Expression and Functional Analysis of CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7 Isozymes

Abstract

ABSTRACT:The objectives of this study were to compare the drug-metabolizing activity of human CYP2D6.24 (I297L), CYP2D6.26 (I369T), and CYP2D6.27 (E410K) allelic isoforms with wild-type CYP2D6.1 and to express the CYP2D7 protein derived from an indel polymorphism (CYP2D7 138delT) and investigate its possible codeine O-demethylase activity. Successful creation of individual cDNAs corresponding to CYP2D6*24 (2853 A>C), CYP2D6*26 (3277 T>C), and CYP2D6*27 (3853 G>A) allelic variants and CYP2D7 was achieved via molecular cloning. The corresponding proteins, CYP2D6.24, CYP2D6.26, CYP2D6.27, and CYP2D7, were expressed in insect cells by using a baculovirus-mediated expression system. All CYP2D proteins showed the empirical carbon monoxide difference spectra. We were surprised to find that the CYP2D7 protein was detected mainly in mitochondrial fractions, whereas all CYP2D6 allelic isoforms were present in the microsomal fraction. Furthermore, CYP2D7 did not produce any morphine from codeine. In contrast, CYP2D6.24, CYP2D6.26, and CYP2D6.27 allelic isoforms all showed active drug-metabolizing activities toward both codeine and dextromethorphan O-demethylation. Whereas CYP2D6.24 exhibited the highest intrinsic clearance in dextromethorphan O-demethylation (ϳ6-fold higher than that by CYP2D6.1), it had the lowest enzyme efficiency in codeine O-demethylation (ϳ50% lower than that by CYP2D6.1). Overall, the enzymatic consequences of CYP2D6 allelic isozymes are substrate dependent. These data would help preclinical and clinical assessments of the metabolic elimination of drugs that are mediated by human CYP2D enzyme.

What factors determine the severity of hepatitis A‐related acute liver failure?

“…Acetaminophen adducts were measured in all 24 for whom sera were available; 20 were undetectable and four demonstrated levels below 1.0 nmol a-cys adducts/mL (range 0.18–0.34). None were considered compatible with liver toxicity due to acetaminophen [16]. A more detailed clinical description of these cases has been published recently.…”

“…Adducts in sera were measured using a high-performance liquid chromatography method with electrochemical detection as previously described [16,17]. …”

Pharmacokinetic Interactions between Monoamine Oxidase A Inhibitor Harmaline and 5-Methoxy-N,N-Dimethyltryptamine, and the Impact of CYP2D6 Status

“…All other reagents or organic solvents were either analytical or high-performance liquid chromatography (HPLC) grade. CYP2D6.1 enzyme was expressed and purified as reported (Yu et al, 2002;Zhang et al, 2009).…”

“…Enzyme incubations were carried out as reported using purified CYP2D6.1 enzyme (Yu et al, 2002;Jiang et al, 2009;Zhang et al, 2009). In particular, each reaction was incubated in 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 200 ml at 37°C for 20 minutes, which consisted of 0.02 mM CYP2D6.1, 0.2 mM cytochrome P450 reductase, 10 mg L-a-dilauroylphosphatidylcholine, and 2 or 10 mM 5-MeO-DMT in the absence or presence of harmaline (2 or 20 mM).…”

Pinoline May be Used as a Probe for CYP2D6 Activity

“…Incubation reactions with CYP2D6 allelic isoforms and HLMs were conducted in 100 mM potassium phosphate buffer, pH 7.4, in a final volume of 200 l at 37°C, as described previously (Yu et al, 2002;Felmlee et al, 2008;Zhang et al, 2009). In particular, each reaction consisted of 0.25 mg/ml microsomal proteins or 0.1 M cDNA-expressed CYP2D6, 0.2 M P450 reductase, and 10 g L-␣-dilaurylphosphatidylcholine.…”