Method for the quantitative analysis of cilostazol and its metabolites in human plasma using LC/MS/MS.

An LC/MS/MS method for the simultaneous determination of cilostazol, a quinolinone derivative, and three active metabolites, OPC-13015, OPC-13213, and OPC-13217, in human plasma was developed and validated. Cilostazol, its metabolites, and the internal standard, OPC-3930 were extracted from human plasma by liquid-liquid partitioning followed by solid-phase extraction (SPE) on a Sep-Pak silica column. The eluent from the SPE column was then evaporated and the residue reconstituted in a mixture of methanol/ammonium acetate buffer (pH 6.5) (2:8 v/v). The analytes in the reconstituted solution were resolved using reversed-phase chromatography on a Supelcosil LC-18-DB HPLC column by an 17.5-min gradient elution. Cilostazol, its metabolites, and the internal standard were detected by tandem mass spectrometry with a Turbo Ionspray interface in the positive ion mode. The method was validated over a linear range of 5.0-1200.0 ng/ml for all the analytes. This method was demonstrated to be specific for the analytes of interest with no interference from endogenous substances in human plasma or from several potential concomitant medications. For cilostazol and its metabolites, the accuracy (relative recovery) of this method was between 92.1 and 106.4%, and the precision (%CV) was between 4.6 and 6.5%. During the validation, standard curve correlation coefficients equalled or exceeded 0.999 for cilostazol and its metabolites. These data demonstrate the reliability and precision of the method. The method was successfully cross-validated with an established HPLC method.

Determination of cilostazol and its metabolites in human urine by high performance liquid chromatography.

A high performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous quantification of cilostazol, and its known metabolites in human urine was developed and validated. Cilostazol, its metabolites and the internal standard OPC-3930 (structural analogue of cilostazol) were extracted from human urine using liquid-liquid extraction with chloroform. The organic extract was then evaporated and the residue was reconstituted in 8% acetonitrile in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto an HPLC system and was subjected to reverse-phase HPLC on a 5-microm ODS column. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the internal standard were well resolved at baseline with adequate resolution from constituents of human urine. The lower limit of quantification was 100 ng/ml for cilostazol and all metabolites. The method was validated for a linear range of 100-3000 ng/ml for all the metabolites and cilostazol. The overall accuracy (% relative recovery) of this method ranged from 86.1 to 116.8% for all the analytes with overall precision (%CV) being 0.8-19.7%. The long-term stability of clinical urine samples was established for at least 3 months at -20 degrees C in a storage freezer. During validation, calibration curves had correlation coefficients greater than or equal to 0.995 for cilostazol and the seven tested metabolites. The method was successfully used for the analysis of cilostazol and its metabolites in urine samples from clinical studies, demonstrating the reliability and robustness of the method.

In vitro metabolism and interaction of cilostazol with human hepatic cytochrome P450 isoforms.

1. Cilostazol (OPC-13013) undergoes extensive hepatic metabolism. The hydroxylation of the quinone moiety of cilostazol to OPC-13326 was the predominant route in all the liver preparations studies. The hydroxylation of the hexane moiety to OPC-13217 was the second most predominant route in vitro. 2. Ketoconazole (1 microM) was the most potent inhibitor of both quinone and hexane hydroxylation. Both the CYP2D6 inhibitor quinidine (0.1 microM) and the CYP2C19 inhibitor omeprazole (10 microM) failed to consistently inhibit metabolism of cilostazol via either of these two predominant routes. 3. Data obtained from a bank of pre-characterized human liver microsomes demonstrated a stronger correlation (r2=0.68, P < 0.01) between metabolism of cilostazol to OPC-13326 and metabolism of felodipine, a CYP3A probe, that with probes for any other isoform. Cimetidine demonstrated concentration-dependent competitive inhibition of the metabolism of cilostazol by both routes. 4. Kinetic data demonstrated a Km value of 101 microM for cilostazol, suggesting a relatively low affinity of cilostazol for CYP3A. While recombinant CYP1A2, CYP2D6 and CYP2C19 were also able to catalyze formation of specific cilostazol metabolites, they did not appear to contribute significantly to cilostazol metabolism in whole human liver microsomes.

The preparation of soft gelatin capsules for a radioactive tracer study.

Clinical doses are developed for the oral coadministration of radiolabeled and nonlabeled forms of a poorly soluble investigational compound: OPC-41061. The release rates of the labeled and nonlabeled forms are equated and matched to the release rate of the polymer spray-dried form of the drug in the proposed market product. The study involves the physicochemical characterization of the powders using thermal analysis and dissolution testing, development and extemporaneous manufacture of liquid-filled soft gelatin capsules, and dissolution and stability testing of the final dosage form. Thermal analysis indicated that the labeled powder was amorphous and that the nonlabeled powder, which had been jet-milled, was crystalline. Dissolution testing of the jet-milled and spray-dried powders indicated that the former was released at a significantly slower rate. A liquid formulation containing 25% dimethyl acetamide and 75% polyethylene glycol 400 (PEG 400) solubilized the desired dose of 60 mg and exhibited a drug profile that was similar to the spray-dried formulation. The final formulation was a soft gelatin capsule containing 60 mg of drug, including 100 microCi radioactivity, dissolved in 0.8 ml of a 25% dimethyl acetamide/75% PEG 400 solution. The formulation was chemically and physically stable for a period greater than the duration of the study.

Effect of disulfiram-mediated CYP2E1 inhibition on the disposition of vesnarinone.

Vesnarinone is an orally administered inotropic agent that is metabolized in vitro by the cytochrome P450 (CYP) isozymes CYP3A4 and CYP2E1. The purpose of this study was to assess the contribution of CYP2E1 activity to the disposition of vesnarinone in humans by characterizing the pharmacokinetics before and after disulfiram-mediated CYP2E1 inhibition. The pharmacokinetics of vesnarinone 60 mg were determined in normal healthy volunteers (N = 7) before and after daily disulfiram administration (250 mg). Chlorzoxazone 250 mg was also administered before, during, and after disulfiram administration to serve as a positive control for CYP2E1 inhibition. Disulfiram treatment decreased 6-hydroxychlorzoxazone formation clearance by nearly 95% but effected only a modest decrease in vesnarinone apparent oral clearance (5.7 +/- 1.0 vs. 5.0 +/- 0.5 ml/min; p = 0.022). In contrast to the modest effect on the parent drug, disulfiram treatment substantially increased plasma concentrations of the primary metabolite OPC-18692. The Cmax of OPC-18692 was increased approximately 7-fold, and the area under the plasma concentration-time curve was increased 18-fold (2.9 +/- 0.9 vs. 53.7 +/- 33.2 micrograms.h/ml; p = 0.006). The results indicate that CYP2E1 inhibition has only a modest, clinically insignificant effect on vesnarinone disposition but markedly increases plasma concentrations of the OPC-18692 metabolite. The pharmacological properties of this metabolite have not been fully defined; thus, the clinical importance of this observation depends on whether this metabolite contributes to any of the toxicity associated with vesnarinone administration.

Simultaneous quantitative determination of cilostazol and its metabolites in human plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of cilostazol, a quinolinone derivative, and its known metabolites OPC-13015, OPC-13213, OPC-13217, OPC-13366, OPC-13269, OPC-13326 and OPC-13388 in human plasma was developed and validated. Cilostazol, its metabolites and two internal standards, OPC-3930 and OPC-13112, were extracted from human plasma by a combination of liquid-liquid and liquid-solid phase extractions, with combined organic solvents of n-butanol, methanol, chloroform, methyl-tert.-butyl ether, and a Sep-Pak silica column. The combined extract was then evaporated and the residue was reconstituted in ammonium acetate buffer (pH 6.5). The reconstituted solution was injected onto a HPLC system and was subjected to reversed-phase HPLC on a 5 microm ODS-80TM column to obtain quality chromatograph and good peak resolution. A gradient mobile phase with different percentages of acetonitrile in acetate buffer (pH 6.5) was used for the resolution of analytes. Cilostazol, its metabolites and the two internal standards were well separated at baseline from each other with resolution factor being 74 and 138. This HPLC method was demonstrated to be specific for all analytes of interest with no significant interference from the endogenous substances of human plasma. The lower limit of quantitation was 20 ng/ml for cilostazol and all metabolites. The method was validated initially for an extended linear range of 20-600 ng/ml for all metabolites and cilostazol, and has been revised later for a linear range of 20-1200 ng/ml for cilostazol and two major and active metabolites OPC-13015 and OPC-13213. The overall accuracy (relative recovery) of this method was established to be 98.5% to 104.9% for analytes with overall precision (CV) being 1.5% to 9.0%. The long-term stability of clinical plasma samples was established for at least one year at -20 degrees C. Two internal standards of OPC-3930 and OPC-13112 were evaluated and validated. However, the data indicated that there was no significant difference for all accuracy and precision obtained by using either OPC-3930 or OPC-13112. OPC-3930 was chosen as the internal standard for the analysis of plasma samples from clinical studies due to its shorter retention time. During the validation standard curves had correlation coefficients greater than or equal to 0.998 for cilostazol and the seven metabolites. These data clearly demonstrate the reliability and reproducibility of the method.

LC/MS/MS analysis of vesnarinone and its principal metabolites in plasma and urine.

An LC/MS/MS assay was developed and successfully used to quantitate vesnarinone and its principal metabolites (OPC-8230, OPC-18136, and OPC-18137) in human plasma and urine. Samples were pre-treated with liquid-solid extraction followed by simultaneous monitoring of primary and daughter ions which were used for the identification and quantitation of the analytes on LC/MS/MS. This assay offers advantages of specificity, speed and greater sensitivity over the previously developed HPLC-UV assay. The lower limit of quantitation is 500 ng x ml(-1) for vesnarinone and 20 ng x ml(-1) for OPC-8230, OPC-18137, and OPC-18136 in plasma. Methodology is similar for the estimation of these analytes in urine with the lower limit of quantitation being 500 ng x ml(-1) for vesnarinone and 100 ng x ml(-1) for each metabolite. Ascorbic acid was added to stabilize the analytes from degradation. This LC/MS/MS method was developed to overcome many practical problems associated with the HPLC method. The LC/MS/MS method offers the flexibility of analyzing additional metabolites and changing the linearity range to accommodate the differences in linear range (200-10000 ng x ml(-1) for vesnarinone and 20-1000 for metabolites) for the analytes.

Cilostazol pharmacokinetics after single and multiple oral doses in healthy males and patients with intermittent claudication resulting from peripheral arterial disease.

OBJECTIVE: To study the pharmacokinetics of cilostazol following single oral administration of 50 to 200 mg in healthy young males, and after repeated oral administration of 100 mg every 12 hours to patients with peripheral arterial disease (PAD). DESIGN: The healthy male single dose study was a single-centre, randomised sequence, open-label, incomplete block, 3-period, 4-treatment, crossover design. The patient study was a single-centre, multiple dose, open-label study. STUDY PARTICIPANTS: 20 healthy nonsmoking male volunteers were enrolled and successfully completed the single dose study. 26 patients (21 males, 5 females) with intermittent claudication resulting from PAD were enrolled and completed the single/multiple dose study. MAIN OUTCOME MEASURES: Noncompartmental pharmacokinetic parameters, the area under the plasma concentration-time curve from zero to the time of last measurable plasma concentration, and maximum plasma concentration. RESULTS: Peak plasma concentrations of cilostazol occurred about 3 hours after drug administration and then declined biexponentially with concentrations detectable (> 20 micrograms/L) in the plasma for at least 36 hours postdose. The apparent elimination half-life of cilostazol (approximately 11 hours) was similar after a single dose or after multiple doses, with steady state being reached within 4 days. Cilostazol accumulated 1.7-fold following multiple dose administration. The apparent volume of distribution (Vz/F; 2.76 L/kg) suggested extensive distribution of cilostazol in the tissues. The oral clearance of cilostazol (CL/F; 0.18 L/h/kg) was much lower than liver blood flow, indicating a low extraction ratio drug, and hence low probability of a significant first-pass effect. None of the administered doses were recovered in the urine as unchanged cilostazol, suggesting that metabolism, rather than urinary excretion, is the major elimination route. Following single oral doses of 50 to 200 mg, the plasma concentrations of cilostazol and its metabolites increased less than proportionally to the dose. The pharmacokinetics of cilostazol in normal healthy volunteers are predictive of those in patients with PAD. Single oral doses of 50 to 200 mg cilostazol as well as 100 mg cilostazol every 12 hours were well tolerated. CONCLUSION: The plasma concentration of cilostazol and its metabolites increased less than proportionally with increasing doses. The relatively low plasma clearance and high volume of distribution of cilostazol suggest a low first-pass effect and extensive distribution. The pharmacokinetics of cilostazol in normal volunteers is predictive of that in patients with PAD. Cilostazol was well tolerated in healthy volunteers and patients with intermittent claudication resulting from PAD.

Relative bioavailability and effects of a high fat meal on single dose cilostazol pharmacokinetics.

OBJECTIVES: The objectives of this research were to (1) assess the relative bioavailability following administration of a 100 mg cilostazol suspension versus 100 mg tablet; (2) assess dosage form equivalency (2 x 50 mg compared with 1 x 100 mg); (3) compare the relative bioavailability following a single 50 mg dose of cilostazol administered as an ethanolic solution versus a 50 mg tablet; and (4) determine the effects of high fat diet on the pharmacokinetics of cilostazol following a single dose of 100 mg cilostazol in the fed or fasted state. Results were compiled from 3 separate studies to address these objectives. DESIGN: All studies involved healthy adult males receiving single oral doses of cilostazol in the fed or fasted state. The fed state consisted of administering cilostazol after ingestion of a high fat meal. One study compared the relative bioavailability of 100 mg suspension and 2 x 50 mg tablet versus 100 mg tablet in a randomised crossover design. The study involving administration of a 50 mg cilostazol ethanolic solution was a single treatment study. The effects of food on the pharmacokinetics of cilostazol after administration of 100 mg cilostazol in the fed or fasted state as well as the pharmacokinetic profile following administration of a single 50 mg oral dose of cilostazol were assessed in a randomised crossover design. STUDY PARTICIPANTS: All participants were healthy nonsmoking males aged between 19 and 48 years whose bodyweight was within 15% of ideal bodyweight. MAIN OUTCOME MEASURES: Noncompartmental pharmacokinetic parameters were determined for each study participant. RESULTS: The area under the plasma concentration-time curve (AUC) parameters were within the 80 to 125% criterion for bioequivalence for the cilostazol and its primary metabolite, OPC-13015. The maximum observed plasma concentrations (Cmax) for these formulations were not equivalent and indicated that the absorption of cilostazol from a suspension is more rapid than from a tablet. The apparent terminal half-lives (t1/2z) of cilostazol and OPC-13015 were shorter after administration of the suspension compared with the tablet. Cmax and AUC following administration of a single 50 mg cilostazol tablet were approximately 80% of that from the same dose administered as an ethanolic solution. The t1/2z of cilostazol decreased from 15.5 hours after a tablet to 2.5 hours after an ethanolic solution. Upon coadministration with a high fat meal, the Cmax of cilostazol increased 90% and AUC infinity increased 25% (p < 0.05). The t1/2z decreased from 15.1 +/- 14.5 hours (mean +/- SD) in the fasted state to 5.4 +/- 2.0 hours in the fed state. Single oral doses of 50 and 100 mg cilostazol were well tolerated. CONCLUSIONS: The relative bioavailability of the 100 mg cilostazol tablet versus an oral 100 mg cilostazol suspension is 100%. The 2 x 50 mg and 1 x 100 mg tablets are considered to be bioequivalent. The absorption following administration of 50 mg cilostazol ethanolic solution is faster and appears to be greater than that after administration of the 50 mg tablet. Coadministration of food increases the rate and extent of cilostazol absorption. The oral pharmacokinetics of cilostazol and metabolites are absorption-rate limited. The significant differences in the t1/2z observed when comparing cilostazol tablet, suspension, and solution as well as the effects of food suggest 'flip-flop' pharmacokinetics.

Effect of hepatic impairment on the pharmacokinetics of a single dose of cilostazol.

OBJECTIVE: The pharmacokinetic profiles of cilostazol and its metabolites following a single oral dose of cilostazol 100 mg were compared between individuals with impaired and normal liver function. DESIGN: The study was conducted as a single-centre, open-label, single dose pharmacokinetic and tolerability trial. STUDY PARTICIPANTS: 12 patients with impaired and compensated liver function were compared with 12 volunteers with normal liver function. Participants in each group were matched for gender, age and weight. Of the 12 patients with hepatic impairment examined in this study, 10 had mild impairment (Child-Pugh class A) and 2 had moderate impairment (Child-Pugh class B). MAIN OUTCOME MEASURES: Blood and urine were collected up to 144 hours after drug administration. Pharmacokinetics were determined by noncompartmental methods. RESULTS: Protein binding did not differ between the groups (95.2% healthy volunteers, 94.6% hepatically impaired patients). Mean +/- SD unbound oral clearance of cilostazol decreased by 8.6% because of hepatic impairment (3380 +/- 1400 ml/min in healthy volunteers, 3260 +/- 2030 ml/min in hepatically impaired patients). Total urinary excretion of metabolites was significantly higher in healthy volunteers (26 vs 17% of dose). Overall, the pharmacokinetics of cilostazol and its metabolites, OPC-13213 and OPC-13015, were not substantially different in those with mild and moderate hepatic disease compared with values in healthy volunteers. Except for terminal-phase disposition half-life and apparent terminal-phase volume of distribution for cilostazol, the ratios of geometric means of pharmacokinetic parameters for plasma cilostazol, OPC-13213 and OPC-13015 in those with hepatic impairment versus healthy volunteers were close to 100%. CONCLUSIONS: Based on the results of the pharmacokinetic analysis, dose adjustment in patients with mild hepatic impairment is not necessary. However, caution should be exercised when cilostazol is administered to patients with moderate or severe hepatic impairment.

Effect of renal impairment on the pharmacokinetics of cilostazol and its metabolites.

OBJECTIVE: The pharmacokinetics of cilostazol were studied in patients with mild, moderate and severe renal impairment and in healthy volunteers after administration of 50 mg single and multiple doses of cilostazol. DESIGN: This was an open-label, single and multiple dose study administering 50 mg cilostazol every 12 hours to healthy volunteers and patients with varying degrees of renal impairment. PARTICIPANTS: 6 normal volunteers [creatinine clearance (CLCR) > or = 90 ml/min]; 6 patients with mild (CLCR 50 to 89 ml/min), 5 with moderate (CLCR 26 to 49 ml/min) and 6 with severe (CLCR 5 to 25 ml/min) renal impairment. OUTCOME MEASURES: Noncompartmental pharmacokinetic parameters were determined for each study participant. RESULTS: At steady state, in the severe renal disease group, cilostazol and OPC-13015 peak concentrations (Cmax) were 29 and 41% lower and the areas under the concentration-time curve over the dosage interval (AUC tau) 39 and 47% lower than in the healthy volunteers. Cmax and AUC tau of OPC-13213 were significantly higher, 173 and 209%, respectively, than those in the healthy volunteers. The accumulation ratios were not significantly different between the various renal function groups for cilostazol and its metabolites. The estimated pharmacological activity of cilostazol and its metabolites was similar between the normal volunteers and those with severe renal impairment. CONCLUSIONS: A dosage reduction in renally impaired patients is not supported by the pharmacokinetics of cilostazol and its metabolites in this patient group.

Inhibition of CYP2D6 by quinidine and its effects on the metabolism of cilostazol.

OBJECTIVE: In vitro results are inconclusive as to whether cilostazol is metabolised by cytochrome P450 isoenzyme 2D6 (CYP2D6). The goals of this study were (1) to assure the dose of quinidine and timing relative to cilostazol used in this study were adequate to cause inhibition of CYP2D6, (2) to evaluate carryover effects of quinidine administration, and (3) to evaluate the effect of CYP2D6 deficiency and administration of quinidine (a CYP2D6 inhibitor) on the pharmacokinetics of a single 100 mg oral dose of cilostazol. DESIGN: This study was conducted as a single-centre, open-label, randomised sequence, 2-period, crossover pharmacokinetic trial. Water alone (treatment without quinidine) or two 200 mg oral doses of quinidine sulfate with water were administered 25 hours and 1 hour prior to a single 100 mg dose of cilostazol in period 1. Study participants were crossed over to opposite treatment in period 2. Metoprolol 25 mg, used as a positive control, was administered 1 hour after quinidine sulfate with water or using water alone to assess the magnitude of CYP2D6 inhibition by quinidine. STUDY PARTICIPANTS: 22 healthy nonsmoking Caucasian (14 male and 8 female) volunteers participated in the study. MAIN OUTCOME MEASURES: Serial blood and urine samples were collected at predose and after cilostazol administration to characterise cilostazol and its metabolite pharmacokinetics. Additional plasma samples were taken to assess the pharmacokinetics of quinidine. Urine samples were collected to measure metoprolol and hydroxymetoprolol. RESULTS: Administration of metoprolol with quinidine caused a significant (p < 0.001) decrease in the urinary 4-hydroxymetoprolol/metoprolol ratio compared with administration of metoprolol alone (42-fold decrease, 0.065 vs 2.707). Hence, quinidine effectively converted extensive metabolisers of CYP2D6 to poor metabolisers of CYP2D6. The 21-day washout period was adequate to have complete recovery from quinidine inhibition of CYP2D6. The analysis of variance demonstrated that the mean maximum plasma concentration (Cmax) for cilostazol, both adjusted and unadjusted for the free fraction, was higher in the control group than in the quinidine group (p = 0.023). However, the time to Cmax (p = 0.669), the area under the plasma concentration-time curve from time zero to infinity (AUC infinity; p = 0.133), and the apparent oral clearance (p = 0.135) were unchanged. The geometric mean ratios (90% confidence interval) comparing with quinidine (test) and without quinidine (reference) coadministration for Cmax and AUC infinity are 0.86 (0.77, 0.95) and 0.92 (0.84, 1.00), respectively. Similar patterns were observed for OPC-13015 and OPC-13213 with regard to Cmax, area under the plasma concentration-time curve from time zero to the last measurable concentration at time t, and AUC infinity (where determinable). The slight decrease in the systemic availability of cilostazol and its metabolites was thought to be a result of the increased gastrointestinal motility secondary to quinidine. CONCLUSIONS: Administration of quinidine sulfate 200 mg profoundly inhibited CYP2D6-mediated metabolism. The effects of quinidine inhibition of CYP2D6 metabolism were completely reversible during the 21-day washout period. Coadministration of quinidine with cilostazol had no substantial effect on cilostazol or its metabolites (OPC-13015 and OPC-13213). Hence, CYP2D6 does not have a significant contribution in the metabolic elimination of cilostazol.

Effect of omeprazole on the metabolism of cilostazol.

OBJECTIVE: In vitro results suggest that cilostazol is metabolised by cytochrome P450 (CYP) isoforms 1A2, 2D6, 3A4 and 2C19. This study was designed to evaluate the effect of concomitant administration of omeprazole (a CYP2C19 inhibitor) on the pharmacokinetics of a single 100 mg oral dose of cilostazol. DESIGN: This study was conducted as a single-centre, open-label, nonrandomised, 2-period, crossover pharmacokinetic trial. A single 100 mg dose of cilostazol was administered orally on days 0 and 14. Oral omeprazole (40 mg every day) was administered on days 7 to 18. STUDY PARTICIPANTS: 20 healthy nonsmoking male and female volunteers. MAIN OUTCOME MEASURES: Serial blood samples were collected before and after cilostazol administration to characterise the pharmacokinetics of cilostazol and its metabolites. RESULTS: Following omeprazole coadministration, the increases in cilostazol maximum plasma concentration (Cmax) and area under the plasma concentration-time curve at time t (AUCt) were 18% (p = 0.062) and 26% (p < 0.001), respectively. For the 2 major circulating metabolites, OPC-13015 and OPC-13213, the OPC-13015 Cmax and AUCt increased by 29 and 69%, respectively (p < 0.001). However, for OPC-13213, the Cmax and AUCt decreased by 22 and 31%, respectively (p < 0.001). The plasma protein binding of cilostazol was unaffected by coadministration of omeprazole. CONCLUSIONS: Coadministration of cilostazol with omeprazole resulted in an increase in the systemic exposure of cilostazol and its active metabolite, OPC-13015, by 26 and 69%, respectively. For the other active metabolite, OPC-13213, systemic exposure decreased by 31% because of inhibition of cilostazol metabolism to this metabolite. These changes in systemic exposure were well tolerated. A dose of 50 mg cilostazol twice a day should be considered during coadministration of inhibitors of CYP2C19, such as omeprazole.

Effects of CYP3A inhibition on the metabolism of cilostazol.

OBJECTIVE: In vitro results suggest that cilostazol is metabolised by cytochrome P450 (CYP) isoforms 1A2, 2D6, 3A and 2C19. This study investigated the role of CYP3A inhibition on the metabolism of cilostazol. DESIGN: The study was conducted as a single-centre, open-label, nonrandomised, 2-period, crossover pharmacokinetic trial. A single dose of cilostazol 100 mg was administered orally on days 1 and 15. Erythromycin (150 mg orally 3 times daily) was administered on days 8 to 20. 14C-erythromycin (3 microns Ci) was administered intravenously on days 1 and 15 one hour before cilostazol administration to determine baseline and the inhibitory effect of erythromycin treatment on CYP3A activity. STUDY PARTICIPANTS: 16 healthy nonsmoking male volunteers. MAIN OUTCOME MEASURES: Serial blood and pooled urine samples were collected before and after cilostazol administration to quantitate cilostazol and its metabolites. Serial exhalation samples were collected after intravenous 14C-erythromycin administration and radioactivity was quantitated by scintillation counting. Pharmacokinetics were determined by noncompartmental methods and compared before and after erythromycin administration. Tolerability assessments included adverse events, laboratory tests, vital signs and electrocardiographs. RESULTS: Following erythromycin coadministration, cilostazol maximum plasma concentration (Cmax), area under the plasma concentration-time curve at time t (AUCt), and area under the curve from zero to infinity (AUC infinity) increased significantly by 47, 87, and 73%, respectively, and an approximately 50% reduction in unbound clearance was observed for the major circulating metabolite of cilostazol, OPC-13015. Cmax decreased significantly (p < 0.001) by 24%, while AUCt increased by 8%; this increase was not significant. For the second major metabolite, OPC-13213, the Cmax and AUCt increased by 29 and 141%, respectively (p < 0.001). CONCLUSIONS: In vivo results are in agreement with previous in vitro human microsome studies, indicating that cilostazol is metabolised to OPC-13015 via CYP3A. In addition, OPC-13213 concentrations increased after inhibition of CYP3A because of inhibition of sequential metabolism of OPC-13213 via CYP3A. A starting dose for cilostazol of 50 mg twice daily should be considered during coadministration of inhibitors of CYP3A.

Effect of multiple cilostazol doses on single dose lovastatin pharmacokinetics in healthy volunteers.

OBJECTIVE: To assess the effects of cilostazol on lovastatin pharmacokinetics. DESIGN: This was a single-centre, open-label, multiple dose, sequential treatment study. Participants received single oral doses of lovastatin 80 mg on days 1, 7 and 9, as well as oral cilostazol 100 mg twice daily on days 2 to 8, followed by a single oral 150 mg cilostazol dose on day 9. STUDY PARTICIPANTS: 15 healthy, nonsmoking male or female volunteers (aged 18 to 60 years) were enrolled, and 12 completed the study. MAIN OUTCOME MEASURES: Pharmacokinetic parameters were calculated using plasma concentrations of lovastatin and its beta-hydroxy metabolite and of cilostazol and its metabolites. Differences in the pharmacokinetics of each drug when given alone or in combination were assessed by analysis of variance. RESULTS: The maximum observed plasma concentration (Cmax) of lovastatin or its metabolite did not differ significantly when lovastatin was given alone and when it was given with 100 mg of cilostazol. The mean ratios of the area under the plasma concentration-time curve from zero to the time of the last measurable concentration (AUCt) for lovastatin coadministered with 100 mg of cilostazol to that with lovastatin given alone were 1.6 for lovastatin and 1.7 for its metabolite. With 150 mg of cilostazol, lovastatin Cmax did not change, whereas Cmax of the metabolite increased 2.2-fold. The mean AUCt ratios for lovastatin given with 150 mg cilostazol/lovastatin given alone were 1.6 and 2.0 for lovastatin and its metabolite, respectively. All increases in lovastatin and metabolite AUC were statistically significant, except for the 1.6-fold increase in lovastatin AUC with 150 mg of cilostazol. Maximum steady-state plasma drug concentration (Cssmax) and AUC during a dosage interval (AUC tau) for cilostazol 100 mg twice daily decreased 14 and 15%, respectively, upon lovastatin coadministration. CONCLUSIONS: Lovastatin and metabolite exposure is increased only by up to 2-fold when cilostazol is coadministered, which is considerably less than that observed for potent CYP3A inhibitors such as itraconazole and grapefruit juice. Absorption of cilostazol decreased approximately 15% when it was given with lovastatin. No dosage adjustments are necessary for cilostazol when coadministered with lovastatin, whereas lovastatin dose reductions may be needed when the 2 drugs are given together.

Effect of cilostazol on the pharmacokinetics and pharmacodynamics of warfarin.

OBJECTIVE: To evaluate the effect of cilostazol administration on warfarin pharmacokinetics and pharmacodynamics following a single 25 mg dose of warfarin. DESIGN: A randomised double-blind 2-period crossover with healthy volunteers receiving either 100 mg cilostazol twice daily for 13 days or matching placebo twice daily for 13 days, and the other treatment 21 days later. A single 25 mg dose of warfarin was given 14 days prior to the start of the study, and 7 days after the cilostazol and placebo treatments. STUDY PARTICIPANTS: 15 normal healthy male volunteers. OUTCOME MEASURES: Noncompartmental pharmacokinetic parameters for (R)- and (S)-warfarin, the area under the curve of the prothrombin time (AUCPT), activated partial thromboplastin time (AUCaPTT), Ivy bleeding times, unbound fraction (fu) of cilostazol, and warfarin were determined for each individual. RESULTS: For (R)- and (S)-warfarin, the 90% confidence intervals for the ratios of the geometric means (90% CI) of the maximum plasma concentration and area under the plasma concentration-time curve were between 0.88 to 1.03. The 90% CI for the AUCPT and AUCaPTT was between 0.95 and 1.06. For Ivy bleeding time, the 90% CI for the ratios of the geometric means ranged between 0.71 and 1.22. The fu of cilostazol did not differ significantly between the 2 treatments. There was a 17% increase in the fu of warfarin (p < 0.05), which was not clinically significant. CONCLUSIONS: Coadministration of warfarin with twice daily administration of cilostazol 100 mg did not alter (R)- and (S)-warfarin pharmacokinetics, prothrombin time, partial thromboplastin time, Ivy bleeding times, or cilostazol protein binding.

Interaction potential and tolerability of the coadministration of cilostazol and aspirin.

OBJECTIVE: This study evaluated the effects of repeated oral drug administration with cilostazol alone and with aspirin (acetylsalicylic acid) on platelet aggregation, coagulation and bleeding time as well as the cilostazol-aspirin pharmacokinetic interaction in healthy males. DESIGN: This was a randomised, double-blind, placebo-controlled, crossover study. Participants received either cilostazol 100 mg or placebo twice a day for 10 days; aspirin 325 mg/day was coadministered for the last 5 days. After a 14-day washout period, participants received the alternative treatment. STUDY PARTICIPANTS: 12 healthy male volunteers were enrolled. MAIN OUTCOME MEASURES: Differences in bleeding times, platelet aggregation, prothrombin time (PT) and activated partial thromboplastin time (aPTT) between cilostazol with aspirin and cilostazol alone. Noncompartmental pharmacokinetic parameters were determined for each study participant. RESULTS: Cilostazol, with or without aspirin, caused no changes in PT, aPTT or bleeding time. There was a 23 to 35% increase in inhibition of ADP-induced ex vivo platelet aggregation by cilostazol plus aspirin when compared with aspirin alone. There was no additive or synergistic effect on arachidonic acid-induced platelet aggregation. Statistically significant but clinically insignificant increases in the area under the plasma concentration-time curve to the last measurable plasma concentration and trough concentrations of cilostazol and its metabolites (OPC-13015 and OPC-13213) occurred after aspirin coadministration, with no differences observed in the maximum plasma concentration Drug-related adverse events were generally mild, the most frequent being headache. CONCLUSIONS: Cilostazol and aspirin coadministration did not cause clinically significant changes in PT, aPTT, bleeding time, platelet aggregation or plasma concentrations of cilostazol and its 2 active metabolites. Cilostazol was generally well tolerated with or without aspirin.

Effect of increasing gastric pH with famotidine on the absorption and oral pharmacokinetics of the inotropic agent vesnarinone.

The effect of famotidine, an H2 receptor blocker, on the oral absorption and pharmacokinetics of the novel agent vesnarinone was investigated after oral administration of 60 mg vesnarinone with and without pretreatment with intravenous famotidine. The single-blind, randomized, two-way crossover study was conducted in 12 volunteers, with a washout period of 7 days between the two treatments. A pH monitor was used to ensure that gastric pH of the subjects was < or = 3 in the absence of and > or = 5 in the presence of famotidine. A significant decrease in maximum concentration (Cmax) and increase in time to Cmax (tmax) was observed for vesnarinone during treatment with famotidine, whereas area under the concentration-time curve (AUC) was similar for both treatments. The physicochemical properties of the drug support the above observations. Therefore, therapies that increase gastric pH will affect the rate but not the extent of absorption of vesnarinone or the safety or efficacy profile of vesnarinone.

Pharmacokinetics of multiple-dose oral cilostazol in middle-age and elderly men and women.

Cilostazol is being developed for the treatment of intermittent claudication due to peripheral arterial disease (PAD). This study was conducted to investigate the effects of age and gender on the pharmacokinetics of cilostazol after multiple-dose administration. It was an open label, multiple-dose study of cilostazol administered to male and female subjects 50 years of age and older at a dose of 100 mg (oral tablet) twice daily for 7 days. Equal numbers of healthy male and female (7 per group), nonsmoking subjects stratified into age groups of 50 to 59 years, 60 to 69 years, and 70 years or older were enrolled. Serial plasma samples were obtained. Data were analyzed by model-independent methods. Cilostazol was absorbed at a moderate rate, with peak plasma concentrations occurring at an overall mean of 2.4 hours after administration. Cilostazol is extensively bound (95%), primarily to albumin. A trend toward increasing cilostazol free fraction with age was observed in the male subjects, which was explained by a decrease in plasma albumin concentration with age. Differences in plasma protein binding between age and gender groups (less than 15%) are not expected to have any clinical significance. Plasma cilostazol concentrations reached steady state by day 4. The pharmacokinetic characteristics of cilostazol were not affected by age or gender.

The quantitative determination of cilostazol and its four metabolites in human liver microsomal incubation mixtures by high-performance liquid chromatography.

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150 x 4.6 mm, 5 microm) column and a Cosmil C-18 column (150 x 4.6 mm, 5 microm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile in acetate buffer (pH = 6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43-17.59. The components of liver microsomal incubation mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml(-1) for cilostazol and 100 ng ml(-1) for each of the metabolites. This method has been validated for linear ranges of 100-4000 ng ml(-1) for OPC-13213, OPC-13217 and OPC-13326; 100-2000 ng ml(-1) for OPC-13015; and 1000-20000 ng ml(-1) for cilostazol. The percent relative recovery of this method was established to be 81.2-101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8-7.7%. The autosampler stability of the analytes was evaluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, accurate and reproducible.