Pharmaceutical compositions for use in the treatment of cardiovascular calcification

Abstract

The inventors demonstrate that the phosphatase and hydrolase domains of soluble epoxide hydrolase (sEH) regulate the cardiovascular calcification process and revealed that inhibition of the phosphatase domain of sEH could represent a new pharmacological target in the prevention of cardiovascular calcification. The present invention thus relates to a therapeutically effective amount of an inhibitor of phosphatase activity of soluble epoxide hydrolase for use in a method of treating cardiovascular calcification in a subject in need thereof.

Claims

1. A method of treating cardiovascular calcification in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an inhibitor of phosphatase activity of soluble epoxide hydrolase, wherein the inhibitor of phosphatase activity of soluble epoxide hydrolase is N-acetyl-S-farnesyl-Lcysteine (AFC) or ebselen.

2. The method of claim 1 wherein the cardiovascular calcification is valvular or vascular calcification.

3. The method of claim 1 wherein the subject is elderly and/or has a mineral imbalance disorder.

4. An in vitro method for screening a plurality of test substances to identity those which are useful for the treatment of cardiovascular calcification in a subject in need thereof comprising i) testing each of the test substances for its ability to inhibit the phosphatase activity of soluble epoxide hydrolase and ii) identifying a test substance which inhibits said phosphatase activity as useful for treating cardiovascular calcification in a subject in need thereof.

5. The method of claim 3, wherein the mineral imbalance disorder is associated with severe renal failure in a subject on hemodialysis, a hemodialysis arteriovenous (AV) graft/shunt, a vein graft, a vascular anastomosis, diabetes, Paget's disease, rheumatoid arthritis, osteoporosis or osteoarthritis.

Description

FIGURES

(1) FIG. 1: Inhibition of the sEH phosphatase domain with N-acetyl-S-farnesyl-L-cysteine (AFC) dose-dependently reduces the calcium content of rat aortas cultured in procalcifying conditions (inorganic phosphate; Pi=3.8 mM) during 7 days.

(2) FIG. 2: Inhibition of the sEH phosphatase domain with N-acetyl-S-farnesyl-L-cysteine (AFC) dose-dependently reduces the calcium content of valvular interstitial cells cultured in procalcifying conditions (inorganic phosphate; Pi=3 mM) during 14 days.

(3) FIG. 3: Inhibition of the sEH phosphatase domain with ebselen dose-dependently reduces the calcium content of aortic rings cultured in procalcifying conditions (inorganic phosphate; Pi=3.8 mM) during 7 days.

(4) FIG. 4: Inhibition of the sEH phosphatase domain with ebselen dose-dependently reduces the calcium content of valvular interstitial cells cultured in procalcifying conditions (inorganic phosphate; Pi=3 mM) during 10 days.

EXAMPLE 1 (DATA NOT SHOWN)

(5) Introduction: Notably expressed in the cardiovascular system either by endothelial, vascular smooth muscle and cardiac cells, soluble epoxide hydrolase (sEH) is a bifunctional enzyme which contributes to the regulation of the endothelial function and maintain of cardiovascular homeostasis. It is hypothesized that the enhanced cardiovascular calcification could be related to a procalcifying effect of sEH phosphatase.

(6) Aim: In this work we assessed ex vivo the role of the sEH hydrolase domain in vascular calcification.

(7) Materials and methods: Rat aortic rings were cultured in procalcifying conditions using inorganic phosphate (Pi: 3.8 mM) for 7 days. Trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB)—a potent and specific inhibitor of sEH hydrolase—was added at concentrations, ranging from 0.1 to 10 μM (n=6-16 per group). Rat aortic calcium content was determined using a calcium colorimetric assay with O-Cresolphthalein.

(8) Results: Aortic calcium content was significantly and dose-dependently increased by the inhibition of the phosphatase domain with t-AUCB (Pi: 100±9%; 0.1 μM: 110±9%; 1 μM: 128±15%; 10 μM: 141±16%). Alizarin Red and VonKossa histochemical staining confirmed these results.

(9) Conclusion: These results demonstrate that the inhibition of the hydrolase domain of sEH increases the aortic calcification.

EXAMPLE 2 (FIG. 1)

(10) Introduction: Notably expressed in the cardiovascular system either by endothelial, vascular smooth muscle and cardiac cells, soluble epoxide hydrolase (sEH) is a bifunctional enzyme which contributes to the regulation of the endothelial function and maintain of cardiovascular homeostasis. Aside from its hydrolase activity, sEH also possesses a phosphatase domain whose function has been poorly investigated.

(11) Aim: In this work we assessed ex vivo the role of the sEH phosphatase domain in vascular calcification.

(12) Materials and methods: Rat aortic rings were cultured in procalcifying conditions using inorganic phosphate (Pi: 3.8 mM) for 7 days. N-acetyl-S-farnesyl-L-cysteine (AFC)—sEH phosphatase domain inhibitor—was added at concentrations, ranging from 0.1 to 10 μM (n=4-21 per group). Rat aortic calcium content was determined using a calcium colorimetric assay with O-Cresolphthalein.

(13) Results: Aortic calcium content was significantly and dose-dependently decreased by the inhibition of the phosphatase domain with AFC (Pi: 100±6.9%; Pi+AFC 0.1 μM: 83.6±9.1%; Pi+AFC 1 μM: 74.2±2.9%; Pi+AFC 10 μM: 54.5±12%). Alizarin Red and VonKossa histochemical staining confirmed these results.

(14) Conclusion: These results demonstrate that the inhibition of the phosphatase domain of sEH may represent a new pharmacological target in the prevention of vascular calcification.

EXAMPLE 3 (FIG. 2)

(15) Introduction: Notably expressed in the cardiovascular system either by endothelial, vascular smooth muscle and cardiac cells, soluble epoxide hydrolase (sEH) is a bifunctional enzyme which contributes to the regulation of the endothelial function and maintain of cardiovascular homeostasis. Aside from its hydrolase activity, sEH also possesses a phosphatase domain whose function has been poorly investigated.

(16) Aim: In this work we assessed in vitro the role of the sEH phosphatase domain in valvular calcification.

(17) Materials and methods: Human valvular interstitial cells (VICs) were cultured in procalcifying conditions using inorganic phosphate (Pi: 3 mM) for 14 days. N-acetyl-S-farnesyl-L-cysteine (AFC)—sEH phosphatase domain inhibitor—was added at concentrations, ranging from 0.1 to 5 μM (n=5-6 per group). VICs aortic calcium content was determined using a calcium colorimetric assay with O-Cresolphthalein.

(18) Results: VICs calcium content was significantly and dose-dependently decreased by the inhibition of the phosphatase domain with AFC (mean±SEM; Pi: 90.3±4.6 μg; Pi+AFC 0.1 μM: 76.7±5.7 μg; Pi+AFC 1 μM: 51.1±2.9 μg; Pi+AFC 5 μM: 45.8±2 μg). Alizarin Red staining confirmed these results.

(19) Conclusion: These results demonstrate that the inhibition of the phosphatase domain of sEH may represent a new pharmacological target in the prevention of valvular calcification.

EXAMPLE 4 (FIG. 3)

(20) Introduction: Notably expressed in the cardiovascular system either by endothelial, vascular smooth muscle and cardiac cells, soluble epoxide hydrolase (sEH) is a bifunctional enzyme which contributes to the regulation of the endothelial function and maintain of cardiovascular homeostasis. Aside from its hydrolase activity, sEH also possesses a phosphatase domain whose function has been poorly investigated.

(21) Aim: In this work we assessed ex vivo the role of the sEH phosphatase domain in vascular calcification.

(22) Materials and methods: Rat aortic rings were cultured in procalcifying conditions using inorganic phosphate (Pi: 3.8 mM) for 7 days in absence and in presence of ebselen at a concentration of 1 and 10 μM (n=6-8 per group).

(23) Results: Relative aortic calcium content was significantly and dose-dependently decreased by the inhibition of the phosphatase domain with ebselen (Pi: 100±10%; Pi+ebselen 1 μM: 77±8%; Pi+ebselen 10 μM: 62±7%).

(24) Conclusion: These results demonstrate that the inhibition of the phosphatase domain of sEH may represent a new pharmacological target in the prevention of valvular calcification.

EXAMPLE 5 (FIG. 4)

(25) Introduction: Notably expressed in the cardiovascular system either by endothelial, vascular smooth muscle and cardiac cells, soluble epoxide hydrolase (sEH) is a bifunctional enzyme which contributes to the regulation of the endothelial function and maintain of cardiovascular homeostasis. Aside from its hydrolase activity, sEH also possesses a phosphatase domain whose function has been poorly investigated.

(26) Aim: In this work we assessed in vitro the role of the sEH phosphatase domain in valvular calcification.

(27) Materials and methods: Human valvular interstitial cells (VICs) were cultured in procalcifying conditions using inorganic phosphate (Pi: 3 mM) for 10 days. Ebselen—sEH phosphatase domain inhibitor—was added at the concentrations of 0.1 and 1 μM (n=4 per group). VICs aortic calcium content was determined using a calcium colorimetric assay with O-Cresolphthalein.

(28) Results: VICs calcium content was significantly and dose-dependently decreased by the inhibition of the phosphatase domain with ebselen (mean±SEM; Pi: 100±5.9%; Pi+ebselen 0.1 μM: 88.8±11%; Pi+ebselen 1 μM: 67.6±7.2%).

(29) Conclusion: These results demonstrate that the inhibition of the phosphatase domain of sEH may represent a new pharmacological target in the prevention of valvular calcification.

REFERENCES

(30) Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.