OSTEOPORESIS

RELATED APPLICATIONS

This application claims the benefit of priority to U. S. Provisional Patent

Application Serial No. (USSN) 62/348,577 filed June 10, 2016. The aforementioned application is expressly incorporated herein by reference in its entirety and for all purposes.

GOVERNMENT RIGHTS

This invention was made with government support under grants AR051300, R21-

AR065658, U01-NS58030, P01-AG007996, P30-CA023100, P30-NS047101, P30- AR04603 and NS058030, awarded by the National Institutes of Health (NIH), DHHS. The government has certain rights in the invention.

TECHNICAL FIELD

This invention generally relates to physiology and bone metabolism. In particular, in alternative embodiments, provided are compounds, including formulations and pharmaceutical compositions, and methods of making and using them, for treating, ameliorating, preventing or reversing osteoporesis, including post-menopausal osteoporosis. In alternative embodiments, provided are methods for, and uses of nitrosyl- cobinamide (NO-Cbi) for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration; increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; increasing osteoblast numbers or regulating osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis.

BACKGROUND

Most FDA-approved treatments for osteoporosis target osteoclastic bone resorption. Only parathyroid hormone (PTH) derivatives improve bone formation, but they have drawbacks, so new bone-anabolic agents are needed. The Nitric Oxide (NO)/cGMP/protein kinase G (PKG) signaling pathway mediates anabolic effects of estrogens and mechanical stimulation in bone cells by increasing osteoblast proliferation and osteocyte survival. Nitrates, which generate NO, reduce bone loss in estrogen- deficient rats and increase bone mineral density in post-menopausal women. However, nitrates are limited by induction of oxidative stress and development of tolerance, and may increase cardiovascular mortality after long-term use.

Organic nitrates generate NO in vivo after mitochondrial biotransformation; nitrates are used to treat coronary insufficiency and heart failure, and epidemiological studies suggest their use may reduce fracture risk. ^19"21 Based on these data and preclinical studies showing a bone-protective effect of organic nitrates in OVX rats, ^{22, 23} several clinical trials have examined the skeletal effects of organic nitrates in post-menopausal women, showing an increase in bone formation markers, a decrease in bone resorption markers, and improved bone mineral density in women with estrogen deficiency. ²⁴

Organic nitrates are the only NO donors currently FDA-approved for long-term use, but clinical benefits of organic nitrates are limited by development of tolerance and induction of oxidative stress. ^27"30 Nitrates require enzymatic activation to release NO, and this reaction generates reactive oxygen species, especially O2 ^" , which can have detrimental effects in the cardiovascular and skeletal systems. ¹⁰ ' ²⁹ ' ³¹ SUMMARY

In alternative embodiments, provided are methods for:

- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;

- preventing bone loss due to estrogen deficiency;

- inhibiting osteoclast differentiation;

- reducing osteoclast numbers;

- preventing estrogen deficiency-induced osteocyte apoptosis;

- increasing serum cGMP concentration;

- increasing bone formation and bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or

- increasing osteoblast number and regulating osteoblastic gene expression, comprising

administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporosis or at risk of developing osteoporosis,

thereby:

- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;

- preventing bone loss due to estrogen deficiency;

- inhibiting osteoclast differentiation;

- reducing osteoclast numbers;

- preventing estrogen deficiency-induced osteocyte apoptosis;

- increasing serum cGMP concentration;

- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or

- increasing osteoblast number and regulating osteoblastic gene expression.

In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, is formulated as a pharmaceutical composition, or is formulated as a pharmaceutical composition comprising a pharmaceutically acceptable carrier or excipient thereof.

In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered or co-administered with another therapeutic agent, wherein optionally therapeutic agent is administered simultaneously, before or after the nitrosyl-cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered in or with an implant or a bone implant.

In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in a device, an implant, a bone implant, a bead, a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, a product of manufacture, a liposome, a particle, a microparticle or a nanoparticle.

In alternative embodiments, the nitrosyl-cobinamide, or the bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof, or the pharmaceutical composition, is administered as or is formulated in or as a kit, a pump, a device, a subcutaneous infusion device, a continuous subcutaneous infusion device, an infusion pen, a needles, a reservoir, an ampoules, a vial, a syringe, a cartridge, a pen, a disposable pen or jet injector, a prefilled pen or a syringe or a cartridge, a cartridge or a disposable pen or jet injector, a two chambered or multi-chambered pump.

In alternative embodiments, provided are Uses of a nitrosyl-cobinamide (NO-

Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof in the manufacture of a medicament for:

- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;

- preventing bone loss due to estrogen deficiency;

- inhibiting osteoclast differentiation;

- reducing osteoclast numbers;

- preventing estrogen deficiency-induced osteocyte apoptosis;

- increasing serum cGMP concentration;

- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or

- increasing osteoblast number and regulating osteoblastic gene

expression.

In alternative embodiments, provided are therapeutic formulations for use in:

- treating, ameliorating, preventing or reversing osteoporosis or postmenopausal osteoporosis;

- preventing bone loss due to estrogen deficiency;

- inhibiting osteoclast differentiation;

- reducing osteoclast numbers;

- preventing estrogen deficiency-induced osteocyte apoptosis;

- increasing serum cGMP concentration;

- increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or - increasing osteoblast number and regulating osteoblastic gene expression,

wherein the therapeutic formulation comprises a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof.

In alternative embodiments, provided are bone implants comprising a nitrosyl- cobinamide, or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof. The details of one or more embodiments as provided herein are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

All publications, patents, patent applications cited herein are hereby expressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings set forth herein are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

FIG. 1A-I illustrate data showing that NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs, as described in detail in Example 1, below:

FIG. 1(A) graphically illustrates data from experiments where POBs were incubated in medium with 0.1% FBS (3 x 10 ⁵ cells/ml) for 2 h prior to receiving 10 μΜ NO-Cbi (NOCbi) for the indicated times, and stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction;

FIG. 1(B) and FIG. 1(C) graphically illustrate data from experiments where POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (FIG. IB);

FIG. 1(D) and FIG. 1(E) graphically illustrate data from experiments where serum- deprived POBs were treated with vehicle or 10 μΜ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies; FIG. 1(F) illustrates immunofluorescence images from experiments where POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μΜ NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC-coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue);

FIG. 1(G) graphically illustrates data from experiments where POBs cultured in medium with 0.1% FBS for 18 h were treated with 10 μΜ NO-Cbi or vehicle for 1 h, and transferred to fresh medium containing ³ H-thymidine for 24 h; and

FIG. 1(H) and FIG. 1(1) graphically illustrate data from experiments where confluent POBs were differentiated in ascorbate-containing medium for 14 d and some cells received 10 μΜ NO-Cbi (open bars) or vehicle (filled bars) for the last 24 h.

FIG. 2A-F illustrate data from experiments showing that NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II, as described in detail in Example 1, below:

FIG. 2A illustrates data from experiments where POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites ("floxed" PRKG2 ^f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE), and forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control;

FIG. 2B illustrates data from experiments where cells were infected as in FIG. 2A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μΜ NO-Cbi or vehicle for 10 min, and Akt and GSK-3P phosphorylation were assessed using antibodies specific for Akt(pSer ⁴⁷³ ) and GSK-3P(pSer ⁹ ), with total GSK-3P serving as a loading control; densitometric quantitation is shown on the right, with relative amounts of pAkt and pGSK-3 found in vehicle-treated control virus-infected cells assigned a value of 1;

FIG. 2C illustrates data from experiments where PRKG2 ^f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in FIG. 2B, they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining, and the bottom panel shows nuclei counterstained with Hoechst 33342, and numbers below indicate the percentage of cells showing nuclear β-catenin; FIG. 2D illustrates data from experiments where cells were infected and cultured as in FIG. 2B, and they were treated with NO-Cbi or vehicle for 1 h prior to measuring ³ H- thymidine incorporation into DNA for 24 h;

FIG. 2E illustrates data from experiments where cells were infected with control or CRE virus as described in FIG. 2 A, and treated with 10 μΜ NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family- la (Wntla), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (bCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1; and

FIG. 2F illustrates data from experiments where cells POBs cultured in 10 % FBS were treated with 10 μΜ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively.

FIG. 3A-D illustrate data showing that NO-Cbi inhibits osteoclast differentiation, as described in detail in Example 1, below:

FIG. 3 A and FIG. 3B illustrate data from experiments where murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations;

FIG. 3C illustrates data from experiments where cells were cultured as in FIG. 3 A, but some cultures received 10 μΜ NO-Cbi, 5 μΜ DETA-NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 μΜ 8-pCPT-cGMP together with RANKL; and

FIG. 3D illustrates data from experiments where expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1.

FIG. 4A-G illustrate data showing that NO-Cbi increases serum cGMP

concentration, bone formation, and osteoblastic gene expression in OVX mice, as described in detail in Example 1, below:

FIG. 4A illustrates data from experiments where werum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi; FIG. 4B illustrates data from experiments where the number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia;

FIG. 4C, FIG. 4D and FIG. 4F illustrate data from experiments where trabecular calcein labeling was assessed at the tibia (FIG. 4C), with quantification of mineral apposition rate (MAR, FIG. 4D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, FIG. 4F); and

FIG. 4G illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of osteocalcin (OCN), osteopontin (Sppl), alkaline

phosphatase (ALP), collagen-al (Collal), low-density lipoprotein receptor- related protein-5 (Lrp5) and tubulin (Tubal) mRNA was quantified by qRT-PCR and normalized to 18S rRNA.

FIG. 5 A-E illustrate data showing that NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis, where mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:

FIG. 5A, FIG. 5B and FIG. 5C illustrate data from experiments where the percentage of apoptotic osteocytes was assessed in trabecular (FIG. 5 A, FIG. 5B) and cortical bone (FIG. 5C) by TUNEL staining (black nuclei) of tibial sections:

FIG. 5D illustrates data from experiments where osteoblast and osteocyte apoptosis was assessed by Western blotting of extracts obtained from tibial bone (after removal of bone marrow), using an antibody specific for cleaved caspase-3, with β-actin serving as a loading control (n =2 mice per group); and

FIG. 5E illustrate data from experiments where Erk activity in cortical (top panel) and trabecular (bottom panel) bone-lining cells was assessed by immunofluorescence staining using a phospho-Erk-specific antibody and horse radish peroxidase-coupled secondary antibody (brown).

FIG. 6A-C illustrate data showing that NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice, and mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4, as described in detail in Example 1, below:

FIG. 6 A and FIG. 6B illustrate data from experiments where osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia; and FIG. 6C illustrates data from experiments where RNA was extracted from femurs, and the relative abundance of RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA.

FIG. 7A-D illustrate data showing that NO-Cbi increases trabecular bone mass in OVX mice. Mice subjected to OVX or sham operation were treated with vehicle or NO- Cbi as described in Fig. 4, as described in detail in Example 1, below:

FIG. 7A illustrates data from experiments where tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown; and

FIG. 7B, FIG. 7C and FIG. 7D illustrate data from experiments where trabecular bone volume/tissue volume (FIG. 7B), trabecular number (FIG. 7C), and trabecular bone mineral density (FIG. 7D) were quantified at the proximal tibia.

FIG. 8 illustrates endocortical calcein labeling, where the endocortical calcein labeling, as illustrated in the FIG. 8A images, was assessed at the tibia, with

quantification of mineral apposition rate (MAR, FIG. 8B), bone formation rate (BFR, FIG. 8C), and mineralizing surface per bone surface (MS/BS, FIG. 8D); data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8, NO-Cbi-treated OVX mice; *p < 0.05 and **p < 0.01 for the indicated pair-wise comparisons.

FIG. 9 illustrates images of a TUNEL staining of apoptotic osteocytes in cortical tibial bone, where the percentage of apoptotic osteocytes was assessed in cortical bone by TUNEL staining (black nuclei) of tibial sections, as described in Fig. 5C.

FIG. 10 graphically illustrates data from a Micro-CT analysis of tibial cortical bone, where tibiae were analyzed by micro-CT, and cortical (cross-sectional) thickness (FIG. 10A) and cortical tissue mineral density (FIG. 10B) were quantified at the mid- tibial diaphysis, as described in Example 1, below; data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8 NO-Cbi-treated OVX mice.

Like reference symbols in the various drawings indicate like elements.

Reference will now be made in detail to various exemplary embodiments of the invention, examples of which are illustrated in the accompanying drawings. The following detailed description is provided to give the reader a better understanding of certain details of aspects and embodiments of the invention, and should not be interpreted as a limitation on the scope of the invention.

DETAILED DESCRIPTION

In alternative embodiments, provided are compounds, including formulations and pharmaceutical compositions, and methods of making and using them, for treating, ameliorating, preventing or reversing osteoporesis, In alternative embodiments, provided are methods for, and uses of nitrosyl-cobinamide (NO-Cbi) for: treating, ameliorating, preventing or reversing osteoporosis; preventing bone loss due to estrogen deficiency; inhibiting osteoclast differentiation; reducing osteoclast numbers; preventing estrogen deficiency-induced osteocyte apoptosis; increasing serum cGMP concentration;

increasing bone formation or bone mass, or increasing trabecular bone volume, trabecular number, and trabecular bone mineral density; or increasing osteoblastic gene expression, comprising administering a nitrosyl-cobinamide (NO-Cbi), or a bioisostere, stereoisomer, tautomer, hydrate, solvate or pharmaceutically acceptable salt thereof to: an individual; an individual with osteoporesis or at risk of developing osteoporesis.

As described in Example 1, below, we examined the skeletal effects of a novel NO donor, nitrosyl-cobinamide (NO-Cbi), derived from the vitamin B12 precursor cobinamide; it directly releases NO without biotransformation or generation of reactive oxygen species. ^32"35 We found that NO-Cbi prevented bone loss in OVX mice by enhancing osteoblast activity and inhibiting osteoclast differentiation.

We tested nitrosyl-cobinamide (NO-Cbi), a novel NO-donor with anti -oxidant properties, in a mouse model of estrogen deficiency-induced osteoporosis (3). Compared with sham-operated mice, ovariectomized mice had lower serum cGMP concentrations, which were largely restored to normal by treatment with NO-Cbi, or low-dose estrogen replacement. Micro-CT analyses of tibiae showed that all three pharmacological interventions significantly improved trabecular bone architecture in ovariectomized animals, with similar effect sizes. NO-Cbi reversed ovariectomy -induced osteocyte apoptosis as efficiently as estradiol, and enhanced bone formation parameters in vivo, consistent with in vitro effects on osteoblast proliferation, differentiation, and survival. Ovariectomy dramatically increased osteoclast numbers, and this effect was completely reversed by estradiol. NO-Cbi significantly decreased the number of osteoclasts in ovariectomized mice, suggesting cGMP-independent effects of NO-Cbi in osteoclasts. We conclude that estrogen deficiency represents a state of relative NO and cGMP deficiency, and that NO-dependent or NO-independent guanylate cyclase stimulation is a novel, anabolic treatment strategy for post-menopausal osteoporosis. These data confirm an important role of NO/cGMP signaling in bone biology.

We have found that the novel nitric oxide (NO) donor nitrosyl-cobinamide (NO-

Cbi) prevents osteoporosis in ovariectomized mice. Most drugs used for treating osteoporosis inhibit osteoclasts (which resorb bone) but do not stimulate osteoblasts (which make new bone). We found that NO-Cbi both inhibits osteoclasts and stimulates osteoblasts, so it is an ideal drug for treating osteoporosis, including post-menopausal osteoporosis, and no drug like it is currently available.

"NO-Cbi" or nitrosyl cobinamide is a compound having the structure:

Methods of making and formulating and dosaging NO-Cbi are described in Boss et al, USPN 8,222,242.

In alternative embodiments, also provided are bioisosteres of compounds of NO-

Cbi. In alternative embodiments, bioisosteres provided herein are compounds comprising one or more substituent and/or group replacements with a substituent and/or group having substantially similar physical or chemical properties which produce substantially similar biological properties to a NO-Cbi, or stereoisomer, racemer or isomer thereof. In one embodiment, the purpose of exchanging one bioisostere for another is to enhance the desired biological or physical properties of NO-Cbi without making significant changes in chemical structures. For example, in one embodiment, bioisosteres of compounds as provided herein are made by replacing one or more hydrogen atom(s) with one or more fluorine atom(s), e.g., at a site of metabolic oxidation; this may prevent metabolism (catabolism) from taking place. Because the fluorine atom is similar in size to the hydrogen atom the overall topology of the molecule is not significantly affected, leaving the desired biological activity unaffected. However, with a blocked pathway for metabolism, the molecule may have a longer half-life or be less toxic, and the like.

In alternative embodiments, NO-Cbi compositions used to practice emboidments described herein are prepared in a wide variety of dosage forms according to any means suitable in the art for preparing a given dosage form. Pharmaceutically acceptable carriers can be either solid or liquid. Non-limiting examples of solid form preparations include a tablet, a pill, a capsule, a liquid, a gel, a geltab, a powder, a spray or aerosol, a cachet, a suppository, a dispersible granule, and the like. A solid carrier can include one or more substances which can also act as diluents, flavoring agents, buffering agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Suitable solid carriers include magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, acacia, tragacanth, methylcellulose, sodium

carboxymethyl-cellulose, polyethylene glycols, vegetable oils, agar, a low melting wax, cocoa butter, and the like. Non-limiting examples of liquid form preparations include solutions, suspensions, syrups, slurries, and emulsions. Suitable liquid carriers include any suitable organic or inorganic solvent, for example, water, alcohol, saline solution, buffered saline solution, physiological saline solution, dextrose solution, water propylene glycol solutions, and the like, preferably in sterile form.

In alternative embodiments, compositions used to practice embodiments described herein are formulated and administered to the subject as pharmaceutically acceptable salts. Non-limiting examples of pharmaceutically acceptable salts include acid addition salts such as those containing hydrochloride, sulfate, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p- toluenesulfonate, cyclohexylsulfamate and quinate. Such salts can be derived using acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid, according to means known and established in the art.

In alternative embodiments, aqueous solutions are prepared by dissolving the active component in water and adding suitable colorants, flavors, stabilizing and thickening agents as desired. Aqueous suspensions can also be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, and other well- known suspending agents.

In alternative embodiments, solid forms are according to any means suitable in the art. For example, capsules are prepared by mixing the composition with a suitable diluent and filling the proper amount of the mixture in capsules. Tablets are prepared by direct compression, by wet granulation, or by dry granulation. Their formulations usually incorporate diluents, binders, lubricants and disintegrators as well as the compound. Non- limiting examples of diluents include various types of starch, cellulose, crystalline cellulose, microcrystalline cellulose, lactose, fructose, sucrose, mannitol, kaolin, calcium phosphate or sulfate, inorganic salts such as sodium chloride and powdered sugar.

Powdered cellulose derivatives are also useful. Non-limiting examples of tablet binders include starches, gelatin and sugars such as lactose, fructose, glucose and the like. Natural and synthetic gums are also convenient, including acacia, alginates, methylcellulose, polyvinylpyrrolidone and the like. Polyethylene glycol, ethylcellulose and waxes can also serve as binders.

In alternative embodiments, compositions used to practice embodiments described herein are formulated with lubricants, which can be used in a tablet formulation to prevent the tablet and punches from sticking in the die. The lubricant can be chosen from such slippery solids as talc, magnesium and calcium stearate, stearic acid and

hydrogenated vegetable oils. Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, guar gum, citrus pulp, carboxymethyl cellulose, and sodium lauryl sulfate. Tablets can be coated with sugar as a flavor and sealant, or with film-forming protecting agents to modify the dissolution properties of the tablet. The compounds can also be formulated as chewable tablets, by using large amounts of pleasant-tasting substances such as mannitol in the formulation, as is now well-established in the art.

In alternative embodiments, compositions used to practice embodiments described herein are formulated as liquid formulations or solid form preparations, e.g., which can be intended to be converted, shortly before use, to liquid form preparations. In alternative embodiments, liquid forms include solutions, suspensions, syrups, slurries, and emulsions. Liquid preparations can be prepared by conventional means with

pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats or oils); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). In alternative embodiments, preparations can contain, in addition to the active agent, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like. In alternative embodiments, compositions can be in powder form for constitution with a suitable vehicle such as sterile water, saline solution, or alcohol, before use.

In alternative embodiments, compositions used to practice embodiments described herein are formulated in solution in both the un-ionized and ionized forms. Generally lipid soluble or lipophilic drugs diffuse most readily across mucosal membranes. In alternative embodiments, compositions used to practice embodiments described herein are formulated with buffering agents, pH-adjusting agents, or ionizing agents to adjust the ratio of unionized:ionized forms of the NO-Cbi.

In alternative embodiments, compositions used to practice embodiments described herein are formulated with permeation enhancers or permeability enhancers, which can significantly enhance the permeability of lipophilic and nonlipophilic drugs, including the NO-Cbi. In alternative embodiment, penetration enhancers as described in Cooper et al. (1987) "Penetration Enhancers", in Transdermal Delivery of Drugs, Vol. II, Kyodonieus et al., Eds., CRC Press, Boca Raton, Fla are used. Additional forms of chemical enhancers also can be used, such as those enhancing lipophilicity, have been developed to improve transport when physically mixed with certain therapeutic agents and provide more predictable absorption, including for example as described in U.S. Pat. Nos.

4,645,502; 4,788,062; 4,816,258; 4,900,555; 3,472,931; 4,006,218; and 5,053,227.

In alternative embodiments, carriers can be coupled to pharmaceutical agents described herein to enhance intracellular transport, as described e.g., by Ames et al. (1973) Proc. Natl. Acad. Sci. USA, 70:456-458 and (1988) Proc. Int. Symp. Cont. Rel. Bioact. Mater., 15: 142. In alternative embodiments, permeation enhancers include bile salts such as sodium cholate, sodium glycocholate, sodium glycodeoxycholate, taurodeoxycholate, sodium deoxycholate, sodium lithocholate chenocholate, chenodeoxycholate, ursocholate, ursodeoxycholate, hydrodeoxycholate, dehydrocholate, glycochenocholate, taurochenocholate, and taurochenodeoxycholate. Other permeation enhancers such as sodium dodecyl sulfate ("SDS"), dimethyl sulfoxide ("DMSO"), sodium lauryl sulfate, salts and other derivatives of saturated and unsaturated fatty acids, surfactants, bile salt analogs, derivatives of bile salts, or such synthetic permeation enhancers as described in U.S. Pat. No. 4,746,508 can be used. In alternative

embodiments, DMSO, SDS, and medium chain fatty acids (about C-8 to about C-14) their salts, derivatives, and analogs are used. In alternative embodiments, the permeation enhancer concentration within the dissolvable matrix material can be varied depending on the potency of the enhancer and rate of dissolution of the dissolvable matrix. Other criteria for determining the enhancer concentration include the potency of the drug and the desired lag time. The upper limit for enhancer concentration is set by toxic effect to or irritation limits of the mucosal membrane.

In alternative embodiments, compositions used to practice embodiments described herein are formulated with a disintegrating agent. Tablet disintegrators are substances which swell when wetted to break up the tablet and release the compound, and include starches such as corn and potato starches, clays, celluloses, aligns, gums, methylcellulose, agar, bentonite, wood cellulose, powdered natural sponge, cation-exchange resins, alginic acid, sodium alginate, guar gum, citrus pulp, carboxymethyl cellulose, polyvinyl- pyrrolidone, and sodium lauryl sulfate. Acrylic type polymers can also advantageously be used as disintegrators, including methacrylic copolymers of type C (as disclosed in U.S. Pat. No. 6,696,085).

The compositions can be formulated for use in topical administration. Such formulations include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.

In alternative embodiments, compositions used to practice embodiments described herein are formulated as creams, drops, liniments, lotions, ointments and pastes are liquid or semi-solid compositions for external application. Such compositions can be prepared by mixing the active ingredient(s) in powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid with a greasy or non-greasy base. The base can comprise complex hydrocarbons such as glycerol, various forms of paraffin, beeswax; a mucilage; a mineral or edible oil or fatty acids; or a macrogel. Such compositions can additionally comprise suitable surface active agents such as surfactants, and suspending agents such as agar, vegetable gums, cellulose derivatives, and other ingredients such as preservatives, antioxidants, and the like.

In alternative embodiments, compositions used to practice embodiments described herein are formulated for injection into the subject or individual. For injection, the compositions can be formulated in aqueous solutions such as water or alcohol, or in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. The solution can contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Injection formulations can also be prepared as solid form preparations which are intended to be converted, shortly before use, to liquid form preparations suitable for injection, for example, by constitution with a suitable vehicle, such as sterile water, saline solution, or alcohol, before use.

In alternative embodiments, compositions used to practice embodiments described herein are formulated in sustained release vehicles or depot preparations. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compositions 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. Liposomes and emulsions are well-known examples of delivery vehicles suitable for use as carriers for hydrophobic drugs.

In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered by systemic administration, e.g., by oral, intravenous, intraperitoneal and intramuscular delivery. In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered by infusion or injection (intravenously, intramuscularly, intracutaneously, subcutaneously, intrathecal, intraduodenally, intraperitoneally, and the like). The compositions can also be administered intranasally, vaginally, rectally, orally, or transdermally. In alternative embodiments, the compositions are administered intravenously. Administration can be at the direction of a physician.

In alternative embodiments, compositions used to practice embodiments described herein are formulated and/or administered via buccal administration, and the

compositions can take the form of spray, liquid, tablets, troche or lozenge formulated in conventional manner. Compositions for oral or buccal administration, can be formulated to give controlled release of the active compound. Such formulations can include one or more sustained-release agents known in the art, such as glyceryl mono-stearate, glyceryl distearate and wax.

In alternative embodiments, compositions used to practice embodiments described herein are formulated for topical administration and/or administered or applied topically. Such administrations include applying the compositions externally to the epidermis, the mouth cavity, eye, ear and nose. Compositions for use in topical administration include, e.g., liquid or gel preparations suitable for penetration through the skin such as creams, liniments, lotions, ointments or pastes, and drops suitable for delivery to the eye, ear or nose.

In alternative embodiments, compositions used to practice embodiments described herein are formulated for and/or administered via inhalation. Compositions can be inhaled through the nose or mouth. In some embodiments, inhalation can occur via a nasal spray, dry powder inhaler, metered-dose inhaler, vaporizer, and nebulizer.

In alternative embodiments, alternative pharmaceutical delivery systems are employed. Non-limiting examples of such systems include liposomes and emulsions. Certain organic solvents such as dimethyl-sulfoxide can also be employed. In alternative embodiments, compounds can be delivered using a sustained-release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. The various sustained-release materials available are well known by those skilled in the art.

Sustained-release capsules can, depending on their chemical nature, release the compounds over a range of several days to several weeks to several months.

In alternative embodiments, nanoparticles, nanolipoparticles, vesicles and liposomal membranes comprising compounds and compositions are used to practice the methods and embodiments as provided herein. Provided are multilayered liposomes comprising compounds used to practice embodiments as provided herein, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070082042. The multilayered liposomes can be prepared using a mixture of oil-phase components comprising squalane, sterols, ceramides, neutral lipids or oils, fatty acids and lecithins, to about 200 to 5000 nm in particle size, to entrap a composition used to practice embodiments as provided herein.

Liposomes can be made using any method, e.g., as described in Park, et al., U.S. Pat. Pub. No. 20070042031, including method of producing a liposome by encapsulating an active agent (e.g., compounds and compositions as provided herein, or a compound used to practice methods as provided herein), the method comprising providing an aqueous solution in a first reservoir; providing an organic lipid solution in a second reservoir, and then mixing the aqueous solution with the organic lipid solution in a first mixing region to produce a liposome solution, where the organic lipid solution mixes with the aqueous solution to substantially instantaneously produce a liposome encapsulating the active agent; and immediately then mixing the liposome solution with a buffer solution to produce a diluted liposome solution.

. In one embodiment, liposome compositions used to practice embodiments as provided herein comprise a substituted ammonium and/or polyanions, e.g., for targeting delivery of a compound as provided herein, or a compound used to practice methods as provided herein, to a desired cell type or organ, e.g., brain, as described e.g., in U.S. Pat. Pub. No. 20070110798.

Provided are nanoparticles comprising compounds as provided herein, e.g., used to practice methods as provided herein in the form of active agent-containing

nanoparticles (e.g., a secondary nanoparticle), as described, e.g., in U.S. Pat. Pub. No. 20070077286. In one embodiment, provided are nanoparticles comprising a fat-soluble active agent used to practice embodiments as provided herein, or a fat-solubilized water- soluble active agent to act with a bivalent or trivalent metal salt.

In one embodiment, solid lipid suspensions can be used to formulate and to deliver compositions used to practice embodiments as provided herein to mammalian cells in vivo, in vitro or ex vivo, as described, e.g., in U.S. Pat. Pub. No. 20050136121.

In alternative embodiments, the effective amount of a composition to be administered can be dependent on any number of variables, including without limitation, the species, breed, size, height, weight, age, overall health of the subject, the type of formulation, the mode or manner or administration, or the severity of the osteoporesis. The appropriate effective amount can be routinely determined by those of skill in the art using routine optimization techniques and the skilled and informed judgment of the practitioner and other factors evident to those skilled in the art. Preferably, a

therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity to the subject.

In alternative embodiments, a concentration of NO-Cbi administered is in a range of about 0.01% to about 90% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 50% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 40% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 30% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 25% of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 20%) of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 15%) of the dry matter weight of the composition. In some aspects, NO-Cbi comprises up to about 10%> of the dry matter weight of the composition.

In alternative embodiments, individuals or subjects are administered NO-Cbi in a daily dose range of about 0.001 mg/kg to about 10 mg/kg of the weight of the subject. The dose administered to the subject can also be measured in terms of total amount of drug administered per day; for example, individuals or subjects are administered NO-Cbi in a dosage range of about 0.01 to about 500 milligrams of NO-Cbi per day: 0.05 milligrams of NO-Cbi per day; about 0.1 milligrams of NO-Cbi per day; about 0.5 milligrams of NO-Cbi per day; about 1 milligrams of NO-Cbi per day; about 5 milligrams of NO-Cbi per day; about 10 milligrams of NO-Cbi per day; about 25 milligrams of NO- Cbi per day; about 50 milligrams of NO-Cbi per day; about 100 milligrams of NO-Cbi per day; about 150 milligrams of NO-Cbi per day; or about 200 milligrams of NO-Cbi per day. Treatment can be initiated with smaller dosages that are less than the optimum dose of NO-Cbi, followed by an increase in dosage over the course of the treatment until the optimum effect under the circumstances is reached. If needed, the total daily dosage can be divided and administered in portions throughout the day.

For effective treatment of osteoporesis, or for stimulation of bone growth, one skilled in the art can recommend a dosage schedule and dosage amount adequate for the subject being treated. In alternative embodiments, dosing can occur one to four or more times daily for as long as needed. The dosing can occur less frequently if the

compositions are formulated in sustained delivery vehicles. The dosage schedule can also vary depending on the active drug concentration, which can depend on the needs of the subject.

In alternative embodiments, compositions used to practice embodiment described herein can be co-administered with other therapeutic agents that are selected for their particular usefulness against the condition that is being treated. For example, such therapeutic agents can be pain relievers, blood thinners/anticoagulants, clot busters, stomach antacids, compounds which lessen untoward effects of the compositions, other known agents that lower blood pressure. The compositions can be co-administered with cobalamin (vitamin B12) to reduce or eliminate potential toxicity of the administered cobinamide.

In alternative embodiments, the administration of these additional compounds can be simultaneous with the administration of NO-Cbi, or can be administered in tandem, either before or after the administration of NO-Cbi, as necessary. Any suitable protocol can be devised whereby the various compounds to be included in the combination treatment are administered within minutes, hours, days, or weeks of each other. In alternative embodiments, repeated administrations are in a cyclic protocol. The invention will be further described with reference to the examples described herein; however, it is to be understood that the invention is not limited to such examples.

EXAMPLES

Example 1 : Nitrosyl-cobinamide (NO-Cbi) is effective for treating osteoporosis

This example describes data demonstrating that exemplary compositions provided herein, including exemplary nitrosyl-cobinamide (NO-Cbi), a direct NO-rel easing agent, is effective for treating osteoporosis in a mouse model of estrogen deficiency-induced osteoporosis.

In murine primary osteoblasts, NO-Cbi increased intracellular cGMP, Wnt/β- catenin signaling, proliferation, and osteoblastic gene expression, and protected cells from apoptosis. Correspondingly, in ovariectomized (OVX) C57B1/6 mice, NO-Cbi increased serum cGMP concentrations, bone formation, and osteoblastic gene expression, and prevented estrogen deficiency-induced osteocyte apoptosis. NO-Cbi additionally prevented an ovariectomy-induced increase in osteoclasts, likely due to a reduction in the RANKL/osteoprotegerin gene expression ratio, which regulates osteoclast differentiation, and due to direct inhibition of osteoclast differentiation, observed in vitro in the presence of excess RANKL. While positive NO effects in osteoblasts were mediated by cGMP/PKG, the osteoclast-inhibitory effects appeared to be largely cGMP-independent. Consistent with its effects on osteoblasts and

osteoclasts, NO-Cbi increased trabecular bone mass in OVX mice. NO-Cbi represents a novel direct NO-releasing agent that, in contrast to nitrates, does not generate superoxide, and combines anabolic and anti-resorptive effects in bone, making it an effective agent for treating osteoporosis.

MATERIALS AND METHODS

Materials. Antibodies against Akt, Akt(pSer ⁴⁷³ ), Erkl(pTyr ²⁰⁴ ), GSK-3p, GSK- 3P(pSer ⁹ ), and cleaved caspase-3 were from Cell Signaling, and antibodies against Erkl/2, and β-actin were from Santa Cruz Biotechnology. A β-catenin-specific antibody and fluorophore-labeled secondary antibodies were from InVitrogen. DETA-NONOate was from Cayman and the cGMP agonist 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) from BioLog.

Preparation of NO-Cbi. NO-Cbi was generated by reducing dinitro-cobinamide under deoxygenated conditions. Ascorbic acid and dinitro-cobinamide were incubated at a ratio of 5: 1 for 1 h at RT, and then the solution was purged with nitrogen to remove any free NO. NO-Cbi was stable at RT for at least 1 month when protected from light and filter- sterilized.

Animal Experiments. Ten week-old female C57B1/6 mice were purchased from Jackson Laboratories. They were maintained in accordance with the "Guide for the Care and Use of Laboratory Animals" (2011, 8 ^th ed., Washington, D.C., Natl. Research Council, Natl. Academies Press), and all experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Diego. Mice were housed at 3-4 animals per cage in a temperature-controlled environment with a 12 h light/dark cycle; they were fed standard Teklad Rodent Diet with ad lib access to food and water. After one week of acclimatization, mice weighing 19.5-22.0 g were randomly divided into three groups— Groups 1 and 2 (eight mice each) underwent bilateral ovariectomy, while Group 3 (six mice) underwent a sham operation. One mouse in Group 1 had to be euthanized post-operatively because of suture failure. Beginning one week post- surgery, mice received daily i.p. injections six days per week for five weeks, either vehicle (0.1 ml 9.25 mM ascorbic acid, Group 1), or NO-Cbi (10 mg/kg/d given as 0.1 ml of 1.85 mM NO-Cbi, Group 2). This NO-Cbi dose did not significantly reduce systolic blood pressure (< 10 mm Hg), consistent with our previous report, ³² and the NO-Cbi- treated mice showed no signs of toxicity, and had similar weight gain during the experiment as vehicle-treated OVX mice. Double calcein labeling was performed by intraperitoneal injection of calcein (25 mg/kg) at seven and three days before euthanasia. Mice were euthanized one hour after the last drug or vehicle injection by CO2 intoxication and exsaguination; blood samples were collected by cardiac puncture and allowed to clot. Femoral and tibial bones were dissected for quantitative RT-PCR, histology, and micro- CT analyses.

Mice carrying floxed prkg2 alleles (PRKG2 ^f/f mice). To generate PRKG2 ^f/f mice, we PCR-amplified genomic DNA fragments encoding prkg2 exon III with flanking sequences from 129/SvJ ES cells using KOD polymerase (EMD Millipore Corporation). The prkg2-f\oxed construct was assembled in the vector pDL L (gift from Ju Chen of UCSD) and consisted of a 4.2 kb 5' arm, a 0.55 kb fragment including exon III flanked by LoxP sites, a 1.3 kb neo cassette flanked by FRT sites, and a 3kb 3' arm. All PCR products and fusion sites were sequenced. The construct was electroporated into Rl ES cells derived from 129/SvJ mice, and G418-resistant clones were isolated and screened by PCR; homologous recombination was confirmed by Southern blot analysis using probes outside of the 5' and 3' arms. A recombinant clone with normal chromosome analysis in 20 metaphase spreads was injected into C57B1/6 blastocysts to establish germline chimeric mice. Heterozygous PRKG2 ^f/+ mice were mated with homozygous FLPeR mice containing FLPe recombinase (from Jackson Laboratories) to remove the neo cassette.

Culture of murine primary osteoblasts (POBs). POBs were isolated from the femurs and tibiae of 8-12 week-old C57/B16 mice, or from PRKG2 ^f/f mice in a mixed

background, and were grown in DMEM supplemented with 10% FBS, as described. ¹⁵ ' ³⁶ In some cases, ascorbate (0.3 mM) and β-glycerolphosphate (10 mM) was added to the medium to induce differentiation. The cells were used at passages 1-5, and were checked for mineralization capacity. ³⁶ To delete exon II of PKG II, cells from PRKG2 ^f/f mice were infected with adenovirus encoding CRE recombinase (or control virus expressing LacZ) at an MOI of 50 and used 48 h later.

Culture of murine primary osteoclasts. Osteoclasts were generated from murine bone marrow as described. ³⁷ Briefly, bone marrow cells were plated at 10 ⁶ cells/cm ² in a- Minimal Essential Medium with 10% FBS and 50 ng/ml M-CSF, and non-adherent cells were discarded after 48 h. RANKL (150 ng/ml) was added on day 3, medium was replaced on days 5 and 7; the indicated drugs were added with fresh medium on days 3,5, and 7. On day 8, cultures were fixed and stained for TRAP using a commercially- available kit (Sigma), or were harvested for RNA isolation.

Quantitative RT-PCR assays. Frozen bone shafts (frozen at -80°C after removal of bone marrow cells) were pulverized with a mortar and pestle in liquid nitrogen. RNA was purified using Trizol-reagent (Molecular Research Center, Inc.) and 1 μg of RNA was reverse-transcribed and quantitative PCR was performed using a MX3005P™ realtime PCR detection system with Brilliant II SYBR® Green QPCR Master Mix™ (Agilent Technologies). ¹⁵ All primers were tested with serial cDNA dilutions. Relative changes in mRNA expression were analyzed using the 2 ^"ΔΔα method, with 18S rRNA serving as an internal reference; we used mean ACT values (gene of interest minus 18S rRNA) measured in the OVX/vehicle-treated group to calculate ΔΔΟΤ values for the OVX/NO- Cbi group.

Preparation of bone cell extracts and Western blotting. Protein extracts were prepared from mouse bones as described previously. ³⁶ Briefly, -50 mg of pulverized bone was incubated for 15 min on ice in 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 2 mM Na ₃ V0 ₄ , 10 mM NaF, and protease inhibitor cocktail. The samples were centrifuged at 13,000 g for 15 min at 4°C. Supernatants were boiled in SDS sample buffer and proteins were resolved by SDS-PAGE and analyzed by Western blotting as described. ¹³ Films were scanned using Image J software.

Quantitation of NOx and cGMP. NO production was measured based on nitrite and nitrate accumulation in the medium with a two-step colorimetric assay, as previously described. ¹³ Serum cGMP concentrations were measured by ELISA using a kit according to the manufacturer's protocol (Biomedical Technologies Inc., MA).

Proliferation Assay. POBs in 6-well dishes were cultured in 0.1% FBS overnight and treated with NO-Cbi for 1 h; cells were then transferred to fresh medium with 0.1% FBS and 10 μθ of [methyl- ³ H]thymidine (20 Ci/mmol, final concentration 0.5 μΜ) for 24 h. Cells were extracted in situ in ice-cold 10% trichloroacetic acid, precipitated DNA was collected on glass microfiber filters, and radioactivity on washed filters was measured by scintillation counting.

Immunofluorescence Staining. POBs were plated on glass coverslips, transferred to medium containing 0.1% FBS or 0.1% BSA, and incubated in the absence or presence of NO-Cbi for the indicated time. Cells were fixed and permeabilized and incubated with cleaved caspase-3- or β-catenin-specific antibodies (both at 1 : 100 dilution), followed by secondary antibodies conjugated to FITC or AlexaFluor 555, respectively; nuclei were counterstained with Hoechst 33342. ³⁶ Images were analyzed with a Keyence BZ-X700™ fluorescence microscope. Bone histomorphometry, TRAP and TUNEL staining. Tibiae were fixed in 70% ethanol, dehydrated, and embedded in methyl-methacrylate and sectioned at the

University of Alabama, Birmingham, Center for Metabolic Bone Disease. Some sections were stained with Masson's tri chrome, or stained for tartrate-resistant acid phosphatase, while unstained sections were used for assessing fluorochrome labeling. ³⁶ TUNEL staining of de-plasticized sections was performed as described. ³⁶ Slides were scanned with a Hamamatsu Nanozoomer 2.0 HT Slide Scanning System™, and image analysis was performed using the Nanozoomer Digital Pathology NDP.view2™ software.

Histomorphometric measurements were performed between 0.25 and 2.25 mm distal to the growth plate as described. ³⁸

Immunohi stochemi stry . Femurs were fixed overnight in 10% neutral formalin solution, decalcified in 10% EDTA (pH 7.5) for 5 days, and embedded in paraffin.

Sections (8 μπι thick) were de-paraffinized in xylene and rehydrated in graded ethanol and water. For antigen retrieval, slides were placed in 10 mM sodium citrate buffer (pH 6.0) at 80-85°C, and allowed to cool to room temperature for 30 min. Endogenous peroxidase activity was quenched in 3% hydrogen peroxide for 10 min. Slides were blocked with 5% normal goat serum and incubated overnight at 4°C with anti-phospho- ERK antibody (1 : 100 in blocking buffer), followed by HRP-conjugated secondary antibody for 1 h at room temperature. After development with 3,3-diaminobenzidine substrate (Vector Laboratories, Inc., Burlingame, CA), slides were counterstained with hematoxylin for 2 min.

Micro-CT. Micro-CT analyses were performed on ethanol-fixed tibiae, using a Skyscan 1076™ (Kontich, Belgium) scanner at 9 μπι voxel size, and applying an electrical potential of 50 kVp and current of 200 μΑ, with a 0.5 mm aluminum filter, as described. ³⁸ Mineral density was determined by calibration of images against 2 mm diameter hydroxyapatite rods (0.25 and 0.75 g /cm ³ ). Cortical bone was analyzed by automatic contouring 3.6 mm-4.5 mm distal to the proximal growth plate, using a global threshold to identify cortical bone, and eroding one pixel to eliminate partial volume effects. Trabecular bone was analyzed by automatic contouring the proximal tibial metaphysis 0.36 -2.1 mm distal to the growth plate and using an adaptive threshold to select the trabecular bone.

Statistical Analyses. Graph Pad Prism 5™ was used for two-tailed Student t-test (to compare two groups) or one-way ANOVA with Bonferroni post-test analysis (to compare more than two groups); p<0.05 was considered significant. For in vivo experiments, we tested our primary hypothesis, that NO-Cbi affects bone architecture and parameters of bone formation in OVX mice, and our secondary hypothesis that ovariectomy induces bone loss compared to sham-operated animals, using the Student t-test to assess for differences between means of two groups. Based on variability data from our published studies, ³⁸ we estimated that a sample size of 6 mice would provide 90% power to detect the difference in bone volume/tissue volume (BV/TV) between OVX and sham-operated mice, whereas 8 mice per group were required to detect an absolute increase of 0.35% in BV/TV in drug-treated OVX mice with 90% power (a error set at 5%).

RESULTS

NO-Cbi enhances cGMP/PKG and Erk/ Akt signaling, gene expression, proliferation, and survival in POBs.

We have shown that cobinamide binds NO with high affinity (K _a ~10 ¹⁰ M ^"1 ), yielding NO-Cbi, where one water molecule on cobinamide is replaced by one NO molecule. ³³ NO-Cbi is stable in aqueous solutions for at least 16 h at room temperature even when exposed to air, but it releases NO rapidly in the presence of NO scavengers or when added to cells in culture medium. ³² Murine POBs produced nitrite and nitrate— stable NO oxidation products— yielding a medium concentration of -10 μΜ over 2 h (Fig. 1A). Adding 10 μΜ NO-Cbi to the cells increased the amount of nitrite and nitrate within 10 min, and by 30 min, the amount of NO oxidation products generated was maximal and accounted for >90% of the NO added to the cells as NO-Cbi (Fig. 1 A). NO-Cbi increased the intracellular cGMP concentration in a dose-dependent fashion, resulting in near- maximal phosphorylation of the PKG substrate vasodilator-stimulated phosphoprotein (VASP) at 3-10 μΜ (Fig. 1B,C).

We previously showed that estrogens activate Erk and Akt in a NO/cGMP/PKG- dependent manner in osteocytes and osteoblasts, and that activation of these signaling proteins is essential for estrogen's anti-apoptotic effects in osteocytes/blasts. ¹⁴ We also showed that the pro-proliferative effects of fluid shear stress on osteoblasts are mediated by NO/cGMP/PKG via Src activation of Erk. ¹⁵ Consistent with increasing intracellular cGMP, NO-Cbi increased Erk and Akt phosphorylation in murine POBs, protecting cells from serum starvation-induced apoptosis and stimulating proliferation (Fig. 1D-G). In the POBs, NO-Cbi raised mRNA expression of osteoblast differentiation-related genes, including osteocalcin (OCN), osteopontin (Sppl), collagenl-al (Collal), alkaline phosphatase (ALP), and low-density lipoprotein receptor-related protein-5 (Lrp5), with tubulin (Tubal) expression serving as a control for RNA quality (Fig. 1H). NO-Cbi decreased osteoblast expression of RANKL mRNA, whereas it increased expression of the RANKL antagonist OPG, suggesting that NO-Cbi could negatively affect osteoclast differentiation (Fig. II).

NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II.

The Wnt/p-catenin pathway controls osteoblast differentiation, proliferation, and survival, and is essential for bone mass acquisition and maintenance; increased or decreased gene expression and gain- or loss-of-function mutations of pathway components cause high versus low bone mass phenotypes— for example, expression and activity of the Wnt co-receptor Lrp5 positively correlate with bone mass in humans and mice. ³⁹ Stability and nuclear translocation of β-catenin are negatively controlled by glycogen synthase kinase-3 (GSK-3), which in turn is negatively controlled by phosphorylation on a site targeted by Akt and PKG II. ⁴⁰ ' ⁴¹ To examine the role of NO and PKG II in osteoblast Wnt signaling, we used POBs isolated from mice which carry floxed PKG II alleles (PRKG2 ^f/f mice) and infected cells with adenovirus encoding CRE recombinase to induce PKG II deficiency (Fig. 2A). In cells infected with control virus encoding LacZ, NO-Cbi induced Akt and GSK-3 phosphorylation and β-catenin nuclear translocation, but these effects were largely lost in CRE virus-infected, PKG Il-deficient cells (Fig. 2B,C). Similarly, NO-Cbi-induced proliferation was prevented by CRE- mediated PKG II knockout (Fig. 2D). NO-Cbi treatment increased Wntla, Lrp5, and β- catenin mRNA expression in the presence, but not in the absence of PKG II (Fig. 2E). Lrp5 protein increased in parallel to mRNA in response to NO-Cbi (Fig. 2F). Transcript levels of the Wnt/p-catenin target genes cyclin D (CycD), ALP, and OCN were increased by NO-Cbi in PKG Il-expressing, but not in PKG Il-deficient cells (Fig. 2E). We conclude that NO-Cbi stimulates Wnt/p-catenin signaling in murine POBs in a PKG II- dependent manner.

NO-Cbi inhibits osteoclast differentiation.

To examine the effect of NO-Cbi on osteoclast differentiation in vitro, we cultured adherent murine bone marrow cells with recombinant M-CSF and RANKL. ³⁷ NO-Cbi dramatically reduced the number of TRAP -positive osteoclasts, with maximal effects observed at 10 μΜ (Fig. 3A,B). Less pronounced inhibition was observed with the NO donor DETA-NONOate at a concentration calculated to release equivalent amounts of NO (Fig. 3C). In contrast, the cGMP analog 8-pCPT-cGMP, at a concentration that maximally activated PKG I and II in intact cells, had a much smaller, non-significant effect on the number of TRAP-positive cells (Fig. 3C). Consistent with the effects of NO-Cbi on osteoclast differentiation, mRNA expression of the osteoclast-specific genes TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) was markedly reduced in NO- Cbi-treated osteoclast cultures (Fig. 3D).

NO-Cbi increases serum cGMP concentration, bone formation, and osteoblastic gene expression in OVX mice.

To test if NO-Cbi could prevent bone manifestations of estrogen deficiency, we subjected mature C57B1/6 mice to bilateral ovariectomy (OVX) or sham operation, and injected the OVX mice with vehicle or NO-Cbi for five weeks, starting one week postoperatively. We used a NO-Cbi dose (10 mg/kg/d) that had no significant effect on systolic blood pressure and resulted in serum cobinamide concentrations below our limits of detection (<1 μΜ). Similar to our previous results, ³⁸ OVX mice had lower serum cGMP concentrations compared to sham-operated mice - but NO-Cbi significantly increased serum cGMP at 1 h post administration (Fig. 4A). NO-Cbi reversed the decrease in osteoblast numbers found in OVX mice, and significantly increased mineral apposition rate (MAR), mineralizing surfaces (MS/BS), and bone formation rate (BFR) on trabecular bone surfaces (Fig. 4B-F). Similarly, NO-Cbi increased MAR and BFR on endocortical surfaces, although endocortical MS/BS was not affected (Fig. 8).

Consistent with these results, mRNA expression of osteoblast differentiation-related genes (OCN, Sppl, ALP, Collal, and Lrp5) was higher in femoral shafts of NO-Cbi- treated OVX mice compared to vehicle-treated OVX mice, whereas tubulin-al mRNA was unchanged (Fig. 4G). Together, these results indicate that NO-Cbi increases osteoblastic activity in OVX mice.

NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis.

Estrogen deficiency-induced bone loss is, in part, due to osteocyte apoptosis. ¹¹ ' ³⁸ ' ⁴² We showed previously that the osteocyte protective effects of estrogen require

NO/cGMP/PKG signaling, and are at least partly dependent on Erk activation. ¹⁴

Consistent with previous reports, ¹⁴ ' ³⁸ ' ⁴² we saw a significant increase in TUNEL-positive, apoptotic osteocytes in trabecular and cortical bone of OVX mice, with NO-Cbi reducing osteocyte apoptosis to values found in sham-operated mice (Fig. 5A-C, Fig. 9, for cortical bone). Similar results were obtained when we examined the amount of cleaved caspase-3 in bone lysates from NO-Cbi- versus vehicle-treated OVX mice (Fig. 5D).

Immunohistochemical staining of pErk was evident in bone-lining osteoblastic cells of sham-operated mice, but was faint or absent in OVX mice; treating OVX mice with NO- Cbi induced prominent pErk staining in bone-lining cells (Fig. 5E; isotype-matched control immunoglobulin produced no staining, not shown).

NO-Cbi regulates RANKL/OPG expression and reduces osteoclasts in OVX mice.

Estrogens reduce osteoclast survival, ^{9, 43} and, as expected, we found an increased number of TRAP-positive osteoclasts on trabecular bone in the OVX mice (Fig. 6 A,B; few osteoclasts were seen on cortical surfaces under all conditions). Treating OVX mice with NO-Cbi reduced osteoclast numbers to values found in sham-operated animals (Fig. 6A,B). Compared to vehicle-treated OVX mice, femurs of NO-Cbi-treated OVX mice contained less RANKL and more OPG mRNA, and mRNA expression of the osteoclast marker genes CTSK and TRAP was decreased (Fig. 6C). Thus, NO-Cbi reduced osteoclast numbers and osteoclastic gene expression in OVX mice, at least, in part, by reducing the RANKL/OPG ratio.

NO-Cbi increases trabecular bone mass in OVX mice.

To determine the effects of NO-Cbi on estrogen deficiency-induced bone loss, we analyzed tibial microarchitecture by micro-CT. Consistent with previous reports, ³⁸ ' ⁴⁴ OVX mice had significantly lower trabecular bone volume, trabecular number, and trabecular bone mineral density compared to sham-operated mice; NO-Cbi partly restored all three parameters (Fig. 7A-D). We found no difference in cortical thickness or cortical tissue mineral density between sham-operated and OVX mice, and NO-Cbi did not affect cortical bone parameters (Fig. 10). Estrogen deficiency-induced bone loss is more pronounced in trabecular than cortical bone, and cortical loss may take longer than trabecular loss and varies with age and strain of mice. ³⁸ ' ^44-46

Discussion

A better understanding of the molecular mechanisms controlling bone homeostasis has led to development of multiple drugs for osteoporosis that inhibit bone resorption by targeting osteoclast differentiation and/or function. ^{2, 5} However, the only clinically- available agents that stimulate osteoblast activity and bone formation are PTH analogs, but they also stimulate osteoclasts, limiting their effectiveness. ² ' ⁵ Therefore, bone- anabolic agents that simultaneously inhibit osteoclast function are needed. We found that the NO donor NO-Cbi improved trabecular bone architecture and increased bone formation markers in OVX mice, while dramatically decreasing osteoclasts. Our results confirm bone-anabolic effects of NO, explore mechanisms of NO actions in bone cells, and demonstrate in vitro and in vivo effectiveness of a novel, direct NO-releasing agent.

An important role of NO in osteoblast biology is supported by rodent studies: First, NOS3-deficient mice have reduced bone mass due to defects in osteoblast number and maturation, and they have exaggerated bone loss after ovariectomy, with a blunted response to estrogens ^47"49 And second, NO-generating organic nitrates reduce bone loss from estrogen deficiency in rats (as measured by DXA), whereas NOS inhibitors block estrogen's bone-protective effects and prevent bone formation induced by mechanical stimulation in rodents. ^16"18 ' ²² ' ²³ In humans and rats, serum concentrations of the NO metabolites nitrite and nitrate correlate with estradiol concentrations, and increase with estrogen administration. ^{23, 24} Estrogen deficiency reduces NOS expression and activity, leading to a state of relative NO- and, consequently, cGMP-deficiency. ³⁸ ' ⁵⁰ NO-Cbi restored serum cGMP concentrations in OVX mice, and the drug's positive effects on osteoblast proliferation, differentiation, and survival were likely mediated via increased intracellular cGMP concentrations and PKG activation, because the drug increased Wnt/p-catenin signaling in a PKG II-dependent fashion. We have previously shown that cGMP-elevating agents and cGMP analogs increase osteoblast proliferation, differentiation, and survival, with PKG II activation of Src required for Erk and Akt activation, and PKG I phosphorylation of Bel -2 contributing to anti-apoptotic effects in osteoblasts and osteocytes. ¹⁴ ' ¹⁵ ' ³⁸ Consistent with its in vitro effects on POBs, NO-Cbi increased osteoblast number, mineral apposition and bone formation rates; it enhanced osteoblastic gene expression, reduced osteocyte apoptosis, and increased trabecular bone volume and BMD in OVX mice. Like NO-Cbi, the NO-independent soluble guanylate cyclase activator cinaciguat increased bone formation and osteoblast marker genes and ameliorates bone loss in OVX mice; however, in contrast to NO-Cbi, cinciguat did not affect osteoclast parameters significantly. ³⁸

NO appears to have dual functions in osteoclast biology: low NO concentrations generated by NOS1 promote, while higher NO concentrations generated by NOS2 inhibit osteoclast differentiation and survival. ⁵¹ ' ⁵² NOS 1 -deficient mice have an increased bone mass with reduced bone turnover due to defects in osteoclast differentiation and function, indicating a positive role of NO in osteoclasts. ^{51, 53} However, NOS2 induction by RANKL serves as a feed-back inhibition of RANKL-induced osteoclast differentiation, and NOS2-deficient osteoclasts show increased differentiation in response to RANKL. Adding NO donors to mature osteoclasts inhibits their resorptive activity. ^54"56

NO-Cbi treatment of OVX mice decreased osteoclast numbers and reduced osteoclast-specific TRAP and CTSK mRNAs in bone; these results correlated with a decrease in RANKL and increase in OPG mRNAs in bone. In contrast, the cGMP- elevating agent cinaciguat did not influence osteoclast numbers, osteoclast-specific genes, RANKL, or OPG in OVX mice. ³⁸ In agreement with these in vivo results, NO- Cbi reduced RANKL and increased OPG mRNA in cultured POBs, whereas cinaciguat did not affect these genes. ³⁸ RANKL and OPG are key regulators of osteoclast differentiation produced primarily by cells of the osteoblastic lineage. ² Consistent with our results in POBs, other workers have shown down-regulation of RANKL by NO, but not by cGMP analogs, in bone marrow stromal cells. ⁵⁷ In addition to reducing the RANKL/OPG ratio in bone and POBs, NO-Cbi directly inhibited osteoclast

differentiation in vitro, in the presence of excess RANKL and M-CSF, whereas a cGMP analog had little effect. cGMP-independent NO effects on osteoclast differentiation have been reported previously. ⁵² ' ⁵⁸ Thus, in addition to inducing anabolic responses in osteoblasts, NO-Cbi inhibited osteoclastogenesis directly and indirectly, by reducing the RANKL/OPG ratio.

Epidemiological data link the use of organic nitrates (i.e., nitroglycerin and iso- sorbide mononitrate) to reduced fracture risk, ^19"21 and four prospective, randomized trials showed a positive effect of these nitrates on bone density, at doses lower than those used for vasodilation. ²⁴ Firs^ in young women who underwent ovariectomy, nitroglycerin was as effective as estrogen replacement in preventing bone loss. ⁵⁹ Second, in post-menopausal women with established osteoporosis, subjects randomized to isosorbide mononitrate showed similar improvement in BMD as subjects who received a bisphosphonate. ⁶⁰ Third, in healthy post-menopausal women, isosorbide mononitrate decreased a bone resorption marker (N-terminal telopeptide) and increased a bone formation marker (alkaline phosphatase) compared to placebo. ⁶¹ And fourth, in osteopenic post-menopausal women, nitroglycerin increased BMD at the lumbar spine and hip, and increased cortical thickness at the radius and tibia; it also decreased N- terminal telopeptide and increased alkaline phosphatase. ²⁵ These results indicate positive effects of nitroglycerin on bone formation and possibly on bone strength— with cortical changes superior to those observed with PTH— but the studies were underpowered to assess fracture risk. ' In contrast, another trial in post-menopausal women failed to show an effect of nitroglycerin on BMD, possibly because treatment adherence was poor. ²⁶

A major problem with organic nitrates is that they must be activated by

mitochondrial aldehyde dehydrogenase, which leads to oxidative stress, development of tolerance, and induction of endothelial dysfunction. ^{28, 29} In fact, several large trials of chronic nitrate use in patients with coronary artery diseases have shown increased mortality in the treated patients, ⁶³ ' ⁶⁴ which may be attributable to increased oxidative stress. ²⁷ Since oxidative stress is implicated in the pathophysiology of estrogen deficiency- and age-related osteoporosis, ¹⁰ and oxidative stress can lead to decreased NO bio-availability and soluble guanylate cyclase desensitization, ³⁰ the beneficial effects of nitrates in post-menopausal osteoporosis are likely limited by their pro-oxidant properties.

Considerable effort has been directed towards developing second generation, direct NO-releasing agents, but to date, none have shown efficacy in clinical trials, and some generate toxic metabolites in the process of NO release, making them unsuitable for clinical use. ³⁰ ' ⁶⁵ We have developed NO-Cbi as a novel, direct NO donor with major advantages over existing, FDA-approved nitrates. NO-Cbi is derived from the penultimate vitamin B12 precursor cobinamide; cobinamide is found at low concentrations in human serum, and we have found in rodents that cobinamide has no toxicity at >50- fold higher doses than those used in this study and those required for delivery of pharmacological amounts of NO (G.R.Boss et al., unpublished data and ³² ). After NO release, cobinamide is generated, and cobinamide can bind O2 ^" and other reactive oxygen species, ³⁵ providing a potential added benefit of protecting cells from oxidative stress. Repeated administration of NO-Cbi does not induce tolerance, and NO-Cbi is stable and can be administered by multiple routes, including oral ingestion (G.R. Boss et al., unpublished data and ³² ).

A limitation of our study is that ovariectomy does not mimic the gradual cessation of ovarian function occurring during menopause, however, OVX mice are an accepted model for bone loss due to estrogen deficiency, and show changes in bone architecture and turnover similar to those observed in post-menopausal women, including increased bone resorption and increased osteoblast/osteocyte apoptosis. ^{9, 42, 44, 46} Some authors have observed increased bone formation markers in parallel with increased resorption in OVX mice, whereas we and others found no change in mineral apposition rate and a trend towards reduced mineralizing surface and bone formation rate after OVX. ^66"69 These differences may be attributable to differences in mouse strain and age, the type of bone examined, and the time interval after OVX. We did not observe cortical bone loss post OVX, but bone loss in OVX mice also varies greatly among different inbred strains, with age of the mice, the site examined, and the time interval after surgery. ³⁸ ' ^{44" 46} Another limitation of our study is that we examined a single dose of NO-Cbi; this dose was chosen based on its lack of effect on systolic blood pressure. Further studies are needed to optimize treatment dose and schedule, and to test the effect of oral NO- Cbi administration.

In conclusion, we have shown that NO-Cbi regulates bone remodeling by promoting osteoblast proliferation, differentiation, and survival, and by simultaneously inhibiting osteoclast differentiation. NO-Cbi improved bone mass in OVX mice, a frequently-used model of post-menopausal osteoporosis. ⁹ ' ⁴⁴ ' ⁷⁰ We are unaware of previous work using a direct NO-releasing agent as a bone-anabolic agent in animals or humans. These studies represent proof of concept for the effectiveness of NO-Cbi as an anabolic agent for treating osteoporosis.

FIGURE LEGENDS

Figure 1 : NO-Cbi enhances cGMP/PKG and Erk/Akt signaling, gene expression, proliferation and survival in POBs. (A) POBs were incubated in medium with 0.1% FBS (3 x 10 ⁵ cells/ml) for 2 h prior to receiving 10 μΜ NO-Cbi (NOCbi) for the indicated times. Stable NO oxidation products (nitrite plus nitrate, NOx) were measured in the medium by the Griess reaction (NOx present in medium without cells was subtracted). (B,C) POBs were treated with vehicle or NO-Cbi at the indicated concentrations for 30 min, and intracellular cGMP concentrations were measured by ELISA (B). VASP phosphorylation was analyzed by Western blot using a phospho-Ser ²⁵⁹ -specific antibody, with densitometric quantitation of pVASP normalized to β-actin shown above. (D,E) Serum-deprived POBs were treated with vehicle or 10 μΜ NO-Cbi for 10 min and ERK and Akt activation were assessed by blotting with phospho-specific antibodies.

Densitometric quantitation of pErk and pAkt normalized to total Erk and Akt, respectively, is shown above. (F) POBs were serum-starved for 36 h in medium containing 1% BSA with 10 μΜ NO-Cbi or vehicle; apoptosis was assessed by immunofluorescence staining with antibodies specific for cleaved caspase-3 and FITC- coupled secondary antibodies (green); nuclei were counterstained with Hoechst 33342 (blue). (G) POBs cultured in medium with 0.1% FBS for 18 h were treated with 10 μΜ NO-Cbi or vehicle for 1 h, and transferred to fresh medium containing ³ H-thymidine for 24 h; thymidine incorporation into DNA was measured as described in Experimental Procedures. (H,I) Confluent POBs were differentiated in ascorbate-containing medium for 14 d and some cells received 10 μΜ NO-Cbi (open bars) or vehicle (filled bars) for the last 24 h. Expression of osteocalcin (OCN), osteopontin (Sppl), collagen 1-Al (Collal), alkaline phosphatase (ALP), low-density lipoprotein receptor-related protein-5 (Lrp5), tubulin (Tubal), receptor activator of nuclear factor kappa-B ligand (RANKL), and osteoprotegerin (OPG) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1. Panels A-I show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between NO-Cbi- and vehicle-treated cells. In panel F, ^### p <0.01 for the comparison between cells in starvation versus control medium.

Figure 2: NO-Cbi stimulates Wnt signaling and mPOB proliferation via PKG II. (A) POBs isolated from mice homozygous for prkg2 alleles flanked by LoxP sites ("floxed" PRKG2 ^f/f ) were infected with adenovirus expressing β-galactosidase (LacZ, control) or CRE recombinase (CRE). Forty-eight h later, relative amounts of prkg2 mRNA were determined by qRT-PCR, and knockdown efficiency of PKG II protein was analyzed by Western blotting, with caveolin-1 serving as a loading control. (B) Cells were infected as in A, but 30 h later were transferred to medium containing 0.1% FBS, and 18 h later were treated with 10 μΜ NO-Cbi or vehicle for 10 min. Akt and GSK-3P phosphorylation were assessed using antibodies specific for Akt(pSer ⁴⁷³ ) and GSK-3P(pSer ⁹ ), with total GSK-3P serving as a loading control; densitometric quantitation is shown on the right, with relative amounts of pAkt and pGSK-3 found in vehicle-treated control virus-infected cells assigned a value of 1. (C) PRKG2 ^f/f POBs were infected with control or Cre virus and transferred to 0.1% FBS as in B; they were treated with NO-Cbi or vehicle for 6 h, prior to detecting β-catenin by immunofluorescence staining. The bottom panel shows nuclei counterstained with Hoechst 33342. Numbers below indicate the percentage of cells showing nuclear β-catenin. (D) Cells were infected and cultured as in B; they were treated with NO-Cbi or vehicle for 1 h prior to measuring ³ H-thymidine incorporation into DNA for 24 h. (E) Cells were infected with control or CRE virus as described in A, and treated with 10 μΜ NO-Cbi or vehicle for 24 h. Expression of Wingless type MMTV-integration site family- la (Wntla), low-density lipoprotein receptor- related protein-5 (Lrp5), β-catenin (bCat), cyclin D (CycD), alkaline phosphatase (ALP), osteocalcin (OCN), and tubulin (Tubal) mRNAs were measured by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in untreated cells assigned a value of 1. (F) POBs cultured in 10 % FBS were treated with 10 μΜ NO-Cbi for the indicated times, and Lrp5 protein (open symbols) and mRNA (filled symbols) were assessed by Western blotting and qRT-PCR, respectively. Panels A-F show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between NO-Cbi-treated versus vehicle-treated cells infected with control virus, and ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 for comparison between NO-Cbi-treated cells infected with control versus CRE virus.

Figure 3 : NO-Cbi inhibits osteoclast differentiation. (A,B) Murine bone marrow mononuclear cells were cultured in the presence of M-CSF, with RANKL added after 3 d; together with RANKL, cells received vehicle or NO-Cbi at the indicated concentrations. Tartrate-resistant acid phosphatase (TRAP)-positive cells (red) were counted on day 8. (C) Cells were cultured as in A, but some cultures received 10 μΜ NO-Cbi, 5 μΜ DETA- NONOate (Deta-NO, which releases 2 moles of NO/mol of drug), or 100 μΜ 8-pCPT- cGMP together with RANKL. (D) Expression of TRAP, cathepsin K (Ctsk), and calcitonin receptor (CalcR) mRNAs were determined by qRT-PCR and normalized to 18S rRNA, with relative mRNA levels in vehicle-treated cells assigned a value of 1.

Panels B-D show means ± SEM of at least three independent experiments; *p < 0.05, **p < 0.01, ***p < 0.001, for the comparison between vehicle-treated versus drug-treated cells.

Figure 4: NO-Cbi increases serum cGMP concentration, bone formation, and osteo- blastic gene expression in OVX mice. Eleven week-old mice were subjected to ovariectomy (OVX) or sham operation, and 7 d later daily i.p. injections were started with either vehicle (Veh) or NO-Cbi (10 mg/kg/d) for 6 d/week for a total of 5 weeks. Mice additionally received calcein 7 d and 4 d prior to euthanasia. (A) Serum cGMP concentrations were measured by ELISA 1 h after the last injection of vehicle or NO-Cbi. (B) The number of trabecular osteoblasts per bone perimeter (N.Ob/B.Pm) was counted at the proximal tibia. (C-F) Trabecular calcein labeling was assessed at the tibia (C), with quantification of mineral apposition rate (MAR, panel D), mineralizing surface per bone surface (MS/BS, panel E), and bone formation rate (BFR, panel F). (G) RNA was extracted from femurs, and the relative abundance of osteocalcin (OCN), osteopontin (Sppl), alkaline phosphatase (ALP), collagen-al (Collal), low-density lipoprotein receptor- related protein-5 (Lrp5) and tubulin (Tubal) mRNA was quantified by qRT- PCR and normalized to 18S rRNA. Data were calculated according to the AACT method, using the mean of the vehicle-treated OVX group. Data for panels A-F are the means ± SEM from n= 6 sham operated mice, n= 7 vehicle treated OVX mice, and n=8 NO-Cbi- treated OVX mice; data in panel G represent 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.

Figure 5: NO-Cbi prevents estrogen deficiency-induced osteocyte apoptosis. Mice were subjected to OVX or sham operation and were treated with vehicle or NO-Cbi as described in Fig. 4. (A-C) The percentage of apoptotic osteocytes was assessed in trabecular (A,B) and cortical bone (C) by TUNEL staining (black nuclei) of tibial sections. Data in B and C represent means ± SEM from n= 6 mice per group. *p < 0.05, **p < 0.01, for the indicated pair-wise comparisons. (D) Osteoblast and osteocyte apoptosis was assessed by Western blotting of extracts obtained from tibial bone (after removal of bone marrow), using an antibody specific for cleaved caspase-3, with β-actin serving as a loading control (n =2 mice per group). (E) Erk activity in cortical (top) and trabecular (bottom) bone-lining cells was assessed by immunofluorescence staining using a phospho-Erk-specific antibody and horse radish peroxidase-coupled secondary antibody (brown); isotype-matched control IgG produced no signal (not shown).

Figure 6: NO-Cbi regulates RANKL/OPG and reduces osteoclasts in OVX mice. Mice subjected to OVX or sham-operation were treated with vehicle or NO-Cbi as described in Fig. 4. (A,B) Osteoclasts were identified by TRAP staining (red), and the number of trabecular osteoclasts per bone perimeter (N.Oc/B.Pm) was counted at the proximal tibia. (C) RNA was extracted from femurs, and the relative abundance of

RANKL, OPG, CTSK, and TRAP mRNA was quantified by qRT-PCR and normalized to 18S rRNA. Data were calculated according to the AACT method using the mean of the vehicle-treated OVX group. Data represent the mean ± SEM from 6 mice per group. *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.

Figure 7: NO-Cbi increases trabecular bone mass in OVX mice. Mice subjected to OVX or sham operation were treated with vehicle or NO-Cbi as described in Fig.4. (A) Tibiae were analyzed by micro-CT imaging, and three-dimensional reconstruction of the trabecular bone at the proximal tibia below the growth plate is shown. (B-E) Trabecular bone volume/tissue volume (B), trabecular number (C), and trabecular bone mineral density (D) were quantified at the proximal tibia as described in Materials and Methods. Data represent means ± SEM from n= 6 sham-operated mice, n= 7 vehicle-treated OVX mice, and n=8 NO-Cbi-treated OVX mice; *p < 0.05, **p < 0.01, ***p < 0.001 for the indicated pair-wise comparisons.

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A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.