a better understanding of the multiple endocrine activity of synthetic progestins is required not only for their more rational use in the prevention and therapy of breast and endometrial cancers as well as endometriois and bone loss but also to avoid side effects caused by interaction with steroid receptors unnecessary for the desired beneficial effect . precise analysis of the biological actions of synthetic &# 34 ; progestins &# 34 ; having affinity for many steroidal receptors would ideally require the selection of in vitro models possessing functional receptors for all major classes of steroids . for this purpose , we have chosen the zr - 75 - 1 human breast cancer cell line , which possesses functional receptors for estrogens , androgens , progesterone and glucocorticoids ( vignon et al ., j . clin . endocrinol . metab . 56 : 1124 - 1130 , 1983 ) in order to compare the relative contribution of the different steroid receptor systems in the control of cell proliferation by synthetic progestins . while estrogens are strongly mitogenic in zr - 75 - 1 cells ( poulin and labrie , cancer res . 46 : 4933 - 4937 , 1986 ) and specifically regulate the expression and / or the secretion of several proteins ( dickson and lippman , endocr . rev . 8 : 29 - 43 , 1987 ), androgens ( poulin et al ., breast cancer res . treatm . 12 : 213 - 225 , 1988 ), as well as progestins ( poulin et al ., breast cancer res . treatm . 13 : 161 - 172 , 1989 ) inhibit their proliferation through specific interactions with their respective receptors . many progestins have been used in the treatment of breast cancer , including mpa ( blossey et al ., cancer 54 : 1208 - 1215 , 1984 ; hortobayyi et al ., breast cancer res . treatm . 5 : 321 - 326 , 1985 ), mga ( johnson et al ., semin . oncol . 13 ( suppl . ): 15 - 19 , 1986 ; tchekmedyan et al ., semin . oncol . 13 ( suppl . ): 20 - 25 , 1986 ) and norethindrone ( clavel et al ., eur . j . cancer clin . oncol . 18 : 821 - 826 , 1982 ; earl et al ., clin . oncol . 10 : 103 - 109 , 1984 ). using the in vitro system of human breast cancer zr - 75 - 1 cells , i have found that the synthetic progestins or anabolic steroids , nor - testosterone , r1881 , dromostanolone , fluoxymesterone , ethisterone , methandrostanolone , oxandrolone , danazol , stanozolol , calusterone , oxymetholone , cyproterone acetate , chlormadinone acetate and norgestrel , possess androgenic activity at low concentrations . in addition to inhibition of cell growth , the secretion of two glycoproteins , namely gross cystic disease fluid protein - 15 ( gcdfp - 15 ) and gcdfp - 24 is markedly stimulated by androgens ( simard et al ., mol . endocrinol . 3 : 694 - 702 , 1989 ; simard et al ., endocrinology 126 : 3223 - 3231 , 1990 ). measurements of gcdfp - 25 or gcdfp - 24 secretion can thus be used as sensitive parameter or marker of androgen action in these cells . in fact , changes in gcdfp - 15 and gcdfp - 24 secretion are opposite to the changes in cell growth under all experimental conditions examined . all the synthetic progestins or anabolic steroids that i have studied exhibit androgenic activity on zr - 75 - 1 breast cancer growth and secretion of gcdfp - 15 and gcdfp - 24 . identification of the receptors ( estrogen , androgen , progesterone and glucocorticoid ) responsible for the action of the compounds is essential in order to assess the potential actions ( including adverse effects ) of such compounds . it is thus especially important to assess the specific interaction at low concentrations with the androgen receptor since such low concentrations do not interact with the glucocorticoid receptor , thus avoiding or minimizing secondary side effects . one method for inhibiting growth of breast and endometrial cells is activation of the androgen receptor with an effective compound having an affinity for the receptor site such that is binds to the androgen receptor at low concentrations while not significantly activating other classes of steroid receptors linked to potential side effects . it is important to select compounds having maximal affinity for the androgen receptor which have minimal or no virilizing effects in women . in order to minimize interaction of such compounds with the glucocorticoid and estrogen receptors , it is important to use low dose of the compounds . it is also important to choose steroids having androgenic activity at low concentrations which are not metabolized into estrogens under in vivo conditions which , at the low concentrations used , will not lead to significant activation of receptors other than the androgen receptors . my research has shown that the compounds used in the invention , particularly anabolic steroids and synthetic progestins , vary markedly , over different concentrations , in their ability to activate different classes of steroidal receptors . hence , by carefully controlling concentration , it is possible to selectively cause activation of desired receptors while not causing significant activation of undesired receptors . for example , at the low concentrations specified herein , mpa can be utilized to desirably activate androgen receptors while substantially avoiding side effects associated with glucocorticoid activation which have plagued prior art treatments . thus , this invention provides a novel method for prevention and therapy of breast and endometrial cancer as well as other diseases responsive to activation of the androgen receptor , e . g . bone loss and endometriosis . in this invention , the amount of the androgenic compounds administered is much lower than previously used in art for the treatment of breast and endometrial cancer . to help in determining the potential effects of the treatment , blood concentrations of the compound can be measured . for example , measurements of plasma medroxyprogesterone acetate ( mpa ) levels can be made by radioimmunoassay following extraction as follows : antibody 144a was raised in rabbits against 17 - hydroxyprogesterone - 3 - 0 - carboxymethyloxime - bsa . the labeled steroid used in the radioimmunoassay ( ria ) was methyl - 17α - hydroxyprogesterone acetate , 6α -[ 1 , 2 - 3 h ( n )]- obtained from nen ( cat no : net 480 ) while the reference preparation was medroxyprogesterone acetate ( mpa ) obtained from steraloids . the assay buffer used was 0 . 1 % gelatin in 0 . 1m sodium phosphate , 0 . 15m sodium chloride , 0 . 1 % sodium azide , ph 7 . 2 . the extraction solvent mixture was ethyl etheracetone ( 9 : 1 , v : v ) [ eea ] while the lh - 20 chromatography solvent mixture was iso - octane : toluene : methanol ( 90 : 5 : 5 ; v : v : v ) [ ioth ]. one ml of plasma was extracted twice with 5 ml of eea . the extracts were evaporated to dryness with nitrogen and the remaining residue was dissolved in one ml of ioth . the extracts were then subjected to lh - 20 chromatography on 10 × 30 cm columns ( corning cat no : 05 722a ) filled with 2 g of lh - 20 ( pharmacia ). the gel was washed with 30 ml of ioth before addition of one ml of sample and elution with ioth . the first 6 ml were discarded . the following 10 , 16 . 5 and 27 . 5 ml of eluent were fraction i ( progesterone ), ii ( mpa ) and iii ( 17 - lh - progesterone ), respectively . fraction ii was evaporated to dryness and reconstituted in 1 . 5 ml of assay buffer . radioimmunoassay to each 12 × 75 mm borosilicate test tube was added : 0 . 2 ml containing 25 , 000 dpm of tritiated steroid , 0 . 5 ml of reference preparation ranging from 5 to 5000 pg / tube or 0 . 5 ml of extracted sample fraction ii , 0 . 2 ml of antiserum 144a diluted 1 / 5000 or 0 . 2 ml of assay buffer to account for non specific binding . the tubes were then incubated overnight at 4 ° c . then , 0 . 2 ml 2 % charcoal norit - a , 0 . 2 % dextran t - 70 diluted in water was added . the tubes were then shaken gently and , after 10 min , they were centrifuged at 2000 × g for 10 min . the . supernatant was mixed with 8 ml of formula - 989 ( nen : nef - 989 ) and the radioactivity was counted in a β - counter . the lower and upper limits of detection of mpa are 10 and 10000 pg / ml , respectively , while the slope ( logit - log ) is - 2 . 2 and the ed 50 value is 315 pg / ml . non - specific and net binding are 1 . 5 and 45 %, respectively . antibody 144a is highly specific for mpa since cross - reactivity with progesterone , 20α - oh - prog , pregnenolone , 17 - oh - pregnenolone , dht , androstenedione , testosterone , 30α - diol , estrone , estradiol and cortisol is less than 0 . 1 %. ria data were analyzed using a program based on model ii of roadbard and lewald ( in : 2nd karolinska symposium , geneva , 1970 , pp . 79 - 103 ). plasma mpa levels are usually shown as the means ± sem ( standard error of the mean ) of duplicate measurements of individual samples . statistical significance is measured according to the multiple - range test of duncan - kramer ( kramer , c . y ., biometrics 12 : 307 - 310 , 1956 ). to assist in determining the activity of the potential compounds on the various steroid receptors , androgen , glucocorticoid , progesterone and estrogen - receptor - mediated activities of synthetic progestins and anabolic steroids can be measured in zr - 75 - 1 human breast cancer cells using cell growth as well as gcdfp - 15 and gcdfp - 24 release as parameters of response ( poulin and labrie , cancer res . 46 : 4933 - 4937 , 1986 ; poulin et al ., breast cancer res . treatm . 12 : 213 - 225 , 1988 ; poulin et al ., breast cancer res . treatm . 13 : 161 - 172 , 1989 ; poulin et al ., breast cancer res . treatm . 13 : 265 - 276 , 1989 ; simard et al ., mol . endocrinol . 3 : 694 - 702 , 1989 ; simard et al ., endocrinology 126 : 3223 - 3231 , 1990 ). the following properties permit measurement of progesterone receptor ( pgr ) activity : 1 ) the addition of insulin completely reverses the inhibition due to the interaction of the progestin r5020 with the pgr in zr - 75 - 1 cells ; and 2 ) the antiproliferative effect of r5020 is observed only under e 1 - stimulated conditions . these two characteristics of zr - 75 - 1 cell growth permit study of the extent to which a tested compound &# 39 ; s effects on zr - 75 - 1 cells are attributated to its interaction with pgr by evaluating the effect of insulin and / or estrogen addition on the growth response measured at the end of a 15 - day incubation of zr - 75 - 1 cells with the test compounds . the contribution of the estrogen receptor ( er ), on the other hand , can be directly measured by incubating zr - 75 - 1 cells in the presence or absence of estrogen in the medium . in order to analyze the interactions of synthetic progestins or anabolic steroids with the androgen receptor ( ar ) and glucocorticoid receptor ( gr ) in their inhibitory action on cell growth , one takes advantage of the additivity of the anti - proliferative effects of androgens and glucocorticoids in this cell line ( poulin et al ., breast cancer res . treatm . 12 : 213 - 225 , 1988 ). thus , one can saturate ar with 5α - dihydrotestosterone ( dht ) and then measure the effect on cell proliferation resulting from the addition of a putative glucocorticoid . on the other hand , the effect of a putative androgen can similarly be measured following saturation of gr by dexamethasone ( dex ). the specificity of the growth - inhibitory activity thus observed with the test compound can also be further assessed by its reversibility using the appropriate antagonist ( i . e . antiglucocorticoid or antiandrogen ). thus , in the presence of excess androgen ( 1 μm dht ) in the presence of e 2 and insulin , glucocorticoid effects can be assessed with precision and with no interference by the other receptors . the same applies to study of the role of ar when the cells are incubated in the presence of excess glucocorticoid ( 3 μm dex ), in the presence of e 2 and insulin . as demonstrated by detailed kinetic studies , 1 μm dht and 3 μm dex exert maximal inhibitory effects on the ar and gr , respectively . in addition , the possible antagonistic activities of &# 34 ; progestins &# 34 ; mediated through the ar and gr can be determined by saturating both receptor systems with dht and dex with one ligand being in far greater excess than the other in order to allow reversal through a single chosen receptor at a time . all experiments are performed with zr - 75 - 1 cells grown in e2 - supplemented media containing insulin in order to prevent the pgr - mediated effect of &# 34 ; progestins &# 34 ; on cell growth . using the foregoing techniques , i have found that numerous androgenic compounds which also activate other receptors ( e . g . glucocorticoid or progesterone receptors ) vary in their relative effects on different receptors as a function of concentration . by staying within concentration ranges defined herein , compounds of the invention may beneficially affect androgen receptors without substantial undesirable effects on other receptors . selection of patients who may benefit from the method &# 39 ; s described herein the appearance of breast cancer is usually detected by self breast examination and / or mammography . endometrial cancer , on the other hand , is usually diagnosed by endometrial biopsy . both cancers can be diagnosed and evaluated by standard physical methods well known to those skilled in the art , e . g . bone scan , chest x - ray , skeletal survey , ultrasonography of the liver and liver scan ( if needed ), cat scan , mri and physical examination . endometriosis can be diagnosed following pains or symptoms associated with menstruations in women while definitive diagnosis can be obtained by laparascopy and , sometimes , biopsy . bone density , on the other hand , can be measured by standard methods well known to those skilled in the art , e . g . qdr ( quantitative digital radiography ), dual photon absorptiometry and computerized tomography . plasma and urinary calcium and phosphate levels , plasma alkaline phosphatase , calcitonin and parathormone concentrations , as well as urinary hydroxyproline and calcium / creatinine ratios . breast or endometrial cancer , osteoporosis or otherwise insufficient bone mass , and other diseases treatable by activating androgen receptor may be treated in accordance with the present invention or prophylactically prevented in accordance herewith . typically suitable androgenic compounds include 6 - alpha - methyl , 17 - alpha - acetoxy progesterone or medroxyprogesterone acetate available , for example , from upjohn and farmitalia carlo erba , s . p . a . under the trade names provera , depoprovera or farlutal , and the acronym mpa . other suitable androgenic compounds include those described in labrie et al . ( fertil . steril . 31 : 29 - 34 , 1979 ) as well as anabolic steroids or progestins ( raynaud and ojasso , in : innovative approaches in drug research , elsevier sci . publishers , amsterdam , pp . 47 - 72 , 1986 ; sandberg and kir doni , pharmac . ther . 36 : 263 - 307 , 1988 ; and vincens , simard and de ligni eres , les androg enes . in : pharmacologie clinique , base de th erapeutique , 2i eme edition , expansion scientifique ( paris ), pp . 2139 - 2158 , 1988 ), as well as calusterone ( 7β , 17α - dimethyltestosterone ), anabolic steroids ( lam , am . j . sports medicine 12 , 31 - 38 , 1984 ; hilf , r ., anabolic - androgenic steroids and experimental tumors . in : ( kochachian , c . d ., eds . ), handbook of experimental pharmacology , vol . 43 , anabolic - androgenic steroids , springer - verlag , berlin , 725 pp , 1976 ), fluoxymesterone ( 9α - fluoro - 11β - hydroxy - 17α - methyltestosterone ), testosterone 17β - cypionate , 17α - methyltestosterone , pantestone ( testosterone undecanoate ), δ 1 - testololactone and andractim . other typical suitable androgenic compounds are cyproterone acetate ( androcur ) available from shering ag , 6 - alpha - methyl , 17 - alpha - acetoxy progesterone or medroxyprogesterone acetate ( mpa ) available from , among others , upjohn and farmitalia , calbo erba , gestodene available from shering , megestrol acetate ( 17α - acetoxy - 6 - methyl - pregna - 4 , 6 - diene - 3 , 20 - dione ) available from mead johnson & amp ; co ., evanswille , ind ., under the trade name of megace . other synthetic progestins include levonorgestrel , norgestimate , desogestrel , 3 - ketodesogestrel , norethindrone , norethisterone , 13α - ethyl - 17 - hydroxy - 18 , 19 - dinor - 17β - pregna - 4 , 9 , 11 - triene - 20 - yn - 3 - one ( r2323 , gestrinone ), demegestone , norgestrienone , gastrinone and others described in raynaud and ojasso , j . steroid biochem . 25 : 811 - 833 , 1986 ; raynaud et al ., j . steroid biochem . 25 : 811 - 833 , 1986 ; raynaud et al ., j . steroid biochem . 12 : 143 - 157 , 1980 ; raynaud , ojasoo and labrie , steroid hormones , agonists and antagonists , in : mechanisms of steroid action ( g . p . lewis and m . ginsburg , eds ), mcmillan press , london pp . 145 - 158 ( 1981 ). any other progestin derivative having the above - described characteristics could also be useful for the invention . the androgenic compound is preferably administered as a pharmaceutical composition via topical , parenteral or oral means . the compound can be administered parenterally , i . e . intramuscularly or subcutaneously by injection of infusion by nasal drops , by suppository , or where applicable intravaginally or transdermally using a gel , a patch or other suitable means . the androgenic compound may also be microencapsulated in or attached to a biocompatible , biodegradable polymer , e . g . poly ( d1 , 1 - lactide - co - glycolide ) and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous , slow release of the compound over a period of 30 days or longer . in addition to the oral route , a preferred route of administration of the compound is subcutaneous depot injection . depoprovera can be released at a relatively constant rate for approximately 3 months after intramuscular administration of an aqueous suppression . the amount of each compound administered is determined by the attending clinician taking into consideration the patient &# 39 ; s condition and age , the potency of each component and other factors . in the prevention of breast and endometrial cancer , as well as bone loss , according to this invention , the following dosage ranges are suitable . the androgenic composition is preferably administered in a daily dosage which delivers less than 25 mg of active androgenic steroid per 50 kg of body weight . a dosage of 1 - 10 mg per 50 kg of body weight , especially 3 - 7 mg ( e . g . 5 mg ) is preferred . the dosage selected preferably maintains serum concentration below 50 nanomoles per liter , preferably between 1 . 0 nanomoles per liter and 10 , 15 or 25 nanomoles per liter depending on patient &# 39 ; s response . the dosage needed to maintain these levels may vary from patient to patient . it is advisable for the attending clinicial to monitor levels by the techniques described herein and optimize dosage accordingly . for prophylactic purposes , administration of the androgen is preferably started in the perimenopausal period for the prevention of breast and endometrial cancer and bone loss in normal women . the androgen may be associated with an accepted dose of an estrogen used to prevent other signs and symptoms of menopause . in women , when estrogen formation and / or action has been blocked for treatment of endometriosis , leiomyomata , breast cancer , uterine cancer , ovarian cancer or other estrogen - sensitive disease , administration of the androgen can be started at any time , preferably at the same time as blockade of estrogens . the androgen for intramuscular or subcutaneous depot injection may be microencapsulated in a biocompatible , biodegradable polymer , e . g ., poly ( d , 1 - lactide - co - glycolide ) by , among other techniques , a phase separation process or formed into a pellet or rod . the microspheres may then be suspended in a carrier to provide an injectable preparation or the depot may be injected in the form of a pellet or rod . see also european patent application epa no . 58 , 481 published aug . 25 , 1982 for solid compositions for subdermal injection or implantation or liquid formulations for intramuscular or subcutaneous injections containing biocompatible , biodegradable polymers such as lactideglycolide copolymer and active compounds . these formulations permit controlled release of the compound . the androgens useful in the present invention can be typically formulated with conventional pharmaceutical excipients , e . g ., spray dried lactose and magnesium stearate into tablets or capsules for oral administration . the active substance can be worked into tablets or dragee cores by being mixed with solid , pulverulent carrier substances , such as sodium citrate , calcium carbonate or dicalcium phosphate , and binders such as polyvinyl pyrrolidone , gelatin or cellulose derivatives , possibly by adding also lubricants such as magnesium stearate , sodium lauryl sulfate , &# 34 ; carbowax &# 34 ; or polyethylene glycol . of course , taste - improving substances can be added in the case of oral administration forms . as further forms , one can use plug capsules , e . g ., of hard gelatin , as well as closed soft - gelatin capsules comprising a softener or plasticizer , e . g . glycerine . the plus capsules contain the active substance preferably in the form of granulate , e . g ., in mixture with fillers , such as lactose , saccharose , mannitol , starches , such as potato starch or amylopectin , cellulose derivatives or highly dispersed silicic acids . in soft - gelatin capsules , the active substance is preferably dissolved or suspended in suitable liquids , such as vegetable oils or liquid polyethylene glycols . in place of oral administration , the active compound may be administered parenterally . in such case , one can use a solution of the active substance , e . g ., in sesame oil or olive oil . the active substance can be microencapsulated in or attached to a biocompatible , biodegradable polymer , e . g . poly ( d , 1 - lactide - co - glycolide ) and subcutaneously or intramuscularly injected by a technique called subcutaneous or intramuscular depot to provide continuous slow release of the compound ( s ) for a period of 2 weeks or longer . the invention also includes kits or single packages containing the pharmaceutical composition active ingredients or means for administering the same for use in the prevention and treatment of breast and endometrial cancer as well as bone loss and treatment of endometriosis as discussed above . the kits or packages may also contain instructions on how to use the pharmaceutical compositions in accordance with the present invention . following the above therapy using the described regimen , tumor growth of breast and endometrial cancer as well as bone loss and endometriosis can be relieved while minimizing adverse side effects . the use of the described regimen can also prevent appearance of the same diseases . prevention of mammary carcinoma induced by dimethylbenz ( a ) anthracene ( dmba ) in the rat , by low dose medroxyprogesterone acetete (&# 34 ; mpa &# 34 ;) to illustrate the efficacy of the present invention in reducing the incidence of mammary carcinoma , fig1 illustrates the effect of a single subcutaneous injection of depo - provera ( medroxyprogesterone acetate ( mpa ) ( 30 mg )) one week before inducing carcinoma with dimethylbenz ( a ) anthracene . fig1 shows the period from 30 to 85 days following administration of dmba . one curve in fig1 shows the average number of tumors per animal in the group protected by depo - provera while the other curve shows the average number of tumors per animal in the unprotected group . it is estimated that the 30 mg . injection of depo - provera would release approximately 0 . 17 mg . of active medroxyprogesterone acetate per day over a six - month period . as may be seen by comparing the two graphs in fig1 the depo - provera - treated group showed much greater resistance to the development of tumors than did the unprotected group . after 85 days an average of 1 . 89 tumors per rat was observed in the unprotected group , . while only 0 . 30 tumor per rat was observed in the depo - provera protected group . tumor number and size measured with calipers were determined weekly . treatment of mammary . carcinoma induced by dimethylbenz ( a ) anthracene in the rat , by low dose medroxyprogesterone acetate fig2 illustrates the inhibition of mammary carcinoma growth which may be achieved in accordance with the methods of the invention . tumors were induced in ovariectomized rats using dimethylbenz ( a ) anthracene . estradiol was used to stimulate growth in both a treatment and control group of rats . each animal in the treatment group received a single subcutaneous administration of 30 mg of depo - provera ( which is estimated to release approximately 0 . 17 mg . per day of active medroxyprogesterone acetate for a period of about six months ). this figure illustrates the average estradiol - stimulated change in total tumor area in each group following treatment . as may be seen in fig2 the group treated with depo - provera exhibited significantly less tumor growth than the untreated group . the terms and descriptions used herein are preferred embodiments set forth by way of illustration only , and are not intended as limitations on the many variations which those of skill in the art will recognize to be possible in practicing the present invention as defined by patent claims based thereon .
a method of treatment or prevention of breast and endometrial cancer , osteoporosis and endometriosis in susceptible warm - blooded animals comprising administering a low dose . of a progestin or other steroid derivative having androgenic activity and low masculinizing activity . pharmaceutical compositions useful for such treatment and pharmaceutical kits containing such compositions are disclosed . an in vitro assay permitting specific measurements of androgenic activity of potentially useful compounds is also disclosed .