TREATING CONDITIONS WITH (+)-LIAROZOLE

Abstract: Methods and compositions are disclosed utilizing optically pure (+)-liarozole for the treatment of conditions supported by estrogen or caused by elevated estrogen levels such as estrogen-dependent breast cancer, gynecomastia, systemic lupus erythematosus and premature labor while substantially reducing the concomitant liability of adverse effects associated with the racemic mixture of liarozole. Methods and compositions are also disclosed utilizing optically pure (+)-liarozole for the treatment of conditions supported by or caused by elevated testosterone levels, such as hormone dependent prostate cancer. Further disclosed are methods and compositions utilizing pure (+)-liarozole in combination with retinoic acid for enhancing therapy with retinoic acid, such as in the treatment of neuroectodermal and neuroepithelial malignancies, including glioma, promyelocytic leukemia and hormone-independent prostate cancer.

Description: TREATING CONDITIONS WITH ( -J- )-LIAROZOLE TECHNICAL FIELD The invention relates to novel compositions of matter containing optically pure (+)-liarozole and methods for using the same. More particularly, the invention relates to the use of (+)-liarozole for treatment in humans for conditions supported by high levels of estrogen or testosterone and/or low levels of retinoic acid. BACKGROUND OF THE INVENTION This invention relates to novel compositions of matter containing optically pure (+)-liarozole. These compositions possess potent activity in treating estrogen-dependent breast cancer, gynecomastia, systemic lupus erythematosus, premature labor and other diseases including those that would benefit from a selective inhibition of aromatase. It is also an effective agent for treating conditions supported by or caused by elevated testosterone levels and for treating conditions that are ameliorated by increased levels of retinoic acid, either endogenous or exogenous. Optically pure (+)-liarozole provides this treatment while substantially reducing adverse effects including, but not limited to, nausea, vomiting, decreased appetite, fatigue, leg cramps, light-headedness, orthostatic hypotension, hot flashes and suppressed serum androgen and aldosterone levels, which may be associated with the administration of the racemic mixture of liarozole. Also disclosed are methods for treating the above described conditions in a human while substantially reducing the adverse effects that are associated with the racemic mixture of liarozole by administering the (+) isomer of liarozole to said human. The active compound of these compositions and methods is an optical isomer of liarozole. The preparation of racemic liarozole is described in U.S. Patent 4,859,684 and U.S. Patent 5,157,046. Chemically, the active compound is the (+) isomer of 5-[ (3-chlorophenyl) (lH-imidazol-1-yl)methyl]-lH- benzimidazole hereinafter referred to as liarozole. The absolute stereochemistry of the (+) isomer is presently unknown. Formula I indicates a single enantiomer of undetermined absolute stereochemistry, here presented arbitrarily as R * : The graphic representation of enantiomerically pure liarozole is taken from Maehr J. Chem. Ed. 62. 114- 120 (1985) . Thus, wedge outlines and dotted or broken lines denote enantiomerically pure compounds of indeterminate absolute configuration. (+)-Liarozole, which is the subject of the present invention, is not presently commercially available. All of the medicinal chemistry that has been reported has utilized the racemic mixture, which is available for research purposes only. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute con iguration of the molecule about its chiral center(s) . The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. There is no correlation between nomenclature for the absolute stereochemistry and for the rotation of an enantiomer. Thus, D- lactic acid is the same as (-) lactic acid, and L- lactic acid is (+) . For a given chemical structure, these chiral compounds exist as a pair of enantiomers which are identical except that they are non- superimposable mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric or racemic mixture. Stereochemical purity is of importance in the field of pharmaceuticals, where 12 of the 20 most prescribed drugs exhibit chirality. A case in point is provided by the L-form of the beta-adrenergic blocking agent, propranolol, which is known to be 100 times more potent than the D-enantiomer. Furthermore, optical purity is important since certain isomers may actually be deleterious rather than simply inert. For example, it has been suggested that the D-enantiomer of thalidomide was a safe and effective sedative when prescribed for the control of morning sickness during pregnancy, while the corresponding L-enantiomer has been believed to be a potent teratogen. Neither the separation of racemic liarozole into (+)-liarozole and (-)-liarozole nor the enantioselective synthesis of (+)-liarozole has been previously described. Racemic liarozole is presently in clinical trials as an adjunct to retinoic acid for the treatment of primary malignant brain tumors and carcinoma of the prostate. Estrogen has been implicated in the progression of several diseases, including human breast cancer, where estrogens appear to provide the major hormonal support for cancer cells. The main source of estrogen production in postmenopausal women is the extraglandular conversion of androstenedione to estrone; the adrenal glands per se produce little or no estrogens. Androstenedione is secreted and converted to estrone in peripheral tissues via the multicomponent aromatase enzyme complex. Estrone can be either conjugated into estrone sulfate to form a slowly revolving storage pool with a potential for back conversion to estrone, or reduced to estradiol (E 2 ) , the major active estrogenic steroid. Fat and muscle contain the majority of extraglandular aromatase activity present in postmenopausal women. It has recently been reported that approximately two thirds of human breast cancers contain measurable aromatase activity. It is generally accepted that estrogen deprivation plays an important role in the endocrine treatment strategy for patients with breast cancer. Inhibitors of the aromatase enzyme system have become of interest during recent years; they are used both to lower systemic estrogen levels and, perhaps more importantly, to inhibit intracellular conversion of androgens to estrogens by tumor-cell aromatase. Aromatase occurs widely in tissues such as adipose tissue, brain, testes, and ovaries. The enzyme is a membrane-bound microsomal complex containing NADPH-cytochrome c reductase and cytochrome P-450 units. The mechanism by which androstenedione is converted to estrone has been studied in detail. This conversion can be competitively inhibited by substrate mimics. Aromatase inhibition can also be achieved by compounds which bind directly to the cytochrome P-450 of the enzyme. The inhibition of 20,22-desmolase, also a cytochrome P-450 containing enzyme, prevents the oxidative side-chain cleavage of 20α,22β- dihydroxycholesterol and results in a blockage of not only estrogen biosynthesis but also the biosynthesis of other important secretory steroids. Since this cleavage is a step common also to cortisol biosynthesis, replacement therapy is necessary when desmolase is inhibited. Aromatization is the final step in the biogenesis of estrone and estradiol and is, consequently, the most effective step at which to selectively interfere without affecting the biogenesis of other steroids. Racemic liarozole has been found to be an effective inhibitor of mammalian aromatase. As a result of its aromatase inhibitory activity racemic liarozole may also be useful to treat gynecomastia, systemic lupus erythematosus, premature labor and other diseases. Racemic liarozole has also been found to be an effective inhibitor of retinoic acid 4-hydroxylase, another p-450 dependent enzyme. As such, it appears to prolong the half-life of exogenously administered retinoic acid, and may therefore be useful for the treatment of neuroectodermal and neuroepithelial malignancies such as glioma, promyelocytic leukemia and hormone-independent prostate cancer. Thus it would be particularly desirable to find a compound with the advantages of the racemic mixture of liarozole which would not have the adverse effects associated with the racemic mixture. SUMMARY OF THE INVENTION It has now been discovered that the optically pure (+) isomer of liarozole is an effective agent for treating estrogen-dependent breast cancer, gynecomastia, systemic lupus erythematosus and premature labor and other conditions including those that would benefit from a selective inhibition of aromatase. It is also an effective agent for enhancing therapy with retinoic acid for treating neuroectodermal and neuroepithelial malignancies such as glio a, promyelocytic leukemia and hormone- independent prostate cancer. It is also an effective agent for treating conditions supported by or caused by elevated testosterone levels. The optically pure (+) isomer of liarozole provides this effective treatment while substantially reducing adverse effects of racemic liarozole including, but not limited to, nausea, vomiting, decreased appetite, fatigue, leg cramps, light-headedness, orthostatic hypotension, hot flashes and suppressed serum aldosterone levels. The present invention also includes methods for treating the above described conditions in a human while substantially reducing adverse effects that are associated with the racemic mixture of liarozole by administering the optically pure (+) isomer. DETAILED DESCRIPTION OF THE INVENTION The present invention encompasses a method of treating breast cancer, which comprises administering to a human in need of such therapy, an amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, said amount being sufficient to retard the growth of the cancer. The method substantially reduces the concomitant liability of adverse effects associated with the administration of the racemic compound by providing an amount which is insufficient to cause adverse effects associated with the racemic mixture of liarozole. The present invention also encompasses a composition for the treatment of a human afflicted with breast cancer, which comprises a therapeutically effective amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, and a pharmaceutically acceptable carrier. A therapeutically effective amount is an amount sufficient to suppress the growth of the cancer but insufficient to cause the adverse effects associated with racemic liarozole. A further aspect of the present invention includes a method of treating a condition supported by estrogen or caused by or contributed to by elevated estrogen levels in a human, which comprises administering to a human in need of such therapy, an amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, sufficient to reduce estrogen levels. The method substantially reduces the concomitant liability of adverse effects associated with the administration of racemic liarozole by providing an amount which is insufficient to cause adverse effects associated with the administration of racemic liarozole. Conditions associated with elevated estrogen levels in humans may include, but are not limited to, gynecomastia, systemic lupus erythematosus, and premature labor. In addition, the invention encompasses a method of treating prostate cancer, which comprises administering to a human in need of such therapy, an amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, said amount being sufficient to retard the growth of the cancer. The (+)-liarozole may optionally be administered in combination with retinoic acid. The method substantially reduces the concomitant liability of adverse effects associated with the administration of racemic liarozole by providing an amount which is insufficient to cause adverse effects associated with the racemic mixture of liarozole. The invention further encompasses a method of treating conditions supported by or caused by elevated testosterone levels in a human which comprises administering to a human in need of such therapy an amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer. The invention also encompasses a method of treating a condition ameliorated by retinoic acid in a human which comprises administering an amount of (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, sufficient to suppress the degradation of retinoic acid. In addition, the invention encompasses a pharmaceutical composition which comprises a retinoic acid, preferably all trans retinoic acid, and a therapeutically effective amount of (+) liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, and a pharmaceutically acceptable carrier. The racemic mixture of liarozole (i.e., a 1:1 mixture of the two enantiomers) exhibits anticancer activity through its potent aromatase and 4-hydroxylase inhibition, thus providing therapy and a reduction of symptoms in a variety of conditions and disorders related to the presence of estrogen or testosterone in disadvantageous amounts or of retinoic acid in inadequate amounts. However, this racemic mixture, while offering the expectation of efficacy, causes adverse effects. Utilizing the optically pure or substantially optically pure isomer of (+)-liarozole results in enhanced efficacy, diminished adverse effects and, accordingly, an improved therapeutic index. It is therefore more desirable to use the (+) isomer of liarozole than to administer the racemic mixture. The term "adverse effects" includes, but is not limited to, nausea, vomiting, decreased appetite, fatigue, leg cramps, light-headedness, orthostatic hypotension, hot flashes and suppressed serum androgen and aldosterone levels, and may also include cheilitis, conjunctivitis and desquamation when liarozole is coad inistered with retinoic acid. The term "substantially free of its (-) stereoisomer" as used herein means that the compositions contain at least 90% by weight of (+)-liarozole and 10% by weight or less of (-)-liarozole. In a more preferred embodiment the term "substantially free of the (-) isomer" means that the composition contains at least 99% by weight of (+)-liarozole, and 1% or less of (-)-liarozole. In the most preferred embodiment, the term "substantially free of its (-) stereoisomer" as used herein means that the composition contains greater than 99% by weight of (+)-liarozole. These percentages are based upon the total amount of liarozole in the composition. The terms "substantially optically pure (+) isomer of liarozole" or "substantially optically pure (+)-liarozole" and "optically pure (+) isomer of liarozole" and "optically pure (+)-liarozole" are also encompassed by the above-described amounts. The term "treating breast cancer" or "treating prostate cancer" as used herein means treating, alleviating or palliating such condition and suppressing the growth of cancerous tissue, thus providing increased survival time. The term "treating a condition supported by estrogen or contributed to by elevated levels of estrogen" as used herein means treating, alleviating or palliating such disorders, thus providing relief from the symptoms of the aforementioned conditions or slowing the progression of the disease. Among such conditions are gynecomastia, systemic lupus erythematosus, and premature labor. The term "treating a condition supported by testosterone or caused by elevated levels of testosterone" as used herein means treating, alleviating or palliating such disorders, thus providing relief from the symptoms of the aforementioned conditions or slowing the progression of the disease. Among such conditions are androgen dependent prostate carcinoma. The term "treating a condition ameliorated by retinoic acid" as used herein means treating, alleviating, or palliating such disorders, thus providing relief from the symptoms of the aforementioned conditions or slowing the progression of the disease. Among such conditions are neuroectodermal and neuroepithelial malignancies, glioma, promyelocytic leukemia and hormone- independent prostate cancer. The chemical synthesis of the racemic mixture of liarozole can be performed by the method described in U.S. Patent 4,859,684 cited above. The (+) isomer of liarozole may be obtained by resolution of the enantiomers of liarozole or of precursors thereto using conventional means such as fractional crystallization of diastereomeric salts with chiral acids. Other standard methods of resolution known to those skilled in the art including, but not limited to, simple crystallization and chromatographic resolution, can also be used. (See for example, E.L. Eliel, Stereochemistry of Carbon Compounds. McGraw Hill (1962) and [ ilen and Lochmuller, "Tables of Resolving Agents", Journal of Chromatography 113. 283-302 (1975)]. In addition, the a ine precursor of structure (VI) of Raeymaekers et al. U.S. Patent No. 4,857,684 may be resolved by fractional crystallization of diastereomeric salts with chiral acid. The magnitude of a prophylactic or therapeutic dose of (+)-liarozole in the acute or chronic management of disease will vary with the severity and nature of the condition to be treated and the route of administration. The dose and perhaps the dose frequency will also vary according to the age, body weight and response of the individual patient. In general, when treating conditions such as prostate cancer, which are supported by elevated testosterone levels, the total daily dose range for (+)-liarozole for such conditions described herein is from about 100 mg to about 900 mg in single or divided doses. Preferably a daily dose range should be from about 200 mg to about 600 mg in single or divided doses, while most preferably a daily dose range should be about 400 mg in single or divided doses. However, in the treatment of conditions such as breast cancer, which are supported by estrogen or caused by elevated estrogen levels, the total daily dose range for (+)- liarozole for such conditions described herein is from about 50 mg to about 300 mg in single or divided doses. Preferably a daily dose range should be about 100 mg to about 300 mg in single or divided doses, while most preferably a daily dose range should be about 200 mg in single or divided doses. In managing the patient, the therapy should be initiated at a lower dose, perhaps at about 50 mg to about 150 mg, and increased up to about 400 mg or higher depending on the patient's global response. It is further recommended that patients over 65 years and those with impaired renal or hepatic function initially receive low doses and that they be titrated based on individual response(s) and blood level(s). It may be necessary to use dosages outside these ranges in some cases, as will be apparent to those skilled in the art. Further, it is noted that the clinician or treating physician will know how and when to interrupt, adjust, or terminate therapy in conjunction with individual patient response. The terms "a therapeutically effective amount," "an amount sufficient to suppress cancer but insufficient to cause said adverse effects" and "an amount sufficient to reduce estrogen levels but insufficient to cause said adverse effects" are encompassed by the above-described dosage amounts and dose frequency schedule. Any suitable route of administration may be employed for providing the patient with an effective dosage of (+)-liarozole. For example, oral, rectal, parenteral (subcutaneous, intramuscular, intravenous) , transdermal, and like forms of administration may be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, patches, and the like. The pharmaceutical compositions of the present invention comprise (+)-liarozole as the active ingredient, or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients. The terms "pharmaceutically acceptable salts" or "a pharmaceutically acceptable salt thereof" refer to salts prepared from pharmaceutically acceptable non- toxic acids. Suitable pharmaceutically acceptable acid addition salts for the compound of the present invention include acetic, benzenesulfonic (besylate) , benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, aleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, and the like. Hydrochloride and fumarate salts are particularly preferred. The compositions of the present invention include suspensions, solutions, elixirs, or solid dosage forms. Carriers such as starches, sugars, and microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like are suitable in the case of oral solid preparations (such as powders, capsules, and tablets) , and oral solid preparations are preferred over the oral liquid preparations. Because of their ease of administration, tablets and capsules represent the most advantageous oral dosage unit forms, in which case solid pharmaceutical carriers are employed. If desired, tablets may be coated by standard aqueous or nonaqueous techniques. In addition to the common dosage forms set out above, the compounds of the present invention may also be administered by controlled release means and delivery devices such as those described in U.S.Patent Nos. : 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, the disclosures of which are hereby incorporated by reference. Pharmaceutical compositions of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient, as a powder or granules, or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil liquid emulsion. Such compositions may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation. For example, a tablet may be prepared by compression or molding, optionally, with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active agent or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent. Desirably, each tablet contains from about 50 mg to about 300 mg of the active ingredient, and each cachet or capsule contains from about 50 mg to about 300 mg of the active ingredient. Most preferably, the tablet, cachet or capsule contains either one of three dosages, about 100 mg, about 200 mg or about 300 mg of (+)-liarozole for oral administration. The invention is further defined by reference to the following examples describing in detail the preparation of the compositions of the present invention, as well as their utility. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the purpose and interest of this invention. The relative activity, potency and specificity of optically pure liarozole and racemic liarozole as an inhibitor of aromatase can be determined by a pharmacological study in vitro according to accepted methods. The assay is performed in a total volume of 1 L at 37° C. Briefly, 120 μg of human placental microsomal protein is incubated with 11 μg of [4- 14 C]androstene-3,17-dione ([4- 14 C]A), 2.4 x 10" 4 M NADPH (tetrasodium salt) , and the appropriate concentration of inhibitor. The [4- 14 C]A is added as a solution in 1.7% ethanol in 0.05M potassium phosphate buffer (pH 7.4) so that the final concentration of ethanol does not exceed 0.02% (v/v) . The reaction is started by the addition of enzyme and stopped after 20 min by the addition of ethyl acetate. Following extraction and centrifugation, the aqueous phase is reextracted with ethyl acetate. The extract is evaporated to dryness and dissolved in acetone prior to being chromatographed on silica gel 60 thin-layer plates using ethyl acetate/isooctane (140:60) or toluene/chloroform/methanol (70:140:20). The radioactive estradiol and estrone peaks are identified by comparison with authentic standards and quantified with a liquid scintillation counter. The tests provide an estimate of relative activity and potency. Differential inhibition by racemic liarozole and its enantiomers of the various enzymes involved in conversion of cholesterol to other steroids can be assessed by the method of Newton et al. [Jj_ Steroid Biochem. Molec. Biol. 39. 723-727 (1991)]. A male guinea pig (Duncan-Hartley, 500-650g) is killed by cervical dislocation and the kidneys and attached adrenal glands are carefully removed. The kidney and fatty tissue are dissected away and the intact adrenal is cut in half, length-wise. The two pieces of each adrenal are placed on a cooled Petri dish (4°C) and cut into cubes of about 1 mm 3 with a fine blade. The divided tissue is then placed into lOmL Minimal Essential Medium (MEM) containing Earl's salts, 20 mM Hepes buffer, but without L-glutamine (MEM-A) (4°C), previously gassed for 5-10 minutes with 95% 0 2 /5% C0 2 . Fragments of tissue are washed (x2) with fresh media (MEM-A, 4°C) and poured into a small gas chamber (50 L with magnetic stirring bar) . MEM (10 mL) containing 2mg/mL collagenase (MEM-B) is added. The chamber is kept at 37° C and gassed with 95% 0 2 /5% C0 2 . Cells are allowed to disperse for 10 minutes following which the medium is removed and discarded. A further 10 mL aliquot of MEM-B is added to fragments and dispersion is carried out for 10 min. Medium containing dispersed cells (supernatant 2) is removed and kept on ice. Cell dispersion is carried out for a further 2 cycles and supernatants 2-4 are finally centrifuged at 4° C for 5 minutes at 400 g. A portion of each supernatant is removed and discarded leaving 5 mL above the cell pellet and 1.5 mL MEM containing 2mM ascorbate 0.5% BSA and 8 mM CaCl 2 (MEM-C, made up just prior to addition) is added. Starting with supernatant 3, cells are carefully resuspended (without air bubbles) and suspensions are filtered through 1 μm nylon mesh. The pooled cell suspension is centrifuged at 400 g for 5 minutes, the supernatant removed and 10 mL MEM- C added to resuspend cells. Centrifugation is again performed for 5 minutes at 400 g and the pellet is resuspended in 3 L MEM-C. Approximately equal aliquots of this suspension are then placed into two chambers of a multi-well plate (24 well) and left for 2 h in an incubation chamber flushed with 95% 0 2 /5% C0 2 . Following this period, cells are resuspended by gentle flushing with covering medium, removed from the wells and centrifuged for 5 minutes at 400 g. The supernatant is removed and the pelleted cells are resuspended in 2 mL MEM containing 4mM ascorbate, 0.5% BSA and 8 mM CaCl 2 (MEM-D) . Cell number is determined with a haemocytometer using trypan blue to exclude counting non-viable cells. The number of cells in the preparation is adjusted to give between 2.5 x 10° and 5 x 10 6 cells/mL by the addition of MEM- D. To determine the basal production of steroids, portions of the cell suspension are added to wells of microtitre plates followed by 25 μL of a 10% solution of dimethylsulphoxide (DMSO) in MEM-A containing 0.5% BSA and 8 mM CaCl 2 (MEM-E) and 25 μL MEM-A alone. To observe the effect of ACTH on steroid output, 25 μL MEM-E containing 10% DMSO is added to 50 μL cell suspension followed by 25 μL ACTH (200 pg/mL in MEM- A) . The effect of (+)-liarozole, (-) liarozole and racemic liarozole on ACTH-stimulated steroidogenesis is determined in wells containing 50 μL cell suspension, 25 μL drug solution in MEM-E and 25 μL ACTH in MEM-A. Incubation is conducted for 90 minutes in an atmosphere of 95% 0 2 /5% C0 2 . 17-Hydroxyprogesterone and andostenedione (A) are measured using direct radioimmunoassays ("RIAs") . Medium from each well is diluted 1:5 in steroid stripped human serum prior to assay. Cortisol is measured in the medium covering each of the cell layers by a direct RIA. The aromatase enzyme is measured by quantifying the amount of tritiated water released from radiolabelled A during aromatization to estrone. Placental microsomes and NADPH (ImM) are added in 0.1 M sodium phosphate buffer (pH 7.4) to assay tubes containing 0.5 μCi [l3- 3 H]A and unlabelled aromatase inhibitors (lO'^-lO^M final concentration) , to give a final volume of l mL. After incubation for 1 hour at 37° C, 0.5 L of trichloroacetic acid is added followed by 1 mL of activated charcoal suspension (5% v/v) . Following incubation for 30 minutes at 37° C, tubes and contents are centrifuged at 1500 g for 15 minutes at 4° C and radioactivity is determined in 1 mL aliquots of each supernatant. In order to determine IC 50 values, counts observed at each concentration are calculated as a percentage of those observed in control tubes. Inhibition of retinoic acid catabolis is measured by the method of Van Wauwe et al. [J.P.E.T. 252. 365 (1990)]. Male istar rats weighing 200-220 g are treated p.o. with test compound or vehicle (PEG 200) in a volume of 0.5 mL per 100 g of body weight. One hour later, the animals are anesthetized with diethylether and injected i.v. with retinoic acid (RA) at a dose of 0.1 mg/kg. At designated times after this injection, rats are sacrificed by decapitation and trunk blood collected on heparin (500 U/mL) . After centrifugation (1000 X g, 15 min) , plasma fractions are recovered and processed immediately. In experiments performed to assess the effects on endogenous plasma levels of RA, rats are treated p.o. with racemic liarozole, (-) liarozole and (+) liarozole or PEG 200 and sacrificed. To avoid isomerization of RA, all extraction experiments are performed under subdued yellow light. Plasma samples (1 L) are diluted with 1 mL of water and mixed with 18 mL of acetonitrile to precipitate proteins. After centrifugation (1000 X g, 5 min) , supernates are recovered, diluted with 18 mL of 40 mM acetic acid and passed through 100-mg Cι g minicolumns. After washing the minicolumns with 4 mL of 40% acetonitrile in water, the absorbed material is eluted with 3 mL of methanol. The eluates are evaporated in vacuo and the residues dissolved in 300 μL of mobile phase for reverse-phase HPLC analysis. Extraction samples (250 μL) are analyzed on a 10-μm C u column. The mobile phase consists of methanol-water-formic acid (85:15:0.05) containing 30 mM ammonium acetate at a flow rate of 1 mL/min. The effluent is monitored by UV absorbance detection at 350 nm and RA (retention time 7-8 min) is quantified by peak-area integration. A standard curve is prepared by extracting rat plasma to which known amounts of RA had been added. Additional experiments are performed on adult, male ferrets to correlate dosage and side effects (i.e. emesis) . The animals are first adapted to wearing a nylon jacket connected to a stainless-steel cable, which in turn is attached to a brass swivel at the cage top. After habituation to the tether- harness, each animal receives a surgically implanted catheter in its right jugular vein. The catheter is flushed daily with heparinized sodium chloride. The drug studies are conducted 1 week after the surgical procedure. Tethered animals are individually housed. Eight to eleven animals are used to evaluate each dose of each test compound. Individual animals are weighed weekly and randomly given, at greater than 48 hour intervals, a single i.v. or p.o. dose of control vehicle, racemic liarozole, (+)-liarozole or (-)-liarozole. At least three dose levels of each test compound are evaluated. Individual animals are observed for 30 min following administration of test substance. The frequency of, and latency to all expulsions, retches and defecations are recorded. Data obtained from dose-response curves is tested for statistical significance by chi-square analysis. ED 50 values are determined for each compound. EXAMPLES Example 1 ORAL FORMULATION Capsules: Formula Quant: Lty per capsule in mg A B C (+) -Liarozole 100 200 300 Lactose 330 230 330 Cornstarch 65 65 65 Magnesium Stearate 5 5 5 Fill Weight 500 500 700 The (+)-liarozole, lactose and cornstarch are blended until uniform and then the magnesium stearate is blended into the resulting powder, which is sieved and filled into suitably sized, two-piece, hard gelatin capsules using conventional machinery. Other doses may be prepared by altering the fill weight and, if necessary, changing the capsule size to suit. Example 2 ORAL FORMULATION Tablets: Formula Quantity per tablet in mg A B C (+) -Liarozole 100 200 300 Lactose 309 209 309 Cornstarch 30 30 30 Water (per thousand Tablets)* 300 mL 300 mL 300 mL Cornstarch 60 60 60 Magnesium Stearate 1 1 1 Compression Weight 500 500 700 *The water evaporates during manufacture The (+)-liarozole is blended with the lactose until a uniform blend is formed. The smaller quantity of cornstarch is blended with the water to form the resulting corn starch paste. This is then mixed with the uniform blend until a uniform wet mass is formed. The remaining cornstarch is added to the resulting wet mass and mixed until uniform granules are obtained. The granules are then screened through a suitable milling machine, using a 1/4 inch stainless steel screen. The milled granules are dried in a suitable drying oven until the desired moisture content is obtained. The dried granules are then milled through a suitable milling machine, magnesium stearate is blended in, and the resulting mixture is compressed into tablets of the desired shape, thickness, hardness and disintegration. Tablets of other strengths may be prepared by altering the ratio of active ingredient to the excipients or to the final weight of the tablet.

Claims: What is claimed is: 1. A method of treating breast cancer in a human which comprises administering to said human a therapeutically effective amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer. 2. The method of claim 1 wherein (+)-liarozole is administered by parenteral, transdermal, or oral administration. 3. The method of claim 2 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof administered is from about 50 mg to about 300 mg per day. 4. The method of claim 3 wherein the amount administered is from about 100 mg to about 300 mg per day. 5. The method of claim 4 wherein the amount administered is about 200 mg per day. 6. The method of claim 1 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof is greater than approximately 90% by weight of the total amount of liarozole. 7. The method of claim 1 wherein the amount of said (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, is administered together with a pharmaceutically acceptable carrier. 8. A pharmaceutical composition which comprises a therapeutically effective amount of (+)- liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, and a pharmaceutically acceptable carrier. 9. The composition according to claim 8 adapted for oral administration. 10. The composition according to claim 8 adapted for parenteral delivery. 11. A method of treating a condition supported by estrogen or caused by elevated estrogen levels in a human which comprises administering to said human a therapeutically effective amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, said amount being sufficient to reduce estrogen. 12. The method according to claim 11 wherein said condition is chosen from the group consisting of gynecomastia, systemic lupus erythematosus, and premature labor. 13. The method of claim 11 wherein (+)-liarozole is administered by parenteral, transdermal, or oral administration. 14. The method of claim 13 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof administered is from about 50 mg to about 300 mg per day. 15. The method of claim 14 wherein the amount administered is from about 100 mg to about 300 mg per day. 16. The method of claim 15 wherein the amount administered is about 200 mg per day. 17. The method of claim 11 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof is greater than approximately 90% by weight of the total amount of liarozole. 18. The method of claim 11 wherein the amount of said (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, is administered together with a pharmaceutically acceptable carrier. 19. The composition according to claim 10 further comprising a retinoic acid. 20. The composition according to claim 19 wherein said retinoic acid is substantially all trans-retinoic acid. 21. A method of treating prostate cancer in a human which comprises administering to said human a therapeutically effective amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer. 22. The method of claim 21 wherein (+)- liarozole is administered by parenteral, transdermal, or oral administration. 23. The method of claim 22 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof administered is from about 100 mg to about 900 mg per day. 24. The method of claim 22 wherein the amount administered is from about 200 mg to about 600 mg per day. 25. The method of claim 24 wherein the amount administered is about 400 mg per day. 26. The method of claim 21 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof is greater than approximately 90% by weight of the total amount of liarozole. 27. The method of claim 21 wherein the amount of said (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, is administered together with a therapeutically effective amount of retinoic acid. 28. A method of treating a condition supported by testostrone or caused by elevated testosterone levels in a human which comprises administering to said human a therapeutically effective amount of (+)-liarozole, or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer. 29. The method of claim 28 wherein (+)-liarozole is administered by parenteral, transdermal, or oral administration. 30. The method of claim 29 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof administered is from about 100 mg to about 900 mg per day. 31. The method of claim 30 wherein the amount administered is from about 200 mg to about 600 mg per day. 32. The method of claim 31 wherein the amount administered is about 400 mg per day. 33. The method of claim 28 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof is greater than approximately 90% by weight of the total amount of liarozole. 34. The method of claim 28 wherein the amount of said (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, is administered together with a pharmaceutically acceptable carrier. 35. A method of treating a condition ameliorated by retinoic acid in a human which comprises administering a therapeutically effective amount of (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-)-stereoisomer, said amount being sufficient to suppress the degradation of retinoic acid. 36. A method according to claim 35 wherein said condition is ameliorated by the inhibition of retinoic acid 4-hydroxylase. 37. A method according to claim 35 wherein said condition is chosen from the group consisting of neuroectodermal and neuroepithelial malignancies. 38. A method according to claim 35 wherein said condition is chosen from the group consisting of glioma, promyelocytic leukemia and hormone- independent prostate cancer. 39. The method of claim 35 wherein (+)-liarozole is administered by parenteral, transdermal, or oral administration. 40. The method of claim 39 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof administered is from about 100 mg to about 600 mg per day. 41. The method of claim 40 wherein the amount administered is from about 200 mg to about 400 mg per day. 42. The method of claim 41 wherein the amount administered is about 400 mg per day. 43. The method of claim 35 wherein the amount of (+)-liarozole or a pharmaceutically acceptable salt thereof is greater than approximately 90% by weight of the total amount of liarozole. 44. The method of claim 35 wherein the amount of said (+)-liarozole or a pharmaceutically acceptable salt thereof, substantially free of its (-) stereoisomer, is administered together with a pharmaceutically acceptable carrier.

Inventors: GRAY NANCY M, GRAY, NANCY, M.
Applicants: SEPRACOR INC, SEPRACOR, INC.
Application year: 1995
Main classification: A61K31
IPC codes: A61K31, A61P35, C07D403