Andrea Gaedigk

Clinical pharmacogenetics implementation consortium guidelines for cytochrome P450 2D6 genotype and codeine therapy: 2014 Update

Codeine is bioactivated to morphine, a strong opioid agonist, by the hepatic cytochrome P450 2D6 (CYP2D6); hence, the efficacy and safety of codeine are governed by CYP2D6 activity. Polymorphisms are a major cause of CYP2D6 variability. We summarize evidence from the literature supporting this association and provide therapeutic recommendations for codeine based on CYP2D6 genotype. This document is an update to the 2012 Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2D6 genotype and codeine therapy.

Optimization of cytochrome P4502D6 (CYP2D6) phenotype assignment using a genotyping algorithm based on allele frequency data.

Cytochrome P4502D6 (CYP2D6) is a highly polymorphic gene locus with > 50 variant alleles which lead to a wide range in enzymatic activity. So called poor metabolizers are carriers of any two non-functional alleles of the CYP2D6 gene. CYP2D6 genotyping is cumbersome and the question of how much genotyping is necessary for an accurate phenotype prediction is still debated. The goal of this study was to determine the optimum amount of genotyping required to accurately predict the phenotype at a reasonable cost in a white North American population. To address this issue, we designed a polymerase chain reaction (PCR)/restriction fragment length polymorphism-based genotyping strategy to detect key mutations linked to extensive metabolizer or poor metabolizer associated alleles in combination with extra-long PCR (XL-PCR). All mutations with the exception of gene deletions and duplications are detectable by simple restriction digestion analysis and agarose gel electrophoresis. In addition, we utilized a genotyping algorithm based on our own and published allele frequency data and phenotype analysis to calculate the probability of a correct genotype (and thus, phenotype) assignment. As little as one XL-PCR reaction followed by a maximum of six reamplification reactions allows an accurate prediction of an individuals genotype to 99.15%. As few as four reamplification reactions identify 97.9% of poor metabolizer individuals. We evaluated our model in 208 white North Americans by testing for the presence of key mutations linked to CYP2D6*2, *3, *4, *6, *7, *8, *9, *10, *11, *12, *15, *17 and *18 alleles and the *5, *13 and *16 gene deletions. For all individuals, the correct phenotype has been predicted. Discordant phenotype assignment occurred in only two individuals which subsequently was attributed to CYP2D6 inhibition by concomitant drug therapy.

Combined phenotypic assessment of CYP1A2, CYP2C19, CYP2D6, CYP3A, N‐acetyltransferase‐2, and xanthine oxidase with the “Cooperstown cocktail”

Simultaneous administration of several probes enhances the utility of phenotyping, but poor specificity, side effects, and use of drugs not approved by the Food and Drug Administration limit the usefulness of prior phenotyping cocktails.

Concordance between Tramadol and Dextromethorphan Parent/Metabolite Ratios: The Influence of CYP2D6 and Non‐CYP2D6 Pathways on Biotransformation

Cytochrome P4502D6 (CYP2D6) activity has been shown to be a determinant of both the pharmacokinetics and pharmacodynamics of tramadol in adults. This study evaluated the association between CYP2D6 activity, as determined by dextromethorphan (DM) urinary metabolite ratio, and tramadol biotransformation in 13 children (7–16 years). CYP2D6 genotype was determined by XL‐PCR and PCR/RFLP. Phenotype was assessed by HPLC quantitation of DM and its metabolites from a 12‐ to 24‐hour urine collection following a single oral dose of DM. There was only a modest correlation between tramadol/M1 (metabolite 1)plasma concentration or AUC and the DM/dextrorphan (DX) urinary molar ratio in the study cohort; however, when subjects were segregated based on the number of functional CYP2D6 alleles, a much stronger relationship was observed for subjects with two functional alleles, with essentially no relationship evident in those individuals with one functional allele. Further evaluation of these data suggested that the CYP2D6‐mediated metabolite (M1) is formed to a lesser extent, and the formation of the non‐CYP2D6 product (M2) is more pronounced in subjects with one versus two functional alleles. Thus, the number of functional CYP2D6 alleles and the availability of alternative cytochromes P450 capable of metabolizing tramadol may explain the poor association between DM, a well‐characterized CYP2D6 probe, and tramadol in a population of CYP2D6 extensive metabolizers.

Quantification of intraindividual variability and the influence of menstrual cycle phase on CYP2D6 activity as measured by dextromethorphan phenotyping

Intraindividual variability and the effects of menstrual cycle phase on CYP2D6 activity were evaluated by dextromethorphan phenotyping in 20 Caucasian normal volunteers. Dextromethorphan 30 mg was administered to 10 men every 14 days for 3 months, and to 10 premenopausal women during the mid-follicular and mid-luteal phases of each menstrual cycle for three complete cycles. Urinary dextromethorphan/dextrorphan molar ratios were obtained after an overnight urine collection. Ten women and nine men were extensive metabolizer phenotypes, and one man was a poor metabolizer phenotype (confirmed by genotyping). There was no difference in dextromethorphan metabolic ratios between the mid-follicular (mean +/- SD: 0.00728+/-0.00717) and mid-luteal (0.00745+/-0.00815) phases of the menstrual cycle (P = 0.88). Also, no significant difference was found in the intraindividual variability of the metabolic ratios between the two phases (P = 0.80). No statistically significant sex difference in CYP2D6 activity was found between men (0.00537+/-0.00431) and women (0.00737+/-0.00983) extensive metabolizers (P = 0.84). For all individuals, intraindividual variability in dextromethorphan ratios ranged from 12.1-136.6% with a median of 36.7%. Because hormonal fluctuations within the mid-follicular and mid-luteal phases of the menstrual cycle do not appear to affect CYP2D6 activity, pharmacokinetic or clinical investigations of CYP2D6 substrate activity may not require menstrual cycle phase stratification. Because baseline metabolic ratios may fluctuate an average of 37%, repeat baseline and treatment phenotyping assessments should be obtained for accurate determination of a given drugs effect on CYP2D6 activity when measured by dextromethorphan.

Pharmacogenetic allele nomenclature: International workgroup recommendations for test result reporting

This article provides nomenclature recommendations developed by an international workgroup to increase transparency and standardization of pharmacogenetic (PGx) result reporting. Presently, sequence variants identified by PGx tests are described using different nomenclature systems. In addition, PGx analysis may detect different sets of variants for each gene, which can affect interpretation of results. This practice has caused confusion and may thereby impede the adoption of clinical PGx testing. Standardization is critical to move PGx forward.

Ontogeny of Hepatic Drug Transporters as Quantified by LC-MS/MS Proteomics.

Protein expression of major hepatic uptake and efflux drug transporters in human pediatric (n = 69) and adult (n = 41) livers was quantified by liquid chromatography / tandem mass spectroscopy (LC‐MS/MS). Transporter protein expression of OCT1, OATP1B3, P‐gp, and MRP3 was age‐dependent. Particularly, significant differences were observed in transporter expression (P < 0.05) between the following age groups: neonates vs. adults (OCT1, OATP1B3, P‐gp), neonates or infants vs. adolescents and/or adults (OCT1, OATP1B3, and P‐gp), infants vs. children (OATP1B3 and P‐gp), and adolescents vs. adults (MRP3). OCT1 showed the largest increase, of almost 5‐fold, in protein expression with age. Ontogenic expression of OATP1B1 was confounded by genotype and was revealed only in livers harboring SLCO1B1*1A/*1A. In livers >1 year, tissues harboring SLCO1B1*14/*1A showed 2.5‐fold higher (P < 0.05) protein expression than SLCO1B1*15/*1A. Integration of these ontogeny data in physiologically based pharmacokinetic (PBPK) models will be a crucial step in predicting hepatic drug disposition in children.

Long‐Read Single Molecule Real‐Time Full Gene Sequencing of Cytochrome P450‐2D6

The cytochrome P450‐2D6 (CYP2D6) enzyme metabolizes ∼25% of common medications, yet homologous pseudogenes and copy number variants (CNVs) make interrogating the polymorphic CYP2D6 gene with short‐read sequencing challenging. Therefore, we developed a novel long‐read, full gene CYP2D6 single molecule real‐time (SMRT) sequencing method using the Pacific Biosciences platform. Long‐range PCR and CYP2D6 SMRT sequencing of 10 previously genotyped controls identified expected star (*) alleles, but also enabled suballele resolution, diplotype refinement, and discovery of novel alleles. Coupled with an optimized variant‐calling pipeline, CYP2D6 SMRT sequencing was highly reproducible as triplicate intra‐ and inter‐run nonreference genotype results were completely concordant. Importantly, targeted SMRT sequencing of upstream and downstream CYP2D6 gene copies characterized the duplicated allele in 15 control samples with CYP2D6 CNVs. The utility of CYP2D6 SMRT sequencing was further underscored by identifying the diplotypes of 14 samples with discordant or unclear CYP2D6 configurations from previous targeted genotyping, which again included suballele resolution, duplicated allele characterization, and discovery of a novel allele and tandem arrangement. Taken together, long‐read CYP2D6 SMRT sequencing is an innovative, reproducible, and validated method for full‐gene characterization, duplication allele‐specific analysis, and novel allele discovery, which will likely improve CYP2D6 metabolizer phenotype prediction for both research and clinical testing applications.

Evaluation of a [13C]-Dextromethorphan Breath Test to Assess CYP2D6 Phenotype

A [13C]‐dextromethorphan ([13C]‐DM) breath test was evaluated to assess its feasibility as a rapid, phenotyping assay for CYP2D6 activity. [13C]‐DM (0.5 mg/kg) was administered orally with water or potassium bicarbonate‐sodium bicarbonate to 30 adult Caucasian volunteers (n = 1 each): CYP2D6 poor metabolizers (2 null alleles; PM‐0) and extensive metabolizers with 1 (EM‐1) or 2 functional alleles (EM‐2). CYP2D6 phenotype was determined by 13CO2 enrichment measured by infrared spectrometry (delta‐over‐baseline [DOB] value) in expired breath samples collected before and up to 240 minutes after [13C]‐DM ingestion and by 4‐hour urinary metabolite ratio. The PM‐0 group was readily distinguishable from either EM group by both the breath test and urinary metabolite ratio. Using a single point determination of phenotype at 40 minutes and defining PMs as subjects with a DOB ≤0.5, the sensitivity of the method was 100%; specificity was 95% with 95% accuracy and resulted in the misclassification of 1 EM‐1 individual as a PM. Modification of the initial protocol (timing of potassium bicarbonate‐sodium bicarbonate administration relative to dose) yielded comparable results, but there was a tendency toward increased DOB values. Although further development is required, these studies suggest that the [13C]‐DM breath test offers promise as a rapid, minimally invasive phenotyping assay for CYP2D6 activity.

Single-Dose Pharmacokinetics of Oral and Intravenous Pantoprazole in Children and Adolescents

The primary objective was to determine the pharmacokinetics of single oral and intravenous doses of pantoprazole in children 2 to 16 years of age. The secondary objective was to assess the safety and tolerability of these doses. Male and female hospitalized and nonhospitalized patients from ages 5 to 16 years received single oral doses (20 mg or 40 mg), and those from ages 2 to 16 years received single intravenous doses (0.8 mg/kg or 1.6 mg/kg) of pantoprazole. The plasma concentration‐time data for each patient were analyzed using noncompartmental methods. Routine safety and tolerability assessments were also obtained. The mean values for peak plasma concentration and total area under the plasma concentration‐time curve increased with increasing dose. Pharmacokinetic values were similar in patients from ages 2 to 16 years and to those previously obtained in adults. Statistically significant differences were observed for dose‐normalized pantoprazole area under the plasma concentration‐time curve when compared between CYP2C19 extensive metabolizers with 1 versus 2 functional alleles. All adverse events were mild in severity and considered to be unrelated to study drug. The pharmacokinetic profile of oral and intravenous pantoprazole was similar in children ages 2 to 16 years. The doses used here were safe and well tolerated in this population.