| Background:Roxithromycin(RM) has been widely used clinically to treat infection caused by Gram-positive bacteria and gram-negative bacteria.Ambroxol (Amb) is used for treating acute and chronic lung disease associated with abnormal secretion and expectoration of sputum dysfunction,promoting mucus discharge and improving lung function;Amb can enhance RM lung tissue penetration, increase the concentration of RM in lung. The two drugs often combined in clinical treatment of bronchitis and lung bacterial infection. RM was mainly metabolied by liver drug enzyme, It has been reported that Amb may affect the liver drug enzyme. Up to now, no researches indicated Amb caused the changes of RM pharmacokinetic parameters. This study mean to refer the theory and experimental gist whether Amb affect the RM pharmacokinetic parameters and clinical rational drug combination.Objectives:To investigate the effects of Amb on pharmacokinetic parameters of RM in isolated hepatocyte incubation system and rats,and to evaluate the interaction between the two drugs and the rationality of drug combination used in clinic.Methods:â‘ Set up steady and reliable separation technique of isolated rat hepatocytes. Determined the yield,vigor,cellular morphous and fine structure of hepatocytes by normal microscope,transmission electron microscope and HE dyeing.â‘¡Set up isolated hepatocyte incubation solution.Quantify the toxicity of RM and Amb on isolated hepatocytes by a MTT method.â‘¢Study the effects of Amb on RM metabolism in isolated hepatocyte incubation solution: 4μg·mg-1 of RM was alone added in the control group; Experimental groupâ… ,â… andâ…¢were added 4μg·mg-1 RM and different amounts of Amb.The Amb concentration was 2,4,8μg·mg-1 respectively. Each group was incubated for 60 min at 37℃, the concentration of RM in hepatocyte incubation solution were detected by HPLC-UV, to investigate the effects of Amb on RM metabolism.â‘£Study the effects of Amb on RM pharmacokinetic parameters: 72 SD rats were randomly divided into 4 groups.Control group was only given RM; experimental groupâ… ,â… andâ…¢were given 2.7mg·kg-1, 5.4 mg·kg-1, 10.8 mg·kg-1 dose of Amb, tid,for 6 days, and were given both the Amb and RM in the seventh day. Serila blood samples were collected by orbital plexus. The RM plasma concentrations were determinated by HPLC-UV, and the pharmacokinetic parameters of RM were calculated by DAS2.1.1 .Results:â‘ The well-rounded separation technique of rat hepatocytes and isolated rat hepatocyte incubation system was set up.The yield of rat hepatocytes was 12×108 cell·liver-1.Both the vigor and purity of hepatocytes were about 90%.â‘¡In the RM concentration range of 112μg·mg-1 and Amb concentration range of 0.516μg·mg-1, the effect of RM and Amb on the viability of isolated rat hepatocytes did not have significant difference.The hepatocyte viability was>80%.â‘¢The RP-HPLC determination of RM: mobile phase was acetonitrile:water(include 0.02mol·L-1 ammonium dihydrogen phosphate)=35:65 (V/V) , which was adjusted pH value to 7.1 by triethylamine.The flow rate was 1ml·min-1 .The wave length was 210 nm and the sensitivity was 0.004 AUFS.â‘£Compared with the control group, Amb did not have effect on RM metabolism in isolated rat hepatocyte incubation solution (P>0.05).⑤In vivo experiments,the results showed that RM main pharmacokinetic parameters such as Tmax,T1/2z,Cmax,Vz,CLz,AUC0～t,AUC0～∞were no significant difference (P>0.05) between the experimental groups and the control group.Conclusions : We have established the separation technique of isolated rat hepatocytes and isolated hepatocyte incubation system which were steady, mature, satisfied the requirement of the metabolism study of RM and Amb.Through studying in vitro and in vivo, Amb did not have effect on RM metabolism in rats. It is safe to combine the two drugs and there is no need to adjust the dosage of RM. This study provided the certain experimental evidences for clinical use of RM when co-administrated with Amb.
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