Influence of Nitrates on Bone Remodeling and Endothelial Function in Patients With Type 2 Diabetes Mellitus

Diabetes mellitus (DM) is a chronic debilitating disease that affects almost all the systems in the body with accompanying diabetic complications (neuropathy, retinopathy and nephropathy) adding to the global burden of DM. These diabetic complications arise as a manifestation of hyperglycaemic damage induced at the cellular level particularly the endothelial cells (EC). Exposure to hyperglycaemic stress causes endothelial dysfunction, which is the major perpetrator of microvascular disease. Disruption of endothelial function also marks the advent of the subsequent development of cardiovascular disease (CVD) that includes atherosclerosis and coronary artery disease (CAD). Microvascular disease occurs as a result of deficiency of a potent endothelial vasodilator, nitric oxide (NO). NO plays a fundamental role in vascular regulation which is mediated through vasodilation. Other favourable characteristics of NO include antiplatelet activity and prevention of vascular smooth muscle cell (VSMC) proliferation. Unsurprisingly, diminished NO levels in DM will eventually lead to occlusion of the microvasculature in the retina, glomerulus and peripheral nerve (vasa nervorum).

Of late, the skeletal system has also emerged as another vulnerable target of diabetic microvascular disease. Certain predisposing factors like diabetic neuropathy (DN), visual disturbances (retinopathy and cataract), gait abnormalities and hypoglycemia increase the likelihood of fractures in DM. Among these factors DN contributes to a major extent in the development of fractures especially fractures of the lower extremities.

Current antiresorptive therapies include estrogen/ HRT (Hormone replacement therapy), bisphosphonates (BPP), selective estrogen receptor modulators (SERM) and denosumab (DMB). These agents mainly retard bone resorption in the trabecular bone, with minimal effects on cortical bone and are associated with only a 20-30% reduction in the risk of non-vertebral fractures. Moreover, they decrease bone formation and are not devoid of adverse effects. Some of these agents are also expensive and at times unavailable in some countries. All these limitations challenge the discovery of an ideal agent that can prevent bone resorption, increase bone formation and also reduce the risk of non-vertebral fractures.

Lately the therapeutic role of organic nitrates (source of NO) in bone metabolism has led to a breakthrough in this field of research. Nitrates are commonly used in the treatment of angina. Since nitrates are widely available, inexpensive and associated with limited side effects, it would be practical to exploit these characteristic features in the treatment of bone disease and endothelial dysfunction in DM.

AIMS

This pilot study aims to investigate the effect of Isosorbide mononitrate in patients with type 2 DM with respect to:

• Its influence on bone remodelling by assessing the levels of bone markers and its ability to improve BMD.
• Its role in modulating endothelial dysfunction.

HYPOTHESIS

In this randomised pilot study, patients with type 2 DM who receive 20 mg of Isosorbide mononitrate will show:

• An improvement in bone formation as measured by serum procollagen type 1 amino terminal propeptide (P1NP)
• Suppression of bone resorption as assessed by measurement of serum C-terminal cross-linked telopeptide of type-I collagen (CTX)
• Improvement in calcaneal BMD
• Improvement in endothelial mediated vasodilation.

PATIENTS 40 female/ male subjects with type 2 DM will be considered eligible for the study. They will be recruited from the Tameside Diabetes Centre and appropriate GP practices and a written consent will be obtained from them prior to participation in the study.

Since there is no placebo in this study, the research fellow and research nurse will be blinded to the treatment the patient is receiving. As it is known that the duration of diabetes can affect microvascular circulation and possibly bone metabolism, the two groups will be matched for the following variables: age, gender, duration of diabetes and severity of neuropathy.

Diabetes self-education and management along with dietary advice will be provided to all the participants in the study.

RANDOMISATION

With the help of a computer-generated program the study participants will be randomised into 2 groups:

• Group 1: 20 subjects will receive 20 mg/day of isosorbide mononitrate orally
• Group 2: 20 subjects will receive standard care

MEASUREMENTS Biochemical parameters at baseline and 6 months later

• Serum procollagen type 1 amino terminal propeptide (P1NP), a marker of bone formation which indicates osteoblastic activity.
• Serum C-terminal cross-linked telopeptide of type-I collagen (CTX), a marker of bone resorption Clinical parameters at baseline
• Laser Doppler imaging
• Calcaneal BMD (Sahara clinical bone sonometer; Hologic, Waltham, MA) Blood samples will be obtained from the antecubital vein following an overnight fast and after resting for 15 minutes in the supine position.

Assessment of the microcirculation with Laser Doppler Iontophoresis at baseline and 6 months A standard measurement of microcirculation is laser Doppler iontophoresis, which is used by several research institutes. In this trial the skin microcirculation will be measured on the ventral aspect of the forearm using a Perimed Laser Doppler imager and iontophoresis system.

Endothelial-mediated vasodilation will be measured by the iontophoresis of acetylcholine, while sodium nitroprusside will be used to measure endothelium-independent vasodilation. The iontophoresis system consists of an ION chamber (iontophoresis delivery vehicle device) that sticks firmly to the skin and a reference electrode. The response in blood flow will be imaged and quantified using the Perimed Laser Doppler Imager (Sweden).

Assessment of calcaneal BMD at baseline and at 6 months This is a simple and convenient method to assess peripheral BMD and assess fracture risk. The device used is a quantitative ultrasound called Sahara Clinical Bone Sonometer (Sahara Clinical Bone Sonometer; Hologic, Waltham, MA). The calcaneus is the preferred peripheral site to assess fracture risk. This device uses ultrasound waves to determine the BMD of the calcaneus. In this procedure, once the bare heel is placed in the device, the BMD is calculated within 30 seconds and the results are then generated on paper by the device.

STATISTICAL ANALYSIS The results obtained from this study will be reported as a difference between the baseline and 6 months later following a single daily oral dose of 20 mg isosorbide mononitrate. The difference between the 2 groups will also be inferred at the end of 6 months. The changes in skeletal parameters (serum P1NP and CTX) and microcirculation will be compared between the 2 groups using t-tests to determine post hoc differences. A p-value of < 0.05 will be considered significant. All analyses will be conducted using the Statistical Package for Social Sciences (SPSS Inc., Chicago, Illinois, USA).

DURATION OF THE STUDY The estimated time for enrolment of patients will be within 6 months. The duration of the study is 6 months and the last follow up will be at the end of 12 months.