Exploring the potential of low-dose sulfasalazine in stable coronary artery disease patients: randomized, double-blind, placebo-controlled study

Recent statistics reports by Centre for Disease Control and Prevention (CDC) and World Health Organization (WHO) have presented the facts that cardiovascular disease (CVD) accounts for 30% of all deaths.1 Genetic polymorphism, age, gender, smoking, inflammation, systolic blood pressure (SBP), cholesterol, physical inactivity, low socio-economic status, and psychosocial risk factors contribute to development of CVD.2 Inflammation-induced cardiac and vascular remodelling, oxidative stress, unregulated synthesis of nitric oxide, impaired immune cells signalling, and cytokines cascade contribute to cardiovascular damage. Sulfasalazine, an anti-cytokine drug, is reasonably accepted clinically for its efficacy in inflammatory diseases due to potential NF-kB and TNF-α inhibitory activity.3 However, none of the anti-cytokine drugs has been incorporated in a standard regimen for treatment of coronary artery disease (CAD). Our study aimed at controlling inflammation using sulfasalazine 500 mg, once a day for 24 weeks in stable CAD patients. A randomized, double-blind, placebo-controlled, parallel group study was carried out, under the Department of Pharmaceutical Sciences and Drug Research, after due approval from Institutional Ethics Committee of Punjabi University, Patiala. The study was performed in accordance with the declaration of Helsinki and the code of Good Clinical Practice. Written informed consent was obtained from each patient prior to recruitment in the study. Inclusion criteria were: male/female patients (n = 50, ≥18 years of age) with positive angiographic (n = 47) and/or echocardiographic evidence of CAD (n = 3) as described by Chang et al. 2009.4 Patients with concomitantly present other cardio-metabolic disorders and low-grade systemic inflammation (high-sensitivity C-reactive protein level ≥1 but ≤10 mg/L) were also included. Exclusion criteria were: any major surgery or stroke within the last 3 months, presence of active infection(s), congestive heart failure (New York Heart Association (NYHA) class III–IV), allergy to sulfa drugs, history of autoimmune or chronic inflammatory diseases, chronic use of non-steroidal anti-inflammatory agents, abnormal renal and liver function enzyme levels. All women of child-bearing potential, pregnant and lactating women or those currently on investigational study medication within 30 days prior to the screening visit were also included. Eligible patients (n = 50) who met the inclusion criteria were randomized (1:1 randomization using a random number table) into two groups (n = 25 each). Group A received sulfasalazine 500 mg tablets and group B matched placebo tablets, once a day for 24 weeks. Assessment of eligible patients was carried out at baseline (before starting drug therapy) and after 24 weeks of treatment. The sample size was estimated by considering high-sensitivity C-reactive protein as the primary response variable (n = 25/group). To detect a mean change <3 mg/L (high-sensitivity C-reactive protein cut-off level ≤3.0), a sample size of 25 per group was calculated at 80% power and 95% significance level (α = 0.05).5 Results were expressed as mean ± standard error of mean and number/percentage wherever applicable and considered statistical significant at P ≤ 0.05. Results were compared between the two groups using an unpaired t-test for normally distributed data and Mann–Whitney U test for skewed distributed data. Pearson's correlation followed by multivariate regression analysis was applied to find independent predictors/variables. Intra- and interobserver variability for cardiovascular Doppler profiling was 1.85 and 2.55% for the brachial artery and 2.35 and 3.10% for the carotid artery, respectively. Assessment of endothelial-dependent flow-mediated dilatation (FMD) of the brachial artery was carried out by two cardiologists, who were blinded to the treatment protocol, in accordance with the guidelines detailed by Corretti et al., 2002.6 Flow-mediated dilatation was calculated in percentage as FMD (%) = 100 × {(post-hyperaemia diameter – basal diameter)/basal diameter}. Intima-media thickness (IMT), diastolic (Dd), and systolic (Ds) diameter (D) of the common carotid artery were recorded using carotid ultrasonography. Diastolic, Ds, and brachial BP measurements were used to derive parameters of arterial stiffness, i.e. Young's elastic modulus (YEM), carotid arterial compliance (CAC), and stiffness index (SI) as described by Cipolli et al.7 Serum TNF-α and high-sensitivity C-reactive protein (by ELISA technique); haemoglobin, total and differential leucocyte count, and platelets counts (automated cell counter); creatinine, serum glutamate oxaloacetate transaminase, and glutamate pyruvate transaminase (biochemical kits) were analysed. Table 1 presents the baseline profile and post-treatment effect on inflammatory markers, and brachial and carotid artery variables. Age ‘per se’ is a natural independent predictor of endothelial function.8 However, we could not find significant correlation of age with FMD (r = −0.13, P = 0.36). All patients had both hypertension and CAD, reflecting high pervasiveness of hypertension in CAD subjects and involvement of defective vascular tone in producing endothelial dysfunction resulting from imbalance between α1-adrenergic receptor (AR) and β2-AR-meditated vasodilatation.9 Moreover, the SBP was found to be an independent predictor of endothelial dysfunction. Both high-sensitivity C-reactive protein (P = 0.04) and SBP (P = 0.01), but not age and BMI (P > 0.05), were found to be independent predictors of FMD (FMD = 37.742 − (3.058 × high-sensitivity C-reactive protein) − (0.125 × SBP). None of the variables (age, high-sensitivity C-reactive protein, SBP) was found to be an independent predictor of IMT, except BMI (IMT = 1.510 − (0.0291 × BMI, P ≤ 0.01). Patients' biochemical and clinical profile (at baseline and after treatment) BMI, body mass index; TNF-α, tumour necrosis factor-alpha; NTG, nitroglycerine; IMT, intima-media thickness; YEM, Young's elastic modulus. aStatistically significant values (P ≤ 0.05). Data presented as mean ± SEM. Patients' biochemical and clinical profile (at baseline and after treatment) BMI, body mass index; TNF-α, tumour necrosis factor-alpha; NTG, nitroglycerine; IMT, intima-media thickness; YEM, Young's elastic modulus. aStatistically significant values (P ≤ 0.05). Data presented as mean ± SEM. Serum levels of safety parameters were not found to be out of normal range (Table 2). There were no drop-outs due to adverse effects or any other reason. Patients' self-reported adverse effects were mainly fatigue and nausea (11 vs. 6), and itching of skin (5 vs. 3) in Group A and B, respectively. Patients' tolerability profile of drugs TLC, total leucocyte count; DLC, differential leucocyte count; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate transaminase. aStatistically significant P values (<0.05). Patients' tolerability profile of drugs TLC, total leucocyte count; DLC, differential leucocyte count; SGOT, serum glutamate oxaloacetate transaminase; SGPT, serum glutamate pyruvate transaminase. aStatistically significant P values (<0.05). Thus, controlling inflammation-induced endothelial dysfunction and carotid arterial remodelling with sulfasalazine may help in improving the therapeutic outcome of the disease. Although sulfasalazine has been well explored in rheumatic diseases, its mechanism of action involved in curtailing the atherosclerotic inflammatory process is still not clear. There is a need to further elucidate the mechanism in large randomized controlled trials to establish the efficacy and safety of sulfasalazine in CAD patients. In the present study, the effect of sulfasalazine on inhibiting NF-kappa B-regulated gene expression and gene polymorphisms such as CaMK410 linked to endothelial dysfunction were not evaluated. We derived arterial stiffness indices based on brachial BP rather than central aortic BP, which is considered less appropriate due to differences in pulse pressure amplification at the central vs. brachial artery. Moreover, only one dose level (500 mg/day) was evaluated on a small number of subjects due to institutional constraints such as lack of sufficient resources, time, and funding.