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Introduction
Tibolone, [7a,17a-7-methyl-17-hydroxyl-19-norpregn-5(10)-en-20-yn-3-one], also called 7-methyl-norethynodrel
(MN), is a synthetic steroid (Figure 1) used in hormone
replacement therapy (HRT) for postmenopausal women.
Clinical data indicates that tibolone can produce the hormonal
effects needed to treat climacteric symptoms and to prevent
the long-term effects of menopause without stimulating the
breast or endometrial tissues[1-3]. After administration to
women, the compound is quickly metabolized into a
3a-hydroxyl metabolite (3a-hydroxyl-7-methyl- norethynodrel;
3a-HMN) and a 3b-hydroxyl metabolite (3b-hydroxyl-7-methyl-
norethynodrel; 3b-HMN) by the enzymes
3a/b-hydroxyste-roid dehydrogenase in the intestine and the
liver[4,5]. Tibolone itself has weak binding affinities to human estrogen,
progesterone and androgen receptors, whereas the two 3-hydroxyl
metabolites bind solely to the estrogen receptor, and
3a-HMN is approximately 3 times as effective as
3b-HMN[6]. The clinical merits of tibolone are thought to be a result of
the tissue-specific activity of the drug and its main
metabolites[7, 8].
The pharmacokinetics of tibolone in female patients have
been studied recently, and the plasma concentrations of
3a-HMN and 3b-HMN were determined by using a gas
chromatography-mass spectrometry (GC-MS)
method[9]. However, the pharmacokinetics of tibolone metabolism in a Chinese
human population have not been studied. The present study
was undertaken to investigate stereoselectivity in the
pharmacokinetics of tibolone metabolism in healthy Chinese
female subjects using a validated liquid chromatography-mass
spectrometry (LC-MS) method.
Materials and methods
Subjects Twenty healthy Chinese female subjects
ranging in age from 30 to 40 years (36.4±3.6 years), in weight from
47 to 67 kg (56.1±5.8 kg), and in height from 143 to 170 cm
(157.1±6.4 cm) were enrolled in the study. All subjects were
in good health and underwent a pre-enrolment screening
that included ascertaining the subjects¡¯ medical history, a
physical examination, an electrocardiogram (ECG),
laboratory tests and urinalysis. No medication was used by the
subjects for at least 2 weeks before the study and alcohol
was forbidden within the 72 h before drug administration.
Approval was obtained from the Institutional Review Board
of the Obstetrics and Gynecology Hospital, Medical Center
of Fudan University, Shanghai, and all subjects gave written
informed consent.
Clinical protocol After an overnight fast of at least 10 h,
subjects took one tablet of Livial, which contained 2.5 mg
tibolone, with 200 mL water and continued fasting for 2 h.
Blood samples obtained from an antecubital vein prior to
treatment and at 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0, and
18.0 h after treatment were placed in heparinized tubes. The
samples were immediately centrifuged at
3000×g for 15 min, and the plasma was separated and frozen at -20 °C until
analysis.
LC-MS analysis of the metabolites in plasma
Plasma concentrations of 3a-HMN and 3b-HMN were determined
by using a LC-MS method described
elsewhere[10]. Plasma samples were derivatized with
p-toluenesulfonyl isocyanate after extraction with ethyl acetate. Separation of the two
derivatized 3-hydroxyl metabolites was carried out on a
Diamonsil C18 column with a linear gradient elution of
mixtures of methanol and ammonia acetate aqueous solution.
The analytes were detected with a mass spectrometry
detector in the negative selected ion monitoring (SIM) mode.
The limit of quantitation for both 3a-HMN and
3b-HMN was 100 ng/L, and the accuracy was 91.6%-116% for
3b-HMN and 90.4%-104% for 3b-HMN. The inter-day and
intra-day coefficients of variation for 3a-HMN and
3b-HMN were less than 13.8% and 11.3%, respectively, over the
concentration range 0.1-30 μg/L.
Pharmacokinetic analysis Pharmacokinetic parameters
were estimated by using non-compartmental analysis of
curves of 3a/b-HMN isomer plasma concentrations versus
time. Maximum plasma concentrations
(Cmax) and the corresponding times
(Tmax) were read as the coordinates of the
highest raw data point for each volunteer. The area under
the plasma concentration versus time curve
(AUC0-t) was estimated by using the linear trapezoidal rule up to the last
measurable time. AUC0-¥ was obtained by adding the part of
the area extrapolated to infinity (last measurable
concentration/ke) to
AUC0-t, where ke is the slope of the linear
regression analysis of natural log concentrations against time. The
elimination half-life (T½) was estimated from the plasma data
by using the equation
T½=0.693/ke
. The values were expressed as mean±SD. Differences between
3a-HMN and 3b-HMN in the pharmacokinetic data were evaluated
statistically by using the independent-samples
t-test. P<0.05 was considered statistically significant.
Results
Plasma drug concentration-time curves After subjects
were given tibolone, the mean plasma concentrations of
3a-HMN and 3b-HMN were all below 10 ng/mL, and the mean
plasma concentrations of 3a-HMN were much greater than
those of 3b-HMN (Figure 2). The last time point for which all
subjects had measurable concentrations of 3a-HMN and
3b-HMN was 18 h.
Pharmacokinetic parameters of the
metabolites After subjects were given tibolone at a dose of 2.5 mg,
Cmax values for 3a-HMN and 3b-HMN were estimated to be 8.75 and 3.59
μg/L, and the mean AUC0-t values were 26.30 and 9.89
μg·h-1· L-1, respectively.
There was no significant difference in
Tmax and
T½ between 3a-HMN and 3b-HMN
(P>0.05, Table 1). After subjects were given tibolone, the two metabolites both reached
peak concentrations in the blood at 1.5 h, and
T½ values for 3a-HMN and 3b-HMN were 7.45±2.03 h and 7.50±1.83 h,
respectively.
Discussion
The mean pharmacokinetic parameters of 3b-HMN were
similar to those found by Timmer et
al[9], whereas the peak plasma concentration of
3a-HMN in our study (8.75±4.36 μg/L) was much lower than that reported elsewhere for early
(14.6±5.4 μg/L)[9] or late postmenopausal women (16.7±6.6
μg/L)[9]. Moreover, the
AUC0-¥ of 3a-HMN in the present study (30.97±14.10
μg·h-1·L-1) was also lower than that
reported elsewhere for early (49.6±14.6
μg·h-1·L-1) or late
postmenopausal women (62.6±17.3
μg·h-1·L-1)
[9].
There was a significant difference between the two
3-hydroxyl metabolites in all mean pharmacokinetic parameters
except for Tmax and
T½ (Table 1). The mean
Cmax and AUC0-t values of
3b-HMN were approximately 59% and 62% lower than that of
3a-HMN, respectively. Mean 3a/3b stereoisomer ratios for plasma concentrations of the two metabolites
in 20 subjects ranged from 1.5 to 3.0 after administration
(Figure 3). These results indicate that a stereoselective
difference exists in the 3-hydroxylization metabolism of tibolone.
Although a 3a-hydroxylization intensive metabolism was
found for the metabolism pharmacokinetics of tibolone in
the 20 subjects as a whole (with a mean 3a/3b
AUC0-T ratio of 3.11), two 3a-hydroxyl-poor metabolizers were found in
the present study (with 3a/3b AUC0-t ratios of 0.71 and 0.99;
Figure 4). Differences in the 3a/b-hydroxylization
metabolism of tibolone, as well as desogestrel, by different species
of animals have been confirmed by Verhoeven et
al[11,12]: both drugs had mainly 3a-hydroxyl metabolites in rats, but
3b-hydroxyl metabolites in dogs. Because 3a-hydroxysteroid
dehydrogenase and 3b-hydroxysteroids dehydrogenase
reduce 3-keto steroids, polymorphism for 3a/b-hydroxylization
metabolism can be explained by individual differences in
3a-hydroxysteroid dehydrogenase activity and 3b-hydro-xysteroid dehydrogenase activity.
Acknowledgement
The authors express their thanks to Prof Shao-fen
ZHANG (Department of Gynecology, The Obstetrics and
Gynecology Hospital, Medical Center of Fudan University,
Shanghai) and her group for their help in subject selection
and drug administration, and assistance in the medical ward.
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