Comparative Study On Pharmacokinetics Of Cephradine In Diabetic And Normal Rats

Objective: To study effects of diabetes mellitus on pharmacokinetics of cephradine (CED) by comparing the difference in pharmacokinetics (PK) behaviour of CED between diabetic and normal rats in order to provide experiment basis for clinical rational usages of CED in patients with diabetes mellitus.Methods: Diabetes was induced by a single injection of freshly prepared Alloxan solution, 60 mg/kg body weight through the tail vein. Rats whose blood glucose was over 16mmol/l were taken as diabetes mellitus group and the consumption of food and water, as well as body weight, urine output of rats were measured at same time. Experiment rats were divided into two large groups: normal control (CTL) large group and diabetes mellitus (DM) large group. Each of large groups was subdivided into 2 groups, based on the different routes of administration, namely, intravenous (iv) dosing group and intragastric (ig) dosing group, each group including high dose (180mg/kg) and low dose (90mg/kg) small groups. In iv group, blood samples (0.4ml) were collected from angular vein at time zero and 5,15,30,50,80,120,180,240,360,450 and 600 min with heparinized Eppendorff tubes. In ig group, blood samples (0.4ml) were collected similarly at time zero and 15,30,45,60,75,105,150,240,330,450 and 600 min and subsequently centrifuged at 3000rpm for 15 min (4℃) to yield plasma samples. CED concentration in plasma was determined by a reversed phase HPLC. Chromatographic separation was achived on a Kromasil C₁₈ column (250×4.6mm ID, 5μm) equipped with a Kromasil C₁₈ pre-column (20×4.6mm ID, 10μm), using a mobile phase composed of 0.025mol/L KH₂PO₄-MeOH-CH₃CN (87:6:7 v/v) running at a flow rate of 1.0mL/min. The UV detector was set at 261nm. 200μL of plasma sample was added with internal standard cephalexin (CEX) 10μL(400μg/mL), 10μL water and 80μL of 10% Trichloracetic acid (TCA) solution and vortexed for 0.5min, and then centrifuged at 12000rpm for 10 min (4℃). The resultant 50μL supernatant was injected into the column. The validation of the method including specificity, precision, recovery, linearity and detection limit was conducted by routine procedures. The stability test included the stability of CED plasma samples kept frozen at–20℃and at room temperature, the stability of CED standard stock solution and CEX stock solution stored frozen at–20℃, the stability of the TCA-deproteinized plasma samples at 2-4℃. At the end of the experiment, the kidneys were excised and weighed as wet weight (KW) for calculation of ratio of kidney weight to body weight (KW/BW).Results: The calibration curve of CED had a good linearity over the range from 1μg/mL to 100μg/mL (r>0.9999). The quantification limit was 1μg/mL. The method developed in present paper showed a good precision of RSD<9.40 %, a high method recovery of 96.00%～100.33% and a high extraction recovery of 83.98%～87.88%. It had been demonstrated that CED plasma samples kept frozen at–20℃and at room temperature were stable for at least 1 month and 8h, respectively. CED standard stock solution and CEX stock solution stored frozen at–20℃was also stable for at least 1 month, the TCA-deproteinized plasma samples at 2-4℃were stable for at least 8h. The PK behaviour of CED in plasma after iv administration could be described by two-compartment model and first-order kinetics. Compared with the CTL groups, t_1/2β and MRT were decreased remarkably and CL_t was increased significantly in DM groups (p<0.05). These results indicate that the elimination of CED in DM groups was faster than that in the CTL groups. After oral administration, the PK behaviour of CED in plasma could be described by one-compartment model and first-order kinetics. Comparing with CTL groups, a significant decrease in t_1/2k and t_max and a remarkable increase in CL_t and C_max were observed for DM groups (p<0.05). These results demonstrated that the absorption and elimination of CED in DM groups were faster than those in the CTL groups. There was a decreased trend of t_1/2ka in DM rats, in spite of p>0.05 vs CTL rats. No significant difference in the bioavailability (F) between the two groups was observed (p>0.05). KW and KW/BW in DM groups were increased remarkably compared with in CTL groups (p<0.05).Conclusion: The method developed by us is validated to fully meet requirements for CED PK study in rats. Diabetic rats showed a decreased t_1/2β and MRT and an increased CL_t when CED was iv administered, and a decreased t_max, increased C_max and almost identical F when CED was orally administered in relation to CTL group. These results indicate that in DM pathological status, CED eliminates from body more quickly than in normal status and intestinal absorption of CED in DM groups was faster than that in the CTL groups. The KW and KW/BW were increased remarkably, suggesting that kidney become compensatory hypertrophy and hyperfunction in early-stage diabetes. This might constitute one of causes of quick elimination of CED in diabetic rats.