The effect of organic nitrates on osteoporosis

Pilot Study:
Mean headache score associated with each of intermittent NTG and ISMO use. Subjects will be given four seven day diaries, with a visual analogue scale (VAS) for each day of both treatment periods and the wash out period. Subjects will rate their headache upon awakening on a daily basis; our previous study demonstrated that subjects randomised to ISMO who developed headaches reported that the headache was most severe upon awakening and gradually subsided over the day. The VAS uses a 100 mm continuous horizontal line with the left pole labeled 'no headache' and the right pole labeled 'terrible headache'; subjects will be instructed to draw a vertical line intersecting the VAS indicating the severity of their morning headache. We will calculate the mean headache score with intermittent NTG and intermittent ISMO for each subject. Then we will calculate the mean headache score for all subjects when taking ISMO and when taking NTG. The nitrate that gives the lowest mean headache score will be used in our main study. Our earlier work indicates that the nitrate formulation associated with the most severe headaches will result in the greatest number of drop outs. The VAS can detect subtle changes in subjective complaints (i.e. headache), has documented reliability, and has been used in several studies designed to study the effects of NTG ointment on headache in healthy women.

Main Study:
Change from baseline in BMD at the lumbar spine over 24 months among women randomised to treatment compared with women randomised to placebo. BMD will be measured (on the same machine in all subjects) at the lumbar spine (L1 to L4) using a Lunar DPX-L bone densitometer (Lunar Corporation, Madison WI). A single experienced technician, certified by the International Society of Clinical Densitometry (ISCD) and blinded to the treatment assignment, will perform all BMD measurements. The intraclass correlation coefficient for BMD measurements is 0.98. The PI will be blinded to treatment assignment and will report the BMD. BMD reporting is objective and based on standard ISCD criteria.

Main study:
1. Change from baseline in total hip BMD. This will provide information about the effects of nitrates on cortical and trabecular bone and ultimately on the ability of nitrates to prevent hip fractures.
2. Change from baseline in bone formation and bone resorption markers. Measuring markers of bone turnover will increase our understanding of the mechanisms by which intermittent nitrates increase BMD (i.e. a decrease in bone resorption and/or an increase in bone formation). We will measure bone turnover markers three months after initiating calcium and vitamin D because we expect a 10% decrease in markers with the use of supplements, and the maximal change in bone markers is typically observed at three months. Thus, we will be assessing any additional effects of intermittent nitrates on markers of bone turnover in women receiving calcium and vitamin D supplements. Urinary NTx will be measured on a second morning urine sample using a monoclonal antibody technique (Osteomark). The intra-assay variability is 7.6% and the inter-assay variability is 4.0%. Serum BSAP will be measured on a fasting serum sample using a monoclonal antibody technique (Metra Biosystems). The intra-assay variability is 5.8% and the inter-assay variability is 5.2%. To minimise variability, subjects will be given written and verbal instructions on how to collect the second morning urine sample; for each subject we will collect all the samples (baseline, three, 12 and 24 months post-randomisation) at the same time, and we will store the blood and urine at -70°C and analyze all the samples together in a single laboratory. We will compare measures of bone turnover markers at 3, 12, and 24 months after randomisation using a repeated measure of analysis of variance. By measuring markers at three months we will be able to directly compare these results to the findings in our earlier study. By measuring markers at 12 and 24 months we will be able to evaluate to a limited degree, the question of tachyphylaxis with nitrates. Recall that in our earlier study we found a decrease in markers of bone resorption and an increase in markers of bone formation after 90 days of treatment with intermittent ISMO. Measuring markers after one and two years of treatment will allow us to determine if there is still a marked decrease in bone resorption and increase in bone formation; no further changes in markers may indicate that the effects of nitrates on bone is transient.
3. Adverse events. Adverse events will be assessed using a standardized, validated interviewer administered questionnaire on a monthly basis by telephone. We will assess nitrate and non nitrate related adverse events. Documentation of adverse events is critical before proceeding to a larger, longer study of intermittent nitrates and fractures.
4. Bone Microarchitecture. Fracture risk is influenced by both the quantity of bone (assessed by BMD) and quality of bone. Features of bone quality include trabecular and cortical volumetric bone density, measures of bone geometry, and trabecular texture. We will measure trabecular and cortical volumetric bone densities at the radius and tibia using an XCT2000 pQCT scanner at Hamilton Health Sciences. Transaxial images will also be processed to yield values of bone geometry and trabecular bone texture. Measurements of trabecular bone structure discriminate women with low BMD and fracture from women with low BMD but no fracture. As such, they may be particularly important when assessing the effect of a new treatment on fractures. The various measurements produced by pQCT will allow us to describe the effects of nitrates on the various compartments of bone.