CA2335134A1 - Therapeutic combinations of (selective) estrogen receptor modulators (serm) and growth hormone secretagogues (ghs) for treating musculoskeletal frailty - Google Patents

Abstract

This invention is directed to pharmaceutical combination compositions and methods containing (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)- 5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt thereof and 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahyd ro- pyrazolo-[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramid e or a pharmaceutically acceptable salt thereof, methods of using such compositions and kits containing such compositions. The compositions are useful for treating musculoskeletal frailty, including osteoporosis, osteoporotic fracture, low bone mass, frailty and low muscle mass.

Description

THERAPEUTIC COM$INATIONS OF (SELECTIVE) ESTROGEN RECEPTOR MODULATORS (SERM) AND GROWTH
HORMONE SECRETAGOGUES (GHS) FOR TREATING MUSCULOSKELETAL FRAILTY
BACKGROUND OF THE INI~rENTION
This invention relates to a pharmaceutical! combination of a selective estrogen receptor modulator (SERM) and a growth hormone secretagogue (GHS) that stimulates bone formation, increases bone mass, decreases serum lipid levels and increases muscle mass. The invention also r~alates to kits containing such combinations and the use of such combinations to treat musculoskeletal frailty, including osteoporosis, osteoporotic fracture, low bone mass, frailty, low muscle mass and the like in mammals, including humans.. tn particular, this invention relates to a combination of (-)-cis-6-phenyl-5-{4-(2-ipyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of or a pharrnace~rticatly acceptable salt thereof and 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrazoto-[4,3-c]pyridin-5-yl)-9(R)-benzyloxymethyl-2-~oxo-ethyt)-isobutyramide or a pharmaceutically acceptable salt thereof, kits contaiining such a combination and the use of such a combination to treat mus~,~.uloskeletal frailty, including osteoporosis, osteoporotic fracture, low bone mass, frailty; low muscle mass and the like in mammals, including humans.
Osteoporosis is' a systemic skeletal disease, characterized by t~nr bone mass and deterioration of bone tissue, with a conseduent increase in bone fragility and susceptibility to fracture. In the U.S., the condition affects more than million people and causes more than 1.3 million fractures each year, including 500,000 spine, 250,000 hip and 240,000 wrist fractures annually. Hip fractures are the most serious, with 5-20% of patients dying vwithin one year, and over 50%
of survivors being incapacitated.
The elderly are at greatest r7sk of osteoiparosis, and the problem is therefore predicted to increase significantly with tlhe aging of the population.
Worldwide fracture incidence is forecast to increase three-fold over the next years, and one study estimates that there will be 4.5 million hip fractures worldwide in 2050.
Although both men and women are susceptible to musculoskeletal frailty, including osteoporosis, women are at greater risk of osteoporosis than men.
Women experience a sharp acceleration of bone loss immediately following menopause. Other factors that increase bone loss leading to osteoporosis include smoking, alcohol abuse, a sedentary lifestyle and low calcium intake.
Estrogen is the agent of choice in preventing osteoporosis or post menopausal bone loss in women. in addition, Black, et al. in EP 0605193A1 report that estrogen, particularly when taken orally, lowers plasma levels of LDL
and raises those of the beneficial high density lipoproteins (HDL's). Long-term estrogen therapy, fiowever, has been implicated in a variety of disorders, including an increase in the risk of uterine cancer, endometrial cancer and possibly breast cancer, causing many women to either avoid this treatment or take the medica#ion for only a short period of time. Although the risk of endometrial cancer is thought to be reduced by a concurrent use of a progesterone, there is still concern about possible increased risk of breast cancer with the use of estrogen. Recently suggested therapeutic regimens, which seek to lessen the cancer risk, such as administering combinations of progesterone and estrogen, cause the patient to experience unacceptable bleeding. Furthermore, combining progesterone with estrogen seems to blunt the serum cholesterol lowering effects of estrogen.
The sign~cant undesirable side effects associated with estrogen therapy support the need to develop alternative Therapies for osteoporosis that have the desirable benefiaal effect on serum LDL but do not cause undesirable side effects.
Recently, a number of selective estrogen receptor modulators have been proposed for treatment of osteoporosis. It has been reported (Osteoporosis Conference Scrip No. 1812113 April 16!20, 1993, p. 29) that raloxifene, 6-hydroxy-2-(4-hydroxyphenyl)-3-[4-(2-piperidinoethoxy} benzoyl] benzo[bj thiophene, mimics the favorable action of estrogen on bone and lipids but, unlike estrogen, has minimal uterine stimulatory effect. [Black, L.J. ei: al., Raloxifene (LY139481 Hd) Prevents Bone Loss and Reduces Serum Cholesterol Without Causing Uterine Hypertrophy in Ovariectomized Rats, J. Clin. lnveat., 1994, 93:63-fig and Delmas, P.D. et al., Effects of Raioxifene on Bone Mineral Density, Serum Cholesterol Concentration, and Uterine Endometrium in Postmenopausal Women, New England Journal of Medicine, 1997, 337:1641-164.7j.
Agents such as droioxifene, U.S. pat. no. 6,254,595, prevent bone loss and thereby reduce the risk of fracture without estrogen's side effects. However, estrogen and estrogen agonists alone are only expected to reduce The fracture risk by about 50% leaving approximately 50°/° of osteopenic women still at risk for an osteoporotic fracture.
Commonly assigned U.S. P at.No . 5,552,4'I2, -discloses SERM compounds of the formula ,Y
wherein the variables are defined as set forth therein.
Growth hormone (GH), which is secreted from the pituitary gland, stimulates growth of alt tissues of the body that are capable of growing. tn addition, GH is known to have the following basic effects on the metabolic process of the body:
1. increased rate of protein synthesis in substantially all cells of the body;
2. Decreased rate of carbohydrate utilization in cells of the body;
3. Increased mobilization of free fatty acids and use of fatty acids for energy.
Defiaency in GH results in a variety of medical disorders. In children, it causes dwarfism. tn adults, the consequences of acquired GH deficiency inGude profound reduction in lean body mass and concomitant increase in total body fat, particularly in. the tFUncal region: . Decreased- stceletat .and cardiac ntus~e~-mass and muscle strength lead to a significant reduction in exerase capacity. Bone density is also reduced. Administration of exogenous. GH has been shovm to reverse many of the metabolic changes. Additional. benefits of therapy have included reduction in LDL cholesterol and improved psychological well-being.
In cases where increased levels of GH were desired, the problem was generailx~sotved. by providing exage~ous...Gk -or- by administering arr agent-wpyi~y.
stimulated GH production andlor release. In either case the peptidyl nature of the compound necessitated that it be administered by injectiion. Initially the source of 4$6 PCT/IB99/01117 GH was the extraction of the pituitary glands of cadavers. This resulted in an expensive product, and carried with it the risk that a disease associated with the source of the pituitary gland could be transmitted to the recipient of the GH
(e.g., Jacob-Creutzfetd disease). Recently, recombinant GH has become available which, while no longer carrying any risk of disease transmission, is still a very expensive product which must be given by injection or by~a nasal spray.
Most GH defiaencies are caused by defects in GH release, not primary defects in pituitary synthesis of GH. Therefore, an alternative strategy for normalixing serum GH levels is by stimulating il:s release from somatotrophs.
1~ Increasing GH secretion can be achieved by :stimulating or inhibiting various neurotransmitter systems in the brain and hypothalamus. As a resutt, the development of synthetic GH-releasing agents to stimulate pituitary GH
secretion are being pursued, and may have several advantages over expensive arid inconvenient GH replacement therapy. By acting along physiologic regulatory pathways, the most desirable agents would stimulate pulsatile GH secretion, and excessive Levels of GH that have been associated with the undesirable side effects of exogenous GH administration would !be avoided by virtue of intact negative feedback loops.
Physiologic and pharmacologtc stimulators of GH secretion include 2t7 arginine, L-3,4dihydroxyphenylalanine (L-DOPA), glucagan, vasopressin, and insulin induced hypoglycemia, as welt as activities such as sleep and exercise, indirectly cause GH to be released from the pituttairy by acting in some fashion on the hypothalamus pefiaps either to decrease somatos#atin secretion or to increase the secretion of the known secretagogue GH releasing factor (GHRF) or an~ unknown endogenous GH-reteasing hormone or' all of these.
Commonly assigned International Patent Application Publication Number W0971243fi9, designating, inter olio, the United States; discloses GH
secretagogues of the formula Rt ~~ Rs Y (Cf"!?~e (C1"!?~n C ~ ~ R6 R~
.C/ \N/
'R$
R2/~ ~ N~ (Cti~w R O
wherein the variables are defined as set forth therein.
Tang et at., Restorino and Maintainino Bone in Osteogenic Female Rat 5 Skeleton: t_ Chanoes in Bone Mass and Strvctmrp, ~, gone Mineral Research 7 (9), p1093-'1104, 1992 disUoses data for the lose, restore and maintain (LRM) concept, a practical approach for reversing existing osteoporosis. The LRM
concept uses anabolic agents to restore bone mass and architecture (+ phase) and then switches to an agent with the established abiliify to maintain bone mass, to keep the new bone (+I phase). The rat study utilized PGEz and risedronate, a bisphosphonate, to show that most of the new canc;ettous and cortical bone induced by PGEz can be maintained for at least 60 days after discontinuing PGEz by administering risedronate.
Shen et al., Effects of Reciprocal Treatment with Estrogen and Estrogen 1 ~ plus Parathyroid Hormone on Bone Structure and Sh~ength in Ovariectomi2ed Rats, J. Clinical Investigation, 1995, 96:2331-2338 disGoses data for the combination andJor sequential use of anti-resorptive agents and anabolic agents for the treatment of osteoporosis.
Commonly assigned lntemational Patent Applicaation Publication Number WOg7/31640, designating, inter olio, the United States, discloses the use of certain GH secretagogues in combination with cE:rtain SERMS to treat osteoporosis.

This invention is directed to a pharmaceutical composition comprising:
a. a first compound, the first compound beinci ( ~cs$phenyl-5,.(ø(2-PY~tidin-~l-y1-ethoxy~phenyt)-5,fi,7.8-tetrahydronaphthaie~ne-2-of or a pharmaceutically acceptable salt thereof; and b. a second compound, which is 2-amino-N-(2-{3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexa:hydropyrazolo[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxoethyl)isobutyramide or a pharmaceutically acceptable salt thereof.
This invention is further directed to a pharmaceutical composition as recited i:n the immediately preceding paragraph additionally comprising a pharmaceutical carrier.
This invention is also directed to a pharmaceutical composition as described in either of t:he first two paragraphs of this summary, wherein the first compound is {-)-cis-6 phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-Lohenyl)-5,6,7,8-tetrahydronaphthalene-2-of D-tartrate a:nd the second compound is 2-amino-N-(2-(3a(R)-benzyl-2-methyl-.3-oxo-2,3,3a,4,6,7-hexahydropyrazolo[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxoethyl)isobutyramide L-tartrate.
This invention is still further directed to a method, designated Method A, for treating a mammal suffering from musculoskeletal frailty, comprising administering to the mammal a pharmaceutical composition as recited in any of the first three paragraphs of this summary.
A preferred method within Method A, designated Method B, is wherein the mammal is suffering from osteoporosis.
Another preferred method within Method A, designated Method C, is wherein mammal is suffering from osteotomy, childhood idiopathic bone loss or bone :Loss associated with periodontitis.
This invention is still further directed to a method, designated Method Al, for treating a ma~unal suffering from r , CA 02335134 2001-O1-18 musculoskeletal frailty, comprising administering to the mamma 1:
a. a first compound, which is (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-0l or a pharmaceutically acceptable salt thereof; and b. a second compound, which is 2~-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexalzydropyrazolo[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxoethyl)isobutyramide or a pharmaceutically acceptable salt thereof.
This invention is particularly directed to a method of Method A1 wherein the first compound and the second compound are administered substantially simultaneously.
This invention is also particularly directed to a method of Method A1, hereinafter termed Method D, wherein the second compound is administered for a pESriod of from about three months to about three years.
This invention is more particularly directed to a method of Method D followed by administration of the first compound for a period of from about three months to about three years without the administration of the second compound during the period of from about three months to about three years.
This invention is also more particularly directed to a method of Method D followed by administration of the first compound for a period greater than about. three years without the administration of the second compound during the greater than about three year period.
This invention is also directed to a method, hereinafter termed Method E, for treating musculoskeletal frailty in a mammal suffering therefrom, comprising administering to the mammal a therapeutically effective amount of a composition as recited in any of the first three paragraphs of this summary.
A preferred method within Method E is wherein bone healing following facial reconstruction,, maxillary reconstruction or mandibular reconstruction is enhanced, vertebral synostosis is induced, long bone extension is enhanced, the healing rate of a bone gra ft or a long bone fracture is enhanced or prosthetic ingrowth is enhanced.
Additionally preferred is a method comprising administering to the mammal:
a. a first compound which is (-)--cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8--tetrahydronaphthalene-2-0l or a pharmaceutically acceptable s<~lt thereof; and b. a second compound, which is 2--amino-N- (2- (3a (R) -benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydropyrazolo[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxoethyl)isobutyramide or a pharmaceutically acceptable salt thereof.
This invention is also directed to a method for increasing muscle mass in a mammal, comprising administering to the mammal a muscle mass increasing effective amount of a composition as recited in any of the first three paragraphs of this summary. Additionally preferred is a method comprising administering to the mammal:
a. a first compound which is (-)--cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8--tetrahydronaphthalene-2-0l or a pharmaceuticaly acceptable sa7_t thereof; and b. a second compound, which is 2--amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydropyrazolo[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxoethyl)isobutyramide or a pharmaceutically acceptable salt thereof.

8a In all of the methods of this invention, it is particularly preferred that the mammal is a human.
This invention is also directed to a kit comprising a treatment for a mammal suffering from musculoskeletal frailty comprising:
a. a therapeutically effective amount of (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-;phenyl)-5,6,7,8-tetrahydonaphthalene-2-of or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in a first unit dosage form;
b. a therapeutically effective amount of 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1(R)-benzyloxymeth;yl-2-oxo-ethyl)-isobutyramide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier in a second unit dosage form; and c. a container.
This invention is particularly directed to a kit as described in the immediately preceding paragraph, wherein the first unit dosage form comprises (-)-cia-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-0l D-tartrate and the second unit dosage form comprises 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo--2,3,3a,4,6,7-hexahydro-pyrazolo [4, 3-c] pyridin-5-yl) -1 (R) -benzy:Loxymethyl-2-oxoethyl)isobutyramide L-tartrate.
In all of the composition, mei~hods and kits of this invention, it is particularly preferred that the D-tartrate salt-of (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2--of is used and that the L-WO 99/6548b 9 PCT/IB99/OI I I7 tartrate salt of 2-amino-N-{2-(3a(R)-benzyl-2:-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrazolo-[4, 3-cjpyridin-5-yl)-1 {R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide is used.
The phrase "condition which presents wiith low bone mass" refers to a condition where the level of bone mass is below the age specfic normal as defined in standards by the World Health Organization "Assessment of Frachrre Risk and its Application to Screening for Postmenopausal Osteoporosis (9994), Report of a World Health Organization Study Group. World Health Organization Technical Series 843". . Childhood idiopathic an<i primary osteoporosis are also included. Included in the treatment of osteoporosis is the prevention or attenuation of long term complications such as curvature of the spine, toss of height, prosthetic surgery, and prevention of prostate malfunctioning. Also included is increasing the bone fracture healing rate and enhancing the rate of successful bone grafts. Also included is periodontal disease and alveolar bone loss.
The phrase "condition which presents with tow bone mass" also refers to a mammal known to have a significantly higher than average chance of developing such diseases as are described above inGu~ding osteoporosis {e.g., post-menopausai women, men over the age of 60, <~nd persons being treated with drugs known to cause osteoporosis as a side effect (such as glucocorticoid)).
Those skilled in the art will recognize that the term bone mass actually refers to bone mass per unit area which is sometimes (although not strictly correctly) referred to as bone mineral density.
The phrase "musculoskeletal frailty refers to a condition wherein a subject has low bone mass andlor low muscle mass, and inGudes such diseases, disorders and conditions such as, but not limited to, conditions which present with low bone mass, osteoporosis, conditions which present with low muscle mass, osteotomy, childhood idiopathic bone toss, bone loss associated with periodontitES, bone healing following facial reconstruction, maxillary reconstruction, mandibular reconstruction and bone fracture. Further, musculoskeleta# frailty encompasses such conditions as intertaces between newly attached prostheses and bone which require bone ingrowth.

WO 99/65486 PCT/IB991(t1117 The term "treating", "treat" or "treatment" as used herein includes curative, preventative (e.g., prophylactic) and palliative treatrnent.
The parenthetical negative or positive sign used herein in the nomenclature denotes the direction ptane polarized light is rotates! by the particular stereoisomer.
5 The compositions of this invention may include hydrates of the~compounds used therein.
The pharmaceutical compositions and methods of this invention result in a more rapid and higher magnitude bone mass gain than is achievable with the same doses of (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-10 tetrahydro-naphthalene-2-of as described above alone or 2-amino-N-j2-(3a-(R)-benzy!-2-methyl-3-oxo-2, 3,3a,4,6,7-hexahydro-pyra;zoto-[4, 3-cjpyridin-5-yl)_ 1.(R~.
benzyloxyrriethyl-2-oxo-ethyl]-isobutyr~amide as described above alone.
Further, these combinations increase bone density and muscle mass while at the same time reducing fat mass and total serum cholesterol. Thus, these combinations increase bone mass and decrease fracture rates to a greater extent than is achievable through use of either agent alone. This invention makes a significant contribution to the art by providing compositions and methods that increase and maintain bone mass resulting in prevention, retardation, and/or regression of osteoporosis and related bone disorders.
Other features and advantages will be appairent from the speciftcation and claims which describe the invention.
DETAILED DESCRIPTION OF TFIE INVENTION
The first compound of this invention is (-)-cns-6-phenyl-5-j4-(2-pyrrolidin-1 yl-ethoxyrphenyl]-5,fi,7,8-tetrahydro-naphthalene-2-of or a pharmaceutically acceptable salt thereof, which has the structure of Formula I:

HO

(-~-Cis-6-phenyl-5-[4-{2-pyrroiidin-1-yl-ethoxy~-pheny!)-5,6,7,8-tetrahydto-naphthalene-2-of and the pharmaceutically acceptable sall;s thereof are prepared as described in commonly assigned US Patent Number 5,:552,412, which is referenced above.
{-~-Cis-~6-phenyl-5-[4-{2-pyrrotidin-1-yI-ethoxy}-phenyi~-5,6,7, 8-tetrahydro-naphthalene-2-of D-tartrate is prepared as set forth in the immediately preceding paragraph or, altemativeiy, as set forth in lntema~onal Patent Application Publication Number W097116434 .
The second compound of this invention is 2-amino-N-[2-(3a-{R~-benzyl-2-methyf-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazofo-[4,3-cJpyrid!in-5-yl)-1-(RJ-benzyioxymethyt-2-oxo-e#hyt]-isabutyramide or a pharmaceutically acceptable salt thereo#, which has the structure of Formula ti:
Me nne she -.

2-Amino-N-[2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazoto-[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl]-is~obutyramide and pharmaceutically acceptable salts thereof are prep<~red as set forth in commonly assigned International Patent Application Publica6a~n Number W097/24369, which is referenced above.
In addition, when the compounds or the pham~aceutically acceptable salts thereof of this invention form hydrates or solvates they are also within the scope of the invention.
The pharmaceutical combinations and methods of this invention are all adapted to therapeutic use as agents that either activate bone turnover or prevent bone resorption or increase bone formation in m~ammais, particularly humans.
Since these functions are dosely related to the development of osteoporosis and bone related disorders, these combinations, by virtue of their action on bone, prevent, arrest, regress or reverse osteoporosis.
The utility of the compositions and methods of the present invention as medical agents in the treatment of muscutoskeletal frailty (e.g., conditions which present with low bone mass or low muscle ma~,ss including osteoporosis} in mammals (e.g. humans) is demonstrated by the activity of the compounds of this invention in conventional assays as set forth in U.S. Patent Number 5,552,412 and International Patent Application Publication Number W097I24369. Further evidence of the utility of the instant combination is sEa forth in Example One below.
Such assays also provide a means whereby' the activities of the compounds of this invention can be compared between themselves and with the activities of other knovm compounds. The results of these comparisons are useful for determining dosage levels in mammals, including humans, for the treatment of such diseases.
Administration of the compounds of this invE:ntion can be via any method which delivers a compound of the combination of thi:> invention systemically and/or locally. These methods include oral routes, parenteral, intraduodenal routes, etc.
Generally, the compounds of this invention are administered orally, but parenteral administration (e.g., intravenous, intramuscular, transcutaneous, subcutaneous or intramedullary) may be utilized, for example, vvhere oral administration is inappropriate for the instant target or where the patient is unable to ingest the drug. The two different compounds of this invention can be co-administered simultaneously or sequentially in any order, or ,a single phamtaceutical composition comprising a first compound as described above and a second compound as described above in a pharmaceutically acceptable carrier can be administered.
In any event the amount and timing of compounds administered wilt, of course, be dependent on the subject being treated, on the severity of the afFlic~ion, on the manner of administration and on the judgrnent of the prescribing physician.
Thus, because of patient to patient variability, the dosages given. below are a guideline and the physician may titrate doses of the drug to achieve the activity (e.g., bone mass augmentation) that the physician considers appropriate for the individual patient. In considering the degree of activity desired, the physician must balance a variety of factors such as bone mass starting level, age of the patient, presence of preexisting disease, as well as pnesence of other diseases (e.g., cardiovascular). For example, the administraition of (-)-cis-6-phenyt-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl}-5,6,7,&tetrahydron~aphthalene-2-of can provide cardiovascular benefits, par6culacly for post menopausal women. The following paragraphs provide preferred dosage ranges for the various components of this invention.
An effective dosage for ~ (-)-cis-6-phenyl-5-[4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7;&tetrahydro-naphthalene-2-of is in the range of 0.0001 to 100 mg/kglday, preferably 0.001 to 10 mg!!cg/day.
An effective dosage for 2-amino-N-(2-(3a(Ft)'benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1 (R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide is in the range of 0.0001 to 100 mg/kglday, preferably 0.01 to 5 mg/kgJday.
Where the tartrate salt or other pharmaceutically acceptable salt of either of the above compounds is used in this invention, the skilled person will be able to calculate effective dosage amounts by calculating the molecular weight of the salt form and performing simple stoichiometric ratios.
The' compounds of the present invention acre generally administered in the form of a pharmaceutical composition comprising <~t least one of the compounds or pharmaceutically acceptable salts thereof of i;his invention together with a pham~aceu~cally acceptable vehicle or diluent. Thus, the compounds and pharmaceutically acceptable salts thereof of this invention can be administered separately or together in any conventional oral, p;arenteral or transdennal dosage form. When administered separately, the administration of the other compound or a pharmaceutically acceptable salt thereof of the invention follows.
For oral administration a pharmaceutical c~~mposition can take the form of solutions, suspensions, tablets, pills, capsules, powders, and the tike.
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 polyvinytpyrrolidone, sucrose, gelatin and acacia.
Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often useful for tabletting purposes. Solid compositions of a similar type are also employed as fillers in soft and hard-filled gelatin capsules;
preferred 'I5 materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aquE:ous suspensions and/or elixirs are desired for oral administration, the compaunds~ or pharmaceutically a~p~bte salts thereof of this invention can be combined with various sweetening agents, flavoring agents, coloring agents, emulsifying agents andlor suspending agents, as wail as such diluents as water, ethanol, propylene glycol, glycerin and various tike combina#ions thereof.
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 dilueryt 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'/o to 5%
concentration), otherwise similar to the above parenteral solutions, are prepared.

Methods of preparing various pharmaceutical compositions with a certain amount of each active ingredient are known, or will be apparent in light of this .disclosure, to those skilled in this art. For examples, see Remington_'s Pharmaceutical Saences, Mack Publishing Company, Easton, Pa., 19th ~ Edition 5 {1990).
Pharmaceutical compositions according to the invention may contain 0.1 %-95% of a combination of the compounds or -pharmaceutically acceptable salts thereof of this invention, preferably 1~°-70%. In any event, the composition or formulation to be administered will contain a nquantity of the compounds or 10 pharmaceutically acceptable salts thereof of the invention in an amount effective to treat the diseaselcondition of the subject being treated.
Since the present invention relates to treai;ment with a combination of the two active ingredients which may be administered separately, the invention also relates to combining separate pharmaceutical compositions in kit form. The kit 15 inGudes two separate pharmaceutical compositions: (-)-cis-6..phenyt-5-{4-(2-pyn-olidin-1-yl-ethoxy~.phenyl)-5,6,7,8-tetrahydronaphthalene-2-o! or a pharmaceutically acceptable salt thereof and 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-F~yrazolo-j4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxo-ethylrisobutyramide or a pharmaceutically acceptable salt thereof. The kit includes a container 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 inGudes directions for the administration of the ,>eparate 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 wiidely 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 pfaced in the recesses and the sheet of relatively stiff material is sealed aigainst 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 seated in tihe 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 is desirable to provide a memory aid on a card insert, 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 tablets or capsules :>o speafied 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 spills or capsules to be taken on a given day. Also a daily dose of SERM can consi:>t of one tablet or capsule white a daily dose of a GH secretagogue can consist of s~everat tablets or capsules.
The memory aid should retied this.
In another speafic 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 which indicates the number of daily doses that has been dispensed. Another example of such a memory-aid is a battery-powered micro chip 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 takE:n.
The following assay is used to show that the combination and methods of this invention increases lean body mass and decr~aases fat body mass whereas the GH secretagogue alone would be expected to decrease #at body mass with no change in lean body mass and the SERM alone would be expected to increase both lean and fat body mass. Further, the combination increases bone density and decreases total serum cholesterol.

Example One Female S-D rats (Harian) were sham-operated or ovariectomized (OVX) at 3.5 months of age. Drug administration started v~~fien the rats were 9 months of age and 5.5 months post surgery. The sham-opeu~ated rats received daily gavage of vehicle (10% ethanol in water), while the OV:~C rats received daily gavage of vehide, or 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c)pyridin-5-yt)-1 (R)-benzyloxymethyt-2-oxoethyl~.isobutyramide at 5 mg/kgJd alone, or (-)cis-6-phenyl-5-(4-(2-PYrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,$-tetrahydronaphthalene-2-of at 0.1 mg/kgld atone, or co-treatment of 2-amino-N-(2-(3a(R}-benzyt-2-methyl-3-oxo-2,3,3a,4,6,7-hexahycfro-pyrazolo-[4,3-c]pyndin-5-yI)-1 (R~benzyioxymethyt-2-oxoethylrisobutyramide and (-)cis-6-phenyl-5-(4-(2_ pyrrolidin-1-yl-ethoxy)-phenyly-5,6,7,&tetrahydronaphthalene-2-o! for 4 weeks.
In the combination group, 2-amino-N-{2-(3a(R)-ben;zyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-cjpyridin-5-yl)-1 (R)-benzyloxymethyt-2-oxoethyt)-isobutyramide was given 2 hours prior to {-)cis~-6-phenyl-5-(4-(2-py~olidin-1-y!-ethoxyrphenytr5,6,7,8-tetrahydronaphthalene-2-ol. There were $ to 10 rats per each subgroup. Ail rats were given subcutaneous injections. with 10 mg/kg of calcein (Sigma Chemical Co., St. Louis, MO) on 13 and 3 days before autopsy.
It wits be recognized by those skilled in the art that the compounds used in this assay may be administered in the form of a pharmaceutically acceptable salt and that the dosage amount can be readily determined by calculating the molecular weight of the salt and performing simple ratios.
Before autopsy on the terminal day of the assay, all rats under ketaminelxylazine anesthesia underwent dual-enercty X ray absorptiornetry (DXA, QDR-10001W, Hologic Inc., Waltham, MA) equipped with Rat Whole Body Scan software (Hotogic Inc.; Waltham, MA) for lean and 'fat body mass determination.
The rats were then autopsied and blood was obtained by cardiac puncture. Total serum cholesterol was determined using a high pertormance cholesterol colorometic assay (Boehringer Mannheim Biochennicals, fndianapolis, IN). Tlie body weight gain was calculated as body weight at .autopsy minus body weight at day 0. The 'uterine wet weight was determined immediately at autopsy.
The right femur from each rat was removed at autopsy and scanned using dual energy x-ray absorptiometry (DXA, QDR 1000N'd, Hologic inc., Wattham, MA) WO 99165486 PCT/IB99/Oll1'7 equipped with "Regional High Resolution Scan" software (Holcgic Inc., Wattham, MA). The scan field size was 5.08 x 1.902 cm, rE~solution is 0.0254 x 0.0127 cm and scan speed was 7.25 mmlsecond. The femoral scan images were analyzed and total femoral bone area, bone mineral conteni~ and bone mineral density were determined according to the method described in H. Z. Ke et al., Droloxifene, a New Estrogen AntagonistlAgonist, Prevents Bone Loss in Ovariectomized Rats.
ENDOCRINOLOGY 136;2435-2441, 1995.
Study Results and Discussion Compared to the controls, 2-amino-N-{2-(3a(R)-benzyi-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-y!;)-1 (R)-benzybxymethyl-2-oxoethyl)-isobutyramide alone increased both tean and fat body mass; while ( )cis-6-phenyl-5-{4-{2-pyrrolidin-1-yt-ethoxy)-phenyl)-5,6"7,8-tetrahydronaphthalene-2-al alone decreased fat body mass with no change in lean body mass. Combination of 2-amino-N-(2-(3a(Rj-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1 (R)-benzyloxymethyl-2-oxoethy!)-isobutyramide and ( )cis.6-phenyl-5-{4-(2-pynofidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-of increased lean body mass and decreased fat body mass. Therefore, combination of both compounds have a better body composiition profile than 2-amino-N-(2-{3a{R)-benzy!-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydlro-pyrazolo-[4,3-c]pyridin-5-yl)-1 (R)-benzyloxymethyl-2-oxoethyl)-isobutyramide alone or (-)as-6-phenyl-5..(4.(2-pyn-olidin-1-yl-ethoxy)-phenyl)-5,fi,7,8-tetrahydronaphthalene-2-of alone.
A 5-6% increase in total femoral bone area was observed in subjects receiving the combination relative to those subjects who received placebo.
This result is similar to the increase in total femoral bone area which was observed in subjects receiving either (-)cis-6-phenyl-5-(4-(c!-pYrrolidin-1-yl-ethoxy)-phenyl)_ 5,6,7,8-tetrahydronaphthalene-2-o! or 2-amino-N-(:?-(3a(R)-benzyl-2-methyl_3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)..1 (R)-benzyloxymethyl-2-oxoethyl)-isobutyramide and {-)cis-6-phenyl-5-(4.(2;-pyrrofidin-1-yl-ethoxy)-phenyt)-5,6,7,8-tetrahydronaphthalene-2-of alone. Total fiernoral bone mineral content increased by 8.5% in 2-amino-N-(2-(3a(R)-bent:yl-2-methyl-3-oxo..2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1 (R)-benzyfa~xymethy-2-oxoethyt)-isobutyramide alone and 7.7°~ in (-)cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl_ethoxy}-phenyl)-5,6,7,8-tetrahydronaphthaiene-2-of alone. However; in the ~
combination WO 99!65486 group, total femoral bane mineral content increased by 12.5%, which was a significant increase compared to either alone. A similar pattern was found in the tots! femoral bone mineral density.
Total serum cholesterol decreased in (-)cis-6-phenyl-5-(4-(2-pyrrblidin-1-yl ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-o1 alone group and the combination group.
These data indicated that combination of 2-amino-N-(2-(3a(R~benzyt-2-methyt-3-axo-2,3,3a,4,6,7-hexahydro-pyrazolo-j4,3-cjpyridin-5-yl)-1 (R)-benzyloxymethyl-2-oxoethyl)-isobutyramide and ( )cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,fi,7,8-tetrahydronaphthaiene-2-of, have multiple benefts.
These benefits include an increase in Lean mass and a decrease in fat mass and senrm lipid. Further, an increase in bone mass was observed.
It should be understood that the invention is not limited to the particular embodiments described herein, but that various changes and modifications may be made vri~thout departing from the spirit and scope: of this invention as defined by the following clairris.

Claims (30)

CLAIM

1. A pharmaceutical composition comprising:
a. a first compound, said first compound being (-)-cis-8-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt thereof; and b. a second compound, said second compound being 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrrazolo-[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition of claim 1 additionally comprising a pharmaceutical carrier.

3. A pharmaceutical composition of claim 1 wherein said first compound is (-)cis-6-phenyl-5-(4-(2-pyrrolidine-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-ol D-tartrate and said second compound is 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-yl)-1(R)-benzyloxymethyI-2-oxo-ethyl)-isobutyramide L-tartrate.

4. Use of a pharmaceutical composition of claim 1 to prepare a medicament for treating a mammal suffering from musculoskeletal frailty.

5. A use of claim 4 wherein said first compound is (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl-5,6,7,8-tetrahydronaphthalene-2-ol D-tartrate and said second compound is 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3, 3a, 4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide L-tartrate.

6. A use of claim 4 wherein said mammal is suffering from osteoporosis.

7. A use of claim 4 wherein osteotomy, childhood idiopathic bone loss or bone loss associated with periodontitis is treated.

8. The use of claim 4 wherein bone healing following facial reconstruction, maxillary reconstruction or mandibular reconstruction is treated, vertebral synostosis is induced or long bone extension is enhanced, the healing rate of a bone graft is enhanced or prosthetic ingrowth is enhanced.

9. The use of claim 8 wherein a bone fracture is treated in a human.

10. A use of claim 6 wherein osteoporosis is treated in a human.

11. A kit comprising:

a. (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-ol or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent in a first unit dosage form;

b. 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent in a second unit dosage form; and c. a container.

12. A kit of claim 11 wherein the first unit dosage form comprises (-)-cis-6-phenyl-5-(4-(2-pyrrolidin-1-yl-ethoxy)-phenyl)-5,6,7,8-tetrahydronaphthalene-2-ol D-tartrate and the second unit dosage form comprises 2-amino-N-(2-(3a(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7-hexahydro-pyrazolo-[4,3-c]pyridin-5-yl)-1-(R)-benzyloxymethyl-2-oxo-ethyl)-isobutyramide L-tartrate.

13. A pharmaceutical composition according to any one of claims 1 to 3, which is for treating a mammal suffering from musculoskeletal frailty.

14. A pharmaceutical composition according to claim 13 wherein the mammal is suffering from osteoporosis.

15. A pharmaceutical composition according to claim 13, which is for treating osteotomy, childhood idiopathic bone loss or bone loss associated with periodontitis.

16. A pharmaceutical composition according to claim 13, which is for treating reconstruction, maxillary reconstruction or mandibular reconstruction; for inducing vertebral synostosis; for enhancing long bone extension; for enhancing the healing rate of a bone graft; or for enhancing prosthetic ingrowth.

17. A pharmaceutical composition according to claim 13, which is for treating a bone fracture in a human.

18. A pharmaceutical composition according to claim 14, which is for treating osteoporosis in a human.

19. A pharmaceutical composition according to any one of claims 1 to 3 or any one of claims 13 to 18, which contains the ingredient a. at a dosage of 0.001 to 10 mg/kg/day and the ingredient b. at a dosage of 0.01 to 5 mg/kg/day.

20. A kit according to any one of claims 11 to 13, which is for treating a mammal suffering from musculoskeletal frailty.

21. A kit of claim 20, which is adapted so that the first and second unit dosages are administered substantially simultaneously.

22. A kit of claim 20, which is adapted so that the second unit dosage is administered for a period of from about three months to about three years.

23. A kit of claim 22 which is adapted so that, after the administration of the second unit dosage, the first compound is administered for a period of from about three months to about three years without the administration of the second compound during the period of from about three months to about three years.

24. A kit of claim 22, which is adapted so that, after the administration of the second unit dosage, the first compound is administered for a period greater than about three years without the administration of the second compound during the greater than about three year period.

25. A kit of any one of claims 20 to 24, wherein the mammal is suffering from osteoporosis.

26. A kit of any one of claims 20 to 24, wherein the mammal is suffering from osteotomy, childhood idiopathic bone loss or bone loss associated with periodontitis.

27. A kit of any one of claims 20 to 24, which is for treating bone healing following facial reconstruction, maxillary reconstruction or mandibular reconstruction; for inducing vertebral synostosis; for enhancing long bone extension; for enhancing the healing rate of a bone graft; or for enhancing prosthetic ingrowth.

28. A kit of any one of claims 20 to 24, which is for treating a bone fracture in a human.

29. A kit of claim 20, which is adapted so that the second dosage unit is given to the mammal about 2 hours prior to the first dosage unit.

30. A method for increasing muscle mass in a mammal in need thereof, which comprises administering to the mammal a muscle mass increasing effective amount of a composition of claims 1, 2 or 3.