Development Of Compound Glycyrrhizin Tablets And Studies On In Vitro Metabolism In Rat Liver Microsomes

Compound glycyrrhizin tablets is compose of glycyrrhizinate (GL), glycine (GLY) and DL-methionine (MET). It is used clinically for the treatment of allergic disorder, inflammation and several virus diseases including chronic hepatitis, anaphylactic reaction,systemic lupus erythematosus,hemorrhagic fever with renal syndrome and atypical pneumonia (SAS) and so on. Preclinical studies finished the study of prescription and preparation technology, quality criterion studies, stability and bioequivalence evaluation in rabbits. As well as we obtained the clinical ratify document from the State Food and Drug Administration (No: 2006 L01472). On the basis of preclinical studies, the aim of this work was, therefore, to development of compound glycyrrhizin tablets for industrial production, revise the quality criterion, evaluate the bioequivalence of self-made compound glycyrrhizin tablets and SNMC in healthy Chinese male volunteers and studies on in vitro metabolism in rat liver microsomes. This study was divided into three parts:Part one The purpose of the research is to obtain optimizing parameters of prescription and technology for industrial production and quality evaluation. A HPLC (PDA dectator) method was developed to simultaneously determinate content, related substances and dissolution of the three principal compositions in compound glycyrrhizin tablet at the first. After method investigation and destroyed test by acid, alkali, oxidation, high temperature showed that the method was sensitive, accurate, reproducible, simple and specificity. It can be used as a reliable, effective method for the quality control of compound glycyrrhizin tablet. The analysis method is more advanced and reliable than the analysis method of SNMC (Stronger Neo-Minophagen C tablet, SNMC), which is imported from Janpan as market product. Trial production of compound glycyrrhizin tablets were finished and optimizing parameters of prescription and technology, which is suitable for industrialized production, were be obtained. According to requirements of clinical ratify document and Chinese Pharmacopoeia (2005 version) to revise quality criterion, quality and stability of three batch sample of trial production were investigated and compared with SNMC. The results shows that the prescription and technology of industrial production are reasonable and stable.Part two According to the formulation of requirements of"Drug Registration and Management Method"and GCP (good clinical practice), to evaluate the bioequivalence of self-made compound glycyrrhizin tablets and SNMC in healthy Chinese male volunteers. The concentration of glycyrrhetic acid (GA) in plasma was determined by LC/MS/MS. SNMC as reference preparation, twenty two volunteers were randomly divided into two groups (test and reference), with double cross-over design and single-dose oral administration. The sample was prepared for injection using a liquid-liquid extraction method, with ginsenoside Rh2 as the internal standard (IS). The method was proved to be suitable for clinical investigation and bioequivalence evaluation of different formulations containing GL, which offered advantages of convenience, accurate, sensitivity and specificity. The main pharmacokinetic parameters were calculated and the bioequivalence was evaluated with DAS2.0 practical pharmacokinetic program. The main pharmacokinetic parameters of test and reference were as follows: AUC0-∞(5794±3129)ng·h·ml-1and (5621±2752)ng·h·ml-1, AUC0-t (5084±2191) ng·h·ml-1 and (5187±2367)ng·h·ml-1, Cmax (288±249)ng·ml-1and (287±160)ng·ml-1, tmax (13.5±6.0) h and (16.7±7.2) h, t1/2 (13.1±8.5)h and (10.3±5.65)h, respectively. The two-one side t-test analysis showed that the confidence intervals of Cmax, AUC0-t and AUC0-∞were (80.9%-106.0%), (85.4%-122.3%) and (86.8%-127.3%), respectively. The relative bioavailahilitv of the test preparation was (95.8±20.8)%. Results of variance analysis and two one-side t-test showed that there were no statistical significant difference between the two preparations in the AUC and Cmax. The two-one side t-test analysis showed that the confidence intervals of Cmax, AUC0-t and AUC0-∞were (80.9%～106.0%), (85.4%～122.3%) and (86.8%～127.3%), respectively. In the case of Tmax was carried out by the Wiloxon-ranked sign test for the matched pairs, and the statistical result showed that there was no significant difference between the two preparations. The test and reference preparations were bioequivalentPart three Studies on in vitro mtabolism of GL and GA in rat liver microsomes. The Sprague-Dawley rats were induced for 3d with Phenobarbital (ip, 80mg/kg) and sacrificed after fast 12h. The liver microsomes were prepared by applying ultracentrifuga- tion approach. The protein concentration was determined by Bradford method. The in vitro metabolism study model of liver microsomes was established for the investigation of the enzyme activites. Phenacetin, Clivorine and Propofol as probe substrate of Cytochrome p450 (CYP450) isozymes, esterase and phase II transferase (UGT), respectively. The results verified the in vitro metabolism study model of Sprague-Dawley rat liver microsomes and enzyme activites are available. A UPLC/MS method was developed and validated for the simultaneous determination of GL, GA and its metabolites for the first time. The method is convenience, accurate, sensitivity and specificity. Metabolites were identified by UPLC/MS spectrum in rat liver microsomes incubates. GL, as probe substrate is in incubation with rat liver microsomes. The results showed that no metabolites of GL, such as 18β-glycyrrhetic acid-3-O-β-D- monoglucuronide (GAMG, 3-MGA), were detected in rat liver microsomes incubations. CYP450, esterase and phase II transferase (UGT ) had no effect on the metabolism of GL. The results provided direct evidence for the metabolic pathway of GL in liver microsomes at first time. GA as probe substrate is in incubation with rat liver microsomes. It was found that no metabolites of GA were detected in rat liver microsomes esterase incubations. NADPH as cofactor had catalytic effect on the metabolism of GA. Six metabolites (M1, M2, M3, M4, M5 were hyhroxy-GA, m/z 487; M6 was unknown metabolite, m/z 501) of GA were detected in rat liver microsomes CYP450 incubations inβ-NADPH presence at first time. It was shown that GA metabolism and M1, M2, M3 formation exhibited NADPH-dependent oxidation mechanism. GAMG and GL were detected in rat liver microsomes phase II transferase incubations in UGT presence at first time. It was shown that GAMG formation exhibited UGT-oxidation mechanism.At the same time, Three metabolites (M1', M2', M3') of hydroxy-GA were detected in rat liver microsomes phase II transferase incubations in UGT presence at first time. Selective chemical inhibitors to various CYP450 isoforms (CYP1A2?CYP3A?CYP2C19?CYP2D6?CYP2E1 and FMO ) were employed to conduct inhibition experiments. Ketoconazole reduced hydroxylation of GA in a dose-dependent manner. The kinetic behaviors of M1, M2, M3 were described well by a single-enzyme Michaelis-Menten equation, respectively. Methimazole, as FMO inhibitors, displayed partly inhibition for hydroxylation of GA.Inhibition experiments showed that Furafylline, Ticlopidine, Quinidine and 4-Methlpyrazole Methimazole, which were inhibitors as CYP1A2?CYP2C19?CYP2D6 and CYP2E1, respectively, did not display significant inhibition. The present study indicates that hydroxylation of GA in rat liver microsomes is mediated by CYP3A. To observe the effects of GLY and MET on the metabolism of GA. GA combine with GLY or MET as substrate is in incubation with rat liver microsomes, respectively. GLY can enhance metabolism of GA.The formation of M1, M2, M3 were decrease, while The formation of unknomn metabolite M6 were increase. GLY and MET on the metabolism of GA in CYP450 incubation had no significant effects. At same time, GLY and MET can both increase active component GA 90% and increase formation of GAMG and GL 35% in II transferase incubation. The present study indicates that GLY and MET probably enhance the effects of GA and accelerate the excrete of metabolites of GA.In this thesis, the prescription and technology of compound glycyrrhizin tablet for industrial production was optimized. We finished three batch trial production and revised quality criterion to investigate production quality. The study of bioequivalence evaluation in heathy volunteers was finished and the result showed the test and reference preparations were bioequivalence. In vitro mtabolism of GL and GA in Sprague-Dawley rat liver microsomes was systemic studied. To observe the effects of GLY and MET on the metabolism pathway of GA. The metabolism studies of the GL and GA in vitro will laid a experiments foundation for safety evaluation and in vivo metabolism studies of GA and GL in further. Meanwhile, it will provide some ideas and methods to investigate metabolism of other compound preparation.