United States Patent (19) 11) Patent Number: 4,937,238 Lemon 45 Date of Patent: Jun. 26, 1990

(54) PREVENTION OF MAMMARY Chemical Abstracts; vol. 100 (1984) #186065M; Dehen CARCINOMA ninet al. Chemical Abstracts; vol. 101 (1984) #488096; Leclercq 75 Inventor: Henry M. Lemon, Omaha, Nebr. et al. 73) Assignee: The Board of Regents of the Chemical Abstracts; vol. 101 (1984) #48830b; Katzenel University of Nebraska, Lincoln, lenbogen. Nebr. Primary Examiner-Joseph A. Lipovsky Attorney, Agent, or Firm-Vincent L. Carney (21 Appl. No.: 253,358 57 ABSTRACT (22 Filed: Sep. 30, 1988 To reduce the risk of breast cancer, a drug is periodi cally administered to young mammals before pregnancy Related U.S. Application Data in doses of about 1 microgram to 50 micrograms per 63 Continuation of Ser. No. 836,104, Mar. 4, 1986, aban kilogram of body weight. The drug: (1) competes with doned. and displaces 17beta from the mammary gland cells in an effective manner to prevent the possible 51) int. C...... A61 K31/56

formation of DNA-damaging epoxide estradiol metaboo 52 U.S. C...... 514/178; 514/182 lites; (2) binds to breast tissue to a greater extent than 58) Field of Search ...... 514/178, 182 estradiol 17 beta; (3) induces terminal nonlactation dif ferentiation of the mammary gland; (4) is nontoxic and (56) References Cited noncarcinogenic; and (5) preferably does not cause U.S. PATENT DOCUMENTS anti-ovulatory activity. The drug is selected from a 3,868,452 2/1975 Kraay et al...... 514/182 group of drugs including: (1) 4-OH estradiol; (2) d ; and (3) 17 alpha ethynyl . OTHER PUBLICATIONS The Merck Index; 10th Ed. (1983); #3579, Equilenin. 5 Claims, No Drawings 1. 4,937,238 2 It is a still further object of the invention to provide a PREVENTION OF MAMMARY CARCINOMA novel method of reducing the risk of breast carcinoma using a drug with minimum undesirable effects. This application is a continuation of application Ser. It is a still further object of the invention to provide a No. 836,104 filed Mar. 4, 1986, now abandoned. 5 method for reducing the risk of breast cancer through the use of a drug which does not inhibit ovulation, BACKGROUND OF THE INVENTION uterine hypertrophy, or body growth. This invention relates to drugs for reducing the risk It is a still further object of the invention to provide a of breast cancer and methods of using them. method of reducing the risk of breast cancer through It is known that the risk of breast cancer is related to O treatment with a drug not having cell transforming pregnancy and that certain compounds affect activity or exhibiting damage to DNA. the risk of cancer. Such compounds have been adminis In accordance with the above and further objects of tered to mice and to women. the invention, a hormone or a drug which creates a One compound that has been tested is estriol as de hormone is induced in young female mammals in a scribed in "Estriol Prevention of Mammary Carcinoma 15 dosage which competes with and displaces cell trans Induced by 7, 12-dimenthylbenzanthracene and Procar forming hormones to prevent the formation of DNA bazine' by Henry M. Lemon, Cancer Research, 35, damaging epoxides from hormone metabolites and in 1341-1353, May 1975. Estriol, a naturally occurring duces pre-lactational terminal differentiation of the major human pregnancy hormone, was shown in that mammary gland in sufficient quantities to reduce the article to be moderately effective in inhibiting the de 20 incidence of carcinoma in undifferentiated cells which velopment of breast cancers induced by a carcinogenic might otherwise be caused by other natural occurring chemical. hormones. The drug is administered in quantities which Two other compounds and their method of use were do not have undesirable effects on the mammal such as described in "Pathophysiologic Considerations in the the inhibition of ovulation or inhibiting of growth. Treatment of Menopausal Patients with Oestrogens; the 25 Preferably, the drug is one with long retention time Role of Ostriol in the Prevention of Mammary Carci within the breast tissue at the effective dosage. Small noma’ by Henry M. Lemon, Acta Endocrinologica, doses are applied between 1 ug (micrograms) for each 1980, Suppl. 233:17-27. Those drugs are 6 keto estradiol kilogram of body weight and 50 ug for each kilogram of and estriol 3-methyl ether. They were shown to have a body weight between the ages in humans of 15 years to 30-50 percent lifetime effect in reducing the appearance 30 20 years. of breast carcinoma in treated rats as compared to other As an active ingredient, the drug is selected from a rats receiving carcinogen at the same time. group of drugs including: (1) 4-OH estradiol; (2) d In one test: (1) 26 percent of the rats developed can equilenin; and (3) 17 alpha ethynyl estriol. The most cer in 7 months and 63 percent in one year when treated satisfactory of these drugs is believed at this time to be with estriol and a carcinogen; and (2) 31 percent of rats 35 17 alpha ethynyl estriol. The drugs d-equilenin and developed cancer in 7 months and 61 percent in one 4-OH estradiol may be conjugated with 17 alpha ethy year when treated with 6 keto estradiol. Several other nyl to increase their effectiveness and/or reduce the related drugs tested at the same did not provide the dose. same beneficial results. At first testing, 17 alpha ethynyl estriol appeared not In tests with the other drugs the rats: (a) were treated 40 to have anti-mammary carcinogenic activity but in this with the drug and the carcinogen; and (b) examined for test the 17alpha ethynyl estriol was administered within percentage incidence of cancer at 7 months and one 2 to 3 days of administering a carcinogen to the same year. Some of the other drugs in the test and their re rats and it is believed that the ethynyl estriol provided sults were: (1) with 78 percent incidence in 7 stimulation of cell proliferation during the time of the months and 89 percent in one year; (2) 45 maximum damage by the carcinogen resulting in in with 88 percent incidence in 7 months and 100 percent creased cancer. Subsequent testing which avoided the in one year; (3) 17 alpha ethynyl estradiol 17 beta with large scale proliferation of DNA damaged cells has 90 percent in 7 months and 100 percent in one year; and shown ethynyl estriol to be effective in rats. (4) 2-OH estradiol 17 beta with 78 percent in 7 months As can be understood from the above summary of the and 94 percent in one year. 50 invention, the method of treatment and drugs of this The three drugs which were effective and the meth invention have several advantages, such as: (1) they ods of treatment using them have the disadvantages of reduce the risk of esdridial dependent mammary carci low effectiveness and/or toxicity. noma; (2) they do not inhibit body growth; (3) they do It is known to increase retention in uterus tissue by not inhibit ovulation; (4) they do not stimulate conjugating drugs with 17 alpha ethynyl which has a 55 induction of secretory activity in the mammary gland; high rate of retention in uterus nuclei. Moreover, estro (5) they have a high maturation activity for immature gen, 11-beta methoxy 17alpha ethynyl estradiol 17beta, mammary glands; and (6) they do not cause uterine which is retained in breast tissue, is known in biochemi hypertrophy. cal studies of breast cancer tissue. However, this estro gen has the disadvantage of being carcinogenic in ani DETALED DESCRIPTION mals. It is also known that some cause cell Broadly, the drug is a hormone or compound which transformation in tissue culture. causes production of the hormone in mammals, includ ing humans, which hormone or compound: (1) does not SUMMARY OF THE INVENTION transform cells into malignant cell clones; (2) competes It is an object of the invention to provide a novel 65 with and displaces cell-transforming hormones; (3) pre method for preventing breast cancer. vents the formation of DNA-damaging epoxides from It is a further object of the invention to provide a hormone metabolites; (4) induces prelactational termi novel drug for the prevention of breast cancer. nal differentiation of the mammary gland; (5) is retained 4,937,238 3 4. for a substantial amount of time in breast tissue; and (6) administered in sufficient quantity to'compete with and has minimum undesirable effects. Preferably it should not induce lactation when administered in the quantities displace hormones which are likely to induce cell trans used to reduce the risk of cancer. formation and their metabolites, and to increase the More specifically, the hormone should: (1) compete 5 speed of differentiation of the mammary gland cells but with and displace estradiol 17 beta from the mammary in small enough quantities to avoid undesirable effects. gland cells in an effective manner to prevent the possi ble formation of DNA-damaging epoxide estradiol me tabolites; (2) bind to breast tissue to a greater extent than O R6 R FORMULA estradiol 17 beta; (3) induce terminal differentiation of the mammary gland; (4) be nontoxic and noncarcino genic; and (5) preferably not have anti-ovulatory activ ity. The treatment may also be used in larger quantities as a combined cancer preventative means and contra ceptive. The drug is selected from a group of drugs 15 including: (1) 4-OH estradiol; (2) d-equilenin; and (3) 17 alpha ethynyl estriol. The drug is preferably 17 alpha ethynyl estriol. Suitable compounds: (1) have the general formula shown in formula 1 where R2 is a hydroxy group, R8 is an oxygen function comprising a 16 alpha hydroxy R6 O FORMULA2 group or a keto group (=0), R1, and R9-R11 are hy droxy groups or nonreactive hydrogen groups and R7 is a methyl group; or (2) have the general formula shown in formula 2 where R2 is a hydroxy group and R1 and 25 R9-R11 are hydroxy groups or nonreactive hydrogen groups, R7 is a methyl group, and R8 may be a 17 beta hydroxy group. In either formula 1 or 2: (1) R8 can be a 17 alpha hydroxy group and (2) R4 is a non-reactive hydrogen. The general structures of formulae 1 and 2 will resemble the peripheral structures of ethynyl es FORMULA 3 triol, d-equilenin, and 4-OH estradiol. Formula 1 identi fies the four rings by using a convention that will be followed throughout this specification. To provide significant cancer inhibition, a proper 35 dose is between 2 ug (micrograms) each 24 hours for 28 days and lug each 24 hours for 84 days. A single 638 ug dose provides a full year of protection, as does the 1.0 ug/24 hour dose, something not yet demonstrated as OH FORMULA 4 well for any other estrogen. CH3 Five specific suitable active ingredients are: (1) 17 alpha ethynyl estriol shown in formula 3; (2) 4-OH estradiol 17 beta shown in formula 4; (3) d-equilenin shown in formula 6, (4) 17alpha ethynyl 4-OHestradiol 17 beta; and (5) 17 alpha ethynyl d-equilenin. 45 Some of the specific compounds which are the active HO ingredient in the dose administered to patients for pre OH c vention of mammary carcinogenesis are known com pounds and are not part of this invention in general. FORMULAS Ethynyl estriol is obtainable from Eli Lilly, Inc., Green field, Ind., and the other estrogens from Sigma, Inc., St. Louis, Mo., or Steraloids, Inc., Wilton, N.H. Generally, the compounds are estrogens which have an additional oxygen function (EO, or -OH) on the bottom and right side of the molecule (R1, R2 or D 55 ring) and R8-R9 or A ring of the molecules, or would be expected to undergo selective oxidation at this part of the molecule in the tissues in vivo. For example, (1) estriol, ethynyl estriol, 4-OH estradiol, 6-keto estradiol O FORMULA 6 have this oxygen function; (2) can only be 60 oxidized in vivo at the 4C position, since the ethynyl group blocks 16 beta oxidation, and the 11 beta methoxy group blocks 2-hydroxylation and R3; and (3) d-equilenin has been shown to be oxidized in vitro by tissues almost exclusively at the C4 (R1) position. 65 Young virgin female mammals have the drug of this HO invention administered to them either in one large dose OH or periodically to reduce the risk of breast cancer. It is 4,937,238 6 The inventidin is illustrated by the following non FORMULA 7 limiting examples: EXAMPLES General In tests for anti-mammary carcinogenic activity fol lowing the feeding of carcinogen to mammals, ten dif ferent estrogenic compounds were implanted in female O rats fed a carcinogen. The incidence of breast cancer for each compound was determined. Rats receiving only OH FORMULA 8 the carcinogen were used as controls. Ethynyl estriol CH was obtained from Eli Lilly, Inc., Greenfield, Indiana; the other estrogens were obtained from Sigma, Inc., St. 15 Louis, Mo., or Steraloids, Inc., Wilton, N.H. Rats and their Maintainence

HO In all of the tests, intact female Sprague-Dawley rats, obtained from Sasco, Inc., Omaha, Nebr., at 50 to 55 OH O 20 days of age were used. FORMULA 9 They were: (1) randomized upon receipt; (2) housed individually in suspended cages; (3) fed Purina Lab Chow and distilled water; and (4) the temperature was 25 maintained within 22-23 degrees and 40-50 percent relative humidity. Formation of Pelets The hormones were formed into pellets weighing OH FORMULA 10 30 6.38SD mg (milligrams) having 1 millimeter diameters C3H and being between 1 and 2 millimeters in length, gener ally being cylindrical in shape. Each pellet contained 5-10 percent of the hormone to be administered in crys HO talline sodium chloride w/w (The weight of the hor 35 mone was 5-10 percent of the total weight of the pel lets). The pellets were compressed into solid form using HO a Forbes manual pellet press as described in Forbes, T. HsC2 or FORMULA 11 R., “Absorption of Pellets of Crystalline , C , Methyl Testosterone, Proges 40 terone, Desoxycorti-costerone, and Im NoSna planted in the Rat." Endocrinology, 29: 7071, 1941. Hormones HO CHs 45 The hormones used in examples 1-10 are shown in CH3 FORMULA 12 formulas 3-12 respectively and are: (1) 17alpha ethynyl O-meC-C-N estriol shown in formula3; (2) 4-OH estriadiol 17beta shown in formula 4; (3) 11 beta methoxy 17 alpha ethy Nos nyl estradiol 17 beta shown in formula 5; (4) d-equilenin 50 shown in formula 6; (5) estriol shown in formula 7; (6) 6-keto estradiol 17 beta shown in formula 8; (7) 17 ethy nyl estradiol 17 beta shown in formula 9; (8) 2-hydroxy estradiol 17 beta shown in formula 10; (9) diethylstilbese trol shown in formula 11; and (10) tamoxifen shown in 55 Generally, drugs which compete with and displace formula 12. The ethynyl estriol was obtained from Ely estradiol 17beta from mammary gland cells and prevent Lilly, Inc., Greenfield, Ind., moxestrol was obtained the possible formation of DNA-damaging epoxides from Southwest Foundation for Biomedical Research from estradiol metabolites are used in sufficient quanti in San Antonio, Tex., and the remainder of the hor ties to increase pre-lactational differentiation of the mones were obtained either from Sigma, Inc., St. Louis, post-pubertal mammary gland cells. They are adminis Mo. or Steraloids, Inc., Wilton, N.H. tered to female humans at ages younger than 20 and Carcinogen prior to pregnancy, usually in periodic doses. This treat The carcinogen DMBA (7, 12-dimethylbenz (a) an ment may be most useful soon after (within five years in thracene) was obtained from Eastman Kodak Co., the case of humans) the onset of menarche and the date Rochester, N.Y., and was dissolved in sesame oil imme of pregnancy in doses and quantities too small to induce diately before administration, in a concentration of 20 lactation or toxicity. mg for each 1.5 ml of oil for each dose. 4,937,238 7 8 biopsied at necropsy to obtain evidence of estrogenic Administration of DMBA stimulation of the virgin ductal system. Significant dif DMBA was administered under light etheranesthesia ferences in cancer incidence between treated and con using a flexible 12 cm garage tube. trolled groups were determined. Administration of Hormones Calculations of Differences in Cancer Incidence Hormonal treatment of one-half to two-thirds of each The differences in cancer incidence between treated batch of age-matched rats was begun 14-21 days prior and controlled groups in each experiment were deter to DMBA therapy or 13-14 days following DMBA mined by X2 using the Yate's correction. When the therapy. Two methods were used, hereinafter referred 10 number of rats was small, the Fisher exact probability to as: (1) pellet method; and (2) minipump method. test for four-fold tables with cells containing less than five rats was used. The significance of difference in Pellet Method mammary-carcinoma-free survival in rate-days was Pellets were implanted monthly, subcutaneously. The determined for these groups at 210 days after DMBA dynamics of release from such pellets has been de- 15 administration using the Mann-Whitney U Test. Differ scribed by Forbes, T. R. in "Further Observation on the ences in mean values of uterine weights were evaluated Relative Absorption Rates of Pellets of Various Crys using Students t test of significance. talline Compounds Implanted Subcutaneously in Rats, “i Endocrinology,' 30:761-769, 1942. EXAMPLE ONE Minipump Method 20 Ethynyl Estriol Alzet Osmotic Minipumps from Alzet Corporation in The ethynyl estriol was administered either two to California were s.c. (subcutaneously) implanted in each three weeks before DMBA feeding or two weeks after. rat and the designated hormone dose was pumped con The results of this treatment are shown in Tables 1-5, 9 tained in a vehicle composed of 5 percent ETOH, 25 25 and 10. percent distilled water and 70 percent propylene glycol. EXAMPLE TWO Experiment Design Tamoxifen The randomized rats were divided into groups of Tamoxifen was administered in doses of 1.5 to 2.5 mg 4-15 rats each and each group was assigned to a differ- 30 each month starting 13 to 14 days after feeding DMBA. ent one of: (1) untreated controls; (2) carcinogen-treated The results of this treatment are shown in Table 6. rats; and (3) carcinogen-untreated rats. The rats were examined for neoplasm every 10 to 14 EXAMPLE THREE days after feeding DMBA. At least one mammary neo Diethylstibestrol plasm was verified pathologically in each tumor-bear- 35 Diethylstibestrol was administered in doses of micro ing rat which was killed at the time of biopsy. Wet and grams each month starting 13 to 14 days after feeding dry uterine weights were obtained at necropsy. DMBA. The results of this treatment are shown in Rats free of neoplasms were followed for one year. Table 6. At the end of the one year, the remaining rats were necropsied. Non-neoplastic mammary glands were also

TABLE 1. INHIBITION OF MAMMARY CARCINOGENESIS BY ETHINYL ESTRIOL PROR TO CARCINOGEN Mammary Carcinoma Incidence CONTROLS EHDNY ESROL Ratio of rats with Ratio of rats with carcenogenesis to carcenogensis to SIGNIFICANT Treatment total rats observed total rats observed OFFERENCE Dosage and time of after after after after U X2 administration 210 days 365 days 210 days 365 days method method 638 ug monthly for 7 months. 8/10 9/10 O/8 1/8 <001 a.001 starting at 35 d (days) of age (14 days before DMBA) 638 ug implanted at 50 7/11 1/13 S/3 <05 role days of age (21 days before DMBA) continuous infusion start ing at 35 days of age (15 days before DMBA) 2 ug/24 h (hrs) x 28 d A12 M12 6/12 <.02 6 ug/24 h x 28 d Sv12 <001 4,937,238 9 10 TABLE 2 INHIBITION OF MAMMARY CARCINOGENESIS BY ETHNYL ESTROL TWO WEEKSAFTER CARCINOGEN Mammary Carcinoma Incidence CONTROLS ETHINYLESTROL Ratio of rats with Ratio of rats with carcenogenesis to carcenogensis to SIGNIFICANT Treatment total rats observed total rats observed DIFFERENCE Dosage and time of after after after after U X2 administration 210 days 365 days 210 days 365 days method method 638 ug monthly X 19 9/12 10/12 Ov 9/11 <.001 Ole 638 ug monthly X 7 14/14 14/14 1/5 5/15 <.001 <.02 continuous infusion 9/12 12/12 4/9 5/9 none <.025 1 ug/24 h x 84 d 638 ug implanted once 7/11 1/12 <.05

TABLE 3 UTERNE WEIGHTS OBTANEd AT THE TIME OF NECROPSY FOR NEOPLASMS (IN GRAMS) - HORMONE-TREATED CONTROLS - - Number Number Significant Treatinent of Rats Wet Uterine Weight of Rats Wet Uterine Weight Difference monthly EE3 pellet 7 452.07 15 526.139 Oe implants monthly EE3 pellet 4 .513 .076 10 530.119 One implants single EE3 pellet 5 .66 .122 9 547,159 One at age 50 d. - 2 ug/24 h infused 8 471 .136 12 534.124 none 28d 6 ug/24 h infused 7 . .546.090 12 534.124 One 28 d.

TABLE 4 UTERINE WEIGHTS OBTAINED AT THE END OF HORMONAL STIMULATION HORMONE-TREATED CONTROLS Number Number Significant Treatment of Rats Wet Uterine Weight of Rats Wet Uterine Weight Difference 1.2 ug/24 h. .426 452 -6 infused 28 days 2.0 ug/24 h. 2 502 2 .34 --47 infused 28 days 3.4 ug/24h 1 .556 1. 323 --42 infused 28 days 6.0 ug/24 h. 2 592 2 .341 --74 infused 28 days 6.0 ug/24h 1 .683 1 339 - 101 infused 28 days o 2.0 ug/24h 2 791 2 341 - 132 infused 28 days 6.0 ug/24 h 2 925 2 .341 -- 172 infused 28 days

TABLE 5 UERINE WEIGHTS OBTANED A THE END OF HORMONAL STIMULATION HORMONE-TREATED CONTROLS Number Number Significant Treatment of Rats Wet Uterine Weight of Rats Wet Uterine Weight Difference 30 dafter 467 ug 1 589 .346 --70 S.C. 60 dafter 645,469 604 1. 620 -3 ug s.c. each month 60 d after 615,577 1 636 l .393 -62 untreated females 4. .423 c.102 80 days of age (all of the above variations in weight are within the normal spontaneous weight variations plus or minus 3 times the standard deviation XSD) 4,937,238 11 12 TABLE 6 INFLUENCE OF OHERESTROGENS AND OF THE ANTESTROGEN TAMOXFENUPON DMBA-INDUCED MAMMARY CARCINOGENSIS (ALL TREATMENT INITIATED 13-14 DAYS AFTER FEEDING DMBA) Mammary Carcinoma Indicidence CONTROLS REATED Ratio of rats with car- Ratio of rats with car cenogenic to total rats cenogenic to total rats and percentage with and percentage with SIGNIFICANT Treatment carcenogenic: carcenogenic: OFFERENCE Dosage and time of observed observed observed observed U X2 administration for 7 mos. for 1 year for 7 mos. for 1 year method method Tamoxifen 18/19 14/18 16/18 ore Oc 1.5-2.5 mg/mo (month) (95%) (78%) (89%) Diethylstilbestrol 10/10 7/8 8/8 ce One 320 ug/no (100) (88) (100) 7a ethynyl estra- 1474 9/10 O/O de dio 17B (100) (90) (100) 638 ug/mo X 7 mo 2-OH estradiol 78 15/19 18/19 14/18 7/18 O role 638 ug/no X 9 mo (79) (95) (78) (94)

TABLE 7 INFLUENCE OF OTHERESTROGENS AND OF THE ANT-ESROGEN TAMOXFENUPON DMBA-INDUCE MAMMARY CARCINOGENSIS (ALL TREATMENT INITIATED 13-14 DAYS AFTER FEEDING DMBA) - Mammary Carcinoma Indicidence - CONTROLS TREATED Ratio of rats with car- Ratio of rats with car cenogenic to total rats cenogenic to total rats and percentage with and percentage with SIGNIFICANT Treatment carcenogenic: carcenogenic: DFFERENCE Dosage and time of observed observed observed observed U x2 administration for 7 mos. for 1 year for 7 mos. for 1 year method method Estriol 18/9 5/19 2/19 (.001 <05 638 ug/mo x 10 mo (95) (26) (63) 4-OH estradiol 16/20 19/20 7/20 3/20 <001 <.05 638 ug/mo x 12 mo (80) (95) (35) (65) 6-keto estradiol 10/12 4/3 8/13 <.01 one 638 ug/mo x 4 no (83) (31) (61) d-equilenin 11/1 4/O 4/10 <001 <.01 638 ug/mo X 4 mo (100) (40) (40)

TABLE 8 INFLUENCE OF OTHER ESTROGENS AND OF THE ANTI-ESTROGEN TAMOXFENUPON OMBA-INDUCED MAMMARY CARCINOGENSIS (ALL TREATMENT INITIATED 13-14 DAYSAFTER FEEDING DMBA) - MAYM CarcinonaSCOCC Indicidence CONTROLS TREATED Ratio of rats with car- Ratio of rats with car cenogenic to total rats cenogenic to total rats and percentage with and percentage with SIGNIFICAN Treatment carcenogenic; carcenogenic: DIFFERENCE Dosage and time of observed observed observed observed U x2 administration for 7 mos. for 1 year for 7 mos. for 1 year method method Moxestrol (11 beta 2/30 26/30 3/29 14/29 <001 <05 methoxy 17a ethynyl 17B estradiol) 638-320 ug/mo X no

EXAMPLE FOUR EXAMPLE FIVE Alpha Ethynyl Estradiol 17 Beta 2-OH Estradio 17 Beta 17 alpha ethynyl estradiol 17 beta was administered in 65 2-OH estradiol 17 beta was administered in doses of doses of 638 micrograms each month starting 13 to 14 630 micrograms each month for 9 months starting 13 to days after feeding DMBA. The results of this treatment 14 days after feeding DMBA. The results of this treat are shown in Table 6. ment are shown in Table 6. 4,937,238 13 A- 14 EXAMPLE SIX TABLE 10-continued Estriol ESTRUS CYCLES NOTED DURING A 21-DAY PERIOD OF Estriol was administered in doses of 638 micrograms ESTROGEN INFUSION each month for 10 months starting 13 to days after 5 Number of Cycles/ feeding DMBA. The results of this treatment are shown Treatment 21 Days in Table 6. 2.0 mg/d 0 2.0 mg/d 2 EXAMPLE SEVEN 6.0 mg/d O } mean 1/rat 4-OH Estradio 10 6.0 mg/d 2 Control - no infusions 4. a mean S/rat 4-OH estradiol was administered in doses of 638 mi Control o no infusions 6 crograms each month starting 13 to 14 days after feed Ethynyl estriol infused continuously ing DMBA. The results of this treatment are shown in 2.0 mg/d Table 7. 15 2.0 mg/d EXAMPLE EIGHT 6.0 mg/d } mean 2.5/rat 6.0 mg/d : 6-Keto Estradiol R 6-keto estradiol was administered in doses of 638 Certain drugs such as 17 alpha ethynyl estriol reduce micrograms each month starting 13 to 14 days after 20 the risk of mammary carcinoma when administered in feeding DMBA. The results of this treatment are shown doses that do not have undesirable side effects even in Table 7. though other similar drugs are inactive or carcinogenic EXAMPLE NINE or toxic. 25 Although a preferred embodiment of the invention D-Equilenin has been described in some detail, many modifications d-equilenin was administered in doses of 638 ug (mi and variations are possible in the preferred embodiment crograms) each month starting 13 to 14 days after feed without deviating from the invention. Accordingly, it is ing DMBA. The results of this treatment are shown in to be understood, that, within the scope of the appended Table 7. 30 claims, the invention may be practiced other than as specifically described. EXAMPLETEN What is claimed is: Moxestrol (17 Beta Methoxy 17 Alpha Ethynyl 17 Beta 1. A method of preventing breast cancer in female Estradiol) mammals comprising the steps of administering to the Moxestrol (17 beta methoxy 17alpha ethynyl 17 beta 35 female mammal a compound which competes with and estradiol) was administered in doses of 638 to 320 mi displaces cell-transforming hormones in the mammary crograms each month starting 13 to 14 days after feed gland without forming DNA-damaging epoxides from ing DMBA. The results of this treatment are shown in hormone metabolites in a dose of I microgram to 50 Table 8. micrograms per kilogram of body weight once every 24 TABLE 9 hours for 20 days for each month from between 3 to 6 MAMMARY GLAND PROLIFERATIONS months after the onset of menarche in sufficiently low AND LACTATION dosages to avoid interfering with ovarium function, INDUCED BY ETHYNYL ESTROL whereby the immature mammary glands are converted AND ETHYNYLESTRADIOL 17 Mammary 45 to mature differentiated cells in the glands without lac Alveolar tation; wherein the composition of matter administered Treatment A. Proliferations Lactation is selected from the group of estrogen compounds com Continuous sub-cutaneous infusion prising 4-hydroxy estradiol, 17 alpha ethinyl estriol and by mini-pump into virgin 50 day d-equilenin. old S-D females for 4 weeks: 50 2. A method of preventing breast cancer in female Ethynyl estradiol 2 mg/d ------mammals according to claim 1 in which the steps of Ethynyl estradiol 2 mg/d - - - -- Ethynyl estradiol 6 mg/d ------administering to the female mammal a compound com Ethynyl estradiol 6 mg/d ------prises administering the compound to human females Ethynyl estriol 2 mg/d ------0 between the age of 15 and 21 years. Ethynyl estriol 2 mg/d ---- O 55 Ethynyl estriol 6 mg/d ---- 0. 3. A method according to claim 2 wherein the com Ethynyl estriol 6 mg/d ---- 0. pound has the formula: No treatment given 0. O No treatment given 0. O OH (1 plus (--) barely detected; 4 pluses (--) up to 50% replacement of fat tissue by f's-C=CH -OH TABLE 10 ESTRUS CYCLESNOTEld DURING A 21-DAYPERIOD OF ESTROGEN INFUSION 65 HO Number of Cycles/ Treatment 2 Days 4. A method according to claim 2 wherein the com Ethynyl estradiol infused continuously pound has the formula: 4,937,238 15 16 5. A method according to claim 2 wherein the com pound has the formula:

OH CH3

O HO HO OH

15

20

25

30

35

45

50

55

65