Studies On In Vitro And In Vivo Biotransformation Of AF-5 And HH07A

The metabolism of a drug has important consequences on its therapeutic efficacy or on its toxicity.The investigation of drug biotransformation therefore plays an important role in the process of new drug research .By investigating biotransformation of a drug , the structures of metabolites are identified,and metabolic pathways of the drug can also be clarified.moreover the relationship can be established between the structure.metabolism and toxicity. These are very useful to improve the drug structure and select the proper pharmaceutical preparation form . This dissertation is mainly concerned with the studies on in vitro and in vivo biotransformation of AF-5 and HH07A, two compounds with new chemical structures.The dissertation is divided into two parts . The first part is entitled'The study on in vitro and in vivo biostransformation of AF-5".Chapter1:A GC-MS method is developed for'the analysis of small amounts of AF-5 in tissue samples.and the distribution and excretion of AF-5 are investigated, moreover the concentration variation with time in rat brain is discussed in detail.Chapter 2: The in vitro metabolism of AF-5 was studied by rat hepatic microsomes system.Both GC-MS and TLC methods were developed for the on-line analysis of AF-5 and fourteen metabolites.then several factors affecting the metabolism of AF-5 in vitro such as inducer ,inhibitor. AF-5 concentration and so on were investigated in detail ,and the enzyme kinetic characteristics of metabolism of AF-5 in vitro were confirmed.Chapter 3: According to the enzyme kinetic study of metabolism of AF-5 in vitro, the constitution of the incubation system was optimized. Column chromatography .thin layer chromatography and preparative high performace liquid chromatography were then used for the isolation and purification of the eight major metabolites , and their structures were elucidated by UV.IR, MS and NMR data.The metabolic sites and stereoselectivity of AF-5 in vitro were also discussed.Chapter4:A solid-phase extraction method with Amberlite XAD-2 was developed to extract and separate the metabolites from the biological fluids of rat such as plasma .urine, bile and brain . The in vivo metabolites of AF-5 in biological fluids were investigated by GC-MS. The mass spectra of the metabolites are presented and major fragmention pathways are discussed. Furthermore .the in vivo characteristics of the metabolites such as elimination , excretion, existing form , dymamic behavior and metabolic stereoselectivity were investigated.The second part of this dissertation is entitled "The study on in vivo and in vitro biotransformation of HH07A".Chapter 1 :The chromatographic behavior of HH07A was investigated in detail on different reversed-phase media such as C2, C8, C18 bonded silica. A HPLC method for determining HH07A in biological samples was developed, and the excretion characteristics of HH07A were discussed.Chapter 2: The in vitro metabolism of HH07A was studied by rat hepatic microsomes system, The constitution of the incubation system was optimized and a RP-HPLC- DAD method was developed for the on-line analysis of HH07A and its two metabolites. The thin layer chromatography, column chromatography and preparative HPLC were used for the isolation and purification of a major metabolite.and its structure was identified by UV,IR,MS and NMR data.Chapter 3:A method for the separation of HH07A enantiomers by high performance capillary electrophoresis was developed. The working solution is composed of 0.04mol/LTris-H3PO4(pH=3)-0.0065mol/Lβ-cyclodextrin. The method has been used to examine the HH07A metabolism in rat hepatic microsomes system.Chapter 4:The metabolism of HH07A In vlvo has been Investigated by GC-MS.After oral administration of HH07A, the rat urine was passed through a macroporous resin XAD-2 column, which was then eluted with methanol After hydrolyzed with glucuronidase, the eluate was extracted with dichloromethane,concentrated,TMS derivatized and analyzed with GC-MS. The mass spectra of the metabolites and their derivatives were presented.The structures of the metabolites were proposed and the in vivo metabolic pathway was discussed.The above experimental results suggested that according to the chemical and physical characteristics of the drug, specific and sensitive chromatographic methods were first developed to detect the small amount of the drug in biological samples, and the ADME of the drug in vitro was investigated.Chromatographic technologies were used to isolate and purify the metabolites in vitro,and their structures were identified by UV,IR,MS and NMR data. On these bases, the metabolism of the drug in vivo was investigated .The metabolites in biological fluids were identified and detected by GC-MS or HPLC-DAD method.Thus the in vivo metabolic characteristics of the drug were elucidated.This is a convenient and effective way in the investigation of the biotransformation of drugs with new chemical structures.