Pegylated menaquinol composition and method of treatment

Abstract

This application discloses, in part, isolated, stable and bioactive pegylated menaquinol derivatives for treating various diseases, and methods of using them. The present invention relates to bioactive menaquinol derivatives, compositions and formulations, and combinations thereof, their reduced and bioactive forms of menaquinol and salts thereof for treating vitamin K-related diseases including osteoporosis and osteopenia. In one embodiment, this application discloses novel biologically effective menaquinol derivatives (including their salts) such as menaquinol-7 derivatives and menaquinol-9 derivatives, and methods of using them for treating various diseases.

Description

Technical Field

[0001] Related Applications This application is related to Provisional Patent Application No. 63 / 347,007, filed May 30, 2022; Provisional Patent Application No. 63 / 347,008, filed May 30, 2022; Provisional Patent Application No. 63 / 413,495, filed Oct. 5, 2022; Provisional Patent Application No. 63 / 413,506, filed Oct. 5, 2022; Provisional Patent Application No. 63 / 447,310, filed Feb. 21, 2023; and Provisional Patent Application No. 63 / 447,314, filed Feb. 21, 2023.

[0002] Field of the Invention The present invention relates to bioactive menaquinol derivatives, compositions and formulations, and combinations thereof, the reduced and bioactive forms of menaquinol, and salts thereof, for treating diseases related to vitamin K, including osteoporosis and osteopenia.

Background Art

[0003] Background of the Invention Vitamin K is known as a group of structurally similar fat-soluble vitamins. Vitamin K2 or menaquinone has nine related compounds that can be classified into short-chain menaquinones (such as menaquinone-4 or MK-4) and long-chain menaquinones (such as MK-7, MK-8, and MK-9 to MK-12). Vitamins include phylloquinone (K1), menaquinone (K2), and menadione (K3). Plants synthesize vitamin K1, while bacteria can produce a range of vitamin K2 forms, including the conversion of K1 to K2 by intestinal bacteria. Vitamin K3 is a synthetic version of the vitamin and is prohibited for human use by the U.S. FDA due to its toxicity.

[0004] Taking broad-spectrum antibiotics has been shown to potentially reduce vitamin K production in the gut by nearly 74% in humans compared to those not taking these antibiotics. A diet low in vitamin K also reduces the body's vitamin K concentration. Vitamin K1 is preferentially used by the liver as a coagulation factor. Vitamin K2 is preferentially used in the brain, vasculature, chest, and kidneys. Vitamin K2 contributes to the production of myelin and sphingolipids (fats essential for brain health) and provides protection from oxidative damage in the brain. Vitamin K2, such as MK-4, promotes bone health by stimulating the production of connective tissue in bone.

[0005] In animals, the major storage form, vitamin K2, has several subtypes, which differ in the chain length of the isoprenoid group or the residues in the side chain. These vitamin K2 homologs are called menaquinones and are characterized by the number of isoprenoid residues in their side chains. For example, MK-4 has four isoprene residues in its side chain and is the most common type of vitamin K2 in animal products. MK-4 is usually synthesized from vitamin K1 in certain animal tissues (arterial wall, pancreas, and testis) 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 as dietary supplements for bone health in the United States. MK-4 has been shown to reduce the incidence of fractures. In Japan, since 1995, a dose of 45 mg per day of MK-4 has been approved by the Ministry of Health, Labour and Welfare for the prevention and treatment of osteoporosis.

Chemical formula

[0006] Vitamin K2 has usually been proposed and evaluated both in vitro and in vivo as MK-4 for treating various cancers. For example, (1) Lamson et al., "The Anticancer Effects of Vitamin K", Altern. Med. Rev., 8(3), 303-318 (2003) presented a review of vitamin K therapy. According to Lamson et al., "Both in vitro and in vivo studies have shown that vitamin K2 also exhibits anticancer effects. Several cancer cell lines were screened (including liver, colon, leukemia, lung, stomach, lymphocytes, nasopharynx, thoracic and oral squamous epithelia)", and activity was observed. Lamson et al. also mentioned the inhibition of proliferation in the HOS TE85 human osteosarcoma and MC3T3-E1 mouse osteoblast cell lines, C6 rat and RBR17T and T98G human glioma cell lines, myeloblastic (ML1) and promyelocytic (HL60) lung cancer cell lines, and stated that vitamin K2 analogs, especially MK-4, induced apoptosis in isolated leukemia cells (post myelodysplastic syndrome (MDS) and acute myeloid leukemia). Lamson et al. reported two individual cases and also mentioned a pilot study on the treatment of MDS and post-MDS acute myeloid leukemia (post-MDS AML) with K2 (MK-4) in Japan and a Japanese trial in hepatocellular carcinoma, both of which showed promising results. (2) Yoshida et al., "Apoptosis induction of vitamin K2 in lung carcinoma cell lines: The possibility of vitamin K2 therapy for lung cancer", Int. J. Oncol., 23, 627-632 (2003) stated that "vitamin K2 (menaquinone-4, VK2) has been reported to exhibit apoptosis and differentiation-inducing effects on leukemia cells", and that "the clinical advantages of using VK2 have been demonstrated for the treatment of patients with acute leukemia and myelodysplastic syndrome".They demonstrated that MK-4 was active in vitro against seven lung cancer cell lines: small cell lung cancer (LU-139, LU-I30), squamous cell lung cancer (LC-AI, LC-l / sq), adenocarcinoma of the lung (PC-14, CCL185), and large cell carcinoma (IA-LM). Similarly, they also showed that a combination of MK-4 at the optimal lower dose and cisplatin had growth inhibitory activity against LU-139 cells in vitro. (3) Ohtsuka et al., "Vitamin K2 Inhibits the Growth and Invasiveness of Hepatocellular Carcinoma Cells via Protein Kinase A Activation", Hepatology, 40, 243-251 (2004) stated that MK-4 inhibits hepatocellular carcinoma cells (HepG2 and PRF / PLC / 5) in vitro and in vivo (mouse xenografts including PRF / PLC / 5). They also mentioned an ongoing human clinical trial with intermediate results that MK-4 treatment works favorably for the inhibition of portal vein invasion of liver cancer. (4) Tokita et al., "Vitamin K2-induced antitumor effects via cell-cycle arrest and apoptosis in gastric cancer cell lines", Int. J. Molecular Med., 17, 235-243 (2006) showed that when four types of gastric cancer cells (KATO III (signet ring cell carcinoma), MKN7 (well-differentiated tubular adenocarcinoma), MKN74 (moderately differentiated tubular adenocarcinoma, liver metastasis), and FU97 (poorly differentiated adenocarcinoma, AFP-producing)) were exposed to MK-4 in vitro, cell growth was inhibited in a dose-dependent manner, and when the same cell lines were treated with up to 10 mg / L of 5-fluorouracil or docetaxel, cell growth was inhibited in a dose-dependent manner in the MK-4-added group compared to the control.(5) According to Kawakita et al., "Growth inhibitory effects of vitamin K2 on colon cancer cell lines via different types of cell death including autophagy and apoptosis", Int. J. Molecular Med., 23, 709-716 (2009), it was shown that MK-4 exhibited a dose-dependent inhibitory effect in vitro in the COLO201 and PMC01 colon cancer cell lines, but not in the DLD-1 cell line. (6) Li et al., "Induction of apoptosis in hepatocellular carcinoma Smmc-7721 cells by vitamin K2 is associated with p53 and independent of the intrinsic apoptotic pathway", Mol. Cell Biochem., 342, 125-131 (2010) states that "vitamin K2 (VK2) can exert a cell growth inhibitory effect in various human cancer cells", and shows that vitamin K2 inhibited cell proliferation in vitro in a dose-dependent manner in Smmc-7721 hepatocellular carcinoma cells. (7) According to Samykutty et al., "Vitamin K2, a Naturally Occurring Menaquinone, Exerts Therapeutic Effects on Both Hormone-Dependent and Hormone-Independent Prostate Cancer Cells", Evid.-based Complement. Altern. Med., 287358 (2013), it has been shown that vitamin K2 can suppress the survival of androgen-dependent and androgen-independent prostate cancer cells in both in vitro (LNCaP, DU145 and 22RV1 cells) and in vivo (mouse xenografts containing LNCaP, DU145, and 22RV1 cells).(8) Kiely et al., "Real-time cell analysis of the inhibitory effect of vitamin K2 on adhesion and proliferation of breast cancer cells", Nutr. Res., 35(8), 736-743 (2015) states that "the antitumor properties of VK derivatives have been reported in both hepatocellular carcinoma and glioblastoma", and shows that in triple-negative breast cancer cell line MDA-MB-231 and HER2+ breast cancer cell line MDA-MB-453, MK-4 exhibited a dose-dependent growth inhibitory effect in vitro. (9) Duan et al., "Vitamin K2 Induces Mitochondria-Related Apoptosis in Human Bladder Cancer Cells via ROS and JNKp38 MAPK Signal Pathways", PLoS ONE, 11(8), e0161886 (2016) states that "numerous studies have shown that vitamin K2 can exhibit anticancer activity in various cancer cell lines, including leukemia, lung cancer, ovarian cancer, prostate cancer, and hepatocellular carcinoma", and in their study, MK-4 inhibited human bladder cancer cell lines (T24, J82, EJ) in vitro and EJ in vivo (mouse xenograft). (10) Mishima, "A non-canonical vitamin K cycle is a potent ferroptosis suppressor", Nature, 608, 708-790 (2022) states that "ferroptosis, a non-apoptotic form of cell death characterized by iron-dependent lipid peroxidation, plays an important role in organ injury, degenerative diseases, and the vulnerability of treatment-resistant cancers", and that "the fully reduced forms of vitamin K (a group of naphthoquinones including menaquinone and phylloquinone) confer a potent ferroptosis-suppressing function" and "protect cells from harmful lipid peroxidation and ferroptosis".

[0007] Osteoporosis is a bone disease that leads to an increased risk of fractures. In osteoporosis, bone mineral density (BMD) decreases, the bone microstructure is disrupted, and the amount and type of non-collagenous proteins in the bone change. The World Health Organization defines osteoporosis (in women) as a state where the bone mineral density is 2.5 standard deviations lower than the peak bone mass, that is, in the case of an average healthy 30-year-old woman. Osteoporosis is most common in postmenopausal women (referred to as postmenopausal osteoporosis). Osteoporosis can also occur in men, in the presence of certain hormonal disorders and other chronic diseases, or as a result of medication (specifically glucocorticoids) (when the disease is referred to as steroid or glucocorticoid-induced osteoporosis), and as a result of nutritional deficiencies or other metabolic disorders, such as hyponatremia, or as a secondary result of cancer. Osteopenia is a condition where the bone mineral density is lower than normal and is considered by many physicians to be a precursor to osteoporosis.

[0008] The underlying mechanism in most cases of osteoporosis is an imbalance between bone resorption and bone formation. The three main mechanisms by which osteoporosis develops include inappropriate peak bone mass (during growth, the skeleton develops with insufficient mass and strength), excessive bone resorption during remodeling, and inappropriate formation of new bone. Hormonal factors strongly determine the rate of bone resorption. The lack of estrogen (for example, as a result of menopause) increases bone resorption and reduces the deposition of new bone that normally occurs in weight-bearing bones. In addition to estrogen, calcium metabolism plays an important role in bone metabolic turnover, and deficiencies in calcium and vitamin D lead to disorders of bone deposition. Furthermore, the parathyroid gland responds to low calcium levels by secreting parathyroid hormone, which increases bone resorption to ensure sufficient calcium in the blood. Medications used in the treatment of osteoporosis include calcium, vitamin D, vitamin K, bisphosphonates, calcitonin, teriparatide, strontium ranelate, hormone replacement, and selective estrogen receptor modulators.

[0009] Cardiovascular disease (CVD) has been proven to be the most frequent cause of death in patients with chronic kidney disease (CKD). When compared to the general population, the cause of death due to CVD is about 10 to 20 times higher in CKD patients who are undergoing hemodialysis treatment. Furthermore, it has been demonstrated that vascular calcification and associated arteriosclerosis are frequently seen in the occurrence of CVD. Therefore, the disclosed treatment method can be applied to the treatment of peripheral arterial disease. Additionally, CKD patients undergoing dialysis treatment have a three-fold higher risk of fractures such as spinal fractures and other types of fractures.

[0010] It has been proven that there is a direct correlation between the level of vitamin K in a patient's blood and the incidence of vascular calcification, bone density, and bone strength. Therefore, the auxiliary use of vitamin K such as MK-7 and its fat-soluble hydroquinone (menadione) derivatives disclosed herein partially realizes an important clinical advantage of reducing vascular calcification observed in arteriosclerosis, and either increases bone mineralization or improves bone mineral density, which helps in treating or preventing CVD and treating or preventing bone diseases in patients with CKD. In humans, a nutritional intake of MK-7 and MK-9 that is sufficiently absorbed can be established, and as a result, this nutritional intake leads to a significant increase in the serum in the case of MK-7 levels.

Chemical formula

Prior art documents

Non-patent documents

[0011]

Non-patent document 1

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Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

[0012] In one embodiment, the present application discloses novel menaquinol derivatives (including their salts) that are biologically active, such as menaquinol-7 derivatives and menaquinol-9 derivatives, and methods of using them for the treatment of various diseases.

[0013] The above examples of related art and limitations are illustrative and are not intended to be exclusionary. Other limitations of the related art will be apparent to those of ordinary skill in the art upon reading this specification and examining the drawings or figures presented herein. SUMMARY OF THE INVENTION

[0014] The inventors recognize that there is a continuing need to design novel compounds and their formulations that are effective for the indications disclosed above. The following embodiments, aspects, and variations are examples and are not intended to limit the scope.

[0015] The inventors further anticipate that the novel pegylated menaquinol derivatives of the following paragraphs will exhibit the same vitamin K2-like (or MK-4-like) activity in diseases where vitamin K2 and MK-4 are active, and will have similar utility in the treatment of these diseases.

[0016] In one embodiment, the present application relates to Formula I, II, or III: [Chemical formula] (wherein m is an integer from 2 to 15, and m 1 is, if present, an integer from 3 to 15, and n is 7, 8, 9 or 10, R is H or -C(O)C1-C6 alkyl, -C(O)(CH2) q -O-C(O)C1-C6 alkyl and -Z-(C=Y)(CH2) s -(OCH2CH2)-O-R 1 selected from the group consisting of q is 1, 2 or 3, R 1 is H or -CH3, R 2 is H or C1-C3 alkyl, s is 1, 2 or 3, Y is O, NR 2 or S, Z is -C(O)CH2O-, -C(O)CH2NH- or absent, Z 1 is -C(O)CH2O-, -C(O)CH2NH- or absent) discloses an isolated, stable and bioactive menaquinol derivative.

[0017] In each variant form of the menaquinol derivative of formula I, II or III, m or m 1 are each independently 2-30. In another variation, m or m 1 are each independently 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In another variation, m and m 1 are each independently 6, 7, 8, 9, 10 or 11. In another variation, m and m 1 are each independently 8, 9 or 10. In another variation, m or m 1 are independently 12, 14 or 15. In another variation, C1-C6 alkyl is -CH3 or -CH2CH3. In another variation, the PEG group (i.e., -(OCH2CH2) m - or -(CH2OCH2) mThe polyethylene glycol group represented by (-) has an average molecular weight of 200 Da (dalton), 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1 kDa (kilodalton), 1.5 kDa, 2 kDa, 3 kDa, 4 kDa or 5 kDa.

[0018] In one aspect, the menaquinol derivative is of formula Ia, Ib or Ic:

Chemical formula

[0019] In one variant form of the menaquinol derivative of formula Ia, R is CH3C(O)-, m is 7, R 1 is -CH3 and n is 7. In another variant form of formula Ia, R is CH3C(O)-, m is 7, R 1 is -CH3 and n is 9.

[0020] In another aspect, the menaquinol derivative is of formula Ia.1, Ib.1 or Ic.1:

Chemical formula

[0021] In another aspect, the menaquinol derivative is of formula IIa, IIb or IIc: [Chemical formula] wherein m is an integer from 8 to 12, n is 7, 8, 9 or 10, s is 1, 2 or 3, and R 1 is H or -CH3. In another variant, m is from 2 to 7.

[0022] In yet another aspect, the menaquinol derivative is of formula Ia.1, Ib.1, Ic.1, Ia.2, Ib.2, Ic.2, IIa, IIb, IIc, IIa.1, IIb.1, IIc.1, IIa.1.1, IIb.1.1, IIc.1.1, IIa.1.2, IIb.1.2, IIc.1.2, IIa.2, IIb.2, IIc.2, IIa.3, IIb.3, IIc.3, IIa.4, IIb.4, IIc.4, IIa.5, IIb.5 and IIc.5. In one variant, R 2 is H or CH3. In another variant, R 2 is CH3. In another aspect of the above menaquinol derivatives, n is 7. In another aspect, there is provided a pharmaceutical composition comprising a therapeutically effective amount of the disclosed menaquinol derivative or a mixture thereof, and a pharmaceutically acceptable additive, which is effective for the treatment of conditions related to vitamin K selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification, including soft tissue calcification.

[0023] In another variant of each of the above embodiments and aspects, n is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Treatment of COPD:

[0024] Chronic obstructive pulmonary disease (COPD) is a term used to describe a progressive lung disease that makes breathing difficult. The two main forms of COPD are emphysema and chronic bronchitis. Additionally, elastolysis (proteolytic degradation of elastin) is an important feature of COPD. It contributes to the loss of arterial flexibility and promotes calcification of the vascular intima-media. 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 that promote elastolysis. Vitamin D may be an important determinant of the rate of elastin degradation, and low vitamin D levels result in low MGP activity, which is inadequate for protection against elastolysis (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, the increased production of activated (carboxylated) MGP by administration of vitamin K2 disclosed herein acts to suppress the deleterious effects of elastolysis in subjects with COPD, thereby preventing, or decelerating, or reversing one or more symptoms of COPD. Further, treatment of elastin degradation may be effective in the treatment of Covid, such as Covid-19 and its variants. Accordingly, the nanoparticle formulations disclosed herein may be administered to treat or prevent elastin degradation and diseases associated with elastin degradation.

[0025] In one embodiment, a method for treating, preventing, decelerating the progression of, arresting, and / or reversing tissue calcification or calciphylaxis in a mammal (or subject), the method comprising administering to the mammal at least 0.1 mg per day of the above composition to prevent, decelerate the progression of, and / or arrest tissue calcification, and the above composition being administered as a pharmaceutical composition, is provided. In another embodiment, the present application discloses a method for improving the tissue concentration of menaquinol as a cofactor for gamma-glutamyl carboxylase (GGCX) for catalyzing the carboxylation of vitamin K-dependent proteins associated with the treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification in a patient in need of treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification, the method comprising administering a therapeutically effective amount of a menaquinol derivative, or a pharmaceutical composition comprising an effective amount of the menaquinol derivative or a mixture thereof cited above.

[0026] In yet another aspect, a method for treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with skin aging and senescence in tissues, pathologies associated with mitochondrial dysfunction, and cachexia associated with malnutrition in a mammal, the treatment being accompanied by an increase in the lifespan of the mammal, the method comprising administering a therapeutically effective amount of a compound, or a composition comprising the menaquinol derivative or a mixture thereof cited above, is provided.

[0027] A method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative diseases, and cardiovascular diseases. In yet another aspect, the method comprises administering to the mammal a therapeutically effective amount of the menaquinol derivative or a mixture thereof cited above. In another aspect of the method, the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

[0028] In another aspect, there is provided a method of treating or preventing osteoporosis and / or osteopenia, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a composition comprising the menaquinol derivative or a mixture thereof cited above. In yet another aspect, there is provided a method of treating, preventing, slowing the progression of, arresting, and / or reversing calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising the substantially pure menaquinol derivative or a mixture thereof disclosed above and a pharmaceutically acceptable additive to prevent, slow the progression of, arrest, or reverse calciphylaxis. In another aspect of the method, the mammal has distal calciphylaxis and / or central calciphylaxis. In yet another aspect of the method, the mammal has diabetes, chronic kidney disease or end-stage renal disease. In another aspect of the method, the mammal has stage 3, stage 4 or stage 5 chronic kidney disease. In another aspect above, the mammal is undergoing hemodialysis. In yet another aspect, the mammal is receiving non-warfarin based anticoagulant therapy. In yet another aspect, the anticoagulant therapy is oral anticoagulant therapy. In another aspect, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eliquixaban or fondaparinux, or an inhibitor of factor IIa activity selected from dabigratran or argatroban.

[0029] In another aspect of the above method, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect, the mammal has calciphylaxis-related skin lesions. In another aspect of the method, administration of the composition results in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the total surface area of the skin lesions. In yet another aspect of the method, when the mammal is administered the compound or mixture thereof disclosed above, the T50 value of the serum of the mammal is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% improved compared to the T50 value of the serum of the mammal before administration of the compound or mixture thereof disclosed above. In yet another aspect, administration of the above compound or mixture thereof results in an improvement in the ratio of the carboxylated form to the non-carboxylated form of the vitamin K-dependent protein in the plasma of the mammal, and the ratio after administration of the composition is higher than before administration of the composition.

[0030] In another aspect, there is provided a method for treating, preventing, decelerating the progression of, 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, decelerating the progression of, halting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 50 mg per day of the disclosed compound or a mixture thereof to prevent, decelerate the progression of, and / or halt tissue calcification, wherein the disclosed compound or a mixture thereof is administered in a pharmaceutical composition. In one aspect of the method, the mammal has diabetes. In another aspect of the method, the mammal has type II diabetes or the mammal is diagnosed as prediabetic. In yet another aspect, the mammal has chronic kidney disease or the mammal has stage 4 or 5 chronic kidney disease / end-stage renal disease. In another aspect of the method, the mammal is undergoing hemodialysis or the mammal is receiving non-warfarin based anticoagulant therapy. In one variant, the anticoagulant therapy is oral anticoagulant therapy. In another aspect of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0031] In another aspect, there is provided a method for treating, preventing, decelerating the progression of, halting, and / or reversing tissue calcification in a mammal undergoing hemodialysis and in need of treating, preventing, decelerating the progression of, halting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 5 mg per day of any one of the compounds cited above or a mixture thereof, thereby preventing, decelerating the progression of, halting, and / or reversing tissue calcification, wherein the compound or mixture thereof is administered in a pharmaceutical composition. In another aspect of the method, the mammal has diabetes.

[0032] In another aspect, there is provided a method for increasing at least one of the bioavailability and serum half-life of menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9, or menaquinone-10 / menadiol-10, the method comprising administering or supplementing the above-cited compound or composition thereof to a mammal in need of increasing at least one of the bioavailability and serum half-life, such that at least one of the bioavailability and serum half-life of menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9, or menaquinone-10 / menadiol-10 is improved by at least 5% compared to administration or supplementation using each of menaquinone-7, menaquinone-8, menaquinone-9, or menaquinone-10.

[0033] In yet another aspect, a method of treating at least one of a wound or area of pain and inflammation in a subject in need of treating at least one of a wound or area of pain and inflammation due to calciphylaxis or tissue calcification, the method comprising topical application of a skin structure or transdermal patch to the subject's wound or area of pain and inflammation, the skin structure or transdermal patch comprising any of the above-quoted compounds for treating the wound, pain and inflammation area, is provided. In one variant of the method, the topical application of the skin structure or transdermal patch is performed before, together with (or simultaneously with), or after the above-quoted method.

[0034] In another embodiment, a method of treating cancer is provided, wherein the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervical cancer, liver cancer (hepatocellular carcinoma, HCC), colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer and head and neck cancer.

[0035] In another embodiment, there is provided a pharmaceutical composition comprising a therapeutically effective amount of a menaquinol derivative of any one of Formulas I, II, and III of the above embodiments, aspects, and variations, or a mixture of those menaquinol derivatives, and a pharmaceutically acceptable additive, which is effective for treating a vitamin K-related condition selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification. As used herein, unless specifically stated otherwise, the menaquinol compounds or derivatives of Formulas I, II, and III include the menaquinol derivatives (or compounds) disclosed herein, including Formulas Ia.1, Ib.1, Ic.1, Ia.2, Ib.2, Ic.2, IIa, IIb, IIc, IIa.1, IIb.1, IIc.1, IIa.1.1, IIb.1.1, IIc.1.1, IIa.1.2, IIb.1.2, IIc.1.2, IIa.2, IIb.2, IIc.2, IIa.3, IIb.3, IIc.3, IIa.4, IIb.4, IIc.4, IIa.5, IIb.5, and IIc.5; and III. In one variation of the pharmaceutical composition, the composition comprises sunflower oil. In another variation of the pharmaceutical composition, the composition comprises an oil selected from the group consisting of sunflower oil, corn oil, vegetable oil (such as jojoba oil), or a combination thereof. In another variation, the composition comprises an oil selected from the group consisting of canola oil, coconut oil, cottonseed oil, olive oil, palm oil, peanut oil, rapeseed oil, safflower oil, sesame oil, soybean oil, heliotrope oil, almond oil, cashew nut oil, hazelnut oil, walnut oil, hickory nut oil, pine nut oil, pistachio oil, castor oil, and combinations thereof.

[0036] In another embodiment, a method for increasing the tissue concentration of menaquinol as a cofactor for gamma-glutamic acid carboxylase (GGCX) to catalyze the carboxylation of vitamin K-dependent proteins in patients in need of treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification, the method comprising administering a therapeutically effective amount of a menaquinol derivative, or a pharmaceutical composition comprising an effective amount of any one of the above menaquinol derivatives or a mixture thereof, is provided.

[0037] In one variant, by this method, the tissue concentration of menaquinol is increased by at least 20%, 30%, 40%, 50%, 60%, 70% or higher compared to the administration of the corresponding menaquinone. In another variant, by this method, the tissue concentration of menaquinol is increased by at least 40% or 50% compared to the administration of the corresponding menaquinone. In another variant, by this method, the tissue concentration of menaquinol is increased by at least 20%, 30%, 40%, 50%, 60%, 70% or higher compared to the administration of the corresponding menaquinone, and optimal benefits are realized at the tissue level, such as in at least one of the skin or dermal tissue, mitral valve, aortic valve and blood vessels.

[0038] In another embodiment, a method for treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid arthritis, atherosclerotic arteriosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with aging and senescence of the skin in tissues, pathologies associated with mitochondrial dysfunction, cachexia associated with malnutrition in a mammal, the treatment being accompanied by an increase in the lifespan of the mammal, the method comprising administering a therapeutically effective amount of a compound, or a composition comprising a menaquinol derivative of any one of the above embodiments, aspects and variants or a mixture of the above menaquinol derivatives, is provided.

[0039] In another embodiment, there is provided a method of treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative diseases, and cardiovascular diseases, the method comprising administering to the mammal a therapeutically effective amount of any one of the above menaquinol derivatives or a mixture thereof. In one aspect of the method, the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

[0040] In another embodiment, there is provided a method of treating or preventing osteoporosis and / or osteopenia, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a composition comprising a menaquinol derivative of any one of the above embodiments, aspects, and variations or a mixture of the menaquinol derivatives cited above.

[0041] In another embodiment, a method for treating, preventing, slowing the progression of, arresting, and / or reversing calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising any substantially pure menaquinol derivative or a mixture thereof of the above menaquinol derivatives and a pharmaceutically acceptable additive to prevent, slow the progression of, arrest, or reverse calciphylaxis is provided. In one aspect of the above method, the mammal has distal calciphylaxis and / or central calciphylaxis. In another aspect of the method, the mammal has diabetes, chronic kidney disease or end-stage kidney disease. In another aspect of the method, the mammal has stage 3, stage 4 or stage 5 chronic kidney disease. In yet another aspect of the method, the mammal is undergoing hemodialysis. In another aspect of the method, the mammal is receiving non-warfarin based anticoagulant therapy. In yet another aspect of the method, the anticoagulant therapy is oral anticoagulant therapy. In another aspect of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eribaxaban or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0042] In yet another aspect of the above method, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect of the method, the mammal has a calciphylaxis-related skin lesion. In yet another aspect of the method, upon administration of the composition, the total surface area of the skin lesion is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. As used herein, the method results in a reduction in the surface area of the skin lesion, which constitutes healing of the skin or wound. Thus, or rather, the method enhances or accelerates wound healing. Thus, such methods are generally applicable to wound healing such as burns (or scalds) of the skin, wounds that result in ulcers, and generally wounds resulting from trauma, skin cancer, infection and cosmetic surgery. Further, as disclosed below, the above method can be used in combination with or simultaneously with topical administration of the composition to further increase wound healing.

[0043] In another aspect of the above method, when any one compound (or a plurality thereof) of the menaquinol derivatives or a mixture of the above embodiments, aspects and variations is administered to a mammal, the T50 value of the serum of the mammal is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher than the T50 value of the serum of the mammal before administration of the compound or mixture thereof disclosed above.

[0044] In yet another aspect of the method, when any one menaquinol compound of the above embodiments, aspects and variations is administered, the ratio of the carboxylated form to the non-carboxylated form of the vitamin K-dependent protein in the plasma of the mammal is improved, and the ratio after administration of the composition is higher than before administration of the composition.

[0045] In yet another embodiment, there is provided a method for treating, preventing, decelerating the progression of, arresting, 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, decelerating the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 50 mg per day of a compound of any one of the above embodiments, aspects, or variations thereof to prevent, decelerate the progression of, and / or arrest tissue calcification, wherein the compound or mixture thereof listed above is administered in a pharmaceutical composition. In another aspect of the method, the mammal has diabetes. In another aspect of the method, the mammal has type II diabetes.

[0046] In yet another aspect of the above method, the mammal is diagnosed with prediabetes. In another aspect of the method, the mammal has chronic kidney disease. In yet another aspect of the method, the mammal has stage 4 or 5 chronic kidney disease / end-stage renal disease. In yet another aspect of the method, the mammal is undergoing hemodialysis. In another aspect of the method, the mammal is undergoing non-warfarin based anticoagulant therapy. In another aspect of the method, the anticoagulant therapy is oral anticoagulant therapy. In another aspect, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0047] In another embodiment, a method for treating, preventing, decelerating the progression of, arresting, and / or reversing tissue calcification in a mammal undergoing hemodialysis and in need of treating, preventing, decelerating the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 5 mg per day of a compound of any one of the above embodiments, aspects, and variations thereof, thereby preventing, decelerating the progression of, arresting, and / or reversing tissue calcification, wherein the compound or mixture thereof of any one of the above embodiments, aspects, and variations thereof is administered in a pharmaceutical composition, is provided. In another aspect, the mammal has diabetes.

[0048] In another embodiment, a method for increasing at least one of the bioavailability and serum half-life of menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9, or menaquinone-10 / menadiol-10, the method comprising administering or supplementing to a mammal in need of increasing at least one of the bioavailability and serum half-life a compound or composition of any one of the above embodiments, aspects, and variations thereof, whereby at least one of the bioavailability and serum half-life of menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9, or menaquinone-10 / menadiol-10 is improved by at least 5% compared to administration or supplementation using menaquinone-7, menaquinone-8, menaquinone-9, or menaquinone-10, is provided. Treatment of Alzheimer's disease, apoptosis, and cancer:

[0049] Alzheimer's disease (AD) is a severe neurodegenerative disorder. Its sporadic form affects the elderly population (the incidence significantly increases in those >75 years old), and there are also various familial forms that occur in the 40s or 50s of life. AD is characterized by the presence of extracellular senile plaques and intracellular neurofibrillary changes in the patient's brain. The core component of senile plaques is a small 4 kDa amyloid peptide, which is produced by proteolytic processing of the amyloid precursor protein (APP), a large transmembrane protein. Cleavage of APP by beta-secretase (BACE-1) releases a soluble APP-beta fragment, while the 99 amino acid-long C-terminus remains membrane-bound. This C-terminal fragment is subsequently proteolytically processed by gamma-secretase (a membrane multi-enzyme complex) to produce amyloid peptides of various lengths, mainly 40 and 42 amino acids long (Hardy J. et al. (2002) Science; 297 (5580):353-356). In one embodiment, the treatment of diseases or conditions such as Alzheimer's disease, mild cognitive impairment, impaired glucose tolerance, or type 2 diabetes using the compositions disclosed herein is mediated by BACE-1, BACE-2, or cathepsin D activity. In one variant, the disclosed method may be used to treat calcification in the brain.

[0050] β-Amyloid (Aβ) has been shown to cause neuron death by promoting apoptosis and also directly by 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 PC12 cells derived from rat pheochromocytoma, vitamin K2 blocked neuron 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 significant apoptosis. Pretreatment with vitamin K2 also decreased the amount of apoptosis signaling proteins, including a lower Bax / Bcl-2 ratio, decreased the presence of reactive oxygen species (ROS), and increased the amount of the antioxidant glutathione. Yagami, T. Gas6 Rescues Cortical Neurons from Amyloid β Protein-Induced Apoptosis Neuropharmacology 2002, 43, 1289-1296. The researchers determined that inactivation of the p38MAP kinase pathway is the mechanism regarding the potential preventive role of VK2 in Alzheimer's disease (AD).

[0051] The researchers observed that by increasing the concentration of vitamin K2 (VK2), cells showed an extended survival, probably because the cells were protected from Aβ-induced neuronal death. This effect was reversible upon addition of warfarin, which also inhibited vitamin K-dependent carboxylation. VK2 was observed to dose-dependently decrease the number of ROS and to decrease the activity of caspase-3, an enzyme that mediates Aβ-induced apoptosis, to one-quarter at a concentration of 10 μmol / L. The authors also found that Gas6 plays a role in the protection by VK2 from Aβ cytotoxicity, which was supported by measurements of Ca(2+) influx, chromatin condensation, and DNA fragmentation as markers for Aβ neurotoxicity and apoptosis in rat fetal neuronal cultures. Gas6 dose-dependently inhibits Ca(2+) influx and significantly reduces chromatin condensation and the amount of DNA fragmentation induced by Aβ. Thus, there is a clear correlation between the antioxidant and anti-apoptotic properties of vitamin K2, which is relevant to the process of programmed cell death as an effective mechanism related to the treatment of cancer.

[0052] Targeting apoptosis is also effective for all types of cancer because apoptosis avoidance is a prominent feature of cancer. Apoptosis is also non-specific to the cause or type of cancer. Villa-Pulgarin J.A. et al. Mitochondria and lipid raft-located F oSee F1-ATP synthase as major therapeutic targets in the antileishmanial and anticancer activities of 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.

[0053] Vitamin K2 has also been used for clinical applications to assist in the treatment of cancer. XV, F. et al, Research Progress on the anticancer effects of vitamin K2 (Review). Oncol. Lett. 2018, 15, 8926-8934. Furthermore, it has been found that supplementation with vitamin K2 inhibits the growth and metastasis of multiple cancer lines.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. 2012m 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.

[0054] In fact, the intake of MK-4 and MK5-MK-9 was found to be inversely correlated with cancer mortality. In particular, in the male cohort study, an inverse correlation was found between the incidence of progressive prostate cancer and the intake of MK, especially MK5-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, the undercarboxylation of osteocalcin (a marker of inappropriate vitamin K status) was significantly higher in cases of progressive or high-grade prostate cancer compared to 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 diseases, triple-negative diseases, and early-stage diseases. 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 of treating cancer, such as prostate cancer, by administering a therapeutically effective amount of the disclosed composition to control or reduce the hypocarboxylation of osteocalcin at high levels. Stability of the compounds in SGF, FaSSIF, and FeSSIF:

[0055] The stability of the disclosed compounds, such as the tested disclosed compounds, can be determined in simulated gastric fluid (SGF, pH - 1.2), fasting simulated intestinal fluid (FaSSIF, pH - 6.5), and fed-state simulated intestinal fluid (FeSSIF, pH - 5) at 0, 30, 60, 120, and 240 minutes. The percentage of compound disappearance over time can be calculated by comparing, by HPLC / LCMS analysis, the peak area of the analyte at "0" minutes. The formation of menaquinones, such as menaquinone-7 (i.e., MK-7) derived from the tested compounds disclosed herein, was observed.

[0056] Regarding the stability of the tested disclosed compounds of formula I, II, or III; SGF (pH 1.2); FaSSIF (pH 6.5), and FeSSIF (pH 5.0), attention was paid to the percentage of residual compounds compared to 0 minutes at time intervals of t = 0, 30 minutes, 60 minutes, 120 minutes, and 240 minutes.

[0057] Stability of the disclosed compounds of formula I, II or III tested; attention was paid to SGF (pH 1.2), FaSSIF (pH 6.5) and FeSSIF (pH 5.0), and to the formation of menaquinone-7 with respect to the fold change compared to 0 minutes at time intervals of t = 0, 30 minutes, 60 minutes, 120 minutes and 240 minutes.

[0058] The stability of the disclosed compounds of formula I, II or III tested was determined in simulated gastric fluid (SGF, pH - 1.20), fasting simulated intestinal fluid (FaSSIF, pH - 6.5) and fed state simulated intestinal fluid (FeSSIF, pH - 5) at 0, 30, 60, 120 and 240 minutes. The percentage of compound disappearance over time was calculated by comparing the peak area of the analyte at "0" minutes by HPLC / LCMS analysis. The formation of menaquinone-7 from the test article was observed. Stability in human plasma and rat plasma: The disclosed compounds are stable over time.

[0059] The stability of the disclosed compounds of formula I, II or III tested was determined in human plasma and rat plasma (K2EDTA) at 0, 15, 30, 60 and 120 minutes. By HPLC / LC-MS-MS analysis, the percentage of the compound that disappeared over time was calculated by comparison with 0 minutes. The formation of menaquinone-7 from the test compound was monitored at time intervals of t = 0, 30 minutes, 60 minutes and 120 minutes.

[0060] The stability of the disclosed compounds of formula I, II or III tested was determined in human plasma and rat plasma (K2EDTA) at 0, 15, 30, 60 and 120 minutes. By comparison with 0 minutes by HPLC / LC-MS-MS analysis, the amount of menaquinone-7 formed based on the fold change compared to 0 minutes was calculated. The percentage of the remaining test compound was observed at time intervals of t = 0, 30 minutes, 60 minutes and 120 minutes.

[0061] The stability of the disclosed compounds of formula I, II or III tested is determined in human plasma and rat plasma (K2EDTA) at 0, 15, 30, 60 and 120 minutes. The stability of the test compounds, based on the amount of menaquinone-7 formed and the fold change compared to 0 minutes, is calculated by HPLC / LC-MS-MS analysis by comparing to 0 minutes and observed at time intervals of t = 0, 30 minutes, 60 minutes and 120 minutes.

[0062] The stability of the disclosed compounds of formula I, II or III tested was determined in human plasma and rat plasma (K2EDTA) at 0, 15, 30, 60 and 120 minutes. The % of the compound that disappeared over time was calculated by HPLC / LC-MS-MS analysis by comparison to 0 minutes. Attention is paid to the formation of menaquinone-7 from the test compound. In vivo test:

[0063] Male Sprague-Dawley rats, 8 - 10 weeks old and weighing approximately 202 - 223 grams, are used for the in vivo test. The animals are fasted overnight and allowed free access to water. The animals are divided into 4 groups, G1 - G4, with each group containing 3 rats. To determine the bioavailability, the test article is individually dissolved in corn oil to obtain a uniform formulation. The test article is administered to the animals by forced oral administration, with the doses of compound Ia.1 (m = 9, n = 7) (G1), compound Ia.1 (m = 9, n = 9) (G2), menaquinone-7 (G3) and menaquinone-9 (G4) being, per kg of body weight, 1088 μg for the animals in G1, 1073 μg for G2, and 1000 μg for G3 and G4. The doses of Ia.1 (n = 7) (G1) and Ia.1 (n = 9) were adjusted according to the content of menaquinol. Blood samples are collected at various time points during the next 48 hours after administration.

[0064] A blood sample was collected and transferred to a tube containing K2EDTA and immediately placed on ice for plasma preparation. Plasma was prepared by centrifugation at 3500 g for 10 minutes, and aliquots were frozen at -80 °C until analysis. Quantification of the analyte in plasma was determined by LC-MS-MS analysis. Plasma PK parameters were calculated using WinNonlin software.

[0065] In another embodiment, the present application is a pharmaceutical composition comprising a therapeutically effective amount of a menaquinol derivative or a mixture thereof according to any one of the above embodiments and aspects, and a pharmaceutically acceptable additive, which is effective for the treatment of a vitamin K-related condition selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification. A pharmaceutical composition is disclosed.

[0066] In another embodiment, the present application provides a method for improving the tissue concentration of menaquinol as a cofactor for gamma-glutamate carboxylase (GGCX) to catalyze the carboxylation of vitamin K-dependent proteins associated with the treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification in patients in need of treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification. A method is disclosed that includes administration of a therapeutically effective amount of a menaquinol derivative, or a pharmaceutical composition comprising an effective amount of the menaquinol derivative or a mixture thereof disclosed above.

[0067] In one variant of the method, administration of a menaquinol derivative overcomes oxidative blockade in patients with CKD and in patients undergoing hemodialysis, achieving maximum levels of menaquinol and maximum benefit at the tissue level. In one variant, the tissue is skin or dermal tissue. In another variant, the tissue is at least the mitral valve, the aortic valve of the patient, and blood vessels of the patient. In another variant of the method, the menaquinol derivative is a compound of formula I, II or III, or a mixture thereof. In another variant, by the method, the tissue concentration of the corresponding menaquinol is improved by at least 20%, 30%, 40%, 50%, 100%, 150%, 200%, 250%, 300%, 400% or 500% or higher compared to administration of the corresponding menaquinone. In another variant, by the method, the tissue concentration of menaquinol is improved by at least 40% or 50% compared to administration of the corresponding menaquinone. The term "corresponding menaquinone" means, for example, when the menaquinol derivative is an MK-7 derivative, the "corresponding menaquinone" is MK-7, and when the menaquinol derivative is an MK-10 derivative, the corresponding menaquinone is MK-10.

[0068] In another embodiment, the present application discloses a method for treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid arthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with aging and senescence of the skin in tissue, pathologies associated with mitochondrial dysfunction, cachexia associated with malnutrition in mammals, wherein the treatment is accompanied by an increase in the lifespan of the mammal, and the method comprises administration of a therapeutically effective amount of a compound, or a composition comprising a menaquinol derivative of formula I, II or III, or a mixture thereof, comprising all of the disclosed compounds. As specified above, the menaquinol compounds or derivatives of formula I, II or III are the menaquinol derivatives (or compounds) disclosed herein, including the menaquinol derivatives (or compounds) of formula Ia, Ib, Ic, Ia.1, Ib.1, Ic.1, IIA, IIb and IIc, and the compounds thereof disclosed herein.

[0069] In another aspect, the present application discloses a method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative diseases, and cardiovascular diseases, the method comprising administering to the mammal a therapeutically effective amount of the menaquinol derivatives or mixtures thereof disclosed above. In another aspect of the method, the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory diseases. In one variant of the method, the cancer is melanoma and lung cancer. In another variant, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical, liver (hepatocellular carcinoma, HCC), colorectal, pancreas, bladder, renal, and melanoma. In another aspect of the method, the cancer is selected from the group consisting of lung, chest, prostate, ovary, and head and neck.

[0070] In another aspect, the present application discloses a method for treating or preventing osteoporosis and / or osteopenia, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a composition comprising a menaquinol derivative of formula I, II, or III, or mixtures thereof.

[0071] In another aspect, the present application discloses a method for treating, preventing, decelerating the progression of, arresting, and / or reversing calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising a substantially pure menaquinol derivative of formula I, II or III or a mixture thereof and a pharmaceutically acceptable additive to prevent, decelerate the progression of, arrest, or reverse calciphylaxis. In another aspect of the method, the mammal has distal calciphylaxis and / or central calciphylaxis. In another aspect of the method, the mammal has diabetes, chronic kidney disease or end-stage renal disease. In another aspect of the method, the mammal has stage 3, stage 4 or stage 5 chronic kidney disease. In another aspect of the method, the mammal is undergoing hemodialysis. In yet another aspect of the invention, the mammal is undergoing non-warfarin based anticoagulant therapy. In another aspect of the method, the anticoagulant therapy is oral anticoagulant therapy. In yet another aspect of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eribaxaban or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0072] In another aspect of the above method, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect of the method, the mammal has calciphylaxis-related skin lesions. In another aspect, upon administration of the composition, the total surface area of the skin lesions decreases by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In another aspect of the method, when a compound of formula I, II or III or a mixture thereof is administered to a mammal, the T50 value of the serum of the mammal is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% improved compared to the T50 value of the serum of the mammal before administration of the above compound or mixture thereof. In yet another aspect of the method, when a compound of formula I, II or III or a mixture thereof is administered, the ratio of the carboxylated form to the non-carboxylated form of the vitamin K-dependent protein in the plasma of the mammal is improved, and the ratio after administration of the composition is higher than before administration of the composition.

[0073] In another embodiment, a method for treating, preventing, decelerating the progression of, arresting, 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, decelerating the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 50 mg per day of a compound of formula I, II or III or a mixture thereof to prevent, decelerate the progression of, and / or arrest tissue calcification, wherein the above compound or mixture thereof is administered in a pharmaceutical composition, is provided.

[0074] In each of the above-listed methods, the present specification also discloses the listed compounds or compositions thereof for use as a medicament in the treatment of the listed medical conditions or diseases, and the present specification discloses the use of the listed compounds in the manufacture of a formulation or medicament for treating the disclosed medical conditions or diseases.

[0075] In one variant form, the menaquinol derivative is administered at a dose of at least 1 mg, 3 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg or 50 mg. In another variant form, the menaquinol derivative is administered at a dose of at least 100 mg, 150 mg, 200 mg, 25 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg. In another variant form, the menaquinol derivative is administered at a dose of at least 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1,000 mg or more.

[0076] In one aspect of the above method, the mammal has diabetes. In another aspect, the mammal has type II diabetes. In another aspect, the mammal is diagnosed with prediabetes. In another aspect of the above method, the mammal has chronic kidney disease. In yet another aspect of the method, the mammal has stage 4 or 5 chronic kidney disease / end-stage renal disease. In another aspect of the method, the mammal is undergoing hemodialysis. In another aspect of the method, the mammal is undergoing non-warfarin-based anticoagulant therapy. In another aspect of the method, the anticoagulant therapy is oral anticoagulant therapy. In yet another aspect of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eliquis or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0077] In another embodiment, the present application is directed to a method for treating, preventing, slowing the progression of, arresting, and / or reversing tissue calcification in a mammal that is undergoing hemodialysis and in need of treating, preventing, slowing the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 5 mg per day of a compound of formula I, II, or III, or a mixture thereof, thereby preventing, slowing the progression of, arresting, and / or reversing tissue calcification, wherein the compound or mixture thereof is administered in a pharmaceutical composition.

[0078] In one variant, the menaquinol derivative is administered at a dose of at least 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg or 50 mg. In another variant, the menaquinol derivative is administered at a dose of at least 100 mg, 150 mg, 200 mg, 25 mg, 300 mg, 350 mg, 400 mg, 450 mg or 500 mg. In another variant, the menaquinol derivative is administered at a dose of at least 500 mg, 600 mg, 700 mg, 800 mg, 900 mg or 1,000 mg or more. In another aspect of the above method, the mammal has diabetes.

[0079] In another aspect, the present application discloses a pharmaceutical composition comprising a therapeutically effective amount of the menaquinol derivative (also referred to as the "compound" or "disclosed compound") or a mixture thereof as disclosed above and a pharmaceutically acceptable additive, which is effective for the treatment of conditions related to vitamin K selected from the treatment of osteoporosis and atherosclerosis.

[0080] In another aspect, the present application discloses a method for treating a disease selected from the group consisting of neurodegenerative diseases, retinopathies, rheumatoid arthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, pathologies associated with aging and senescence of the skin in tissues, pathologies associated with mitochondrial dysfunction, and cachexia associated with malnutrition in mammals, 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 compound, or a composition comprising a menaquinol compound or a mixture thereof as disclosed above.

[0081] In another embodiment, there is provided a method for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative diseases, and cardiovascular diseases, the method comprising administering to the mammal a therapeutically effective amount of a compound or a mixture thereof as disclosed herein. In another aspect of the method, the proliferative disease is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

[0082] In another embodiment, there is provided a method for treating and / or preventing osteoporosis and / or osteopenia, the method comprising administering to a patient in need of treatment a therapeutically effective amount of a composition comprising a compound or a mixture thereof as disclosed above. The disclosed methods for administering the present compounds and compositions, or combinations thereof, can be used for the treatment or alleviation of vascular calcification, improvement of bone mineral density, and treatment, alleviation, or prevention of bone diseases, such as in patients with CKD.

[0083] In another embodiment, a method for treating, preventing, slowing the progression of, halting, and / or reversing calciphylaxis in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a composition comprising a substantially pure menaquinol compound disclosed herein and a pharmaceutically acceptable additive to prevent, slow the progression of, halt, or reverse calciphylaxis. In one aspect of the method, the mammal has distal calciphylaxis and / or central calciphylaxis. In another aspect, the mammal has diabetes, chronic kidney disease or end-stage renal disease. In another aspect, the mammal has stage 3, stage 4 or stage 5 chronic kidney disease. In another aspect of the method, the mammal is undergoing hemodialysis. In yet another aspect, the mammal is undergoing non-warfarin based anticoagulant therapy.

[0084] In another aspect of the above method, the anticoagulant therapy is oral anticoagulant therapy. In another aspect, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eliquixaban or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban. In another aspect, the mammal has chronic obstructive pulmonary disease (COPD). In another aspect, the mammal has calciphylaxis-related skin lesions. In another aspect of the method, administration of the composition results in at least a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reduction in the total surface area of the skin lesions. In another aspect of the method, when a substantially pure compound disclosed herein is administered to a mammal, the T50 value of the mammal's serum is improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% compared to the T50 value of the mammal's serum before administration of the disclosed compound. In another aspect, administration of the disclosed compound improves the ratio of carboxylated to non-carboxylated vitamin K-dependent proteins in the plasma of the mammal, and the ratio after administration of the composition is higher than before administration of the composition. In one aspect of the method, the improvement in the ratio of carboxylated to non-carboxylated 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.

[0085] In certain embodiments described above, upon administration of the disclosed compound, the amount of non-carboxylated vitamin K-dependent protein in the plasma of the subject is decreased 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 amount prior to administration of the compound. In certain variants, 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.

[0086] In certain variants of the method described above, upon administration of the compound, the plasma level of osteoprotegerin or fetuin A is increased 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 prior to administration of the compound. In other variants, upon administration of the compound, the plasma level of D-dimer or high-sensitivity C-reactive peptide (hs-CRP) is decreased 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 D-dimer or high-sensitivity C-reactive peptide (hs-CRP) prior to administration of the compound.

[0087] In certain embodiments, the method may include administering to the subject from about 10 mg to about 750 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 50 mg to about 750 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 20 mg to about 500 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 50 mg to about 500 mg of the compound per day. In certain embodiments, the method may include administering to the subject from about 20 mg to about 250 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 5 mg to about 250 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 20 mg to about 100 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 50 mg to about 100 mg of the compound per day. In other embodiments, the method may include administering to the subject from about 5 mg to about 75 mg of the compound per day, such as administering to the subject 5, 10, 25, 50, 75, 100, 200, 300, 400 or 500 mg of the compound per day.

[0088] In certain embodiments, the compound 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 compound may be administered to the subject for a period that includes at least the duration of hemodialysis.

[0089] In another embodiment of the method of treating calciphylaxis, in addition to measuring the change / reduction in lesion size after administration of the disclosed compound, pre-drug administration and post-drug administration, a biopsy of the relevant lesion can be taken using von Kassa staining to determine the tissue level of PTH and evidence of changes in calcium and phosphate deposition in dermal arterioles.

[0090] The presence of uremic acidification is determined, for example, by an increase in lipid peroxidation in plasma, detected by an increase in F2 isoprostanes, an increase in isolevuglandin - protein adducts in plasma; an increase in the oxidation of proteins and amino acids, detected, for example, by the oxidation of tyrosine residues, cysteine residues or methionine residues, lysine oxidation and threonine oxidation, thiol oxidation and carbonyl formation in plasma proteins; the formation of reactive aldehydes, detected, for example, by detecting glyoxal, methylglyoxal, acrolein, glycolaldehyde and parahydroxy phenacetaldehyde; an increase in reactive carbonyl compounds, measured, for example, by measuring the amount of hydrazine formed after reaction with 2,4 - dinitrophenylhydrazine; a decrease in plasma glutathione levels and glutathione peroxidase function; and an increase in the ratio of oxidized thiol to reduced thiol measured.

[0091] In another embodiment, a method for treating, preventing, decelerating the progression of, arresting, 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, decelerating the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 0.2 mg or at least 2 mg per day of a substantially pure compound disclosed herein to prevent, decelerate the progression of, and / or arrest tissue calcification, wherein the compound is administered in a pharmaceutical composition, is provided. In another aspect of the method, the mammal has diabetes. In yet another aspect, the mammal has type II diabetes or the mammal is diagnosed with prediabetes. In another aspect, the mammal has chronic kidney disease. In another aspect of the above method, the mammal has stage 4 or 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 aspect, the anticoagulant therapy is oral anticoagulant therapy. In another aspect of the method, the anticoagulant therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban, eliquixaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

[0092] In another embodiment, there is provided a method for treating, preventing, decelerating the progression of, arresting, and / or reversing tissue calcification in a mammal undergoing hemodialysis and in need of treating, preventing, decelerating the progression of, arresting, and / or reversing tissue calcification, the method comprising administering to the mammal at least 0.2 mg or at least 2 mg per day of a substantially pure compound disclosed herein, thereby preventing, decelerating the progression of, arresting, and / or reversing tissue calcification, wherein the disclosed compound is administered in a pharmaceutical composition. In another aspect, the mammal has diabetes.

[0093] Metabolism of Vitamin K: Development of vascular and soft tissue calcification after the inability to regenerate the reduced form of vitamin K: Vitamin K is an essential cofactor enzyme required for the post-translational modification of vitamin K-dependent (VKD) proteins. There are a number of VKD proteins, many of which are clinically relevant in patients with ESRD. They include central coagulation factors such as factor II, factor VII, factor IX, and factor X, as well as extracellular matrix proteins including matrix GLA-1 and osteocalcin. Under normal conditions, vitamin K is reduced by the enzyme NADPH oxidase to vitamin K hydroquinone (KH2). 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. Further oxidation of vitamin KH2 to 2-3 epoxide vitamin K occurs by enzymatic carboxylation of glutamate residues (Figure 2). In the final step of the vitamin K cycle, vitamin K 2-3 epoxide must be oxidized by an enzyme to return to its native structure. This step, which is catalyzed by vitamin K epoxide reductase (VKOR), is also a component of the vitamin K cycle that is 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 to explain the high incidence of calciphylaxis and other forms of dystrophic calcification among patients receiving warfarin therapy.

[0094] In one variant, administration or supplementation with the disclosed compounds and compositions reduces the risk of vascular and soft tissue calcification by increasing the amount of primary calcium phosphate protein particles (CPP) consisting of fetuin A and carboxylated matrix GLA-1 protein. Under normal physiological conditions, plasma calcium and phosphate concentrations are nearly supersaturated and thus are expected to precipitate as crystalline hydroxyapatite in blood vessels and soft tissues. The observation that this process does not occur suggests that there are effective chemical and biological means to block pathological calcification. Recent studies have shown that circulating calcium phosphate crystals form complexes with two calcification-inhibiting proteins to form primary calcium phosphate protein particles (CPP). These protein-inorganic complexes are mainly composed of fetuin A, a liver-derived protein that has been shown to prevent vascular calcification. A lesser amount of a second protein is matrix Gla-1 protein, which also functions to prevent pathological calcification. Matrix Gla-1 is a vitamin K-dependent protein, and initial studies by Price et al. have shown that the formation of fetuin-matrix Gla-1 inorganic nanoparticles (primary calcium phosphate CPP) depends on the gamma-carboxylation of matrix Gla-1. Preclinical studies have suggested that the calciprotein system functions as an alternative means to prevent pathological calcification when humoral defense lines such as pyrophosphate, magnesium, and albumin are breached. The "absorption" of calcium-phosphate crystals by primary CCP occurs in a cooperative and time-dependent process.

[0095] The time (T 50 ) to 50% saturation of primary CCP is accurate and is a sensitive means to determine the ability of plasma to "precipitate" or "absorb" excess calcium phosphate crystals. Patients with short T 50 times suggest a reduced ability to absorb calcium phosphate crystals, while patients with long T 50 times are consistent with a high ability. Recent clinical studies have shown that T 50The validity of the test has been proven, and low T 50 Time has been shown to be associated with an increased risk of myocardial infarction, heart failure, and all-cause mortality. Therefore, any clinical intervention that can increase the synthesis of circulating primary CCP will improve the ability to prevent pathological calcification. Patients with CKD and ESRD show a decrease in the levels of carboxylated matrix Gla-1 protein, and it is noted that this process is essential for the formation of primary CPP. Therefore, supplementation or administration of the disclosed compounds and compositions in patients with CKD or ESRD reduces the risk of pathological calcification and prevents the development of vascular and soft tissue calcification. By restoring the production of carboxylated matrix Gla-1 and GAS-6 by supplementation or administration of the disclosed compounds or compositions, the development of soft tissue and vascular calcification in skin tissue can be prevented or delayed.

[0096] The regeneration of vitamin K involves two key enzymes: vitamin K 2,3-epoxide reductase (VKOR) and NAD(P)H:quinone oxidoreductase (NQO1). As shown in the figure (FIG.), VKOR reduces 2,3-vitamin K epoxide to vitamin K quinone, while NADPH reduces vitamin K quinone to its hydroquinone form (KH2). Recent studies have shown that VKOR has two different isoforms (VKORC-1 and VKORC1-like-1 [VKORC1-L1]) that differ in both enzymatic properties and tissue distribution. For example, Westhofen et al. showed that VKORC-L1 has one-third the affinity for 2,3-epoxide vitamin K compared to VKORC1. Subsequent studies have supported the hypothesis that VKOR-L1 is a specialized isoform that provides protection from oxidative damage through the regeneration of vitamin K. Incubating cultured HEK293T cells with H2O2 increased the expression level of VKOR-L1 and reduced the evidence of oxidative damage to the membrane. Clinical observations that calciphylaxis and vitamin K-dependent vascular calcification are more common in the dermis raise the question of whether there is differential expression of VKOR enzymes in the skin. Casper et al. determined that the mRNA expression of key enzymes is involved in the regeneration of vitamin K. Furthermore, the expression of NADPH in the dermis was below the detection level. These observations suggest that any condition or procedure that blocks the reconstitution of vitamin K (i.e., hemodialysis) predisposes that tissue to pathologic calcification.

[0097] The oxidative properties of uremic plasma and the oxidative effects of dialysis itself result in "metabolic inhibition" and accumulation of 2,3-epoxide vitamin K, as well as a decrease in intracellular levels of vitamin K2. The "downstream" effects of this blockade include the inability of gamma-carboxylated proteins to participate in the prevention of soft tissue and vascular calcification. The oxidative effects of hemodialysis exacerbate this effect, which can partly explain the tendency of ESRD patients to develop calciphylaxis and vascular calcification.

[0098] Relationship between vitamin K and circulating vitamin K-dependent proteins in CKD-ESRD patients: It is widely recognized that in ESRD patients, vitamin K levels may not decline despite malnutrition. For example, Holder et al. examined 172 stable dialysis patients and found that only 6% of the patients showed clinically significant vitamin K deficiency. However, when testing the levels of carboxylated osteocalcin in the patients, the levels were reduced in 60% of the patients. To confirm that this is a general effect of reduced vitamin K activity, the authors also measured PIVKA-II, another vitamin K-dependent protein. In fact, it was found that the levels of carboxylated prothrombin were reduced in 90% of both CKD and ESRD patients. In a similar study, Pilkey et al. measured vitamin K1 levels in 142 ESRD patients and found that most of the patients had adequate vitamin K stores, but 93% of the patients had levels of undercarboxylated osteocalcin exceeding 20% of the total level. There was no correlation between the total amount of vitamin K1 and the circulating levels of undercarboxylated osteocalcin. Such unexpected findings are consistent with the hypothesis that in uremic patients, total vitamin K levels can be normal, but the production of the reduced form is blocked by the oxidative properties of uremia.

[0099] In one variant form, administration or supplementation or dosing of the disclosed compounds and compositions reverses hemodialysis-induced inhibition of vitamin K-dependent proteins by normalization of functional reduced vitamin K. Based on the observation that oxidative conditions can disrupt the vitamin K cycle, it is suggested that the oxidative burden that occurs during hemodialysis contributes to the high rates of vascular and soft tissue calcification observed in the ESRD population. Studies by Himmelfarb et al. have confirmed that mere provision of hemodialysis can result in oxidation of numerous tissue proteins. For example, hydroxyl amino acid side chains are oxidized to carbonyl groups. In studies of CKD and ESRD patients, Himmelfarb et al. demonstrated the use of carbonyl side chain oxidation as a measure of overall oxidative burden, and demonstrated that both CKD and ESRD patients exhibit a much higher percentage (15-fold) (see FIG. 5) of carbonyl proteins compared to normal controls. The percentage of carbonyl proteins is even higher in patients undergoing dialysis, demonstrating that dialysis not only fails to reduce the oxidative burden but also appears to contribute to it. As shown in FIG. 5, patients with uremia were found to have carboxylated protein levels up to 15-fold higher. Thus, the oxidative burden generated by provision of hemodialysis results in oxidation of functional vitamin K hydroquinone (KH2) to non-functional native vitamin K. Oxidation of KH2 by hemodialysis blocks the ability to function as a cofactor for GGCX, which downstream results in reduced gamma-carboxylation of vitamin K-dependent proteins.

[0100] To confirm that uremia and hemodialysis disrupt the vitamin K cycle, the ratio of vitamin K quinone to 2-3 epoxide vitamin K and vitamin K hydroquinone (KH2) can be determined in patients with normal renal function, CKD patients (stages IV and V), and ESRD patients. To determine whether the hemodialysis process itself further disrupts the vitamin K cycle, the inventors can measure the levels of oxidized vitamin K immediately before hemodialysis, then during dialysis (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 measurable. Patients with CKD and ESRD have higher levels of 2-3 epoxide vitamin K and lower levels of vitamin hydroquinone (KH2) compared to controls. To determine whether the loss of the reduced form of vitamin K (KH2) results in a decrease in the carboxylation of vitamin K-dependent proteins, the inventors can measure the levels of the following biomarkers in controls, CKD (stages IV and V), and ESRD (before dialysis, after dialysis). Matrix GLA-1 protein; growth arrest-specific gene 6 (Gas-6) protein; PIVAK-II protein; osteocalcin; protein C; protein S; fetuin A; and osteoprotegerin (plasma level during dialysis: 6.7 ± 2.2 pmol / L). The inventors expand these studies by including patients undergoing stable hemodialysis three times a week. The levels of carboxylated vitamin K-dependent proteins and uncarboxylated vitamin K-dependent proteins in pre-dialysis serum can be compared to the levels obtained at 2 hours and at the end of the dialysis session. The oxidative effect of dialysis itself results in a decrease in the levels of carboxylated vitamin K2-dependent proteins.

[0101] In one variant, administration or supplementation with the disclosed compounds and compositions in ESRD patients with uremic arteriolosclerosis (calciphylaxis) shortens the time to wound healing by preventing new blood vessel calcification and restoring blood flow: Skin biopsy: To confirm that supplementation with the disclosed compounds and compositions prevents the development of small vessel calcification and skin ischemia, the inventors identified patients with calciphylaxis confirmed by dermal biopsy and randomized the patients to treatment with menaquinone-7 or placebo. In another variant of the above procedure, this treatment uses menaquinone-9 or placebo. Clinical endpoints can include: 1) the time to removal of vacuum therapy for wounds, and 2) the time to wound healing defined as the time required for a 50% reduction in the sum of the surface areas of all calciphylaxis wounds.

[0102] In another variant, administration or supplementation with the disclosed compounds and compositions results in a significant improvement in the bioavailability of the compounds, including menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9 and menaquinone-10 / menadiol-10 and their individual mixtures, compared to administration or supplementation. In one variant, the bioavailability is improved by at least 5%, 10%, 15%, 20%, 30%, 40%, 50% or higher, respectively, compared to administration or supplementation using menaquinone-7, menaquinone-8, menaquinone-9 and menaquinone-10.

[0103] In another variant, administration or supplementation with the disclosed compounds and compositions results in a significant improvement in the serum half-life of the compounds, including menaquinone-7 / menadiol-7, menaquinone-8 / menadiol-8, menaquinone-9 / menadiol-9 and menaquinone-10 / menadiol-10 and their individual mixtures, compared to administration or supplementation. In one variant, the serum half-life is improved by at least 5%, 10%, 15%, 20%, 30%, 40%, 50% or more compared to administration or supplementation using menaquinone-7, menaquinone-8, menaquinone-9 and menaquinone-10, respectively.

[0104] Histopathological endpoint: Comparison of diagnostic skin biopsies after 12 weeks of menaquinone-7 treatment with skin biopsies at protocol repeat. Changes in the level of interstitial calcium deposition were defined as changes in Von Kossa staining that could be quantified by digital image color analysis. The inventors were able to validate biomarkers at the tissue level using skin biopsies. This process allows for the confirmation of the preventive properties of the disclosed menadiol derivatives against early vascular calcification. Validation of these biomarkers in tissue also allows clinicians to utilize the biomarkers as a means of tracking clinical response. Microvascular calcification precedes the development of CUA lesions. The level of calcification can be quantified by Von Kossa calcium staining in peripheral tissue and normalized as calcium content per unit area. Von Kossa can be used as a means of confirming the preventive properties of the disclosed menadiol derivatives against the development of vascular calcification.

[0105] In one variant, in ESRD patients with uremic arteriolosclerosis (calcinosis; CUA), replenishing the disclosed compounds and compositions normalizes carboxyprotein C levels in the dermis and prevents primary thrombosis of skin blood vessels, reducing the time to wound healing. Thus, in one variant, administration or supplementation with the disclosed compounds or compositions in diabetic patients prevents the development of vascular dementia by preventing the development of calcification and microvascular damage.

[0106] In yet another embodiment, there is provided a formulation of a fortified food or beverage comprising the step of adding to the food or beverage a composition comprising any one of the above compounds or a mixture thereof.

[0107] Similarly, salts of amino acids such as alginates, gluconates and galacturonates are included in the above embodiments, aspects and variants. Certain compounds of the invention may exist in unsolvated forms, as well as solvated forms including hydrated forms, and are intended to be within the scope of the invention. Similarly, pharmaceutical compositions are provided that contain pharmaceutically acceptable additives and a therapeutically effective amount of at least one compound of the invention.

[0108] Pharmaceutical compositions comprising a compound of the invention or a derivative thereof may be formulated as solutions or lyophilized powders for parenteral administration. The powder can be reconstituted prior to use by the addition of a suitable diluent or other pharmaceutically acceptable carrier. Liquid formulations are generally buffered isotonic aqueous solutions. Examples of suitable diluents are normal isotonic saline aqueous solutions, 5% dextrose in water or buffered sodium or ammonium acetate solutions. Such formulations are particularly suitable for parenteral administration but can also be used for oral administration. Additives such as polyvinylpyrrolidone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride or sodium citrate may also be added. Alternatively, these compounds may be encapsulated, tableted or prepared as emulsions or syrups for oral administration.

[0109] A pharmaceutically acceptable solid or liquid carrier can be added to enhance or stabilize the composition, or to facilitate the preparation of the composition. Examples of liquid carriers include syrups, peanut oil, olive oil, glycerin, physiological saline, alcohol, or water. Solid carriers include starch, lactose, calcium sulfate dihydrate, clay, magnesium stearate or stearic acid, talc, pectin, acacia, agar, or gelatin. The carrier can contain a sustained release substance such as glyceryl monostearate or glyceryl distearate alone or together with wax. The amount of the solid carrier varies, but can be about 20 mg to about 1 g per dosage unit. Pharmaceutical preparations are prepared according to conventional pharmaceutical techniques including, for example, milling, mixing, granulating, and compressing for tablet form, or milling, mixing, and filling for hard gelatin capsule form, as required. When a liquid carrier is used, the preparation is in the form of a syrup, elixir, emulsion, or aqueous or non-aqueous suspension. Such liquid preparations may be administered directly p.o. 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.

[0110] The disclosed compounds and compositions may contain oleic acid, Kolliphor® EL (polyoxyl castor oil or Cremophor EL), vitamin E TPGS (D-α-tocopherol polyethylene glycol-1000 succinate), Savie (an emulsifier derived from polysarcosine), Maisine® CC (glyceryl monolaurate), Gelucire® 44 / 14 (lauroyl polyoxyl-32 glyceride), Miglyol® 812N (ester of capric fatty acid and glycerin derived from saturated coconut oil and palm kernel oil), Plurol® Oleique (polyglyceryl-6 dioleate), Lauroglycol™ 90 (propylene glycol monolaurate (type II), Labrasol® (caprylocaprylyl polyoxyl-8 glyceride), Captisol® (SBE-β-cyclodextrin), Encapsin™ HPB (hydroxypropyl-β-cyclodextrin), Peceol™ (glycerol monooleate / glyceryl (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil® M2125CS (linoleoyl polyoxyl-6 glyceride), and a solubility enhancer or solubilizer selected from medium-chain monoglycerides and diglycerides.

[0111] In one variant form, the compounds or pharmaceutically acceptable salts thereof disclosed herein are provided, optionally, in the form of their single stereoisomers or mixtures of stereoisomers, and compositions containing the compounds.

[0112] In addition to the above exemplary embodiments, aspects, and variant forms, further embodiments, aspects, and variant forms will become apparent by reference to the drawings and figures and by consideration of the following description.

Mode for Carrying Out the Invention

[0113] Detailed Description of the Invention Definition: Unless specifically stated otherwise herein, the definitions of the terms used are the standard definitions used in the fields of organic synthesis and pharmaceutical science. Exemplary embodiments, aspects, and variations are illustrated (illustratived) in the figures and drawings, and it is intended that the embodiments, aspects, and variations, as well as the figures and drawings, disclosed herein be considered illustrative and not limiting.

[0114] "Pharmaceutically acceptable salt" means 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 use. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, or organic acids such as acetic acid, propionic acid, hexanoic acid, malonic acid, succinic acid, malic acid, citric acid, gluconic acid, salicylic acid, etc.

[0115] "Therapeutically effective amount" means the amount of a compound or drug that exerts any of the biological effects listed herein.

Brief Description of the Drawings

[0116]

Figure 1

[0117]

Figure 2

[0118]

Figure 3

[0119]

Figure 4

[0120]

Figure 5

Example

[0121] Experiment: The following procedures can be used for the preparation of the compounds of the present invention. The starting materials and reagents used in the preparation of these compounds are available from commercial suppliers such as Aldrich Chemical Company (Milwaukee, Wis.), Bachem (Torrance, Calif.), Sigma (St. Louis, Mo.), or can be prepared by methods well known to those skilled in the art according to the procedures described in reference works such as Fieser and Fieser's Reagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps., Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: Advanced Organic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

[0122] In some cases, a protecting group may be introduced and finally removed. Suitable protecting groups for amino, hydroxy and carboxy groups are described in Greene et al., Protective Groups in Organic Synthesis, Second Edition, John Wiley and Sons, New York, 1991. Standard organic chemical reactions can be achieved by using several different reagents, for example, as described in Larock: Comprehensive Organic Transformations, VCH Publishers, New York, 1989. Preparation of menaquinol derivatives General reaction scheme:

Chem.

Chem.

[0123] Alternatively, the preparation of menaquinol mono - esters and diester derivatives can be carried out using HATU (1 - [bis(dimethylamino)methylene] - 1H - 1,2,3 - triazolo[4,5 - b]pyridinium 3 - oxide hexafluorophosphate, hexafluorophosphate azabenzotriazolium tetramethyluronium) as a coupling reagent. In this case, the corresponding pegylated carboxylic acid A (X = - OH) is treated with HATU to form an - OAt - active ester, and then reacted with the corresponding menaquinol formed from menaquinone. See, for example, Carpino L A et al. The uronium / guanidinium peptide coupling reagents: Finally the true uranium salts. Angewandte Chemie International Edition, 2002, 41(3): 441 - 445; Carpino L A et al.; Comparison of the Effects of 5 - and 6 - HOAt on Model Peptide Coupling Reactions Relative to the Cases for the 4 - and 7 - Isomers. Organic letters, 2000, 2(15): 2253 - 2256. Selective hydrolysis of the diester:

Chemical formula

[0124] Furthermore, the selective formation of monoester menaquinol at the 1- or 4-position can be carried out by first preparing a diester and then selectively hydrolyzing one of the ester groups to form the desired monoester derivative. For example, under certain controlled conditions, it has been demonstrated that the menaquinol ester formed at the 4-position of the quinone (the ketone adjacent to the isoprenoid side chain) is more readily hydrolyzed than the menaquinol ester group at the 1-position. Standard hydrolysis conditions include aqueous NaOH in the presence of an organic solvent such as THF or diethyl ether, or NaOMe in THF and / or methanol or ethanol. Alternatively, hydrolysis can be carried out in an organic solvent or a mixture of organic solvents such as THF or ether, at an elevated temperature such as about 30 °C to 50 °C, in dilute acidic water such as HCl or H2SO4, if necessary. If a mixture of monoester and diester is formed from the hydrolysis reaction, i.e., if the selective hydrolysis is incomplete, the desired monoester can be readily separated from the diester by column chromatography or preparative chromatography.

[0125] Under various standard conditions known in the art, the acylation reaction and the selective hydrolysis reaction result in the desired monoacylated derivative or diacylated derivative having the expected 1 1H-NMR spectral display. General reaction scheme:

Chemical formula

Chemical formula

[0126] To a stirred solution of Na2S2O4 (42.4 g, 0.24 mol) in water (160 mL, 8V) was added 2-methyl-1,4-dihydronaphthalene-1,4-dione 1 (20 g, 0.116 mol) in ethyl acetate (160 mL, 8V) at room temperature (25 - 30 °C). The reaction mixture was stirred at room temperature for 2 h. Completion of the reaction was observed by the color change from dark brown to light brown solution. The reaction mixture was transferred to a separatory funnel and the separated organic layer was dried over sodium sulfate and concentrated. The resulting crude product was taken up in acetic anhydride (66 mL, 0.69 mol), cooled to 0 °C, and concentrated H2SO4 (4 mL, 0.2V) was added. The reaction mixture was stirred at room temperature for 2 h. Completion of the reaction was monitored by TLC R f = 0.4 (20% ethyl acetate / hexane).

[0127] The reaction mixture was slowly added to water (500 mL) at ambient temperature (25 - 30 °C) and stirred at the same temperature (25 - 30 °C) for 10 min. The resulting solid was filtered and dried to give compound 2 as a brown solid (26.0 g, 87%). R f = 0.4 (20% ethyl acetate / hexane). 1 1H NMR (400 MHz, CDCl3) δ (ppm): 7.86 - 7.80 (m, 1H), 7.80 - 7.72 (m, 1H), 7.57 - 7.44 (m, 2H), 7.15 (s, 1H), 2.47 (d, J = 11.0 Hz, 6H), 2.33 (s, 3H). LC-MS m / z(M+1): 259.8 (calculated molecular weight - 258.26). Step - 2: Preparation of 4-hydroxy-2-methylnaphthalen-1-yl acetate (3):

Chemical formula

[0128] To a stirred solution of compound 2 (25 g, 0.096 mol) in ethanol (500 mL, 20 V) was added sodium hydroxide (3.8 g, 0.096 mol) and Na2S2O4 (5.0 g, 0.029 mol) in water (50 mL, 2 V) at room temperature (25 - 30 °C). The reaction mixture was stirred at room temperature for 2 hours. The completion of this reaction was monitored by TLC. The reaction mixture was neutralized with 1.5 N HCl (pH - 3 to 4), and the resulting reaction mixture was filtered and the filtrate was concentrated (ethanol was removed). The crude product was diluted with ethyl acetate (500 mL) and washed with water (2 × 200 mL) and brine solution (200 mL). The separated organic layer was dried over sodium sulfate and concentrated. Washing the crude product with n - hexane (3 × 100 mL) gave compound 3 as a brown solid (18.2 g, 87%). R f = 0.3 (20% ethyl acetate / hexane). 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.03 (dt, J = 8.3, 1.1 Hz, 1H), 7.70 - 7.61 (m, 1H), 7.49 (ddd, J = 8.4, 6.8, 1.4 Hz, 1H), 7.41 (ddd, J = 8.3, 6.8, 1.3 Hz, 1H), 6.48 (s, 1H), 5.54 (d, J = 1.5 Hz, 1H), 2.49 (s, 3H), 2.21 (s, 3H). LC - MS m / z(M + 1): 217.6 (calculated molecular weight - 216.23). Synthesis of MK - 7 - monoacetate: Step - 3: Preparation of 3 - [(2E,6E,10E,14E,18E,22E) - 3,7,11,15,19,23,27 - heptamethyloctacosa - 2,6,10,14,18,22,26 - heptaen - 1 - yl] - 4 - hydroxy - 2 - methylnaphthalen - 1 - yl acetate (4):

Chem.

[0129] To a stirred solution of Compound 3 (10.0 g, 0.046 mol) in toluene (100 mL, 10 V) at room temperature (25 - 30 °C), heptaprenol (16.0 g, 0.032 mol) and benzenesulfonic acid (0.72 g, 0.0046 mol) were added. The reaction mixture was stirred at room temperature for 16 - 24 hours. The completion of this reaction was monitored by TLC. The reaction mixture was diluted with ethyl acetate (500 mL) and washed with water (2 × 250 mL) and brine solution (200 mL). The separated organic layer was dried over sodium sulfate and concentrated. The obtained crude product was purified by column chromatography (2 - 3% ethyl acetate / hexane) and crystallized from a combination of acetone and N - heptane (1:4 V), and Compound 4 was obtained as an off - white solid (4.1 g, 13%). R f = 0.5 (15% ethyl acetate / hexane). 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.13 - 8.10 (m, 1H), 7.64 - 7.62 (m, 1H), 7.46 - 7.39 (m, 2H), 5.70 (s, 1H), 5.27 - 5.24 (m, 1H), 5.11 (tp, J = 6.8, 4.4, 2.9 Hz, 6H), 3.53 (d, J = 6.8 Hz, 2H), 2.47 (s, 3H), 2.26 (s, 3H), 2.17 - 1.93 (m, 24H), 1.88 (d, J = 1.3 Hz, 3H), 1.68 (d, J = 1.6 Hz, 3H), 1.59 (d, J = 9.7 Hz, 18H). LC - MS m / z(M + 1): 693.39 (calculated value of monoisotopic mass - 692.51). Synthesis of MK - 7 - PEG2 - monoacetate: Step - 4: Preparation of 4 - (acetyloxy) - 2 - [(2E,6E,10E,14E,18E,22E) - 3,7,11,15,19,23,27 - heptamethyloctacosa - 2,6,10,14,18,22,26 - heptaen - 1 - yl] - 3 - methylnaphthalen - 1 - yl 2 - [2 - (2 - methoxyethoxy)ethoxy]acetate:

Chemical Structure

[0130] To a solution of Compound 5 (0.41 g, 0.00230 mol) in DCM (10 mL, 10 V) was added N,N'-dicyclohexylcarbodiimide (0.534 g, 0.0025 mol) and 4-dimethylaminopyridine (17 mg, 0.00014 mol) at 0 - 5 °C, and the reaction mixture was stirred at the same temperature (0 - 5 °C) for 10 minutes. To this reaction mixture was added Compound 4 (1.0 g, 0.0014 mol) in DCM (10 mL) at 0 - 5 °C, and the mixture was slowly stirred at ambient temperature over 2 hours and then at 25 - 30 °C for 2 hours. The completion of this reaction was monitored by TLC. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (2 × 50 mL) and brine solution (100 mL). The separated organic layer was dried over sodium sulfate and concentrated. The obtained crude product was purified by column chromatography (20% ethyl acetate / hexane) to give Compound MK-7-PEG2-monoacetate as a thick pale yellow liquid (738 mg, 60%). R f = 0.4 (20% ethyl acetate / hexane). 1 H NMR (400 MHz, CDCl3) δ (ppm): 7.74 - 7.60 (m, 2H), 7.51 - 7.41 (m, 2H), 5.15 - 5.01 (m, 7H), 4.62 (s, 2H), 3.93 - 3.86 (m, 2H), 3.80 - 3.65 (m, 4H), 3.61 - 3.54 (m, 2H), 3.39 (s, 5H), 2.48 (s, 3H), 2.24 (s, 3H), 2.10 - 1.90 (m, 24H), 1.72 (dd, J = 35.3, 1.3 Hz, 6H), 1.57 (d, J = 1.7 Hz, 18H). LC-MS m / z(M+1): 853.39 (calculated value of monoisotopic mass - 852.59). HPLC purity: 99.58%. Synthesis of MK-7-PEG4-monoacetate: Preparation of Engineering-4: 4-(acetyloxy)-2-[(2E,6E,10E,14E,18E,22E)-3,7,11,15,19,23,27-heptamethyloctacosa-2,6,10,14,18,22,26-heptaen-1-yl]-3-methylnaphthalen-1-yl 2,5,8,11,14-pentaoxahexadecane-16-oate: [Chemical formula]

[0131] To a solution of Compound 5 (0.614 g, 0.00230 mol) in DCM (10 mL, 10V) was added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.497 g, 0.0025 mol) and 4-dimethylaminopyridine (17 mg, 0.00014 mol) at 0 - 5 °C, and the reaction mixture was stirred at the same temperature (0 - 5 °C) for 10 minutes. To this reaction mixture was added Compound 4 (1.0 g, 0.0014 mol) in DCM (10 mL) at 0 - 5 °C, and the mixture was slowly stirred at ambient temperature over 2 hours and then at 25 - 30 °C for 2 hours. The completion of this reaction was monitored by TLC. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (2 × 50 mL) and brine solution (100 mL). The separated organic layer was dried over sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (40% ethyl acetate / hexane) to give Compound MK-7-PEG4-monoacetate as a thick pale yellow liquid (842 mg, 62%). R f = 0.4 (40% ethyl acetate / hexane). 11H NMR (400 MHz, CDCl3) δ (ppm): 7.76 - 7.59 (m, 2H), 7.55 - 7.41 (m, 2H), 5.19 - 4.99 (m, 7H), 4.62 (s, 2H), 3.88 (dd, J = 5.8, 3.5 Hz, 2H), 3.75 (dd, J = 5.7, 3.5 Hz, 2H), 3.66 (ddt, J = 15.1, 9.2, 3.6 Hz, 10H), 3.53 (dd, J = 5.8, 3.6 Hz, 2H), 3.36 (s, 5H), 2.48 (s, 3H), 2.24 (s, 3H), 2.01 (dtd, J = 31.6, 8.5, 7.9, 4.8 Hz, 24H), 1.76 (d, J = 1.3 Hz, 3H), 1.68 (d, J = 1.5 Hz, 3H), 1.57 (s, 18H). LC-MS m / z(M+1): 941.31 (Calculated value of monoisotopic mass - 940.64). HPLC purity: 99.62%. Synthesis of MK-9-monoacetate: General reaction scheme:

Chemical formula

Chemical formula

[0132] To a stirred solution of Compound 3 (10.0 g, 0.046 mol) in toluene (100 mL, 10 V) at room temperature (25 - 30 °C), solanesol (20.45 g, 0.032 mol) and benzenesulfonic acid (0.72 g, 0.0046 mol) were added. The reaction mixture was stirred at room temperature for 16 - 24 h. Completion of the reaction was monitored by TLC (15% ethyl acetate / hexane). The reaction mixture was diluted with ethyl acetate (500 mL) and washed with water (2 × 250 mL) and brine solution (200 mL). The separated organic layer was dried over sodium sulfate and concentrated. The resulting crude product was purified by column chromatography (2 - 3% ethyl acetate / hexane) and crystallized from a combination of acetone and n - heptane to give Compound 4 as an off - white solid (4.1 g, 13%). R f = 0.5 (15% ethyl acetate / hexane). 1 H NMR (400 MHz, CDCl3) δ (ppm): 8.13 - 8.10 (m, 1H), 7.64 - 7.62 (m, 1H), 7.46 - 7.26 (m, 2H), 5.70 (S, 1H), 5.28 - 5.24 (m, 1H), 5.11 (tp, J = 6.8, 4.4, 2.9 Hz, 9H), 3.53 (d, J = 6.8 Hz, 2H), 2.47 (s, 3H), 2.26 (s, 3H), 2.17 - 1.93 (m, 32H), 1.88 (d, J = 1.3 Hz, 3H), 1.68 (d, J = 1.6 Hz, 3H), 1.59 (d, J = 9.7 Hz, 24H). LC - MS m / z(M + 1): 829.36 (calculated value of monoisotopic mass - 828.64). Synthesis of MK - 9 - PEG2 - monoacetate: Step - 4: Preparation of 4 - (acetyloxy) - 3 - methyl - 2 - [(2E,6E,10E,14E,18E,22E,26E,30E) - 3,7,11,15,19,23,27,31,35 - nonamethylhexatriaconta - 2,6,10,14,18,22,26,30,34 - nonaen - 1 - yl]naphthalen - 1 - yl 2 - [2 - (2 - methoxyethoxy)ethoxy]acetate: [Chemical]

[0133] To a solution of compound 5 (0.513 g, 0.00192 mol) in DCM (10 mL, 10V) at 0 - 5 °C, N,N'-dicyclohexylcarbodiimide (0.447 g, 0.0021 mol) and 4-dimethylaminopyridine (14 mg, 0.00012 mol) were added, and the reaction mixture was stirred at the same temperature (0 - 5 °C) for 10 minutes. To this mixture, compound 4 (1.0 g, 0.0012 mol) in DCM (10 mL) was added at 0 - 5 °C. The reaction mixture was stirred slowly at ambient temperature over 2 hours at 25 - 30 °C. The completion of this reaction was monitored by TLC. The reaction product was diluted with dichloromethane (100 mL) and washed with water (2 × 50 mL) and brine solution (100 mL). The separated organic layer was dried over sodium sulfate and concentrated. The obtained crude product was purified by column chromatography (20% ethyl acetate / hexane) to give compound MK-9-PEG2-monoacetate as an off-white solid (738 mg, 60%). R f = 0.4 (20% ethyl acetate / hexane). 1 H NMR (400 MHz, CDCl3) δ (ppm): 7.71 - 7.63 (m, 2H), 7.47 - 7.44 (m, 2H), 5.12 - 5.00 (m, 9H), 4.62 (s, 2H), 3.90 - 3.86 (m, 2H), 3.80 - 3.65 (m, 4H), 3.61 - 3.54 (m, 2H), 3.39 (s, 5H), 2.48 (s, 3H), 2.24 (s, 3H), 2.10 - 1.91 (m, 32H), 1.77 (d, J = 1.4 Hz, 3H), 1.68 (t, J = 1.4 Hz, 3H), 1.60 - 1.53 (m, 24H). LC-MS m / z(M+1): 989.75 (calculated value of monoisotopic mass - 988.71). HPLC purity: 99.18%. Synthesis of MK-9-PEG4-monoacetate: Preparation of Engineering-4: 4-(acetyloxy)-3-methyl-2-[(2E,6E,10E,14E,18E,22E,26E,30E)-3,7,11,15,19,23,27,31,35-nonamethyloctatriaconta-2,6,10,14,18,22,26,30,34-nonaen-1-yl]naphthalen-1-yl 2,5,8,11,14-pentaoxahexadecane-16-oate:

Chemical formula

[0134] To a solution of Compound 5 (0.513 g, 0.00192 mol) in DCM (2.0 L, 400 V) at 0 - 5 °C, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.416 g, 0.00217 mol) and 4-dimethylaminopyridine (17 mg, 0.00014 mol) were added, and the reaction mixture was stirred at the same temperature (0 - 5 °C) for 10 minutes. To the above reaction mixture, Compound 4 (1.0 g, 0.0012 mol) in DCM (10 mL) was added at 0 - 5 °C. The reaction mixture was stirred slowly at ambient temperature for 2 hours at 25 - 30 °C. The completion of this reaction was monitored by TLC. The reaction mixture was diluted with dichloromethane (100 mL) and washed with water (2 × 50 mL) and brine solution (100 mL). The separated organic layer was dried over sodium sulfate and concentrated. The obtained crude product was purified by column chromatography (40% ethyl acetate / hexane) to give Compound MK-9-PEG4-monoacetate as a thick pale yellow liquid (779 mg, 60%). R f = 0.4 (40% ethyl acetate / hexane). 11H NMR (400 MHz, CDCl3) δ (ppm): 7.71 - 7.63 (m, 2H), δ 7.47 - 7.45 (m, 2H), 5.09 (dt, J = 20.5, 7.7 Hz, 9H), 4.61 (s, 2H), 3.88 (dd, J = 5.7, 3.6 Hz, 2H), 3.74 (dd, J = 5.7, 3.6 Hz, 2H), 3.70 - 3.54 (m, 10H), 3.52 (dd, J = 5.9, 3.4 Hz, 2H), 3.37 (s, 5H), 2.48 (s, 3H), 2.24 (s, 3H), 2.02 (dt, J = 33.0, 7.5 Hz, 32H), 1.77 (s, 3H), 1.68 (d, J = 1.5 Hz, 3H), 1.60 (s, 24H). LC-MS m / z(M+1):1077.75(calculated value of monoisotopic mass - 1076.76). HPLC purity: 98.75% Synthesis of MK-9: Step-1: Preparation of 2-methyl-3-[(2E,6E,10E,14E,18E,22E,26E,30E)-3,7,11,15,19,23,27,31,35-nonamethylhexatriaconta-2,6,10,14,18,22,26,30,34-nonaen-1-yl]-1,4-dihydronaphthalene-1,4-dione) [Chemical formula]

[0135] To a stirred solution of Compound 1 (1.0 g, 0.0012 mol) in a mixture of acetonitrile:diethyl ether:water (1:1:1) (12 mL, 12 V) at room temperature (25 - 30 °C), cerium(IV) ammonium nitrate (1.9 g, 0.0036 mol) was added. The reaction mixture was stirred at room temperature for 1 - 2 hours. The completion of this reaction was monitored by TLC. The reaction mixture was diluted with ethyl acetate (100 mL) and washed with water (2 × 50 mL) and brine solution (100 mL). The separated organic layer was dried over sodium sulfate and concentrated. The obtained crude product was purified by column chromatography (2 - 3% ethyl acetate / hexane) and crystallized from a combination of acetone and n - heptane (1:4 V), and Compound MK9 was obtained as a yellow solid (580 mg, 51%). R f = 0.7 (15% ethyl acetate / hexane). 1 1H NMR (400 MHz, CDCl3) δ (ppm): 8.08 (ddd, J = 6.2, 3.1, 1.8 Hz, 2H), 7.71 - 7.66 (m, 2H), 5.20 - 4.99 (m, 9H), 3.37 (d, J = 7.0 Hz, 2H), 2.19 (s, 3H), 2.12 - 1.89 (m, 32H), 1.68 (d, J = 1.4 Hz, 3H), 1.66 - 1.51 (m, 24H). LC - MS m / z(M + 1): 785.27 (calculated value of monoisotopic mass - 784.61). HPLC purity: 98.0%.

[0136] The following representative compounds can be prepared using the above process. [Chemical formula] [Chemical formula]

[0137] Menadione - 7 (MK - 7) and Compound Ia.1, m = 9, n = 7: Comparison of single - dose oral pharmacokinetics in male Sprague - Dawley rats

Table 1-1

Table 1-2

[0138] Approximately 5 mg of menaquinone-7 was weighed and transferred to a graduated tube. Next, 10 mL of sunflower oil was added and vortexed thoroughly until the test article was completely dissolved. The formulation was prepared in a dark room under monochromatic light. The final strength of the formulation was 500 μg / mL. The formulation was freshly prepared before administration. Oral PK study of menaquinone-7 in male Sprague-Dawley rats: Clinical observation:

Table 2

Table 3

Chemical formula

Table 4

Table 5

[0139] Single-dose oral pharmacokinetics of compound Ia.1, m = 9, n = 7 at an equivalent dose of 1000 μg of menaquinone-7 per kg of body weight in male Sprague-Dawley rats: Oral PK study of compound Ia.1, m = 9, n = 7 in male Sprague-Dawley rats:

Table 6

[0140] Weigh approximately 6 mg of Compound Ia.1, m = 9, n = 7 and transfer it to a graduated tube. Next, add 9.836 mL of sunflower oil and vortex mix thoroughly until the test article is completely dissolved. The formulation was prepared in a dark room under monochromatic light. The final strength of the formulation is 610 μg / mL (equivalent to 500 μg / mL of MK-7). The formulation is freshly prepared before administration. Oral PK test of Compound Ia.1, m = 9, n = 7 in male SD rats: Clinical observation: [Table 7-1] [Table 7-2]

[0141] Results: Oral PK test of Compound Ia.1, m = 9, n = 7 in male SD rats - Plasma concentration of Compound Ia.1, m = 9, n = 7: [Table 8] Oral PK test of Compound Ia.1, m = 9, n = 7 in male SD rats - Plasma concentration of menaquinone-7: [Table 9] Results: Oral PK test of Compound Ia.1, m = 9, n = 7 in male SD rats: PK parameters in plasma of MK-7: [Table 10] [Table 11] Results: Comparative data - Plasma concentration of menaquinone-7: [Table 12] Comparative Data - Plasma PK Parameters of Menadione-7:

Table 13

[0142] Based on the above results, in rats, after oral administration of 1 mg / kg b.w. of menadione-7 using sunflower oil as the formulation vehicle, the maximum plasma concentration is 19 ng / mL. When compound Ia.1, m = 9, n = 7 is administered at the same dose, the observed maximum plasma concentration is approximately 40% higher. Depending on the compound tested, the maximum plasma concentration ranges from approximately 40% to 60% higher.

[0143] The observed plasma exposure of menadione-7 after administration of menadione-7 was 279.43 ± 74.91, while the plasma exposure of menadione-7 after compound Ia.1, m = 9, n = 7 was approximately 45% higher. It can be seen that the plasma exposure of menadione-7 is higher after oral administration of compound Ia.1, m = 9, n = 7 compared to oral administration of menadione-7 alone at a similar dose of 1000 μg per kg of body weight. The concentration of compound Ia.1, m = 9, n = 7 was not observed. Administration of the Compounds in Subjects at Risk of Developing Calciphylaxis:

[0144] This example describes the administration of the compounds of the present application to subjects at risk of developing calciphylaxis but who have not yet developed the characteristic skin lesions of calciphylaxis. The possible risk factors include, but are not limited to, diabetes, obesity, hemodialysis, and previous 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 compounds can result in protection of the subjects from skin lesions and changes in certain biomarker levels that serve as indicators for the prevention of the development of calciphylaxis.

[0145] Subjects at risk of developing calciphylaxis are administered orally once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year or indefinitely with the selected compound of the present application at 5 mg, 10 mg, 20 mg, 30 mg, 50 mg or 100 mg. The dosage form is a soft gel capsule of 5 mg, 10 mg, 20 mg, 50 mg or 100 mg. Two 50 mg capsules will be administered once daily to the 100 mg dosage cohort. All subjects with a high risk factor for calciphylaxis will develop characteristic skin lesions of calciphylaxis. The intention of active treatment with the compound of the present application (prior to clinical diagnosis of calciphylaxis) is to prevent the appearance of the lesions. A decrease or elimination in the frequency of appearance of the lesions is contemplated as the success of the treatment.

[0146] It is possible to evaluate several biomarkers to determine the efficacy of a compound administered at three dosage levels. Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein (MGP), and total 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. Obtain a blood sample and measure the biomarkers according to the following schedule. Blood sampling can be performed during treatment on a weekly or monthly basis. Administration of the disclosed compound is intended to result in (i) a decrease in PIVKA-II, which is an indicator of deceleration, arrest or reversal of the progression of calciphylaxis, (ii) a decrease in uncarboxylated MGP, uncarboxylated osteocalcin and / or uncarboxylated protein C, which are indicators of deceleration, arrest or reversal of the progression of calciphylaxis. Furthermore, pulse wave velocity (PWV) can be measured to evaluate arterial compliance. Improvement in vascular compliance is an indicator of deceleration, arrest or reversal of the progression of calciphylaxis. Administration of the disclosed compounds of the present application to a subject diagnosed with calciphylaxis:

[0147] This example describes the administration of the disclosed compounds to a subject diagnosed with calciphylaxis. Typical symptoms include the appearance of skin lesions with characteristic pain (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. Additional conditions are thought to be required for an accurate diagnosis.

[0148] Subjects diagnosed with calciphylaxis receive the disclosed compound orally once daily at 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is a soft gel capsule of 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg. Two 50 mg capsules are administered once daily to the 100 mg dosage cohort.

[0149] Cessation or reduction of lesion size and frequency is intended to be an indicator of treatment success. Administration of the disclosed compound as described above results in cessation or reduction of lesion size and frequency. Further, since calciphylaxis has a high risk of lethality, an increase in the overall survival period of the diagnosed subject will be an indicator of treatment success. Further, administration of the disclosed compound as described above results in an increase in the overall survival period of the diagnosed subject. Administration of the disclosed compound in subjects with end-stage renal disease (ESRD) reverses or decelerates the progression of tissue calcification:

[0150] This example describes the administration of the disclosed compound to subjects in a stable hemodialysis state with ESRD. Administration of the disclosed compound results in changes in certain biomarker levels that are indicators of deceleration, cessation, or reversal of the progression of aortic compliance (by plethysmography), vascular calcification, and tissue calcification.

[0151] ESRD subjects in a stable hemodialysis state receive the disclosed compound orally once daily at 5 mg, 10 mg, 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, or 500 mg for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely. The dosage form is a soft gel capsule of 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, or 100 mg. Two 50 mg capsules are administered once daily to the 100 mg dosage cohort.

[0152] A 50-year-old, 65-kg male patient diagnosed with typical symptoms associated with moderate calciphylaxis is treated for 8 weeks with 100 mg of compound Ia.1, m = 9, n = 7. After the treatment period, the patient is hospitalized and evaluated. The patient is found to have a significant change in the tested biomarker levels, suggesting a reduction of approximately 10% in vascular calcification and also showing a 10% reduction in tissue calcification.

[0153] A 65-year-old, 45-kg female patient diagnosed with typical symptoms and diagnosis associated with moderate calciphylaxis is treated for 10 weeks with 20 mg of compound Ia.1, m = 9, n = 7. After the treatment period, the patient is hospitalized and evaluated. The patient is found to have a significant change in the tested biomarker levels, suggesting a reduction of approximately 20% in vascular calcification and also showing a 15% reduction in tissue calcification.

[0154] A 55-year-old, 70-kg male patient diagnosed with typical symptoms and diagnosis associated with moderate calciphylaxis is treated for 3 months with 50 mg of compound Ia.1, m = 9, n = 7. After the treatment period, the patient is hospitalized and evaluated. The patient is found to have a significant change in the tested biomarker levels, suggesting a reduction of approximately 25% in vascular calcification and also showing a 20% reduction in tissue calcification.

[0155] The coronary artery calcium score (CAC) is used to estimate the degree of calcification of the thoracic artery. A high CAC score is an indicator of calcification, and the treatment aims to halt the long-term improvement of the CAC score, or its reversal or deceleration of the acceleration rate.

[0156] Aortic pre-pulse plethysmography is also used to measure arterial compliance, which decreases as calcification increases. Pulse wave velocity (PWV) is also measured to assess arterial compliance. The above-mentioned metrics are useful for estimating the usefulness of treatments intended to prevent, slow the progression of, halt or reverse vascular calcification. These measurements are used before and after treatment with the disclosed compounds to evaluate the value of the treatment.

[0157] Several biomarkers are evaluated to determine the efficacy of the disclosed compounds at three dose levels. Exemplary biomarkers include PIVKA-II; uncarboxylated matrix Gla protein (MGP), and total MGP; uncarboxylated osteocalcin protein, carboxylated osteocalcin protein, and total osteocalcin protein; uncarboxylated protein C, carboxylated protein C, and total protein C and hs-CRP. Blood samples are obtained and, most conveniently, the biomarkers are measured while the patient is present at the hospital for hemodialysis.

[0158] Administration of the disclosed compounds can result in (i) a decrease in PIVKA-II, which is an indicator of deceleration, arrest, or reversal of the progression of tissue calcification; (ii) a decrease in undercarboxylated MGP, undercarboxylated osteocalcin, and / or undercarboxylated protein C, which are indicators of deceleration, arrest, or reversal of the progression of tissue calcification; and / or (iii) a decrease in hs-CRP, which is an indicator of deceleration, arrest, or reversal of the progression of tissue calcification, and / or reduction of inflammation. After administration of 0.2 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 300 mg, 400 mg, or 500 mg, or more of the disclosed compounds and compositions per day, at least one of PIVKA-II, undercarboxylated matrix Gla protein (MGP), and undercarboxylated osteocalcin protein shows a change that is an indicator of deceleration, arrest, or reversal of the progression of tissue calcification. The biological activities of the above-mentioned compounds containing menaquinol derivatives of formula I, II, or III and the compounds disclosed herein are tested using 13 various representative cell lines, both in cell stocks and mouse cell models, and also using the HL-60 human leukemia cell line disclosed in the following experimental items. Other cell lines for testing various cancers can be obtained from the American Type Culture Collection (ATCC). Study design: Cells are seeded in 96-well microplates, and after 3 days of exposure to the above-mentioned disclosed compounds, the CellTiter-Glo Luminescent Cell Viability kit is used to determine the number of viable cells in the culture based on the quantification of ATP present, which represents the presence of metabolically active cells. The OD signal is proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells present in the culture. This method is used to monitor the drug-induced effect on cell proliferation. Substances: Reagents and antibodies [Table 14] Description of cell lines and culture medium components:

Table 15-1

Table 15-2

[0159] Test 10 mg of the compound in DMSO at a concentration of 100 μM in a 20 mM stock solution using 1 mL of the stock solution. Preparation of plates for cell assays: Day 1

[0160] 1) Using a 37°C water bath, pre-warm the cell medium, 0.25% trypsin, and the medium.

[0161] 2) Evaluate the degree of confluence and observe the cells under a microscope to confirm the absence of bacterial and fungal contaminants.

[0162] 3) Remove the medium and wash the cells twice with 10 mL of PBS. Add 5 mL of 0.25% trypsin / EDTA reagent to a T-75 flask. Place the flask in the incubator for several minutes or until the cells detach. Add 10 mL of fresh cell medium containing FBS, rinse the cells, and transfer them to a centrifuge tube. In the case of suspension cells, collect the cells and centrifuge them directly.

[0163] 4) Centrifuge the collected cells at 1200 rpm for 5 minutes.

[0164] 5) After centrifugation, discard the supernatant. Resuspend the cell pellet using 10 mL of cell medium containing FBS.

[0165] 6) Remove 20 μL of the resuspended cells and count the cells. Count the cells using a Cell Counter Star by adding 20 μL of the cell suspension to 20 μL of the dye, and record the number of live cells and the survival rate in the cell tracking sheet.

[0166] 7) Use a cell culture medium containing FBS, adjust the volume of the suspension, and achieve the cell concentration. The seeding density for each adherent cell line is 3000 cells / well. For HL-60, the seeding density is 5000 cells / well.

[0167] 8) Transfer 90 μL of the cell suspension to each well of a 96-well white plate according to the plate map. For the blank control, add 100 μL of complete medium.

[0168] 9) Incubate the cells overnight at 37 °C / 5% CO2. Preparation of the compound dosage gradient solution: Day 2

[0169] 1) Dissolve the solid compound in DMSO to make a stock solution.

[0170] 2) Dilute this compound appropriately.

[0171] 3) Add 10 μL of the diluted compound at each concentration to 90 μL of the cells. For the vehicle group, add 10 μL of the corresponding vehicle to 90 μL of the cells.

[0172] 4) Incubate the cells at 37 °C / 5% CO2 for 3 days for each of the test compounds and cisplatin. CTG detection: Day 5

[0173] 1) Incubate the plate at room temperature away from light for 30 minutes.

[0174] 2) Thaw three vials of CellTiter-Glo® reagent at room temperature and equilibrate these vials to room temperature before use. Avoid light.

[0175] 3) Add 100 μL of CellTiter-Glo® reagent per well to each well. Avoid light.

[0176] 4) Mix the contents on an orbital shaker for 2 minutes.

[0177] 5) Incubate the plate at room temperature for 10 minutes to stabilize the luminescence signal.

[0178] 6) Read the plate with Envision.

[0179] Map the plates for a total of 52 plates, with 4 plates for each cell line.

[0180] The results of the cell assays showed that the disclosed compounds were positive for changes in viable cells in cultures and cell growths, and it was demonstrated that at various concentrations, certain compounds were significantly more active than other compounds. Topical formulations for treating wounds:

[0181] Compounds of formula I, II or III, and compositions containing these compounds can be effectively used as topical compositions or wound dressing compositions for treating wounds or damaged skin, such as wounds associated with tissue calcification and calciphylaxis. The composition can heal, restore and revitalize the patient's wounds and skin. In one variant, the composition contains from about 1.25 mg to 500 mg of a compound of formula I, II or III or a mixture thereof. In another variant, the composition can contain from about 0.01% w / w to 20% w / w or about 1%, 5%, 10%, 15% or about 20% of a compound of formula I, II or III or a mixture thereof in the formulation. The wound can be an acute wound or a chronic wound. Such wounds can include ulcers such as diabetic ulcers, pressure ulcers and venous ulcers.

[0182] In one variant, topical application of the composition to damaged skin reduces inflammation and promotes wound healing.Without being bound by any theory asserted herein, it is noted that the composition can stimulate proteasome function to promote normal fibroblast activity with the formation of new collagen (i.e., reverse aging).This function helps proteasome to absorb fragmented protein particles, including collagen, thus minimizing the increase in the formation of undesirable clumped collagen.

[0183] In one variation, the composition can be prepared in the form of a gel, cream or ointment. In another variation, the topical composition further comprises at least one component selected from the group consisting of water, solvent, preservative, surfactant, gelling agent and pH balancing agent. The compound or mixture of compounds may be formulated for administration in a pharmaceutical carrier using methods known in the art. For example, see Remington, The Science and Practice of Pharmacy (9th Edition), incorporated herein by reference. th See, Ed. 1995). The compound may be mixed with an acceptable carrier. Carriers that can be used include petrolatum, lanolin, polyethylene glycol, alcohol, transdermal enhancers, and combinations of two or more thereof. In another variation, the topical composition may be partially or entirely incorporated into a delivery vehicle such as a microsphere or nanoparticle, or encapsulated, such as in a liposome. In another variation, the topical composition may be pre-impregnated into / on a support structure such as a tape, patch, or bandage.

[0184] In another embodiment, the composition is formulated and delivered using a transdermal patch. As provided herein, the transdermal patch achieves a metered sustained release or delivery of a compound of Formula I, II or III, or a mixture thereof, over a desired period of time. The transdermal patch can also be easily removed in the event that the patient experiences an undesirable side effect associated with the patch. As currently formulated, the transdermal patch can deliver a more accurate dosage of the compound over time. In one variant, the transdermal patch delivers an amount of a compound of Formula I, II or III, or a mixture thereof, in the range of about 1 μg / cm 2 / day to about 600 μg / cm 2 / day, about 1 μg / cm 2 / day to about 280 μg / cm 2 / day, about 10 μg / cm 2 / day to about 280 μg / cm 2 / day or about 50 μg / cm 2 / day to about 250 μg / cm 2 / day to the drug delivery region or area (i.e., the region where the active agent-containing composition contacts the intact human skin surface or wound). In one variant, the composition is formulated to achieve a sustained delivery of a compound of Formula I, II or III, or a mixture thereof, over a continuous period of about 5 hours to 7 days. In another variant, the transdermal patch is formulated to deliver the cited compound over a continuous period of about 12 hours to 24 hours or about 1 day to 3 days. The transdermal patch may be administered continuously over an administration period of about 1 week to about 1 month.

[0185] The composition for institutional use may contain, in addition to the compounds of formula I, II or III or mixtures thereof, other additives such as acids such as citric acid or lactic acid, bases or buffering agents such as sodium lactate, sodium acetate, sodium phosphate, sodium citrate, sodium borate or sodium gluconate, etc. for pH adjustment. Further, in some embodiments, the composition may contain an antibacterial preservative. Antibacterial preservatives include, for example, methylparaben, propylparaben, benzyl alcohol, ethylhexylglycerin, potassium sorbate, phenoxyethanol, EDTA, grapefruit seed extract, tea tree oil, sodium benzoate, dehydroacetic acid and combinations thereof. In another variant, the composition may contain an antifungal preservative, optionally in combination with an antibacterial preservative. In one variant, the composition may contain an anti-biofilm antimicrobial agent such as lactoferrin, xylitol, farnesol gallium, dispersin B and EDTA, or combinations thereof.

[0186] In one embodiment, the skin patch can be prepared as follows: 1) Dissolve a measured amount of the compound or mixture of compounds of formula I, II or III in a solvent such as THF, EtOAc or methanol, or mixtures thereof, and add it to DuroTak 87-2194 adhesive to form a solution containing the above compound. 2) Use a mechanical coater to form a solution of the blend of the compound and the adhesive on the release paper surface. 3) Next, pass the coated release paper through an oven that causes evaporation of the solvent (e.g., ethyl acetate and any solvent present in DuroTak liquid) to form a solid adhesive matrix containing the compound of formula I, II or III or mixtures thereof, which is a solid mucosal layer dispersed in the adhesive matrix. 4) Next, laminate a polyethylene film to the adhesive matrix. 5) Next, use a die cutter to cut the laminate of the compound-containing patch to the specified dimensions, and then individually pouch-pack this patch into a sealed foil-lined material.

[0187] In another variant, the composition may be used without a microporous tape or other support structure. In one variant, the composition is sufficiently viscous or adhesive to create an occlusive dressing without a support structure such as a tape. In another variant, the composition may be applied to the lower part of the support structure or the upper part of the support structure.

[0188] In yet another variant, when the composition is formulated into a microporous support structure such as a tape for receiving the composition on the outer surface of the tape, the composition is delivered to contact the damaged skin through pores, the skin or damaged skin tissue.

[0189] In one embodiment, a tape (or other support structure) is adapted to the length of the wound or scar, and the topical composition is applied to the tape. The application of the tape and the gel can be started immediately when convenient, after the wound has been observed or before the subcutaneous wound breaks the skin. In one variant, the tape is a microporous tape that allows contact between the topical composition and the skin when the topical composition is applied on the tape. In one variant, the topical composition may be applied to the skin before the tape is placed on the composition.

[0190] Accordingly, the present application discloses a method for accelerating wound healing, the method comprising administering a therapeutically effective amount of a compound of formula I, II or III to a site in need of accelerated healing and promoting wound healing. The method of topical administration may be carried out in conjunction with oral administration of a composition consisting of formula I, II or III so as to accelerate wound healing, which means that the topical administration may be administered before, during and / or after oral administration of the compounds of formula I, II or III disclosed herein.

[0191] As provided herein, the acceleration of wound healing involves the rate of healing of treated wounds being somewhat improved compared to the rate of healing that occurs in untreated wounds, with or without oral administration of a compound of Formula I, II or III as disclosed herein. The acceleration of wound healing achieved using the disclosed method results in the rate of healing of treated wounds being at least 5% faster than that of untreated wounds, the rate can be at least 10% faster, 15%, 20%, 25% faster; or at least 50%, 75% or 100% faster. Such an improvement or acceleration in the rate of wound healing of at least 5% - 100% can be compared to the case where the patient or subject is concomitantly treated by oral administration of a compound of Formula I, II or III.

[0192] Some exemplary embodiments, aspects and variations are presented herein, and those skilled in the art will recognize certain modifications, alterations, additions and combinations, as well as certain sub - combinations of embodiments, aspects and variations. The following claims are to be construed as including all such modifications, alterations, additions and combinations, and it is intended that certain sub - combinations of embodiments, aspects and variations are within their scope. The entire disclosure of all documents cited throughout this application is hereby incorporated by reference herein. References: 1) Rachel M. Holden et al. Vitamins K and D Status in Stages 3-5 Chronic Kidney Disease; Clin J Am Soc Nephrol 5: 590-597, 2010. 2) Pilkey, R.M. MD et al. Subclinical Vitamin K Deficiency in Hemodialysis Patients Am J Kidney Dis 49:432-439, 2007. 3) Westhofen P et al. Human vitamin K 2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediates vitamin K-dependent intracellular antioxidant function. J Biol Chem 2011;286: 15085-94. 4) Caspers, M. et al., Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain. Thrombosis Research 135:977-983, 2015. 5) Himmelfarb, J. et al., Plasma protein thiol oxidation and carbonyl formation in chronic renal failure. Kidney International, Vol. 58: 2571-2578 2000.6) Price, P.A. et al., Discovery of a High Molecular Weight Complex of Calcium, Phosphate, Fetuin, and Matrix-Carboxyglutamic Acid Protein in the Serum of Etidronate-treated Rats. J Biol Chem. 277 (6): 3926-3934, 2002. 7) Pasch, A. et al. Nanoparticle-Based Test Measures Overall Propensity for Calcification in Serum J Am Soc Nephrol 23: 1744-1752, 2012. 8) Nigwekar, S.U. et al. Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis. J Am Soc Nephrol 28: 1717-1722, 2017.

Claims

1. Bioactive menaquinol derivatives of formulas I, II, or III: 【Chemistry 24】 (In the formula, m is an integer between 2 and 15. I understand １ If present, it is an integer between 3 and 15. n is 7, 8, 9, or 10. R is H or -C(O)C １ ~C ６ alkyl, -C(O)(CH ２ ) ｑ -O-C(O)C １ ~C ６ alkyl and -Z-(C=Y)(CH ２ ) ｓ -(OCH ２ CH ２ )-O-R １ selected from the group consisting of R １ is H or -CH ３ And, R ２ is H or C １ ~C ３ It is alkyl, q is 1, 2, or 3. s is 1, 2, or 3. Y is O, NR ２ or S, Z is -C(O)CH ２ O-, -C(O)CH ２ NH- or not present, Z １ is -C(O)CH ２ O-, -C(O)CH ２ (either NH- or non-existent).

2. Formulas Ia, Ib, or Ic: 【Chemistry 25】 (In the formula, m is an integer between 2 and 15. n is 7, 8, 9, or 10. R is either H or -C(O)C １ ~C ６ Alkyl, -C(O)(CH ２ ) ｑ -O-C(O)C １ ~C ６ Alkyl and -Z-(C=Y)(CH ２ ) ｓ - (OCH ２ CH ２ )-O-R １ Selected from the group consisting of, R １ is H or -CH ３ And, R ２ is H or C １ ~C ３ It is alkyl, q is 1, 2, or 3. s is 1, 2, or 3. Y is O, NR ２ or S, Z is -C(O)CH ２ O-, -C(O)CH ２ The menaquinol derivative according to claim 1, which is NH- or does not exist.

3. Formula Ia. 1, Ib. 1, or Ic. 1: 【Chemistry 26】 (In the formula, m is an integer between 8 and 12. n is 7, 8, 9, or 10. R １ is H or -CH ３ (is) The menaquinol derivative according to claim 1, wherein the menaquinol derivative is as described above.

4. Formulas IIa, IIb, or IIc: 【Chemistry 27】 (In the formula, m is an integer between 8 and 12. n is 7, 8, 9, or 10. s is 1, 2, or 3. R １ is H or -CH ３ (is) The menaquinol derivative according to claim 3.

5. Formula Ia. 1, Ib. 1, Ic.

1. Ia. 2, Ib. 2, Ic. 2, IIa, IIb, IIc, IIa. 1, IIb. 1, IIc. 1, IIa. 1.1, IIb. 1.1, IIc. 1.1, IIa. 1.2, IIb. 1.2, IIc. 1.2, IIa. 2, IIb. 2, IIc. 2, IIa. 3, IIb. 3, IIc. 3, IIa. 4, IIb. 4, IIc. 4, IIa. 5, IIb. 5 and IIc. 5: 【Chemistry 28】 【Chemistry 29】 【Transformation 30】 The menaquinol derivative according to claim 1.

6. The menaquinol derivative according to claim 1, wherein n is 7.

7. A therapeutically effective amount of the menaquinol derivative or mixture thereof according to claim 1 and pharmaceutically acceptable additives A pharmaceutical composition comprising a vitamin K-related condition selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification.

8. A composition comprising a menaquinol derivative or mixture thereof according to claim 1 for improving the tissue concentration of menaquinol as a cofactor of gamma glutamate carboxylase (GGCX) for catalyzing the carboxylation of vitamin K-dependent proteins in connection with the treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification in patients requiring treatment or prevention of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification, or a pharmaceutical composition comprising a menaquinol derivative or mixture thereof according to claim 1 and a pharmaceutically acceptable additive, wherein the pharmaceutical composition is effective for the treatment of vitamin K-related conditions selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification.

9. A composition comprising a menaquinol derivative or a mixture thereof according to claim 1 for treating diseases selected from the group consisting of neurodegenerative diseases, proliferative disorders, retinopathy, polyarthritis, atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia, cataracts, systemic infections, age-related and aging pathologies of the skin in tissues, pathologies related to mitochondrial dysfunction, and cachexia associated with nutritional deficiencies, or a pharmaceutical composition comprising a menaquinol derivative or a mixture thereof according to claim 1 and a pharmaceutically acceptable additive, wherein the pharmaceutical composition is effective in treating vitamin K-related conditions selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification. A composition or pharmaceutical composition in which the treatment results in an increase in the lifespan of a mammal.

10. A composition comprising a menaquinol derivative or a mixture thereof according to claim 1 for treating a mammal having a disease selected from the group consisting of vitamin K deficiency, osteoporosis, proliferative disorders and cardiovascular diseases, or a pharmaceutical composition comprising a menaquinol derivative or a mixture thereof according to claim 1 and a pharmaceutically acceptable additive, wherein the pharmaceutical composition is effective in treating a vitamin K-related condition selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification.

11. The composition or pharmaceutical composition according to claim 9, wherein the proliferative disorder is selected from the group consisting of cancer, leukemia, and inflammatory diseases.

12. A composition comprising a menaquinol derivative or a mixture thereof according to Claim 1, or a pharmaceutical composition comprising a menaquinol derivative or a mixture thereof according to Claim 1 and a pharmaceutically acceptable additive, for treating or preventing osteoporosis and / or osteopenia in a patient requiring treatment for osteoporosis and / or osteopenia, wherein the pharmaceutical composition is effective in treating a vitamin K-related condition selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis or tissue calcification.

13. A composition comprising a substantially pure menaquinol derivative or mixture thereof according to claim 1 for treating, preventing, slowing, stopping, and / or reversing the progression of calciphylaxis in mammals requiring treatment, prevention, slowing, stopping, and / or reversing its progression, or a pharmaceutical composition comprising a menaquinol derivative or mixture thereof according to claim 1 and a pharmaceutically acceptable additive, wherein the pharmaceutical composition is effective for treating conditions related to vitamin K selected for the treatment of osteoporosis, arteriosclerosis, calciphylaxis, or tissue calcification.

14. The composition or pharmaceutical composition according to claim 13, wherein the mammal has distal calciphylaxis and / or central calciphylaxis.

15. The composition or pharmaceutical composition according to claim 13, wherein the mammal has diabetes, chronic kidney disease, or end-stage renal disease.

16. The composition or pharmaceutical composition according to claim 15, wherein the mammal has a stage 3, stage 4, or stage 5 chronic kidney disease.

17. The composition or pharmaceutical composition according to any one of claims 9 to 16, wherein the mammal is undergoing hemodialysis.

18. The composition or pharmaceutical composition according to any one of claims 9 to 16, wherein the mammal is receiving non-warfarin-based anticoagulant therapy.

19. The composition or pharmaceutical composition according to claim 18, wherein the anticoagulant therapy is oral anticoagulant therapy.

20. The composition or pharmaceutical composition according to claim 18, wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

21. The composition or pharmaceutical composition according to any one of claims 8 to 16, wherein the mammal has chronic obstructive pulmonary disease (COPD).

22. The composition or pharmaceutical composition according to any one of claims 8 to 16, wherein the mammal has a calciphylaxis-related skin lesion.

23. The composition or pharmaceutical composition according to claim 22, wherein administration of the composition reduces the total surface area of ​​the skin lesion by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

24. The composition or pharmaceutical composition according to any one of claims 8 to 16, wherein when the mammal is administered the compound or mixture thereof according to any one of claims 1 to 6, the T50 value of the mammal's serum is improved by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to the T50 value of the mammal's serum before administration of the compound or mixture thereof according to any one of claims 1 to 6.

25. The composition or pharmaceutical composition according to any one of claims 13 to 16, wherein administration of the compound or mixture thereof according to any one of claims 1 to 6 improves the ratio of carboxylated to non-carboxylated vitamin K-dependent proteins in the plasma of the mammal, and the ratio after administration of the composition is higher than that before administration of the composition.

26. A pharmaceutical composition for treating, preventing, slowing, stopping, and / or reversing tissue calcification in prediabetic mammals (or subjects) having diabetes mellitus, chronic kidney disease or a combination thereof, and requiring treatment, prevention, slowing, cessation, and / or reversal of its progression, comprising at least 50 mg per day of the compound or a mixture thereof described in claim 1.

27. The pharmaceutical composition according to claim 26, wherein the mammal has diabetes.

28. The pharmaceutical composition according to claim 27, wherein the mammal has type II diabetes.

29. The pharmaceutical composition according to claim 26, wherein the mammal has been diagnosed with prediabetes.

30. The pharmaceutical composition according to any one of claims 26 to 29, wherein the mammal has a chronic kidney disease.

31. The pharmaceutical composition according to any one of claims 26 to 29, wherein the mammal has a stage 4 or 5 chronic kidney disease / end-stage kidney disease.

32. The pharmaceutical composition according to any one of claims 26 to 29, wherein the mammal is undergoing hemodialysis.

33. The pharmaceutical composition according to any one of claims 26 to 29, wherein the mammal is receiving non-warfarin-based anticoagulant therapy.

34. The pharmaceutical composition according to claim 33, wherein the anticoagulant therapy is oral anticoagulant therapy.

35. The pharmaceutical composition according to claim 33, wherein the anticoagulation therapy comprises an inhibitor of factor Xa activity selected from apixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, retaxaban, eribaxaban, or fondaparinux, or an inhibitor of factor IIa activity selected from dabigatran or argatroban.

36. A pharmaceutical composition for treating, preventing, slowing, stopping, and / or reversing tissue calcification in mammals undergoing hemodialysis and requiring treatment of, prevention of, slowing, stopping, and / or reversal of its progression, comprising at least 5 mg per day of the compound or a mixture thereof according to claim 1.

37. The pharmaceutical composition according to claim 36, wherein the mammal has diabetes.

38. A composition for use in a method for increasing at least one of the bioavailability and serum half-life of menaquinone-7 / menaquinol-7, menaquinone-8 / menaquinol-8, menaquinone-9 / menaquinol-9, or menaquinone-10 / menaquinol-10, comprising the compound described in claim 1, wherein the method comprises administering or supplementing the composition to a mammal that requires an increase in at least one of the bioavailability and serum half-life, and the method improves at least one of the bioavailability and serum half-life of menaquinone-7 / menaquinol-7, menaquinone-8 / menaquinol-8, menaquinone-9 / menaquinol-9, or menaquinone-10 / menaquinol-10 by at least 5% compared to administration or supplementation using menaquinone-7, menaquinone-8, menaquinone-9, or menaquinone-10.

39. A skin structure or transdermal patch for treating at least one of areas of wound or pain and inflammation caused by calciphylaxis or tissue calcification in a subject that requires treatment of at least one of areas of wound or pain and inflammation caused by calciphylaxis or tissue calcification, comprising the compound described in Claim 1, wherein the skin structure or transdermal patch is applied topically to the area of ​​wound or pain and inflammation in the subject.

40. The skin structure or transdermal patch according to claim 39, wherein the topical application of the skin structure or transdermal patch is performed before, together with (or simultaneously with) or after the administration of the composition according to any one of claims 8 to 10, 12, 13, or 38 or the pharmaceutical composition according to any one of claims 8 to 10, 12, 13, 26, or 36.

41. The composition or pharmaceutical composition according to claim 11, wherein the cancer is selected from the group consisting of melanoma, lung cancer, breast cancer, leukemia, neuroblastoma, glioblastoma, cervical cancer, liver cancer (hepatocellular carcinoma, HCC), colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, prostate cancer, ovarian cancer, and head and neck cancer.

42. A pharmaceutical composition for treating, preventing, slowing, stopping, and / or reversing the progression of Alzheimer's disease (AD) in a mammal or subject that requires treatment of Alzheimer's disease (AD), comprising at least 5 mg per day of a compound or mixture thereof according to any one of claims 1 to 6.

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