WO2002098353A2

WO2002098353A2 - Compositions and methods for inhibiting bone resorption

Info

Publication number: WO2002098353A2
Application number: PCT/US2002/017141
Authority: WO
Inventors: Alfred A. Reszka; John E. Fisher
Original assignee: Merck and Co Inc
Current assignee: Merck and Co Inc
Priority date: 2001-06-05
Filing date: 2002-05-31
Publication date: 2002-12-12
Anticipated expiration: 2003-12-05
Also published as: AU2002345434A1; WO2002098353A3

Abstract

The present invention relates to parenteral compositions and methods for inhibiting bone resorption, stimulating bone formation and/or treating or preventing elevated blood cholesterol levels in a mammal while counteracting potential local irritation at injection sites. The compositions useful herein comprise the combination of a pharmaceutically effective amount of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a pharmaceutically effective amount of a squalene synthase inhibitor.

Description

TITLE OF THE INVENTION

COMPOSITIONS AND METHODS FOR INHIBITING BONE RESORPTION

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to parenteral compositions and methods for inhibiting bone resorption, stimulating bone formation or suppressing serum cholesterol (LDL) levels in a mammal while counteracting potential local irritation at injection sites. The compositions useful herein comprise the combination of a pharmaceutically effective amount of a 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor (hereafter "HMG-CoA reductase inhibitor") and a pharmaceutically effective amount of a squalene synthase inhibitor.

BACKGROUND OF THE INVENTION

It has been clear for several decades that elevated blood cholesterol is a major risk factor for coronary heart disease, and many studies have shown that the risk of coronary heart disease (CHD) events can be reduced by lipid-lowering therapy. Prior to 1987, the lipid-lowering armamentarium was limited essentially to a low saturated fat and cholesterol diet, the bile acid sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), the fibrates and probucol. Unfortunately, all of these treatments have limited efficacy or tolerability, or both. Substantial reductions in LDL (low density lipoprotein) cholesterol accompanied by increases in HDL (high density lipoprotein) cholesterol could be achieved by the combination of a lipid- lowering diet and a bile acid sequestrant, with or without the addition of nicotinic acid. However, this therapy is not easy to administer or tolerate and was therefore often unsuccessful except in specialist lipid clinics. The fibrates produce a moderate reduction in LDL cholesterol accompanied by increased HDL cholesterol and a substantial reduction in triglycerides, and because they are well tolerated these drugs have been more widely used. Probucol produces only a small reduction in LDL cholesterol and also reduces HDL cholesterol, which, because of the strong inverse relationship between HDL cholesterol level and CHD risk, is generally considered undesirable. With the introduction of lovastatin, the first inhibitor of HMG-CoA reductase to become available for prescription in 1987, for the first time physicians were able to obtain large reductions in plasma cholesterol with very few adverse effects. Recent studies have unequivocally demonstrated that lovastatin, simvastatin and pravastatin, all members of the HMG-CoA reductase inhibitor class, slow the progression of atherosclerotic lesions in the coronary and carotid arteries. Simvastatin and pravastatin have also been shown to reduce the risk of coronary heart disease events, and in the case of simvastatin a highly significant reduction in the risk of coronary death and total mortality has been shown by the Scandinavian Simvastatin Survival Study. This study also provided some evidence for a reduction in cerebrovascular events.

A variety of disorders in humans and other mammals involve or are associated with abnormal bone resorption. Such disorders include, but are not limited to, osteoporosis, Paget's disease, periprosthetic bone loss or osteolysis, and hyper- calcemia of malignancy. The most common of these disorders is osteoporosis, which in its most frequent manifestation occurs in postmenopausal women. Osteoporosis is a systemic skeletal disease characterized by a low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. Osteoporotic fractures are a major cause of morbidity and mortality in the elderly population. As many as 70% of women and a third of men will experience an osteoporotic fracture. A large segment of the older population already has low bone density and a high risk of fractures. There is a significant need to both prevent and treat osteoporosis and other conditions associated with bone resorption. Because osteoporosis, as well as other disorders associated with bone loss, are generally chronic conditions, it is believed that appropriate therapy will typically require chronic treatment.

Multinucleated cells called osteoclasts are responsible for causing bone loss through a process known as bone resorption. It is well known that bisphosphonates are selective inhibitors of osteoclastic bone resorption, making these compounds important therapeutic agents in the treatment or prevention of a variety of generalized or localized bone disorders caused by or associated with abnormal bone resorption. See H. Fleisch, Bisphosphonates In Bone Disease, From The Laboratory To The Patient, 4th Edition, Academic Press (2000), which is incorporated by reference herein in its entirety. Without being limited by theory, it is believed that bisphosphonates inhibit osteoclast function by triggering apoptosis, i.e. programmed cell death. See D.E. Hughes et al., "Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo", Journal of Bone and Mineral Research, 10 (10), 1478-1487 (1995), which is incorporated by reference herein in its entirety. At present, it is believed that suggests that HMG-CoA reductase inhibitors can be useful as inhibitors of bone resorption and/or stimulants of bone formation, and thus useful as a treatment for osteoporosis. Oral dosing with simvastatin was shown to increase bone mass in rats after oral administration, and this was attributed to its bone anabolic effects (Mundy G, Garrett R, Harris S, Chan J, Chen D, Rossini G, Boyce B, Zhao M, Gutierrez G. Stimulation of bone formation in vitro and in rodents by statins. Science. 1999 Dec 3 ;286(5446): 1946-9). A case-control study suggests that statin use might be associated with reduced fracture risk in the elderly population (Wang PS, Solomon DH, Mogun H, Avorn J. HMG-CoA reductase inhibitors and the risk of hip fractures in elderly patients. JAMA. 2000 Jun 28;283(24):3211-6).

However, along with their benefits, HMG-CoA reductase inhibitors can cause potentially adverse side effects such as myopathy, rhabdomyolysis, liver dysfunction, alopecia and pruritus. A variety of skin changes (e.g., nodules, discolor- ation, dryness of skin/mucous membranes, changes to hair/nails) have been reported. In animal studies, local inflammatory irritation in the subcutis has been reported with subcutaneous administration of statin (Joles, J; Willekes-Koolschijn, N; Koomans, H; Van Tol, A; Geelhoed Mieras, T; Crommelin, D; Van Bloois- L; Krajnc Franken, M; Cohen, L; Griffioen, M; et-al. Subcutaneous administration of HMG-CoA reductase inhibitors in hyperlipidaemic and normal rats. Lab-Anim. 1992 Oct; 26(4): 269-80). Intravenous injection might be indicated in patients that are intolerant to oral dosing or incapable of or uncomfortable with swallowing solid oral dosing forms. In addition, intravenous administration is costly and inconvenient, especially when the patient must be given an intravenous infusion lasting several hours on repeated occasions.

A quicker and simpler means of administration could be achieved through other types of parenteral administration such as subcutaneous injection. The patient could perform the injection at home, much like with insulin, thus avoiding the need for costly and time-consuming visits to the doctor's office or clinic. It may also be of use for the development of continuous injection pumps. Dermal application of lovastatin has also been shown to allow greater increases in bone formation than with oral administration in the rat (G Gutierrez, I. R., Garrett, G, Rossini, M., Castano, G, Chapa, A., Escobedo, J., Esparza, D., Horn, M., Qiao, S., Taylor, D., Lalka, G. R. Mundy. Dermal Application of Lovastatin to Rats Causes Greater Increases in Bone Formation and Plasma Concentrations than when Administered by Oral Gavage. J. Bone Miner. Res. 15: S427, 2000). Absence of first-pass metabolism was attributed to the greater effectiveness of dermal application. Parenteral administration is an alternate dosing method to dermal application that can be used to bypass first-pass metabolism.

It is found in the present invention that squalene synthase inhibitors can block the apoptosis, i.e. programmed cell death, that can be induced by the HMG- CoA reductase inhibitors in vitro. Further it is found that squalene synthase inhibitors can block the inflammatory response in the subcutis that can be induced by the HMG- CoA reductase inhibitors after parenteral injection.

In the present invention, the combination of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a squalene synthase inhibitor is highly effective for inducing bone formation while mitigating the potential local irritation at injection sites that can be associated with HMG-CoA reductase inhibitor therapy. The combination of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a squalene synthase inhibitor is highly effective for normalizing serum cholesterol levels while mitigating the potential local irritation at injection sites that can be associated with HMG-CoA reductase inhibitor therapy. The combination has the advantage of providing increased safety and better patient compliance, which should maximize therapeutic efficacy. It is believed that the squalene synthase inhibitor blocks the potentially harmful effect of the HMG-CoA reductase inhibitor on the cells in and adjacent to the subcutis by causing accumulation of protein isoprenylation precursors, which when added exogenously block the induction of apoptosis by the HMG-CoA reductase inhibitor. By selecting an appropriate dosage of the squalene synthase inhibitor it is possible to parenterally deliver a sufficiently high local concentration of the squalene synthase inhibitor to the desired site to block the potentially harmful effects of the HMG-CoA reductase inhibitor, while still allowing the HMG-CoA reductase inhibitor to act at desired sites within the liver, to normalize serum cholesterol levels, and within the bone environment, to promote bone anabolism and/or suppress bone resorption.

It is an object of the present invention to provide compositions comprising the combination of an HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a squalene synthase inhibitor. It is another object of the present invention to provide improved parenteral methods for inhibiting bone resorption and the conditions associated therewith in a mammal, particularly wherein said mammal is a human.

It is another object of the present invention to provide improved parenteral methods for stimulating bone formation and the conditions associated therewith.

It is another object of the present invention to treat or prevent elevated blood cholesterol.

It is another object of the present invention to provide such methods wherein the dosing is maintained until the desired therapeutic effect is achieved.

These and other objects will become readily apparent from the detailed description which follows.

SUMMARY OF THE INVENTION The present invention relates to a pharmaceutical composition comprising a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

In further embodiments, the present invention relates to a pharmaceutical composition comprising a pharmaceutically-effective amount of a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and an amount of a squalene synthase inhibitor effective to counteract HMG-CoA reductase inhibitor-associated local irritation at injection sites.

In further embodiments, the present invention relates to a method for inhibiting bone resorption and/or stimulating bone formation in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

In further embodiments, the present invention relates to a method for treating or preventing elevated blood cholesterol in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

In further embodiments, the present invention relates to a method for inhibiting bone resorption and/or stimulating bone formation in a mammal in need thereof comprising sequentially administering a squalene synthase inhibitor and a

HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof. In further embodiments, the present invention relates to a method for treating or preventing elevated blood cholesterol in a mammal in need thereof comprising sequentially administering a squalene synthase inhibitor and a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof.

In further embodiments, the present invention relates to the use of a composition in the manufacture of a medicament for inhibiting bone resorption and/or stimulating bone formation in a mammal in need thereof, said composition comprising HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

In further embodiments, the present invention relates to the use of a composition in the manufacture of a medicament for treating or preventing elevated blood cholesterol in a mammal in need thereof, said composition comprising HMG- CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

In further embodiments, the present invention relates to the use of a composition comprising a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor for inhibiting bone resorption and/or stimulating bone formation in a mammal in need thereof.

In further embodiments, the present invention relates to the use of a composition comprising a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor for treating or preventing elevated blood cholesterol and/or stimulating bone formation in a mammal in need thereof.

All percentages and ratios used herein, unless otherwise indicated, are by weight. The invention hereof can comprise, consist of, or consist essentially of the essential as well as optional ingredients, components, and methods described herein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Skin irritation induced by alendronate and risedronate, which inhibit farnesyl diphosphate synthase, is similar to that induced by simvastatin in vivo. Rats were injected subcutaneously for a period of 5 days (vehicle, alendronate [ALN] at 0.5 mg/kg or risedronate [RIS] at 0.5 mg kg) or 2 days (vehicle or simvastatin [SIM] at 10 mg/kg). Skin was harvested after necropsy, fixed with formalin, thin-sectioned and stained with hematoxylin and eosin. Note the thickened region in the subcutis (between arrows) after treatment with alendronate [ALN], risedronate [RIS] or simvastatin [SIM] versus vehicle controls. Area was determined by measuring the thickness and length of each affected area and is represented vs. the H2O control.

Figure 2

A squalene synthase inhibitor in the absence of lipoprotein maximally suppresses lovastatin-induced apoptosis.

Chl.Es cells were grown for 24 hours in either fetal bovine serum (FBS) (top panels) or lipoprotein-deficient FBS (LPDS) in the absence (middle) or presence (bottom) of zaragozic acid a (Zara-A) at 10 μM. Continuing for an additional 24 hours, cells were treated without lovastatin (Control) or with lovastatin (10 μM). Apoptosis is manifest as cells that are rounded and / or detached from the surface of the petri dish. The suppression is complete after inclusion of Zara-A.

Figure 3

Schematic of cholesterol biosynthetic pathway and interactions with agents used in this study.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions and methods for inhibiting bone resorption, stimulating bone formation and/or treating or preventing elevated blood cholesterol in a mammal in need of such treatment, while counteracting the occurrence of local irritation at injection sites. The compositions comprise a pharmaceutically effective amount of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a pharmaceutically effective amount of a squalene synthase inhibitor.

The term "pharmaceutically effective amount", as used herein, means that amount of the HMG-CoA reductase inhibitor compound or squalene synthase inhibitor, that will elicit the desired therapeutic effect or response or provide the desired benefit when administered in accordance with the desired treatment regimen. A preferred pharmaceutically effective amount of the HMG-CoA reductase inhibitor is a bone resorption inhibiting amount, a bone formation stimulating amount and/or an amount sufficient to treat or prevent elevated blood cholesterol levels. A preferred pharmaceutically effective amount of the squalene synthase inhibitor is an amount that will counteract, i.e. block or mitigate, the occurrence of local irritation at injection sites, while not counteracting, or only minimally counteracting, the therapeutic bone resorption effects of the farnesyl diphosphate synthase inhibiting bisphosphonate. The term "counteracting the occurrence of local irritation at injection sites", as used herein, means to prevent, block, decrease, or lessen the occurrence of unwanted side effects local irritation at injection sites, relative to treatment with a HMG-CoA reductase inhibitor alone.

In the present invention it is an object to inhibit bone resorption, or more specifically to inhibit undesired or abnormal bone resorption. The term "abnormal bone resorption", as used herein means a degree of bone resorption that exceeds the degree of bone formation, either locally, or in the skeleton as a whole. Alternatively, "abnormal bone resorption" can be associated with the formation of bone having an abnormal structure, as in Paget's disease. The term "bone resorption inhibiting", as used herein, means preventing bone resorption by the direct or indirect alteration of osteoclast formation or activity. Inhibition of bone resorption refers to prevention of bone loss, especially the inhibition of removal of existing bone either from the mineral phase and/or the organic matrix phase, through direct or indirect alteration of osteoclast formation or activity.

The term "bone formation stimulating", as used herein, means a dose that enhances osteoblast proliferation, differentiation, and/or osteoblastic bone formation, leading to a net increase in the bone formation rate.

The term "until the desired therapeutic effect is achieved", as used herein, means that the therapeutic agent or agents are continuously administered, according to the dosing schedule chosen, up to the time that the clinical or medical effect sought for the disease or condition being treated is observed by the clinician or researcher. For methods of treatment of the present invention, the pharmaceutical composition is continuously administered until the desired change in bone mass or cholesterol levels is observed. In such instances, achieving an increase in bone mass or a replacement of abnormal bone structure with normal bone structure are the desired objectives. Likewise, a lowering of blood cholesterol levels is also a desired objective. For methods of prevention of the present invention, the pharmaceutical composition is continuously administered for as long as necessary to prevent the undesired condition. In such instances, maintenance of bone mass density is often the objective. Nonlimiting examples of administration periods can range from about 2 weeks to the remaining lifespan of the mammal. For humans, administration periods can range from about 2 weeks to the remaining lifespan of the human, preferably from about 2 weeks to about 20 years, more preferably from about 1 month to about 20 years, more preferably from about 6 months to about 10 years, and most preferably from about 1 year to about 10 years.

The term "parenteral administration" as used herein means taken into the body or administered in a manner other than through the digestive tract, as by intravenous, subcutaneous or intramuscular injection.

Compositions of the Present Invention

The pharmaceutical compositions of the present invention comprise a pharmaceutically effective amount of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and a pharmaceutically effective amount of a squalene synthase inhibitor. These compositions are useful for inhibiting bone resorption, stimulating bone formation and/or treating or preventing elevated blood cholesterol levels in a mammal in need thereof while counteracting the potentially adverse effects, such as local irritation at injection sites, that can be associated with the parenteral administration of the HMG-CoA reductase inhibitor.

HMG-CoA Reductase Inhibitor

The compositions herein comprise a compound which inhibits the enzyme, HMG-CoA reductase. Compounds which have inhibitory activity for HMG-CoA reductase can be readily identified by using assays well-known in the art. See U.S. Patent No. 4,231,938, to Monoghan et al., issued November 4, 1980 and U.S. Patent No. 5,354,772, to Kathawal, issued October 11, 1994, both of which are incorporated by reference herein in their entirety.

Examples of HMG-CoA reductase inhibitors that are useful herein include but are not limited to lovastatin (MEVACOR®; see U.S. Patent No. 4,231,938, already cited above and incorporated by reference herein), simvastatin

(ZOCOR®; see U.S. Patent No. 4,444,784, to Hoffman et al., issued April 24, 1984), pravastatin (PRAVACHOL®; see U.S. Patent No. 4,346,227, to Terahara et al., issued August 24, 1982), fluvastatin (LESCOL®; see U.S. Patent No. 5,354,772, already cited above and incorporated by reference herein), atorvastatin (LIPITOR®; see U.S. Patent No. 5,273,995, to Roth, issued December 28, 1993) and cerivastatin (also known as rivastatin; see U.S. Patent No. 5,177,080, to Angerbauer et al., issued January 5, 1993); and mevastatin (compactin, see U.S. Patent No. 3,983,140, to Endo et al, issued September 28, 1976. The patents cited in the previous sentence not already incorporated by reference are also incorporated by reference herein in their entirety. The structural formulas of these and additional HMG-CoA reductase inhibitors that can be used in the present invention are described at page 87 of M. Yalpani, "Cholesterol Lowering Drugs", Chemistry & Industry, pp. 85-89 (5 February 1996), which is incorporated by reference herein in its entirety. The term HMG-CoA reductase inhibitor is intended to include all pharmaceutically acceptable lactone and open acid (that is where the lactone ring is opened to form the free acid), as well as salt and ester forms of compounds which have HMG-CoA reductase inhibitory activity, and therefor the use of such lactone, open acid, salt, and ester forms is included within the scope of this invention. Preferably, the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, mevastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof. More preferably, the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof. More preferably, the HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof.

Preferred HMG-CoA reductase inhibitors can be represented by the chemical formula

wherein Z is selected from the group consisting of:

wherein

Rl is Ci-Cio alkyl;

R2 is selected from the group consisting of C1-C3 alkyl, hydroxy, oxo, and C1-C3 hydroxy substituted alkyl;

R3 is selected from the group consisting of hydrogen, hydroxy, C1-C3 alkyl, and C1-C3 hydroxy substituted alkyl;

a, b, c, and d are all single bonds, or a and c are double bonds, or b and d are double bonds, or one of a, b, c, and d is a double bond; and

n is O, 1, or 2;

wherein

X is selected from the group consisting of N[CH(CH3)2] and CH(CH2)3CH3

and

wherein R4 and R5 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, C1-C4 alkoxy, and trifluoromethyl, and Rβ, R7, R8, and R9 are each independently selected from the group consisting of hydrogen, fluorine, chlorine, bromine, iodine, C1-C4 alkyl, and C1-C4 alkoxy. SeeU.S. Patent No. 5,650,523, to DeCamp et al., issued July 22, 1997, which is incorporated by reference herein in its entirety. The pharmaceutically acceptable lactone, open acid, salt, and ester forms of the compounds depicted by the preceding chemical formulas are intended to be within the scope of the present invention.

The term "pharmaceutically acceptable salts" as used herein in referring to the HMG-CoA reductase inhibitors shall mean non-toxic salts of the compounds employed in this invention which are generally prepared by reacting the free acid with a suitable organic or inorganic base. Examples of salt forms of HMG- CoA reductase inhibitors include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium, calcium edetate, camsylate, carbonate, chloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutamate, glycoUylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynapthoate, iodide, isothionate, lactate, lactobionate, laurate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphate/diphosphate, polygalacturonate, potassium, salicylate, sodium, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, valerate, and mixtures thereof. The term "esters" as used herein in referring to the HMG-CoA reductase inhibitors is used in its standard meaning to denote the condensation product of a carboxylic acid and an alcohol. Ester derivatives of the described compounds can function as prodrugs which, when absorbed into the bloodstream of a warm-blooded animal, can cleave in such a manner as to release the drug form and permit the drug to afford improved therapeutic efficacy. The term "lactones" is used herein in referring to the HMG-CoA reductase inhibitors is used in its standard meaning to denote a cyclic condensation product of a carboxylic acid and an alcohol, i.e. a cyclic ester.

The term "open acid" is used herein in referring to the HMG-CoA reductase inhibitors to denote that the lactone ring is open, i.e. uncyclized, to form the free acid.

It is recognized that mixtures of two or more HMG-CoA reductase inhibitors can be utilized.

The dosage regimen utilizing a HMG-CoA reductase inhibitor is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt or ester thereof employed. A consideration of these factors is well within the purview of the ordinarily skilled clinician for the purpose of determining the therapeutically effective or prophylactically effective dosage amounts needed to prevent, counter, or arrest the progress of the condition. The term "patient" includes mammals, especially humans. Administering of the drug or drugs to the patient includes both self-administration and administration to the patient by another person.

The precise dosage of the HMG-CoA reductase inhibitor will vary with the dosing schedule, the particular compound chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies.

In particular, for daily dosing, the amounts of the HMG-CoA reductase inhibitor can be the same or similar to those amounts which are employed for anti- hypercholesterolemic treatment and which are described in the Physicians' Desk Reference (PDR), 52nd Ed. of the PDR, 1998 (Medical Economics Co), which is incorporated by reference herein in its entirety. For the additional active agents, the doses can be the same or similar to those amounts which are known in the art.

Parenteral dosage amounts of the HMG-CoA reductase inhibitor are from about 1 to 200 mg/day, and more preferably from about 5 to 160 mg/day. However, dosage amounts will vary depending on the potency of the specific HMG- CoA reductase inhibitor used as well as other factors as noted above. An HMG-CoA reductase inhibitor which has sufficiently greater potency may be given in sub- milligram daily dosages. The HMG-CoA reductase inhibitor may be administered from 1 to 4 times per day, and preferably once per day.

For example, the daily dosage amount for simvastatin can be selected from 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg, and 80 mg; for lovastatin, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 40 mg and 80 mg; for fluvastatin sodium, 0.1 mg, 0.5 mg, 1 mg, 5 mg„ 10 mg, 20 mg, 40 mg and 80 mg; for pravastatin sodium, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, and 40 mg; and for atorvastatin calcium, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, and 40 mg.

Squalene Synthase Inhibitors

The compositions of the present invention comprise a pharmaceutically effective amount of a squalene synthase inhibitor.

The squalene synthase inhibitors of the present invention are useful to block local irritation at injection sites when a HMG-CoA reductase inhibitor is administered parenterally. Non-limiting examples of squalene synthase inhibitors of the present invention can be categorized into four groups: zaragozic acid squalestatins, phosphate-derived substrate analogues, carboxylic acid-derived compounds, and quinuclidines and related amines. Naturally occurring isoforms of the zaragozic acid/squalestatins are characterized by the 2,8-dioxobicyclo[3.2.1]octane-3,4,5-tricarboxylic acid ring system. These naturally occurring forms can be isolated from fungal fermentations and natural products, and also serve as starting materials for diverse semisynthetic and synthetic analogues. Zaragozic acid/squalestatins of the present invention include compounds described in U.S. Patent Nos. 5,506,262 and 5,369,125 to Merck & Co., Inc.; JP-7173166 to Takeda; JP-9124655 to Sankyo Co. Ltd.; U.S. Patent No. 5,430,055 to Pfizer, Inc.; U.S. Patent No. 5,409,950 to Glaxo Group Ltd.; and JP 9227566 to Sagami Chuo Kagaku Kenkyujo. Non-limiting examples of zaragozic acid/squlaestatins include: a) Zaragozic Acid A

CO₂H b) Zaragozic Acid B

c) Zaragozic Acid C

C0₂H d) Squalestatin SI

CO₂H Phosphate-derived inhibitors were originally designed as substrate analogues for squalene synthase, and include compounds described in U.S. Patent Nos. 5,374,628, 5,428,028, 5,470,845 and 5,447,922 to ER Squibb & Sons, Inc. and U.S. Patent No. 5,441,946 to Rhone-Poulenc Rorer Pharm., Inc. Non-limiting examples of phosphate-derived inhibitors include:

Representative carboxylic acid-derived inhibitors can be characterized as having a lipophilic group(s) couple to one or more carboxylic acid residues. Carboxylic acid-derived residues of the present invention compounds disclosed in JP 7041454 to Sankyo Co. Ltd.; WO 9504025 to Chugai Seiyaku Kabushiki Kaisha; WO 9740006 to Cancer Res. Campaign Tech. Ltd.; JP 7173120 to Banyu; WO 9633159 and WO 9521815 to Abbott Lab; EP 645377, EP 645378 and WO 9521834 to Takeda Chem. Ind. Ltd.; WO 9748701 and EP 814080 to Pfizer, Inc. Non-limiting examples of carboxylic acid-derivatives inhibitors include:

Biaryl quinuclidine, quinuclidine dervivatives and related amines are squlaene synthase inhibitors, and include compounds described in WO 9403541, WO 9405660 and WO 9535295 to Zeneca Ltd.; WO 9626938 and JP 8134067 to Yamanouchi Pharm. Co. Ltd.; U.S. Patent Nos. 5,385,912, 5,494,918, 5,395,846, 5,451,596, and WO 9531458 and WO 9500146 to Rhone-Poulenc Rorer Pharm., Inc. Nonlimiting examples of quinucidines and related amines include:

It is recognized that mixtures of two or more of the squalene synthase inhibitors can be utilized.

The precise dosage of the squalene synthase inhibitor will vary with the dosing schedule, the particular compound chosen, the age, size, sex and condition of the mammal or human, the nature and severity of the disorder to be treated, and other relevant medical and physical factors. Thus, a precise pharmaceutically effective amount cannot be specified in advance and can be readily determined by the caregiver or clinician. Appropriate amounts can be determined by routine experimentation from animal models and human clinical studies. Generally, an appropriate amount is chosen to obtain an inhibition of the potentially adverse effects of the HMG-CoA reductase inhibitors. For the squalene synthase inhibitor, human doses which can be administered are generally in the range of about 25 ng/day to about 10 mg/day, preferably from about 25 nanograms to about 1 milligram. A typical nonlimiting exemplary dose is about 1 microgram, for a human subject.

Other Components of the Pharmaceutical Compositions

The HMG-CoA reductase inhibitors and the squalene synthase inhibitors of the present invention are typically administered in admixture with suitable pharmaceutical diluents, excipients, or carriers, collectively referred to herein as "carrier materials", suitably selected with respect to parenteral administration, consistent with conventional pharmaceutical practices. For parenteral dosing, in particular, subcutaneous administration, the agents are typically combined in aqueous vehicle, such as sterile water or sterile isotonic (0.9 %) sodium chloride.

The compounds used in the present method can also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinyl- pyrrolidone, pyran copolymer, polyhydroxylpropyl-methacrylamide, and the like.

Methods of the Present Invention

The present invention comprises methods for inhibiting bone resorption in mammals. The present invention also comprises methods for stimulating bone formation in mammals. The present invention also comprises methods for treating and/or preventing elevated blood cholesterol in mammals. The present invention also comprises methods for treating abnormal bone resorption in mammals. The present invention also comprises methods for preventing abnormal bone resorption in mammals. In preferred embodiments of the present invention, the mammal is a human.

The methods and compositions of the present invention are useful for both treating and preventing abnormal bone resorption and conditions associated therewith. Conditions associated with abnormal bone resorption include both generalized and localized bone loss. Also, the creation of bone having an abnormal structure, as in Paget's disease, can be associated with abnormal bone resorption. The term "generalized bone loss" means bone loss at multiple skeletal sites or throughout the skeletal system. The term "localized bone loss" means bone loss at one or more specific, defined skeletal sites. Generalized boss loss is often associated with osteoporosis. Osteoporosis is most common in post-menopausal women, wherein estrogen production has been greatly diminished. However, osteoporosis can also be steroid-induced and has been observed in males due to age. Osteoporosis can be induced by disease, e.g. rheumatoid arthritis, it can be induced by secondary causes, e.g. glucocorticoid therapy, or it can come about with no identifiable cause, i.e. idiopathic osteoporosis. In the present invention, preferred methods include the treatment or prevention of abnormal bone resorption in osteoporotic humans.

Localized bone loss has been associated with periodontal disease, with bone fractures, and with periprosthetic osteolysis (in other words where bone resorption has occurred in proximity to a prosthetic implant). Generalized or localized bone loss can occur from disuse, which is often a problem for those confined to a bed or a wheelchair, or for those who have an immobilized limb set in a cast or in traction.

The methods and compositions of the present invention are useful for treating and or preventing the following conditions or disease states: osteoporosis, which can include post-menopausal osteoporosis, corticosteroid- induced osteoporosis, male osteoporosis, disease-induced osteoporosis, idiopathic osteoporosis; Paget's disease; abnormally increased bone turnover; osteomalacia; periodontal disease; localized bone loss associated with periprosthetic osteolysis; bone fractures; coronary heart disease; and elevated blood cholesterol.

The compositions and methods of the present invention are administered and carried out until the desired therapeutic effect is achieved.

In the methods of the present invention the HMG-CoA reductase inhibitor and the squalene synthase inhibitor are generally administered concurrently. In alternate embodiments, the HMG-CoA reductase inhibitor and the squalene synthase inhibitor can be adminsitered sequentially.

The following Examples are presented to better illustrate the invention.

EXAMPLE 1

Chl.Es fibroblasts are cultured in petri dishes and pretreated for 24 hours at 37°C with (a) nothing or (b) the squalene synthase inhibitor, zaragozic acid A, at a concentration of 10 μM. The cells are then continued in culture for an additional 24 hours in the presence or absence of an HMG-CoA reductase inhibitor. At the end of this incubation period the cells are scored for the presence or absence of apoptosis. Absence of apoptosis is indicated by the presence of cells that are adherent to the petri dish surface and spread into a normal fibroblastic shape (e.g. triangular or otherwise spread over a two dimensional area and with little or no brightness at the cell periphery under phase contrast microscopy). Presence of apoptosis is scored as the rounding of cells, typically coupled with enhanced brightness at the cell periphery under phase contrast microscopy. Apoptotic cells may also detach partially or fully from the dish. Quantitative analyses may be performed by assessing the number of adherent and spread cells as a percent of the population. Trypan blue exclusion may also be used as a score for cell viability. Quantitative analyses may include determin- ing the percent of cells that can exclude trypan blue (viable cells). EXAMPLE 2

Rats are administered an inhibitor of HMG-CoA reductase inhibitor, such as simvastatin in the absence or presence of several doses of a squalene synthase inhibitor (e.g. 0.1-30 micrograms). Dosing by subcutaneous injection occurs daily at 24 hour intervals and continues for a period of 2-5 days. Each successive injection is administered into the same site in the animal. Animals are then euthanized and the skin area surrounding the injection site is excised and fixed in a solution of 10% formalin for 7 days at 4°C. Affected area is then measured using a caliper. Measurements include the thickness of the skin sample (subcutis to stratum corneum) and the diameter of the affected area (measured within the plane of the skin surface). Thickened volume is calculated as the skin thickness multiplied by the area (πr2), where r is radius or 0.5 x diameter.

EXAMPLE 3

As a specific embodiment of the present invention, 70 mg of a HMG-CoA reductase inhibitor and 5 micrograms of a squalene synthase inhibitor are dissolved in sterile isotonic (0.9%) sodium chloride to a total volume of 0.5 mL.

Claims

WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising an HMG-CoA reductase inhibitor or a pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

2. The pharmaceutical composition according to Claim 1 wherein said a HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, mevastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof.

3. The pharmaceutical composition according to Claim 2 wherein said squalene synthase inhibitor is selected from the group consisting of:

a) Zaragozic Acid A

c) Zaragozic Acid C

d) SqualestatinSl

and the pharmaceutically acceptable salts and mixtures thereof.

4. The pharmaceutical composition according to Claim 3 wherein said squalene synthase inhibitor is selected from the group consisting of:

a) Zaragozic Acid A

b) Zaragozic Acid B

c) Zaragozic Acid C

d) Squalestatin Sl

and the pharmaceutically acceptable salts and mixtures thereof.

5. The pharmaceutical composition according to Claim 4 wherein said squalene synthase inhibitor is zaragozic acid A and the pharmaceutically acceptable salts thereof.

6. The pharmaceutical composition according to Claim 5 wherein said a HMG-CoA reductase inhibitor is selected from the group consisting of lovastatin, simvastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereo .

7. The pharmaceutical composition according to Claim 6 wherein said a HMG-CoA reductase inhibitor is simvastatin.

8. The pharmaceutical composition according to Claim 6 wherein said a HMG-CoA reductase inhibitor is lovastatin.

9. A pharmaceutical composition comprising from 0.1 to 100 mg of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and from 25 nanograms to 1 milligram of a squalene synthase inhibitor.

10. The pharmaceutical composition according to Claim 9 comprising from 2 to 70 mg of a HMG-CoA reductase inhibitor or a pharmaceutically-acceptable salt thereof and from 0.1 microgram to 1 milligram of a squalene synthase inhibitor.

11. A pharmaceutical composition comprising from 1 to 100 mg of simvastatin, and from 0.1 microgram to 1 milligram of zaragozic acid A.

12. The pharmaceutical composition according to Claim 11 comprising from about 2 to about 70 mg of simvastatin, and from about and from about 0.1 microgram to about 1 milligram of zaragozic acid A.

13. A pharmaceutical composition comprising from 1 to 100 mg of lovastatin, and from 0.1 microgram to 1 milligram of zaragozic acid A.

14. The pharmaceutical composition according to Claim 13 comprising from about 2 to about 70 mg of lovastatin, and from about and from about 0.1 microgram to about 1 milligram of zaragozic acid A.

15. The pharmaceutical composition which is prepared by combining a HMG-CoA reductase inhibitor and a squalene synthase inhibitor.

16. A method for inhibiting bone resorption in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

17. The method according to Claim 16 wherein said mammal is a human.

18. A parenteral method for treating or preventing osteoporosis in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

19. A method according to Claim 18 wherein said mammal is a human.

20. A method for stimulating bone formation in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

21. A method according to Claim 20 wherein said mammal is a human.

22. A method for treating and/or preventing elevated blood cholesterol in a mammal in need thereof comprising administering a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof and a squalene synthase inhibitor.

23. A method according to Claim 22 wherein said mammal is a human.

24. A method according to Claim 16 wherein said HMG-CoA reductase inhibitor or pharmaceutically-acceptable salt thereof is selected from the group consisting of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, cerivastatin, mevastatin, and the pharmaceutically acceptable lactones, open acids, salts, and esters thereof, and mixtures thereof.

25. A method according to Claim 24 wherein said squalene synthase inhibitor is selected from the group consisting zaragozic acid A, zaragozic acid B, zaragozic acid C, squalestatin S, and pharmaceutically acceptable salts and mixtures thereof.

26. A method for inhibiting bone resorption in a mammal in need thereof comprising sequentially administering a squalene synthase inhibitor and a HMG-CoA reductase inhibitor or pharmaceutically acceptable salt thereof.