CA 02265697 l999-03- llwg 93/10771 PCT/US97/16601- 1 -PREVENTION OF OVARIAN CANCERBY ADMINISTRATION OF PROGESTIN PRODUCTSFIELD OF THE ].NVENTION5 The present invention relates generally to methods of preventing1015202530the development of ovarian cancer by administering progestin products, aloneor in association with other hormones such as estrogen products.BACKGROUND OF THE INVENTIONOvarian cancer is the fourth leading cause of cancer deathsamong women in the United States and causes more deaths than all othergynecologic malignancies combined. In the United States, a womanâs lifetimerisk of developing ovarian cancer is l in 70. In 1992, about 21,000 cases ofovarian cancer were reported, and about 13,000 women died from the disease.[Chapter 321, Ovarian Cancer, ,13th ed., Isselbacher et al., eds., McGrawâHill, New York (1994), pages1853-1858; American Cancer Society Statistics, Cancer J. Clinicians, 45:30 (1995). Epithelial ovarian cancer, the most common ovarian cancer, has adistinctive pattern of spread: in addition to metastasis through the lymphaticand blood vessels to areas such as the liver, lung and brain, cancer cells mayalso migrate through the peritoneum to produce multiple metastatic nodules inthe visceral and parietal peritoneum âand the hemidiaphragms. Early stageovarian cancer is often asymptomatic and is detected coincidentally bypalpating an ovarian mass on pelvic examination. In premenopausal patients, _about 95% of these masses are benign. Even after menopause, 70% of massesare benign but detection of any enlargement requires exploratory surgery. Inpostmenopausal women with a pelvic mass, a markedly elevated serum CAâ125 level of greater than 95 U/ml indicates malignancy with a 96% positivepredictive value. [Chapter 321, Ovarian Cancer, I-Iarr-isonâs Principles ofInternal Medicine, supra.]Epithelial ovarian cancer is seldom encountered in women lessW0 98/1077]1015202530CA 02265697 l999-03- llPCT/US97/16601- 2 _than 35 years of age. Its incidence increases sharply with advancing age andpeaks at ages 75 to 80, with the median age being 60 years. The single mostimportant risk factor for this cancer is a strong family history of breast orovarian cancer. In families in which ovarian, breast, endometrial or coloncancer can be tracked as an apparent autosomal dominant trait, the risk of thiscancer can be as high as 50%. Having a single first-degree relative withovarian cancer increases a womanâs risk by at least three-fold, and having apersonal history of breast or colorectal cancer increases the risk ofsubsequently developing ovarian cancer by twoâfold. [Chapter 321, OvarianCancer, Harrisonâs Principles of Internal Medicine, supra.] In addition, thosewith identifiable genetic mutations in genes such as BRCA1 also have anincreased risk. Baker et al., Etiology, Biology, and Epidemiology of OvarianCancer, Seminars in Surgical Oncology 10: 242-248, 1994; Amus et al.,Genetic Epidemiology ofEpithelial Ovarian Cancer, Cancer 71: 566-72,1993; Whitmore, Characteristics Relating To Ovarian Cancer Risk:Implications for Preventing and Detection, Gynecologic Oncology 55, 515-19,1994. Oncogenes associated with ovarian cancers include the HER-2/neu (c-erbB-2) gene, which is overexpressed in a third of ovarian cancers, the fmsoncogene, and abnormalities in the p53 gene, which are seen in about half ofovarian cancers. A number of environmental factors have also been associatedwith a higher risk of epithelial ovarian cancer, including a high fat diet andintake of lactose in subjects with relatively low tissue levels of galactose-1-phosphate uridyl transferase.In epidemiological studies, behavior associated with decreasedovulation, such as pregnancy, breastfeeding and use of estrogen-progestincombination oral contraceptive medications, decrease the risk of ovariancancer; use of estrogen-progestin combination oral contraceptives for as longas 5 years can reduce the risk of ovarian cancer by 50%. Greene et al., TheEpidemiology of Ovarian Cancer, Seminars Oncology, 11: 209-225, 1984;Whitmore et al., Characteristics Relating To Ovarian Cancer Risk:Collaborative Analysis of 12 US Case-Control Studies, American LWO 98/1077]10152030CA 02265697 l999-03- llPCT/US97/16601- 3 _Epidemiology 136: 1212-20, 1992. Conversely, early menarche, latemenopause and nulliparity (no pregnancies) have been shown to increase therisk of ovarian cancer. The risk has been shown to positively correlate withthe number of ovulatory cycles in a womanâs lifetime. Wu et al., Personaland Environmental Characteristics Related To Epithelial Ovarian Cancer,âAmerican J. Epidemiology, Vol. 108(6) 1216-1227. The long-term use ofovulation-inducing ovarian hyperstimulants such as clomiphene has been shownto be associated with an increased risk of ovarian cancer in some women.Rossary et al., Ovarian Tumors in a Cohort 0fInfem'le Women, New Engl. J.Med., 331: 771-6, 1994. Thus, some factors that favor prolonged and persistent ovulation have been thought to increase ovarian cancer risk, whereassome factors that suppress ovulation have been thought to decrease risk.[Chapter 321, Ovarian Cancer, Harrisonâs Principles of Internal Medicine,supra.] These data have led to the "incessant ovulation" hypothesis for thedevelopment of ovarian cancer. Casagrande et al., âIncessant Ovulation â andOvarian Cancer, Lancet at 170-73 (July 28, 1979). This hypothesis is thatrepeated ovulation cycles, each of which involves the disruption and repair ofthe ovarian surface epithelium, may cause neoplastic transformation of theovarian epithelium in susceptible individuals and that the risk of ovarian canceris associated with the number of ovulation cycles in a womanâs lifetime.There is no established pharmaceutical approach to theprevention of ovarian cancer. For all women, especially those at high risk ofdeveloping this disease, the only option available at this time is surgicalremoval of the ovaries, with all of the attendant risks and subsequent adversehealth consequences due to resulting estrogen deï¬ciency.Although epidemiological evidence suggests that the use ofcombination oral contraceptives, which contain both an estrogen and aprogestin, is associated with a subsequent reduced risk of developing epithelialovarian cancer, the mechanism for this protective effect is unknown, and oralcontraceptive preparations are not currently approved for this purpose. Thereduction in risk of ovarian cancer in women who have used estrogen-WO 98/107711015202530CA 02265697 l999-03- llPCT/U S97/ 16601- 4 _progestin combination oral contraceptives for at least three years isapproximately 40 percent. Moreover, this protective effect increases with theduration of use and persists for up to two decades after discontinuation of use.Rosenberg et al., A Case Control Study of Oral Contraceptive Use andInvasive Epithelial Ovarian Cancer, _1'_h_e WHO Collaborative % gfNeoplasia @ ï¬eiï¬ Contraceptives; Epithelial Ovarian Cancer andCombined Oral Contraceptives, In_tâl g 18: 538-45, 1989; Leeet al., The Reduction in Risk of Ovarian Cancer Associated with OralContraceptive Use, New Engl. J. Med. 316: 650-51, 1987; Thomas P.Gross, James J. Schlesselman, Yhe Estimated Ejjfect of Oral ContraceptiveUse on the Cumulative Risk of Epithelial Ovarian Cancer, Obstetrics .Gynecology 83: 4l9â24, 1994; Franceschi et al., Pooled Analysis of 3European Case-Control Studies of Epithelial Ovarian Cancer: III. OralContraceptive Use, I_nLâ_l J. Cancer 49: 61-65, 1991.It is commonly believed that the protective effect of oralcontraceptives is related to the ability of these drugs to inhibit ovulation.Estrogen-progestin combination oral contraceptives act primarily bysuppressing the pituitary glandâs production of gonadotropins, therebyinhibiting the hormonal stimulus for ovulation. These combination drugs alsohave direct inhibitory effects on the reproductive tract, including inducingchanges in the cervical mucus that decrease the ability of sperm to enter theuterus, as well as changes in the endometrium that reduce the likelihood ofimplantation, and reducing fallopian tube motility and uterine secretions.The epidemiological studies showing the protective effect ofcombination oral contraceptives evaluated older combination preparationswhich typically contained higher doses of drug than most contraceptiveregimens used today. Common older regimens contained 50 micrograms ormore of ethinyl estradiol (an estrogen) or 100 micrograms or more ofmestranol (an estrogen) and greater than 1 mg of norethindrone, norethindroneacetate or norethynodrel (a progestin). Table 1 infra lists the progestin andestrogen content of some older regimens. All of the currently used 1owâdoseW0 98/107711015202530CA 02265697 l999-03- llPCT/US97/16601- 5 _combination oral contraceptives contain lower doses of both progestin andestrogen, as well as a lower ratio of progestin to estrogen. Consequently, ithas not been deï¬nitively established that the newer low-dose combination oralcontraceptives are associated with the same protective effect as the older high-dose combination contraceptives. Rosenblatt et al. , High Dose and Low DoseCombined Oral Contraceptives: Protective Against Epithelial Ovarian Cancerand The Length of the Protective Effect, Eur. J. Cancer, 28: 1870-76, 1992.Despite the overall safety of combination oral contraceptives,their use is not recommended for women smokers older than age 35, forwomen of all ages who are at increased risk for myocardial infarction, forwomen with liver disease, and for women older than age 40. Serious andpotentially fatal side effects include deep vein thrombosis, pulmonary emboli,myocardial infarction, thromboembolic stroke, hemorrhagic stroke, and highblood pressure. In the 35-39 year old age group, the use of oralcontraceptives among women smokers doubles their risk of death. After age40, the mortality rate even in non-smoker women using oral contraceptives(32.0 per 100,000) is greater than women using no contraception (28.2 per100,000), while the mortality rate for smoker women is quadnipled (117.6 vs.28.2 per 100,000). [Chapter 340, Disorders of the Ovary and FemaleReproductive Tract, , supra, pages2017-2036.]Progestin-only contraceptives do not reliably inhibit ovulation,but are nevertheless contraceptively effective, presumably due to direct effectson the reproductive tract. The actual contraceptive mechanism of action is _unclear. Prior epidemiological studies have exhibited no consistent pattern ofeither increasing or decreasing risk of ovarian cancer according to duration ofuse. The WHO Collaborative Study of Neoplasia and Steroid ContraceptivesDepot-Medroxyprogesterone Acetate(DMPA) and Risk of Epithelial OvarianCancer, I_;1_t_â_l J_._C_anc_er. 492191-195 (1991); Liam et al., Risk ofBreast,Uterine, Corpus, and Ovarian Cancer in WomenReceiving/Medroxyprogesterone Injections, J. Am. Med. Assân 24912909-2912W0 98/10771101520CA 02265697 l999-03- llPCT/US97l1660l- 5 -(1983). Thus, unlike the data available for progestin-estrogen combinationcontraceptives, the prior art relating to progestin-only contraceptives does notsuggest that the use of a progestin reduces the risk of epithelial ovarian cancer.Estrogen, alone or with low doses of progestin, is also used ashormonal replacement therapy in menopausal women. For long term use,Premarin® (conjugated equine estrogen) is generally given at a dose of 0.625mg orally daily (equivalent to 10 to 20 pg ethinyl estradiol orally per day) oran equivalent dose transdermally. Other regimens add cyclic progestins orcontinuous low-dose progestins, typically 2.5 to 10 mg per day of Provera®(medroxyprogesterone acetate). One epidemiologic study has suggested thathormone replacement therapy with estrogen alone may be associated with anincreased risk of developing ovarian cancer. Rodriguez et al., EstrogenReplacement Therapy and Fatal Ovarian Cancer, Am. J. Epidemiology,14l:828-835 (1995).SUMMARY OF THE INVENTIONThe present invention provides a method for preventing thedevelopment of epithelial ovarian cancer by administering progestin products,either alone or in combination with other agents, such as estrogen products. Amethod is provided of preventing ovarian cancer comprising administering to afemale subject an amount of progestin product effective to increase apoptosisin ovarian epithelial cells of the female subject.It is further the object of this invention to expand the clinicalusage of progestin drugs beyond the current use of these drugs as oralcontraceptive agents in young women or as part of estrogen-progestin hormonereplacement regimens in postmenopausal women. One aspect of the inventionprovides a method for preventing the development of ovarian cancercomprising administering to a female subject a composition consistingessentially of a progestin product (i. e. , a progestin product alone without anestrogen product).WO 98/1077110152030CA 02265697 l999-03- llPCT/US97/16601_ 7 _The invention also provides a method for preventing thedevelopment of ovarian cancer comprising administering a progestin product toa female subject according to a regimen that is not effective for contraception.This can be accomplished in a number of ways, including altering the dosageof progestin product, the type of progestin product, the ratio of progestinproduct to estrogen product, or the timing of administration.With regard to infertile female subjects, the present inventionfurther provides a method for preventing the development of ovarian cancercomprising administering a progestin product according to a regimen that isdifferent from that currently used for hormone replacement therapy. Again,this can be accomplished in a number of ways, including altering the dosage,timing, ratio of progestin product to estrogen product, or the type of progestinâproduct.It is contemplated that the progestin product may beconcurrently administered in combination with additional agent(s), such as anestrogen product, a second progestin product, an androgenic agent, anandrogen agonist, a progestin agonist, an estrogen antagonist, or anotherhormone product, or with other agents that induce apoptosis of ovarianepithelial cells. Such additional agent(s) may be selected to improve theactivity of the progestin agent for preventing ovarian cancer or to reduce anyside effects of the progestin agent. Preferably if estrogen is used as the secondagent, it is used in doses lower than those currently used in combination oralcontraceptive regimens or in doses selected to provide a progestin/estrogen"product ratio that is higher than the ratio currently used in combination oralcontraceptives.The present invention is based on the discovery thatadministration of progestin alone induced an accelerated rate of apoptosis invivo in ovarian epithelial cells of monkeys. Apoptosis is one of the mostimportant mechanisms used for the elimination of cells that have sustainedDNA damage and which are thus prone to transformation into malignantneoplasms. This novel explanation for the association between estrogen-W0 98/1077110152530CA 02265697 l999-03- llPCT/US97/16601- 3 _progestin combination oral contraceptive use and a reduced risk of ovariancancer is a complete departure from the widely accepted theory thatsuppression of "incessant ovulation" is responsible for this reduced risk. Thisï¬nding thus relates to the discovery that progestin alone or estrogenâprogestincombinations may be administered in ways that do not effectively inhibitovulation or otherwise inhibit contraception, yet which still prevent ovariancancer.The invention further relates to the discovery that progestinalone induced a greater rate of apoptosis than a combination of âestrogen andprogestin, which in turn induced a greater rate of apoptosis than estrogenalone. The invention thus contemplates that administration of progestin alonebe effective for preventing the development of ovarian cancer, contrary to thesuggestions of the prior art that progestin has no effect on risk of ovarianCi1IlC6I'.DETAILED DESCRIPTION OF THE INVEN'I'IONThe present invention generally relates to methods forpreventing the development of epithelial ovarian cancer by administering aprogestin product. either alone or in combination with other agents, such as anestrogen product. The invention provides a method of preventing ovariancancer comprising administering to a female subject an amount of progestinproduct effective to increase apoptosis in ovarian epithelial cells of the femalesubject. The invention also provides a method of increasing apoptosis inovarian epithelial cells of a female subject comprising administering to afemale subject an amount of progestin product effective to increase apoptosisin ovarian epithelial cells of the female subject. In particular, the methods ofthe present invention will be particularly advantageous when applied to femalesat high risk of developing ovarian cancer.In a second aspect of the invention, a method is provided forpreventing the development of ovarian cancer comprising administering to afemale subject a composition consisting essentially of a progestin product (i. e.,W0 98/ 107711015202530CA 02265697 l999-03- llPCT/US97/16601_ 9 -a progestin product alone without an estrogen product). The female subjectmay be a fertile female or an infertile female, including perimenopausal andpostmenopausal women. The most preferred product for administration wouldbe an agent that provides the greatest rate of apoptosis of ovarian epithelialcells with the least side effects. Use of a progestin product for longerdurations, or at higher doses, at appropriate intervals, and/or use of an agentthat maximizes apoptosis, without creating unacceptable side effects, in fertileor infertile women may reduce the risk of ovarian cancer further than thatpreviously achieved by combination oral contraceptive use, potentially by asmuch as 60% to 80%.The invention further contemplates expanding the clinical âusageof progestin drugs beyond the current use of these drugs as oral contraceptiveagents in young women or as part of estrogen-progestin hormone replacementregimens in postmenopausal women. Specifically, a third aspect of the presentinvention provides a method for preventing the development of ovarian cancercomprising administering a progestin product to a fertile female subjectaccording to a regimen that is not effective for contraception. This can beaccomplished in a number of ways, including altering the dosage of progestinproduct, the type of progestin product, the ratio of progestin product toestrogen product, or the timing of administration. Also speciï¬callycontemplated is administration of a progestin product in doses higher thanthose currently used for contraception.Oral contraceptive administration regimens are selected tosimulate the normal menstrual cycle,â which averages 28 days in women ofreproductive age. The menstrual cycle begins at the onset of a menstrualbleeding episode and lasts until the onset of the next. Thus, day 1 of a cyclewould be the first day of menstruation, and day 28 would be the day beforethe onset of the next menstrual bleeding episode. Oral contraceptives aretypically taken daily, at the same time each day, for 21 days, followed by aplacebo for the next 7 days. The female generally experiences a menstrualbleeding episode during the sevenâday placebo period. Thus, a woman first. .,.,....W............»..........»w.......................».,........t,......,.. ...,. .....-.r.._W0 98l107711015202530CA 02265697 l999-03- llPCT/US97/16601_ 10 -starting on oral contraceptives is generally instructed to begin taking them atsome time between day 1 and 7.The oral contraceptives must be taken according to the dailyregimen for a full menstrual cycle before they are effective for contraception.A woman beginning an oral contraceptive regimen is not effectively protectedagainst conception if the oral contraceptives are taken for less than the fullmenstrual cycle, if they are not taken daily, and if they are not taken for 21consecutive days. A minimum blood level of the exogenously administeredestrogen or progestin hormones must be maintained daily in order to suppressovulation. If the blood level drops too low, ovulation may occur and the otherinhibitory mechanisms on the reproductive tract may fail to preventconception.Thus, according to this third aspect of present invention, aregimen of progestin product administration that is not effective forcontraception would include, for example, administering or delivering(regardless of whether the route of administration is oral or via injection orimplant) progestin product in doses lower than those effective for contraceptiveuse and/or lower than those previously used in contraceptives; administeringprogestin product with estrogen product at a progestin/estrogen ratio that ishigher than that previously used in contraceptives; administering the drug forless than one menstrual cycle; administering the drug for nonconsecutivemenstrual cycles, e. g. , every other cycle; administering the drug for one ormore menstrual cycles for fewer than 21 consecutive days in each cycle;delivering the drug (regardless of whether the route of administration is oral orvia injection or implant) with a less than daily frequency; or administering thedrug for one or more menstrual cycles according to a regimen that fails tomaintain a contraceptive blood level of the drug or its active metabolite for 21consecutive days in each cycle. A regimen of progestin product administrationthat is different from that currently used for contraception would also includeadministering the progestin product at a daily dose higher than that currentlyused for contraception.WO 98/107711015202530CA 02265697 l999-03- llPCT/US97/16601- 11 _Exemplary regimens according to this third aspect of theinvention include administering progestin product at a dose less than a doseequivalent to 1 mg daily of norethindrone, more preferably less than 0.2 mgdaily, or less than 0.05 mg daily, and possibly as low as 0.025 mg daily of anorethindrone equivalent dose. Another exemplary regimen includesadministering progestin product at a dose higher than 10 mg daily of anorethindrone equivalent dose. A further exemplary regimen includesadministering a progestin product with an estrogen product at a ratio of greaterthan 239:1 by weight in norethindrone/ethinyl estradiol equivalent doses.Additional exemplary regimens include administering any dose of progestinproduct with a less than daily frequency; or administering any close ofprogestin product for a brief time, e. g. , one week only, during the menstrualâcycle. It is contemplated that the most desirable mode of administration maybe administering the progestin product for a brief period sufficient to produceapoptotic turnover of damaged ovarian cells, followed by repeated dosingperiods at intervals, for example 1, 3, 5 or 10 years, selected to provideapoptotic turnover adequate to prevent malignant transformations. The mostpreferable progestin product for administration would be a product thatmaximizes the apoptotic tumover of ovarian epithelial cells and minimizes anyside effects.The fourth aspect of the present invention provides a method forpreventing the development of ovarian cancer in infertile female subjects,comprising administering a progestin product according to a regimen that isdifferent from that cunently used for hormone replacement therapy. Again,this can be accomplished in a number of ways, including altering the dosage,timing, ratio of progestin product to estrogen product, or the type of progestinproduct. Other contemplated regimens would include, for example,administering or delivering progestin product in doses lower or higher thanthose previously used in hormone replacement therapy; or administeringprogestin product with estrogen product at a progestin/estrogen ratio that ishigher than that previously used in hormone replacement therapy.W0 98/1077110152030CA 02265697 l999-03- llPCT/US97/16601- 12 _Estrogen is the primary agent in hormone replacement therapy.Postmenopausal women are generally given estrogen alone, or with low dosesof progestins. The hormones may be administered continuously or cyclically.Continuous administration is typically 0.625 mg Premarin® (a conjugatedequine estrogen) daily or its equivalent, with 2.5 mg Provera°(medroxyprogesterone acetate) daily. Cyclical administration is typically 25consecutive days of 0.625 mg Premarin® daily, with 10 mg Provera® daily ondays 16 through 25, followed by 5 days of no hormone treatment (duringwhich time these women will menstruate).Exemplary regimens according to the fourth aspect of thepresent invention include doses of progestin product less than a dose equivalentto 2.5 mg of medroxyprogesterone acetate daily (equivalent to about 1.25 mgof norethindrone), or less than 0.5 mg daily of a norethindrone equivalentdose. Another exemplary regimen includes a dose of progestin product greaterthan a dose equivalent to 10 mg of medroxyprogesterone acetate daily(equivalent to about 5 mg of norethindrone) for 10 days every month. Afurther exemplary regimen includes doses of progestin product with estrogenproduct at a ratio of greater than 1:1 by weight in norethindrone/ethinylestradiol equivalent doses, or a ratio of greater than 50:1 or 100:1. It is alsocontemplated that the most desirable mode of administration may beadministering the progestin product for a brief period sufficient to produceapoptotic turnover followed by repeated dosing periods at selected intervalsadequate to prevent malignant transformations. A presently preferredprogestin product is levonorgestrel or other 19âno1testosterone derivatives.The most preferable progestin product for administration would be a productthat maximizes the apoptotic turnover of ovarian epithelial cells and minimizesany side effects.The present invention yet further provides a novel use ofprogestin product in preparation of a non-contraceptive medicament forprevention of ovarian cancer in female subjects, as well as a novel use ofprogestin product in preparation of a medicament for prevention of ovarianW0 98ll07711015202530CA 02265697 l999-03- llPCT/US97/16601_ 13 _cancer in infertile female subjects.All doses given herein are appropriate for a female subject ofabout 60 kg weight; the dosages naturally will vary more or less depending onthe weight of the subject. The doses may be increased or decreased, and theduration of treatment may be shortened or lengthened as determined by thetreating physician. The frequency of dosing will depend on thepharmacokinetic parameters of the agents and the route of administration. Theoptimal pharmaceutical formulation will be determined by one skilled in the artdepending upon the route of administration and desired dosage. See forexample, Remingtonâs Pharmaceutical Sciences, 18th Ed. (1990, MackPublishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of whichis hereby incorporated by reference. Such formulations may inï¬uence thephysical state, stability, rate of in vivo release, and rate of in vivo clearance ofthe administered agents.Those of ordinary skill in the art will readily optimize effectivedosages and concurrent administration regimens as determined by goodmedical practice and the clinical condition of the individual patient.Regardless of the manner of administration, the speciï¬c dose may becalculated according to body weight, body surface area or organ size. Furtherreï¬nement of the calculations necessary to determine the appropriate dosagefor treatment involving each of the above mentioned formulations is routinelymade by those of ordinary skill in the art and is within the ambit of tasksroutinely performed by them without undue experimentation, especially in lightof the dosage information and assays disclosed herein. Appropriate dosagesmay be ascertained through use of established assays for determining dosagesin conjunction with appropriate dose-response data. The ï¬nal dosage regimenwill be determined by the attending physician, considering various factorswhich modify the action of drugs, e.g. the drugâs specific activity, the severityof the damage and the responsiveness of the patient, the age, condition, bodyweight, sex and diet of the patient, the severity of any infection, time ofadministration and other clinical factors. As studies are conducted, furtherW0 98/ 107711015202530CA 02265697 l999-03- llPCT/U S97/ 16601- 14 -information will emerge regarding the appropriate dosage levels for thetreatment of various diseases and conditions.It is contemplated that the routes of delivery of progestinproducts (either alone or in combination with other pharmaceuticals) couldinclude oral, sublingual, injectable (including short-acting, depot, implant andpellet forms injected subcutaneously or intramuscularly), vaginal creams,suppositories, pessaries, rings, rectal suppositories, intrauterine devices, andtransdermal forms such as patches and creams.The present invention is related to the discovery thatadministration of progestin alone induced an accelerated rate of apoptosis invivo in ovarian epithelial cells of monkeys. Apoptosis is a process whereby agenetic program within the cell is activated to trigger aâ speciï¬c series ofevents within the cell eventually leading to the death and efficient disposal ofthe cell. Richard Lockshin, Zahm Zakeri, The Biology of Cell Death and ItsRelationship to Aging in Cellular Aging and Cell Death, pp. 167-180, 1996.WileyâLiss Inc., Editors: N.J. Holbrook, G. Martin, R. Lockshin. C.Miligan, L. Schwartz, Programmed Cell Death During Development ofAnimals in Cellular Agingâ and Cell Death, pp. 181-208, 1996. WileyâLissInc. P53-Dependent Apoptosis in Tumor Progression and in Cancer Therapy,Scott W. Lowe, H. Earl Ruley in Cellular Aging and Cell Death, pp. 209-234,1996. WileyâLiss, Inc.For cells that have sustained DNA damage, apoptosis is one ofthe most important mechanisms used for the elimination of these cells, thepreservation of which could otherwise lead to the development of malignantneoplasms. Canman et al., DNA Damage Responses: P-53 Induction, CellCycle Pertubations, and Apoptosis, Cold Spring Harbor Symp. Quant. Biol.,59:277-286 (1994). Thus, the apoptosis pathway is a virtually universalsafeguard to prevent the persistence and proliferation of damaged cells that canbe lethal to the organism. For normal tissues, the processes of cellproliferation and cell death are usually in a steadyâstate balance, and theapoptosis mechanism not only serves to prevent overgrowth of tissue, but alsoWO 98/107711015202530CA 02265697 l999-03- llPCT/US97/16601- -to eliminate those cells that are aberrant and therefore prone to resist normalgrowth regulatory controls.An accelerated rate of apoptosis would facilitate the destructionand thereby removal of ovarian surface epithelial cells which have defectiveDNA and which have the potential to transform into malignant neoplasms.Given the importance of the apoptotic pathway for removal of abnormal cellsfrom tissues, and thus the protection of normal tissues from neoplastictransformation, it is likely that the induction of apoptosis by progestins is oneof the major (if not the major) mechanism underlying the effect of combinationoral contraceptives in reducing the risk of ovarian cancer.This novel explanation for the association between estrogen-progestin combination oral contraceptive use and a reduced risk of ovariancancer is a complete departure from the widely accepted theory thatsuppression of "incessant ovulation" is responsible for this reduced risk. Thisfinding thus leads to the discovery that progestin alone or estrogenâprogestincombinations may be administered in ways that do not effectively inhibitovulation or otherwise inhibit contraception, yet which still prevent ovariancancer. Since the protective mechanism for progestin containing compounds isrelated to a direct biological effect on the ovarian epithelium, it is likely thatthe use of progestin drugs in postmenopausal women who are not ovulatingwill also be protective against the development of epithelial ovarian carcinoma.The invention is further based on the discovery that use ofprogestin alone induces a more accelerated rate of apoptosis in vivo in ovarianepithelial cells of monkeys compared to the combination of estrogen andprogestin, which in turn induced a greater rate of apoptosis than estrogenalone. The implications of this discovery are that the progestin component ofthe oral contraceptive is responsible for this effect, and that administration ofprogestin alone may be effective for preventing the development of ovariancancer, contrary to established reports that it has no effect on risk of ovariancancer. Since the human-equivalent dose of the progestin only dose given themonkeys is insufficient to reliably block ovulation in women, yet showed theW0 98/ 107711015202530CA 02265697 l999-03- llPCT/US97/16601_ 16 -greatest degree of apoptosis (and thus protection), this indicates that ovulatoryblockade per se is not essential for the protective effect, and that progestinproduct only (or with estrogen product) in doses less than sufficient to preventovulation is effective in preventing ovarian cancer.The term "progestin product" or "progestogenic agent" as usedherein includes any drug which binds to the progestin receptor and induces aprogestational effect. This deï¬nition thus includes all of the knownprogestins, derivatives of progesterone or testosterone that have progestinactivity, progestin agonists, and any other agent that increases the rate ofapoptosisâ in ovarian epithelial cells. It is contemplated that not only presentlyavailable progestins but also progestins introduced in the future will be usefulaccording to the present invention. The known synthetic progestins are mainlyderivatives of 17âalphaâhydroxy-progesterone or 19ânortestosterone. Theseprogestins can be classiï¬ed into three groups: the pregnane, estrane, andgonane derivatives. The pregnane progestins, derived from 17 alphahydroxy-progesterone, include, for example, medroxyprogesterone acetate,chlormadinone acetate, megestrol acetate, and cyproterone acetate. All ofthese are roughly 20% to 50% of the potency of norethindrone. The estranes,derived from 19-nortestosterone include norethindrone, norethynodrel,lynestrenol, norethindrone acetate, ethynodiol acetate, and norethindroneenanthate. All of these are metabolized to norethindrone and are roughlyequivalent to the same dosage of norethindrone. The gonanes are derivedfrom the basic estrane structure, with the addition of an ethyl group of position13 of the molecule. This additional ethyl group confers augmentedprogestogenic activity, and also signiï¬cant androgenic effects. Drugs in thisgroup include, for example, norgestrel (-d and -1), norgestimate, desogestrel,and gestodene. All of these are roughly equivalent to four times the dose ofnorethindrone. The oral preparations currently on the market are: norgestrel0.075 mg, medroxyprogesterone acetate 2.5 mg, 5.0 mg, and 10.0 mg,norethindrone 0.35 mg, and norethindrone acetate 0.50 mg.Progestogenic agents have a variety of biological effectsW0 98/107711015202530CA 02265697 l999-03- 11PC T/U S97] 1 6601-17-including antifertility, inhibition of midcycle luteinizing hormone surge,inhibition of ovulation, inhibition of corpus lutetium function and development,and production of a secretory endometrium. In addition, the progestins haveimportant effects on carbohydrate metabolism, lipid and lipoproteinmetabolism and have cardiovascular effects.Progestogenic potency can be measured by other biologicaloutcomes, including the ability of these agents to bind to the progesteronereceptor. The progestogenic activity of the various progestin derivatives canvary. In a review of the literature, Dorflinger has noted that the progestogenicpotency of all these estrane drugs is equivalent, and exhibit only 5-10 percentof the progestogenic activity of levonorgestrel.In addition to their progestogenic effects, the syntheticprogestins have the ability to bind to both estrogen and androgen receptors, toa varying degree. These drugs can therefore have estrogenic, androgenic,antiestrogenic or antiandrogenic effects. For example, the estrane progestinsare weak estrogen agonists, and therefor have slight estrogen activity. Incontrast, the gonane levonorgestrel has no estrogenic activity, but does haveandrogenic activity. The 19-noitestosterone derivatives have androgenicactivity mediated by variable binding to the androgen receptor.Given the diverse binding patterns of the different syntheticprogestins to various receptors (progestin, androgen and estrogen receptors),the estrogenic, progestogenic and androgenic activity can vary among thedifferent synthetic progestin formulations, thus leading to varying degrees ofprogestational activity and androgenic side effects. For example, theprogestational binding activity of norethindrone is less than 20% that oflevonorgestrel and less than 10% that of 3-ketodesogestrel, the activemetabolite of the progestin desogestrel, while the binding affinity ofnorethindrone to the androgen receptor is similar to that of 3-ketodesogestrel,and yet both compounds have less than 50% of the nuclear cell androgenicactivity of levonorgestrel.It is contemplated that the progestins with more androgenicW0 98/107711015202530CA 02265697 l999-03- llPCT/U S97] 16601- -activity-and less estrogenic activity, such as levonorgestrel, may be preferredas more potent for preventing the development of ovarian cancer. Such agentswould include the 19ânortestosterone derivatives, such as norethindrone,norethynodrel, lynestrenol, norethindrone acetate, ethynodiol acetate, andnorethindrone enanthate.The term "estrogen product" as used herein includes ethinylestradiol, mestranol (a 50 mg dosage of which is equivalent to 35 mg ofethinyl estradiol), conjugated equine estrogen, estrone, estradiol, esteriï¬edestrogens, estropipate, and other estrogen equivalents and estrogen agonists."Concurrent administration" or "co-administration" as usedherein includes administration of the agents together, or before or after eachother. The agents may be administered by different routes. For example, oneagent may be administered intravenously while the second agent isadministered intramuscularly, intravenously or orally. They may beadministered simultaneously or sequentially, as long as they are given in amanner sufficient to allow both agents to achieve effective concentrations inthe body.The term "infertile female" as used herein includesperimenopausal and postmenopausal females past the age of reproduction andyounger women not capable of conception, including ovulation, fertilizationand implantation.The term "effective for contraception" as used herein includessufficient inhibition of fertility, including ovulation or implantation.The term "contraceptive blood level'' as used herein includes ablood level sufficient to inhibit fertility, including ovulation or implantation.The term "females at high risk of developing ovarian cancer"includes females with a family history of breast or ovarian cancer, femaleswith a prior history of breast or ovarian cancer, or females with a mutation inthe BRCAI gene or any other mutation shown to be associated with a high riskof developing ovarian cancer.Various combinations of progestin and estrogen that have beenCA 02265697 l999-03- 11W0 98/10771 PCT/U S97/ 16601-19..used in oral contraceptives are shown in Table 1.Table 1: Previously Used Combinations of Progestin and EstrogenNorethindrone EE EquivalentDose Equivalent Dose DoseProgestin (mg) Dose Estrogen (mg) , (mg) P/E RatioNorethynodrel 9.85 9.85 Mestranol 0.150 0.105 93.8105.00 5.00 0.075 0.053 95.2382.50 2.50 0.036 0.025 99.2062.50 2.50 O. 100 0.070 35.7145 Norethindrone 10.00 10.00 Mestranol 0.060 0.042 238.0952.00 2.00 0.100 0.070 28.5711.00 1.00 0.050 0.035 28.5711.00 1.00 0.080 0.056 17.857Norethindrone 1.00 1.00 Ethinyl 0.050 0.050 20.0000.50 0.50 estradiol 0.035 0.035 Iâ 14.2860.40 0.40 0.035 0.035 11.429/Norethindrone 2.50 2.50 EB 0.050 0.050 50.000acetate 1.00 1.00 0.050 0.050 20.0000.60 0.60 0.030 0.030 20.0001.50 1.50 0.030 0.030 50.0001.00 1.00 0.020 0.020 50.000Ethynodiol 1.00 1.00 Mestranol 0.100 0.070 - 14.28610 diacetateEthynodiol 1.00 1.00 EE 0.050 0.050 20.000diacetatedl-Norgestrel 0.50 2.00 E 0.050 0.050 10.0000.30 1.20 0.030 0.030 10.000Equivalencies15 50 mg Mestranol = 35 mg Ethinyl estradiol (BE)0.5 mg dl-Norgestrel = 2 mg NorethindroneW0 98/ 107711015202530CA 02265697 l999-03- llPCT/US97/16601_ 20 -Each block describes a speciï¬c combination of progestin and estrogen, e.g.,norethynodrel and mestranol, and within each block older combinations arelisted first, with successively newer combinations following. Two trends areevident. First, over time the size and ratio of the dosages has decreased, 1'. e. ,the downward trend of the progestin component is steeper than the downwardtrend of the estrogen component. On a relative scale, therefore, estrogen hasbecome more important over time. Second, with this downward trend indosage, it is apparent that the relative ratio of progestin to estrogen is alsotrending downward. By contrast, the present invention emphasizes the greaterimportance of progestin in combination with estrogen, and thus emphasizescombination ratios even higher than those ratios, e. g., 100-1, that have longsince been abandoned.Other aspects and advantages of the present invention will beunderstood upon consideration of the following illustrative examples. Example1 addresses the effect of administration of progestin or estrogen products,alone or in combination, on the ovarian epithelial cells of monkeys. Example2 addresses the effect of progestin and estrogen products, alone or incombination, on the ovaries of humans. Example 3 addresses the effect ofhormonally active agents, alone or in combination, in vitro on human ovariantissue. Example 4 addresses the effect of gonadal hypertrophy on rodentovaries. Example 5 addresses the effect of various hormonally activeproducts, alone or in combination, on monkey ovaries. Example 6 addressesthe effect of various hormonally active agents on the ovarian tissue oftransgenic mice that have been altered to have altered expression of receptors,growth factors, integrins or protooncogenes.EXAMPLE 1EFFECT OF ESTROGEN AND PROGESTIN IN VIVOON MONKEY OVARIESYoung female adult cynomolgus monkeys were fed a diet fortwo years that contained either no hormones, the oral combinationW0 98/107711015202530CA 02265697 l999-03- llPCT/US97/16601_ 21 _contraceptive "Tn'phasil®," the estrogenic component of "Triphasil®" (ethinylestradiol) alone, or the progestin component of "Triphasil®" (levonorgestrel)alone, each administered in the same pattern that occurs in a "Tn'phasil®"regimen. Doses were scaled on the basis of caloric intake, which is theaccepted way to achieve humanâequivalent doses. The human-equivalent doseswere thus: six days of 0.030 mg ethinyl estradiol + 0.050 mg levonorgestrel,followed by 5 days of 0.040 mg ethinyl estradiol + 0.075 mg levonorgestrel,followed by 10 days of 0.030 mg ethinyl estradiol + 0.125 mg levonorgestrel,followed by 7 days of no treatment. This cyclic regimen was repeated every28 days continuously for 2 years.At the completion of the two years of the study, the animalswere sacriï¬ced, and their ovaries were removed and both formalin ï¬xedandparaffin embedded as well as ï¬ash frozen and stored at minus 70 degreesCelsius. Five-micron ovarian sections were mounted on coated slides, andstained with the Apoptag-plus kit (Oncor, Gaithersburg, MD), whichspecifically labels the 3â end of free DNA fragments in cells undergoing DNAfragmentation, a characteristic of apoptosis. After staining, cells undergoingapoptosis were easily identiï¬ed by their dark brown nuclear discoloration.The ovarian surface epithelium was examined histologically to assess ovarianepithelial morphology and to determine the percentage of ovarian cellsundergoing apoptosis. To calculate the percentage of ovarian epithelial cellsundergoing apoptosis, both the total number of ovarian epithelial cells and thenumber undergoing apoptosis were counted on each ï¬ve-micron ovariansection. At each step, the investigators were completely blinded with regard to _which treatment group was associated with each ovary.The ovarian surface epithelium is comprised of a single layer ofepithelial cells that rests on a basement membrane overlying the ovariancortex. In the control and nonâprogestin treated monkeys, the ovarian surfaceepithelium typically had a lush appearance with the epithelial cells containingabundant cytoplasm and visible microvilli at the surface with apoptotic cellsrarely seen. In the progestin treated monkeys, the ovarian surface epitheliumWO 98/10771CA 02265697 l999-03- llPCT/US97ll6601-22-was observed to contain numerous brownâstaining apoptotic cells.The median percentage of ovarian epithelial cells undergoingapoptosis for each of the treatment groups is shown below in Table 2.Table 2: Apoptotic Effect of Four Treatments On Monkey EpitheliaMedian Percent of Range of Percent of5 Treatment Number Apoptotic Cell Counts Apoptotic Cell CountsControl 20 3.8% 0.1 - 33.0%Ethinyl-estn'adol- 20 1.8% 0.1 - 28.6%onlyCombination Pill 17 â 14.5% 3.0 - 61.0%10 Levonorgestrel 18 24.9% 3.5 â 61.8%15202530Multiple Comparisons: Control - Levonorgestrel (p < 0.001)Combination Pill - Ethinylâestradiol (p < 0.001)Ethinyl-estradiol - Levonorgestrel (p < 0.001)Control â Combination Pill (p< 0.05)From Table 2, the median percentage of apoptosis in the controlgroup of monkeys not receiving any hormonal therapy was approximately3.8%. Statistically, this was not significantly different from the rate ofapoptosis seen in the ovarian epithelium in monkeys receiving only theestrogen component of "Triphasil®," ethinyl estradiol, in which the medianpercentage of apoptosis was 1.8%.A marked and significantly greater level of apoptosis. was notedin the other two groups of monkeysâthose that received the combination pill(containing both ethinyl estradiol and levonorgestrel) and those that receivedlevonorgestrel (the progestin) alone. In this latter group (progestin alone), theobserved median percentage of cells undergoing apoptosis was over six timesgreater than the level of apoptosis observed in the control, untreated monkeys.Because the only difference between the combination pill group and estrogen-alone group is the presence of the levonorgestrel component of thecombination pill, and because the degree of apoptosis of the ovarian epitheliumin the estrogen-alone group was no different than that of the control group,W0 98/ 1077 110152025CA 02265697 l999-03- llPCT/US97/ 16601- -these data demonstrate that the accelerated rate of apoptosis in the ovarianepithelium in combination pill treated monkeys is due to the effects of theprogestational component (levonorgestrel) of the combination pill. Moreover,the higher rate of apoptosis among the monkeys that received a progestationalagent alone than in the monkeys that received the combination pill, althoughnot statistically significant, indicates that progestin-only treatment is moreeffective at inducing apoptosis of the ovarian surface epithelium than aprogestin/estrogen combination treatment.EXAMPLE 2EFFECT OF PROGESTIN AND ESTROGEN IN VIVOON HUMAN OVARIESVarious progestins alone, including pregnanes, estranes andgonanes, various estrogens alone, or various progestinâestrogen combinationsat varying doses are administered to women for at least one month prior to ascheduled surgery for removal of the ovaries and uterus. In particular,regimens of estrogen alone, estrogen with medroxyprogesterone acetate (oranother 17-hydroxy-progesterone derivative), and estrogen with levonorgestrel(or another 19-nortestosterone derivative) are evaluated. To evaluate theeffects of the different dosage regimens, the ovaries are examined for variousmarkers, including apoptosis, proliferation, expression of growth factors,expression of steroid hormone receptors, and expression of other enzymes orgenes.EXAMPLE 3EFFECT OF HORMONALLY ACTIVE AGENTS IN VITROON HUMAN OVARIAN TISSUEOvarian epithelia cultured from ovaries removed from normalwomen or women with epithelial ovarian cancer are treated with variousprogestins alone, including pregnanes, estranes and gonanes, various estrogensalone, various progestin-estrogen combinations, progesterone receptor agonists,W0 98/1077]10152025CA 02265697 l999-03- llPCT/U S97/ 16601-24-progesterone receptor antagonists, estrogen receptor agonists, or estrogenreceptor antagonists, each at varying doses and varying durations, from e.g.,24 hours to 7 days. The ovarian tissue is then examined for various markers,including apoptosis, proliferation, expression of growth factors, expression ofsteroid hormone receptors, and expression of other enzymes or genes. Themost potent agent for inducing apoptosis is determined.EXAMPLE 4EFFECT OF GONADAL HYPERTROPHY ON RODENT OVARIESThe ovaries of mice or rats are modestly "hyperstimulated" bycompensatory gonadal hypertrophy after unilateral oophorectomy. The ovariesof the control animals, which received no treatment, are removed andexamined at age 4, 4.5, 5 and 6 months. One ovary of the test animals isremoved and examined at age 4 months, and the remaining ovary of each testanimal is removed and examined at either age 4.5, 5 or 6 months. Theovarian tissue is examined for various markets, including apoptosis,proliferation, expression of growth factors, expression of steroid hormonereceptors, and expression of other enzymes or genes.EXAMPLE 5 AEFFECT OF HORMONALLY ACTIVE AGENTS IN VIVOON MONKEY OVARIESMature young female monkeys are treated with one of thefollowing: control, leuprolide acetate (a gonadotropin releasing hormone[GnRH or LHRH} agonist), various oral contraceptives, levonorgestrel,norethindrone, medroxyprogesterone acetate, ethinyl estradiol, testosterone,testosterone derivatives, RU-486, progestin agonists, progestin antagonists,estrogen agonists and estrogen antagonists, each at varying doses. The ovariantissue is removed and examined for various markers, including apoptosis,proliferation, expression of growth factors, expression of steroid hormonereceptors, and expression of other enzymes or genes.CA 02265697 l999-03- 11W0 98/ 10771 PCT/US97/ 16601_ 25 -EXAMPLE 6EFFECT OF HORMONALLY ACTIVE AGENTS IN VIVOON OVARIES OF TRANSGENIC MICEThe effect of various progestins, estrogens or androgens, each at5 varying doses, is evaluated on the ovarian tissue of transgenic mice that havebeen altered to "knockout" their progcstin receptor, to have an alteredexpression of the estrogen receptor, to express BRCA1, or to have alteredexpression of growth factors, integrins or protooncogenes._ Numerous modiï¬cations and variations in the practice of the10 invention are expected to occur to those skilled in the art upon consideration ofthe foregoing description on the presently preferred embodiments thereof.Consequently the only limitations which should be placed upon the scope ofthe present invention are those that appear in the appended claims.