Potent effects of novel anti-platelet aggregatory cilostamide analogues on recombinant cyclic nucleotide phosphodiesterase isozyme activity

Abstract

The inhibitory potential of novel anti-platelet aggregatory cilostamide analogues on phosphodiesterase (PDE) isozyme activities was investigated with recombinant PDE isozymes expressed in a baculovirus/Sf9 expression system. The recombinant enzymes (PDE1–PDE5 and PDE7) showed K m values and sensitivities to selective inhibitors similar to those reported previously for native enzymes purified from tissues. The cyclooctylurea derivative OPC-33540 (6-[3-[3-cyclooctyl-3-[(1 R∗,2 R∗)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1 H)-quinolinone) inhibited recombinant PDE3A ( ic 50 = 0.32 nM) more potently and selectively than the classical PDE3 inhibitors cilostamide, cilostazol, milrinone, and amrinone. The cyclopropylurea derivative OPC-33509 [(-)-6-[3-[3-cyclopropyl-3-[(1 R,2 R)-2-hydroxycyclohexyl]ureido]-propoxy]-2(1 H)-quinolinone] was less potent ( ic 50 = 0.10 μM) than OPC-33540, demonstrating that the cyclooctyl moiety was important for a potent inhibitory effect. In platelets, OPC-33540 potentiated cyclic AMP accumulation concentration-dependently in both the absence and the presence of 3 nM prostaglandin E 1 (PGE 1) (doubling concentrations: 32.5 and 6.2 nM, respectively). OPC-33540 inhibited thrombin-induced platelet aggregation potently ( ic 50 = 27.8 nM). The anti-platelet aggregation effect also was stimulated in the presence of 3 nM PGE 1 ( ic 50 = 6.0 nM). There was a good correlation between the ic 50 values of PDE3 inhibitors in this study for recombinant PDE3A activity and their ic 50 values for thrombin-induced platelet aggregation ( r = 0.998). These data demonstrated that OPC-33540 is a highly selective and potent PDE3 inhibitor and a useful probe for identification of the intracellular functions of PDE3.