WO2013034098A1

WO2013034098A1 - Combination use of vitamin d and hmg coa reductase inhibitor for treating osteoporosis

Info

Publication number: WO2013034098A1
Application number: PCT/CN2012/081126
Authority: WO
Inventors: Yiu Wa KWAN
Original assignee: The Chinese University Of Hong Kong
Priority date: 2011-09-07
Filing date: 2012-09-07
Publication date: 2013-03-14

Abstract

The present invention provides a method for treating osteoporosis by co- administration of a vitamin D and a 3-hydroxy-3-methyl-glutaryl-Co A reductase (HMG Co A reductase) inhibitor. Also provided are compositions and kits useful for treating osteoporosis comprising a vitamin D and an HMG Co A reductase inhibitor.

Description

COMBINATION USE OF VITAMIN D AND HMG COA REDUCTASE INHIBITOR FOR TREATING OSTEOPOROSIS

BACKGROUND OF THE INVENTION

[0001] This application claims priority to U.S. Provisional Patent Application No. 61/531 ,963, filed September 7, 201 1 , the contents of which are incorporated by reference in the entirety.

BACKGROUND OF THE INVENTION

[0002] Osteoporosis, a silent disease, is a skeletal disorder characterized by compromised bone strength predisposing to an increased risk of fractures. The global burden of osteoporosis is significant nowadays - over 200 million people worldwide and approximately 30% of all postmenopausal women in the USA and Europe are affected by this disease. Osteoporosis occurs in both men and women because of age and hormonal factors. With an increase in life expectancy of the general population, it is anticipated that number of patients with this disease will be escalated within this decade and beyond. Despite recent successes with drugs that inhibit bone resorption, there is a clear need for anabolic agents that can substantially increase bone formation in patients who have already suffered substantial bone loss. Current therapeutic strategies such as hormone replacement therapy and bisphosphonates are effective but are associated with serious side effects, particularly after long-term uses. Because of the prevalence of this disease and its significant implications on patients' quality of life, there exists a need for new methods to treat and reduce the risk of osteoporosis. This invention fulfills this and other related needs.

BRIEF SUMMARY OF THE INVENTION

[0003] In one aspect, the present invention provides a method for treating osteoporosis in a subject. The method includes the step of administering to the subject an effective amount of a vitamin D (e.g., vitamin D, vitamin D₃, or la,25-dihydroxyvitamin D) and a 3-hydroxy-3- methyl-glutaryl-CoA reductase (HMG CoA reductase) inhibitor (or statin, such as simvastatin). In some cases, the subject being treated by this method is not one otherwise in need of treatment by the HMG CoA reductase inhibitor. In some embodiments, the vitamin D and the HMG CoA reductase inhibitor are administered in one composition, whereas in other embodiments, the vitamin D and the HMG CoA reductase inhibitor are administered in two separate compositions. Typically, the amount of the vitamin D administered is no more than 2000 IU daily, whereas the amount of the HMG CoA reductase inhibitor administered is no more than 100 mg daily.

[0004] In another aspect, the present invention provides a composition useful for the treatment of osteoporosis in a subject. The composition contains a vitamin D (e.g., l a,25- dihydroxyvitamin D), a 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG CoA reductase) inhibitor (or a statin, such as simvastatin), and a pharmaceutically acceptable carrier. In some cases, the composition is packaged in daily dose units, with each unit comprising no more than 2000 IU of the vitamin D and no more than 100 mg of the HMG CoA reductase inhibitor. In some embodiments, the composition is formulated for oral administration. [0005] In yet another aspect, the present invention provides a kit comprising compositions for treatment of osteoporosis. Typically, the kit contains a first composition that comprises a vitamin D (such as l a,25 -dihydroxyvitamin D) and a second composition that comprises an HMG CoA reductase inhibitor (such as simvastatin). In some embodiments, the first composition is packaged in daily dose units, with each unit having no more than 2000 IU of the vitamin D. In other embodiments, the second composition is packaged in daily dose units, with each unit having no more than 100 mg of the HMG CoA reductase inhibitor. Either or both of the first and second compositions may be formulated for oral administration.

BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 : illustration of biochemical pathways by which cholesterol is synthesized from acetyl-CoA. The involvement of 3-hydroxy-3-methylglutaryl-CoA (HMG CoA) reductase and its inhibitors, statins, are shown in the synthesis scheme.

[0007] Figure 2: expression levels of calcium-sensing receptor (CaSR) as determined in immunocytochemistry and Western blot analysis. CaSR is a sensor of [Ca²⁺]₀ and a regulator for CaSR activities, which changes the [Ca²⁺]₀ stimulus into multiple cellular signaling pathways. It is possible that there is an alteration of the activities / expression of CaSR in osteoblasts, during and after menopause, which hinders the continuous bone formation and repairing, resulting in a progressive weakening of skeleton structures over time and development of osteoporosis. The immunocytochemistry results shown here illustrate that the expression of CaSR is lower in osteoblasts of ovariectomized (OVX) rats compared to age-matched normal rats. DMSO (solvent for simvastatin) or Ethanol (solvent for l a,25 dihydroxyvitamin D3) alone was tested and they had no effects on CaSR expression of OVX and normal rats. Simvastatin (10 nM) or 1α,25 dihydroxyvitamin D3 (10 nM), applied alone for 7 days, failed to modify the expression of CaSR of normal and OVX rats. After combination treatment of ΙΟηΜ simvastatin plus 10 nM la,25 dihydroxyvitamin D3, the expression of CaSR in osteoblasts of OVX rats increased and the "difference" in CaSR expression as observed in controls (drug-free conditions) was ameliorated, i.e., there was no significant difference in CaSR expression in osteoblasts between normal and OVX rats.

[0008] Figure 3: expression levels of F-actin cytoskeleton as shown in immunocytochemistry and Western blot analysis. The cytoskeleton plays important roles in cells functions, e.g., transduction of signals and transport of cargo synthesis in the cytoplasm to plasma membrane for secretion. Thus, an alteration of cytoskeleton levels (occurs over time in the continuous decrease of estrogen levels during / after menopause) inevitably modifies cellular signals transduction processes and the secretory functions of osteoblasts which resulted in a progressive deterioration of skeleton structures and fracture of bone occurred. The immunocytochemistry results shown here illustrate that the expression of F-actin cytoskeleton is lower in osteoblasts of

ovariectomized (OVX) rats compared to age-matched normal rats. DMSO (solvent for simvastatin) or Ethanol (solvent for 1 a,25 dihydroxyvitamin D3) alone was tested and they had no effects on F-actin cytoskeleton expression of OVX and normal rats. Simvastatin (10 nM) or la,25 dihydroxyvitamin D3 (10 nM), applied alone for 7 days, enhanced the expression of F- actin cytoskeleton of normal and OVX rats. However, there was a significant difference in F- actin cytoskeleton expression between normal rats and OVX rats after treatments with simvastatin (10 nM) or la,25 dihydroxyvitamin D3 (10 nM) alone. After combination treatment of ΙΟηΜ simvastatin plus 10 nM la,25 dihydroxyvitamin D3, the expression of F-actin cytoskeleton in osteoblasts of OVX rats increased, and the "difference" in F-actin cytoskeleton expression as observed in controls (drug-free conditions) and treated with simvastatin (10 nM) and la,25 dihydroxyvitamin D3 (10 nM) alone was ameliorated, i.e., there was no significant difference in F-actin cytoskeleton expression in osteoblasts between normal and OVX rats.

DEFINITIONS

[0009] The term "Osteoporosis," as used in this application, refers to a condition involving thinning of bone structure, loss of bone density, and an increased tendency of bone fractures. Risk factors for osteoporosis include aging, gender (being female), low body weight, reduced sex hormone levels (as seen in women during menopause), smoking, and certain medications. [0010] The term "vitamin D" encompasses a group of steroid vitamin or fat-soluble

prohormones. There are five forms of vitamin D, vitamin Di, D₂, D₃, D4, D5. Human vitamins D₂ (ergocalciferol) and D₃ (cholecalciferol) are of particular significance. As used in this application, "vitamin D" also encompasses certain compounds derived from vitamin D during metabolism of vitamin D, such as the active metabolite of vitamin D, la,25-dihydroxyvitamin D₃, or calcitriol.

[0011] The term "treat" or "treating," as used in this application, describes to an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a relevant condition. In other words, "treating" a condition encompasses both therapeutic and prophylactic intervention against the condition.

[0012] "3-hydroxy-3-methylglutary-CoA reductase inhibitors" or "HMG CoA reductase inhibitors" are also known as statins. They are a class of compounds used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase, which plays a central role in the de novo production of cholesterol in the liver, see Figure 1. As increased cholesterol levels have been associated with cardiovascular diseases, statins are frequently prescribed drugs for the treatment and prevention of these diseases. Statins approved for such use include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin, and they are within the meaning of "HMG CoA reductase inhibitor" in this application.

[0013] The term "effective amount" as used herein refers to an amount of a given substance that is sufficient in quantity to produce a desired effect. For example, an effective amount of a vitamin D or an HMG CoA reductase inhibitor is the amount of one compound, when coadministered with other, to achieve a therapeutic or prophylactic effect such that the symptoms of osteoporosis are reduced, reversed, eliminated, or prevented in a patient who has been given the combination of the two compounds for the purposes of treating or reducing the risk of developing osteoporosis in a subject. An amount adequate to accomplish this is defined as the

"therapeutically effective dose." The dosing range varies with the nature of the therapeutic agent being administered and other factors such as the route of administration and the severity of a patient's condition.

[0014] The term "subject" or "subject in need of treatment," as used herein, includes individuals who seek medical attention due to risk of, or actual suffering from, osteoporosis. Subjects also include individuals currently undergoing therapy that seek manipulation of the therapeutic regimen. Subjects or individuals in need of treatment include those that demonstrate symptoms of osteoporosis or are at risk of suffering from its symptoms. For example, a subject in need of treatment includes individuals with a genetic predisposition or family history for osteoporosis, those that have suffered relevant symptoms in the past, those that have been exposed to a triggering substance or event, as well as those suffering from chronic or acute symptoms of the condition. A "subject in need of treatment" may be of either gender but are typically of more advanced age, e.g., 60 years or older.

[0015] A "subject not otherwise in need of treatment by an HMG CoA reductase inhibitor " is a subject who has not been diagnosed with a condition or is known to be at risk of developing the condition, to which administration of an HMG CoA reductase inhibitor is known to confer clinically discernable beneficial effects. For instance, statins are known to be prescribed by physicians to patients who have abnormally high cholesterol levels for the purpose of treating various cardiovascular diseases and related conditions either therapeutically or prophylactically, and a patient suffering from such a disease or condition is one "otherwise in need of treatment by an HMG CoA reductase inhibitor."

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[0016] Osteoporosis is a disease that impacts a significant portion of the human population, especially among women and the elderly. Today, over 200 million people worldwide are affected by osteoporosis. Approximately 33% of women aged between 60 to 70 and 66% of those over 80 have osteoporosis. About 40% of all affected women suffer a bone fracture during their lifetime with serious consequences such as decreased quality of life and heavy hospital costs. With the increased life expectancy, it is anticipated that number of aging-associated bone loss will be escalated exponentially. [0017] The currently available therapies for treating osteoporosis are monotherapies and have numerous drawbacks such as undesirable side effects, especially in long term use. In contrast to monotherapy, combination drug therapy utilizes more than one medication with which individual agent is given at the lowest possible therapeutic dosage (i.e., minimal side effects are anticipated) and with additive/synergistic therapeutic outcomes resulted. As reported herein, the inventor studied the bone anabolic properties of two therapeutic agents administered in combination, which has not been proposed before, by evaluating the levels of bone formation biomarkers, e.g., alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and osteocalcin (OCN), Alizarin red (for mineralized extracellular matrix), and a novel protein (which is important in altering cellular Ca²⁺ levels) recently discovered by the inventor's research group as being expressed at a lower level in bone cells of ovariectomised (OVX) rats as compared to normal rats. Similar to other diseases (e.g., AIDS, peptic ulcer) using similar treatment regime, combination drug therapy in this study has provided desirable enhanced bone anabolic effects, ex vivo, in bone cells of ovariectomized rats, an animal model for human estrogen-deficiency / postmenopausal associated osteoporosis.

[0018] This invention provides a novel combination drug therapy using Vitamin D or its derivatives, plus any HMG CoA reductase inhibitors (including statins such as simvastatin, as well as other agents such as bisphosphonates that affect the same cellular pathways in a similar manner to statins), in the treatment of hormone deficiency-related (i.e., post-menopausal) and aging-associated osteoporosis. Vitamin D is important in maintaining bone strength. The readily-available source of vitamin D is from the exposure to sunlight. Osteoporosis associated with vitamin D deficiency due to inadequate sunlight exposure is particularly prevalent in women certain ethnic groups due to their culture preference for lighter skin color.

[0019] The original clinical use of HMG CoA reductase inhibitors (so-called statins) is for reduction of cholesterol levels. More than a decade ago, it has been briefly reported that statins may stimulate bone formation in vitro and in rodents. A retrospective analysis of older women taking statins for lowering lipid in blood is accompanied with a greater hip bone mineral density and a lower risk of hip fracture. However, HMG CoA reductase inhibitors have not been put into clinical uses for this purpose, which may be related to dose-related side effects of statins.

[0020] As disclosed herein, the present inventor discovered for the first time the a combination treatment strategy of co-administering a vitamin D and a HMG CoA reductase inhibitor (or a bisphosphonate) has a surprising synergistic effect on treating osteoporosis, which could not have been gleaned from the previously known use of vitamin D or statins (or bisphosphonates).

II. Patient Population

[0021] Practicing this invention utilizes co-administration of a vitamin D compound and an HMG CoA reductase inhibitor (statin). Typically, patients to receive this treatment are individuals who have received a diagnosis of osteoporosis or are known to have an elevated risk of developing osteoporosis, due to genetic or other risk factors (i.e., gender, age, medical history, nutrition, physical activity, and the like). In some cases, the patient to receive the treatment method of this invention does not by coincidence also suffer from or have an increased risk of any condition for which a statin may be prescribed. Known conditions for which physicians prescribe statin drugs for therapeutic or prophylactic treatment include various cardiovascular diseases associated with hypercholesterolemia such as coronary artery diseases, hypertension, and atherosclerosis. In rare cases, statins are also known for their use to confer benefits to conditions such as certain types of cancer and conditions caused or exacerbated by inflammation and oxidative stress.

[0022] Diagnosis of osteoporosis can be made by utilizing methodology well known and widely accepted in the medical community. Typically, bone density of a patient being examined is measured and then compared to the average peak bone density of young adults of the same sex and race. This score is called the "T score," and it expresses the bone density in terms of the number of standard deviations (SD) below peak young adult bone mass. Osteoporosis is defined as a bone density T score of -2.5 or below, and osteopenia (between normal and osteoporosis) is defined as bone density T score between -1 and -2.5. A patient having a diagnosis of osteopenia is considered within the meaning of this application a subject known to have an elevated risk of developing osteoporosis and is therefore an appropriate recipient for the treatment method of this invention.

[0023] Various methods are known for measuring bone density. For instance, a routine X-ray can reveal osteoporosis as the bones appear much thinner and lighter than normal bones. A dual- energy X-ray absorptiometry scan (DXA, formerly known as DEXA) is particularly useful for diagnosing osteoporosis. DXA measures bone density in the hip and the spine, offering advantages such as fast administration time (hence exposing patients to very little radiation) and a relatively high precision level. III. Vitamin D and HMG CoA Reductase Inhibitors

[0024] The present invention is based on the surprising discovery that, even at relatively low dosages, the combination of a vitamin D (such as vitamin D, vitamin D3, or la,25- dihydroxyvitamin D₃) and an HMG CoA reductase inhibitor (such as simvastatin) can yield an unexpected, synergistic effect when co-administered in an effective amount to a patient suffering from or at risk of developing osteoporosis for the purpose of treating, preventing, eliminating, alleviating, reducing, reversing, or delaying the onset of symptoms of osteoporosis. The surprising results of this combination treatment have been shown in isolated osteoblasts from normal and OVX rats. Compositions containing either or both of a vitamin D and an HMB CoA reductase inhibitor are therefore one aspect of this invention.

A. Vitamin D and Related Compounds

[0025] For the purpose of treating osteoporosis by co-administration of a vitamin D and an HMG CoA reductase inhibitor, a composition comprising a vitamin D and a physiologically or pharmaceutically acceptable carrier or excipient is used in accordance with the present invention. Vitamin D and its derivatives, including compounds produced from vitamin D metabolites, such as vitamin D, vitamin D3, or la,25-dihydroxyvitamin D, can be used along or in combination as the active therapeutic agent in the composition.

B. HMG CoA Reductase Inhibitors

[0026] According to the present invention, an HMG CoA reductase inhibitor, or statin, is coadministered with a vitamin D compound for treating or preventing osteoporosis. Any compound that has the same or similar biological activity, i.e., capable of inhibiting enzymatic activity of HMG CoA reductase and thus suppressing the biosynthesis of cholesterol, can be used for this purpose. Statins approved for medicinal use include atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin. Typically, one or more HMG CoA reductase inhibitor is formulated with at least one physiological or

pharmaceutical acceptable carrier or excipient to produce a pharmaceutical composition.

[0027] Bisphosphonates, which are also prescribed for treating osteoporosis, may serve as alternatives or substitutes to statins for the combination treatment scheme of this invention.

Bisphosphonates (also called diphospho nates) are a class of drugs that prevent the loss of bone mass and are therefore used to treat osteoporosis and similar diseases. These compounds have two phosphonate (P0₃) groups and are similar in structure to pyrophosphate. IV. Composition and Administration for Treating Osteoporosis

[0028] By illustrating the synergistic effect of a vitamin D and an HMG CoA reductase inhibitor (or a bisphosphonate), the present invention further provides compositions for treating patients suffering from osteoporosis in the combination scheme. As used herein, treatment of narcolepsy encompasses reducing, lessening, eliminating, or reversing one or more of the symptoms of osteoporosis, as well as preventing or delaying the onset of one or more of the relevant symptoms. B. Pharmaceutical Compositions

1. Formulations

[0029] Compounds of the present invention, e.g., a vitamin D and an HMG Co A reductase inhibitor (or a bisphosphonate), are useful in the manufacture of a pharmaceutical composition or a medicament comprising an effective amount thereof in conjunction or mixture with excipients or carriers suitable for application. A pharmaceutical composition or medicament can be administered to a subject for the treatment of osteoporosis.

[0030] An therapeutically active ingredient, e.g., a vitamin D, an HMG CoA reductase inhibitor (or a bisphosphonate), either alone or in combination, can be administered via liposomes, which serve to target the conjugates to a particular tissue, as well as increase the half- life of the composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the inhibitor to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among the targeted cells (e.g., osteocytes), or with other therapeutic compositions. Thus, liposomes filled with a desired active agent can be directed to the site of delivery, where the liposomes then deliver the selected active agents.

Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9: 467, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.

[0031] Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in "Remington's

Pharmaceutical Sciences" by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally, or rectally.

[0032] Typical formulations for topical administration include creams, ointments, sprays, lotions, and patches. The pharmaceutical composition can, however, be formulated for any type of administration, e.g., intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices. Formulation for administration by inhalation (e.g., aerosol), or for oral, rectal, or vaginal administration is also contemplated.

2. Routes of administration

[0033] Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

[0034] Suitable formulations for transdermal application include an effective amount of an active agent of the present invention with a carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used.

[0035] For oral administration, a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a

pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient, e.g., a vitamin D, an HMG Co A reductase inhibitor (or a bisphosphonate), alone or in combination, together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or

polyethyleneglycol; for tablets also (c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired (d) disintegrants, e.g., starches (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate, and/or (f) absorbents, colorants, flavors and sweeteners. [0036] Tablets may be either film coated or enteric coated according to methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p- hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral

administration can be suitably formulated to give controlled release of the active compound.

[0037] Compounds and agents used in the present invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

[0038] For administration by inhalation, the active agent may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane,

dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0039] The active agent used in the present invention can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides. [0040] Furthermore, the active agents can be formulated as a depot preparation. Such long- acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0041] A pharmaceutical composition or medicament of the present invention comprises (i) an effective amount of a therapeutic agent, and (ii) another therapeutic agent, e.g., a vitamin D being the first agent, and an HMG CoA reductase inhibitor (or a bisphosphonate) being the second. In a combination treatment scheme of the present invention, the two therapeutic agents may be formulated in a single composition for administration together, or they may be used individually or sequentially in two separate compositions. Administration of the separate compositions may be by the same or different route of administration or at the same time or in a staggered fashion. 3. Dosage

[0042] Pharmaceutical compositions or medicaments can be administered to a subject at a therapeutically effective dose to prevent, treat, control or reverse the symptoms of osteoporosis as described herein. The pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject.

[0043] The dosage of active agents administered is dependent on the subject's body weight, age, individual condition, surface area or volume of the area to be treated and on the form of administration. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. As discussed herein, the combination treatment method of this invention takes advantage of the surprising effectiveness of two therapeutically active agents (a vitamin D and a statin or a bisphosphonate) even at a low dosage. A patient receiving the combination treatment for osteoporosis is typically given no more than 2000 IU per day of a vitamin D (such as vitamin D, vitamin D3, or la,25-dihydroxyvitamin D₃) and no more than 100 mg per day of a statin (such as simvastatin), although dosage as high as 2500 IU, 3000 IU, 4000 IU, or up to 5000 IU per day of vitamin D and 200 mg, 300 mg, 400 mg, or up to 500 mg per day of an HMG CoA reductase inhibitor (or a bisphosphonate) may be used in some cases. Possible daily doses include 100, 200, 300, 400, 500, 600, 700, 800, 1000, 1500, or 2000 IU for a vitamin D in combination with 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg for an HMG CoA reductase inhibitor (or a bisphosphonate).

[0044] The dosage of a pharmaceutical composition of the invention can be adjusted throughout treatment, depending on severity of symptoms, frequency of recurrence, and physiological response to the therapeutic regimen. Those of skill in the art commonly engage in such adjustments in therapeutic regimen.

VII. KITS

[0045] The invention provides compositions and kits for practicing the methods described herein for treating osteoporosis in a subject.

[0046] Kits for treating osteoporosis typically include at least two separate compositions kept in two separate containers: the first comprising a vitamin D (such as vitamin D, vitamin D3, or l a,25-dihydroxyvitamin D) and the second comprising an HMG CoA reductase inhibitor (such as simvastatin) or in the alternative a bisphosphonate. Typically, the two compositions are formulated for oral administration. In some cases, either or both of the two compositions are packaged in daily dose units, with each unit containing a pre-determined amount of vitamin D or HMG CoA reductase inhibitor, for example, no more than 2000 IU of vitamin D, vitamin D3, or l a,25-dihydroxyvitamin D₃ per unit or no more than 100 mg of statin (e.g., simvastatin) or in the alternative a bisphophonate per unit.

[0047] Kits of this invention may provide instruction manuals to guide users in properly administering the two compositions to a subject suffering from or at risk of developing osteoporosis.

EXAMPLES

[0048] The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed or modified to yield essentially the same or similar results.

Example 1

[0049] In this study, primarily osteoblasts (osteoblasts-like) cells were harvested from the trabecular bones in the iliac crests of Sprague-Dawley (normal and ovariectomized, OVX) rats. These cells demonstrated the activities of alkaline phosphatase (ALP; an enzyme / biomarker for bone formation), indicating that they are genuine osteoblasts (bone forming cells). In these cells, the inventor demonstrated the expression of Ca²⁺-sensing receptors (CaSR), and for the first time reported in the literature in osteoblasts of OVX rats with a lower expression using

immunocytochemistry methods (see Figure 2). A lower expression of CaSR indicated that one

2__|_

of the bone building ions (i.e., Ca ) was lower inside the osteoblasts of OVX rats with symptoms of osteoporosis developed. Incubation (7 days) with a combination of la,25- dihydroxyvitamin D₃ (10 nM, the most biologically active vitamin D metabolite) plus simvastatin (10 nM) resulted in an increase in the expression of osteocalcin (OCN, a bone formation biomarker) and CaSR in osteoblasts of OVX rats. Strontium ranelate (1 mM, i.e. , 100,000-fold higher than simvastatin / 1 a,25-dihydroxyvitamin D₃ used), served as a positive control on CaSR activation, increased OCN expression of osteoblasts of OVX rats. It is anticipated that an increase in CaSR expression (or activation) leads to an increase in cellular Ca²⁺ levels, which are important for mineralization of extracellular matrix (bone structure building) of osteoblasts, and for the cellular signaling cascades that are important for bone cells functions.

[0050] In the combination treatment scheme of this invention, the lowest possible therapeutic dosages are used for treating hormone-deficiency related and aging-associated osteoporosis: vitamin D at no more than 50mg daily and simvastatin at no more than 100 mg daily. Since the current and common therapeutic strategies for treating osteoporosis is "monotherapy" treatment using an active agent mainly to deal with the bone structure, it is inevitable that a relatively high dose of drug is used (e.g., strontium ranelate, 2 g per day; alendronate, 10 mg per day) for a long time. The main disadvantage in such monotherapy is side-effects such as nausea, diarrhea, memory loss, drug rash with eosinophilia, and an increased risk of venous thromboembolism / pulmonary embolism. Interestingly, vitamin D and Ca²⁺ supplements (either one or both together) are also prescribed to some patients simply based on the general belief that these supplements are good for the bone structure, while no consideration is given to the possible beneficial interactions and the underlying mechanisms involved between these agents prescribed. Ironically, it has been suggested in some studies that either vitamin D or Ca supplement alone (although both are important for bone health) provided no obvious overall beneficial effects to bone structure especially after osteoporosis has begun. In contrast, a combination drug therapy, as shown by the results of this study, with detailed underlying mechanisms delineated not only offers the patients with effective treatment with solid scientific proof, it also minimizes the side effects as associated with high doses of drags currently prescribed and a reduction of cost for drag prescriptions.

[0051] In this study, the inventor determined and compared the effects of a combination of la,25-dihydroxyvitamin D₃ and simvastatin (both at 10 nM for 7 days incubation) on bone anabolic responses, ex vivo, of osteoblasts isolated from normal and ovariectomized (OVX) Sprague-Dawley rats. OVX rats are animal models used for mimicking hormone-deficiency-

2__|_ related (i.e., post-menopausal) osteoporosis. Before drug treatments, a lower expression of Ca - sensing receptor (CaSR) was detected in osteoblasts harvested from OVX rats, which was reported by the inventor's research team. A lower expression of CaSR implying that a lower influx of extracellular Ca²⁺ into the osteoblasts occurred, which accounts for the porous / brittle properties of bone commonly observed in osteoporosis as less Ca²⁺ ions are available for bone formation / building. Seven days after the incubation with la,25-dihydroxy vitamin D₃ (calcitriol) and simvastatin, an increase in mRNA expression of osteocalcin (OCN, a bone forming biomarker) and protein expression of CaSR was observed in osteoblasts (bathed in culture medium with physiological Ca²⁺ level, 2 mM) of OVX rats. These results indicate that a combination of la,25-dihydroxyvitamin D₃ (calcitriol) and simvastatin produces, ex vivo, bone anabolic effects especially in osteoblasts of OVX rats. More importantly, the bone anabolic

2__|_

effects of this drag combination were observed under normal physiological Ca levels, which suggests that Ca²⁺ supplement is probably not needed and it can reduce the potential adverse effects (e.g., heart diseases development) associated with extra Ca²⁺ consumption.

[0052] It is anticipated that patients with osteoporosis will take drag treatments for a long period of time. Beneficial results have so far been shown in the combination of la,25- dihydroxyvitamin D₃ (calcitriol) and simvastatin 7 days after drag treatments, as evaluated in the bone anabolic effects, ex vivo. Further testing is to be performed to determine the bone anabolic effects, ex vivo, of this drag combination for 14, 21 , and 28 days. Rats are sacrificed 3 months (i.e., 12 weeks) after ovariectomized, and osteoblasts are harvested and cultured for another month before performing different assays / drag treatments for the indicated periods. In addition, the in vivo bone anabolic effects will be determined after the consumption of vitamin D and simvastatin for 4 weeks on bone's strength and density of OVX rats before, during (i.e., 6 weeks after ovariectomy and a certain degree of osteoporosis has developed) and after ovariectomy. The levels of Ca²⁺ present in urine (hypercalciuria is associated with bone resorption) of individual rat (OVX rats) before and after drag administration will be measured and compared. Example 2

[0053] In this study, the inventors monitored and evaluated for a period of over 9 months since September 201 1 the synergistic bone anabolic effects of vitamin D3 and simvastatin (1 , 3, and 10 nM each) (vitamin D3 alone, simvastatin alone, and in combination for 7, 14, and 21 days treatment schedules) using osteoblasts (bone building cells) harvested from the iliac crest of the age-matched normal (control) and ovariectomized (OVX) (i.e., estrogen deficient / postmenopausal osteoporosis) rats. The protein expression of various bone anabolic markers, e.g., osteopontin, osteocalcin, and p-cortactin (important for bone cells migration), alkaline phosphatase activity (ALP, an estimate of the rate of bone formation), Ca²⁺-sensing receptor (CaSR) and F-actin cytoskeleton (both are important for bone structure / development) before and after drug treatments was measured and compared using Western immune-blotting analysis. In general, osteoblasts harvested from OVX rats have a lower level of osteopontin, osteocalcin, p-cortactin, ALP, CaSR and F-actin cytoskeleton compared with normal (control) rats. Vitamin D3 plus simvastatin, compared to either drug applied alone, resulted in a greater magnitude increase of all bone anabolic markers measured especially in osteoblasts of OVX rats compared to normal (control) rats. In addition, a relatively greater increase of all bone anabolic markers measured was observed after Vitamin D3 plus Simvastatin combination treatments at a lower concentration (e.g. , 1 nM) compared to higher concentrations (e.g. , 3 nM and 10 nM). This is possibly due to the "ceiling effects" occurred at higher concentrations of drugs. Moreover, for the same bone anabolic marker, e.g., CaSR, a greater increase was observed after 21 days compared to 7 days treatment in both strains of rat. Immuno-staining of sections of femur head collected from both strains of rats illustrated that a lower level of matrix extracellular phosphoglycoprotein (MEPE, important in bone mineralization) and CaSR was observed in OVX rats compared with normal (control) rats. Therefore, these results strongly indicate that vitamin D3 plus Simvastatin administered together provided a synergistic bone anabolic effect especially in OVX rats. Additional study is underway to determine levels of other bone anabolic marker / parameter such as collagen I and Ca²⁺ deposits in osteoblasts of both strains of rats before and after drug treatments. Moreover, drugs (alone and in combination) will be administered at the later stage of this project to both strains of rats, and bone will be collected after drug treatment for bone strength and mineral density measurements.

[0054] All patents, patent applications, and other publications, including GenBank Accession Numbers, cited in this application are incorporated by reference in the entirety for all purposes.

Claims

WHAT IS CLAIMED IS: 1. A method for treating osteoporosis in a subject in need thereof, comprising administering to the subject an effective amount of a vitamin D and a 3-hydroxy-3-methyl- glutaryl-CoA reductase (HMG CoA reductase) inhibitor.

2. The method of claim 1 , wherein the subject is not otherwise in need of treatment by the HMG CoA reductase inhibitor.

3. The method of claim 1 , wherein the vitamin D is 1 a,25-dihydroxyvitamin D.

4. The method of claim 1 , wherein the HMG CoA reductase inhibitor is simvastatin.

5. The method of claim 1 , wherein the vitamin D and the HMG CoA reductase inhibitor are administered in one composition.

6. The method of claim 1 , wherein the vitamin D and the HMG CoA reductase inhibitor are administered in two separate compositions.

7. The method of claim 1 , wherein the amount of the vitamin D administered is no more than 2000 IU daily.

8. The method of claim 1 , wherein the amount of the HMG CoA reductase inhibitor administered is no more than 100 mg daily.

9. A composition comprising a vitamin D, a 3-hydroxy-3-methyl-glutaryl- CoA reductase (HMG CoA reductase) inhibitor, and a pharmaceutically acceptable carrier.

10. The composition of claim 9, wherein the vitamin D is la,25- dihydroxy vitamin D.

11. The composition of claim 9, wherein the HMG CoA reductase inhibitor is simvastatin.

12. The composition of claim 9, which is packaged in daily dose units, each unit comprising no more than 2000 IU of the vitamin D and no more than 100 mg of the HMG CoA reductase inhibitor.

13. The composition of claim 9, which is formulated for oral administration.

14. A kit for treating osteoporosis, comprising a first composition that comprises a vitamin D and a second composition that comprises an HMG CoA reductase inhibitor.

15. The kit of claim 14, wherein the vitamin D is la,25-dihydroxyvitamin D.

16. The kit of claim 14, wherein the HMG CoA reductase inhibitor is simvastatin.

17. The kit of claim 14, wherein the first composition is packaged in daily dose units, each unit comprising no more than 2000 IU of the vitamin D.

18. The kit of claim 14, wherein the second composition is packaged in daily dose units, each unit comprising no more than 100 mg of the HMG CoA reductase inhibitor.

19. The kit of claim 14, wherein each of the first and second compositions is formulated for oral administration.