Ann. N.Y. Acad. Sci. ISSN 0077-8923 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES Issue: Skeletal Biology and Medicine Nitric oxide and bone Sunil J. Wimalawansa Professor of Medicine, Division of Endocrinology, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, New Jersey, USA Address for correspondence: Prof. Sunil J. Wimalawansa, M.D., Ph.D., M.B.A., D.Sc., University Professor, Division of Endocrinology, Metabolism & Nutrition, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ 08903-0019. Voice: 732-235-9584; fax: 732-235-8892. [email protected]Age-associated decrease in nitric oxide (NO) production may be related to an increase in cardiovascular events, sexual dysfunction, and osteoporosis. Relative NO deficiency is a plausible biological basis for NO replacement therapy. Hormone replacement therapy (HRT) enhances local NO production and rectifies NO deficiency in postmenopausal women. However, excess local production of NO aggravates bone destruction in inflammatory arthropathies. In addition to its use in alleviating angina and erectile dysfunction, NO compounds could be a valuable supplemental therapy for chronic conditions including osteoporosis. Estrogen mediates its beneficial effects in bone, in part via the NO/cGMP pathway; hence NO donor therapy is an alternative to estrogen, estrogen agonists-antagonists, and androgen receptor modulator therapy in the prevention and treatment of osteoporosis. Large numbers of animal studies and human pilot studies support the concept of using NO donors for preventing bone loss. Administration of exogenous NO or prolonging endogenous NO activity are practical ways to supplement NO. Key words: glyceryl trinitrate; cGMP; hormone replacement therapy; menopause; nitric oxide donors; nitric oxide synthase inhibitors; nitroglycerin; RANK; osteopenia; osteoporosis; bone mineral density; postmenopausal women Introduction Menopause-associated decreased estrogen levels in- crease osteoclast activity and bone turnover, re- sulting in bone loss. 1,2 Nitric oxide (NO) has an estrogen-like beneficial effect in bone, but without estrogenic adverse effects. Therefore, NO donors could be an attractive alternative to estrogen ther- apy for osteoporosis. 3–7 Beneficial therapeutic ef- fects of estrogen on bone mineral density (BMD) are in part mediated through the NO/cyclic guanosine monophosphate pathway (cGMP) 8 and insulin-like growth factor (IGF-1). 9–11 Nitric oxide has been shown to have a variety of effects on bone; 8,12–23 at medium doses, it sup- pressed osteoclastic bone resorption and promoted growth of osteoblasts. 24,25 In aqueous buffers and culture conditions, nitrite is the principal oxidation product of NO, 26 whereas in vivo NO is almost com- pletely oxidized to nitrate. These end products have proven to be useful markers for NO biosynthesis in biological samples. 25 Nitric oxide is enzymatically produced by oxi- dation and cleavage of the amino-terminal nitro- gen atom of amino acid l-arginine. The reaction is dependent on electrons donated by the cofac- tor NADPH, which requires oxygen, and yields l- citrulline as a co-product. 27 A family of three related enzymes, the NO syn- thases (NOS), regulates the synthesis of NO. These are characterized as a neuronal form (type 1; nNOS) originally isolated from brain; an endothelial form (type 3; eNOS) originally isolated from bovine aortic endothelial cells; 28 and an inducible form (type 2; iNOS) originally isolated from murine macrophages. 29 eNOS and nNOS are expressed con- stitutively and are characterized by highly regulated, rapid and controlled manner, but low-output NO production that imposes a tonic physiological func- tion. 30,31 Table 1 illustrates the differences and com- monalities of three NOS isoenzymes. Role of nitric oxide in bone metabolism During the past two decades, significant advances have been made in understanding cellular mech- anisms involved in bone metabolism leading to identification of novel therapeutic agents. However, doi: 10.1111/j.1749-6632.2009.05230.x 394 Ann. N.Y. Acad. Sci. 1192 (2010) 394–406 c 2010 New York Academy of Sciences.
Transcript
Ann. N.Y. Acad. Sci. ISSN 0077-8923
ANNALS OF THE NEW YORK ACADEMY OF SCIENCESIssue: Skeletal Biology and Medicine
Nitric oxide and bone
Sunil J. WimalawansaProfessor of Medicine, Division of Endocrinology, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School,New Brunswick, New Jersey, USA
Address for correspondence: Prof. Sunil J. Wimalawansa, M.D., Ph.D., M.B.A., D.Sc., University Professor, Division ofEndocrinology, Metabolism & Nutrition, Department of Medicine, UMDNJ-Robert Wood Johnson Medical School, NewBrunswick, NJ 08903-0019. Voice: 732-235-9584; fax: 732-235-8892. [email protected]
Age-associated decrease in nitric oxide (NO) production may be related to an increase in cardiovascular events, sexualdysfunction, and osteoporosis. Relative NO deficiency is a plausible biological basis for NO replacement therapy.Hormone replacement therapy (HRT) enhances local NO production and rectifies NO deficiency in postmenopausalwomen. However, excess local production of NO aggravates bone destruction in inflammatory arthropathies. Inaddition to its use in alleviating angina and erectile dysfunction, NO compounds could be a valuable supplementaltherapy for chronic conditions including osteoporosis. Estrogen mediates its beneficial effects in bone, in part viathe NO/cGMP pathway; hence NO donor therapy is an alternative to estrogen, estrogen agonists-antagonists, andandrogen receptor modulator therapy in the prevention and treatment of osteoporosis. Large numbers of animalstudies and human pilot studies support the concept of using NO donors for preventing bone loss. Administrationof exogenous NO or prolonging endogenous NO activity are practical ways to supplement NO.
synthase inhibitors; nitroglycerin; RANK; osteopenia; osteoporosis; bone mineral density; postmenopausal women
Introduction
Menopause-associated decreased estrogen levels in-crease osteoclast activity and bone turnover, re-sulting in bone loss.1,2 Nitric oxide (NO) has anestrogen-like beneficial effect in bone, but withoutestrogenic adverse effects. Therefore, NO donorscould be an attractive alternative to estrogen ther-apy for osteoporosis.3–7 Beneficial therapeutic ef-fects of estrogen on bone mineral density (BMD) arein part mediated through the NO/cyclic guanosinemonophosphate pathway (cGMP)8 and insulin-likegrowth factor (IGF-1).9–11
Nitric oxide has been shown to have a varietyof effects on bone;8,12–23 at medium doses, it sup-pressed osteoclastic bone resorption and promotedgrowth of osteoblasts.24,25 In aqueous buffers andculture conditions, nitrite is the principal oxidationproduct of NO,26 whereas in vivo NO is almost com-pletely oxidized to nitrate. These end products haveproven to be useful markers for NO biosynthesis inbiological samples.25
Nitric oxide is enzymatically produced by oxi-dation and cleavage of the amino-terminal nitro-
gen atom of amino acid l-arginine. The reactionis dependent on electrons donated by the cofac-tor NADPH, which requires oxygen, and yields l-citrulline as a co-product.27
A family of three related enzymes, the NO syn-thases (NOS), regulates the synthesis of NO. Theseare characterized as a neuronal form (type 1; nNOS)originally isolated from brain; an endothelial form(type 3; eNOS) originally isolated from bovineaortic endothelial cells;28 and an inducible form(type 2; iNOS) originally isolated from murinemacrophages.29 eNOS and nNOS are expressed con-stitutively and are characterized by highly regulated,rapid and controlled manner, but low-output NOproduction that imposes a tonic physiological func-tion.30,31 Table 1 illustrates the differences and com-monalities of three NOS isoenzymes.
Role of nitric oxide in bone metabolismDuring the past two decades, significant advanceshave been made in understanding cellular mech-anisms involved in bone metabolism leading toidentification of novel therapeutic agents. However,
for proper understanding, it is important to con-sider multiple interactions of these chemical enti-ties in vivo as well as during the development ofnew therapeutic agents. Basic interactions betweencytokines, hormones, and NO in bone metabolismare illustrated in Figure 1.
Nitric oxide has been shown to regulate os-teoclasts, which are responsible for bone resorp-tion.32,33 Bone formation and resorption are keyprocesses that are necessary for the constant remod-eling that occurs in bone tissue to keep it healthy,and to repair bone micro-damage. The NO donors,have beneficial effects on controlling bone resorp-tion (decreased osteoclastic activity), and have amilder anabolic action on bone formation (i.e., en-hanced osteoblastic activity).25,27 Hence, for post-menopausal women who cannot tolerate or affordhormone replacement therapy (HRT) treatment, ni-troglycerin therapy may become a cost-effective op-tion in the future.25
Furthermore, studies have demonstrated thatsupplementary nitroglycerin has beneficial additiveeffects on BMD when co-administered with vita-min D, calcitonin, and bisphosphonates.27 Takentogether, these data suggest that long-term therapywith NO will not only increase BMD, albeit less effi-ciently than bisphosphonates, but may also decreasefracture rates.8
Nitric oxide donor compounds, such as ni-troglycerin, glyceryl trinitrate, and nitrates, aresafe and cost-effective medications used in clinicalpractice for several decades.34 These compounds arewell tolerated; headaches are the main adverse ef-fect.35,36 These compounds are effective in the pre-vention or reversal of estrogen-depleted osteoporo-sis in rodent models and in human subjects.7,8,22,35
In the past, many postmenopausal women relied onHRT after menopause to reduce the risk of heart
disease and osteoporosis. However, the Women’sHealth Initiative (WHI) study37 demonstrated that,while HRT is effective in preventing fractures, itcould slightly increase the risk of stroke, heart dis-ease, and breast cancer. Thus, finding a cost-effectivealternative therapy for osteoporosis with little or noadverse effects would be very useful. One such groupof agents is NO donors.
In vitro cellular biological studiesNitric oxide appears to play an autocrine andparacrine regulatory role in bone metabolism. Inaddition, NO modulates the activity of both os-teoblasts and osteoclasts in vitro. A number of stud-ies suggest that, NO donors increase osteoblast cellproliferation, osteocalcin synthesis, and in vitro for-mation of a mineralized matrix.25
On the other hand, NOS inhibitors have an an-tiproliferative effect on osteoblastic cells in vitroand lead to enhanced bone resorption in vivo.21,38
The NO-cGMP pathway also seems to be in-volved in the mechanism of bone resorption by cy-closporin.39 Moreover, release of large amounts ofNO from iNOS in cytokine-stimulated cells also hasan antiproliferative effect on osteoblasts,6 increasesosteoblast apoptosis, and enhances osteoclast-mediated bone resorption.40,41 Therefore, NO ap-pears to have a biphasic effect on bone formations:19
in low concentrations, it promotes bone forma-tion, whereas it inhibits effect at higher concentra-tions. Figure 2 indicates the dose-dependant effect ofthree NO donor compounds on osteoblast-like cellproliferation.
In primary human and rat osteoblast cul-tures, 17�-estradiol dose-dependently stimulatesosteoblast cell proliferation and differentiation asassessed by alkaline phosphatase activity and bonenodule formation.42 This effect is abolished after
Figure 1. Role of NO in bone: Interactions of cytokines, estrogen, growth factors, mechanical stresses, and stimulatingeNOS and iNOS in bone homeostasis and bone metabolism.25
NOS = nitric oxide synthase enzyme.
inhibition of NOS activity.43 Moreover, osteoblastscultured from eNOS gene knockout mice donot respond to 17�-estradiol. Additionally, 17�-estradiol enhances eNOS enzyme expression andNO metabolite levels in osteoblasts.23 The latter arealso abolished in the presence of NOS inhibitors.Collectively, these observations suggest that thestimulatory effect of estrogen on osteoblast prolifer-ation and differentiation relies on local productionof NO via stimulating the eNOS isoform.20,25
NO compounds have been shown to de-crease bone resorption in vitro and similar ef-fects have been demonstrated with l-arginine.44
Studies have demonstrated production of NO byosteoclasts in response to a rise in intracellu-lar Ca2+, leading to a retraction of pseudopo-dia and subsequent inhibition of bone resorp-tion.45 Estrogen is known to regulate eNOS inosteoblasts,4 and osteoblasts produce NO,15 whereascytokine-induced inflammation16 enhances iNOSactivity.41
Nitric oxide is also involved in isoprenylation(geranylgeranylation) of the Rho GTPase, Rho-PK,which in turn inactivates a factor that would oth-erwise turn on the BMP-2/Cbfa1-Runx-2 cycle.25,46
Largely the site and rate of NO synthesis, the quan-tity generated, and the nature of the environmentinto which it is released determine these broad-
ranging actions of NO and the tissue specificity.Meanwhile, the activity of NO is quenched by thepresence of free hemoglobin or reactive oxygen in-termediates, and the activity of antioxidant defensesystems.30,31,47
Nitric oxide is a mediator in mechanicalstimulationNitric oxide is a key mediator in osteoblastic stimu-lation following exercise, shear-strain, and mechan-ical stimulation.48–50 Bone marrow cells51 as wellas mechanical strain of bone cells produce NO.52–55
Pulsating fluid flow simulating canaliculi and shear-stress releases NO, which leads to osteoblastic stim-ulation.56,57 NO enhances and/or modulates the ac-tions of the locally released cytokines.
In addition to releasing NO, fluid shear-stressesalso release prostaglandins,58 perhaps via pre-osteoclast cells.59 The mechanisms involved in thetransduction of the effects of mechanical forces havenot been fully elucidated, but data suggest the in-fluence of both prostaglandins and NO. Indeed, theinhibition of NO synthesis in vivo impairs the boneformation induced by mechanical loading.55 Fur-thermore, the exogenous NO donors can potentiatethe osteogenic effect of loading. Upregulationof eNOS activity is critical in mechanical load-induced bone growth,52,55,56,60 as well as in fracture
Figure 2. Dose-dependant effects of NO donors on osteoblastic-like cells proliferation. These data demonstrate abiphasic effects of NO on cell proliferation in response to NO.25 ∗P < 0.01.24
healing.61–64 All data suggest that NO is involved as asecond messenger in mechanical and stress-inducedbone formation, and mediates the osteogenic effectsof sex-steroid hormones.8,25
NO is involved in fracture healingThere is ample evidence to show that NO is involvedin fracture healing. After a fracture, a marked iNOSexpression is observed within 24 hours, which isconsistent with the initial inflammatory phase.61
iNOS was localized principally to endosteal os-teoblasts, and the expression is transient. This signalseems to attract bone cells to the site of injury to ac-tivate the repair process. This is followed by increasein eNOS expression in osteocytes and in endothe-lial cells within the local environment.61 This makessense, as the increase of blood supply is required forthe repair process.
The presence of large numbers of vascular en-dothelial cells,65 and the overexpression of eNOSthat generates NO locally, play a role in frac-ture healing.62,63 This is supported by the demon-stration of time-dependent differential expressionof NOS isoenzymes at the fracture sites. In ad-dition, NOs also activate other growth factorsas well as cyclooxygenase enzymes.66 The above-mentioned scenarios are consistent with eNOS’sbeing involved in the vascular response and neo-vascularization that are crucial to fracture repair.Moreover, the NOS inhibitors significantly impairfracture healing; this is reversed by local delivery ofNO donors.
Estrogen and NO connectionThe effects of estrogen in bone cells are mediated viaestrogen receptor alpha,67 and it upregulates eNOSgene in bone. A beneficial effect of estrogen on boneis abolished in the presence of NO synthase enzymeinhibitors, such as l-NAME.8,22 This suggests thatat least in part, the effects of estrogen in bone aremediated via a NO pathway.8,42 Subsequent studiesdemonstrated upregulation of eNOS expression andactivity in human osteoblast cells after stimulationby 17�-estradiol.4
A pilot human study demonstrated thatnitroglycerin ointment applied once daily inoophorectomized women prevented the estrogendepletion-induced bone loss.7,24,25 The effect ofnitroglycerin was equivalent to that of estrogen(Premarin), which prevented estrogen-deficiency-induced bone loss. This indirect evidence furthersupports HRT working via NO in the skeleton.7,8
These data suggest that NO therapy could serveas a safer alternative therapy in preventing post-menopausal bone loss.25
Nitroglycerin has beneficial additive effects onBMD when co-administered with bisphosphonates.This is not surprising because the mechanisms ofactions of these two classes of agents are different.NO donors are potential therapies to control boneloss and fragility,8,42 and long-term therapy withNO not only could increase BMD, but also maydecrease fracture rates. However, the multiple in-teractions that affect bone cell activity and effectsof various compounds in vivo, as well as the right
Figure 3. Changes of lumbar spine BMD (predominantly tubercular bone) and femur weight (predominantly corticalbone) over the 6-week treatment period in 12-week-old female ovariectomized (OVX) Wistar rats in various treatmentgroups: E2 = 17�-estradiol-treated; LN = l-NAME-treated, NG = nitroglycerin-treated. In vivo study demonstratedthat the actions of estrogen in bone are mediated via the NO/cGMP pathway. Treatments were started immediatelyafter ovariectomy. There was no additive effects when NO was co-administered with estrogen. Values are mean ± ofSEM for five animals per group. Comparisons were made against ovariectomized rats – OVX-control.8
∗P < 0.005; ∗∗P < 0.02.
dosage, must be taken into account when designingclinical studies and development of new therapeuticagents.
NO and bone and joint inflammationEnhanced expression of NO via iNOS in inflamma-tory conditions (e.g., lipopolysasccharide-inducedbone resorption) is alleviated with NOS inhibitors.This inflammation-associated bone loss has beenpredominantly due to augmenting the cytokine-induced matrix metalloproteinase 1 (MMP-1) prod-uct of osteoblasts, subsequently activating osteo-clasts.68 Nevertheless, the inhibition of osteoclastactivity may be the predominant effect of NO undernormal conditions. In vitro studies show that usinghigh doses of NO also leads to rapid osteoclast celldeath. NO donors also inhibit osteoclast formationin mouse bone-marrow cultures, an experimentalsystem frequently used to study factors regulatingosteoclastogenesis.42 The effect is likely to mediateby the NO-induced apoptosis of osteoclast progen-itors that occure at physiological doses.
NO prevents ovariectomy-induced bone lossIn vivo studies suggest that the predominant ef-fects of NOs are on osteoclast cells, but its an-
abolic effects on osteoblast cells are also impor-tant.25 Although the actions of NO on osteoblasts,osteoclasts, and osteocytes are weaker than thoseseen with bisphosphonates or parathyroid hormonetherapy, the persistent gentle actions of NOs arelikely to generate healthier and more physiologicalbone tissues in the longer term. The use of NOS in-hibitors such as aminoguanidine or l-NAME leadsto osteopenia in rats, while eNOS-deficient micehave reduced bone formation.5 Whereas iNOS ac-tivation enhances bone resorption41,69 and is re-ported to be one of the main mechanisms of actionin inflammation-induced osteoporosis.70 Moreover,the inhibition of NOS activity negatively affect thebone metabolism.71
We have demonstrated that the NO donornitroglycerin prevents bone loss in ovariec-tomy as well as corticosteroid-induced boneloss models, as assessed by BMD, bone weight,and bone histomorphometry in rats.7,8,12,25,72
Figure 3 illustrates the effects of nitroglycerin onprevention of ovariectomy-induced bone loss, withNO donor therapy, in comparison to the positivecontrol group (those receiving estrogen). Further-more, animal models have demonstrated that al-most all beneficial effects of estrogen on bone are
blocked in the presence of NO synthase inhibitorssuch as l-NAME,8 (Fig. 3).
Animal studies have demonstrated a similar ef-ficacy of NO donor therapy in male rats. Amongcastrated rats, preservation of BMD by testosteroneor estrogen is blocked by concomitant administra-tion of the NOS blocker, l-NAME. These data sug-gest that NO therapy is also likely to help males,especially those with hypogonadism, as well as mit-igating age-related bone loss in older males. Due tothe milder nature of adverse effects, NO therapy willhave a significant advantage over estrogen replace-ment therapy or selective estrogen receptor mod-ulators (SERMs) in postmenopausal women, andtestosterone and selective androgen receptor mod-ulator (SARMs) in men.
Rationale for using NO donor compoundsfor osteoporosisThe rationale for using NO for osteoporosis hasbeen developed after many in vitro and in vivo stud-ies conducted over the past 20 years.7,12,22,25,73 Post-menopausal women have low circulatory levels ofNO, and this is increased following HRT admin-istration,74 androgen replacement (Wimalawansa,unpublished material), and calcitonin therapy.75
Overall results from these animal and human stud-ies indicate that nitroglycerin has wide benefi-cial effects on bone metabolism. A cross-sectionalclinical study carried out in 1989 in 450 cardiacpatients who used various doses of nitrates (arbi-trarily divided into <35 mg vs. >45 mg per day)in comparison with an age- and sex-matched con-trol group (290 persons), demonstrated a signifi-cant dose-dependent effect of NO therapy on theBMD.25,42
Human clinical trials
Several previous clinical studies confirmed that ni-trate use has favorable effects on the skeleton inpostmenopausal osteoporosis.7,76–79 For example,a recent randomized controlled clinical study re-ported an equivalent effect on increasing lumbarBMD after isosorbide mononitrate 20 mg daily, orwith alendronate, 70 mg once a week over a 12-month period.78 There was a 10.8% and a 12.1%increase in BMD after 12 months on isosorbidemononitrate and alendronate, respectively. The au-thors concluded that effects of isosorbide mononi-
trate are comparable to those of alendronate in im-proving BMD.
The first randomized human study, based on an-imal data, was conducted in the mid-1990s to assessthe efficacy of topically administered nitroglycerinointment in comparison with standard oral estro-gen therapy (Premarin), using BMD and biochem-ical markers as end points.7 Data from this pilotstudy established an equipotent effect of nitroglyc-erin to estrogen (HRT), preventing oophorectomy-induced bone loss in women (i.e., effectively pre-venting the early menopause-associated acceleratedbone loss).
Figure 4 illustrates the efficacy as well asthe equipotency of NO donor, nitroglycerin(30 mg/day) to estrogen, in maintaining the BMDin oophorectomized women.7 While this dose islower than that taken by cardiac patients for re-lief of angina, dose is almost double the one that isused in the NOVEL clinical study (taking adherenceinto account). Hence, the optimum dose (assumingthat patients are adhering to therapy) is likely to bebetween 30 and 40 mg of nitroglycerin (or equiva-lence) per day. Figure 4B illustrates the biochemicalmarkers urinary N-telopeptide (NTx) (bone resorp-tion) and serum osteocalcin (bone formation) levelsin this study in women treated with NO donor, ni-troglycerin versus estrogen.7
Although both drugs equally decreased the uri-nary NTx levels, only nitroglycerin increased serumosteocalcin and bone-specific alkaline phosphatase(BS-ALP). This supports the notion that increase inbone formation in response to nitroglycerin ther-apy in humans is similar to that we previously ob-served with animal studies.8,12,27 Although changesin bone biomarkers were not extraordinarily high, a∼30% decrease in urinary NTx with a 20% increaseof osteocalcin and 25% increase of BS-ALP suggesta positive effect on bone balance and consequentpotential to increase BMD in the longer term.42
A cross-sectional study also supported the roleof NO in enhancing BMD.80 Adequate supplemen-tation in animals or humans with NO precursorl-arginine could also be effective in prevention ofbone losses,81 but the amount of l-arginine nec-essary to injest to achieve these biological effectsis high, making this approach impractical.27 Inaddition, the role of combination therapies82–84
needs to be investigated, especially with the com-bination of NO donors with bisphosphonates.25
Figure 4. Equipotency of NO donor nitroglycerin with estrogen therapy in maintaining BMD in early post-menopausal women. (A) Mean BMD (g/cm2) in lumbar spine, and (B) in the total hip in estrogen (Premarin,0.625 mg/day) versus nitroglycerin-treated (30 mg of nitroglycerin per day) group of oophorectomized women(n = 7 per group; mean ± SEM) at baseline (open columns), at 6 months (hatched columns), and at 12 months (solid
NO dose-response, bone mineral density,and fracture reductionA recently published case-controlled study compar-ing 124,655 subjects with fractures with 373,962sex-matched controls reported that those who used(vs. not used) nitrates had a lower risk of any frac-ture (OR = 0.89; 95% CI = 0.86–0.92), and forhip fractures (OR = 0.85; 95% CI = 0.79–0.92);thus a 15% reduction of hip fractures was seen inthose who used nitrates.77 This observational studyfurther clarified that the medium doses of nitratescould be more effective than the very low or veryhigh doses in fracture prevention.
Retrospective analysis of the Canadian Multi-Center Osteoporosis Study (CaMOS) reported thatnitrate users had significantly higher BMD and
lower fracture rates;79 but the frequency of the frac-tures or the doses of nitrates used were not docu-mented. Additional, a 3-month prospective studyhas indicated that treatment with 20 mg isosor-bide mononitrate in healthy women decreased N-telopeptide by 45.4% (95% CI = 25.8–64.9), andincreased serum bone-specific alkaline phosphataseby 23.3% (95% CI = 8.9–37.8).85 Both actions favorbone metabolism, and suggest a degree of disassoci-ation of bone resorption from formation. These dataare similiar to the previously published data.3,7,22
Across many studies, intermediate doses of NOhave been shown to promote skeletal health,24,25,77
while high doses such as those used in angina pec-toris may promote bone loss.24,25 Higher doses (aswith NO levels generated after iNOS activation)
lead to bone loss through osteoclast activation andosteoblast suppression. Lower-to-medium concen-trations of NO (as with NO-generated via eNOSactivity) stimulate osteoblast and osteocyte ac-tivity, while controlling osteoclast-mediated boneresorption.25,64
NOVEL clinical studyOn the basis of the pilot human study data thatare described in the foregoing section, a single-center, large randomized, double-blind, placebo-controlled clinical study funded by the NationalInstitutes of Health was conducted to assess the ef-fectiveness of topically administered nitroglycerin inprevention of early postmenopausal bone loss. Thisclinical trial, known as the NOVEL [Nitroglycerinas an Option: Value in Early Bone Loss] study, wasdesigned to answer the questions: Can nitroglyc-erin 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 com-pare the effects of nitroglycerin with HRT and SERM(raloxifene). Because of the WHI study data,37 theNOVEL protocol was modified to compare treat-ment with nitroglycerin to that with inactive oint-ment, calcium, and vitamin D.86,87
This 3-year clinical study in early postmenopausalsubjects evaluated the use of 22.5 mg of transdermalnitroglycerin (however, the average dose received bysubjects was ∼16 mg/day) and calcium plus vitaminD (n = 88, each group). The primary end-point, thelumbar spine BMD, was not significantly different inthe patients taking nitroglycerin compared to thosewho received calcium plus vitamin D only. Theseresults, together with those of the four other studiespublished to date, show that higher dosages shouldbe studied.
Although the primary end point was negative,several other measurement variables demonstrateda positive trend in the group treated with nitro-glycerin, in comparison to the control group. Theseinclude total body bone mineral content (DXA)[−31.1 g vs. −35.3 g (P = 0.80), percent changes−1.3 vs. −1.43% (P = 0.85)] and the rate of de-crease of BMD [the annual lumbar spine BMD de-crease was 0.70% in the active arm and 0.83% in theplacebo arm] (Fig. 5) observed over a 3-year period.
Taken together, the high adherence to calcium(630 mg) plus vitamin D (400 IU) in both groups(>85%) and the active lifestyle of the study par-
ticipants may have blunted the anticipated BMDdecrease in the placebo group (expected 1.5% vs.observed 0.8% per year of bone loss), further de-creasing the difference between the two treatmentgroups. Since several studies using nitrate productsdemonstrated positive outcomes on BMD (and/orpositive biochemical markers of bone turnover),further studies are warranted using higher dosesof NO donors (nitroglycerin or nitrates) in bothprevention and treatment of bone loss in post-menopausal women as well as in men.
There was a somewhat lower-than-expected com-pliance and the dose used was considered sub-therapeutic (22.5 mg of nitroglycerin a day); as aresult most subjects in this clinical study appliedan average of about 16 mg of nitroglycerin daily.Nevertheless, those who did adhere to the therapydemonstrated a beneficial effect that was reflectedin increased BMD in relation to the biochemicalmarker end points.89 Nitroglycerin therapy has thepotential to become a highly cost-effective treat-ment option for prevention and treatment of post-menopausal osteoporosis in the future.
Currently, it is not clear whether the sub-therapeutic dose used, the less-than-optimal adher-ence to therapy, or the fact that this was conductedduring the early menopausal period when a higherdose of NO is required are factors responsible for thelack of efficacy demonstrated in the NOVEL clinicalstudy.
Nitroglycerin seems to have a relatively narrowtherapeutic window for treatment of osteoporo-sis.3,24,25,34,64 in view of the potential lack of effectsat lower dosage and a possible harm from higherdosage, perhaps the optimal dose is expected to bebetween 30 and 40 mg a day.24,42 The right dose isnecessary to obtain positive BMD results.
Conclusions
We previously demonstrated that a NO donor inhumans is as effective as estrogen in inhibitingoophorectomy-induced bone loss,7 suggesting animportant therapeutic implication in the preven-tion and treatment of osteoporosis. It may bepossible that additional opportunities exist for thetreatment of osteoporosis, such as a NO donorin combination with an osteoclast inhibitor suchas calcitonin or bisphosphonates.24,25 Since thebone turnover is relatively high during the early
postmenopausal period, it is possible that the dose ofnitroglycerin required to prevent bone loss in earlypostmenopausal women could be higher. Moreover,different nitrate preparations have widely varyingPK/PD, which must be taken into account in doseadjustments. These findings suggest that NO dose-response needs to be studied further.
The actions of NO on the skeletal system are dose-dependent and biphasic. At low concentrations (re-sult of eNOS activity), NO stimulate osteoblast andosteocyte activity and keep the osteoclast-mediatedbone resorption under control. At higher concen-trations (after iNOS activation or exogenous ad-ministration of higher doses of NO donors), it maylead to bone loss.42 Imbalances of skeletal bioavail-able NO can enhance bone turnover, bone loss, andconsequent fractures. Nitric oxide can also facil-itate fracture healing, is a key second messengerin mechanical stress-induced bone formation, andmediates the osteogenic-like effects of sex-steroidhormones.25 In view of this wide range of func-tions, there are multiple opportunities for therapeu-tic interventions using the NO-cGMP pathway forosteoporosis.
Bioavailability is likely to vary between differentNO donors and nitrate preparations. The actions ofNO on the skeletal system are related to dose, route,and mode and frequency of application,3 whereasNO insufficiency leads to bone loss.24 Nitric oxidehas an estrogen-like effect on bone, but withoutestrogenic adverse effects. Therefore, NO donorscould become a highly cost-effective and attractivenew class of therapy for osteoporosis.3–6,87
However, its therapeutic window is narrow, andhence the right dose must be given to achieve bene-ficial effects.24,42,88 Since the NOVEL clinical studydata are nonconclusive, additional dose-rangingstudies are necessary. Additional opportunities existto treat osteoporosis such as administration of a NOdonor in combination with an osteoclast inhibitorsuch as calcitonin or bisphosphonates; this may fur-ther enhance bone quality and strength in patientswith osteoporosis.
Supporting Information
Additional Supporting Information may be foundin the online version of this article:
Figure S1. Mean percent change from baselineto the given follow-up visit in BMD of the spine
(A), total hips (B), femoral neck (C), and totalbody calcium (BMC) (D) by intention to treat anal-ysis. Nitroglycerin-treated group; solid line, closedcircles (active treatment group); calcium plus vi-tamin D group; dashed line, open circles (controlgroup). There is no statistical difference betweenthe two groups. Error bars represent standard errorof the mean. The number of subjects in the activeand placebo arms, respectively, are as follows: at6 months, 92 and 90; at 12 months, 90 and 90; at 24months, 88 and 86; at 36 months, 88 and 82.88
Figure S2. On the basis of animal studies, the pre-dicted representation of BMD responses to varyingdoses of nitrates and nitroglycerin in current clinicalpractice. This graph demonstrates the narrow ther-apeutic window for the use of NO donor treatmentsin improving human skeletal health (i.e., enhanceBMD and decrease fractures).24,64
Figure S3. Schematic global representation of theeffects on the skeleton of too little and too highlevels of bioavailable NO within the bone cellularenvironment. Either extreme can lead to increasedbone turnover and decreased bone mass and bonequality, which leads to fractures.
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Conflict of interest
The author declares no conflicts of interest.
References
1. Liberman, U.A., S.R. Weiss, J. Broll, et al. 1995. Effect
of oral alendronate on bone mineral density and the
incidence of fractures in postmenopausal osteoporosis:
the Alendronate Phase III Osteoporosis Treatment Study
Group. N. Engl. J. Med. 333: 1437–1443.
2. Horsman, A., M. Jones, R. Francis & C. Nordin. 1983.
The effect of estrogen dose on postmenopausal bone loss.
N. Engl. J. Med. 309: 1405–1407.
3. Wimalawansa, S., T. Chapa, L. Fang, et al. 2000.
Frequency-dependent effect of nitric oxide donor ni-
troglycerin on bone. J. Bone Miner. Res. 15: 1119–1125.
