Endothelial action of thienopyridines and thienopyrimidinones in the isolated guinea pig heart

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Summary

Antiplatelet thienopyridines (ticlopidine, clopidogrel) and their thienopyrimidinone congeners, induce prostacyclin-dependent thrombolysis in vivo. Here we tested whether thienopyridines (ticlopidine, clopidogrel, and its enantiomer without antiplatelet properties) and structurally related thienopyrimidinones release NO from coronary endothelium in the isolated guinea pig heart, perfused according to Langendorff technique. The involvement of endothelium-derived NO in coronary vasodilation induced by these agents was assessed by effect of l-NG-nitro-arginine methyl ester (L-NAME). In addition, effect of thienopyridines or thienopyrimidinones on nitrite accumulation in cultured endothelium was assayed.

Tienopyridines (10–100 μmol L−1) and thienopyrimidinones (10–30 μmol L−1) produced concentration-dependent increase in coronary flow comparable to that induced by acetylcholine (0.1 μmol L−1) or bradykinin (3 nmol L−1) which was inhibited by L-NAME (by 50–70%) but not by indomethacin. Furthermore, thienopyridines and thienopyrimidinones caused NO release from cultured endothelial cells.

In conclusion, both thienopyridines independently from their antiplatelet action and their thienopyrimidinone congeners that are devoid of antiplatelet action stimulate coronary endothelium to release NO. Endothelial action of these compounds merits further investigation.

Introduction

Ticlopidine [5-(2-chlorophenyl-methyl)-4,5,6,7-tetrahydro-thieno [3,2c] pyridine hydrochloride] and clopidogrel [thieno[3,2c]pyridine(4H)acetic acid, alpha (2chlorophenyl)-6,7-dihydro-, methyl ester, S-(7)] are members of the thienopyridine group of antiplatelet drugs [1], [2], [3], [4]. These drugs are increasingly used in prevention and treatment of myocardial infarction (CAPRIE-R) [5], cerebro-vascular diseases (CATS) [6] or diabetic microangiopathy (TIMAD-R) [7]. Their antiplatelet action is related to the inhibition of P2Y12 subtype of ADP receptors on platelets [8], [9], [10], [11], [12], [13]. The P2Y12 receptor is linked to Gi protein and its activation leads to a fall in intraplatelet cAMP level [14], [15], [16], [17], [18]. Importantly, antiplatelet effects of ticlopidine or clopidogrel are not detected in vitro and appear at least 24 h after oral administration [19], since they are mediated by active metabolites of ticlopidine or clopidogrel formed in the liver [18], [20]. Unidentified thienopyridine metabolite modulates also vascular contractile responses [21], [22]. Recently, it was demonstrated that thienopyridines display also a direct thrombolytic effect in vivo [23], [24] and a direct effect on endothelial cells in vitro [25], both associated with the release of endothelial prostacyclin (PGI2). Importantly, endothelial action of thienopyridines did not depend on their antiaggregatory properties [23], [26], [27]. Furthermore, Dupin et al. carried out different structural modifications of thienopyridines and by adding a second N atom in the pyridine cycle and additional cycle fused to the thienyl ring synthesized benzothienopyrimidinone derivatives (Fig. 1, Table 1), which were characterized by more potent PGI2-dependent thrombolytic activity as compared to thienopyridines [28]. Although PGI2 and NO are released from the endothelium in a coupled manner [29], PGI2-dependent thrombolysis induced by thienopyridines or thienopyrimidinones in vivo in rats was not associated with NO-dependent hypotension [23], [26], [28]. Accordingly, the aim of the present study was to analyse whether endothelial action of thienopyridines and thienopyrimidinones is limited to the stimulation of endothelial PGI2 production or these compounds possess also the ability to stimulate the production of endothelial NO. As a bioassay, we used the model of isolated guinea pig heart. We have shown previously that this model is suitable for the detection of the NO-dependent endothelial action of cardiovascular drugs [30], [31] while in vivo model of thrombolysis uncovers their endothelial action mediated by PGI2 [30].

Section snippets

Materials and methods

Hydrochlorides of thienopyridines, i.e. ticlopidine, clopidogrel (SR 25990C) or its enantiomer deprived of antiplatelet properties (SR25989C) were kindly donated by Sanofi Laboratories (Toulouse, France) and icatibant (HOE 140, an antagonist of kinin B2 receptor) by Hoechst Marion Roussel Company (Frankfurt am Main, Germany). Thienopyrimidinone derivatives (D2, D7, D9, D12, D15) were synthesized by professor J.P. Dupin from Laboratory of Pharmaceutical Organic Chemistry, University Victor

Coronary vasodilation induced by thienopyridines and thienopyrimidinones in the isolated guinea pig heart

In the isolated guinea pig heart basal coronary flow was 7.04±0.31 ml min−1 and basal dp/dtmax was 981±23.8 mm Hg s−1 (n=66). Thienopyridines (ticlopidine, clopidogrel and clopidogrel enantiomer without antiplatelet properties) as well as thienopyrimidinone derivatives (D2, D7, D9, D12, D15, Fig. 1, Table 1) produced an increase in coronary flow in a concentration-dependent manner (Fig. 2). Among thienopyridines, ticlopidine, clopidogrel and enantiomer of clopidogrel produced nearly equipotent

Discussion and conclusions

In the present study, we demonstrated that thienopyridines known as antiplatelet drugs and their thienopyrimidinone derivatives [28] displayed direct endothelial action in the isolated guinea pig heart. Endothelial action of thienopyridines was independent from their antiplatelet activity since it was immediate, present in platelet-free experimental model and shared by ticlopidine, clopidogrel and clopidogrel enantiomer lacking the antiaggregatory activity [34]. Furthermore, not only

Acknowledgments

This article was supported by the grants from Polish State Committee for Scientific Research (KBN) 2 PO5A 084 26 (J.J.), 4 PO5A 061 25 (R.O.) and 4 PO5A 00325 (S.C.). Associate Professor Stefan Chlopicki is a recipient of a Professorial grant from Foundation for Polish Science.

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