| Objective:To investigate the effect of diazepam?DI?on the pharmacodynamic and pharmacokinetic of tramadol?TR?in mice,which provides the theoretical foundation for the joint clinical application of the two drugs.Methods:1.Pharmacodynamic study?1?The analgesic effects of tramadol and diazepam alone or in combination on the acetic acid-induced writhing test in mice.Healthy female ICR mice,8 weeks old,weighing 20±2 g,were used.The animal was fasted overnight?12 h?with free access to water before the experiments.And they were divided into 6 groups,including normal saline group?NS,n=10?,TR-H group?25 mg/kg,n=10?,TR-L group?12.5 mg/kg,n=10?,DI group?0.5 mg/kg,n=10?,TR-H+DI group?25 mg/kg+0.5 mg/kg,n=10?,and TR-L+DI group?12.5 mg/kg+0.5 mg/kg,n=10?.Sixty minutes after the intraperitoneally injection of the drugs?alone or combined?,the animals received 0.1mL/10g i.p.of a 0.6%acetic acid solution.And the number of writhes was counted within 15 minutes after the administration of the acetic acid solution?A writhe standard was definded as contraction of the abdominal muscles accompanied by elongating the body and extension of the hind limbs??2?The analgesic effects of tramadol and diazepam alone or in combination on the hot-plate test in mice.Healthy female mice?20±2 g?,8 weeks old,were placed on a hot plate preheated to 55±0.5?.The latency was defined as the period from the time when the animal was placed on the surface of the hot plate to the time when the animal licked its back paws or jumped off avoid thermal pain.After repeated tests,mice with latency of 10-30 s were selected for formal experiments.The mice were divided into 4groups,including normal saline group?NS,n=10?,TR group?25 mg/kg,n=10?,DI group?0.5 mg/kg,n=10?,and TR+DI group?25 mg/kg+0.5 mg/kg,n=10?.Back-paw licking latency was measured after 10,15,30,and 60 min after administrstion of the test drugs.If the lantency exceeds 60s,stop the test and record it for 60 s to avoid thermal pain.2.Pharmacokinetic study?3?Dialysis sample collection:mice were divided into TR+DI?25 mg/kg+0.5mg/kg?group and TR?25 mg/kg?group,with 6 mice in each group.The mice were anaesthetized and secured in a stereotaxic frame,then a microdialysis probe was implanted into the frontal cortex?FrCx?and perfused with normal saline?NS?at a flow rate of 2?L/min for dialysate sample collecting.After 1 h of preconditioning,the drug was administered,and then the dialysate?about 20?L?was collected continuously at10-min intervals for 5 h.?4?Plasma sample collection:mice were divided into TR+DI?25 mg/kg+0.5mg/kg?group and TR?25 mg/kg?group,with 54 mice in each group.After anesthetized with urethane?1.8 g/kg,i.p.?for 1h,the drug was administered.And 500?l blood was collected through removal of eyeballs at 0.033,0.08,0.17,0.33,0.5,1,2 and 4 h,respectively after administration.The HPLC-FLD method was established to determine the concentration of tramadol in mice dialysate samples and plasma samples.The DAS3.1.5 software was used to caculate the drug concentration-time profile and pharmacokinetic parameters.SPSS13.0 was used to compared the pharmacokinetic parameters of the two groups.Results:1.Pharmacodynamic study?1?The acetic acid-induced writhing test:Administration of tramadol?25 mg/kg/12.5 mg/kg?produced a significant antinociceptive effect and compared to the control group animals,the number of writhes was significant reduction.Although the number of writhes in mice after administration of diazepam?0.5 mg/kg?was reduced,there was no significant difference.The number of writhes of the TR-H+DI group?25 mg/kg+0.5 mg/kg?reduced significantly compare to the TR-H group?25 mg/kg TR??P<0.01?.And there was no significant difference in the number of writhes between the TR-L+DI group?12.5 mg/kg+0.5 mg/kg?and the TR-H group?25 mg/kg??P>0.05?.?2?The hot-plat test:Diazepam?0.5 mg/kg,i.p.?did not show any antinociceptive activity after administration?P>0.05?.The TR+DI group?25 mg/kg+0.5 mg/kg?produced a significant antinociceptive effect and the back-paw licking latency in 15,30,and 60min after administration of combination was longer than that obtained from the TR group?25 mg/kg?.Compared with the control group,there was no significant difference of the latency of the TR group?25 mg/kg?at 60 min after administration?P>0.05?,while the TR+DI group?25 mg/kg+0.5 mg/kg?had significant difference?P<0.05?.2.Pharmacokinetic study?3?The main tramadol pharmacokinetic parameters of TR+DI group and TR group in FrCx were obtained:Cmax was?940.22±92.63??g/L and?726.72±185.50??g/L,Tmax was?0.25±0.00?h and?0.42±0.00?h,t1/2/2 was?1.07±0.60?h and?1.39±0.56?h,AUC?0-??</sub>was?1199.04±266.99??g/L*h and?1026.76±135.22??g/L*h.Compared with TR group,the Cmax of tramadol in the FrCx extracellular fluid of mice in TR+DI group was increased?P<0.05?,while Tmax was shorten?P<0.05?,and there was no significant difference in other pharmacokinetics parameters?p>0.05?.?4?The main tramadol pharmacokinetic parameters of TR+DI group and TR group in plasma were obtained:Cmax was?6415.94±942.56??g/L and?6328.78±824.12??g/L,Tmax was?0.17±0.00?h and?0.17±0.00?h,t1/2/2 was?0.73±0.14?h and?0.88±0.10?h,AUC?0-??was?5987.84±593.41??g/L*h and?6820.00±1558.26??g/L*h.Ths results showed that TR+DI group of tramadol pharmacokinetics parameters in mice plasma has no siginicant difference compared with TR group?P>0.05?.Conclusion:?1?The combination of tramadol and diazepam has an antinociceptive synergism,which not only enhances the analgesic effect,but also prolongs the time of analgesic effect.?2?The pharmacokinetics of tramadol in mice brain had significant difference between single administration and co-administration with diazepam.?3?The antinociceptive synergism mechanism produced by the combination of tramadol and diazepam may be related to the increase of unbound drug concentration of tramadol in the target organ. |
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