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Division of Clinical Pharmacology, Department of Medicine, School of Medicine, Indiana University, Indianapolis, Indiana
(Received December 22, 2004; accepted April 7, 2005)
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Abstract |
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). The determination of CYP2D6 phenotype is widely used in drug interaction and pharmacogenetic clinical studies. Dextromethorphan (DTM) has been traditionally used as a probe to measure in vivo and in vitro CYP2D6 activity (Dayer et al., 1989). The primary enzyme catalyzing the O-demethylation of dextromethorphan is CYP2D6 (Schmid et al., 1985; Kupfer et al., 1986). Although the parent compound and its metabolite concentrations can be determined in different biological fluids (e.g., serum and saliva), their ratio in urine is the most frequently used method to assess CYP2D6 status in vivo. Indeed, the dextromethorphan to dextrorphan urinary metabolic ratio (UMR) is routinely applied to segregate populations into two major phenotypic groups: extensive and poor metabolizers (Hou et al., 1996; Sachse et al., 1997; Tateishi et al., 1999). Although noninvasive and easy to perform, the utility of the urinary metabolic ratio to assess moderate alterations in CYP2D6 activity has not been established (Kohler et al., 1997; Tenneze et al., 1999; Chladek et al., 2000; Yeh et al., 2003). The aim of this report is to compare DMT/DT urinary metabolic ratio, DMT/DT area under the concentration-time curve (AUC) ratio, and DTM oral clearance to assess the ability of UMR to determine the activity of CYP2D6 in vivo.
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Materials and Methods |
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). For the purpose of this report, we focus exclusively on the evaluation of CYP2D6 activity. Subjects. After approval by the Clarian Health Partners, Inc. (Indianapolis, IN) and Indiana University, Purdue University, Indianapolis Institutional Review Board, and Research Involving Human Subjects Committee of the Food and Drug Administration, 12 volunteers participated in the study conducted at the Indiana University General Clinical Research Center after giving written informed consent. Participants (six men and six women, age 31 ± 6 years, weight 79 ± 10 kg) were nonsmokers and had no significant medical conditions as assessed by medical history; physical examination, including electrocardiography; and blood and urine chemistry screens.
Study Design. In the first phase of the study (part 1), the volunteers received a standard light breakfast in the morning after an overnight fast. One hour later, they received 30 mg of dextromethorphan hydrobromide as part of a cocktail of drugs (Gorski et al., 2004) administered orally with 240 ml of water. Blood samples were collected from an indwelling venous catheter at the following times: 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 10, 12, 24, 36, 48, and 72 h after drug ingestion. Urine sampling occurred over the following intervals: 0 to 12, 12 to 24, 24 to 36, 36 to 48, 48 to 60, and 60 to 72 h after oral drug administration. Subjects remained sitting until 4 h after oral dosing and were allowed to eat 2 h after dosing. Part 2 of the study took place 5 to 7 days after completing study part 1; the volunteers began a course of echinacea and on the sixth day of dosing, the study design of part 1 was repeated.
Sample Analysis. The urinary concentrations of dextromethorphan and its metabolites were determined in the 0- to 24-h pooled sample by high-performance liquid chromatography with fluorescence detection (Jones et al., 1996a,b). The serum concentrations of dextromethorphan and dextrorphan were determined by liquid chromatography-mass spectrometry (Gorski et al., 2004).
Pharmacokinetic Analysis. Standard noncompartment pharmacokinetics analyses were used to determine the pharmacokinetic parameters of interest (WinNonlin version 4.0; Pharsight, Mountain View, CA). The terminal elimination rate constant (ß) was determined by log linear regression. The AUC after oral drug administration was determined by a combination of linear and logarithmic trapezoidal methods with extrapolation to infinity [AUC(0-
)]. The oral clearance of DTM was calculated by dividing the oral dose by the DTM AUC.
Statistical Analysis. Data were normally distributed after log transformation and were analyzed using paired t test or by linear regression analysis (SAS/STAT software version 8.2; SAS Institute, Cary, NC). The comparisons between urinary metabolic ratio and other putative CYP2D6 indices were based on the derived slope estimates of the relationship and their corresponding SE, and p values were calculated from a standard normal distribution. In addition, linear mixed models were used to estimate within-subject and between-subject variations. The total sample size for two-group comparisons was calculated assuming the effect size is 30% change, type I error rate is 5%, and the required power is 80%. All the differences were judged significant when p was less than 0.05.
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Results |
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0.3) (Schmid et al.1985) and one as a poor metabolizer. Urine data for one EM was not available. Echinacea did not significantly alter the dextromethorphan oral clearance in the EM (Gorski et al., 2004), and we therefore considered EM dextromethorphan data as replicate determinations and excluded the poor metabolizer data from the analysis. The individual AUC(0-), the oral clearance, and the DTM/DT UMR for the two study phases are presented in Table 1.
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The relationship between the UMR and the oral clearance of DTM and various serum DTM/DT ratios were examined. A poor correlation (r = 0.24; p = 0.04) between the urinary metabolic ratio and the dextromethorphan oral clearance was observed (Fig. 1A). The correlations between the UMR and serum concentration ratios at 3, 4, 6, and 8 h after DTM administration were also low (0.001, 0.10, 0.21, and 0.19, respectively). The DTM/DT AUC ratio was significantly (r = 0.84; p = 0.005) correlated with the dextromethorphan oral clearance (Fig. 1B). Likewise, significant correlations were observed between the oral clearance of DTM and the 3-h (r = 0.60; p = 0.003), 4-h (r = 0.72; p < 0.001), 6-h (r = 0.67; p < 0.001), and 8-h (r = 0.74; p < 0.001) DTM/DT serum concentration ratios. There was also a high correlation between AUC of DTM and the 3-, 4-, 6-, and 8-h serum ratios (r = 0.84, 0.88, 0.79, and 0.82, respectively).
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Discussion |
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; Gorski et al., 2004). The dextromethorphan urinary metabolic ratio is a commonly used method for quantifying CYP2D6 activity in vivo. However, the capability of the urinary metabolic ratio to predict the oral clearance of dextromethorphan has not been evaluated previously. Nevertheless, the correlation of dextromethorphan to dextrorphan ratios in urine and plasma has been examined in several studies, but the results are contradictory. For example, Tenneze et al. (1999) showed that the log DTM/DT metabolic ratio in 24-h urine correlated with the log DTM/DT metabolic ratio in plasma 3 h after DTM administration in CYP2D6 EMs (r = 0.92). In contrast, the analysis of EM healthy subjects data from Kohler et al. (1997) showed a very low correlation between the UMR in 8-h urine collection and in serum 1 h after DTM administration (r = 0.19). Chladek et al. (2000) found good correlation between UMR and 3-h plasma ratio (r = 0.88), and Capon et al. (1996) observed a similar correlation between the UMR and partial clearance of DTM to DT (r2 = 0.82). However, these studies included poor metabolizers in the analysis, and the correlations among EMs alone would be significantly lower.
We assessed the capacity of different parameters to reflect dextromethorphan oral clearance and found that the UMR had the lowest performance compared with the ratio of DTM/DT AUC and other serum concentration ratios. This poor correlation presumably reflects the previously recognized contributions of phenomenon other than CYP2D6 activity to the UMR (Labbe et al., 2000). The phenomena that modulate the UMR, such as urine pH, may also contribute to the increased intra (35 versus 26%) and intersubject (100 versus 68%) variability in UMR with respect to DTM/DT AUC ratio.
The poor correlation between dextromethorphan oral clearance and UMR (r = 0.24) compared with DTM/DT AUC ratio (r = 0.84) makes the UMR a questionable quantitative measure of in vivo CYP2D6 catalytic activity in EMs. This deficiency precludes the use of the UMR in drug-interaction studies that focus on modest changes in CYP2D6 activity. On the other hand, the UMR remains a suitable index for the identification of poor and extensive metabolizers.
The UMR has been the preferred CYP2D6 phenotyping method over plasma sampling because it is easier and less expensive. Nonetheless, taking into account the required sample sizes, the estimation of DTM CL PO seems to be the most efficient strategy.
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Footnotes |
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
ABBREVIATIONS: DTM, dextromethorphan; UMR, urinary metabolic ratio; DT, dextrorphan; AUC, area under the concentration-time curve; EM, extensive metabolizer.
Address correspondence to: Dr. J. Christopher Gorski, Division of Clinical Pharmacology, Indiana University School of Medicine, Wishard Memorial Hospital, W.D. Myers Bldg. W7123, 1001 West 10th St., Indianapolis, IN 46202. E-mail: jcgorski{at}iupui.edu
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