HK1208871B - Oxysterol analogue, oxy149, induces osteogenesis and hedgehog signaling and inhibits adipogenesis - Google Patents

Description

Oxysterol analog oxysterol compound 149 inducing osteogenesis and HEDGEHOG signaling and inhibiting adipogenesis

Priority of U.S. provisional application 61/643,776 filed on 7/5/2012, which is incorporated herein by reference in its entirety, is claimed.

The present invention was made with government support (Grant No. ar059794) and received the premium of National institutes Health. The government has certain rights in the invention.

Background

Biologicals are commonly used in the medical field to promote bone growth, including fracture healing and surgical treatment of spinal disorders (1-4). Spinal fusion procedures are generally performed by orthopedic surgeons, neurosurgeons, and the like to address degenerative disc disease and arthritis affecting the lumbar and cervical vertebrae. Historically, autologous bone grafts, typically taken from the iliac crest of a patient, have been used to increase fusion between vertebral body segments (vertebral levels). However, the associated donor site morbidity, increased surgical time, and increased blood loss associated with harvesting autologous bone grafts (5-7) provide motivation for finding safe and effective alternatives.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is commonly used to promote spinal fusion in humans. Its use was approved by the U.S. Food and Drug Administration (FDA) for single level anterior interbody fusion in 2002 (8). The use of rhBMP-2 has since increased dramatically and indications for its use have expanded to include posterior lumbar spinal fusion and cervical spinal fusion. Despite the efficacy of rhBMP-2, recent reports have questioned its safety when used in spinal fusion surgery. Reported complications include subcutaneous fluid accumulation, soft tissue swelling, vertebral osteolysis, ectopic bone formation, retrograde ejaculation, and carcinogenesis (9-12). Moreover, airway edema is observed when it is applied to the cervical spine, prompting the FDA to issue a public health notice warning of its use in cervical spine surgery. No suitable substitute has been found to date with similar efficacy in inducing fusion without the adverse effects of rhBMP-2 (12).

Oxysterol (oxysterol) forms a large family of oxygenated derivatives of cholesterol found in the circulatory system as well as in human and animal tissues. Oxysterols have been found to be present in atherosclerotic lesions and play a role in a variety of physiological processes, such as cell differentiation, inflammation, apoptosis, and steroid production. Some of the present inventors have previously reported that specific naturally occurring oxysterols have robust osteogenic properties (13). The most potent osteogenic naturally occurring oxysterol, 20(S) -hydroxycholesterol ("20S") (14), is osteogenic and anti-adipogenic when applied to multipotent mesenchymal cells that can differentiate into osteoblasts and adipocytes. Structural modifications of 20S have previously been performed to synthesize more potent analogs of 20S, including oxysterol compound 34 and oxysterol compound 49, which have been shown to induce osteogenic differentiation and inhibit adipogenic differentiation of bone Marrow Stromal Cells (MSCs) by activating hedgehog (hh) signaling (15). In addition, oxysterol compound 34 and oxysterol compound 49 stimulated spinal fusion in vivo in a rat model of posterolateral spinal fusion (15). Oxysterol molecules of the prior art have widely and unpredictably varying properties. There remains a need for new and improved oxysterols, compared to rhBMP-2 and prior art oxysterols, that provide increased potency and enhanced efficacy, are convenient to synthesize, and have lower manufacturing costs. The novel oxysterols may provide physicians with a more viable clinical choice for treating, for example, long bone fractures, spinal disorders, and osteoporosis.

The osteogenic oxysterols described above are particularly suitable for direct, topical administration to a target cell, tissue or organ of interest. At present, there is no commercial combinationAnabolic drugs are used for systemic delivery and intervention in bone diseases such as osteoporosis, a disease of bone loss in elderly men and women, as well as in postmenopausal women. The only currently available systemically delivered drug that induces bone formation is(teriparatide [ rDNA origin)]Injections) which are expensive, have adverse effects and are required by the FDA to be used for no more than 24 months. There is a need for osteogenic agents, such as osteogenic oxysterols, that induce systemic bone formation more safely and efficiently after systemic administration in, for example, osteoporotic patients.

Drawings

FIG. 1 shows the molecular structure of osteogenic oxysterol. The molecular structures of 20(S) -hydroxycholesterol (20S), oxysterol compound 34, oxysterol compound 49, and oxysterol compound 133 are shown. Oxysterol compound 34 differed from 20S in having an additional OH group at C6 and the double bond between C5 and C6 was eliminated. Oxysterol compound 49 has a similar structure to oxysterol compound 34 and includes a double bond between C25 and C27. Oxysterol compound 133 differed from oxysterol compounds 34 and 49 by the absence of C27 and an increase in side chain length by one carbon.

Figure 2 shows dose-dependent activation of alkaline phosphatase activity by oxysterol. Fused (FIG. 2A) C3HT101/2 cells or (FIG. 2B) M2-10B4 cells were treated with control vehicle or 0.125-10. mu.M oxysterol compound 133. For direct comparison with oxysterol compound 133, C3H cells were also treated with oxysterol compound 34 and oxysterol compound 49 (fig. 2A). After 4 days, alkaline phosphatase (ALP) activity was measured in whole cell extracts. Data from representative three separate experiments are reported as mean ± SD of triplicate determinations and normalized to protein concentration. (p <0.0001 for cells treated with all oxysterol at a dose of 0.25 μ M or higher versus control vehicle).

Fig. 3 shows that oxysterol compound 133 induced osteogenic differentiation. (FIG. 3A) fused C3HT101/2 cells were treated with control vehicle or 2.5. mu.M oxysterol 133 in osteogenic medium. Expression of osteogenic genes Runx2, ALP, BSP, OSX and OCN was measured by quantitative real-time PCR after 48 hours (48h), 4, 7 and 14 days of treatment. Results from representative experiments are reported as mean ± SD of triplicate determinations. (p <0.005 for ALP, BSP and OSX at all time points and Runx2 and OCN at 4, 7 and 14 days for control vs. (FIG. 3B) C3H10T1/2 cells were treated with control vehicle or 2.5. mu.M oxysterol 133 for 3 weeks. To detect extracellular mineralization, von Kossa staining was performed and the mineralized matrix showed dark black staining under light microscope (10X). (FIG. 3C) in cultures in parallel to those described in (B), mineralization was quantified using the 45Ca incorporation test (p <0.005 for control vs. all concentrations of oxysterol 133). (fig. 3D) primary human MSCs were treated with control vehicle or 5 μ M oxysterol compound 133 in osteogenic medium for 4 weeks. Expression of osteogenic genes OSX, BSP and OCN was measured by quantitative real-time PCR. Results from representative experiments are reported as mean ± SD of triplicate determinations (p <0.05 for all genes in control vs. oxysterol 133 treated cells). (FIG. 3E) Primary human MSCs were treated with control vehicle or 0.5, 1 and 5 μ M oxysterol compound 133 in osteogenic media for 5 weeks. To detect extracellular mineralization, von Kossa staining was performed and the mineralized matrix showed dark black staining under light microscope (10X).

FIG. 4 shows the role of the hedgehog pathway in oxysterol compound 133-induced osteogenic differentiation. (FIG. 4A) C3H10T1/2 fused cells were treated in osteogenic media with either a control vehicle or oxysterol 133 in the presence or absence of 4. mu.M cyclopamine (Cyc). ALP activity after 4 days, and expression of the osteogenic genes ALP, BSP and OSX after 7 days were measured by quantitative real-time PCR (p <0.001 for ALP activity and expression of all indicated genes for control vs. oxysterol compound 133, and for oxysterol compound 133vs. oxysterol compound 133+ Cyc). (FIG. 4B) C3H10T1/2 cells were transfected with a control plasmid (pGL3B) or a plasmid containing an 8X-Gli luciferase reporter and treated with either a control vector or oxysterol compound 133 and luciferase activity was determined after 48 hours. Results from representative experiments are reported as mean ± SD of triplicate determinations. (p <0.001 for controls vs.100nM, 250nM oxysterol 133 and 1. mu.M oxysterol 133). (FIG. 4C) the amount of YFP-Smo captured by 20S beads or control beads was compared in samples containing no competitor or 50 μ M free competitor sterol (20S, oxysterol compound 133 or oxysterol compound 16). YFP-Smo captured by the beads was measured by western blot (top) and plotted against the amount captured in the binding reaction without competitor (bottom).

FIG. 5 shows a plain radiograph of the fused mass formed by BMP2 and oxysterol compound 133. Faxitron images of two representative animals of the indicated group are shown 8 weeks after surgery. Arrows (Arrowheads) indicate lack of bone formation; arrow (arrows) indicates bone formation. Group I (control); there is no interapophyseal space for bone formation. Group II (BMP 2); bridging bone mass and bilateral fusion at L4-L5. Group III (oxysterol compound 133, 20 mg); bridging bone mass and bilateral fusion at L4-L5. Group IV (oxysterol compound 133, 2 mg); bridging bone mass in animals shown to induce fusion by oxysterol compound 133 and bilateral fusion at L4-L5.

Figure 6 shows the micro CT of the fusion mass formed by BMP2 and oxysterol compound 133. micro-CTs of two representative animals of the indicated group are shown. Arrows indicate lack of bone formation; bone formation is shown in arrows group I (control); there is no interapophyseal space for bone formation. Group II (BMP 2); bone mass bridging the intertransverse space and bilateral fusion at L4-L5. Group III (oxysterol compound 133, 20 mg); bone mass bridging the intertransverse space and bilateral fusion at L4-L5. Group IV (oxysterol compound 133, 2 mg); bone mass bridging the intertransverse space in animals shown to be fused by oxysterol compound 133 and bilateral fusion at L4-L5. Group V (oxysterol compound 133, 0.2 mg); the arrow at the distal right indicates a small amount of bone formation from the L5 transverse process.

Fig. 7 shows a histological analysis of the effect of oxysterol compound 133 on spinal fusion. (FIG. 7A) shows coronal histological sections (10X) of two individual representative animals of each group. Group I (control) had no significant bone formation in the intertransverse process space (arrows). Group II (BMP2) demonstrated bridging bones at L4-L5 (arrowheads), clearly demonstrating trabecular and cortical bone formation of the fused mass. The group III (oxysterol compound 133-20 mg) and group IV (oxysterol compound 133, 2mg) samples demonstrated significant bone formation in the intertransverse process space (arrow) and trabecular and cortical bone formation comparable to that induced by BMP 2. (FIG. 7B) coronal histological sections from two animals from group II (BMP2) and group III (oxysterol compound 133, 20mg) demonstrated significant adipocyte formation in the fusion mass of BMP2 treated animals and significantly fewer adipocytes in the fusion mass from oxysterol treated animals (arrow, magnification 20X).

FIG. 8 shows that osteogenic differentiation markers, alkaline phosphatase activity, were induced by oxysterol compound 133 and oxysterol compound 149 in (FIG. 8A) M2-10B4 bone marrow stromal cells, and in (FIG. 8B) C3H10T1/2 embryonic fibroblasts. The fused cells are treated with a carrier, oxysterol compound 133 or oxysterol compound 149. After 4 days, alkaline phosphatase (ALP) activity was measured in whole cell extracts. Data from representative three separate experiments are reported as the mean of triplicate determinations ≠ SD, and normalized to protein concentration.

Fig. 9 shows that oxysterol compound 133 and oxysterol compound 149 induced osteogenic differentiation and expression of osteogenic differentiation marker genes. Fused C3HT101/2 cells were treated with vehicle, oxysterol compound 133 or oxysterol compound 149 in an osteogenic medium. Expression of osteogenic genes Runx2 (fig. 9E), ALP (fig. 9A), Bone Saliva Protein (BSP) (fig. 9B), zinc finger transcription factor (Osterix) (OSX) (fig. 9C), and Osteocalcin (OCN) (fig. 9D) was determined by quantitative real-time PCR 8 days after treatment. Results from representative experiments are reported as mean ± SD of triplicate determinations.

Figure 10 shows that oxysterol compound 133 and oxysterol compound 149 induce hedgehog pathway signaling. Fused C3H10T1/2 cells were treated with control vehicle, oxysterol compound 133 or oxysterol compound 149 in osteogenic media in the presence or absence of 4. mu.M cyclopamine (Cyc). Expression of hedgehog pathway target genes Gli1 (fig. 10A), Ptch1 (fig. 10B), and HIP (fig. 10C) was determined by quantitative real-time PCR after 72 hours. Results from representative experiments are reported as mean ± SD of triplicate determinations.

Disclosure of Invention

The inventors herein describe and characterize a molecule (compound) that is a hybrid of a newly identified, particularly potent oxysterol molecule (oxysterol compound 133) and a tetracycline-derived bone-targeting moiety. This hybrid molecule is referred to as oxysterol compound 149. Due to its ability to be selectively and specifically delivered to bone, oxysterol compound 149 is particularly suitable for systemic delivery to a subject, e.g., for targeting osteoporosis.

The inventors herein first identified an osteogenic oxysterol, oxysterol compound 133, which is well suited for a variety of clinical uses and described its ability to promote osteogenic differentiation in vitro and spinal fusion in vivo in a rat model. Of the large number of oxysterol analogues synthesized and tested, oxysterol compound 133 was unexpectedly particularly effective and easy to synthesize. Oxysterol compound 133 induced significant expression of osteogenic markers Runx2, osterix (osx), alkaline phosphatase (ALP), Bone Sialoprotein (BSP), and Osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts. Activation of oxysterol compound 133-induced 8X-Gli luciferase reporter, which binds directly to Smoothened, and oxysterol compound 133-induced osteogenesis was inhibited by the hedgehog (Hh) pathway inhibitor cyclopamine, confirming the role of the Hh pathway in mediating the osteogenic response to oxysterol compound 133. Furthermore, oxysterol compound 133 induced the expression of OSX, BSP and OCN and stimulated robust mineralization in primary human mesenchymal stem cells. Bilateral spinal fusion was observed by X-ray at the fusion site in animals treated with oxysterol compound 133 after only 4 weeks in vivo, and it was confirmed by manual evaluation, micro-CT and histology after 8 weeks to have efficacy equivalent to bone morphogenic protein-2 (BMP 2). However, unlike BMP2, oxysterol compound 133 did not induce adipogenesis in the fused mass and resulted in denser bone formation as evidenced by a greater BV/TV ratio and smaller trabecular detachment. Oxysterol compound 133 is therefore useful in the treatment of diseases that would benefit from localized stimulation of bone formation, including, for example, spinal fusion, fracture repair, bone regeneration/tissue engineering applications, increasing mandibular density for dental implants, osteoporosis, and the like.

The present inventors also demonstrated that oxysterol compound 133 inhibited adipogenesis of multipotent MSC cells. Oxysterol compound 133 is therefore useful for treating diseases such as xanthoma formation, local accumulation of fat pads, and obesity.

Advantages of oxysterol compound 133 include, for example, greater ease of synthesis and improved fusion times when compared to other osteogenic oxysterols studied by the present inventors.

Furthermore, the inventors herein describe a modified form of oxysterol compound 133 having attached a tetracycline-derived molecule as a bone-targeting moiety. This hybrid molecule, termed oxysterol compound 149, is selectively and specifically delivered to bone (selectively delivered to bone) due to its linkage with agents that target bone. Without wishing to be bound by any particular theory, it is suggested that oxysterol compound 149 selectively aggregates in bone and stimulates mesenchymal stem cells to undergo osteogenic differentiation and make new bone, and that this stimulation of osteogenic differentiation is mediated by activation of hedgehog signaling in bone cells. Whatever its mechanism of action, oxysterol compound 149 is effective for osteogenesis after systemic delivery to a subject because it is selectively and specifically delivered to bone. The ability to deliver systemically represents a significant advantage, for example for the treatment of osteoporotic subjects. Oxysterol compound 149 is a small molecule osteogenic oxysterol that can be used as a member of next generation bone synthesis therapeutics, as well as useful drugs for the treatment of a variety of other diseases, including diseases that would benefit from stimulation of Hh pathway activity.

One aspect of the present invention is a compound, designated oxysterol compound 149(Oxy 149), having the formula

Or a pharmaceutically acceptable salt or solvate thereof.

One component of oxysterol compound 149 is oxysterol compound 133 having the formula

Another aspect of the invention is a bioactive or pharmaceutical composition comprising an oxyphenol compound 149, or a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable carrier. The terms "bioactive" composition or "pharmaceutical" composition are used interchangeably herein. Both terms refer to compositions that can be administered to a subject, used for coating, or present in a medical device (which is introduced to the subject), and the like. These biologically active compositions or pharmaceutical compositions are sometimes referred to herein as "pharmaceutical compositions or biologically active compositions of the invention". The phrase "administering oxysterol compound 149" is sometimes used herein in the context of administering the compound to a subject (e.g., contacting the subject with the compound). It will be appreciated that the compound for this use may generally be in the form of a pharmaceutical or biologically active composition comprising the oxyphenol compound 149.

Another aspect of the invention is a method of inducing (stimulating, enhancing) a hedgehog (hh) pathway mediated response in a cell or tissue (e.g., in a subject) comprising contacting the cell or tissue with an effective amount (e.g., a therapeutically effective amount) of an oxysterol compound 149, wherein the hedgehog (hh) pathway mediated response is stimulation of osteoblast differentiation, bone morphogenesis, and/or hyperosteogeny. Hh-mediated responses are useful in regenerative medicine.

Another aspect of the invention is a method of treating a subject having a bone disease, osteopenia, osteoporosis, or bone fracture, comprising administering to the subject an effective amount of a biologically active composition or pharmaceutical composition comprising an oxyphenol compound 149. The subject can administer the bioactive or pharmaceutical composition in a therapeutically effective dose in an effective dosage form at selected intervals, for example, to increase bone mass, ameliorate symptoms of osteoporosis, or reduce, eliminate, prevent, or treat other symptoms that may benefit from increased bone morphogenesis and/or hyperosteogeny. The subject can administer the bioactive or pharmaceutical composition in a therapeutically effective dose in an effective dosage form at selected intervals to ameliorate the symptoms of osteoporosis. In one embodiment, the subject is treated to induce bone formation by collecting mammalian mesenchymal stem cells (e.g., from the subject or from a suitable mammal, or from a tissue or cell bank), treating the mammalian mesenchymal cells with an oxysterol compound 149 to induce osteoblast differentiation of the cells, and administering the differentiated cells to the subject.

In any of the methods of the present invention, the oxysterol compound 149 may be administered to a cell, a tissue, or an organ by local administration. For example, the oxysterol compound 149 may be applied topically with a cream or the like, or it may be injected or otherwise introduced directly into cells, tissues, or organs, or it may be introduced using a suitable medical device (e.g., an implant). Alternatively, the oxysterol compound 149 may be administered systemically, e.g., orally, intravenously (via IV) or by injection such as Intraperitoneal (IP) injection.

Another aspect of the invention is a kit for performing one or more of the methods described herein. The kit optionally can comprise an effective amount (e.g., a therapeutically effective amount) of oxysterol compound 149 in a container.

Another aspect of the invention is an implant for use in a subject (e.g., an animal such as a human) comprising a substrate having a surface. The bioactive or pharmaceutical composition of the oxysterol compound 149 is included on the surface or inside of the implant in a sufficient amount to induce bone formation in the surrounding bone tissue.

Optionally, the bioactive compositions, methods, kits, or medical devices of the invention may comprise one or more other suitable therapeutic agents, for example, parathyroid hormone, sodium fluoride, insulin-like growth factor I (ILGF-I), insulin-like growth factor II (ILGF-II), transforming growth factor beta (TGF- β), a cytochrome P450 inhibitor, an osteogenic prostanoid, BMP2, BMP 4, BMP 7, BMP 14, and/or an anti-resorptive agent, for example, a bisphosphonate.

Oxysterol compound 149 has the following structure

The chemical name is (3S,5S,6S,8R,9S,10R,13S,14S,17S) -3-hydroxy-17- ((S) -2-hydroxyoct-2-yl) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a ] phenanthren-6-yl 4- ((2- (2- (2- ((3-carbamoyl-2-hydroxy-4-methoxyphenyl) amino) -2-oxoethoxy) ethoxy) ethyl) amino) -4-oxobutanoate.

Example II describes the steps of design and synthesis of the oxysterol compound 133, as well as the synthetic steps of attaching the oxysterol compound 133 to a bone-targeting moiety to generate the hybrid molecular oxysterol compound 149. The tetracycline derivative fused to oxysterol compound 133 to form oxysterol compound 149 was originally designed and characterized to function as a bone delivery system when linked to estradiol. See, for example, USP 8,071,575, which is incorporated herein by reference in its entirety. The present application is primarily directed to specific bone targeting moieties that are linked to oxysterol compound 133 to produce oxysterol compound 149. However, variants of the bone targeting moiety, or variants in the region of attachment between the bone targeting moiety and oxysterol compound 133, as described in USP 8,071,575, are also included.

In addition to the compound oxysterol compound 149 of formula I, other embodiments of the present invention include any and all individual stereoisomers at the stereocenter shown in the formula, including diastereomers, racemates, enantiomers, and other isomers of the compound. In embodiments of the invention, "oxysterol compound 149" or "compound having formula I" or "oxysterol compound 149 or a pharmaceutically acceptable salt thereof" may include all polymorphs and solvates of the compound, such as hydrates and those formed with organic solvents. A "solvate" is a complex or aggregate formed by one or more molecules of a solute, e.g., a compound or a pharmaceutically acceptable salt thereof, and one or more molecules of a solvent. The solvate may be a crystalline solid having a substantially fixed molar ratio of solute to solvent. Suitable solvents are known to the person skilled in the art, for example water, ethanol or dimethyl sulfoxide. The isomers, polymorphs and solvates can be prepared by methods known in the art, such as by regiospecific and/or enantioselective synthesis and resolution.

The ability to prepare salts depends on the acidity or basicity of the compound. Suitable salts of the compounds include, but are not limited to, acid addition salts such as those formed with hydrochloric, hydrobromic, hydroiodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, pyruvic, malonic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, carbonic, cinnamic, mandelic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, p-aminosalicylic, 2-phenoxybenzoic, and 2-acetoxybenzoic acids; salts with saccharin; alkali metal salts, such as sodium and potassium salts; alkaline earth metal salts, such as calcium and magnesium salts; and salts with organic or inorganic ligands, such as quaternary ammonium salts.

Other suitable salts include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, calcium ethylenediaminetetraacetate, camphorsulfonate, carbonate, hydrochloride, clavulanate, citrate, dihydrochloride, edetate, edisylate, propionate laurylsulfate, methanesulfonate, fumarate, glucoheptonate, gluconate, glutamate, glycollylarasanate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, laurate, malate, maleate, mandelate, methanesulfonate, methylbromide, methylnitrate, methylsulfate, mucate, salts of said compounds, Naphthalenesulfonate (napsylate), nitrates, ammonium salts of N-methylglucamine, oleates, pamoate (pamoate), palmitates, pantothenate, phosphate/diphosphate (diphosphonate), polygalacturonate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, theachlorate (teoclate), tosylate, triethyliodide (triethyliodide), and valerate.

It is to be understood that reference herein to "oxysterol compound 149" includes pharmaceutically acceptable salts or solvates thereof.

In any of the methods, compositions or kits of the invention, particularly for treating a subject, the compositions of the invention may optionally be combined with one or more other suitable therapeutic agents. Any therapeutic agent suitable for treating a particular disease may be used. Suitable such agents or drugs will be apparent to those skilled in the art. For example, for the treatment of bone diseases, conventional therapeutic agents may be used in combination with the compositions of the present invention. Some such agents include, for example, parathyroid hormone, sodium fluoride, insulin-like growth factor I (ILGF-I), insulin-like growth factor II (ILGF-II), transforming growth factor beta (TGF-beta), cytochrome P450 inhibitors, osteogenic prostanoids, BMP2, BMP 4, BMP 7, BMP 14 and/or bisphosphonates or other inhibitors of bone resorption.

The compositions or compounds of the invention can be formulated as pharmaceutical compositions comprising a composition of the invention and a pharmaceutically acceptable carrier. "pharmaceutical preparationBy "an acceptable carrier" is meant that the material is not biologically or otherwise undesirable, i.e., the material can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with other components included in the pharmaceutical composition. The carrier is naturally selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art. For a discussion of pharmaceutically acceptable carriers and other components of Pharmaceutical compositions, see, e.g., Remington's Pharmaceutical Sciences, 18^thed., Mack Publishing Company, 1990. Some suitable pharmaceutical carriers will be apparent to those skilled in the art and include, for example, water (including sterile and/or deionized water), suitable buffers (e.g., PBS), physiological saline, cell culture medium (e.g., DMEM), artificial cerebrospinal fluid, dimethyl sulfoxide (DMSO), and the like.

Those skilled in the art will appreciate that the particular formulation of the present invention will depend, at least in part, on the particular drug or combination of drugs used and the route of administration chosen. Thus, there are a wide variety of suitable formulations of the compositions of the present invention. Some representative formulations are discussed below. Others will be apparent to those skilled in the art. Oxysterol compound 149 may be administered topically or directly to a cell, tissue, or organ in need of treatment, or it may be administered systemically.

Formulations or compositions suitable for oral administration may consist of: a liquid solution, such as an effective amount of oxysterol compound 149 dissolved in a diluent such as water, saline, or fruit juice; capsules, sachets or tablets, each containing a predetermined amount of active ingredient, as a solid, granular or lyophilized unit; solutions or suspensions in aqueous liquids; and oil-in-water emulsions or water-in-oil emulsions. Tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, diluents, buffering agents, wetting agents, preservatives, flavoring agents, and pharmacologically compatible carriers. Suitable formulations for oral delivery may also incorporate synthetic and natural polymeric microspheres, or other devices to prevent degradation of the drug of the invention in the gastrointestinal tract.

Formulations suitable for parenteral administration (e.g., intravenous) include aqueous and non-aqueous, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (i.e., lyophilized) condition requiring only the addition of the sterile liquid carrier for injections, for example, water, immediately prior to use. Ready-to-use injection solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described.

Oxysterol compound 149, alone or in combination with other therapeutic agents, can be prepared as an aerosol formulation for administration by inhalation. These aerosol formulations may be placed in a pressurized acceptable propellant, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for topical administration include lozenges (comprising the active ingredient in a flavor, usually sucrose and acacia or tragacanth); pastilles (comprising the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and acacia); mouthwash (containing the active ingredient in a suitable liquid carrier); or a cream, emulsion, suspension, solution, gel, cream, paste, foam, lubricant, spray, suppository, and the like.

Other suitable formulations include, for example, hydrogels and polymers suitable for timed release of the oxohydrin compound 149, or nanoparticles for small dose delivery of the oxohydrin compound 149, which formulations are well known to those skilled in the art.

Those skilled in the art will appreciate that an appropriate or suitable formulation may be selected, modified or developed based on the particular application of interest. In addition, the pharmaceutical compositions of the present invention can be prepared for administration by a variety of different routes, whether systemic, local, or both. Examples include, but are not limited to, intra-articular, intracranial, intradermal, intrahepatic, intramuscular, intraocular, intraperitoneal, intrathecal, intravenous, subcutaneous, transdermal administration, or administration directly to the atherosclerotic site in the bony region, such as by direct injection, introduction using a catheter or other pharmaceutical device, topical application, direct application, and/or by implantation of a device into an artery or other suitable tissue site.

Oxysterol compound 149 may be formulated for inclusion in, or suitable for release by, a surgical or medical device or implant. In certain aspects, the implant may be coated or otherwise treated with the oxysterol compound 149. For example, hydrogels, or other polymers, such as biocompatible and/or biodegradable polymers, can be used to coat the implant with the compositions of the present invention (i.e., the compositions can be adapted for use in medical devices through the use of hydrogels or other polymers). Polymers and copolymers for coating medical devices with agents are well known in the art. Examples of medical devices and implants include, but are not limited to, sutures and prostheses such as prosthetic joints, and may be, for example, in the shape of a needle, screw, plate, or prosthetic joint.

An "effective amount" of oxysterol compound 149, as described herein, refers to an amount that brings about at least a detectable effect. By "therapeutically effective amount," as used herein, is meant an amount that will result in at least a detectable therapeutic response (e.g., an improvement in one or more symptoms) in the treated subject over a reasonable period of time.

In embodiments of the invention, oxysterol compound 149 can stimulate or inhibit a therapeutic response by a degree of stimulation or inhibition of about 1%, 5%, 10%, 20%, 30%, 40%, 50%, 150%, 200% or more of an untreated control sample as measured by any of a variety of conventional tests. Intermediate values of these ranges are also included.

The dose of oxysterol compound 149 may be in a unit dosage form, such as a tablet or capsule. The term "unit dosage form," as used herein, refers to physically discrete units suitable as unitary dosages for animal (e.g., human) subjects, each unit containing a predetermined quantity of an agent of the invention, alone or in combination with other therapeutic agents, calculated to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier, or vehicle.

One skilled in the art can routinely determine the appropriate dosage, regimen and method of administration for a particular formulation of the composition to be used to achieve a desired effective amount or concentration of the drug in an individual patient. One skilled in the art can also readily determine and use an appropriate indicator of an "effective concentration" of a compound (e.g., oxysterol compound 149) by direct or indirect analysis of an appropriate patient sample (e.g., blood and/or tissue) in addition to analysis of an appropriate clinical symptom of a disease, disorder, or condition.

In the context of the present invention, the precise dosage of oxysterol compound 149 or a composition thereof administered to an animal such as a human will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity or mechanism of any disease being treated, the particular drug or carrier used, the manner of administration thereof, other drugs the patient takes and other factors generally considered by the attending physician, etc., when determining the individual regimen and dosage level appropriate for a particular patient. The dose used to achieve the desired concentration in vivo will be determined by the potency of the oxy steroid compound 149 form, the pharmacodynamics associated with oxy steroid compound 149 in the host, whether other drugs are taken, the severity of the disease state of the infected individual, and, in the case of systemic administration, the weight and age of the individual. The size of the dose may also be determined by any adverse side effects which may accompany the particular drug or composition used. It is generally desirable to keep adverse side effects to a minimum whenever possible.

For example, a dosage range of about 5ng (nanogram) to about 1000mg (milligram), or about 100ng to about 600mg, or about 1mg to about 500mg, or about 20mg to about 400mg can be administered. For example, the dose can be selected to achieve a dose to body weight ratio of about 0.0001mg/kg to about 1500mg/kg, or about 1mg/kg to about 1000mg/kg, or about 5mg/kg to about 150mg/kg, or about 20mg/kg to about 100 mg/kg. For example, a dosage unit range can be from about 1ng to about 5000mg, or from about 5ng to about 1000mg, or from about 100ng to about 600mg, or from about 1mg to about 500mg, or from about 20mg to about 400mg, or from about 40mg to about 200mg of oxysterol compound 149 or a composition comprising oxysterol compound 149. In one embodiment of the invention, the amount of oxysterol compound 149 described above (e.g., a few grams) is administered topically, such as part of a stent during spinal fusion.

One dose may be administered once daily, twice daily, four times daily, or more than four times daily as needed to elicit the desired therapeutic effect. For example, a dosing regimen may be selected to achieve a serum concentration of a compound of the invention in the range of about 0.01 to about 1000nM, or about 0.1 to about 750nM, or about 1 to about 500nM, or about 20 to about 500nM, or about 100 to about 500nM, or about 200 to about 400 nM. For example, a dosing regimen may be selected to achieve a mean serum concentration of half the maximum dose of a compound of the invention in the range of about 1 μ g/L (micrograms per liter) to about 2000 μ g/L, or about 2 μ g/L to about 1000 μ g/L, or about 5 μ g/L to about 500 μ g/L, or about 10 μ g/L to about 400 μ g/L, or about 20 μ g/L to about 200 μ g/L, or about 40 μ g/L to about 100 μ g/L.

Certain embodiments of the invention may also include treatment with additional drugs (either independently or in synergy with oxysterol compound 149) to improve treatment outcomes. When administered as a combination therapy, the drug other than oxysterol compound 149 may be administered simultaneously with oxysterol compound 149, or may be administered separately as needed. Two (or more) drugs may also be combined in the composition. The dosages of each drug in combination may be lower than the dosages used alone. Suitable dosages can be determined by one skilled in the art using standard dosage parameters.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "subject" includes any animal having disease symptoms treatable with an oxyphenol compound 149. Suitable subjects (patients) include laboratory animals (e.g., mice, rats, rabbits, or guinea pigs), farm animals, and domestic or pet animals (e.g., cats, dogs, or horses). Non-human primates, including human patients, are included. Typical subjects include animals that exhibit an abnormal amount (a lower amount than a "normal" or "healthy" subject) of one or more physiological activities stimulated by hedgehog signaling. Aberrant activity may be modulated by any of a variety of mechanisms, including activation of hedgehog activity. Abnormal activity can lead to pathological conditions.

One embodiment of the invention is a kit for use in any of the methods disclosed herein, including in vitro or in vivo kits. The kit comprises oxysterol compound 149 or a biologically active or pharmaceutical composition thereof, and may comprise one or more other oxysterols, such as an oxysterol that results in an increase in Hh pathway-mediated activity, or other suitable therapeutic agent. Optionally, the kit comprises instructions for performing the method. Optional elements of the kits of the invention include suitable buffers, pharmaceutically acceptable carriers, and the like, containers, or packaging materials. The reagents of the kit may be in containers in which the reagents are stable, e.g., in lyophilized form or stable liquid. The agent may also be in a single use form, for example, in a single dosage form. One skilled in the art will appreciate the kit components suitable for carrying out any of the methods of the present invention.

A variety of diseases can be treated with oxysterol compound 149 alone or in combination with other therapeutic agents.

For example, as shown in the examples herein, oxysterol compound 149 results in an increase in hedgehog pathway activity.

One effect of the oxysterol compound 149 is to target pluripotent cells to induce their lineage-specific differentiation into various cell types, e.g., osteoblasts, e.g., as shown in the examples, mesenchymal stem cells treated with the oxysterol compound 149 show inducible expression of osteoblast differentiation markers. Without wishing to be bound by any particular mechanism, it is suggested that this lineage specific differentiation is due to induction of hedgehog signaling in these cells. Regardless of the mechanism by which oxysterol compound 149 acts, however, the therapeutic methods discussed herein are encompassed by the present invention. Oxysterol compounds 149 are useful for treating diseases that would benefit from stimulation of bone formation, osteoblast differentiation, bone morphogenesis, and/or hyperosteogeny. Such diseases or treatments include, for example, osteoinductive treatments to stimulate localized bone formation in spinal fusion or osteoporosis, fracture repair or healing, dental surgery where increased bone formation in the mandible is of clinical benefit, repair of craniofacial bone defects such as cleft palate/lip due to trauma or congenital defects, and a variety of other musculoskeletal diseases where natural bone growth is insufficient, as will be apparent to those skilled in the art. Treatment can be administered to treat open fractures and fractures at high risk of non-union, and to treat subjects with spinal disorders, including subjects in need of spinal fusion (e.g., anterior interbody fusion, posterior lumbar spinal fusion, and cervical spinal fusion) or subjects with degenerative disc disease or arthritis affecting the lumbar and cervical spine. Moreover, oxysterol compound 149 is useful for treating osteoporosis, particularly in the elderly and postmenopausal population, caused by increased bone resorption by osteoclasts and decreased bone formation by osteoblasts.

More specifically, the following types of bone-related treatments may be performed:

1. oxysterol compound 149 is delivered locally in vivo as a osteogenic agent to stimulate local bone formation using a scaffold composed of compatible molecules such as, but not limited to, collagen I, which absorbs oxysterol compound 149 and is then placed in vivo. For example, a scaffold comprising oxysterol compound 149 may be placed between transverse processes or in an intervertebral disc in which fusion of two or more vertebrae is indicated, for example, in spinal fusion, prosthetic joints, and non-joint fusion. In other embodiments, a scaffold comprising oxysterol compound 149 is placed in the fractured bone to stimulate bone formation and healing of the fracture; placed in a bone defect such as a calvarial bone or maxillofacial bone defect in which bone regeneration by an oxyphenol compound 149 is indicated; or placed in the jaw bone to stimulate bone formation as a means of regenerating bone prior to dental surgery such as dental implants.

2. Oxysterol compound 149 is useful as an in vitro osteogenic agent. For example, it is administered to osteoprogenitor cells, such as mesenchymal stem cells, to stimulate osteogenic differentiation thereof, and then the cells are administered in plastic surgery and other procedures as described in 1) above to stimulate local bone formation.

3. Oxysterol compound 149 is used in vitro to stimulate the hedgehog signaling pathway in osteoprogenitor cells, resulting in osteogenic differentiation of the cells in vitro or in vivo.

Another embodiment of the invention relates to hybrid molecules comprising an oxysterol compound 133 or other osteogenic oxysterol previously described by some inventors of the present invention, wherein the oxysterol is linked to other forms of tetracycline-derived bone targeting moieties described by some inventors of the present invention. Some of the moieties are described, for example, in USP 7,196,220 and USP 7,196,220.

Any osteogenic oxysterol molecule can be linked (conjugated) to such tetracycline derivatives and used as described herein. Representative such oxysterols include oxysterol compounds 8, 34, 40, and 49, or other suitable oxysterols previously described by the present inventors or others. Some such hybrid molecules include the following:

in the above and in the following examples, all temperatures are described in unmodified degrees celsius; and all parts and percentages are by weight unless otherwise indicated.

Examples

Example I-materials and methods

Cell culture and reagents

Mouse pluripotent bone Marrow Stromal Cell (MSC) line M2-10B4(M2) and embryonic fibroblast line C3H10T1/2(C3H) were purchased from American Type Culture Collection (Rockville, Md.) and cultured as we previously reported (14, 15). Treatment was performed in RPMI (for M2 cells) or DMEM (for C3H cells) containing 5% fetal bovine serum, 50 μ g/ml ascorbate and 3mM beta-glycerophosphate (β GP) (differentiation media) to induce osteogenic differentiation. Cyclopamine was purchased from EMDBiosciences, Inc (La Jolla, CA). Primary Human Mesenchymal Stem Cells (HMSC) were purchased from Lonza (walker, MD), cultured and passaged in growth medium purchased from StemCell Technologies (Vancouver, Canada) according to the manufacturer's instructions. Osteogenic differentiation of HMSCs was induced by treating cells in low glucose DMEM containing antibiotics and 10% heat-inactivated FBS, 10-8M dexamethasone, 10mM β GP and 0.2mM ascorbate.

Alkaline phosphatase Activity and Von Kossa staining

Whole cell extracts were tested for alkaline phosphatase (ALP) activity as previously described (13,14) and cell monolayers were subjected to von Kossa staining to effect mineralization (16).

Quantitative RT-PCR

Total RNA was extracted using RNA isolation Trizol reagent from Ambion, inc. (Austin, TX) according to the manufacturer's instructions. RNA (1. mu.g) was reverse transcribed using reverse transcriptase from Bio-Rad (Hercules, Calif.) to prepare single-stranded cDNA. The Q-RT-PCR reaction was performed using the iQ SYBR Green Supermix and the iCycler RT-PCR detection System (Bio-Rad). The primer sequences for mouse genes Gli-1, Patched1(Ptch1), bone-liver-kidney isozyme of alkaline phosphatase (ALP), Bone Sialoprotein (BSP), Runx2, osterix (osx), Osteocalcin (OCN) and GAPDH were used as described previously (14). Human primer sequences were GAPDH 5 '-CCT CAA GAT CAT CAG CAA TGC CTC CT (SEQ ID NO:1) and 3' -GGTCAT GAG TCC TTC CAC GAT ACC AA (SEQ ID NO:2), BSP 5 '-AGA AGA GGA GGA GGA AGAAGA GG (SEQ ID NO:3) and 3' -CAG TGT TGT AGC AGA AAG TGT GG (SEQ ID NO:4), OSX 5 '-GCG GCA AGA GGT TCA CTC GTT CG (SEQ ID NO:5) and 3' -CAG GTC TGC GAA ACT TCT TAGAT (SEQ ID NO: 6); relative expression levels were calculated as previously described using the 2 Δ Δ CT method (15).

Transient transfection and Gli-dependent reporter assay

70% of the fused cells in 24-well plates were transiently transfected with Gli-dependent firefly luciferase and renilla luciferase vectors according to our previous description (17, 18). FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) was used IN a ratio of 3:1 to nuclease-free water, and total DNA per well did not exceed 500 ng. Luciferase activity was assessed 48 hours after cell treatment using a dual luciferase reporter assay system (Promega Corporation, Madison, WI) according to the manufacturer's instructions.

Synthesis and molecular characterization of oxysterol compound 133

The material was obtained from a commercial supplier and used without further purification. Air or moisture sensitive reactions were performed under argon atmosphere using oven-dried glassware and standard syringe/septum technology. The reaction was detected on silica gel TLC plates under UV light (254nm) and then developed using Hanessian's staining solution. Column chromatography was performed on silica gel 60. The 1H NMR spectrum was measured in CDCl 3. The data obtained are reported in ppm from an internal standard (TMS, 0.0ppm) as chemical shifts (multiplicity, integration, coupling constants in Hz.). Stepwise details of the synthetic schemes and characterization of intermediates and final products are provided in supplementary materials.

Animal(s) production

38 male Lewis rats, 8 weeks old, were purchased from Charles River Laboratories (Wilmington, Mass.) and maintained and housed in a UCLA zoo according to the rules set by UCLA Office of Protection of Research Subjects. The study was conducted according to a protocol approved by the UCLA Animal Research Council (ARC). All animals were euthanized using a standard CO2 room 8 weeks after spinal fusion surgery, and their spines were excised and stored in 40% ethanol.

Surgical procedure

Animals were pre-dosed for 30 minutes with extended release buprenorphine, followed by surgery and anesthesia with 2% isoflurane in oxygen (1L/min). The surgical site was shaved and disinfected with Betadine and 70% ethanol. Spinal fusion between the posterolateral transverse processes at L4-L5 was performed as in previous studies (21, 22). The L6 vertebral body was identified using the iliac crest as a landmark. A4-cm longitudinal median incision was made through the skin and subcutaneous tissue down to the lumbar dorsal fascia via L4-L5. A2-cm longitudinal median paravertebral incision was then made bilaterally in the paravertebral muscles to expose the transverse processes of L4-L5, which were then stripped with a high speed rasp. The surgical site was then rinsed with sterile saline, and 5mm x 13mm pieces of collagen sponge (Helistat, Integra Life Sciences) containing either dimethyl sulfoxide (DMSO) control, rhBMP-2, or oxysterol compound 149 were placed bilaterally with each implant spanning the transverse process. The implant was then covered with overlying paravertebral muscles and the lumbar dorsal fascia and skin were sutured with 4-0 Prolene sutures (Ethicon, inc., Somerville, NJ). Immediately after surgery, animals were allowed to walk, eat and drink water ad libitum.

Radiographic analysis

Posterior anterior radiographs of the lumbar spine of each animal were taken using a Faxitron LX60 box radiography system at 4, 6, and 8 weeks post surgery and evaluated blindly by two independent observers using the following standardized measures 0, no fusion; 1, unilateral fusion; and 2, complete bilateral fusion. The scores of the observers were added together and only a score of 4 was considered a complete fusion.

Manual assessment of fusion

After 8 weeks of surgery, animals were euthanized and surgically removed from the spine, and movements between the segments were assessed blindly by two independent observers (motion between levels). Bone non-healing was recorded if movement was observed between the segments or transverse processes on either side. Complete fusion was recorded if no movement was observed on either side. Spinal scores were either fused or unfused. Consensus was required to consider complete fusion.

Micro-computer tomography

Each removed spine was analyzed by high resolution micro-computed tomography (micro-CT) using a SkyScan 1172 scanner (SkyScan, Belgium) with a voxel isotropic resolution of 20 μm and X-ray energies of 55kVp and 181mA to further assess fusion rates and observe fusion masses as we previously reported (15). 360 bumps were obtained with an angular range of 180 ° and a step size of 0.5 and an exposure time of 220 milliseconds per slice. The 5 scaffolds (frames) were averaged at each rotation step to get better signal-to-noise ratio. A 0.5mm aluminum filter was used to limit the X-ray frequency to minimize radiation hardening artifacts. The virtual image slices were reconstructed based on the Feldkamp algorithm (SkyScan) using cone-beam reconstruction software version 2.6. These settings produce 1024 x 1024 pixel images of a continuous cross-section. Sample reorientation and 2D visualization were performed using dataviewer (skyscan). 3D visualization was performed using Dolphin Imaging version 11(Dolphin Imaging & Management Solutions, Chatsworth, Calif.). Fusion is defined as the bilateral presence of bridging bone between the L4 and L5 transverse processes. The reconstructed image is judged by two experienced independent observers as being fused or unfused. To quantify the bone density formed in each fusion block, the tissue volume in the block (TV), the trabecular bone volume in the Block (BV), the BV/TV ratio, trabecular thickness and trabecular separation were calculated. It was performed using DataViewer software using measurements covering 501 axial slices (20 um/slice, 10.02mm length) that were clustered at the interbody level of L4-5 within each fusion block.

Histology

After undergoing micro-CT, two representative specimens of each surgical group were treated by dehydration without decalcification, washed in xylene and embedded in methyl methacrylate as we reported previously (15, 23). The continuous coronal sections were cut to a thickness of 5um and stained with toluidine blue pH 6.4. Micrographs of the sections were obtained as previously reported using a ScanScope XTSystem (Aperio Technologies, inc., Vista, CA) at 10X magnification in fig. 7A and 20X (24) in fig. 7B.

Statistical analysis

Statistical analysis was performed using the StatView 5 program. All p values were calculated using ANOVA and Fisher's Predicted Least Significant Difference (PLSD) significance tests. Values with p <0.05 were considered significant.

Example II-synthetic scheme for Synthesis of oxysterol Compound 133 and its attachment to bone targeting Agents to produce Hybrid molecular oxysterol compound 149

The material was obtained from a commercial supplier and used without further purification. Air or moisture sensitive reactions were performed under argon atmosphere using oven-dried glassware and standard syringe/septum technology. The reaction was monitored by silica gel TLC plates under UV light (254nm) and then developed using Hanessian's staining solution. Column chromatography was performed on silica gel 60.¹H NMR spectra in CDCl₃Is measured. The data obtained are reported in ppm from an internal standard (TMS, 0.0ppm) as chemical shifts (multiplicity, integration, coupling constants in Hz.). The following is a step-by-step description of the scheme. Showing the structures of oxysterol compound 34 and oxysterol compound 49 for comparison with the structure of oxysterol compound 133, the present inventors have previously reported the synthesis of oxysterol compound 34 and oxysterol compound 49 [ Johnson et al (2011), Journal of Cellular Biochemistry112，1673-1684]。

1- ((3S,5S,6S,8R,9S,10R,13S,14S,17S) -3, 6-bis ((tert-butyldimethylsilyl) silaneAlkyl) oxygen Yl) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a]Phenanthren-17-yl) ethanones(1)

Prepared according to published patent procedures [ Parhami et al (2009), WO 2009/07386, pp.52]

¹H NMR(CDCl3，400MHZ):3.47(1H，dddd，J＝11.0，11.0，4.8，4,8 Hz)，3.36(1H，ddd，J＝10.4，10.4，4.4Hz)，2.53(1H，d，J＝8.8，8.8Hz)，2.20-2.14(1H，m)，2.10(3H，s)，2.01-1.97(1H，m)，1.88-1.82(1H，m)，1.73-0.89(17H，m)，0.88，18H，s)，0.79(3H，s)，0.59(3H，s)，0.043(3H，s)，0.04(3H，s)，0.03(3H，s)，0.02(3H，s)。¹³C NMR(CDCl3，100MHZ):209.5，72.2，70.1，63.7，56.4，53.7，51.8，44.2，41.9，38.9，37.6，36.3，34.3，33.2，31.7，31.5，25.94，25.92，24.4，22.7，21.1，18.3，18.1，13.5，13.4，-4.1，-4.6，-4.7.

(R) -2- ((3S,5S,6S,8R,9S,10R,13S,14S,17S) -3, 6-bis ((tert-butyldimethylsilyl) Oxy) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a]Phenanthren-17-yl) oct-3-yn-2-ol(2)

To a cold (0 ℃ C.) solution of n-hexyne (1.5mL, 12mmol) in THF (6mL) was added a 1.6M solution of n-BuLi in hexane (3.75 mL). The resulting solution was stirred for 30 minutes until a solution of compound 1(1.27g, 2.2mmol) in THF (10mL) was added via cannula. The mixture was warmed to room temperature over 3 hours and diluted with water (40mL) and the crude product was isolated by extraction with ethyl acetate (3 × 30 mL). The combined organic layers were washed with brine and Na₂SO₄And (5) drying. Concentration gave an oily product which was purified by silica gel (hexanes, EtOAc, gradient). There was 1.30g of product 2 (92%).

¹H NMR(CDCl3，300MHZ):3.50(1H，ddd，J＝15.9，11.0，4.8Hz)，3.36(1H，dt，J＝10.6，4.3Hz)，2.18(1H，t，t＝6.9Hz)，2.10(1H，m)，1.91-1.62(4H，m)，1.53-1.31(2H，m，3H，s)，1.31-0.93(22H，m)，0.93(3H，s)，0.92(3H，m)，0.90(18H，s)，0.88(3H，s)，0.61(1H，m)，0.04(6H，s)，0.03(6H，s)。¹³C NMR(CDCl3，75MHZ):85.9，83.9，72.4，71.4，70.3，60.5，55.8，53.8，51.8，43.5，36.3，33.7，33.0，30.7，25.9，22.0，18.4，18.3，18.1，13.6，13.5，-4.7，-4.7.

(S) -2- ((3S,5S,6S,8R,9S,10R,13S,14S,17S) -3, 6-bis ((tert-butyldimethylsilyl) Oxy) -10, 13-dimethylhexadecahydro-1H-cyclopenta [ a]Phenanthren-17-yl) octan-2-ol(3)

Compound 2(1.3g, 2.0mmol) was dissolved in EtOAc (5mL) and MeOH (5mL) and Pd/C (10%, 0.1g) were added to the solution. The mixture was degassed repeatedly under vacuum and then exposed to hydrogen at atmospheric pressure (balloon). After 18 hours at room temperature, the mixture was diluted with EtOAc (20mL) and filtered through celite to remove the catalyst. The filter was washed with EtOAc and the combined filtrates were evaporated. There was 1.3g of reduced product 3, which was used without further purification.

¹H NMR(CDCl3，300MHZ):3.50(1H，ddd，J＝15.9，11.0，4.8Hz)，3.36(1H，dt，J＝10.6，4.3Hz)，2.1-1.95(2H，m)，1.75-1.35(10H，m)，1.32-1.29(10H，m，3H，s)，0.91-1.21(10H，m)，0.89(18H，s)，0.82(3H，s)，0.79(3H，s)，0.63(3H，m)，0.04(6H，s)，0.03(6H，s)¹³CNMR(CDCl3，75MHZ):75.2，72.3，57.6，56.4，53.8，51.8，42.9，37.6，36.3，33.7，31.9，30.0，25.9，22.6，18.3，18.1，14.1，13.8，13.5，-4.6，-4.7.

(3S,5S,6S,8R,9S,10R,13S,14S,17S) -17- ((S) -2-hydroxyoct-2-yl) -10, 13-dimethyl hexadecahydro-1H-cyclopenta [ a]Phenanthrene-3, 6-diols(oxysterol compound 133)

A 1M solution of TBAF in THF (8mL, 8mmol, 4 equiv.) was added directly to compound 3(1.3g, 2.0mmol, 1.0 equiv.) and the resulting solution was diluted with THF (1mL) and stirred at rt for 72 h. The mixture was then diluted with water (50mL) and extracted repeatedly with EtOAc (4 × 40 mL). The combined organic layers were washed with brine, washed with Na₂SO₄Dry and evaporate the solvent. The crude product was purified by silica gel chromatography (hexanes, EtOAc, gradient then 10% MeOH in EtOAc) to give a white solid (0.6g, 70%) which was triturated in aqueous acetone (acetone, water, 3: 1).

¹H NMR(CDCl3，300MHZ):3.50(1H，ddd，J＝15.9，11.0，4.8Hz)，3.36(1H，dt，J＝10.6，4.3Hz)，2.19(1H，m)，2.10-1.90(3H，m)，1.85-1.60(7H，m)，1.55-1.38(7H，m)，1.25(11H，brs)，1.20-0.95(4H，m)，0.90(3H，m)，0.86(3H，s)，0.80(3H，s)0.62(2H，m)。¹³C NMR(CDCl3，75MHZ):75.1，71.1，69.3，57.5，56.2，53.6，51.6，44.0，42.8，41.4，40.1，37.2，36.2，33.5，32.1，31.8，30.9，29.9，26.3，24.2，23.6，22.5，22.2，20.9，14.0，13.6，13.3。MS:M+H＝420.36.HRMS(ESI)m/z[M-2H₂O H]⁺C₂₇H₄₄Calculated OH 385.3470, found 385.3478.

4- (((3S,5S,6S,8R,9S,10R,13S,14S,17S) -3-hydroxy-17- ((S) -2-hydroxyoct-2-yl) - 10, 13-dimethylhexadecahydro-1H-cyclopenta [ a]Phenanthren-6-yl) oxy) -4-oxobutanoic acid(6)

To oxysterol compound 133(80mg, 0.2mmol) in CH₂Cl₂Et was added to the solution (2mL)₃N(0.08mL)、DMAP (. about.1 mg, 5 mol%) and succinic anhydride (20mg, 1 eq). The mixture was stirred at room temperature for 6 hours, then a second portion of succinic anhydride (20mg, 1eq) was added. After 18 hours at room temperature, the reaction mixture was taken up with saturated NaHCO₃Solution (20mL) and CH₂Cl₂(10mL) dilution. Separating the layers and using CH for the aqueous layer₂Cl₂(3 × 10 mL). The combined organic layers were washed with 0.5MHCl solution and water, washed with Na₂SO₄Dry and evaporate the solvent. The crude product was purified by silica gel chromatography (EtOAc followed by 10% MeOH in EtOAc) to give a fraction enriched in recovered starting material, a fraction enriched in the desired compound 6(40mg, 38%) and a mixed fraction containing 6 and its regioisomers.

¹H NMR(CDCl3，300MHZ):4.71(1H，ddd，J＝15.9，11.0，4.8Hz)，3.36(1H，dt，J＝10.6，4.3Hz)，2.65(4H，m)，2.19(1H，m)，2.10-1.90(3H，m)，1.85-1.60(7H，m)，1.55-1.38(7H，m)，1.25(11H，brs)，1.20-0.95 (4H，m)，0.90(3H，m)，0.86(3H，s)，0.80(3H，s)0.62(2H，m).

(3S,5S,6S,8R,9S,10R,13S,14S,17S) -3-hydroxy-17- ((S) -2-hydroxyoct-2-yl) -10,13- dimethylhexadecahydro-1H-cyclopenta [ a]Phenanthren-6-yl 4- ((2- (2- (2- ((3-carbamoyl-2-hydroxy-4-methoxy) Phenylphenyl) amino) -2-oxoethoxy) ethoxy) ethyl) amino) -4-oxobutanoic acid ester(oxysterol Compound 149)

To compound 6(0.1g, 0.19mmol) in CH₂Cl₂Et was added to the solution (2mL)₃N (0.1mL), then BTA amine HCl salt 4(0.15g, 0.41mmol, 1.7eq) was added and the mixture was stirred for 10 min. EDCI (140mg, 3eq) was then added to the mixture once. The mixture was stirred at room temperature for 72h under an inert atmosphere (a viscous slurry formed as the solvent partially evaporated). The reaction mixture was then washed with saturated NaHCO₃Solution (20mL) and CH₂Cl₂(10mL) And (6) diluting. Separating the layers and using CH for the aqueous layer₂Cl₂(3 × 10 mL). The combined organic layers were washed with 0.5M HCl solution and water, Na₂SO₄Dry and evaporate the solvent. The crude product was purified by silica gel chromatography twice (first with 10% MeOH in EtOAc, then with CH₂Cl₂MeOH 1-3%) to give oxysterol compound 149(50mg, 32%) with an estimated purity of 90%.¹H NMR(CDCl3，300MHZ):14.62(1H，m)，8.39(1H，d，j＝9Hz)，6.40(3H，d，J＝9Hz)，4.73(1H，m)，4.14(2H，s)，3.92(3H，s)，3.76-3.34(9H，m)，2.57-2.42(4H，m)，2.19(1H，m)，2.10-1.90(3H，m)，1.85-1.60(8H，m)，1.55-1.38(8H，m)，1.25(12H，brs)，1.20-0.95(4H，m)，0.90(3H，m)，0.86(3H，s)，0.80(3H，s)0.62(2H，m)。¹³C NMR (CDCl3, 75MHZ) 172.5, 172.4, 171.7, 168.2, 154.7, 154.3, 124.2, 121.1, 102.7, 100.2, 75.1, 73.9, 71.3, 70.3, 70.1, 69.4, 69.3, 57.7, 56.3, 53.7, 51.7, 51.6, 44.1, 42.9, 41.4, 40.1, 39.3, 37.0, 36.3, 33.6, 32.3, 31.9, 31.1, 30.0, 29.7, 28.3, 27.1, 26.4, 24.3, 23.7, 22.7, 21.0, 14.1, 13.7, 13.4. Analytical HPLC Phenomenex C-18 column (Gemini, 3X100mm, 5 μm). A is water, formic acid (999:1), B is acetonitrile, formic acid (999: 1). 0, 0.5, 8, 10, 20, 20, 100, 100% after run time per minute. Retention time 8.1 min, MS: M + H831.4.

Example III-results of experiments bone formation by oxy 133 and spinal fusion Internal stimulation

Oxysterol compound 133 induced osteogenic differentiation of bone marrow stromal cells, embryonic fibroblasts, and human mesenchymal stem cells

To achieve the goal of developing molecules that induce osteogenic differentiation of osteoprogenitors, we modified the molecular structure of the most potent naturally occurring osteogenic oxysterol, 20(S) -hydroxycholesterol (20S), based on an understanding of the structural activity relationships observed in over 100 previously synthesized analogs. We previously reported that robust osteogenic differentiation was achieved using two structural analogs of 20S, oxysterol compound 34 and oxysterol compound 49 (15). These molecules are formed by adding an alpha hydroxy (OH) group on carbon 6(C6) of both oxysterol compounds 34 and 49, and a double bond between C25 and C27 in oxysterol compound 49 (fig. 1) (15). In the studies reported herein, we attempted to further improve these two molecules by developing a more easily synthesized and more potent analog to fit the scale-up for future preclinical and clinical studies in large animals and humans, respectively. This molecule would be a candidate for therapeutic development and clinical use to increase localized bone formation to stimulate spinal fusion and fracture healing, and perhaps also for systemic administration to treat diseases such as osteopenia and osteoporosis. Through a structure activity relationship study, a new analog, oxysterol compound 133, was synthesized according to the protocol described in example II and tested for osteoinductive activity. Oxysterol compound 133 differed from oxysterol compounds 34 and 49 by the absence of C27 and the addition of a carbon length in the side chain (fig. 1). Importantly, oxysterol compound 133 can be more easily prepared on a large scale due to the significantly lower cost of the resulting products compared to oxysterol compound 34 and oxysterol compound 49 as a result of the inexpensive commercially available raw materials. Moreover, the alkyne addition used in the preparation of oxysterol compound 133 is superior to the Grignard chemistry used in the synthesis of oxysterol compound 34 and oxysterol compound 49 in terms of yield, purity (diastereoselectivity) and scalability of the product.

Oxysterol compound 133 had unexpectedly improved efficacy in inducing alkaline phosphatase (ALP) activity, as compared to other structural analogs of 20S, as measured by ALP enzyme activity assays in C3H and M2 cells. This is a useful model for osteogenic activity, as we have previously reported other oxysterol analogues (15). A dose-dependent increase in ALP activity was observed at low micromolar (μ M) concentrations of oxysterol compound 133 (fig. 2A, 2B). EC50 for oxysterol compound 133 was found to be about 0.5 μ M in C3H (fig. 2A) and 0.44 μ M in M2 cells (fig. 2B). EC50 of oxysterol compound 34 and oxysterol compound 49 in C3H cells was found to be similar to the values previously reported in M2 cells, 0.8 and 0.9 μ M, respectively, and significantly higher than EC50 of oxysterol compound 133 (fig. 2A). Moreover, high doses of oxysterol compound 133 induced higher levels of ALP activity compared to similar doses of oxysterol compound 34 and oxysterol compound 49 in C3H cells (fig. 2A). Oxysterol compound 133 was found to have other beneficial effects in inducing osteogenic differentiation of cells by analyzing the expression of osteogenic differentiation marker genes Runx2, osterix (osx), ALP, Bone Sialoprotein (BSP), and Osteocalcin (OCN). Runx2 expression was induced 2-fold and 3.2-fold, respectively, in C3H cells after 4 days and 7 days of treatment with 2.5 μ M oxysterol compound 133, which returned to baseline levels at 14 days (fig. 3A). OSX expression was significantly induced 3-fold after 2 days and remained elevated throughout the experiment, reaching a maximum induction of 4.5-fold (fig. 3A). Treatment of C3H cells with oxysterol compound 133 induced expression of ALP 18-fold after 2 days, which reached a maximum of 120-fold after 4 days, and then decreased to 22-fold after 7 days and 14 days, respectively (fig. 3A). BSP expression was maximally induced 9-fold on day 4 and remained induced throughout the experiment, although levels decreased with prolonged exposure of the cells to oxysterol 133 (fig. 3A). Oxysterol compound 133 treatment also induced osteoblast-specific gene osteocalcin expression by 2.8-fold after 4 days, and by a maximum of 4.2-fold after 14 days of treatment (fig. 3A). Oxysterol compound 133 induced robust matrix mineralization in C3H cell cultures after 21 days of treatment as determined by von Kossa staining (fig. 3B) and quantitative extracellular matrix 45Ca test (fig. 3C). These data demonstrate the efficacy and potency of oxysterol compound 133 as an osteoinductive oxysterol.

Osteogenesis effect of oxysterol compound 133 was also tested by evaluating the expression of osteogenic genes in primary human Mesenchymal Stem Cells (MSCs) after 1 week, 2 weeks, and 4 weeks of treatment. ALP expression was high at all time points in untreated cells and treatment with oxysterol compound 133 was unchanged (data not shown). After 1 week, a significant 2-fold increase in BSP expression was observed, which further increased to 4-fold after 2 and 4 weeks (fig. 3D). Oxysterol compound 133 also had significant OSX induction (3 fold) and OCN induction (2 fold) after 4 weeks (fig. 3D). Furthermore, oxysterol compound 133 stimulated robust extracellular matrix mineralization in primary human MSC cell cultures after 5 weeks of treatment, as evidenced by von Kossa staining (fig. 3E).

Oxysterol compound 133 induces osteogenic differentiation by activating hedgehog pathway signaling

Previous studies have demonstrated that oxysterol compounds 34 and oxysterol compounds 49 of 20S and their structural analogs induce osteogenic differentiation by activating Hh pathway signaling (15). However, the molecular mechanism of activation of the osteogenic oxysterol-mediated Hh pathway signaling was previously unknown. Due to its greater osteogenic activity, oxysterol compound 133 is a useful tool for identifying the molecular mechanisms by which Hh pathway activation and osteogenesis are achieved by semisynthetic oxysterols. To determine whether and how oxysterol compound 133 was induced by Hh pathway, the effect of cyclopamine, a selective Hh pathway inhibitor, on the activity of oxysterol compound 133-induced ALP and the expression of osteogenic differentiation markers ALP, BSP, and OSX was examined. Cyclopamine completely inhibited the activity of oxyphenol compound 133-induced ALP and the expression of osteogenic markers ALP, BSP, and OSX in C3H cells (fig. 4A), as well as in M2 cells (data not shown), indicating that oxyphenol compound 133 did not function through Hh signaling pathway. To further analyze the activation of Hh signaling by oxysterol compound 133, activation of Gli-dependent luciferase reporter transfected into C3H cells was detected using previously reported methods (15, 17). Oxysterol compound 133 induced a dose-dependent increase in Gli-dependent reporter activity, reaching a 5-fold induction at 100nM and a 17-fold induction at 1 μ M oxysterol compound 133 (fig. 4B).

Oxysterol compound 133 activates the hedgehog signaling pathway by binding to smooth receptors

We previously reported that 20S selectively activates Hh signaling by binding to Smo receptors (19). To determine whether the oxysterol compound 133 activated Hh signaling by the same mechanism, we tested the ability of the oxysterol compound 133 to compete for YFP-labeled Smo (YFP-Smo) bound to the 20S analog (coupled to magnetic beads). As we reported previously, this analogue, nat-20S-yne, contains an alkyne moiety on the isooctyl chain, allowing click chemistry-mediated coupling to magnetic beads (20S-beads) (19). Using these beads for sterol-binding assays, the amount of YFP-Smo retained on the beads relative to the competitor-free sample was measured by western blot. Compounds that bind Smo at the same site as 20S compete with the 20S-beads and reduce the amount of protein in the eluate. We have tested a number of other sterols both in the Smo binding assay and in the Hh signaling assay, binding to Smo in all cases being associated with changes in Hh pathway activity (19). Oxysterol compounds 133 and 20S (positive control) both reduced the amount of YFP-Smo captured on 20S-coupled beads (fig. 4C). In an important control, a structurally related analog oxysterol compound 16, which did not activate Hh signaling or osteogenesis (unpublished observation by Parhami et al), did not prevent YFP-Smo and 20S-bead interactions (fig. 4C). This reduction in the amount of YFP-Smo captured by the 20S-beads in the presence of free oxysterol compound 133 indicated that oxysterol compound 133 bound to the same site on Smo as 20S. It is important to emphasize that our assay is semi-quantitative and cannot be used to derive the Kd for the interaction, mainly because we do not know the concentration of YFP-Smo in the extract and the amount of 20S that is heavily immobilized on the beads.

Oxysterol compound 133 stimulates bone formation and spinal fusion in vivo

The 8-week old Lewis rats were divided into 5 treatment groups, which differed only in the reagents contained in the collagen sponges at the site of surgery, group 1-control vehicle (DMSO) only (n ═ 7), group II-5 μ g rhBMP-2(n ═ 8), group III-20mg oxysterol compound 133(n ═ 7), group IV-2mg oxysterol compound 133(n ═ 8), and group V-0.2mg oxysterol compound 133(n ═ 8). Bone formation and spinal fusion were assessed post-operatively by radiographic analysis at various time points, and at sacrifice using manual assessment, microscopic computer tomography and histological assessment. The fusion rates at sacrifice are summarized in table 1.

TABLE 1 fusion rate (%)

	X-ray radiation	micro-CT	Hand touch test
				Control	0	0	0
BMP2	100	100	100
				Oxysterol Compound 13320mg	100	100	86
Oxysterol Compound 1332mg	50	50	50
				Oxysterol compound 1330.2mg	0	0	0

Radiographic analysis

The first set of radiographs was taken 4 weeks after surgery. At this time point, bilateral fusion was observed in 8 of 8 animals in the BMP2 group, in 6 of 7 animals in the oxysterol compound 133-20mg group, in 3 of 8 animals in the oxysterol compound 133-2mg group, and no fusion in the control and oxysterol compound 133-0.2mg groups. Unilateral fusion was observed in the remaining animals treated with 133-20mg oxysterol compound and three animals treated with 133-2mg oxysterol compound. This is in contrast to previous studies with oxysterol compounds 34 and 49, in which no fusion was observed at the 4-week time point (15). By week 6, all animals were bilaterally fused in the oxysterol compound 133-20mg group. At week 8, fusion was again noted in all animals in the BMP2 and oxysterol compound 133-20mg group and in animals in 4/8 in the oxysterol compound 133-2mg group (FIG. 5). No fusion cake was observed in the final 8-week radiograph in the DMSO or oxysterol compound 133-0.2mg (data not shown) group (fig. 5).

Manual and visual assessment of bone formation

After sacrifice, the spine was removed from each animal and manually evaluated as we previously described (15, 25-27). The results of the visual evaluation and the manual evaluation were similar to those of the radiographs at 8 weeks. No unilateral or bilateral fusion was observed in DMSO or oxysterol compound 133-0.2mg groups. Some bone formation was observed in both animals in the oxysterol compound 133-0.2mg group. Bilateral fusion was observed in all animals in the BMP2 group and in animals in the 6/7 in the oxysterol compound 133-20mg group. The remaining animals in the oxysterol compound 133-20mg group had unilateral movement despite significant bilateral fusion mass. The hand-touch test confirmed that half (4/8) of the animals in the oxysterol compound 133-2mg group had bilateral fusion, two other animals had unilateral fusion, and two other animals had no signs of fusion.

Microcomputer tomography and histological evaluation

Evaluation of the bridged trabeculae using micro-CT analysis confirmed the results observed with the radiographs, gross observation and hand-touch test (figure 6). Although some bone formation was observed in both animals in the oxysterol compound 133-0.2mg group, no bilateral fusion was observed in this group or the DMSO group. Bilaterally bridged trabeculae were observed in all animals in the BMP2 group and the oxysterol compound 133-20mg group. Bilateral fusion was also observed in 4/8 animals in the oxysterol compound 133-2mg group, and unilateral fusion was observed in the other two animals. The results of the microstructural analysis derived from the micro-CT images are shown in table 2. The total volume of the BMP2 fusion mass was significantly greater than both the oxysterol compound 133-2mg and 20-mg samples. However, the average BV/TV ratio of the oxysterol compounds 133-2mg and 20-mg fusion blocks was significantly greater than that of the BMP2 group, indicating denser bone in the blocks. There was no significant difference in trabecular thickness between BMP2 and oxysterol compounds 133-2mg or oxysterol compounds 133-20 mg. Trabecular separation was significantly greater in the BMP2 fusion mass compared to oxysterol compound 133-2mg and oxysterol compound 133-20mg, also indicating a lower bone density in the BMP2 fusion mass.

TABLE 2 quantitative assessment of bone microstructures by micro-CT imaging

Statistical significant differences in total tissue volume, bone volume to tissue volume ratio and trabecular separation between BMP2 and oxysterol compounds 13320mg and 2mg (p <0.01) were demonstrated. No difference in bone volume or trabecular thickness was observed.

Histological analysis was then performed in the DMSO group, BMP2 group, oxysterol compound 133-20mg group, and oxysterol compound 133-2mg group of two representative animals. Histological evaluation confirmed the formation of trabeculae in the fusion mass and continuous cortical bone connecting the transverse processes of the fully fused lumbar vertebrae in rats treated with BMP2, or with oxysterol compound 133 at 2 or 20mg doses (figure 7A). There was no bone formation in the samples of the control rats. The size of the fusion mass was increased in rats treated with BMP2 compared to rats treated with 20mg or 2mg of oxysterol compound 133. However, visual examination of the histological specimens showed that BMP2 also induced robust formation of adipocytes in the fusion mass, which was significantly less in the group treated with oxy 133 (fig. 7B). Furthermore, visual observation showed that trabecular bone formation was more robust in the 133-20mg oxysterol compound group than in the BMP2 group.

Example IV-study showing the comparison of the Activity of oxysterol Compound 149 with the Activity of oxysterol Compound 133

Oxysterol compound 149 was tested as described above for oxysterol compound 133 and found to stimulate osteoblastic differentiation of cells in vitro. The data are shown in FIGS. 8-10, and some details of the experiments are summarized in the figure description.

Additional experiments in vitro and in vivo with oxysterol compound 149 will also be performed following the experiments described herein for oxysterol compound 133. It is expected that oxysterol compound 149 will exhibit the desired efficacy and biological effects, for example, when administered to a cell, tissue or organ of interest.

EXAMPLE V Effect of oxysterol Compound 149 after systemic administration

Conventional procedures were used to test the beneficial properties of oxysterol compound 149 following systemic administration to animal models. Oxysterol compound 149 was tested for its ability to prevent or reverse osteoporosis in animal models of osteoporosis. Such animal models include, but are not limited to, ovariectomized mice and rats, glucocorticoid-or other drug-induced osteoporosis in rodents, and osteoporosis with age in rodents and non-human primates. In these studies, oxysterol compound 149 was administered systemically by subcutaneous, i.v., i.p., or oral administration, or by administration of a vaporized formulation of oxysterol compound 149 via the nasal passage. The improvement following treatment with oxysterol compound 149 compared to placebo or anti-resorptive drugs was assessed by: measuring factors that change in blood with the induction of bone formation (e.g., alkaline phosphatase and osteocalcin), factors that decrease bone resorption (e.g., C-and N-terminal peptides of collagen I), and measuring bone density, bone mineral content, and other bone parameters using radiographs of CT imaging that determine improvements in bone microstructure. Oxysterol compound 149 is expected to selectively accumulate in bone due to its bone-targeting properties, and, for example, stimulate mesenchymal stem cells to undergo osteogenic differentiation and generate new bone. Oxysterol compound 149 is useful for healing bone fractures and preventing and/or treating osteoporosis because it stimulates bone formation when administered systemically to a subject.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions and to use the invention to its fullest extent. The foregoing preferred embodiments are illustrative only, and are not intended to limit the scope of the invention in any way. The entire contents of all applications, patents and publications cited above, including U.S. provisional application 61/643,746 filed on 7/5/2012, are hereby incorporated by reference in their entirety, particularly with respect to the contents of the present application citations. Also incorporated herein by reference in their entirety are other applications for oxysterols derived from the present inventors' laboratories, including International application publications WO/2008/115469, WO/2008/082520, WO/2007/098281, WO/2007/028101, WO/2006/110490, WO/2005/020928, WO/2004/019884, and PCT applications filed on the same day as the present application (having attorney docket No. 58086-342052, filed on 7/5/2012, based on U.S. provisional application 61/643,746).

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Claims (18)

1. A compound having the structure:

or a pharmaceutically acceptable salt thereof.

2. A biologically active composition comprising a compound having the structure:

3. the bioactive composition of claim 2, further comprising at least one additional agent selected from the group consisting of parathyroid hormone, sodium fluoride, insulin-like growth factor I (ILGF-I), insulin-like growth factor II (ILGF-II), transforming growth factor beta (TGF- β), a cytochrome P450 inhibitor, an osteogenic prostanoid, BMP2, BMP 4, BMP 7, BMP 14, and an anti-resorptive agent.

4. Use of a compound according to claim 1 or a biologically active composition according to claim 2 in the manufacture of a medicament for the treatment of bone disorders.

5. Use of a compound according to claim 1 or a biologically active composition according to claim 2 in the manufacture of a medicament for increasing bone mass.

6. The use of claim 4, wherein the bone disorder is osteoporosis.

7. Use of a compound according to claim 1 or a biologically active composition according to claim 2 in the manufacture of a medicament for increasing bone morphogenesis and/or hyperosteogeny.

8. Use of a compound of claim 1 or a biologically active composition of claim 2 in the manufacture of a medicament for inducing bone formation to increase bone mass.

9. Use of the compound of claim 1 or the composition of claim 2 in the manufacture of a medicament for inducing osteoblastic differentiation of mesenchymal stem cells of a mammal, wherein the mesenchymal stem cells of the mammal are bone marrow stromal cells of a subject.

10. Use of the compound of claim 1 or the composition of claim 2 in the preparation of a medicament for stimulating a hedgehog (Hh) pathway mediated response in a cell or tissue of a subject, wherein the Hh pathway mediated response is stimulation of osteoblast differentiation, bone morphogenesis, and/or hyperosteogeny.

11. The use according to any one of claims 4 to 10, wherein the medicament is formulated for topical administration.

12. The use of any one of claims 4-10, wherein the medicament is formulated for systemic administration.

13. Use of a compound of claim 1 or a composition of claim 2 in the manufacture of a medicament for stimulating mammalian cells in a mammal to express a level of a biomarker for osteoblast differentiation greater than the level of the biomarker in untreated cells.

14. The use of claim 13, wherein the biomarker is alkaline phosphatase activity, calcium incorporation, mineralization and/or expression of osteocalcin mRNA.

15. The use of claim 13, wherein the mammalian cell is a mesenchymal stem cell, an osteoprogenitor cell, or a cell in a skull injury, disruption or defect.

16. An implant for the human or animal body comprising a substrate having a surface, wherein the surface or interior of the implant comprises the bioactive composition of claim 2 in an amount sufficient to induce bone formation in surrounding bone tissue.

17. The implant of claim 16, wherein the substrate forms the shape of a needle, screw, plate, or prosthetic joint.

18. The use of claim 4, wherein the bone disorder is osteoporosis or a bone fracture.