Influence of Nitrates on Bone Remodeling and Endothelial Function in Patients With Type 2 Diabetes Mellitus
Influence of Nitrates on Bone Remodeling and Endothelial Function in Patients With Type 2 Diabetes Mellitus
Type 2 diabetes mellitus (DM) is becoming a leading global epidemic. DM affects several
 systems in the body. Most of the complications encountered in DM are attributed to
 uncontrolled hyperglycemia or poor glycemic control. Hyperglycemic stress tends to damage the
 inner lining of the small blood vessels (endothelium). Normally, the endothelium releases a
 chemical substance called nitric oxide (NO) which relaxes the blood vessels and also prevents
 blockade of these vessels. Therefore damage to the endothelium (endothelial dysfunction)
 results in diminished levels of NO which ultimately leads to occlusion of these small blood
 vessels (microvascular occlusion). Microvascular occlusion of vessels supplying the eyes,
 kidneys and nerves leads to serious complications like diabetic retinopathy, nephropathy and
 neuropathy.

 Of late, the skeletal system has emerged as another vulnerable target of diabetic
 microvascular disease. Patients with DM have an increased risk of developing fractures.
 Certain predisposing factors like diabetic neuropathy and visual disturbances (retinopathy
 and cataract) increases the likelihood of fractures in DM. More recently, evolving research
 has demonstrated NO's prospective role in bone preservation. Earlier studies have also
 validated the use of nitrates (donor of NO) in improving bone strength and reducing the risk
 of fractures.

 So far no study has investigated the effect of nitrates on endothelial function and bone
 microarchitecture in patients with diabetes. The investigators therefore propose to
 investigate the influence of nitrates on endothelial dysfunction and bone integrity in
 patients with type 2 diabetes. 40 patients with type 2 DM will be recruited into the study;
 20 patients will receive 20 mg of oral isosorbide mononitrate daily and the other 20 will not
 receive the study drug. The investigators hope to demonstrate an improvement in endothelial
 function (by measuring skin blood flow) and bone integrity (by measuring markers of bone
 formation and bone resorption and bone mineral density - BMD) following 6 months of nitrate
 therapy.
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.
Inclusion Criteria:

 -  Females and males aged between 40-75 years

 -  A diagnosis of type 2 DM based on one of the following criteria (ADA - 2010):

 -  Fasting plasma glucose (FPG) >= 126 mg/dL (7.0 mmol/L) or

 -  2-h plasma glucose >= 200 mg/dl (11.1 mmol/L) during an OGTT or

 -  Classic symptoms of hyperglycemia or hyperglycemic crisis with a random plasma glucose
 >= 200 mg/dL (11.1 mmol/L).

 -  Known history of type 2 diabetes mellitus on treatment

 Exclusion Criteria:

 -  At screening, age below 40 years and above 75 years.

 -  Pregnancy or lactation

 -  Type 1 diabetes mellitus (patients with a history of ketoacidosis, age of onset of DM
 before 25 years of age, BMI <21 kg/m2 and use of insulin without a concomitant oral
 hypoglycemic agent)

 -  Patients with uncontrolled hypertension (systolic blood pressure [SBP] > 160/90 mmHg)
 or hypotension (SBP of <=100 mm Hg) at screening.

 -  History of hypersensitivity to nitrates

 -  History of low blood pressure

 -  History of raised intracranial pressure (from cerebral haemorrhage or head trauma)

 -  History of cardiovascular disease (ischaemic heart disease, previous stroke and severe
 peripheral vascular disease [Ankle brachial pressure index - ABPI< 0.7])

 -  History of acute circulatory failure (shock), circulatory collapse, cardiogenic shock

 -  History of hypertrophic obstructive cardiomyopathy, constrictive pericarditis, cardiac
 tamponade, low cardiac filling pressures, aortic/ mitral valve stenosis

 -  History of general systemic illness including cardiac, hepatic or renal insufficiency

 -  Patients with clinical nephropathy (24 hour protein > 0.5g or dipstix protein +) or
 renal failure (serum creatinine > 130 µmol/l).

 -  History of anaemia

 -  History of closed angle glaucoma

 -  History of migraine headaches

 -  History of hypothyroidism

 -  History of hypothermia

 -  History of malnutrition

 -  History of Paget's disease and other metabolic bone disorders

 -  History of coeliac or inflammatory bowel disease

 -  History of multiple myeloma or cancer

 -  History of nitrate use for cardiac conditions

 -  History of treatment with phosphodiesterase type-5 inhibitors

 -  History of foot ulcers

 -  History of active foot deformities e.g. Charcot foot

 -  History of glucocorticoid intake within the last 3 months

 -  History of hormone replacement therapy in the last 12 months

 -  History of treatment with SERM (selective estrogen receptor modulator)

 -  History of treatment with thiazolidinedione

 -  History of anticonvulsant use

 -  History of past or current treatment for osteoporosis

 -  History of bisphosphonate therapy within the last 3 years