Detection of an Endogenous Urinary Biomarker Associated With CYP2D6 Activity Using Global Metabolomics

Jessica Tay-Sontheimer; Laura M Shireman; Richard P Beyer; Taurence Senn; Daniela Witten; Robin E Pearce; Andrea Gaedigk; Cletus L Gana Fomban; Justin D Lutz; Nina Isoherranen; Kenneth E Thummel; Oliver Fiehn; J Steven Leeder; Yvonne S Lin

Disclosures

Pharmacogenomics. 2014;15(16):1947-1962. 

In This Article

Results

Demographic Characteristics of Pediatric Subjects

Healthy pediatric volunteers (n = 189), some of whom were related, were recruited for a longitudinal study of CYP2D6 activity. A spot urine sample was collected predose and a timed urine sample was collected from 0–4 h after DM administration for determination of DM/DX. Based on DM/DX, ten subjects were phenotypic CYP2D6 PMs and the remaining 179 were classified as non-PMs (intermediate, extensive or ultrarapid metabolizers). PMs and non-PMs were randomly assigned to training or validation sets. Demographics for the training (n = 94, 5 PMs) and validation sets (n = 95, 5 PMs) are presented in Table 1. The subjects ranged from 7 to 16 years of age. Pubertal development was determined by the assignment of Tanner stage scores on a scale of 1 (prepubertal) to 5 or 6 (full maturation) for breast size and pubic hair, respectively. Log(DM/DX) did not differ by gender, age or measures of pubertal development (data not shown [GAEDIGK A, PEARCE RE, TAY-SONTHEIMER J ET AL . EFFECT OF AGE AND GENOTYPE ON CYP2D6 ACTIVITY IN CHILDREN AND ADOLESCENTS (2014), MANUSCRIPT IN PREPARATION]). However, there was a negative correlation between log(DM/DX) and urinary pH (p < 0.0001, n = 189).

Selection of CYP2D6 Endogenous Biomarkers in Pediatric Subjects

Identification of CYP2D6 Biomarker Candidates by Global Metabolomics in the Pediatric Training Set. Spot and timed urine collection samples in the pediatric training set were analyzed by global metabolomic profiling using LC-QTOF MS in ESI+ and ESI- modes. Ions were aligned, normalized and log-transformed as described in the 'Patients & methods' section. The LC-QTOF data contained 3839 and 2883 ions in ESI+ and ESI- modes, respectively. For selecting ions associated with CYP2D6 phenotype, a generalized-estimating equation approach was used to linearly regress log ion intensities against log(DM/DX). An independent correlation structure was used in this approach to account for subject relatedness. As summarized in Figure 1 & Table 2, in ESI+ data, one and six ions (Benjamini–Hochberg [BH] corrected p-value <0.01) were found in the spot and timed urine samples, respectively; in ESI- data, eight ions (BH corrected p < 0.01) were observed in the timed urine samples. As listed in Table 2, some significant ions found only in timed urine samples are likely DM metabolites, such as DX glucuronide and oxo-dextrorphan-glucuronide.[45] Significant ions found in both urine-collection time points would likely result in fewer false positives and would exclude DM metabolites. For these reasons, only significant ions found both before and after DM administration (spot and timed urine samples, respectively) were further investigated. One ESI+ ion with a m/z of 444.3102 and eluting at 6.5 min, which we will refer to as M1, was found to be significant in both spot and timed urine samples. We focused on this candidate biomarker in subsequent experiments. A negative correlation existed between M1 abundance and log(DM/DX) (Table 2) suggesting that it may be the product of a reaction catalyzed by CYP2D6.

Figure 1.

Manhattan plots of pediatric training dataset ions. The ion abundances were regressed against log(dextromethorphan-to-dextrorphan metabolic ratio). Corresponding p-values were obtained and corrected using the BH method. The corresponding -log(BH corrected p-values) are shown for ESI+ mode (A & B) and ESI- mode (C & D) from LC quadrupole TOF analysis. Results from spot and 0–4 h timed urine samples are presented in (A & C) and (B & D), respectively. Ion m/z and retention time are explicitly shown for significant ions. The BH corrected significance threshold is indicated by the dashed line (p = 0.01).
BH: Benjamini–Hochberg.

Fragmentation of M1. Fragmentation of M1 in a representative urine sample at 20 V by LC-QTOF yielded the MS/MS spectrum shown in Figure 2A. The most abundant product ions were, in order of decreasing abundance, m/z 98.0964 > 370.2732 > 206.1883 > 56.0494 > 55.0550 > 150.1259 > 81.0692. Major metabolomics databases were queried for the potential identity of M1 based on the parent mass and product ions, but no matches were found.

Figure 2.

Product ion spectra of M1 (m/z 444.3102) from a representative pediatric spot urine sample. Spectra were obtained in ESI+ mode via (A) LC-QTOF, precursor mass indicated by a diamond and (B) LC-QqQ, precursor mass not shown. A peak for the mass transition of m/z 444.3→98.1, the most abundant product ion, was (C) undetectable in a representative PM subject after a 15 µl injection and (D) clearly observable in a urine sample in a representative non-PM subject after a 5 µl injection.
LC-QqQ: LC triple-quadrupole; LC-QTOF: LC quadrupole TOF; PM: Poor metabolizer.

Association & Validation of Endogenous Biomarkers With CYP2D6 Activity in Pediatric Subjects

Semiquantitative Analysis of M1 in Pediatric Training Set Samples. To improve sensitivity compared with the LC-QTOF method, we developed a targeted semiquantitative LC-QqQ based assay. Using multiple-reaction monitoring (MRM), we monitored the mass transitions (precursor to product ions) of m/z 444.3→370.3, 444.3→206.2, 444.3→150.1, 444.3→98.1 and 444.3→56.1 (Figure 2B). The mass transition of m/z 444.3→98.1, the most abundant product ion, was selected to determine the relative abundance of M1 in the urine samples. All training set non-PM subjects analyzed had quantifiable levels of M1 in both spot and timed urine samples (Figure 2D). In contrast, M1 levels were undetectable in all but one timed PM sample, even with an injection volume threefold higher than that used for non-PM samples (Figure 2C). For reference, when urine with approximately the mean M1 abundance was diluted 264-fold, a peak was still visually observable (data not shown). In samples with undetectable M1 signals, a low value was assigned as the M1 abundance. M1 peak intensities were normalized by urinary creatinine concentration to account for differences in urine concentration for each sample. As M1 may be the product of a reaction catalyzed by CYP2D6, we expressed the ratio as creatinine/M1 to match the DM/DX, where the parent (DM) is normalized to the metabolite (DX). Compared to non-PM subjects, PM subjects had 105-fold higher DM/DX and 123- and 147-fold higher creatinine/M1 ratios in spot and timed urine samples, respectively (Figure 3).

Figure 3.

Phenotypic measures of CYP2D6 in the pediatric training set for poor metabolizer (n = 5) and non-poor metabolizer (n = 89) groups. (A) DM/DX was determined in timed urine samples. Creatinine/M1 was measured by LC-MS/MS in (B) spot and (C) timed urine samples. A low value was assigned for samples with undetectable M1 abundances. Lines depict the median and boxes represent the 25th and 75th percentiles. The 10th and 90th percentiles are indicated by the whiskers. As PM and non-PM status was determined by DM/DX, the p-value is not reported in (A). Similarly, as M1 was identified as a marker of CYP2D6 using DM/DX in this same set of urine samples, the p-values are not reported in (B &C). Open boxes show spot urine samples and gray shaded boxes show timed urine samples.
DM/DX: Dextromethorphan-to-dextrorphan metabolic ratio; PM: Poor metabolizer.

Confirmation of CYP2D6 Biomarker Candidate in the Pediatric Validation Set. Similar results were found in the pediatric validation set. M1 abundance was undetectable in all PM subjects and a low value was assigned to M1 in these cases. A significant and positive relationship was observed between log(creatinine/M1) and log(DM/DX) in spot and timed urine samples (r2 = 0.31 and 0.28, respectively; p < 0.0001 for each). Compared to non-PM subjects, PM subjects had 82-fold higher DM/DX and 167- and 120-fold higher creatinine/M1 (p < 0.0001) in spot and timed urine samples, respectively (Figure 4).

Figure 4.

Phenotypic measures of CYP2D6 in the pediatric validation set for poor metabolizer (n = 5) and non-poor metabolizer (n = 90) subjects. (A) DM/DX was determined in timed urine samples. Creatinine/M1 was measured by LC-MS/MS in (B) spot and (C) timed urine samples. A low value was assigned for samples with undetectable M1 abundances. Lines depict the median and boxes represent the 25th and 75th percentiles. The 10th and 90th percentiles are indicated by the whiskers. Statistical analyses were performed using unpaired t-tests. As PM and non-PM status was determined by DM/DX, the p-value is not reported in (A). Open boxes show spot urine samples and gray shaded boxes show timed urine samples.
DM/DX: Dextromethorphan-to-dextrorphan metabolic ratio; PM: Poor metabolizer.

Relationship Between CYP2D6 Activity Score & Phenotypic Activity as Determined by DM/DX and M1 in Pediatric Subjects. Assigning an activity score to each child allowed us to group individuals by genotypes with comparable levels of function. In both the training and validation sets, 5% of the subjects had an activity score of 0 (PMs), 91–92% of the subjects had activity scores between 0.5 and 2 owing to the presence of partially or fully functional alleles and 3% of subjects had activity scores greater than 2. Despite the large interindividual variability observed in each activity score group, both DM/DX and creatinine/M1 differed among the activity score groups (p < 0.0001 for all sets, Figure 5).

Figure 5.

The relationship between phenotypic measures of CYP2D6 activity and CYP2D6 activity score. DM/DX and creatinine/M1 as measured by LC triple-quadrupole in subjects from the (A & C) training (n = 94) and (B & D) validation (n = 95) datasets are shown. Lines depict the median and boxes represent the 25th and 75th percentiles. The 10th and 90th percentiles are indicated by the whiskers. Spot and timed urine samples are indicated with open and shaded boxes, respectively. DM/DX and creatinine/M1 ratios differed by activity score (p < 0.0001) as determined by one-way ANOVA.
DM/DX: Dextromethorphan-to-dextrorphan metabolic ratio; PM: Poor metabolizer.

Biomarker Response to CYP2D6 Inhibition in Adult Subjects

Effect of CYP2D6 Inhibition on M1 in Adult Subjects. The ability of M1 to reflect alterations in CYP2D6 activity was tested in adults. As part of a DDI study, 10 subjects received multiple doses of oral fluoxetine, a potent CYP2D6 inhibitor. Urine was collected before and during fluoxetine treatment. The urinary DM/DX increased 218-fold during fluoxetine treatment (p < 0.0001, Figure 6A) compared with baseline. Creatinine/M1 ratios were increased by 9.56-fold during the treatment phase (p = 0.029, Figure 6B). In addition, log(DM/DX) and log(creatinine/M1) were correlated (Spearman r = 0.55, p = 0.012; Figure 6C).

Figure 6.

The effect of CYP2D6 inhibition by fluoxetine treatment on phenotypic markers of CYP2D6 activity in adult subjects. CYP2D6 activity was measured by (A) DM/DX or (B) creatinine/M1 in subject urines. (C) The relationship between log(DM/DX) and log(creatinine/M1). Open diamonds indicate subjects without fluoxetine treatment, gray shaded diamonds show subjects with fluoxetine treatment. Statistical analyses were performed using paired t-tests or the Spearman rank correlation.
DM/DX: Dextromethorphan-to-dextrorphan metabolic ratio.

Comments

3090D553-9492-4563-8681-AD288FA52ACE

processing....