Cloning and expression of CYP2D6*1 and CYP2D6*10 and its application in vitro drug-herbs interaction studies
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Date
2013-04
Authors
Lee, Wee Leng
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
CYP2D6 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.
Description
Keywords
Drug-herbs