Drug Metabolism and Disposition Fast Forward
First published on April 9, 2007; DOI: 10.1124/dmd.106.014159
0090-9556/07/3507-1112-1118$20.00
DMD 35:1112-1118, 2007
Pharmacokinetic Parameters of Tibolone and Metabolites in Plasma, Urine, Feces, and Bile from Ovariectomized Cynomolgus Monkeys after a Single Dose or Multiple Doses of Tibolone
H. A. M. Verheul,
C. J. Timmer,
M. L. P. S. van Iersel,
L. P. C. Delbressine, and
H. J. Kloosterboer
NV Organon, Oss, The Netherlands
(Received December 4, 2006;
accepted April 5, 2007)
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Abstract
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Levels of nonsulfated and sulfated tibolone metabolites were determined in plasma, urine, and feces from six ovariectomized, mature female cynomolgus monkeys after a single dose and multiple p.o. doses (including bile) of tibolone using validated gas chromatography/mass spectrometry and liquid chromatography/tandem mass spectrometry assays. In plasma, the predominant nonsulfated metabolite after single and multiple dosing was the estrogenic 3
-hydroxytibolone; levels of the estrogenic 3ß-hydroxytibolone were 10-fold lower and of progestagenic/androgenic 
4-tibolone, 5-fold lower. Tibolone was undetectable. The predominant sulfated metabolite was 3S,17ßS-tibolone; levels of 3ßS,17ßS-tibolone were about 2-fold lower, and monosulfated 3-hydroxymetabolites were about 10-fold lower. After multiple doses, areas under the curve of nonsulfated metabolites were lower (2-fold), and those of sulfated metabolites were 25% higher. In plasma, >95% metabolites were disulfated. In urine, levels of all the metabolites after single and multiple doses were low. After a single dose, high levels of 3ß-hydroxytibolone and the 3-monosulfated metabolites (3ßS,17ßOH-tibolone and 3S,17ßOH-tibolone) were found in feces. After multiple dosing, 3-hydroxytibolone increased, and the ratio of 3/3ß-hydroxytibolone became about 1. The predominant sulfated metabolite was 3S,17ßS-tibolone. Levels of all the metabolites in feces were higher after multiple doses than after a single dose. Levels of nonsulfated and 3-monosulfated metabolites were higher in feces than in plasma. Bile contained very high metabolite levels, except monosulfates. This may contribute to the metabolite content of the feces after multiple doses. 3ß-Hydroxytibolone and 3S,17ßS-tibolone predominated. In conclusion, tibolone had different metabolite patterns in plasma, urine, feces, and bile in monkeys. The bile contributed to the metabolite pattern in feces after multiple doses. The major excretion route was in feces.
In randomized clinical trials, tibolone (Livial, Org OD14, Organon, Roseland, NJ and Oss, The Netherlands) has estrogenic effects on vasomotor symptoms, vagina, and bone in early postmenopausal women. No or only transient estrogenic effects on breast and endometrium (Rymer et al., 2002
; Kloosterboer, 2004; Landgren et al., 2005) are found as indicated by its neutral effects on mammographic density, a low incidence of tenderness, and no or only initial, transient vaginal bleeding. These tissue-specific effects may result from metabolism to different active metabolites and from a balance between activation by sulfatase and deactivation by the sulfotransferases (SULT) (see Fig. 1). This defines tibolone as a selective tissue estrogenic activity regulator (STEAR) (Kloosterboer, 2004). Previously reported (Timmer and Doorstam, 2002; Timmer and Houwing, 2002; Timmer and Huisman, 2002; Timmer et al., 2002; Vos et al., 2002) pharmacokinetic (PK) data in postmenopausal women have been limited to tibolone and its active, nonsulfated metabolites [3-hydroxytibolone (3OH-tib), 3ß-hydroxytibolone (3ßOH-tib), and 4-tibolone (4-tib)]. The predominant metabolite in blood is 3OH-tib, followed by 3ßOH-tib. Levels of tibolone and 4-tib after a single dose (sd) and after multiple doses (md) are low in postmenopausal women and become undetectable after 4 to 6 h. The 3OH-tib and 3ßOH-tib bind to the estrogen receptors, and tibolone and 4-tib bind to the androgen and progestagen receptors (de Gooyer et al., 2003; Kloosterboer, 2004). In a PK analysis (Vos et al., 2002) using radiolabeled tibolone, qualitative metabolite patterns have been determined with high-performance liquid chromatography in plasma, urine, and feces from three healthy, postmenopausal subjects, indicating that more than 75% of the metabolites in the circulation are monosulfated (mono-S) or disulfated (di-S) and that more than 80% of the radiolabel is excreted after 192 h, predominantly in the feces. Whereas sulfation renders compounds inactive at receptors, the enzyme sulfatase may readily reconvert 3-mono-S but not 17-mono-S metabolites into active receptor binding metabolites (Goldzieher et al., 1988; de Gooyer et al., 2001; Takanashi et al., 2003; Simoncini et al., 2004). As in postmenopausal women, pharmacological studies with tibolone in monkeys have shown that tibolone reduces hot flushes (Jelinek et al., 1984) and that 0.2 mg/kg/day tibolone for 2 years protects ovariectomized cynomolgus monkeys against bone loss, without stimulation of breast and endometrium (Clarkson et al., 2002, 2004; Cline et al., 2002; Williams et al., 2002). These results show a high similarity with the effects in postmenopausal women. In monkeys, only a very limited PK profile of tibolone has been determined: plasma levels of 3OH-tib, 3ßOH-tib, and 4-tib 1 h after dosing with 0.2 mg/kg/day tibolone were shown to be comparable with those after 2.5 mg/day in postmenopausal women (Clarkson et al., 2001; Timmer and Houwing, 2002). To determine the PK profile of nonsulfated and sulfated tibolone metabolites, a study was conducted in ovariectomized cynomolgus monkeys: an sd of 0.5 mg/kg tibolone followed, after a washout of 7 days, by md from day 8 to day 44. Blood, urine, and feces were collected for 7 days after the dose on day 1 and 36. At necropsy (day 44), tissues, blood, and bile were collected after the final dose of tibolone for their kinetic profiles. Because the bile is an excretion route for steroids and may also contribute to the steroid metabolite content of the feces, the results for the tibolone metabolites in the bile are reported here. Tissue results at necropsy will be reported elsewhere (Verheul et al., 2007). After development of analytically validated assays, blood, urine, feces, and bile have been assayed for their content of nonsulfated metabolites, 3-mono-S metabolites, and di-S metabolites. This article presents the levels of tibolone and its sulfated and nonsulfated metabolites in plasma, urine, bile (md only), and feces from ovariectomized monkeys after sd and md of 0.5 mg/kg/day tibolone.
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Materials and Methods
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Collection of Samples. Six mature, healthy, female cynomolgus monkeys (weighing 2.0–2.8 kg) were ovariectomized. After at least 1 month postovariectomy, the monkeys received an sd, and after a washout of 1 week, they received repeated daily p.o. doses of tibolone (Org OD14; 0.5 mg/kg/day) from days 8 through 44 via nasogastric gavage. Cynomolgus monkeys were selected in view of the high similarity to humans with respect to effects on target and safety tissues. A dose of 0.5 mg/kg/day was chosen for this study to enhance the chance to measure levels of tibolone's metabolites in plasma, urine, feces, bile, and tissues at the later time points. On days 1 and 36, plasma was collected at 0.5, 1, 2, 4, 6, 12, and 24 h after dosing into tubes containing K3EDTA, centrifuged (10 min at 2000g within 1 h after collection), and stored below –20°C until analysis. The volume required for the determination of the nonsulfated metabolites exceeded the available sample volume of a particular monkey at a particular time point. Because the means of the individual plasma levels for the sulfated metabolites were shown to be comparable with those of pooled samples, plasma samples were pooled per time point, and these pools were used for the analysis of the nonsulfated metabolites. Urine and feces were collected in daily pools per individual for 7 days after day 1 (sd) and during steady state after day 36 (md), respectively. On day 44, at 1, 1.25, 2.25, 4, 6, and 24 h after the final dose, one animal was necropsied, and the bile was collected. This design was used to establish plasma, bile, and tissue concentration-time curves, rather than to determine the concentrations at one single time point in six animals. The study was conducted at Huntingdon Life Sciences (East Millstone, NJ and Huntingdon, Cambridgeshire, UK) and complied with the Animal Welfare Act regulations and Good Laboratory Practice standards.
Preparation of Feces and Bile. Feces were weighed and homogenized with internal standards in 70% ethanol during 5 to 15 min using an ultraturrax at room temperature in cold buffers (0–4°C). Bile was weighed, and the analytes with internal standards were extracted in 70% ethanol during 5 to 15 min. The homogenates of feces and bile samples were divided into aliquots and stored at 0–4°C for at least 24 h before further sample processing. The aliquots were centrifuged; the supernatant was evaporated, redissolved in water, and stored at –20°C until analyzed as described below.
Assays. Tibolone, related reference compounds, and deuterated tibolone standards D5-tib, D5-3OH-tib, and D3-4-tib were supplied by Organon. Other compounds were obtained from Sigma-Aldrich (St. Louis, MO).
For the determination of tibolone, 3OH-tib, and 3ßOH-tib, the analytes and internal standard (D5-tib, D3-4-tib, D5-3OH-tib) were extracted from plasma, urine, or from the homogenates of bile or feces by solid-phase extraction using 100-mg, 3-ml Isolute C18 (EC) columns (Sopachem, Wageningen, The Netherlands) and eluted with acetonitrile. The analytes were quantified without derivatization (tibolone) and with derivatization (3OH-tib and 3ßOH-tib) using Tri-Sil reagent (Pierce, Etten-Leur, The Netherlands) under alkaline conditions at room temperature with gas chromatography/mass spectrometry (GC/MS) [Agilent Technologies (Palo Alto, CA) 6890/5973 GC-MSD] and operated in the positive mode (ABL, Assen, The Netherlands). The concentration of tibolone was corrected for conversion to 4-tib during the analytical procedure using the ratio D5-tib/D5-4-tib as correction factor. All the other tibolone metabolites were quantified (at Xendo, Groningen, The Netherlands) by high-performance liquid chromatography (PerkinElmer Instruments, Munich, Germany), with tandem mass spectrometry detection (LC/MS/MS) (API4000; Applied Biosystems, Nieuwekerk aan de IJssel, The Netherlands). For the analysis of 4-tib, samples were mixed with an equal volume of ammonium formate buffer (pH 5.0) and extracted by liquid-liquid extraction using 5 ml of ethyl acetate/hexane (50:50 v/v%). The resulting organic phase was evaporated, redissolved in water/methanol (50:50 v/v%), and subjected to LC/MS/MS analysis using a Synergi MAX-RP 80A column (Phenomenex, Torrance, CA) under isocratic conditions (35% ammonium acetate, pH 3.0, 65% methanol) and positive ionization (turbo ion spray).
The selection of the sulfated metabolites to be assessed in validated assays was based on a pilot study with plasma pools, urine, liver, and myometrium using semiquantitative and nonvalidated LC-MS assays (Organon, Schaijk, The Netherlands). The di-S metabolites (3S,17ßS-tib and 3ßS,17ßS-tib) proved to be present in high levels in plasma, urine, and tissues. Both 3-mono-S metabolites (3S,17ßOH-tib and 3ßS,17ßOH-tib) and 17ß-mono-S metabolites (3OH,17ßS-tib, 3ßOH,17ßS-tib, and 17ßS-tib) were evaluated. The 3-mono-S metabolites were readily detected. However, 17ß-mono-S metabolites could only be detected in plasma pools and not in urine and tissues: the areas under the curve (AUC) in plasma were 143 and 255 ng/ml · h after sd and 201 and 273 ng/ml · h after md for 3OH,17ßS-tib and 3ßOH,17ßS-tib, respectively. Low (<5 ng/ml) levels of 17ßS-tib were detected only at 0.5 and 1 h. This indicates that the levels of 17ß-mono-S metabolites were low, presumably because they are readily sulfated to di-S metabolites. In a previous study (Vos et al., 2002) with radiolabeled tibolone, 17ß-mono-S metabolites constituted less than 10% of the radiolabel administered. Based on these results, the limited sample volumes, and because 17-mono-S compounds cannot readily be desulfated (Goldzieher et al., 1988; de Gooyer et al., 2001; Takanashi et al., 2003; Simoncini et al., 2004) to receptor-binding metabolites, it was decided to develop validated assays for the di-S metabolites and only for 3-mono-S metabolites. The results obtained with the semiquantitative analysis proved to be similar to those obtained with the validated assays.
For the analysis of the 3-mono-S metabolites in the quantitative and validated assays at Xendo, samples were extracted with ethyl acetate/hexane (50:50 v/v%) as described for 4-tib. The resulting water phase was subjected to on-line solid-phase extraction using a Prospect 2 system with a HySphere C18 HD cartridge (Spark Holland B.V., Emmen, The Netherlands), followed by LC/MS/MS analysis using a Synergi MAX-RP 80A column (Phenomenex) under isocratic conditions (100% ammonium acetate, pH 6.0) and negative ionization (turbo ion spray). The di-S metabolites were isolated from the samples by protein precipitation with 2 volumes of acetonitrile. The supernatant was evaporated, redissolved in 100 µl of ammonium acetate (pH 6.0)/methanol (70:30 v/v), and analyzed by LC/MS/MS using a Zorbax SB Phenyl column (Agilent Technologies through Bester, Amstelveen, The Netherlands) with an ammonium acetate (pH 6.0)/methanol gradient and negative ionization.
The GC/MS procedures have been validated for human serum, and LC/MS/MS procedures have been validated for human serum, myometrium, and breast tissue with regard to selectivity, sensitivity, calibration curves, accuracy, precision, stability, dilution, and carryover. Validation procedures were guided by Shah et al. (2000) and the U.S. Department of Health and Human Services, Food and Drug Administration Guidance for Industry (2001). The procedures have been used for monkey plasma, urine, feces, and bile without further validation. Detection limits were 0.1 to 0.5 ng/ml (for plasma and urine) and 0.5 to 2 ng/g (for feces and bile). Analytes were determined with acceptable precision (coefficient of variance <20% for overall, within-batch, and between-batch variation) and accuracy (bias <20%), except for the mono-S metabolites. Quality control samples for the 3-mono-S metabolites showed that the bias was >20% for low and medium concentrations, resulting in a maximal 60% overestimation. Despite this overestimation, the levels of the mono-S were very low, and the mono-S metabolites seem to contribute little to the metabolite patterns. Therefore, the potential overestimation at lower levels was accepted. For metabolites with concentrations outside the calibration range, a "best estimate" of the concentration is given, provided that the peak exceeded the background by at least 3-fold; if lower, a best estimate of "0" was assigned.
Calculations. AUC0-tlast, AUC0–24 h, or AUC0.5–24 h, Cmax, and Tmax were calculated using WinNonlin version 4.1 on SAS version 8.2 (SAS Institute, Cary, NC). If a value is missing, a best estimate was made by intrapolation of the results at adjacent time points. Means per metabolite and per time point were calculated. Ratios and percentages of metabolites were calculated by matrix, sampling time, and AUC.
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Results
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Plasma. The predominant nonsulfated metabolite after sd and md was 3OH-tib, followed by 4-tib and 3ßOH-tib with 5- and 10-fold lower levels, respectively; see Fig. 2, left top and bottom, for concentration-time curves and Table 1 for the PK parameters (AUC, Cmax, Tmax, and t1/2). The levels rapidly decreased over time. Tibolone was undetectable. After md, AUC of all the nonsulfated metabolites were approximately 2-fold lower than after sd. At 20- to 60-fold higher levels than the nonsulfated 3OH-tib, the predominant sulfated metabolite after sd and md was 3S,17ßS-tib, followed by 3ßS,17ßS-tib and the 3-mono-S, 2- and 10-fold lower, respectively, than the corresponding di-S metabolites (Fig. 2, right top and bottom; Table 1). AUC and Cmax of di-S metabolites after md were about 25% increased compared with sd, whereas the AUC of the mono-S metabolites remained the same. The Tmax for the nonsulfated tibolone metabolites was 0.5 h after both sd and md; the Tmax for the mono-S metabolites changed from 1 h after sd to 0.5 h after md, whereas the Tmax for the di-S metabolites remained 2 h. The plasma concentrations of all the metabolites, except for 3S,17ßS-tib, returned to baseline at 24 h after sd and md, indicating that these metabolites did not accumulate. The levels of the di-S metabolites at 24 h after sd were similar to those after md. Compared with sd, t1/2 of 3OH-tib and 3ßOH-tib after md was reduced, whereas the t1/2 of the di-S metabolites was higher (Table 1). To further characterize the metabolite profile in plasma independent of the actual levels and to allow comparison with the profiles in urine, feces, and bile, we examined various ratios and percentages. The progestagen/estrogen ratio [(tibolone + 4-tib)/(3OH-tib + 3ßOH-tib)] ranged from 0.1 to 0.4 after sd and md, indicating that the balance is toward the 3OH metabolites in plasma. The 3/3ß ratio (3OH-tib/3ßOH-tib) was >10. The 3 and 3ß metabolites were predominantly present in their sulfated forms, and percentages of sulfated compounds increased in time from 80% at 0.5 h to 98% at 24 h and from 96 to 99% for sd and md, respectively, which is in line with the sulfated percentages calculated using the AUC (Table 1). The percentage of 3-mono-S metabolites decreased both after sd (18% at 0.5 h to 4% at 24 h) and md (9% to 1%). Based on AUC, tibolone metabolites were present as disulfates for more than 95 and 98% after sd and md, respectively.
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FIG. 2. Levels in plasma pools after sd and md of tibolone. Concentration-time plots of nonsulfated (A and C) and sulfated (B and D) tibolone metabolites after sd (A and B) and md (C and D) of tibolone are shown. It should be noted that y-axes have different scales. Plasma pools were made by mixing equal volumes from individual samples.
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Urine. Table 2 shows that after sd and md, 3ßOH-tib tended to be the nonsulfated tibolone metabolite in urine with the highest levels. The levels of tibolone were undetectable. The predominant sulfated metabolite was 3S,17ßS-tib. Levels of mono-S metabolites were 10- to 20-fold lower. Metabolite levels in urine after sd became undetectable after 3 to 4 days. It should be realized that after md, levels of the metabolites should be comparable for each of the 24-h collection periods because monkeys received a new dose of tibolone each morning. Compared with the total amount of metabolites excreted during 0 to 168 h after sd, the levels of nonsulfated metabolites in 24-h urine samples after md were 2 to 4 times higher, and the levels of the sulfated metabolites were similar. In view of these low levels and a urinary volume of 100 to 120 ml/day, urine seemed to contribute very little to the excretion of tibolone. The 3/3ß ratio in urine was 0.8, clearly different from the ratios found in plasma.
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TABLE 2 Concentrations (ng/ml) of tibolone and nonsulfated and sulfated metabolites and percentages in monkey urine samples
See Table 1 footnotes. The mean (±S.D.) urine volume is 101 ± 6 ml and 123 ± 11 ml after sd and md, respectively.
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Feces. The predominant nonsulfated and sulfated metabolites after sd in feces were 3ßOH-tib and the mono-S metabolite 3ßS,17ßOH-tib, respectively (Table 3). As indicated previously, the levels after md should be comparable for each of the 24-h collection periods. After md, the levels of the nonsulfated metabolites, 3OH-tib and 3ßOH-tib, were equally high, and 4-tib and tibolone were still present in considerable amounts (Table 3). The predominant sulfated metabolite after md was the di-S metabolite 3S,17ßS-tib, followed by 3ßS,17ßS-tib. The mono-S metabolites were 30 to 40% of all the sulfated metabolites. During all the 24-h collection periods after sd, the levels of all the nonsulfated and sulfated metabolites were lower than after md. The concentrationtime curves of the combined estrogenic (3OH-tib and 3ßOH-tib), progestagenic (tib +4-tib), mono-S, and di-S metabolites are presented in Fig. 3 (left, sd; right, md) and show that the levels of all the metabolites after sd decreased to <10 ng/g after 96 h. The levels of the nonsulfated 3OH metabolites (3OH-tib + 3ßOH-tib) were relatively high compared with those of the mono-S and di-S metabolites (Fig. 3, left). Figure 3 (right) shows that the pattern of the three 24-h collection periods after md was comparable. It also shows that the amount of the nonsulfated metabolites after md during a 24-h period is about equal to the total amount (0–168 h) after sd, whereas that of the di-S metabolites was 3- to 4-fold higher and that of 3ßS,17ßOH-tib was 3-fold lower. Comparing the results in urine (Table 2) with those in feces (Table 3) and taking the average urine volume (100–120 ml) and feces weight (about 60 g) into account, it is clear that the major route of excretion for tibolone and its metabolites was via the feces. As in plasma, the progestagen/estrogen ratio after sd and md in feces was low (<0.4), whereas, in contrast, the ratio of 3OH-tib/3ßOH-tib was about 1. The percentage of sulfated metabolites after md was comparable with sd, whereas the percentage of mono-S was about 2-fold lower after md. Compared with plasma, the percentage of sulfated metabolites was about 2-fold lower in feces, whereas the percentage of mono-S metabolites was more than 10-fold higher.
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TABLE 3 Concentrations of tibolone and nonsulfated and sulfated metabolites and percentages in monkey feces samples
See Table 1 footnotes. The mean (± S.D.) weight of feces is 57 ± 4 g and 61 ± 4 g after sd and md, respectively.
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Bile. Bile was collected at necropsy after multiple doses of tibolone with one animal per time point. The levels of all the nonsulfated metabolites were high at 1 h and decreased to baseline levels at 24 h, whereas the levels of di-S metabolites increased about 2-fold from 1 to 1.25 h and were at 24 h above 20,000 ng/g and 2500 ng/g for 3S,17ßS-tib and 3ßS,17ßS-tib, respectively. Based on AUC, 3ßOH-tib and 3S,17ßS-tib were the predominant nonsulfated and sulfated metabolites, respectively (Table 4). The progestagen/estrogen ratio was very low (<0.05), and the ratio of 3OH-tib/3ßOH-tib ranged from 0.2 to 0.6. The percentage of the mono-S metabolites was about zero in the bile. Compared with plasma, the AUC and Cmax of all the metabolites in bile were considerably higher except for the 3-mono-S metabolites.
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TABLE 4 PK parameters in monkey bile after multiple doses of tibolone
See Table 1 footnotes. Note: data from the monkey necropsied at 6 h after final dose have been excluded from the calculation for the PK parameters because the total weight of the bile was very low. Mean (±S.D.) weight bile: 0.72 ± 0.34 g.
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Discussion
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This report presents for the first time the kinetic profiles of tibolone and its nonsulfated and sulfated metabolites in plasma, urine, and feces from cynomolgus monkeys after sd and md of tibolone. At necropsy, after md, the metabolite profile is also determined in bile.
As in humans (Timmer and Doorstam, 2002; Timmer and Houwing, 2002; Timmer and Huisman, 2002; Timmer et al., 2002), the predominant, nonsulfated metabolite in monkey plasma is the estrogenic 3OH-tib with a Tmax of about 0.5 h; the levels of tibolone and the progestagenic/androgenic 4-tib rapidly decrease with time. The lower or comparable levels and AUC in monkey plasma after md and sd suggest that no accumulation of tibolone and its metabolites occurs in plasma, except for the di-S metabolites, which have higher Cmax (20–30%) and AUC (about 50%) after md. However, the levels of the di-S metabolites at 24 h after dosing were comparable between sd and md, suggesting no accumulation. A clear difference with humans is the ratio of 3OH-tib/3ßOH-tib in monkey plasma (>10 times versus 3 times in humans) (Timmer and Doorstam, 2002; Timmer and Houwing, 2002; Timmer and Huisman, 2002; Timmer et al., 2002; Kloosterboer, 2004). This may be attributed to species differences or the study design (e.g., dose or formulation).
The data in plasma indicate that the most important phase I metabolic reaction is the rapid reduction of the 3-keto group to 3OH-tib. In vitro studies have shown that the aldoketoreductase (AKR) 1C4 enzyme expressed in liver (Steckelbroeck et al., 2004) predominantly catalyzes the formation of 3OH-tib. Nonsulfated OH groups are rapidly sulfated, the 3-mono-S appearing slightly faster in plasma than the 3,17-di-S metabolites both after sd and md. In vitro studies have shown that tibolone and its metabolites can readily be sulfated at the C3 position by SULT2A1 (all the tibolone metabolites, with a high affinity for 3OH-tib), SULT1E1 (all the tibolone metabolites, except 4-tib), and SULT2B1b (3OH-tib and 3ßOH-tib only) (Falany et al., 2004). SULT2A1, expressed in liver, was shown to be able to produce 3,17-di-S metabolites (Falany et al., 2004), explaining the high levels of the di-S in the circulation. Compared with sd, levels of nonsulfated metabolites in plasma after md are lower and those of sulfated metabolites, in particular the di-S metabolites, are higher. A reduced expression of the AKR1C family members to explain the lower levels of the nonsulfated metabolites is less likely because the consequent increase in levels of tibolone or 4-tib has not been found. A more efficient sulfation (e.g., by induction of SULT) explains the lower levels of the nonsulfated metabolites and the shift toward higher levels of the di-S metabolites. However, it does not explain the extent of the increase; the reduction in the AUC of the nonsulfated metabolites is, by far, exceeded by the increase in the AUC of the sulfated metabolites. In addition, the induction of SULT has only been described for SULT1E1 in endometrial tissues by progestagens, including tibolone (Falany and Falany, 1996). Other explanations for the extent of the increase in the di-S metabolites after md may be the contribution of the enterohepatic circulation, which is obviously not yet present after sd. Another explanation may be the slower elimination of the di-S metabolites, which is supported by the relatively high elimination half-life for di-S metabolites found in plasma.
Tibolone metabolites may be excreted via urine and feces. After sd, the metabolite pattern in feces shows relatively high levels of nonsulfated 3-hydroxymetabolites, tibolone, 4-tib, and of the mono-S metabolites, whereas levels of di-S metabolites are relatively low. This pattern is different from that in plasma. Because the contribution of the bile to the feces is expected to be low in the early phase after sd, the metabolic pattern after an sd seems to reflect the metabolic capacity of the gastrointestinal tract. This pattern could be explained as follows: part of the tibolone dose is not absorbed but chemically converted in the stomach to 4-tib, explaining the high levels of 4-tib in the feces; part of tibolone is metabolized in the intestine to 3-hydroxymetabolites by the AKR1C family present in the intestine (Penning et al., 2000; Steckelbroeck et al., 2004). The nonsulfated hydroxy groups can then be sulfated by SULT, such as SULT2A1, SULT1E1, or SULT2B1b (Falany et al., 2004). Recently, SULT2A1 was shown to be present in the small intestine of postmenopausal women (Wang et al., 2006). The initial sulfation capacity in the intestine seems to be low in view of the high mono-S and the low di-S metabolite levels after sd in feces. After md, the concentrations of all the metabolites during a 24-h period are higher than those during the 0- to 24-h period after sd. However, assuming that the total excreted levels after sd (period 0–168 h) are equivalent to a 24-h period after md, the metabolite levels in the feces after sd are similar to those after md for all the nonsulfated metabolites and 3S,17ßOH-tib. After md, however, the levels of the di-S metabolites (3S,17ßS-tib and 3ßS,17ßS-tib) were about 3-fold higher, and the levels of the mono-S metabolite (3ßS,17ßOH-tib) were about 3-fold lower than after sd. The reduction in the mono-S level after md may be explained by a more efficient sulfation. In addition, the bile may significantly contribute to the metabolite pattern in the feces after md, in view of its high concentrations of tibolone metabolites and because the bile weight at a particular moment (0.7 g) only reflects a part of the daily production and of the volume emptied in the intestine. Compared with the feces, the levels of metabolites in urine are much lower. Taking into account the mean weight of the feces (about 60 g) and the urine volume (100–120 ml), it is clear that the major route of excretion of tibolone is via the feces.
In the bile, higher levels of 3ßOH-tib than of 3OH-tib are found after md, which is opposite to the situation in plasma. This may indicate that the 3ßOH-tib is more efficiently absorbed from plasma or excreted by the liver, thus explaining the plasma predominance of the 3OH-tib and the bile predominance of 3ß-OH-tib. However, local metabolization in liver and bile of tibolone to the 3ßOH-tib cannot be excluded.
It is concluded that after sd and md, 3OH-tib and 3S,17ßS-tib are the predominant nonsulfated and sulfated metabolites in plasma, respectively. Different tibolone metabolite patterns are observed with high levels of di-S metabolites in plasma, bile, and urine, especially after md, whereas feces contained high levels of nonsulfated and mono-S metabolites. The bile contributes to the metabolite pattern in the feces. The major route of excretion for tibolone metabolites is via the feces.
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Footnotes
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.106.014159.
ABBREVIATIONS: tibolone, (7,17)-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one; SULT, sulfotransferase(s); PK, pharmacokinetic; 3OH-tib, 3-hydroxytibolone; 3ßOH-tib, 3ß-hydroxytibolone; 4-tib, 4-tibolone; sd, single dose; md, multiple doses; mono-S, monosulfated; di-S, disulfated; GC/MS, gas chromatography/mass spectrometry; LC/MS/MS, liquid chromatography/tandem mass spectrometry; AUC, area under the curve; AKR, AKR1C, aldoketoreductase 1C family.
Address correspondence to: Herman A. M. Verheul, NV Organon, P.O. Box 20, 5340BH Oss, The Netherlands. E-mail: herman.verheul{at}organon.com
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H. A. M. Verheul, M. L. P. S. van Iersel, L. P. C. Delbressine, and H. J. Kloosterboer
Selective Tissue Distribution of Tibolone Metabolites in Mature Ovariectomized Female Cynomolgus Monkeys after Multiple Doses of Tibolone
Drug Metab. Dispos.,
July 1, 2007;
35(7):
1105 - 1111.
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Abstract
Materials and Methods
) of (tibolone +