Pharmacokinetics Of Shuanghuanglian Injection Combined With Azithromycin Injection

Infectious disease is characterized by acute onset and rapid transformation. In oder to have efficient work, injection is usually applied to treat this kind of disease in the clinical practice. Azithromycin injection as chemical medicine and Chinese medicine Shuanghuanglian injection (SHL) are two major medicine and always matched together to treat infectious disease. Whether the metabolite of their chemical ingredients interacted after taking drugs simultaneously was not reported by far. The research discussed that the primary influencing factor of the content measurement of forsythiaside (FTA) in the plasma sample pretreatment, and built a method of the plasma sample pretreatment. Based on it, the metabolism process of two medicines combination in rats are studied preliminarily, the pharmacokinetic process of the major antibacterial active ingredient for FTA and Azithromycin (Azi) in rats was observed after the combination of Azithromycin injection and Forsythia extract (FE) or SHL. By comparing the differences of pharmacokinetics parameters, this study aims to explore the interactions between traditional Chinese medicine and chemical medicine, and to produce the basic ideas for the reasonable combination of the traditional Chinese medicine and chemical medicine in the clinical application.The study is mainly divided into four parts.Part one-the research of the effects to FTA determination by the difference of the pretreatment processing of plasma samples.High performance liquid chromatography (HPLC) method was established to the determination of FTA. This study examined the altering FTA concentrations in each pretreatment step and controlled the stability of FTA in plasma samples, including the evaluation of the storage temperature and time before plasma treatment, plasma vortexing with the IS and extraction reagent, centrifugation and evaporation conditions, reconstitution times and sample storage conditions before injection. The results were processed by a mathematical statistical analysis for SPSS17.0.The research of the effects indicated that the pretreatment processing was necessary to maintain the stability of FTA in plasma samples. The plasma samples should be stored in an ice water mixture immediately after collection and be treated within 4 h. Plasma samples vortexed with IS and ethyl acetate must be placed at 25℃ for less than 2 h or at -20℃ for less than 4 h, and be centrifuged at 4℃ or 25℃. The samples must be also evaporated to dry at 25℃ in water bath under nitrogen stream, the treated plasma sample residues should be reconstituted within 24 h, and the supernatant was better to be stored in an auto-sampler at 4℃ and injected into the HPLC within 60 h.Part two-the pharmacokinetic study of FTA after the use of FE alone and the combination of Azithromycin injection.In order to achieve the target, the SPF SD rats were utilized as the experimental objects. Firstly, we respectively gave these rats the FE and the FE combined with Azithromycin by intraperitoneal injection and intravenous injection, and then blood samples were collected from the retrobulbar capillary plexus at different times after administration. Secondly, the samples of plasma were obtained by the method of part-one and then the concentration of FTA in plasma samples of each time point were determined by HPLC. Finally, through the analysis of all pharmacokinetic parameters by non-compartmental analysis using Kinetica 4.4 software, the differences between the pharmacokinetic parameters were processed by SPSS17.0.The study established the HPLC methods in the determination of FTA of FE in rat plasma samples, this method showed good performance in terms of specificity, precision, accuracy, and recovery rate, which met all requirements.In the way of intraperitoneal injection, compared with the administration of FE alone, there is a significant increasing of FTA in Cmax (P<0.05), Tmax (P<0.05) and AUC (P<0.05), and a dramatic decline in MRT (P<0.05), and no obvious difference inT1/2 (P>0.05) by the administration of FE combined with Azithromycin injection.In the way of intravenous injection, compared with the administration of FE alone, there is a significant growth of FTA in T1/2 (P<0.05) and AUC (P<0.05), and a dramatic decrease in CL (P<0.05), and no obvious difference in MRT (P>0.05) and Vd (P>0.05) by the administration of FE combined with Azithromycin injection.Part three-the pharmacokinetic study of FTA after the use of SHL alone and the combination of Azithromycin injection.SPF SD rats were utilized as the experimental objects. Firstly, we respectively gave these rats SHL alone and combined with Azithromycin by intravenous injection and then blood samples were collected from the retrobulbar capillary plexus at different times after administration. Secondly, the samples of plasma were obtained by the method of part-one and then the concentration of FTA in plasma samples of each time point were determined by HPLC. Finally, all pharmacokinetic parameters were processed by non-compartmental analysis using Kinetica 4.4 software and the differences between data were processed by SPSS17.0.The study established the HPLC methods in the determination of FTA of SHL in rat plasma samples. This method showed good performance in terms of specificity, precision, accuracy, and recovery rate, which satisfied all requirements.In the way of intravenous injection, compared with the use of SHL alone, there is a significant increase of FTA in T1/2 (P<0.05) and AUC (P<0.05), and a dramatic decline in CL (P<0.05), and no obvious difference in MRT (P>0.05) and Vd (P>0.05) by the administration of SHL combined with Azithromycin injection.Part four-the pharmacokinetic study of Azi after the injection of Azithromycin Lactobionate alone, the Azithromycin Lactobionate combined with FE, the Azithromycin Lactobionate combined with SHL.SPF SD rats were utilized as experimental objects. We firstly gave these rats Azithromycin Lactobionate, the combination of Azithromycin Lactobionate and FE, the combination of Azithromycin Lactobionate and SHL by intravenous injection and then blood samples were collected from the retrobulbar capillary plexus at different times after administration. Secondly, the samples of plasma were obtained by acetonitrile protein precipitation method and then the concentration of Azi from the plasma samples of each time point were determined by LC-MS/MS. Finally, all pharmacokinetic parameters were processed by non-compartmental analysis using Kinetica 4.4 software and the differences between data were processed by SPSS17.0.The study established the LC-ESI-MS/MS methods in the measurement of the injection of Azithromycin Lactobionate in rat plasma samples, this kind of method showed good performance in terms of specificity, precision, accuracy, matrix effects and recovery rate, which satisfied all the requirements.In the way of intravenous injection, compared with the administration of the injection of Azithromycin Lactobionate alone, there is a significant increase of Azi in T1/2 (P<0.05), a extramarked growth in AUC (P<0.01) and Vd (P<0.01), there is an extra obvious decline in CL (P<0.01) after the use of the Azithromycin Lactobionate combined with FE, there is a dramatic reduce in CL (P<0.05) after the use of the Azithromycin Lactobionate combined with SHL, and there is no obvious differences in MRT (P>0.05) and Cmax (P>0.05) by administration of the Azithromycin Lactobionate combined with FE and the Azithromycin Lactobionate combined with SHL.ConclusionIn conclusion, for the first time, the research discussed the influence of the relative factors for the measurement of FTA in the plasma sample pretreatment, confirmed that the temperature and storage time was the major factors of content transformation, and built the plasma sample pretreatment method of FTA. The plasma sample which contained the Forsythia suspensa or FTA was pretreated on the basis of the method obtained from the research, which confirmed that the experimental data, results, parameters of pharmacokinetics was stable and controllable. Moreover, the plasma pretreatment method will help to study the FTA stability of other bio-matrices in the future. The research established the HPLC methods in the determination of the FTA of FE and the FTA of SHL in rat plasma samples, and established the LC-ESI-MS/MS methods in the measurement of Azi in rat plasma samples, these kinds of methods showed good performance in terms of specificity, precision, accuracy, and recovery rate, which satisfied all the requirements.The results showed that the Azi had significant influence to FTA in FE and SHL in vivo pharmacokinetic process, and the trend of effect is consistent. It indicated that the Azi could be affected significantly after the combination with FE and SHL in pharmacokinetics behaviors, and the trend of effect is consistent. It is prompted that there is significant synergistic interactions in the clinical application of Azi combined with FE and SHL. When the Azi is combined with Forsythia suspensa and SHL containing Forsythia suspensa in the clinical practice, the interaction of drugs and the decrease of dosage provide the evidence of decreasing side effect of Azi. It provides informations for the future study of traditional Chinese medicine and chemical medicine in combination.In addition, the study has preliminarily inferred the drug properties of FTA according to the interactions of the pharmacokinetics after the combination of traditional Chinese medicine and chemical medicine. The results deduced that the drug properties of FTA are cold.