Bioequivalence Study With Cephradine Capsules In Volunteers

Objective: To study pharmacokinetics and relative bioavailability in humans of test and reference cephradine capsules and evaluate the equivalence of the two preparations.Methods and Results: Cephradine in plasma was chromatographed with a reversed phase Hypersil BDS C18 column (200×4.6mm ID, 5μm) equipped with a BDS C18 pre-column (20×4.6mm ID, 10μm), and a mobile phase composed of 0.025mol/L KH2PO4: MeOH: CH3CN (87:5:8 v/v) running at a flow rate of 1.2ml/min. Detection wavelength was set at 261nm. It had been demonstrated that cephradine plasma samples kept frozen at–20℃were stable for at least 1 month and at room temperature for at least 8h.Cephradine standard stock solution stored frozen at–20℃was also stable for at least 1 month, the TCA-deproteinized plasma samples at 2-4℃for at least 8h. 200μL of plasma sample was added with 100μL of 10% TCA solution and vortexed for 5min, and then centrifuged (12000r/min×10min). The resultant 50μL supernatant was injected onto the column.18 male healthy volunteers were given orally a single dose 500 mg of test (T) and reference (R) cephradine capsules in an open randomized two-preparation double–period cross-over design experiment. Pharmaco- kinetic parameters were obtained from the plasma concentration-time data. AUC0-5h was 1496.68±272.38μg/mL?min and 1421.75±268.09μg/mL?min; Cmax 14.41±3.46μg/mL and 14.39±3.62μg/mL; Tmax 59.17±15.84min and 63.33±20.92min;t1/2 56.69±9.68min and 54.21±7.53min, respectively. The relative bioavailability of T to R was 106.3±15.8%. The analysis of variance of lnAUC and lnCmax indicated no significant difference between the two preparations and between the two periods, but showed significant difference between individuals (P<0.05). The two-way one-side t-test and 1-2αconfident interval test of lnAUC and lnCmax displayed that tL and tH were all greater than 1.746 (critical value), 90% confident intervals were within 80～125% for AUC and 70～143% for Cmax, indicating that the two preparations are of bioequivalence. Non-parameter test for tmax by wilcoxon method exhibited U0.05 and U0.01 were 1.96 and 2.58, respectively, the values larger than Ucalculation of 1.20, implying no significant difference in tmax between T and R.Conclusions: The cephradine T and R capsules are of equivalence.Objective: To develop a HPLC method for determination of cephradine (CED) in plasma and urine of rabbits and thereby to study its pharmacokinetics (PK) and preliminarily investigate urinary excretion mechanism of CED in rabbits.Methods:CED in plasma and urine was chromatographed on a reversed phase Kromasil C18 column (250×4.6mm ID, 5μm) equipped with a BDS C18 pre-column (20×4.6mm ID, 10μm), using a mobile phase composed of 0.025mol/L KH2PO4: MeOH: CH3CN (87:5:8 v/v) running at a flow rate of 1.2mL/min. Detection wavelength was set at 261nm. The validation of the method including linearity, precision and recovery was conducted at high, middle and low concentrations by routine procedures (n=5) as described in guiding principles of PK study issued by SFDA. Peak area ratio of CED to CEX was plotted against CED concentration to construct calibration curve.The stability test was carried out by determine concentration of standard plasma sample of CED at different time post-preparation.Fifteen rabbits were randomly divided into 3 groups(each group containing 5 animals) receiving iv administration of CED 100,50 and 25 mg/kg, respectively. 200μL of plasma or urine sample was added with 10μL cefalexin(CEX) ( 400μg/mL ) , 10μL water and 80μL of 10% TCA(Trichloracetic acid) solution and vortexed for 0.5min, and then centrifuged (12000r/min×10min). The resultant 50μL supernatant was injected onto the column. Creatinine concentration in plasma and urine were assayed with reference to Jaffe's picrate method and used to calculated CLCR and thereby estimate GFR.Results:The calibration curve of CED had a good linearity over therange from 2μg/mL to 100μg/mL (plasma) and 1μg/mL to 100μg/mL (urine) (r>0.9999). It showed a good precision of RSD<9.53 % as well as a method recovery of 90.12%～91.31% (plasma) and 95.95%～99.54% (urine) and a resume recovery of 79.40%～88.01% (plasma) and 78.22%～90.89% (urine). The PK behaviour of CED in plasma and urine after iv administration could be described by two-compartment model. The main pharmacokinetic parameters were t1/2β88.01～91.83min, MRT 57.49～83.84min, CLt 10.22～12.89ml/min/kg (plasma pK study) and t1/2β76.93～79.56min, MRT 61.41～74.01min, CLR 5.20～6.66mL/min/kg (urinary pK study), as well as CLCR 2.95～3.30mL/min/kg.Conclusion:The method developed by us is validated to fully meet requirements for CED PK study in rabbits. The t1/2,MRT determined using urinary data are in good agreement with those using plasma data, and all indicate a rapid elimination of CED in rabbits. Both plasma and urine data demonstrate that CED follows linear pharmacokinetics. It is estimated that approximately 50.9～51.8% of CED dosed is cleared through renal routes. Since CLCR is smaller than CLR, it is reasoned that CED excretes into urine by both renal glomerular filtration and glomerular tubule secretion.