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DRUG METABOLISM

Bioequivalence Studies of Tibolone in Premenopausal Women and Effects on Expression of the Tibolone-Metabolizing Enzyme AKR1C (Aldo-Keto Reductase) Family Caused by Estradiol

Keon W. Kang, PhD and Yoon G. Kim, PhD

From the BK21 Project Team, the College of Pharmacy, Chosun University, Kwangju, Republic of Korea (Dr Kang), and the Department of Pharmacology, College of Medicine, Dankook University, Chonan, Republic of Korea (Dr Kim).

Address for reprints: Yoon G. Kim, PhD, Department of Pharmacology, College of Medicine, Dankook University, San 29, Anseo-Dong, Chonan-Si, Choungnam 330-714, Republic of Korea; e-mail: kyg90{at}dankook.ac.kr.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study aimed to investigate the bioequivalence of a test formulation of tibolone with the marketed reference formulation in 24 young healthy female volunteers. Tibolone is a synthetic steroid hormone for menopausal women. Volunteers were treated with the 2 formulations of tibolone (total dose of active ingredient 2.5 mg) according to a 2 x 2 crossover design with a 1-week washout period. Plasma concentrations of 3{alpha}- and 3β-hydroxytibolone, which are major metabolites of tibolone, were assayed in timed samples over a 24-hour period with a validated gas chromatography/mass spectrometry (GC/MS) method that had a lower limit of quantification of 0.5 ng/mL. The reference and test formulations gave a mean 3{alpha}-hydroxytibolone Cmax of 5.0 and 5.2 ng/mL, respectively, and a mean 3β-hydroxytibolone Cmax of 16.4 and 16.5 ng/mL, respectively. The mean AUCt of 3{alpha}-hydroxytibolone was 24.7 and 24.3 ng h/mL, whereas the mean AUCt of 3β-hydroxytibolone was 57.6 and 54.8 ng h/mL for the test and reference formulations, respectively. The authors did not find significant differences in pharmacokinetic parameters between the 2 formulations, but metabolite formation was different from reports in postmenopausal women. The authors therefore measured the effects of estradiol on the expression of the tibolone-metabolizing enzymes, from the aldo-keto reductase (AKR1C) family, using HepG2 cell (human hepatoma cells) and MCF-7 cell (human breast cancer cells). Estradiol increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 and protein levels of total AKR1C in HepG2 cells. Estradiol selectively enhanced levels of AKR1C2 mRNA in MCF-7 cells. Thus, changes in the major metabolites of tibolone might result from changes in AKR1C family expression by patient estrogen status.

Key Words: Tibolone • pharmacokinetics • metabolite • AKR1C • post-menopause • estradiol

Tibolone ([7{alpha}, 17{alpha}]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one) is a synthetic steroid hormone that improves postmenopausal climacteric symptoms and prevents postmenopausal osteoporosis without stimulating the endometrium or breast.1-4 Tibolone is a selective tissue estrogenic activity regulator (STEAR) that is distinctive from selective estrogen receptor modulators (SERMs) such as tamoxifen and raloxifene.5,6

Tissue-specific effects of tibolone are due to 3 active metabolites: 3{alpha}-hydroxytibolone, 3β-hydroxytibolone, and a {Delta}4-isomer.5,7 3{alpha}- and 3β-hydroxytibolone are weak ligands for the estrogen receptor, which could explain the beneficial effects of tibolone.8 Tibolone and its {Delta}4-isomer prevent endometrial stimulation by their progestogenic activity.8

Most pharmacokinetic studies of tibolone have been conducted on postmenopausal women.9-12 After oral administration to postmenopausal women, tibolone shows fast absorption (mean tmax is within 1 hour) and metabolism to 3 metabolites.9,13 Plasma concentrations of tibolone could be measured until 6 hours after oral administration, and only Cmax and tmax are the available pharmacokinetic parameters.9,13 3{alpha}-Hydroxytibolone is the main metabolite in plasma,13 but 3β-hydroxytibolone is the main metabolite in peripheral tissue.14 Steckelbroeck et al15 reported that tibolone is metabolized by 4 human isozymes of the aldo-keto reductase (AKR) 1C subfamily. AKR1C1 and AKR1C2 mediate tibolone metabolism to 3β-hydroxytibolone, AKR1C4 mediates metabolism to 3{alpha}-hydroxytibolone, and AKR1C3 mediates metabolism to both metabolites but its activity is weak compared with other subtypes.15 Steckelbroeck et al suggest that liver-specific expression of AKR1C4 could explain why 3{alpha}-hydroxytibolone is the major circulating metabolite.15

We assessed the bioequivalence of a novel tibolone formulation in 24 premenopausal women by 2 active metabolites, 3{alpha}- and 3β-hydroxytibolone, because they cause the beneficial effects of tibolone, and we could not get the plasma concentration-time profile of tibolone for the bioequivalence test with available assay methods. Interestingly, we found that the main metabolite of tibolone in young women is 3β-hydroxytibolone rather than 3{alpha}-hydroxytibolone. As previous reports focused on metabolism in postmenopausal women, this difference in metabolites might be due to changes in metabolizing enzyme expression by different hormone levels in post- and premenopausal women.

In this study, we report different proportions of major tibolone metabolites in premenopausal women. We also measure estrogen-induced changes in AKR1C family expression in HepG2 and MCF-7 cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Test and Reference Medications
The test medication, Livlone (2.5 mg of tibolone, Myungmoon Pharm Co, Ltd, Seoul, Korea), and the reference medication, Livial (2.5 mg of tibolone, Organon Korea, Seoul, Korea), were supplied as tablets from Myungmoon Pharm Co, Ltd.

Participants
Twenty-four healthy premenopausal Korean women ranging in age from 21 to 35 years (23.8 ± 3.2 years), in weight from 43 to 63 kg (53.7 ± 5.1 kg), and in height from 150 to 170 cm (160.8 ± 5.5 cm) were enrolled in the study. The sample size of n = 24 participants was sufficient to ensure a power of 90% for correctly concluding bioequivalence under the following assumptions: {alpha}= 0.05, 0.95 <µT/µR < 1.05, and an intrasubject coefficient of variation of 15.0% (intrasubject coefficient of variation for AUCt and Cmax of 3{alpha}-hydroxytibolone was 7.49% and 11.70%, respectively).16 Volunteers were selected after completing a clinical screening protocol with physical examinations and laboratory tests (blood analysis: hemoglobin, hematocrit, white blood cell [WBC], platelet, differential counting of WBC, blood urea nitrogen, total bilirubin, cholesterol, total protein, albumin, alkaline phosphatase, glucose fasting, aspartate aminotransferase, and alanine aminotransferase; urine analysis: specific gravity, color, pH, sugar, albumin, bilirubin, red blood cell [RBC], WBC, and cast).

Participants were excluded if they had a history or clinical evidence of significant respiratory, cardiovascular, gastrointestinal, endocrine, hematologic (including thromboembolic disorders), neurologic, immunologic, psychiatric, or other chronic disease or active alcoholism or drug abuse. Pregnant women were excluded by an hCG test. Participants were not permitted to take any medication, including any herbal medicines or oral contraceptives, for 14 days before study initiation. They also refrained from alcoholic beverages and xanthine-containing foods and beverages for 48 hours prior to each dosing and until the collection of the last blood sample. This study was performed according to the revised Declaration of Helsinki for biomedical research involving human participants and the rules of good clinical practice. The study protocol was approved by the Institutional Review Board of Dankook University Hospital, Chonan, Korea. All participants signed a written informed consent after they had been informed about the nature and details of the study.

Study Design
Each volunteer received an oral dose of 2.5 mg (1 tablet) of tibolone in a standard 2 x 2 crossover model in a randomized order. There was a 1-week washout period between the doses. Volunteers were admitted to the hospital (Dankook University Hospital, Choungnam, Korea) the evening before each treatment session and were confined to the hospital unit until completion of the session. Medication was administered after 10 hours of fasting. At 7:00 AM, the median cubital vein was cannulated (D&B-CATH, Seoul, Korea), and 1 mL of heparinized normal saline injectable solution (20 units/mL) was flushed into the cannula to prevent blood clotting. The medication was taken at 8:00 AM of each dosing day with 240 mL of tap water. After 4 hours of medication, all participants were given a standardized meal. They were restricted to the supine position or sleeping up to 8 hours after medication. Approximately 10 mL of blood was collected via the cannula at the following time intervals: predose and 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 15, and 24 hours after administration. Then, 1 mL of a heparinized normal saline injectable solution was flushed after each blood sampling. The blood samples were centrifuged immediately, and plasma samples were frozen at -70°C until analysis began.

Assay of 3{alpha}- and 3β-Hydroxytibolone Levels in Plasma
The concentrations of 3{alpha}- and 3β-hydroxytibolone in plasma were analyzed by a gas chromatographic/mass spectrometry (GC/MS) method17 with slight modifications. Briefly, 10 µL of internal standard (1,2-d2-testosterone, 10 µg/mL in methanol) was added to 500-µL aliquots of plasma in a polypropylene tube. After vigorous mixing, the plasma sample was loaded onto an HLB Oasis SPE cartridge that was activated by serial elution with 1 mL of methanol and 1 mL of buffer solution (pH 4.2). After eluting loaded plasma samples with a light vacuum, the cartridge was washed 2 times with 1 mL of buffer solution. The cartridge was eluted with 1 mL of methanol and collected in a clean Eppendorf tube. After evaporation under a gentle nitrogen stream, 40 µL of MSTFA/NH4I/DTE (100:10:1, v/v/v) was added and incubated at 60°C for 20 minutes. After cooling at ambient temperature, a 2-µL aliquot was injected onto the GC/MS system. The lower limit of quantification (LLOQ) for 3{alpha}- and 3β-hydroxytibolone was 0.5 ng/mL. At this concentration, the accuracy and precision were 104% and 7%, respectively, for 3{alpha}-hydroxytibolone and 97% and 9%, respectively, for 3β-hydroxytibolone (data not shown).

Pharmacokinetic Analysis
Noncompartmental pharmacokinetic characteristics were derived by standard methods. The maximum plasma concentration, Cmax, and the time of its occurrence, tmax, were compiled from the concentration-time data. The AUCt was calculated using the linear trapezoidal rule and was extrapolated to infinity according to the following relationship:

Formula
where AUC{infty} is the area under the plasma concentration-time curve from 0 to time infinity, Ct is the last concentration evaluated in plasma greater than the LLOQ, and β is the elimination rate constant at the terminal phase.

Cell Culture
HepG2 (human hepatoma cell line) and MCF-7 (human breast cancer cell line) cells were cultured at 37°C in 5% CO2/95% air in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated from cells using a total RNA isolation kit (RNAgents, Promega, Madison, Wisconsin). The total RNA (1.0 µg) was reverse-transcribed using an oligo (dT) 18mer as a primer and M-MLV reverse transcriptase (Bioneer, Eumsung, Korea) to produce the cDNA. Polymerase chain reaction (PCR) was performed using selective primers for human AKR1C1, AKR1C2, AKR1C3, and AKR1C4.18 As an mRNA loading control, we used selective primers for S16 ribosomal protein (S16r) genes (sense: 5'-TCCAA GGGTCCGCTGCAGTC-3', antisense: 5'-CGTTCACCTTGATGAGCCCATT-3'). PCR was performed for 35 cycles under the following conditions: denaturation at 98°C for 10 seconds, annealing at 60°C (AKR1C subtypes) or 51°C (MDR1) for 0.5 minutes, and elongation at 72°C for 1 minute. The band intensities of the amplified DNAs were compared after visualization using a UV transilluminator.

Immunoblot Analysis
After washing with sterile PBS, HepG2 or MCF-7 cells were lysed in EBC lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 137 mM sodium chloride, 10% glycerol, 2 mM EDTA, 1 mM sodium orthovanadate, 25 mM β-glycerophosphate, 2 mM sodium pyrophosphate, 1 mM phenylmethylsulfonylfluoride, and 1 µg/mL leupeptin. The cell lysates were centrifuged at 10 000 g for 10 minutes to remove the debris, and the proteins were fractionated using a 10% separating gel. The fractionated proteins were then transferred electrophoretically to nitrocellulose paper, and the proteins were immunoblotted with specific antibodies. Horseradish peroxidase- or alkaline phosphatase-conjugated anti-IgG antibodies were used as the secondary antibodies. The nitrocellulose blots were developed using a 5-bromo-4-chloro-3-indolylphosphate (BCIP)/4-nitroblue tetrazolium (NBT) or an ECL chemiluminescence system.

Statistical Analysis
We used the following tests to compare AUCt, AUC{infty}, Cmax, and tmax: analysis of variance (ANOVA) was performed using logarithmically transformed AUCt and Cmax, as well as original scaled values of tmax. The range of bioequivalence for parametric analysis was set to the commonly accepted 80% to 125% of the pharmacokinetic parameters obtained from the reference medication, and the range of equivalence for nonparametric analysis was set to 20% of the reference mean. All statistical comparisons were made using EquivTest Version 1.0 (Statistical Solutions Ltd, Saugus, Massachusetts) and the Bioeqv80 program.19


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
All 24 volunteers completed the study. The tolerability of both tibolone medications was satisfactory, with no clinically relevant or drug-related side effects observed.

Pharmacokinetic Characteristics
The mean plasma 3{alpha}- and 3β-hydroxytibolone concentration-time profiles after a single oral administration of the medications are shown in Figure 1. The pharmacokinetic parameters of 3{alpha}- and 3β-hydroxytibolone are summarized in Table I. The concentrations of the 2 tibolone metabolites at 24 hours were less than the LLOQ, and the last sampling time at which all participants showed measurable concentrations of 3{alpha}- and 3β-hydroxytibolone was 10 and 15 hours, respectively. Almost identical plasma 3{alpha}- and 3β-hydroxytibolone concentration profiles were obtained from both formulations (Figure 1). The mean terminal half-life of 3{alpha}- and 3β-hydroxytibolone was 6.0 ± 2.7 h and 3.2 ± 1.2 hours, respectively. With 3{alpha}-hydroxytibolone, the mean terminal half-life was similar to previously reported values but was shorter for 3β-hydroxytibolone.9,10 Interestingly, the AUCt, AUC{infty}, and Cmax of 3{alpha}- and 3β-hydroxytibolone for both medications showed different patterns than previously reported. For example, the Cmax of 3{alpha}- and 3β-hydroxytibolone for the reference medication was 5.04 ± 1.78 ng/mL and 16.39 ± 6.23 ng/mL, whereas the Cmax of 3{alpha}-hydroxytibolone was much higher than that of 3β-hydroxytibolone in postmenopausal women.9,10 The AUCt and AUC{infty} of 3β-hydroxytibolone were about 2-fold larger than those of 3{alpha}-hydroxytibolone (Table I). The AUCt and AUC{infty} of 3{alpha}-hydroxytibolone were also much higher than those of 3β-hydroxytibolone in previous reports.9,10


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  		Table I Mean ± SD Pharmacokinetic Parameters of 3{alpha}- and 3β-Hydroxytibolone After Single Oral Administration of 2 Different Formulations of Tibolone (2.5 mg) to Healthy, Young Women (n = 24)

 
Standard Bioequivalence Analysis
No significant sequence effect was found for the bioavailability parameters, indicating that the crossover design was properly performed (data not shown). No significant period effect in AUCt and Cmax was detected.

The standard bioequivalence analyses for AUCt and Cmax are shown in Table II. The 90% confidence intervals of the test/reference ratio for AUCt and Cmax for 3{alpha}-hydroxytibolone were 0.956 to 1.030 and 0.976 to 1.096; for 3β-hydroxytibolone, they were 0.864 to 1.043 and 0.951 to 1.135 (Table II), respectively, which were all within the commonly accepted bioequivalence range of 0.80 to 1.25. These results showed that the 2 formulations of tibolone are bioequivalent and thus may be prescribed interchangeably.


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  		Table II Standard Bioequivalence Analysis for AUCt (ng·h/mL) and Cmax (ng/mL) Between 2 Formulations

 
Immunoblast Analysis in Human Hepatoma Cells
We tested whether estradiol could change AKR1C subtype expression in HepG2 cells. HepG2 cells were incubated with 10 nM 17-β-estradiol (E2) for 6 or 10 hours. AKR1C2 and AKR1C4 were expressed in untreated HepG2 cells, but AKR1C1 and AKR1C3 were not. E2 treatment increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 at 6 hours but did not affect AKR1C4 expression. Because subtype-specific antibodies are not commercially available, we performed Western blot analyses using an AKR1C-specific antibody. E2 treatment increased total AKR1C levels from 24 to 48 hours after exposure. These results demonstrated that the main metabolic enzyme of tibolone could be affected by estrogen exposure.

Selective Upregulation of AKR1C2 by Estrogen in Human Breast Cancer Cells
We determined the effects of E2 on the expression of AKR1C subtypes in MCF-7 cells because it is also metabolized in breast tissue. E2 selectively enhanced the level of AKR1C2 mRNA at 6 hours, and other AKR1C subtypes were not detected in MCF-7 cells. Western blot analyses confirmed that AKR1C protein was induced at 12 to 24 hours after E2 treatment.


Figure 1
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  		Figure 1. Mean plasma concentration-time profiles of (A) 3{alpha}-hydroxytibolone and (B) 3β-hydroxytibolone after single oral administration of reference medication ({circ}) or test medication (bullet) to 24 young women. Vertical bar represents standard deviation.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGEMENTS
 REFERENCES
 
In this study, we found that 2 different formulations of tibolone were bioequivalent and could be prescribed interchangeably. Interestingly, we also found that the 2 measured hydroxyl-metabolites of tibolone showed different patterns than those reported in postmenopausal women. We found that the main metabolite of tibolone was 3β-hydroxytibolone, but in previous reports, it was 3{alpha}-hydroxytibolone.9-12

Tibolone is metabolized to 3β-hydroxytibolone by AKR1C1 and AKR1C2 and to 3{alpha}-hydroxytibolone by AKR1C4.15 AKR1C4 is expressed in the liver specifically, but AKR1C1 and AKR1C2 are expressed in the target tissue of tibolone, including the mammary gland and uterus.18 These facts explain how the main metabolite is 3{alpha}-hydroxytibolone in blood and 3β-hydroxytibolone in the target tissue. However, in human liver autopsy samples, HepG2 cells, and primary human hepatocytes, the main hepatic metabolite is 3β-hydroxytibolone.20 Interestingly, this report strongly supports our results but is inconsistent with previous results in postmenopausal women.9-12 Previous reports also indicated the existence of an unknown, extrahepatic metabolizing enzyme to explain this contradiction.20


Figure 2
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  		Figure 2. Effect of 17-β-estradiol (E2) on expression of AKR1C family in human cell lines. (A) Expression of AKR1C in human hepatoma cells (HepG2) after treatment with E2 (10 nM). Upper panel: mRNA level determined by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Lower panel: Immunoblot analysis of AKR1C. (B) Expression of AKR1C in human breast cancer cells (MCF-7) after treatment with E2 (10 nM). Upper panel: mRNA level determined by RT-PCR analysis. Lower panel: Immunoblot analysis of AKR1C.

 
An additional difference is our measurement in young, premenopausal women, whereas previous studies were performed in postmenopausal women. Although age alone could influence our results (we recruited 20 women younger than 25 years old), estrogen levels would also be different in these 2 populations. Estrogen affects the activity and expression of drug-metabolizing enzymes, including phase I and phase II enzymes,21,22 and also affects pharmacokinetics through hormone replacement therapy or oral contraceptives.23-26 Estrogen increases AKR1C (3{alpha}-hydroxysteroid dehydrogenase) expression in the liver and mesenteric arteries,27,28 but there are no direct data showing the effects of estrogen on the expression of each AKR1C subtype. We therefore evaluated the effects of estradiol on AKR1C expression using human HepG2 and MCF-7 cells. E2 treatment increased mRNA levels of AKR1C1, AKR1C2, and AKR1C3 but not AKR1C4 in HepG2 cells, and it increased total AKR1C protein levels. E2 treatment increased AKR1C2 expression only in MCF-7 cells. These data explain how young, healthy women could express more 3β-hydroxytibolone-producing enzyme than postmenopausal women in both elimination organs (HepG2 cells) and drug target organs (MCF-7 cells).

However, we still cannot explain why the main metabolite in postmenopausal women is 3{alpha}-hydroxytibolone, except that the existence of an additional metabolizing enzyme for tibolone20 could also be affected by estrogen levels. Another possibility could be the considerable differences in sulfotransferase (SULT) activity on the hydroxyl-metabolite of tibolone between pre- and postmenopausal women, which is about 4- to 5-fold higher in premenopausal women.29 However, SULT activities for each hydroxyl-metabolite were not significantly different.29 Because most of the hydroxyl-metabolite of tibolone exists as a sulfated form that forms a reservoir of tibolone activity, the effects of changed SULT activity between pre- and postmenopausal women should not be totally ruled out.

Another considerable factor is ethnic difference. Even though there was no description about race for enrolled participants, most of previous tibolone pharmacokinetic studies were conducted in Europe, which means the majority of enrolled participants in previous studies might have been Caucasian or at least more heterogeneous (multiracial). Interestingly, Zuo et al30 reported that 3{alpha}-hydroxytibolone is the main metabolite after oral administration of tibolone in Chinese women, and this seems to be similar to the results of previous studies conducted in Europe. In this study, the Chinese women ranged in age from 30 to 40 years (mean 36.4 years), but their menstrual status was not described (however, most of them would be premenopausal considering their age). The data with Chinese women are controversial compared with our data. The reason for the ethnic difference of tibolone metabolism might be due to the metabolizing enzyme. Until now, there has been no report about ethnic differences in the AKR1C family. However, the human AKRs are highly polymorphic, and some single-nucleotide polymorphisms (SNPs) of high penetrance exist.31 This characteristic of AKRs might be carefully considered as a cause for the ethnic differences of tibolone pharmacokinetics that may exist.

In conclusion, this study presents novel data that the main metabolite of tibolone in premenopausal women is 3β-hydroxytibolone, not 3{alpha}-hydroxytibolone, as reported in postmenopausal women. We also show that estrogen treatment increases AKR1C1-3 expression in human cell lines. These results suggest that changes in major tibolone metabolites might be due to changes in AKR1C family expression by estrogen status in patients. However, more detailed experiments will be needed to explain why the main metabolite of tibolone in postmenopausal women is 3{alpha}-hydroxytibolone and to confirm differences between tibolone pharmacokinetics in pre- and postmenopausal women.


   ACKNOWLEDGEMENTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
REFERENCES
 
The authors thank Dr Yong Lim Ko in the Seoul Health College for helping assay tibolone metabolites.

Financial disclosure: This study was supported by a Korea Research Foundation Grant funded by the Korean government (MOEHRD, Basic Research Promotion Fund; KRF-2006-003-E00067).

DOI: 10.1177/0091270008323262


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 

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