COMPETITIVE PROGESTERONE ANTAGONISTS: RECEPTOR BINDING AND BIOLOGIC ACTIVITY OF TESTOSTERONE AND 19-NORTESTOSTERONE DERIVATIVES

Transcription

1 FERTILITY AND STERILITY Copyright 1979 The American Fertility Society Vol. 31, No.5, May 1979 Printed in U.SA. COMPETITIVE PROGESTERONE ANTAGONISTS: RECEPTOR BINDING AND BIOLOGIC ACTIVITY OF TESTOSTERONE AND 19-NORTESTOSTERONE DERIVATIVES JERRY R. REEL, PH.D.* RONALD R. HUMPHREY, M.S. YU-HSIN SHIH, M.S. BARBARA L. WINDSOR, B.S. RAY SAKOWSKI PAUL L. CREGER, PH.D. RICHARD A. EDGREN, PH.D.t Departments of Pharmacology and Chemistry, Warner-Lambert/Parke Davis, Pharmaceutical Research Division, Ann Arbor, Michigan Testosterone and 19-nortestosterone derivatives were evaluated in a developmental scheme designed to identify competitive progesterone antagonists having abortifacient activity. Compounds that displayed significant binding to the rabbit uterine progesterone receptor were followed in biologic tests for progestational, antiprogestational, and abortifacient activities. Of the seven compounds tested for both progestational and antiprogestational activity, only one, 5 crdihydronorethindrone, behaved exclusively as an antagonist. Five other 19-nortestosterones (19-nortestosterone, 17 {3-hydroxyestra-4, 9(10)-dien-3-one, norethindrone, norethindrone acetate, and R 2323) proved to be mixed agonistslantagonists. 5crDihydronorethindrone, norethindrone, and 19-nortestosterone terminated pregnancy during the postnidatory period in rats; in addition, the latter two compounds inhibited progesterone-supported pregnancy in spayed rats and curtailed pregnancy during the postnidatory period in hamsters. These results demonstrate that several 19-nortestosterone derivatives bind to the uterine progesterone receptor and behave either as antagonists or mixed agonistslantagonists. Fertil SteriI31:552, 1979 Ovarian progesterone plays an obligatory role during early pregnancy in many species, and its deficiency can lead to failure of implantation, abortion, or resorption of the conceptus. 1 Removal of the source of progesterone will terminate pregnancy in most species, but the period during which ovariectomy is effective varies. 1 Humans, as Csapo and Pulkkinen 2 have shown, require luteal pro- Received September 18, 1978; revised November 13, 1978; accepted December 11, *Present address and adress for reprint requests: Research Triangle Institute, P.O. Box 12194,Research Triangle Park, N. C tpresent address: Syntex Laboratories, Inc., Medical Department, Palo Alto, Calif gesterone during the first 5 or 6 weeks of pregnancy as judged by the effects of oophorectomy and luteectomy. The termination of pregnancy through induced functional luteolysis has been an attractive approach to human population control that has eluded its pursuers. The prostaglandins, which are effective luteolytic agents in various domestic and laboratory species, have proved ineffective in man, whereas luteinizing hormone-releasing hormone (LHRH) and its agonistic analogs are active in laboratory rodents and possibly in man.a Progesterone antibodies, by combining with circulating progesterone, also terminate early gestation in the rat. Thus, progesterone deficiency, whether effected by elimination of the source of the hormone 552.

2 Vol. 31, No.5 COMPETITIVE PROGESTERONE ANTAGONISTS 553. ~ (luteectomy, luteolysis) or by making circulating hormone unavailable (antibodies), results in the curtailment of pregnancy. The goal of preventing progesterone from functioning can, theoretically, be achieved by preventing the hormone from binding to its uterine receptor. One such drug, R 2323, has been reported.4, 5 In humans, its effects are equivocal; it appears not to act as an abortifacient when administered after a missed menstrual period,4 and its effects at midcycle are either anti-implantational,s presumably as a progesterone antagonist, or antiovulatory,4 owing to suppression of pituitary gonadotropin secretion. The search for compounds that will compete with progesterone for binding to its receptor sites continues in many laboratories. We have reasoned that the scheme shown should identify such an agent (Fig. 1). Binding to the receptor, the first phase of the sequence, is not conclusive since such binding characterizes either an agonist or an antagonist. Therefore, compounds with defined binding affinity were followed in biologic tests for progestational and antiprogestational activities, namely, stimulation or inhibition of glandular arborization in rabbit uteri, inhibition of progesterone-supported pregnancies in spayed rats, and postnidatory termination of pregnancies in rats and hamsters. MATERIALS AND METHODS Compounds. The steroids evaluated were obtained from the Warner-Lambert/Parke-Davis chemical files, except as follows: testosterone and 17 a-ethynylestradiol (Steraloids, Wilton, N. H.); 19-nortestosterone (Schering A. G., Berlin, Germany); norethindrone and norethindrone acetate (Syntex Laboratories, Palo Alto, Calif.); SC-9022 (Searle Laboratories, Chicago, Ill.); d-norgestrel (levonorgestrel)* and dl-norgestrel (Wyeth Laboratories, Philadelphia, Pa.); R 1881, R 2323, and 17,8-hydroxyestra-4,9(10)dien-3-one (Roussel Uclaf, Romainville, France); and 17-allylestr-4- en-17,8-01 (Organon Pharmaceuticals, West Orange, N. J.). [1,2-3 H1Progesterone (48-52 Cil *The nomenclature for the enantiomers ofnorgestrel is confused in the literature. The World Health Organization has recently designated the biologically active enantiomorph levonorgestrel, which heretofore has been known as d norgestrel. According to Fieser's convention, the active form (-)-norgestrel, or levonorgestrel, is that enantiomer with absolute configuration at all asymmetric centers identical with estrone, the basis for comparison. This form was erroneously called (+ )-norgestrel by Edgren et al.6 o Preliminary High Dose Testing o Quantification ~~ln~vi~tro~~1 1~ ~I~nV~iv~o ~ ~,----, Progesterone Progesterone Supported - Supported Pregnancy I Pregnancr FIG. 1. Preclinical developmental scheme for competitive progesterone antagonists having abortifacient activity. The assays indicated within the circles are performed at a single high concentration (in vitro) or dose (in vivo) of the compound, whereas those within the squares are performed at multiple concentrations or doses of the compound in order to quantify the results. mmole) was purchased from New England Nuclear Corporation, Boston, Mass. Progesterone Receptor Binding Assays. In each assay 10 to 15 immature New Zealand White rabbits (0.8 to 1.1 kg) received subcutaneous (sc) injections daily for 3 days of 15 IJ.f5 of 17,8-estradiol in peanut oil. Twenty-four hours after the final injection, the rabbits were asphyxiated with CO2, and the uteri were excised. The pooled uteri were rinsed six times in ice-cold 0.9% NaCl, minced with scissors in 3 volumes of ice-cold 10 mm N tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid, 1 mm ethylenediamine tetraacetic acid disodium salt, 12 mm thioglycerol, and 30% glycerol, ph 7.4, and homogenized using a Brinkman Polytron homogenizer, Pt. 10. The homogenate was centrifuged at 105,000 x g for 1 hour at 0 C and the supernatant fraction was filtered through a double layer of presoaked gauze (Parke-Davis, type IV, 24/20, 8-ply) to remove the buoyant fatty layer. The filtrate (cytosol) was employed as the source of sol uble progesterone receptor. In the competitive binding assay, a series ofpolypropilene tubes was prepared in duplicate to contain 0.2 ml of cytosol or buffer, 0.1 ml of various concentrations of unlabeled test compound, progesterone (or buffer), 0.2 ml of homogenization buffer, and 0.05 ml of [1,2-3Hlprogesterone, such that the final concentration was 2.5 x 10-8 M. All tubes were incubated for 16 hours at 0 C. To remove free and weakly bound [3Hlprogesterone, 0.5 ml of dextran-coated charcoal suspension (0.1% Dextran T-40 [Pharmacial and 1% acid-washed Norit A charcoal [Pfanstiehll in homogenization buffer) then was

3 554 REELETAL. May 1979 added to the incubation mixture. This mixture was agitated briefly in a Vortex mixer and incubated in an ice bath for 15 minutes. The tubes were centrifugedat1500 xgfor20minutesato C.Aportion ofthe supernatant fraction (0.2 ml) was counted in 10 ml of AmershamlSearle PCS, using a Packard 3375 spectrometer. Receptor-bound [3H]progesterone was determined by subtracting the assay blank cpm (0.3% of the total radioactivity) from the sample cpm. Linear competitive binding curves were obtained by plotting the percentage of bound [3H]progesterone versus the concentration of the competitor, using logit-log graph paper. The relative binding affinity (RBA) of each compound was calculated by using the following equation: RBA = ---- x 100 [Clsoo/c where [Plso% = molar concentration of unlabeled progesterone required to decrease bound [3H]progesterone to 50% of the buffer control (100% bound [3H]progesterone) and [C]50% = molar concentration of test compound required to decrease bound [3H]progesterone to 50% of the buffer control (100% bound [3H]progesterone). Progestational and Antiprogestational Activity. Progestational and antiprogestational activity was assessed by the McPhail modification 7 of the Clauberg assay. Immature New Zealand White rabbits (0.8 to 1.1 kg) received sc injections of 5 J-Lg of estrone in peanut oil on days 1,3, and 5. Progesterone and/or test compound or the peanut oil vehicle then were administered sc on days 7,8,9, and 10. On day 11 the rabbits were asphyxiated with CO 2 and the uteri were excised, weighed, and fixed in 10% formalin. The fixed tissues were embedded in paraffin, sectioned at 6 J-Lm, and stained with hematoxylin and eosin. Endometrial proliferation was scored from 0 (estrone-primed controls) to 4 (maximal proliferation) according to the grading system of McPhail.7 I nhibition of Progesterone-Maintained Pregnancy in Ovariectomized Rats. Charles River CD rats (Portage, Mich.) were ovariectomized on day 7 of gestation (day 1 = sperm-positive vaginal smear) and treated daily with progesterone (8 mg) or test compound and progesterone (8 mg) from days 7 through 21. Progesterone and test compounds were suspended in aqueous vehicle 8 and injected at separate subcutaneous sites. Autopsy was performed as above on the morning of day 22. Animals with clear-cut indications of resorption at all implantation sites were scored as "terminated," whereas those with one or more normal fetuses remaining were considered "pregnant." Postnidatory Termination of Pregnancy in Rats. Charles River CD rats received daily sc injections of test compound or aqueous vehicle from day 7 through 12 of pregnancy. The animals were killed with CO2 on day 21 and the uteri were removed, trimmed, and weighed. The number of normal and abnormal term fetuses was recorded and the outcome of the pregnancy was classified as described above. Postnidatory Termination of Pregnancy in Hamsters. Golden hamsters were received from Charles River Lakeview (Newfield, N. J.) on day 2 of gestation (day 1 = sperm-positive vaginal smear). Test compounds were suspended in aqueous vehicle and inj ected sc daily from days 6 through 10 of pregnancy. The animals were killed for autopsy on day 15 and the outcome of the pregnancies was classified as described above. Androgenic and Antiandrogenic Activity. Androgenic and antiandrogenic activity was assessed by the method of Hershberger et al. 9 Charles River male rats were castrated at 22 days of age. Each animal was given daily sc injections oftestosterone and/or 5a-dihydronorethindrone in aqueous vehicle for 7 days, beginning on the day of castration. Twenty-four hours after the final injection, the animals were killed with CO 2 and the seminal vesicles, ventral prostates, and levator ani muscles were excised and weighed. RESULTS Progesterone Receptor Binding Affinity. As a first step toward finding a competitive progesterone antagonist, relatives of testosterone and 19-nortestosterone were tested for their ability to compete with [3H]progesterone for receptor sites in the rabbit uterine cytosol. For the series of compounds tested, the RBAs ranged from 0.5 to 101% (Table 1). As reported earlier by McGuire et ai.,10 two modifications of testosterone [1] that significantly enhanced binding affinity were the removal of the C-10 methyl group to evolve 19-nortestosterone [4] and the introduction of alkyl or alkynyl groups in the 17 a-position of testosterone [2,3]. Combina tions of both types of modification tn the same molecule [7, 8, 16] led to compounds with even greater binding affinity than those with the single alteration. Chlorine addition on the 17 a ethynyl group [14] further increased binding affinity, whereas phenyl substitution [15) decreased

4 Vol. 31, No.5 COMPETITIVE PROGESTERONE ANTAGONISTS 555 TABLE 1. Relative Binding Affinities (RBA) of a Series of Testosterones and 19-Nortestosterones R2 R1 Compound Unsaturation R, R, R, R. R, Other RBA no OH H CH 3 CH OH CH 3 CH3 CH OH C=CH CH 3 CH 3 19 (3)a OH H H CH 3 6 (2) 5 4,9(10) 0 OH H H CH3 26 (2) 6 4,9(10),11 0 OH CH 3 H CH3 101 (3) OH C=CH. H CH OH C=CH H CH3 57 (2) ON C=CH H CH 3 48 (2) OAcF 3 C=CH H CH 3 17 (2) OH C=CH H CH3 6~-F 62 (3) OH C=CH H CH 3 2a-F OH C=CH H CH 3 2=C-C-OEt 6 0 II H OH C=CI H CH 3 84 (3) OH C=CC.Hs H CH OH H.C-C=CH. H CH3 48 (2) I CH 3 17 (-)4 0 OH C=CH H C.Hs 92 (3) 18 (±)4 0 OH C=CH H C.Hs ,9(10),11 0 OH C=CH H C.Hs OH H CH 3 CH 3 5a-H OH C=CH CH 3 CH 3 5a-H 0.5 (2) 22 0 OH C=CH H CH 3 5a-H 10 (3) 23 0 OH C=CH H H CH3 5a-H;2=C 2 (2) 0 H H 24 0 OH CH 3 H CH3 5a-H;2=C 0.7 (2) 0 25 H Anti 4 NOH OH C=CH H CH Syn4 NOH OH C=CH H 27 CH Anti (54%) 3 21 NOH OH C=CH H CH 3 Syn (46%) ~-OH OH C=CH H CH ,3,5(10).B-OH OH C=CH H CH 3 2 (2) 30 3,5 OH C=CH H CH OH H.C-C=CH. H CH 3 6 H 32 4 CH. OH CH 3 H CH 3 4 anumber of assays is indicated in parentheses. binding. Unsaturation at C-9(10) [5] or C-9(10) for R 2323 [19] as compared with (-)-norgestrel and C-ll [6] also increased the binding affinity of [17] and (±)-norgestrel [18]. 19-nortestosterone; however, this was not the case Ring A reduction of 17 a-ethynyltestosterone [3j

5 556 REELETAL. May 1979 and 17a-ethynyl-19-nortestosterone [8] to their sterically similar (AlB trans) 5a-dihydro counterparts, [21] and [22], respectively, resulted in diminished affinity for the receptor. Various substituents at the C-2 position of 17 a-ethynyl- or 17a-methyl-19-nortestosterone [12, 13, 23, 24] or esterification at the 17 {3-position [9, 10] led to decreased binding activity as compared with 17 a ethynyl-19-nortestosterone [8]. A C-6 fluorine substituent appeared to have no influence on binding affinity as compared with [8]. Compounds with modifications at the C-3 and/or C-4 positions [25-32] also were studied. The C-3 oximino derivatives [25, 26, 27] retained a significant degree of binding activity, but their RBAs were only one-third to one-half that of the corresponding 3-oxo compound [8]. Furthermore, the C-3 hydroxy derivative [28] had about one-fifth the binding affinity of [8]. 17 a-ethynylestradiol [29], which contains a 3{3-hydroxy function and an aromatic ring A, had very low, yet detectable, binding activity. Finally, the derivatives lacking a 3-oxo function with unsaturation at either C-3, 5 [30], or C-4 [31] or the 17a-methyl-19-nortestosterone derivative with a C-3 methylene group [32] exhibited little affinity for the receptor. Progestational and Antiprogestational Activity. Since progesterone receptor binding assays do not distinguish between agonistic and antagonistic activities, representative compounds from the testosterone and 19-nortestosterone series were tested for progestational and antiprogestational activity using endometrial gland proliferation in estrogen-primed immature rabbits as the biologic end-point (Table 2). Because of limited quantities, a number of the compounds could only be tested at low and/or variable doses in one or the other assay; some compounds were not available in sufficient amounts for either test. Testosterone [1], 19-nortestosterone [4], and 19-nortestosterone with a double bond at C-9(10) [5] were active progestagens at doses of20mg/day; 17a-methyl-19-nortestosterone with double bonds at C-9(10) and C-ll [6] displayed progestational activity comparable TABLE 2. Progestational and Antiprogestational Activity of a Series of Testosterone and 19-Nortestosterones Compound no." Progestational' Antiprogestationalc Daily dose McPhail hllex Daily dose McPhail index (compoundjprogesterone) (comprundlprogesterone) "'II / / / / / / / / / d / / d / / / / / / / / / / / / / / / / / / / / /2.65 mg ~ See Table 1 for chemical structures. ~ lii'he first value presented under the McPhail index is that for the test compound at the daily dose indicated. The second McPhail index value is for 200 pg of progesterone/day tested in the same assay. 'y cthe first value presented under the McPhail index is that for the test compound at the daily dose indicated in combination with progesterone (200 f.lg/day). The second value is for 200 f.lg of progesterone/day tested in the same assay. dcompounds were injected intramuscularly at the daily doses indicated. There is no second set of values, since progesterone was not tested in these particular assays.

6 "( Vol. 31, No.5 COMPETITIVE PROGESTERONE ANTAGONISTS 557 TABLE 3. Postnidatory Termination of Pregnancy and Inhibition of Progesterone-Maintained Pregnancy by a Series of Testosterones and 19-Nortestosterones Compound no." > >5 16 >5 17 >5 18 > asee Table 1 for chemical structures. Postnidatory tennination rf pregnancy, ED", Inhibition of l'rogesterone-maintained pregnancy m ovariectomized rats Rat Hamster Daily dose Proportion of pregnancies inhibited mgldny > >10 mg 5 3/5 10 2/5 5 0/5 5 5/5 to [4) and [5) at 1J20th the dose. As anticipated, norethindrone [8) and norethindrone acetate [9) stimulated endometrial proliferation at daily doses of 0.1, 0.3, and 0.5 mg. At 12 mg/day, R2323 [19) exhibited measurable agonistic activity. In contradistinction, 5a-dihydronorethindrone [22) showed little or no agonistic activity at daily doses up to 10 mg. When administered simultaneously with progesterone, testosterone [1) was inactive as an antagonist at 20 mg/day, whereas two 19-nortestosterones, [4) and [5), were active at daily doses of 10 or 20 mg. In regard to the latter observation, several of the relatives of 17 a-ethynyl-19- nortestosterone also exhibited antagonistic activity. Hence, norethindrone [8), norethindrone acetate [9), norethindrone trifluoroacetate [10), norethindrone having a glycolic acid ethyl ester substitution at C-2 [13) or chlorine on the 17 a ethynyl group [14), R 2323 [19), and the 3-desoxo derivative with unsaturation at C-3 and C-5 [30) were active at doses of 20 mg/day or less. In contrast, norethindrone bearing a phenyl substituent on the 17 a-ethynyl group [15) and 5a-reduced 17 a-ethynyltestosterone [21) were inactive at and 14-mg daily doses, respectively. In addition, such known progestogens as SC-9022 [16) and racemic norgestrel [18) did not show antiprogestational activity. Of greater interest was the observation that 5a-dihydronorethindrone [22) and its C-2 carboxaldehyde substituted derivative [24] possessed antiprogestational activity. Significantly, 5adihydronorethindrone inhibited endometrial proliferation in a dose-dependent manner when given over a range of doses from 1 to 20 mg daily. Inhibition of Progesterone-Maintained Pregnancy. Because of limitations of time and compound, only three materials were studied in this assay (Table 3). Testosterone at 5 mg/day induced resorption in three offive ovariectomized rats maintained with 8 mg of progesterone; two offive pregnancies resorbed in animals receiving 19- nortestosterone at 10 mg, while 5 mg had no effect. Norethindrone, at 5 mg/day completely inhibited the effect of progesterone in five of five rats. Postnidatory Termination of Pregnancy. Testosterone [1), 19-nortestosterone [4), 19-nortestosterone with unsaturation of C-9(10) [5), norethindrone [8), norethindrone acetate [9), and 5Q-dihydronorethindrone [22) effectively interrupted normal pregnancy in 50% (ED 50) of the rats at daily doses of 5 mg or less. On the other hand, 17 Q-ethynyltestosterone [3), norethindrone with a phenyl substituent on the 17 a-ethynyl group [15), SC-9022 [16), (-)-norgestrel [17), and racemic norgestrel [18] were inactive in the pregnant rat at doses of 5 mg/day (Table 3). In marked contrast to its activity in the rat, testosterone [1) was ineffective in the pregnant hamster at doses as high as 100 mg/day. 19-Nortestosterone [4], norethindrone [8), and norethindrone acetate [9], although not as potent as in the pregnant rat, did terminate pregnancy in the hamster. As in the rat, racemic norgestrel [18) was inactive in the pregnant hamster at doses as high as 10 mg/day (Table 3). Androgenic and Antiandrogenic Activity of 5a Dihydronorethindrone 122]. Since 5a-dihydronorethindrone [22] exhibited progesterone receptor binding affinity (RBA = 10%), possessed antiprogestational activity with little or no proges-

7 558 REEL ET AL. May 1979 ANDROGEN ASSAY SEII\INAl VESiClES flo 80 _70 '".!.60 VENTRAL PROSTATE... LEVATOR ANI *... non ii:30 ;:: 20.LC.LJ50~5OO~I..l.,S.LJIOL..L C I 5 10.JIg Jig mg mg mg ~ JIg mg mg mg, TESTOS- 5&-_ TESTOS- 5&_ mias- 5&-_ TElIOIIE HE TERON NE TEROIIE ANTI-ANDROGEN ASSAY SEMINAL VENTRAL LEVATOR VESICLES PROSTATE ANI flo % ;60 '"... SO ii:!! o ~ ~ J Im9 5mg 10mg ~-DIIIYORO-NE + 5OOp9 T STOSTERONE & rf ~ mg ~-DIIIYORO-NE '9 TESTOSTERONE DAILY DOSE W ling ~-DIHYDAO-NE '9 TES70STERONE FIG. 2. Bioassay of 5a-dihydronorethindrone [22] for androgenic activity (A) and antiandrogenic activity (8). Assay procedures are described under "Materials and Methods." Bars and brackets denote means ± standard error. Asterisks above bars indicate treatment groups that are significantly different from the corresponding control group. The levels of statistical significance were determined by Student's t-test. *p < 0.001; **p < tational activity, and terminated pregnancy in rats, its hormonal activity was further investigated. In particular, androgenic and antiandrogenic activity ofthe compound was of interest since 5a-dihydronorethindrone is structurally related to androgens and because norethindrone [8] and R 2323 [19] are known to be androgenic. Furthermore, various progestogens' are antiandrogenic. Accordingly, 5a-dihydronorethindrone was tested for androgenic and antiandrogenic activity in the immature castrated rat. 5a-Dihydronorethindrone at 1, 5, and 10 mg/day was weakly androgenic as judged by the increase in wet weights of the seminal vesicles and ventral prostate (Fig. 2A). Moreover, the weight increases in both target organs were similar and submaximal, at the 5 and 10 mg doses, suggesting an impeded androgenic response. 5 a-dihydronorethindrone also displayed weak myotrophic activity as indicated by small, but significant, increases in levator ani weights. However, this compound exhibited no anti-androgenic or anti-myotrophic activity at doses ofl, 5, or 10 mg versus 500 p.g of testosterone per day (Fig. 2B). DISCUSSION By following a logical scheme of study, this preliminary paper establishes a rational basis for preclinical development of compounds that are antagonists of progesterone at the uterine receptor level. In vitro study alone cannot establish efficacy in this regard because binding affinity fails to discriminate between agonists, antagonists, and what may be called partial or incomplete agonists. In vivo studies, in part because of the high variability of most appropriate assays, have failed in the past to demonstrate the antagonistic effects of such well-studied compounds as norethindrone and its close relatives. The present study suggests that compounds related to testosterone and 19-nortestosterone, which bind to the progesterone receptor, may secondarily inhibit the full expression of a progestational effect. As with the various estrogen antagonists, 8 these substances have agonistic effects that differ from the natural substances. Typically, with respect to both the anti estrogens and the present group of progesterone antagonists, the blockers as agonists have dose-response curves with shallow slopes and produce less than characteristically maximal responses. For example, norethindrone 6 and R neither induce maximal arborization of endometrial glands in estrogenprimed, immature rabbits nor maintain pregnancy in spayed rats or rabbits. 12 The structural features and molecular interactions of various testosterone and 19-nortestosterone relatives that determine progesterone receptor affinity recently have been summarized by Janne et a1.,13 Lee et a1.,14 and Duax et al. 1S Therefore, these aspects are not considered further here. Most investigators, including ourselves, have usually noted a positive correlation between binding affinity and biologic activity. Deviations from this relationship have generally been attributed to differences in the pharmacokinetics of a given steroid with respect to that of progesterone. In most instances, binding affinity has been corre~ lated with progestational activity as judged by en-...

8 Vol. 31, No.5 COMPETITIVE PROGESTERONE ANTAGONISTS 559 dometrial proliferation in the estrogen-primed rabbit. With the exception ofr 2323 and certain of its relatives, antiprogestational and abortifacient activity have largely been ignored. The results of the Roussel-Uclafgroupll. 16 and those ofthe present study now amply demonstrate the mixed agonistic/antagonistic profile of 19-nortestosterone and a number ofits derivatives. In addition, at least one member of our series, 5a-dihydronorethindrone, was shown to behave as a competitive progesterone antagonist. Of the seven compounds tested for both progestational and antiprogestational activity, only one, 5a-dihydronorethindrone, did not show some degree of correlation between binding affinity and progestational activity. Although it had an RBA of 10%, this compound was virtually devoid of progestational activity in the McPhail assay. In support, Kontula et alp reported an RBA of 17% for 5a-dihydronorethindrone utilizing rabbit and human myometrial cytosols, and Edgren et ai.18 found the steroid to be inactive in both Clauberg and estrogen antagonist assays. Seventeen members of the testosterone and 19- nortestosterone series were tested for antiprogestational activity. Eleven, all of which were 19- nortestosterones, exhibited activity. Significantly, 5a-dihydronorethindrone inhibited progesteroneinduced endometrial proliferation in a dose-dependent manner; thus this 19-nortestosterone derivative had the characteristics of competitive progesterone antagonist. Five compounds, namely, 19-nortestosterone, 17,8-hydroxyestra-4, 9(10)-dien-3-one, norethindrone, norethindrone acetate, and R 2323 possessed both progestational and antiprogestational activity and therefore behaved as partial agonists/antagonists. The five remaining compounds were not tested for progestational activity and therefore cannot yet be precisely classified. 19-Nortestosterone, norethindrone, and norethindrone acetate terminated pregnancy in the rat and hamster, with rats being the more sensitive in each instance. Since these effects might be due to estrogenic activity, the daily EDso dose of 17 {3- estradiol for terminating pregnancy in rat and hamster was determined. When administered subcutaneously from days 7 through 12 in the rat or days 6 through 10 in the hamster, the daily EDso dose of 17 {3-estradiol was approximately 10 pg/ animal in both species. Therefore, the marked difference in sensitivity between the rat and hamster seems inexplicable on the basis of intrinsic estrogenicity of the 19-nortestosterones. This contention finds support in the recent work oftamaya et ai.,19 who employed rabbit uterine cytosols to determine the estrogen and progesterone receptor affinities of a small series of 19-nortestosterone derivatives. As indicated by the inhibitor binding constants, norethindrone, norethindrone acetate, and 5a-dihydronorethindrone had only weak affinity for the estrogen receptor, whereas their affinities for the progesterone receptor were nearly two orders of magnitude greater. Thus, although these particular 19-nortestosterones probably do not possess a significant degree of intrinsic estrogenicity, species differences in estrogenic conversion might underlie the differential sensitivity between the rat and hamster. The interruption of progesterone-maintained pregnancy in ovariectomized rats by testosterone and 19-nortestosterone also might involve estrogenic conversion. The failure of norethindrone to maintain pregnancy in ovariectomized rats and rabbits12 has been suggested to involve estrogenic conversion, since estrogens will block progestogens in this and various other tests. When a number of C-18 or C-19 steroids was incubated with a human placental aromatizing enzyme system or perfused through fresh human placenta, testosterone, 19-nortestosterone, and 17a-methyl-19-nortestosterone were aromatized to the corresponding phenolic products in a yield of 35% to 53%, whereas neither 17 a-ethynyltestosterone nor norethindrone was converted to 17 a ethynylestradiol to any measurable extent.20 It was suggested that the 17a-ethynyl side-chain prevents aromatization ofc-18 or C-19 steroids to estrogenic metabolites. Following oral administration of norethindrone to women, norethindrone itself, 17 a-ethynylestradiol, four ring A-reduced metabolites (3a, 5a; 3a, 5,8; 3,8, 5a; 3,8, 5,8), and an unidentified hydroxylated ring A-reduced metabolite were detected as urinary constituents by mass fragmentography.21 An additional intermediate (17,8-hydroxy-17 -ethynyl-5,8-estran-3-one) was found in the blood. 21 In view of the antiprogestational and abortifacient activity of 5 a-dihydronorethindrone, it seems possible that one or more of the ring A-reduced norethindrone metabolites might possess similar activities. Jones and Edgren22 previously found that norethindrone failed to produce vaginal keratinization (estrogenic response) in ovariectomized rats when administered parenterally, whereas it was effective when given orally. Apparently after oral administration norethindrone is transformed to biologically significant amounts of estrogenic metabolites in the

9 560 REELETAL. gastrointestinal tract or in the liver of rats. Since all compounds in the present series were given by the subcutaneous route, estrogenic conversion, as might occur following oral administration, cannot be invoked to explain our results. However, the effects of testosterone, 19-nortestosterone, and non-ethynylated 19-nortestosterones could still be mediated through conversion to their corresponding estrogenic metabolites by organs not necessarily involving the gut; 17 a-ethynylated C 1S and C 19 steroids might be expected to be less subject to this latter type of metabolism. 5a-dihydronorethindrone has the additional advantage of having a saturated ring A. Since mammalian tissues in vitro have not shown the capacity to perform the necessary metabolic steps for saturated ring A oxidation, ring A-reduced steroids should be resistant to aromatization. Though in vitro studies have shown that cultured gut flora can oxidize and aromatize ring A-reduced steroids, N aftolin et al.2 3 have been unable to find aromatized or aromatizable products following incubation of 5 a-dihydrotestosterone with bowel or bowel contents from adult male rats. It will be of interest to learn whether 5a-dihydronorethindrone, like norethindrone, undergoes estrogenic conversion after oral administration. In any case, 5a-dihydronorethindrone probably does not undergo significant aromatization after subcutaneous administration as in the present study. Hence, the antiprogestational and abortifacient properties ofthis compound are unlikely to be due to intrinsic estrogenicity or estrogenic conversion. As might be anticipated by its structural similarity to dihydrotestosterone, norethindrone, and R 2323, 5a-dihydronorethindrone displayed weak androgenicity, but lacked antiandrogenic activity. Since tesosterone lacked anti progestational activity at daily doses of 20 mg/rabbit, the weak androgenic activity of 5a-dihydronorethindrone could not account for its antiprogestational activity. The relationship between the androgenic and abortifacient activity of 5 a-dihydronorethindrone, if any, is not yet clear. However, data from the pregnant rat suggest that if classic androgenic activity were directly related to abortifacient activity, then testosterone should have been some 10 to 20 times more potent than 5a-dihydronorethindrone in terminating pregnancy. That this was not the case argues against a direct relationship between androgenic and abortifacient activity. Finally, assessment of the abortifacient activity of 5a-dihydronorethindrone in the hamster is of immediate interest since testosterone was inactive at May 1979 large doses in this species. A possible antiprogestational effect ofnorethindrone in humans has been reported by Seidl et al.,24 who attempted to salvage pregnancies in habitual aborters with newer progestogens. Of the eleven patients who received norethindrone, only one delivered normally at term, whereas there were one premature delivery, one obstetric accident, and seven abortions during therapy. (The remaining patient was still in therapy when the paper by Seidl et ap4 was published.) These, and similar data from other sources, led to the conclusion that norethindrone (and other 19-norsteroids with progestational activity) did not support human pregnancy. This conclusion and data on masculinization of fetuses led to the abandonment of this therapeutic approach. Such early studies were uncontrolled and the assumption of efficacy, or lack thereof, was based on calculations that purported to show a likelihood approximating zero that the fourth pregnancy after three spontaneous abortions would carry normally to term. However, Goldzieher and Benigno25 have shown these calculations to be erroneous and that a considerable proportion of such pregnancies will proceed to term. If correct, these data and calculations would suggest that an antifertility effect was exerted by the norethindrone; in particular it may be playing a role as an abortifacient, as would be predicted from our data. Since Seidl et al. 24 administered the drug well after the luteal-placental shift, norethindrone may be effective against placental progesterone. The biologic data, and the earlier clinical data, suggest that norethindrone, and particularly 5adihydronortestosterone, warrant reevaluation as possible abortifacients during early pregnancy. Acknowledg17J,ents. 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