Vitamin k2 for treating coronary artery calcification (CAC)

Abstract

The present invention relates to high doses of vitamin K2, in particular menadione-7 (MK-7), for use in the treatment of coronary heart disease in high risk subjects having a coronary artery calcification (CAC) score > = 400 (e.g., at the beginning of treatment), where the treatment results in a slowing progression of calcification in the coronary artery.

Description

Technical Field

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

[0002] Coronary artery calcification (CAC) and CAC progression are strong predictors of acute myocardial infarction (AMI) and cardiovascular death. Menaquinone-7 (MK-7), a form of vitamin K2, is a cofactor involved in protein carboxylation that inhibits arterial calcification and is thought to reduce the rate of progression of aortic valve calcification (AVC) in patients with aortic stenosis.

[0003] Additionally, ischemic heart disease causes 19% and 20% of deaths in men and women, respectively, so prevention is critical. Ischemic heart disease is often unnoticed until symptoms of a myocardial infarction occur. However, subclinical coronary artery disease is easily detected by non-contrast cardiac CT scans for coronary artery calcification (CAC). CAC increases with age, and men have higher CAC scores than women. In people without CAC, the risk of future cardiovascular disease (CVD) is very low, but as the CAC score increases, the risk of ischemic heart disease also increases. Therefore, in order to prevent CVD, it is important to identify and treat individuals with severe CAC.

[0004] Vascular calcification is a slowly progressive process caused by an imbalance between mechanisms that promote and inhibit calcium deposition in the vessel wall, with vitamin K-dependent proteins playing a crucial role in this inhibition. The most common K vitamin is phylloquinone (vitamin K1), as it is essential for the activation of several coagulation factors. Menaquinone (vitamin K2) is another very important vitamin K species. Vitamin K2 is thought to be required for the gamma-carboxylation of proteins involved in the inhibition of arterial calcification, namely matrix-Gla protein (MGP). Without these activated proteins, the balance of cellular calcium absorption and the mineralization process in bones and blood vessels are impaired.

[0005] Vitamin K2 enters the circulation via lymph associated with chylomicrons, which travel directly to the liver. Extrahepatic distribution is facilitated by LDL particles, in which the presence of long-chain menaquinones is higher than that of short-chain menaquinones. All tissues expressing LDL receptors will be targets for vitamin K2, especially long-chain menaquinones such as MK-7 (Schurgers, LJ and C. Vermeer (2002) and Shearer, MJ and P. Newman (2008)).

[0006] There are 20 known vitamin K-dependent proteins (VKDPs) in the body, with vitamin K2 acting as a cofactor to activate these proteins through a carboxylation process (Simes, DC, et al. (2020)). Matrix Gla protein (MGP) is expressed in vascular smooth muscle cells and is a potent inhibitor of vascular calcification. The key to how matrix Gla protein works lies in its carboxyl group. Matrix Gla must be carboxylated to function properly, and vitamin K2 acts as a cofactor for this enzymatic reaction together with gamma-glutamyl carboxylase. In the presence of vitamin K2, matrix Gla protein is carboxylated, meaning it is "turned on" to repel calcium penetration. Insufficient vitamin K2 results in insufficient or "turned off" matrix Gla carboxylation, meaning it cannot inhibit calcium penetration into soft tissues. A commonly used biomarker of vitamin K2 status in the body is dephosphorylated uncarboxylated MGP (dp-ucMGP). This biomarker is the inactive form of MGP, and its high levels reflect low vitamin K2 status and vice versa.

[0007] Several VKDPs have anti-inflammatory functions, such as protein C, protein S, Gas 6, and GRP (Simes, DC, et al. (2020)). In addition, there are data showing the anti-inflammatory regulatory effects of vitamin K2 that are independent of γ-glutamyl carboxylase. Using human monocyte-derived macrophages, Pan and colleagues found that when the cells were pretreated with vitamin K2 (menadione-7), cytokine release (TNF-α, IL-1α, IL-1β) was inhibited, and the scientists were able to show a dose-response relationship (Pan, MH, et al. (2016)). Other research groups (Ohsaki, Y., et al. (2006) and Reddi, K., et al. (1995)) found that IL-6 also had a similar inhibitory effect. The release of proinflammatory cytokines is mainly regulated by the NF-kB signaling pathway, and vitamin K has been shown to inhibit the release of IkB by NF-kB, thereby allowing it to enter the cell 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 controversial hypothesis was proposed that the action of statins is not only to inhibit cholesterol synthesis, but also to inhibit the levels of isopentenyl intermediates by inhibiting HMG-CoA reductase, thereby also inhibiting the conversion of vitamin K1 to K2 in the body (Okuyama, H., et al. (2015)). Since vitamin K2 has a higher preference for LDL particles directed to extrahepatic tissues, the use of statins may lead to extrahepatic vitamin K deficiency, resulting in reduced activation of VKDP in different tissues. A cross-sectional clinical trial investigated the association between statin use, CAC, and VKDP activation levels. The authors found that statin users had higher CAC scores. They did not find any differences in the carboxylation status of MGP. However, they found that the levels of the non-carboxylated form of VKDP osteocalcin were significantly higher in statin users (Zhelyazkova-Savova, MD, et al. (2021)).

[0009] Currently, there are no recommendations for vitamin K2 supplementation. Furthermore, it is known that the daily intake of vitamin K2 in the Western world is insufficient to meet the requirements for full activation of MGP. Furthermore, there is no documented evidence of toxicity for either vitamin K1 or vitamin K2, and the World Health Organization has not set an upper tolerance limit for vitamin K intake.

[0010] The recent AVADEC trial, which investigated the effect of high-dose vitamin K2 supplementation on the progression of aortic valve calcification, was published in Diederichsen ACP et al. (2022). The publication disclosed the treatment of male subjects with an aortic valve calcification score of more than 300 arbitrary units. However, in this study, the progression of aortic valve calcification was not significantly reduced.

[0011] EP1728507 A1 discloses in a study on rats that high intake of vitamin K can remove calcified deposits from blood vessels already affected by pre-existing calcification.

[0012] EP2558084 B1 discloses a pharmaceutical composition comprising vitamin K and nicotinamide in a study on rats for preventing or treating diseases accompanied by extra-osseous calcification.

[0013] WO19021232 A1 relates to a composition comprising vitamin K or its analogs and derivatives, inorganic magnesium salts and iron (III) oxides, complexes or salts, pharmaceutical or food grade excipients, additives and / or co-formulations for use in a preventive or therapeutic method for treating vascular calcification in an in vitro experiment (the experimental model consists of calcified vascular smooth muscle cells removed from rat aorta).

[0014] WO19191773 A1 discloses administering vitamin K, including vitamin K2, to mammals to rapidly reverse vascular calcification. WO19191773 A1 further discloses a clinical study conducted on kidney transplant patients to study the effectiveness and safety of vitamin K2 supplementation on arteriosclerosis.

[0015] EP1728507 B1 discloses the use of vitamin K2 (eg in combination with vitamin D) to reverse vascular calcification in a rat model.

[0016] Geleijnse JM et al. (2004) Effects of dietary menaquinone on aortic calcification and coronary heart disease (CHD) in men and women aged 55 years and older with no history of myocardial infarction at baseline.

[0017] Shea MK et al. (2009) reported the effects of vitamin K1 (phylloquinone) supplementation on CAC progression in elderly men and women aged 60–80 years who had no known coronary heart disease (asymptomatic) at baseline and concluded that vitamin K1 supplementation, when taken with recommended amounts of calcium and vitamin D, can reduce the progression of existing CAC in asymptomatic elderly men and women.

[0018] None of the above prior art documents discloses or suggests administering or supplementing vitamin K2 to subjects (eg, human patients) with severe CAC (defined as a CAC score ≥ 400, ie, patients at higher risk of acute myocardial infarction (AMI)).

[0019] Therefore, it would be advantageous to provide a treatment approach that reduces the further progression of coronary artery calcification in the subgroup of patients with severe CAC (defined as a CAC score ≥ 400). Summary of the invention

[0020] A previous population-based study (Diederichsen ACP et al. (2012)) investigated the prevalence of CAC scores over 400 in the Danish population and showed that 2% of men aged 50 years had a CAC score over 400, rising to 14% of men aged 60 years. 1% of women aged 50 years had a CAC score over 400, rising to 4% of women aged 60 years. Furthermore, in a random selection of Danish men and women aged 65-74 years, 37.8% of men and 11.3% of women were found to have a CAC score > 400 (Kvist TV et al. (2017)).

[0021] The present invention was conceived in this context and is based on a surprising observation that vitamin K2 supplementation reduces CAC progression in subjects (e.g., human subjects) with severe CAC (defined as a CAC score ≥ 400) compared to placebo. Specifically, high-dose supplementation with vitamin K2 and vitamin D reduces CAC progression in subjects (e.g., human subjects) with severe CAC (defined as a CAC score ≥ 400).

[0022] Therefore, one object of the present invention relates to treating this specific subgroup of subjects (e.g. patients) defined as having a CAC score ≥ 400 as determined by cardiac CT scan and by using the so-called Agatston method or similar methods for determining CAC.

[0023] In a first aspect, the present invention provides vitamin K2 for use in preventing or treating coronary heart disease in a subject with a coronary artery calcification (CAC) score ≥400.

[0024] The present invention also relates to the use of vitamin K2 for preventing or treating coronary heart disease in a subject with a CAC score ≥ 400, by administering to the subject 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg vitamin K2 per day, more preferably 720 μg vitamin K2 per day, preferably wherein vitamin K2 is administered in combination with vitamin D, preferably 1-50 μg vitamin D per day, 10-50 μg vitamin D per day, preferably 25 μg vitamin D per day, for example wherein the prevention or treatment results in a slowing of the progression of calcification of the coronary arteries.

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

[0026] In yet another aspect, the present invention provides vitamin K2 for use in slowing the progression of calcification in the coronary arteries, wherein slowing the progression of calcification in the coronary arteries is a reduction in the CAC score as determined by cardiac CT scanning and using the Agatston method or a similar method for determining CAC 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 another aspect, the present invention provides vitamin K2 for use in slowing the progression of calcification in coronary arteries, wherein the slowing of the progression of calcification in coronary arteries is a decrease in calcified plaque (mm 3 ) was reduced by at least 1%, at least 3%, at least 9%, at least 12%, and at least 12.7%.

[0028] In another aspect, the present invention provides vitamin K2 for use in slowing the progression of calcification in coronary arteries, wherein the slowing of the progression of calcification in coronary arteries is a decrease in non-calcified plaque (mm) in the intervention group. 3 ) progression is reduced by at least 1%, at least 3%, at least 5%, at least 7%, and at least 8%.

[0029] In another aspect, the invention provides vitamin K2 for use in reducing calcification in the coronary arteries as measured by CAC score, as measured by cardiac CT scan and by using the Agatston method or a similar method for determining 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 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 baseline.

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

[0032] Another aspect of the invention relates to a method of preventing or treating coronary artery calcification in a subject having a CAC score ≥ 400, wherein the method involves administering vitamin K2 to the subject.

[0033] The present invention may relate to a method for treating coronary artery calcification in a subject with a CAC score ≥ 400, by administering to the subject 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg vitamin K2 per day, preferably 720 μg vitamin K2 per day, preferably in combination with vitamin D, preferably 1-50 μg vitamin D per day, preferably 10-50 μg vitamin D per day, preferably 25 μg vitamin D per day.

[0034] Another aspect relates to the use of vitamin K2 in the manufacture of a medicament for preventing or treating coronary heart disease in a subject with a coronary artery calcification (CAC) score ≥400.

[0035] Another aspect relates to a kit of parts comprising:

[0036] (A) Vitamin K2, and

[0037] (B) Vitamin D;

[0038] For the prevention or treatment of coronary heart disease in subjects with a coronary artery calcification (CAC) score ≥ 400.

[0039] BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 CAC progression from baseline to 24 months follow-up is shown for all subjects in the intervention and placebo groups (entire cohort) (203 AU vs. 254 AU, p=0.089).

[0041] Figure 2 CAC progression is shown in the subgroup with a baseline CAC score <400AU. There was no difference in progression in subjects with a baseline CAC score <400AU (77AU vs. 81AU, p=0.849)

[0042] Figure 3 The progression of CAC in the subgroup with a baseline CAC score of ≥400 AU is shown. In subjects with a baseline CAC score of ≥400 AU, the progression of CAC score was slowed at 24 months follow-up (288 AU vs. 380 AU, p=0.047).

[0043] Figure 4 The composition of plaques in this cohort is shown. Specifically, Figure 4 The total plaque composition divided into two subgroups is shown: "calcified plaque burden" and "non-calcified plaque burden". The latter is further described as "low-attenuation plaque burden". (See Table 3 for more details)

[0044] Figure 5 showed that after 24 months of vitamin K2 ( Delta tablets, 720 μg / day) supplementation therapy, supplemented with the recommended daily dose of vitamin D (25 μg / day) (oral tablets), the average increase in dp-ucMGP in the placebo group was 36.95 pmol / L, while that in the intervention group was -229,4 pmol / L (P<0.0001).

[0045] Figure 6a -c shows that after 24 months, vitamin K2 ( Delta tablets, 720 μg / day) supplemented with the recommended daily dose of vitamin D (25 μg / day) (oral tablet form), no difference in the carboxylation status of dp-ucMGP was found between patients taking statins and those not taking statins at baseline ( Figure 6a In the placebo group, there was no difference in the carboxylation status of dp-ucMGP between patients taking statins during supplementation and those not taking statins ( Figure 6b In the intervention group, the mean reduction in dp-ucMGP was -203.2 pmol / L in patients not taking statins, compared with -239.8 pmol / L in the statin group ( Figure 6c ).

[0046] Figure 7 showed that after 24 months of vitamin K2 ( Delta tablets, 720 μg / day) supplementation therapy, supplemented with the recommended daily dose of vitamin D (25 μg / day) (oral tablets), the non-calcified plaque volume progression in the placebo group was 46 mm 3 , while the intervention group was -6mm 3 (p=0.172). The volume of calcified plaque in the placebo group progressed to 20 mm 3 , while the intervention group was 2mm 3 (p=0.179). The total plaque volume progression in the placebo group was 66 mm 3 , while the intervention group was -4mm 3 (p=0.146).

[0047] Figure 8a -b shows that in the whole population, vitamin K2 ( Delta tablets, 720 μg / day) supplementation therapy, supplemented with the recommended daily dose of vitamin D (25 μg / day) (oral tablets), from baseline to 24 months, CAC scores decreased in 7 of 132 people in the placebo group and 13 of 143 people in the intervention group (p = 0.2533) ( Figure 8a Among patients with a CAC score ≥400, from baseline to 24 months, 3 of 76 patients in the placebo group had a reduction in CAC score, while 10 of 85 patients in the intervention group had a reduction in CAC score (p = 0.0858) ( Figure 8b ).

[0048] Fig. 9 showed that after 24 months of vitamin K2 supplementation ( Delta tablets, 720 μg / day) supplemented with the recommended daily dose of vitamin D (25 μg / day) (oral tablets), many patients had a reduction in total plaque volume from baseline to 24 months. Total plaque volume decreased in 23 of 98 people in the placebo group and in 34 of 109 people in the intervention group.

[0049] The present invention will be described in more detail below. Summary of the invention

[0051] Before discussing the present invention in further detail, the following terms and conventions are first defined:

[0052] Agatston method: The CAC scoring quantification system used in this article in CT images is the Agatston method, which uses the weighted sum of lesions with density above 130HU (Hounsfield scale), multiplying the calcification area by a factor related to the maximum plaque attenuation: 130-199HU, factor 1; 200-299HU, factor 2; 300-399HU, factor 3; ≥400HU, factor 4 (see, e.g., Neves PO et al. (2017), which is incorporated herein by reference). The Agatston method is well known in the art.

[0053] CAC score: As used herein, CAC refers to "coronary artery calcification." To determine the CAC score, a non-contrast CT scan must be performed. The CAC score reflects the amount of calcium in the walls of the heart's arteries. This test uses a special type of imaging test called a cardiac computed tomography (CT). This scan produces multiple pictures to check if calcium is present and, if so, how much. The calcium score is calculated based on the amount of plaque seen on the CT scan, quantified by the Agatston method (see also Neves PO et al. (2017)). CAC scores are well known in the art.

[0054] Vitamin K2: Vitamin K2 as used herein refers to any of the 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. The number of isoprenyl units n in their side chains is different, ranging from 4 to 13, so vitamin K2 consists of various forms. It is indicated by the suffix (-n), for example, MK-7 contains seven isoprenyl units.

[0055] Vitamin K2 is also referred to as prodrugs of vitamin K2. Suitable prodrugs are described, for example, in WO 2013 / 128037.

[0056] Coronary heart disease may be referred to herein as ischemic heart disease or coronary artery disease.

[0057] Embodiments of the invention

[0058] One embodiment of the invention relates to vitamin K2 for use in preventing or treating coronary heart disease, or a method for preventing or treating coronary heart disease in a subject having a coronary artery calcification (CAC) score of ≥ 400 as determined by cardiac CT scan and using the Agatston method or a similar method for determining CAC. Generally, the prevention or treatment described herein slows the progression of calcification in the coronary arteries.

[0059] In embodiments and certain aspects of the invention, when referring to a CAC score ≥ 400, it generally refers to a CAC score ≥ 400 at the start of treatment. As discussed in the Examples, beneficial effects can be seen in subjects who initially exhibit high CAC levels.

[0060] Vitamin K2 (e.g., MK-7) can be administered to a subject at a dose of 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg vitamin K2 per day, and most preferably 720 μg vitamin K2 per day. Other suitable doses of vitamin K2 include at least 100 μg vitamin K2 per day, preferably at least 360 μg vitamin K2 per day, for example at least 500 μg vitamin K2 per day.

[0061] Therefore, another embodiment of the present invention relates to a method for the use of vitamin K2 to prevent or treat coronary heart disease in a subject with a coronary artery calcification (CAC) score of ≥400 (e.g., at the start of treatment), as determined by cardiac CT scan and using the Agatston method or a similar method for determining CAC, wherein the subject is administered 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg vitamin K2 per day, and most preferably 720 μg vitamin K2 per day. Other suitable doses of vitamin K2 include at least 100 μg vitamin K2 per day, preferably at least 360 μg vitamin K2 per day, such as at least 500 μg vitamin K2 per day.

[0062] Another embodiment of the present invention relates to vitamin K2 for use in treating coronary heart disease in a subject, or a method of treating coronary heart disease in a subject, wherein the subject has a coronary artery calcification (CAC) score as determined by cardiac CT scan and using the Agatston method or a similar method for determining CAC.

[0063] ≥400 (e.g. at the start of treatment), wherein the subject is administered 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg vitamin K2 per day, most preferably 720 μg vitamin K2 per day, in combination with vitamin D, preferably 1-50 μg vitamin D per day, preferably 10-50 μg vitamin D per day, preferably 25 μg vitamin D per day, preferably wherein the treatment results in a slowing of the progression of calcification in the coronary arteries.

[0064] Another embodiment of the present invention relates to vitamin K2 for use in preventing or treating coronary heart disease in a subject with a coronary artery calcification (CAC) score ≥ 400, as determined by cardiac CT scan and using the Agatston method or a similar method, wherein the subject's blood concentration of dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) is 433 to 2179 pmol / L, 433 to 500 pmol / L, 475 to 600 pmol / L, 550 to 700 pmol / L, 650 to 800 pmol / L, 750 to 900 pmol / L, 850 to 1000 pmol / L, 950 to 1250 pmol / L, 1200 to 1750 pmol / L, 1700 to 2000 pmol / L, and 1950 to 2179 pmol / L.

[0065] Another embodiment of the present invention relates to the use of vitamin K2 for preventing or treating coronary heart disease in a subject with a coronary artery calcification (CAC) score ≥ 400, as determined by cardiac CT scan and using the Agatston method or a similar method, wherein the subject has a blood concentration of dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) of 433 to 2179 pmol / L, 400 to 2000 pmol / L, preferably 425 to 1500 pmol / L, preferably 450 to 1250 pmol / L, preferably 475 to 1000 pmol / L, and more preferably 500 to 900 pmol / L.

[0066] In another aspect, the present invention provides vitamin K2 for use in slowing the progression of coronary artery calcification.

[0067] In yet another aspect, the present invention provides vitamin K2 for use in slowing the progression of coronary artery calcification, wherein slowing the progression of coronary artery calcification refers to a reduction in CAC score of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, or at least 8.9% as measured by cardiac CT scanning and using the Agatston method or a similar method for measuring CAC.

[0068] In another aspect, the present invention provides vitamin K2 for slowing down the progression of coronary artery calcification, wherein slowing down the progression of coronary artery calcification refers to the reduction of calcified plaque (mm 3 ) was reduced by at least 1%, at least 3%, at least 9%, at least 12%, and at least 12.7%.

[0069] In another aspect, the present invention provides vitamin K2 for slowing the progression of coronary artery calcification, wherein slowing the progression of coronary artery calcification refers to a decrease in non-calcified plaque (mm 3 ) is reduced by at least 1%, at least 3%, at least 5%, at least 7%, and at least 8%.

[0070] In another aspect, the invention provides vitamin K2 for use in reducing calcification in the coronary arteries as measured by CAC score, the CAC score determined by cardiac CT scan and by using the Agatston method or a similar method of determining 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.

[0071] In another aspect, the present invention provides vitamin K2 for use in reducing total plaque volume in a coronary artery 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%, or at least 13.1% compared to baseline.

[0072] Another embodiment of the present invention is directed to a method for preventing or treating coronary heart disease in a subject having a CAC score ≥ 400, wherein the CAC score is determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method comprises administering vitamin K2 to the subject.

[0073] Another embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject having a CAC score ≥ 400, the score being determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method involves administering vitamin K2 to the subject, wherein the dose of vitamin K2 administered is 100 μg-100 mg vitamin K2 per day, preferably 200 μg-50 mg vitamin K2 per day, preferably 360 μg-10 mg vitamin K2 per day, preferably 360 μg-5 mg vitamin K2 per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg per day, and more preferably 720 μg vitamin K2 per day.

[0074] Another embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject with a CAC score of ≥ 400 as determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method comprises administering vitamin K2 to the subject, wherein the vitamin K2 is administered at a dose of 720 μg vitamin K2 per day. Wherein the vitamin K2 is administered in combination with vitamin D, preferably 1-50 μg vitamin D per day, preferably 10-50 μg vitamin D per day, preferably 25 μg vitamin D per day.

[0075] Another embodiment of the present invention relates to a method for preventing or treating coronary heart disease in a subject having a CAC score of ≥ 400 as determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method comprises administering vitamin K2 to the subject, wherein the vitamin K2 is administered at a dose of 720 μg vitamin K2 per day. Wherein the vitamin K2 is administered in combination with 25 μg vitamin D per day, wherein the vitamin K2 is selected from any one of vitamin K2 homologues, 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.

[0076] Another embodiment of the present invention is directed to a method of preventing or treating coronary heart disease in a subject having a CAC score of ≥ 400 as determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method comprises administering vitamin K2 to the subject, wherein the vitamin K2 is administered at a dose of 720 μg vitamin K2 per day. Wherein the vitamin K2 is administered in combination with 25 μg vitamin D per day. Wherein the vitamin K2 is menaquinone-7 (MK-7).

[0077] Another embodiment of the present invention is directed to a method for preventing or treating coronary heart disease in a subject with a CAC score of ≥400 as determined by cardiac CT scanning and using the Agatston method or a similar CAC determination method, wherein the method comprises administering vitamin K2 to the subject, wherein the vitamin K2 is administered at a dose of 720 μg vitamin K2 per day. wherein the vitamin K2 is administered in combination with 25 μg vitamin D per day. wherein the vitamin K2 is menaquinone-7 (MK-7), wherein the vitamin K2 is administered in an injectable or oral formulation.

[0078] Another embodiment of the present invention is directed to a method of slowing the progression of coronary artery calcification in a subject by administering to the subject vitamin K2, preferably 720 μg vitamin K2 per day in the form of menaquinone-7 (MK-7).

[0079] Another embodiment of the present invention is directed to a method of reducing total non-calcified plaque volume in a coronary artery of a subject by administering to the subject vitamin K2, preferably 720 μg vitamin K2 per day in the form of menaquinone-7 (MK-7).

[0080] Another embodiment of the present invention is directed to a method of reducing the blood concentration of dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) in a subject by administering vitamin K2 to the subject, preferably 720 μg of vitamin K2 per day in the form of menaquinone-7 (MK-7).

[0081] Another embodiment of the present invention relates to a method for reducing cardiovascular events (e.g., myocardial infarction, revascularization, and death), which is suitable for subjects who have not had a myocardial infarction, percutaneous coronary intervention, or coronary artery bypass surgery, by administering vitamin K2 to the subject, preferably 720 μg of vitamin K2 per day in the form of menaquinone-7 (MK-7).

[0082] The vitamin K2 administered according to the present invention may be any 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, preferably menaquinone-7 (MK-7).

[0083] The vitamin D used in the embodiment of the present invention can be any one of vitamin D1, vitamin D2, vitamin D3, vitamin D4 or vitamin D5, or a combination thereof. The dosage of vitamin D is generally 1-50 μg per day, preferably 10-50 μg per day, and more preferably 25 μg per day. Other suitable vitamin D administration ranges include at least 1 μg vitamin D per day, preferably at least 5 μg vitamin D per day, and preferably at least 10 μg vitamin D per day.

[0084] 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 in the form of a microencapsulated product, such as described in WO2015 / 169816.

[0085] According to another embodiment of the present invention, the vitamin K2 source and / or the vitamin D source can be administered to a subject (eg, a patient) as an injection or oral preparation. Oral administration / preparation of vitamin K2 and vitamin D is preferred.

[0086] If vitamin K2 and vitamin D are administered simultaneously, they may be administered simultaneously, separately or sequentially. This applies to all embodiments / aspects of the invention, whether compounds used, kits used, methods, uses, etc.

[0087] The compounds or combinations of the present invention can be used in any animal subject, in particular mammals, more specifically humans or animals used as disease models (eg, mice, monkeys, etc.), preferably humans. Therefore, preferably, the subject is a human subject.

[0088] The present invention also provides a combination product comprising vitamin K2 and vitamin D for use in preventing or treating coronary heart disease in a subject having a coronary artery calcification (CAC) score ≥ 400 (eg at the start of treatment).

[0089] The present invention also provides a kit of parts (e.g., a multi-component drug kit), comprising

[0090] (A) Vitamin K2 and

[0091] (B) Vitamin D;

[0092] For preventing or treating coronary heart disease in a subject with a coronary artery calcification (CAC) score ≥ 400 (e.g. at the start of treatment), e.g., where the treatment results in a slowing of the progression of calcification in the coronary arteries. For example, the two parts of the kit ((A) and (B)) can be separate oral formulations or separate injection formulations.

[0093] According to another embodiment of the present invention, the total time of applying the dosage of vitamin K2 and / or vitamin D of the present invention is 0.1-15 years, preferably 0.5-10 years, more preferably 1-5 years, and most preferably 2 years. Other suitable times include at least 0.1 years, such as at least 0.5 years, such as at least 1 year, such as at least 2 years.

[0094] It should be noted that embodiments and features described in one aspect of the invention are also applicable to other aspects of the invention.

[0095] All patent and non-patent references cited in this application are incorporated herein by reference in their entirety.

[0096] The present invention will now be described in further detail in the following non-limiting examples. Example

[0097] Example 1 (Unpublished sub-study of the so-called AVADEC trial)

[0098] The present invention is based on a sub-study of the so-called AVADEC trial. In this sub-study, the inventors examined the progression of CAC in participants without coronary heart disease (without myocardial infarction and / or revascularization) at baseline.

[0099] Changes in CAC were assessed in the entire group and in two prespecified subgroups (low risk: CAC score <400 AU and high risk: CAC ≥400 AU at baseline).

[0100] 304 participants (male, mean age 71 years) with no history of coronary artery disease and an aortic valve calcification score ≥ 300 were randomized (1:1) to receive vitamin K2 ( Delta tablets, 720 μg / day), supplemented with the recommended daily dose of vitamin D (25 μg / day), oral tablets or placebo treatment (inactive treatment) for two years. Delta tablets are a MK-7 tablet formulation. Exclusion criteria were treatment with vitamin K antagonists or coagulopathy.

[0101] Non-contrast CT scans were performed at baseline (0 months), 12 months, and 24 months of follow-up. Contrast CT scans were performed at baseline (0 months) and 24 months. CAC scores were measured using sophisticated software and expressed in AU. In contrast CT scans, quantitative coronary plaque composition assessment was performed using Autoplaque. For more details, see Example 2.

[0102] The intervention and placebo groups were similar in all traditional cardiovascular risk factors except for familial susceptibility to cardiovascular disease (14.4% vs. 6.7%, p = 0.046). The inventors found that CAC progressed from baseline (0 months) to 24 months of follow-up in both the intervention and placebo groups (203 AU vs. 254 AU, p = 0.089) (see Figure 1-3 ).

[0103] Patients with a CAC score <400 AU at baseline (0 months) had similar progression (77 AU vs. 81 AU, p=0.846). Among patients with a CAC score ≥400, CAC progression was significantly lower in the intervention group (288 AU vs. 380 AU, p=0.047). However, a preliminary analysis of the comparison CT scans of 180 participants showed no difference in non-calcified plaque volume progression (10 mm 3 vs.37mm 3, p=0.276). In addition, the number of events (all-cause death, myocardial infarction, and coronary revascularization) was significantly lower in participants who received vitamin K2 and D (1.9% vs. 6.7%, p=0.048).

[0104] Table 1a. Patient characteristics at baseline (month 0)

[0105]

[0106]

[0107] Table 1b. Characteristics of CAC scores at baseline (month 0)

[0108] Baseline N Minimum (AU) Maximum (AU) Average(AU) Median (AU) All groups 304 0 6269 1034 641.5 Placebo group 149 0 5687 1031 654.7 Intervention Group 155 0 6269 1037 636.4

[0109] Table 2. Progression of CAC score. Mean change in CAC score from 0 to 24 months

[0110] Table 2a. Whole group

[0111]

[0112] The CAC score increased by 253.95 in the placebo group, while the CAC score increased by 202.68 in the vitamin K2+D group.

[0113] Table 2b. Subgroups with baseline CAC score less than 400

[0114]

[0115] The CAC score increased by 80.71 in the placebo group, while it increased by 77.12 in the vitamin K2+D group, with no significant difference between the two groups.

[0116] Table 2c. Subgroups with baseline CAC score ≥400.

[0117]

[0118] The placebo group had an increase in CAC score of 379.95, while the vitamin K2+D group had an increase in CAC score of 288.07. This equated to a significant decrease in CAC score progression of 91.88 in the intervention group.

[0119] Table 3. Plaque composition analysis of the entire cohort

[0120]

[0121] The analysis in Table 3 was not divided into CAC scores <400 and CAC scores ≥400. For more information, see Figure 4 .

[0122] Example 2 (Determination of CAC score by multi-slice computed tomography)

[0123] Cardiac CT scans were performed using a dedicated cardiac CT scanner. Standard non-contrast and contrast scans were performed according to routine clinical care.

[0124] To assess the CAC score, the following CT settings were used: 120 kV tube voltage, and prospective scanning 300 ms after the QRS complex. The scanning protocol during contrast scanning depended on the local CT scanner and the patient's heart rate. For patients with a stable heart rate above 60 beats per minute (bpm), oral or intravenous beta-blockers were administered until the heart rate was appropriate (if possible below 60), and a prospective gating protocol was used. For patients with a heart rate >70 bpm or arrhythmias despite pretreatment with beta-blockers, prospective scanning was performed 200-400 ms after the QRS complex.

[0125] In addition, nitrates were injected sublingually before the scan. Using a dual-head power injector, 50-80 mL of contrast medium was injected into the antecubital vein at a rate of 6.0 mL / s, followed by 60 mL of intravenous saline (6.0 mL / s). Data acquisition parameters depended on the local CT scanner, but the slice collimation would be less than 0.6 mm, the gantry rotation time would be as fast as possible, and the tube voltage would be 70 or 120 kV (depending on the patient's weight).

[0126] All scans were sent to the core laboratory, Odense University Hospital, for analysis. CAC score was measured using the Agatston method, which sums all calcification points in the coronary arteries.

[0127] The coronary artery tree will be analyzed for the presence and severity of coronary artery disease (CAD) according to the American Heart Association 16-segment model classification. Coronary plaques are defined as visible structures within or near the lumen of a coronary artery that can be clearly distinguished from the vessel lumen and surrounding pericardial tissue. All coronary artery segments with a diameter ≥ 2 mm and plaque will be analyzed using semi-automated software. The scans will be analyzed by experienced cardiologists.

[0128] Example 3 (Measurement of Vitamin K2 Status by dp-ucMGP)

[0129] For the entire population, the carboxylation status of dp-ucMGP ranged from 387 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean of 773 pmol / L and a median of 732 pmol / L. In the placebo group, dp-ucMGP ranged from 387 pmol / L (minimum) to 1323 pmol / L (maximum), with a mean of 763 pmol / L and a median of 718 pmol / L. In the intervention group, dp-ucMGP ranged from 456 pmol / L (minimum) to 2179 pmol / L (maximum), with a mean of 783 pmol / L and a median of 736 pmol / L.

[0130] For patients with a CAC score ≥ 400, the carboxylation status of dp-ucMGP ranged from 433 pmol / L (min) to 2179 pmol / L (max), with a mean of 789 pmol / L and a median of 746 pmol / L. In the placebo group, dp-ucMGP ranged from 433 pmol / L (min) to 1323 pmol / L (max), with a mean of 769 pmol / L and a median of 728 pmol / L. In the intervention group, dp-ucMGP ranged from 456 pmol / L (min) to 2179 pmol / L (max), with a mean of 808 pmol / L and a median of 751 pmol / L.

[0131] Table 4

[0132]

[0133] After 24 months of supplementation, the placebo group had a mean increase of 36.95 pmol / L in dp-ucMGP, while the intervention group had an increase of -229.4 pmol / L (P<0.0001). Figure 5 ).

[0134] Table 5

[0135]

[0136] Example 4 (Use of statins)

[0137] In stratified analysis of study treatment and subgroup interaction, CAC score progression in participants receiving statins was significantly reduced by the intervention (p = 0.048).

[0138] Table 6

[0139]

[0140] At baseline, there was no difference in dp-ucMGP carboxylation status between patients taking statins and those not taking statins ( Figure 6a In the placebo group, there was no difference in dp-ucMGP carboxylation status between patients taking statins during supplementation and those not taking statins ( Figure 6b In the intervention group, the average dp-ucMGP reduction in patients not taking statins was -203.2 pmol / L, while in the statin group it was -239.8 pmol / L ( Figure 6c ).

[0141] Table 7

[0142]

[0143] Example 5 (Plaque Volume and Composition)

[0144] Plaque volume and composition were measured by contrast-enhanced CT for the entire cohort. Only good and excellent images (at baseline and 24 months) were included in the analysis. Non-calcified plaque volume progression was 46 mm in the placebo group. 3 , while the intervention group was -6mm 3 (p=0.172). The volume of calcified plaque in the placebo group progressed to 20 mm 3 , while the intervention group was 2mm 3 (p=0.179). The total plaque volume progression in the placebo group was 66 mm 3 , while the intervention group was -4mm 3 (p=0.146)(see Figure 7 ).

[0145] Table 8 Total plaque volume at baseline

[0146]

[0147] Table 9 Calcified plaque volume at baseline

[0148]

[0149]

[0150] Table 10 Non-calcified plaque volume at baseline

[0151]

[0152] Table 11

[0153]

[0154] Example 6 (CAC score and total plaque volume reduction)

[0155] A categorical analysis was performed, counting the number of patients with a reduction in CAC score from baseline to 24 months. For the entire population, 7 of 132 patients in the placebo group had a reduction in CAC score, while 13 of 143 patients in the intervention group had a reduction in CAC score (p = 0.2533). Among patients with a CAC score ≥ 400, 3 of 76 patients in the placebo group had a reduction in CAC score, while 10 of 85 patients in the intervention group had a reduction in CAC score (p = 0.0858) (see Figure 8a -b).

[0156] In addition, a categorical analysis was performed, counting the number of patients with a reduction in total plaque volume (all image qualities) from baseline to 24 months. Total plaque volume decreased in 23 of 98 patients in the placebo group and in 34 of 109 patients in the intervention group (p = 0.2753) (see Fig. 9 ).

[0157] Example 7 (Incidence of Cardiovascular Events)

[0158] A total of 13 participants experienced clinical safety events during the follow-up period. The event rate (composite event rate of myocardial infarction, revascularization, and all-cause death) was 10 (6.7%) in the placebo group and 3 (1.9%) in the vitamin K2 and D group (p=0.048).

[0159] Table 12

[0160]

[0161] References:

[0162] Diederichsen ACPet al. (2012): (DanRisk study) "Discrepancy between coronary arterycalcium score and HeartScore inmiddle-aged Danes: the DanRiskstudy", (Eur J Prev Cardiol.2012Jun; 19(3):558-64)

[0163] Diederichsen ACPet al. (2022): "Vitamin K2 and D in Patients WithAortic Valve Calcification: A Randomized Double-Blinded Clinical Trial", Circulation.25April 2022;145:1387-1397.

[0164] Geleijnse J.M.et al.(2004):”Dietary intake of menaquinone isassociated with reduced risk of coronary heart disease:The Rotterdam study”,American Society for Nutritional Sciences:3100-3105.

[0165] Kvist T.V.et al.(2017):(DANCAVAS pilot study),“The DanCavas PilotStudy of Multifaceted Screening for SubclinicalCardiovascular Disease in Menand Women Aged 65-74Years”,Eur J Vasc Endovasc Surg(2017)53,123-131.

[0166] Ohsaki,Y.,et al.,Vitamin K suppresses the lipopolysaccharide-inducedexpression of inflammatory cytokines in cultured macrophage-like cells viathe inhibition of the activation of nuclear factor kappaB through therepression ofIKKalpha / beta phosphorylation.J Nutr Biochem,2010.21(11):p.1120-6.

[0167] Okuyama,H.,et al.,Statins stimulate atherosclerosis and heartfailure:pharmacological mechanisms.Expert Rev Clin Pharmacol,2015.8(2):p.189-99.

[0168] Ozaki,I.,et al.,Menatetrenone,a vitamin K2 analogue,inhibitshepatocellular carcinoma cell growth by suppressing cyclin D1 expressionthrough inhibition of nuclear factor kappaB activation.Clin Cancer Res,2007.13(7):p.2236-45.

[0169] Pan,M.H.,et al.,Inhibition of TNF-alpha,IL-1alpha,and IL-1beta byPretreatment of Human Monocyte-Derived Macrophages with Menaquinone-7 andCell Activation with TLR Agonists In Vitro.J Med Food,2016.19(7):p.663-9.

[0170] Schurgers,L.J.and C.Vermeer,Differential lipoprotein transportpathways of K-vitamins in healthy subjects.Biochim Biophys Acta,2002.1570(1):p.27-32.

[0171] Shea M.K.et al.(2009):“Vitamin K supplementation and progression ofcoronary artery calcium in older men and women”,Am J Clin Nutr 2009;89:1799-1807.

[0172] Shearer,M.J.and P.Newman,Metabolism and cell biology of vitaminK.Thromb Haemost,2008.100(4):p.530-47.

[0173] Simes,D.C.,et al.,Vitamin K as a Diet Supplement with Impact in HumanHealth:Current Evidence in Age-Related Diseases.Nutrients,2020.12(1).

[0174] Xia,J.,et al.,The role of PKC isoforms in the inhibition ofNF-kappaBactivation by vitamin K2 in human hepatocellular carcinoma cells.J NutrBiochem,2012.23(12):p.1668-75.

[0175] Zhelyazkova-Savova,M.D.,et al.,Statins,vascular calcification,andvitamin 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 with a coronary artery calcification (CAC) score of ≥ 400 as determined by cardiac CT scan and CAC determined by using the Agatston method or a similar method.

2. Vitamin K2 is used to prevent or treat coronary heart disease in a subject whose blood concentration of dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) is 433 to 2179 pmol / L.

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

4. Vitamin K2 according to claim 2, wherein the blood concentration of dephosphorylated uncarboxylated matrix Gla protein (dp-ucMGP) is 400 to 2000pmol / L, preferably 425 to 1500pmol / L, preferably 450 to 1250pmol / L, preferably 475 to 1000pmol / L, more preferably 500 to 900pmol / L.

5. Vitamin K2 for the prevention or treatment of coronary heart disease in subjects receiving statin therapy.

6. Vitamin K2 for use according to any one of claims 1 to 5, wherein the subject is administered 100 μg-100 mg per day, preferably 200 μg-50 mg per day, preferably 360 μg-10 mg per day, preferably 360 μg-5 mg per day, preferably 360-1440 μg vitamin K2 per day, preferably 500-1000 μg per day, more preferably 720 μg vitamin K2 per day.

7. Vitamin K2 for use according to any one of claims 1 to 6, wherein vitamin K2 is administered in combination with vitamin D, preferably 1-50 μg vitamin D per day, more preferably 10-50 μg vitamin D per day, most preferably 25 μg vitamin D per day.

8. Vitamin K2 for use according to any one of claims 1 to 7, wherein vitamin K2 is selected from vitamin K2 homologs, any one of 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. Vitamin K2 for use according to any one of claims 1 to 8, wherein the vitamin D administered in combination with vitamin K2 is selected from any one of vitamin D1, vitamin D2, vitamin D3, vitamin D4 or vitamin D5, or a combination thereof.

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

11. Vitamin K2 is used to slow the progression of calcification in the coronary arteries.

12. Vitamin K2 for use according to claim 11, wherein the slowing of the progression of calcification in the coronary arteries refers to a decrease in the CAC score measured by cardiac CT scanning and using the Agatston method or a similar CAC measurement method 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%.

13. Vitamin K2 for use according to claim 11, wherein the slowing down of the progression of calcification in the coronary artery refers to the reduction of calcified plaques (mm 3 ) was reduced by at least 1%, at least 3%, at least 9%, at least 12%, and at least 12.7%.

14. Vitamin K2 for use according to claim 11, wherein the slowing down of the progression of coronary artery calcification is the reduction of non-calcified plaques (mm 3 ) is reduced by at least 1%, at least 3%, at least 5%, at least 7%, and at least 8%.

15. Vitamin K2 for use in reducing coronary artery calcification by at least 0.5%, at least 1%, at least 1.5%, at least 2%, at least 2.5%, at least 4.8% reduction compared to baseline as measured by cardiac CT scan and CAC score using the Agatston method or a similar CAC determination method.

16. Vitamin K2 is used to reduce 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%, or at least 13.1% compared to baseline.

Images