This work was presented in part as an abstract at the 25th Annual Meeting of the American Society for Bone and Mineral Research, Minneapolis, Minnesota, USA, September 18-23, 2003.
The authors have no conflict of interest
NO regulates bone remodeling in cellular and animal models. We examined the effect of administering ISMO, a NO donor, on bone turnover in 144 postmenopausal women. After 3 months, women randomized to ISMO had a greater decrease in bone resorption and a greater increase in bone formation compared with placebo. NO donors may prevent postmenopausal bone loss.
Introduction: NO both stimulates bone formation and inhibits bone resorption in vitro. NO donors (nitrates) are inexpensive and widely available, but their value for postmenopausal osteoporosis has never been evaluated in a randomized trial.
Materials and Methods: We randomly assigned 144 healthy postmenopausal women with a hip BMD T score between 0 and −2.5 to 5 or 20 mg/day of isosorbide mononitrate (ISMO) or placebo for 12 weeks. We measured urine N-telopeptide (NTx), a marker of bone resorption, and serum bone-specific alkaline phosphatase (BSALP), a marker of bone formation. Markers were measured immediately before randomization and after 12 weeks of treatment. We calculated the percent change in NTx and BSALP for each of the treatment groups (placebo, 5 mg ISMO, and 20 mg ISMO). Our primary outcome was the percent change in NTx and BSALP in the 5- and 20-mg ISMO groups compared with placebo.
Results and Conclusions: Compared with women randomized to placebo, women randomized to 20 mg of ISMO had a 45.4% decrease in NTx (95% CI, 25.8-64.9) and a 23.3% increase (95% CI, 8.9-37.8) in BSALP. Women randomized to 5 mg of ISMO had a 36.3% decrease in NTx (95% CI, 14.8-57.8) and a 15.9% increase in BSALP (95% CI, 1.1-30.7). ISMO decreases bone resorption and increases bone formation. These findings suggest that nitrates may be useful for the prevention of postmenopausal osteoporosis.
EVIDENCE FROM SEVERAL sources suggests that NO can mediate bone loss. First, NO decreases bone resorption by decreasing osteoclast formation, motility, and function in vitro.(1–4) Second, administration of organic nitrates to oophorectomized rats prevents bone loss.(5) Third, organic nitrates alleviate bone loss induced by corticosteroid administration in rats.(6) Fourth, plasma NO is positively correlated with estradiol levels and increases with exogenous estrogen administration in humans.(7–9) Finally, laboratory studies indicate that local production of NO is an important mediator of the bone-forming effects of parathyroid hormone (PTH).(10)
Organic nitrates, such as isosorbide dinitrate, isosorbide mononitrate (ISMO), and nitroglycerin (NTG), are NO donors.(11) Treatment with these agents increases levels of NO, which in turn, produce vasodilation. Thus, nitrates are useful in the treatment of myocardial ischemia, although continuous administration of organic nitrates results in tolerance to its anti-ischemic effects.(12) Tolerance to nitrates may also occur in bone. In a previous observational study, we found that women who were taking organic nitrates once daily, or less often, had greater hip and heel BMD than nonusers or those taking nitrates more frequently.(13)
Only one published study has examined the effects of organic nitrates on postmenopausal bone loss. An open-label trial randomized 16 oophorectomized women, 36-45 years of age, to transdermal NTG (15 mg/day) or conjugated estrogen (0.625 mg orally).(14) After 6 months, women taking transdermal NTG had a 40% decrease in urinary N-telopeptide (NTx), a marker of bone resorption, and a 25% increase in bone-specific alkaline phosphatase (BSALP), a marker of bone formation, compared with baseline. There was no difference in hip and spine BMD at 12 months between women taking estrogen and those taking transdermal NTG, suggesting that estrogen and NTG had similar effects in preventing bone loss after oophorectomy. The limitations of this study included the open-label design, lack of a placebo control, the small number of participants, and the inability to generalize these findings to older postmenopausal women.
To test the hypothesis that administration of organic nitrates would decrease bone resorption and increase bone formation, we conducted a randomized double-blind trial comparing the effects of placebo and 5 and 20 mg of ISMO on markers of bone turnover in postmenopausal women. We administered ISMO once daily, instead of two times per day as it is usually prescribed, because our earlier findings indicated that less frequent administration is associated with a greater increase in BMD.(13)
MATERIALS AND METHODS
We enrolled ambulatory women, 50-80 years old, who were at least 3 years postmenopausal and had a BMD value at the femoral neck between 0 and 2.5 SD below the mean for young adults. We excluded women with a history of hip, wrist, or spine fracture, women with active metabolic bone disease (such as Paget's disease), women who reported use of calcitonin, conjugated estrogen, progestin, raloxifene, or tamoxifen within 6 months of study entry, and women who were treated with corticosteroids (prednisone equivalent of 5 mg/day) within 3 months of study entry. We also excluded women who reported use of a bisphosphonate within 3 years of study entry. Finally, we excluded women with a self-reported history of migraine headaches, myocardial infarction, angina, valvular, or congenital heart disease, a systolic blood pressure ≤100 mm Hg, a diastolic blood pressure ≥110 mm Hg, or an abnormal 12-lead electrocardiogram (ECG). This study was approved by the appropriate institutional review boards, and written informed consent was obtained from each study participant.
All participants completed a baseline interview that asked about potential risk factors for osteoporosis, dietary calcium intake, and medical history. Participants also had a 12-lead ECG. BMD of the hip and spine were measured with DXA using a Lunar DPX-L machine (Lunar Corp., Madison, WI, USA).
Potential participants were given 90 tablets of calcium carbonate (500 mg of elemental calcium) and 90 tablets of vitamin D (400 IU). We randomized all women who returned after 12 weeks using computer-generated random numbers to receive 5 or 20 mg of ISMO or identical placebo. To escalate the dose of ISMO among women randomized to receive 20 mg and to maintain blinding, all participants were given a blister pack containing 9 capsules and a bottle containing 90 capsules (placebo or 5 or 20 mg ISMO capsules). All participants were instructed to take three capsules for the first 3 days from the blister pack and then take one capsule per day from the bottle. For placebo assignments, capsules in the blister pack and bottle were placebo. Participants randomized to 5 mg of ISMO took one 5-mg ISMO capsule and two placebo capsules from the blister pack for the first 3 days and then 5-mg ISMO capsules from the bottle. Participants randomized to 20 mg of ISMO took one 5-mg ISMO capsule and two placebo capsules on day 1, two 5-mg ISMO capsules and one placebo capsule on day 2, and three 5-mg ISMO capsules on day 3 from the blister pack, and then took 20-mg ISMO capsules from the bottle. All participants were instructed to take the study medication at bedtime with a glass of water and to continue taking their calcium and vitamin D supplements.
We telephoned participants 1 week after randomization to ask about adherence and adverse events. We specifically asked about headache, nausea, and dizziness. We then phoned participants on a monthly basis to ask the same questions.
We advised participants who reported headaches to take acetaminophen, and we called participants on a daily basis until the headache resolved or they discontinued the study medication. If participants discontinued the study medication, we asked them to return to the study center to provide blood and urine samples. We defined adherence as having taken 85% or more of the study medication by pill count.
Collection and measurements of blood and urine
We obtained fasting serum samples and a second morning urine sample at randomization and at the end of 12 weeks or as soon as possible after discontinuation of study treatment. Specimens were stored at −70°C and analyzed in the same batch at the end of the study. All laboratory personnel were masked to the treatment assignment.
Urinary NTx, a marker of bone resorption, was measured using a monoclonal antibody technique (Osteomark).(15,16) The manufacturer's interassay CV is 7.6%, and the intra-assay CV is 4.0%. Serum BSALP, a marker of bone formation, was also measured using a monoclonal antibody technique (Metra Biosystems).(17,18) The manufacturer's interassay CV is 5-8%, and the intra-assay CV is 4-6%.
We used least-squares regression models to analyze the association between treatment assignment and percent mean change in NTx or BSALP. For example, we calculated the percent change in NTx for each participant as:
We averaged the percent change over all the study participants. Similar calculations were performed for BSALP.
We examined correlations between BMD at the femoral neck and lumbar spine at study entry and changes in NTx and BSALP, correlations between NTx and BSALP at the start of randomization and changes in each of these markers, and the correlation between change in NTx and the change in BSALP.
The primary intention-to-treat analysis included all participants in whom blood and urine samples were available, regardless of adherence to study medication. We also analyzed results among women who completed the trial and who were adherent to the study medication on treatment analysis. We present results adjusted for baseline difference in age; however, unadjusted results were very similar. Analyses were performed using STATA software (Version 7.0; STATA, College Station, TX, USA).
Table Table 1.. Characteristics of the 144 Study Participants Randomized to Treatment*
Of the 451 women screened for the study, 146 were eligible for participation and were assigned calcium and vitamin D. Of these women, 144 (98.6%) returned after 12 weeks and were randomized to one of three groups, placebo, 5 mg of ISMO, or 20 mg of ISMO (Fig. 1). The mean age of study participants was 60 years; women randomized to the treatment groups (5 and 20 mg of ISMO) were 3 years younger than the women randomized to the placebo group (p = 0.006), but there were no other significant differences between the groups (Table 1).
Changes in markers of bone turnover
Intention to treat analysis:
Compared with women randomized to placebo, women assigned to ISMO (5 and 20 mg) had a 40.1% decrease in NTx (95% CI, 24.1-56.1) and a 19.7% increase in BSALP (95% CI, 8.2-31.2). Compared with women randomized to placebo, those assigned to 20 mg of ISMO had a 45.4% (95% CI, 25.8-64.9) decrease in NTx, whereas women randomized to 5 mg of ISMO had a 36.3% (95% CI, 14.8-57.8) decrease in NTx. Compared with women randomized to placebo, those assigned to 20 mg of ISMO had a 23.3% (95% CI, 8.9-37.8) increase in BSALP, and women randomized to 5 mg of ISMO had a 15.9% (95% CI, 1.1-30.7) increase in BSALP (Figs. 2A and 2B).
Of all women randomized, 84 (58.3%) completed the trial and took at least 85% of the study medication. The on-treatment analysis showed that, compared with women who adhered to placebo, those adhering to 20 mg of ISMO had a 46.9% (95% CI, 20.1-73.7) reduction in NTx and a 29.4% (95% CI, 10.8-48.4) increase in BSALP, and women adhering to 5 mg of ISMO had a 38.2% (95% CI, 8.3-68.2) reduction in NTx and a 28.2% (95% CI, 4.6-52.1) increase in BSALP.
BMD at study entry was not correlated with the decrease in NTx (r = 0.16 for spine BMD and r = 0.08 for hip BMD) or the increase in BSALP (r = 0.17 for spine BMD and r = 0.05 for hip BMD). NTx at the start of randomization was modestly correlated with the change in NTx (r = 0.43), and BSALP at the start of randomization was modestly correlated with the change in BSALP (r = 0.48). The change in NTx was strongly correlated with the change in BSALP (r = 0.79).
Nine of the 47 women (19%) randomized to the 20-mg ISMO group and 7 of the 49 women (14%) in the 5-mg ISMO group discontinued treatment because of headache compared with 2 of the 48 women (4%) in the placebo group (p = 0.04; Table 2). All discontinuations caused by headache occurred within 7 days of randomization, and all headaches resolved within 48 h of discontinuing the study drug. There were no significant differences in the occurrence of any other adverse events.
Table Table 2.. Disposition of the 144 Study Participants Randomized to Treatment
This is the first randomized trial to show that daily oral administration of the organic nitrate ISMO to postmenopausal women significantly decreases bone resorption while increasing bone formation. Compared with placebo, ISMO at 20 mg/day decreased NTx, a marker of bone resorption, by 45%, an effect that is similar to the decreases in NTx, also compared with placebo, observed with alendronate (∼50%), risedronate (∼40%), and estrogen (∼50%), and greater than the 25% decreases that have been reported with raloxifene.(19,20) However, all the antiresorptive agents concomitantly decrease rates of bone formation. In contrast, we observed that treatment with ISMO resulted in significant increases (16-23%) in BSALP, a marker of bone formation. The decrease in bone resorption, coupled with an increase in bone formation, suggests that ISMO may improve bone strength and reduce fracture risk to a degree that is similar to or greater than what is seen with the current antiresorptive agents. Of note, the changes we observed in bone turnover markers are similar to those reported in a small open-label trial of nitroglycerin compared with estrogen in women with surgical menopause.(14)
The women randomized to treatment were, on average, 3 years younger that women randomized to placebo. However, there are several reasons why this difference in age did not influence our findings. First, the baseline values of NTx and BSALP were not statistically different between the treatment and placebo groups. Second, a substantial determinant of differences in bone turnover markers is years since menopause,(21) which was not statistically different between groups, and third, all our analyses were adjusted for age.
Strontium ranelate is the only other agent currently available that, in a large trial, has shown increases in bone formation and decreases in resorption. A phase 2 trial reports that women randomized to 2 g/day of strontium ranelate for 2 years have an 8.6% decrease in NTx and an 11% increase in BSALP compared with baseline.(22) We observed substantially greater changes in bone turnover markers in women randomized to ISMO. Furthermore, women randomized to strontium had a 3% increase in lumbar spine BMD and a reduction by almost one-half in the incidence of vertebral deformities compared with women randomized to placebo. This supports the notion that use of ISMO may increase BMD and decrease fractures.
Although we did not measure BMD in this trial, several studies have found that decreases in bone resorption are correlated with increases in BMD.(20,23,24) For example, the correlation between the decrease in urinary NTx and increase in spine BMD among women treated with alendronate is 0.53.(23) Increased bone resorption may increase fracture risk by increasing the rate of bone loss, by disrupting trabecular connectivity, or by increasing the number of sites that could act as areas of weakness and enable fracture propagation. Prospective studies have found that women with high levels of bone resorption have about a 1.5- to 3-fold increase risk of hip or nonspine fractures that is independent of BMD.(20) Furthermore, prospective studies and a recent meta-analysis have shown that decreases in markers of bone resorption are predictive of both increased BMD and decreased fracture risk.(19,20) Based on this meta-analysis, we would expect a 40% reduction in markers of bone resorption, such as we observed in women taking 20 mg of ISMO, to be associated with a 30% decrease in the risk of nonvertebral fractures. However, because there are no data on which to base estimates of fracture reduction from a combination of decreased resorption and increased bone formation, the true effect may be even larger.
Headaches were about four times more common among women randomized to ISMO compared with women randomized to placebo and may limit the use of this type of nitrate in the treatment of postmenopausal osteoporosis. The prevalence of mild to moderately severe headaches in our study (12.5%) was lower than what has been reported in the cardiovascular literature (36-52%), although the rates of discontinuation were higher (17% and 10%, respectively).(25) We attempted to minimize the development of nitrate-induced headaches by giving the study medication at bedtime, gradually increasing the dose among women randomized to 20 mg of ISMO, and encouraging the use of acetaminophen if headaches occurred. Of note, transdermal NTG may be better tolerated.(14)
Our trial has limitations. Participants in our study were healthy women and most were white, but it seems unlikely that nitrates will have materially different effects in men or women of other racial groups. We measured only one marker of bone formation and one marker of bone resorption. Our study lasted only 12 weeks, and trials are needed to assess the long-term effects of nitrates. However, data from the cardiovascular literature suggest that the long-term use of nitrates (up to 20 years) is safe and not associated with any adverse long-term affects.(25)
Our study confirms that once daily ISMO, an NO donor, significantly decreases bone resorption but, unlike other antiresorptive treatments for osteoporosis, ISMO also significantly increases bone formation. In contrast to the currently available treatments for osteoporosis, preparations of organic nitrates are very inexpensive, universally available, and can be used without concerns of long-term adverse effects. If randomized trials show that they substantially reduce fracture risk, intermittent use of nitrates may be an attractive and cost-effective approach to the worldwide prevention and treatment of osteoporosis.
We thank Drs Richard Eastell, Susan Jaglal, Murray Krahn, Donald Redelmeier, Susan Ross, Edward Sellers, Scott Walker, and Reinhold Vieth and the women who generously gave their time to participate in this study. SAJ is supported by a Canadian Institute of Health Research (CIHR) phase 1 Clinician Scientist Award. GAH is supported by a CIHR Scientist Award and is the F.M. Hill Professor in Academic Women's Medicine. This research was funded by grants from Physicians Services and the Department of Medicine, Sunnybrook and Women's College Health Sciences Centre.