PHARMACEUTICAL COMPOSITIONS AND METHODS COMPRISING A COMBINATION OF A SELECTIVE ESTROGEN RECEPTOR MODULATOR AND AN AROMATASE INHIBITOR

Field of the Invention The present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof a combination of a selective estrogen receptor modulator and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof. Particularly, the present invention relates to pharmaceutical compositions and methods of treatment comprising administering to a patient in need thereof (-)-cis-6-phenyl-5-[4-(2- pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalen e-2-ol or a pharmaceutically acceptable salt or prodrug thereof and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof.

Background of the Invention U.S. Patent No. 5,552,412, incorporated herein by reference, discloses SERM compounds of the formula

wherein the variables are defined as set forth therein. Cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl- ethoxy)-phenyl)-5,6,7,8,-tetrahydronaphthalene-2-ol, and more particularly (-)-cis-6-phenyl-5-(4- (2-pyrrolidin-1 -yl-ethoxy)-phenyl)-5,6,7,8,-tetrahydronaphthalene-2-ol is an orally active, highly potent SERM which prevents bone loss, decreases total serum cholesterol, and does not have estrogen-like uterine stimulating effects in OVX rats. U.S. Patent No. 6,204,286, also incorporated herein by reference, discloses substantially pure (-)-cis-6-phenyl-5-(4-(2-pyrrolidin- 1-yl-ethoxy)-phenyl)-5,6,7,8,-tetrahydronaphthalene-2-ol. U.S. Patent No. 5,948,809, also incorporated herein by reference, discloses (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)- phenyl)-5,6,7,8,-tetrahydronaphthalene-2-ol, tartrate salt. U.S. Patent Application No. 09/833,169, filed April 11 , 2001 , discloses methods of treating female sexual dysfunction with compounds such as cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8 ,- tetrahydronaphthalene-2-ol, of the Formula immediately hereinabove.

The present invention provides for pharmaceutical compositions and methods of treatment using a combination of the selective estrogen receptor modulator (-)-cis-6-phenyl-5-[4- (2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphtha lene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor, or a pharmaceutically acceptable salt or prodrug thereof.

Summary of the Invention

The present invention provides pharmaceutical compositions comprising the compound (- )-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7 ,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof. Particular embodiments of this invention are pharmaceutical compositions comprising (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)- phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof wherein the aromatase inhibitor is selected from aminoglutethimide; formestane; atamestane; anastrazole; fadrozole; finrozole; letrozole; vorozole; 4-[N-(4-bromobenzyl)-N-(4- cyanophenyl)amino]-4H-1 ,2,4-triazole or 6-methyleneandrosta-1 ,4-diene-3,17-dione, also known as exemestane, or a pharmaceutically acceptable salt or prodrug thereof. More particularly, the present invention provides pharmaceutical compositions comprising (-)-cis-6-phenyl-5-[4-(2- pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and exemestane or a pharmaceutically acceptable salt or prodrug thereof.

The present invention also provides a method of treating metabolic bone disease, senile osteoporosis, postmenopausal osteoporosis, steroid induced osteoporosis, low bone turnover osteoporosis, osteomalacia, renal osteodystrophy, osteoarthritis, rheumatoid arthritis, bone fractures, bone grafts, wrinkles, ovarian cancer, glioma, renal cell cancer, liver cancer, colon cancer, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, hypogonadism, andropause, frailty, muscle damage, sarcopenia, osteosarcoma, anorexia, low bone mass resulting from aggressive athletic behavior, prevention of second hip fracture, vaginal atrophy, endometrial hyperplasia and female sexual dysfunction, the method comprising administering to a patient in need thereof a therapeutically effective amount of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 - yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and a therapeutically effective amount of an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof. A particular embodiment of the present method of treatment is the method wherein the (-

)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5, 6,7,8-tetrahydronaphthalene-2-ol or a

pharmaceutically acceptable salt or prodrug thereof and aromatase inhibitor are administered orally. Additional embodiments of this invention are methods of treatment as described above wherein the (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and aromatase inhibitor are administered parenterally or transdermally. Further embodiments of this invention are methods of treatment wherein the aromatase inhibitor is selected as described hereinabove for the pharmaceutical compositions of the invention. A particular method of treatment is a method of treating breast cancer, the method comprising administering to a patient in need thereof a therapeutically effective amount of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl- ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and a therapeutically effective amount of exemestane.

Detailed Description of the Invention The present invention relates to pharmaceutical compositions and methods of treating metabolic bone disease, senile osteoporosis, postmenopausal osteoporosis, steroid induced osteoporosis, low bone turnover osteoporosis, osteomalacia, renal osteodystrophy, osteoarthritis, rheumatoid arthritis, bone fractures, bone grafts, wrinkles, ovarian cancer, glioma, renal cell cancer, liver cancer, colon cancer, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, hypogonadism, andropause, frailty, muscle damage, sarcopenia, osteosarcoma, anorexia, low bone mass resulting from aggressive athletic behavior, prevention of second hip fracture, vaginal atrophy, endometrial hyperplasia and female sexual dysfunction using a combination of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5, 6,7,8- tetrahydronaphthalene-2-olor a pharmaceutically acceptable salt or prodrug thereof and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof. In a preferred embodiment, the present invention relates to a method of treating metabolic bone disease, senile osteoporosis, postmenopausal osteoporosis, steroid induced osteoporosis, low bone turnover osteoporosis, osteomalacia, renal osteodystrophy, osteoarthritis, rheumatoid arthritis, bone fractures, bone grafts, wrinkles, ovarian cancer, glioma, renal cell cancer, liver cancer, colon cancer, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, hypogonadism, andropause, frailty, muscle damage, sarcopenia, osteosarcoma, anorexia, low bone mass resulting from aggressive athletic behavior, prevention of second hip fracture, vaginal atrophy, endometrial hyperplasia and female sexual dysfunction using (-)-cis-6- phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetra hydronaphthalene-2-ol, D-tartrate salt and exemestane.

-A-

In a preferred embodiment, the methods of treatment using the combination are senile osteoporosis, postmenopausal osteoporosis, bone fractures, bone grafts, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, frailty, muscle damage and sarcopenia.

Osteopenia is a thinning of the bones, but less than is seen with osteoporosis and is the stage before true osteoporosis. The World Health Organization has developed diagnostic categories based on bone mass density (BMD) to indicate if a person has normal bones, has osteopenia or has osteoporosis. Normal bone density is within one standard deviation (+1 or -1) of the young adult mean bone density. Osteopenia (low bone mass) is defined as bone density of 1 to 2.5 standard deviations below the young adult mean (-1 to -2.5), and osteoporosis is defined as a bone density that is 2.5 standard deviations or more below the young adult mean (>-2.5).

Hypogonadism is generally defined as inadequate gonadal function, as manifested by deficiencies in gametogenesis and/or the secretion of gonadal hormones, which can result in retardation of puberty and/or reproductive insufficiency. There are three main types of hypogonadism: 1) primary hypogonadism; 2) secondary hypogonadism and 3) resistance hypogonadism. In primary hypogonadism damage to the Leydig cells impairs androgen production. In secondary hypogonadism disorder of the hypothalamus or pituitary impairs gonadotropin secretion and in resistance hypogonadism, the body response to androgen is inadequate. Anorexia is a disease that has the following characterisitcs: refusal to maintain body weight at or above a minimally normal weight for age and height (e.g., weight loss leading to maintenance of body weight less than 85% of that expected; or failure to make expected weight gain during period of growth, leading to body weight less than 85% of that expected);intense fear of gaining weight or becoming fat, even though underweight; and disturbance in the way in which one's body weight or shape is experienced, undue influence of body weight or shape on self- evaluation, or denial of the seriousness of the current low body weight. The compounds and combinations of the present invention can be used to treat anorexia and can be used to treat bone loss associated with anorexia.

Another condition that can be treated using the combinations of the present invention is bone loss associated with aggressive athletic behavior, particularly in women. Aggressive participation in exercise, athletics or sports such as ballet and distance running can result in bone loss, which is usually accompanied in women by ammenorhea. Men who also exhibit aggressive athletic behavior also exhibit bone loss.

Andropause (also called male menopause or viropause) is a natural occurrence in men that typically happens between the age of forty and fifty-five. Andropause is a decline in the level of the hormone testosterone. As testosterone levels decline, and men enter andropause, various

changes or conditions may be observed including decreased energy and strength, increased body fat, osteoporosis, depression, decreased mental acuity, inability to maintain muscle, cardiovascular disease, atherosclerosis, decreased libido, decreased strength of orgasms, erectile dysfunction, increased irritability, and aching and stiff joints, particularly in the hands and feet. In addition, males undergoing or having undergone andropause can have gynecomastia, serum lipid disorders, including hypercholesterolemia, reduced vascular reactivity, hypogonadism, and benign prostatic hyperplasia.

Frailty is characterized by the progressive and relentless loss of skeletal muscle mass resulting in a high risk of injury from fall, difficulty in recovery from illness, prolongation of hospitalization, and long-term disability requiring assistance in daily living. The reduction of muscle mass, physical strength and physical performance typically leads to diminished quality of life, loss of independence, and mortality. Frailty is normally associated with aging, but may also result when muscle loss and reduced strength occur due to other factors, such as disease- induced cachexia, immobilization, or drug-induced sarcopenia. Another term that has been used to denote frailty is sarcopenia, which is a generic term for the loss of skeletal muscle mass, or quality. Examples of skeletal muscle properties that contribute to its overall quality include contractility, fiber size and type, fatiguability, hormone responsiveness, glucose uptake/metabolism, and capillary density. Loss of muscle quality, even in the absence of loss of muscle mass, can result in loss of physical strength and impaired physical performance. The term 'muscle damage' as used herein is damage to any muscle tissue. Muscle damage can result from physical trauma to the muscle tissue as the result of accidents, athletic injuries, endocrine disorders, disease, wounds or surgical procedures. The methods of the present invention are useful for treating muscle damage by facilitating muscle damage repair. Osteoporosis in the elderly woman is determined by the amount of peak bone mass gained in adolescence leading to adulthood, the premenopausal maintenance of such peak bone mass, and the rate of postmenopausal bone mass loss. Determinants of peak bone mass include genetic, nutritional, weight loading (exercise), and environmental factors. Enhancement of peak bone mass in adolescence is therefore desirable in order to maximize the skeletal mass in order to prevent the development of osteoporosis later in life. Likewise, enhancement of peak bone mass in adolescence for males is also desirable.

Hip fracture has a significant impact on medical resources and patient morbidity and mortality. Few patients admitted with a hip fracture are considered for prophylactic measures aimed at the reduction of further fracture risk. Currently, 10-13% of patients will later sustain a second hip fracture. Of patients who suffered a second hip fracture, fewer patients maintained their ability to walk independently after the second fracture than did so after the first (53 and 91 % respectively, P<0.0005). Pearse E.O. et al., Injury, 2003, 34(7), 518-521. Following second hip

fracture, patients' level of mobility determined their future social independence. Older patients and those with a history of multiple falls had a shorter time interval between fractures. Second hip fracture has a significant further impact on patients' mobility and social independence. It is therefore desirable to have new methods for the prevention of second hip fracture. Osteosarcoma is a relatively common, highly malignant primary bone tumor that has a tendency to metastasize to the lungs. Osteosarcoma is most common in persons 10 to 20, though it can occur at any age. About half of all osteosarcomas are located in the region of the knee but it can be found in any bone. Pain and a mass are the usual symptoms of osteosarcoma. Typical treatment for osteosarcoma is chemotherapy in combination with surgery. Either preoperative or postoperative chemotherapy with agents such as methotrexate, doxorubicin, cisplatin or carboplatin can be used to treat the osteosarcoma.

Endometrial hyperplasia is an increase in the number of cells in the endometrium whereby the bulk of the endometrium is increased. The endometrium is the mucous membrane comprising the inner layer of the uterine wall and consists of a simple columnar epithelium and a lamina propria that contains simple tubular uterine glands. The structure, thickness, and state of the endometrium undergo marked change with the menstrual cycle.

Female sexual dysfunction (FSD) is characterized by psychological and/or physiological changes associated with the sexual response cycle. The current description of the sexual response cycle was introduced by Basson and is a modified version of the models introduced by Masters and Johnson in 1966 and Kaplan in 1979. Basson's model consists of 5 non-linear elements including intimacy needs, sexual stimuli, sexual arousal, sexual desire and enhanced intimacy. The model suggests a receptive desire stemming from sexual arousal (Basson FL J Sex Marital Then 2001 , 27(5). 395-403). The 4 major subtypes of FSD based on the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV; American Psychiatric Association, 1994) classification with recent modifications by the American Foundation for Urologic Disease (AFUD) are described below. All of the definitions include the element of personal distress.

Hypoactive sexual desire disorder (HSDD): persistent or recurrent deficiency (or absence) of sexual fantasies/thoughts and/or desire for or receptivity to sexual activity, which causes marked distress or interpersonal difficulty. This category contains a sub-category of sexual aversion disorder which is the persistent or recurring phobic aversion to and avoidance of sexual contact with a sexual partner, which causes personal distress.

Female sexual arousal disorder (FSAD): persistent or recurrent inability to attain or maintain sufficient excitement, causing personal distress, which may be expressed as a lack of subjective excitement, or genital (lubrication/swelling) or other somatic responses.

Female orgasmic disorder (FOD): persistent or recurrent difficulty, delay in, or absence of attaining orgasm following sufficient sexual stimulation and arousal, which causes personal distress.

Sexual pain disorder: (dyspareunia, vaginismus, and other sexual pain disorders): recurrent or persistent genital pain associated with sexual intercourse or the recurrent or persistent involuntary spasm of the musculature of the outer third of the vagina that interferes with vaginal penetration, which causes personal distress.

Most experts believe that the subtypes are not necessarily discrete and frequently coexist in the same woman such that the first 3 disorders (desire, arousal, and orgasm) are often closely related. The most common FSD subtypes are HSDD and FSAD. Isolated FOD is far less common. The co-morbidity of these diagnoses makes differentiating primary from secondary diagnoses quite difficult. The sexual response cycle is mediated by a delicate, balanced interplay between the sympathetic and parasympathetic nervous systems. For example, vasocongestion is largely mediated by parasympathetic (cholinergic) outflow; orgasm is predominantly sympathetic (adrenergic). The response cycle is easily inhibited by cortical influences or by impaired hormonal, neural, or vascular mechanisms, by medical or surgical interventions, by psychological or emotional problems, or by combinations of these factors. Sexual dysfunctions may be lifelong (no effective performance ever, generally due to intrapsychic conflicts) or acquired (after a period of normal function); generalized (global) or situational (limited to certain situations or certain partners); and total or partial.

HSDD is often characterized by complaints of a lack of interest in sex, even in ordinarily erotic situations. The disorder can be associated with infrequent sexual activity, often causing serious marital or relationship conflict. However, some patients have sexual encounters fairly often to please their partners and may have no difficulty with performance but continue to have sexual apathy. The acquired form of HSDD is commonly caused by boredom or unhappiness in a long-standing relationship (often situational where sexual desire may be normal or even intense with others), depression (which leads more often to decreased interest in sex than it does to impotence in the male or to inhibited excitement in the female), anxiety, dependence on alcohol or psychoactive drugs, illness, fatigue, side effects from prescription drugs (e.g., antihypertensives, antidepressants), surgical interventions, and hormonal deficiencies such as low testosterone levels, due either to natural menopause or to surgical menopause. This disorder can be secondary to FSAD, FOD, or pain disorders.

Sexual excitement and pleasure are accompanied by a combination of vascular and neuromuscular events which lead to engorgement of the clitoris, labia and vaginal wall, increased vaginal lubrication and dilatation of the vaginal lumen (Levin, R.J., Clin. Obstet. Gvnecol.. 1980:7; 213-252; Ottesen, B., Gerstenberg, T., Ulrichsen, H. et al., Eur. J. Clin. Invest., 1983:13;

321-324; Levin, R.J.. Exp. Clin. Endocrinol., 1991 :98; 61-69; Levin, R.J., Ann. Rev. Sex Res.. 1992:3; 1-48; Masters, W. H., Johnson, V. E. Human Sexual Response. Little, Brown: Boston, 1996; Berman, J.R., Berman, L & Goldstein, L, Urology, 1999:54; 385-391). Vaginal engorgement enables transudation to occur and this process is responsible for increased vaginal lubrication. Transudation allows a flow of plasma through the epithelium and onto the vaginal surface, the driving force for which is increased blood flow in the vaginal capillary bed during the aroused state. In addition engorgement leads to an increase in vaginal length and luminal diameter, especially in the distal 2/3 of the vaginal canal. The luminal dilatation of the vagina is due to a combination of smooth muscle relaxation of its wall and skeletal muscle relaxation of the pelvic floor muscles. Some sexual pain disorders such as vaginismus are thought to be due, at least in part, by inadequate relaxation preventing dilatation of the vagina; it has yet to be ascertained if this is primarily a smooth or skeletal muscle problem. (Masters, W. H., Johnson, V. E. Human Sexual Response. Little, Brown: Boston, 1996; Berman, J.R., Berman, L. & Goldstein, L, Urology, 1999:54; 385-391). FSAD may be experienced as a lack of subjective excitement or lack of genital response such as vaginal lubrication, vaginal engorgement, clitoral sensation or smooth muscle relaxation. Orgasms may be impeded. Sexual dysfunction due to a physical disorder is usually generalized (not specific to a given partner or situation) and can be due to a number of factors including medications (e.g., diuretics, antihistamines, antidepressants, e.g., Selective Serotonin Reuptake Inhibitors [SSRIs] or antihypertensive agents), pelvic surgery, prior pelvic trauma, decreased vaginal or clitoral blood flow, reduced estrogen at menopause or after childbirth and during lactation, or by illnesses with vascular components such as diabetes and atherosclerosis. Psychological factors may underlie FSAD as they may inhibit a woman's response and prevent her from relaxing into the arousal process (Walsh K. & Berman, J., 2004, Drugs Aging 21 (10), 655-675). This disorder can be secondary to HSDD, FOD, or pain disorders.

In women, orgasm is accompanied by contractions (not always subjectively experienced as such) of the muscles of the outer third of the vagina. Generalized muscular tension, perineal contractions, and involuntary pelvic thrusting (every 0.8 sec) usually occur. Orgasm can be followed by a sense of general pleasure, well-being, and muscular relaxation. Although women can be orgasmic throughout their lives, sexual activity often decreases after age 60 because of the relative lack of partners and untreated physiologic changes (e.g., atrophy of the vaginal mucosa, with resultant dryness and painful coitus).

Lifelong FOD is often associated with emotional trauma or sexual abuse. Acquired FOD can be the result of surgery, trauma, hormone deficiencies, emotional stressors, relationship conflicts, or medications (e.g, SSRIs) (Walsh K. & Berman, J., 2004, Drugs Aging. 21 (10), 655- 675). FOD can be secondary to HSDD, FSAD, or pain disorders.

Sexual pain disorders include dyspareunia [pain with intercourse], vaginismus [involuntary muscle spasms of the outer third of the vagina, and other genital pain caused by non-coital sexual stimulation] . Dyspareunia is usually introital but may also occur before, during, or after intercourse. Pain during or after coitus is the chief complaint. Pain disorders may be caused by pelvic floor disorders such as cystocele, rectocycle, and uterine prolapse, medications which reduce lubrication, endometriosis, pelvic inflammatory disease, inflammatory bowel diseases urinary tract problems, previous OB/GYN trauma, radiation atrophy, inflammation of the rectum or vagina, or menopausal involution with dryness and thinning of the mucosa. Psychological issues can also play a significant role. Vaginal atrophy is one of the most common and unpleasant conditions of menopause that women experience. Beginning in perimenopause, the vaginal epithelium becomes increasingly avascular, thin, and nonkeratinized; the number of superficial mature cells significantly decreases and may disappear altogether; intermediate and parabasal cells, which contain much less glycogen, increase and predominate. The combination of these effects causes an increase in vaginal pH (often > 5.0), vaginal dryness and an increased susceptibility to urinary and vaginal infections.

The present invention provides methods of treatment that ameliorate potential adverse side effects associated with the administration of aromatase inhibitors alone and provide certain synergistic effects. The present methods of treatment comprising combination therapy with (-)- cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof provide the advantages of decreased incidence and/or severity of bone loss, bone fracture, osteoarthritis, arthralgia, dyspareunia, vaginal atrophy and cardiovascular events when compared with aromatase monotherapy. In the treatment of breast cancer the combination therapy with (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl- ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof is particularly advantageous and produces a synergistic effect in treating breast cancer when compared to the administration of either agent alone. The present invention is also concerned with pharmaceutical compositions comprising (-

)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5, 6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor or a pharmaceutically acceptable salt or prodrug thereof and a carrier, solvent, diluent and the like for treating metabolic bone disease, senile osteoporosis, postmenopausal osteoporosis, steroid induced osteoporosis, low bone turnover osteoporosis, osteomalacia, renal osteodystrophy, osteoarthritis, rheumatoid arthritis, bone fractures, bone grafts, wrinkles, ovarian cancer, glioma,

renal cell cancer, liver cancer, colon cancer, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, hypogonadism, andropause, frailty, muscle damage, sarcopenia, osteosarcoma, anorexia, low bone mass resulting from aggressive athletic behavior, prevention of second hip fracture, vaginal atrophy, endometrial hyperplasia and female sexual dysfunction. A preferred combination is a combination of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl- ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and exemestane. A particularly preferred combination is a combination of (-)-cis- 6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tet rahydronaphthalene-2-ol, D-tartrate salt and exemestane. As used herein, the term "hydroxy-protecting group" signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups (hereinafter referred to simply as "silyl" groups), and alkoxyalkyl groups. Alkoxycarbonyl protecting groups are alkyl-O-CO- groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert- butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term "acyl" signifies an alkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, malonyl, succinyl, or glutaryl group, or an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl substituted benzoyl group. The word "alkyl" as used in the description or the claims, denotes a straight-chain or branched alkyl radical of 1 to 10 carbons, in all its isomeric forms. Alkoxyalkyl protecting groups are groupings such as methoxymethyl, ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred silyl- protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl, diphenyl-t-butylsilyl and analogous alkylated silyl radicals. The term "aryl" specifies a phenyl-, or any alkyl-, nitro- or halo- substituted phenyl group.

A "protected hydroxy" group is a hydroxy group derivatized or protected by any of the above groups commonly used for the temporary or permanent protection of hydroxy functions, e.g., the silyl, alkoxyalkyl, acyl or alkoxycarbonyl groups, as previously defined. The terms "hydroxyalkyl", "deuteroalkyl" and "fluoroalkyl" refer to any alkyl radical substituted by one or more hydroxy, deuterium or fluoro groups respectively.

The second compound used in the combinations and methods of the present invention is an aromatase inhibitor. Aromatase inhibitors act by inhibition or inactivation of aromatase, an enzyme of the cytochrome P-450 superfamily and the product of the CYP19 gene. Aromatase is responsible for the synthesis of estrogens from androgenic substrates, such as the synthesis of estrone from the preferred substrate androstenedione and estradiol from testosterone. Aromatase inhibitors have been classified as first-, second- and third-generation inhibitors

according to the chronologic order or their clinical development and as type 1 or type 2 inhibitors according to their mechanism of action. Smith, M.D., I.E. and Dowsett, Ph.D., M, N Engl J Med 2003, 348, 2431-2442. The type 1 aromatase inhibitors are generally steroidal analogues of androstenedione that bind irreversibly to aromatase, thereby inactivating the enzyme. The type 2 aromatase inhibitors are generally nonsteroidal and bind reversibly to the heme group of the enzyme by way of a basic nitrogen.

Preferred aromatase inhibitors for use in the combinations and methods of the present invention include the compounds described in U.S. Patent Nos. 4,808,616 and 4,904,650. Those compounds are of formula (A) as described herein and as given below:

wherein: each of R and R ₂ , independently, is hydrogen or CrC ₆ alkyl and Ri is hydrogen, halogen or C ₁ -

C ₆ alkyl. Preferred compounds within formula (A) immediately above are those wherein R is hydrogen or C _r C ₄ alkyl; R ₁ is hydrogen, fluorine, chlorine or C _r C ₄ alkyl and R ₂ is hydrogen or CrC ₄ alkyl. More preferred compounds within this group are those of formula (A) wherein R is hydrogen, methyl or ethyl; R ₁ is hydrogen, fluorine or chlorine and R ₂ is hydrogen.

Examples of specific aromatase inhibitors include: 6-methylenandrosta-1 ,4-diene-3,17- dione; 1-methyl-6-methylenandrosta-1 ,4-diene-3,17-dione; 1-ethyl-6-methylenandrosta-1 ,4- diene-3,17-dione; 4-methyl-6-methylenandrosta-1 ,4-diene-3,17-dione; 4-ethyl-6- methylenandrosta-1 ,4-diene-3,17-dione; 4-fluoro-6-methylenandrosta-1 ,4-diene-3,17-dione; 4- chloro-6-methylenandrosta-1 ,4-diene-3,17-dione; 6-ethylidenandrosta-1 ,4-diene-3,17-dione; 6- propylidenandrosta-1 ,4-diene-3,17-dione; 4-fluoro-1-methyl-6-methylenandrosta-1 ,4-diene- 3,17-dione; 4-chloro-1-methyl-6-methylenandrosta-1 ,4-diene-3,17-dione; 1-methyl-6- ethylidenandrosta-1 ,4-diene-3,17-dione; and 4-fluoro-6-ethylidenandrosta-1 ,4-diene-3,17- dione.

A most preferred compound within the above group of aromatase inhibitors is 6- methylenandrosta-1 ,4-diene-3,17-dione, also known as exemestane, and marketed as Aromasin ^® . 6-Methylenandrosta-1 ,4-diene-3,17-dione can be prepared according to the

procedure of Example 1 in U.S. Patent Nos. 4,808,616 and 4,904,650 and which procedures are reproduced as follows:

0.50 g of 6-methylenandrost-4-ene-3,17-dione and 0.57 g of dichlorodicyanobenzoquinone (DDQ), were refluxed in 20 ml of anhydrous dioxane for about 15 hours. To remove the DDQ the suspension was filtered through alumina. After evaporation of the solvent the residue was dissolved in ethyl acetate, the organic layer washed with water, dried over sodium sulfate and the solvent removed under vacuum. The crude product was chromatographed on silica gel using hexane/ethyl acetate 40% to yield 0.25 g of pure 6- methylenandrosta-1 ,4-diene-3,17-dione, m.p. 188 ^Q - 191 ^s C, λ _ma *247 mp(ε 13.750). Found: C 81.01 , H 8.05. C ₂₀ H ₂₄ O ₂ requires: C 81.04, H 8.16.

Other exemplary aromatase inhibitors useful in the combinations and methods of the present invention include: 3-(4-aminophenyl)-3-ethyl-2,6-piperidinedione which is commonly called aminoglutethimide and is marketed as Cytadren ^® ; 4-hydroxyandrostenedione, also known as formestane; 1-methylandrosta-1 ,4-diene-3,17-dione, also known as atamestane; α,α,α,α'-tetramethyl-5-(1 H-1 ,2,4-triazol-1 -ylmethyl)-1 ,3-benzenediacetonitrile, also known as anastrazole, is described in U.S. RE36617 and is marketed as Arimidex ^® ; 4-(5,6,7,8- tetrahydroimidazo[1 ,5-a]pyridin-5-yl)-benzonitrile, monohydrochloride, also known as fadrozole; 4-(3-(4-Fluorophenyl)-2-hydroxy-1-(1 H-1 ,2,4-triazol-1-yl)-propyl)-benzonitrile, also known as finrozole; 4,4'-(1 H-1 ,2,4-triazol-1-ylmethylene)bis-benzonitrile, also known as letrozole, is described in U.S. Patent 4,978,672 and is marketed as Femara ^® ; 6-[(4-chlorophenyl)-1 H-1 ,2,4- triazol-1-ylmethyl]-1-methyl-1 H-benzotriazole, also known as vorozole; and 4-[N-(4- bromobenzyl)-N-(4-cyanophenyl)amino]-4H-1 ,2,4-triazole, also known as YM-511 which is being developed by Yamanouchi.

It is noted that when compounds are discussed herein, it is contemplated that the compounds may be administered to a patient as a pharmaceutically acceptable salt, prodrug, or a salt of a prodrug. All such variations are intended to be included in the invention.

The term "patient in need thereof" means humans and other animals who have or are at risk of having metabolic bone disease, senile osteoporosis, postmenopausal osteoporosis, steroid induced osteoporosis, low bone turnover osteoporosis, osteomalacia, renal osteodystrophy, osteoarthritis, rheumatoid arthritis, bone fractures, bone grafts, wrinkles, ovarian cancer, glioma, renal cell cancer, liver cancer, colon cancer, breast cancer, prostate cancer, obesity, osteopenia, male osteoporosis, hypogonadism, andropause, frailty, muscle damage, sarcopenia, osteosarcoma, anorexia, low bone mass resulting from aggressive athletic behavior, prevention of second hip fracture, vaginal atrophy, arthralgia, endometrial hyperplasia and female sexual dysfunction. As certain of the conditions being treated affect only females it is to ,

be understood that in such cases the patient is female. A preferred patient is a postmenopausal female human.

The term "treating", "treat" or "treatment" as used herein includes preventative (e.g., prophylactic), palliative, adjuvant and curative treatment. For example, the treatment of breast cancer, as used herein means that a patient having breast cancer or at risk of having breast cancer can be treated according to the methods described herein. For patients undergoing preventative treatment, a resulting reduction in the incidence of the disease state being preventively treated is the measurable outcome of the preventative treatment.

By "pharmaceutically acceptable" it is meant the carrier, diluent, excipients, and/or salts or prodrugs must be compatible with the other ingredients of the formulation, and not deleterious to the patient.

The term "prodrug" means a compound that is transformed in vivo to yield a compound of the present invention. The transformation may occur by various mechanisms, such as through hydrolysis in blood. A discussion of the use of prodrugs is provided by T. Higuchi and W. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

For example, when a compound used in the compositions and methods of the present invention contains a carboxylic acid functional group, a prodrug can comprise an ester formed by the replacement of the hydrogen atom of the acid group with a group such as (C _r C ₈ )alkyl, (C ₂ -C ₁₂ )alkanoyloxymethyl, 1 -(alkanoyloxy)ethyl having from 4 to 9 carbon atoms, 1-methyl-1- (alkanoyloxy)-ethyl having from 5 to 10 carbon atoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms, 1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms, 1-methyl-1- (alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms, N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms, 1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms, 3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl, di-N,N-(C _r C ₂ )alkylamino(C ₂ - C ₃ )alkyl (such as β-dimethylaminoethyl), carbamoyl-(CrC ₂ )alkyl, N,N-di(CrC ₂ )alkylcarbamoyl- (C _r C ₂ )alkyl and piperidino-, pyrrolidino- or morpholino(C ₂ -C ₃ )alkyl.

Similarly, when a compound used in the compositions and methods of the present invention comprises an alcohol functional group, a prodrug can be formed by the replacement of the hydrogen atom of the alcohol group with a group such as (C _r C ₆ )alkanoyloxymethyl, 1- ((Ci-C ₆ )alkanoyloxy)ethyl, 1 -methyl-1 -((C _r C ₆ )alkanoyloxy)ethyl, (C _r

C ₆ )alkoxycarbonyloxymethyl, N-(CrC ₆ )alkoxycarbonylaminomethyl, succinoyl, (CrC ₆ )alkanoyl, α-amino(CrC ₄ )alkanoyl, arylacyl and oc-aminoacyl, or α-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independently selected from the naturally occurring L-amino acids,

P(O)(OH) ₂ , -P(O)(O(C ₁ -C ₆ )HlRyI) ₂ or glycosyl (the radical resulting from the removal of a hydroxyl group of the hemiacetal form of a carbohydrate).

When a compound used in the compositions and methods of the present invention comprises an amine functional group, a prodrug can be formed by the replacement of a hydrogen atom in the amine group with a group such as R ^x -carbonyl, R ^x O-carbonyl, NR ^x R ^Xl - carbonyl where R ^x and R ^X| are each independently (Ci-C ₁₀ )alkyl, (C ₃ -C ₇ )cycloalkyl, benzyl, or R ^x -carbonyl is a natural α-aminoacyl or natural α-aminoacyl-natural α-aminoacyl, -C(OH)C(O)OY ^X wherein Y ^x is H, (C _r C ₆ )alkyl or benzyl), -C(OY ^X0 ) Y ^X1 wherein Y ^xo is (C ₁ -C ₄ ) alkyl and Y ^X1 is (CrC ₆ )alkyl, carboxy(CrC ₆ )alkyl, amino(C _r C ₄ )alkyl or mono-N- or di-N, N-(C ₁ - C ₆ )alkylaminoalkyl, -C(Y ^X2 ) Y ^X3 wherein Y^ is H or methyl and Y ^X3 is mono-N- or di-N,N-(C _r C ₆ )alkylamino, morpholino, piperidin-1-yl or pyrrolidin-1-yl.

The expression "pharmaceutically acceptable salt" refers to nontoxic anionic salts containing anions such as (but not limited to) chloride, bromide, iodide, sulfate, bisulfate, phosphate, acetate, maleate, fumarate, oxalate, lactate, tartrate, citrate, gluconate, methanesulfonate and 4-toluene-sulfonate. The expression also refers to nontoxic cationic salts such as (but not limited to) sodium, potassium, calcium, magnesium, ammonium or protonated benzathine (N,N'-dibenzylethylenediamine), choline, ethanolamine, diethanolamine, ethylenediamine, meglamine (N-methyl-glucamine), benethamine (N-benzylphenethylamine), piperazine or tromethamine (2-amino-2-hydroxymethyl-1 ,3-propanediol). It will be recognized that the compounds used in the compositions and methods of this invention can exist in radiolabeled form, i.e., said compounds may contain one or more atoms containing an atomic mass or mass number different from the atomic mass or mass number ordinarily found in nature. Radioisotopes of hydrogen, carbon, phosphorous, fluorine and chlorine include ³ H, ¹⁴ C, ³² P, ³⁵ S, ¹⁸ F and ³⁶ CI, respectively. Compounds used in the compositions and methods of this invention which contain those radioisotopes and/or other radioisotopes of other atoms are within the scope of this invention. Tritiated, i.e., ³ H, and carbon- 14, i.e., ¹⁴ C, radioisotopes are particularly preferred for their ease of preparation and detectability. Radiolabeled compounds of this invention can generally be prepared by methods well known to those skilled in the art. Conveniently, such radiolabeled compounds can be prepared by carrying out the procedures disclosed herein except substituting a readily available radiolabeled reagent for a non-radiolabelled reagent.

It will be recognized by persons of ordinary skill in the art that some of the compounds used in the compositions and methods of this invention have at least one asymmetric carbon atom and therefore are enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physicochemical differences by methods known perse as, for example, chromatography and/or fractional crystallization. Enantiomers can

be separated by converting the enantiomeric mixture into a diasteromeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing, including both chemical hydrolysis methods and microbial lipase hydrolysis methods, e.g., enzyme catalyzed hydrolysis) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers and mixtures thereof are considered as part of this invention. Also, some of the compounds used in the compositions and methods of this invention are atropisomers (e.g., substituted biaryls) and are considered as part of this invention.

In addition, when the compound (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]- 5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof or the aromatase inhibitors, form hydrates or solvates, they are also within the scope of the invention.

Administration of the compounds used in the compositions and methods of this invention can be via any method that delivers a compound of this invention systemically and/or locally. These methods include oral, parenteral, and intraduodenal routes, etc. Generally, the compounds of this invention are administered orally, but parenteral administration (e.g., intravenous, intramuscular, transdermal, subcutaneous, rectal or intramedullary) may be utilized, for example, where oral administration is inappropriate for the target or where the patient is unable to ingest the drug. The compounds used in the compositions and methods of this invention may also be applied locally to a site in or on a patient in a suitable carrier or diluent. In general an effective dosage for cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)- 5,6,7,8-tetrahydronapthalene-2-ol or a salt thereof, used in the pharmaceutical compositions and methods of this invention is in the range of 0.001 to 100 mg/kg/day, preferably 0.01 to 10 mg/kg/day. The aromatase inhibitors used in the combinations and methods of the present invention can be administered to a human patient in a dosage range of about 0.01 mg/kg to about 50 mg/kg. For example, aminoglutethimide can be administered initially at in a dosage of 250 mg given at 8-hour intervals and the dosage may be increased to a daily dose of 2 grams. Aminoglutethimide, marketed as Cytadren ^® , is commercially available as a 250 mg oral tablet. Other commercially available dosage forms of aromatase inhibitors include letrozole, marketed as Femara ^® , which is available as a 2.5 mg oral tablet, and anastrazole, marketed as Arimidex ^® , which is available as a 1 mg oral tablet. Exemestane, marketed as Aromasin ^® , is available as a 25 mg oral tablet. A preferred therapeutically effective amount of the pharmaceutical composition of this invention is about 0.01 mg/kg/day to about 20 mg/kg/day. The amount and timing of administration will, of course, be dependent on the subject being treated, on the severity of the affliction, on the manner of administration and on the

judgment of the prescribing physician. Thus, because of patient to patient variability, the dosages given herein are guidelines and the physician may titrate doses of the drug to achieve the treatment that the physician considers appropriate for the patient. In considering the degree of treatment desired, the physician must balance a variety of factors such as age of the patient, presence of preexisting disease, as well as presence of other diseases. The dose may be given once a day or more than once a day and may be given in a sustained release or controlled release formulation. It is also possible to administer the compounds using a combination of an immediate release and a controlled release and/or sustained release formulation. The administration of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5, 6,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and an aromatase inhibitor or the combination thereof can be according to any continuous or intermittent dosing schedule. Once a day, multiple times a day, once a week, multiple times a week, once every two weeks, multiple times every two weeks, once a month, multiple times a month, once every two months, once every three months, once every six months and once a year dosing are non-limiting examples of dosing schedules for (-)-cis-6-phenyl-5-[4-(2- pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and an aromatase inhibitor or the combination thereof. The compounds used in the compositions and methods of the present invention are generally administered in the form of a pharmaceutical composition comprising at least one of the compounds together with a pharmaceutically acceptable vehicle or diluent. Thus, the compounds used in the compositions and methods of this invention can be administered in any conventional oral, parenteral, rectal or transdermal dosage form.

For oral administration a pharmaceutical composition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the like. Tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate are employed along with various disintegrants such as starch and preferably potato or tapioca starch and certain complex silicates, together with binding agents such as polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the compounds of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents and/or suspending agents, as well as such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof. Acceptable dosage forms for (-

)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5, 6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and an aromatase inhibitor include tablets, capsules, solutions and suspensions. Other suitable formulations will be apparent to those skilled in the art. For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. In this connection, the sterile aqueous media employed are all readily obtainable by standard techniques well known to those skilled in the art.

For purposes of transdermal (e.g., topical) administration, dilute sterile, aqueous or partially aqueous solutions (usually in about 0.1% to 5% concentration), otherwise similar to the above parenteral solutions, are prepared. Methods of preparing various pharmaceutical compositions with a certain amount of active ingredient are known, or will be apparent in light of this disclosure, to those skilled in this art. For examples of methods of preparing pharmaceutical compositions, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 19th Edition (1995). The pharmaceutical combinations of this invention generally will be administered in a convenient formulation. The following formulation examples only are illustrative and are not intended to limit the scope of the present invention.

In the formulations that follow, "active ingredient" means a combination of a first compound, (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6 ,7,8- tetrahydronapthalene-2-ol, or a pharmaceutically acceptable salt or prodrug thereof and a second compound, an aromatase inhibitor. In a preferred embodiment, the first compound of the "active ingredient" used in the formulation is (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl- ethoxy)-phenyl)-5,6,7,8-tetrahydronapthalene-2-ol, D-tartrate salt and the second compound is exemestane.

Formulation 1 : Gelatin Capsules

Hard gelatin capsules are prepared using the following:

Ingredient Quantity (mg/capsule)

Active ingredient 0.25-100

Starch, NF 0-650

Starch flowable powder 0-50

Silicone fluid 350 centistokes 0-15

A tablet formulation is prepared using the ingredients below: Formulation 2: Tablets

Ingredient Quantity (mg/tablet)

Active ingredient 0.25-100 Cellulose, microcrystalline 200-650 Silicon dioxide, fumed 10-650 Stearate acid 5-15

The components are blended and compressed to form tablets.

Alternatively, tablets each containing 0.25-100 mg of active ingredients are made up as follows: Formulation 3: Tablets

Ingredient Quantity (mg/tablet)

Active ingredient 0.25-100

Starch 45

Cellulose, microcrystalline 35

Polyvinylpyrrolidone (as 10% solution in water) 4

Sodium carboxymethyl cellulose 4.5

Magnesium stearate 0.5

Talc 1

The active ingredient, starch, and cellulose are passed through a No. 45 mesh U.S. sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through a No. 14 mesh U.S. sieve. The granules so produced are dried at 50° - 60 ⁰ C and passed through a No. 18 mesh U.S. sieve. The sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through a No. 60 U.S. sieve, are then added to the granules which, after mixing, are compressed on a tablet machine to yield tablets.

Suspensions each containing 0.25-100 mg of active ingredient per 5 ml dose are made as follows:

Formulation 4: Suspensions

Ingredient Quantity (mg/5 ml)

Active ingredient 0.25-100 mg

Sodium carboxymethyl cellulose 50 mg

Syrup 1.25 mg

Benzoic acid solution 0.10 mL

Flavor q.v.

Color q.v.

Purified Water to 5 mL

The active ingredient is passed through a No. 45 mesh U.S. sieve and mixed with the sodium carboxymethyl cellulose and syrup to form smooth paste. The benzoic acid solution, flavor, and color are diluted with some of the water and added, with stirring. Sufficient water is then added to produce the required volume.

An aerosol solution is prepared containing the following ingredients: Formulation 5: Aerosol

Ingredient Quantity (% by weight)

Active ingredient 0.25

Ethanol 25.75

Propellant 22 (Chlorodifluoromethane) 70.00 The active ingredient is mixed with ethanol and the mixture added to a portion of the propellant 22, cooled to 30 ⁰ C, and transferred to a filling device. The required amount is then fed to a stainless steel container and diluted with the remaining propellant. The valve units are then fitted to the container.

Suppositories are prepared as follows: Formulation 6: Suppositories

Ingredient Quantity

(mg/suppository)

Active ingredient 250

Saturated fatty acid glycerides 2,000

The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in the saturated fatty acid glycerides previously melted using the minimal necessary heat. The mixture is then poured into a suppository mold of nominal 2 g capacity and allowed to cool.

An intravenous formulation is prepared as follows: Formulation 7: Intravenous Solution

Ingredient Quantity

Active ingredient dissolved in ethanol 1% 20 mg

Intralipid™ emulsion 1 ,00O mL

The solution of the above ingredients is intravenously administered to a patient at a rate of about 1 ml_ per minute.

Soft gelatin capsules are prepared using the following: Formulation 8: Soft Gelatin Capsule with Oil Formulation

Ingredient Quantity (mg/capsule)

Active ingredient 10-500

Olive Oil or Miglyol® Oil 500-1000

Another aspect of the present invention is a kit comprising: a. an amount of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6 ,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and a pharmaceutically acceptable carrier or diluent in a first unit dosage form; b. an amount of an aromatase inhibitor, and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; and c. a container.

The kit comprises two separate pharmaceutical compositions: (-)-cis-6-phenyl-5-[4-(2- pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and a second compound as described above. The kit comprises container means for containing the separate compositions such as a divided bottle or a divided foil packet, however, the separate compositions may also be contained within a single, undivided container. Typically, the kit comprises directions for the administration of the separate components. The kit form is particularly advantageous when the separate components are preferably administered in different dosage forms (e.g., oral and parenteral), are administered at different dosage intervals, or when titration of the individual components of the combination is desired by the prescribing physician.

An example of such a kit is a so-called blister pack. Blister packs are well known in the packaging industry and are being widely used for the packaging of pharmaceutical unit dosage

forms (tablets, capsules, and the like). Blister packs generally consist of a sheet of relatively stiff material covered with a foil of a preferably transparent plastic material. During the packaging process recesses are formed in the plastic foil. The recesses have the size and shape of the tablets or capsules to be packed. Next, the tablets or capsules are placed in the recesses and the sheet of relatively stiff material is sealed against the plastic foil at the face of the foil which is opposite from the direction in which the recesses were formed. As a result, the tablets or capsules are sealed in the recesses between the plastic foil and the sheet. Preferably the strength of the sheet is such that the tablets or capsules can be removed from the blister pack by manually applying pressure on the recesses whereby an opening is formed in the sheet at the place of the recess. The tablet or capsule can then be removed via said opening.

It may be desirable to provide a memory aid on the kit, e.g., in the form of numbers next to the tablets or capsules whereby the numbers correspond with the days of the regimen which the dosage form so specified should be ingested. Another example of such a memory aid is a calendar printed on the card e.g., as follows "First Week, Monday, Tuesday, ...etc.... Second Week, Monday, Tuesday,..." etc. Other variations of memory aids will be readily apparent. A "daily dose" can be a single tablet or capsule or several tablets or capsules to be taken on a given day. Also, a daily dose of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof can consist of one tablet or capsule while a daily dose of the aromatase inhibitor compound can consist of several tablets or capsules and vice versa. The memory aid should reflect this.

In another specific embodiment of the invention, a dispenser designed to dispense the daily doses one at a time in the order of their intended use is provided. Preferably, the dispenser is equipped with a memory-aid, so as to further facilitate compliance with the regimen. An example of such a memory-aid is a mechanical counter that indicates the number of daily doses that have been dispensed. Another example of such a memory-aid is a battery-powered microchip memory coupled with a liquid crystal readout, or audible reminder signal which, for example, reads out the date that the last daily dose has been taken and/or reminds one when the next dose is to be taken.

The (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6 ,7,8-tetrahydronaphthalene- 2-ol or a pharmaceutically acceptable salt or prodrug thereof and the aromatase inhibitor can be administered in the same dosage form or in different dosage forms at the same time or at different times. All variations of administration methods are contemplated. A preferred method of administration is to administer the combination in the same dosage form at the same time. Another preferred administration method is to administer the (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 - yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or

prodrug thereof in one dosage form and the aromatase inhibitor in another, both of which are taken at the same time.

The (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6 ,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof used in the compositions and methods of this invention are all adapted to therapeutic use as agents that mediate estrogen receptors in mammals, particularly humans. The aromatase inhibitors or a pharmaceutically acceptable salt or prodrug thereof used in the compositions and methods of this invention are all adapted to therapeutic use as agents that inhibit or inactivate aromatase thereby effecting the synthesis of estrogens from androgenic substrates (specifically, the synthesis of estrone from the preferred substrate androstenedione and estradiol from testosterone). By virtue of these activities, the combinations of this invention are useful for treating breast cancer and conditions that present with low bone mass and for improving frailty and other disease/conditions detailed above. The combinations of this invention are particularly advantageous and provide synergistic activity in the treatment of breast cancer.

The utility of the compositions of the invention and the salts thereof as medical agents in the treatment of the above described disease/conditions in mammals (e.g., humans, male or female) is demonstrated by the activity of the combinations of this invention in conventional assays and the in vitro and in vivo assays described below. The in vitro and in vivo assays (with appropriate modifications within the skill in the art) may be used to determine the activity of analogous agents as well as the combinations of this invention. Such assays also provide a means whereby the activities of the compositions of this invention can be compared with the activities of other known compounds. The results of these comparisons are useful for determining dosage levels in mammals, including humans, for the treatment of such diseases.

The following protocols can be varied when appropriate by those skilled in the art. The use of the combinations of this invention in prophylactic treatment of breast cancer can be assessed by methods known in the art such as those described in Cohen et al., LAS, a Novel Selective Estrogen Receptor Modulator with Chemopreventive and Therapeutic Activity in the N-Nitroso-N-methylurea-induced Rat Mammary Tumor Model, Cancer Research, 61 , 8683- 8688, December 15, 2001. The combination of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)- phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof and aromatase inhibitor are administered according to the protocol as described in the Cohen reference. When the Al compound employed is exemestane, a representative therapeutically effective dose is approximately 0.3 mg/kg/day. The efficacy of the combination is

determined by measuring the reduction in incidence or delay in incidence of the NMU induced tumors in the rat.

Effect of Selective Estrogen Receptor Modulator (SERM) and/or Aromatase Inhibitor (Al) on Body Weight, Body Composition and Bone Density in the Aged Intact and Ovariectomized Female Rat

The purpose of this study is to test the effects of test compositions in aged intact or ovariectomized (OVX) female rat model. In the following protocol the SERM compound employed is (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8- tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof. When the Al compound employed is exemestane, a representative therapeutically effective dose is approximately 0.3 mg/kg/day. Study Protocol

Sprague-Dawley female rats are sham-operated or OVX at 18 months of age, while a group of rats is necropsied at day O to serve as baseline controls. One day post-surgery, the rats are treated with either vehicle or aromatase inhibitor (Al) test compound, SERM test compound or a combination of Al and SERM test compound for 59 days. The vehicle or Al test compound is administered either orally or by subcutaneous injection (s.c), with the Al compound being administered at a therapeutically effective dose. The SERM test compound is administered daily at a dose of 0.1 milligrams per kilogram of body weight by oral gavage

(P.O.)-

All rats are given s.c. injection of 10 mg/kg of calcein (Sigma, St.Louis, MO) for fluorescent bone label 2 and 12 days before necropsy. On the day of necropsy, all rats under ketamine/xylazine anesthesia are weighed and undergoe dual-energy X-ray absorptiometry (DXA, QDR-4500/W, Hologic Inc., Waltham, MA) equipped with Rat Whole Body Scan software for lean and fat body mass determination. The rats are necropsied, then autopsied and blood is obtained by cardiac puncture. The distal femoral metaphysis and femoral shafts from each rat are analyzed by peripheral quantitative computerized tomography (pQCT), and volumetric total, trabecular and cortical bone mineral content and density are determined.

Peripheral Quantitative Computerized Tomography (pQCT) Analysis: Excised femurs are scanned by a pQCT X-ray machine (Stratec XCT Research M, Norland Medical Systems, Fort Atkinson, Wl.) with software version 5.40. A 1 millimeter (mm) thick cross section of the femur metaphysis is taken at 5.0 mm (proximal femoral metaphysis, a primary cancellous bone site) and 13 mm (femoral shafts, a cortical bone site) proximal from the distal end with a voxel size of 0.10 mm. Cortical bone is defined and analyzed using contour mode 2 and cortical

mode 4. An outer threshold setting of 340 mg/cm ³ is used to distinguish the cortical shell from soft tissue and an inner threshold of 529 mg/cm ³ to distinguish cortical bone along the endocortical surface. Trabecular bone is determined using peel mode 4 with a threshold of

655 mg/cm ³ to distinguish (sub)cortical from cancellous bone. An additional concentric peel of 1 % of the defined cancellous bone is used to ensure that (sub)cortical bone was eliminated from the analysis. Volumetric content, density, and area are determined for both trabecular and cortical bone (Jamsa T. et al., Scv7e 23:155-161 , 1998; Ke, H.Z. et al., Journal of Bone and

Mineral Research, 16:765-773, 2001).

Vaginal histology: Vaginal tissue is fixed and embedded in paraffin. Five micron sections are cut and stained with Alcian Blue staining. Histology examination of vaginal luminal epithelial thickness and mucopolysaccharide (secreted cells) is performed. The experimental groups for the protocol are as follows:

Group I: Baseline controls

Group II: Sham + Vehicle Group III: OVX + VeNcIe

Group IV: OVX + Al Test Compound (in Vehicle)

Group V: OVX + SERM Test Compound

Group Vl: OVX + Al Test Compound and SERM Test Compound.

Fracture Healing Assays

In the following protocols the pharmaceutical composition employed comprises (-)-cis-6- phenyl-5-[4-(2-pyrrolidin-1 -yl-ethoxy)-phenyl]-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt or prodrug thereof, and an aromatase inhibitor.

Assay For Effects On Fracture Healing After Systemic Administration Fracture Technigue: Sprague-Dawley rats at 3 months of age are anesthetized with

Ketamine. A 1 cm incision is made on the anteromedial aspect of the proximal part of the right tibia or femur. The following describes the tibial surgical technique. The incision is carried through to the bone, and a 1 mm hole is drilled 4 mm proximal to the distal aspect of the tibial tuberosity 2 mm medial to the anterior ridge. Intramedullary nailing is performed with a 0.8 mm stainless steel tube (maximum load 36.3 N, maximum stiffness 61.8 N/mm, tested under the same conditions as the bones). No reaming of the medullary canal is performed. A standardized closed fracture is produced 2 mm above the tibiofibular junction by three-point bending using specially designed adjustable forceps with blunt jaws. To minimize soft tissue damage, care is taken not to displace the fracture. The skin is closed with monofilament nylon sutures. The operation is performed under sterile conditions. Radiographs of all fractures are taken immediately after nailing, and rats with fractures outside the specified diaphyseal area or

with displaced nails are excluded. The remaining animals are divided randomly into the following groups with 10 - 12 animals per each subgroup per time point for testing the fracture healing. The first group receives daily gavage of vehicle (water : 100% Ethanol = 95 : 5) at 1 ml/rat, while the others receive daily gavage from 0.01 to 100 mg/kg/day of the pharmaceutical composition to be tested (1 ml/rat) for 10, 20, 40 and 80 days.

At 10, 20, 40 and 80 days, 10 - 12 rats from each group are anesthetized with Ketamine and sacrificed by exsangυination. Both tibiofibular bones are removed by dissection and all soft tissue is stripped. Bones from 5 - 6 rats for each group are stored in 70% ethanol for histological analysis, and bones from another 5 - 6 rats for each group are stored in a buffered Ringer's solution (+4°C, pH 7.4) for radiographs and biomechanical testing which is performed.

Histological Analysis: The methods for histologic analysis of fractured bone have been previously published by Mosekilde and Bak (The Effects of Growth Hormone on Fracture Healing in Rats: A Histological Description. Bone, 14:19-27, 1993). Briefly, the fracture site is sawed 8 mm to each side of the fracture line, embedded undecalcified in methymethacrylate, and cut frontals sections on a Reichert-Jung Polycut microtome in 8 μm thick. Masson-Trichrome stained mid-frontal sections (including both tibia and fibula) are used for visualization of the cellullar and tissue response to fracture healing with and without treatment. Sirius red stained sections are used to demonstrate the characteristics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site. The following measurements are performed: (1 ) fracture gap - measured as the shortest distance between the cortical bone ends in the fracture, (2) callus length and callus diameter, (3) total bone volume area of callus, (4) bony tissue per tissue area inside the callus area, (5) fibrous tissue in the callus, and (6) cartilage area in the callus.

Biomechanical Analysis: The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue lnt 45:292-297, 1989). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedure. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.

Assay for Effects on Fracture Healing After Local Administration Fracture Technigue: Female or male beagle dogs at approximately 2 years of age are used under anesthesia in the study. Transverse radial fractures are produced by slow continuous loading in three-point bending as described by Lenehan et al. (Lenehan, T. M.; Balligand, M.; Nunamaker, D.M.; Wood, F.E.: Effects of EHDP on Fracture Healing in Dogs. J Orthop Res 3:499-507; 1985). A wire is pulled through the fracture site to ensure complete

anatomical disruption of the bone. Thereafter, local delivery of the test pharmaceutical composition to the fracture site is achieved by slow release of compound delivered by slow release pellets or by administration of the compounds in a suitable formulation such as a paste gel solution or suspension for 10, 15, or 20 weeks. Histological Analysis: The methods for histologic analysis of fractured bone have been previously published by Peter et al. (Peter, C.P.; Cook, W.O.; Nunamaker, D. M.; Provost, M. T.; Seedor, J.G.; Rodan, G.A. Effects of alendronate on fracture healing and bone remodeling in dogs. J. Orthop. Res. 14:74-70, 1996) and Mosekilde and Bak (The Effects of Growth Hormone on Fracture Healing in Rats: A Histological Description. Bone, 14:19-27, 1993). Briefly, after sacrifice, the fracture site is sawed 3 cm to each side of the fracture line, embedded undecalcified in methymethacrylate, and cut on a Reichert-Jung Polycut microtome in 8 μm thick of frontal sections. Masson-Trichrome stained mid-frontal sections (including both tibia and fibula) are used for visualization of the cellullar and tissue response to fracture healing with and without treatment. Sirius red stained sections are used to demonstrate the characteristics of the callus structure and to differentiate between woven bone and lamellar bone at the fracture site. The following measurements are performed: (1) fracture gap - measured as the shortest distance between the cortical bone ends in the fracture, (2) callus length and callus diameter, (3) total bone volume area of callus, (4) bony tissue per tissue area inside the callus area, (5) fibrous tissue in the callus, (6) cartilage area in the callus. Biomechanical Analysis: The methods for biomechanical analysis have been previously published by Bak and Andreassen (The Effects of Aging on Fracture Healing in Rats. Calcif Tissue lnt 45:292-297, 1989) and Peter et al. (Peter, C.P.; Cook, W.O.; Nunamaker, D.M.; Provost, M. T.; Seedor, J.G.; Rodan, G.A. Effects of Alendronate On Fracture Healing And Bone Remodeling In Dogs. J. Orthop. Res. 14:74-70, 1996). Briefly, radiographs of all fractures are taken prior to the biomechanical test. The mechanical properties of the healing fractures are analyzed by a destructive three- or four-point bending procedures. Maximum load, stiffness, energy at maximum load, deflection at maximum load, and maximum stress are determined.

All documents cited in this application, including patents and patent applications, are hereby incorporated by reference.