K belongs to a group of fat-soluble vitamins, including, among others, vitamin K1 (also known as phylloquinone), which is produced by plants, and vitamin K2 (also known as menaquinone), which is produced 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, while 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 oxidoreductase). Only the reduced form of MK-7 (i.e., MKH2-7) functions as a cofactor for γ-glutamate carboxylase (GGCX), an enzyme that catalyzes the carboxylation of vitamin K-dependent proteins (see Figures 1 and 2). Enzymatic carboxylation of the glutamate residue 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 known as VKOR). In some tissues, the paralog VKORC1L1 (VKORC1-Like-1) can also catalyze this reaction. Warfarin is thought to block both the production 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 cause 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. In particular, new clinical approaches are needed in subjects with diabetes, chronic kidney disease (CKD), or endoscopy-associated vascular disease (ESRD), and in subjects receiving anticoagulant and / or statin therapy. [Overview of the project]

[0007] We have discovered that menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) can be effectively used in subjects, such as those with diabetes and / or chronic kidney disease, at high doses, for example, at least 2 mg per day, to prevent, slow, halt, and / or reverse tissue calcification.

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

[0009] In another embodiment, the present invention provides a method for preventing or treating tissue calcification (e.g., slowing, stopping, and / or reversing its progression) in a subject undergoing hemodialysis who requires prevention or treatment of tissue calcification, the method comprising administering to the subject at least 2 mg per day of substantially pure MK-7, MKH2-7 or a combination thereof to prevent or treat tissue calcification (e.g., slowing, stopping, and / or reversing its progression), 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 any particular embodiment of the above configuration, the subject has diabetes, for example, type 2 diabetes. In any particular embodiment, 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 any particular embodiment, the subject is receiving non-warfarin-based anticoagulation therapy, for example, oral anticoagulation therapy.

[0011] In another embodiment, the present invention provides a method for preventing or treating tissue calcification (e.g., slowing, stopping, and / or reversing its progression) in a subject having stage 5 chronic kidney disease requiring prevention or treatment of tissue calcification and simultaneously receiving oral non-warfarin-based anticoagulation therapy. The method comprises administering to the subject at least 2 mg per day of substantially pure MK-7, MKH2-7, or a combination thereof, thereby preventing or treating tissue calcification in the subject (e.g., slowing, stopping, and / or reversing its progression), 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. In certain embodiments, the subject has diabetes. In certain embodiments, the subject is undergoing hemodialysis.

[0012] In any particular embodiment of the above aspects, administration of MK-7 and / or MKH2-7 to a 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%, or more) compared to the subject's serum T50 level before administration of MK-7 and / or MKH2-7, respectively.

[0013] In any particular embodiment 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 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%, or more) compared to the ratio before administration of MK-7 and / or MKH2-7, respectively. In a particular embodiment of any of the above aspects, administration of MK-7 and / or MKH2-7 reduces the amount of 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%, or more) compared to the pre-administration amount of MK-7 and / or MKH2-7. In a particular embodiment, the vitamin K-dependent proteins are 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 any particular embodiment of the above aspects, administration of MK-7 and / or MKH2-7 increases the plasma levels 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%, or more) compared to the plasma concentrations of osteoprotegerin or fetuin A prior to each administration of MK-7 and / or MKH2-7.

[0015] In any particular embodiment of the above aspects, administration of MK-7 and / or MKH2-7 reduces the plasma levels of D-dimer or highly sensitive 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%, or more) compared to the plasma concentrations of D-dimer or highly sensitive C-reactive protein (hs-CRP) prior to each administration of MK-7 and / or MKH2-7.

[0016] In a particular embodiment of any of the above aspects, the subject has lesions of skin and / or blood vessels, and administration of MK-7 and / or MKH2-7 reduces the size of the lesions of skin and / or blood vessels compared to the size of the lesions before the initiation of the treatment regimen. In a particular embodiment, 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 embodiment, the present invention relates to a method for preventing or treating tissue calcification (e.g., slowing, halting, and / or reversing its progression) in a subject that requires prevention or treatment of tissue calcification, wherein the method involves administering at least 2 mg per day of substantially pure menaquinone-7 (MK-7), menaquinol-7 (MKH2-7), or a combination thereof, to increase the serum T50 level 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%, or more) compared to the serum T50 level of the subject before administration of MK-7 and / or MKH2-7. (ii) Increase 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%, or more) compared to the ratio before administration of MK-7 and / or MKH2-7; (iv) Increase 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%, or more) compared to the ratio before administration of MK-7 and / or MKH2-7. (v) Increase the plasma levels 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% or more) compared to the plasma concentration of theoprotegerin or fetuin A, or (v) D-dimer or highly sensitive C-reactive protein (hs) prior to administration of MK-7 and / or MKH2-7. To cause at least one or a combination of the following, which reduces the plasma level of D-dimer or highly sensitive C-reactive protein (hs-CRP) compared to the plasma concentration of D-dimer (-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%), thereby preventing or treating tissue calcification in the subject (e.g., slowing its progression),The present invention provides a method comprising administering to a subject (to stop and / or reverse growth), 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. The vitamin K-dependent protein may 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 embodiment, the present invention provides a method for preventing or treating tissue calcification (e.g., slowing, halting, and / or reversing its progression) in a subject that requires prevention or treatment of tissue calcification, 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, MK-7 and / or MKH2-7 are administered in the same dosage form as the statin. In other embodiments, MK-7 and / or MKH2-7 are administered in a separate dosage form from the statin. In certain embodiments, administration of MK-7 and / or MKH2-7 prevents or reduces the vitamin K depletion effect of the statin.

[0019] In another embodiment, the present invention provides a method for improving aortic compliance in a subject that requires improvement of aortic compliance, the method comprising administering to the subject an effective daily dose of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).

[0020] In another aspect, the present invention provides a method of preventing or treating (e.g., slowing, halting and / or reversing progression of) peripheral vascular disorder in a subject that needs to prevent or treat peripheral vascular disorder, wherein the subject has ESRD or CKD, and the method comprises administering to the subject a therapeutically effective amount of substantially pure menaquinone-7 (MK-7) and / or menadiol-7 (MKH2-7) per day.

[0021] In another aspect, the present invention provides a method of preventing or treating (e.g., slowing, halting and / or reversing progression of) one or more symptoms of chronic obstructive pulmonary disease (COPD) in a subject that needs to prevent or treat one or more symptoms of COPD, the method comprising administering to the subject at least 2 mg of substantially pure menaquinone-7 (MK-7) and / or menadiol-7 (MKH2-7) per day, thereby preventing or treating (e.g., slowing, halting or reversing progression of) 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, such as type II diabetes, or is diagnosed with prediabetes. In certain embodiments, the subject has chronic kidney disease, such as stage 1, stage 2, stage 3, or end stage renal disease (ESRD), such as stage 4 or stage 5. In certain embodiments, the subject is undergoing non-warfarin based anticoagulation therapy, such as oral anticoagulation therapy.

[0023] In certain embodiments, when a non-warfarin-based anticoagulant therapy is used, the anticoagulant therapy may include an inhibitor 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 a warfarin-based anticoagulant therapy.

[0024] In certain embodiments, the subject is receiving a statin. For example, the statin may 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 may be calcification of blood vessels or skin.

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

[0027] In a particular embodiment of any of the above embodiments, the method may include administering approximately 2 mg to approximately 750 mg of MK-7 and / or MKH2-7 to the subject per day. In another embodiment, the method may include administering approximately 5 mg to approximately 750 mg of MK-7 and / or MKH2-7 to the subject per day. In a particular embodiment of any of the above embodiments, the method may include administering approximately 2 mg to approximately 500 mg of MK-7 and / or MKH2-7 to the subject per day. In another embodiment, the method may include administering approximately 5 mg to approximately 500 mg of MK-7 and / or MKH2-7 to the subject per day. In a particular embodiment of any of the above embodiments, the method may include administering approximately 2 mg to approximately 250 mg of MK-7 and / or MKH2-7 to the subject per day. In another embodiment, the method may include administering approximately 5 mg to approximately 250 mg of MK-7 and / or MKH2-7 to the subject per day. In any particular embodiment of the above configuration, the method may include administering approximately 2 mg to approximately 100 mg of MK-7 and / or MKH2-7 to the subject per day. In other embodiments, the method may include administering approximately 5 mg to approximately 100 mg of MK-7 and / or MKH2-7 to the subject per day. In other embodiments, the method may include administering approximately 10 mg to approximately 75 mg of MK-7 and / or MKH2-7 to the subject per day, for example, 10, 25, 50, or 75 mg of MK-7 and / or MKH2-7 to the subject 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 may be administered to the subject for a certain period, including at least the duration of hemodialysis.

[0029] In certain embodiments, MK-7 and / or MKH2-7 are administered orally. MK-7 and / or MKH2-7 may be encapsulated in tablets, caplets, or capsules.

[0030] The above description illustrates several aspects and embodiments of the present invention. The patent application specifically considers all combinations and variations of the aspects and embodiments. These and other aspects and features of the present invention are described in the following detailed description and claims. [Brief explanation of the drawing]

[0031] The aforementioned and other objects, features and advantages of the present invention will become apparent from the following description of preferred embodiments as shown in the accompanying drawings. Similarly, the elements referenced will identify common features in the corresponding drawings.

[0032] [Figure 1] Figure 1 is a schematic diagram illustrating the effects of the vitamin K cycle and the oxidation of vitamin K hydroquinone induced by uremic activity and hemodialysis.

[0033] [Figure 2] Figure 2 is a schematic diagram showing the carboxylation of vitamin K-dependent proteins by gamma glutamate carboxylase (GGCX). [Modes for carrying out the invention]

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

[0035] While we do not wish to be bound by theory, oxidative stress induced by the retention of uremic toxins depletes functional vitamin K2 in the vascular endothelium, bringing certain vitamin K-dependent proteins, including carboxylated MGP and activated protein C, to suboptimal concentrations. The subsequent decrease in the calcium-binding capacity of these and other vitamin-dependent proteins contributes to abnormal tissue calcification. Using high doses of substantially pure MK-7 and / or MKH2-7, it is possible to increase the 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 pharmaceutical compositions. MK-7 and / or MKH2-7 may be administered in single-dose units (e.g., one capsule) or multiple-dose 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 composition of the present invention contains menaquinone-7 (MK-7), a form of vitamin K2. The IUPAC name of 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] (Equation I)

[0037] Furthermore, the product intended for use according to the methods described herein is menaquinol-7 (MKH2-7), which is the reduced form of MK-7. The IUPAC name of MKH2-7 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-diol, and the chemical structure of MKH2-7 is shown in formula II. [ka] (Formula II)

[0038] While we do not wish to be bound by theory, it is thought that the supplemental MK-7 and / or MKH2-7 forms of vitamin K may increase protection against nonspecific oxidation of vitamin K intermediates caused by uremic and / or dialysis.

[0039] The long aliphatic chains of MK-7 are not synthesized by humans but are synthesized by bacteria in the colon. Dietary sources of MK-7 include fermented foods such as natto (soybeans fermented with Bacillus subtilis), cheese, sauerkraut, buttermilk, pork, eel, prey, and buckwheat bread. Furthermore, MK-7 is found in nutritional supplements (e.g., Rejuvenation Therapeutics®; Bio-Tech). TM Pharmacal MK-7 Vitamin K2, Fayetteville AR; NOW® Foods MK-7 Vitamin K-2 (Vitamin K2 MK-7) is available for use as Vitamin K2 MK-7.

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

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

[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 filtrate is concentrated under vacuum to obtain a pure reduced product. The procedure is carried out under argon. To stabilize MKH2-7 in its reduced form, it may be helpful to mix it with one or more antioxidants, e.g., vitamin C, vitamin C esters (e.g., ascorbyl palmitate), and / or vitamin E, for example, in capsules or softgels. Alternatively, a prodrug of MKH2-7 may be prepared, for example, by esterifying one or both hydroxyl groups with various groups (e.g., acetates) to produce a stable prodrug that is metabolized in the target body to produce MKH2-7.

[0044] II. Dosage Form and Administration In certain embodiments, a useful composition according to the present invention comprises, essentially consists of, or comprises substantially pure MK-7 and / or MKH2-7. "Substantially pure" refers to a composition of the active ingredient comprising 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 terms “effective dose” and “therapeutic effective dose” refer to an amount of one or more compounds (e.g., MK-7 and / or MKH2-7) sufficient to produce a beneficial or desired effect. An effective dose may be administered in one or more administrations, applications, or doses and is not intended to be limited to a specific formulation or route of administration.

[0046] In another embodiment, the invention provides 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 mg 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,The present invention provides dosage forms containing 000 mg, approximately 100 mg to approximately 750 mg, approximately 100 mg to approximately 500 mg, approximately 100 mg to approximately 250 mg, approximately 100 mg to approximately 200 mg, or approximately 100 mg to approximately 150 mg, such as oral dosage forms. In certain embodiments, substantially pure MK-7 and / or MKH2-7 in amounts of 2 mg, 5 mg, 10 mg, 25 mg, 75 mg, or 100 mg is administered to the subject in a suitable dosage form, such as tablets, caplets, or capsules (e.g., liquid or gel capsules).

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

[0048] MK-7 and / or MKH2-7 are understood to be administered in single-dose units (e.g., one capsule) or multiple-dose units (e.g., two, three, or four times). The compositions for use according to the present invention may be formulated as one or more dose units. Such dose units may be administered once daily 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 induce a therapeutic response. The dose units may be packaged in a kit, for example, a kit containing one or more blister packages of about 1 to about 20 dose units (e.g., capsules) per sheet, or in a bottle containing multiple dose units (e.g., capsules).

[0049] In certain embodiments, pharmaceutical compositions comprising MK-7 and / or MKH2-7 may be formulated for administration in solid or liquid form, including aqueous drugs (aqueous or non-aqueous solutions or suspensions), tablets (e.g., buccal, sublingual, and / or targeting systemic absorption), boluses, powders, granules, pastes for tongue application, and / or topical creams. As used herein, “pharmaceutical composition” refers to a combination of an activator and one or more pharmaceutically acceptable carriers (inactive or active) to make a composition particularly suitable for in vivo or ex vivo diagnostic or therapeutic use. As used herein, “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers and additives, e.g., phosphate-buffered saline solution, water, emulsions (e.g., oil / water or water / oil emulsions), and various types of wetting agents. The composition may also contain 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 composition may be formulated in oils such as castor oil, sesame oil, medium-chain triglyceride (MCT) oil, olive oil, soybean oil, or coffee bean oil. Suitable additives for use with the MK-7 and / or MKH2-7 composition include antioxidants, bioavailability enhancers, solubility enhancers or solubilizers, and stabilizers.

[0051] In certain embodiments, the MK-7 and / or MKH2-7 composition comprises one or more antioxidants, for example, 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 composition contains 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 acid), lecithin, and lauroglycol. TM Contains 90 (propylene glycol monolaurate (type II)), deoxycholic acid, Phosal® 50 PG (lecithin in propylene glycol, sunflower mono-diglyceride, ascorbyl palmitate), and an emulsifier selected from beeswax and wax flakes.

[0053] In certain embodiments, the MK-7 and / or MKH2-7 composition contains 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), and Lauroglycol TM 90 (contains a bioavailability enhancer selected from propylene glycol monolaurate (type II), Labrasol® (caprylocaproyl polyoxyl-8 glyceride), Captisol® (SBE-β-cyclodextrin), Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride), and Phosal® 50 PG (lecithin in propylene glycol, sunflower mono-diglyceride, and ascorbyl palmitate).

[0054] In certain embodiments, the MK-7 and / or MKH2-7 composition includes 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 oil), Plurol® Oleique (polyglyceryl-6 dioleate), and Lauroglycol TM 90 (Propylene glycol monolaurate (Type II), Labrasol® (Caprylocaproyl polyoxyl-8 glyceride), Kolliphor® EL (Polyoxyl castor oil), Captisol® (SBE-β-cyclodextrin), Encapsin TM HPB (Hydroxypropyl-β-Cyclodextrin), Peceol TMIt contains (glycerol / glyceryl monooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride), and a solubility enhancer or solubilizer selected from medium-chain mono- and di-glycerides.

[0055] In certain embodiments, the MK-7 and / or MKH2-7 composition contains isopropyl myristate, Capmul® PG-8 NF (propylene glycol monoester of caprylic acid), and Lauroglycol TM It contains a solvent selected from the group consisting of 90 (propylene glycol monolaurate (type II)), polyethylene glycol (PEG), and propylene glycol.

[0056] In certain embodiments, the MK-7 and / or MKH2-7 composition includes 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 composition contains sodium oleate, Span 20 (sorbitan laurate), Span® 80 (sorbitan oleate), vitamin E TPGS (D-α-tocopherol polyethylene glycol-1000 succinate), and Lauroglycol TM It contains a surfactant selected from the group consisting of 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 their esters.

[0059] In certain embodiments, compositions useful for carrying out the present invention are orally deliverable. The terms “orally deliverable” or “oral administration” as used herein include all forms of delivering the compositions described herein to an object, where the composition is placed in the mouth of the object, whether or not the composition is swallowed. Therefore, “oral administration” includes buccal, sublingual, and esophageal administration.

[0060] In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least two weeks, for example, two, three, four, five weeks or longer. In certain embodiments, MK-7 and / or MKH2-7 are administered for at least six weeks. In certain embodiments, MK-7 and / or MKH2-7 are administered to the subject for at least two months, at least three months, at least six months, at least one year, at least two years, at least three years or indefinitely. In certain embodiments, MK-7 and / or MKH2-7 are administered for at least as long as the subject is receiving anticoagulation therapy other than warfarin or drugs with a similar mechanism of action to warfarin (e.g., asenocoumarol and fenprocumone), and / or statin therapy. If the subject is undergoing hemodialysis, MK-7 and / or MKH2-7 may be administered to the subject for a period including the duration of hemodialysis.

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

[0062] The terms “subject” and “patient” as used herein are used interchangeably and refer to organisms to be treated by the methods and compositions of the present invention. Such organisms are preferably mammals (e.g., humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, or non-human primates, e.g., monkeys, chimpanzees, baboons, and rhesus macaques), more preferably humans.

[0063] III. Method This disclosure relates, in part, to methods for preventing or treating tissue calcification in subjects where it is needed, for example, by slowing, stopping, and / or reversing its progression. In certain embodiments, the method relates to preventing tissue calcification. In certain embodiments, the method relates to slowing the progression of tissue calcification. In certain embodiments, the method relates to stopping tissue calcification. In certain embodiments, the method relates to reversing tissue calcification.

[0064] In certain embodiments, the Disclosure relates to a method for improving aortic compliance in a subject requiring improvement of aortic compliance, the method comprising administering to the subject an effective daily dose of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7). In certain embodiments, the Disclosure relates to a method for slowing, stopping and / or reversing the progression of peripheral vascular disease in a subject requiring slowing, stopping and / or reversing the progression of peripheral vascular disease, the method comprising administering to the subject an effective daily dose of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).

[0065] As used herein, the term "treating" includes any effect that results in an improvement in a condition, disease, disorder, etc., or an improvement in its symptoms, such as alleviation, reduction, delay in progression, arrest, regulation, improvement, or elimination. Treating can be curing, ameliorating, or at least partially improving a disorder. In certain embodiments, treating is curing a disease. Unless otherwise indicated, the term "disorder" refers to, and is used interchangeably with, the terms disease, pathologic condition, or illness.

[0066] The method can include administering to a subject a menaquinone-7 (MK-7) and / or menadiol-7 (MKH2-7) composition as 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., delaying progression, arresting, and / or reversing) tissue calcification. Tissue calcification refers to the accumulation of calcium salts in body tissues. In certain embodiments, the tissue calcification is soft tissue calcification. The term "soft tissue" includes any tissue other than bone or teeth. 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, such as type I or II diabetes, or is diagnosed with prediabetes. Diabetes often results in chronic kidney disease (CKD) because uncontrolled blood sugar and blood pressure damage the capillaries in the kidneys. CKD often results in the occurrence of uremia (urea in the blood), which inhibits the cyclic regeneration of vitamin K and can cause tissue calcification. Thus, in certain embodiments, the subject has CKD.

[0068] Patients with chronic kidney disease are classified into stages based on glomerular filtration rate (GFR). The GFR of stage 1 patients is ≧90 mL / min / 1.73 m 2Stage 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 kidney disease, such as polycystic kidney disease, can also cause ESRD. In certain embodiments, the subject has ESRD.

[0069] Patients with ESRD require hemodialysis. However, hemodialysis often leads to oxidation of numerous tissue proteins, and both CKD and ESRD patients often have a higher percentage of carbonyl proteins (up to 15 times) 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, the oxidative load caused by hemodialysis is thought to lead to the oxidation of KH2 to MK-7. Oxidation of KH2 by hemodialysis is thought to block its ability to function as a cofactor of GGCX and reduce the γ-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. Accordingly, in certain embodiments of this specification, the subject is undergoing hemodialysis.

[0071] 2. Anticoagulant therapy Vitamin K is necessary for the production of coagulation factors. However, certain anticoagulants, such as certain oral anticoagulants, function as vitamin K antagonists that prevent blood clotting. Given that tissue calcification can occur when vitamin K activity is not regulated, the methods described herein relate to preventing or treating tissue calcification (e.g., slowing, halting, and / or reversing its progression) in subjects receiving anticoagulant therapy and requiring prevention or treatment of tissue calcification, and include administering menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), comprising the compositions described herein, to a subject, thereby preventing or treating tissue calcification (e.g., slowing, halting, and / or reversing its progression).

[0072] Exemplary anticoagulant therapies include inhibitors of factor Xa or factor IIa activity. Factor Xa inhibitors include oral 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 fondaparinux (e.g., Arixtra®, GlaxoSmithKline). Factor IIa (thrombin) inhibitors include oral dabigatran (e.g., Pradaxa®, Boehringer Ingelheim) and intravenous argatroban (e.g., Pfizer).

[0073] In certain embodiments, the subject is concurrently receiving non-warfarin-based anticoagulation therapy. Non-warfarin-based anticoagulation therapy may be based on an anticoagulant that does not primarily act to block the conversion of vitamin K to vitamin K hydroquinone and / or the conversion of vitamin K2,3-epoxide to vitamin K. Exemplary non-warfarin-based anticoagulants may act as inhibitors of factor Xa activity or factor IIa activity. Exemplary non-warfarin-based anticoagulants include apixaban, rivaroxaban, betrixaban, edoxaban, dabigatran, fondaparinux, and argatroban. In certain embodiments, the subject has been previously exposed to warfarin-based anticoagulation therapy.

[0074] In certain embodiments, the methods described herein relate to preventing or treating (e.g., slowing, halting, and / or reversing) tissue calcification in a subject having CKD or ESRD and receiving oral non-warfarin-based anticoagulation therapy. The method may include administering to the subject at least 2 mg per day of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to prevent or treat (e.g., slowing, halting, or reversing) tissue calcification. The subject may also have diabetes mellitus, prediabetes, and / or be undergoing hemodialysis.

[0075] 3. Statin therapy Coronary artery calcification has been observed to 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 suggest 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] While we do not wish to be bound by theory, it is conceivable that HMG coA reductase inhibitors directly inhibit MK-4 production in coronary 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 patients with CKD and ESRD are functionally deficient in vitamin K, and blocking vitamin K (MK-4) production in endogenous vascular smooth muscle cells would only exacerbate calcification in the vascular wall, 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] Therefore, the present invention provides a method for preventing or treating tissue calcification (e.g., slowing, stopping, and / or reversing its progression) in a subject that requires it, wherein the subject is receiving a statin. The method may include administering the menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) compositions described herein to the subject. In certain embodiments, the subject is 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] Furthermore, the present invention provides a method for preventing or treating tissue calcification (e.g., slowing, halting, and / or reversing its progression) in subjects requiring such treatment, the method comprising administering to a subject the 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. Menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) and the statin may be administered as separate dosage forms or in the same dosage form.

[0079] 4. Improved aortic compliance and cessation and / or reversal of peripheral vascular complications. In certain embodiments, the Disclosure relates to a method for stopping and / or reversing peripheral vascular disease in a subject having ESRD or CKD, wherein the method comprises administering an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to the subject. In certain embodiments, the Disclosure relates to a method for improving aortic compliance in a subject having ESRD or CKD, wherein the method comprises administering an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to the subject.

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

[0081] One of the clinical consequences of vascular calcification is arteriosclerosis, which reduces vascular compliance (e.g., vascular elasticity), such as aortic compliance. Therefore, according to the method described herein, administration of an effective amount of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) may improve aortic compliance. The decrease in aortic compliance can be assessed using aortic plethysmography (i.e., the ability of blood vessels to dilate), see, for example, Inuma et al. (2012) HONG KONG JOURNAL OF NEPHROLOGY 14(2):48-53. The decrease in aortic compliance can also be assessed by measuring pulse wave velocity (PWV). PWV is the speed at which an arterial pulse travels through the circulatory system and is used as a measure of arteriosclerosis. Higher PWV indicates higher arteriosclerosis and therefore lower 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, e.g., carotid-femoral PWV or local PWV. Methods for measuring regional PWV are known in the art and include, for example, the use of pulse transducer probes, TY-360 pressure transducers, electrocardiogram (ECG) synchronized Doppler units, pulse transducer probes, photoplethysmography, Complior® (piezoelectric pressure transducer), SphygmoCor®, Arteriograph®, and PulsePen® (tonometer and integrated ECG unit). Methods for measuring local 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 vascular aortic compliance 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 vascular aortic compliance by approximately 10% to 500%, approximately 50% to 200%, and approximately 75% to 125%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases vascular aortic compliance 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 before administration of MK-7 and / or MKH2-7. In certain embodiments, administration of MK-7 and / or MKH2-7 increases vascular aortic compliance by approximately 10% to 500%, approximately 50% to 200%, or approximately 75% to 125% compared to the vascular 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 aims to stop or reverse the long-term increase in the CAC score, or to slow the rate of increase.

[0084] 5. Chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a term referring to a progressive lung disease that causes difficulty breathing. The two main forms of COPD are emphysema and chronic bronchitis. Symptoms of COPD include dyspnea, cough, mucus production, and wheezing. Elastin degradation (protein breakdown of elastin) is also a key feature of COPD. This contributes to the loss of arterial flexibility and promotes calcification of the vascular intima. 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. Therefore, it has been hypothesized that vitamin D is a critical determinant of elastin degradation rate, and that low vitamin D levels lead to low MGP activity and insufficient protection against elastin degradation (Piscaer et al., (2017) "Vitamin D deficiency: the linking pin between COPD and cardiovascular diseases?" RESP. RES. 18:189). While we do not wish to be bound by theory, the increased production of activated (carboxylated) MGP by administration of MK-7 and / or MKH2-7 suppresses the harmful effects of elastin degradation in subjects with COPD, thereby preventing, slowing, halting, or reversing one or more symptoms of COPD.

[0085] A reduction in COPD symptoms, including dyspnea, cough, mucus production, and wheezing, can be measured by any means known in the art. A slowing of the rate of increase of one or more COP symptoms over time indicates a slowing of the progression of one or more COPD symptoms, or a reduction of one or more symptoms by, for example, 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 COPD symptoms.

[0086] Elastin degradation can be measured by any method known in the art, including, for example, measuring the blood isodesmosine level in a subject. A slowing rate of increase in isodesmosine levels over time indicates a slowing of the progression of one or more symptoms of COPD, or a decrease in isodesmosine levels of, for example, 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, delay, cessation, and / or reversal of tissue calcification in the subject organism. In certain embodiments, biomarkers may 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 proteins to uncarboxylated proteins, as described in more detail below.

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

[0089] The time required for 50% conversion of primary CPP to secondary CPP (T50) is an accurate and sensitive means of determining the plasma's ability to sequestrate excess calcium phosphate crystals. The conversion from primary to secondary forms involves the formation of more elongated crystals. Elongation results in a more turbid solution, which can be detected by turbidimetric methods. Under conditions of high calcium phosphate loading, the time to reach 50% conversion is shortened because the reserves for calcium phosphate absorption are reduced. Therefore, a short T50 time suggests a decreased ability to absorb calcium phosphate crystals, while a long T50 time is consistent with high ability. Short T50 times are often associated with warfarin treatment or hemodialysis, as well as uremic oxidative loading, and are generally associated with an increased risk of myocardial infarction, heart failure, and all-cause mortality.

[0090] While we do not wish to be bound by theory, administration of MK-7 and / or MKH2-7 is thought to increase T50 hours and improve the ability of the subject body to prevent pathological calcification. Since 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 CPP, administration of MK-7 and / or MKH2-7 according to the methods of this disclosure may reduce the risk of pathological calcification and prevent the development of soft tissue (e.g., vascular) calcification.

[0091] Accordingly, in the specific embodiments described herein, administration of MK-7 and / or MKH2-7 to a subject increases the subject's serum T50 level (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 subject's serum T50 level before administration of MK-7 and / or MKH2-7. The method for measuring the serum T50 value of the subject is publicly 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 for increasing serum T50 levels 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., substantially pure MK-7 and / or MKH2-7 in amounts of 10, 15, 25, 50 or 75 mg per day) of MK-7 and / or MKH2-7 to the subject, wherein the administration of MK-7 and / or MKH2-7 to the subject increases the serum T50 level 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 serum T50 level of the subject before administration of MK-7 and / or MKH2-7.

[0093] 2. Carboxylation of vitamin K-dependent proteins Despite dietary deficiencies, vitamin K levels may not decrease in ESRD patients. For example, in a study of 172 stable hemodialysis patients, only 6% showed clinically significant vitamin K deficiency (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, levels were reduced in approximately 60% of patients (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 uremia patients, total vitamin K levels are normal, but the production of reduced vitamin K is blocked by the oxidative properties of uremic substances (ibid.).

[0094] The observation that oxidative states can disrupt the vitamin K cycle suggests that the oxidative load generated during hemodialysis further contributes to the high rate of soft tissue (e.g., blood vessel) calcification observed in 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 percentage of carbonyl proteins (15 times) compared to normal controls.

[0095] While we do not wish to be bound by theory, it is thought that the oxidative load resulting from uremic and / or hemodialysis leads to the oxidation of functional vitamin K hydroquinone (KH2) to non-functional natural vitamins. Oxidation of KH2 can block its ability to function as a cofactor for GGCX and reduce the amount of gamma-carboxylated vitamin K-dependent proteins. Therefore, administration of MK-7 and / or MKH2-7 to subjects with diabetes, CKD and / or ESRD and / or subjects undergoing hemodialysis may increase the ratio of one or more carboxylated vitamin K-dependent proteins to uncarboxylated vitamin K-dependent proteins in these subjects, or decrease the amount of one or more uncarboxylated vitamin K-dependent proteins in these subjects.

[0096] Therefore, 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 (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 subject's plasma by about 10% to 500% or more, about 30% to about 500%, about 30% to about 200%, or about 30% to about 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the carboxylated to uncarboxylated ratio of vitamin K-dependent proteins in the plasma of the subject by about 50% to about 500%, about 50% to about 200%, and about 50% to about 100%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the carboxylated to uncarboxylated ratio of vitamin K-dependent proteins in the plasma of the subject by about 70% to about 500%, about 70% to about 200%, and 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 proteins in the subject's plasma (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 proteins in the subject's plasma 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 carboxylated to uncarboxylated ratio of vitamin K-dependent proteins in the plasma of an object having the impairment described herein, wherein the method involves 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). The present invention relates to a method comprising administering substantially pure MK-7 and / or MKH2-7 (mg) to a subject, wherein the administration of MK-7 and / or MKH2-7 to the subject increases the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins 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 ratio before administration of MK-7 and / or MKH2-7.

[0099] In certain embodiments, the Disclosure relates to a method for reducing the amount of noncarboxylated vitamin K-dependent protein in the plasma of a subject having the impairment described herein, the method comprising administering to the subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., substantially pure MK-7 and / or MKH2-7 in amounts of 10, 15, 25, 50 or 75 mg per day) of MK-7 and / or MKH2-7, wherein the administration of MK-7 and / or MKH2-7 to the subject reduces the amount of noncarboxylated vitamin K-dependent protein in the plasma of the subject by (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 of MK-7 and / or MKH2-7 before administration.

[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. The carboxylated forms of MGP, Gas-6, PIVKA-II, osteocalcin, activated protein C, and activated protein S can be measured using sandwich assays, as is 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 carboxylated to uncarboxylated ratio of vitamin K-dependent proteins in the subject after administration of MK-7 indicates the success of the rescue of the GGCX carboxylation system. Therefore, effective administration of MK-7 and / or MKH2-7 may increase the ratio of carboxylated to uncarboxylated proteins in the subject.

[0101] PIVKA-II is an uncarboxylated, abnormal form of prothrombin, also known as des-γ carboxyprothrombin (DCP). Therefore, elevated levels of PIVKA-II correspond to a more poor vitamin K state. As a biomarker of vitamin K state, 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 method described herein, 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, 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 considered a potent inhibitor of calcification in arterial walls and other soft tissues in non-human animals, and its activity depends on vitamin K-dependent carboxylation of gamma-glutamic acid (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). Non-carboxylated MGP, formed as a result of vitamin K deficiency, is associated with cardiovascular disease. Recent studies suggest that hemodialysis patients have poor vitamin K status (Westenfeld et al. (2012), see above).Circulating inactive form of matrix gla protein (dp-ucMGP) has been shown to gradually increase 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 hemodialysis patients (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). Therefore, plasma dp-ucMGP has been proposed as a surrogate marker for vascular calcification in CKD (Schurgers et al. (2010), see above). Furthermore, lower levels of circulating desphosphocarboxylated MGP (dp-cMGP) have been suggested 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 the regulation of bone formation and regelation (Elliott et al. 2014, above; Marles et al. 2017, above). The percentage of uncarboxylated osteocalcin is a sensitive marker of vitamin K status in bone, and asymptomatic vitamin K deficiency is defined by a greater than 20% increase in the percentage of uncarboxylated osteocalcin (Elliott et al. 2014, above). Supplementation with MK-7 at a dose of 100-200 μg / d for 4-12 weeks has been shown to significantly increase the ratio of carboxylated to uncarboxylated 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-glutamate protein is synthesized in the liver and helps maintain normal blood clotting through the 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, see above). Protein C regulates the clotting process by inactivating the procoagulant activity of factors V and VIII in the presence of 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, such as protein C and / or protein S deficiencies, 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 experiencing hemolysis (N=53) showed 4.5-fold higher dp-ucMGP and 8.4-fold higher noncarboxylated osteocalcin levels compared to healthy controls of the corresponding age (N=50). PIVKA-II levels were evaluated in 49 hemodialysis patients.Vitamin K2 supplementation induced dose- and time-dependent decreases in circulating dp-ucMGP, uncarboxylated osteocalcin, and PIVKA-II levels. The response rates for the decrease in dp-ucMGP levels were 77% and 93% in the groups administered 135 μg and 360 μg of menaquinone-7, respectively (Westenfeld et al. (2012), see above).

[0105] 3. Levels of other biomarkers of calcification In certain embodiments, administration of MK-7 and / or MKH2-7 increases the plasma levels 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 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 levels of osteoprotegerin or fetuin A by approximately 10–50%, 50–100%, 100–200%, or 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. Therefore, the increase in levels of osteoprotegerin and / or fetuin A indicates that administration of MK-7 and / or MKH2-7 can prevent, slow, halt, and / or reverse tissue calcification.

[0106] In certain embodiments, the Disclosure provides a method for increasing plasma levels of osteoprotegerin or fetuin A in an object having the disorder described herein, wherein the method involves 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). The present invention relates to a method comprising administering substantially pure MK-7 and / or MKH2-7 (mg) to a subject, wherein the administration of MK-7 and / or MKH2-7 to the subject increases the plasma levels 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 concentration of osteoprotegerin or fetuin A prior to the administration of MK-7 and / or MKH2-7.

[0107] In a particular embodiment of any of the above embodiments, administration of MK-7 and / or MKH2-7 reduces the plasma level of D-dimer (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 D-dimer before administration of MK-7 and / or MKH2-7. In a particular embodiment of any of the above embodiments, 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 indicates the functional state of vitamin D metabolism. Proteins C and S prevent the production of thrombin and fibrin, and functional vitamin K deficiency contributes to the loss of proteins C and S, which can lead to microthrombosis through fibrin breakdown and D-dimer formation. Therefore, the presence of D-dimer is an indicator of asymptomatic thrombosis. Thus, a decrease in plasma levels of D-dimer may indicate that administration of MK-7 and / or MKH2-7 can restore the GGCX carboxylation system, which may lead to the prevention, slowing, halting, and / or reversing of tissue calcification.

[0108] In certain embodiments, the Disclosure relates to a method for reducing plasma levels of D-dimer in a subject having the impairment described herein, the method comprising administering to the subject at least 2 mg / day (e.g., at least 2 mg per day, e.g., substantially pure MK-7 and / or MKH2-7 in amounts of 10, 15, 25, 50 or 75 mg per day) of MK-7 and / or MKH2-7, wherein the 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 before administration of MK-7 and / or MKH2-7.

[0109] In any particular embodiment 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., 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 plasma levels of high-sensitivity C-reactive protein (hs-CRP) by approximately 10–50%, 50–100%, or 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., blood vessel) calcification. Therefore, a decrease in plasma levels 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 highly sensitive C-reactive protein (hs-CRP) in an object having the disorder described herein, wherein the method involves 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). The present invention relates to a method comprising administering substantially pure MK-7 and / or MKH2-7 (mg) to a subject, wherein the administration of MK-7 and / or MKH2-7 to the subject reduces 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) before administration of MK-7 and / or MKH2-7.

[0111] V. Lesions The methods and compositions described herein may be useful for slowing or stopping, or stopping and / or reversing, the progression of 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 epoxydoreductase complex subunit 1 (VKORC1, also known as VKOR) (see Figure 1). In some tissues, the paralog VKORC1L1 (VKORC1-Like-1) can also carry out this reaction. Skin exhibits the lowest expression of VKOR-C1, consistent with the clinical observation that vitamin K-dependent vascular calcification is more common in the skin. Low cutaneous expression of VKOR-C1 suggests that any condition or treatment that blocks vitamin K production, such as hemodialysis, makes the skin more susceptible to pathological calcification.

[0113] Therefore, 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 administration of MK-7 and / or MKH2-7 reduces the size of the skin lesions. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area of ​​the 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 total volume of skin lesions by approximately 30% to approximately 100%, approximately 30% to approximately 90%, approximately 30% to approximately 80%, approximately 30% to approximately 70%, or approximately 30% to approximately 60%. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area or total volume of skin lesions by approximately 50% to approximately 100%, approximately 50% to approximately 90%, approximately 50% to approximately 80%, approximately 50% to approximately 70%, or approximately 50% to approximately 60%. In certain embodiments, administration of MK-7 and / or MKH2-7 reduces the total surface area or total volume of skin lesions by approximately 70% to approximately 100%, approximately 70% to approximately 90%, or approximately 70% to approximately 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 there are multiple lesions. The volume of a lesion can be calculated from a three-dimensional model of the lesion constructed from digital images.

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

[0117] In certain embodiments, administration of MK-7 and / or MKH2-7 reduces 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 (i.e., eliminates the skin lesions).

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

[0119] The reduction in 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 arteriosclerosis. Higher PWV indicates higher arteriosclerosis and therefore lower vascular compliance. Methods for measuring PWV are well known in the art and are described, for example, in Pereira et al. (2015). Briefly, PWV can be assessed by measuring regional PWV, e.g., 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, electrocardiogram (ECG) synchronized Doppler units, pulse transducer probes, photoplethysmography, Complior® (piezoelectric pressure transducer), SphygmoCor®, Arteriograph®, and PulsePen® (tonometer and integrated ECG unit). Ibid. Methods for measuring local 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 a 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 pre-treatment vascular compliance. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of a vessel by approximately 10–50%, approximately 50–10%, approximately 100–200%, or approximately 200–500%. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of a 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 pre-treatment vascular compliance. In certain embodiments, administration of MK-7 and / or MKH2-7 increases the vascular compliance of a vessel by approximately 10–50%, approximately 50–100%, approximately 100–200%, or approximately 200–500% compared to pre-treatment vascular compliance.

[0121] VI. Kits for medical use Another aspect of the present invention provides a kit for treating one or more of the disorders or conditions described herein. The kit may comprise i) instructions for treating the medical disorder; and ii) one or more dose units comprising substantially pure MK-7 and / or MKH2-7.

[0122] Throughout this specification, where a composition is described as having, including, or comprising a particular component, or where a process and method is described as having, including, or comprising a particular step, it is further intended that there exist compositions of the present invention that are essentially composed of or consist of the described component, and processes and methods according to the present invention that are essentially composed of or consist of the described process step.

[0123] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0124] Throughout this specification, where compositions and kits are described as having, including, or comprising certain components, or where processes and methods are described as having, including, or comprising certain steps, it is intended that, additionally, there exist compositions and kits of the present invention that are essentially composed of or consist of the described components, and processes and methods according to the present invention that are essentially composed of or consist of the described process steps.

[0125] In this application, when an element or component is included in and / or selected from a list of elements or components described, it should be understood that the element or component may be any one of the lists of elements or components described, or that the element or component may be selected from a group consisting of two or more elements or components described.

[0126] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly described herein, may be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is referred to, 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, while embodiments within this application are described and depicted in a manner that enables a clear and concise application to be described and depicted, it should be understood that embodiments may be combined or separated in various ways without departing from the teachings and the invention. For example, it should be understood that all features described and depicted herein may be applicable to all embodiments of the invention described and depicted herein.

[0127] As used in this disclosure, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of the article, unless the context is appropriate. For example, “element” means one or more elements.

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

[0129] The expression "at least one of" includes each of the objects listed after the expression and any combination of two or more of the listed objects, unless otherwise understood from the context and usage. Expressions "and / or" relating to three or more listed objects should be understood to have the same meaning, unless otherwise understood from the context.

[0130] The use of the terms “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including their grammatical equivalents, should generally be understood as open-ended and non-restrictive, not excluding, for example, further additional unspecified elements or steps, unless otherwise explicitly stated or understood from the context.

[0131] Where the term “about” is used before a quantitative value, the present invention also includes the specific quantitative value itself unless otherwise explicitly stated. As used herein, the term “about” refers to ±10% of the nominal value unless otherwise specified or inferred.

[0132] For example, when the molecular weight of a polymer is given, rather than its absolute value, it should be understood as the average molecular weight unless otherwise specified or understood from the context.

[0133] As a general rule, percentages and specified compositions are given by weight unless otherwise specified. Furthermore, if a variable is not defined, the definition preceding the variable takes precedence.

[0134] It should be understood that, as long as the present invention remains operable, the order of the steps or the order in which specific actions are performed is not important. Furthermore, two or more steps or actions may be performed simultaneously.

[0135] Any and all examples herein, or any use of exemplary terms herein, such as “etc.” or “including,” are intended merely to better illustrate the invention and, unless otherwise requested, do not limit the scope of the invention. Nothing in this specification should be construed as indicating an element not claimed to be essential to the practice of the invention. [Examples]

[0136] This disclosure is further illustrated by the following embodiments, which should not be construed as limiting the scope or spirit of this disclosure to the specific procedures described herein. The embodiments are provided to illustrate specific embodiments and should not be understood as limiting the scope of this disclosure.

[0137] Example 1 - Administration of MK-7 and / or MKH2-7 to subjects with end-stage renal disease (ESRD) to reverse tissue calcification or slow its progression. This example describes the 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 thought to result in changes in aortic compliance (as measured by plethysmography), vascular calcification, and specific biomarker levels, indicating a delay, cessation, or reversal of the progression of tissue calcification.

[0138] Patients with ESRD receiving stable hemodialysis should be orally administered MK-7 and / or MKH2-7 at 10 mg, 25 mg, or 50 mg doses once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is 10 mg or 25 mg softgel capsules. Two 25 mg capsules are administered once daily to the 50 mg cohort.

[0139] The degree of calcification of the thoracic arteries is estimated using the coronary artery calcification score (CAC). A high CAC score indicates calcification, and treatment aims to stop or reverse the long-term increase in the CAC score, or slow the rate of increase. In addition, arterial compliance, which decreases as calcification increases, is measured using aortic plethysmography. Furthermore, pulse wave velocity (PWV) is measured to assess arterial compliance. These measurements are useful in estimating the effectiveness 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 therapeutic value.

[0140] In addition, several biomarkers will be evaluated to determine the efficacy 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 will be collected and biomarkers measured during patient visits for hemodialysis.

[0141] Administration of MK-7 and / or MKH2-7 may result in (i) increased PIVKA-II, osteoprotegerin, or fetuin A, indicating a delay, cessation, or reversal of tissue calcification progression; (ii) decreased noncarboxylated MGP, noncarboxylated osteocalcin, and / or noncarboxylated protein C, indicating a delay, cessation, or reversal of tissue calcification progression; and / or (iii) decreased hs-CRP, indicating a delay, cessation, or reversal of tissue calcification progression and / or decreased inflammation.

[0142] After daily administration of 10 mg, 25 mg, or 50 mg of MK-7 and / or MKH2-7, at least one of PIVKA-II, hypocarboxylated matrix Gla protein (MGP), noncarboxylated osteocalcin protein, noncarboxylated protein C, osteoprotegerin, fetuin A, and hs-CRP is thought to show delayed, halted, or reversed progression of tissue calcification.

[0143] Example 2 - Administration of MK-7 and / or MKH2-7 to subjects with stable end-stage renal disease (ESRD) undergoing hemodialysis. This example describes the administration of MK-7 to subjects with ESRD undergoing hemodialysis who are at risk of developing tissue calcification. Administration of MK-7 may result in changes in specific biomarker levels indicating prevention of tissue calcification or cessation or delay of its development.

[0144] Many subjects with stable ESRD but without calciphylaxis were enrolled in the study and administered 10 mg capsules of MK-7 orally daily for 14 days. Levels of specific biomarkers, including uncarboxylated matrix Gla protein (MGP), uncarboxylated osteocalcin, osteoprotegerin, fetuin A, and hs-CRP, were assessed on day 1 and day 15 (the end of treatment), and the changes between day 1 and day 15 were calculated. Between day 1 and day 15, the mean decrease in uncarboxylated MGP was -21.9%, and the mean decrease in uncarboxylated osteocalcin was -55.5%. Unexpectedly, subjects also showed mean increases in osteoprotegerin and fetuin A, as well as decreases in hs-CRP. Osteoprotegerin, fetuin A, and hs-CRP are not vitamin K-dependent proteins, and changes in these proteins are not considered to be previously 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 the studies, administration of MK-7 can result in a decrease in non-carboxylated MGP, non-carboxylated osteocalcin, and hs-CRP, as well as an increase in fetuin A and osteoprotegerin, which may indicate that administration of MK-7 prevents tissue calcification or halts or slows its progression in the subject.

[0146] Embedding with proper attribution Each of the patent and scientific document disclosures referenced herein is incorporated in its entirety by attribution for all purposes.

[0147] Equal portions The present invention may be embodied in other specific forms without departing from its spirit or essential features. Therefore, the embodiments described herein should be considered in all respects as illustrative and not limiting the invention described herein. Accordingly, the scope of the invention is indicated not by the foregoing description but by the appended claims, and all modifications within the meaning and equivalent scope of the claims are intended to be encompassed therein. Furthermore, the present invention includes the following aspects and embodiments. [1] A method for preventing, slowing, stopping and / or reversing tissue calcification in subjects with prediabetes or diabetes, chronic kidney disease or combination thereof who require prevention, slowing, stopping and / or reversing the progression of tissue calcification, the method comprising administering to a subject at least 2 mg per day of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), thereby preventing, slowing and / or stopping tissue calcification, wherein MK7 and / or MKH2-7 are administered in a pharmaceutical composition. [2] The method according to [1], wherein the subject has diabetes. [3] The method according to [2], wherein the subject has type 2 diabetes. [4] The method described in [1], wherein the subject has been diagnosed with prediabetes. [5] The method according to any one of [1] to [4], wherein the subject has chronic kidney disease. [6] The method according to any one of [1] to [4], wherein the subject has stage 4 or 5 chronic kidney disease / end-stage renal disease. [7] The method according to any of [1] to [6], provided the subject is undergoing hemodialysis. [8] The method according to any of [1] to [7], wherein the subject is receiving non-warfarin-based anticoagulation therapy. [9] The method according to [8], wherein the anticoagulation therapy is oral anticoagulation therapy.

[10] The method according to [9], wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux) or factor IIa activity (e.g., dabigatran, or argatroban).

[11] A method for preventing, slowing, stopping and / or reversing tissue calcification in a subject undergoing hemodialysis who requires prevention, slowing, stopping and / or reversing the progression of tissue calcification, 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), thereby preventing, slowing, stopping and / or reversing tissue calcification, wherein MK7 and / or MKH2-7 are administered in a pharmaceutical composition.

[12] The method according to

[11] , wherein the subject has diabetes.

[13] The method by which diabetes is determined to be type 2 diabetes, as described in

[12] .

[14] The method described in

[11] , wherein the subject has been diagnosed with prediabetes.

[15] The method according to any of

[11] to

[14] , wherein the subject has chronic kidney disease.

[16] The method according to

[15] , wherein the subject has stage 4 or stage 5 chronic kidney disease / end-stage renal disease.

[17] The method described in any of

[11] to

[16] , wherein the subject is receiving non-warfarin-based anticoagulation therapy.

[18] The method according to

[17] , wherein the anticoagulation therapy is oral anticoagulation therapy.

[19] The method according to

[17] , wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban, or fondaparinux) or factor IIa activity (e.g., dabigatran, or argatroban).

[20] A method for preventing, slowing, stopping or reversing tissue calcification in a subject having stage 5 chronic kidney disease requiring prevention, slowing, stopping or reversing of progression, and receiving oral nonwarfarin-based anticoagulation therapy, wherein the method comprises administering to the subject at least 2 mg per day of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), thereby preventing, slowing, stopping and / or reversing tissue calcification in the subject, wherein MK7 and / or MKH2-7 are administered in a pharmaceutical composition.

[21] The method according to

[20] , wherein the subject is diabetic.

[22] The method described in

[20] , wherein the subject has been diagnosed with prediabetes.

[23] The method according to

[21] or

[22] , wherein the subject is undergoing hemodialysis.

[24] The method according to any one of

[21] to

[23] , wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban, or fondaparinux) or factor IIa activity (e.g., dabigatran, or argatroban).

[25] The method according to any of [1] to

[24] , wherein administration of MK-7 and / or MKH2-7 to a subject increases the subject's serum T50 level (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 level before administration of MK-7 and / or MKH2-7.

[26] The method according to any one of [1] to

[25] , wherein administration of MK-7 and / or MKH2-7 increases (a) the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the plasma of the subject, or (b) the amount of uncarboxylated vitamin K-dependent proteins in the plasma of the subject, compared to the ratio or amount before administration of MK-7 and / or MKH2-7.

[27] The method according to

[26] , wherein the vitamin K-dependent protein is selected from matrix Gla protein, growth arrest-specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, or activated protein S.

[28] The method according to any one of [1] to

[27] , wherein administration of MK-7 and / or MKH2-7 to a subject increases the plasma levels of osteoprotegerin or fetuin A compared to the plasma levels of osteoprotegerin or fetuin A prior to administration of MK-7 and / or MKH2-7.

[29] The method according to any one of [1] to

[28] , wherein administration of MK-7 and / or MKH2-7 to a subject reduces the plasma level of D-dimer or highly sensitive C-reactive protein (hs-CRP) compared to the plasma level of D-dimer or highly sensitive C-reactive protein (hs-CRP) before administration of MK-7 and / or MKH2-7.

[30] The method according to any one of [1] to

[29] , wherein administration of MK-7 and / or MKH2-7 reduces the size of skin and / or vascular lesions in a subject having skin lesions.

[31] The method according to

[30] , wherein administration of MK-7 and / or MKH2-7 reduces the total surface area of ​​the lesion by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[32] A method for preventing, slowing, stopping and / or reversing tissue calcification in a subject requiring prevention, slowing, cessation and / or reversal of tissue calcification, comprising administering to the subject at least 2 mg per day of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), wherein MK7 and / or MKH2-7 are as follows: (i) Increase the serum T50 level 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 subject's serum T50 level before administration of MK-7 and / or MKH2-7. (ii) Increase 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%, or more) compared to the ratio before administration of MK-7 and / or MKH2-7. (iii) Increasing the plasma levels 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%, or more) compared to the plasma concentration of osteoprotegerin or fetuin A prior to administration of MK-7 and / or MKH2-7, or (iv) Reduce the plasma levels of D-dimer or highly sensitive C-reactive protein (hs-CRP) compared to the plasma concentration of D-dimer or highly sensitive C-reactive protein (hs-CRP) before administration of MK-7 and / or MKH2-7 (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%). A method, administered in a pharmaceutical composition, to cause at least one of the following, thereby preventing, slowing, stopping, and / or reversing tissue calcification in a subject body.

[33] The method according to

[32] , wherein the subject has diabetes.

[34] The method described in

[32] , wherein the subject has been diagnosed with prediabetes.

[35] The method according to

[32] or

[33] , wherein the subject has chronic kidney disease.

[36] The method according to any of

[32] to

[35] , provided the subject is undergoing hemodialysis.

[37] The method according to any of

[32] to

[36] , wherein the subject is receiving non-warfarin-based anticoagulation therapy.

[38] The method according to

[37] , wherein the anticoagulant therapy is oral anticoagulant therapy.

[39] The method according to

[38] , wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity (e.g., apixaban, rivaroxaban, betrixaban, edoxaban, or fondaparinux) or factor IIa activity (e.g., dabigatran, or argatroban).

[40] The method according to any one of

[33] -

[39] , wherein the vitamin K-dependent protein is selected from matrix Gla protein, growth arrest-specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, or activated protein S.

[41] The method according to any one of [1] to

[40] , comprising administering to a subject approximately 2 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[42] The method according to any one of [1] to

[41] , comprising administering to a subject approximately 5 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[43] The method according to any one of [1] to

[42] , comprising administering to a subject approximately 10 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[44] The method according to any one of [1] to

[43] , comprising administering 10, 25, 50, 75, 100 mg or more of MK-7 and / or MKH2-7 per day to a subject.

[45] The method according to any one of [1] to

[44] , wherein MK-7 and / or MKH2-7 are administered to the subject for at least two weeks.

[46] The method according to any of [1] to

[45] , wherein MK-7 and / or MKH2-7 are administered to the subject for at least 6 weeks.

[47] The method according to any of [1] to

[46] , wherein if the subject is undergoing hemodialysis, MK-7 and / or MKH2-7 are administered to the subject for a certain period of time, including the duration of hemodialysis.

[48] ​​The method according to any of [1] to

[47] , wherein MK-7 and / or MKH2-7 are administered orally.

[49] The method according to any one of [1] to

[48] , wherein MK-7 and / or MKH2-7 are placed in a tablet, a caplet, or within a caplet.

[50] The method according to any of [1] to

[49] , wherein the subject has been previously exposed to warfarin-based anticoagulation therapy.

[51] The method according to any of [1] to

[50] , wherein the subject is receiving a statin.

[52] The method according to

[51] , wherein the statin is selected from simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin.

[53] The method according to any of [1] to

[52] , wherein the tissue calcification is vascular calcification.

[54] The method according to any of [1] to

[53] , wherein the tissue calcification is cutaneous calcification.

[55] A method for preventing, slowing, stopping and / or reversing tissue calcification in an object that requires prevention, slowing, stopping and / or reversal of tissue calcification, wherein the method is (a) at least 2 mg of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) per day; and (b) statins A method comprising administering to a target body.

[56] The method according to

[55] , wherein the statin is selected from simvastatin, lovastatin, atorvastatin, pravastatin, pitavastatin, rosuvastatin and fluvastatin.

[57] The method according to

[55] or

[56] , wherein MK-7 and / or MKH2-7 are administered in the same dosage form as the statin.

[58] The method according to

[55] or

[56] , wherein MK-7 and / or MKH2-7 are administered in a dosage form separate from the statin.

[59] A method for improving aortic compliance in a subject requiring improvement of aortic compliance, the method comprising administering to the subject an effective daily dose of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).

[60] A method for halting and / or reversing peripheral vascular disease in a subject that requires halting and / or reversing peripheral vascular disease, wherein the subject has ESRD or CKD, and the method comprises administering to the subject an effective daily dose of substantially pure menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7).

[61] A method for preventing, slowing the progression of, stopping and / or reversing one or more symptoms of chronic obstructive pulmonary disease (COPD) in a subject who requires prevention, slowing the progression of, stopping and / or reversing one or more symptoms of chronic obstructive pulmonary disease (COPD), 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), thereby preventing, slowing the progression of, stopping and / or reversing one or more symptoms of COPD, wherein MK7 and / or MKH2-7 are administered in a pharmaceutical composition.

[62] The method according to

[61] , wherein one or more symptoms are selected from the group consisting of dyspnea, cough, mucus production, wheezing, and elastin breakdown.

[63] The method according to any one of

[59] to

[62] , comprising administering to a subject approximately 2 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[64] The method according to any one of

[59] to

[63] , comprising administering to a subject approximately 5 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[65] The method according to any one of

[59] to

[64] , comprising administering to a subject approximately 10 mg to approximately 100 mg of MK-7 and / or MKH2-7 per day.

[66] The method according to any one of

[59] to

[65] , comprising administering 10, 25, 50, 75 or 100 mg of MK-7 and / or MKH2-7 per day to a subject.

[67] The method according to any of

[59] to

[66] , wherein MK-7 and / or MKH2-7 are administered to the subject for at least two weeks.

[68] The method according to any of

[59] to

[67] , wherein MK-7 and / or MKH2-7 are administered to the subject for at least 6 weeks.

[69] The method according to any of

[59] to

[67] , wherein MK-7 and / or MKH2-7 are administered to the subject indefinitely.

[70] The method according to any of

[59] -

[69] , wherein MK-7 and / or MKH2-7 are administered orally.

[71] The method according to any of

[59] to

[70] , wherein MK-7 and / or MKH2-7 are placed in a tablet, a caplet, or within a caplet.

[72] The method according to any of

[59] to

[71] , wherein the subject is receiving a statin.

[73] The method according to

[72] , wherein administration of MK-7 and / or MKH2-7 prevents or reduces the vitamin K depletion effect of statins.

Claims

1. A pharmaceutical product for treating tissue calcification in subjects with prediabetes or diabetes, chronic kidney disease or a combination thereof who require treatment of tissue calcification, wherein the pharmaceutical product comprises 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 the plasma levels of osteoprotegerin or fetuin A compared to the plasma levels of osteoprotegerin or fetuin A before administration of MK-7 and / or MKH2-7.

2. The pharmaceutical product according to claim 1, wherein the subject has diabetes.

3. The pharmaceutical product according to claim 2, wherein the subject has type II diabetes.

4. The pharmaceutical product according to claim 1, wherein the subject has been diagnosed with prediabetes.

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

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

8. A pharmaceutical product according to any one of claims 1 to 7, wherein the subject is receiving non-warfarin-based anticoagulation therapy.

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

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

11. The pharmaceutical product according to claim 10, wherein the factor Xa activity inhibitor is apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux.

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

13. A pharmaceutical agent for treating tissue calcification in a subject undergoing hemodialysis who requires treatment of tissue calcification, wherein the pharmaceutical agent comprises at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7), and the administration of at least 10 mg of menaquinone-7 (MK-7) and / or menaquinol-7 (MKH2-7) to the subject increases the plasma levels of osteoprotegerin or fetuin A compared to the plasma levels of osteoprotegerin or fetuin A before administration of the pharmaceutical agent.

14. The pharmaceutical product according to claim 13, wherein the subject has diabetes.

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

16. The pharmaceutical product according to claim 13, wherein the subject has been diagnosed with prediabetes.

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

18. The pharmaceutical product according to claim 17, wherein the subject has stage 4 or 5 chronic kidney disease / end-stage renal disease.

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

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

21. The pharmaceutical product according to claim 20, wherein the anticoagulant therapy comprises an inhibitor of factor Xa activity or factor IIa activity.

22. The pharmaceutical product according to claim 21, wherein the factor Xa activity inhibitor is apixaban, rivaroxaban, betrixaban, edoxaban, or fondaparinux.

23. The pharmaceutical product according to claim 21, wherein the factor IIa activity inhibitor is dabigatran or argatroban.

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