A Rapid and Simple CYP2D6 Genotyping Assay—Case Study With the Analgetic Tramadol Ju ¨ rgen Borlak, Robert Hermann, Katharina Erb, and Thomas Thum There is substantial evidence for a causal relationship between genetic variability of the CYP2D6 gene and changes in the pharmacokinetics of drugs. Therefore, knowledge of single-nucleotide polymorphisms (SNPs) prior to drug administration is highly desired for assisting in the development of individualized pharmacotherapy. We therefore developed a robust assay that detects common CYP2D6 alleles within 60 minutes of blood withdrawal and links carriers of the variant CYP2D6*3 and *4 alleles to the pharmacokinetics of tramadol. This new genotyping assay employs fluorescence resonance energy transfer (FRET) analysis, which permits parallel identification of the CYP2D6*3 and CYP2D6*4 alleles within 60 minutes of blood withdrawal. We determined the genotypes of 100 healthy unrelated individuals and studied the pharmacokinetics of tramadol in 24 CYP2D6 genotyped healthy subjects. The total allelic frequencies of homozygote carriers were 0.015 and 0.25 for the CYP2D6*3 and *4 alleles, respectively, and the plasma area under the curve (AUC) was 84% above those of exten- sive metabolizers (homozygous EM group): 3,941.2 ng/mL  h (95% confidence interval [CI], 2,928.9 ng/mL  h to 4,953.5 ng/mL  h) versus 2,142.6 ng/mL  h (95% CI, 1,829.6 ng/mL  h to 2,455.7 ng/mL  h). Likewise, the AUC for the O-desmethyl- tramadol metabolite (M1) was significantly reduced in poor metabolizers (PMs): 300.2 ng/mL  h (95% CI, 260.3 ng/mL  h to 340.0 ng/mL  h) versus 842,6 ng/mL  h (95% CI, 715.1 ng/mL  h to 970.0 ng/mL  h). We observed a statistically significant correlation between plasma tramadol AUC and production of the O-desmethyl metabolite in CYP2D6 genotyped healthy volunteers. Our assay can be used reliably in clinical pharmacology studies and may be used for dose adjustment. © 2003 Elsevier Inc. All rights reserved. I DIOSYNCRATIC RESPONSES to drug therapy may be linked to genetic polymorphisms and indeed, more than 40 drugs and environmental chemicals and as many as 20% of all commonly prescribed drugs are subjected to CYP2D6-medi- ated metabolism. 1,2 Importantly, more than 77 variant alleles have been identified for this particular mono-oxygenase (see http://www.imm.ki.se/CYPalleles/), but only certain nucleotide changes of the CYP2D6 gene are of functional importance and result in the poor or ultrarapid metabolizer phenotype, which leads to significant changes in the pharmacokinetics of CYP2D6 substrates. 1,3 From a practical point of view it is literally impossible to screen for all possible allelic variants of drug metabolising enzymes on a routine basis. Nonetheless, genotype information is highly desirable, particularly when the prescribed drugs are substrates for enzymes with clinically relevant genetic poly- morphisms. Thus, genetic information is required to secure therapeutic efficacy and safety based on dose adjustments, especially when drugs with narrow therapeutic windows or cocktails of medicines are given. 1 We therefore developed a robust assay that would detect within 60 minutes of blood withdrawal, 2 common CYP2D6 alleles that usually confer the poor metabolizer (PM) pheno- type. We developed a new genotyping protocol employing fluorescence resonance energy transfer (FRET) analysis and applied the assay to the Roche Diagnostics (Mannheim, Ger- many) LightCycler to allow polymerase chain reaction (PCR) amplification and product analysis simultaneously. Our PCR assay monitors fluorescence quantification of DNA binding dyes, which hybridize to selected target sequences; using this approach, we can specifically assay nucleotide polymorphisms of the CYP2D6*3 and CYP2D6*4 allele. We report the geno- types of 100 healthy unrelated individuals from Southern Ger- many enrolled in clinical phase I trials, of which 24 subjects participated in a clinical trial with tramadol. This drug is a synthetic 4-phenyl-piperidine analogue of codeine and is widely prescribed as a centrally acting analgesic with a dual mechanism of action that includes low agonistic effects for the -opioid receptor, as well as inhibition of monoamine (seroto- nine, norepinephrine) reuptake. 4 Furthermore, tramadol is mainly metabolized to the N- and/or O-desmethyl-metabolite. CYP2D6 is the major isoform responsible for tramadol’s oxi- dation; nonetheless CYP2B6 and CYP3A4 are minor contrib- utors. 5 We studied the relationship between carriers of the CYP2D6*3 and *4 alleles and the production of the O-des- methyl-tramadol metabolite (M1). This metabolite is exclu- sively produced by CYP2D6 and thus allows genotype/pheno- type correlation. Overall, we report an assay that enables swift, large-scale, and cost-effective determination of major CYP2D6 polymorphisms for clinical pharmacology studies and provides guidance for dose adjustment within 60 minutes of blood sample withdrawal. MATERIALS AND METHODS Human Subjects General CYP2D6 screen. One hundred healthy male and female subjects from a Human Pharmacology Unit were examined for partic- ipation in various clinical research studies. These subjects were geno- typed for CYP2D6*3 and CYP2D6*4 after having given written in- formed consent. From the Fraunhofer Institute of Toxicology and Experimental Med- icine, Center of Drug Research and Medical Biotechnology, Hannover, Germany; and Human Pharmacology, Corporate Research, Viatris GmbH, Frankfurt, Germany. Submitted January 27, 2003; accepted April 30, 2003. Address reprint requests to Prof Dr Ju ¨rgen Borlak, Fraunhofer Institute of Toxicology and Experimental Medicine, Center of Drug Research and Medical Biotechnology, Nicolai-Fuchs-Str. 1, D-30659, Hannover, Germany. © 2003 Elsevier Inc. All rights reserved. 0026-0495/03/5211-0042$30.00/0 doi:10.1016/S0026-0495(03)00256-7 1439 Metabolism, Vol 52, No 11 (November), 2003: pp 1439-1443