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.

Future Perspective

The findings of this study extend our current knowledge of CYP2D6 and demonstrate that metabolomics methods may be useful in revealing new biomarkers of drug metabolizing enzymes. In this study, the exploration of urinary metabolites led to the detection of a novel ion, M1, which may, lead to its use as an endogenous marker alone or in combination with other biomarkers of CYP2D6 activity. Future studies of M1 could include validation in different ethnic groups, additional pediatric and adult studies and may be useful in studying CYP2D6 activity in pregnant women and patients with kidney or liver disease. Upon further in vitro and in vivo validation, the clinical implementation of a noninvasive, endogenous biomarker test predictive of CYP2D6 phenotype could advance personalized medicine by potentially replacing current phenotyping or genotyping methods. Furthermore, validated endogenous biomarkers would make retrospective analyses of clinical samples possible and may aid in first-in-man studies where there is a concern for DDIs.

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Acknowledgements
The authors would like to thank K Kerr (University of Washington, Seattle, WA, USA) for her statistical consultation.

Ethical conduct of research
The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Table 1. Demographics of the pediatric study population.
Characteristic	All subjects (n = 189)	Training set (n = 94)		Validation set (n = 95)
Characteristic	All subjects (n = 189)	PM (n = 5)	Non-PM (n = 89)	PM (n = 5)	Non-PM (n = 90)
Age (years)	11.2 ± 2.5 (7–16)	11.3 ± 2.4 (9–14)	11.2 ± 2.4 (7–16)	11.0 ± 1.6 (9–14)	11.1 ± 2.5 (7–16)
Tanner stage
– Breast size	2.4 ± 1.5 (1–5)	2.4 ± 1.7 (1–5)	2.4 ± 1.5 (1–5)	1.6 ± 0.5 (1–2)	2.5 ± 1.5 (1–5)
– Pubic hair	2.4 ± 1.5 (1–6)	2.4 ± 1.7 (1–5)	2.3 ± 1.5 (1–6)	1.4 ± 0.5 (1–2)	2.5 ± 1.5 (1–5)
Gender
– Male (%)	61	60	66	80	56
Race (%)
– African–American	43	0	43	20	47
– Caucasian (Hispanic)	49 (5)	100 (0)	46 (4)	80 (0)	47 (6)
– Mixed	8	0	11	0	7
Urinary DM/DX ratio	0.078 ± 0.3 (9.0 × 10⁻⁵–3.3)	1.4 ± 1 (0.63–3.3)	0.014 ± 0.03 (9.0 × 10⁻⁵–0.20)	1.1 ± 0.7 (0.28–1.8)	0.013 ± 0.02 (3.0 × 10⁻⁴–0.14)

Age at visit 1, Tanner stage at enrollment and DM/DX are shown as the mean ± standard deviation (range).
DM/DX: Dextromethorphan-to-dextrorphan metabolic ratio; Non-PM: CYP2D6 intermediate, extensive or ultrarapid metabolizer phenotype subjects; PM: CYP2D6 poor metabolizer phenotype subjects.

Table 2. Summary of significant (Benjamini–Hochberg corrected p-value <0.01) m/z ions by ionization mode associated with log(dextromethorphan-to-dextrorphan metabolic ratio) following global metabolomics analysis of pediatric training set samples by LC-QTOF mass spectrometry.
m/z	RT (min)	Possible identity [Ref.]	Slope	r²	p-value	BH corrected p-value
ESI+ using spot urine samples
444.3102	6.5	–^†	-0.79	0.34	1.69 × 10⁻⁷	6.50 × 10⁻⁴
ESI+ using timed urine samples
421.2056	2.5	–^†	-1.70	0.67	1.31 × 10⁻⁴⁴	5.03 × 10⁻⁴¹
444.3102	6.5	–^†	-0.79	0.27	2.33 × 10⁻⁶	3.80 × 10⁻³
464.2283	3.2	–^†	-0.78	0.40	2.97 × 10⁻⁶	3.80 × 10⁻³
273.1668	5.6	–^†	0.73	0.24	3.97 × 10⁻⁶	3.81 × 10⁻³
434.2196	2.6	Dextrorphan glucuronide [49]	-1.51	0.56	1.09 × 10⁻⁵	7.85 × 10⁻³
445.3135	6.5	–^†	-0.65	0.25	1.23 × 10⁻⁵	7.85 × 10⁻³
ESI- using timed urine samples ^‡
447.1866	6.3	–^†	-2.05	0.70	9.55 × 10⁻⁵²	2.75 × 10⁻⁴⁸
448.1884	6.3	Hydroxy-dextrorphan-glucuronide [41]	-2.23	0.68	1.87 × 10⁻⁴⁴	2.70 × 10⁻⁴¹
446.1827	6.3	Oxo-dextrorphan-glucuronide [41]	-1.91	0.71	1.49 × 10⁻⁴¹	1.43 × 10⁻³⁸
336.1275	3.5	Dextrorphan sulfate [49]	-1.49	0.57	1.63 × 10⁻²⁹	1.18 × 10⁻²⁶
432.1668	4.5	Oxo-hydroxymorphinan-glucuronide [41]	-1.60	0.56	5.77 × 10⁻²³	3.33 × 10⁻²⁰
433.1697	4.5	–^†	-1.73	0.51	1.47 × 10⁻¹⁴	7.08 × 10⁻¹²
418.1878	2.5	3-hydroxymorphinan-glucuronide [49]	-1.14	0.41	1.21 × 10⁻¹⁰	4.97 × 10⁻⁸
193.0358	2.6	–^†	1.03	0.093	2.52 × 10⁻⁵	9.08 × 10⁻³

The possible identity of m/z ions was assigned based on mass.
^†Identity unknown.
^‡No ions were found to be significant in the spot urine collection samples in ESI- mode.
BH: Benjamini-Hochberg; RT: Retention time.

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Executive Summary

Current knowledge on CYP2D6 phenotyping

CYP2D6 is an important drug-metabolizing enzyme encoded by a highly polymorphic and complex gene locus. Commonly used methods of CYP2D6 phenotyping require administration of a probe drug, such as dextromethorphan (DM).

Aim

In this study, global metabolomics was used to identify an endogenous biomarker of CYP2D6 activity in pediatric subjects.

Association & validation of endogenous biomarkers with CYP2D6 activity in pediatric subjects

Spot and timed urine samples were collected in pediatric subjects before and after a single oral dose of DM, respectively.
Following global metabolomics analysis of the urine samples in the training set (n = 94, five poor metabolizers), over 6500 mass features were regressed against the urinary DM metabolic ratio.
An unknown endogenous ion (with m/z 444.3102 at 6.5 min, referred to as M1) detected in ESI+ mode was significantly correlated with the urinary DM metabolic ratio (Benjamini–Hochberg corrected p < 0.01).

Biomarker relationship with CYP2D6 activity in pediatric subjects

Targeted LC-MS/MS analysis of M1 showed that the ion was able to discriminate poor metabolizers from intermediate, extensive and ultrarapid metabolizers in separate training and validation sets of pediatric subjects.
M1 differed among CYP2D6 activity scores (p < 0.0001 for all sets in a one-way ANOVA), albeit with large interindividual variability in each group.
Major metabolomics databases were queried for parent and product ions (following fragmentation), but no matches were found.

Biomarker response to CYP2D6 inhibition in adult subjects

In adult subjects, CYP2D6 inhibition by fluoxetine resulted in over ninefold reduction of urinary abundance of M1 along with decreased dextrorphan metabolite formation from DM.

Conclusion

M1 may be a product of a reaction catalyzed by CYP2D6.
The current study is limited to the ability of M1 to discriminate poor metabolizers from other phenotypes.
Future structural elucidation needs to be performed to identify M1 and its parent compound. The urinary ratio of the parent to the metabolite may provide a more sensitive measure of CYP2D6 activity than M1 alone.
Upon validation as a biomarker, this endogenous metabolite may be used to improve personalized medicine by conveniently and noninvasively ascertaining CYP2D6 phenotype without the use of an exogenous probe in various populations.

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

Future Perspective

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Authors and Disclosures

Authors and Disclosures

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