Journal of
Personalized
Medicine
Article
Pharmacokinetics of Tamoxifen and Its Major Metabolites and
the Effect of the African Ancestry Specific CYP2D6*17 Variant
on the Formation of the Active Metabolite, Endoxifen
Comfort Ropafadzo Kanji 1,2, * , Georginah Nyabadza 1 , Charles Nhachi 2 and Collen Masimirembwa 1,3, *
1
2
3
*
Citation: Kanji, C.R.; Nyabadza, G.;
Nhachi, C.; Masimirembwa, C.
Pharmacokinetics of Tamoxifen and
Its Major Metabolites and the Effect
African Institute of Biomedical Science and Technology (AIBST), Harare P.O. Box 2294, Zimbabwe
Department of Clinical Pharmacology, University of Zimbabwe College of Health Sciences,
Harare P.O. Box 2294, Zimbabwe
Sydney Brenner Institute for Molecular Bioscience (SBIMB), University of the Witwatersrand,
Johannesburg 2000, South Africa
Correspondence: ckanji@aibst.edu.zw (C.R.K.); cmasimirembwa@aibst.edu.zw (C.M.)
Abstract: Tamoxifen (TAM) is widely used in the treatment of hormone receptor-positive breast
cancer. TAM is metabolized into the active secondary metabolite endoxifen (ENDO), primarily by
CYP2D6. We aimed to investigate the effects of an African-specific CYP2D6 variant allele, CYP2D6*17,
on the pharmacokinetics (PK) of TAM and its active metabolites in 42 healthy black Zimbabweans.
Subjects were grouped based on CYP2D6 genotypes as CYP2D6*1/*1 or *1/*2 or *2/*2 (CYP2D6*1 or
*2), CYP2D6*1/*17 or 2*/*17, and CYP2D6*17/*17. PK parameters for TAM and three metabolites
were determined. The pharmacokinetics of ENDO showed statistically significant differences among
the three groups. The mean ENDO AUC0-∞ in CYP2D6*17/*17 subjects was 452.01 (196.94) h·*ng/mL,
and the AUC0-∞ in CYP2D6*1/*17 subjects was 889.74 h·ng/mL, which was 5-fold and 2.8-fold lower
than in CYP2D6*1 or *2 subjects, respectively. Individuals who were heterozygous or homozygous for
CYP2D6*17 alleles showed a 2- and 5-fold decrease in Cmax, respectively, compared to the CYP2D6*1
or *2 genotype. CYP2D6*17 gene carriers have significantly lower ENDO exposure levels than
CYP2D6*1 or *2 gene carriers. Pharmacokinetic parameters of TAM and the two primary metabolites,
N-desmethyl tamoxifen (NDT) and 4-hydroxy tamoxifen (4OHT), did not show any significant
difference in the three genotype groups. The African-specific CYP2D6*17 variant had effects on
ENDO exposure levels that could potentially have clinical implications for patients homozygous for
this variant.
of the African Ancestry Specific
CYP2D6*17 Variant on the Formation
of the Active Metabolite, Endoxifen. J.
Pers. Med. 2023, 13, 272. https://
Keywords: tamoxifen; pharmacogenetics; CYP2D6*17; genetic polymorphism; pharmacokinetics; metabolism
doi.org/10.3390/jpm13020272
Academic Editor: Youssef Daali
Received: 6 December 2022
Revised: 13 January 2023
Accepted: 25 January 2023
Published: 31 January 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1. Introduction
Tamoxifen (TAM) has been the mainstay, adjuvant, and neoadjuvant treatment for
estrogen receptor-positive (ER+) breast cancer for more than 40 years [1]. TAM is a selective
oestrogen receptor modulator (SERM) that works by inhibiting estrogen binding at the
receptor site, thereby inhibiting the hormone’s cell proliferation role. Five-year treatment
with TAM in ER+ breast cancer patients has been observed to reduce the mortality rate by
a third [2] and the recurrence rate by 30–50% [3]. However, about 30–50% of women on
TAM therapy experience disease recurrence [4,5]. Determinants for TAM treatment efficacy
includes genetics [6], drug-drug interactions [7], and poor treatment compliance [8].
TAM is a prodrug that requires extensive metabolism via the cytochrome P450 (CYP)
enzymes to its metabolites N-desmethyl tamoxifen (NDT), 4-hydroxy tamoxifen (4OHT)
and endoxifen (ENDO) to elicit anti-tumor activity [9]. The metabolites 4OHT and ENDO
have 100-fold more affinity for the target oestrogen receptor and 30-to-100-fold more
potency than TAM the parent drug. The secondary metabolite of TAM, ENDO, which is
J. Pers. Med. 2023, 13, 272. https://doi.org/10.3390/jpm13020272
https://www.mdpi.com/journal/jpm
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responsible for most of TAM’s anti-tumour effects, is mainly produced by the action of
CYP2D6 on NDT [10,11]. Genetic variants in the gene coding for the CYP2D6 enzyme
may lead to reduced enzyme activity (12). CYP2D6 activity determines ENDO levels, and
genetic variants or drug interactions that affect CYP2D6 activity affect the ENDO exposure
levels in vivo (13). Several studies have observed impaired formation of ENDO in carriers
of CYP2D6 null or reduced function enzyme variants [12–14].
Drug-gene or drug-drug interactions may influence TAM treatment outcomes [15]. The
role of TAM pharmacogenetics in breast cancer treatment outcomes has been extensively
studied in Caucasian populations [16] The majority of the studies found that patients with
the poor CYP2D6 metabolizer status (PM) responded poorly to TAM. The observed poor
response was due to reduced capacity to produce the active metabolite ENDO [14,17,18].
The CYP2D6*4 variant is associated with majority of PM enzyme status in Caucasians, with
a frequency of 20% [19]). This variant is less prevalent in Asian and African populations,
where its frequency distribution is less than 2% [20]. However, Asian populations have a
high prevalence of the low activity variant CYP2D6*10, which confers reduced metabolic
activity. The frequency of CYP2D6*10 in the Asian population is 41.17% [21].
Seven [5,22–25] retrospective studies of patients of Asian ethnicity, reported the association of the CYP2D6 genotype with clinical outcomes in Asian populations. CYP2D6*10
is the major variant in the Asian population. Four of the seven studies reported a significant reduction in progression-free survival, disease-free survival or time to disease
progression odds ratio in patients homozygous for CYP2D6*10 compared with wild-type
homozygotes. Recent studies in Japanese patients have demonstrated the potential utility
of this knowledge to be applied in dose adjustment, where the dose of TAM was increased
from 20 mg/day to 30 and 40 mg/day with improved efficacy and no apparent increase
in adverse drug effects. The higher dose led to the intermediate metabolizer’s (IM) patients producing the active metabolite, ENDO, comparable to that produced by extensive
metabolizers when given the standard dose of 20 mg/day.
CYP2D6*4 and CYP2D6*10 variants have been observed at very low to intermediate
frequencies in African populations, with reported allele frequencies ranging from 0–12%
for CYP2D6*4 and 0–19% for CYP2D6*10 [26–29]. However, African populations have a
high frequency of a CYP2D6 variant unique to people of African origin, CYP2D6*17, that
exists at a frequency of more than 34% in these populations [30]. As there are limited
human in vivo single-dose studies assessing the effect of CYP2D6*17 carrier status on the
metabolism of TAM, we conducted a single-dose pharmacokinetic study to determine the
effect of the CYP2D6*17 variant on the pharmacokinetics of TAM and its metabolites.
2. Materials and Methods
2.1. Trial Design
We conducted an open label, three parallel arms, single treatment, single oral dose
clinical study in healthy subjects at the African Institute of Biomedical Science and Technology, Harare, Zimbabwe. The study design and workflow are shown in Figure 1. Forty-two
eligible, healthy males and females were enrolled sequentially between April 2019 and
August 2021. Participant demographics are presented in Table 1. Each individual signed
an informed consent form. The study was approved by the Medical Research Council
of Zimbabwe (MRCZ) and the Medicines Control Authority of Zimbabwe (MCAZ). Two
weeks prior dose administration until study completion, subjects were not allowed to
smoke, take alcohol, take prescribed or over the counter medication, or recreational drugs.
During the screening process, blood samples were obtained to determine the CYP2D6
genotype. Subjects eligible for inclusion in the study were carriers of the following CYP2D6
genotypes: CYP2D6*1/*1 or *1/*2 or *2/*2 (CYP2D6*1 or *2), CYP2D6*1/*17 or *2/*17 and
CYP2D6*17/*17. Subjects were grouped equally into three arms of 14 subjects based on the
CYP2D6 genotypes: Arm1 *1/*1 or *1/*2 or *2/*2, Arm 2 *1/*17 or *2/*17 and Arm 3 *17/*17.
Each participant received a single oral dose of 20 mg TAM. Blood samples were collected
prior to the dose, followed by extensive sampling in the first 24 h, during which subjects
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were housed at the clinical trial unit for 44 h. The subjects were housed at the clinical trial
unit from 18 h before dose administration to 26 h after administration. Daily sampling at
24-h intervals until 504 h post-dose administration was conducted were subjects visited the
clinical trial unit daily. This resulted in a total of 34 samples per participant. Blood samples
were collected to measure the plasma concentrations of TAM and its metabolites.
Figure 1. Study design and sequence flow chart for the pharmacokinetic study to determine the effect
of CYP2D6*17 on the metabolism of TAM to ENDO.
Table 1. Baseline characteristics of subjects enrolled in the study.
Participant Baseline Characteristics
N1
Female (N, [%])
Male (N, [%])
Sex
Race
Age: years (median, [range])
Weight: kg (median, [range])
Height: cm (median, [range])
BMI: kg/m2 (median,] [range])
1
N is the number of subjects.
42
3 [7]
39 [93]
Black
23.4 [19.1–30.2]
65.0 [53–89]
173.0 [155–192]
22.5 [19.2−26.7]
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2.2. CYP2D6 Genotype
A 4-mL blood sample was collected in an EDTA tube (standard clinical purple-top
tube) and kept on ice. Within 1 h of collection, the samples were processed and aliquots
stored at −20 ◦ C until analysis. The CYP2D6 genotype for the *1 (wild-type) or *2(rs16947,
normal activity) and *17 (rs28371706, reduced activity) was determined using TaqMan
chemistry on the GenoPharm® custom open array. Supplementary Table S1 contains the
variants and SNP ids that were investigated. In brief, the CYP2D6 enzyme was tested
for genetic variation. As per manufacturers protocol DNA was extracted from 200 µL of
peripheral whole blood using the MagMAX™ DNA Multi-Sample Ultra 2.0 Kit on the Thermofisher KingFisher™ Flex Purification System with the MagMAX Ultra 2.0–200 µL script
for KingFisher Flex. Extracted DNA was quantified with the Qubit 4 fluorometer using the
Qubit dsDNA BR Assay Kit and stored at −20 ◦ C short term before analysis. Genotyping
for CYP2D6 was performed on the GenoPharm® custom open array panel as per the manufacturer’s protocol. In brief, a reaction mixture of 5 µL genomic DNA and 5 µL of TaqMan™
Genotyping master mix (Cat. No. 4462164) was prepared per sample. The PCR mix was
transferred to the GenoPharm® custom open array panel using the automated Applied
Biosystems™ QuantStudio™ 12K Flex OpenArray™ AccuFill™ System according to the
manufacturer’s instructions. A no template control (reaction mixture with all reagents but
no template DNA) was included in each run. The 33 nl reaction mix was run per data point
on the Applied Biosystems™ QuantStudio™ 12K Flex Real-Time PCR System (Thermo
Fisher Scientific, Marsiling Industrial Estaste, Singapore). Genotypes for the samples were
determined by the TaqMan™ Genotyper Software as per the manufacturer’s instructions.
Genotype calls were generated with TaqMan® Genotyper Software. The CYP2D6 copy
number was determined using the Applied Biosystems TaqMan copy number assays for
exon 9, the primary copy number assay (Assay ID: Hs00010001_cn) to quantify CYP2D6
duplications or identify CYP2D6 gene deletions (CYP2D6*5) in the samples. AlleleTyper™
software was used to convert sample genotype information for the CYP genes interrogated
to the star (*) allele nomenclature using a predefined allele translation table that maps a
specified allele pattern to the star allele call.
2.3. Measurement of Plasma Tamoxifen and Metabolite Concentrations
At each sampling time point, a 4-mL blood sample was collected in an EDTA tube
(standard clinical purple-top tube) and stored on ice. The following time points were used
in this study, 0, 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 12, 16, 24, 48, 72, 96, 120, 144, 168, 192, 216, 240,
264, 288, 312, 336, 360, 384, 408, 432, 456, 480 and 504 h. Within 1 h of collection, the blood
was centrifuged (3000 rpm for 10 min at 4 ◦ C), and the plasma was isolated and stored at
−80 ◦ C.
2.4. Bioanalysis of Tamoxifen and Metabolites
Plasma samples were extracted using protein precipitation with ice cold acetonitrile
as the extraction solvent. Briefly, 200 µL of plasma was spiked with 10 µL of 2 µg/mL
propranolol (internal standard) followed by addition of 590 µL of ice-cold acetonitrile. The
mixture was vortexed for 30 s, sonicated for 2 min before centrifugation at 16,000× g for
10 min. A volume of 700 µL of supernatant was collected and was evaporated to dryness
under a gentle stream of nitrogen. The dried residue was reconstituted in 50 µL of mobile
phase and 10 µL was injected into the LC/MS-MS for analysis.
2.5. LC-MS/MS Conditions
A 3200 Q TRAP Series triple quadrupole (Applied Biosystems MDS SCIEX, Toronto,
Canada) liquid chromatography- mass spectrometry (MS/MS) system coupled to an Agilent 1100 series HPLC system (Agilent Technologies, Waldbronn, Germany) was used to
carry out all the analysis operated using Analyst software version 1.6 (AB SCIEX, Toronto,
Canada). The compounds of interest were separated using Zorbax C18 2.1 X100 mm,
3.5 µm column (Zorbax Agilent, Santa Clara, CA, USA). The mobile phase consisted of
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0.1% formic acid in 10mM ammonium formate solution as mobile phase A and 0.1% formic
acid in acetonitrile as mobile phase B delivered using a gradient elution: 0–9 min, B 30%,
9.01–9.5 min, B 52%- and 9.5–13-min B 30%. The column was maintained at a temperature
of 40 ◦ C.
Analytes were followed using multiple reaction monitoring (m/z 372.5→ 72.2, 374.4→
58.1, 358.4→ 58.0, 388.4→ 72.2 and 260.3→ 183.3 for TAM, ENDO, NDT, 4OHT and propranolol as internal standard (IS) respectively). MS/MS analyses were performed in positive
ionization mode, ion source temperature 500 ◦ C, curtain gas 25, ion spray voltage 5500,
GS1 and GS2 gas 50 and 30 respectively. The lower limit of quantification was 0.05 ng/mL
for 4OHT and 0.1 ng/mL for TAM, ENDO and NDT with the standard curve linear in the
range between 0.01–1000 ng/mL.
2.6. Pharmacokinetic Analysis
Pharmacokinetic parameters were estimated from plasma concentrations using Non
compartmental analysis (NCA) in WinNonlin software version 8.2 (Certara). The area
under the curve (AUC) from time of dosing to the last quantifiable concentration (AUClast )
and infinity (AUC0-∞ ) was estimated using the linear and logarithmic trapezoidal rule.
The linear up and log down method was used. The elimination rate constant (Kel) was
determined by the program using nonlinear regression of the natural logarithm of concentration values in the elimination phase. The terminal half-life (Thalf ) was calculated using
the equation Thalf = ln2/λ. The apparent clearance, (CL/F) was determined from the equation CL/F = Dose/AUCinf. PK profiles were plotted as graphs of TAM and metabolites
concentrations vs. time.
TAM dose increase estimation to predict the ENDO steady-state plasma concentrations
(Css ) and achieve therapeutic levels of ENDO in patients carrying the CYP2D6*17/*17
was done in Phoenix WinNonlin using the non-parametric superposition tool. As per
Phoenix WinNonlin users guide, the non-parametric superposition object in Phoenix is
based on non-compartmental results representing single-dose data in order to forecast
drug concentrations after numerous doses at steady state. The predictions are based
on an accumulation ratio estimated from the terminal slope, which can be utilized for
simple (constant dose) or elaborate dosing plans (based on the Phoenix WinNonlin User’s
Guide). The simulated TAM dose was increased from 20 mg/day to 30 mg/day to finally
40 mg/mL.
2.7. CYP2D6*17 Activity Score and Calibration Curve
Metabolic ratio (MR) for NDT to ENDO were determined as concentration of ENDO
divided by concentration of NDT. Standard curve for the activity as (MR) of ENDO/NDT
vs. predicted activity score based on the consensus activity score was performed using
external calibration as reported by L. Thorén and colleagues [12]. Linearity was assessed
by linear regression of the calibration curve. Metabolic ratios from this study were used to
interpolate the predicted activity score.
3. Statistical Analysis
To detect a 25% difference in the Cmax AUC0-∞ of ENDO between CYP2D6 normal
metabolizer (NM) phenotype and CYP2D6 IM phenotype, with a two-sided 5% significance
level, power of 80%, and allowing 20% subject dropout. The study required 14 subjects per
group. This was based on a within-patient variation of 35% in the pharmacokinetics of TAM
and ENDO. The Schumann’s two, one-sided t test was used for sample size determination.
Statistical analysis was performed using the SPSS® software package, version 22.0
(IBM, North Castle, NY, USA) and visualization in GraphPad prism version 8.4.3. Analyses
included descriptive statistics, paired t tests. Analysis of variance (ANOVA) was performed on the AUC and Cmax after transformation of the data to their natural logarithmic
(ln) values.
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4. Results
4.1. Baseline Characteristics
A total of 42 subjects were enrolled in the study, 3 females and 39 males as shown
in Table 1. Out of the initial 153 subjects screened for CYP2D6 genotype 85 subjects were
eligible. From the 85 subjects, 31 were lost to follow up, 54 were screened for study eligibility and 42 were dosed with single oral dose of 20 mg TAM. No concomitant medicines
were taken by the subjects. Amongst the three CYP2D6 genotype groups, there were no
significant differences in participant demographic characteristics (data not shown). TAM
was generally well tolerated by the subjects across all groups and there were no significant
differences in physical evaluation, vital signs or lab tests observed.
4.2. Pharmacokinetic Analysis
Mean plasma concentration time profiles following oral administration of 20 mg TAM
are shown in Figure 2. TAM was rapidly absorbed and was quantifiable at 0.5 h post
dose administration with mean Tmax at 4.25 h. PK parameters of TAM and metabolites
are summarized in Table 2. TAM shows inter individual variation in the three groups
however there was no significant difference in the mean PK parameters of TAM among
the three studied CYP2D6 groups all (p > 0.05). The mean ENDO AUC0-∞ of 452.01
(196.94) hr·ng/mL, in CYP2D6*17/*17 subjects and AUC0-∞ 930.69 (212.41) h·ng/mL in
CYP2D6*1/*17 or *2/*17, subjects were 5.8-fold and 2.8-fold lower than in CYP2D6*1 or *2
subjects with AUC0-∞ of 2625.5(1167.68) h·ng/mL. This observed difference in AUC was
statistically significant (p < 0.001). A similar trend was observed for mean Cmax , where
we observed a 5-fold and 2-fold statistically significant difference for Cmax in CYP2D6*1
or *2 subjects compared to CYP2D6*17/*17 and CYP2D6*1/*17 (p < 0.001) respectively.
Analysis by CYP2D6 genotype, for the primary metabolites NDT showed no difference
between the different groups. Table 2 shows that the presence of CYP2D*17 resulted in
a statistically significant difference in Cmax and Tmax when compared to CY2D6*1 or *2
(p < 0.05). However, there was no statistically significant difference for 4OHT AUC within
the three study arms.
Table 2. Effect of CYP2D6 genotypes on the Pharmacokinetics of TAM and its main metabolites
following a single oral dose administration of 20 mg TAM.
Tamoxifen
Cmax (ng/mL)
Tmax (h)
AUC0-last (h·ng/mL)
AUC0-∞ (h·ng/mL)
Thalf (h)
V/f (L)
CL (L/h)
N-desmethyl tamoxifen
Cmax (ng/mL)
Tmax (h)
AUC0-last (h·ng/mL)
AUC0-∞ (h·ng/mL)
Thalf (h)
V/f (L)
CL (L/h)
4-Hydroxy-tamoxifen
Cmax (ng/mL)
Tmax (h)
AUC0-last (h·ng/mL)
AUC0-∞ (h·ng/mL)
CYP2D6*1/*1, *1/*2 or *2/*2
CYP2D6*1/*17 or *2/*17
CYP2D6*17/*17
p Value
51.88 (18.25)
4.154 (1.97)
3650.75 (1203.75)
4560.56 (2030.04)
152.81 (44.15)
1530.42 (833.69)
4.88 (1.38)
62.96 (25.68)
3.94 (1.27)
4246.94 (2068.27)
4853.29 (2285.58)
160.90 (34.58)
1166.25 (585.18)
4.84 (1.90)
54.23 (14.13)
4.67 (2.22)
3520.99 (1874.01)
4673.49 (2036.21)
165.27 (60.73)
1344.75 (790.65)
4.97 (1.84)
0.3803
0.0503
0.5116
0.9345
0.7834
0.4404
0.9793
33.98 (16.01)
39 (27.09)
8542.90 (2863.16)
10,680.76 (4546.64)
262.00 (114.97)
666.231 (299.65)
2.30 (1.04)
39.66 (20.33)
25.25 (19.15)
7742.71 (2072.57)
9944.45 (2567.0)
297.06 (179.08
578.21 (353.64)
1.79 (0.4)
31.37 (9.447)
49.78 (31.44)
7232.27 (1387.74)
9372.26 (2088.38)
256.24 (125.26)
603.4 (240.53)
2.39 (0.55)
0.3791
0.0593
0.2918
0.5693
0.7165
0.7306
0.0701
1.518 (0.57)
29.33 (14.89)
245.33 (142)
321.067 (129.19)
1.06 (0.41)
24 (16.49)
218.72 (153.63)
299.35 (168.81)
0.724 (0.360
44.8 (28.06)
132.318 (121.7)
245.27 (170.76)
0.0002 *
0.0340 *
0.0950
0.4305
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Table 2. Cont.
Thalf (h)
V/f (L)
CL (L/h)
Endoxifen
Cmax (ng/mL)
Tmax (h)
AUC0-last (h·ng/mL)
AUC0-∞ (h·ng/mL)
Thalf (h)
V/f (L)
CL (L/h)
CYP2D6*1/*1, *1/*2 or *2/*2
CYP2D6*1/*17 or *2/*17
CYP2D6*17/*17
p Value
98.57 (79.17)
4229.37 (2072.66)
103.64 (32.79)
122.99 (50.12)
4629.26 (1752.04)
128.48 (69.80)
103.14 (65.24)
30,269.62 (7873.91)
288.16 (257.62)
0.6036
<0.001 *
0.070
5.26 (2.14)
124.8 (40.8)
1857.62 (854.39)
2625.5 (1167.68)
246.05 (102.01)
3324.79 (1825)
12.57 (9.99)
2.95 (1.0)
132 (18.14)
808.42 (180.82)
930.69 (212.41)
171.87 (35.49)
3940.09 (2330.34)
20.35 (6.2)
1.034 (0.50)
144 (26.83)
355.51 (132.36)
452.01 (196.94)
243.71 (99.46)
11,931.32 (4261.43)
38.6 (17.63)
<0.001 *
0.2517
<0.001 *
<0.001 *
0.0508
<0.001 *
<0.001 *
Data represented as mean (standard deviation). Analysis of variance (ANOVA) was used to test for the differences
between the means of the key pharmacokinetic (PK) parameters: area under the plasma concentration–time
curve (AUC), peak plasma concentration of the compound (Cmax), time needed to achieve Cmax (Tmax ), Thalf ,
elimination half-life, hours (hr), clearance (CL), the apparent volume of distribution (V/f), AUC0-last, area under
the plasma concentration–time curve from time zero to the last sampled time point; AUC0-∞, AUC from time
zero to infinity. * Statistically significant difference is defined as p < 0.05.
Figure 2. The PK profiles are for the three genotype groups (CYP2D6*1 or *2, CYP2D6*1/*17 or *2/*17,
and CYP2D6*17/*17) showing log transformed plasma concentration versus time for (a) TAM, (b)
NDT, (c) 4OHT and (d) ENDO after single oral 20 mg dose of TAM.
4.3. Activity Score for CYP2D6*17
CYP2D6 mediates the major steps in the formation of ENDO from NDT. We therefore
investigated the relationship between CYP2D6 genotypes and the MR of ENDO/NDT.
The mean concentrations were significantly different between genotype groups (p < 0.05)
showing increased MR with CYP2D6 activity despite considerable interindividual variability between the different arms (Figure 3). Using the ENDO/NDT MR to capture the
J. Pers. Med. 2023, 13, 272
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role of CYP2D6 for allele variants *17/*17 and *1/*17 and *2/*17 on the calibration curve we
interpolated the predicted activity score. The median interpolated predicted activity score
for homozygous CYP2D6*17 was 0.667 vs. the activity score of 1 in the current CPIC current
guidelines as shown in Figure 3. Therefore, the activity score of CYP2D6*17 was estimated
at 0.334. This is lower than the activity score of 0.5 in the current guidelines [31,32].
Figure 3. CYP2D6 activity calibration curve plotted as observed MR [12] vs. activity score obtained
from the CPIC guidelines [32]. The interpolated predicted activity scores for the three arms are
presented on the graph as the median and interquartile range (IQR). The predicted activity score
for our study is shown in grey for CYP2D6*1 or *2, CYP2D6*1/*17 or *2/*17, and CYP2D6*17/*17.
Predicted activity scores on the y axis were based on the current CPIC guidelines.
4.4. Dose Escalation and Predicted Endoxifen Css
As shown in Figure 4, the simulated median ENDO Css in arms 1 and 2 was higher
than the reported ENDO therapeutic threshold of 5.9 ng/ml [14]. However, 42% of subjects
in the homozygous CYP2D6*17 arm were below the threshold, with a group median of
7 ng/mL.
Figure 4. Simulated steady state concentrations of ENDO in CYP2D6 genotype arms, with horizontal
line highlighting ENDO therapeutic threshold as reported by Madlensky [14]. Boxes interquartile
range (IQR) including median.
Simulating TAM dose escalation in the CYP2D6 *17/*17 arm from 20 to 30 and
40 mg/day resulted in a significant increase in the median plasma ENDO concentra-
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tion. At a dose of 30 mg/day, n = 12 subjects were above the putative therapeutic ENDO
threshold of 5.97 ng/mL, and a further increase in TAM dose to 40 mg/day resulted in all
14 subjects in the homozygous CYP2D6*17 arm having ENDO concentrations above the
putative ENDO threshold of 5.97 ng/mL. The mean ENDO concentrations in the simulated
dose escalation are shown in Figure 5.
Figure 5. Dose escalation simulation plot of TAM from 20 to 40 mg/day with predicted change
of mean steady state ENDO concentration in the CYP2D6*17/*17 arm. The dotted red line shows
putative reported ENDO therapeutic threshold at 5.97 ng/mL.
5. Discussion
TAM is important as an adjuvant or neoadjuvant in the management of oestrogen
receptor positive breast cancer [33]. The role of pharmacogenetics on the pharmacokinetics
and efficacy of TAM have been subjects to extensive research efforts [34,35]. Multiple
enzymes are involved in TAM biotransformation, suggesting that there could be more than
one enzyme polymorphism that can influence the pharmacokinetics of TAM [36]. Several
TAM pharmacogenetic studies have been published in breast cancer patient cohorts [37]
but to our knowledge, this is the first pharmacogenetic study evaluating the effect of
CYP2D6*17 on the pharmacokinetics and metabolism of TAM and its metabolites in a
controlled black healthy subjects’ population.
TAM metabolism involves several CYP enzymes that include but not limited to
CYP2B6, CYP2C19, CYP3A4, CYP3A5, and CYP2D6 [36] and CYP mediated metabolism
pathways are subject to genetic variability that can affect exposure levels of TAM and
metabolites. Genetic variation in CYP3A4, CYP3A5, and CYP2C19 may also affect TAM
metabolism. However, it is less clear how these enzymes influence ENDO plasma levels [38–42]. Studies have shown that variability in CYP2D6 results in different exposure
levels of ENDO between wild type and variant alleles, Studies have reported a gene dose
effect for CYP2D6 and ENDO formation for patients on TAM therapy [43].
In this study, TAM pharmacokinetics showed high interindividual variability and
did not show any statistically significant differences in TAM PK parameters across the
3 different CYP2D6 genotype arms. TAM was rapidly absorbed, with a mean Tmax of
4.25 h. A high overall mean Cmax of 55 ng/mL was observed, which is comparable to
what has been reported by Adam et al. [44] of 42 ng/mL, and earlier studies with radio
labelled TAM showed peak TAM concentrations ranging from 60–100 ng/mL [45]. The
observed long terminal half-life of 6.6 days was within the reported TAM half-life range
of 5–9 days [44–46]. As reported by Etienne [47] all the three studied metabolites were
detectable from a single 20 mg dose of TAM. NDT was quantitatively the predominant
metabolite and 4OHT was quantitatively the minor metabolite. This agrees with what has
already been published, where NDT is a product in the major metabolic pathway of TAM
metabolism [47]. NDT had a higher AUC than the parent drug as has been observed before
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in a bioequivalence study in female volunteers [48]. In general, the major metabolites NDT
and ENDO had longer half-lives than TAM, indicating increased exposure and circulation
duration of the metabolites.
Previous studies have shown that CYP2D6 activity accounts for 39–58% of ENDO
interindividual variability [49–51]. In this study, we have described an association between expression of CYP2D6*17, a reduced activity enzyme variant, and pharmacokinetic
parameters of TAM and its major active metabolite, ENDO. The CYP2D6*17 allele has a
clear impact on ENDO pharmacokinetics in homozygous carriers, resulting in a 5.8-fold
reduction in AUC0-∞ and 5-fold reduction in Cmax in homozygous individuals compared
to the wild type. In a study of TAM-treated Algerian breast cancer patients, including those
genotyped as CYP2D6*17/*17. The median ENDO concentration for IMs was 2.4 times
lower than that of CYP2D6*1 carriers, clearly showing that the reduced activity variant
in the homozygous state results in reduced levels of ENDO compared to the NM and
ultra-rapid metabolizer phenotypes [52].
The debate on the clinical relevance of the TAM-CYP2D6 drug-gene interaction has
not yet been resolved as there are conflicting results from different studies on the role of the
CYP2D6 genotype on the clinical outcomes of breast cancer treatment. Recent studies [14,18]
have suggested a plasma ENDO therapeutic threshold of 5.9 ng/mL, which results in
improved treatment outcomes by reducing the risk of breast cancer recurrence by 30% [14].
About 40% of CYP2D6*17 homozygous subjects in our study were below the reported
putative therapeutic threshold for ENDO. Demonstrating that some patients homozygous
for CYP2D6*17 will still achieve therapeutic levels of ENDO whilst others would be below
the therapeutic range. Studies have shown that patients below this threshold could be
at a higher risk of disease relapse or death. Thus, in clinical practice, clinicians should
consider a dose increase in patients carrying the CYP2D6*17 variant who do not respond to
standard dosing of 20 mg daily of TAM. A dose increase would reduce the potential risk
of therapeutic failure, as what has been observed in the clinical setup, where PM patients
with low ENDO levels had higher risk of disease recurrence [5,40,53–55].
Several dose escalation studies with individuals with reduced CYP2D6 metabolic
activity, such as the CYP2D6*10, have shown that individuals with sub-therapeutic plasma
ENDO levels can achieve therapeutic effective ENDO concentrations by increasing TAM
doses from 20 to 40 or 60 mg [56–59]. With dose escalation, a linear increase in ENDO
plasma concentrations has been observed in some studies within the 20 to 60 mg/day
TAM dose range [56,57,60]. We simulated dose increases in IM subjects homozygous for
CYP2D6*17 based on the assumption of linear PK. The simulation indicated that doses
of 40 mg/day would ensure that all subjects homozygous for CYP2D6*17 would reach
the proposed putative threshold of 5.97 ng/mL. Our results align with what has been
reported by Puszkiel and colleagues, who, using a population pharmacokinetic (PopPK)
model, showed that CYP2D6 IMs and PMS would require a dose increase of 40 and
80 mg per day, respectively [61]. Genotyping of clinically significant CYP2D6 alleles and
subsequent dose adjustment in IM and PM patients has been observed to result in increased
ENDO plasma levels [59]. However, some have proposed the use of therapeutic drug
monitoring for ENDO to determine the dose adjustment [62,63]. Some studies have used
sub-therapeutic baseline ENDO concentrations to determine the dose of TAM. The dose
adjustments resulted in increased levels of END and improved treatment outcomes [51,60].
However, despite several dose-escalation attempts, it is important to note that increasing
TAM dose has been shown to benefit IM patients more than PM phenotype patients in
terms of increasing ENDO plasma levels [51,58,60]. Dezentjé and colleagues used both
ENDO concentration and CYP2D6 genotype in a dose escalation study that resulted in all
patients, including PMs, having a concentration above the threshold of 5.97 ng/mL [64].
More studies need to be done to reach a consensus on how best to use CYP2D6 genotypes
as predictors for patients who can benefit from dose adjustment.
TAM metabolism is complex, involving several enzymes that can be induced or
inhibited. This can result in CYP-mediated drug-drug interactions that can influence the
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levels of TAM and its metabolite. Current CPIC guidelines for TAM therapy highlight
the need to avoid moderate and strong CYP2D6 inhibitors [32]. Binkhorst and colleagues
demonstrated that strong CYP2D6 inhibitors such as paroxetine and fluoxetine reduced
ENDO to sub-therapeutic levels, posing a risk for poor treatment outcomes in patients,
including those who are CYP2D6 NMs [65]. Rifampicin, a very strong CYP450 enzyme
inducer was shown to result in reduced plasma levels of TAM and its metabolites, including
ENDO. This phenomenon was explained by the ability of rifampicin to induce CYP3A4
and potentially UDP-glucuronosyltransferase (UGT) [66], resulting in increased clearance
of ENDO, as UGTs play a pivotal role of converting ENDO to an inactive glucuronide
metabolite. Hence, due to the drug interactions with TAM, there is a risk of enzyme
phenocopying, as has been observed where NM phenotypes after taking CYP2D6 inhibitors
had low ENDO exposure levels that were comparable to PM and IM phenotypes. However,
a recent study found that taking probenecid with TAM resulted in an increase in ENDO
exposure levels, even in PM patients with limited or no side effects. This reported drugdrug interaction could be important in increasing the exposure levels of ENDO in IM
and PM patients who have been observed to have sub-therapeutic ENDO levels. The
observed TAM-probenecid drug interaction was proposed to be through CYP induction
and inhibition of the glucuronidation pathway [67] Such interactions if proved to have no
or low adverse drug effects with long-term repeated use, could be useful as boosters, as the
case with ritonavir in Antiretroviral therapy for HIV patients [68].
When the activities of CYP2D6 variants were compared in vitro, CYP2D6*17 activity
was less than 15% that of CYP2D6*1 [69]. Bodies such as the Clinical Pharmacogenetics
Implementation Consortium (CPIC), the Dutch Pharmacogenetics Working Group (DPWG),
and the Canadian Pharmacogenomics Network for Drug Safety (CPNDS) have come up
with pharmacogenetic guidelines to assist with dosing for patients on TAM therapy [32,70].
CPIC and DPWG guidelines have assigned an activity score of 0.5 to the CYP2D6*17
variant [71]. In this study, however, we predicted a lower activity score value of 0.34 with
respect to the CYP2D6*17 allele and TAM metabolism. Efforts to understand the functional
status of CYP2D6*17 have demonstrated that CYP2D6*17 activity is substrate-specific,
and the calculated activity score values ranged from 0.09 to 0.54 with different CYP2D6
substrates [32,72,73]. A recent study [74] reported comparable in vitro activity between
CYP2D6*17 and CYP2D6*10. This review article highlighted that the CYP2D6 activity
was below the assigned activity score value of 0.5 [74]. The lower value we predicted
underscores the potential need to re-evaluate the activity scoring of CYP2D6*17 with TAM
to accurately capture the phenotype group, as there is a risk of underestimating the necessity
for dose adjustment and reducing the predictive performance of CYP2D6. Re-evaluation
of the CYP2D6*10 variant from the previous activity score value of 0.5 to 0.25 resulted in
improved prediction of ENDO by CYP2D6 in Asian populations [50].
Limitations
Limitations of our study include having more male subjects (93%) compared to females,
yet >98% of breast cancer is female breast cancer; hence, most patients who are prescribed
TAM are female breast cancer patients. TAM is still an important drug for hormonal therapy
in the treatment of ER+ male breast cancer [75]. Pharmacokinetics has been shown to differ
between male and female patients due to different physiological variables. Gender has
been associated with differences in steady state endoxifen concentrations [76], but the
clinical significance of gender has not been fully established [35]. The second limitation is
that our evaluation on the effect of CYP2D6 variants was on a single dose administration
on a drug that is given on a daily basis for up to 5-years. However, we performed dose
simulations to predict steady-state plasma levels of ENDO. The study controlled for the
demographic parameters, resulting in demographic homogeneity for age, weight, and
BMI, which may partially explain the lack of associations between plasma levels and the
demographic parameters of age, weight, and BMI. Thirdly the study had a small number
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12 of 16
of subjects. These findings should therefore be confirmed in different cohorts with a larger
number of subjects.
6. Conclusions
The presence of the CYP2D6*17 allele in a homozygous state can significantly influence
the generation of ENDO from TAM. Our study shows statistically significant findings that
the reduced activity variant CYP2D*17 is associated with low levels of the active metabolite
ENDO. Through simulated dose escalations, it further shows that increasing the dose of
TAM to 40 mg/day in subjects homozygous for CYP2D6*17 would result in all the subjects
attaining therapeutic levels of ENDO that are above the putative 5.97 ng/mL threshold. Our
study proposes a revised activity score demonstrating a lower capacity to generate ENDO.
Owing to the high prevalence of the CYP2D6*17 variant among the black Zimbabwean
population, the results of this study might provide benefit for breast cancer patients upon
the introduction of personalised TAM therapy in Zimbabwe. Further studies are warranted
to confirm our findings.
Supplementary Materials: The following supporting information can be downloaded at https:
//www.mdpi.com/article/10.3390/jpm13020272/s1, Table S1: CYP2D6 SNP variants that were
tested to determine the subjects CYP2D6 genotypes prior to dosing.
Author Contributions: Conceptualization, C.N. and C.M.; Formal analysis, C.R.K. and C.M.; Funding
acquisition, C.M.; Investigation, C.R.K. and C.M.; Methodology, C.R.K. and C.M.; Project administration, G.N.; Patient selection and recruitment, G.N.; Resources, C.M.; Supervision, C.N. and C.M.;
Writing—original draft, C.R.K.; Writing—review & editing, C.N. and C.M. All authors have read and
agreed to the published version of the manuscript.
Funding: Funding and drug supply for this study were supplied by Novartis Pharmaceutical
Company through an Investigator Initiated grant.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the Medical Research Council of Zimbabwe (MRCZ) study approval
number (MRCZ/A/2386) and the Medicines control Authority of Zimbabwe (MCAZ) (CT170/2018).
Informed Consent Statement: Written informed consent was obtained from all subjects involved in
the study.
Data Availability Statement: The data that support the findings of this study are available upon
reasonable request.
Acknowledgments: The authors would like to acknowledge all the study subjects, the clinical trial
team at AiBST-Chitungwiza Clinical Trial Unit, supporting staff at AiBST, Roslyn Thelingwani and
Nyasha Kapungu for assistance with drug analysis and the International Science Program (ISP) for
funding Comfort Kanji’s masters’ work.
Conflicts of Interest: The authors declare no conflict of interest.
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