CONTRACEPTION QUANTITATION OF RU 486 IN HUMAN PLASMA BY HPLC AND RIA AFTER COLUMN CHROMATOGRAPHY Oskari Heikinheimo, Marjatta Tevilin, Croxatto** and Pekka L~hteenm~ki Donna Shoupe*, Horaclo Steroid Research Laboratory, Department of Medical Chemistry, University of Helsinki, Finland Department of Obstetrics and Gynecology, Section of Reproductive Endocrinology, University of Southern California, Los Angeles, California, USA Institute Chileno De Medicina Reproductiva, Santiago, Chile ABSTRACT Chromosorb R column chromatography was used for separation of RU 486 from its immunologically cross-reacting metabolites prior to quantitative analysis by radioimmunoassay (RIA) or high-performance- liquid chromatography (HPLC). The results of the two assay methods were in good agreement with each other (r=0.99, n=29). The retention time of RU 486 in our HPLC system was 2.5 min. Plasma concentrations of RU 486 were measured by HPLC up to 48 h following single oral administration of 100, 400, 600 and 800 mg of RU 486 to female volunteers. The plasma peak concentrations (2.0 - 2.5 ~g/ml) were reached within the first hour. After redistribution, the plasma concentrations of RU 486 were not significantly affected by the doses studied but remained in the same range throughout the 48 hours. The plasma half-life between 24 and 48 hours was 27 hours or more. We conclude that HPLC is valuable in studies on the metabolism and pharmacokinetics of RU 486, but a less laborious RIA method after Chromosorb R column chromatography is suitable and gives reliable results in large-scale clinical studies. Correspondence: Pekka LEhteenm~ki, M.D Steroid Research Laboratory Department of Medical Chemistry University of Helsinki Siltavuorenpenger 10 A SF-00170 He]sinki, Finland Submitted for publication January 14, 1986 Accepted for publication December 4, 1986 DECEMBER !986 VOL. 34 NO. 6 613
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`Corcept Therapeutics, Inc.
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`Neptune Generics, LLC v. Corcept Therapeutics, Inc.
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`CONTRACEPTION INTRODUCTION RU 486 is a new 19-norsteroid derivative with potent antl- progestational (1) and anti-glucocorticoid activity (2,3). A preliminary clinical study showed that RU 486, given in early pregnancy, resulted in abortion in 9 of 11 women (4). It also induced menses at the midsecretory phase of the cycle (4). Antiprogestational effect of RU 486 is mediated via its binding to the human progesterone receptor with higher affinity than progesterone (5). Results of studies with RU 486 as a new antifertility agent are very promising. Since the metabolites of synthetic stero~s are often biologically active, their concentrations in bloo~ can also have clinical signlficance. We have utilized a human plasma pool, collected after oral intake of RU 486, as a source of a mixture of RU 486 and its metabolites. The putative metabolites of RU 486 were separated bY thin layer chromatography an~ characterized by RIA, UV-spectrophotometry and HPLC. The need for a specific and reliable assay method in evaluation of RU 486 is obvious. The RIA for RU 486 developed by Salmon and Mouren (6) is nonspecific for its metabolites (7). Our assay methods developed for RU 486 utilize Chromosorb R column chromatography to eliminate those putative metabolites cross-reacting in the direct RIA. The quantitative analysis of RU 486 is performed by HPLC or RIA. Plasma concentrations of RU 486 were measured for 48 hours after oral administration of 100, 400, 600 and 800 mg of RU 486. MATERIALS AND METHODS Chemicals: RU 486 (17#-hydroxy-ll #~(4-dimethylaminophenyl)-17~-(1- propyny])-estra-4,9-dien-3-one), (6,7-°H)-RU 486 (specific activity 37 Ci/mmol) and the corresponding antibody were provided by the Roussel- Uclaf Research Center (Romainville, France). n-Hexane, ethyl acetate, ethylene glycol, diethyl ether, methanol, triethanolamine, chloroform and acetone were purchased from Merck (Darmstadt, West Germany). Chromosorb n W-NAW 60-80 Mesh was from Sigma (St. Louis, Missouri, USA) and Macherey~Nagel (Duren, West Germany); Kieselgel 60 F 264 and the octadecyl reversed phase HPLC column Hibar LiChrosorb RP-18 (250 mm x 4 mm internal diameter) were from Merck. 614 DECEMBER 1986 VOL. 34 NO. 6
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`CONTRACEPTION Laboratory equipment: HPLC system consisted of Solvent programmer ~odel 660, two chromatography pumps, Model M-6000 A and liquid chromatography injector Model U 6 K from Waters Associates Inc. (Milford, Massachusetts, USA). The spectrophotometric detector LC-55B and deuterium power supply for the HPLC system were purchased from Perkin-Elmer (Oak Brook, Illinois, USA); the recorder was a Servogor RE 511 from Goerz Electric (Vienna, Austria). Spectrophotometric studies were performed with a Graphicord UV-240 and Option program, interface OPI-2 from Shimadzu (Kyoto, Japan). A liquid scintillation counter 1212 Minibeta from Wallac (Turku, Finland) and Teleprinter 43 from Teletype Corporation (Skokie, Illinois, USA) were used in our radioimmunoassay studies. Plasma samples: Plasma samples were collected from healthy female v--o-lunteers, aged from 25 to 32 years, in the mid]urea] phase following oral ingestion of 100, 600 and 800 mg of RU 486 by five subjects and 400 mg by four subjects (50 mg tablets provided by Roussel-Uclaf, Paris, France). In the group receiving 100 mg of RU 486 blood samples were collected at -1/2, O, 1, 2, 4, 6, 24 and 48 hours. Samples were collected at -1/2, O, 1, 2, 4, 6, 10, 24 and 48 hours in groups receiving 400, 600 and 800 mg, these groups were part of RU 486 tolerance study (8). Detection of immunoreactlve metabolites was carried out as follows: * Diethy] ether extraction of a plasma pool collected from female volunteers after oral intake of 200 or 400 mg of RU 486. * Thin layer chromatography (TLC) of standard RU 486, extracts of blank plasma and female plasma pool on Kieselgel 60 F 264 with chloroform:acetone [90:10] (v/v). * Elution of 5 mm TLC-slices with 2 ml of ethanol. * RIA of the eluates as described below. * UV-absorption spectra of the RIA-positive eluates. * HPLC (Hibar LiChrosorb RP-18 column) fractionation of the immunoreactive eluates with methanol:water ~0:10] (v/v); flow rate 1 ml/min. * UV-absorption spectra of the HPLC-fractions. Separation ofRU 486 from its metabolites on Chromosorb R columns: Column chromatography was performed by pac-king Pas't'eur pipettes under vibration with 3 ml of Chromosorb R W-NAN 60-80 mesh/20% ethylene glycol (w/w). A patient plasma sample, blank plasma pool containing RU 486 and DECEMBER 1986 VOL. 34 NO. 6 615
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`CONTRACEPTION blank plasma containing aH-RU 486 were applied to the columns, left for 30 minutes and then eluted as follows: (I) 5 ml of ethyl acetate:n-hexane [5:95] (v/v), (II) 6 ml of ethyl acetate:n-hexane [30:7~] (v/v), (Ill) 5 ml of ethyl acetate:n-hexane [75:25-~ (v/v) and (IV) 5 ml of ethyl acetate. The eluates were assessed by RIA as described below or the radioactivity was counted. Quantitation of RU 486: I Chromosorb a column ~: 0.2 - 0.4 ml of plasma/serum was applleo on the column described above, left for 30 min, eTuted with 5 m7 of ethy7 acetate:n-hexane ~:9~](v/v) and evaporated under nitrogen until dry. For the analysis of plasma samples a standard curve was prepared by running plasma pools (prepared by addition of known amounts of RU 486 into blank plasma) through the columns. IIa IIb For HPLC of RU 486 samples were redissolved into appropriate volum---~f-~he-H--~C eluent used routinely: methanol:water: triethanolamine F~0:10:0.05] (v/v/v), pumped at 1.5 ml/min; the injection volume was 100 pl. For RIA of RU 486 samples were redissolved into appropriate volume of--~.T--M phosphate-gelatin buffer containing 20g methanol (v/v). RIA was performed as described below. Evaluation of the ChromosorbR-HPLC method: Plasma pools, containing 0.~-5, 1.0 and 5.0 ~g of P~--~/ml were prepared for the recovery studies. Five 0.2 and 0.4 ml samples of each pool were chromatographed in the Chromosorb a columns. The eluates were redissolved into corresponding volume of methanol. Fifty microlitres was evapor ted until dry, redissolved into buffer used for RIA as described below. The rest was evapor ted until dry and redissolved into HPLC eluent. RU 486 concentrations were measured both by RIA and the routine HPLC system and compared with dilution series to assess the recovery. The sensitivity of the routine HPLC'method was controlled by assaying an RU 486 dilution series. With the injection volume of 100 ~I, the amount of RU 486 injected varied from 30 ng to 2.0 ng. RadioimmunoassaY of RU 486 without chromatography: A phosphate buffer con-tainingO, l% ge-Ta~Tn--'TW/V) was prepared by dissolving 9 g NaCl, 1 ~H sodium azide and 1 g ~elatin in 1 liter of 0.1M phosphate buffer, 7.0 and stored at + 4 C. The antiserum against RU 486 was donated by Roussel-Uclaf, diluted 1/100 (v/v) into phosphate-gelatin buffer and stored at -20°C. A'siock solution of RU 486 at 1.0 mg/ml was stored at +4°C. For the standard curve, solutions of RU 486 were prepared just before use by pipetting a dilution series containing 2 - 2000 pg of RU 486 in ethanol per plastic test tube. The standards were evaporated until 616 DECEMBER 1986 VOL. 34 NO. 6
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`CONTRACEPTION dry and dissolved in 100 ~l of phosphate-gelatin buffer containing 20% methanol (v/v). Tritiated RU 486 was stored at -20°Ci in toluene. Prior to use (6,7- 3H)-RU 486 was purified using the ChromosorbB-method followed by dilution in phosphate-gelatin buffer containing 0.025% Triton X-tO0 (w/v) (Koch-Light Laboratories Ltd, Colnbrook, Bucks, England) at a concentration of 50,000 counts per 1 ml. A charcoal suspension was prepared, composed of 0.25% charcoal (w/v) (Norit A, from Sigma) and 0.025% dextran T70 (w/v) (Pharmacia, Uppsala, Sweden) in phosphate-gelatin buffer. Scintillation fluid YA-riatuike (70% pseudochumene) was obtained from Yliopiston Apteekki (Helsinki, Finland). Radioimmunoassay was performed according to Salmon and Mouren (6) as follows: Plasma samples of 0.1 ml were diluted with 0.4 ml of physiological saline containing 0.025% Triton X-IO0 (w/v). The samples were vortex- mixed and extracted once with 5 ml of diethy] ether for 10 minutes. The aqueous phases were frozen in -70°C cold ethanol and the extracts were evaporated to dryness with nitrogen in a +40°C water bath. The samples were dissolved in 0.1 ml phosphate-gelatin buffer containing 20% methanol. They were then mixed with 0.1 ml of antibody diluted in phosphate-gelatin buffer and left for 30 minutes at room temperature. Tritiated RU 486 (0.1 ml) was added, vortexed and left to incubate overnight in an ice-bath. Ice-cold charcoal suspension (0.5 ml) was added together with 0.1 ml of 0.5% phosphate-gelatin buffer (w/v), left for 5 min on ice and then centrifuged for 10 minutes at 3000 x g. A Finnfuge centrifuge (Labsystems Oy, Helsinki, Finland) was used. Supernatants were transferred to polyethylene counting vials together with 2.5 ml of scintillation fluid and were counted for 5 minutes. Statistical analysis: Welch paired t-test was used for statistical analysis. The half-life of RU 486 was calculated graphically from the RU 486 concentrations measured at 24 and 48 hours. RESULTS Figure 1 shows the TLC of RU 486 standard, as well as the extracts of blank plasma and plasma pools obtained by combining samples collected from female volunteers four or forty-eight hours after oral administration of 200 or 400 mg of RU 486. RIA was performed on the eluates of the sliced TLC in order to find the cross-reacting steroids. Four distinguishable spots were found with the RIA, viz. authentic RU 486 with an Rf-value of 0.56, two UV-visible spots with Rf-va]ues of 0.38 and 0.19 and a spot with an Rf-value of 0.11. UV- absorption spectra of the 0.56, 0.38 and 0.19 spots revealed absorption maximum at 304 rim. The UV-absorption spectrum of RU 486 had absorption maxima at 304 and 262 nm. By using a wave-length of 304 nm in the fractionation HPLC DECEMBER 1986 VOL. 34 NO. 6 617
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`CONTRACEPTION system, analysis of the TLC-spot extracts revealed material with three different retention times, 4.33 min, 3.50 min and 3.17 min, the first one being identical to that of RU 486. The UV-absorptlon spectra were determined for the fractionated HPLC-peaks. In addition to the common absorption maximum at 304 nm, each of them had a separate absorption maximum at 262, 250 and 235 nm, respectively. The percentage of cross-reacting material in the lowest TLC-spot (Rf=O.11), increased from 9% at 4 hours to 22% at 48 hours, of the total RIA-measurable material. This material had an UV-absorption maximum at wave-length 270 nm with the retention ~ime of 3 min in our HPLC fractionation system. . 9/~t.,F,~ .~ / r.t~.t,,~_ awl I ! O,] II 4 O~ 11 6 03 ~0 14 (,,a ~4 s 04 ~o "~ 03 ~ ~O, } 33% ],l } -- 0.4 " 6~1 '5~l ...... 0;~ s~, " ~ 33% 03 14~ 4~4 o 4~ t*1 o.1 1 o s "6. -~" 3~% ~i- %" .4 ~ 22% .... o 2-"~ 02 711 ZlJT o ~" ] 9% :99-'T'~'J 5% --T'--" ]~ J ~09 o.~ 47 szl o ~ ~':'" 9 % 22% blank plasma RU ~g6 ~,h plasma t.8 h plasma poo( pool. Figure 1. TLC-chromatography.of RU 486 standard, diethyl ether extracts of pooled plasma samples collected 4 and 48 hours after ora] intake of 200 or 400 mg of RU 486 and diethy] ether extract of blank plasma. Percentages of the total RIA measurable material were c~]cul~ted for each distinct spot. Figure 2 shows the ChromosorbRelution profi]es of a patient sample collected after oral intake of RU 486, a standard plasma pool of RU 486 ~ and a plasma sample containi~Ig (6,7- H)-RU 486. 0.25-m1 fractions were collected, and the radioactivity was counted or the concentration of RIA-measurabIe RU 486 assayed. It appeared that most if not all unchanged RU 486 came out with the first eluate. The metabolites came out with the second and third eluates. 618 DECEMBER 1986 VOL. 34 NO. 6
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`CONTRACEPTION P. lOx 10 4 ' 5xt04o - 600 a -5oo I -4oo ; ~ ~l '4 t l~, 1~' I : , • ! 5 Fr~ction number, CHROMOSORB W-NAW 60-BO MESH//20°Io ETHYLENEGLYCOL I 5rr~ ~tl~l ~cct~t~ , n-h~x~n~ ~g5 ~" 6 ml " " 30,70 ][~ 5rn! " " 75~25 J~ 5 ml ethyl acetate 100 jl ,o ; :. 3H-RU 486standal'd RU 4B6 standard ~----o Patic~nt plasma 4'o Figure 2. Chromo~orb ®elution profiles of a plasma sample containin-g (6,7- H)-RU 486 (o), a standard plasma-pool of RU 486 (o) and patient plasma collected 48 h after oral intake of RU 486 (~). Figure 3 shows an example of a standard curve of RIA for RU 486. The optimal part of the assay ranges from 15 pg to 800 pg per tube. The sensitivity was 10 pg per tube. The interassay coefficient of variation varied from 20.6 to 25.9%. The intraassay C.V. ranged from 7% to 33~. Due to high concentrations of RU 486 in plasma after oral intake of therapeutic doses, dilution factors up to 1:25000 had to be used in clinical samples. Figure 4 shows the evaluation and comparison of HPLC and RIA after Chromosorb R column chromatography in the assay of plasma RU 486. Five aliquots of 0.2 and 0.4 ml from standard plasma pools containing 0.25, 1.0 and 5.0 ~g RU 486/m1 were chromatographed with 5 ml of eluate I and subsequently analyzed by HPLC and RIA. HPLC showed a better precision with the coefficients of variation (C.V.) from 1% to 11%. Accprdingly, the C.V. for RIA varied from 7% to 33%. The accuracy was calculated from the recoveries, which varied from 78% to 118%. There was very highcorrelation between RiA and HPLC results (r=0.99, n=29). The system was unaffected by the plasma volume. The sensitivity of the HPLC syst~ was 4 ng of RU 486 injected in 100 ~1 of methanol. DECEMBER 1986 VOL. 34 NO. 6 619
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`CONTRA CEPTION LO~it C- o) -3 .2 -1 :O -1 -2 "3- 10 -- .... J .......... i ........... • ........... 50 160 ~250 500 1000 Pg / tube 2 5 RU 486 Figure 3. A representative standard curve radloimmuno---assay for RU 486. of the 5000, 4000, E " 3OO0- (- u .J o_ 2000- -r 1000- / o 2001JI samples / A 4001JI samples / r = 0.99 1000 2000 3000 4000 5000 RIA ng /rnl ~ 4. R Evaluation and comparison of HPLC and RIA after hromoso-~b chromatography. Five aliquots of 0.2 ml and 0.4 ml from plasma pools containing 0.25 ~g/ml, 1.0 #g/ml .and 5.0 #g/ml of RU 486 were analyzed. The equation for the linear regression line was Y = 0.94X + 0,07. 620 DECEMBER 1986 VOL. 34 NO. 6
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`CONTRACEPTION Figure 5 showsaplasma concentrations of RU 486 as measured by HPLC after Chromosorb chromatography in samples collected 1, 2, 4, 6, 10, 24 and 48 hours after oral intake of 100, 400, 600 and 800 mg of RU 486. The peak concentrations were reached within the first hour after ingestion of each dosage. Interestingly, even though there was an eight-fold difference in the oral dose, the mean plasma RU 486 concentration did not differ significantly at any point measured between the 800 mg and 100 mg group; except at 2 hours (p<O.05). Evidently 48 hours of plasma sampling was not enough for calculation of the disappearance of RU 486 from the circulation. The half-life between Z4 and 48 hours always exceeded 27 hours. E IGI (D ::) I:E 5000- 2500- 1000- 500 - 250- 800 mg o--o 600 rng ~-.~ 400 rng A-~ 100 rng e-.a "% ~., 1 ~, 1C) 24 4~ hours TIME Figure 5. Plasma concentrations of RU 486 (mean+S.E.M.) within 48 hours--after oral intake of 100, 400, 600 and 800 mg of RU 486, as measured by HPLC after Chromosorb R chromatography. DECEMBER 1986 VOL. 34 NO. 6 621
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`CONTRACEPTION DISCUSSION RU 486 has both antlglucocortlcoid and antiprogestational activity of high potency. This Is mediated by high affinity binding of the steroid to glucocorticoid and progestin receptors (9). RU 486 can therefore be applied in two distinct clinical directions: the symptomatic treatment of hypercortisolemia (10) and the regulation of fertility (4). The metabolltes of RU 486 can b~ biologically active also in human as suggested by Peraedt et al. (11), with different affinities to progestin and glucocorticoid receptors (11). Hence, knowledge of the metabolism of RU 486 and thus the plasma concentrations of the drug and its metabolites may be of clinical importance. The direct RIA lacks the specificity to measure RU 486. Making use of the cross-reactivity of the putative metabolites of RU 486 in the RIA (6), we verified three of them in a pool of plasma collected from human female volunteers after oral intake of RU 486. These three metabolites were extracted with diethyl ether and separated by thin layer chromatography. Two of the metabolites shared a common UV-absorption maximum with RU 486 at 304 nm, which makes it possible to assay them simultaneously by HPLC, if known amounts of reference metabolites were available. The common absorption maximum suggests that these two putative metabolites share common features and most likely are menD- and didemethylated metabolltes of the parent steroid, as suggested by previous studies on rats (11). The verification of these putative metabolites allowed us to develop a column chromatographic method to purify RU 486. Chromosorb B columns present a simple and efficient method for separation of RU 486 from its cross-reacting metabolites in plasma. It can be employed prior to RIA when only the parent steroid is measured in large-scale clinical samples. The more precise HPLC method is needed for measuring RU 486 when metabolism and pharmacokinetics of RU 486 are studied. The retention times in our system were 2.5 minutes, thus allowing for a substantial number of samples to be analyzed. In our previous report (8) plasma concentrations of RU 486 were measured by a nonspecific RIA which includes a pool of cross-reacting metabolites. Our present data indicates that the peak plasma concentrations of RU 486 are reached rapidly. During an initial six- hour redistribution period,RU 486 concentrations decrease into about half of the peak levels (Fig. 5). After that a steady-state plasma RU 486 concentration is reached for a long time; the longer the higher the dose (Fig. 5). This may indicate a cessation of RU 486 metabolism for a while, or more probably, a return of RU 486 into circulation from other compartments of the body (such as fat). Oral administration of ~H-RU 486 resulted in remarkable extravascular diffusion in rats (11). RU 486 is well tolerated also in high doses (8). This together with our observation on the long half-life of RU 486 gives a good rationale for a single dose administration. At high doses the progestomimetic 622 DECEMBER 1986 VOL. 34 NO. 6
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`CONTRACEPTION properties of RU 486 may decrease its biological efficacy (5). It is of interest to see that both central and peripheral anti- glucocorticoid activity of RU 486 is strongly dose-dependent occurring only with doses above 400 mg (12,13)o Yet our studies indicate that the circulating plasma levels of RU 486 are not sfgnificantly affected by increasing the oral dose of RU 486 from 100 mg to 800~ mg; thus suggesting that other mechanisms must be partly involved in the anti- steroidal action of RU 486. This justifies further studies on the effect of various oral doses on metabolism of RU 486. ACKNOWLEDGEMENTS This work was undertaken as part of the contraceptive development program sponsored and coordinated by the International Committee for Contraception Research of the Population Council, Inc., New York, New York. The financial support provided by the Ford Foundation and the Mellon Foundation is gratefully acknowledged. The content does not necessarily reflect the policy of any of the funding sources. RU 486, H-RU 486 and the corresponding antibody were kindly provided by Roussel-Uclaf, Paris, France. REFERENCES . . . . 5. Philibert, D., Deraedt, R., Teutsch, G., Tournemine, C. and Sakiz, E.: RU 486: a new lead for steroidal anti-hormones. Sixty-Fourth Annual Meeting of the Endocrine Society, San Francisco, June 16 to 18, 1982. Abstract No. 668. Philibert, D., Deraedt, R. and Teutsch, G.: RU 38486 - A potent antiglucocorticoid in vivo. VIII International Congress of Pharmacology, Tokyo, duly 19 to 24, 1981. Abstract No. 1463. Moguilewsky, M. and Philibert, D.: RU 38486: Potent anti- glucocorticoid activity correlated with strong binding to the cytosolic glucocorticoid receptor fo}lowed by an impaired activation, d. Steroid Biochem. 20:271-276 (1984). Herrmann, W., Wyss, R., Riondel, A., Philibert, D., Teutsch, G., Sakiz, E. and Baulieu, E.E.: The effects of an antiprogesterone steroid on women: interruption of the menstrual cycle and of early pregnancy. C.R. Acad. Sc. Paris 294:933-938 (1982). Gravanis, A., Schaison, G., George, M., de Brux, d., Satyaswaroop, G., Baulieu, E.E. and Robel, P.: Endometrial and pituitary reponses to the steroidal antiprogestin RU 486 in postmenopausal women, d. Clin. Endocrinol. Metab. 60:156-163 (1985). DECEMBER 1986 VOL. 34 NO. 6 623
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`CONTRACEPTION . . . . 10. 11. 12. 13. Salmon, J. and Mouren, M.: Radioimmunoassay of RU 486. In: The Antiprogesterone Steroid RU 486 and Human Fertility Control (Segal, S.j. and Baulieu, E.E., eds.). Plenum Press, New York, 1985, p. 99-101. Pbilibert, D. In: Proceedings on RU 486 (Segal, S.J. and Bau]ieu, E.E., eds.). Bellagio, Italy, 1984. Shoupe, D., Mishell, D., Lacarra, M., Gutierrez, E., L~hteenm~ki, P. and Spitz, I.: Endocrinologic effects of the antiprogesterone RU 486 in the luteal phase of normal women. In: The Antiprogesterone Steroid RU 486 and Human Fertility Control (Segal, S.J. and Baulieu, E.E., eds.). Plenum Press, New York, 1985, p. 285-293. Schreiber, J., Hsueh, A. and Baulieu, E.E.: Binding of the anti- progestin RU-486 to rat ovary steroid receptors. Contraception 28:77-85 (1983). Chrousos, G., Cutler, G., Simons, S., Pons, M., Moriarty, R. and Loriaux, D.: Development of antiglucocorticoids with potential clinical usefulness. In: Progress in Research and Clinical Applications of Corticosteroids. Heyden and Son Inc., Philadelphia, 1982, p. 552-176. Deraedt, R., Bonnat, C., Busigny, M., Chatelet, P., Cousty, C., Mouren, M., Philibert, D., Pottier, J. and Salmon, J.: Pharmacokinetics of RU 486. In: The Antiprogesterone Steroid RU 486 and Human Fertility Control (Segal S.J. and Baulieu E.E., eds.). °]enum Press, New York, 1985, p. 103-122. Bertagna, X., Bertagna, C., Luton, J-P., Husson, J-M and Girard, F.: The new steroid analog RU 486 inhibits glucocorticoid action in man. J. Olin. Endocrinol. Metab. 59:25-28 (1984). Galliard, R., Poffet, D., Riondel, A. and Saurat J-H.: RU 486 inhibits peripheral effects of glucocorticoids in humans. J. Clin. Endocrinol. Metab. 61:1009-1011 (1985). 624 DECEMBER 1986 VOL. 34 NO. 6
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