1 BIOQUAL, Inc., Rockville, MD, 20850, USA and 2 Contraception and Reproductive Health Branch, National Institute of Child Health and Human Development, Rockville, MD, 20892, USA
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Abstract |
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Key words: antiprogestin/bioavailability/contraception/protein binding/rhesus monkey
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Introduction |
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The present study was undertaken to characterize the circulating concentrations of CDB-2914 equivalents in female rhesus monkeys following a single 50 mg dose of CDB-2914 administered in aqueous or oily formulations by the i.v., oral or i.m. routes, and to compare the serum concentrations of CDB-2914 and mifepristone given orally in aqueous suspending vehicle (ASV) or gelatin capsules. In conjunction with the kinetic studies, the binding of CDB-2914 and mifepristone by purified human AAG and by human and rhesus monkey corticosteroid binding globulin (CBG) and sex hormone binding globulin (SHBG) was also investigated. Furthermore, the high-affinity binding of these two antiprogestins by proteins in diluted rhesus monkey serum was studied in an attempt to determine whether this phenomenon influenced circulating concentrations.
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Materials and methods |
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[125I]CDB-2914 and [125I]mifepristone were prepared by iodination of the respective 3-carboxymethyloximehistamine conjugates using a modified chloramine-T method (Niswender et al., 1975) and purified twice by HPLC using a 5 µm LiChrosorb RP-18 column (4.6x250 mm; Merck, Darmstadt, Germany) and a gradient of acetonitrile:water (40:60) to 100% acetonitrile over 40 min at a flow rate of 1 ml/min.
Animals
Nine intact, regular cycling, female rhesus monkeys (Macaca mulatta) of mean (± SE) bodyweight 7.6 ± 0.5 kg were used in these studies. Menstrual cycles were monitored daily by vaginal swabs to detect the presence and quantity of blood. Male New Zealand White rabbits weighing 34 kg were employed for antisera production. Animals were maintained in facilities accredited by the Association for Assessment and Accreditation of Laboratory Animal Care Inter- national, and the environmental conditions of the animal rooms were maintained as recommended by the National Research Council (National Research Council, 1996) to the maximum extent possible. The Institutional Animal Care and Use Committee of BIOQUAL approved all study protocols.
The same three to four monkeys were used for both CDB-2914 and mifepristone pharmacokinetic studies. The monkeys were not anaesthetized during dosing or blood collection. The animals were held in a chair restraint during oral and i.v. dosing, and for an additional hour after dosing. In the i.v. study, monkeys were injected at random times in their menstrual cycles. In all other studies, the monkeys were dosed at midcycle (days 1316; day 1 = first day of menses) and fasted overnight before dosing and for 4 h after oral dosing, which was performed via a gastric catheter. When test compounds were administered as aqueous suspensions the catheter was flushed with additional vehicle to ensure complete delivery. Gelatin capsules containing test compounds were forced through the catheter using a bolus of air. Blood samples were collected by venepuncture from the cephalic, saphenous or femoral veins immediately before and at various times after dosing. Serum was obtained by centrifugation at 2600 g for 15 min, and stored at 20°C until assayed for CDB-2914 or mifepristone and their immunoreactive metabolites.
Antisera
Polyclonal antisera against CDB-2914 3-carboxymethyloximeBSA were raised in four male rabbits using a previously described method (Vaitukaitis et al., 1971). Initially, each rabbit was immunized with divided intradermal injections of 4 mg of the immunogen emulsified in Freund's complete adjuvant:saline (1:1, v/v). This was followed by booster immunizations in Freund's incomplete adjuvant:saline (1:1, v/v) at 4-week intervals until no further increases in antibody titres were obtained. Sera from bleeds nos. 35 from the rabbit (#67192) with the highest antibody titre and the greatest CDB-2914 specificity were pooled and used for assay development and validation. This antiserum was titrated with each new preparation of [125I]CDB-2914 to determine the final dilution that resulted in 3040% specifically bound radioligand. The N-mono- and -didemethylated putative metabolites of CDB-2914 exhibited 76% and 59% cross-reactivity with the antiserum respectively (Table I
). Progesterone, mifepristone, oestradiol, oestrone, cortisol, testosterone and the putative 17
-hydroxylated metabolite of CDB-2914 exhibited <1% cross-reactivity.
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Serum samples from five monkeys dosed orally with 5 mg CDB-2914/kg were assayed for CDB-2914 and its putative immunoreactive metabolites over a series of dilutions. Using a semi-log plot of percent bound versus concentration, the diluted serum samples decreased in a linear and parallel fashion to the CDB-2914 standard curve. Parallelism was assessed by comparison of the mean slopes calculated by RiaSmartTM for the regression lines (Altman, 1991). The mean slope of the standard curves (n = 6) was 0.86 [95% confidence intervals (CI) 0.76 to 0.96], and the mean slope of the diluted serum samples (n = 5) was 0.80 (95% CI 0.66 to 0.94).
The study serum samples were assayed for CDB-2914 or mifepristone equivalents as follows. CDB-2914 and mifepristone equivalents were extracted from sera with absolute methanol (1:4, v/v). Following centrifugation to remove the protein precipitate, the supernatant extracts were diluted to 20% methanol with assay buffer (50 mmol/l phosphate, pH 7.2, containing 0.1% gelatin), and then diluted further as required with assay buffer containing 20% methanol. Duplicate 200 µl aliquots were incubated overnight with 30 000 counts per min (c.p.m.) [125I]CDB-2914 or [125I]mifepristone and the respective antibody at 26°C. The final methanol concentration in the incubation mixture was 5%. Bound and free radioligand were separated using a dextran-coated charcoal method (Reel et al., 1979). Following precipitation of the charcoal by centrifugation (2600 g, 15 min), the supernatants were decanted and counted for 2 min each in a Packard CobraTM II gamma counter. The serum equivalents for CDB-2914 or mifepristone were derived by correcting for aliquot volume and serum dilution factor. Two of the early metabolites of mifepristone in women, the N-mono- and -didemethylated products (Deraedt et al., 1985
; Heikinheimo et al., 1987a
), cross-reacted with the antibody at 86% and 57% respectively (Table I
). It seems likely that these two metabolites were also produced in female monkeys, though no attempt was made at their isolation and identification. The serum concentrations were expressed as ng equivalents of CDB-2914 and mifepristone per ml in order to reflect the unknown contribution of their proximal metabolites which have been found to cross-react with the antisera used.
Serum protein binding
The relative binding affinities (RBA) of SHBG and CBG for CDB-2914, mifepristone, progesterone, oestradiol (SHBG only) and dexamethasone (CBG only) were determined using a published method (Hammond and Lähteenmäki, 1983). Briefly, aliquots of pooled human serum and pooled female rhesus monkey serum were charcoal-stripped and incubated with 5 nmol/l [3H]5
-dihydrotestosterone (5
-DHT) or 5 nmol/l [3H]cortisol and reference standard or competitor concentrations from 1 to 800 nmol/l. Non-specific binding was measured in the presence of a 200-fold molar excess of unlabelled 5
-DHT or cortisol in the SHBG and CBG assays respectively. The SHBG assay also contained 120 nmol/l cortisol to block binding of the competitors to serum CBG. Samples were incubated for 2 h at 4°C. Bound and free radioligands were separated using a dextran-coated charcoal method (Reel et al., 1979
). Following precipitation of the charcoal by centrifugation at 2000 g for 20 min, the supernatants were decanted and counted. The EC50 values for the 5
-DHT and cortisol standard curves and the competitor curves were calculated using RiaSmartTM. The RBA (%) of the competitors were calculated using the following equation: (EC50 of standard ÷ EC50 of competitor)x100. Equilibrium dialysis was used to assess the binding by other serum proteins. Aliquots of female monkey serum diluted 1:100 in phosphate-buffered saline (PBS) (10 mmol/l NaHPO4, 144 mmol/l NaCl, pH 7.2) containing 5% ethanol (PBS/5% ethanol) and 12.2 µmol/l purified human AAG (in PBS/5% ethanol) were dialysed against 5 nmol/l [3H]CDB-2914 or 5 nmol/l [3H]mifepristone. Incubations were carried out at 37°C, and both dialysis chambers were sampled over time until equilibrium was reached. At equilibrium (8 h), the percent bound radioligand was calculated using the following equation: ([3H] c.p.m. in serum protein chamber) ([3H] c.p.m. in radioligand chamber) ÷ ([3H] c.p.m. in serum protein chamber)x100.
Pharmacokinetic parameters and statistical analysis
Parameters were estimated using the National Institutes of Health PROPHET Public Procedures drug modelling program (Holford, 1990). The PROPHET `drugmodel' procedure was used to determine the mean peak serum concentration (Cmax; ng/ml) and time to Cmax (h) values. The area under the curve (AUC) values (ng/mlh) were calculated using the trapezoidal rule. Statistical comparisons of Cmax, time to Cmax, and AUC were performed using Student's t-test (SigmaStat, version 2.0; Jandel Scientific, San Raphael, CA, USA). Graphs were prepared using Sigma Plot, version 4.0 (Jandel Scientific).
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Results |
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In order to make estimates of bioavailability, the mean AUC were calculated for circulating concentrations of CDB-2914 equivalents following i.v., oral and i.m. administration (Table II). The ratio of the oral AUC072 h to the i.v. AUC072 h x 100 gave an oral bioavailability of 56% (Table II
). In comparison, the ratio of the i.m. AUC0168 h to the i.v. AUC072 h x 100 yielded an i.m. bioavailability of 62% (Table II
).
Circulating concentrations of CDB-2914 and mifepristone equivalents after oral administration in aqueous suspending vehicle (ASV) or gelatin capsules
The oral pharmacokinetics of mifepristone have been studied extensively in women. Therefore, the results of a comparative study of CDB-2914 and mifepristone in female rhesus monkeys might provide some insight into what doses of CDB-2914 would be appropriate for phase I clinical trials pursuant to the treatment of endometriosis and uterine fibroids (Passaro et al., 1997). Oral administration of 5 mg CDB-2914/kg in ASV resulted in a mean Cmax of 192 mg/ml, and a mean time to Cmax of 5 h, whereas 5 mg mifepristone/kg in ASV gave a serum Cmax of 82 ng/ml and a time to Cmax of 3 h (Figure 3A
, Table III
). Although Cmax and time to Cmax were not significantly different between the two groups, the AUC0120 h for CDB-2914 was significantly greater (P < 0.05) than that for mifepristone. Hence, the AUC0120 h ratio of CDB-2914 was 4.7-fold greater than that of mifepristone (Table III
).
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Serum protein binding
Neither CDB-2914 nor mifepristone bound to monkey or human SHBG or CBG. The protein binding assays for each species were performed on two separate occasions. The RBA values of SHBG for oestradiol, which served as a positive control, were 10 ± 1% and 3 ± 1% (mean ± SD, n = 2) for monkey and human respectively. The negative control, progesterone, did not compete with [3H]5-DHT for binding to either the monkey or human SHBG. For CBG, the mean RBA values for progesterone, the positive control, were 40 ± 6% and 10 ± 3% (mean ± SD, n = 2), for monkey and human respectively. As expected, dexamethasone did not compete with [3H]cortisol for binding to CBG from either species.
By equilibrium dialysis, neither [3H]CDB-2914 nor [3H]mifepristone bound to serum proteins in 100-fold diluted monkey serum. The positive control, purified human AAG, bound 53 ± 10% (mean ± SD, n = 4) and 89 ± 5% (mean ± SD, n = 2) of the added [3H]CDB-2914 and [3H]mifepristone respectively. In addition, there was no evidence of specific binding of either antiprogestin by charcoal-stripped 75-fold diluted monkey serum using the same dextran-coated charcoal method as was employed to assess binding by SHBG and CBG.
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Discussion |
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Although the metabolites of CDB-2914 were not isolated and characterized in the present study, it appears likely that two of the earliest metabolites would be the monodemethylated and didemethylated products, based on an analogy to mifepristone (Heikinheimo et al., 1989, 1994
). Noteworthy, the proximal metabolites of mifepristone were biologically active to varying degrees in the rat (Deraedt et al., 1985
). The monodemethylated and didemethylated derivatives of CDB-2914 have been chemically synthesized and tested. The synthetic monodemethylated metabolite of CDB-2914 exhibited considerable affinity for the rabbit uterine PR and thymic GR, as well as antiprogestational, anti-ovulatory and postcoital antifertility activity in the rat (unpublished data).
The absorption of orally dosed mifepristone has been shown to be rapid in humans (Lähteenmäki et al., 1987; Kekkonen et al., 1996
) and rats (Deraedt et al., 1985
; Heikinheimo et al., 1994
), and this was also observed in female rhesus monkeys in the present study. In contrast, results from the earlier of these studies (Deraedt et al., 1985
) described slow and irregular absorption of mifepristone in the cynomolgus monkey. However, a different vehicle [polyethylene glycol (PEG) 300] was employed in this study, and this may account for the difference seen between rhesus and cynomolgus monkeys. In contrast, the absorption of CDB-2914 was slower than mifepristone following administration in either gelatin capsules or in an ASV, as indicated by the time to Cmax. The difference in absorption of the two antiprogestins given in these two formulations may reflect the solubility and the extent of aggregation of drug particles in the gastrointestinal tract. The AUC0120 h values for CDB-2914 and mifepristone administered orally in gelatin capsules were only 67% and 38% respectively that of the AUC values when these drugs were given in ASV. Interestingly, the oral bioavailability of mifepristone ranged from 15% in cynomolgus monkeys to 40% in humans and rats when administered in PEG 300 (monkeys), tablets (humans) and methylacetamide (rats) respectively (Deraedt et al., 1985
). In addition, when the AUC for CDB-2914 in ASV or gelatin capsule were compared with the AUC for mifepristone in ASV or gelatin capsule, the serum concentrations of CDB-2914 were 4.7- and 5.3-fold greater than those of mifepristone. Hence, CDB-2914 appeared to be more efficiently absorbed than did mifepristone in the rhesus monkey, regardless of the oral dosing formulation.
The persistence of detectable concentrations of CDB-2914 and its immunoreactive metabolites in serum following oral administration of CDB-2914 in ASV or gelatin capsules, or by i.m. injection, suggests significant extravasation of CDB-2914 and its putative immunoreactive metabolites. It is also possible that the profile of metabolites is different following oral administration as compared with i.m. injection. Early studies on the pharmacokinetics and metabolism of mifepristone indicated that, due to first-pass metabolism, the volume of distribution in humans was only 10% of body weight, whereas in rats and cynomolgus monkeys the volume of distribution represented 135% and 200% respectively of body weight (Deraedt et al., 1985). Of note was the finding that if the elimination half-life for mifepristone and its immunoreactive metabolites was calculated for this study in rhesus monkeys (data not shown), the value was identical to the 15 h value reported earlier (Deraedt et al., 1985
) for cynomolgus monkeys. In human subjects, orally dosed mifepristone exhibits a longer elimination half-life that ranges from 25 to 30 h (Heikinheimo et al., 1989
; Kekkonen et al., 1996
). The longer half-life in humans appears to be the result of mifepristone binding by serum AAG rather than extensive extravascular diffusion, thereby sequestering the drug in the central compartment for a longer period. The most likely explanation for the difference between humans and laboratory species is the lack of a high-affinity serum binding protein for mifepristone in rats and monkeys (Moguilewsky and Philibert, 1985
; Heikinheimo et al., 1994
). Based on our preliminary serum protein binding studies, the same appears to hold true for CDB-2914.
Because of the structural similarity of CDB-2914 to mifepristone, it was considered likely that the slow elimination and increased bioavailability of CDB-2914 in the rhesus monkey might reflect high-affinity binding to one or more serum binding proteins. On the contrary, the current studies did not detect high-affinity binding of either CDB-2914 or mifepristone in rhesus monkey serum. The results for mifepristone agree with those published earlier (Moguilewsky and Philibert, 1985), which suggested that there was no specific binding of mifepristone by cynomolgus monkey serum. AAG is present in monkey serum, but the data suggest that its steroid binding characteristics differ from that of human AAG. In this regard, the results of the present study and other studies carried out using equilibrium dialysis demonstrated that purified human AAG and diluted human serum (unpublished data) bind CDB-2914, but to a lesser extent than mifepristone. Neither SHBG nor CBG appears to serve as a serum carrier protein for CDB-2914 or mifepristone in monkeys or in humans. The lack of mifepristone binding to human SHBG and CBG has been reported previously (Heubner et al., 1985
; Heikinheimo et al., 1987b
). Thus, the pharmacokinetics of CDB-2914 and mifepristone do not appear to be influenced by high-affinity binding to specific serum proteins in the rhesus monkey. The lack of specific binding by monkey AAG, SHBG and CBG suggests that the pharmacokinetics of CDB-2914 and mifepristone in the rhesus monkey may differ significantly from that in the human.
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Acknowledgments |
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Notes |
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4 To whom correspondence should be addressed
* Presented in part at the 10th International Congress of Endocrinology, San Francisco, California,1996, Abstract P2-691
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References |
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Submitted on October 25, 1999; accepted on January 31, 2000.