Establishments Of The Microbial Models With The Activity Of CYP2C9 And CYP2D6 Respectively

The microbial models with the activity of CYP2C9 and CYP2D6 were established respectively by the methods of screening strains, optimizing systems, studying inhibitors and drug-drug interactions. The metabolites control of glibenclamide and indomethacin were prepared by microbial transformations.1 Establishment the microbial model with the activity of CYP2C91.1 The model strain was screened out from Cunninghamella elegans AS 3.156, C. elegans AS 3.2028, C. echinulata AS 3.2004, C. blakesleeana AS 3.153 and C. blakesleeana AS 3.910 by investigating their ability to transform CYP2C9 substrates glibenclamide, indometacin and diclofenac, respectively. The structures and yields of metabolites were determined by liquid chromatography-mass spectrometry. Orthogonal designs were applied to optimize main factors affecting yields of target products in transformation system and it was found that C. blakesleeana AS 3.910 possessed the greatest capability of transforming three substrates on the condition of wheat-bran medium, initial pH 6.5. Therefore, the microbial model with the activity of CYP2C9 was established primary.1.2 The ability of C. blakesleeana AS 3.910 transforming CYP2C9 substrates tolbutamide, indometacin and diclofenac to target products declined sharply when CYP2C9 inhibitors sulfaphenazole, benzbromarone and valproic acid were respectively added in transformation system. It was validated that there existed CYP2C9 isoenzyme in the model strain at cell level.1.3 Every two of tolbutamide, indometacin and diclofenac were added to the C. blakesleeana AS 3.910 transformation system simultaneously to evaluate drug-drug interactions among three substrates. The results show the substrates inhibite each other, which prove there is CYP2C9 isoenzyme in C. blakesleeana AS 3.910.2 Establishment the microbial model with the activity of CYP2D62.1 Fifty-nine filamentous fungi were screened for their abilities to transform CYP2D6 probe drug dextromethorphan to O-demthyldextromethorphan (dextrorphan). The yield of dextrorphan was 82.7% transformed by C. blakesleeana AS 3.153 that was selected for further investigation. Orthogonal designs were applied to optimize main factors which may affect target products yield of CYP2D6 substrates dextromethorphan, propafenone and metoprolol transformed by C. blakesleeana AS 3.153. Finally, it was found that the model strain showed the greatest transformation capability on the conditions of wheat-bran medium, initial pH 6.5. As a result, the microbial model with activity of CYP2D6 was established primary.2.2 The target products yield of dextromethorphan, propafenone and metoprolol transformed by C. blakesleeana AS 3.153 decreased sharply with CYP2D6 inhibitor quinidine in transforme system. Every two of dextromethorphan, propafenone and metoprolol were added to the C. blakesleeana AS 3.153 stransformation system simultaneously to investigate drug-drug interactions among three substrates. The results show the substrates inhibite each other, which prove there is CYP2D6 isoenzyme in C. blakesleeana AS 3.153.3 Preparation metabolites control by microbial transformationThe transformation products of glibenclamide and indometacin were prepared by CYP2C9 microbial model established in our study. The target metabolites were isolated by colum chromatography and semi-preparative HPLC, the structures of which were identified by ESI-MSⁿ, ¹H NMR and ¹³C-NMR. The results show glibenclamide is metabolized to 4'-trans-hydroxyglibenclamide, 3'-cis-hydroxyglibenclamide and indomethacin to O-desmethylindomethacin, N-deschlorobenzoylindomethacin and O-desmethy- N-deschlorobenzoylindomethacin, which displays the special superiority of microbial model in preparing human drug metabolites control.