the instant invention relates to the use of 5 - androstene 3β , 17α diol ( herein referred to as αaed or 17 αaed ), its esters and ethers , to inhibit growth and accelerate cell aging , induce apoptosis and death of tumor cells as a means of treating malignancies . the active agents of the invention may also be used as contraceptives and abortifacients . the active agents are of the structure : wherein r may be h , alkenyl of 2 - 8 carbons , alkyl of 1 - 8 carbons , phenylalkyl of 1 - 4 carbons , phenyl or cor 2 , wherein r 2 is h ; alkyl of 1 - 8 carbons , alkenyl of 2 - 8 carbons , phenylalkyl wherein the alkyl has 1 - 4 carbons ( including benzyl ) or phenyl . any phenyl moiety may have up to three substituents chosen from among hydroxy , carboxy of 1 - 4 carbons , halo , alkoxy of 1 - 4 carbons , alkyl of 1 - 4 carbons , or alkenyl of 2 - 4 carbons and wherein any alkyl may be a straight chain , branched chain , or the alkyl may be wholly or partially cyclized . it has been found that these active agents , when administered as described herein , inhibit cell growth . the αaed is administered in sufficient dosages to provide a blood concentration of from 5 to 10 , 000 ηm when given systemically . a more preferred blood or tissue fluid concentration is in the rang of 10 to 10 , 000 ηm . the dosage will vary with the type of cell to be inhibited . the method of administration will depend on the location of the target cells . such means as parenteral or oral administration are also appropriate . the αaed may also be administered by applicator or in a spray to tissue during surgery . compositions containing the active agents taught herein may be administered vaginally or rectally either by instillation of a liquid composition or on supports such as sponges . other preferred methods of administration include buccal , sublingual , nasal or endotracheal routes . sprays or mists may be useful for such administration . furthermore , sprays may be useful for administration to the operative area during surgery . for example , sprays may be used to contact the peritoneal cavity or the thoracic cavity during surgery . compositions of the invention may also be administered to the intestinal mucosa by oral or rectal routes . suppositories , solutions for use as retention enemas and creams or jellies are appropriate means for use in rectal administration . compositions of the invention may also be applied to the vaginal mucosa using creams , jellies suppositories or douching solutions . the compositions may be in the form of prophylactic vaginal preparations or may be used in lubricants on condoms . jellies and creams may also be administered by application in a cervical diaphragm which , when in place , will provide for prolonged contact with the cervix . for purposes of administration into an orifice of the body , the compositions of the invention may be administered via a flexible tube into the target site . as indicated previously , the compositions containing αaed may be administered as a douche or retention enema . other target sites include the bladder , uterus , trachea , nasopharynx , sinus or ( via the nasal passage ) the pituitary . the carrier system used in a given instance will depend on the mode of administration . the active agents are lipophilic compounds . solvents and carriers for lipophilic steroids known in the art are appropriate for use in the compositions containing αaed or the esters and ethers of αaed . examples of such carriers are glycols such as polypropylene glycol , polyethylene glycol , ethanol , dmso and cyclodextrins ( especially the amorphous cyclodextrins ). cyclodextrins will pass through the buccal mucosa into the circulation easily . this method is particularly appropriate for administration as a means of avoiding intravenous administration while bypassing the liver . other vehicles that should be considered include fatty acid esters of polyoxyethylene sorbitol ( tweens ) or sorbitan ( spans ) for preparation of emulsions . the compositions taught herein may be used to treat most neoplasms , including for , for example , use in treatment of neoplasms , including those of the blood - forming organs , the liver , pancreas , thyroid , andrenals , pituitary , ovaries , testicles , breast , central nervous system ( including brain , spinal column ), bone , connective tissue , lungs , liver , the gastro - intestinal system , uterus , mucous membranes , mouth and tongue , the lining of the peritoneum , the lymphatics and sensory organs . the βaed , 17 beta - oestradiol and tamoxifen were obtained from sigma chemical company ( st . louis , mo .). the αaed and flutamide were obtained from steraloid , inc . ( walton , n . h .) and schering corporation ( kenilworth , n . j . ), respectively . all steroids except βaed were dissolved in ethanol . βaed was dissolved in dmso : ethol ( 1 : 1 v / v ). stock solutions were filtered and kept at 4 ° c . for testing , stock solutions were diluted in media immediately before use . the final concentration of vehicle was always ≦ 0 . 2 % in all samples , and this concentration had no significant cytotoxic effect on the human breast cell cancer cell line designated zr - 75 - 1 ( american type culture collection ) as determined by trypan blue exclusion . the zr - 75 - 1 cell line ( passage 89 ) was obtained from the american type culture collection ( rockville , md .). the cells were cultured in rpmi - 1640 medium containing 10 % heat - inactivated fetal calf serum , 200 μm l - glutamine , 10 nm hepes , 1 . 5 u / ml penicillin , and 1 . 5 μg / ml streptomycin in 5 % co 2 at 37 ° c . the cells were passaged twice weekly . cells were first seeded at initial density of 1 × 10 5 cells per ml in quadruplicates in 24 multi - well flat bottom plates ( costar ). cells were then allowed to adhere and grow in phenol red - free rpmi 64 media supplemented with 10 % heat - inactivated fetal calf serum ( fcs ), 200 μm l - glutamine , 10 mm hepes , 2 . 5 u / ml penicillin and 2 . 5 μg / ml streptomycin in 5 % co 2 at 37 ° c . after 48 hours , four wells were sacrificed and counted to determine plating efficiency . in the remaining wells , medium was removed by aspiration and cells in each well were exposed to the media containing the specified steroid . the controls containing only medium or medium with vehicle were also prepared . in all samples , medium was changed every 48 hours . at the pre - established time - point . cells were removed by trypsinization and washed . cell number and viability were determined by trypan blue exclusion using a hemocytometer . parallel cultures were also run to determine cell proliferation . for cell proliferation assays , cell suspensions were prepared by trypsinization of cells from cultures prepared in accord with methods described above . cell viability was determined by trypan blue exclusion . the cells were then seeded in flat - bottom 96 - well microtiter plates at a density of 2 × 10 3 cells / well and were allowed to rest for 48 hours in order to adhere . non - adherent dead cells were removed by aspiration . cells were then grown in media without phenol red . some of the media contained supplements as indicated above . the active agents or vehicle control were added to the media . the cells were then grown for six days . media was changed on the samples every 48 hours . on day 6 , cells were pulsed with 1 μci [ 3 j ]- thymidine for the last 6 hours of incubation before harvesting onto glass filter using a hpd cell harvester ( cambridge technology , watertown , mass .) and counted on a klb scintillation counter . initial tests were carried out to determine the optimal ( maximal ) dose of αaed required to inhibit growth of the zr - 75 - 1 cells in in vitro as determined by tritiated tymidine incorporation . at concentration of 12 . 5 ηm or greater , the αaed significantly inhibited the growth of zr - 75 - 1 cells , and this anti - proliferative effect occurred in a dose and time - dependent manner at half - maximal ( 50 ηm ) and maximal dose ( 100 ηm ) levels . to ensure that inhibition was not due to cytotoxicity , cell count and viability were assessed by trypan blue exclusion . the addition of the 17αaed was not toxic to the cells . as opposed to αaed , the βaed ( an epimere ) alone at 100 ηm concentrations did not have any antiproliferative effect on the growth of the zr - 75 - 1 cells . zr - 75 - 1 cells ( 2 × 10 3 ) were treated as described above over a six day period with differing concentrations of αaed or with vehicle - only cultures . the medium was changed every 48 hours . cells were pulsed with 3 h - thymidine for the last six hours of incubation . results showed increasing proliferation at 6 . 25 ηm with decreasing cell proliferation at 12 . 5 ηm and marked decrease at concentrations of 50 ηm concentrations . cells were treated as in example 1 , except that in some samples a combination of 17αaed and βaed were used . the concentration of the αaed varied while the concentration of the βaed in the samples containing the combination of agents remained constant at 2 . 5 ηm . cells were pulsed with 3 h - thymidine for the last six hours . the proliferation of cells in cultures containing αaed in the presence of βaed showed decreased proliferation at all concentrations of αaed . effects of αaed on growth of zr - 75 - 1 cells in the presence of estradiol was studied . cells treated with increasing concentration of αaed in the presence or absence of 1 ηm concentration of estradiol over a 6 day period were studied . cells were treated with increasing concentration of αaed in the presence of 1 ηm concentration of estradiol over a 6 day period , with medium changed every 48 hours . cells were pulsed with 3 h - thymidine for the last six hours of incubation . at higher doses of αaed the αaed suppressed proliferation even in the presence of estradiol . hence , estradiol can not effectively overcome the antiproliferative activity of αaed on this human breast cancer cell line . ( this indicates that αaed may be useful in prevention of estrogen - dependent breast cancer . effects of αaed on growth of zr - 75 - 1 cells in the presence of flutamide , an antiandrogen , were studied using the process of example 3 except that estradiol was replaced with flutamide . the administration of αaed with antiandrogens , especially in treating estrogen - dependent malignancies such as breast cancer , should be considered particularly advantageous treatment option . example 5 was again studied in the presence of ru486 . again , it was shown that at effective concentrations it was possible to lower dosage of ru486 in the presence of effective amounts of 0 . 1 μl concentrations of αaed with 0 . 5 μm concentration of ru486 there was synergistic action to decrease proliferation of cells . this synergism was shown to be even greater at ru486 concentrations of 1 μm . this combination of active agents would be especially useful for treatment of tumors which are dependent on estrogen or progesterone . effect of αaed on growth of lymphoid neoplasm ( p388d1 cells obtained from the american type culture collection ) in the presence of ru486 at concentration of 0 . 5 μm and 1 . 0 μm was studied in accord with the methods described above . it was found that the use of αaed in combination with ru486 resulted in increased effectiveness over use of one agent . effects of αaed at doses of 50 ηm and 100 ηm doses on murine macrophage myeloma cells ( raw 264 . 7 , obtained from the american type culture collection ) was studied . at both 50 ηm and 100 ηm levels there was significant inhibition of proliferation . water , 100 ml , is mixed with 7 g . β - hydroxypropyl cyclodextrin and 1 mg αaed . fill ampules with the solution and sterilize . this preparation may be added to solutions for administration to the mucosa , for oral administration , or for parenteral administration . the cyclodextrin / αaed preparation is prepared as above . the material is freeze - dried and placed in sterile ampules . the resulting powder may be placed in vials . the contents of the vials may then be snorted into the nasal cavity . it is also appropriate to dissolve the contents of the vials and place in solution for intravenous or topical application , including for infusion into a wound site . it may also be applied by spraying or sponging into the operative site such as the abdominal or thoracic cavity . the preparation of example 12 is diluted with 100 ml water . the preparation is sprayed into the abdominal cavity during and after removal of a colon malignancy . αaed may be delivered to or through the skin by any means , including subcutaneous or intradermal injection or topical application . one means of topical application is the use of skin patches impregnated with the active agent . this means of delivery is advantageous since it is non - invasive and easily administered by relatively unskilled care providers . capsules of a formulation of αaed for oral administration is prepared by mixing 2 mg . αaed , 15 mg . starch and 5 mg . magnesium stearate . the capsules are administered twice a day to achieve a daily dosage of 1 - 50 mg ./ da . the compositions of the invention may be administered intrathecally either at the spinal level or into the cisterna magna . when αaed , its esters or ethers are administered orally , it is necessary that the active agents be protected from destruction and absorption in the upper gastro - intestinal tract . the active agents are most effective when the period of exposure to the mucosa of the intestinal tract is increased . hence use of capsules containing the active agents in formulations that effect slow release in the intestine are appropriate . the active agents may also be used in veterinary medicine for treatment of animals suffering from tumors . for purposes of such treatment , the αaed may be added to the chow of the animals . the sterile solutions may be administered to the lung either by bronchioscopic means or by mist which may be under pressure . patched for administration of αaed may be formulated as adhesive patches containing the active agent . for example , the patch may be a discoid in which a pressure - sensitive silicone adhesive matrix containing the active agent may be covered with a non - permeable backing . the discoid may either contain the active agent in the adhesive or may be attached to a support made of material such as polyurethane foam or gauze that will hold the active agent . when patches are used in treating animals , the area must be shaved or plucked . in all instances , the area to which the patch is applied should be cleaned carefully before application . a patch composed of trilaminate of an adhesive matrix sandwiched between a non - permeable backing and a protective covering layer is prepared in the following manner : to a pressure - sensitive silicone adhesive composition biopsa ™ q7 - 2920 ( dow corning corporation , midland , mich ., u . s . a .) in cyclohexane ( 50 % w / v ) is added sufficient αaed to provide a 0 . 5 % αaed composition . the adhesive is applied to a polyester film to provide in successive layers about 2 mg . of active agent per cm 2 . patches should be covered with a protective layer which will be removed before application . patches may be prepared containing permeation enhancers such as cyclodextrin , butylated hydroxyanisole , or butylated hydroxytoluene . the active agents may be administered to the mucosa of oral , pharyngeal or nasal cavity by tablet or lozenge . the antiproliferative agents taught herein may be used in conjunction with other active agents such as vinca alkaloids , nucleic acid inhibitors , platinum agents , interleukin - 2 , interferons , alkylating agents , antimetabolites , corticosteroids , dna intercalating agents , anthracyclines and ureas . examples of specific agents , in addition to those exemplified herein , include hydroxyurea , 5 - fluorouracil , anthramycin , asparaginase , bleomycin , dactinomycin , dacabazine , cytarabine , busulfan , thiotepa , lomustine , mechlorehamine , cyclophosphamide , melphalan , mechlorethamine , chlorambucil , carmustine , 6 - thioguanine , methotrexate , etc .
the instant application provides means of accelerating cell aging and programed cell death in tumor cells by administration of 3β , 17α androstenediol or its ethers or esters .