Masuhiro Yoshitake

Cilostazol as a unique antithrombotic agent.

Cilostazol (CLZ) was originally developed as a selective inhibitor of cyclic nucleotide phosphodiesterase 3 (PDE3). PDE3 inhibition in platelets and vascular smooth muscle cells (VSMC) was expected to provide an antiplatelet effect and vasodilation. Recent preclinical studies have demonstrated that CLZ also possesses the ability to inhibit adenosine uptake by various cells, a property that distinguishes CLZ from other PDE3 inhibitors, such as milrinone. After extensive preclinical and clinical studies, CLZ has been shown to have unique antithrombotic and vasodilatory properties based upon these novel mechanisms of action. CLZ was approved in 1988 for the treatment of symptoms related to peripheral arterial occlusive disease in Japan (Pletaal) and in 1999 in the U.S. and in 2001 in the U.K. (Pletal) for the treatment of intermittent claudication symptoms. Despite its remarkable antiplatelet properties, CLZ is not generally considered an antithrombotic agent in Western countries, perhaps due to the bulk of its antithrombotic preclinical and clinical development being conducted in Japan. In this review, the unique properties of CLZ are reviewed with the focus on CLZ as a unique antiplatelet agent targeting platelets and VSMC, demonstrating synergy with endogenous mediators and showing lowered risk of bleeding risk compared to other antiplatelet drugs.

Comparison of the effects of cilostazol and milrinone on intracellular cAMP levels and cellular function in platelets and cardiac cells.

Cilostazol is a potent cyclic nucleotide phosphodiesterase (PDE) type 3 (PDE3) inhibitor that was recently approved by the Food and Drug Administration (FDA) for the treatment of intermittent claudication. Its efficacy is presumed to be due to its vasodilatory and platelet activation inhibitory activities. Compared with those treated with placebo, patients treated with cilostazol showed a minimal increase in cardiac adverse events. Because of its PDE3 inhibitory activity, however, the possibility that cilostazol exerts positive cardiac inotropic effects is a safety concern. Therefore we compared the effects of cilostazol with those of milrinone, a selective PDE3 inhibitor, on intracellular cyclic adenosine monophosphate (cAMP) levels in platelets, cardiac ventricular myocytes, and coronary smooth muscle cells. We also compared the corresponding functional changes in these cells. Cilostazol and milrinone both caused a concentration-dependent increase in the cAMP level in rabbit and human platelets with similar potency. Furthermore, cilostazol and milrinone were equally effective in inhibiting human platelet aggregation with a median inhibitory concentration (IC50) of 0.9 and 2 microM, respectively. In rabbit ventricular myocytes, however, cilostazol elevated cAMP levels to a significantly lesser extent (p < 0.05 vs. milrinone). By using isolated rabbit hearts with a Langendorff preparation, we showed that milrinone is a very potent cardiotonic agent; it concentration-dependently increased left ventricular developed pressure (LVDP) and contractility. Cilostazol was less effective in increasing LVDP and contractility (p < 0.05 vs. milrinone), which is consistent with the cardiac cAMP levels. The cardiac effect of OPC-13015, a metabolite of cilostazol with about sevenfold higher PDE3 inhibition, was similar to cilostazol. Whereas milrinone concentration-dependently increased cAMP in rabbit coronary smooth muscle cells, cilostazol did not have such an effect. However, both compounds increased coronary flow equally in rabbit hearts. Our results show that although cilostazol and milrinone both inhibit PDE3, cilostazol preferentially acts on vascular elements (platelets and flow). This unique profile of cilostazol is consistent with its beneficial and safe clinical outcomes in patients with intermittent claudication.

Comparison of the Effects of Cilostazol and Milrinone on cAMP-PDE Activity, Intracellular cAMP and Calcium in the Heart

We investigated the basis for the difference in the cardiotonic effects of the PDE3 inhibitors cilostazol and milrinone in the rabbit heart. Cilostazol displayed greater selectivity than milrinone for inhibition of cAMP-PDE activity in microsomal vs cytosolic fractions from rabbit heart. This difference was due to the inhibition of significantly less cytosolic cAMP-PDE activity by cilostazol compared to milrinone. A combination of cilostazol (>15 μM) and the PDE4 selective inhibitor, rolipram (5 μM), inhibited levels of cytosolic cAMP-PDE activity similar to those inhibited by milrinone on its own. This suggested that milrinone inhibited PDE4 in addition to PDE3 activity. In isolated rabbit cardiomyocytes, milrinone (>10 μM) caused greater elevations in intracellular cAMP and calcium than cilostazol. In the presence of rolipram, however, the cAMP and calcium elevating effects of cilostazol and milrinone were similar. Therefore, in rabbit heart, partial inhibition of PDE4 by milrinone contributed to greater increases in cardiomyocyte cAMP and calcium levels than cilostazol. PDE4 activity in failing human heart was lower than in rabbit heart and there was no significant difference in the inhibition of human cytosolic cAMP-PDE by cilostazol and milrinone. Our results suggest that in normal rabbit heart inhibition of PDE4 by milrinone may partly contribute to the greater cardiotonic effect of milrinone when compared to cilostazol. However, the lower level of PDE4 activity in failing human heart suggests that factors other than inhibition of PDE4 by milrinone may contribute to differences in cardiotonic action when compared to cilostazol.

Inhibition of adenosine uptake and augmentation of ischemia-induced increase of interstitial adenosine by cilostazol, an agent to treat intermittent claudication.

Cilostazol (Pletal), a quinolinone derivative with a cyclic nucleotide phosphodiesterase type 3 (PDE3) inhibitory activity, was recently approved by the Food and Drug Administration for treatment of symptoms of intermittent claudication (IC). However, the underlying mechanisms of action are not entirely clear. In this study, we showed that cilostazol inhibited adenosine uptake into cardiac ventricular myocytes, coronary artery smooth muscle, and endothelial cells with a median effective concentration (EC50) approximately 10 microM. In vivo, cilostazol increased cardiac interstitial adenosine levels after a 2-min ischemia in rabbit hearts (329 +/- 92% increase vs. 102 +/- 29% ischemia alone). The combination of cilostazol and 2-min ischemia reduced infarction from subsequent 30-min regional ischemia and 3 h of reperfusion (infarct size was 18 +/- 4% vs. 53 +/- 3% in the hearts with 2-min ischemia alone or 48 +/- 2% in the hearts treated with cilostazol alone). In contrast, milrinone had no effect on either adenosine uptake or interstitial adenosine levels. These data show that cilostazol, unlike milrinone, inhibits adenosine uptake, and thus potentiates adenosine accumulation from a 2-min ischemia. Future studies are needed to investigate the role of adenosine in the treatment of IC by cilostazol.

New mechanism of action for cilostazol: Interplay between adenosine and cilostazol in inhibiting platelet activation

Cilostazol, a potent phosphodiesterase 3 inhibitor and anti-thrombotic agent, was recently shown to inhibit adenosine uptake into cardiac myocytes and vascular cells. In the present studies, cilostazol inhibited [3H]-adenosine uptake in both platelets and erythrocytes with a median inhibitory concentration (IC50) of 7 &mgr;M. Next collagen-induced platelet aggregation was studied and it was found that adenosine (1 &mgr;M), having no effect by itself, shifted the IC50 of cilostazol from 2.66 &mgr;M to 0.38 &mgr;M (p < 0.01). This shifting was due to an enhanced accumulation of cAMP in platelets and was significantly larger than that by the combination of adenosine and milrinone, which has no effect on adenosine uptake. Similarly, cilostazol, by blocking adenosine uptake, enhanced the adenosine-mediated cAMP increase in Chinese hamster ovary cells that overexpress human A2A receptor. Furthermore, the inhibitory effect of cilostazol on platelet aggregation in whole blood was significantly reversed by ZM241385 (100 n M), an A2A adenosine receptor antagonist, and by adenosine deaminase (2 U/ml). These data suggest that the inhibitory effects of cilostazol on adenosine uptake and phosphodiesterase 3 together elevate intracellular cAMP, resulting in greater inhibition of agonist-induced platelet activation.

Interplay between inhibition of adenosine uptake and phosphodiesterase type 3 on cardiac function by cilostazol, an agent to treat intermittent claudication.

The authors have recently shown that cilostazol, a type 3 cyclic nucleotide phosphodiesterase (PDE3) inhibitor, has a much weaker positive inotropic effect than milrinone, a PDE3 inhibitor of similar potency. They have also shown that cilostazol inhibits adenosine uptake, whereas milrinone has no such effect. This study investigated the possible cardiac functional significance of cilostazol on adenosine uptake inhibition. In isolated rabbit hearts, 10 &mgr;M of cilostazol elevated adenosine concentration in interstitial dialysate (0.16 ± 0.01 &mgr;M, or ∼0.81 &mgr;M in the interstitial space when adjusted for recovery rate of microdialysis) and coronary effluent (0.69 ± 0.03 &mgr;M). The values are significantly higher than those for 10 &mgr;M of milrinone (0.11 ± 0.1 &mgr;M in interstitial dialysate and 0.2 ± 0.04 &mgr;M in coronary effluent). Although cilostazol increased contractility, heart rate, and coronary flow in isolated rabbit hearts, the effect on contractility and heart rate was significantly augmented in the presence of an adenosine A 1 receptor antagonist. Conversely, an adenosine A 1 receptor agonist or an adenosine uptake inhibitor attenuated the positive inotropic effect of milrinone. These results indicate that adenosine uptake inhibition by cilostazol increases interstitial and circulatory adenosine concentration, and antagonizes PDE3 inhibition-induced contractility and heart rate increases through an adenosine A 1 receptor-mediated mechanism.

Cilostazol and dipyridamole synergistically inhibit human platelet aggregation.

It has been previously shown that cilostazol (Pletal®), a drug for relief of symptoms of intermittent claudication, potently inhibits cyclic nucleotide phosphodiesterase type 3 (PDE3) and moderately inhibits adenosine uptake. It elevates extracellular adenosine concentration, by inhibiting adenosine uptake, and combines with PDE3 inhibition to augment inhibition of platelet aggregation and vasodilation while attenuating positive chronotropic and inotropic effects on the heart. In the present study, we tested the hypothesis that cilostazol combined with a more potent adenosine uptake inhibitor, dipyridamole, synergistically inhibited platelet aggregation in human blood. In the presence of exogenous adenosine (1 μM), the combination of cilostazol and dipyridamole synergistically increased intra-platelet cAMP. Furthermore, cilostazol inhibited platelet aggregation in a washed platelet assay concentration-dependently with IC50s of 0.17 ± 0.04 μM (P < 0.05 versus plus adenosine alone of 0.38 ± 0.05 μM), 0.11 ± 0.06 μM (P < 0.05), and 0.01 ± 0.01 μM (P < 0.005) when combined with 1, 3, or 10 μM dipyridamole, respectively (n = 5). In whole blood, cilostazol (0.3 to 3 μM) and dipyridamole (1 or 3 μM) synergistically inhibited collagen- and ADP-induced platelet aggregation in vitro. Furthermore, the synergism was confirmed in an open-label, sequential study in healthy human subjects using ex vivo whole-blood collagen-induced platelet aggregation. Four hours after oral co-administration of cilostazol (100 mg) and dipyridamole (200 mg), platelet aggregation was inhibited by 45 ± 17%, while no significant inhibition was observed from subjects treated with either drug alone. The combination may provide a potential treatment of arterial thrombotic disorders.

Antiplatelet and Antithrombotic Activity of Cilostazol is Potentiated by Dipyridamole in Rabbits and Dissociated from Bleeding Time Prolongation

Purpose: To determine the antiplatelet effect of cilostazol (Pletal®) and its interaction with dipyridamole in in vitro and in vivo rabbit models, and to see if it can be dissociated from bleeding time prolongation.Methods:In vitro collagen-induced platelet aggregation was measured by an impedance-based aggregometer. The in vivo antithrombotic effect was evaluated in a rabbit carotid artery cyclic flow reduction (CFR) model, in which repetitive thrombosis was induced by mechanical injuries of the artery and stenosis. Template bleeding time was determined in rabbit ear arterioles and hindlimb nail cuticles.Results:In vitro platelet aggregation was slightly inhibited by 4 μ M cilostazol (22 ± 6%), and modestly by 13 μ M (57 ± 3% of aggregation). While dipyridamole itself up to 13 μ M had no significant inhibition, it potentiated the effect from cilostazol: in the presence of 4 μ M dipyridamole, 4 μ M cilostazol inhibited aggregation by 47 ± 6%. Dipyridamole also potentiated the CFR reducing effect of cilostazol: combination of dipyridamole (no effect by itself) and cilostazol at 1 μ M decreased CFRs to levels achieved by 3–4 μ M cilostazol alone. Bleeding times were similar in controls and animals treated with cilostazol, or with cilostazol and dipyridamole. In contrast, aspirin (4 mg/kg), while reducing CFRs, significantly increased bleeding time.Conclusion: These results suggest that dipyridamole potentiates the antiplatelet effect of cilostazol without prolongation of the bleeding time, implying a potential novel combination antithrombotic therapy.

Expression of glycoprotein VI in vascular endothelial cells

Glycoprotein (GP) VI, a collagen receptor, plays an important role in collagen-mediated platelet aggregation and adhesion. To date, GPVI expression has been found only in platelets and megakaryocytes. In the present studies, we have demonstrated that GPVI was also expressed in cultured human umbilical vein endothelial cells (HUVEC) at both transcript and protein levels. Using a GPVI-specific probe, a ˜6-kb band was detected in HUVEC as well as in platelets and megakaryoblastic cell lines by Northern blotting. Using polyclonal antibodies raised against platelet GPVI peptides, the same size band (57 kDa) was labeled with convulxin (CVX) after immuno-precipitation in both HUVEC and platelet lysates. In addition, a ˜70-kDa band was also labeled in HUVEC. Surface expression of GPVI in HUVEC was confirmed by flow cytometry with GPVI-specific IgG or by direct labeling with FITC-conjugated CVX. Since HUVEC lack FcRγ chain that forms complex with GPVI in platelets for signaling process, the function of GPVI in vascular endothelial cells remains to be determined.

Single embryonic stem cell-derived embryoid bodies for gene screening

Shake(speare) While those embroiled in the debate over the use of embryonic stem (ES) cells in scientific research “take arms against a sea of troubles” in state and federal legislatures, down in the labyrinthine halls of many research institutions, the research continues unabated: to figure out what gives ES cells their unique potency and how it can be manipulated to treat the many frailties “that flesh is heir to.” Important and essential in vitro studies attempt to shed light on the elusive and mysterious pluripotency of these cells and elucidate the molecular triggers that signal any one particular cell to travel down any one particular differentiation pathway. Using a variety of approaches, such as gene trapping, those genes involved in regulation of differentiation can be identified and targeted. In order to perform such experiments, however, it is necessary to be able to track the molecular changes of the clonal progeny of a single cell as they differentiate and divide to form so-called embryoid bo...