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Genetic polymorphism in dextromethorphan metabolism by CYP2D6 and CYP3A4 enzyme isoforms / Mthokozisi Muziwandile Nkosingiphile Mgwabi

Most administered drugs are metabolised in the liver by Phase I enzymes and more
importantly by the cytochrome P450 (CYP) system. The extent of first-pass metabolism is
important in determining whether the drug will have therapeutic or adverse effects after being
administered to a patient. To date the CYP family has been shown to consist of 74 families
denoted as CYPl to CYP118, and only a few families are significantly involved in drug
metabolism. CYP3A4 is the most important isoenzyme followed by CYP2D6, CYP2C9, and
CYP2C19 with a small contribution by CYP2E1, CYP2A6, and CYPlA4. CYP2D6 and
CYP3A4 enzyme isoforms have been well established to exhibit interethnic and interindividual
variability with regard to drug metabolising capacity.
Mutation on the gene coding for a metabolising enzyme is a major cause of variation in drug
metabolism. This mutation gives rise to allelic variants producing enzymes with altered
metabolising activity. The presence of an allele with decreased metabolic activity in an
individual gives rise to the poor metabolising (PM) phenotype. When the PM phenotype
occurs at a frequency of more than 1% within a given population, then the term genetic
polymorphism applies. The aberrant metabolic capacity translates into variable drug
responses of more than 20-fold, leading to different susceptibility to sub-therapeutic effects or
adverse drug reactions. A significant number of drugs, such as the B-adrenergic blockers,
antidepressants, antipsychotic and antiarrhythmic agents, are entirely or partly metabolised by
CYP2D6 and CYP3A4. Genetic polymorphism is especially important for drugs with a narrow
therapeutic/toxicity window.
Phenotyping involves the use of a probe drug that is administered to the subject, followed by
determination of the parent drug and its metabolites in the urine. The aim of this study was to
develop and validate an HPLC method for phenotypic determination of the CYP3A4 and
CYP2D6 enzymes, followed by the application of the assay in a random heterogeneous
population of males.
Dextromethorphan (DXM) was used as an in vivo probe for simultaneous determination of the
phenotypic expression of CYP2D6 and CYP3A4. An HPLC method coupled with a
fluorescence detector was developed for the phenotypic determination of CYP2D6 and
CYP3A4 iso-enzymes as determined by the concentration of dextromethorphan/dextrophan
(DXM/DX) and dextromethorphan/3methoxy-morphinan (DXM/3MM) metabolic ratios
respectively. The compounds were separated on a phenyl column (150 x 4,6 mm, 5-um
particle size) serially connected to nitrile column (250 x 4,6 mm, 5-um particle size) using
mobile phase of 80% (1.5% glacial acetic acid and 0.1% triethyl amine in distilled water) and
20% acetonitrile. Solid phase extraction was used to extract the analytes from urine samples
using silica cartridges. The suitability of the method was demonstrated in a preliminary study
with sixteen healthy Caucasian males. After a single oral 30 mg DXM dose, the volunteers
were required to collect all urine samples voided 8 hours post oral dose. DXM/3HM and
DXM/DX metabolic ratios were determined from collected urine samples.
The method was validated for DXM and DX at a concentration range of 0.25 - 30 ug/ml, and at
0.025 - 3 ug/ml for 3MM. Calibration curves were linear with R2 values of at-least 0.999 for all
compounds of interest. Recoveries were 97%, 93%, and 65% for DX, DXM and 3MM,
respectively. The method was reproducible with intra-day precision having coefficients of
variation percentage (CV%) of less than 17% for all analytes. Inter-day precision had a CV%
of less than 14% for all analytes. The limit of detection was 30 ug/ml for all compounds. All
volunteers were classified with an extensive metaboliser (EM) phenotype. In conclusion the
method described is suitable for polymorphic determination of CYP2D6 and CYP3A4 in a
population study, and may have value in further studies planned at investigating the critical
issue of racial genetic polymorphism in ethnic groups in South Africa. / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Identiferoai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nwu/oai:dspace.nwu.ac.za:10394/163
Date January 2003
CreatorsMgwabi, Mthokozisi Muziwandile Nkosingiphile
PublisherNorth-West University
Source SetsSouth African National ETD Portal
Detected LanguageEnglish
TypeThesis

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