Cloning and expression of CYP2D6*1 and CYP2D6*10 and its application in vitro drug-herbs interaction studies

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dc.contributor.authorLee, Wee Leng
dc.date.accessioned2017-09-08T01:04:06Z
dc.date.available2017-09-08T01:04:06Z
dc.date.issued2013-04
dc.description.abstractCYP2D6 is one of the maJor CYPs enzymes and is responsible for the metabolism of about 20% of cunently available therapeutic chugs. CYP2D6 is polymorphic and CYP2D6* 10 is the common allele in the Malaysian population. There is lack of data on local herbal-drug interaction. The objective of this study was to generate an in vitro drug-herbs int1eraction assay and use it to analyse local herb interaction. Recombinant CYP2D6* 1 was generated and co-expressed with CYPreductase in E. coli. Recombinant CYP2D6*10 was generated using site-directed mutagenesis and expressed as CYP2D6* 1. The HPLC method was developed to measure bufuralol 1 '-hydroxylase activity of CYP2D6. The kinetic parameters, Km and Vmax for CYP2D6*1 were 9.686::1:: 0.674 JlM and 6.867 ± 0.119 pmoVminlpmol of CYP2D6*1 respectively. For CYP2D6*10, the Km and Vmax were 41.02 ± 0.674 JlM and 0.351 ± 0.0013 pmoVmint!pmol of CYP2D6*10 respectively. Kinetic parameters from in vitro CYP2D6 catalytic activity analysis were in accordance with others published studies. Extracts of EU inhibited CYP2D6* 1 by non-competitive mechanism with Ki and IC50 of 302.03 ± 10.59 ng/ml and 178.38 ± 12.29 ng/ml, respectively. However EU inhibited CYP2D6* 10 to a lesser extent by competitive mechanism with Ki of215.29 ± 11.76 ng/ml and IC50 was 490.90 ± 13.88 ng/ml. ELJ did not show any mechanism-based inhibition. It also suggested the presence of chug-herb and chug-food interactions when certain natural products were consumed concunently with conventional medi,cines. This study have successfully expressed recombinant CYP2D6*1 and CYP2D6*10 protein. A specific and sensitive HPLC method was developed and validated to analysis the in vitro catalytic activity of enzymes and ELJ inhibition effect. Future studies are required to investigate the inhibitmy effects of more local herbs on CYP2D6 activity for an improved understanding of dug-herb interactions.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/4531
dc.language.isoenen_US
dc.publisherUniversiti Sains Malaysiaen_US
dc.subjectDrug-herbsen_US
dc.titleCloning and expression of CYP2D6*1 and CYP2D6*10 and its application in vitro drug-herbs interaction studiesen_US
dc.typeThesisen_US
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