Menaquinone nanoparticles and methods of treatment

Abstract

This application discloses compositions comprising nanoparticles of vitamin K2 and methods of use thereof. The present invention relates to nanoparticles of menaquinones, such as MK-7, compositions and formulations thereof for treating vitamin K-related diseases. This specification further discloses menaquinols, such as menaquinol-4 (MKH2-4) through menaquinol-14 (MKH2-14), compositions and formulations thereof, and methods of preparation and use thereof for treating vitamin K-related diseases.

Description

[Technical Field]

[0001] This application is related to U.S. Application No. 63 / 447,305, filed February 21, 2023, entitled "Nano-Particles of Menaquinone and Methods of Treatment."

[0002] FIELD OF THE INVENTION The present invention relates to nanoparticles of menaquinones, such as MK-7, compositions and formulations thereof, for treating diseases associated with vitamin K. The present specification further discloses menaquinols, such as menaquinol-4 (MKH2-4) through menaquinol-14 (MKH2-14), compositions and formulations thereof, and methods of preparation and use, for treating diseases associated with vitamin K. [Background technology]

[0003] Background of the Invention Vitamin K is known as a group of structurally similar fat-soluble vitamins. Vitamin K2 (or vitamin K2) or menaquinones have nine related compounds that can be subdivided into short-chain menaquinones (e.g., menaquinone-4 or MK-4) and long-chain menaquinones (e.g., MK-7, MK-8, MK-9-14). Vitamins include phylloquinone (K1), menaquinone (K2), and menadione (K3). Plants synthesize vitamin K1, while bacteria can produce a range of vitamin K2 forms, including conversion of K1 to K2 by bacteria in the small intestine. Vitamin K3 is a synthetic version of the vitamin and is prohibited for human use by the U.S. Food and Drug Administration due to its toxicity.

[0004] It has been established that taking broad-spectrum antibiotics can reduce vitamin K production in the intestine by nearly 74% in humans compared to those not taking these antibiotics. A diet low in vitamin K also reduces vitamin K levels in the body. Vitamin K1 is used preferentially by the liver as a clotting factor. Vitamin K2 is used preferentially in the brain, vasculature, breast, and kidney. Vitamin K2 contributes to the production of myelin and sphingolipids (fats essential for brain health) and protects against oxidative damage in the brain. Vitamin K2, like MK-4, promotes bone health by stimulating the production of connective tissue in bone.

[0005] In animals, vitamin K2, the main storage form, has several subtypes that differ in the length of the isoprenoid group or the number of residues in the side chain. These vitamin K2 homologs, called menaquinones, are characterized by the number of isoprenoid residues in their side chains. For example, MK-4, which has four isoprene residues in its side chain, is the most common type of vitamin K2 in animal products. MK-4 is typically synthesized from vitamin K1 in certain animal tissues (arterial walls, pancreas, and testes) by replacing the phytyl group with an unsaturated geranyl group containing four isoprene units. Unlike MK-4, MK-7 is not produced by human tissues. MK-7 can be converted from phylloquinone (K1) in the colon by E. coli bacteria. MK-4 and MK-7 are sold in the United States as dietary supplements for bone health. MK-4 has been shown to reduce the incidence of fractures. In Japan, since 1995, MK-4 at a dose of 45 mg per day has been approved by the Ministry of Health, Labor and Welfare for the prevention and treatment of osteoporosis.

[0006] Cardiovascular disease (CVD) is established as the most frequent cause of death in patients with chronic kidney disease (CKD). Compared to the general population, mortality due to CVD is approximately 10-20 times higher in CKD patients receiving hemodialysis treatment. Furthermore, vascular calcification and associated arterial stiffness have been demonstrated to be common in the development of CVD. Therefore, the disclosed methods of treatment can be applied to the treatment of peripheral arterial disease. Furthermore, CKD patients receiving dialysis treatment have a three-fold increased risk of fractures, including vertebral fractures and other types of fractures.

[0007] Vitamin K, including MK-7, is present in low concentrations in the normal diet. It has also been established that there is a direct correlation between the level of vitamin K in a patient's blood and the incidence of vascular calcification, bone mineral density, and bone strength. Therefore, the supplemental use of vitamin K, such as MK-7 and its lipid-soluble hydroquinone (menaquinol), also disclosed herein as a nanoparticle formulation, can provide important clinical benefits, in part, by reducing vascular calcification, as seen by arteriosclerosis, and can increase bone mineral density, which aids in the treatment or prevention of CVD and bone disease in patients with CKD. In one aspect, the disclosed method for administering MK-7 as a nanoparticle composition or formulation disclosed herein can be used to treat or reduce vascular calcification, increase bone mineral density, and treat, alleviate, or prevent bone disease, such as in patients with CKD.

[0008] In foods, vitamin K1 has also been established to be bound to the chloroplast membranes of green leafy vegetables. MK-4, derived from the conversion of menadione, a synthetic analog of vitamin K, is found in animal products such as eggs and meat. Long-chain menaquinones, such as MK-7, MK-8, and MK-9, are found in fermented foods such as cheese, curd, and sauerkraut. It has also been established that the effects of long-chain MK-ns, such as MK-7, on normal blood clotting are greater and longer-lasting than those of vitamin K1 and MK-4. MK-7 has also been shown to have a longer serum half-life compared to MK-4 and provides a superior carboxylated grade of osteocalcin compared to vitamin K1. See Sato et al., Nutrition Journal, 2012, 11:93.

[0009] In humans, nutritional doses of MK-7 can be established as being well absorbed, resulting in significant elevations in serum MK-7 levels. [ka]

[0010] More than one-third of drugs listed in the United States Pharmacopoeia have been found to be water-insoluble or poorly water-soluble. Furthermore, more than 40% of drugs are insoluble in the human body, which is significant considering that there are more than 5,000 small molecules under development. Solubility and stability issues are two of the challenging properties that hinder drug development. Furthermore, aqueous solubility is required for formulating many organic compounds under development as pharmaceuticals. Traditional formulation systems for highly insoluble drugs have required the application of a combination of organic solvents, surfactants, and emulsions, among other methods. For example, poorly water-soluble drugs, such as vitamin K2 or MK-7, are typically excreted from the gastrointestinal tract before they can be absorbed into the blood circulation. It is known that the dissolution rate of a particular compound or drug can increase as the surface area increases or the particle size decreases. Therefore, significant emphasis has been placed on the development of nanoparticles for delivering insoluble, low-solubility, or poorly soluble drugs. Nanoparticles are generally considered to be solid particles having diameters between about 1 nm and 1000 nm. The above examples of the related art and limitations are intended to be illustrative only and not exhaustive. Other limitations of the related art will become apparent to those skilled in the art upon reading this specification and examining the figures or drawings presented herein. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Sato et al., Nutrition Journal, 2012, 11:93 Summary of the Invention [Means for solving the problem]

[0012] Summary of the Invention There is a continuing need for new formulations that are effective for these indications. The following embodiments, aspects and variations thereof are exemplary and not intended to be limiting in scope. [ka]

[0013] In one embodiment, a composition is provided comprising nanoparticles (or nano-sized particles) of water-soluble vitamin K2, wherein the nanoparticles have an average particle size of about 0.1 nm to 1,000 nm. In one embodiment, the vitamin K2 is selected from the group consisting of MK-4 (menaquinone-4), MK-5 (menaquinone-5), MK-6 (menaquinone-6), MK-7 (menaquinone-7), MK-8 (menaquinone-8), MK-10 (menaquinone-10), MK-11 (menaquinone-11), MK-12 (menaquinone-12), MK-13 (menaquinone-13), and MK-14 (menaquinone-14). In another embodiment, the vitamin K2 is MK-7. In another embodiment, the vitamin K2 compounds, including MK-4 through MK-14, and also MKH2-4 through MKH2-14, are selected from the group consisting of MKH2-4 (menaquinol-4), MKH2-5 (menaquinol-5), MKH2-6 (menaquinol-6), MKH2-7 (menaquinol-7), MKH2-8 (menaquinol-8), MKH2-9 (menaquinol-9), MKH2-10 (menaquinol-10), MKH2-11 (menaquinol-11), MKH2-12 (menaquinol-12), MKH2-13 (menaquinol-13), and MKH2-14 (menaquinol-14), and the compounds are stabilized for storage and administration. In another embodiment, the nanoparticles have an average particle size of less than 200 nm, 175 nm, 150 nm, 125 nm, 115 nm, 100 nm, 90 nm, 80 nm, or less than 75 nm. In another embodiment, the composition is stable upon storage at room temperature.

[0014] In one variation, the nanoparticles have an average particle size range of about 10 nm to 750 nm, 20 nm to 700 nm, 40 nm to 600 nm, 50 nm to 500 nm, 60 nm to 400 nm, or about 45 nm to 95 nm. In another variation, the nanoparticles have an average particle size of about 70 nm to 300 nm, 80 nm to 200 nm, 90 nm to 175 nm, 100 nm to 150 nm, or about 120 nm to 130 nm. In another variation, the nanoparticles have an average particle size of about 75 nm to 175 nm, 85 nm to 165 nm, 95 nm to 155 nm, 105 nm to 145 nm, 145 nm to 175 nm, or about 155 nm to 165 nm. In another variation, the nanoparticles have an average particle size range of about 75 nm to 105 nm, or 85 nm to 90 nm. In another variation, the average particle size may be about 155 nm.

[0015] In another embodiment of the above composition, the nanoparticles are prepared using a homogenizer selected from the group consisting of a rotor-stator homogenizer, a bead mill homogenizer, or a mortar and pestle homogenizer. In one variation, the nanoparticles are prepared using a milling process such as wet milling, wet milling using a high-pressure homogenizer, a dry milling process, or jet milling. See T. Niwa et al., "Universal wet-milling technique to prepare oral nanosuspension focused on discovery and preclinical animal studies - Development of particle design method," International Journal of Pharmaceutics, Vol. 405, 1-2, 28 February 2011, 218-227; T. Niwa et al., "Design of dry nanosuspension with highly spontaneous dispersible characteristics to develop soluble formulations for poorly water-soluble drugs," Pharmaceutical Research, 28, 2339-2349, 2011.

[0016] In another embodiment, the composition includes an additive or additive selected from the group consisting of Poloxamer 188, Polysorbate 80, Polysorbate 20, Vit E-TPGS (TPGS), TPGS-1000, TPGS-750-M, Solutol HS15, PEG-40 Hydrogenated Castor Oil, Kolliphor RH40, PEG-35 Castor Oil, PEG-8-Glyceryl Caprylate / Caprate, PEG-32-Glyceryl Laurate, PEG-32-Glyceryl Palmitostearate, Polysorbate 85, Polyglyceryl-6-Dioleate, Sorbitan Monooleate, Capmul The composition further comprises at least one emulsifier (or solubilizer) selected from the group consisting of MCM, Maisine 35-1, glyceryl monooleate, glyceryl monolinoleate, PEG-6-glyceryl oleate, PEG-6-glyceryl linoleate, oleic acid, linoleic acid, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, polyglyceryl-3 diisostearate, carboxymethylcellulose (CMC), polysorbate 80 (P80), and lecithin; or a mixture thereof. In another embodiment, the emulsifier is selected from polysorbate 80, Vitamin E-TPGS, Solutol HS15, PEG-40 hydrogenated castor oil, and PEG-35 castor oil.

[0017] In one embodiment, the composition further comprises at least one bioavailability enhancer selected from the group consisting of medium chain fatty acids, omega-3 fatty acids, capric acid, caprylic acid, alkyl glycosides, chitosan, trimethylated chitosan, ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, salicylic acid, genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one)) and pharmaceutically acceptable salts thereof.

[0018] In another embodiment, the composition is a nanosuspension in water or a nanoparticle emulsion in water. In yet another embodiment, the composition, nanosuspension, or nanoparticle emulsion is at least 5 times more soluble than commercially available (by chemical synthesis or fermentation) non-homogenized vitamin K2 or vitamin K-2 that is not formed or prepared as nanoparticles, such as MK-7. In one variation, the composition is at least 2, 3, 5, 7, 10, 15, 20, or more times more soluble than commercially available non-homogenized vitamin K2. As used herein, "non-homogenized" vitamin K2 refers to commercially available vitamin K2, such as MK-7, MK-8, MK-9, etc., that is not homogenized, milled, or otherwise prepared as nanoparticles or nanosuspensions as described herein. In another embodiment, the vitamin K2 is MK-7. In another embodiment, the vitamin K2 nanosuspension is prepared or performed at a concentration of 0.01 mg / mL in water. In yet another embodiment, the nanosuspension is in aqueous solution in Fed State Simulated Intestinal Fluid (FeSSIF). In yet another embodiment, the solubility is determined after 10 minutes in FeSSIF.

[0019] In another embodiment, the composition further comprises a pharmaceutically acceptable excipient, and the composition is effective in treating a vitamin K-related condition, or in treating osteoporosis or arteriosclerosis.

[0020] In another embodiment, there is provided a method for the treatment of a disease in a mammal selected from the group consisting of neurodegenerative diseases, retinopathy, rheumatoid polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with skin aging and aging in tissues, pathologies associated with mitochondrial dysfunction, and cachexia associated with nutritional deficiencies, wherein the treatment is accompanied by an increase in the lifespan of the mammal, the method comprising administering a therapeutically effective amount of any of the above compositions.

[0021] In another embodiment, a method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative disorders, and cardiovascular disorders is provided, comprising administering a therapeutically effective amount of any of the above-described compositions to the mammal. In yet another embodiment, a method for treating or preventing osteoporosis and / or osteopenia is provided, comprising administering a therapeutically effective amount of the above-described compositions to a patient in need of treatment. In yet another embodiment, a method for treating, preventing, slowing, halting, and / or reversing calciphylaxis in a mammal in need thereof is provided, comprising administering to the mammal a therapeutically effective amount of any of the above-described compositions and a pharmaceutically acceptable excipient to prevent, slow, halt, or reverse calciphylaxis. In one aspect, the mammal has distal calciphylaxis and / or central calciphylaxis. In another aspect, the mammal has diabetes, chronic kidney disease or end-stage renal disease.

[0022] In one variation of the above method, the mammal has stage 3, stage 4, or stage 5 chronic kidney disease. In another variation of this method, the mammal is undergoing hemodialysis. In another variation of this method, the mammal is undergoing non-warfarin-based anticoagulant therapy. In another variation of this method, the anticoagulant therapy is oral anticoagulant therapy. In yet another variation of this method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, elibaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigratran or argatroban.

[0023] COPD Treatment: Chronic obstructive pulmonary disease (COPD) is a term used to describe progressive lung disease that makes breathing difficult. The two main forms of COPD are emphysema and chronic bronchitis. Furthermore, elastinolysis (proteolysis of elastin) is a key feature of COPD. COPD contributes to loss of arterial flexibility and promotes calcification of the intimal media of blood vessels. It has also been shown to be a strong predictor of mortality in COPD patients (Rabinovich et al., (2016) Circulating desmosine levels do not predict emphysema progression but are associated with cardiovascular risk and mortality in COPD, ERJ Express doi: 10.1183 / 13993003.01824-2015). MGP has been demonstrated to inhibit the production of matrix metalloproteinases, which promote elastinolysis. Vitamin D may be an important determinant of the rate of elastin degradation, and low vitamin D levels result in inadequate MGP activity to protect against elastin degradation (Piscaer et al., (2017) Vitamin D deficiency: the linking pin between COPD and cardiovascular diseases? RESP. RES. 18:189). Without wishing to be bound by theory, administration of vitamin K2 as disclosed herein increases the production of activated (carboxylated) MGP, thereby acting to inhibit the deleterious effects of elastin degradation in subjects with COPD, thereby preventing, slowing the progression of, or reversing one or more symptoms of COPD. Furthermore, treating elastin degradation may be effective in treating Covid, such as Covid-19 and its variants. Thus, the nanoparticle formulations disclosed herein can be administered to treat or prevent elastin degradation and diseases associated with elastin degradation.

[0024] In one embodiment of any of the above methods, the mammal has chronic obstructive pulmonary disease (COPD). In another embodiment of the method, the mammal has calciphylaxis-associated skin lesions. In one variation of the method, administration of the composition reduces the total surface area of ​​skin lesions by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another variation of the method, administration of the composition to the mammal increases the serum T50 value of the mammal by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the serum T50 value of the mammal before administration of the composition. In another variation of the method, administration of the composition increases the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in the plasma of the mammal after administration of the composition, which is higher than before administration of the composition.

[0025] In one embodiment, a method is provided for treating, preventing, slowing the progression of, halting, and / or reversing tissue calcification or calciphylaxis in a mammal (or subject), the method comprising administering at least 0.1 mg per day of the composition described above to the mammal to prevent, slow the progression of, and / or halt tissue calcification, including soft tissue calcification, wherein the composition is administered in a pharmaceutical composition. In another embodiment, a method for treating, preventing, slowing, halting, and / or reversing tissue calcification in a prediabetic mammal (or subject) having diabetes, chronic kidney disease, or a combination thereof and in need of treating, preventing, slowing, halting, and / or reversing tissue calcification is provided, the method comprising administering to the mammal at least 0.01 mg or at least 0.1 mg per day of any one of the compositions described above to prevent, slow, and / or halt tissue calcification, wherein the composition is administered in a pharmaceutical composition. In one variation of this method, the mammal has diabetes. In another variation, the mammal has type II diabetes. In another variation, the mammal has been diagnosed as prediabetic. In one aspect, the mammal has chronic kidney disease. In one variation of the above method, the mammal has stage 4 or stage 5 chronic kidney disease / end-stage renal disease. In another variation of this method, the mammal is undergoing hemodialysis. In another variation, the mammal is receiving an anticoagulant therapy that is not warfarin-based. In another variation, the anticoagulant therapy is oral anticoagulant therapy. In another variation, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, elibaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0026] In another variation, there is provided a method for treating, preventing, slowing, stopping, and / or reversing tissue calcification in a mammal undergoing hemodialysis and in need thereof, the method comprising administering at least 0.01 mg or at least 0.1 mg of a composition to the mammal per day, thereby preventing, slowing, stopping, and / or reversing tissue calcification, the composition being administered in a pharmaceutical formulation. In one variation of this method, the mammal has diabetes. In another aspect, the present application discloses a fortified food or beverage formulation comprising adding to food or beverage a composition comprising any one of the compositions disclosed herein.

[0027] In another aspect of the method, the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory disease. In another embodiment, a method is provided for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, a proliferative disease, and a cardiovascular disease, comprising administering to the mammal a therapeutically effective amount of any of the above-described compositions. In one aspect, the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervical cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer, and head and neck cancer.

[0028] In another embodiment, there is provided a method for treating, preventing, slowing, halting, and / or reversing Alzheimer's disease (AD) in a mammal or subject in need thereof, comprising administering to the mammal or subject at least 0.1 mg per day of any one of the compositions described above to prevent, slow and / or halt, or reverse Alzheimer's disease.

[0029] In another aspect, the present application discloses a pharmaceutical composition comprising a therapeutically effective amount of the menaquinone disclosed above and a pharmaceutically acceptable excipient, wherein the pharmaceutical composition is effective for treating a vitamin K-related condition selected from the treatment of osteoporosis and atherosclerosis.

[0030] In another embodiment of the above method, the anticoagulant therapy is oral anticoagulant therapy. In another embodiment, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, elibaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban. In another embodiment, the mammal has chronic obstructive pulmonary disease (COPD). In another embodiment, the mammal has calciphylaxis-associated skin lesions. In another embodiment of the method, administration of the composition reduces the total surface area of ​​skin lesions by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In another embodiment of the method, administering a composition disclosed herein to a mammal increases the serum T50 value of the mammal by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the serum T50 value of the mammal before administration of the disclosed composition. In another embodiment, administration of the disclosed composition increases the ratio of carboxylated to uncarboxylated forms of vitamin K-dependent proteins in the plasma of the mammal after administration of the composition, which is higher than before administration of the composition. In one embodiment of the method, the increase in the ratio of carboxylated to uncarboxylated vitamin K-dependent proteins in the plasma of the mammal after administration of the composition is 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.

[0031] In certain embodiments of the above, administration of the disclosed compositions reduces the amount of uncarboxylated vitamin K-dependent protein in the subject's plasma, e.g., by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, compared to the amount prior to administration of the composition. In certain variations, the vitamin K-dependent protein is selected from matrix Gla protein (MGP), growth arrest-specific gene 6 (Gas-6) protein, PIVKA-II protein, osteocalcin, activated protein C, activated protein S, factor II, factor VII, factor IX, and factor X.

[0032] In certain variations of the above methods, administration of the composition increases the plasma level of osteoprotegerin or fetuin-A by, for example, 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 before administration of the composition. In other variations, administration of the composition reduces plasma levels of D-dimer or high sensitivity C-reactive peptide (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 D-dimer or high sensitivity C-reactive peptide (hs-CRP) before administration of the composition.

[0033] In certain variations of the above methods, the method may comprise administering to the subject about 0.1 mg to about 200 mg of the composition per day. In other variations, the method may comprise administering to the subject about 0.1 mg to about 150 mg of the composition per day. In other variations, the method may comprise administering to the subject about 0.1 mg to about 100 mg of the composition per day. In other variations, the method may comprise administering to the subject about 2 mg to about 200 mg of the composition per day. In certain variations, the method may comprise administering to the subject about 2 mg to about 250 mg of the composition per day. In other variations, the method may comprise administering to the subject about 2 mg to about 250 mg of the composition per day. In other variations, the method may comprise administering to the subject about 2 mg to about 100 mg of the composition per day. In other variations, the method may include administering to the subject about 3 mg to about 100 mg of the composition per day. In other variations, the method may include administering to the subject about 0.5 mg to about 75 mg of the composition per day, for example, administering to the subject 0.1 mg, 1 mg, 2 mg, 3 mg, or 10 mg of the composition per day.

[0034] In certain variations, the composition is administered to the subject for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely, as needed. If the subject is undergoing hemodialysis, the composition may be administered to the subject for a period that includes at least the duration of the hemodialysis.

[0035] In another variation of the method for treating calciphylaxis, after administration of the disclosed compositions, in addition to measuring the change / reduction in lesion size before and after drug administration, biopsies of relevant lesions can be taken using von Kossa staining to determine tissue levels of PTH, and evidence of changes in calcium and phosphate deposition in cutaneous arterioles.

[0036] As disclosed herein, the presence of uremic oxidative blockade can be detected, for example, by detecting an increase in F2 isoprostanes, resulting in an increase in plasma lipid peroxidation (Morrow et al. (1990) A series of prostaglandin F2-like compounds are produced in vivo by humans by a non-cyclooxygenase, free radical-catalyzed mechanism, PROC. NATL. ACAD. SCI. USA 87:9383-9387), an increase in isolevuglandin-plasma protein adducts (Salomon et al. (2000) Isolevuglandin-protein adducts in humans: Products of free radical induced lipid oxidation through the isoprostane pathway, BIOCHIM BIOPHYS ACTA 1485:225-235), an increase in exhaled ethane (Handelman et al. (2000) J AM. SOC. NEPHROL. 11:271A); for example, increased oxidation of proteins and amino acids by detecting oxidation of tyrosine residues (Heinecke et al. (1999) Detecting oxidative modification of biomolecules with isotope dilution mass spectrometry: Sensitive and quantitative assays for oxidized amino acids in proteins and tissues, METHODS ENZYMOL. 300:124-144), oxidation of cysteine ​​or methionine residues in plasma proteins, lysine oxidation and threonine oxidation, thiol oxidation and carbonyl formation (Himmelfarb et al. (2000) Plasma protein thiol oxidation and carbonyl formation in chronic renal failure, KIDNEY INT.58:2571-2578); reactive aldehyde formation, e.g., by detecting glyoxal, methylglyoxal, acrolein, glycoaldehyde, and parahydroxyphenylacetaldehyde (Miyata et al. (1999) Alterations in nonenzymatic biochemistry in uremia: Origin and significance of 'carbonyl stress' in long-term uremic complications. KIDNEY INT. 55:389-399); increased reactive carbonyl compounds, e.g., by measuring hydrazine formation after reaction with 2,4-dinitrophenylhydrazine; decreased plasma glutathione levels and glutathione peroxidase function (Ceballos-Picot et al. (1996) Glutathione antioxidant system as a marker of oxidative stress in chronic renal failure, FREE RADIC. BIOL. MED. 21:845-853); and an elevated ratio of oxidized to reduced thiols (Hultberg et al. (1995) Reduced, free, and total fractions of homocysteine ​​and other thiol compounds in plasma from patients with renal failure, NEPHRON 70:62-67; Himmelfarb et al. (2002) Plasma aminothiol oxidation in chronic renal failure, KIDNEY INT 61:705-716; Ward et al. Polymorphonuclear leukocyte oxidative burst is enhanced in patients with chronic renal insufficiency, J AM. SOC. NEPHROL. 5:1697-1702).

[0037] In another embodiment, a method for treating, preventing, slowing, halting, and / or reversing tissue calcification in a prediabetic mammal (or subject) having diabetes, chronic kidney disease, or a combination thereof and in need of treating, preventing, slowing, halting, and / or reversing tissue calcification is provided, the method comprising administering at least 0.01 mg of a disclosed composition to the mammal to prevent, slow, and / or halt tissue calcification, wherein the composition is administered in a pharmaceutical composition. In another aspect of this method, the mammal has diabetes. In yet another aspect, the mammal has type II diabetes, or the mammal has been diagnosed as prediabetic. In another aspect, the mammal has chronic kidney disease. In another aspect of the above method, the mammal has stage 4 or stage 5 chronic kidney disease / end-stage renal disease. In yet another aspect, the mammal is undergoing hemodialysis. In another aspect, the mammal is undergoing non-warfarin-based anticoagulant therapy. In another embodiment, the anticoagulant therapy is oral anticoagulant therapy. In another embodiment of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, elibaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0038] In another embodiment, there is provided a method for treating, preventing, slowing, halting, and / or reversing tissue calcification in a mammal undergoing hemodialysis and in need thereof, the method comprising administering to the mammal at least 0.01 mg or 0.1 mg per day of a substantially pure composition disclosed herein, thereby preventing, slowing, halting, and / or reversing tissue calcification, wherein the disclosed composition is administered in a pharmaceutical composition. In another aspect, the mammal has diabetes.

[0039] Vitamin K Metabolism: Development of vascular and soft tissue calcification after failure to regenerate reduced forms of vitamin K: Vitamin K is an essential enzyme cofactor required for the post-translational modification of vitamin K-dependent (VKD) proteins. Although several VKD proteins exist, many are clinically relevant in ESRD patients. These include core coagulation factors such as factors II, VII, IX, and X, as well as extracellular matrix proteins including matrix GLA-1 and osteocalcin. Under normal conditions, vitamin K is reduced to vitamin K hydroquinone (KH2) by the enzyme NADPH oxidase. Only the reduced form of vitamin K can function as a cofactor for gamma-glutamate carboxylase (GGCX), which catalyzes the carboxylation of vitamin K-dependent proteins. Warfarin blocks the production of vitamin K hydroquinone by acting as a reductive sink. Enzymatic carboxylation of the glutamic acid residue further oxidizes vitamin KH2 to the 2-3 epoxide vitamin K (Figure 2). The final step of the vitamin K cycle requires enzymatic oxidation of the vitamin K2-3 epoxide back to its native structure. This step is catalyzed by vitamin K oxidative reductase (VKOR), a component of the vitamin K cycle that is also blocked by the oxidative action of warfarin. The observation that warfarin blocks both the production of vitamin K hydroxyquinone (KH2) and the regeneration of vitamin K2 2-3 epoxide helps explain why the incidence of calciphylaxis and other forms of dystrophic calcification is higher among patients receiving warfarin therapy.

[0040] In one variation, supplementation with the disclosed composition reduces the risk of vascular and soft tissue calcification by increasing the formation of primary calciprotein particles (CPPs) composed of fetuin A and carboxylated matrix GLA-1 protein. Under normal physiological conditions, plasma calcium and phosphate concentrations are near supersaturation, and therefore, precipitation as crystalline hydroxyapatite is expected in blood vessels and soft tissues. The observation that this process does not occur suggests the existence of powerful chemical and biological means to block pathological calcification. Recent studies have shown that circulating calcium phosphate crystals form complexes with two calcification-inhibitory proteins to form primary calciprotein particles (CPPs). These protein-mineral complexes are primarily composed of fetuin A, a liver-derived protein shown to prevent vascular calcification. The second, less abundant protein is matrix GLA-1 protein, which also functions to prevent pathological calcification. Matrix Gla-1 is a vitamin K-dependent protein, and early studies have shown that the formation of fetuin-matrix Gla-1 inorganic nanoparticles (primary calciprotein CPPs) depends on gamma-carboxylation of matrix Gla-1. Preclinical studies suggest that the calciprotein system functions as an alternative means of preventing pathological calcification when humoral defenses such as pyrophosphate, magnesium, and albumin are breached. The "absorption" of calcium-phosphate crystals by primary CCPs occurs in a coordinated and time-dependent process.

[0041] Time to 50% saturation of the primary CCP (T 50 ) is an accurate and sensitive means of determining the ability of plasma to "precipitate" or "absorb" excess calcium phosphate crystals. 50 Patients with long T suggest a decreased ability to absorb calcium phosphate crystals. 50 Patients with more time are more likely to have high performance. 50 The test is validated and has a low T50 It has been confirmed that time is associated with the increase of myocardial infarction, heart failure and all-cause mortality.Therefore, any clinical intervention that can increase the synthesis of circulating primary CPP will improve the ability to prevent pathological calcification.It is noted that patients with CKD and ESRD show a decrease in the level of carboxylated matrix Gla-1 protein, so this process is essential for the formation of primary CPP.Therefore, supplementation or administration of the disclosed composition and composition in patients with CKD or ESRD reduces the risk of pathological calcification and prevents the occurrence of calcification of blood vessels and soft tissues.

[0042] Supplementation or administration of the disclosed compositions can prevent or slow the development of soft tissue and vascular calcification in skin tissue by restoring the production of carboxylated matrix Gla-1 and GAS-6.

[0043] Vitamin K regeneration involves two key enzymes: vitamin K 2-3 epoxide oxidative reductase (VKOR) and NAD(P)H:quinone oxidoreductase (NQO1). As shown in the diagram, VKOR reduces 2-3 vitamin K epoxide to vitamin K quinone, while NADPH reduces vitamin K quinone to its hydroxyquinone form (KH2). Recent studies have demonstrated that VKOR has two distinct isoforms (VKORC-1 and VKORC1-like-1 [VKORC1-L1]) that differ in both enzymatic properties and tissue distribution. For example, Westofen et al. demonstrated that VKORC-L1 has one-third the affinity for 2-3 epoxide vitamin K compared with VKORC1. Subsequent studies supported the hypothesis that VKOR-L1 is a specialized isoform that protects against oxidant injury through vitamin K regeneration. Incubation of cultured HEK293T cells with H2O2 increased VKOR-L1 expression and reduced evidence of membrane oxidant damage. The clinical observation that calciphylaxis and vitamin K-dependent vascular calcification are more prevalent in the dermis raises the question of whether there is differential expression of VKOR enzymes in the skin. To address this question, Casper et al. determined that mRNA expression of key enzymes involved in vitamin K regeneration. As shown in Figures 3 and 4, skin exhibited the lowest levels of VKOR-C1 compared with any other tissue. Furthermore, NADPH expression in the dermis was below detection levels. These observations suggest that any condition or procedure that blocks vitamin K regeneration (i.e., hemodialysis) predisposes that tissue to pathological calcification.

[0044] The oxidative properties of uremic plasma and the oxidative effects of dialysis itself result in a "metabolic blockade" and accumulation of 2-3 epoxide vitamin K and reduced intracellular levels of vitamin K2. The "downstream" effects of this blockade include the inability of gamma-carboxylate key proteins to participate in the prevention of soft tissue and vascular calcification. The oxidative effects of hemodialysis exacerbate this effect, which may partially explain the propensity of ESRD patients to develop calciphylaxis and vascular calcification.

[0045] Relationship between vitamin K and circulating vitamin K-dependent proteins in CKD-ESRD patients: It is widely recognized that vitamin K levels may not be reduced among ESRD patients despite dietary deficiency. For example, Holder et al. studied 172 stable dialysis patients and found that only 6% of patients exhibited clinically significant vitamin K deficiency. However, when the patients' carboxylated osteocalcin levels were tested, up to 60% had reduced levels. To confirm that this was a general effect of reduced vitamin K activity, the authors also measured another vitamin K-dependent protein, PIVKA-II. Indeed, up to 90% of both CKD and ESRD patients were found to have reduced levels of carboxylated prothrombin. In a similar study, Pilkey et al. measured vitamin K1 levels in 142 ESRD patients and found that while the majority of patients had adequate vitamin K reserves, 93% had uncarboxylated osteocalcin levels exceeding 20% ​​of the total. Note that there was no correlation between total vitamin K1 and circulating levels of uncarboxylated osteocalcin. This unexpected finding is consistent with the hypothesis that in uremic patients, total vitamin K levels may be normal, but production of the reduced form is blocked by the oxidative properties of uremia.

[0046] In one variation, supplementation or administration of the disclosed compositions reverses hemodialysis-induced inhibition of vitamin K-dependent proteins by normalizing the functional reduced form of vitamin K. The observation that oxidant conditions can disrupt the vitamin K cycle suggests that the oxidant load generated during hemodialysis may contribute to the high rates of vascular and soft tissue calcification observed in ESRD populations. Studies by Himmelfarb et al. and others have confirmed that the simple delivery of hemodialysis can result in the oxidation of some tissue proteins. For example, hydroxyl amino acid side chains are oxidized to carbonyl groups. In a study of CKD and ESRD patients, Himmelfarb et al. demonstrated the use of carbonyl side chain oxidation as a measure of overall oxidant load, and demonstrated that both CKD and ESRD patients exhibited a higher percentage (15-fold) of carbonyl proteins compared to normal controls (see Figure 5). The proportion of carbonylated proteins was even higher among patients undergoing dialysis, demonstrating that dialysis not only reduces oxidant load but also appears to contribute to it. As shown in Figure 5, patients with uremia were found to have up to 15-fold higher levels of carbonylated proteins. Thus, the oxidative load generated by hemodialysis delivery results in the oxidation of functional vitamin K hydroquinone (KH2) to non-functional natural vitamin K. The oxidation of KH2 by hemodialysis blocks its ability to function as a cofactor for GGCX, which downstream results in decreased gamma-carboxylation of vitamin K-dependent proteins.

[0047] To confirm that uremia and hemodialysis disrupt the vitamin K cycle, we can determine the ratios of vitamin K quinone to 2-3 epoxide vitamin K and vitamin K hydroxyquinone (KH2) in patients with normal renal function, CKD patients (stages IV and V), and ESRD patients. To determine whether the very process of hemodialysis further disrupts the vitamin K cycle, we can measure the levels of oxidized vitamin K immediately before hemodialysis, then midway through (2 hours), and 30 minutes after dialysis. Previous studies examining the interaction between warfarin and vitamin K metabolism have shown that 2-3 epoxide vitamin K is easily measured. Patients with CKD and ESRD have higher levels of 2-3 epoxide vitamin K and lower levels of vitamin K hydroxyquinone (KH2) compared with controls. To determine whether loss of the reduced form of vitamin K (KH2) leads to decreased carboxylation of vitamin K-dependent proteins, we measured the levels of the following biomarkers in control, CKD (stages IV and V), and ESRD (pre- and post-hemodialysis): matrix GLA-1 protein; growth arrest-specific gene 6 (Gas-6) protein; PIVAK-II protein; osteocalcin; protein C; protein S; fetuin A; and osteoprotegerin (dialysis plasma level: 6.7 ± 2.2 pmol / L). We expanded these studies by including patients undergoing stable 3x / week hemodialysis. Pre-dialysis serum levels of carboxylated and uncarboxylated vitamin K-dependent proteins can be compared with those obtained 2 hours and at the end of the dialysis session. The oxidative effect of dialysis itself leads to decreased levels of carboxylated vitamin K-dependent proteins.

[0048] In one variation, supplementation with the disclosed compositions in ESRD patients with uremic arteriolar calcification (calciphylaxis) reduces wound healing time by preventing new blood vessel calcification and restoring blood flow: Skin biopsy: To confirm that supplementation with the disclosed compositions prevents the development of small vessel calcification and cutaneous ischemia, the inventors can identify patients with calciphylaxis confirmed by dermal biopsy and randomize them to treatment with menaquinone-7 or placebo. Clinical endpoints can include the following: 1) time to weaning of the wound from vacuum therapy, and 2) time to wound healing, defined as the time required for a 50% reduction in the sum of the surface area of ​​all calciphylaxis wounds.

[0049] Histopathological endpoints were compared between diagnostic skin biopsies after 12 weeks of menaquinone-7 treatment and skin biopsies at protocol repeats. Changes in interstitial calcium deposition levels were defined as changes in von Kossa staining, which can be quantified by digital image color analysis. Skin biopsies can be used to validate biomarkers at the tissue level, thereby confirming the preventive properties of MK-7 against early vascular calcification. Validation of these biomarkers in tissue also allows clinicians to utilize biomarkers as a means of tracking clinical response. Microvascular calcification precedes the development of CUA lesions. Calcification levels were quantified by von Kossa calcium staining in peripheral tissues and normalized as calcium content per unit area. We may use von Kossa as a means of confirming the preventive properties of MK-7 against the development of vascular calcification.

[0050] In one variation, in ESRD patients with uremic arteriolar calciphylaxis, supplementation with the disclosed composition reduces wound healing time by normalizing carboxyprotein C levels in the dermis and preventing primary thrombosis of dermal blood vessels. Thus, in one variation, supplementation or administration of the disclosed composition in diabetic patients prevents the onset of vascular dementia by preventing the onset of calcification and small vessel disease.

[0051] Treatment of Alzheimer's disease, apoptosis and cancer: Alzheimer's disease (AD) is a devastating neurodegenerative disorder. Its sporadic form affects the elderly population (with a significant increase in incidence in those >75 years of age), and various familial forms exist, with disease onset in the fourth or fifth decade of life. AD is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary tangles in the brains of patients. The core component of senile plaques is a small 4-kDa amyloid peptide, which is generated by proteolytic processing of the large transmembrane protein amyloid precursor protein (APP). Cleavage of APP by beta-secretase (BACE-1) releases a soluble APP-beta fragment, while the 99-amino acid C-terminus remains membrane-tethered. This C-terminal fragment is subsequently proteolytically processed by gamma-secretase (a membrane multienzyme complex) to produce amyloid peptides of various lengths, primarily 40 and 42 amino acids in length (Hardy J. et al. (2002) Science; 297 (5580):353-356). In one embodiment, the compositions disclosed herein are used to treat diseases or conditions such as Alzheimer's disease, mild cognitive impairment, impaired glucose tolerance, or type 2 diabetes, which are mediated by BACE-1, BACE-2, or cathepsin D activity. In one variation, the disclosed methods may be used to treat calcification in the brain.

[0052] Beta-amyloid (Aβ) has been shown to cause neuronal death by promoting apoptosis and also through direct toxicity. Hadipour, E. et al. Vitamin K2 Protects PC12 Cells Against Aβ (1-42) and H2O2-Induced Apoptosis via P38 MAP Kinase Pathway. Nutr. Neurosci. 2020, 23, 343-352. Neurotoxicity occurs through various mechanisms, including disruption of calcium homeostasis, oxidative stress, and mitochondrial dysfunction. In rat pheochromocytoma-derived PC12 cells, vitamin K2 prevented neuronal death caused by Aβ(1-42), the most neurotoxic form of Aβ. Experiments also demonstrated that when cells were exposed to either hydrogen peroxide (H2O2) or Aβ(1-42), cells pretreated with vitamin K2 showed less pronounced apoptosis. Pretreatment with vitamin K2 also reduced the levels of apoptosis signaling proteins, including a lower Bax / Bcl-2 ratio, reduced the presence of reactive oxygen species (ROS), and increased the levels of the antioxidant glutathione. Yagami, T. Gas6 Rescues Cortical Neurons from Amyloid β Protein-Induced Apoptosis Neuropharmacology 2002, 43, 1289-1296. Researchers have determined that inactivation of the p38 MAP kinase pathway is a mechanism for the potential preventive role of vitamin K2 in Alzheimer's disease (AD).

[0053] The researchers observed that increasing concentrations of vitamin K2 (VK2) prolonged cell survival, likely due to protection from Aβ-induced neuronal death. This effect was reversible upon the addition of warfarin, which also blocked vitamin K-dependent carboxylation. VK2 was observed to dose-dependently reduce the number of reactive oxygen species (ROS) and, at a concentration of 10 mcmol / L, reduce the activity of caspase-3, an enzyme that mediates Aβ-induced apoptosis, by 2.5-fold. The authors also found that Gas6 plays a role in VK2 protection from Aβ cytotoxicity, supported by measurements of Ca(2+) influx, chromatin condensation, and DNA fragmentation as markers of Aβ neurotoxicity and apoptosis in fetal rat neuronal cell cultures. Gas6 dose-dependently inhibited Ca(2+) influx and significantly reduced the amount of chromatin condensation and DNA fragmentation caused by Aβ. Thus, a clear correlation exists between the antioxidant and anti-apoptotic properties of vitamin K2, which relate to the process of programmed cell death as a relevant effective mechanism in the treatment of cancer.

[0054] Targeting apoptosis is also effective in various types of cancer, as apoptosis evasion is a hallmark of cancer. Apoptosis is also non-specific to the cause or type of cancer. Villa-Pulgarin JA et al. Mitochondria and lipid raft-located F oSee F1-ATP synthase as a major therapeutic target in the anti-inflammatory and anticancer activities of the ether lipid edelfosine. PLoS Negl. Trop. Dis. 2017;11:e0005805. doi: 10.1371 / journal.pntd.0005805; Elmore S. Apoptosis: A review of programmed cell death. Toxicol. Pathol. 2007;35:495-516. doi: 10.1080 / 01926230701320337.

[0055] Vitamin K2 has also been used in clinical applications to supplement cancer treatment. XV, F. et al., Research Progress on the anticancer effects of vitamin K2 (Review). Oncol. Lett. 2018, 15, 8926-8934. Furthermore, vitamin K2 supplementation has been found to inhibit the growth and metastasis of multiple cancer types.See Xia, J. et al., The role of PKC isoforms in the inhibition of NF-κB activation by vitamin K2 in human hepatocellular carcinoma cells. J. Nutr. Biochem. 2012, 23, 1668-1675; Showalter, S.L. et al., Naturally occurring K vitamins inhibit pancreatic cancer cell survival through a caspase-dependent pathway. J. Gastroenterol. Hepatol. 2010, 25, 738-744. Enomoto, M. et al. Vitamin K2-induced cell growth inhibition via autophagy formation in cholangiocellular carcinoma cell lines. Int. J. Mol. Med. 2007, 20, 801-808; Jinghe, X., Vitamin K and hepatocellular carcinoma: The basic and clinic. World J. Clin. Cases 2015, 3, 757-764. Sada, E. et al. Vitamin K2 modulates differentiation and apoptosis of both myeloid and erythroid lineages. Eur. J. Haematol. 2010, 85, 538-548; Yaguchi, M. et al., Vitamin K2 and its derivatives induce apoptosis in leukemia cells and enhance the effect of all-trans retinoic acid. Leukemia 1997, 11, 779-787.

[0056] In fact, intake of MK-4 and MK-5 to MK-9 has been determined to be inversely correlated with cancer mortality. In particular, in a cohort study of men, the incidence of advanced prostate cancer was inversely correlated with intake of MK, particularly MK-5 to MK-9. Nimptsch, K. et al. (2008) Dietary intake of vitamin K and risk of prostate cancer in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg). Am. J. Clin. Nutr. 87, 985-992. Nimptsch, K. et al. (2010) Dietary vitamin K intake in relation to cancer incidence and mortality: results from the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition (EPIC-Heidelberg). Am. J. Clin. Nutr. 91, 1348-1358. In a nested case-control follow-up study, osteocalcin undercarboxylation (a marker of inadequate vitamin K status) was significantly higher in cases of advanced or high-grade prostate cancer compared with controls. Nimptsch, K. et al. (2009) Serum undercarboxylated osteocalcin as biomarker of vitamin K intake and risk of prostate cancer: a nested case-control study in the Heidelberg cohort of the European Prospective Investigation into Cancer and Nutrition. Cancer Epidemiol. Biomark. Prev. 18, 49-56.Wang et al. also reported a positive correlation between MK intake and the risk of breast cancer incidence and death, particularly in the case of luminal-like triple-negative disease and early-stage disease. Wang, K. et al. (2021) Vitamin K intake and breast cancer incidence and death: results from a prospective cohort study. Clin. Nutr. 40, 3370-3378. Thus, in one variation, a method is provided for treating cancer, such as prostate cancer, by administering a therapeutically effective amount of the disclosed composition to control or reduce high levels of osteocalcin undercarboxylation.

[0057] The pharmaceutical compositions disclosed herein may be formulated as liquids or lyophilized powders for parenteral administration. Powders can be reconstituted by adding a suitable diluent or other pharmaceutically acceptable carrier before use. Liquid formulations are generally buffered, isotonic aqueous solutions. Examples of suitable diluents include normal isotonic saline solution, 5% dextrose in water, or buffered sodium or ammonium acetate solutions. While such formulations are particularly suitable for parenteral administration, they can also be used for oral administration. Additives such as polyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride, or sodium citrate may also be added. Alternatively, these compositions may be encapsulated, tableted, or prepared in emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers can be added to enhance or stabilize the compositions or to facilitate their preparation. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, or water. Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. The carrier may also include a sustained-release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but can be from about 20 mg to about 1 g per dosage unit. Pharmaceutical preparations are made according to conventional techniques of pharmacy, including milling, mixing, granulating, and compressing, if necessary, into tablet form, or milling, mixing, and filling into hard gelatin capsule form. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid formulations may be administered directly in a po or filled into soft gelatin capsules.Suitable formulations for each of these methods of administration can be found, for example, in Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0058] As disclosed herein, the disclosed compositions may contain a variety of ingredients, including oleic acid, Kolliphor® EL (polyoxyl castor oil or 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 fatty acid and glycerin derived from saturated coconut and palm kernel oils), Plurol® Oleique (polyglyceryl-6 dioleate), Lauroglycol™ 90 (propylene glycol monolaurate (type II)), Labrasol® (caprylocaproyl polyoxyethylene glycol), and PEG-1000 (caprylic / capric triglyceride). The composition may also include a solubility enhancer or solubilizer (or emulsifier) ​​selected from: glycerol / glyceryl monooleate (type 40), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride), Kolliphor® EL (polyoxyl castor oil), Captisol® (SBE-beta-cyclodextrin), Encapsin™ HPB (hydroxypropyl beta-cyclodextrin), Peceol™ (glycerol / glyceryl monooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride), and medium chain mono- and diglycerides.

[0059] In addition to the exemplary embodiments, aspects and variations described above, further embodiments, aspects and variations will become apparent by reference to the drawings and figures (FIG) and by study of the following descriptions. DETAILED DESCRIPTION OF THE INVENTION

[0060] Detailed Description of the Invention Definition: Unless specifically stated otherwise herein, the definitions of terms used are standard definitions used in the fields of organic synthesis and pharmaceutical science. Exemplary embodiments, aspects, and variations are illustrated in the figures and drawings, and it is intended that the embodiments, aspects, and variations disclosed herein, and the figures and drawings, be considered illustrative and not limiting.

[0061] As used herein, the term "composition" is used interchangeably with "formulation" and refers to the nanoparticles described herein.

[0062] As used herein, the term "emulsion" generally refers to a mixture of two normally immiscible liquids, one colloidally suspended in the other (defining the dispersed phase). The particle size of the dispersed phase in an emulsion is generally between hundreds of nanometers and tens of micrometers. If the micelles are sufficiently small, the emulsion is essentially transparent. Typically, if the micelles have a median particle size of less than 100 nm, the emulsion appears transparent to the human eye. In one example, the micelles in the emulsions of the present application have a median particle size of less than 60 nm. In typical examples, the micelles formed in the emulsion have a median particle size of between about 30-40 nm, 20-30 nm, 15-20 nm, or about 10-15 nm. In another example, the emulsion is stable, meaning that essentially no separation between the aqueous phase and the lipophilic components occurs (e.g., the emulsion remains clear). A typical aqueous medium used in the emulsions of the present application is water, which may optionally contain other solubilizing agents, such as pharmaceutically acceptable excipients.

[0063] As used herein, the term "emulsifier" is used interchangeably with the terms "surfactant" and "solubilizer," as defined herein.

[0064] As used herein, the term "nanoparticle" refers to a particle having at least one dimension that is less than 1,000 nm, as determined, for example, by dynamic light scattering particle size measurement. Other methods known in the art include disk centrifugation, nanoparticle tracking analysis, tunable resistive pulse sensing, or electron microscopy.

[0065] The term "micelle" is used herein in accordance with its art-recognized meaning and includes all forms of micelles, including, for example, spherical micelles, cylindrical micelles, worm-like micelles, and sheet-like micelles, and vesicles, that are formed in water or mostly in water.

[0066] "Pharmaceutically acceptable salt" refers to a salt composition that is generally considered to have the desired pharmacological activity, is considered safe and non-toxic, and is acceptable for veterinary and human pharmaceutical applications. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like, or organic acids such as acetic acid, propionic acid, hexanoic acid, malonic acid, succinic acid, malic acid, citric acid, gluconic acid, salicylic acid, and the like.

[0067] Generally, as used herein, a "poorly soluble drug" refers to a drug that has a solubility of 100 mg / mL or less in water. An alternative definition can include drugs that have a solubility of less than 1 mg / mL over the physiological pH range.

[0068] A "stable" formulation or composition is one in which vitamin K2 essentially retains at least one of its physical stability, chemical stability, and biological activity for at least 6 months or at least 12 months upon storage at room temperature. Various analytical techniques for measuring the stability of drug particles are available in the art, such as those set forth in Jones, A. et al. Adv. Drug Delivery Rev. 10: 29-90 (1993). In one aspect, stability can be measured for a selected period of time at a selected temperature.

[0069] As used herein, the term "suspension" generally refers to a dispersion of fine particles in a liquid.

[0070] "Therapeutically effective amount" means an amount of a compound or composition that exerts any of the biological effects listed herein. [Brief explanation of the drawings]

[0071] [Figure 1] FIG. 1 is a representation of the chromatogram of menaquinone-7 and its positional isomers, shown in a 3:1 ratio, as determined by 1H NMR.

[0072] [Figure 2] FIG. 2 is a scheme showing the oxidation of KH2 functional carboxylation of vitamin K-dependent proteins induced by uremia and dialysis.

[0073] [Figure 3] FIG. 3 is a graph showing VKORC1 in arbitrary units and in specific tissues.

[0074] [Figure 4] FIG. 4 is a graph showing NADPH in arbitrary units and in specific tissues.

[0075] [Figure 5]FIG. 5 is a graph showing that CKD and ESRD patients exhibit a higher proportion of carbonyl proteins compared to normal controls. [Figure 6] Not specified. [Figure 7] Not specified. [Figure 8] Not specified. [Figure 9] Not specified. [Figure 10] Not specified. [Example]

[0076] experiment: 1. Dissolution test of MK-7 nanosuspension in fed-state simulated intestinal fluid (FeSSIF) material MK-7 - Anthem Bioscience Lot A012220081 MK-7 Nanosuspension - F-10 Lot LPI461-1-10 Sodium deoxycholate - SAFC Lot SLBT2846 Deionized Water - Barnstead E-Pure Filtration System Zirconium Milling Beads, 0.4mm - Netzsch Lot #1310508 FeSSIF, pH 5 - prepared by LPI Polypropylene tube, 1.5 mL, with screw cap; supplied by General Lab Device Bead impact homogenizer (bench scale) Vortex mixer (bench scale) ·Chemical balance HPLC with variable wavelength UV detector - Agilent 1100 series Dynamic Light Scattering Particle Size Analyzer - Malvern Zetasizer ·Melting equipment – ​​Wankel7000

[0077] MK-7 (menaquinone-7, API) was suspended in an aqueous vehicle containing the wetting agent sodium deoxycholate. The API concentration was 4%, and the wetting agent concentration was 0.4% w / w. The API particles were milled to a particle size of 155 nm using zirconium milling beads and high-shear bead impactor mixing. Dissolution of the nanosuspension was tested in triplicate at a concentration of 0.01 mg / mL in fed-state simulated intestinal fluid (FeSSIF) at pH 5. These results were compared to the dissolution of the API "as is" (MK-7 - Anthem Bioscience), tested under identical conditions. [Table 1]

[0078] procedure Part 1. Nanosuspension preparation process 1. Set up the equipment and materials in the room by irradiating with low-wavelength yellow light. 2. Add 0.040 g of MK-7 and 0.005 g of sodium deoxycholate to a 1.5 mL polypropylene tube with a screw cap. 3. Drain to 1 g with deionized water. 4. Vortex for approximately 1 minute to completely dissolve the sodium deoxycholate and uniformly suspend the MK-7. 5. Add 0.4 g of 0.4 mm zirconium beads to the suspension. 6. Cover the suspension tube with aluminum foil to protect the MK-7 from light. 7. Place the tube in an ice bath. 8. Set up a bead impactor mixer in a -20°C freezer. 9. Mix the suspension using the bead impactor for 12 5-minute intervals in a -20°C freezer. Monitor the temperature of the suspension using a laser thermometer and maintain the suspension at a temperature below 30°C. 10. Measure particle size using dynamic light scattering by diluting 10 μL of suspension in 950 μL of 0.5% sodium deoxycholate vehicle in a clear cuvette. 11. Ensure particle size is less than 300 nm. 12. Measure assay and impurities in triplicate by RP-HPLC using amber glassware. 14. Store nanosuspension at 2-8°C. [Table 2-1] [Table 2-2] result [Table 3-1] FIG. 6 shows an example chromatogram of F-10 nanosuspension, with all impurities detected being less than 0.05% total peak area. [Table 3-2] FIG. 7 shows an example chromatogram of F-10 nanosuspension, with all impurities detected being less than 0.05% total peak area.

[0079] Part 2. In vitro release testing 1. Clean the dissolution system with DI water. Preparation of FeSSIF: 2. Fill three vessels with 500 mL of FeSSIF medium and heat to 37°C. 3. Set the following dissolution conditions:

[0080] [Table 4]

[0081] 4. Vortex the suspension for approximately 1 minute to ensure uniform suspension. Weigh three 0.15g aliquots of the 4% nanosuspension into tared syringes to achieve a target 100% dissolved concentration of 0.01mg / mL. Record the weight. 5. Add the weighed suspension directly to each container. 6. Start the timer. 7. After 5 minutes, manually withdraw 3mL from each dissolution container with a syringe. 8. Attach a 0.2µm filter to the syringe. 9. Transfer the 2mL dissolution sample back to the container. 10. Transfer the final 1mL sample to an amber HPLC vial and crimp the seal. 11. Repeat steps 8-11 at 10, 15, 30, 45, and 60 minutes. 12. After the 60 minute mark, mix each container in a high-shear mixer for 5 minutes. 13. Repeat steps 8-11 and label this sample "infinite." 14. Run the sample on the HPLC method for 10 minutes using the duplicate HPLC standard solution.

[0082] [Table 5] 8A and 8B show the % dissolution profiles of MK-7 API and nanosuspension in FeSSIF.

[0083] The nanomilled particles dissolve at a significantly faster rate and in greater amounts than the "as is" API particles or the MK-7 API.

[0084] 2. Summary of dissolution studies of MK-7 nanosuspension in fed-state simulated intestinal fluid (FeSSIF).

[0085] MK-7 (menaquinone-7, API) was suspended in an aqueous vehicle containing the wetting agent sodium deoxycholate. The API concentration was 4%, and the wetting agent concentration was 0.4% w / w. The API particles were milled to a particle size of 156 nm using zirconium milling beads and high-shear bead impactor mixing. The dissolution of the nanosuspension was tested in triplicate at a concentration of 0.01 mg / mL in FeSSIF at pH 5. These results were compared to the dissolution of the "as is" API tested under the same conditions.

[0086] material MK-7 - Pharmaquinone lot API2203-01; MK-7 nanosuspension - F-10 lot LPI461-1-10; sodium deoxycholate - SAFC lot SLBT2846; deionized water - Barnstead E-Pure filtration system; zirconium milling beads, 0.4 mm - Netzsch lot number 1310508; FeSSIF, pH 5 - prepared by LPI. Composition of MK-7 nanosuspension (F-10) [Table 6]

[0087] procedure Clean the dissolution system with DI water. Fill three vessels with 500 mL of FeSSIF medium and heat to 37°C. Set the following dissolution conditions: [Table 7]

[0088] Vortex the suspension for 1 minute to ensure a uniform suspension. Weigh 3 x 0.15 g of nanosuspension into tared syringes to achieve a target 100% dissolved concentration of 0.01 mg / mL. Record the weight. Add the weighed suspension directly to each container. Start the timer.

[0089] At 5 minutes, manually withdraw 3 mL from each vial with a syringe. Attach a 0.2 μm filter to the syringe. Transfer a 2 mL aliquot back into the vial. Transfer a final 1 mL aliquot to an HPLC vial and crimp the seal. Repeat steps 8-11 at 10, 15, 30, 45, and 60 minutes. After the 60 minute time point, mix each vial in a high shear mixer for 5 minutes. Repeat steps 8-11 and label this sample as "infinite."

[0090] Run the sample on a 10 minute HPLC method using the STD of the replicate HPLC.

[0091] Initial MK-7 nanosuspension data [Table 8] FIG. 9 shows an example chromatogram of F-10 nanosuspension, with all impurities detected being less than 0.05% total peak area.

[0092] % dissolution data of MK-7 API and nanosuspension in FeSSIF. [Table 9] FIG. 10 shows the % dissolution profiles of MK-7 API and nanosuspension in FeSSIF.

[0093] The nanomilled particles dissolve at a significantly faster rate and in greater amounts than the "as is" API particles. The nanomilling process is scaled up, and the suspended nanoparticles are removed from the suspension vehicle, dried, formulated into fast-dissolving tablets, and tested.

[0094] Menaquinols, such as MKH2-7, can be prepared from the corresponding menaquinones, such as MK-7, by reduction of the menaquinone using conventional methods of reduction known in the art, including, for example, by reduction with a metal such as zinc and acetic acid, as described by Marchand et al. "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]

[0095] Thus, MK-7 (1 g, 1.54 mmol) may be dissolved in glacial acetic acid (15 mL), and powdered zinc (0.8 g, 12.3 mmol) may be added. The resulting mixture may be sonicated for 0.5 hours, or the reaction mixture may be heated under reflux until the reaction is complete. The resulting mixture is filtered, and the residue is washed with dichloromethane. The combined filtrate is concentrated in vacuo to yield the pure reduction product. This procedure is carried out under an argon or nitrogen atmosphere. To stabilize the reduced form of MKH2-7, MKH2-7 may be mixed with one or more antioxidants, such as vitamin C, vitamin C esters (e.g., ascorbyl palmitate), and / or vitamin E. The resulting product may be processed under an inert atmosphere, such as nitrogen or argon, or formulated in an inert or oxygen-free medium, such as a capsule or softgel.

[0096] Similarly, MK-4 to MK-14 can be reduced to the corresponding MKH2-4 to MKH2-14 compounds in a similar manner.

[0097] General process for preparing nanoparticulate emulsions of menaquinone and menaquinol in water: NOTE: As used herein, the term "menaquinone and menaquinol" used to describe an aqueous solution of menaquinone and / or menaquinol may include or contain primarily or mostly menaquinone; may include or contain primarily or mostly menaquinol; or may include or contain a mixture of both menaquinone and menaquinol in various ratios, which may contain a higher concentration of menaquinone or a higher concentration of menaquinol.

[0098] A 50 mL glass RBF (or glass pressure reactor, or Fisher or Porter bottle) equipped with a large magnetic stir bar (or overhead stirrer) and a pressure valve and inlet valve was charged with the solvent or solvent mixture under nitrogen gas. The valve was closed so that the contents remained under a nitrogen atmosphere. The RBF was heated in an oil bath at approximately 55-100 °C, depending on the desired concentration of MK-7, emulsifier or emulsifier mixture, and solvent or solvent mixture. Once the solvent had heated to the desired temperature for at least 5 minutes, the emulsifier or co-emulsifier was added to the solvent through the inlet valve, and the inlet valve was closed. The resulting mixture was stirred and allowed to reheat to the desired initial temperature for at least 5 minutes.

[0099] The reaction mixture is reheated to the initial temperature, and the desired amount of antioxidant, if desired, and the desired amount of preservative, if desired, are added to the mixture. The resulting mixture is stirred and heated to the desired initial temperature for at least 5 minutes.

[0100] Once the reaction mixture is stirred and reheated to the initial temperature, add MK-7 powder to the reaction mixture under nitrogen through the inlet valve, close the inlet valve, stir the resulting mixture for at least 5 minutes, and reheat to the desired initial temperature for approximately 5 to 60 minutes, depending on the amount or volume of the mixture in the RBF.

[0101] Depending on the desired heating temperature and the solvent or solvent mixture used, the pressure in the glass RBF at the desired initial temperature may be reached to approximately 1-5 psi. After the desired amount of heating time, the resulting mixture is held at the desired temperature, and a sample of the solution is removed from the RBF via a sampling port to confirm the desired NTU or clarity of the solution. Once the desired NTU value is reached, the resulting stirred mixture is slowly cooled to room temperature over approximately 5-15 minutes, depending on the volume of the reaction mixture. Once the mixture has cooled to room temperature, the resulting clear, slightly yellow-amber solution is stored at room temperature or in a refrigerator at approximately 10-15°C.

[0102] The solvent used may be pure water (distilled or deionized water), a mixture of water and an organic solvent (such as DMSO, methanol, ethanol, isopropanol, methyl isobutyl ketone, cyclodextrin and N,N-dimethylacetamide, or a mixture thereof). In one variation, the organic solvent is DMSO. In another variation, the solvent mixture is water and DMSO.

[0103] Process for preparing emulsion of nanoparticle solids of MK-7 in water: A 100 mL glass RBF equipped with a large magnetic stir bar and a pressure valve and inlet / outlet valves was charged with 22.0 g of water under nitrogen gas. With the valve closed, the contents were heated to a temperature of approximately 80°C and stirred for 5 minutes. Kolliphor RH40 (3.0 g) was added to the solvent through the inlet valve, and the inlet valve was closed. The resulting mixture was stirred for at least 5 minutes until it reached 80°C. Under nitrogen, MK-7 was added to the reaction mixture through the inlet valve, and the inlet valve was closed. The mixture was heated to 80°C, stirred for approximately 5 minutes, and held at that temperature for approximately 10 minutes.

[0104] Visual inspection of the reaction mixture indicates completion of the reaction process, as the solution turns clear and slightly yellow. A 1 mL sample of the solution was withdrawn from the reaction mixture via a sampling port, and DLS (dynamic light scattering) analysis indicated a particle size range of approximately 10-20 nm. The resulting stirred mixture was slowly cooled to room temperature over approximately 5 minutes. The resulting solution was transferred to a stoppered glass container under nitrogen and stored at approximately 15°C. Weekly 0.5 mL samples of the solution indicated that the solution remained clear and the particle size (by DLS) remained virtually unchanged after 1 month at 32°C, although some visible precipitation of approximately 1% was present, as determined by quantitative HPLC.

[0105] Process for preparing nanoparticle emulsion of MK-7 in water and DMSO: To a 100 mL glass RBF equipped with a large magnetic stir bar and a pressure valve and inlet / outlet valves, 11.0 g of water and 11 g of DMSO were added under nitrogen gas. With the valve closed, the contents were heated to a temperature of approximately 80°C and stirred for 5 minutes. Kolliphor RH40 (3.0 g) was added to the solvent through the inlet valve, and the inlet valve was closed. The resulting mixture was stirred for at least 5 minutes until it reached 80°C. Under nitrogen, MK-7 was added to the reaction mixture through the inlet valve, and the inlet valve was closed. The mixture was heated to 80°C, stirred for approximately 5 minutes, and held at that temperature for approximately 10 minutes.

[0106] Visual inspection of the reaction mixture indicates completion of the reaction process, as the solution turns clear and slightly yellow. A 1 mL sample of the solution was withdrawn from the reaction mixture via a sampling port, and DLS (dynamic light scattering) analysis indicates a particle size range of approximately 10-20 nm. The resulting stirred mixture was slowly cooled to room temperature over approximately 5 minutes. The resulting solution was transferred to a stoppered glass container under nitrogen and stored at approximately 15°C. 0.5 mL samples of the solution stored at approximately 32°C, taken monthly over a 3-month period, indicate that the slightly yellow solution remains clear and that the particle size (by DLS) remains virtually unchanged.

[0107] Process for preparing nanoparticle emulsion of MK-9 in water: To a 100 mL glass RBF equipped with a large magnetic stir bar and a pressure valve and inlet / outlet valves, add 22.0 g of water under nitrogen gas. Close the valve and heat the contents to a temperature of approximately 80°C and stir for 5 minutes. Add Kolliphor RH40 (3.0 g) to the solvent through the inlet valve and close the inlet valve. Stir the resulting mixture for at least 5 minutes until it reaches 80°C. Add MK-9 to the reaction mixture through the inlet valve under nitrogen and close the inlet valve. Heat the resulting mixture and stir until it reaches 80°C, approximately 5 minutes, and hold at that temperature for approximately 10 minutes.

[0108] Visual inspection of the reaction mixture indicates completion of the reaction process, as the solution turns clear and slightly yellow. A 1 mL sample of the solution is withdrawn from the reaction mixture via a sampling port, and DLS (dynamic light scattering) analysis indicates a particle size range of approximately 10-20 nm. The resulting stirred mixture is slowly cooled to room temperature over approximately 5 minutes. The resulting solution is transferred to a stoppered glass container under nitrogen and stored at approximately 15°C. Weekly 0.5 mL samples of the solution indicate that the solution remains clear and the particle size (by DLS) remains virtually unchanged after 1 month at 32°C, although some visible precipitation of approximately 1% is present, as determined by quantitative HPLC.

[0109] Preparation and stability of nanoparticles of MK-7 in water prepared by microwave heating: Under a nitrogen atmosphere, 5.00 g of MK-7 was added to a 50 mL conical flask equipped with a pressure stopper, followed by 15 mL of Kolliphor EL. The resulting mixture formed a yellow, cloudy suspension. The conical flask was then stoppered and sealed, and placed in a microwave oven. With the microwave oven set to HIGH, the conical flask was heated to 80°C for 2 minutes, at which point the yellow solution became homogenous and formed a substantially clear yellow solution. The conical flask was removed from the microwave, and under a nitrogen atmosphere, the pressure stopper was slowly removed. While the solution was still warm, 35 mL of distilled water was added at a temperature above 50°C, all under a nitrogen atmosphere. The conical flask was resealed and heated again in the microwave oven to approximately 80°C for an additional 2 minutes, forming a clear, homogenous yellow solution. DLS analysis indicated a particle size distribution of approximately 10-15 nm. HPLC analysis indicated that the MK-7 remained unchanged after 7 days at room temperature.

[0110] Administration of nanoparticle compositions in subjects at risk of developing calciphylaxis: This example describes the administration of compositions of the present application to subjects at risk for developing calciphylaxis but who have not yet developed the characteristic skin lesions of calciphylaxis. Possible risk factors include, but are not limited to, diabetes, obesity, hemodialysis, and prior treatment with warfarin (Nigwekar et al. (2016) A Nationally Representative Study of Calcific Uremic Arteriolopathy Risk Factors, J. AM. SOC. NEPHROL. 27(11):3421-9). Administration of these compositions can result in changes in the levels of certain biomarkers that are indicative of protection from skin lesions and prevention of the onset of calciphylaxis.

[0111] Subjects at risk of developing calciphylaxis orally receive 0.1 mg, 3 mg, 5 mg, or 10 mg of a selected composition of the present application 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 a 0.1 mg, 3 mg, 5 mg, or 10 mg pill or soft gel capsule. Two 25 mg capsules will be administered once daily in 50 mg dose cohorts. It should be noted that not all subjects with high risk factors for calciphylaxis will develop the characteristic skin lesions of calciphylaxis. The intent of active treatment (before clinical diagnosis of calciphylaxis) with the compositions of the present application is to prevent the appearance of lesions. Therefore, a reduction in the frequency or elimination of lesion appearance is contemplated as successful treatment.

[0112] Several biomarkers can be evaluated to determine the efficacy of the compositions administered at three dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein and total matrix Gla protein (MGP); uncarboxylated osteocalcin protein, carboxylated osteocalcin protein, and total osteocalcin protein; uncarboxylated protein C, carboxylated protein C, and total protein C, osteoprotegerin, fetuin A, and hs-CRP. Blood samples are obtained and biomarkers are measured according to the following schedule: Blood sampling can be performed weekly or monthly during treatment. Administration of the disclosed compositions results in (i) a decrease in PIVKA-II, which indicates a slowing, halting, or reversal of the progression of calciphylaxis; and (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin, and / or uncarboxylated protein C, which indicates a slowing, halting, or reversal of the progression of calciphylaxis. Additionally, pulse wave velocity (PWV) can be measured to assess arterial compliance. Improvement in vascular compliance is an indication of slowing, halting or reversing the progression of calciphylaxis.

[0113] Administration of the disclosed compositions of the present application in subjects diagnosed with calciphylaxis: This example describes the administration of the disclosed compositions to a subject diagnosed with calciphylaxis. Typical symptoms include the presentation of characteristic painful skin lesions (Nigwekar et al. (2015) Calciphylaxis: Risk Factors, Diagnosis, and Treatment. Am. J. Kidney Dis. 66:133-46). A definitive diagnosis of calciphylaxis is made by skin biopsy.

[0114] Subjects diagnosed with calciphylaxis receive 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed composition orally 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 a 0.1 mg, 3 mg, 5 mg, or 10 mg tablet or soft gel capsule. Two 25 mg capsules are administered once daily in 50 mg dose cohorts.

[0115] It is contemplated that a cessation or reduction in lesion size and frequency is an indicator of successful treatment. Administration of the disclosed compositions as described above results in a cessation or reduction in lesion size and frequency. Furthermore, because calciphylaxis carries a significant risk of death, an increase in the overall survival of a diagnosed subject would be an indicator of successful treatment. Furthermore, administration of the disclosed compositions as described above results in an increase in the overall survival of a diagnosed subject.

[0116] Administration of the disclosed compositions in a subject with end stage renal disease (ESRD) to reverse or slow the progression of tissue calcification: This example describes the administration of the disclosed compositions to subjects with ESRD undergoing stable hemodialysis. Administration of the disclosed compositions results in changes in aortic compliance (by plethysmography), vascular calcification, and levels of certain biomarkers indicative of slowing, halting, or reversing the progression of tissue calcification.

[0117] ESRD subjects on stable hemodialysis receive 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed composition orally once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. Dosage forms are 0.1 mg, 3 mg, 5 mg, or 10 mg tablets or soft gel capsules. Two 25 mg capsules are administered once daily in 50 mg dose cohorts.

[0118] A 50-year-old, 65 kg male patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated with 0.1 mg of the composition for 8 weeks. After the treatment period, the patient is admitted to the hospital and evaluated. The patient is found to have significant changes in the levels of the tested biomarkers, suggesting an approximately 10% reduction in vascular calcification, and also showing a 10% reduction in tissue calcification.

[0119] A 65-year-old, 45 kg female patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated with 0.1 mg of the composition for 10 weeks. After the treatment period, the patient is admitted to the hospital and evaluated. The patient is found to have significant changes in the levels of the tested biomarkers, suggesting an approximately 20% reduction in vascular calcification and also showing a 15% reduction in tissue calcification.

[0120] A 55-year-old, 70 kg male patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated with 0.2 mg of the composition for three months. After the treatment period, the patient is admitted to the hospital and evaluated. The patient is found to have significant changes in the levels of the tested biomarkers, suggesting an approximately 25% reduction in vascular calcification and also showing a 20% reduction in tissue calcification.

[0121] The coronary artery calcium score (CAC) is used to estimate the degree of calcification in the thoracic arteries. A high CAC score is an indicator of calcification, and treatment has the goal of halting or reversing the long-term increase in the CAC score, or slowing the rate of increase.

[0122] Aortic plethysmography is also used to measure arterial compliance, which decreases with increasing calcification. Pulse wave velocity (PWV) is also measured to assess arterial compliance. The above-mentioned measures are useful for estimating the effectiveness of treatments intended to prevent, slow down, stop, or reverse vascular calcification. These measurements are used before and after treatment with the disclosed compositions to assess the value of the treatment.

[0123] Furthermore, several biomarkers are evaluated to determine the efficacy of the disclosed compositions at three dose levels.Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein and total matrix Gla protein (MGP); uncarboxylated osteocalcin protein, carboxylated osteocalcin protein and total osteocalcin protein; uncarboxylated protein C, carboxylated protein C and total protein C and hs-CRP.Most conveniently, blood samples are obtained to measure biomarkers during patients' visits for hemodialysis.

[0124] Administration of the disclosed compositions can result in (i) a decrease in PIVKA-II, which is indicative of a slowing, halting, or reversal of the progression of tissue calcification; (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin, and / or uncarboxylated protein C, which are indicative of a slowing, halting, or reversal of the progression of tissue calcification; and / or (iii) a decrease in hs-CRP, which is indicative of a slowing, halting, or reversal of the progression of tissue calcification, and / or a reduction in inflammation. Following daily administration of 0.01 mg, 0.1 mg, 3 mg, 5 mg, or 10 mg of the disclosed compositions, at least one of PIVKA-II, undercarboxylated matrix Gla protein (MGP), and undercarboxylated osteocalcin protein shows a change indicative of a slowing, halting, or reversal of the progression of tissue calcification.

[0125] While several exemplary embodiments, aspects, and variations are presented herein, those skilled in the art will recognize certain modifications, alterations, additions, and combinations, as well as certain subcombinations of the embodiments, aspects, and variations. The following claims are intended to include all such modifications, alterations, additions, and combinations, and certain subcombinations of the embodiments, aspects, and variations are intended to be within their scope.

[0126] The entire disclosures of all documents cited throughout this application are hereby incorporated by reference.

Claims

1. A composition comprising nanoparticles of water-soluble vitamin K2, said nanoparticles having an average particle size of about 0.1 nm to 1,000 nm.

2. The vitamin K2 is selected from the group consisting of MK-4 (menaquinone-4), MK-5 (menaquinone-5), MK-6 (menaquinone-6), MK-7 (menaquinone-7), MK-8 (menaquinone-8), MK-10 (menaquinone-10), MK-11 (menaquinone-11), MK-12 (menaquinone-12), MK-13 (menaquinone-13) and MK-14 (menaquinone-14), or the vitamin K2 is selected from the group consisting of MKH2-4 (menaquinol-4), MKH2 10. The composition of claim 1, wherein the menaquinone is selected from the group consisting of MKH2-10 (menaquinol-10), MKH2-11 (menaquinol-11), MKH2-12 (menaquinol-12), MKH2-13 (menaquinol-13), and MKH2-14 (menaquinol-14); or a mixture of said menaquinones and menaquinols.

3. 3. The composition of claim 2, wherein the vitamin K2 is MK-7.

4. 4. The composition of claim 1, wherein the nanoparticles have an average particle size of less than 200 nm, 175 nm, 150 nm, 125 nm, 115 nm, 100 nm, 90 nm, 80 nm, or less than 75 nm.

5. 5. The composition of claim 1, wherein the nanoparticles are prepared using a homogenizer selected from the group consisting of a rotor-stator homogenizer, a bead mill homogenizer, or a mortar and pestle homogenizer.

6. Poloxamer 188, Polysorbate 80, Polysorbate 20, Vit E-TPGS (TPGS), TPGS-1000, TPGS-750-M, Solutol HS15, PEG-40 Hydrogenated Castor Oil, Kolliphor RH40, PEG-35 Castor Oil, PEG-8-Glyceryl Caprylate / Caprate, PEG-32-Glyceryl Laurate, PEG-32-Glyceryl Palmitostearate, Polysorbate 85, Polyglyceryl-6-Dioleate, Sorbitan Monooleate, Capmul 6. The composition of any one of claims 1 to 5, further comprising at least one emulsifier selected from the group consisting of MCM, Maisine 35-1, glyceryl monooleate, glyceryl monolinoleate, PEG-6-glyceryl oleate, PEG-6-glyceryl linoleate, oleic acid, linoleic acid, propylene glycol monocaprylate, propylene glycol monolaurate, polyglyceryl-3 dioleate, polyglyceryl-3 diisostearate, and lecithin.

7. 7. The composition of claim 6, wherein the emulsifier is selected from Polysorbate 80, Vit E-TPGS, Solutol HS15, PEG-40 hydrogenated castor oil, Kolliphor RH40 and PEG-35 castor oil or mixtures thereof.

8. 8. The composition of any one of claims 1 to 7, further comprising at least one bioavailability enhancer selected from the group consisting of medium chain fatty acids, omega-3 fatty acids, capric acid, caprylic acid, alkyl glycosides, chitosan, trimethylated chitosan, ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, salicylic acid, genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)chromen-4-one)) and pharmaceutically acceptable salts thereof.

9. 9. The composition of claim 1, which is a nanosuspension in water.

10. 10. The composition of any one of claims 1 to 9, wherein the nanosuspension is at least 5 times more soluble than commercially available non-homogenized vitamin K2.

11. 11. The composition of claim 10, wherein the vitamin K2 is MK-7.

12. 12. The composition of claim 10 or 11, wherein the nanosuspension of vitamin K2 is carried out at a concentration of 0.01 mg / mL in water.

13. 12. The composition of claim 10 or 11, wherein the nanosuspension is in an aqueous solution in fed-state simulated intestinal fluid (FeSSIF).

14. 14. The composition of claim 13, wherein the solubility is determined after 10 minutes in FeSSIF.

15. 15. The composition of any one of claims 1 to 14, further comprising a pharmaceutically acceptable additive, and which is effective in treating a vitamin K-related condition, such as the treatment of osteoporosis or arteriosclerosis.

16. 16. A method for the treatment of a disease in a mammal selected from the group consisting of neurodegenerative diseases, retinopathy, rheumatoid polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with skin aging and aging in tissues, pathologies associated with mitochondrial dysfunction, and cachexia associated with nutritional deficiencies, wherein the treatment is accompanied by an increase in the lifespan of the mammal, and the method comprises administering a therapeutically effective amount of a composition described in any one of claims 1 to 15.

17. 16. A method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, a proliferative disease, and a cardiovascular disease, comprising administering to the mammal a therapeutically effective amount of a composition described in any one of claims 1 to 15.

18. 16. A method for the treatment or prevention of osteoporosis and / or osteopenia, comprising administering to a patient in need of treatment a therapeutically effective amount of a composition according to any one of claims 1 to 15.

19. 19. A method for treating, preventing, slowing, halting, and / or reversing calciphylaxis in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a composition described in any one of claims 1 to 15 and a pharmaceutically acceptable excipient to prevent, slow, halt, or reverse calciphylaxis.

20. 20. The method of claim 19, wherein the mammal has distal calciphylaxis and / or central calciphylaxis.

21. 21. The method of claim 19 or 20, wherein the mammal has diabetes, chronic kidney disease or end-stage renal disease.

22. 22. The method of any one of claims 19 to 21, wherein the mammal has chronic obstructive pulmonary disease (COPD).

23. 23. The method of any one of claims 19 to 22, wherein the mammal has calciphylaxis-associated skin lesions.

24. 16. A method of treating, preventing, slowing the progression of, halting and / or reversing tissue calcification in a pre-diabetic mammal (or subject) having diabetes, chronic kidney disease or a combination thereof and in need of treating, preventing, slowing the progression of, halting and / or reversing tissue calcification, said method comprising the step of administering to said mammal at least 0.1 mg per day of any one of the compositions of any one of claims 1 to 15 to prevent, slow the progression of and / or halt tissue calcification, wherein the composition of any one of claims 1 to 15 is administered in a pharmaceutical composition.

25. 25. The method of claim 24, wherein the mammal has chronic kidney disease.

26. 18. The method of claim 17, wherein the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

27. 16. A method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, a proliferative disease, and a cardiovascular disease, comprising administering to the mammal a therapeutically effective amount of a composition described in any one of claims 1 to 15.

28. 27. The method of claim 26, wherein the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervical, colorectal, pancreatic, bladder, kidney, prostate, ovarian, and head and neck.

29. 16. A method for treating, preventing, slowing, halting, and / or reversing Alzheimer's disease (AD) in a mammal or subject in need thereof, comprising administering to said mammal or subject at least 0.1 mg per day of any one of the compositions of any one of claims 1 to 15 to prevent, slow down, and / or halt, or reverse Alzheimer's disease.

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