In Vitro Expression Of Recombinant Human Cyp2d6 Alleles And Their Comparative Study Of Drug Metabolism And Drug - Drug Interactions

Cytochrome P450 is the major enzyme responsible for the clearing of the exnobitics in human body,such as metabolism the most of the drugs,active the carcinogen,clear the toxicant,endue the liver the function of detoxication.CYP2D6 is the most polymorphism enzyme in CYP450 superfamily.The molecular basis of the genetic polymorphism of CYP2D6 has been elucidated,gene deletion,duplication,single nuclear polymorphism et al., is the primary molecular basis of the polyphorphism.The genetic polymorphism is the cause of the phnotype polymorphism,the disparity of metabolism of the CYP2D6 substrates of the individual,from poor metabolizers(PMs) to utrarapid metabolizers(UMs),are common in Caucansian and Oriental.The disparity of the metabolism ability of individual lead the heterogeneity of treatment effects(HTE) when taking the same dose.The CYP450 mediated drug-drug interaction is one of the main causes of adverse drug reaction(ADRs),many institution have carried out a lot of research in vivo and in vitro in order to avid DDI mediated ADRs.The polymorphism and the proportion in drug metabolism make CYP2D6 be the one of the most impotant object in DDI research.So far,there are over 50 CYP2D6 allele have been identified,CYP2D6^*2,CYP2D6^*10, CYP2D6^*17 are the three most important allele among them.CYP2D6^*2 has two missense mutation which lead to two amino acid change(R296C,S486T),has a high frequence of 27.1～32.4%in Caucansian.CYP2D6^*10 also lead two amino acid change(P34S,S486T),has a high frequence of 40.8～49.5%in Oriental.CYP2D6^*17 lead three amino acid change (T107I,R296C,S486T),has a high frequence of 15～34%in African.In order to study the effect of single nuclear polymorphism on the metabolism ability and DDI property,Four single nuclear mutagenesis(P34S,T107I,R296C and S486T) and three alleles (CYP2D6^*2,CYP2D6^*10 and CYP2D6^*17) were yielded with CYP2D6^*1(WT) cDNA as template using PCR-based site directed mutagenesis introducing the mutagenesis.The cDNA of CYP2D6^*land other mutant were constructed into an eukaryotic expression vector pYES2/CT,the recombinant cDNA were expressed in yeast using galactose as inducer,the microsome were prepared by differential centrifugation,the expression ststus of CYP2D6 enzymes were determined by Western Blot,the quantity of functional CYP2D6 were determined by carbon monoxide(CO) difference spectroscopy.The kenetic analysis were carried out using fluorescent substrate AMMC and drug probe substrate bufuralol,and the Michaelis-Menton constant parameter were caculated,respectively.Inhibition asssy were carried out using fluorescent method and drug probe substrate combine HPLC detection method to test 23 kind of drugs to estimate the difference of inhibition properties between CYP2D6 variants and CYP2D6^*1.In our experiment,the V_max of CYP2D6^*1 for AMMC O-demethylation and bufuralol 1'-hydroxylation were 31.14pmol/min/mg and 175.31pmol/min/mg.Among the four single site mutangenesis,The V_max of P34S for AMMC O-demethylation and bufuralol 1'-hydroxylation compared with those of CYP2D6^*1 reduced to 36.97～51.44%and 14.85～23.43%,while the V_max of the other single site mutangenesis for AMMC O-demethylation and bufuralol 1'-hydroxylation were no conspicuous change compared with that of CYP2D6^*1.The V_max of CYP2D6^*2 for AMMC O-demethylation and bufuralol 1-'hydroxylation were similar compared with that of CYP2D6^*1,R296C and S486T along or combination both have no effect on the metabolism ability of CYP2D6 enzyme.The Vmax of CYP2D6^*10 for AMMC O-demethylation and bufuralol 1'-hydroxylation compared with those of CYP2D6^*1 decrease to 16.13～32.25%and 10.28～12.57%,P34S amino acid change is the main reason for the reduce metabolism ability of CYP2D6^*10,while the S486T amino acid change have no contribute to the decrease of CYP2D6^*10.The V_max of CYP2D6^*17 were decrease to 49.35～51.61%and 41.14-58.85%,T107I,R296C and S486T along have no effect od the metabolism ability of CYP2D6 enzyme,while,the combinant of the three were reduce the metabolism ability of CYP2D6 enzyme notably.The inhibition assays were carried out using AMMC and bufuralol as substrates and 23 drugs as inhibitor to calculate the IC₅₀ values.There are no significant fluctuations in the IC₅₀ values of each drug toward CYP2D6^*1,^*2,^*10,and ^*17.However,the inhibition potency of each drug toward CYP2D6 have cospcious disparity,such as antipsychotic drug:thioridazone＞clomipra mine＞amitriptyline,and antidepressant drug:fluoxetine＞clorpramazine＞sertraline.Correlation analysis of the IC₅₀ values of 11 drugs toward CYP2D6^*1 using two methods,fluorescent and drug probe substrate,the correlation is good with R²=0.9824.