Vitamin K2 for use in the treatment of coronary artery calcification (CAC)

Abstract

The present invention relates to the use of high-dose vitamin K2, particularly menaquinone-7 (MK-7), in the treatment of coronary heart disease in high-risk subjects with a coronary artery calcification (CAC) score of ≧400 (e.g., at the start of treatment), and the treatment slows down the progression of coronary artery calcification.

Description

Technical Field

[0001] The present invention relates to the novel and surprising observation that vitamin K2 supplementation reduces the progression of further coronary artery calcification (CAC) in patients with severe coronary artery calcification.

Background Art

[0002] Coronary artery calcification (CAC) and its progression are strong predictors of acute myocardial infarction (AMI) and death from cardiovascular disease. Menadione-7 (MK-7), a form of vitamin K2, is a coenzyme for the carboxylation of proteins involved in arterial calcification inhibition, and it has been suggested to reduce the progression rate of aortic valve calcification (AVC) in patients with aortic valve stenosis.

[0003] Furthermore, ischemic heart disease accounts for 19% and 20% of all causes of death in men and women, respectively, and prevention is of utmost importance. Ischemic heart disease often remains silent until the symptoms of myocardial infarction appear. However, latent coronary artery disease can be easily detected as coronary artery calcification (CAC) by non-contrast cardiac CT examination. CAC increases with age, and men have a higher CAC score than women. In populations without CAC, the risk of future cardiovascular disease (CVD) is very low, but the risk of ischemic heart disease increases as the CAC score rises. Therefore, it is important to identify and treat patients with severe CAC in order to prevent CVD.

[0004] Vascular calcification is a slowly progressing process, caused by an imbalance between mechanisms that promote and suppress calcium deposition in the vascular wall. Vitamin K-dependent proteins play an essential role in this suppression. The most well-known vitamin K is phylloquinone (vitamin K1), which is essential for the activation of several coagulation factors. Menadione (vitamin K2) is also a very important type of vitamin K. Vitamin K2 is thought to be necessary for the γ-carboxylation of matrix Gla protein (MGP), a protein related to the inhibition of arterial calcification. Without these activated proteins, the balance between cellular calcium uptake and the mineralization processes of bone and blood vessels is disrupted.

[0005] Vitamin K2 enters the circulation through lymph associated with chylomicrons and travels towards the liver. Its extrahepatic distribution is promoted via LDL particles, and long-chain menaquinones have been shown to have a higher prevalence compared to short-chain menaquinones. All tissues expressing the LDL receptor are targeted by vitamin K2, particularly long-chain menaquinones such as MK-7 (Schurgers, L.J. and C. Vermeer (2002), Shearer, M.J. and P. Newman (2008)).

[0006] There are 20 types of vitamin K-dependent proteins (VKDP) in the body, and vitamin K2 functions as a coenzyme that activates these proteins through the carboxylation process (Simes, D.C., et al. (2020)). Matrix-Gla protein (MGP) is expressed in vascular smooth muscle cells and potently inhibits vascular calcification. The key to the function of matrix Gla protein lies in its carboxyl group. Matrix Gla needs to be carboxylated in order to function properly, and vitamin K2 functions as a coenzyme for this enzymatic reaction together with γ-glutamyl carboxylase. When vitamin K2 is present, the matrix Gla protein is carboxylated, that is, it is "on" to prevent the invasion of calcium. When vitamin K2 is deficient, the carboxylation of matrix Gla becomes insufficient or "off", that is, it cannot suppress the infiltration of calcium into soft tissues. A biomarker often used to indicate the state of vitamin K2 in the body is dephosphorylated uncarboxylated MGP (dp-ucMGP). This biomarker is an inactive form of MGP, and a high value reflects a low state of vitamin K2, and vice versa.

[0007] Some VKDPs exhibit anti-inflammatory functions, such as protein C, protein S, Gas6, and GRP (Simes, D.C., et al. (2020)). Additionally, there is data showing anti-inflammatory regulation by vitamin K2 independent of gamma-glutamyl carboxylase. Using human monocyte-derived macrophages, Pan et al. showed that cytokine release (TNF-α, IL-1α, IL-1β) was suppressed when cells were pretreated with vitamin K2 (menaquinone-7), and a dose-response relationship could be demonstrated (Pan, M.H., et al. (2016)). Similar inhibitory effects on IL-6 have also been observed by other research groups (Ohsaki, Y., et al. (2006), Reddi, K., et al. (1995)). The release of pro-inflammatory cytokines is mainly controlled through the NF-kB signaling pathway, and vitamin K has been shown to inhibit the release of IkB from NF-kB and enable its entry into the nucleus (Ohsaki, Y., et al. (2010), Ozaki, I., et al. (2009), Xia, J., et al. (2012)).

[0008] Statins are widely used for their lipid-lowering effects and prevention of cardiovascular events. In 2015, a hypothesis was proposed for discussion that the action of statins not only inhibits cholesterol synthesis but also inhibits prenyl intermediate levels through inhibition of HMG-CoA reductase, thereby inhibiting the conversion of vitamin K1 to K2 in the body (Okuyama, H., et al. (2015)). Since vitamin K2 prefers LDL particles towards extrahepatic tissues, the use of statins may lead to extrahepatic vitamin K deficiency, and as a result, the activation of VKDPs in different tissues may decrease. In a cross-sectional clinical trial, the relationship between statin use and CAC and the activation levels of VKDPs was investigated. The authors found that the CAC score was high in statin users, but no difference was observed in the carboxylation status of MGP. However, the carboxylated form of VKDP osteocalcin showed significantly higher values in statin users (Zhelyazkova-Savova, M.D. et al. (2021)).

[0009] Currently, there is no recommendation for vitamin K2 supplementation. Also, it is well known that the daily intake of vitamin K2 in Western countries is not sufficient to meet the requirements for the full activation of MGP. Furthermore, the toxicity of vitamin K1 and vitamin K2 has not been reported, and the WHO has not set an upper tolerable limit for vitamin K intake.

[0010] The effect of high-dose vitamin K2 supplementation on the progression of aortic valve calcification was examined in the recently published AVADEC trial and reported by Diederichsen ACP et al. (2022). The literature describes the treatment of male subjects with an aortic valve calcification score of 300 arbitrary units or more. However, in this study, the progression of aortic valve calcification decreased, although not significantly.

[0011] European Patent Application Publication No. 1728507 discloses a study using rats that taking a large amount of vitamin K may remove calcified deposits from blood vessels that have already calcified.

[0012] European Patent No. 2558084 discloses a pharmaceutical composition consisting of a vitamin K component and a nicotinamide component for use in the prevention or treatment of diseases accompanied by extraosseous calcification in a study using rats.

[0013] International Publication No. 19 / 021232 relates to a composition for use in a prophylactic or therapeutic method for treating vascular calcification in an in vitro experiment (an experimental model consisting of calcified vascular smooth muscle cells isolated from the aorta of rats), the composition comprising a vitamin K group, or an analogue and derivative thereof, an inorganic magnesium salt and iron(III) oxide, a complex or salt, a pharmaceutical or food-grade excipient, additive and / or co-formulant.

[0014] European Patent No. 1728507 discloses the administration of vitamin K, including vitamin K2, to mammals for the rapid restoration of vascular calcification. European Patent No. 1728507 further discloses a clinical study conducted on kidney transplant patients to study the efficacy and safety of vitamin K2 supplementation for atherosclerosis.

[0015] European Patent No. 1728507 discloses the use of vitamin K2, for example, vitamin K2 in combination with vitamin D, for the restoration of vascular calcification in a rat model.

[0016] Geleijnse J.M. et al. (2004) disclose the effect of dietary menaquinone at baseline on aortic calcification and coronary heart disease (CHD), particularly in men and women aged 55 years and older without a history of myocardial infarction.

[0017] Shea M.K. et al. (2009) disclose the effect of vitamin K1 (phylloquinone) supplementation at baseline on the progression of CAC, particularly in asymptomatic elderly men and women aged 60 - 80 years without known coronary heart disease, and conclude that vitamin K1 supplementation, when taken together with recommended amounts of calcium and vitamin D, suppressed the progression of existing CAC in asymptomatic elderly men and women.

[0018] None of the above prior art documents disclose or suggest the administration or supplementation of vitamin K2 to subjects (e.g., human patients) with severe CAC defined by a CAC score ≧ 400, i.e., subjects at high risk of developing acute myocardial infarction (AMI).

[0019] Therefore, it is beneficial to provide a treatment method for suppressing the progression of further coronary artery calcification in a subgroup of patients with severe CAC defined by a CAC score ≧ 400.

[0020] The prevalence of CAC score of 400 or more in the Danish population has been investigated in a population-based study (Diederichsen A.C.P. et al. (2012)), where 2% of 50-year-old men had a CAC score of 400 or more, which increased to 14% in 60-year-old men. 1% of 50-year-old elderly women had a CAC score of 400 or more, which increased to 4% in 60-year-old elderly women. Furthermore, in randomly selected Danes aged 65 - 74 years, 37.8% of men and 11.3% of women had a CAC score > 400 (Kvist T.V. et al. (2017)).

Summary of the Invention

[0021] The present invention was conceived in such a background and is based on the surprising observation that vitamin K2 supplementation reduces the progression of CAC in severely CAC subjects (e.g., human subjects) defined by a CAC score ≧ 400, compared to placebo. In particular, high-dose supplementation with vitamin K2 and vitamin D suppresses the progression of CAC in subjects with severe CAC defined by a CAC score ≧ 400 (e.g., human subjects).

[0022] Accordingly, the object of the present invention relates to the treatment of a specific subgroup of such subjects (e.g., patients) defined as having a CAC score ≧ 400 measured by cardiac CT scan using the so-called Agatston method or a similar method for measuring CAC.

[0023] In a first aspect, the present invention provides vitamin K2 for use in the prevention or treatment of coronary heart disease in subjects with a coronary artery calcium (CAC) score ≧ 400.

[0024] The present invention also relates to vitamin K2 for use in preventing or treating coronary heart disease in subjects with a coronary artery calcium (CAC) score ≥ 400 by administering to the subject from 100 μg to 100 mg of vitamin K2 per day, preferably from 200 μg to 50 mg of vitamin K2 per day, preferably from 360 μg to 10 mg of vitamin K2 per day, preferably from 360 μg to 5 mg of vitamin K2 per day, preferably from 360 μg to 1440 μg of vitamin K2 per day, preferably from 500 μg to 1000 μg of vitamin K2 per day, more preferably 720 μg of vitamin K2 per day. Preferably, the vitamin K2 is preferably combined with the administration of from 1 μg to 50 μg of vitamin D per day, from 10 μg to 50 μg of vitamin D per day, preferably 25 μg of vitamin D per day, and by prevention or treatment, delays the progression of coronary artery calcification.

[0025] In another aspect, the present invention provides vitamin K2 for use in delaying the progression of coronary artery calcification.

[0026] In yet another aspect, the present invention provides vitamin K2 for use in delaying the progression of coronary artery calcification, and delaying the progression of coronary artery calcification means that the CAC score measured by cardiac CT scan using the Agaston method or a similar method for measuring CAC is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8.9%.

[0027] In yet another aspect, the present invention provides vitamin K2 for use in delaying the progression of coronary artery calcification, and delaying the progression of coronary artery calcification means that the progression of calcified plaque (mm 3 ) in the intervention group is reduced by at least 1%, at least 3%, at least 9%, at least 12%, at least 12.7%.

[0028] In yet another aspect, the present invention provides vitamin K2 for use in retarding the progression of coronary artery calcification, where retarding the progression of coronary artery calcification means that the progression of non-calcified plaque (mm 3 ) in the intervention group is reduced by at least 1%, at least 3%, at least 5%, at least 7%, at least 8%.

[0029] In yet another aspect, the present invention provides vitamin K2 for use in reducing coronary artery calcification, as measured by the CAC score measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC, by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 4.8% compared to baseline.

[0030] In yet another aspect, the present invention provides vitamin K2 for use in reducing the total plaque volume in the coronary arteries by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13.1% compared to baseline.

[0031] In another aspect, the present invention provides a method for treating coronary heart disease in a subject having a CAC score of ≧400, the method comprising administering vitamin K2 to the subject.

[0032] In another aspect, the present invention relates to a method for preventing or treating coronary artery calcification in a subject having a CAC score of ≧400, the method comprising administering vitamin K2 to the subject.

[0033] The present invention also relates to a method for preventing or treating coronary heart disease in a subject with a coronary artery calcium (CAC) score of ≧400 by administering to the subject 100 μg to 100 mg of vitamin K2 per day, preferably 200 μg to 50 mg of vitamin K2 per day, preferably 360 μg to 10 mg of vitamin K2 per day, preferably 360 μg to 5 mg of vitamin K2 per day, preferably 360 μg to 1440 μg of vitamin K2 per day, preferably 500 μg to 1000 μg of vitamin K2 per day, preferably 720 μg of vitamin K2 per day, in combination with the administration of preferably 1 μg to 50 μg of vitamin D per day, preferably 10 μg to 50 μg of vitamin D per day, preferably 25 μg of vitamin D per day.

[0034] Another aspect relates to the use of vitamin K2 in the manufacture of a medicament for the prevention or treatment of coronary heart disease in a subject having a coronary artery calcium (CAC) score of ≧400.

[0035] Another aspect relates to a kit of parts for use in the prevention or treatment of coronary heart disease in a subject with a coronary artery calcium (CAC) score of ≧400, (A) vitamin K2, and (B) vitamin D. BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

Figure 1

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Figure 7

Figure 8

Figure 9

Mode for Carrying Out the Invention

[0037] Hereinafter, the present invention will be described in more detail.

[0038] Before explaining the present invention in more detail, first the following terms and conventions are defined.

[0039] Agatston method: As used herein, the system for quantifying the CAC score in CT images is the Agatston method, which uses the sum of the burdens of lesions having a density of 130 HU (Hounsfield scale) or more and multiplies the area of calcium by a factor related to the maximum plaque attenuation: 130 HU to 199 HU has a coefficient of 1, 200 HU to 299 HU has a coefficient of 2, 300 HU to 399 HU has a coefficient of 3, and ≧400 HU has a coefficient of 4 (see Neves PO et al. (2017) et al. incorporated herein by reference). The Agatston method is well known in the art.

[0040] CAC score: As used herein, CAC means "coronary artery calcification". To measure the CAC score, it is necessary to perform a non-contrast CT scan. The CAC score reflects the amount of calcium in the walls of the arteries of the heart. In this test, a special imaging test called a cardiac CT scan is used. In this scan, multiple images are created to check whether calcium is present and, if so, to what extent. The calcium score is calculated based on the amount of plaque observed in the CT scan (quantified by the Agatston method) (see also Neves PO et al. (2017) et al.). The CAC score is well known in the art.

[0041] Vitamin K2: As used herein, vitamin K 2 homologs, menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone-12 (MK-12) or menaquinone-13 (MK-13), or any combination thereof. Vitamin K2 is composed of various forms because the number n of isoprenyl units in the side chain is different and ranges from 4 to 13. This n is indicated as a suffix (-n), for example, MK-7 contains 7 isoprenyl units.

[0042] Vitamin K2 also means a prodrug of vitamin K2. Suitable prodrugs are described, for example, in International Publication No. WO 2013 / 128037.

[0043] Coronary heart disease may be referred to herein as ischemic heart disease or coronary artery disease.

[0044] One embodiment of the present invention relates to K2 or a method for use in the prevention or treatment of coronary heart disease in a subject having a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC. Typically, the progression of coronary artery calcification can be slowed by the prevention or treatment described herein.

[0045] In embodiments and aspects of the present invention, when the CAC score is referred to as ≧400, it typically means that the CAC score at the start of treatment is ≧400. As described in the examples, beneficial effects are seen in subjects with initially high CAC values.

[0046] Vitamin K2 (e.g., MK-7) may be administered to a subject at 100 μg to 100 mg per day, preferably 200 μg to 50 mg per day, preferably 360 μg to 10 mg per day, preferably 360 μg to 5 mg per day, preferably 360 μg to 1440 μg per day, preferably 500 μg to 1000 μg per day, more preferably 720 μg per day. Other suitable dosages of vitamin K2 include at least 100 μg per day, preferably at least 360 μg per day, for example, at least 500 μg per day.

[0047] One embodiment of the present invention relates to K2 or a method for use in the treatment of coronary heart disease in a subject in which the coronary artery calcium (CAC) score measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC is ≧400 (e.g., at the start of treatment), wherein the subject is administered 100 μg to 100 mg of vitamin K2 per day, preferably 200 μg to 50 mg of vitamin K2 per day, preferably 360 μg to 10 mg of vitamin K2 per day, preferably 360 μg to 5 mg of vitamin K2 per day, preferably 360 μg to 1440 μg of vitamin K2 per day, preferably 500 μg to 1000 μg of vitamin K2 per day, more preferably 720 μg of vitamin K2 per day. Other suitable dosages of vitamin K2 include at least 100 μg per day, preferably at least 360 μg per day, for example at least 500 μg per day.

[0048] Yet another embodiment of the present invention relates to K2 or a method for use in the treatment of coronary heart disease in a subject in which the coronary artery calcium (CAC) score measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC is ≧400 (e.g., at the start of treatment), wherein 100 μg to 100 mg of vitamin K2 per day, preferably 200 μg to 50 mg of vitamin K2 per day, preferably 360 μg to 10 mg of vitamin K2 per day, preferably 360 μg to 5 mg of vitamin K2 per day, preferably 360 μg to 1440 μg of vitamin K2 per day, preferably 500 μg to 1000 μg of vitamin K2 per day, more preferably 720 μg of vitamin K2 per day is administered to the subject, and the vitamin K2 is preferably administered in combination with 1 μg to 50 μg of vitamin D per day, more preferably 10 μg to 50 μg of vitamin D per day, most preferably 25 μg of vitamin D per day, and the treatment slows the progression of coronary artery calcification.

[0049] Yet another embodiment of the present invention relates to vitamin K2 for use in the prevention or treatment of coronary heart disease in subjects with a coronary artery calcium (CAC) score of ≧400 measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC, wherein the subject has a blood concentration of dephosphorylated uncarboxylated matrix-Gla-protein (dp-ucMGP) of 433 pmol / L to 500 pmol / L, 475 pmol / L to 600 pmol / L, 550 pmol / L to 700 pmol / L, 650 pmol / L to 800 pmol / L, 750 pmol / L to 900 pmol / L, 850 pmol / L to 1000 pmol / L, 950 pmol / L to 1250 pmol / L, 1200 pmol / L to 1750 pmol / L, 1700 pmol / L to 2000 pmol / L, and 1950 pmol / L to 2179 pmol / L.

[0050] Yet another embodiment of the present invention relates to vitamin K2 for use in the prevention or treatment of coronary heart disease in subjects with a coronary artery calcium (CAC) score of ≧400 measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC, wherein the subject has a blood concentration of dephosphorylated uncarboxylated matrix-Gla-protein (dp-ucMGP) of 433 pmol / L to 2179 pmol / L, 400 pmol / L to 2000 pmol / L, preferably 425 pmol / L to 1500 pmol / L, preferably 450 pmol / L to 1250 pmol / L, preferably 475 pmol / L to 1000 pmol / L, more preferably 500 pmol / L to 900 pmol / L.

[0051] In another aspect, the present invention provides vitamin K2 for use in retarding the progression of coronary artery calcification.

[0052] In yet another aspect, the present invention provides vitamin K2 for use in retarding the progression of coronary artery calcification, where retarding the progression of coronary artery calcification means that the CAC score measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8.9%.

[0053] In yet another aspect, the present invention provides vitamin K2 for use in retarding the progression of coronary artery calcification, where retarding the progression of coronary artery calcification means that the progression of calcified plaque (mm 3 ) in the intervention group is reduced by at least 1%, at least 3%, at least 9%, at least 12%, at least 12.7%.

[0054] In yet another aspect, the present invention provides vitamin K2 for use in retarding the progression of coronary artery calcification, where retarding the progression of coronary artery calcification means that the progression of non-calcified plaque (mm 3 ) in the intervention group is reduced by at least 1%, at least 3%, at least 5%, at least 7%, at least 8%.

[0055] In yet another aspect, the present invention provides vitamin K2 for use in reducing coronary artery calcification, as measured by the CAC score measured by cardiac CT scan using the Agatston method or a similar method for measuring CAC, by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 4.8% compared to baseline.

[0056] In yet another aspect, the present invention provides vitamin K2 for use in reducing total plaque volume in coronary arteries by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13.1% compared to a baseline.

[0057] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject having a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC, the method comprising administering vitamin K2 to the subject.

[0058] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject having a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC, the method comprising administering vitamin K2 to the subject, and the subject is administered 100 μg to 100 mg of vitamin K2 per day, preferably 200 μg to 50 mg of vitamin K2 per day, preferably 360 μg to 10 mg of vitamin K2 per day, preferably 360 μg to 5 mg of vitamin K2 per day, preferably 360 μg to 1440 μg of vitamin K2 per day, preferably 500 μg to 1000 μg of vitamin K2 per day, more preferably 720 μg of vitamin K2 per day.

[0059] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject having a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC, the method comprising administering vitamin K2 to the subject, vitamin K2 is administered at 720 μg per day, and vitamin K2 is preferably administered in combination with 1 μg to 50 μg of vitamin D per day, more preferably 10 μg to 50 μg of vitamin D per day, most preferably 25 μg of vitamin D per day.

[0060] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject with a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC. The method includes administering vitamin K2 to the subject, with vitamin K2 being administered at 720 μg per day. Vitamin K2 is administered in combination with 25 μg of vitamin D per day, and vitamin K2 is any one of vitamin K2 homologs, menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone-12 (MK-12), or menaquinone-13 (MK-13), or a combination thereof.

[0061] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject with a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC. The method includes administering vitamin K2 to the subject, with vitamin K2 being administered at 720 μg per day. Vitamin K2 is administered in combination with 25 μg of vitamin D per day, and vitamin K2 is menaquinone-7 (MK-7).

[0062] One embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject with a coronary artery calcium (CAC) score of ≧400 measured by a cardiac CT scan using the Agatston method or a similar method for measuring CAC. The method includes administering vitamin K2 to the subject, with vitamin K2 being administered at 720 μg per day. Vitamin K2 is administered in combination with 25 μg of vitamin D per day, and vitamin K2 is menaquinone-7 (MK-7). Vitamin K2 is administered as an injection or an oral preparation.

[0063] Yet another embodiment of the present invention relates to a method of retarding the progression of coronary artery calcification in a subject by administering 720 μg of vitamin K2 per day to the subject, preferably in the form of menaquinone-7 (MK-7).

[0064] Yet another embodiment of the present invention relates to a method of reducing the total amount of non-calcified plaque in the coronary arteries of a subject by administering 720 μg of vitamin K2 per day to the subject, preferably in the form of menaquinone-7 (MK-7).

[0065] Yet another embodiment of the present invention relates to a method of reducing the blood concentration of dephosphorylated uncarboxylated matrix-Gla protein (dp-ucMGP) in a subject by administering 720 μg of vitamin K2 per day to the subject, preferably in the form of menaquinone-7 (MK-7).

[0066] Yet another embodiment of the present invention relates to a method of reducing cardiovascular events such as myocardial infarction, revascularization, and death in subjects without a history of myocardial infarction, percutaneous coronary intervention, or coronary artery bypass surgery by administering 720 μg of vitamin K2 per day to the subject, preferably in the form of menaquinone-7 (MK-7).

[0067] According to the present invention, the vitamin K2 administered is any of vitamin K 2 homologs, menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone-12 (MK-12), or menaquinone-13 (MK-13), or a combination thereof, preferably menaquinone-7 (MK-7).

[0068] According to one embodiment of the present invention, the administered vitamin D may be selected from any of vitamin D1, vitamin D2, vitamin D3, vitamin D4 or vitamin D5, or a combination thereof. Vitamin D is topically administered at 1 μg to 50 μg per day, preferably 10 μg to 50 μg per day, more preferably 25 μg per day. Other suitable ranges of vitamin D administration include at least 1 μg of vitamin D per day, preferably at least 5 μg of vitamin D per day, preferably at least 10 μg of vitamin D per day.

[0069] Vitamin K2 (and optionally vitamin D) can be administered as a pharmaceutically acceptable composition, for example, containing at least one excipient. Vitamin K2 can also be administered, for example, in the form of a microencapsulated product as described in WO 2015 / 169816.

[0070] According to another embodiment of the present invention, both a vitamin K2 source and / or a vitamin D source can be administered to a subject (e.g., a patient) as an injection or an oral preparation. Oral administration / formulation of vitamin K2 and vitamin D is desirable.

[0071] When administering both vitamin K2 and vitamin D, they can be administered simultaneously, separately, or sequentially. This applies to all embodiments / aspects of the present invention, such as the compounds, kits, methods, uses, etc. used.

[0072] The compounds or combinations thereof of the present invention can be used in any animal subject, particularly mammals, more particularly humans or animals that function as disease models (such as mice, monkeys, etc.), preferably humans. Preferably, the subject is a human subject.

[0073] The present invention also provides a combination product comprising vitamin K2 and vitamin D for use in the prevention or treatment of coronary heart disease in subjects with a coronary artery calcium (CAC) score ≧ 400 (e.g., at the start of treatment).

[0074] The present invention also relates to a composition for use in the prevention or treatment of coronary heart disease in a subject with a coronary artery calcification (CAC) score ≧ 400 (e.g., at the start of treatment), comprising: (A) vitamin K2, and (B) vitamin D, in the form of a kit (e.g., a pharmaceutical kit), for example, slowing the progression of coronary artery calcification by treatment. The two parts ((A) and (B)) of the kit may be in the form of, for example, separate oral preparations or separate injection preparations.

[0075] According to yet another embodiment of the present invention, the total duration of administration of vitamin K2 and / or vitamin D in the dosage according to the present invention is from 0.1 year to 15 years, preferably from 0.5 year to 10 years, more preferably from 1 year to 5 years, and most preferably 2 years. Other suitable durations include at least 0.1 year, for example, at least 0.5 year, for example, at least 1 year, for example, at least 2 years.

[0076] It should be noted that the embodiments and features described in the context of one aspect of the present invention are also applicable to other aspects of the present invention.

[0077] All patents and non-patent documents cited in this application are hereby incorporated by reference in their entirety.

[0078] Next, the present invention will be described in more detail in the following non-limiting examples.

Example

[0079] Example 1 (Unpublished sub-study of the so-called AVADEC trial) The present invention is based on a sub-study of the so-called AVADEC trial. In the sub-study, the inventors examined the progression of CAC in participants without a history of coronary artery disease (not having had a myocardial infarction and / or revascularization) at baseline.

[0080] The changes in CAC were evaluated in the overall group and in two predefined subgroups (low risk: CAC score < 400 AU at baseline, high risk: CAC ≥ 400 AU).

[0081] 304 participants (male, mean age 71 years) without a history of coronary artery disease and with an aortic valve calcification score ≥ 300 were randomly assigned (1:1) to a group receiving treatment with oral tablets of vitamin K2 (K2VITAL® Delta tablets, 720 μg / day) and the recommended daily amount of vitamin D (25 μg / day) for 2 years, or to a group receiving placebo treatment (no active treatment) for 2 years. K2VITAL® Delta tablets are MK-7 tablet formulations. Exclusion criteria were treatment with vitamin K antagonists or coagulation disorders.

[0082] Non-contrast CT scans were performed at baseline (0 months), 12 months, and 24 months. Contrast CT scans were performed at baseline (0 months) and 24 months. The CAC score was measured with established software and expressed in AU. On contrast CT scans, quantitative coronary plaque composition evaluation was performed using Autoplaque. See Example 2 for details.

[0083] The intervention group and the placebo group were equivalent in all traditional cardiovascular risk factors except for a family history of cardiovascular disease predisposition (14.4% vs. 6.7%, p = 0.046). The inventors found that CAC progressed in both the intervention group and the placebo group from baseline (0 months) to 24-month follow-up (203 AU vs. 254 AU, p = 0.089) (see Figures 1-3).

[0084] The degree of progression was equivalent in patients with a CAC score < 400 AU at baseline (0 months) (77 AU vs. 81 AU, p = 0.846). In patients with a CAC score ≥ 400, the progression of CAC was significantly lower in the intervention group (288 AU vs. 380 AU, p = 0.047). However, in a preliminary analysis of contrast CT scans with 180 participants as subjects, there was no difference in the progression of non-calcified plaque volume (10 mm 3 vs. 37 mm 3, p = 0.276). Furthermore, the number of occurrences of events (total mortality, myocardial infarction, coronary revascularization) was significantly lower in patients administered vitamin K2 and vitamin D (1.9% vs. 6.7%, p = 0.048).

[0085]

Table 1aA

[0086]

Table 1aB

[0087]

Table 1b

[0088]

Table 2a

[0089] The CAC score increased by 253.95 in the placebo group, while it increased by 202.68 in the vitamin K2 + D group.

[0090]

Table 2b

[0091] The CAC score increased by 80.71 points in the placebo group, while it increased by 77.12 points in the vitamin K2 + D group. There was no significant difference.

[0092]

Table 2c

[0093] The CAC score increased by 379.95 points in the placebo group, while it increased by 288.07 points in the vitamin K2 + D group. This corresponds to a significantly lower progression of the CAC score of 91.88 in the intervention group.

[0094]

Table 3

[0095] The analysis in Table 3 was not separated according to CAC score < 400 vs. CAC score ≥ 400. For details, refer to Example 4.

[0096] Example 2 (Measurement of CAC score by multislice CT examination) The cardiac CT scan was performed using a dedicated cardiac CT scanner. Standard non-contrast and contrast examinations were performed according to normal medical practice.

[0097] To evaluate the CAC score, the following CT settings were used. A prospective scan was performed at a tube voltage of 120 kV, 300 ms after the QRS complex. The scan protocol during the contrast scan depends on the local CT scanner and the patient's heart rate. For patients with a stable heart rate of 60 beats per minute (bpm) or more, β-blockers were administered orally or intravenously until the heart rate reached an appropriate value (60 or less if possible), and a prospective gated protocol was used. For patients with a heart rate exceeding 70 bpm despite pretreatment with β-blockers, or in cases of arrhythmia, a prospective scan was performed 200 - 400 ms after the QRS complex.

[0098] Furthermore, sublingual nitrates were administered before the scan. Using a dual-head power injector, 50 - 80 mL of contrast agent was injected into the anterior inferior vena cava at a rate of 6.0 mL / s, followed by 60 mL of physiological saline intravenously (6.0 mL / s). The data acquisition parameters depend on the local CT scanner, but the slice collimation is 0.6 mm or less, the gantry rotation time is as fast as possible, and the tube voltage is 70 kV or 120 kV according to the patient's weight.

[0099] All scans were sent to the Core Lab at Odense University Hospital for analysis there. The CAC score was measured by the Agatston method, i.e., by summing all the calcified spots in the coronary arteries.

[0100] The coronary artery tree is analyzed for the presence and severity of coronary artery disease (CAD) according to the American Heart Association's 16-segment model classification. A coronary plaque is defined as a visible structure adjacent to or within the lumen of the coronary artery that can be clearly distinguished from the vascular lumen and the surrounding pericardial tissue. All coronary artery segments with plaques having a diameter ≥ 2 mm are analyzed using semi-automated software. The scans were analyzed by experienced cardiologists.

[0101] Example 3 (Status of vitamin K2 measured by dp-ucMGP) In the entire population, the carboxylation status of dp-ucMGP ranged from 387 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean value of 773 pmol / L and a median value of 732 pmol / L. In the placebo group, dp-ucMGP ranged from 387 pmol / L (minimum) to 1323 pmol / L (maximum), with a mean value of 763 pmol / L and a median value of 718 pmol / L. In the intervention group, dp-ucMGP ranged from 456 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean value of 783 pmol / L and a median value of 736 pmol / L.

[0102] The carboxylation status of dp-ucMGP in patients with a CAC score ≥ 400 ranged from 433 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean value of 789 pmol / L and a median value of 746 pmol / L. In the placebo group, dp-ucMGP ranged from 433 pmol / L (minimum) to 1323 pmol / L (maximum), with a mean value of 769 pmol / L and a median value of 728 pmol / L. In the intervention group, dp-ucMGP ranged from 456 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean value of 808 pmol / L and a median value of 751 pmol / L.

[0103] [Table 4]

[0104] After 24 hours of supplementation, the mean increase in dp-ucMGP was 36.95 pmol / L in the placebo group, while it was -229.4 pmol / L in the intervention group (P<0.0001) (see Figure 5).

[0105]

Table 5

[0106] Example 4 (Use of statin) In a stratified analysis focusing on treatment by subgroup interaction, participants receiving statin treatment had a significantly reduced progression of CAC score by the intervention (p = 0.048).

[0107]

Table 6

[0108] At baseline, there was no difference in the carboxylation status of dp-ucMGP between statin users and non-users (Figure 6a). In the placebo group, there was no difference in the carboxylation status of dp-ucMGP between statin users and non-users during the supplementation period (Figure 6b). In the intervention group, the decrease in dp-ucMGP was -203.2 pmol / L on average in non-statin users, while it was -239.8 pmol / L in the statin user group (Figure 6c).

[0109]

Table 7

[0110] Example 5 (Plaque volume and composition) Plaque volume and composition were measured for the entire cohort by contrast-enhanced CT. Only Good and Excellent images (at both baseline and 24 months) were subjects for analysis. The progression of non-calcified plaque volume was 46 mm in the placebo group 3 whereas it was -6 mm in the intervention group 3It was (p = 0.172). The progression of calcified plaque volume was 20 mm in the placebo group 3 whereas it was 2 mm in the intervention group 3 (p = 0.179). The progression of total plaque volume was 66 mm in the placebo group 3 whereas it was -4 mm in the intervention group 3 (p = 0.146) (see Figure 7).

[0111] [Table 8]

[0112] [Table 9]

[0113] [Table 10]

[0114] [Table 11]

[0115] Example 6 (Reduction in CAC score and total plaque volume) A categorical analysis was performed to count the number of patients in whom the CAC score decreased 24 months after baseline. In the entire population, the CAC score decreased in 7 out of 132 patients in the placebo group, whereas it decreased in 13 out of 143 patients in the intervention group (p = 0.2533). Among patients with a CAC score of ≥400, the CAC score decreased in 3 out of 76 patients in the placebo group, whereas it decreased in 10 out of 85 patients in the intervention group (p = 0.0858) (see Figures 8a and 8b).

[0116] Furthermore, a categorical analysis was performed to count the number of patients in whom the total plaque volume decreased until 24 months after the baseline (all image quality). In the placebo group, the total plaque volume decreased in 23 out of 98 cases, while in the intervention group, it was 34 out of 109 cases (p = 0.2753) (see Figure 9).

[0117] Example 7 (Cardiovascular event rate) A total of 13 participants had clinical safety events during the follow-up period. The event rate (the composite event rate of myocardial infarction, revascularization, and all-cause death) was 3 (1.9%) in the vitamin K2·D group compared to 10 (6.7%) in the placebo group (p = 0.048).

[0118] [Table 12]

[0119] Reference Diederichsen A.C.P. et al. (2012): (DanRisk study) “Discrepancy between coronary artery calcium score and HeartScore in middle-aged Danes: the DanRisk study”, (Eur J Prev Cardiol. 2012 Jun;19(3):558-64) Diederichsen A.C.P. et al. (2022): “Vitamin K2 and D in Patients With Aortic Valve Calcification: A Randomized Double-Blinded Clinical Trial”, Circulation. 25 April 2022;145: 1387-1397. Geleijnse J.M. et al. (2004):”Dietary intake of menaquinone is associated with reduced risk of coronary heart disease: The Rotterdam study”, American Society for Nutritional Sciences: 3100-3105. Kvist T.V. et al. (2017): (DANCAVAS pilot study), “The DanCavas Pilot Study of Multifaceted Screening for Subclinical Cardiovascular Disease in Men and Women Aged 65-74 Years”, Eur J Vasc Endovasc Surg (2017) 53, 123-131. Ohsaki, Y., et al., Vitamin K suppresses the lipopolysaccharide-induced expression of inflammatory cytokines in cultured macrophage-like cells via the inhibition of the activation of nuclear factor kappaB through the repression of IKKalpha / beta phosphorylation. J Nutr Biochem, 2010. 21(11): p. 1120-6. Okuyama, H., et al., Statins stimulate atherosclerosis and heart failure: pharmacological mechanisms. Expert Rev Clin Pharmacol, 2015. 8(2): p. 189-99. Ozaki, I., et al., Menatetrenone, a vitamin K2 analogue, inhibits hepatocellular carcinoma cell growth by suppressing cyclin D1 expression through inhibition of nuclear factor kappaB activation. Clin Cancer Res, 2007. 13(7): p. 2236-45. Pan, M.H., et al., Inhibition of TNF-alpha, IL-1alpha, and IL-1beta by Pretreatment of Human Monocyte-Derived Macrophages with Menaquinone-7 and Cell Activation with TLR Agonists In Vitro. J Med Food, 2016. 19(7): p. 663-9. Schurgers, L.J. and C. Vermeer, Differential lipoprotein transport pathways of K-vitamins in healthy subjects. Biochim Biophys Acta, 2002. 1570(1): p. 27-32. Shea M.K. et al. (2009): “Vitamin K supplementation and progression of coronary artery calcium in older men and women”, Am J Clin Nutr 2009; 89: 1799-1807. Shearer, M.J. and P. Newman, Metabolism and cell biology of vitamin K. Thromb Haemost, 2008. 100(4): p. 530-47. Simes, D.C., et al., Vitamin K as a Diet Supplement with Impact in Human Health: Current Evidence in Age-Related Diseases. Nutrients, 2020. 12(1). Xia, J., et al., The role of PKC isoforms in the inhibition of NF-kappaB activation by vitamin K2 in human hepatocellular carcinoma cells. J Nutr Biochem, 2012. 23(12): p. 1668-75. Zhelyazkova-Savova, M.D., et al., Statins, vascular calcification, and vitamin K-dependent proteins: Is there a relation? Kaohsiung J Med Sci, 2021. 37(7): p. 624-631.

Claims

1. Vitamin K2 for use in the prevention or treatment of coronary heart disease in subjects whose coronary artery calcification (CAC) score, measured by cardiac CT scan using the Agaston method for CAC measurement, is ≥400. The subject is administered 360 μg to 10 mg of vitamin K2 per day.

2. The vitamin K2 according to claim 1, wherein the blood concentration of dephosphorylated noncarboxylated matrix-Gla-protein (dp-ucMGP) in the subject is 433 pmol / L to 2179 pmol / L.

3. Vitamin K2 according to claim 2, wherein the blood concentration of the dephosphorylated noncarboxylated matrix-Gla-protein (dp-ucMGP) is 433 pmol / L to 500 pmol / L, 475 pmol / L to 600 pmol / L, 550 pmol / L to 700 pmol / L, 650 pmol / L to 800 pmol / L, 750 pmol / L to 900 pmol / L, 850 pmol / L to 1000 pmol / L, 950 pmol / L to 1250 pmol / L, 1200 pmol / L to 1750 pmol / L, 1700 pmol / L to 2000 pmol / L, and 1950 pmol / L to 2179 pmol / L.

4. Vitamin K2 according to claim 1, wherein the blood concentration of dephosphorylated noncarboxylated matrix-Gla-protein (dp-ucMGP) is 400 pmol / L to 2000 pmol / L, preferably 425 pmol / L to 1500 pmol / L, preferably 450 pmol / L to 1250 pmol / L, preferably 475 pmol / L to 1000 pmol / L, and more preferably 500 pmol / L to 900 pmol / L.

5. The vitamin K2 according to claim 1, wherein the subject receives statin treatment.

6. The vitamin K2 according to claim 1, wherein the subject is administered 360 μg to 5 mg of vitamin K2 per day, preferably 360 μg to 1440 μg of vitamin K2 per day, preferably 500 μg to 1000 μg of vitamin K2 per day, and more preferably 720 μg of vitamin K2 per day.

7. The vitamin K2 according to claim 1, wherein the vitamin K2 is preferably administered in combination with 1 μg to 50 μg of vitamin D per day, more preferably 10 μg to 50 μg of vitamin D per day, and most preferably 25 μg of vitamin D per day.

8. The vitamin K2 according to claim 1, wherein the vitamin K2 is any of the following: a vitamin K2 homolog, menaquinone-4 (MK-4), menaquinone-5 (MK-5), menaquinone-6 (MK-6), menaquinone-7 (MK-7), menaquinone-8 (MK-8), menaquinone-9 (MK-9), menaquinone-10 (MK-10), menaquinone-11 (MK-11), menaquinone-12 (MK-12), or menaquinone-13 (MK-13), or a combination thereof.

9. The vitamin K2 according to claim 7, wherein the vitamin D administered in combination with the vitamin K2 is selected from any of the following: vitamin D1, vitamin D2, vitamin D3, vitamin D4, or vitamin D5, or any combination thereof.

10. Vitamin K2 according to any one of claims 1 to 9, wherein vitamin K2 is administered as an injection or oral preparation.

11. Vitamin K2 for use in the prevention or treatment of coronary heart disease in subjects to reduce the progression of non-calcified plaque volume in the coronary arteries, wherein the subjects are administered 200 μg to 50 mg of vitamin K2 per day.

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