Ascorbate in the prevention of statin induced vascular calcification

Abstract

A method of treating or preventing vascular calcification in a patient by administering L-ascorbic acid or ascorbate to the patient and a pharmaceutical composition containing at least one statin and L-ascorbic acid or ascorbate in a dosage form that allows for the concomitant administering of the at least one statin and L-ascorbic acid or ascorbate to a patient.

Claims

1. A method of treating or preventing vascular calcification in a patient by administering an effective amount of L-ascorbic acid or ascorbate to the patient, wherein the patient is concomitantly treated with at least one statin, wherein a daily dosage form consisting of a pharmaceutical composition consisting of at least one statin, L-ascorbic acid or ascorbate, vitamin B12, vitamin B5, vitamin B1, vitamin B2, vitamin B6, vitamin B7, vitamin B9, vitamin E, and optionally niacin and/or coenzyme Q10 is administered to the patient, wherein the amount of the L-ascorbic acid or ascorbate is from 100 mg to 10 g and the amount of the at least one statin is from 5 mg to 100 mg.

2. A method of treating or preventing vascular calcification in a patient treated with statins by administering an effective amount of L-ascorbic acid or ascorbate to the patient, wherein the patient is concomitantly treated with at least one statin, wherein a daily dosage form consisting of a pharmaceutical composition consisting of at least one statin, L-ascorbic acid or ascorbate, vitamin B12, vitamin B5, vitamin B1, vitamin B2, vitamin B6, vitamin B7, vitamin B9, vitamin E, and optionally niacin and/or coenzyme Q10 is administered to the patient, wherein the amount of the L-ascorbic acid or ascorbate is from 100 mg to 10 g and the amount of the at least one statin is from 5 mg to 100 mg.

3. The method according to claim 1, wherein the vascular calcification is induced by statins.

4. The method according to claim 2, wherein the vascular calcification is induced by statins.

5. The method according to claim 1, wherein the vascular calcification is coronary vascular calcification, cerebrovascular calcification or peripheral vascular calcification.

6. The method according to claim 2, wherein the vascular calcification is coronary vascular calcification, cerebrovascular calcification or peripheral vascular calcification.

7. The method according to claim 1, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, or mixtures thereof.

8. The method according to claim 2, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, or mixtures thereof.

9. The method according to claim 1, wherein the niacin is present.

10. The method according to claim 2, wherein the niacin is present.

11. The method according to claim 1, wherein the amount of the L-ascorbic acid or ascorbate is from 200 mg to 5 g and the amount of the at least one statin is from 10 mg to 80 mg.

12. The method according to claim 2, wherein the amount of the L-ascorbic acid or ascorbate is from 200 mg to 5 g and the amount of the at least one statin is from 10 mg to 80 mg.

13. The method according to claim 1, wherein the amount of the L-ascorbic acid or ascorbate is from 200 mg to 5 g and the amount of the at least one statin is from 10 mg to 40 mg.

14. The method according to claim 2, wherein the amount of the L-ascorbic acid or ascorbate is from 200 mg to 5 g and the amount of the at least one statin is from 10 mg to 40 mg.

15. The method according to claim 1, wherein the coenzyme Q10 is present.

16. The method according to claim 2, wherein the coenzyme Q10 is present.

17. The method according to claim 1, wherein both the niacin and the coenzyme Q10 are present.

18. The method according to claim 2, wherein both the niacin and the coenzyme Q10 are present.

19. The method according to claim 1, wherein the at least one statin and L-ascorbic acid or ascorbate are present as a physical mixture.

20. The method according to claim 2, wherein the at least one statin and L-ascorbic acid or ascorbate are present as a physical mixture.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the effects of treatment with ascorbic acid on calcification of extracellular matrix in cultured human aortic smooth muscle cells.

(2) FIG. 2A shows the effects of Simvastatin on Ca accumulation in AoSMC culture without forskolin.

(3) FIG. 2B shows the effects of mevastatin on Ca accumulation in AoSMC culture with 25 mcM forskolin.

(4) FIG. 3A shows the effects of 200 mcM ascorbate on osteoblast markers expression in human aortic SMC incubated for 4 weeks in osteogenic medium supplemented with 5 mM beta-glycerophosphate and 25 mcM forskolin.

(5) FIG. 3B shows effects of 200 mcM ascorbate on osteoblast markers expression in human dermal fibroblasts incubated for 4 weeks in osteogenic medium supplemented with 5 mM beta-glycerophosphate and 25 mcM forskolin.

(6) Cellular calcification process was investigated in human AoSMC cultured in a regular cell growth medium (5% FBS/DMEM) in the absence and presence of various amounts of ascorbic acid. The calcification process of AoSMC was evaluated by the activity of cellular alkaline phosphatase and calcium accumulation in the cell-produced extracellular matrix (FIG. 1).

(7) The results show that supplementation of AoSMC medium with ascorbic acid up to 300 mcM resulted in a significant decrease in the level of extracellular calcium and lower activity of cellular alkaline phosphatase in dose-dependent manner. In the presence of 300 mcM ascorbate the extracellular Ca accumulation by AoSMC decreased by 20% and alkaline phosphatase activity by 80%.

(8) The results presented in FIG. 2A show that calcium accumulation in AoSMC layers was increased in the presence of simvastatin by 23%. However, concomitant presence of 100 mcM ascorbate calcium resulted in a 54% decrease of accumulated calcium to the value 0.2 mcg/well, which correlated with the values observed in cells not exposed to simvastatin.

(9) The effect of ascorbate on calcium accumulation in AoSMC under enhanced pro-calcification condition (with forskolin) and in the presence of a statin (mevastatin) is presented in FIG. 2B. The results show that in the presence of 1 mM mevastatin calcium accumulation increased from 1.35 mcg/well in control to 1.8 mcg/well with mevastatin. However, when 100 mcM ascorbate was added calcium accumulation decreased by 19% to below control (non-supplemented) values.

(10) In addition to SMC we studied the effect of ascorbate on cellular calcification process in human dermal fibroblasts (DF) and immortalized human fetal osteoblasts (FOB) by evaluating changes in the expression of different pro-osteogenic markers in these cells. The effects of ascorbate in different types of cells challenged with pro-osteogenic conditions such as by growing them in the medium supplemented with 5 mM beta-glycerophosphate and 25 mcM forskolin. The results show that expression of all tested osteogenic markers was significantly reduced by 100 mcM ascorbic acid supplementation in both AoSMC and DF cultures (FIG. 3). Ascorbic acid supplementation of hFOS osteoblasts in pro-osteogenic medium over four week period was cytotoxic. Corresponding data were omitted from the presentation.

(11) We compared the levels of osteogenic markers expression in the test human cell types as presented in Table 1. The results indicate that in a regular growth medium, the expression of osteocalcin, osteoadherin, dentin matrix protein 1 (DMP-1) and sclerostin (SOST) were most prominent in osteoblasts cells (FOB) closely followed by fibroblasts (DF), except of DMP-1, expression of which in fibroblasts slightly overcame that of FOB cultures. Cellular expression of these four osteogenic markers in AoSMC cultured in regular growth medium was significantly (2-4) fold less prominent than in FOB and DF cultures.

(12) In the present tests we demonstrated that ascorbic acid tested up to 300 mcM concentrations can reduce calcium accumulation in ECM produced by AoSMC. This effect was accompanied by the blockage of SMC osteogenic transformation as indicated by changes in specific metabolic parameters, such as reduction in cellular alkaline phosphatase activity, and cellular expression of osteoblast marker proteins. A high level of serum alkaline phosphatase (ALP) is associated with an increased risk of mortality and myocardial infarction. ALP hydrolyses inorganic pyrophosphate, which is a strong inhibitor of calcium phosphate deposition.

(13) TABLE-US-00001 TABLE 1 Osteogenic Marker Osteoadherin/ SOST/ Osteocalcin OSAD DMP-1 Sclerostin Cell type mean sd mean sd mean sd mean sd AoSMC Plain 0.288 0.047 0.259 0.025 0.412 0.063 0.212 0.030 Medium Osteogenic Medium 0.429 0.086 0.315 0.061 0.569 0.111 0.289 0.063 hDF Plain 1.087 0.051 0.889 0.093 1.137 0.089 0.657 0.058 Medium Osteogenic 0.614 0.242 0.403 0.119 0.851 0.116 0.374 0.051 Medium FOS Plain 1.206 0.288 1.493 0.147 0.819 0.307 0.956 0.197 Medium Osteogenic 1.003 0.207 1.049 0.213 0.786 0.078 0.633 0.126 Medium

(14) Under physiological conditions (cells incubated in regular cell culture medium) expression of osteocalcin, osteoadherin and SOST/sclerostin were the highest in hFOS cultures and the lowest in hAoSMC cultures. Expression of these markers were intermediate in hDF cultures. Under physiological conditions (cells incubated in regular cell culture medium) expression of DMP-1 was the highest in hDF cultures and the lowest in hAoSMC cultures. Expression of DMP-1 was intermediate in hFOS cultures. Cell supplementation with pro-osteogenic medium as compared to regular medium caused stimulation of all tested osteomarkers in AoSMC cultures. In contrast, pro-osteogenic medium supplementation caused an inhibition of all tested osteogenic markers in hDF and hFOS cultures.