Supplementary Material For

Supplementary Material for

CYP3A4 and seasonal variation in vitamin D status in addition to CYP2D6 contribute to therapeutic endoxifen level during tamoxifen therapy

Wendy A. Teft1, Inna Y. Gong1,5, Brian Dingle2, Kylea Potvin2, Jawaid Younus2, Theodore A. Vandenberg2, Muriel Brackstone2,3, Francisco E. Perera2, Yun-Hee Choi4, Guangyong Zou4, Robin M. Legan1, Rommel G. Tirona1,5, and Richard. B. Kim1,2,5

1Department of Medicine, Division of Clinical Pharmacology, 2Department of Oncology, 3Department of Surgery, 4Department of Epidemiology and Biostatistics and 5Department of Physiology and Pharmacology, University of Western Ontario

Supplementary Methods

Tamoxifen and Metabolite Measurement

Plasma concentrations of tamoxifen and metabolites (all standards were obtained from Toronto Research Chemicals, Toronto, ON, Canada) were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Plasma samples (100 µl) were precipitated by the addition of 3 volumes of acetonitrile spiked with 15 µl of internal standard (Tamoxifen-D5, 500 ng/ml; Toronto Research Chemicals). Samples were mixed, centrifuged and diluted in 5mM ammonium acetate, pH 4. Upon injection into the liquid chromatograph (Agilent 1200) analytes were separated on a reverse-phase column (Hypersil Gold, 50 x 5 mm, 5 µM particle size) using gradient elution with 5mM ammonium acetate, pH 4 and acetonitrile (30-80%) with an 8 minute run time. Standard curves and quality control samples were prepared in drug free plasma. The mass spectrometer (Thermo TSQ Vantage) with heated electrospray ionization source was set in positive mode for detection of tamoxifen, N-desmethyl-tamoxifen, (Z)-4-OH-tamoxifen, (Z)-3-OH-tamoxifen, (Z)-α-OH-tamoxifen, (Z)-endoxifen, and tamoxifen-D5 with transitions 372 → 72 m/z, 399 → 58 m/z, 388 → 72 m/z, 388 → 72 m/z, 388 → 72 m/z, 374 → 58 m/z and 377 → 72 m/z, respectively. The co-efficient of variation (%) for quality controls were as follows: low QC (9.9%), Medium QC (9.5%) and High QC (2.5%). We note that the measurement of (Z)-3-OH-tamoxifen, (Z)-α-OH-tamoxifen was conducted by relative quantification rather than absolute quantification.

Normalized 4-β-OH-cholesterol Measurement

The cholesterol metabolite 4-β-OH-cholesterol (4-β-HC) has been proposed as a endogenous marker of CYP3A4 activity.1 Thus, the normalized ratio of cholesterol-based metrics, 4-β-HC to total cholesterol (4-β-HC/Total-C) was determined in patients. Free plasma cholesterol concentrations were quantified using an in vitro colorimetric method as per manufacture’s protocol (Wako Ltd, Osaka, Japan). Plasma concentrations of 4-β-OH-cholesterol (Avanti Polar Lipids, Alabaster, AL) were determined using ultra high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, Agilent 1290 UPLC coupled with ThermoScientific TSQ Quantum). Briefly, saponification of plasma samples with added internal standard (4-β-OH-cholesterol-D7) was carried out with 0.5 mL of 1 M ethanolic KOH at 37°C for 1 hr. Following addition of 0.15 mL of water, sterols were extracted twice with 1 mL of hexane and allowed to evaporate to dryness. Subsequently, the reagent mixture composed of 2-methyl-6-nitrobenzoic anhydride, 4-dimethylaminopyridine, picolinic acid, pyridine, and triethylamine, was added to the dried extract and incubated at 80°C for 1 hr to derivatize the sterols. The resulting mixture was extracted with hexane and allowed to to evaporate to dryness. The residue was reconstituted in 100 μL of acetonitrile and aqueous solution (4:1 v/v) and 20 μL was injected into the UPLC-MS/MS. Upon injection into the liquid chromatograph, analytes were separated with the reverse-phase Hypersil Gold Column (100 × 2mm, 1.9 µM) using mobile phases of water and 1:1 v/v acetonitrile:methanol in a 22.5 min run time. The MS was set in positive ionization mode for detection of 4-β-OH-cholesterol and 4-β-OH-cholesterol-D7 of respective transitions 635 à 146 m/z and 642 à 146 m/z. The co-efficient of variation (%) for quality controls were as follows: low QC (14.4%), Medium QC (8.9%) and High QC (12.4%).

Plasma Vitamin D Measurement

Plasma 25-hydroxy-Vitamin D (D2 and D3) levels were measured by ELISA as per manufacture’s protocol (BioVendor, Candler, NC).

Statistical Analysis

CYP2D6 inhibitory medications taken concurrently with tamoxifen at the time of blood sampling, including selective serotonin reuptake inhibitors (SSRIs) and serotonin-norepinephrine reuptake inhibitors (SNRIs) were classified as strong (buproprion, paroxetine, fluoxetine), moderate (duloxetine, sertraline) and mild (citalopram, escitalopram, venlafaxine, desvenlafaxine) inhibitors. Patients were classified into CYP2D6 genotype categories according to CYP2D6 genotype as follows: ultrarapid metabolizers: UM, (greater than two CYP2D6 copies lacking variant alleles), extensive metabolizers: EM/EM (lacking variant alleles), intermediate metabolizers: EM/IM (one intermediate allele), EM/PM (one poor allele), IM/IM (two intermediate alleles), IM/PM (one intermediate and one poor allele) and poor metabolizers: PM/PM (two poor alleles). Dates of blood draw for each patient were categorized into seasons: Winter (January-March); Spring (April-June), Summer (July-September), Fall (October-December).

Supplementary Table S1 Mean Drug Concentrations of Tamoxifen and Metabolites (n=189)
CYP2D6
Genotype (n) / Mean Drug Level ng/ml (SD)
Tamoxifen / N-desmethyl-tamoxifen / (Z)-4-OH-tamoxifen / (Z)-Endoxifen / 3-OH-tamoxifen / α-OH-tamoxifen
All / 265.1 (133.4) / 635.8 (264.6) / 5.55 (2.34) / 31.8 (16.2) / 0.0021 (8.9e-4) / 2.08 (1.0)
UM (3) / 263.8 (128.7) / 521.6 (279.0) / 8.23 (2.31) / 49.0 (25.4) / 0.0021 (8.8e-4) / 1.61 (0.8)
EM/EM (68) / 268.1 (144.0) / 585.1 (227.7) / 6.27 (2.56) / 39.2 (17.4) / 0.0020 (8.5e-4) / 1.99 (0.8)
EM/IM (37) / 234.1 (95.0) / 547.6 (198.0) / 5.50 (2.26) / 33.6 (13.5) / 0.0016 (5.8e-4) / 1.77 (0.8)
EM/PM (54) / 261.6 (106.8) / 678.2 (219.5) / 5.42 (1.75) / 26.8 (11.3) / 0.0022 (9.6e-4) / 2.16 (0.9)
IM/IM (6) / 304.7 (63.6) / 807.1 (209.2) / 5.07 (2.39) / 29.2 (19.1) / 0.0024 (6.4e-4) / 2.46 (0.6)
IM/PM (11) / 253.0 (181.0) / 675.0 (413.3) / 3.73 (1.42) / 16.5 (11.0) / 0.0024 (1.1e-4) / 2.25 (1.2)
PM/PM (10) / 369.0 (241.6) / 958.3 (461.9) / 5.16 (2.66) / 15.3 (6.9) / 0.0030 (1.2e-4) / 3.08 (2.1)
Abbreviations: UM, ultrarapid metabolizer; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer
Supplementary Table S2 Logistic Model for Prediction of Endoxifen Concentrations in Therapeutic Range (C-statistic = 89%)
Predictor Variable / Log Odds Ratio (β) / Standard Error / P
Intercept / 6.087 / 2.307 / 0.084
CYP2D6, UM/EM or EM/EM† / - 5.572 / 1.042 / <0.001
CYP2D6, EM/IM or EM/PM† / -3.838 / 0.763 / <0.001
SSRI/SNRI, Strong† / 7.020 / 1.657 / <0.001
SSRI/SNRI, Moderate or Mild† / 2.178 / 0.867 / 0.012
Venlafaxine Use† / 2.039 / 0.815 / 0.012
Season, Spring or Summer† / 1.799 / 0.720 / 0.012
CYP3A4*22, C/C† / - 1.900 / 0.616 / 0.002
Age, years / - 0.045 / 0.028 / 0.106
BMI / - 0.113 / 0.053 / 0.033
Area under the ROC curve is termed C-statistic
† True = 1, False = 0
P (Falling in Therapeutic Endoxifen Range) = eXβ /(1 + eXβ)
X is a vector of predictor variables, β is a vector of corresponding coefficients
BMI, body mass index; EM, extensive metabolizer; IM, intermediate metabolizer; PM, poor metabolizer; SSRI, selective serotonin reuptake inhibitor; SNRI, serotonin-norepinephrine reuptake inhibitor; UM, ultrarapid metabolizer
Supplementary Table S3 The Observed and the Predicted Frequencies for Therapeutic Endoxifen Levels by Logistic Regression with the Cut-off Value of 0.8
Predicted
Observed / No / Yes / % Correct
No / 30 / 9 / 81%
Yes / 28 / 121 / 77%
Sensitivity = 121/(121+28)% = 81%
Specificity = 30/(30+9)% = 77%
False positive = 9/(9+30)% = 23%
False negative = 28/(28+121)% = 19%

Supplementary Figure S1 Endoxifen concentrations at primary and follow up visits. Trough plasma levels of endoxifen were measured at initial visit and at 6 and 12 month follow-up visits. The top and bottom of the box-and-whisker plots represents 25th and 75th percentile, respectively; median is represented by the middle line, whiskers are the 95% CI.

Supplementary Figure S2 Influence of CYP2D6 genotype on tamoxifen metabolite concentrations. Trough plasma levels of tamoxifen (A), NDM-tamoxifen (B) 4-OH-tamoxifen (C) α-OH-tamoxifen (D), and 3-OH-tamoxifen (E) stratified by CYP2D6 genotype. The top and bottom of the box-and-whisker plots represents 25th and 75th percentile, respectively; median is represented by the middle line, whiskers are the 95% CI. *P<0.05

Supplementary Figure S3 Influence of CYP2D6 genotype on endoxifen to parent metabolic ratios. Metabolic ratios of endoxifen to tamoxifen (A), N-desmethyl-tamoxifen (B), and 4-OH-tamoxifen (C) stratified by CYP2D6 genotype. The top and bottom of the box-and-whisker plots represents 25th and 75th percentile, respectively; median is represented by the middle line, whiskers are the 95% CI. ***P<0.0001

Supplementary Figure S4 Correlation between endoxifen and primary metabolites. Scatter plot and linear regression of plasma concentrations of endoxifen and metabolites 4-OH-tamoxifen (A) and N-desmethyl-tamoxifen (B) in CYP2D6 EM/EM patients.

Supplementary Figure S5 Influence of additional enzymes and transporters on endoxifen concentration. Trough plasma concentrations of endoxifen were not significantly associated with CYP3A5*3 (A), P450 oxidoreductase (B) CYP2C9*2 or *3 (C) CYP2B6*4, *5 or *6 (D), BCRP 34 G>A, 421 C>A (E) or MDR1 3435 C>T (F). The top and bottom of the box-and-whisker plots represents 25th and 75th percentile, respectively; median is represented by the middle line, whiskers are the 95% confidence interval (CI).

Supplementary Figure S6 Influence of CYP3A4 genotype on tamoxifen and metabolite concentrations. Trough plasma levels of NDM-tamoxifen (A), 4-OH-tamoxifen (B) α-OH-tamoxifen (C), and 3-OH-tamoxifen (D) stratified by CYP3A4*22 genotype. The top and bottom of the box-and-whisker plots represents 25th and 75th percentile, respectively; median is represented by the middle line, whiskers are the 95% CI. Scatter plot and linear regression between the metabolic ratio of NDM-tamoxifen/tamoxifen (E) and 4-OH-tamoxifen/tamoxifen (F) to 4-β-OH-cholesterol/total cholesterol.

Supplementary Figure S7 Correlation between endoxifen and Vitamin D. Scatter plot and linear regression of endoxifen and 25-OH-Vitamin D levels.