2-methyl-3-phytyl-1,4-naphthoquinone for use in the treatment or prevention of diseases with dysfunction of vascular endothelium

Abstract

The invention relate to a compound of the general formula presented in the description of the invention for use in the treatment or prevention of diseases associated with vascular endothelial dysfunction, wherein it is provided in nanocapsules having a diameter of no more than 1 pm, containing a lipophilic core and a hydrophilic shell, wherein the nanocapsules are intended for oral administration.

Description

[0001] 2-METHYL-3-PHYTYL-1 ,4-NAPHTHOQUINONE FOR USE IN THE TREATMENT OR PREVENTION OF DISEASES WITH DYSFUNCTION OF VASCULAR ENDOTHELIUM

[0002] The invention relate to 2-methyl-3-phytyl-l,4-naphthoquinone (vitamin Ki, phylloquinone, PK) for use in the treatment or prevention of diseases involving dysfunction of vascular endothelium. The invention belongs to the field of medicine.

[0003] From European patent application EP3937916A1, a method of rapidly improving cardiovascular function, reducing arterial stiffness and reversing vascular calcification in mammals, involving the administration of an effective amount of vitamin K2 for a period of 2 weeks to less than 6 months, is known. Furthermore, a way to increase endothelial nitric oxide (NO) production in mammals involving the administration of effective amounts of vitamin K2 over a period of 2 to 8 weeks has also been disclosed.

[0004] From international patent application WO2017014655A1, a biocompatible nanocapsule containing a polysaccharide and an oil core with a diameter not exceeding 1 pm, stabilised without low molecular weight surfactants, is known. The nanocapsule exhibits long-term stability in aqueous suspension and is capable of highly efficient encapsulation of hydrophobic compounds (1-3).

[0005] In the literature publication entitled. 'Effects of vitamin K on vascular health and physical function in older adults with the vascular disease - a controlled study' by authors R. L. Fulton et al (J. Nutr. Health Aging. 2016 Mar;20(3):325-33; doi: 10.1007 / sl2603-015-0619-4) revealed that oral vitamin K2 use by patients, over a six- month period, did not improve brachial artery endothelial function, measured as the endothelium-dependent response of the vessel to a sudden increase in flow-mediated dilatation (FMD). The authors of the invention also noted that the trend of reduced arterial stiffness only appeared after a period of six months, suggesting that long-term vitamin K2 supplementation is necessary to achieve a therapeutic effect on the functional state of the blood vessels. It was also concluded that the results presented in the publication may still be consistent with a biological effect of vitamin K2 that inhibits calcification but does not directly improve endothelial function.

[0006] However, there is clinical, experimental and epidemiological evidence indicating that vitamin K2 (menaquinone, MK) has vasoprotective effects not only reducing coronary artery calcification but also reducing incident coronary artery disease (4-10). Additionally, in healthy postmenopausal women and patients with renal disease, treatment with vitamin K2, (MK-7), reduced arterial stiffness (11-12). In an experimental study by Bar A. et al. it was shown, for the first time directly, that MK-7 treatment improved endothelial function in hyperlipidaemic ApoE / LDLR- / - mice, measured as acetylcholine (Ach)-dependent vasodilation in the aorta or vasodilation induced by increased femoral artery (FA) blood flow after artery occlusion (13).

[0007] However, the evidence supporting the claim that vitamin Ki (phylloquinone PK) has a protective effect on the cardiovascular system is still not accepted. Yet, there is supporting evidence. For example, high dietary PK intake in elderly patients at high cardiovascular risk reduced the incidence of cardiovascular disease, cancer, and overall mortality, although no effect of MK was observed (14). In addition, PK supplementation in diabetic patients was found to prevent the formation of new calcifications in the aorta and coronary arteries, suggesting that vitamin Ki may also have a vasoprotective effect (15). Doubts about the vasoprotective role of PK also arise from studies in which PK has been shown to have negative effects on the endothelium, including induction of oxidative stress and reduction of nitric oxide (NO) bioavailability and inhibition of prostacyclin (PGI2) production (16-17).

[0008] Indeed, in the publication entitled. 'Vitamin Ki (PK) induces vascular endothelial dysfunction: the role of oxidative stress' by authors C.R. Tirapelli et al. (Toxicol. Appl. Pharmacol. 2006 May 15;213(l):10-7; doi: 10.1016 / j.taap.2005.09.001) revealed that oxidative stress induced by vitamin Ki, through the production of superoxide anion decreased NO activity, and altered vascular reactivity. According to the authors, the results obtained suggest that vitamin Ki (PK) may increase the risk of developing vascular disease by inducing oxidative stress and endothelial dysfunction.

[0009] In the current state of the art, there are several unresolved technical issues regarding the vasoprotective effect of vitamin Ki, with a significant problem being the limited bioavailability of vitamin Ki due to its hydrophobic nature, the conversion of vitamin Ki to endogenous vitamin K2, i.e. MK-4 in the intestine, the uptake of vitamin Ki by the liver, and the dependence of vitamin Ki transport on plasma lipoproteins. Vascular endothelial dysfunction may be caused by limited bioavailability of vitamin Ki, which would require long-lasting supplementation with vitamin Ki. The aim of the present, invention is to rapidly restore endothelial function by increasing the bioavailability of vitamin Ki in the blood vessels.

[0010] An effective replenishment of vitamin !(■ in the aorta was achieved after only 7 days of supplementation with orally administered vitamin Ki. Unexpectedly, all the above-mentioned technical problems have been solved by the present invention.

[0011] The invention relate a compound with the chemical formula: for the oral treatment of diseases or the prevention of diseases associated with endothelial dysfunction, being supplied in a nanocapsule no larger than 1 pm in diameter, having: a) a liquid oil core for the transfer of this compound containing an oil selected from the group comprising: oleic acid, isopropyl palmitate, fatty acids, extracts, and oils of natural origin, in particular linseed oil, soybean oil, argan oil, corn oil, or mixtures thereof, preferably oleic acid or corn oil, b) a stabilising shell consisting of a hydrophobically modified polysaccharide selected from the group comprising: derivatives of chitosan, oligochitosan, dextran, carrageenan, amylose, starch, hydroxypropyl cellulose, pullulan, and glycosaminoglycans, especially hyaluronic acid, heparan sulfate, keratan sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, preferably hyaluronic acid derivatives, chitosan and oligochitosan containing a lipophilic core and a hydrophilic shell.

[0012] Advantageously, the compound for use according to the invention is intended to be used for at least 7 days.

[0013] Preferably, the compound for use according to the invention is intended to be used at a dose of at least 0.5 mg / kg body weight.

[0014] Preferably, the compound for use according to the invention is used to prevent or treat endothelial dysfunction that is a consequence of vitamin Ki deficiency. Preferably, the compound for use according to the invention reverses vascular endothelial dysfunction resulting from Ki deficiency.

[0015] Preferably, the compound for use according to the invention is characterised in that the nanocapsule shell comprises hydrophobically modified hyaluronic acid.

[0016] Preferably, the compound for use according to the invention is characterised in that the shell of the nanocapsule is modified with an unbranched alkyl chain, in particular containing 12 carbon atoms.

[0017] Preferably, the compound for use according to the invention is provided in nanocapsules containing: a lipophilic core containing maize oil with vitamin Ki dissolved in it, preferably at a concentration of 25 g / l to 77 g / l, and a hydrophilic shell containing a hyaluronic acid derivative (HyC12) containing 2 mol-% attached dodecyl chains.

[0018] A further object of the invention is a pharmaceutical composition containing the compound defined above for use according to the invention.

[0019] According to the present invention, a disease resulting from or associated with dysfunction of vascular endothelium is any cardiovascular disease in which endothelial dysfunction plays a pathophysiological role selected from the group comprising diseases such as: vascular bed atherosclerosis of any type, including chronic coronary artery disease, ischemic cerebrovascular episode or limb atherosclerosis, including Buerger's disease (Buerger's disease), thromboangiitis obliterans), a condition or disease selected from various atherosclerosis risk factors: hypercholesterolaemia and other dyslipidaemias including hypertriglyceridaemia, high blood pressure, ageing, smoking, hyperhomocysteinemia, insulin resistance, diabetes, menopause, obesity, mental stress, air pollution, traffic noise, infections, inflammation, including periodontal disease, rheumatoid arthritis, allograft vasculopathy, chronic heart failure, pulmonary hypertension, microvascular diabetic complications such as diabetic retinopathy and nephropathy, diabetic neuropathy chronic obstructive pulmonary disease (COPD), pre- eclampsia / eclampsia, erectile dysfunction, Stein-Leventhal syndrome, sleep apnoea, inflammatory bowel disease, neurodegenerative diseases, in particular neurodegenerative disease selected from vascular dementia, Alzheimer's disease, Parkinson's disease, viral diseases such as influenza virus infection, SARS-CoV-2, Dengue and other infectious diseases that present with vascular endothelial dysfunction.

[0020] Figure description:

[0021] Fig.l - Shows the particle size distribution in a Ki-oil emulsion.

[0022] Fig.2 - Shows the particle size distribution in the Ki-oil-HyC12 emulsion.

[0023] Fig.3 - shows the stability assessment during storage at 4°C based on the values of the zeta potential and nanoparticle diameter over a period of one month.

[0024] Fig.4 (A-D) - shows the reversal of endothelial dysfunction induced by a vitamin Ki-deficient diet in C57BL / 6J mice in vivo after 7 days supplementation of vitamin Ki using nanocapsules based on alkyl-modified hyaluronan, where functional responses to acetylcholine (Ach), (fig. 4A, 4C) or sodium nitroprusside (SNR) (fig. 4B, 4D) were measured in the thoracic (fig. 4A, 4B) and abdominal (fig. 4C, 4D) aorta by magnetic resonance imaging (MRI), and data are presented as mean ± SD (n=6). Statistical significance was assessed by one-way ANOVA followed by Dennett's multiple comparisons test using GraphPad Prism 9. ***p < 0.001.

[0025] Fig.5 - shows vascular-targeted in viva delivery of vitamin Ki using alkyl-modi fied hyaluronan-based nanocapsules, whereby in vivo delivery of vitamin K-;to the aortic wall was measured as aortic Ki concentration after 2 or 7 days of administration. Data are presented as median ± ICQ (n=6), where statistical significance was assessed by Kruskal Wallis test followed by Dunnett's post-hoc test for specific pairwise comparisons using GraphPad Prism 9 software. *p < 0.05, ***p < 0.001; ND (not detected).

[0026] The subject of the invention is shown in the examples, which do not constitute a limitation of the invention.

[0027] Example 1 - Method of preparing an aqueous emulsion with vitamin Ki (Ki-oil, control trial)

[0028] An emulsion of vitamin Ki dissolved in corn oil (Ki-oil) was prepared without polymer or other stabilising agent and at a concentration of 25 g / l for the in vivo experiment. Corn oil was chosen for the dissolution of vitamin Ki because it contained the lowest concentration of vitamin Ki (0.443 mg / kg) compared to sunflower oil (18.811 mg / kg) or soybean oil (10.340 mg / kg) as measured by the LC-APCI-MS / MS technique. The choice of a suitable oil source was important in order not to introduce vitamin Ki in an uncontrolled manner from the oil solvent itself.

[0029] Exam^ ■■Da.Dg.gapsyles containing y

[0030] The aqueous dispersion of the nanocapsules was prepared directly by ultrasound- assisted emulsification using a hydrophobic hyaluronic acid derivative (HyC12) obtained according to a previously described protocol (1-3), namely, 500 mg of hyaluronic acid sodium salt was dissolved in 100 ml of water, followed by the addition of 30 mg of EDC (l-ethyl-3-(3-dimethylaminopropyl) carbodiimide) and 18 mg of NHS (N- hydroxysuccinimide). Then 10 ml of water was added and vigorously stirred for 30 minutes using a magnetic stirrer. Then 80 ml DMF (dimethylformamide), 20 ml chloroform, and 1.55 ml dodecylamine solution (0.1 M in chloroform) were added to the solution. The reaction was carried out at 37°C for 24 hours with vigorous stirring. The resulting mixture was dialysed against a 1:1 (v / v) mixture of tert-butyl alcohol and PBS (pH=7.4, C= 0.1M) for 3 days and then against water for 4 days. The resulting purified HyC12 derivative was then lyophilised. A content of 2 mol-% dodecyl groups (with respect to hyaluronic acid mers) was determined.

[0031] An aqueous solution of HyC12 (1 g / l in 0.01 M PBS) was mixed with an oil phase containing dissolved vitamin Ki (Sigma-Aldrich, Poznan, Poland) in a volume ratio of 1000:3 (v:v). The mixture was first homogenised using a vortex shaker and then sonicated leading to the formation of a dispersion of nanocapsules with vitamin Ki (Ki- oil-HyC12).

[0032] The obtained nanocapsules (Ki-oil-HyC12) had:

[0033] - A lipophilic core with the following composition: corn oil with 25 g / l of vitamin Ki dissolved in it for the in vivo experiment, and

[0034] - a hydrophilic shell with the following composition: hyaluronic acid derivative (HyC12) containing 2 mol-% attached dodecyl chains (C12).

[0035] A comparison of the homogeneity of the size-weighted, volume-weighted diameter distributions of the oil phase in Ki-oil (fig. 1) and Ki-oil-HyC12 nanocapsules (fig. 2) indicated that the dispersion of the nanocapsules was homogeneous in contrast to the unstabilised Ki-oil emulsion. The stability of the nanocapsules was assessed by analysing changes in size and zeta potential using dynamic light scattering (fig. 3), and these parameters remained stable even after 29 days of storage at 4°C for Ki-oil-HyC12. example 3 ■ on of an applied model of vascular endothehai dysfunctior- .nduced

[0036] Male C57BL / 6J mice were fed with two types of modified diets for 5 weeks: a control diet and a vitamin Ki-deficient diet (VKdef). The VKdef diet was based on the standard AIN-93M diet where vitamin Ki-rich ingredients were replaced with ingredients containing less vitamin Ki: soybean oil was replaced with corn oil and corn starch was partially replaced with rice flakes (50% / 50%), additionally the amount of casein in the feed was reduced. Vitamin Ki deficiency in the VKdef diet was confirmed by quantification of vitamin K levels by ultraperformance liquid chromatography coupled to atmospheric pressure chemical ionisation tandem mass spectrometry (LC-APCI- MS / MS) technique as described previously (A. Bar et al. Vascul Pharmacol. 2019 Nov- Dec; 122-123:106581. doi: 10.1016 / j.vph.2019.106581, A. Kieronska-Rudek et al. Cells. 2021 Jun 22;10(7):1571. doi: 10.3390 / cellsl0071571).

[0037] The concentration of vitamin Ki and the vitamin K? homologue (i.e. MK-4) was determined in chow, plasma, liver and aorta. For this purpose, an internal standard solution containing Ki-d7 was added to ground feed samples, tissue homogenates and plasma to obtain its final concentration of 100 ng / ml. Then, the samples were gently mixed (10 min) and ethanol (500 ul) was added. The analytes were extracted with hexane (3 ml). After 10 min of vigorous shaking, the samples were centrifuged (4500 G, 10 min,4°C), and the upper, organic layer was transferred to a fresh glass tube. The extraction step was repeated, and the entire collected organic layer was evaporated to dryness under a stream of nitrogen (37°C). The residual sample was dissolved in 50 pl of 2-propanol and 5 pl of the resulting sample was subjected to LC APCI-MS / MS analysis. Chromatographic separation was performed using an Ultimate 3000 HPLC system (Dionex, Sunnyvale, CA, USA) equipped with a PPP analytical column (Kinetex 2.6 pm PPP, 100 A, 100.0x3.3 mm; Phenomenex, Torrance, CA, USAj.The mobile phase consisted of 0.1% formic acid in 2-propanol and 0.1% formic acid in 5 mlM ammonium

[0038] SUBSTITUTE SHEET (RULE 26) formate, which was delivered in gradient elution mode. The signal from the analytes and internal standard was recorded using a TSQ Quantum Ultra triple quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham MA, USA) in positive ionisation mode. The following ion transitions were monitored for further quantitative analysis, in selected fragmentation reaction monitoring (SRM) modes: Ky 451.4-»187.2; Kj-d?: 458.0 -->194.2 and MK-4: 445.4 -->187.2. The operating parameters of the mass spectrometer were as follows: corona discharge needle voltage: 4 kV; ion source temperature: 325°C; shielding gas pressure: 50 arb; auxiliary gas pressure: 30 arb; transfer capillary temperature: 325°C; collision chamber pressure 1.5 mTorr, collision gas: argon. Calibration curves were drawn as the relationship between the ratio of the analyte / internal standard peak area to the nominal analyte concentration (A. Bar et al. Vascul Pharmacol. 2019 Nov-Dec;122-123:106581. doi: 10.1016 / j.vph.2019.106581: A. Kieronska-Rudek et al. Cells. 2021 Jun 22;10(7):1571. doi: 10.3390 / cellsl0071571).

[0039] The control diet was a modified AIN-93M feed, that was enriched with vitamin Ki at a dose of 0.75 rng / kg feed, resulting in an intake of approximately 90 pg / kg body weight per day as assessed by LC-APCI / S / MS measurements in the feed. Vitamin K? homologues such as MK-4 and MK-7 were not detectable in any of the diets. The dose of vitamin Ki used to reverse the deficiency resulting from VKdef feeding was chosen based on our previous work demonstrating the effect of MK-7 on endothelial function in vivo (Bar et al., 2019).

[0040] Experimental groups:

[0041] 1. Control group - a diet enriched with vitamin Ki to cover the daily requirement (approx. 0.09 rng / kg body weight),

[0042] 2. VKdef group ■ vitamin Ki deficient diet prepared without Ki supplementation.

[0043] Then, after 5 weeks of feeding the VKdef diet, the following Ki supplementation was implemented to reverse K ■ deficiency and endothelial impairment resulting from KI deficiency: a) Ki-Oil - Intragastric administration of Ki-oil with a KI dose of 0.5 mg / kg body weight for the two following days (1 administration / day), b) Ki-Oil-HyC12 - intragastric administration of Ki-oil -HyC12 dose of Ki 0.5 mg / kg mg / kg body weight for the two following days (1 administration / day), c) Ki-Ofi ~ I ntragastric administration of Kj-oil dose of Kj. 0.5 mg / kg body weight for the seven following days (1 administration / day), d) Ki Oil HyC12 - intragastric administration of Ki-oil-HyC12 dose of Ki O.5 mg / kg body weight for the seven following days (1 administration / day).

[0044] All formulations were administered intragastrically, once a day, in equal volumes of 200 qL due to the very similar body weights of the mice participating in the study. After the last administration of vitamin Ki, endothelial function was measured using MRI technology. Subsequently, following the completion of the measurements, the mice were sacrificed and the plasma, aorta, and liver were collected and frozen for further analysis of the vitamin Ki and MK-4 levels in the tissues. The time between the last administration of Ki and the MRI measurement with followed tissue collection ranged from 22 to 26 hours.

[0045] Example 4 - / n vivo studies showing the restoration of normal endothelial function in C57BL / 6J mice

[0046] Mice fed the VKdef diet for 5 weeks showed impaired endothelium-dependent vasodilation in the thoracic and abdominal aorta, in response to Ach administration (of -1.83% and -3.81%, respectively), compared to mice fed with the control diet (fig. 4A, 4C) as assessed by in vivo MRI. The endothelium-independent vasodilatory response to SNP administration remained unchanged in the thoracic and abdominal aorta regardless of diet type, confirming endothelium-specific vascular dysfunction in VKdef mice induced by vitamin Ki deficiency (fig. 4B, 4D).

[0047] Vitamin Ki supplementation for 2 days in nanocapsules (Ki -oil -HyC12), as in the nonencapsulated form suspended in corn oil alone (Ki-oi I) in VKdef mice, was ineffective in reversing the endothelial dysfunction induced by vitamin Ki deficiency. Both endothelium-dependent and endothelium-independent vasodilatory responses in the thoracic and abdominal portions of the aorta remained unaltered (fig. 4A-D).

[0048] Prolongation of vitamin Kj. supplementation for up to 7 days in the form of Ki-oil-HyC12 reversed the impairment of vasodilation in response to Ach in the thoracic and abdominal portions of the aorta. In the thoracic aorta, the impairment of endothelium- dependent vasodilation was completely reversed to the level observed in control mice (6.21% vs. 7.12%, respectively) (fig. 4A), while in the abdominal aorta, the impairment of the response to Ach was partially reversed (2.66% vs. 6.75%, respectively) (fig. 4C). SNP-induced vascular responses remained unchanged, confirming the specific endothelial effect of Ki-oi!-HyC12 supplementation (fig. 4B, 4D). In contrast to Ki-oil- HyC12, administration of vitamin Ki in corn oil alone, for 7 days, did not reverse the impairment of the endothelium-dependent vascular response. Moreover, Ach-induced vasoconstriction was recorded in Ki-oil-supplemented VKdef mice as in nonsupplemented VKdef mice at similar levels (-2.72% vs -1.83% in the thoracic aorta, (fig. 1A) and -4.84% vs -3.81% in the abdominal aorta (fig. 4C), indicating that vitamin Ki administered as Ki-oil did not affect endothelial function even after 7 days of administration.

[0049] Example 5 - Delivery of vitamin Ki in nanocapsules effectively increases vitamin Ki concentration in blood vessels

[0050] Vitamin Ki concentration in the aorta was not detectable in the control groups, in the VKdef group, and was not detectable after: a) 2 days of Ki-oil, administration, b) 2 days of Ki-oil-HyC12 administration, c) 7 days of Ki-oil administration.

[0051] In contrast to all other experimental groups, in which aortic vitamin Ki levels were undetectable, animals given Ki-oil-HyC12 for 7 days showed relatively high aortic vitamin Ki levels (fig. 4A).

[0052] References:

[0053] 1. Czyzynska-Cichon, I.; Janik-Hazuka, M.; Szafraniec-Szcz^sny, J.; Jasinski, K.; W^glarz, W.P.; Zapotoczny, S.; Chlopicki, S. Low dose curcumin administered in hyaluronic acid-based nanocapsules induces hypotensive effect in hypertensive rats. / nt. J. Nanomedicine 2021, 16, 1377-1390, doi:10.2147 / IJN.S291945.

[0054] 2. Janik-Hazuka, M.; Kaminski, K.; Kaczor-Kamiriska, M.; Szafraniec-Szcz^sny, J.; Kmak, A.; Kassassir, H.; Watata, C.; Wrobel, M.; Zapotoczny, S. Hyaluronic acidbased nanocapsules as efficient delivery systems of garlic oil active components with anticancer activity. Nanomaterials 2021, 11, doi:10.3390 / nanoll051354.

[0055] 3. Szafraniec, J.; Btazejczyk, A.; Kus, E.; Janik, M.; Zajac, G.; Wietrzyk, J.; Chlopicki, S.; Zapotoczny, S. Robust oil-core nanocapsules with hyaluronate-based shells as promising nanovehicles for lipophilic compounds. Nanoscale 2017, 9, 18867- 18880, doi:10.1039 / c7nr05851a. Hariri E, Kassis N, Iskandar JP, Schurgers U, Saad A, Abdelfattah O, et al. Vitamin K 2-a neglected player in cardiovascular health: A narrative review. Open Hear. 2021;8(2). Wen L, Chen J, Duan L, Li S. Vitamin K-dependent proteins involved in bone and cardiovascular health (Review). Mol Med Rep. 2018;18(l):3-15. Kurnatowska I, Grzelak P, Masajtis-Zagajewska A, Kaczmarska M, Stefanczyk L, Vermeer C, et al. Effect of Vitamin K2 on progression of atherosclerosis and vascular calcification in nondialyzed patients with chronic kidney disease stages 3-5. Pol Arch Med Wewn. 2015;125(9):631-40. Geleijnse JM, Vermeer C, Grobbee DE, Schurgers U, Knapen MHJ, Van Der Meer IM, et al. Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: The Rotterdam Study. J Nutr. 2004;134(ll):3100-5. Gast GCM, de Roos NM, Sluijs I, Bots ML, Beulens JWJ, Geleijnse JM, et al. A high menaquinone intake reduces the incidence of coronary heart disease. Nutr Metab Cardiovasc Dis. 2009;19(7):504-10. Beulens JWJ, Bots ML, Atsma F, Bartelink MLEL, Prokop M, Geleijnse JM, et al. High dietary menaquinone intake is associated with reduced coronary calcification. Atherosclerosis. 2009;203(2):489-93. Schurgers LJ, Barreto D V., Barreto FC, Liabeuf S, Renard C, Magdeleyns EJ, et al. The circulating inactive form of matrix gla protein is a surrogate marker for vascular calcification in chronic kidney disease: A preliminary report. Clin J Am Soc Nephrol. 2010;5(4):568-75. Knapen MHJ, Braam LAJLM, Drummen NE, Bekers O, Hoeks APG, Vermeer C. Menaquinone-7 supplementation improves arterial stiffness in healthy postmenopausal women: A double-blind randomised clinical trial. Thromb Haemost. 2015;113(5):1135-44. Mansour AG, Hariri E, Daaboul Y, Korjian S, El Alam A, Protogerou AD, et al. Vitamin K2 supplementation and arterial stiffness among renal transplant recipients - a single-arm, single-center clinical trial. J Am Soc Hypertens. 2017;ll(9):589-97. Bar A, Kus K, Manterys A, Proniewski B, Sternak M, Przyborowski K, et al. Vitamin K2-MK-7 improves nitric oxide-dependent endothelial function in ApoE / LDLR- / - mice. Vascul Pharmacol. 2019;122-123(August):106581. Juanola-Falgarona M, Salas-Salvado J, Martinez-Gonzalez MA, Corella D, Estruch R, Ros E, et al. Dietary intake of vitamin K is inversely associated with mortality risk. J Nutr. 2014;144(5):743-50. Bellinge JW, Francis RJ, Lee SC, Bondonno NP, Sim M, Lewis JR. The effect of vitamin K 1 on arterial calcification activity in subjects with diabetes mellitus : a post hoc analysis of a double-blind, randomized, placebo-controlled trial. Am J Clin Nutr. 2021; 1-8. Tirapelli CR, De Andrade CR, Lieberman M, Laurindo FR, De Souza HP, de Oliveira AM. Vitamin KI (phylloquinone) induces vascular endothelial dysfunction: Role of oxidative stress. Toxicol Appl Pharmacol. 2006;213(l):10-7. Nolan RD, Eling TE. Inhibition of prostacyclin synthesis in cultured bovine aortic endothelial cells by vitamin KI. Biochem Pharmacol. 1986 Dec 1;35(23):4273-81.

Claims

Patent claims1. A Compound with the chemical formula:for the oral treatment or prevention of diseases with vascular endothelial dysfunction, whereby it is supplied in a nanocapsule no larger than 1 pm in diameter, having: a) a liquid oil core for the delivery of this compound containing an oil selected from the group comprising: oleic acid, isopropyl palmitate, fatty acids, extracts, and oils of natural origin, in particular linseed oil, soybean oil, argan oil, corn oil, or mixtures thereof, preferably oleic acid or corn oil, b) a stabilising shell composed of a hydrophobically modified polysaccharide selected from the group comprising: derivatives of chitosan, oligochitosan, dextran, carrageenan, amylose, starch, hydroxypropyl cellulose, pullulan and glycosaminoglycans, especially hyaluronic acid, heparan sulfate, keratan sulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, preferably derivatives of hyaluronic acid, chitosan, and oligochitosan.

2. The compound for use according to claim. 1, characterised in that it is intended to be used for at least 7 days.

3. The compound for use according to claim. 1 or 2, characterised in that it is for use at a dose of at least 0.5 mg / kg body weight.

4. The compound for use according to claim. 1, characterised in that endothelial dysfunction is a consequence of vitamin Ki deficiency.

5. The compound for use according to claim. 1, characterised in that it reverses the dysfunction resulting from vitamin Ki deficiency.

6. The compound for use according to claim. 1 or 4, characterised in that the disease resulting from endothelial dysfunction is a cardiovascular disease selected from the group comprising the following diseases: atherosclerosis of the vascular bed, including chronic coronary artery disease, ischaemic cerebrovascular episode, atherosclerosis of the extremities, including Buerger's disease, acute cardiovascular event related to atherosclerosis, in particular sudden cardiac death, acute coronary syndrome, including unstable angina, myocardial infarction, the need for coronary angioplasty, coronary artery bypass surgery, ischemic stroke or revascularisation of the peripheral circulation, or the condition or disease is selected from a group of atherosclerosis risk factors including the following: hypercholesterolaemia and other dyslipidaemias, including hypertriglyceridaemia, high blood pressure, ageing, smoking, hyperhomocysteinemia, insulin resistance, diabetes mellitus, menopause, obesity, mental stress, air pollution, traffic and aircraft noise, infections, inflammatory conditions including periodontal disease, rheumatoid arthritis, allograft vasculopathy or nitrate tolerance, thrombosis that is not directly related to atherosclerosis, in particular thrombosis related to implantation of metal vascular prostheses (stents), coronary artery bypass graft surgery (CABG), extracorporeal circulation surgery, haemodialysis, venous thromboembolism, chronic heart failure, pulmonary hypertension, microvascular complications of diabetes, such as diabetic retinopathy and nephropathy, diabetic neuropathy, nephrotic syndrome, chronic renal failure, adult respiratory distress syndrome (ARDS), cystic fibrosis, chronic obstructive pulmonary disease (COPD), pre-eclampsia / eclampsia, erectile dysfunction, Stein-Leventhal syndrome, sleep apnoea, systemic lupus erythematosus, sickle cell anaemia, inflammatory bowel disease, peptic ulcer disease of the stomach or duodenum, glaucoma, chronic liver disease, primary amyloidosis, neurodegenerative diseases, in particular neurodegenerative disease selected from vascular dementia, Alzheimer's disease, Parkinson's disease, viral diseases such as influenza virus infection, SARS-CoV-2, Dengue and other infectious diseases that present with vascular endothelial dysfunction.

7. The compound for use according to claim. 1, characterised in that the shell of nanocapsule contains hydrophobically modified hyaluronic acid.

8. The compound for use according to claim. 7, characterised in that shell of the nanocapsule is modified with an unbranched alkyl chains, advantageously containing 12 carbon atoms.

9. The compound for use according to claim. 1, characterised in that it is supplied in nanocapsules containing: a lipophilic core containing corn oil with vitamin Ki dissolved in it, preferably at a concentration of 25 g / l to 77 g / l, and a hydrophilic shell containing a hyaluronic acid derivative (HyC12) containing 2 mol-% attached dodecyl chains.

10. A pharmaceutical composition containing a compound as defined according to claims 1-9 for use in the oral treatment or prevention of diseases involving dysfunction of vascular endothelium.

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