Miranda Fong

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.

The role of interleukin-6 in lipopolysaccharide-induced weight loss, hypoglycemia and fibrinogen production, in vivo.

It was recently shown that interleukin (IL)-6 is an important mediator involved in the Colon (C)-26 model of experimental cancer cachexia. In this study, we wished to determine whether IL-6 is also involved in several metabolic changes associated with lipopolysaccharide (LPS) challenge. Administration of a relatively high amount of LPS to mice induced a transient weight loss, hypoglycemia, hypertriglyceridemia and an increase in the hepatic acute phase reactant, fibrinogen. Pretreatment of mice with the rat anti-murine IL-6 antibody (20F3), but not with a control antibody, resulted in a significant improvement of LPS-induced hypoglycemia and weight loss as well as a significant decrease of plasma fibrinogen. Anti-IL-6 antibody had no effect on LPS-induced hypertriglyceridemia. On the other hand, the pretreatment of mice with anti-murine TNF (TN3.19) antibody was able to completely inhibit elevation of triglycerides and modestly improve LPS-induced weight loss although it had no effect on hypoglycemia and fibrinogen production. Taken together, these results suggest that IL-6 plays a role in some of the metabolic changes associated with both an acute (i.e. LPS challenge) and chronic (C-26 cachexia) inflammatory conditions.

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.

Suramin blocks the binding of interleukin-1 to its receptor and neutralizes IL-1 biological activities

This report demonstrates the ability of the anti-cancer drug suramin to interfere with the binding of interleukin (IL)-1 to its receptor and to inhibit IL-1-induced biological activities. In a radioreceptor cell based assay, suramin inhibits the binding of IL-1 alpha to several murine cell lines expressing predominantly type I and type II IL-1 receptors. Affinity cross-linking experiments using IL-1 alpha and EL-4.6.1 cells confirms that suramin inhibits the binding of the ligand to the 80 kDa IL-1 type I receptor. In contrast, suramin fails to displace significantly prebound IL-1. In a cell-free system, suramin prevents the binding of IL-1 alpha and IL-1 beta to murine and human recombinant soluble type I IL-1 receptors. For example, the IC50 for suramin inhibiting IL-1 alpha and IL-1 beta binding to soluble human IL-1 receptor were 204 microM and 186 microM, respectively. The suramin analogues, NF-058 and NF-103 (which bear the same number of sulfate groups as suramin), are between three- and ten-fold less active than suramin in inhibiting IL-1 binding to EL-4.6.1 cells, and to recombinant soluble IL-1 receptor. Furthermore, in a dose-dependent manner suramin prevents several IL-1 mediated biological responses, including thymocyte proliferation, PGE-2 synthesis and IL-6 production. The inhibitory effect of the drug can be significantly reversed by the addition of excess cytokine. Taken together, the results indicate that suramin is a competitive IL-1 receptor antagonist. Because IL-1 participates in a broad range of immunological and inflammatory functions, the data suggest that suramin administration may influence important activities beyond those associated strictly with tumor inhibition.

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.

Vesnarinone is a selective inhibitor of macrophage TNFα release

Abstract Vesnarinone is an experimental drug that has been used successfully in the treatment of congestive heart failure patients. In this report we investigate the effect of vesnarinone on the cytokine secretory products of mononuclear phagocytes. In a concentration-dependent manner, the drug inhibits the endotoxin(LPS)stimulated release of tumor necrosis factor (TNF)α and suppresses interleukin(IL)-6 release, but does not affect the release of IL-lα, IL-10 and leukemia inhibitory factor (LIF) by mouse peritoneal macrophages. Using competitive polymerase chain reaction (PCR) analyses, we find that vesnarinone significantly reduces TNFα, but not IL-10 mRNA. In addition to LPS, the drug inhibits TNFα release induced by several other stimuli. The inhibitory effect of the drug on the TNFα biosynthesis can be observed in differentiated human monocytes, in macrophage cell lines, and in synovial adherent cells from rheumatoid arthritis patients. Although the precise mode of action of vesnarinone in the signal transduction pathway leading to the selective inhibition of TNFα is not known, the drug might be useful in the treatment of diseases involving that cytokine.

Tumor cell IL-6 gene expression is regulated by IL-1 alpha/beta and TNF alpha: proposed feedback mechanisms induced by the interaction of tumor cells and macrophages.

In the present report, we show that progressive growth of the immunogenic C57BL/6J sarcoma, MCA/76‐9, was accompanied by an increase in serum interleukin‐6 (IL‐6) activity. The possible pathways leading to the induction of IL‐6 release by the tumor cells are described. It was shown that macrophage products IL‐lα, IL‐lβ, and to a lesser extent, TNFα, induced the tumor cells in vitro to transcribe the IL‐6 gene and release the gene product. IL‐1 induced significantly more IL‐6 mRNA and bioactivity than TNFα, although both cytokines induced a cumulative increase of bioactivity in the supernates over a period of 24 h. The tumor cells were shown to express receptors for IL‐la, which could be blocked with anti‐IL‐1 receptor antibody. Given the previous reports that tumor‐associated macrophages expressed both IL‐lα/β and TNFα, the data suggest, first, that the mutual interaction of tumor cells and macrophages in situ may contribute to the observed increase in circulating IL‐6 activity, and second, that the release of IL‐6 in vivo may serve to regulate both anti‐tumor immune responses and suppressor mechanisms during significant net loss of body weight, even when tumors reach more than 2 g in weight by 3‐4 weeks of growth (unpublished results). The same tumor cells were routinely grown in vitro in RPMI 1640 medium containing 10% fetal bovine serum (HyClone, Logan, UT), which was routinely tested for endotoxin contamination by the Limulus assay (Whittaker Bioproducts, Walkersville, MD) and shown to be negative at the extinction point of the assay (<0.015 endotoxin units/ml). Mouse serum was obtained at defined intervals after tumor cell implantation by bleeding mice under anesthesia from the brachial plexus. There were four mice in each group for each time point.