Research methodology and study population

This proof-of-concept randomized, double-blind placebo-controlled parallel trial included 70 patients with active rheumatoid arthritis who were diagnosed according to the American College of Rheumatology (ACR)/European League Against Rheumatism "EULAR, 2010" (Felson DT et al. 2011). The patients were recruited from Rheumatology, Rehabilitation and Physical Medicine Department, Mansoura University Hospital, Mansoura, Egypt. The blindness was maintained by the similarity between the placebo and Cilostazol tablets. The patients were allocated randomly into two groups: group 1 (control group; n = 35) which was given IM Methotrexate (7.5 mg weekly plus placebo tablet twice daily 2 h after meal for 3 months and group 2 (Cilostazol group; n = 35) which received IM Methotrexate (7.5 mg weekly) plus Cilostazol (Cilosort™, Multicare, Pharmaceutical, Egypt) 50 mg twice daily 2 h after meal for 3 months. The study protocol was approved from the Research Ethics Committee of Tanta University (Approval code: 35930\10\2) and the Medical Research Ethics Committee of Mansoura University (Approval code: MS.22.10.2163). The research followed the 1964 Declaration of Helsinki ethical criteria. The patients gave their written informed consented. The research was registered on ClinicalTrials.gov with an identification code: NCT05594680.

The inclusion criteria encompassed individuals diagnosed with active rheumatoid arthritis (RA) with 28-joint disease activity score (DAS-28) exceeds 2.6, age 18–60 years, and both genders. The exclusion criteria ruled out individuals with diabetes, cardiovascular conditions (e.g., arrhythmia, congestive heart failure), hematological diseases (severe anemia, coagulation disorders), and inflammatory conditions Additionally, individuals using low doses of aspirin, anticoagulants, antioxidants, and biological DMARDs, as well as those with renal and hepatic dysfunction, and those with hypersensitivity to the study medications were excluded. Pregnant and lactating women were also excluded.

Methods. History, clinical assessment, and anthropometric data collection

Medical history was taken, clinical assessment of morning stiffness duration and the presence of joint pain or swelling were done, demographic parameters (age, sex,) were collected, and anthropometric measurements were done including weight and height with subsequent calculation of body mass index (BMI) = [Weight (kg)] ÷ [Height2 (m2)].

Blood sample collection and assessment of biological markers

Before and 3 months following the intervention, 5 ml of venous blood was withdrawn from each patient between 8:30 am and 10:30 am after 12-h overnight fasting. Blood samples were placed in plain tubes and then centrifuged for 10 min at 3000 rpm. The biological markers were measured in the separated sera using enzyme-linked immune sorbent assay (ELISA) according to the manufacturer’s guidelines for assessing serum levels of human CRP (SunRed, China; Cat#201–12-1799), NF-κBp65 (SunRed, China; Cat#201–12-0667), HO-1 (DL-Develop, China; Cat#DL-HO-1-Hu), and cAMP (DL-Develop, China, Cat#DL-cAMP-Ge).

Disease activity assessment:

Disease activity was measured before and 3 months after treatment using the formula: DAS28-CRP = [0.56 √(tender joint count) + 0.28√(swollen joint count) + 0.36*ln (CRP + 1)] * 1.10 + 1.15. Disease activity was categorized as follows: high disease activity (≥ 5.1), low disease activity (≤ 3.2), and remission (< 2.6) (Wells et al. 2009).

Functional assessment

Before and 3 months after intervention, the validated Arabic Multidimensional Health Assessment Questionnaire (MDHAQ) that includes 14-items Health Assessment Questionnaire (HAQ) was implicated to assess function. The score of the HAQ Questionnaire is calculated from the mean of the sum of the responses of the items where each item is scored from 0–3, where 0 = without any difficulty, 1 = with some difficulty, 2 = with much difficulty, and 3 = unable to do (Aflaki et al. 2024). In addition, morning stiffness (MS) duration in minutes was also assessed.

Assessment of participants' compliance, and drug tolerability

Cilostazol and placebo were given monthly, and pill returned were counted to assess compliance. Participants were followed up weekly by phone calls and monthly through planned appointments to assess adherence and report drug-related side events. Standardized adverse effect checklists were implicated to report adverse effects.

Primary and secondary outcomes

The main outcome is the variation in the biological marker serum concentration. The secondary outcome is the variation in DAS-28-CRP and MDHAQ scores.

Sample size calculation

The sample size was determined using IBM® SPSS® Statistics version 28 software, assuming a 60% improvement in DAS-28 for the Cilostazol group and a 25% improvement for the group given the placebo, with a statistical power of 80% (Van Gestel et al.1999). A sample size of 31 patients per group has been suggested. In this context, a total sample size of 70 patients with 35 in each group is established as the initial target for the current study, assuming that the attrition rate is 10%.

Statistical analysis

The data collected were organized using Microsoft® Office Excel 2019 (Microsoft Corporation). Statistical analyses were performed using IBM® SPSS® Statistics version 28 (IBM Corp., Armonk, NY, USA). Graphical representations were created using GraphPad Prism version 6.01 (GraphPad Software, La Jolla, CA, USA). Data were assessed for normality using t Shapiro–Wilk test or the Kolmogorov–Smirnov test. For parametric data, paired t test was used to compare means within the same group, while unpaired t test was employed to compare means between two groups. Non-parametric data were analyzed using the Mann–Whitney U test for both intra-group and inter-group comparisons. Categorical data were evaluated using Chi-square test, and Fisher’s exact test was applied to analyze adverse effect reports. Results were expressed as mean ± standard deviation (SD), medians, range, and percentages, as appropriate. Statistical significance was set at p ≤ 0.05.