| ObjectiveTo preliminary explore the mechanism of pravastatin and enalapril oral co-administration induced rhabdomyolysis.Methods1. To analysis a clinical case of pravastatin and enalapril oral co-administration induced rhabdomyolysis.2. To develop a method for simultaneous quantification of pravastatin, enalapril, enalaprilat in rat plasma by HPLC-MS/MS. Take 30 μL rat plasma sample, and the protein in plasma was precipitated with methanol. The supernatant was separated by elution of Agilent HC-C18 (4.6x250 mm,5 μm) column. The mobile phase was methanol-0.1% formic acid aqueous solution (75:25, V:V). The flow rate is 1 mL·min-1. An Agelent 6460 triple quadrupole mass spectrometer, equipped with an electrospray ionization interface was operated in the multiple reaction monitoring (MRM) and positive ion mode. The ion pair of quantitative analysis were:m/z 447.0â†'326.8 (Pravastatin sodium), m/z 377.2â†'234.2 (Enalapril), m/z 349.2â†'206.2 (Enalaprilat), m/z 285.0â†'192.9 (Diazepam, internal standard).3. Pharmacokinetic Experiments:A total of 18 male rats were randomly divided into 3 groups (each group contain 6 rats), they are Pravastatin (PV) group (pravastatin sodium,20 mg-kg-1, i.g.), Enalapril (EN) group (enalapril maleate,30 mg-kg-1, i.g.), and Pravastatin plus Enalapril (PV+EN) group (pravastatin sodium,20 mg-kg-1; enalapril maleate,30 mg-kg-1, i.g.). After a single drug treatment, blood samples were collected at different time points for the determination of pravastatin enalapril, and enalaprilat concentration by LC-MS/MS. DAS 2.0 software was used to calculate the pharmacokinetic parameters of pravastatin, enalapril, and enalaprilat.4. Tissue Distribution Experiment:A total of 24 male rats were randomly divided into 2 groups (each group contain 12 rats), they are Pravastatin (PV) group (pravastatin sodium,20 mg-kg-1, i.g.), Pravastatin plus Enalapril group (PV+EN, pravastatin sodium,20 mg-kg-1, i.g.; enalapril maleate,30 mg·kg-1, i.g.). At7 and 14 days after drugs treatment, blood samples, liver, kidney, and muscle were collected at 2 h post pravastatin and/or enalapril administration for the determination of pravastatin concentration by LC-MS/MS. The data was used to calculate the parameter Kp and evaluate the effect of enalapril on uptake of pravastatin in liver, kidney, and muscle tissue.5. Western Blotting Experiment:A total of 36 male rats were randomly divided into 3 groups (each group contain 12 rats), they are set as Control group (saline,3 mL, ig.), Pravastatin (PV) group (pravastatin sodium,20 mg·kg-1, i.g.), and Enalapril (EN) group (enalapril maleate,30 mg·kg-1, i.g.). At 7 and 14 days after drugs treatment, liver tissue were collected in order to quantitatively analyse the expression of Organic anion transporting polypeptide 1a4 (Oatp1a4) and Multidrug resistance associated protein 2 (Mrp2) by Western blotting technique.Result1. Co-administration of pravastatin and enalapril may induce rhabdomyolysis in patients.2. It was feasible that simultaneous quantification of pravastatin, enalapril, and enalaprilat in rat plasma by HPLC-MS/MS. The linear range of pravastatin was 1.47～1500 ng·kg-1in rats plasma; and the limit of quantitation was 1.47 ng·mL-1; the intra-day and inter-day precision values (RSD) were not above 8%; and the accuracy (RE) was between -2.37% and 4.16%. The linear range of enalapril was 11.72-12000 ng·mL-1 in rats plasma; and the limit of quantitation was 2.93 ng·mL-1; the intra-day and inter-day precision values (RSD) were not above 6%; and the accuracy (RE) was between-1.62% and 1.56%. The linear range of enalaprilat was 11.72-12000 ng·mL-1 in rats plasma; and the limit of quantitation was 3.91 ng·mL-1; the intra-day and inter-day precision values (RSD) were not above 6%; and the accuracy (RE) was between -0.94% and 4.71%. The stability (RSD) of pravastatin, enalapril, and enalaprilat were not above 5%; and all of the extraction recovery were above 90%. It was unaffected by matrix effects.3. Pharmacokinetic Experiments:The pharmacokinetic parameters of pravastatin had significantly changed in the PV+EN group compared to that of the PV group. For example, Gnax was increased by 15.28%, AUCo-36h was increased by 32.11%, AUCo-∞ was increased by 32.40%, MRT0-36hwas increased by 13.41%, MRTo-∞was increased by 13.59%(P<0.05). CL was decreased by 24.98%, and V was decreased by 18.72%(P<0.05). Compared to that of the EN group, Tmax of enalapri had significantly delayed by 21.52% in the PV+EN group, and the other pharmacokinetic parameters of enalapri did not show significant alteration (p>0.05). Compared to that of the EN group, the pharmacokinetic parameters of enalaprilat did not show significant alteration in the PV+EN group (p>0.05).4. Tissue Distribution Experiment:At 7 days after drugs treatment, compared to that of the PV group, the plasma concentration of pravastatin at 2 h was significantly increased by 30.34%in PV+EN group (p<0.05); the liver concentration of pravastatin at 2 h was significantly decreased by 21.91% in PV+EN group (p<0.05); the concentration of pravastatin in kidney and muscle tissue at 2 h did not show significant alteration in PV+EN group (p>0.05); Kp value of pravastatin at 2 h in liver tissue was decreased by 33.59% in PV+EN group (p<0.05) while the Kp value of pravastatin at 2 h in kidney and muscle tissue did not show significant alteration in PV+EN group (p>0.05).At 14 days after drugs treatment, compared to that of the PV group, the plasma concentration of pravastatin at 2 h was significantly increased bu 25.50% in PV+EN group (P<0.05); while the liver concentration of pravastatin at 2 h was significantly decreased by 35.82% in PV+EN group (p<0.05); the kidney concentration of pravastatin at 2 h did not show significant alteration in PV+EN group (p>0.05); the muscle concentration of pravastatin at 2 h was significantly increased by 76.41% in PV+EN group (p<0.05). the Kp value of pravastatin at 2 h in liver was decreased by 52.18% in PV+EN group (p<0.05); the Kp value of pravastatin in kidney and muscle tissue at 2 h did not show significant alteration in PV+EN group (p>0.05).5. Western Blotting Experiment:A 7 and 14 days after drugs treatment, compared to that of the Control group, the expression of transporters Oatpla4 and Mrp2 in liver did not show significant alteration in PV group (P>0.05). At 7 and 14 days after drugs treatment, compared to that of the Control group, the expression of transporters Mrp2 in liver also did not she w significant alteration in EN group (P>0.05). At 7 days after drugs treatment, compared to that of the Control group, the expression of transporter Oatpla4 in liver tissue had a trend of reduction in EN group, but did not show statistical significance (p>0.05). At 14 days after drugs treatment, compared to that of the Control group, the expression of transporter Oatpla4 in liver was significantly decreased by 34.10% in EN group (p<0.05).Conclusion1. The HPLC-MS/MS method is sensitive and specific for determination, and had a simple operation. It is suitable for the pharmacokinetic study of pravastatin, enalapril and enalaprilat in rat plasma.2. Pravastatin and enalapril may have drug-drug interaction in rats. The co-administration may increase the plasma concentrations of pravastatin, and decrease the clearance as well as theapparent volume of distribution.3. Oral administration of enalaprilat for several times can decrease the protein expression of Oatpla4 in liver, reduce the uptake of pravastatin in liver, and thus increase the pravastatin concentration in plasma and muscle tissue, which may be one of the causes of pravastatin and enalapril co-administration induced rhabdomyolysis. |
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