1 2 Nitric oxide: novel therapy 3 for osteoporosis · therapies for chronic conditions such as osteoporosis. Keywords: glyceryl trinitrate , hormone eplacement r therapy menopause

Review

10.1517/14656560802197162 © 2008 Informa UK Ltd ISSN 1465-6566 1All rights reserved: reproduction in whole or in part not permitted

Nitric oxide: novel therapy for osteoporosis Sunil J Wimalawansa Robert Wood Johnson Medical School, Department of Medicine, New Brunswick, NJ 08903, USA

Background : Relative nitric oxide (NO) deficiency is responsible for many pathophysiological processes, including in postmenopausal women providing a plausible biological basis for use of NO replacement therapy in humans. Excess or inappropriate local production of NO aggravates bone destruction in some diseases such as septic shock, rheumatoid and other inflammatory arthropathies. Results : A variety of in vitro and in vivo data have revealed the efficacy of nitroglycerin and nitrates on bone cells. Since some part of the beneficial effects of estrogen on bone is mediated via the NO–cGMP pathway, NO donor therapy is an attractive alternative to estrogen therapy to prevent and treat osteoporosis. When the body cannot generate adequate amounts of NO for biological homeostasis, administration of exogenous NO or prolongation of the actions of endogenous NO are practical ways to supplement NO, especially in postmenopausal women. Conclusion : Postmenopausal NO deficiency is rectified with hormone replacement therapy, which enhances local production of NO. Declining local NO production secondary to estrogen deficiency in postmenopausal women, and perhaps in older men, could be one of the key reasons for age-related increased incidences of cardiovascular events, sexual dysfunction as well as osteoporosis. Thus, in addition to supplementation of NO compounds in acute situations such as alleviating angina and erectile dysfunction, it could be a valuable addition to the armamentarium of therapies for chronic conditions such as osteoporosis.

Keywords: glyceryl trinitrate , hormone replacement therapy , menopause , nitric oxide donors , nitric oxide synthase inhibitors , nitroglycerin , RANK , osteopoenia , osteoporosis , vascular system

Expert Opin. Pharmacother. (2008) 9(17):1-20

1. Introduction

Nitric oxide (NO) is a small, diatomic gas radical, highly reactive molecule conveying biochemical signals that result in a wide spectrum of effects on different biological systems [1,2] . It also acts as an important hemodynamic regulator [3] . NO is a widely distributed chemical second messenger [4] , having both intracellular and intercellular actions. It activates guanyl cyclase enzyme and modulates actions of several bioactive peptides as well as neuronal transmitters [1,5] .

NO also has other roles in cells, such as in isoprenylation (geranylgeranylation) of the Rho GTPase that activates Rho-PK, which in turn inactivates a factor that would otherwise turn on the bone morphogenetic protein (BMP)-2/Cbfa1-Runx-2 cycle [5,6] . The broad ranging actions of NO are determined largely by the site and rate of NO synthesis, the quantity generated, and the nature of the environment into which it is released. The reactivity of NO is affected by the presence of reactive oxygen intermediates and the activity of antioxidant defense systems [7-9] . NO is enzymatically produced by oxidation and cleavage of the amino-terminal nitrogen atom of amino acid L -arginine. The reaction is dependent on electrons donated by the cofactor NADPH that requires oxygen, and yields L-citrulline as a coproduct [10] .

1. Introduction

2. Nitric oxide and bone

3. In vitro bone cell cultures and

in vivo animal studies

4. Human studies

5. Conclusion

6. Expert opinion

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Nitric oxide: novel therapy for osteoporosis

2 Expert Opin. Pharmacother. (2008) 9(17)

Discovery of NO as the endothelium-derived relaxing factor opened the door to a wide research arena [3,11,12] . NO was identified as a significant biological signaling molecule that regulates a wide range of important cellular and physiological functions. NO deficiency has been implicated in many pathological and physiological processes within the mammalian body [1] . While deficiency of endogenous NO may cause several disorders including osteoporosis, erectile dysfunction and angina, excess of NO has been implicated in pathological conditions such as inflammatory arthritis [13] .

The NO donor nitroglycerin has been used for many years in clinical practice. Circulating NO products are significantly lower in postmenopausal women than in premenopausal women [14,15] as well as during a certain part of the menstrual cycle [16] . This is rectified after initiation of hormone replacement therapy (HRT) [15,17] . Since menopause can lead to NO deficiency, there is a plausible biological basis for use of NO replacement or supplementation therapy [5] . Furthermore, studies have shown that the beneficial effects of estrogen on bone are mediated via NO [18] . Since HRT has potential adverse effects in postmenopausal women, as reported in the Women’s Health Initiative (WHI) study [19] , it makes sense to supplement NO directly.

2. Nitric oxide and bone

2.1 Osteoporosis Osteoporosis, literally ‘porous bone’, is the most common metabolic bone disease affecting humans. Characterized by bone loss and deterioration of bone quality, it leads to bone fragility and an increased risk for fractures. Osteoporosis is the reduction in the amount of bone mass leading to fractures with minimal trauma [20] . It affects one out of every two women and one out of every five men over age 65. Osteoporosis is a major public health threat for an estimated 40 million in the United States. Every year this disease is responsible for approximately 1.8 million fractures, primarily of the hip, spine, and wrist [21,22] . Over $20 billion is spent annually to treat this bone disease in the USA, and the costs continue to escalate. Identifying a cost-effective, novel medication to combat this common disorder can produce tremendous benefits to the society [13] .

Generally, a balance is maintained between the bone-resorbing osteoclasts and bone-forming osteoblasts. In the absence of estrogen, bone turnover increases, eventually weakening bone and sustaining fractures. Osteoporotic fractures are a significant cause of morbidity and mortality in elderly people. A third of patients with broken hips die within 1 year and a high percentage of the remainder require long-term assistance and nursing care. Until a decade ago, little could be offered to slow or halt the progression of osteoporosis and related fractures. Today, an increasing number of effective medications are available to treat osteoporosis and to reduce risk of fractures. However, these

agents are costly, and all have significant adverse effects, such as upper gastrointestinal irritation and ulceration, constipation, enhanced arterial and venous thrombosis, biochemical abnormalities, and increase fracture incidence after prolonged treatment.

2.2 Potential use of nitric oxide donors for osteoporosis Unlike some other diseases, osteoporosis is easy to diagnose and treat. Prevention of fractures can be achieved by the identification and elimination of risk factors including low bone mineral density (BMD), a bone-healthy lifestyle, avoiding medications that contribute to bone loss, identifying and elimination of secondary causes of bone loss, prevention of falls and injuries, and use of effective antiosteoporotic agents [23] . In the past, many postmenopausal women relied on HRT after menopause to reduce the risk of heart disease and osteoporosis. However, the WHI study [19] demonstrated that while it is effective in preventing fractures, HRT could increase the risk of stroke, heart disease, and breast cancer. Another barrier to HRT and other antiosteoporosis therapies is the high cost. Thus, finding a cost-effective alternative therapy for osteoporosis with little or no adverse effects is essential. One such exciting opportunity is to use NO donor therapy. The original postulation of the role of NO in bone homeostasis was in 1988 ( Figure 1 ) [24] .

Significant advances have been made in understanding cellular mechanisms involved in bone metabolism and generation of novel therapeutic agents, including those with antiresorptive (also known as anticatabolic) and anabolic actions. There is now an in-depth understanding of cellular mechanisms of actions, signal transduction, cell–cell interactions, and interactions among many key factors regulating bone metabolism [6] . Many proinflammatory cytokines can upregulate inducible nitric oxide synthase (iNOS) [13,25] . Key interactions between cytokines, hormones and NO in bone are illustrated in Figure 2 .

The human skeleton is finely constructed and the labile framework supports the vertebrate organism [26] . The skeleton’s ability to change over time and rejuvenate itself is wondrous; this is in part dependent on coupling of bone formation and bone resorption [26] . From birth to young adulthood, the skeleton reaches a state of dynamic stasis by achieving peak bone mass that depends on genetics, mechanical stresses, and appropriate exposure to hormones. This is followed by a decline in bone mass as part of the normal ageing process. These regulated schema are subjected to changes in activity, diet, hormonal status and disease processes. Hormonal, paracrine, and autocrine regulation are involved in maintenance as well as the growth and decay of the skeleton. These complex interactions allow new interventions to stabilize bone mass or restore the age- or disease-depleted skeleton [6,13] .

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Expert Opin. Pharmacother. (2008) 9(17) 3

2.3 Rationale for using nitric oxide donor compounds for osteoporosis Small molecules such as H[+] and NO are actively involved in regulation of bone metabolism ( Figure 1 ), in particular the control of osteoclastic bone resorption [24] . Many in vitro and in vivo animal studies [27] have shown that the NO donor nitroglycerin has a beneficial effect on controlling bone resorption [28] , while also having a positive effect on bone formation [18,23,29] . Treatment with NO may also prevent cell death in vital organs such as the heart, brain, pancreatic β cells, as well as osteoblasts and osteocytes.

2.4 Estrogen and nitric oxide Effects of estrogen in bone cells are mediated via estrogen receptor α [30] , and estrogen also upregulates endothelial NOS (eNOS) gene in bone. A beneficial effect of estrogen on bone is abolished in the presence of NOS enzyme inhibitors such as L -NAME [18,29] . This suggests that some of the effects of estrogen in bone are mediated via the NO pathway [13] . A pilot human study demonstrated similar efficacy of nitroglycerin and estrogen, Premarin ® (Wyeth-Ayerst), in preventing estrogen deficiency induced bone loss. This indirect evidence further supports HRT

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Figure 1 . Schematic representation of interactions between various cells, cytokines, calcium-regulating peptide/hormones, and small molecules such as nitric oxide (NO) and hydrogen in bone formation and bone resorption. Reproduced with permission from [24] . APD: Pamidronate; CGRP: Calcitonin gene-related peptide; CT: Calcitonin; EHDP: Etidronate; OAF: Osteoclast activating factor; PGE: Prostaglandins; PTH: Parathyroid hormone; PTH-rP: PTH-related peptide.

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Figure 2 . Role of nitric oxide in bone. Interactions of cytokines, estrogen, growth factors, mechanical stresses, stimulating endothelial and inducible nitric oxide synthase (NOS) in bone homeostasis and bone metabolism.

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working via NO in the skeleton [31] . These data suggest that NO therapy could serve as a safer alternative therapy in preventing postmenopausal bone loss [5] .

Mechanical loading and fluid flow, as well as estrogen, generate NO locally via bone cells, which is important in mediating the observed anabolic effects. While the major effect of estrogen is inhibition of osteoclastic bone resorption, estrogen also has stimulatory effect on osteoblasts [32] . Studies in vitro have shown 17 β -estradiol enhances both proliferation and differentiation of cultured osteoblasts [33-35] . Osteoblasts have functional estrogen receptors [36,37] , further supporting the indication that 17 β -estradiol stimulates bone formation via direct action [38-40] .

3. In vitro bone cell cultures and in vivo animal studies

3.1 Estrogen, nitric oxide and bone cells Although a link between estrogen and stimulation of eNOS has been established in the vascular system [41,42] , a link between the osteogenic effects of estrogen and NO was first reported by Wimalawansa et al. in 1996 [18] . They demonstrated that the NO donor nitroglycerin prevented the ovariectomy-induced bone loss in a rat model.

Subsequent studies demonstrated upregulation of eNOS expression and activity in human osteoblast cells following stimulation by 17 β -estradiol [43] . In primary human and rat osteoblast cultures, 17 β -estradiol dose dependently stimulates osteoblast cell proliferation and differentiation as assessed by alkaline phosphatase activity and bone nodule formation [13] . The effect is abolished following inhibition of NOS activity [44] . Moreover, osteoblasts cultured from eNOS gene knockout mice do not respond to 17 β -estradiol. Additionally, 17 β -estradiol enhances eNOS enzyme expression and NO metabolite levels in rat osteoblasts [45] . The latter is abolished with NOS inhibitors. Collectively, these observations suggest

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that the stimulatory effect of estrogen on osteoblast proliferation and differentiation relies on local production of NO via the eNOS isoform [5,46] .

NO regulates osteoclasts, the bone cells belonging to the monocyte lineage that are responsible for bone resorption. Bone formation and resorption are key processes working together during remodeling to repair bone microdamage. The NO donor nitroglycerin has a beneficial effect on controlling bone resorption by decreasing osteoclastic activity while improving bone formation by enhancing osteoblast activity [5] . For postmenopausal women who cannot tolerate or afford HRT treatment, nitroglycerin therapy may become an affordable and cost-effective option, costing about a third of current therapies.

Our supplementary studies demonstrated that nitroglycerin has beneficial additive effects on BMD when administered with bisphosphonates [47] . This is not surprising as the mechanisms of action of these two agents are different. NO donors are potential therapies to control bone loss and fragility [13,18] , and long-term therapy with NO will not only increase BMD but may also decrease fracture rates. Multiple interactions exist between various compounds that affect bone cell activity. Figure 3 illustrates the possible final common pathway of mediation of cellular actions of NO, fatty acids, statins and sex-steroid hormones [48] .

NOS enzymes are expressed in osteocytes, osteoblasts, and osteoclasts [45] . Since osteocytes and osteoblasts are abundant in bone and express higher enzyme levels, their NO synthesis may be quantitatively more important. Inflammatory cytokines, however, are potent inducers of iNOS expression in osteoblasts and other cells. Thus, in inflammatory conditions, iNOS-dependent overproduction of NO occurs which leads to bone destruction. In contrast, the eNOS isoform produces constant lower levels of NO generated locally, which has skeletal protective effects [48] .

3.2 In vitro cell biological studies NO appears to play an autocrine/paracrine regulatory role in bone cell metabolism. A number of studies suggest that NO may have an anabolic effect on bone tissues [5,18,29] , and it assists osteoblast-induced mineralization in all cultures [49] . Thus, NO donors increase osteocalcin synthesis and the formation of a mineralized matrix by osteoblasts in vitro , while NOS inhibitors have an antiproliferative effect on osteoblasts ( Figure 4A and 4 B ).

The release of large amounts of NO by cytokine-stimulated cells also has an antiproliferative effect on osteoblasts [50] and enhances osteoclast-mediated bone resorption [51] . Therefore, NO has a biphasic effect on osteoblasts [52] : in low concentrations it promotes bone formation, whereas in higher concentrations it has an inhibitory effect ( Figure 4B ).

Production of NO by osteoclasts in response to a rise in intracellular calcium, leads to cell retraction and inhibition of bone resorption [53] . Some authors have

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suggested that the NO effects on bone cells are not mediated via cGMP [54] , but overwhelming evidence suggests otherwise [5] . Estrogen is known to regulate eNOS activity in osteoblasts [43] , and osteoblasts produce NO [55] . Cytokine-mediated inflammation [56] , however, exhibits enhanced iNOS activity [57] , while NO donor compounds decrease bone resorption [52,58,59] . Similar effects have been demonstrated with L -arginine [60] , while inhibition of NOS activity leads to enhanced bone resorption [59,61] . Interestingly, TNF- α -dependent osteoclastic survival is thought to be iNOS dependent [62] but some researchers have suggested otherwise [63] . Bone cells, in particular osteoclasts [53] , osteoblasts [55] and bone marrow cells, produce NO [64] , as does mechanical strain [65-69] . NO has also been reported to regulate cGMP production in osteoclasts [70] .

Enhanced expression of NO via iNOS in inflammatory conditions (e.g., lipopolysasccharide-induced bone resorption) is alleviated with NOS inhibitors. This inflammation-associated bone loss is predominantly due to augmenting cytokine-induced MMP-1 production by osteoblasts, subsequently activating osteoclasts [71] . Nevertheless, the inhibition of osteoclast activity may be the predominant effect of NO under normal conditions. In vitro studies have shown that using high doses of NO also leads to rapid osteoclast cell death. Indeed, NO donors inhibit osteoclast formation in mouse bone marrow cultures, an experimental system frequently used to study factors regulating osteoclastogenesis [13] . The effect is likely to be mediated by the NO-induced apoptosis of osteoclast progenitors at physiological doses.

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Figure 4 . Osteoblast cell proliferation, generation of nitrite, and effects of nitric oxide enzyme inhibitor L -NAME on bone resorption and the dose-dependent effects of nitric oxide (NO) donors on osteoblastic-like cells. A. Osteoblast cell proliferation in the presence or absence of arginine (Arg), L -NAME, and cGMP inhibitor LY-83583 (*p < 0.01). Generation of nitrite (nmol/5000 bone cells) in osteoblasts (OB) and osteoclasts (OC) alone or in cocultures (*p < 0.01). Effects of nitric oxide enzyme inhibitor L -NAME (10[-2M]) on bone resorption. Arginine by itself was effective in decreasing bone resorption, while the addition of arginine to L -NAME abrogated the bone resorption. B. Dose-dependent effects of nitric oxide (NO) donors on osteoblastic-like cells demonstrating a biphasic response on cell proliferation in response to NO [5] . Prevention of bone loss with NO: in vivo study demonstrating that actions of estrogen in bone are mediated via the NO–cGMP pathway. Treatments started immediately following ovariectomy. There are no additive effects when NO is administered with estrogen. DETA: Diethylenetriamine.

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3.3 In vivo animal studies 3.3.1 Prevention and restoration of ovariectomy-induced bone loss in rats In vivo studies suggest that the predominant effects of NO are on osteoclast cells but its anabolic effects on osteoblast cells are also important [5] . The use of NOS inhibitors such as aminoguanidine or L -NAME leads to osteopoenia in rats. NO donors prevent ovariectomy-induced and glucocorticoid-induced bone loss [29,31] , while eNOS-deficient mice have reduced bone formation [72] . iNOS activation, however, enhances bone resorption [57,58] and is reported to be one of the main mechanisms of action in inflammation-induced osteoporosis [73] , and the inhibition of NOS activity negatively affects bone metabolism [74] .

Mechanical stimuli are important in bone homeostasis, as manifested by the decrease in bone mass following bed rest, inactivity, or microgravity. The mechanisms involved in the transduction of the effect of mechanical forces are not clear but some data suggest the participation of prostaglandins and NO [69,75] . Indeed, the inhibition of NO synthesis in vivo impairs the bone formation induced by mechanical loading of rat tibiae. By contrast, exogenous NO donors potentiate the osteogenic effect of loading. Although the NO donor nitroglycerin by itself has no effect on bones in adult male rats, it prevents the bone loss induced by methylprednisolone [23,76] . These studies suggest that NO-releasing compounds may have a protective effect even on glucocorticoid-induced bone loss [13] . However, the relevance to humans has yet to be examined.

We have demonstrated that the NO donor nitroglycerin prevents both ovariectomy and corticosteroid-induced bone loss, as assessed by BMD, bone weight, and bone histomorphometry in rats [5,18,23] . Further studies using the NO synthase inhibitor L -NAME have shown that at least part of the beneficial effects of estrogen on bone are mediated through NO [18] . Figure 5A illustrates the effects of nitroglycerin on prevention of ovariectomy-induced bone loss. Figure 5B illustrates the restoration of lost BMD after ovariectomy with NO donor therapy, in comparison to the positive control group supplemented with estrogen.

These data indicate that nitroglycerin and estrogen are equally effective in restoration of lost bone. In both prevention and restoration of bone loss in rat models, the beneficial effects of estrogen in bone are completely negated in the presence of NOS inhibitor L -NAME, suggesting that the estrogenic effects of HRT on bone cells are mediated via the NO–cGMP pathway [15,27] .

Estrogen increases synthesis of eNOS [77-79] , and the estrogen-induced antioxidant effect is due to suppression of superoxide anion production via release of NO [80] . Similarly, statins activate protein kinase Akt, leading to increased NO production by eNOS [81] . Furthermore, essential fatty acids and their metabolism also enhance eNOS activity. Therefore, the mechanism of estrogen, statins and essential fatty acids in improving skeletal health

is likely to be mediated by augmenting eNOS activity ( Figure 3 ). This is further supported by the fact that the selective estrogen receptor modulator (SERM) raloxifene is shown to dose dependently increase the release of NO from endothelial cells [82] . The data further support eNOS (which is abandoned in bone) activation and NO release from endothelial cells and osteoblast/osteocytes, inhibiting osteoclastic activity. Unlike estrogen and NO donors, statins and nitroglycerin have different paths of action on osteoblasts. Thus, the combination of these two agents is likely to have an added benefit on the skeleton. NO donors have provided beneficial effects on both trabecular and cortical bone when administered with bisphosphonates, calcitonin, or vitamin D [47] .

Changes in the expression of eNOS have also been shown to influence growth plate chondrocytes as well as osteoblast activity in the metaphyses in the lactation-induced bone loss model [83] . Thus, far, a substantial amount of in vitro and in vivo data have accumulated with respect to the effects of NO on bone cells. In 1996, using these animal and cell culture studies, it was demonstrated that some of the beneficial effects of estrogen on bone cells are in fact mediated through NO [5,18] . Since HRT has significant adverse effects (WHI study) [19] , it is sensible to provide NO directly, without using HRT [13] .

3.3.2 Dose-dependant effects of nitric oxide In several studies using an adult ovariectomized (OVX) rat model, the positive effects of topically applied nitroglycerin (Fougera, 2% ointment) on bone metabolism have been shown by measuring BMD, biochemical markers of bone turnover, and bone histomorphometry [5,18,23] . In these experiments, 17 β -estradiol was used as a positive control and its effects compared with nitroglycerin. The effects of nitroglycerin are equivalent to estrogen in preventing, as well as in treating, OVX-induced bone loss. Although applications of nitroglycerin once daily (0.2 mg/kg) were highly effective in the prevention of OVX-induced bone loss, multiple applications of the same dose were ineffective [84] . The beneficial effects of nitroglycerin are maximal between 0.2 and 0.5 mg/kg. Below 0.2 and above 0.5 mg/kg, the effects were similar to those of placebo. Interestingly, nitroglycerin applied in the morning was more efficacious than when applied in the evening. Figure 6 illustrates the dose- and frequency-dependent effects of nitroglycerin on BMD in female rats.

Our animal studies have demonstrated a similar efficacy of NO donor therapy in male rats. Among castrated rats, preservation of BMD by testosterone or estrogen is blocked by concomitant administration of the NOS blocker L -NAME. These data suggest that NO therapy is also likely to help in males, especially those suffering from hypogonadism and age-related bone loss in older males. Accordingly, it will have a significant advantage over estrogen replacement therapy or SERMs in postmenopausal

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Figure 5 . The effects of nitroglycerin on prevention of ovariectomy-induced bone loss and the restoration of lost bone mineral density (BMD) after ovariectomy (OVX) with nitric oxide (NO) donor therapy. A. Changes in lumbar spine BMD (predominantly tubercular bone) and femur weight (predominantly cortical bone) over the 6-week treatment period in younger, 12-week-old female Wistar rats following OVX in various treatment groups. Values are mean ± of SEM for fi ve animals per group. Comparisons were made against OVX rats (control) (*p < 0.005; ‡ p < 0.02). B. Restoration of lost bone with NO: changes in BMD after 10 weeks of treatment in 36-week-old matured rats. Six weeks were allowed after ovariectomy to set in bone loss. Changes in BMD are expressed as a percentage of BMD at the beginning of the treatment. Values are mean ± SEM with six animals per group. Comparisons were made against OVX rats (*p < 0.01; ‡ p < 0.02). Reprinted from Wimalawansa SJ, De Marco G, Gangula P, Yallampalli C. Nitric oxide donor alleviates ovariectomy-induced bone loss. Bone 1996;18(4):301-4 [18] ,Copyright (1996), with permission from Elsevier and Wimalawansa SJ. Restoration of ovariectomy-induced osteopenia by nitroglycerin. Calcif Tissue Int 2000;66(1):56-60 [29] , with kind permission of Springer Science and Business Media. E 2 : Estrogen treated; LN: L -NAME; NG: Nitroglycerin treated (mg/kg body weight per day); OVX rats: Control; Sham: Sham operated.

women, and testosterone and selective androgen receptor modulators in men.

Nitroglycerin is superior to more than 10 other NO donors examined, and the effects of combining nitroglycerin with vitamin D, calcitonin and bisphosphonates are additive in enhancing BMD [47,85] . Since the final common pathway is the same, no such additive effects were expected or seen when nitroglycerin is administered with sex-steroid hormones such as estrogen, progestrogens, or testosterone. Overall the results indicate that nitroglycerin has marked beneficial

effects on bone metabolism [5] , and bone remodeling [46] . Cyclosporin-A-induced bone loss also involves the NO–cGMP path [84,86,87] .

3.3.3 Comparison of nitric oxide donor therapies We compared the effects of nitroglycerin with other NO donors and the NO substrate ( L -arginine) on the prevention of OVX-induced bone loss. Nitroglycerin is superior to all other donors examined, including the NO substrate L -arginine, on BMD and femur weight in this model

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Figure 6 . Dose- and frequency-dependent effects of nitroglycerin on bone mineral density (BMD) in female rats. A. Dose-dependant changes in BMD and femur weights in 36-week-old female Wistar rats in response to escalating doses [ovariectomy (OVX) placebo: 0.02, 0.1, 0.5, and 2.5 mg/kg body weight of topically applied nitroglycerin ointment]. Changes in BMD after 10 weeks of treatment in sham (sham operated); OVX rats (control); estrogen treated (E 2 ); nitroglycerin treated (NG) (mg/kg body weight per day). Changes in BMD are expressed as a percentage of BMD from the beginning of the treatment (own controls). Data demonstrate a sharp biphasic effect of nitroglycerin on BMD. Values are mean ± SEM with six animals per group. Comparisons were made against OVX rats (*p < 0.05; ‡ p < 0.01). B. Frequency-dependent changes in BMD in response to nitroglycerin in 36-week-old female Wistar rats in response to once a day, twice a day, and three times a day topical application of nitroglycerin ointment (total dose/day remains the same). Changes in lumbar spine BMD (predominantly tubercular bone) and femur weight (predominantly cortical bone) over the 10-week treatment period. Values are mean ± SEM for fi ve animals per group. Comparisons were made against OVX rats (control) (*p < 0.01; ‡ p < 0.02). Reproduced from Wimalawansa S, Chapa T, Fang L, et al. Frequency-dependent effect of nitric oxide donor nitroglycerin on bone. J Bone Miner Res 2000;15(6):1119-25 [84] with permission of the American Society for Bone and Mineral Research.

Wimalawansa


( Figure 7 ) [13] . Furthermore, some donors such as SIN-10, although effective in vitro , were completely ineffective in vivo in the maintenance of the skeleton ( Figure 7 ).

3.3.4 Bone strength after treatment with nitroglycerin The effect of nitroglycerin on bone strength was tested in an OVX rat model. Twelve rats were sham operated and 36 rats ovariectomized. These rats were treated with placebo (sham-operated group and OVX-placebo group), 0.3 mg/kg nitroglycerin once a day (100 µg/kg/week of 17 β -estradiol, subcutaneous injection). After treatment for 8 weeks, bone strength was analyzed using standard techniques in the femoral neck and femur ( Figure 8 ).

Overall these findings indicate that the NO donor, nitroglycerin, not only increased BMD and femur weight but also increased bone strength ( Figure 8 ), as determined by femoral neck strength and three-point bending studies in femur.

3.3.5 Histomorphometric data using nitroglycerin In a glucocorticoid-induced osteopoenia model, we examined the efficacy of nitroglycerin on maintaining bone. Twenty-five, 32-week old rats (n = 5/group) were treated with a placebo, corticosteroid, L -NAME, nitroglycerin, or a combination of corticosteroid and nitroglycerin. Corticosteroid-induced bone loss ( ∼ 10% in 6 weeks) was prevented by co-administration of nitroglycerin [3] . Only trabecular bone volume and cortical bone area data (tibia and femur) are presented here. Nitroglycerin therapy was as effective as estrogen in maintaining trabecular bone volume and cortical bone area ( Figure 9 ). These data are consistent with the BMD and femur weight data, and bone strength study [10,23,84] .

Although this is not a model for estrogen deficiency-induced bone loss, these data are comparable and consistent with the

bone histomorphometric data that were observed in the OVX rat model. Similarly, in OVX studies, we demonstrated that estrogen as well as once daily administration of nitro glycerin prevented loss of trabecular bone volume. In the same study, we also observed that at the doses of nitroglycerin used, there were no significant effects on systolic blood pressure in the rats, as measured by the tail-cuff method. In fact, the higher systolic blood pressure observed in the OVX rats was alleviated with the use of both estrogen and nitroglycerin therapies.

The positive bone balance achieved in our in vivo studies in rat models (i.e., BMD, femur weight, bone histomorpho-metry and bone strength), and in in vitro osteoblast and osteoclast studies suggests that at lower doses, NO mediates osteoblastic cell proliferation and inhibits osteoclastic bone resorption. Prevention of destruction of bone trabeculae secondary to higher bone turnover with nitroglycerin should have positive effects on skeletal tensile strength. If an increase in bone turnover occurring in postmenopausal osteoporosis is prevented with nitroglycerin, its long-term benefits include increased BMD and bone strength, decreased incidence of osteoporotic fractures, and major reduction in the management costs of osteoporosis [13] .

In aged female rats (which are more analogous to postmenopausal women), our data suggest that nitroglycerin applied once a day is effective in improving BMD. The action of nitroglycerin depends on its conversion to NO in tissues, and this conversion may depend upon the availability of thiol groups, cofactors and a cascade of enzymes [10,88,89] . Continuous or frequent exposure to nitroglycerin leads to depletion of these rate-limiting factors, causing rapid loss of nitroglycerin effectiveness [90-92] .

Following the first postulation of NO’s role in osteoclasts and osteoblasts in 1988 [24] , several studies reported that the NO donor nitroglycerin prevented ovariectomy and

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Figure 7 . Percentage change of bone mineral density (BMD) and the mean femur weight during the fi rst 6 weeks after ovariectomy (OVX) (solid bars) and 6 weeks of therapy (hatched bars) with various nitric oxide donors in comparison with sham and placebo (p < 0.01). Nitroglycerin (NG) was equivalent to estrogen and superior to other donors examined in restoration of ovariectomy-induced bone loss. Arg: Arginine.

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Figure 8 . The effect of nitroglycerin on bone strength. A. Ultimate load (force) (N) in the femoral neck: sham (n = 8), ovariectomized (OVX)-placebo (n = 9), estrogen treated (n = 7), nitroglycerin treated (n = 10) (*p < 0.05). B. Slope of load–displacement (N/mm) in the femoral neck: sham (n = 8), OVX-placebo (n = 9), estrogen treated (n = 7), nitroglycerin treated (n = 10) (*p < 0.05). C. Three-point bending of rat femur ultimate force (load) (N) in the femoral neck: sham (n = 8), OVX-placebo (n = 9), estrogen treated (n = 7), nitroglycerin treated (n = 10) (NS). D. Three-point bending of rat femur – slope of load–displacement (N/mm) in the femoral neck (*p < 0.05).

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Figure 9 . Bone histomorphometric data. Change in cortical bone area (mm 2 ) in A. distal femoral metaphysis and B. proximal tibial metaphysis. Values are mean ± SEM (n = 3 – 4/group). Statistical comparisons were made against the control group (ANOVA, *p < 0.05). Trabecular bone volume (TBV %) data in C. the distal femur and D. the proximal tibia of rats. Rats were treated with prednisolone, L -NAME, nitroglycerin or prednisolone plus nitroglycerin, in comparison to placebo (control). CS: Corticosteroid; L -NAME: ; NG: Nitroglycerin.

Wimalawansa


corticosteroid-induced bone losses as determined by improvements in bone mass, biochemical variables of bone turnover and bone histomorphometry in rats [22,84] . Further-more, L -NAME, the NOS inhibitor, prevented synthesis, and release of NO via a NOS enzyme abrogating the beneficial effects of estrogen in bone [18] . The effects of NO on bone cells are concentration dependent; at low concen-trations (equivalent to activation of eNOS or neuronal NOS) NO promotes osteoblast activity and cell proliferation [93] , while higher concentrations (as with induction of iNOS activity with inflammatory cytokines or administration of high-dose exogenous NO) lead to bone loss [5] .

3.4 Adverse effect and drug interactions Orally administered nitrates give the highest incidence of headaches ( ∼ 50%), while around 20% of patients receiving transdermal nitrates may complain of headaches; it is the most common reported adverse effect with nitrate therapy [5] . Therefore, for a chronic condition such as osteoporosis, percutaneous treatment with nitroglycerin is the best choice [10,13] . Headaches are throbbing or pulsating, may be persistent or intermittent, and range in severity from mild to severe. Tolerance could develop within days to weeks.

Although nitroglycerin at the doses used in cardiovascular patients may cause hypotension, proposed smaller doses do not lower blood pressure significantly [94] . Reflex tachycardia may occur in those who are volume depleted; hence, nitrate therapy is initiated with caution in patients with heart rates greater than 100 beats/min. This is especially important in patients with underlying ischemic heart disease or tachycardia, as these may precipitate an acute coronary event. Combination use of nitro-vasodilators with anti-inflammatory drugs may increase gastrointestinal bleeding [1] .

No significant known metabolic drug interactions occur with transdermal nitrate therapy. However, several pharmacodynamic interactions exist with other drugs, causing vasodilatation or reduced blood pressure. Sildenafil is one such potent vasodilatory agent that is used for erectile dysfunction and can interact with nitrates. Hence, the concomitant use of sildenafil and nitrate products is contraindicated, as the additive vasodilatory effects may precipitously drop blood pressure or may cause ischemia in patients with heart disease. Alcohol is another agent that decreases blood pressure, and therefore concomitant use with nitrates is discouraged. Another class of drugs that may interfere with nitrates is PPAR γ compounds.

4. Human studies

4.1 Phase II studies using nitric oxide donor compounds The rationale for using NO compounds for osteoporosis has been developed after many in vitro and in vivo studies

conducted over the past 20 years [5,23,24,29,31] . Circulating nitrate levels increase in the presence of HRT [95] , androgen replacement therapy (Wimalawansa, unpublished data), as well as calcitonin therapy [96] . The overall results from these animal and human studies indicate that nitroglycerin has marked beneficial effects on bone metabolism. Some of these beneficial effects of established agents are likely to be mediated via the cGMP–NO pathway. A cross-sectional clinical study carried out in 1989 on 450 cardiac patients who were on various doses of nitrates (arbitrarily divided into < 35 versus > 45 mg/day) in comparison with an age- and sex-matched control group of 290 individuals demonstrated a dose-dependent effect of nitrates on the BMD ( Figure 10 ) [5,13] .

The first human controlled clinical study to evaluate the beneficial effects of the NO donor nitroglycerin in prevention of oophorectomy-induced bone loss was conducted in the mid 1990s [31] . Based on animal data, this 1-year controlled, randomized human study was conducted to assess the efficacy of topically administered nitroglycerin ointment in comparison with oral estrogen using BMD and biochemical markers as end points [31] . Data from this pilot study established that there was an equipotent effect of nitroglycerin and estrogen (HRT) in preventing oophorectomy-induced bone loss in women (i.e., effectively prevented accelerated bone loss in early menopause).

Figure 11A illustrates the efficacy as well as the equiv-potency of nitroglycerin and estrogen in maintaining the BMD in oophorectomized women [31] . Retrospective analysis revealed that study participants received instructions to apply a 30 mg dosage of ointment once per day, still lower than that taken by cardiac patients for relief of angina. The optimum dose, assuming 100% adherence to therapy, is between 35 and 40 mg of nitroglycerin per day. Figure 11B illustrates the levels of the biochemical markers urinary N -telopeptide (bone resorption) and serum osteocalcin (bone formation) in this study in women treated with nitroglycerin versus estrogen [31] . Since calcium plus vitamin D can suppress bone markers in some patients, this may have dampened the basal levels of biomarkers observed in this study. Although both drugs equally decreased the urinary N -telopeptide levels, only nitroglycerin increased serum osteocalcin and BS-ALP ( Figure 11B ). This suggests the possibility of an increase in bone formation in response to nitroglycerin therapy, and corroborates data from our in vitro studies and in vivo animal studies.

These findings are similar to those previously observed with animal studies [10,18,23] . Although changes in bone biomarkers were not extraordinarily high, a ∼ 30% decrease in urinary N -telopeptide with a 50% increase in osteocalcin and a 27% increase in bone-specific alkaline phosphatase suggest a positive effect on bone balance and a consequent increase in BMD in the long term [13] . The data indicate that once daily transdermal administration of nitroglycerin prevented the expected bone loss of acute estrogen

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Figure 11 . Equipotency of nitric oxide donor nitroglycerin with estrogen therapy in maintaining bone mineral density (BMD) in early postmenopause. A. Mean BMD (g/cm 2 ) in lumbar spine (top) and in total hip (bottom) in estrogen (Premarin, 0.625 mg/day) versus nitroglycerin-treated (30 mg/day) oophorectomized women (n = 7 per group; mean ± SEM) at baseline (open columns), at 6 months (grey columns), and at 12 months (fi lled columns). No statistical differences were observed between the two treatment groups (i.e., the responses in the two groups are comparable). B. Changes in urinary N -telopeptide (NTx; nM BEC/mM creatinine) (top) and serum osteocalcin levels (ng/ml) (bottom) in oophorectomized women treated with estrogen (Premarin, 0.625 mg/day) versus nitroglycerin 30 mg/day (n = 7 per group; mean ± SEM) at baseline, six and at 12 months. Reproduced from Wimalawansa SJ. Nitroglycerin therapy is as effi cacious as standard estrogen replacement therapy (Premarin) in prevention of oophorectomy-induced bone loss: a human pilot clinical study. J Bone Miner Res 2000;15(11):2240-4 [31], with permission of the American Society for Bone and Mineral Research.

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Figure 10 . Data from a cross-sectional study; the author demonstrated a dose-related effect of nitric oxide therapy on bone mineral density (BMD) in clinical practice. Those cardiac patients who received average daily doses of nitrate equivalent to < 35 mg had signifi cantly higher BMD levels ( ‡ p < 0.001) in comparison to those who received higher doses of nitrates (i.e., > 45 mg/day). Those patients who received low-dose nitrate for more than 3 years had signifi cantly higher BMD levels (*p < 0.01) in comparison to an age- and sex-matched population from the same center who also had BMD measurements. Reproduced from Wimalawansa SJ. Rationale for using nitric oxide donor therapy for prevention of bone loss and treatment of osteoporosis in humans. Ann NY Acad Sci 2007;1117:283-97 [5] , with permission of Blackwell Publishing.

Wimalawansa


withdrawal, and had a positive impact on biomarkers in these oophorectomized women.

A cross-sectional study also supported the role of NO in enhancing BMD [98] . Adequate supplementation of animals or humans with the NO precursor L -arginine could also be effective in prevention of bone losses [99] but the amount of L -arginine necessary to have these biological effects is high, making this approach impractical [10] . The role of combination therapies [47,85,97,100] needs to be investigated, especially the combination of NO donors with bisphosphonates [5] .

4.2 Phase III study with nitric oxide donor nitroglycerin Nitroglycerin is a very practical drug and can readily be used in in vivo . It is effective in the prevention and reversal of estrogen-depleted osteoporosis in rodent models and in humans [18,31] . Based on the pilot study data that are reported in the above section, a large single-center randomized double-blind placebo-controlled clinical study funded by the National Institute of Health – Institute of Arthritis, Musculoskeletal and Skin Diseases was conducted to assess the effectiveness of topically administered nitroglycerin. This clinical trial, known as the NOVEL (Nitroglycerin as an Option: Value in Early Bone Loss) study [101,102] , was designed to answer the following questions. Can nitroglycerin stop bone loss in menopausal women? If so, can this be an alternative therapy for estrogen, HRT or SERMs? The original study was designed to compare the effects of nitroglycerin with HRT and the SERM raloxifene. Due to the WHI study data [19] , the NOVEL study protocol was modified to compare nitroglycerin with a group treated with inactive ointment, calcium, and vitamin D [103] .

The primary end point, lumbar spine BMD, of the nitroglycerin-treated group was not statistically different from the control group that received calcium and vitamin D therapies. This was no surprise considering the somewhat

lower than expected compliance and the subtherapeutic dose used (22.5 mg of nitroglycerin a day); most patients in this clinical study applied < 15 mg of nitroglycerin daily ( Figure 12 ). Since this dose is below the therapeutic window necessary for bone homeostasis, intent-to-treat analysis of data from this clinical study were inconclusive on the protective effects on lumbar spine BMD [104] . Nevertheless, thosewho have adhered to therapy are likely to demonstrate a beneficial effect that may reflect in biochemical markers or secondary end points. If the results of studies such as the NOVEL are positive, then nitroglycerin therapy could become a highly cost-effective and attractive treatment option for prevention and treatment of postmenopausal osteoporosis.

A recently published large case–control study conducted in Denmark compared 124,655 patients with fractures with 373,962 sex-matched controls. They reported a 15% reduction in hip fractures in those using organic nitrates ( Figure 13 ) [105] . Furthermore, this study also showed that while medium doses of nitrates are effective, the very low or very high doses are either not effective or detrimental to the skeleton.

Another short-term study data indicated that treatment with isosorbide mononitrate in healthy women decreased N-telopeptide by 36.3% (95% CI = 14.8 – 57.8), and serum bone specific alkaline phosphatase was increased by 15.9% (95% CI = 1.1 – 30.7) [106]. Retrospective analysis of Canadian Multi Center Osteoporosis Study (CaMOS) reported that those who were using nitrates had significantly higher BMD and a tendency to have lower numbers of fractures (Figure 14) [107]. However, the frequency of usage or the doses used by these subjects were not available in this study to make further conclusions. Another randomized controlled clinical study reported an equivalent effect on increasing BMD following isosorbide mononitrate 20 mg daily, or with alendronate, 70 mg once a week over a 12-month period [108]. There were a 10.8 and a 12.1% change in BMD after 12 months in isosorbide mononitrate and alendronate, respectively, and

Figure 12 . Mean percent change from baseline to the given follow up visit in bone mineral density of (A) the lumbar spine, and (B) total body calcium by intention to treat analysis. Active treatment is shown by the solid line, fi lled markers; placebo is shown by the dashed line, open markers. Error bars represent standard error of the mean.

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authors concluded that efficacy of isosorbide mononitrate is comparable to alendronate in improving BMD.

5. Conclusion

NO donor compounds, such as nitroglycerin (glyceryl trinitrate) and nitrates, are safe and cost-effective medications that have been used in clinical practice for many years [94] .

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Figure 13 . Diagrammatic representation of fracture reduction reported in a large nationwide epidemiology study in Denmark. A. Demonstrates the overall effects of three broad dose categories of nitrates used, which showed that the moderate dose of nitrates had the highest effi cacy in fracture reduction, while at higher doses there is no effect on fracture prevention. B. Percentage fracture reduction: any fractures 0.89 (CI = 0.86 – 0.92); hip fractures 0.85 (CI = 0.79 – 0.92); lumbar spine 0.99 (CI = 0.85 – 1.16); and forearm 0.91 (CI = 0.83 – 1.00). Adapted using data from [105] .

These compounds are tolerated well and headaches are the only major adverse effect. NO plays a role in various physiological and pathological conditions. Although new potential indications are continuing to emerge, the most promising and exciting indication is the prevention and treatment of osteoporosis. Recent research and development has also included the use of NO donor compounds and design of agents, as well as combination molecules that are capable of delivering NO directly into the bloodstream and targeting tissues more efficaciously in a sustained, controlled, or pulsatile manner [10] .

The actions of NO on the skeletal system are dose dependent and biphasic. At low concentrations (e.g. as a result of eNOS activity), NO stimulates osteoblast and osteocyte activity and keeps the osteoclast-mediated bone resorption under control. Higher NO concentrations (following iNOS activation or exogenous administration of higher doses of NO donors) may lead to bone loss [13] . Imbalances of skeletal bioavailable NO can lead to enhanced bone turnover, bone loss, and consequent fractures ( Figure 15 ). NO can also facilitate fracture healing, and is involved as a second messenger in mechanical and stress-induced bone formation. Furthermore, NO mediates the osteogenic effects of sex-steroid hormones [5] . With this wide range of functions, there are multiple opportunities for therapeutic interventions using NO donor compounds and the NO–cGMP pathway for osteoporosis.

The progress of therapeutic drug development is somewhat hindered due to the lack of safe and specific NOS inhibitors and the lack of funds to conduct research in this area. The latter is at least in part due to the false belief of pharmaceutical companies which are developing new NO donor therapies may not generate adequate revenue. For example, if a cost-effective and a safe NO donor agent is to be developed for the prevention and treatment of osteoporosis, fracture reduction, or fracture healing, the potential market would be billions of dollars [5,13] . Hence, even though the drug costs less, sheer market size will enable pharmaceutical companies to make healthy profits [10] .

6. Expert opinion

NO donor compounds have a wide market. Given that NO donor agents are economical to use, various forms of these agents are widely available throughout the world. The arena has recently widened following the introduction of various new NO-linked compounds to target NO molecules to specific body tissues. The market for these new agents depends on the adverse effects profiles, the efficacy of these compounds in relation to its parent compound nitroglycerin, and the affordability [13] .

In the area of prevention and treatment of osteoporosis, nitroglycerin can potentially make the largest impact in the worldwide market due to its lower cost, sheer market size

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Figure 14. Annualized percent change in (A) total hip BMD, (B) femoral neck BMD and (C) lumbar spine BMD by nitrate use. These variables have been adjusted for age, weight, baseline BMD, clinical centre and baseline differences. Modifi ed from [107].

(over 400 million potential patients), lack of serious adverse effects, benefits in other systems, and the relative ease of worldwide availability. Furthermore, even though the effects of NO on the skeletal tissue are gentle and moderate in comparison to bisphosphonates or parathyroid hormone injections, NO has favorable effects on both osteoblasts (cell growth and activity) and osteoclasts (suppression of bone resorption). These are somewhat similar to the effects of sclerostin inhibitors and strontium ranelate on bone cells. Therefore, it is logical to assume that the overall effects must be beneficial with long-term nitroglycerin therapy to improve bone quality and fracture reduction. Hence, one could speculate that in the longer term, due to preservation of the normal bone architecture, and the better quality of the new bone formed, nitroglycerin therapy is likely to be superior to other antiosteoclastic classes of therapeutic agents.

6.1 Therapeutic implications NO has an estrogen-like effect on bone but without the estrogenic adverse effects, and therefore NO donors could be an attractive alternative therapy for osteoporosis [43,49,72,84,105-109] .

Wimalawansa also demonstrated that a NO donor in humans is as effective as estrogen in inhibiting postmenopausal bone loss [29,31] , suggesting important therapeutic implications in the treat ment and prevention of osteoporosis. There is also the option of administration of a NO donor in combination with an osteoclast inhibitor, for example calcitonin or bisphosphonates, in the treatment of osteoporosis [47] . Nevertheless, nitroglycerin has a relatively narrow therapeutic window for treatment of skeletal diseases such as osteoporosis [13] . At low to medium doses, NO promotes skeletal health, while at high doses (e.g., the doses of nitrates used in angina pectoris and other cardiovascular diseases) it likely promotes bone loss ( Figure 16 ).

Regulatory guidelines indicate the necessity of carrying out very large, multi-center, controlled clinical studies to demonstrate that a NO donor compound is not inferior to a FDA approved antiosteoporosis therapy, with fracture as the primary end point, in contrast to BMD as a surrogate marker. Conducting an expensive fracture reduction clinical study using a NO donor compound such as nitroglycerin, however, is unrealistic due to the extremely

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Figure 15 . Global schematic representation of the effects of too little and too high levels of bioavailable nitric oxide (NO) within the bone cellular environment in the skeleton. eNOS: Endothelial nitric oxide synthase; iNOS: Inducible nitric oxide synthase; nNOS: Neuronal nitric oxide synthase.

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loss

Figure 16 . Predicted representation of the bone mineral density response to varying doses of nitrates and nitroglycerin use in the current clinical practice (based on animal studies). This demonstrates the narrow therapeutic window for nitric oxide donors in enhancing human skeletal health (i.e., to improve bone mineral density and decrease fractures). This article was published in Wimalawansa SJ. Skeletal effects of nitric oxide: novel agent for osteoporosis. In: Billizikiean JMT, editor, Principles of Bone Biology. 3rd ed. NY: Academic Press; 2008;59:1275-311 [13], Copyright Elsevier (2008).

high cost of such a clinical study, especially when one considers the low cost of the product itself. Regulatory agencies such as the FDA must bear these facts in mind when making decisions. In fact, governments and regulatory agencies should encourage the pharmaceutical industries, universities, and individual researchers to develop economical and efficacious drugs that are devoid of serious adverse effects, especially for managing chronic diseases such as osteoporosis. Developing and marketing such cost-effective therapies would benefit not only governments, but also millions of people worldwide. One such example of this is the use of the NO donor nitroglycerin as a novel therapy in the prevention and treatment of osteoporosis in humans.

Thus, large epidemiological studies, especially multi-million database analysis together with standard non inferiority Phase III studies with BMD, biochemical markers, and bone histomorphometry (finite element analysis, or microMRI for bone quality, and safety) as end points should be considered adequate from a regulatory point of view, taking into consideration the significant benefit of the consumer and the society, massive cost savings to federal governments and to third party payers.

Declaration of interest

The author has no conflict of interest, financial relationships, or received any payment in preparation of this manuscript.

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Affi liation Sunil J Wimalawansa MD PhD MBA FCCP FACP FRCP DSc MRCPath University Professor Professor of Medicine, Physiology, and Pharmacology, Chief, Division of Endocrinology, Metabolism and Nutrition, Director, Regional Osteoporosis Center, Robert Wood Johnson Medical School, Department of Medicine, New Brunswick, NJ 08903, USA Tel: +1 732 235 9584 ; Fax: +1 732 235 8892 ; E-mail: [email protected]

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1 2 Nitric oxide: novel therapy 3 for osteoporosis · therapies for chronic conditions such as osteoporosis. Keywords: glyceryl trinitrate , hormone eplacement r therapy menopause