Establishment Of The Microbial Model With The Activity Of CYP3A4

The microbial model with the activity of CYP3A4 was established by the methods of screening strains, optimizing systems, studying inhibitors and drug-drug interactions.1. The microbial biotransformation studies of midazolam1.1 Six Cunninghamella strains, one Mucor circinelloides and Fifty-four filamentous fungi, twenty-seven Actinomycete strains isolated from soil were selected for a preliminary screening of their ability to metabolize CYP3A4 probe drug dextromethorphan to N-demthyldextromethorphan（3-methoxymorphinan, MEM）. Then Midazolam, which is the most commonly used in vitro/vivo probe substrate for CYP3A4 activity, was used in further screening. The samples were assayed for metabolites by liquid chromatography-mass spectrometr methods. C. blakesleeana AS 3.970 was chosen facilitating the development of the model microorganism. The growth curve of AS 3.970 shows the optimum seed age is 21 h and through different generation study this fungus has the stable transformation capacity.1.2 Orthogonal and single factor designs were applied to optimize main factors affecting yields of target products in transformation system. It was found that C. blakesleeana AS 3.970 could transform midazolam totally（1’-hydroxymidazolam the main metabolite accounts for more than 60％）. The best condition is wheat-bran medium, initial pH 6.5, inoculation 5％, substrate concentration 40μg·mL^-1, and after 96 h incubation period.1.3 The living cell has the superiority in comparison with the resting cell.2. Isolation of midazolam metabolites prepared from microbial transformationThe transformation products of midazolam were prepared by AS 3.970 model microorganism established in our study. The isolation methods of target metabolites were searched by preparative thin layer chromatography, column chromatography and semi-preparative HPLC and identified by TLC, ESI-MSⁿ.3. Establishment the microbial model with the activity of CYP3A43.1 Dextromethorphan and simvastatin are both substrates of CYP3A4. AS 3.970 was used to transform these two drugs and orthogonal designs and single factor were applied to optimize main factors that affect target products. The result was AS 3.970 had great transformation capability. It shows that this strain is suitable to establish the microbial model with CYP3A4 activity.3.2 Evaluated the effect of methanol on AS 3.970 and its transformation capability. The MIC of methanol on AS3.970 is 5％and when methanol at a final concentration less than 1.5％（v/v）, it would have no effect.3.3 Verapamil, the special inhibitor of CYP3A4, was added to the transform system, and the metabolism ratio of midazolam was decreased significant. It validates that there existed CYP3A4 isoenzyme in the model strain at cell level.3.4 The interactions between CYP3A4 substrates were observed. When midazolam was transformed by AS 3.970, added dextromethorphan or simvastatin to the system, the results showed the substrates inhibit. It also proves there is CYP3A4 isoenzyme in C. blakesleeana AS 3.970.