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Objectives: A volunteer trial was performed to compare the pharmacokinetics of 5 drugs--warfarin, ZK253 (Schering), diazepam, midazolam, and erythromycin--when administered at a microdose or pharmacologic dose. Each compound was chosen to represent a situation in which prediction of pharmacokinetics from either animal or in vitro studies (or both) was or is likely to be problematic., Methods: In a crossover design volunteers received (1) 1 of the 5 compounds as a microdose labeled with radioactive carbon (carbon 14) (100 microg), (2) the corresponding (14)C-labeled therapeutic dose on a separate occasion, and (3) simultaneous administration of an intravenous (14)C-labeled microdose and an oral therapeutic dose for ZK253, midazolam, and erythromycin. Analysis of (14)C-labeled drugs in plasma was done by use of HPLC followed by accelerator mass spectrometry. Liquid chromatography-tandem mass spectrometry was used to measure plasma concentrations of ZK253, midazolam, and erythromycin at therapeutic concentrations, whereas HPLC-accelerator mass spectrometry was used to measure warfarin and diazepam concentrations., Results: Good concordance between microdose and therapeutic dose pharmacokinetics was observed for diazepam (half-life [t((1/2))] of 45.1 hours, clearance [CL] of 1.38 L/h, and volume of distribution [V] of 90.1 L for 100 microg and t((1/2)) of 35.7 hours, CL of 1.3 L/h, and V of 123 L for 10 mg), midazolam (t((1/2)) of 4.87 hours, CL of 21.2 L/h, V of 145 L, and oral bioavailability [F] of 0.23 for 100 microg and t((1/2)) of 3.31 hours, CL of 20.4 L/h, V of 75 L, and F of 0.22 for 7.5 mg), and development compound ZK253 (F = <1% for both 100 microg and 50 mg). For warfarin, clearance was reasonably well predicted (0.17 L/h for 100 microg and 0.26 L/h for 5 mg), but the discrepancy observed in distribution (67 L for 100 microg and 17.9 L for 5 mg) was probably a result of high-affinity, low-capacity tissue binding. The oral microdose of erythromycin failed to provide detectable plasma levels as a result of possible acid lability in the stomach. Absolute bioavailability for the 3 compounds examined yielded excellent concordance with data from the literature or data generated in house., Conclusion: Overall, when used appropriately, microdosing offers the potential to aid in early drug candidate selection.

Objective: Contraception is recommended for female patients during ursodeoxycholic acid (UDCA) treatment for the potential teratogenic effect of this bile acid, and the aim of our study was to determine whether this treatment affects the bioavailability of ethinylestradiol (EE2)., Methods: In this double-blind, randomised study, we measured EE2 pharmacokinetics in eight healthy volunteers randomly allocated to receive oral contraceptive (30 microg EE2 and 75 microg gestodene) plus either UDCA (8-10 mg/kg per day) or placebo for 21 days during the first of three consecutive menstrual cycles. After a washout period during the second cycle, the subjects received the alternative treatment during the third menstrual cycle. Serum EE2 and UDCA were measured using radioimmunoassay and gas chromatography-mass spectrometry, respectively., Results: The profile for serum EE2 concentration was similar during UDCA (mean maximum serum concentration 177 pg/ml, SEM 59) and during placebo treatment (153 pg/ml, SEM 62), and mean area under the curve (AUC) was 1374 pg/h per ml (SEM 580) and 1320 pg/h per ml (SEM 551) during the two regimens, respectively. The point estimates and 90% confidence intervals of UDCA/placebo ratios for EE2 AUC and for maximum serum concentration were 1.1 (0.8-1.5) and 1.2 (1.0-1.4), respectively. Mean serum triglycerides concentration increased from 58.3 mg/dl (SEM 6.8) at enrolment to 91.4 mg/dl (SEM 10.7) during placebo (P < 0.01) and to 88.6 mg/dl (SEM 13.7) during UDCA treatment (P < 0.05). During UDCA treatment, serum enrichment with this bile acid and with the metabolite iso-UDCA was 29% (16%) and 3% (2%), respectively., Conclusion: Co-administration with UDCA does not affect the bioavailability of EE2 in healthy volunteers, indicating that contraceptive efficacy is not affected.

This study investigated the pharmacokinetics of a dose-reduced oral contraceptive containing 20 microg ethinylestradiol (EE) + 100 microg levonorgestrel (LNG) in 18 young, healthy females. Serum levels of EE and LNG were determined after single and repeated daily oral administration over three treatment cycles, each consisting of 21 treatment days followed by a 7-day treatment-free period. Additionally, the time courses of sex hormone-binding globulin (SHBG), corticoid-binding globulin (CBG) and total and free testosterone serum levels were analyzed. Both active ingredients were rapidly absorbed and maximum concentrations in serum were reached between, on average, 1 and 2 h after single and multiple administrations, respectively. Concentrations of EE increased during repeated daily administration. An approximate two-fold accumulation was calculated based on the comparison of EE area under the curve (AUC) (0-24 h) values determined after the first and the last tablet administration within a treatment cycle. LNG serum concentrations also increased during repeated daily administration, reaching steady-state levels after about 11 days. Based on the comparison of AUC (0-24 h) values determined after the first and the last tablet administration, LNG accumulated approximately by a factor of 3 within a treatment cycle. Steady-state pharmacokinetics of LNG were similar at the end of the first and the third treatment cycles, indicating no further accumulation of LNG beyond a treatment cycle under long-term use of this combined oral contraceptive. The clearance and volume of distribution of LNG decreased and the terminal half-life increased after repeated daily administration, compared with single administration. These effects have also been reported for other LNG/EE combinations. SHBG serum concentrations increased during repeated daily intake by, on average, 1.5-1.6-fold, and for CBG, an average increase of 1.4-1.8-fold was found. Although free testosterone concentrations declined during repeated daily administration by about 40%, total testosterone remained relatively unchanged at a low level. In conclusion, the pharmacokinetics of EE and LNG determined in the present study were in good agreement with those previously reported for 30 microg EE + 150 microg LNG, taking the 33% dose reduction into account.

In the present study, a new in vitro model combining the short-term incubation of precision-cut human liver slices with DNA-adduct analysis by the (32)P-postlabelling technique is proposed for investigation of the genotoxic potential of xenobiotics. For method validation, the metabolic turnover of testosterone (TES) and the DNA-adduct inducing potential of 2-aminofluorene (2-AF) were used. Precision-cut human liver slices were prepared from a total of 12 human liver samples which were freshly obtained as parts of resectates from liver surgery. The slices were incubated as submersion cultures with TES and 2-AF for up to 6 h in 12-well tissue culture plates at concentrations of 10-50 and 0.06-28 μM, respectively. Slices recovered from the slicing procedure in the 4 °C cold Krebs-Henseleit buffer as indicated by intracellular potassium concentrations which increased for 2 h and then remained stable until the end of the incubation. TES was extensively metabolized by human liver slices with a similar metabolite pattern as observed in vivo. Almost 90% of the metabolites were conjugates. Major phase-I metabolites were androstendione, 6β-OH-androstendione, 6β-OH-TES, and 15β-OHTES. After incubation with 2-AF, substance related DNA-adducts were detected which increased dose-dependently from 12 to 1146 adducts per 10(9) nucleotides. The adduct pattern consisted of one major adduct spot, A, representing 80-90% of the total adduct level and up to four minor adduct spots, B-E. In summary, the present data demonstrate that precision-cut liver slices are a valuable alternative in vitro system for DNA-adduct determination to screen chemicals for potential genotoxicity in humans.

It was the aim of this study to investigate whether assessment of the metabolic stability of selected progestins of the 19-nortestosterone type in human microsomal liver preparations was a suitable approach to predict the oral bioavailability of these drugs in humans. The Michaelis-Menten parameters Vmax and KM,app for norethisterone, levonorgestrel, gestodene, desogestrel, 3-keto-desogestrel, norgestimate, and dienogest were determined in in vitro incubations with human liver microsomes. Using these data, both the in vitro intrinsic clearance (CLint) and, after application of a suitable scaling factor, the scaled in vivo CLint were calculated. For progestins for which human in vivo data were available, the in vitro results were correlated with in vivo CLint values and oral bioavailability. A comparison of the scaled in vivo CLint values with the corresponding in vivo CLint values showed a reasonable correlation, although the latter values were generally approximately 2-fold higher than the former. Excluding desogestrel, which is subject to substantial intestinal metabolism in vivo, there was a linear relationship (r = -0.986) between increasing in vitro CLint values for the progestins and decreasing bioavailability in vivo. Other methods of assessing the metabolic stability of the progestins in vitro, such as evaluation of metabolic half-lives at single initial concentrations, showed either no correlation or a less satisfactory correlation with bioavailability data.

We report investigations into the potential of the steroid hormones chlormadinone acetate (CMA), cyproterone acetate (CPA), dexamethasone (DEX), estradiol (E(2)), ethinylestradiol (EE(2)), gestodene (GEST), levonorgestrel (LNG), megestrol acetate (MGA), medroxy progesterone acetate (MPA), mifepristone (MIFE), norethisterone (NET), prednisolone (PRED), progesterone (P) and testosterone (T) to form DNA adducts in precision-cut human liver slices in vitro from 14 male and female donors using the (32)P-postlabelling technique. The synthetic steroid hormones CPA, CMA and MGA generated DNA adducts in human liver slices obtained from all donors. MPA-related adduct spots were only observed in some of the livers tested. No DNA adduct formation was detectable with DEX, EE(2), E(2), GEST, LNG, MIFE, NET, PRED, P and T. The total DNA adduct levels and adduct patterns were different for each compound. On average, total DNA adduct levels decreased in the following order: CPA>MGA>CMAMPA. The DNA adduct levels varied inter-individually. At a treatment concentration of 1mug/ml, the coefficient of variation of the total adduct levels ranged from 38% to 101%. A sex-specific distribution of the DNA adduct formation was only detected after incubation with MPA. MPA-related adduct spots were observed predominantly in the livers of female donors.

Previous studies with postmenopausal women receiving oral doses of norethisterone-containing preparations have shown that a small fraction of the dose is converted metabolically to ethinyl estradiol and may be detected in the peripheral blood. To investigate the extent and the dose dependence of this conversion in more detail, we performed a study with 24 postmenopausal women who received single oral doses of 5 mg norethisterone as well as 5 and 10 mg norethisterone acetate with a washout phase of 2 weeks between each treatment. After each treatment, blood was collected at regular intervals and the concentrations of norethisterone and ethinyl estradiol were analyzed in the serum samples by a specific radioimmunoassay and by gas chromatography/mass spectrometry, respectively. Ethinyl estradiol was present in the serum samples of all women following treatment with norethisterone acetate and, except for four cases, also after treatment with norethisterone. The conversion ratio of norethisterone acetate to ethinyl estradiol was 0.7 +/- 0.2% and 1.0 +/- 0.4% at doses of 5 and 10 mg, respectively. This corresponded to an oral dose equivalent of about 6 micrograms ethinyl estradiol per milligram of norethisterone acetate. For norethisterone, a conversion ratio of 0.4 +/- 0.4% was found at a dose of 5 mg, which corresponded to an oral dose equivalent of about 4 micrograms ethinyl estradiol per milligram of norethisterone. Although it cannot be excluded that in individual cases, even higher doses of ethinyl estradiol may be produced by conversion, it is concluded that at therapeutic doses of the progestogens, the exposure to metabolically derived ethinyl estradiol is probably of little clinical significance not only in fertile women using oral contraceptive combination preparations containing norethisterone and ethinyl estradiol, but also in postmenopausal women who receive oral doses of estradiol for estrogen replacement. The estrogenic effects of metabolically derived ethinyl estradiol on the liver (eg. synthesis of transport proteins) are very likely more than compensated due to the androgenic activity of norethisterone.

Objective: To investigate a possible age-dependence of the pharmacokinetics of cyproterone acetate following single oral administration., Methods: Twenty eight healthy men between 22 and 74 years of age received a single oral dose of 100 mg cyproterone acetate. The pharmacokinetic parameters, area under the serum concentration-time curve, apparent volume of distribution, apparent clearance, terminal half-life and the concentration ratio of 15 beta-hydroxycyproterone acetate/cyproterone acetate were examined for possible age-dependence using regression analysis., Results: The values of area under the serum level-time curve showed high interindividual variability and were not related to age. With regard to apparent clearance and volume of distribution, decreasing and increasing values, respectively, were observed with increasing age. There was also a clear dependence of the terminal half-life on age. Elderly men had values about two times higher (95 h) than men belonging to the younger age groups (45 h). The mean concentration ratio of 15 beta-hydroxycyproterone acetate/cyproterone acetate was 0.8 (0.3) and showed no age-dependent change., Conclusions: Apparent clearance and apparent volume of distribution of cyproterone acetate showed age-dependent changes. Combined, the two effects cause a clear age-dependence of the terminal half-life of cyproterone acetate. An age-related reduction in liver volume is thought to be mainly responsible for the decrease in hepatic clearance with age. Chronic daily administration of the drug to elderly men may therefore lead to somewhat higher steady-state concentrations in the serum than in young men receiving the same dose.

It was the aim of the study to compare the pharmacokinetic properties of the two new estrogens, ZK 136295 and ZK 115194, with those of ethinylestradiol (EE2) after single intravenous (60 micrograms) and oral (120 and 240 micrograms) administration in 54 postmenopausal women. In particular, our objective was to examine whether one or both compounds were characterized by an improved oral bioavailability with less inter-subject variability than EE2. Drug serum concentrations were determined using specific radioimmunoassays for EE2 and ZK 136295, and a GC/MS/MS-method for ZK 115194. Following i.v. administration of the new estrogens and of EE2, the drugs were rapidly distributed in the body. The mean terminal half-lives were calculated to be 12.3 +/- 12.4, 28.7 +/- 9.6, and 26.1 +/- 11.1 h for ZK 136295, ZK 115194, and EE2 respectively. After oral administration of 120 micrograms, the absolute bioavailability was calculated to be about 40% for ZK 136295 as well as for EE2 with a high inter-individual variation (variation coefficient: 44 and 67%). By doubling the dose, the systemic availability increased dose-dependently for both drugs to about 70% with the same high inter-individual variation. Following single oral administration of 240 micrograms ZK 115194, the absolute bioavailability amounted to 33 +/- 19%. The present study clearly revealed that although the two new estrogens differed considerably in their pharmacokinetic behavior, they demonstrated a reduced and highly variable systemic availability similar to that of EE2.

The effect of a 26 day oestrogen-gestogene sequence therapy on double endometrium thickness, uterus size, blood flow in the uterine blood vessels (pulsatility and resistance index), serum concentration of FSH, LH and SHBG and on vaginal cytology of postmenopausal women was tested double-blind in a placebo-controlled trial. Thirteen postmenopausal women received 0.06 mg ethinyloestradiol (EE2) over 14 days and then a 12 day combination treatment with 0.04 mg EE2 and 0.125 mg levonorgestrel. Eight women received placebo treatment over 26 days. In each group, half the women were less, and half were more than 10 years postmenopausal. The above parameters were unchanged in the placebo group. However, in the verum group, the double endometrium thickness increased from 2 mm to about 6 mm after a 7 day treatment with EE2 and after a further treatment week with EE2, increased another millimeter. There was no obvious difference in the treatment groups between women less than ten years after menopause or more than ten years after menopause. Blood flow in the uterine artery increased significantly (decrease in pulsatility and resistance index by about 50%). Again, there did not seem to be any obvious connection with the menopausal interval. FSH decreased after the first treatment week by about 50% and 65% after the second treatment week. There was no significant decrease in LH. SHBG increased by about a factor of 5. An oestrogen effect could be demonstrated in the vaginal cytology of all cases in the verum group.

Cyproterone acetate (CAS 427-51-0, CPA) is a steroid hormone with antiandrogenic and progestogenic properties, which has been used in the therapy of prostate carcinoma in men, and for the treatment of severe acne and hirsutism in women. The aim of the present study was to compare two equally sensitive analytical methods, a radioimmunoassay (RIA) and a gas chromatographic/mass spectrometric method (GC/MS), for the quantitative determination of CPA in human serum samples and to assess their suitability for bioequivalence trials. To this end, serum samples which had been collected during a study on the bioequivalence oaf two CPA-containing formulations (Androcur 100 and Androcur 50) were analysed using both methods. Basic pharmacokinetic parameters, like Cmax, tmax, t1/2 and AUC were calculated from each data set and corresponding parameters were compared by statistical methods. A comparison of the drug concentration-time curves obtained with both analytical methods revealed that in particular at later sampling times (48 to 120 h post dose) concentration values generated by RIA were slightly higher by about 20-40% than those measured with GC/MS. This indicates the presence of cross-reacting metabolite(s), most likely the 15 beta-hydroxy-cyproterone acetate. Accordingly, the values of Cmax, AUC(0-120 h) and AUC were overestimated by the RIA method by about 10-20%, 5% and 10%, respectively. However, there was a high correlation between corresponding parameters derived from RIA and GC/MS analysis. Although AUC values were slightly overestimated with the RIA method was used, this had no influence on the decision about bioequivalence because the mean ratio of the target variables Cmax and AUC was not affected. The variance of the pharmacokinetic parameters which is relevant for the bioequivalence test were even lower for RIA based values than those calculated after GC/MS analysis. In conclusion, it was found that although the GC/MS method is superior to RIA in terms of specificity, both methods were equally suited to demonstrate the bioequivalence of the two CPA-containing formulations. Thus, in future studies of this kind, the more simple RIA may be used instead of GC/MS.

Precision cut liver slices from humans and rats were used to investigate the covalent binding of xenobiotics to the DNA by means of the (32)P-postlabeling technique. Human liver slices were incubated with the structurally related steroid hormones chlormadinone acetate (5 mu g/ml), cyproterone acetate (0.01-5 mu g/ml), megestrol acetate (5 mu g/ml), and the positive control 2-aminofluorene (0.01-5 mu g/ml), which is known for its marked ability to form DNA-adducts in vivo. Rat liver slices were incubated with cyproterone acetate in concentrations of 0.1, 1, and 5 mu g/ml. The functional viability and metabolic activity of the slices were shown to be sufficiently maintained during the incubation time by measurement of the intracellular K(+)-content and the metabolic turnover of the model substrate 7-ethoxycoumarin, respectively. All three test substances and the control induced DNA-adducts in human liver slices, however, with a different adduct pattern. While the total DNA-adduct levels obtained with cyproterone acetate and megestrol acetate were in the same order of magnitude (on average 1000 DNA-adducts/10(9) nucleotides after incubation with 5 mu g /ml), the relative adduct labeling calculated for chlormadinone acetate was about 400. Following in vitro incubation of rat liver slices with cyproterone acetate, the relative adduct labeling values increased proportionally with increasing concentrations and added linearily to in vivo generated DNA-adducts. At the level of liver slices, different DNA-adduct patterns were induced by cyproterone acetate in rat and man. In contrast to the finding of others, using rat hepatocytes, the relative adduct labeling values of cyproterone acetate and megestrol acetate were in the same order of magnitude after incubation with human liver slices. The present study indicates that liver slices are a useful tool to investigate the in vitro DNA-adduct inducing potential of xenobiotics.

Objective: To examine the influence of daily oral administration of ethinyloestradiol on the total clearance of 13C-labeled ethinyloestradiol in women., Methods: 19, healthy, young women received a single IV dose of 0.06 mg 13C-ethinyloestradiol. Subsequently, they were treated with daily oral doses of 0.06 mg ethinyloestradiol for 8 days. On the last day of oral treatment, they received a further IV dose of 0.06 mg 13C-ethinyloestradiol. The pharmacokinetic parameters clearance, area under the serum level-time curve, terminal half-life, steady-state volume of distribution and mean residence time of 13C-ethinyloestradiol in each volunteer were evaluated after both IV doses, and the corresponding pairs of parameters were examined statistically for the significance of intraindividual differences., Results: Following the first (second) intravenous administration, the mean area under the curve was 2.54 (2.67) ng.h.ml-1. The terminal half-life and mean residence time were 9.7 (9.6) h and 10.5 (10.1) h, respectively. The steady-state volume of distribution was 4.3 (3.9) 1.kg-1 and the clearance was 7.0 (6.6) ml.min-1.kg-1. No significant difference was observed in any of these parameters between the first and the second IV doses of 13C-EE2., Conclusions: Since the clearance in particular remained unchanged after repeated oral administration of ethinyloetradiol, the hypothesis that ethinyloestradiol can inhibit its own metabolism in vivo can be rejected.

The measurement of the urinary excretion ratio of 6 beta-hydroxycortisol (6 beta-OHC)/cortisol was used as a non-invasive method to investigate possible changes in the activity of drug-metabolizing enzymes in women receiving different oral contraceptive formulations for 1 up to 3 treatment cycles. The contraceptive preparations were either levonorgestrel, gestodene or cyproterone acetate, each in combination with ethinyl estradiol, or only the progestogens levonorgestrel or gestodene. There was either no or only a small decrease in the 6 beta-OHC/cortisol ratio. Thus, only a minor inhibitory effect, if any, can be ascribed to the investigated contraceptive steroids in vivo. Previously observed differences between selected contraceptive steroids in vitro were not observed in the same way in vivo. This may be due either to the absence of a marked inhibitory activity in vivo or to the insufficient sensitivity of the marker 6 beta-OHC/cortisol to detect these changes. Another possible reason may be the considerably higher drug concentrations used in the in vitro studies as compared to those present in the serum of women under oral contraceptive therapy.

The study was performed in 14 young women. The combination oral contraceptive contained 75 microgram gestodene (GSD) and 20 microgram ethinyl estradiol (EE2) per dosage unit. The volunteers received a single dose on day 21 of a treatment-free precycle (PCd21) and, after a washout period of 7 days, used the preparation in a 21 d/7 d schedule for three months. Daily drug serum level profiles were taken on PCd21 and on days 1 and 21 of treatment cycles 1 and 3. In addition, trough drug serum levels were followed every other day during treatment cycles 1 and 3. Serum levels of GSD, EE2, CBG, SHBG and testosterone (T) were determined by means of specifically developed or commercially available RIAs. Pharmacokinetic evaluation was carried out with TOPFIT and parameters were evaluated for differences with the t-test. Main target variables were Cmax, tmax and AUC for EE2, GSD and unbound GSD on day 21, cycle 3 vs. PCd21. EE2 pharmacokinetics were in agreement with a dose of 20 microgram/unit. Single-dose Cmax of 65 pg/ml and AUC of 612 pg h ml(-1) increased by 40-60% during treatment cycles as a result of accumulation EE2 induced basal SHBG (102nmol/L) and CBG (42 microgram/ml) serum levels to about 220 nmol/L and 87 microgram/ml, respectively, at the end of treatment cycles 1 and 3. Serum T levels dropped to 50% of baseline levels during treatment cycles and free T concentrations were reduced by 60-70%. GSD pharmacokinetics at the end of treatment cycles 1 and 3 were different from single-dose pharmacokinetics. Single-dose Cmax of 3.5 ng/ml and AUC 0-24 h of 22 ng h ml(-1) increased to steady-state levels of 8-8.7 ng/ml and 90-106 ng h ml(-1), respectively. The increase in GSD levels under treatment is the result of two parallel processes, i.e. accumulation and enlargement of the specific binding compartment. This was shown by protein-binding experiments, demonstrating an increase in specific (SHBG) binding from 69% to 80% and a reduction in the free fraction of GSD by 40% during treatment. The results of GSD and EE2 pharmacokinetics obtained in the present study confirm previous results with Femodene, when the reduction in the EE2 dose by 10 microgram/d is taken into account.

Cyproterone acetate (CPA) is a synthetic steroid hormone used in the therapy of prostate cancer in men and different forms of acne and hirsutism in women. CPA has been shown by 32P-postlabeling analysis to bind covalently to hepatic DNA of rats in vivo and in vitro. A prerequisite for DNA adduct formation of CPA is metabolic activation of the drug to a reactive intermediate. In the present study bile was collected from [3H]CPA-treated female rats and, following chromatographic separation of bile extracts, fractions of the eluate were examined for the presence of reactive metabolites which were able to form adducts with calf thymus DNA in vitro. The formation of adducts was detected by 32P-postlabeling analysis. One major metabolite of CPA present in the bile extracts was isolated and, following a thorough structural elucidation by mass spectrometry and 1H-NMR, this metabolite was identified as 3 alpha-hydroxy-cyproterone acetate (3 alpha-OH-CPA). This metabolite was able to form the same major adduct in vitro which has been observed before in CPA-treated rats in vivo and in rat hepatocytes in vitro. A number of already known or putative metabolites of CPA were available as authentic standards and these were also examined for their propensity to form adducts in vitro. A positive result was obtained for 3-O-acetyl-cyproterone acetate, which formed the same major adduct as 3 alpha-OH-CPA. However, the presence of this putative metabolite in rat bile could not be demonstrated. Besides 3 alpha-OH-CPA, additional reactive metabolites of CPA were present in the bile extracts, however, since these were only minor components, their chemical structures could not be elucidated.

The absence of an effect of high oral doses of vitamin C on the systemic availability of ethinylestradiol in women using a levonorgestrel-containing combination oral contraceptive (0.15 mg levonorgestrel and 0.03 mg ethinylestradiol) was demonstrated in a recent study. However, it is conceivable that the oral administration of gram quantities of vitamin C could also interfere with the sulfation of levonorgestrel (LNG) metabolites during phase II biotransformation, because sulfates represent a major part of the conjugated metabolites of LNG in the serum. This possible interaction was investigated in the aforementioned study, comparing Cmax and AUC(0-12h) values of LNG on the first and 15th day of two successive treatment cycles with and without co-medication of vitamin C. In addition, the serum protein binding of LNG and the concentration of the binding proteins SHBG and CBG were compared between both treatments. Corresponding parameters obtained during treatment with the oral contraceptive alone and during co-administration of vitamin C were evaluated statistically for possible differences. No effect of vitamin C was observed for any of the parameters investigated. Thus, the repeated oral administration of gram quantities of vitamin C does not impair the sulfation of hydroxylated metabolites of LNG. There was also no observable effect on the serum protein binding of LNG and the concentrations of SHBG and CBG in the serum. The results obtained in this study population (American women) for LNG are in good agreement with those obtained from a previous study in European women, who had taken a combination oral contraceptive containing the same doses of LNG and ethinylestradiol.

The progestational activity of norgestimate (NORG), levonorgestrel-oxime (LNG-oxime) and levonorgestrel (LNG) were compared in a pregnancy maintenance study in rats. The compounds were administered subcutaneously to pregnant rats at several doses, blood samples were collected repeatedly, and the concentration of LNG was measured in these samples. It could be demonstrated that following the administration of NORG and LNG-oxime, LNG was a major metabolite present in the serum. The pharmacological response in rats treated with NORG and LNG-oxime could be related to the systemic exposure of these animals to metabolically derived LNG. Thus, both NORG and LNG-oxime can be regarded as pro-drugs of LNG, the latter being almost exclusively responsible for the pharmacological activity of both pro-drugs. This notion was further supported by studies on the comparative binding affinity of these compounds to rabbit and human progesterone receptor (PR). LNG exhibited the highest binding affinity of the compounds studied. Relative binding affinity (RBA) values of LNG using progesterone as reference (100%) were found to be 125% for rabbit PR (rPR), 143% for human uterine PR (hPR) and 125% for recombinant hPR, respectively. In contrast to LNG, NORG exhibited only a low affinity to the PR, which is documented by RBA values of 1.2% for rPR, 3.2% for uterine hPR and 9% for recombinant hPR. The corresponding values of LNG-oxime were 30% (rPR), 20% (uterine hPR) and 18% (recombinant hPR), respectively. Thus, the combined experimental evidence of the present study does not support the view of NORG being a progestogen on its own as has been suggested by others.

In a controlled clinical trial, the elimination of caffeine was examined in 20 healthy women prior to and during one cycle of treatment with either of two oral contraceptive formulations, one containing 0.075 mg gestodene and 0.03 mg ethinylestradiol and one containing 0.125 mg levonorgestrel and 0.03 mg ethinylestradiol. In addition, caffeine clearance was determined 1 month after the last intake of the oral contraceptives. Compared with pretreatment values, the clearance of caffeine was reduced by about 54% and 55% after one treatment cycle with gestodene- and the levonorgestrel-containing oral contraceptive, respectively. Other pharmacokinetic parameters of caffeine, such as tmax and Cmax, were not affected. Clearance values returned to pretreatment values 1 month after the last administration of the oral contraceptives. There was no difference in the reduction of caffeine clearance between contraceptive formulations. A small, but significant difference in the AUC(0-24 h) values of ethinylestradiol was noted between both preparations. There was no correlation between the AUC(model) values of caffeine and the AUC(0-24 h) values of ethinylestradiol. In the present study, a somewhat more pronounced effect on the elimination of caffeine was observed than in previous investigations, where several contraceptive steroids were administered only for a period of 2 weeks.

The pharmacokinetics of levonorgestrel (LNG) and ethinylestradiol (EE2) were determined in 14 healthy women (age 18 to 27 years) during a treatment period of three months with a tri-step combination oral contraceptive (Triquilar). Prior to this treatment period, the same women received a single administration of a coated tablet containing 0.125 mg LNG together with 0.03 mg EE2. There was a washout phase of one week between both treatments. Following single dose administration, a mean terminal half-life of 22 h was observed for LNG. The total clearance was 1.0 ml x min-1 x kg-1 and the volume of distribution was 128 l. During a treatment cycle, LNG levels in the serum accumulated by a factor of about four as compared to single dose administration. Steady-state drug levels were reached during the second half of each cycle. As compared to single dose administration, the following changes were observed for LNG at the end of treatment cycles one and three: reduced total (0.5 ml x min-1 x kg-1) and free clearance (50 ml x min-1 x kg-1) and a reduced volume of distribution (52 l). A concomitant increase in the SHBG concentrations by a factor of two as compared to pretreatment values was observed during treatment and appeared to be mainly responsible for the changes in the pharmacokinetics of LNG. Marked changes were also seen for the serum protein binding of LNG. After single dose administration, the free fraction of LNG was 1.4% and the fractions bound to SHBG and albumin were 55.0% and 43.6%, respectively. At the end of cycle one, the free fraction was only 1.0% and the fractions bound to SHBG and albumin were 69.4% and 30.0%, respectively. There was no difference in corresponding pharmacokinetic parameters and in the serum protein binding of LNG at the end of cycles one and three. On the last day of treatment cycles one and three, the AUC(0-4h) values of EE2 were 331.2 and 369.6 pg x ml-1 x h, respectively, which corresponds to an about 11-24% increase as compared to single dose administration, where an AUC(0-4h) value of 298.3 pg x ml-1 x h was found. Total and free testosterone concentrations decreased during treatment cycles one and three by about 41% and 55%, respectively, compared with the corresponding values measured prior to treatment.

The metabolism of the steroidal aromatase inhibitor atamestane was studied in the rat, the cynomolgus monkey and in the human. Metabolite patterns were recorded in plasma, urine and bile (rat only) before and after enzymatic cleavage of sulfate and glucuronide conjugates. Atamestane was rapidly and extensively metabolized by all three species. Major metabolites which were observed in the human, could be isolated from urine pools of treated monkeys by preparative high performance liquid chromatography and were identified by GC/MS and 1H-NMR analysis. The metabolite patterns observed in the animals and in the human were similar, although some species- and sex-related differences were observed. There seem to be two principal routes by which atamestane is metabolized: one route is characterized by the attack of 17 beta-hydroxysteroid dehydrogenase, the other route includes hydroxylation of the 1-methyl group with subsequent attack by 5 beta-reductase, followed by a hydroxylation at position C-6. Some of the metabolites which were identified still had some pharmacological activity, although less marked than the parent compound.

The serum protein binding of levonorgestrel, gestodene and 3-keto-desogestrel has been determined during several clinical studies with different oral contraceptive formulations and one in vitro study. The results of these studies were combined in order to assess the relation between changes in the concentration of sex hormone-binding globulin (SHBG) and the effect on the free fraction of the progestins as well as on their distribution with respect to the binding proteins albumin and SHBG. Although marked differences in protein binding were seen for the three progestins at low concentrations of SHBG, these differences became less pronounced at high levels of SHBG which were reached during established oral contraceptive therapy. A nonlinear relation could be shown for either the free or the protein-bound fraction of the progestins and the concentration of SHBG in the serum, respectively.

Norgestimate is a novel progestin which undergoes both in vivo and in vitro metabolic conversions to a number of metabolites, of which the most important are levonorgestrel acetate, levonorgestrel oxime and levonorgestrel itself. It has been claimed that the progestogenic activity of norgestimate in clinical studies is almost exclusively based on the parent drug and its major metabolite, levonorgestrel oxime, and that levonorgestrel does not make an important contribution. However, to date, no data on the presence of levonorgestrel in the serum of women who have received oral doses of norgestimate have been presented. In the present study, 12 young female volunteers received single oral doses of 250 micrograms levonorgestrel in combination with 50 micrograms ethinylestradiol and 250 micrograms norgestimate in combination with 35 micrograms ethinylestradiol in an open, randomized, intraindividual comparison. Blood samples were taken at regular time intervals after each treatment, and the serum samples were analyzed for their content of levonorgestrel. Basic pharmacokinetic parameters of levonorgestrel were calculated and from the ratio of the AUC values obtained after both administrations, the bioavailability of norgestimate-derived levonorgestrel was calculated. About 22 +/- 6% of the dose of norgestimate administered became systemically available as levonorgestrel. Thus, it was concluded that levonorgestrel is a major metabolite of orally administered norgestimate, and that at least part of the pharmacologic activity of norgestimate in women is due to the presence of levonorgestrel.

The androgenic and the progestational activity of norgestimate (NORG) and levonorgestrel (LNG) were compared in two animal studies. During a Hershberger test in immature castrated male rats and a pregnancy maintenance test in pregnant rats, NORG and LNG were administered subcutaneously at several doses, serum samples were collected from each animal during the treatment period and the concentration of LNG was measured in these samples. It could be shown in both studies that in those animals which were treated with NORG, LNG was a major metabolite present in the serum. The difference in the pharmacological response in LNG- and NORG-treated rats was equivalent to the difference in the exposure of the animals to either directly administered or metabolically derived LNG. This was true for the androgenic and the progestational activity of NORG. It is concluded that according to the present results, NORG can be described as a pro-drug of LNG and that the latter is probably mainly responsible for the pharmacological effects observed during treatment with NORG. It cannot be excluded, however, that NORG itself or other metabolites of this drug may also contribute to the pharmacodynamic response.

The concept of a weighted pool for estimating the area under the curve (AUC) is presented and set in relationship to the trapezoidal rule. An example from a pharmacokinetic study on ethinyl estradiol is used to demonstrate the use of variance component analysis for relating the intraindividual variance of the AUC, trapezoidal rule and weighted pool to the variance of the determination process. Depending on the sampling times, the theoretical variance of the weighted pool is greater than the theoretical variance of the trapezoidal rule. In the example presented, it was shown that this difference is of no importance in relation to the interindividual variance of the AUC, which dominates the total variance. In the example study, routine quality control samples were also determined in each assay, which allowed independent confirmation of the discussed results on the intraindividual variance of the AUCs.

The pharmacokinetics of cyproterone acetate (CPA) and ethinylestradiol (EE2) were determined in 15 healthy women (age 19 to 34 years), following single dose administration of a combination oral contraceptive, containing 2.0 mg CPA together with 0.035 mg EE2 (Diane-35R). After a wash-out period of one week, the same preparation was administered during a treatment period of three months. After single dose administration, maximum concentrations of CPA in the serum were 15.2 +/- 6.6 ng/ml. Post maximum drug levels declined biphasically with half-lives of 0.8 +/- 0.4 h and 54.0 +/- 26.0 h, respectively. The apparent clearance was calculated to be 3.6 +/- 0.9 ml x min-1 x kg-1 and the volume of distribution (Vz) was 986 +/- 4371. The free fraction of CPA was 3.5 +/- 1.9% and the fractions bound to heat labile proteins and albumin were 4.6 +/- 2.2% and 92.0 +/- 3.5%, respectively. Trough levels of CPA in the serum increased during a treatment cycle, reaching a steady-state around day 16. An about two-fold accumulation of CPA was observed, which was less than expected theoretically. SHBG concentrations in the serum increased by a factor of three during a cycle, without having any effect on the protein binding of CPA. At the end of treatment cycle three, the terminal half-life of CPA had increased to a mean value of 78.6 +/- 16.0 h and the volume of distribution to a value of 1304 +/- 427 1. The apparent clearance showed a small, although significant decrease to a value of 3.0 +/- 0.4 ml x min-1 x kg-1. The observed changes Vz and t 1/2 during the treatment period were attributed to the distribution of CPA into a deep compartment and the slow release of the drug from this compartment. The AUC(0-4h) values of EE2 following single dose administration of the combination oral contraceptive were found to be 187.5 +/- 79.7 pg x ml-1 x h. On the last day of cycles one and three, the AUC(0-4h) values were 311.2 +/- 109.3 and 304.8 +/- 121.5 pg x ml-1 x h, respectively, which corresponds to an about 60% increase as compared to single dose administration. Total and free testosterone concentrations decreased during treatment cycles one and three by about 39% and 62%, respectively, compared with the corresponding values measured prior to treatment.

The effects of daily oral administration of a high dose of 10 mg norethisterone acetate (NET-Ac.)/kg/day over 14 weeks on serum lipid and lipoprotein parameters as well as on blood coagulation were investigated in female monkeys (M. fascicularis). Measurements of lipids and lipoprotein cholesterol were performed in weeks--5 and -1 before treatment and in weeks 4, 8 and 12 after treatment. In addition, various blood coagulation and fibrinolytic parameters were determined in weeks 11-14 after treatment with NET-Ac. Furthermore, the serum levels of norethisterone (NET) were determined in order to monitor the real systemic compound exposure and revealed that Cmax and AUC (0-3 h) values reached for norethisterone in this experiment in monkeys were about 25 times higher than those obtained after an oral contraceptive dose of NET-Ac. in women. The results of lipid and lipoprotein cholesterol determinations showed decreases in serum total lipids, phospholipids, triglycerides and total cholesterol associated with similar decreases in HDL-, LDL- and VLDL-cholesterol fractions after NET-Ac.-treatment in monkeys. These effects were observed from week 4 onwards and maintained their magnitude up to week 12 after treatment. Since both HDL- and LDL-cholesterol fractions decreased, the HDL/LDL-ratio remained almost unchanged. Thus, the results obtained in this study after high-dose treatment with NET-Ac. in monkeys did not indicate any changes of lipid and lipoprotein parameters which in humans are supposed to be associated with an increased risk of cardiovascular lesions, namely a decrease in HDL- and increase in LDL-cholesterol fractions. The results of blood coagulation and fibrinolytic parameters showed increased antithrombin-III and plasminogen levels besides minor changes in other parameters, thus indicating that NET-Ac.-treatment does not contribute to an increased risk of cardiovascular thrombotic events in the cynomolgus monkey.

Previous studies in small numbers of women have suggested that the administration of gram quantities of ascorbic acid interferes with the conversion of ethinyl estradiol (EE2) to its sulfates, leading to higher blood levels of EE2. The possibility of such potentiation has been investigated in 37 women using a combination monophasic oral contraceptive (30 micrograms EE2 and 150 micrograms levonorgestrel) for two consecutive cycles. Concomitant daily administration of 1 g ascorbic acid taken 1/2 hour before OC intake, was randomly assigned to the first or second cycle of OC use. On the first and 15th day of OC intake, blood samples were drawn 11 times over a 12-hour interval and Cmax and AUC(0-12 h) calculated. On pill days 10 and 21, only 6-hour post-intake samples were obtained. Samples were analyzed for levels of ascorbic acid, free and sulfated ethinyl estradiol (and a number of other parameters). Cmax and AUC values for EE2 and EE2-sulfate in cycles with and without ascorbic acid were evaluated statistically by the Grizzle model for days 1 and 15 and the ratios of day 15/day 1 for each of the substances. No effect of ascorbic acid was observed (alpha = 0.05, 1-beta = 0.9). Only on day 15 was there a significantly lower AUC for EE2-sulfate in the presence of ascorbic acid intake. Thus, the competition between ascorbic acid and EE2 for sulfation does not lead to an increased systemic availability of EE2 and is, therefore, unlikely to be of any clinical importance. Ascorbic acid can, therefore, be removed from the list of drugs interfering with the pharmacokinetics of ethinyl estradiol.

The pharmacokinetics of gestodene (GEST) and ethinylestradiol (EE2) were determined in 14 healthy women (age 18 to 32 years) during a treatment period of three months with a new tri-step combination oral contraceptive (Milvane). Prior to this treatment period, the same women received a single administration of a coated tablet containing 0.1 mg GEST together with 0.03 mg EE2. There was a wash-out phase of one week between both treatments. Following single dose administration, a mean terminal half-life of 18 h was observed for GEST. The total clearance was 0.9 ml x min-1 x kg-1 and the volume of distribution was 84 l. During a treatment cycle, GEST levels in the serum accumulated by a factor of 8 as compared to single dose administration. Steady-state drug levels were reached during the second half of each cycle. As compared to single dose administration, the following changes were observed for GEST at the end of treatment cycles one and three: prolonged terminal half-life (20 to 22 h), reduced total (0.16 ml x min-1 x kg-1) and free clearance (ca. 27 ml x min-1 x kg-1), reduced volume of distribution (ca. 18 l). A concomitant EE2-induced increase in the SHBG concentrations by a factor of three as compared to pretreatment values was observed during a treatment cycle and appeared to be mainly responsible for the changes in the pharmacokinetics of GEST. Marked changes were also seen for the serum protein binding of GEST. After single dose administration, the free fraction of GEST was 1.3% and the fractions bound to SHBG and albumin were 69.4% and 29.3%, respectively. At the end of cycle one, the free fraction was only 0.6% and the fractions bound to SHBG and albumin were 81.4% and 18.0%, respectively. There was no difference in corresponding pharmacokinetic parameters and in the serum protein binding of GEST at the end of cycles one and three. On the last day of treatment cycles one and three, the AUC(0-4h) values of EE2 were 299.2 and 278.1 pg x ml-1 x h, respectively, which corresponds to an about 30% increase as compared to single dose administration, where an AUC(0-4h) value of 216.1 pg x ml-1 x h was found. Total and free testosterone concentrations decreased during treatment cycles one and three by about 36% and 60%, respectively, compared with the corresponding values measured prior to treatment. The fraction of unbound testosterone thus decreased from 0.5% to 0.3% during treatment.

The pharmacokinetic parameters of estradiol (E2, CAS 50-28-2), free and total estrone (E1, CAS 53-16-7) were determined in 14 young women following a single oral administration of 2, 4 and 8 mg E2 and a single intravenous administration of 0.3 mg E2 in an open, intraindividual comparison with 4 treatments. The purpose of the study was to determine the absolute bioavailability of orally administered E2 in a larger group of women and to assess the inter- and intraindividual variability of basic pharmacokinetic parameters of E2 and metabolically derived E1. In addition, the outcome of this study should provide a basis for the decision whether E2 could potentially be used in a combination oral contraceptive. There was a dose proportional increase in the AUC-values following the oral administration of 2 mg and 4 mg doses of E2. At the high dose of 8 mg, however, only about 76%, 78% and 70% of the expected values were found for E2, free and total E1, respectively. Especially the reduction in total E1 concentrations points to an incomplete absorption of E2 at the high dose level. The absolute bioavailability of orally administered E2 was calculated based on the 4 mg dose and was found to be 4.9 +/- 5.0%. The mean ratio of free E1 and E2 concentrations in the serum, following parenteral and oral administration of E2 was about 1.0 (i.v.) and between 8.8 to 19.8 (p.o.), respectively. Pharmacokinetic parameters, like AUC, derived from serum level-time curves of E2, free and total E1 showed a high intra- and interindividual variability.(ABSTRACT TRUNCATED AT 250 WORDS)

The majority of combination oral contraceptives contain ethinylestradiol (EE2, CAS 57-63-6) as estrogenic component at doses between 50 and 20 micrograms/unit. Since the concentrations of EE2 in the serum of women under oral contraceptive (OC) therapy are in the lower pg-range, highly sensitive and specific analytical methods are required. Radioimmunoassay (RIA) has been the method of choice, but evidence of specificity in the presence of the coadministered progestogens and their metabolites has not always been provided. The present study compares two radioimmunological methods, which use the same antiserum but different sample volumes and standard curves (extracted vs. non-extracted), with a newly developed gas chromatographic/mass spectrometric (GC/MS) method, in order to cross-validate the methods. For that purpose, 51 serum samples obtained from women who had been taking two different combination oral contraceptives were analysed independently by all three methods. The specificity of the antiserum was further examined by submitting ex vivo serum samples obtained from OC-users to a combination of high pressure liquid chromatography (HPLC) and RIA. The results obtained by the three methods were very similar and correlation coefficients (r) obtained from linear regression analysis were about 0.7. There was no interference from the coadministered progestins on the analysis of EE2 as carried out by the three methods. There were no metabolites in the extracts of ex vivo serum samples which showed cross-reactivity with the antiserum used. Modifications of the radioimmunoassay procedure did not markedly affect the results of EE2 determination. If properly validated, radioimmunoassay can be used as an alternative to GC/MS in pharmacokinetic studies.

The pharmacokinetics of levonorgestrel (LNG) and ethinylestradiol (EE2) were determined in 9 healthy women (age 23 to 42 years), during a treatment period of three months with a low-dose oral contraceptive, containing 0.15 mg LNG together with 0.03 mg EE2 (Microgynon). After a wash-out period of 3 months, 8 of these women received a single administration of the same formulation. The results showed that there was an increase in serum trough levels of LNG, reaching steady-state in the second half of each treatment cycle. The LNG levels achieved were about 3 to 4 times higher than anticipated on the basis of single dose administration. At the end of treatment cycles one and three, the terminal half-life of LNG was in the range of 24-26 h, while a mean value of 20 h was observed following single dose administration. An EE2-induced increase in the SHBG concentration of about 50% as compared to pretreatment values was observed during a treatment cycle. Pretreatment values were reached following the drug-free interval of 7 days between two cycles. After single dose administration, the free fraction of LNG was 1.3 +/- 0.2% and the fractions bound to SHBG and albumin were 64.1 +/- 4.2% and 34.6 +/- 4.0%, respectively. Serum protein binding of LNG did not change during chronic treatment. An about 50% reduction in total and unbound clearance of LNG was observed during chronic treatment, as compared to single dose administration. Increased SHBG binding capacity and a reduced hepatic metabolic capacity were discussed as possible causes of accumulating LNG concentrations in the serum. On the last day of treatment cycles one and three, the AUC(0-24h) values of EE2 were 728 +/- 314 and 778 +/- 318 pg x ml-1 x h, respectively, and were in keeping with data reported from others.

The pharmacokinetics of levonorgestrel (LNG) was determined in 12 healthy women (age 21 to 33 years), following single dose administration of 0.15 mg LNG. The same preparation was also administered during one treatment cycle after a washout phase of 1 week. After single dose administration, maximum concentrations of LNG in the serum were 4.3 +/- 1.3 ng/ml. Post maximum drug levels declined biphasically with half-lives of 0.6 +/- 0.2 h and 13.9 +/- 3.2 h, respectively. The clearance was calculated to be 1.5 +/- 0.6 ml x min-1 x kg-1. The free fraction of LNG was 1.1 +/- 0.1% and the fractions bound to SHBG and albumin were 61.8 +/- 6.7% and 37.1 +/- 6.7%, respectively. There was a gradual decrease in serum trough levels of LNG from about 0.5 to 0.3 ng/ml during the cycle, and a concomitant decrease in SHBG concentrations in the serum by about 50%. Serum protein binding of LNG changed markedly during the treatment cycle. The free fraction increased to a value of 1.7 +/- 0.3%, the SHBG-bound fraction decreased to 42.0 +/- 11.4% and the albumin-bound fraction increased to 56.4 +/- 11.2%. Total serum clearance increased during the same time period from a mean value of 1.5 to about 2.5 ml x min-1 x kg-1. The clearance of unbound LNG, however, remained unchanged. An examination of the free LNG concentrations revealed the same time course of LNG trough levels during the cycle as the simulated curve. This was derived from the pharmacokinetic parameters which were obtained after single dose administration. Thus, the present study showed that the pharmacokinetics of LNG can be fully explained on the basis of single dose pharmacokinetics and the changes in serum protein binding which were caused by a reduction of SHBG levels in the serum during chronic treatment with LNG.

The serum concentrations of gestodene (CAS 60282-87-3) as well as the binding of this progestin to serum proteins were studied in 40 women who took a low-dose oral contraceptive (Femovan, Femodene) containing 30 micrograms ethinyl estradiol (CAS 57-63-6) and 75 micrograms gestodene for 3 months. On days 1, 10, and 21 of the first and the third treatment cycle, respectively, 7 blood samples were drawn before and up to 4 h after pill intake; additional samples were taken prior to morning ingestion of pill on days 2, 5, 11, 15 and 22 of these cycles. Gestodene levels were measured by means of a specific radioimmunoassay and were evaluated for Cmax, tmax, and AUC up to 4 and 24 h. Independent of the test day and the treatment cycle studied, mean maximum gestodene serum levels were found about 0.8 to 0.9 h after pill intake. During the first treatment cycle, mean values of Cmax, AUC(0-4h), and AUC(0-24h) amounted to 4.3 ng.ml-1, 9.3 ng.ml-1.h, and 27.3 ng.ml-1.h on test day 1; these values increased by 250-400% and by 300-500%, respectively, when days 10 and 21 were compared to day 1. On day 1 of the third treatment cycle, these pharmacokinetic parameters were higher by almost a factor of two as compared to the corresponding data obtained on the beginning of the first cycle whereas the increase of these values between day 1 and the subsequent test days (200-300%) was slightly lower in cycle 3 as compared to cycle 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Two low-dose oral contraceptives, both containing the same dose of ethinyl estradiol (EE2, CAS 57-63-6) but different progestins--gestodene (CAS 60282-87-3) and desogestrel (CAS 54024-22-5), respectively--were administered to 18 women in a single dose, cross-over study. The serum concentrations of gestodene (GEST, one of the components of Femovan) and 3-keto-desogestrel (KDG) have been measured by specific radioimmuno-assays and the pharmacokinetics of both progestins were assessed. The serum protein binding of both compounds was also investigated and although the free fraction was the same for GEST and KDG, the distribution with respect to the binding proteins albumin and sex hormone binding globulin (SHBG) was slightly different. GEST was mainly bound to SHBG, while KDG was predominantly bound to albumin. Maximum concentrations of GEST were observed after 0.7 +/- 0.2 h and amounted to 4.9 +/- 2.4 ng/ml. A biphasic pattern of disposition was observed, with half lives of 0.13 +/- 0.06 h and 14.6 +/- 4.2 h, respectively. The AUC was 32.9 +/- 18.3 ng.ml-1.h. For KDG, maximum serum levels of 1.7 +/- 0.8 ng/ml were observed 1.5 +/- 0.8 h post administration. Drug levels declined with half-lives of 0.5 +/- 0.2 h and 17.0 +/- 9.3 h, respectively, and the AUC was 15.2 +/- 10.9 ng.ml-1.h.

The serum concentrations of 3-keto-desogestrel have been measured in 43 women who took a low-dose oral contraceptive containing 30 micrograms ethinyl estradiol (CAS 57-63-6) together with 150 micrograms desogestrel (CAS 54024-22-5) for a period of 3 months. Basic pharmacokinetic parameters, like Cmax, tmax and AUC, as well as the serum protein binding of 3-keto-desogestrel were determined on days 1, 10 and 21 of the first and the third treatment cycle, respectively. During cycle one, Cmax, AUC(0-4h) and AUC(0-24h) values on day 1 were 1.9 +/- 0.7 ng/ml, 3.9 +/- 1.3 ng.ml-1.h and 12.4 +/- 5.7 ng.ml-1.h, respectively. These values increased to 4.7 +/- 2.0 ng/ml, 12.1 +/- 5.6 ng.ml-1.h and 47.3 +/- 26.0 ng.ml-1.h on day 21. Within cycle 3, a similar, although less steep increase was observed for these parameters and there was practically no difference in the values of corresponding parameters on day 21 of both cycles. Throughout treatment, there was a redistribution of 3-keto-desogestrel with respect to the binding proteins albumin and sex hormone binding globulin (SHBG). During cycle 1, the free fraction decreased from 1.8% on day 1 to 1.1% on day 21, and the SHBG-bound fraction increased at the same time from 40% to 62%, mainly at the expense of the albumin-bound fraction. During cycle 3, there were only minor changes as compared to cycle one. The observed changes in the serum protein binding were related to an increase in SHBG levels during the treatment period.

The pharmacokinetics of gestodene (GEST) was determined in 12 healthy women (age 22 to 34 years), following single dose administration of 0.075 mg GEST. The same preparation was also administered during one treatment cycle after a wash-out phase of 1 week. After single dose administration, maximum concentrations of GEST in the serum were 4.9 +/- 2.5 ng/ml. Post maximum drug levels declined biphasically with half-lives of 0.2 +/- 0.2 h and 14.9 +/- 6.7 h, respectively. The apparent clearance was calculated to be 0.8 +/- 0.4 ml x min-1 x kg-1. The free fraction of GEST was 1.3 +/- 0.3% and the fractions bound to SHBG and albumin were 64.3 +/- 10.7% and 34.4 +/- 10.4%, respectively. The results showed that there was a gradual decrease in serum trough levels of GEST during the cycle, due to a concomitant and equally high decrease in SHBG concentrations in the serum of about 26%. At the end of one treatment cycle, the free fraction of GEST increased to 1.8 +/- 0.5%, the SHBG-bound fraction decreased to 57.0 +/- 10.2% and the albumin-bound fraction increased to 41.3 +/- 9.9%. Total serum clearance increased during the same time period from a mean value of 0.8 to about 1.2 ml x min-1 x kg-1. The clearance of unbound GEST, however, remained unchanged. An examination of the free GEST concentrations revealed the same time course of GEST trough levels during the cycle as the simulated curve. This was derived from the pharmacokinetic parameters which were obtained after single dose administration. Thus, the present study showed that the pharmacokinetics of GEST can be fully explained on the basis of single dose pharmacokinetics and the changes in serum protein binding which were caused by a reduction of SHBG levels in the serum during chronic treatment with GEST. There was no evidence of GEST inhibiting its own metabolism.

Ethinyl estradiol is part of almost every combined oral contraceptive, and its pharmacokinetic characteristics have been thoroughly investigated in numerous studies. However, little is known about its pharmacokinetics during long-term administration, as compared with single-dose administration. In this study 10 women received a triphasic formulation that contained ethinyl estradiol together with the progestin gestodene over one treatment cycle. Mean area under the curve values of ethinyl estradiol were significantly higher on the last treatment day, as compared with the corresponding values obtained from the same women after single-dose administration. However, the observed increase in area under the curve was within the range of pharmacokinetic accumulation, to be expected on the basis of dosing interval and terminal half-life. Another point of interest was the effect of the triphasic preparation on testosterone concentrations in serum. Both total and free testosterone levels were suppressed by about 60% as compared with pretreatment values, and there was no correlation with corresponding sex hormone-binding globulin levels in the serum.

After the parenteral administration of the 3H-labeled prostaglandin E2-analogue (PGE2-analogue) to three healthy women, a number of metabolites were observed in the plasma, some of them still potentially pharmacologically active. The metabolite pattern in human plasma was very similar to the one observed in the urine of female guinea pigs, which received a total dose of 0.21 mg [3H]sulprostone by repeated sc administration over a period of 5 days. In vitro perfusion of an isolated guinea pig liver with 50 mg of [3H]sulprostone, dissolved in Tyrode's solution, yielded another source for the isolation of those metabolites, which were also present in human plasma. Both urine and perfusion medium were submitted to repeated HPLC-separations and the recovery during the purification procedure was calculated for each metabolite on the basis of recovered radioactivity after each step of purification. Chromatographically pure metabolites were submitted to GC/MS analysis, 1H-NMR and IR spectroscopy for structural elucidation. Besides the unchanged parent compound, four metabolites of sulprostone could be identified in human plasma by co-chromatography with the isolated compounds. One of two major metabolites was the PGA2-analogue of the parent drug, the other was a cyclization product of the beta-side chain of sulprostone with the cyclopentenone ring, preceded by delta 13 reduction. The two minor metabolites were the free acids of sulprostone and the PGA2-analogue.

A triphasic levonorgestrel (LNG)- and ethinylestradiol-containing oral contraceptive was administered to 18 women. Plasma samples were obtained throughout a treatment cycle just before drug administration and on the last treatment day (day 21), several plasma samples were collected from each individual up to 48 h postadministration. LNG was determined by radioimmunoassay in all plasma samples. In addition, the concentration of sex-hormone-binding globulin (SHBG) was determined in plasma samples collected from the same subjects during treatment, as well as during a pre- and a posttreatment cycle. During the treatment cycle, plasma levels of LNG determined just before drug administration increased and reached steady state at about day 16. This increase was due to an increased dose of LNG according to the triphasic dose regimen, a concomitantly ethinylestradiol-induced increase in SHBG and due to pharmacokinetic accumulation, since LNG had a terminal half-life of approximately 28.5 h and the dosing interval was 24 h. Steady-state levels and pharmacokinetic parameters of LNG determined on the last day of treatment were in good accordance with previously published results.

Two low-dose oral contraceptives, both containing the same dose of ethinyl estradiol (EE2) but different progestins (gestodene and desogestrel, respectively), were compared with respect to the relative bioavailability of EE2. The study was conducted with 31 women as an open intraindividual comparison with the ingestion of both preparations for 3 months, respectively. On days 1, 10 and 21 of the 1st, 3rd and 6th cycle, blood was sampled at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h following administration. The concentrations of EE2 were determined in the serum samples of each individual and the area under the serum concentration versus time curves, AUC (0-4 h) and AUC (0-24 h), were calculated. Corresponding parameters obtained on days 1, 10 and 21 of the 3rd and 6th month of treatment were compared on a statistical basis, and no differences were found. This result was in concordance with a previously performed study, where both formulations were administered to 18 women in a single-dose cross-over design. However, the results of the previous and the present study are at variance with the result of one other study, reporting higher EE2 levels in the serum of women taking the gestodene-containing formulation as compared to those taking the desogestrel-containing formulation.

Little is known about the pharmacokinetics of the two progestins levonorgestrel and gestodene during long-term administration compared with single-dose pharmacokinetics. The predictive value of single-dose administration for the pharmacokinetic behavior of a progestin during long-term treatment was investigated for two triphasic oral contraceptives. One contained levonorgestrel and the other gestodene, each in combination with ethinyl estradiol. In eight Japanese women who received the levonorgestrel-containing formulation over a treatment cycle, steady-state trough levels of levonorgestrel were higher than those obtained by computer simulation based on single-dose administration. An analogous observation was made in a group of 10 white women who received the gestodene-containing formulation. A close correlation between gestodene and sex hormone-binding globulin concentrations was demonstrated for eight subjects; the other two patients already had initially high sex hormone-binding globulin levels. Ethinyl estradiol-induced production of sex hormone-binding globulin seems to be a major factor that contributes to the accumulation of the two progestins in the plasma. Computer simulation, based on single-dose pharmacokinetics, allows an estimation of this contribution.

The pharmacokinetic and protein-binding properties of gestodene and ethinyl estradiol have been investigated after single and multiple dosing in several studies in 83 healthy, young women. After oral administration, gestodene is completely absorbed and bioavailable and exhibits dose-linear pharmacokinetics. During long-term pill use, serum levels of gestodene were four to five times higher than after single administration, showing a periodic increase from day 1 to day 10 during each cycle. Ultrafiltration studies revealed that 75.3% of total serum gestodene is bound to sex hormone-binding globulin, 24.1% is bound to albumin, and only 0.6% is not protein bound. Thus gestodene levels during steady state are explained by an increase in sex hormone binding-globulin as a result of concomitant administered ethinyl estradiol and a specific binding of gestodene to this protein. Serum levels of ethinyl estradiol during single and multiple administration were identical and were not different from those observed with another preparation containing 30 micrograms of ethinyl estradiol.

Results from two clinical pharmacokinetic studies are given. The first study was an observational study in oral contraceptive users who took either a combination of gestodene and ethinyl estradiol (pill A, Femovan) or desogestrel and ethinyl estradiol (pill B, Marvelon). A total of 69 women (39 receiving pill A and 30 receiving pill B) were evaluated to determine serum ethinyl estradiol, sex hormone-binding globulin, corticosteroid-binding globulin, and cortisol levels. Samples were obtained on 1 day during the tenth to twenty-first days of pill intake. All women received the respective oral contraceptive for at least 3 months. The test power was such that an 80% difference of 1 standard deviation of each target variable would have been detected (alpha = 0.05; beta = 0.1). No statistically significant differences were found in sex hormone-binding globulin, corticosteroid-binding globulin, or cortisol serum levels between both groups. Time and height of maximum ethinyl estradiol levels were identical as was the area under the curves. Ex vivo protein-binding analysis of the progestins revealed a free portion of 0.6% for gestodene and 2.5% for 3-ketodesogestrel as the active metabolite of desogestrel. Sex hormone-binding globulin-bound portions were much higher for gestodene (75.3% +/- 9.1%) than for 3-ketodesogestrel (31.6% +/- 12%). The remaining fractions were bound to albumin. In a second study, ethinyl estradiol-bioequivalence from pills A and B was investigated in 18 women in a controlled, single-dose, randomized, crossover design. The area under the ethinyl estradiol serum levels were identical up to 4 hours after pill intake between both treatments. According to the relatively low variation in data in this group of women, a 10% difference in ethinyl estradiol-availability could have been detected. Both studies indicate that the pharmacokinetics of ethinyl estradiol were independent of the concomitantly administered progestin, that is, desogestel and gestodene.

The protein binding of ethinylestradiol (EE2), gestodene (GEST) and 3-keto-desogestrel (KDG) has been determined by ultrafiltration in the serum of women who had either taken a gestodene (n = 37) or desogestrel (n = 28) containing oral contraceptive for a time period of at least 3 months. GEST and KDG were analyzed in individual serum pools whereas EE2 was repeatedly measured in two serum pools, each one representing one treatment group. The respective free fractions of the three steroids were 0.6 +/- 0.1% (GEST), 2.5 +/- 0.2% (KDG), 1.7 +/- 0.6% (EE2, in the gestodene-group) and 1.5 +/- 0.2% (EE2, in the desogestrel-group). EE2 was exclusively bound to albumin, whereas GEST and KDG were also bound to sex-hormone-binding globulin (SHBG). The distribution of the two progestins over the serum binding proteins was determined after heat-treatment of serum samples. For GEST, the contribution of albumin and SHBG was 24.1 +/- 9.1 and 75.3 +/- 9.1%, respectively and for KDG it was 65.9 +/- 11.9 and 31.6 +/- 12.0%, respectively. SHBG and corticosteroid-binding globulin (CBG) concentrations were measured in the serum samples obtained from both treatment groups. In the gestodene-group 180 +/- 61 nmol/l (SHBG) and 89 +/- 13 mg/l (CBG) were measured, the corresponding values in the desogestrel-group were 226 +/- 64 nmol/l (SHBG) and 93 +/- 14 mg/l (CBG). SHBG concentrations were correlated with the total concentration of GEST and its free fraction and a positive (r = 0.395) and negative (r = -0.491) correlation respectively was found. Only a weak negative correlation (r = -0.291) was found for SHBG and the free fraction of KDG in the serum. These data demonstrate that the three contraceptive steroids EE2, GEST and KDG were all bound extensively to serum proteins, however, with pronounced differences concerning their distribution over the various binding proteins.

Two low-dose oral contraceptives, both containing the same dose of ethinyl estradiol (EE2) but different progestins (gestodene and desogestrel, respectively), were compared with respect to the relative bioavailability of EE2. After single-dose administration of both formulations to 18 women in an intraindividual cross-over design, there was no difference in the target variables for EE2 (Cmax, tmax and AUC). With respect to EE2, both formulations were bioequivalent. The observation of others, reporting higher EE2 levels in the serum of women taking the gestodene-containing formulation as compared to those taking the desogestrel-containing formulation, was not confirmed.

The study included 69 women taking a desogestrel (n = 30)- or gestodene (n = 39)-containing low-dose combined oral contraceptive for at least 3 months. Group size was calculated to detect a difference in mean values of 80% of 1 standard deviation (alpha = 0.05, beta = 0.1). Seven serum samples were obtained up to 4 h, and 1 sample 24 h, after drug intake on 1 day between the 10th and the 21st day of the cycle. The concentrations of sex-hormone-binding globulin (SHBG), corticoid-binding globulin (CBG) and cortisol were measured in a 0- to 4-hour serum pool by radioimmunoassay. Ethinyl estradiol (EE2) levels were analyzed in single and pooled samples using anti-EE2-6 beta-carboxymethyloxime-bovine serum albumin antiserum. The area under the curves (AUC) up to 4 and 24 h and Cmax and tmax were evaluated. Statistical analysis (analysis of covariance) did not reveal a dependence of values on duration of treatment or day of cycle. Both treatments resulted in almost identical values for all parameters evaluated. The mean levels of SHBG, CBG and cortisol were in the range of 186-226 nmol/l, 89-93 mg/l and 280-281 micrograms/l, respectively. Mean maximum EE2 levels of 106-129 pg/ml were found 1.6-1.8 h after pill intake and AUC0-4 h accounted for 329-374 pg.h.ml-1. The recently reported differences in serum EE2 and CBG levels between two groups of 11 women each treated with desogestrel- and gestodene-containing pills, respectively, could not be confirmed.

A total of 10 epileptic mothers treated with ethosuximide (ES) as well as their 13 newborns were included in this study. At birth foetal/maternal serum concentration ratios were 0.97 +/- 0.02 (n = 7) and ES half-lives in three neonates were 32, 37 and 38 h. The breast milk concentrations of ES were similar to those in maternal serum (milk/serum: 0.86 +/- 0.08, n = 12) and the nursed infants maintained serum levels between 15 and 40 micrograms/ml. Two major malformations (bilateral clefting, hare-lip) were observed in two neonates whose mothers received either ES/PB or ES/PMD comedication. The number of minor anomalies was higher in the ES group (6.2, n = 12) than in the pair-matched control group of infants born to non-epileptic mothers (2.1, n = 10). Neonatal behaviour complications occurred in seven infants, two of them were severely affected.

This ultrafiltration technique allows determination of free drug in 50 microL of serum. We ultrafiltered sera containing the following drugs--valproic acid (and its major metabolites), phenobarbital, diazepam, indomethacin, phenytoin, furosemide, and chloramphenicol--using both the commercially available micropartition system (MPS-1, Amicon), which requires a 200-microL sample, and our modified micro system, which requires only 50 microL. The value for the free fraction of each drug obtained in the two experiments agreed well. The smaller sample requirement makes the micro method particularly suited for pediatric samples and studies on small laboratory animals.