Pharmacokinetic Profiles Of Doxazosin Enantiomers In Rats And Rat Liver Microsomes

Doxazosin, a quinazoline compound, is a long-acting ai-adrenoceptor ntagonist. Doxazosin is a safe and effective treatment option for lower rinary tract symptoms (LUTS) in patients with benign prostatic hyperplasia BPH), and as an add-on drug in combination therapy to achieve target blood ressures. Doxazosin contains a chiral carbon at the 2-position of the 1,-benzodioxan-2-ylcarbonyl ring, consisting of two optical isomers-)doxazosin and (-)doxazosin, and it is used clinically as a racemate. The-)doxazosin and (-)doxazosin do not have the same biological activity.-)Doxazosin was shown to decrease the carotid blood pressure more weakly han (+)doxazosin in anesthetized rats. In addition, it was reported that the lasma concentration of (+)doxazosin for all the time points tested was higher han that of (-)doxazosin, and the area under curve (AUC) of (+)doxazosin vas 2.3 times higher than that of (-)doxazosin after oral administration of herapeutic doses of racemic doxazosin in 2 healthy volunteers.Pharmacokinetic interaction between the two enantiomers might occur vhen a chiral drug is administered in racemic form, resulting in alterations of he pharmacological action of the drug. Although some reports concerning the harmacokinetics of doxazosin have been published, to our knowledge, no )ther study has reported on the stereoselective pharmacokinetic behavior of-)doxazosin and (+)doxazosin after administration of single enantiomers as veil as the pharmacokinetic interaction between doxazosin enantiomers after dministration of rac-doxazosin. Therefore, we used chiral high-pressure ipuid chromatography (HPLC) to investigate the pharmacokinetic behavior of ioxazosin enantiomers after oral or intravenous (IV) administration of 6.0 g/kg rac-doxazosin,3.0 mg/kg (-)doxazosin, and 3.0 mg/kg (+)doxazosin to Sprague Dawley (SD) rats to clarify how one enantiomer competes with the other with respect to the pharmacokinetic profile after the oral administration of rac-doxazosin. Furthermore, we used rat liver microsomes and wanted to know the enantioselectivity of (-)doxazosin and (+)doxazosin in metabolism, the mainly involved P450 enzymes in the metabolism and the interactions between (-)doxazosin and (+)doxazosin.Part I Enantio selective pharmacokinetics of doxazosin and its enantiomers after oral administration in ratsAim:The purpose of this study was to evaluate the enantioselective pharmacokinetics of doxazosin and its enantiomers after oral administration in rats.Methods:We investigated the pharmacokinetic behavior of doxazosin enantiomers after oral administration of 6.0 mg/kg rac-doxazosin,3.0 mg/kg (-)doxazosin, and 3.0 mg/kg (+)doxazosin to rats using HPLC, and the pharmacokinetic parameters of (-)doxazosin and (+)doxazosin after oral administration of each enantiomer and the racemate were determined by a non-compartmental analysis model for the extravascular input using WinNonlin software, version 5.1 (Pharsight Corp., Mountain View, CA).Results:1 Method validationThe chiral HPLC methods for the determination of doxazosin enantiomers were very selective. Prazosin, (-)doxazosin and (+)doxazosin were clearly well resolved from the matrix components under the chromatographic conditions employed, and the two enantiomers of doxazosin were baseline resolved from each other (Fig.1). For determining the plasma concentration, linear regression equations were calculated using the weighted least-squares linear regression method with a weighting factor 1/x in the range 4-2000 ng/mL. The regression equations for (-)doxazosin and (+)doxazosin were:Y=0.7986C+0.0118 (r=0.9991) and Y=0.8025C+0.0046 (r=0.9994), respectively. The limit of quantity for (-)doxazosin or (+)doxazosin was 4 ng/ml. The average recovery for (-)doxazosin was 99.5% with RSD 3.6%, and for (+)doxazosin was 99.3% with RSD 4.3%. This optimized method was used to evaluate plasma samples after doxazosin administration.2 Plasma concentrations of doxazosin enantiomers after oral administration of rac-doxazosinWhen rats were given 6.0 mg/kg rac-doxazosin, the plasma concentration versus time profiles of doxazosin exhibited a double-peak phenomenon for both (-)doxazosin and (+)doxazosin, and the plasma concentration of (+)doxazosin was higher than that of (-)doxazosin at all sampling time points in all rats.3 Plasma concentrations of doxazosin enantiomers after oral administration of (-)doxazosin or (+)doxazosin aloneAfter oral administration of individual enantiomers, no chiral inversion of (-)doxazosin to (+)doxazosin or vice versa was observed in rat plasma. The disappearance of double-peak was observed for both (-)doxazosin and (+)doxazosin after oral administration of (-)doxazosin or (+)doxazosin alone, and the plasma concentration of (+)doxazosin was higher than that of (-)doxazosin at all sampling time points in all rats.4 Pharmacokinetic parameters of doxazosin enantiomers after oral administration of rac-doxazosinThe average Cmax value of (+)doxazosin in plasma was significantly higher than that of (-)doxazosin (P<0.01), and the average AUC value for (+)doxazosin was 7.3 times higher than that of (-)doxazosin (P<0.01). We also found that both the Vd/F and CL/F value for (+)doxazosin were lower than that for (-)doxazosin (P<0.01).5 Pharmacokinetic parameters of doxazosin enantiomers after oral administration of of (-)doxazosin or (+)doxazosin aloneAdministration of (-)doxazosin alone significantly increased the Cmax value of (-)doxazosin as compared with administration of rac-doxazosin, with a 108% increase from 53.2 ng/mL of the control to 110.5 ng/mL (P<0.05). Administration of (+)doxazosin alone significantly decreased the λ value of (+)doxazosin as compared with administration of the rac-doxazosin, with a iecrease of 32%(P<0.05).Part II Enantioselective pharmacokinetics of doxazosin and its enantiomers after IV administration in ratsAim:The purpose of this study was to evaluate the enantioselective harmacokinetics of doxazosin and its enantiomers after IV administration in ats.Methods:The pharmacokinetic behavior of doxazosin enantiomers after V administration of 3.0 mg/kg (-)doxazosin,3.0 mg/kg (+)doxazosin, and 6.0 ag/kg rac-doxazosin to rats was investigated by HPLC, and the harmacokinetic parameters of (-)doxazosin and (+)doxazosin after IV dministration of each enantiomer and the racemate were determined by the wo-compartmental model and non-compartmental analysis model for V-bolus input using WinNonlin software, version 5.1 (Pharsight Corp., Mountain View, CA).Results:1 Plasma concentration of doxazosin enantiomers after IV administration )f rac-doxazosinAfter IV administration of 6.0 mg/kg rac-doxazosin, the plasma oncentration of (+)doxazosin was higher than that of (-)doxazosin at all sampling time points in all rats.2 Plasma concentration of doxazosin enantiomers after IV administration )f (-)doxazosin or (+)doxazosin aloneAfter IV administration of individual enantiomer, no chiral inversion of-)doxazosin to (+)doxazosin or vice versa was observed in the rat plasma. The )lasma concentration of (-)doxazosin administered alone was not significantly lifferent from that of (-)doxazosin after administration of the racemate, and he plasma concentration of (+)doxazosin administered alone was significantly ower than that of (-)doxazosin after administration of the racemate at 5,10,15 and 30 min (P<0.01 and P<0.05).3 Pharmacokinetic parameters of doxazosin enantiomers after IV dministration of rac-doxazosinThe Cmax value of (+)doxazosin in plasma was significantly higher than that of (-)doxazosin (P<0.01), and the AUC value for (+)doxazosin was 3.2 times higher than that of (-)doxazosin (P<0.01). We also found that Vss, K10, and Cl values for (+)doxazosin were significantly lower than those for (-)doxazosin(P<0.01).4 Pharmacokinetic parameters of doxazosin enantiomers after IV administration of of (-)doxazosin or (+)doxazosin aloneAlthough the AUC, Cmax, and Cl values of (-)doxazosin administered alone were not significantly different from those of (-)doxazosin after administration of the racemate, the Vss values of (-)doxazosin decreased by 31% in the absence of (+)doxazosin (P<0.05). The AUC and Cmax values for (+)doxazosin were reduced by 14% and 17%, respectively, and the Cl value of (+)doxazosin increased by 17% in the absence of (-)doxazosin (P<0.01 and P<0.05) as compared with the administration of the racemate.The values for the pharmacokinetic parameters obtained using the noncompartmental approach were similar to the corresponding parameters calculated by the two-compartmental model.Part Ⅲ Enantioselective metabolism of doxazosin enatntiomers and the metabolic interaction between the isomers in rat liver microsomesAim:The purpose of this study was to evaluate the enantioselective metabolism of doxazosin enatntiomers and the metabolic interaction between the isomers in rat liver microsomes.Methods:We used rat liver microsomes and want to know the enantioselectivity of (-)doxazosin and (+)doxazosin in metabolism, the mainly involved P450 enzymes in the metabolism and the interactions between (-)doxazosin and (+)doxazosin. The depletion rate constant (kdep) was determined from substrate-depletion experiments to evaluate the difference between enantiomers in microsomal incubation experiments.Results:1 Method validationThe chiral HPLC methods for the determine of doxazosin enantiomers were very selective. Prazosin, (-)doxazosin and (+)doxazosin were clearly well resolved from the matrix components under the chromatographic conditions employed, and the two enantiomers of doxazosin were baseline-resolved from one other. For achiral HPLC methods, the selectivity assay showed no interfering peak from the bio-logical matrix in the retention time of the doxazosin or the internal standard. Sulfamethaxazole and a-naphthofoavone was eluted at 2.5 min and 5.1 min, respectively, but they were too small to observe.4-Methylpyrazole was not detected in this detection conditions.For determining the microsomes concentration, linear regression equations were calculated using the weighted least-squares linear regression method with a weighting factor 1/X2 in the range of 0.25-12.50 μg/mL. The precision and accuracy of the methods were assessed for both within-day and between-day determination, and relative standard deviations or relative errors did not exceed a value of 10%.2 Depletion study for doxazosin and its enantiomers in rat liver microsomesThe depletion profiles of each isomers after added (±)doxazosin (3.0 μg/mL) in rat liver microsomes were similar to the plasma concentration-time profiles of (-)doxazosin and (+)doxazosin after IV bolus injection of (±) doxazosin, in that (-)doxazosin depleted faster than (+)doxazosin. The kdep for (-)doxazosin (0.0107±0.0007 L/min) was higher than that for (+)doxazosin (0.0088±0.0005 L/min, P<0.05). However, using the (-)doxazosin or the (+)doxazosin as substrates separately (final concentration at 3.0 μg/mL for each enantiomer), (+)doxazosin depleted faster than (-)doxazosin. The value of kdep for (-)doxazosin (0.0097±0.0007 L/min) lower than that for (+)doxazosin (0.0113±0.0003 L/min, P<0.05).3 Chemical inhibition studies in rat liver microsomesAmong five CYP inhibitors examined, ketoconazole, the inhibitor of rat CYP3A1/2 enzymes, exhibited the greatest inhibitory effect on the depletion of (-)doxazosin and (+)doxazosin in rat liver microsomes. The inhibitory potential of ketoconazole was increased with its concentration changed from 0.5 μmol/L to 4.0 μmol/L; and ketoconazole (8.0 μmol/L) suppressed the activity almost completely (100% of the control). When the concentration of ketoconazole was at 4 μmol/L and 8 μmol/L, the depletion rate constant of (-)doxazosin and (+)doxazosin was significantly lower than that of control, respectively (P<0.01). After treatment of 4-methylpyrazole, the depletion level of (-)doxazosin was markedly decreased. When the inhibitory concentration of 4-methylpyrazole increased from 0 to 10 μmol/L, the kdep for (-)doxazosin decreased from 0.00548±0.00013 to 0.00453±0.00012, and significantly lower than that of control (P<0.01). However, the depletion of (+)doxazosin was not affected by 4-methylpyrazole. The kdep of (+)doxazosin incubated in the presence or absence of 4-methylpyrazole was not significantly different (P>0.05). At their working concentration ranges, a-naphthofoavone, quinidine and sulfamethaxazole did not inhibit (-)doxazosin or (+)doxazosin.4 Enantiomer-enantiomer interaction in rat liver microsomesThere was no significant difference in depletion rate constants between a mixture contained 3.0 μg/mL (-)doxazosin and a mixture contained 3.0 ug/mL (+)doxazosin (P>0.05). The use of a mixture that contained the same concentration of these two isomers (1.5 μg/mL (-)doxazosin:1.5 μg/mL (+)doxazosin) showed that the depletion rate constant of (+)doxazosin was lower than that of (-)doxazosin, which was consistent with the results from (±)doxazosin experiments. Moreover, the depletion rate constants of (+)doxazosin in the 1.0 ug/mL (+)doxazosin:2.0 μg/mL (-)doxazosin and 0.5 μg/mL (+)doxazosin:2.5 μg/mL (-)doxazosin were significantly lower than those of (-)doxazosin in the1.0 μg/mL (-)doxazosin:2.0 μg/mL (+)doxazosin and 0.5 μg/mL (-)doxazosin:2.5 μg/mL (+)doxazosin (P<0.05).Conclusion:Doxazosin pharmacokinetics is substantially stereospecific in rats. AUC and Cmax values for (+)doxazosin after oral or intravenous administration are significantly higher than that of (-)doxazosin, its Cl value is significantly lower. Enantiomer-enantiomer interaction in pharmacokinetics occurs after rac-administration. (+)Doxazosin does not affect the metabolism and excretion of (-)doxazosin, but the presence of (-)doxazosin further increases the plasma concentration of (+)doxazosin by inhibiting the metabolism and excretion of (+)doxazosin. Moreover, (-)doxazosin and (+)doxazosin may competitively affect absorption from the gastrointestinal tract.Doxazosin hepatic metabolism is substantially stereoselective. After incubating rat liver microsomes with (±)doxazosin, (-)doxazosin was depleted faster than (+)doxazosin. However, (-)doxazosin is not depleted faster than (+)doxazosin when a single isomer of doxazosin is incubated with rat liver microsomes. The metabolism of (+)doxazosin is catalysed by CYP3A alone whereas that of (-)doxazosin requires both CYP3A and CYP2E. (-)Doxazosin is depleted much faster than (+)doxazosin when a mixture of the two isomers is used, suggesting a prominent and stereoselective inhibition of the (-)-isomer as compared with the (+)-isomer at the CYP3A enzyme.