Preparation and in vitro-in vivo evaluation of none gastric resident dipyridamole (DIP) sustained-release pellets with enhanced bioavailability.

The objective of this study was to develop none gastric resident sustained-release pellets loaded with dipyridamole with a high bioavailability. Two different kinds of core pellets, one containing citric acid as a pH-modifier (CAP) and, the other without pH-modifier (NCAP) were prepared by extrusion-spheronization and then coated with mixtures of enteric soluble and insoluble polymers (referred to as CAP(1) and NCAP(1)) or insoluble polymer alone (referred to as CAP(2) and NCAP(2)). The relative bioavailability of the sustained-release pellets was studied in fasted beagle dogs after oral administration using a commercially available immediate release tablet (IRT) as a reference. The in vitro release, in vivo absorption and in vitro-in vivo correlation were also evaluated. Results revealed that the plasma drug concentrations after administration of CAP(2), NCAP(1) and NCAP(2) were undetectable, indicating that the drug release was almost zero from the preparations throughout the gastro-intestinal tract. The C(max), T(max) and AUC((0→24)) of CAP(1) were 0.78 ± 0.23 (µg/ml), 3.80 ± 0.30 (h), and 6.74 ± 0.47 (µg/mlh), respectively. While the corresponding values were 2.23 ± 0.32 (µg/ml), 3.00 ± 0.44 (h) and 9.42 ± 0.69 (µg/mlh) for IRT. The relative bioavailability of CAP(1) was 71.55% compared with IRT. By combined incorporation of a pH-modifier into the core of pellets to modify the inner micro-environment and employing mixtures of enteric soluble and insoluble polymers as a retarding layer, drugs with high solubility in stomach and limited solubility in small intestine, such as DIP, could be successfully formulated as sustained release preparations with no pH-dependence in drug release and enhanced bioavailability.

Effect of diallyl trisulfide on the pharmacokinetics of dipyridamole in rats.

This study was aimed to evaluate the effect of diallyl trisulfide (DATS), a major component derived from garlic used to inhibit platelet thromboxane formation, on the pharmacokinetics of dipyridamole. Pharmacokinetic parameters of dipyridamole were determined in rats following intragastric (80 mg/kg suspension or 40 mg/kg solution) or intravenous (3 mg/kg) administration of dipyridamole with coadministration (20 mg/kg) and long-term pretreatment of DATS (10 or 20 mg/kg/day for 15 consecutive days). In addition, everted gut sac models were used to assess transepithelial transport of dipyridamole and the effect of DATS on the intestinal absorption of dipyridamole. After coadministration and long-term pretreatment of DATS, significantly lower Cmax and AUC(0-24 h) were observed for intragastric administration of dipyridamole, whereas little change was noted after intravenous dipyridamole administration. After adding DATS (10 and 50 µg/mL) in the everted gut sacs, absorption of dipyridamole was remarkably decreased in the ileum and jejunum (p < 0.01). In conclusion, DATS reduced the oral exposure of dipyridamole in rats likely by the modification of the dissolution rate and intestinal absorption of dipyridamole, indicating that combined use of DATS or DATS-containing supplements with dipyridamole may require caution as low plasma concentrations of dipyridamole may lead to a subtherapeutic effect of this agent.

Adiponectin is associated with abnormal lipid profile and coronary microvascular dysfunction in patients with dilated cardiomyopathy without overt heart failure.

Reduced plasma adiponectin has been associated with abnormal lipid profile, reduced left ventricle (LV) function, and the extent of coronary atherosclerosis in coronary artery disease. The aim of this study was to assess these relationships in patients with dilated cardiomyopathy (DCM) without overt heart failure. Plasma adiponectin was measured in 55 DCM patients (age, 59 ± 12 years; male, 36; body mass index [BMI], 26.9 ± 0.49 kg/m²; LV ejection fraction, 39.8% ± 1.3%; New York Heart Association class I-II) and in 40 age- and BMI-matched healthy controls. In a subset of 25 patients, myocardial blood flow (MBF) was measured at rest and during intravenous dipyridamole (0.56 mg/kg in 4 minutes) by positron emission tomography and ¹³N-ammonia as a flow tracer. Adiponectin was 6.6 ± 0.34 µg/mL in controls and 10.9 ± 0.85 µg/mL in DCM patients (P < .001), where it was related inversely with BMI (P = .009) and directly with brain natriuretic peptide (P = .017), high-density lipoprotein (HDL) cholesterol (P = .002), and MBF dipyridamole (P = .020). Adiponectin lesser than median value in patients was associated with higher total to HDL cholesterol ratio (4.8 ± 0.24 vs 3.9 ± 0.18, P = .009) and lower MBF reserve (1.76 ± 0.16 vs 2.43 ± 0.19, P = .01). These results could suggest that down-regulation of the adiponectin levels and reduced HDL cholesterol have a key role in causing impaired coronary function and myocardial perfusion in DCM.

Toxicokinetics of a dipyridamole (Persantin) intoxication: case report.

We report a case of a 51-year-old woman who was admitted to the hospital after ingestion of large doses of dipyridamole (12 g), temazepam (1 g) and oxazepam (0.2 g) with suicidal intent. The highest dipyridamole concentration that was measured in serum was 9.2 mg/L, which was paralleled by impaired platelet activation. For temazepam and oxazepam, peak serum concentrations were 8.5 and 1.3 mg/L, respectively. The patient was treated with activated charcoal, magnesium sulfate and aminophylline and could be discharged in good physical condition within 17 hours. This is the first report that provides toxicokinetic data and a corresponding pharmacodynamic effect after an intoxication with dipyridamole.

Predicting in vivo absorption behavior of oral modified release dosage forms containing pH-dependent poorly soluble drugs using a novel pH-adjusted biphasic in vitro dissolution test.

The focus of in vitro dissolution testing during early development of modified release (MR) formulations is to provide predictive estimates of drug release in respect to in vivo performance of a drug product. However, there are enormous challenges in MR drug development to establish proper dissolution conditions for a predictive test. To overcome limitations of dissolution testing at constant pH, a modified USP apparatus 2 was developed, combining biphasic dissolution with a pH-gradient in the aqueous dissolution medium. Quasi sink conditions in the aqueous phase were introduced by the removal of dissolved active via distribution to an organic phase. Results from in vitro drug-release studies and in vivo absorption studies of four MR formulations made by different technologies comprising the pH-dependent poorly soluble drugs, dipyridamole and the investigational drug BIMT 17, indicated that dissolution testing using the biphasic approach enabled an improved forecast of the in vivo behavior and bioavailability of modified release formulations compared to conventional dissolution testing at pH 1, pH 5.5, or pH 6.8. It can be concluded that the novel pH-adjusted dissolution test might be a useful tool in early drug development to develop, select, and optimize MR prototypes of Biopharmaceutical Classification System (BCS) II compounds.

Dipyridamole enhances ischaemia-induced arteriogenesis through an endocrine nitrite/nitric oxide-dependent pathway.

AIMS: Anti-platelet agents, such as dipyridamole, have several clinical benefits for peripheral artery disease with the speculation of angiogenic potential that could preserve ischaemic tissue viability, yet the effect of dipyridamole on ischaemic arteriogenesis or angiogenesis is unknown. Here we test the hypothesis that dipyridamole therapy augments arteriolar vessel development and function during chronic ischaemia. METHODS AND RESULTS: Mice were treated with 200 mg/kg dipyridamole twice daily to achieve therapeutic plasma levels (0.8-1.2 microg/mL). Chronic hindlimb ischaemia was induced by permanent femoral artery ligation followed by measurement of tissue perfusion using laser Doppler blood flow along with quantification of vascular density, cell proliferation, and activation of nitric oxide (NO) metabolism. Dipyridamole treatment quickly restored ischaemic hindlimb blood flow, increased vascular density and cell proliferation, and enhanced collateral artery perfusion compared with control treatments. The beneficial effects of dipyridamole on blood flow and vascular density were dependent on NO production as dipyridamole did not augment ischaemic tissue reperfusion, vascular density, or endothelial cell proliferation in endothelial NO synthase (eNOS)-deficient mice. Blood and tissue nitrite levels were significantly higher in dipyridamole-treated mice compared with controls and eNOS(-/-) mice, verifying increased NO production that was regulated in a PKA-dependent manner. CONCLUSION: Dipyridamole augments nitrite/NO production, leading to enhanced arteriogenesis activity and blood perfusion in ischaemic limbs. Together, these data suggest that dipyridamole can augment ischaemic vessel function and restore blood flow, which may be beneficial in peripheral artery disease.

Quantitative determination of dipyridamole in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study.

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 microL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate XB-C(18) (50 x 2.1 mm i.d, 3 microm) column with the mobile phase consisting of methanol-ammonium acetate (5 mM; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI(+)). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180-4.50 microg/mL (r(2) > or = 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 microg/mL for dipyridamole. The intra- and inter-day precisions (RSD) of the assay at all three QC levels were 1.6-12.7% with an accuracy (RE) of -4.3-1.9%, which meets the requirements of the FDA guidance. The HPLC-MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained-release dipyridamole tablet in volunteers after oral administration.

Distribution of dipyridamole in blood components among post-stroke patients treated with extended release formulation.

Extended release dipyridamole (ERD) is widely used in patients after ischaemic stroke; however, the ability of this antithrombotic agent to be stored in different blood cells has never been explored in post-stroke patients. We hypothesised that since ERD is known to be highly lipophilic, the drug may be present not only in plasma, but also accumulated in platelets, leukocytes, and erythrocytes. Fifteen patients after documented ischaemic stroke were treated with Aggrenox (ERD and low-dose aspirin combination) BID for 30 days, and 12 of them completed the study. ERD concentrations in blood cells and platelet-poor plasma were measured by spectrofluorimetry at Baseline, Day 14, and Day 30 after the initiation of therapy. The background level of spectrofluorometry readings differs slightly among the blood components (132-211 ng/ml) due to the differences in the preparation of samples and cell isolation techniques. As expected, two weeks of ERD therapy produced steady-state plasma concentration of dipyridamole already at Day 14 (1,680 +/- 542 ng/ ml), followed by a slight not significant decrease at one month (1,619 +/- 408 ng/ml). Two weeks of therapy was sufficient to achieve a consistent dipyridamole accumulation in erythrocytes (361 +/- 43 ng/ml), but not in platelets (244 +/- 78 ng/ml), or leukocytes (275 +/- 49 ng/ml). In fact, white blood cells continued dipyridamole intake beyond 14 days period, and this increase (398 +/- 66 ng/ml) was significant (p = 0.02) at 30 days. Treatment with ERD in post-stroke patients resulted not only in achievement of therapeutic plasma dipyridamole concentrations, but also deposition of the drug in erythrocytes and leukocytes, but not in platelets. If confirmed, these data will affect our better understanding of dipyridamole pleiotropy, and may explain long-term benefit of ERD formulation.

Development and validation of a liquid chromatography-tandem mass spectrometry assay for the simultaneous quantitation of prednisolone and dipyridamole in human plasma and its application in a pharmacokinetic study.

We have developed and validated an accurate, sensitive, and robust LC-MS/MS method that determines the concentration of CRx-102 (the combination of prednisolone and dipyridamole) in human plasma. In this method, prednisolone, dipyridamole, and the combined internal standards (IS) prednisolone-d(6) (IS for prednisolone) and dipyridamole-d(20) (IS for dipyridamole) were extracted from 100 microL human EDTA plasma using methylbutyl ether. Calibration curves were linear over a concentration range of 0.4-200 ng/mL for prednisolone and 5-3000 ng/mL for dipyridamole. The analytes were quantitatively determined using tandem mass spectrometry operated in positive electrospray ionization in a multiple reaction monitoring (MRM) mode. This validated method has been used successfully in clinical pharmacokinetic studies of CRx-102 in healthy volunteers.

An exploratory comparative study of volatile compounds in exhaled breath and emitted by skin using selected ion flow tube mass spectrometry.

Selected ion flow tube mass spectrometry (SIFT-MS) has been used to carry out a pilot parallel study on five volunteers to determine changes occurring in several trace compounds present in exhaled breath and emitted from skin into a collection bag surrounding part of the arm, before and after ingesting 75 g of glucose in the fasting state. SIFT-MS enabled real-time quantification of ammonia, methanol, ethanol, propanol, formaldehyde, acetaldehyde, isoprene and acetone. Following glucose ingestion, blood glucose and trace compound levels were measured every 30 min for 2 h. All the above compounds, except formaldehyde, were detected at the expected levels in exhaled breath of all volunteers; all the above compounds, except isoprene, were detected in the collection bag. Ammonia, methanol and ethanol were present at lower levels in the bag than in the breath. The aldehydes were present at higher levels in the bag than in breath. The blood glucose increased to a peak about 1 h post-ingestion, but this change was not obviously correlated with temporal changes in any of the compounds in breath or emitted by skin, except for acetone. The decrease in breath acetone was closely mirrored by skin-emitted acetone in three volunteers. Breath and skin acetone also clearly change with blood glucose and further work may ultimately enable inferences to be drawn of the blood glucose concentration from skin or breath measurements in type 1 diabetes.

Effect of collision-activated dissociation gas and collision energy on the fragmentation of dipyridamole and its rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometric determination in human plasma.

The effect of nitrogen as the collision-activated dissociation (CAD) gas on the fragmentation of dipyridamole was investigated in the range of 10-90 eV collision energy. The results support the collision model reported elsewhere, that the degree of ion fragmentation increases with the increasing mass of the collision gas. A simple, sensitive and high-throughput liquid chromatography/tandem mass spectrometry (LC/MS/MS) method has been developed for the determination of dipyridamole, a platelet aggregation inhibitor in human plasma, using granisetron as internal standard (IS). The method involved liquid-liquid extraction of the analyte and IS from 0.5 mL human plasma with diethyl ether. The chromatographic separation was achieved under isocratic conditions and the ion transitions for dipyridamole (m/z 505.40 --> 429.60) and the IS (m/z 313.10 --> 138.20) were monitored on a triple quadrupole mass spectrometer, operating in positive ion multiple reaction monitoring (MRM) mode. The method was validated over the concentration range 5.1-4499.1 ng/mL for dipyridamole. The method was rugged and rapid with a total run time of 1.2 min. It was successfully applied to a pivotal bioequivalence study in 67 healthy human subjects after oral administration of a 75 mg extended release formulation under fasting condition.

Simultaneous determination of dipyridamole and salicylic acid in human plasma by high performance liquid chromatography-mass spectrometry.

A sensitive, rapid and simple high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for simultaneous determination of dipyridamole and salicylic acid in human plasma has been developed and validated. After the addition of diazepam and rosiglitazone as internal standard (IS), plasma samples were prepared by liquid-liquid extraction followed by an isocratic elution with methanol:2 mM ammonium acetate buffer (pH 4.25; 70/30, v/v) on a Shimadzu VP-ODS C(18) column (5 microm, 150 x 2.0 mm I.D.). Detection was performed on a quadrupole mass spectrometer with ESI interface operating in the positive-ion mode for dipyridamole and negative-ion mode for salicylic acid. Calibration curves were linear (r(2) > 0.99) over the concentration range 10-2500 ng/mL for dipyridamole and 30-4000 ng/mL for salicylic acid with acceptable accuracy and precision, respectively. The intra- and inter-batch precisions were less than 15% of the relative standard deviation. The limits of detection of dipyridamole and salicylic acid were 1 and 15 ng/mL, respectively. The validated HPLC-ESI-MS method was successfully applied to a preliminary pharmacokinetic study of fixed-dose combination of sustained-release dipyridamole/aspirin in Chinese healthy male volunteers.

Redox state of dipyridamole is a critical determinant for its beneficial antioxidant and antiinflammatory effects.

Dipyridamole, a well-known inhibitor of cGMP-dependent phosphodiesterase and the adenosine transporter, reportedly possesses antioxidant properties and attenuates reactive oxygen species (ROS) formation in platelet and endothelial cells. The relevance of the redox status of this compound or the mechanism for its redox-dependent effects is unknown. Oxidation of dipyridamole by horseradish peroxidase and hydrogen peroxide diminished its fluorescence and attenuated dipyridamole-mediated DPPH and ferric ferrozine reduction. Oxidation also led to elimination of dipyridamole's redox-sensitive properties, including inhibiting Cu (II)-induced LDL oxidation and ROS generation. Attenuation of activation- induced platelet release of soluble CD40 ligand (sCD40L) was diminished after dipyridamole oxidation. Dipyridamole but not oxidized dipyridamole effectively inhibited platelet adhesion to collagen-coated slides under flow conditions. By Western blot analysis, dipyridamole enhanced stimulation-induced platelet VASP phosphorylation, whereas oxidized dipyridamole caused attenuation. Using luciferase assays and nuclear translocation studies with confocal microscopy and Western blot analysis, native dipyridamole diminished TNF alpha or thrombin-induced NF kappa B activation and I kappa B alpha phosphorylation. Oxidized dipyridamole had no effect on TNFalpha-mediated NF kappa B activation. These results indicate: (1) the redox state of dipyridamole regulates its antioxidant properties; (2) dipyridamole's platelet inhibitory effects are manifested by enhanced VASP phosphorylation and platelet adhesion on collagen; and (3) dipyridamole's antioxidant effects in vascular cells are at least partially mediated via suppression of inflammatory NF kappa B signaling.

[Detection of dipiridamol in biological fluids].

Acetone is proposed as an isolating agent for dipiridamol isolation from biological fluids. Purification of the isolates was performed with liquid-liquid extraction and colon chromatography with silasorb C-18 sorbent. The technique of dipiridamol detection in the blood and urine is described. The assays results are presented.

Combination treatment with dipyridamole, aspirin, and tPA in an embolic model of stroke in rats.

Antithrombotic therapy has been shown to be effective in preventing secondary strokes. Inhibition of platelet function may reduce formation of thrombi thereby reducing the incidence of stroke. However, stronger inhibition of platelets is correlated with increased risk of bleeding events. The purpose of this study was to test the protective effects of combination therapy with dipyridamole and acetylsalicylic acid (ASA) in comparison to ASA alone, and whether such combination treatment may produce any added benefits when tissue plasminogen activator (tPA) treatment is also used. The study was divided into three parts. In part A, effect of antiplatelets on infarct volume was assessed. In part B, perfusion deficits were measured. In part C, efficacy of antiplatelet therapy in combination with tPA was assessed. In part A, dipyridamole and aspirin treatment significantly reduced infarct volume (P<0.05). In part B, treatment with dipyridamole significantly reduced the perfusion deficits as compared to control (P<0.05). In part C, dipyridamole plus tPA or dipyridamole and aspirin plus tPA significantly decreased infarct volume as compared to tPA alone (P<0.05). The present study suggests that there is significant protection with dipyridamole as both infarct volume and perfusion deficits are significantly reduced. Dipyridamole with tPA also significantly reduced infarct volume as compared to tPA alone. Our data suggests that higher doses of antithrombotic therapy with dipyridamole offer best neuroprotection.

Synergistic effect of cilostazol and dipyridamole mediated by adenosine on shear-induced platelet aggregation.

INTRODUCTION: The Cilostazol Stroke Prevention Study found that cilostazol, a phosphodiesterase 3 inhibitor, can reduce the risk of subsequent stroke in Japanese patients with cerebral infarction. Here, we measured the effects of cilostazol in vitro on shear-induced platelet aggregation, an important mechanism of thrombosis at arterial bifurcations or stenotic lesions. We also evaluated the influences of intrinsic adenosine on the ability of cilostazol to inhibit shear-induced platelet aggregation by investigating the effect of dipyridamole, an inhibitor of cellular adenosine reuptake, in combination with cilostazol in vitro. MATERIALS AND METHODS: We measured platelet aggregation induced by a shear rate of 10,800 s(-1) in whole blood and in platelet-rich plasma from healthy volunteers using a cone-plate streaming chamber. RESULTS: Both cilostazol and adenosine dose-dependently inhibited shear-induced platelet aggregation in platelet-rich plasma samples. Adding a low concentration of adenosine (0.3 microM) did not inhibit shear-induced platelet aggregation, but significantly enhanced the inhibitory effect of cilostazol in platelet-rich plasma. Dipyridamole dose-dependently inhibited shear-induced platelet aggregation in whole blood and significantly enhanced the inhibitory effect of cilostazol on shear-induced platelet aggregation, but did not affect shear-induced platelet aggregation in platelet-rich plasma. The inhibitory effects of cilostazol combined with dipyridamole in whole blood were almost completely reversed by adenosine deaminase. CONCLUSIONS: Dipyridamole appears to synergistically enhance the inhibitory effect of cilostazol on shear-induced platelet aggregation by maintaining high plasma levels of adenosine.

Dipyridamole bioavailability in subjects with reduced gastric acidity.

Dipyridamole (DP) is an antiplatelet agent that shows decreased oral bioavailability with increased gastric pH that occurs with commonly prescribed antacids. An extended-release (ER) formulation of DP that employs tartaric acid to improve bioavailability of DP in the presence of elevated gastric pH was developed as a combination antiplatelet product with immediate-release aspirin. This crossover-designed study examined the relative bioavailability of DP from the composite product compared to conventional DP tablets during reduced gastric acidity. Gastric pH was increased (pH > 4.0) in 20 healthy subjects with lansoprazole (30 mg/d for 5 days). Dipyridamole systemic exposure over 12 hours was compared after oral administration of a single composite ER capsule (200 mg DP + 25 mg aspirin) versus two 100-mg conventional DP tablets given 6 hours apart combined with 81 mg aspirin. DP relative bioavailability was reduced 53% with conventional tablets compared to the composite buffered ER capsule in reduced gastric acid conditions. Peak DP plasma concentrations were 57% lower with immediate-release tablets compared to the composite formulation with high stomach pH. Substituting generic DP plus low-dose aspirin may be less effective than the buffered DP composite product in patients with concomitant antacid therapies.

Criticality of pH for accurate fluorometric measurements of dipyridamole levels in biological fluids.

Extended release dipyridamole (DIP) is widely used in clinical practice as an Aggrenox formulation, which is proven to improve outcomes for secondary stroke prevention in patients after acute vascular events. However, presently established fluorometry techniques are not suitable for trace amount determinations, because of the variable background fluorescence. The authors sought to determine whether biological fluid pH is important for the serial measures of DIP levels in the animal experiments and in patients treated with Aggrenox after ischemic stroke. Post-stroke patient (n = 34) and mice (n = 25) samples were tested to determine DIP levels by established techniques with FluoroMax 3 spectrofluorometer. Both the absorption and emission spectra of DIP were affected by modifications in pH. Fluorescence of DIP was found to be maximal at a wavelength of 490 nm (excitation 420 nm) and the spectral pattern was independent of pH. The intensity of fluorescence, however, was drastically lower at low pH (at pH 2.6, fluorescence was 4% of intensity at pH 9.8). Background plasma fluorescence, however, was completely unaffected by changes in pH. Using these fluorometric characteristics, a regression model that facilitates the efficient and sensitive determination of DIP concentration in biological fluids was formulated. Exploiting pH-dependent characteristics of DIP versus serum fluorescence patterns permits a convenient mathematical model to determine DIP concentration. This relatively inexpensive and time-efficient procedure can quantify drug levels in human/animal plasma/serum, thereby directly determining the level of patient adherence to the prescribed drug regimen, be it in the context of clinical trials or compliance with the animal protocol.

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 microM), 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 microM (P < 0.05 versus plus adenosine alone of 0.38 +/- 0.05 microM), 0.11 +/- 0.06 microM (P < 0.05), and 0.01 +/- 0.01 microM (P < 0.005) when combined with 1, 3, or 10 microM dipyridamole, respectively (n = 5). In whole blood, cilostazol (0.3 to 3 microM) and dipyridamole (1 or 3 microM) 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.