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
1
Department of Medicine, Division of Clinical Pharmacology, 2Department of Oncology,
3
Department 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)-3OH-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)-3OH-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-OHcholesterol-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 UPLCMS/MS. Upon injection into the liquid chromatograph, analytes were separated with the reversephase Hypersil Gold Column (100 × 2 mm, 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  146
m/z and 642  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 (OctoberDecember).
Supplementary Table S1 Mean Drug Concentrations of Tamoxifen and Metabolites (n=189)
Mean Drug Level ng/ml (SD)
CYP2D6
Genotype (n)
All
Tamoxifen
N-desmethyltamoxifen
(Z)-4-OHtamoxifen
(Z)Endoxifen
3-OHtamoxifen
α-OHtamoxifen
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)
-4
1.99 (0.8)
-4
0.0020 (8.5e )
EM/IM (37)
234.1 (95.0)
547.6 (198.0)
5.50 (2.26)
33.6 (13.5)
0.0016 (5.8e )
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)
-4
IM/IM (6)
304.7 (63.6)
807.1 (209.2)
5.07 (2.39)
29.2 (19.1)
0.0024 (6.4e )
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-OHtamoxifen (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-OHtamoxifen (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-andwhisker 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-OHtamoxifen (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.