OVER THE PAST DECADE a large number of new progestational compounds

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1 A Biological Classification of Progestational Agents RICHARD A. EDGREN, PH.D., ROBERT C. JONES, M.S., and DEANN L. PETERSON, B.A. The area of endocrinology concerned with progestational steroids is replete with controversy, as all who read this journal can avow. Even the concept of progestagens is not free from ambiguity. MAXWELL ROLAND 43 OVER THE PAST DECADE a large number of new progestational compounds has become available for use as therapeutic agents and as components of oral contraceptives. 2,50 Initially all such materials were simply characterized as "progestagens," but it soon became apparent that there were marked qualitative differences in their biological effects in laboratory animals. Whereas extensive studies of their clinical potency and incidence of side effects36 have been carried out, only recently has attention been focused on possible differences in their primary effects in women. 35, 42, 44 In an attempt to predict the clinical efficacy of new progestational agents, we have compared the activities of various steroids in a number of laboratory tests and assays. The resulting "spectra" of biological effects permit organization of new compounds into categories that parallel those of the natural, endogenous steroids. In the expectation that this classification may be of value to other endocrinologists, both clinical and experimental, we shall describe, document, and discuss these spectra in this communication. We shall largely restrict our considerations to data generated in our own laboratory or published earlier by the senior author and various associates. Although such a limitation will produce gaps in the spectra of certain compounds, it has the advantage of minimizing the broadly discrepant results reported by various laboratories, even when similar or presumably identical experimental protocols have been employed. From the Research and Development Department. Wyeth Laboratories, Inc., Philadelphia, Pa. 238

2 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 239 CHEMICAL STRUCTURES The chemical structures of the important natural sex steroids fall into two main categories that partially correspond to their primary biological effects (Fig. 1-2). Each of the estrogenic agents has an aromatic A-ring (i.e., a ring with 3 double bonds) and an hydroxyl group at carbon 3 of the 21 I 18 ~O... 12, II , C, 0, ' / I A I B I /7 4 6 Fig. 1. Steroid nucleus showing numbering system for carbon atoms and lettering system for rings. nucleus. Since each lacks an angular methyl grouping at carbon 10 (i.e., carbon 19 and associated hydrogen atoms), they are 19-norsteroids. The three primary estrogens differ among themselves in the nature and distribution of oxygenated groupings at carbons 16 and 17. Estriol differs from estradiol-17,b in having an hydroxyl group in the a-position at carbon 16, whereas estrone bears a ketonic function at carbon 17. Testosterone and progesterone are ~4-3-oxosteroids (i.e., they have double bonds between carbons 4 and 5 of the nucleus and ketones at carbon 3). Both compounds have angular methyl groupings at carbon 10. They differ in the nature of the groupings at the,b-position at carbon 17; testosterone has an hydroxyl and progesterone an acetyl group. The acetoxyprogesterone derivatives ( medroxyprogesterone acetate,,,/n" HO..()()" " ESTRADIOL -17/3,~, rb" o(x), TESTOSTERONE HO(J() "I rb ESTRONE f#.if' CH3.~~'''.' CH ~=o 3 t~fit 'Ii o CH3 AlP PROGESTERONE H ESTRIOL Fig.~. Struct\lral formulas for the important natural estrogens, testosterone, and progesterone.

3 240 EDGREN ET AL. FERTILITY & STERILITY chlormadinone acetate, and megestrol acetate) are acetic acid esters at the a-position of carbon 17 of 17 a-hydroxyprogesterone (Fig. 3). They differ among themselves in substitution and double bonding at carbon 6. Medroxyprogesterone acetate has a methyl group at the a-position of car- Q5P ~H3 CH3 C~?OH CH 3 CH3 o~o_l_'" PROGESTERONE o 17a-HYDROXYPROGESTERONE CH 3 MEDROXYPROGESTERONE ACETATE (Sa-methyl-17a-acetoxyprogesterone) ~H3 WCH3 C:?olcH3 CH 3 o.# ~ CI ~H3 WCH3 C~~O_~_CH3 CH 3 CHLORMADINONE ACETATE MEGESTROL ACETATE (S -chloro-s- de hydro- (S-methyl-S-dehydro- -1.7a-acetoxy progesterone) -17a-acetoxyprogesterone) Fig. 3. Structural fonnulas for progesterone, 17 a-hydroxyprogesterone, and three derivatives of 17 a-hydroxyprogesterone. o bon 6;megestrol acetate has a similar methyl group at carbon 6 and a double bond between carbons 6 and 7 of the nucleus. In chlormadinone acetate, the 6-methyl grouping of megestrol acetate is replaced by a chlorine atom. The remaining compounds are related to testosterone and 19-nortestosterone (Fig. 4). Ethisterone is an acetylenic derivative of testosterone ( 17 a-ethynyltestosterone ); 19-nortestosterone differs from testosterone in that carbon 19 (the angular methyl gl"oup at carbon 10) is missing, and norethisterone (norethindrone) is the 19-norsteroid corresponding to ethisterone in the natural series. Ethynodiol diacetate is the 3,17-diacetate of the reduced, or 3-hydroxy, form of norethisterone. Shifting the A-ring double bond from the 4-5 to the 5-10 position of norethisterone produces norethynodrel. Wy 3707, Wy 4355, and Wy 6343* are IS-homologated steroids related to 19-nortestosterone. Since IS-homologation (i.e., lengthening of the carbon chain at carbon IS) is difficult or impossible to achieve with steroid-starting materials from natural sources, these materials have Compounds manufactured by Wyeth Laboratories, Philadelphia, Pa. Although related to 19-nortestosterone, they are obtained by total synthesis and are not true derivatives. The first, Wy 8707, has recently been assigned the nonproprietary name "norgestrel." CH3

4 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 241 been prepared by total synthesis. 46 Wy 3707 is the IS-homologue of norethisterone; Wy 4355 is derived from Wy 3707 by substitution of chlorine for hydrogen in the ethynyl side chain at the 17 a.-position; and Wy 6343 is the 3-acetoxy derivative of Wy These steroids can exist in two distinct forms. Each of the natural materials has an absolute configuration comparable to that of estrone (the d enantiomer). The totally synthetic materials, however, are racemates composed of equal portions of 1- and d-enantiomers, which are mirror images of each other. Preliminary biological work with the resolved enantiomers has disclosed no biological activities for the l-enantiomers and has indicated that the d-enantiomers are twice as potent as the racemates. 20 BIOLOGICAL ACTIVITIES The biological spectra of the compounds under consideration, summarized in Table 1, will be discussed on a test-by-test basis. ehs ~' ~~~ CHS,~'. 0 0 TESTOSTERONE ETHISTERONE 19-NORTESTOSTERONE (17a-ethynyl-testosterone),095"" ~~~",091'" 0 CH,cO NORETHISTERONE NORETHYNODREL ETHYNODIOL DIACETATE (17a-.thyn~I-19-nortestosterone ) (17a-ethynyl-17-hydroxy- (17a-ethynyl-4-estrene- 5(f0)-estren-3-one) 3, 17- diol diacetate),095"" ~HS ~Hs fh],~'~',~"~' CHS~O WY 3707 WY 4355 WY 6343 (dl-bjf-ethyl-17e- (dl-17e-chloroethynyl- (dl-17cr-chloroethynylethynyl-17jf-hydroxyoon ethyl-1711-hydroxyoon - 13Jf-ethyloon-4-en-3-4-en-3-one) 4-en-3-one) 17Jf-diol-3-acetate) Fig. 4. Structural fonnulas for testosterone, 19-nortestosterone, and their various progestational derivatives.

5 242 EDGREN ET AL. FERTILITY & STERILITY TABLE 1. Comparative Progestational Androgenic Preg. main- Estrogenic Rat Fetal McGinty tenance Delayed ventral mabcu- Olauberg (intra- (Bpayed pa:rturi- Long- prob- liniza- Steroid (systemic) uterine) rat) tion Acute term tate tion Progesterone 100S (I)t 100S AS 100S I I I I Estrone I i I i 100S AS I Testosterone propionate? i i a:j: I I 100S AS Medroxyprogesterone 3500 acetate (3-5:1) A a 300- I I I a 1000 Chlormadinone 3500 a a acetate (3-5:1) Megestrol acetate 2500 A a i (1.5:1) Ethisterone A i a a N orethisterone 8 i I 100 I A 1.6 A (3-10:1) Norethynodrel? I I a 7 A I I Ethynodiol diacetate A a i Wy A A 1000 I I 7.5 A (3:1) Wy A A c3oo0 I I I (2:1) Wy ? I (1.5:1) Code: A-active; I-inactive;?-questionable; S-standard. Activities given in lower-case letters those reported by other laboratories. *Each numerical value given indicates potency in terms of appropriate standard steroid. tparenthetical evaluations, when indicated, are oral: parenteral ratios; i.e., a value of 3:1 indicates times as much must be given by gavage as subcutaneously to produce a given response. essentially inactive when given orally. :J:Tested as the free alcohol.

6 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 243 'vities of Various Steroids* A ntiestr{)genic Pituitary blockade Metrotrophic lifouse uterine growth Hemicasear Mouse Rat trated rat DOle anti- Anti- Anti- Anti- uterine uterine (ED,.., response Clatudo- Adrenal t10n6) estriol estrone androgenic growth growth p.g.) curve genic involution l00s I loos a A? A? 3600 Linear I A? I I a 100S A 18 V-shaped A A 70 A A A Linear A 290 a? I A A 130 a a? I a 60 A A 800 a A A 480 Linear A I I I 2 A 72 V-shaped A I a a 100 a 400 A >3300 I I A 760 Linear I I 11,000 A A I A 211 Linear A? I 2500 A A A 300 Linear

7 244 EDGREN ET AL. FERTILITY & STERILITY Progestational Effects Clauberg Test. This assay is the primary test for animal evaluation of progestagens, although the exact procedure may vary from laboratory to laboratory. Our procedure followed the protocol of Elton and Edgren. 23 Intact immature female Dutch belted rabbits, weighing about 1 kg. ( gm.) were primed daily for 6 days with estradiol-17,8 at a dose of 5 p,g. per day. On the day following the last priming injection, treatment with test compound was begun and continued for a total of 5 days. On the day after the final injection, the rabbits were sacrificed and a segment of the uterus removed for histological examination. Each uterus was scored according to the standard scale of McPhail,37 For reasons that currently are poorly understood, a sophisticated evaluation of slides from the Clauberg test permits a critical discrimination of the activities of various steroids that affect the endometrium. Elton 22 discussed endometrial observations in detail and defined two major categories of progestagens: 1. True progestins-compounds that produce maximal arborization of the endometrial glands (a high mean McPhail index) and low-columnar epithelial cells: e.g., progesterone, 17-acetoxyprogesterone and its derivatives, and various 17 a-substituted derivatives of 19-nortestosterone. 2. Progestagens with effects similar to those of mixtures of progesterone and estrone-those that produce submaximal glandular arborization and tall-columnar epithelial cells, with or without perinuclear vacuoles. There are 2 subclasses of this category: (1) compounds that mimic the effects of mixtures dominated by the progesterone component and produce no perinuclear vacuolization: e.g., 19-nortestosterone and norethisterone (17 a ethynyl-19-nortestosterone); and (2) compounds that act like estrogen alone or estrogen-dominated mixtures in producing marked perinuclear vacuolization: e.g., testosterone propionate and norethynodrel [17 a ethynyl-17 -hydroxy-5( 10) -estren-3-one]. Our own observations largely confirm those of Elton with respect to epithelial morphology and maximal degree of glandular arborization, although our McPhail readings tend to plateau at lower mean values than those he described: e.g., for "pure progestins," about +2 for norethisterone, about for norethynodrel, and < + 1 for testosterone propionate. In addition, the slope of the dose-response curve is quite steep for the "pure progestins" and very shallow for norethynodrel, norethisterone, and testosterone propionate. 21 We have chosen to base our quantitative estimates of potency in this test on the dose that produces a +2 McPhail reading. In Table 1, nor-

8 VOL. 18, l'ro. 2, 1967 PROGESTATIONAL AGENTS 245 ethynodrel and testosterone propionate are indicated as questionable because both materials failed to produce a +2 response by the subcutaneous route at doses up to 1 mg., our normal cut-off point. Estrone was inactive, and progesterone was defined as having a potency of 100%. By this criterion, norethisterone is a very weak progestagen (in agreement with Elton 22 ) and the acetoxyprogesterone derivatives were of a high order of potency. Wy 3707 was roughly intermediate in potency between progesterone and the acetoxyprogesterone derivatives, while the chloroethynyl derivatives of Wy 3707 were more potent than the parent compound. Despite the close chemical relationship of the synthetic materials to norethisterone, they are "pure progestins," having a steep slope of the dose-response curves and producing low-columnar epithelial cells. Since each of these materials is a racemate, the true potencies for the d-enantiomers, the active components, are double those cited in the table, bringing potencies for these materials into the range of those for the acetoxyprogesterone derivatives. Our own testing of ethynodiol diacetate was inadequate for quantitative evaluation; however, the compound is active. In addition to the potency estimates, Table 1 shows the oral:parenteral ratios for certain of the compounds. These ratios indicate the factor by which a subcutaneous dose must be multiplied in order to produce a response equivalent to that obtained by the oral route. The ratio for norethisterone is not comparable to those for the other materials, since the route of administration affects slope and plateau McPhail value. McGinty Test. 34 The McGinty test, which evaluates the intrauterine effects of progestagens, was employed as a secondary test for progestational effects. Virgin female white rabbits were ovariectomized and primed daily with 5 p,g. of estrone per day for 5 days starting 16 days after operation. On the day following the final estrone injection, test compound was introduced into the lumen of a I-inch segment of the uterus. Three days later the rabbits were sacrificed and the treated uterine segments removed for histologic evaluation according to the McPhail index. This test for progestational action is of particular interest in that such materials as norethisterone, norethynodrel, and testosterone propionate, which showed some degree of effect in the Clauberg test, were inactive by local administration. Progesterone and the acetoxyprogesterone derivatives were active, as were Wy 3707 and Wy 4355-despite their close chemical relationship to norethisterone. Pregnancy Maintenance. Spaying of pregnant rats is normally followed by abortion or resorption of the fetuses, although pregnancy may be maintained to term by the administration of adequate doses of progestational

9 246 EDGREN ET AL. FERTILITY & STERILITY agents. For this test we have followed the protocol of Saunders and Elton. Female rats were spayed on Day 8 of pregnancy (Day 1 is defined as the day sperm were found in the vaginal smear); daily supportive treatment was begun immediately and continued to autopsy on Day 18. At the time of ovariectomy the uteri were examined for implantation sites; non gravid rats were discarded. The presence of normal fetuses at autopsy was employed as an index of activity. Among the progestagens, progesterone and the acetoxyprogesterone derivatives were active, whereas norethynodrel and norethisterone were inactive; estrone and testosterone were also inactive. Wy 3707 and Wy 4355 were active, again indicating "true" progestational effects. Occasional positive reports for norethisterone 26 are apparently based on the persistence of placental remnants and resorbing fetuses in animals treated with the compound. Delay of Parturition. Various steroids will delay or prevent parturition in rats and other laboratory animals In our procedure, impregnated female rats were isolated from the breeding colonies, and administration of test compound was initiated on Day 15 of pregnancy and continued until the rats delivered or were sacrificed. Of 36 oil-treated control rats, 34 delivered litters on Day 22 or Day 23 of pregnancy, 1 delivered on Day 24, and 1 was infertile. For comparative purposes, therefore, we considered parturition on Day 25 of pregnancy, or later, to represent significant delay, whereas delivery on Day 24 was considered equivocal. Normally the rats were sacrificed for autopsy on Day 25. All of the progestagens tested in our laboratory have been active. Testosterone propionate 47 and norethynodrel were reported as active, and estrone was reported as inactive. 48 The totally synthetic steroids were the most potent progestagens, with Wy 3707 about 10 times more potent and Wy 4355 about 30 times more potent than progesterone. Medroxyprogesterone acetate was 3-10 times more potent than progesterone, and norethisterone was about equipotent. Estrogenic Activities Acute. Our definition of estrogen is restricted to materials that produce positive responses in some variation of the Allen-Doisy test for vaginal cornification in spayed rats or mice. Uterine growth tests are not qualitatively discriminating, since progestins and androgens are active. For the vaginal cornification test we have employed spayed adult female rats that had been primed every other week with estrone (2.5 p.g. per day for 2

10 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 247 days). Those rats that responded positively were used for testing during the alternate weeks. Two injections were given on 2 successive days, and smears were examined on the afternoon of the third and the morning of the fourth days.ll Progesterone, the acetoxyprogesterone derivatives, testosterone, ethisterone, and norethisterone, and Wy 3707 and Wy 4355 were inactive. Estrone was active. Norethynodrel had about 7% the potency of estrone, and ethynodiol diacetates had about 3%.25 Wy 6343 was equivocal. Long-term. When norethisterone is administered orally to spayed female rats, the character of the vaginal smear changes from mucified to typically estrogenic after 3-5 days of continuous administration. At autopsy, after 2 weeks of treatment, histological examination of the vagina reveals a typically estrogenic response, i.e., a sheath of ke"atinized epithelial cells lining the vaginal lumen. Animals that received norethisterone by the subcutaneous route showed typically mucified vaginas. These data suggest a conversion of the compound to an estrogenic material on oral administration. Estrone, norethynodrel, and norethisterone produce characteristically estrogenic vaginal responses, whereas treatment with progesterone, testosterone, medroxyprogesterone acetate, or Wy 3707 produces mucification or atrophy of the vaginal epithelia. Androgenic Effects Rat Ventral-Prostate Response. Various progestagens are androgenic, and androgenic side effects often complicate therapeutic use of these agents. For comparative and quantitative purposes we employed the growth response of the ventral prostate of young, castrated rats treated for 7 days according to the Hershberger protoco Data on seminal vesicles, although taken at autopsy, were not used because this organ responds to estrogens as well as to androgens and is less sensitive than the prostate. As expected, only testosterone propionate and the compounds related to 19-nortestosterone were markedly androgenic. Although progesterone and progesterone derivatives are often reported as having androgenic effects,6 these activities are trivial and would appear to be of little practical significance. Of the patently androgenic progestagens in the present series, Wy 3707 is the most potent; however, its high progestational potency more than compensates for this greater androgenicity. If the progestational: androgenic ratio of norethisterone is defined as 1, that for Wy 3707 is The chloroethynyl derivatives of Wy 3707 are nonandrogenic by this criterion, although Wy 4355 will stimulate growth of the seminal vesicles at massive doses.

11 248 EDGREN ET AL. FERTILITY & STERILITY Fetal Masculinization. Female fetuses of rats treated with various androgens and progestagens are distinctly virilized,38 and similar effects have been reported in humans by a number of workers.28 Adequate doses of testosterone, medroxyprogesterone acetate,41 ethisterone,49 norethisterone, or Wy 3707 will masculinize rat fetuses, although protocols often vary from laboratory to laboratory. David et al. reported that megestrol acetate is inactive, which is surprising in view of its close chemical relationship to the active medroxyprogesterone acetate.,\vy 4355 was inactive in this test. Early Androgen Syndrome. The administration of androgenic steroids to neonatal female rats produces a syndrome characterized by permanent sterility and persistent estrus, presumably secondary to anovulatory cystic ovaries. These gonadal abnormalities are believed to result from a failure of hypothalamic centers to produce the factors controlling luteinizing hormone secretion.1,31 It seems probable at this writing that all androgenic materials have an effect of this kind.30 Since estrogen,27 progesterone, desoxycorticosterone acetate, and cholesterol51 will also produce states of persistent estrus, the syndrome would appear to be a nonspecific response to many steroids and of questionable comparative significance (at least until differences among the effects of these agents have been elucidated). We have produced the typical "early androgen syndrome" with testosterone propionate and Wy Antiestrogenic Effects Mouse Vaginal Smear. For routine comparative antiestrogenic evaluations we employed a mouse vaginal smear test,9 Spayed adult female mice were selected at random from a colony. Each mouse received a total of 2 p.g. of estrone over a 4-day period, and test compounds were administered mixed with the estrone. Vaginal smears were examined on the afternoon of Day 5; mice in which estrogenic changes were noted were returned to the colony, and the remaining mice were re-examined on the morning of Day 6. Mice in which either smear was keratinized were scored as positive. Normally, % of mice receiving 2 p.g. of estrone show positive responses, but the incidence can be reduced to 0% with adequate doses of estrogen antagonists. Compounds were compared at the ED 5o, the dose estimated to reduce the expected estrogen-stimulated response by 50%. All of the progesterone, testosterone, and nortestosterone derivatives examined were active in this test. The totally synthetic materials were the most potent compounds of the group; norethisterone was less potent than the synthetics but appreciably more potent than the others. The proges-

12 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 249,terone and testosterone derivatives were relatively weak estrogen antagonists. Norethynodrel and, of course, estrone were inactive. Mouse Uterine Growth. ANTIESTRIOL. Estriol-induced uterine growth in immature mice is blocked by testosterone propionate but not by progesterone or the corticoids. 12 In this test 100 p,g. of estriol alone or mixed with test compound was administered over a 3-day period to intact mice days of age. On Day 4 the uteri were removed, cleaned, scored to express contained fluids, and weighed wet on a torsion balance. Wy 3707, Wy 4355, norethisterone, and testosterone propionate diminished the growth expected from the estrogen alone and were therefore active. Progesterone, estrone, and norethynodrel were inactive. ANTIESTRONE. When 0.3 p,g. of estrone was substituted for estriol, a broader range of compounds was active. The quantitative results in this test, based on the dose estimated to reduce expected uterine growth by 50%, closely paralleled those obtained in the vaginal smear test. 9, 13, 14 Again, norethynodrel and estrogen were inactive. Antiandrogenic Effects Estrogens and progestins are known to inhibit the growth responses of various organs to administered androgens. 7 Basing their work on this finding, investigators have employed various procedures' to test steroids for antiandrogenic effects; these procedures have included both topical and systemic administration to chicks for comb-growth studies, and oral and parenteral administration to laboratory rodents for study of the sex accessories. Although too few data are available for critical comparisons, 5 mg. of Wy 3707 failed to prevent the accessory-gland growth induced by 25 p,g. of testosterone in a modified Hershberger test. Metrotrophic Effects Uterine growth in laboratory rodents is not a specific indication of estrogenic activity, as is suggested by the growth induced by androgens and progestational agents. We employ two separate protocols to determine metrotrophic effects. In the first, immature intact female mice received injections of test drug for 3 days and were sacrificed on Day 4. Uterine growth responses were induced by estrone, testosterone propionate, norethisterone,s norethynodrel,s and ethynodiol diacetate. 25 The dose-response curves for testosterone propionate and norethisterone were extremely shallow, thus differing from the typically estrogenic curves of estrone, norethynodrel, and ethynodiol diacetate. Progesterone, Wy 4355, and the acetoxy-

13 250 EDGREN ET AL. FERTILITY & STERILITY progesterone derivatives3 produced a slight, irregular growth of the uterus that was of questionable comparative value. Wy 3707 seemed devoid of such effects. In the second procedure, spayed immature female rats received compound for 3 days and were sacrificed on Day 4. Testosterone propionate produced a typically estrogenic dose-response curve,15 although high dosage levels were required. Similar growth effects were observed with estrone, norethisterone, norethynodrel, Wy 3707, and Wy Again, some irregular uterine growth was stimulated with massive doses of progesterone16 and its derivatives. Pituitary-Blocking Effects Removal of one ovary from an animal is normally followed by "compensatory" hypertrophy of the remaining gonad.39 Although the mechanism of this hypertrophy is not well defined, its prevention by steroids is believed to rehect inhibition of gonadotrophin secretion by the hypophysis. IS Compounds were assayed by estimating the amount necessary to prevent completely (ED1oo ) the hypertrophy obtained in adult female rats 2 weeks after operation. In this test, estrogens (e.g., estrone) were extremely potent materials, whereas progesterone was exceedingly weak. The other materials fell between these extremes. The shapes of the dose-response curves were also instructive. Delta-4-3-oxosteroids produced simple, linear dose-response curves that continued to fall above the EDlOo level; estratrienes, however, had V-shaped dose-response curves that rehected at about the EDloo level and showed a secondary rising cycle. The norethynodrel curve was of this latter, estrogenic type. Doses of chlormadinone acetate up to 1 mg. were totally ineffective in this test; medroxyprogesterone acetate, although not quantified, appeared to be active. Ethynodiol diacetate, Wy 3707, and Wy 4355 were active. Claudogenic Effects The administration of various steroids to pregnant rodents will terminate the pregnancy. In rats, pregnancy is particularly sensitive between the time of mating (Day 0) and the completion of implantation (about Day 7). Petrow's term «claudogen"40 is applicable to active materials, although a number of different mechanisms are unquestionably involved. To assess claudogenic activity we employed the following protocol.19 Adult female rats (Charles River Breeding Farms) were bred in colonies; treatment was begun on the day sperm were found in the vaginal smear (Day 1) and continued until Day 7. The rats were killed for autopsy on Day 14 and

14 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 251 examined for normal fetuses. Progesterone and Wy 3707 were inactive in this test, while estrone, testosterone propionate, norethisterone, norethynodrel, and ethynodiol diacetate 25 were active. Preliminary tests with Wy 4355 suggested activity, but the results were equivocal. Adrenal Involution Acetoxyprogesterone derivatives of various types cause atrophy of the adrenal glands of spayed female ratsp,24 Inconsistent effects have been obtained with progesterone, but occasional atrophy has been seen,n and Brennan and Kraay reported adrenal hypertrophy in male rats that received progesterone and chlormadinone acetate. In spayed females, clear adrenal involution has been reported with medroxyprogesterone acetate,n.24 chlormadinone acetate,3 and megestrol acetate.24 The data available on the remaining compounds failed to reveal atrophy of the adrenal gland. DISCUSSION The data we have presented define for each progestagen a spectrum of activities that may be compared with spectra for such natural steroids as progesterone, estrone, and testosterone propionate. For example, if one defines a "true" or "pure" progestagen as a compound having an activity spectrum similar to progesterone, then, within the limits of available data, the acetoxyprogesterone derivatives, chlormadinone acetate, megestrol acetate, and medroxyprogesterone acetate (except for fetal masculinization) must be considered in this category, although all of these steroids produce a more marked adrenal involution than does progesterone. The data on ethisterone, although fragmentary, suggest a material that is largely androgenic, but that has Clauberg effects that may differentiate it from testosterone. Norethisterone, in acute testing, also appears to be largely androgenic in biological spectrum; however, its marked activity in the Clauberg test differentiates it from testosterone. Moreover, norethisterone appears to be converted significantly to estrogen when administered orally. The activity spectrum for norethynodrel is virtually identical to that of estrone, although again a progestin-like effect is seen in the Clauberg test. Wy 3707 appears to be a "true" progestagen, but its activities are complicated by the retention of some androgenic effects and perhaps by the antiestriol effect demonstrated in the mouse uterine growth test. The chloroethynyl derivatives Wy 4355 and Wy 6343 are, except for their antiestriol effects, "true" progestagens.

15 252 EDGREN ET AL. FERTILITY & STElULITY Ethynodiol diacetate is peculiar in that it has properties of progesterone, testosterone, and the estrogens; the estrogenic effect may result from rapid in-vivo conversion. It would be disingenuous at this time to try to fit ethynodiol diacetate into any fixed grouping. We feel that the following categories define the biological spectra of these materials: (1) «true" progestagens: progesterone, chlormadinone acetate, megestrol acetate, Wy 4355, and "Ty 6343; (2) «true" progestagens with androgenic effects: medroxyprogesterone acetate and Wy 3707; (3) androgens with progestational effects: ethisterone and norethisterone; (4) estrogens with progestational effects: norethynodrel; and (5) progestagens of uncertain position: ethynodiol diacetate. Correlation of these biological categories with clinical potency and efficacy is difficult at the present time. Swyer and Little quantified these materials in a delay-of-menses test and Mears evaluated certain of them as contraceptives (Table 2). Little direct correlation is apparent between these clinical effects and the biological spectra, although the single «estro- TABLE 2. Some Clinical Effects of Various Progestagens ED" for delay of menses Effect (0/0 of patients) (mg./day) Spotting BTB* Decreased Progestational With Dose (all (all A menor- menstrual compound Alone estrogen (mg.) cycles) cycles} rhea flow "True" Megestrol > t With androgenic effects Medroxyprogesterone > Wy Androgens N orethisterone :\: Estrogens Norethynodrel (Enovid-E) :\: t Of uncertain position Ethynodiol >4 1.5 l:j: Data on delay of menses are from Swyer and Little ;50 on clinical effects, when given as contraceptive, from Mears." *Breakthrough bleeding. twith ethynyl estradiol 0.05 mg. :j:with mestranol 0.1 mg.

16 VOL. 18, No.2, 1967 PROGESTATIONAL AGENTS 253 gen with progestational effects" (norethynodrel, as Enovid-E*) had the highest incidence of breakthrough bleeding. The higher dose form (Enovid*) was better in this respect,3g but the incidence seemed higher than one would anticipate from the results with the other materials. Unfortunately, contraceptive studies evaluate commercial products rather than the drugs they contain, and the product dosages may not represent optimal amounts for clinical use. Unfortunately, too, the expense involved in field trials preclude intensive exploration of broad dose ranges. Roland et al. recently attempted a classification of progestagens based on the human endometrial response. Their groupings cut across the biological spectra, since they reported similar maximal effects with medroxyprogesterone acetate, norethynodrel, norethisterone (as the acetate), and ethynodiol diacetate, but not for chlormadinone acetate. Again, whether the doses employed were optimal is uncertain, and patient control cannot be as rigorous as that for laboratory animals. Klopper attempted a classification of progestagens based on both chemical structure and such clinical parameters as uterine hemostasis and histological changes and antiovulatory effects. His four classes were: (I) testosterone derivatives (ethisterone and dimethylethisterone); (II) 19-nortestosterone derivatives (e.g., norethisterone and norethynodrel); (III) 17 tx-hydroxyprogesterone and its derivatives (e.g., 17 tx-hydroxyprogesterone acetate and medroxyprogesterone acetate); and (IV) progesterone (e.g., retroprogesterone). Klopper categorized the nortestosterone derivatives as producing ovulation blockade, atypical endometria, and marked hemostasis but he did not discuss his reasons for isolating the testosterone derivatives. Nor it is clear from his abstract why he separated the hydroxyprogesterone and progesterone derivatives. Again, the biological spectra of these compounds do not correspond to this classification. Maqueo et at. compared the endometrial effects of four progestagens through a single 20-day cycle and found that in spite of the major chemical differences between chlormadinone and the two 19-norsteroids, the effects of 20-day treatment were very similar. Kistner discussed differences in clinical effects of several progestagens, and his evaluations (Table 3) show a crude correlation with our biological classification. Progesterone and medroxyprogesterone acetate were apparently more effective during pregnancy than was norethynodrel or norethisterone, whereas the latter two compounds were better contraceptives. The norsteroids also appeared to be of somewhat greater value in endometriosis -G. D. Searle & Co., Chicago, Ill.

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