A phase III, randomized, two-armed, double-blind, parallel, active controlled, and non-inferiority clinical trial to compare efficacy and safety of biosimilar adalimumab (CinnoRA®) to the reference product (Humira®) in patients with active rheumatoid arthritis

Background This study aimed to compare efficacy and safety of test-adalimumab (CinnoRA®, CinnaGen, Iran) to the innovator product (Humira®, AbbVie, USA) in adult patients with active rheumatoid arthritis (RA). Methods In this randomized, double-blind, active-controlled, non-inferiority trial, a total of 136 patients with active RA were randomized to receive 40 mg subcutaneous injections of either CinnoRA® or Humira® every other week, while receiving methotrexate (15 mg/week), folic acid (1 mg/day), and prednisolone (7.5 mg/day) over a period of 24 weeks. Physical examinations, vital sign evaluations, and laboratory tests were conducted in patients at baseline and at 12-week and 24-week visits. The primary endpoint in this study was the proportion of patients achieving moderate and good disease activity score in 28 joints-erythrocyte sedimentation rate (DAS28-ESR)-based European League Against Rheumatism (EULAR) response. The secondary endpoints were the proportion of patients achieving American College of Rheumatology (ACR) criteria for 20% (ACR20), 50% (ACR50), and 70% (ACR70) responses along with the disability index of health assessment questionnaire (HAQ), and safety. Results Patients who were randomized to CinnoRA® or Humira® arms had comparable demographic information, laboratory results, and disease characteristics at baseline. The proportion of patients achieving good and moderate EULAR responses in the CinnoRA® group was non-inferior to the Humira® group at 12 and 24 weeks based on both intention-to-treat (ITT) and per-protocol (PP) populations (all p values >0.05). No significant difference was noted in the proportion of patients attaining ACR20, ACR50, and ACR70 responses in the CinnoRA® and Humira® groups (all p values >0.05). Further, the difference in HAQ scores and safety outcome measures between treatment arms was not statistically significant. Conclusion CinnoRA® was shown to be non-inferior to Humira® in terms of efficacy at week 24 with a comparable safety profile to the reference product. Trial registration IRCT.ir, IRCT2015030321315N1. Registered on 5 April 2015.


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Results: Patients who were randomized to CinnoRA® or Humira® arms had comparable demographic information, laboratory results, and disease characteristics at baseline. The proportion of patients achieving good and moderate EULAR responses in the CinnoRA® group was non-inferior to the Humira® group at 12 and 24 weeks based on both intention-to-treat (ITT) and per-protocol (PP) populations (all p values >0.05). No significant difference was noted in the proportion of patients attaining ACR20, ACR50, and ACR70 responses in the CinnoRA® and Humira® groups (all p values >0.05). Further, the difference in HAQ scores and safety outcome measures between treatment arms was not statistically significant. Conclusion: CinnoRA® was shown to be non-inferior to Humira® in terms of efficacy at week 24 with a comparable safety profile to the reference product.
Keywords: Adalimumab, Biosimilar, CinnoRA®, Rheumatoid arthritis Background Rheumatoid arthritis (RA) is a chronic, inflammatory, autoimmune disease of unknown pathophysiology leading to peripheral and symmetrical joint synovitis. The primary systemic manifestations are pain, morning stiffness, fatigue, and weight loss [1][2][3]. In progressive forms, it may lead to cartilage damage, joint destruction, and joint swelling resulting in impaired physical function and premature morbidity [4][5][6]. RA mostly develops in the fourth and fifth decades of life, with 80% of the cases occurring between 35 and 50 years of age. The worldwide prevalence of RA is about 0.5-1.0% with a female/ male ratio of 2.5:1.0 [7,8]. Although the presence of chronic inflammation has been proposed as a contributing factor, the exact mechanism of developing RA is still unknown [9].
The management of RA aims primarily at improving patients' quality of life (QoL), achieving low disease activity based on American College of Rheumatology (ACR) and European League Against Rheumatism (EULAR) criteria, and ultimately remission [1,10]. The treatment options for RA include non-steroidal antiinflammatory drugs (NSAIDs), glucocorticoids, conventional synthetic disease-modifying antirheumatic drugs (sDMARDs) and biological DMARDs (bDMARDs). RA treatment has developed considerably in recent years, with the early use of methotrexate (MTX) and the addition of targeted bDMARDs in patients with an inadequate response to MTX [10,11]. In fact, concomitant use of bDMARDs and MTX has been associated with the greatest clinical outcomes in trials and has been approved as the standard of care for patients with moderate-to-severe disease [12]. The stage and severity of the joint condition, the balance between possible adverse effects and expected benefits, and patients' preferences are amongst the influential factors in choosing a DMARD. MTX is the most frequently administered sDMARD and is used either as monotherapy or in combination with other anti-rheumatic drugs. The early onset of action and superior efficacy makes MTX the synthetic agent of choice in the treatment of RA [5,13]. Similarly, biological agents such as anti-tumor necrosis factor-α (anti-TNF-α) monoclonal antibodies are effective in suppressing disease activity, inhibiting structural deterioration and maintaining physical function. Adalimumab, a fully humanized immunoglobulin (IgG1) monoclonal antibody is produced in genetically modified Chinese hamster ovary cells (CHO). Adalimumab consists of two identical heavy and two identical light chains that bind specifically to the transmembrane TNF, thus blocking the interaction of TNF-α with its receptor [4,[14][15][16]. Adalimumab was first approved by the US Food and Drug Administration (FDA) in December 2002 for the treatment of RA and is currently approved for the following indications: RA, juvenile idiopathic arthritis (JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), Crohn's disease (CD), pediatric CD, ulcerative colitis (UC), psoriasis (PsO), pediatric plaque PsO, hidradenitis suppurativa (HS), and non-infectious uveitis [17,18].
Biosimilars are biotherapeutic products that are similar to the licensed biological reference products in terms of quality, efficacy, and safety, but often are provided at a lower price [19,20]. CinnoRA® was developed by CinnaGen Company (Alborz, Iran) as a biosimilar to the innovator adalimumab product (Humira®). This study aimed to evaluate the non-inferiority of test-adalimumab (CinnoRA®) to the reference product in terms of efficacy, tolerability, and safety in patients with active RA.

Study design
In this randomized, double-blind, non-inferiority trial, a total of 136 patients with active RA were randomized in a 1:1 ratio to receive 40-mg subcutaneous injections of either biosimilar adalimumab (CinnoRA®, CinnaGen Co., Iran) or the reference product (Humira®, AbbVie Inc., USA) every other week along with methotrexate (15 mg/week), folic acid (1 mg/day), and prednisolone (7.5 mg/day) over a period of 24 weeks. The study was conducted in accordance with the principles of good clinical practice (GCP) and the declaration of Helsinki across 10 referral hospitals in Iran. All the procedures were approved by the Institutional Review Board (IRB) and Ethics committees of each hospital. Patients provided written informed consent forms before initiation of any study-related procedure. The trial is registered in the Iranian registry of clinical trials with the following identification code IRCT2015030321315N1.
Patients' demographic information was recorded at baseline and a thorough medical examination was performed. Vital signs and laboratory examinations were taken from the patients at baseline and at the 12-week and 24-week visits. All the injections were administered by trained nurses at each study site. After initial screening and assessing eligibility, patients were randomized by permuted balanced block randomization with a block size of four. The randomization was implemented using telephone randomization by an independent contract research organization. Allocated treatments were administered to patients based on their enrollment number. Patients, nurses, and physicians were unaware of the size of the blocks and the allocated treatments, and the blinding was maintained till the end of the intervention. Information relating to demographic characteristics, contact history of subjects diagnosed with active tuberculosis (TB), medical history, and prior medications were collected. Body weight and height were measured and a complete physical examination was performed. Also, vital signs including heart rate and blood pressure were measured. Laboratory examinations, including hematological and blood chemistry assessment, measurement of rheumatoid factor, C-reactive protein (CRP), hepatitis B surface antigen, and hepatitis C antibody, urinalysis, and a pregnancy test in women, were performed during a fasting state.

Participants
Adult subjects of each gender who met the following criteria were included in this study: age between 18 and 75 years; active RA diagnosed by EULAR criteria [21]; moderate-to-severe RA for at least 6 months; lack of response to conventional non-biologic anti-rheumatic drugs after at least 12 months of therapy; and the ability to read, understand, and sign the written informed consent form.
Patients with any of the following criteria were excluded from the study: active or latent TB with a purified protein derivative (PPD) tuberculin test more than 5 mm in size or abnormal chest X-ray (CXR); previous treatment with TNF inhibitors; known hypersensitivity to human immunoglobulin proteins or other components of adalimumab formulation; in women, pregnancy, current nursing, or intention to become pregnant during the study; positive serology test for hepatitis B or C or human immunodeficiency virus antibody; ACR functional class IV or wheelchair/bed bound; taking intravenous antibiotics during the 8 weeks prior to screening or receiving oral antibiotic treatment during the 2 weeks before screening; history of serious, relapsing, or chronic infection; hemoglobin less than 8.5 g/dl; platelet count less than 125,000/μl; leukocyte count less than 3500/μl; serum creatinine more than 2 mg/dl; concomitant use of NSAIDs or more than 10 mg/day of prednisolone; receiving intravenous, intramuscular, intra-articular or oral corticosteroids (prednisolone, more than 7.5 mg/day) in the previous 4 weeks; previous treatment with rituximab, azathioprine, or 6-mercaptopurine (6-MP); history of chronic heart failure (CHF); history of myocardial infarction (MI) or unstable angina pectoris within 12 months prior to screening; history of demyelinating diseases or multiple sclerosis; history of malignancy within 5 years prior to screening; and participation in the study judged by the physician to be potentially harmful to the patient.

Efficacy and safety assessment
The percent of patients achieving good and moderate disease activity score (DAS)-based EULAR response was the primary endpoint of this study. The proportion of patients reaching ACR criteria for 20% (ACR20), 50% (ACR50), and 70% (ACR70) improvements after 24 weeks of treatment with adalimumab along with the disability index of the health assessment questionnaire (HAQ), and safety were the secondary endpoints of this study [22,23]. The incidence of adverse events at each visit was recorded based on patients' reports, vital signs, physical examinations, and laboratory tests.

Statistical analysis
In a study conducted by Broeder et al., 89% of the patients who received adalimumab achieved EULAR response [24]. The sample size of 64 people in each group was estimated by a non-inferiority margin of δ = −0.18 that was based on clinical judgement with 90% power and a 0.025 one-sided significance level. Primary efficacy measures were evaluated using both intention-to-treat (ITT) and per-protocol (PP) populations, while ITT population was used for safety assessment. Data were analyzed using Student's independent samples t test, the Mann-Whitney U test, the normal approximation test, Pearson's chi-square test, and Fisher's exact test. P values <0.05 were considered statistically significant. Data were analyzed using Stata 11.2 software (College Station, TX, USA) and plotted using GraphPad prism version 6.0 (GraphPad Software, USA).

Results
A total of 216 subjects were screened across 10 hospitals in Iran, of whom 136 patients were considered eligible for participation in this study. Patients were enrolled in the CinnoRA® or Humira® arms (68 subjects in each arm) and 64 patients in each group completed the 24week study period. Four patients in the CinnoRA® group withdrew from the study for the following reasons: adverse drug reactions (ADR, n = 2), positive PPD test (n = 1), and poor compliance (n = 1). Similarly, four patients in the Humira® group left the trial because of ADR (n = 3) and poor compliance (n = 1). The study profile is shown in Fig. 1.
Patients who were randomized to the CinnoRA® or Humira® arms had comparable baseline characteristics. The mean age of the participants in the CinnoRA® and Humira® groups was 48.29 ± 12.72 and 47.59 ± 11.48 years, respectively. The mean DAS in 28 joints based on erythrocyte sedimentation rate (DAS28-ESR) was 5.51 ± 1.24 in the CinnoRA® arm and 5.47 ± 1.28 in the Humira® arm. The baseline characteristics are summarized in Table 1.
According to the PP population, the proportion of patients fulfilling moderate and good EULAR response criteria based on the DAS28-ESR at week 12 was 42.42% and 54.55% in the CinnoRA® arm compared to 37.88% and 51.52% in the Humira® group, respectively (cumulative 12week good and moderate EULAR response in the PP population 97% in the CinnoRA® vs. 89% in the Humira® arm; p value = 0.08; CI for the difference = −0.9 to 16). At 24 weeks, 28.13% and 70.31% in the CinnoRA® vs. 31.25% and 67.19% in the Humira® arm met the criteria for moderate and good EULAR response, respectively (cumulative 24-week good and moderate EULAR response in the PP Fig. 1 Trial profile. ITT intention-to-treat, PP per-protocol population 98% in the CinnoRA® vs. 98% in the Humira® arm; p value = 1; CI for the difference = −4 to 4). Similarly, in the ITT population, 41.18% and 36.73% achieved moderate EULAR response at 12 weeks in the CinnoRA® and Humira® arms, respectively, and 52.94% in the CinnoRA® arm and 50.00% in the Humira® arm achieved good EULAR response at 12 weeks (cumulative 12-week good and moderate EULAR response in the ITT population 94% in the CinnoRA® vs. 87% in the Humira® arm; p value = 0.14; CI for the difference = −2 to 17). At 24 weeks, moderate EULAR response was attained in 26.47% of patients in the CinnoRA® arm compared to 29.41% in the Humira® arm. However, good EULAR response was achieved in 66.18% of patients in the CinnoRA® arm and 63.24% in the Humira® arm (cumulative 24-week good and moderate EULAR response in the ITT population 93% in the CinnoRA® vs. 93% in the Humira® arm; p value = 1; CI for the difference = −9 to 9). Based on the prespecified margin of 20%, non-inferiority of test-adalimumab to the reference product in terms of the proportion of patients achieving good or moderate EULAR response was confirmed at both the 12-week and 24-week time points (Fig. 2, Table 2).
The proportion of patients achieving ACR20, ACR50, and ACR70 responses at the 12-week time point were 85%, 61%, and 28% in the CinnoRA® arm compared to respective values of 76%, 48%, and 36% in the Humira® arm. At week 24, 92%, 77%, and 47% of the patients in the CinnoRA® arm achieved ACR20, ACR50, and ACR70 responses, respectively, which was similar to those observed in the Humira® arm (89%, 75%, and 53%, respectively). No statistically significant difference was observed between treatment arms at either the 12-week or the 24week time point (all p values >0.05, Fig. 3).
The incidence of adverse effects was comparable between patients who received test-adalimumab compared to those who took the reference product. Overall, a total of 24 patients (35.29%) in the CinnoRA® arm vs. 30 (44.12%) patients in the Humira® arm reported at least one adverse event. The most prevalent adverse events were local (8.82% with CinnoRA®, 17.65% with Humira®) and respiratory (8.82% with CinnoRA®, 20.59% with Humira®) adverse effects (Table 3).

Discussion
Randomized clinical trials demonstrating comparable efficacy, safety and tolerability of the biosimilar and innovator products are absolutely necessary as well as analytical evidence to establish similar physicochemical and biological actions of the products. Biosimilars improve the availability of more affordable products while offering similar efficacy and safety to the reference product [18,[25][26][27]. The perception of biosimilarity has been changed over recent years, especially in rheumatology. In September 2016, AMJEVITA™ (Amgen®, Thousand Oaks) was approved by the FDA, as a biosimilar adalimumab for treatment of seven inflammatory diseases. Similarly, Exemptia™, another adalimumab biosimilar, was approved in India. Etanercept and infliximab biosimilars were also authorized in various indications. In May 2016, Inflectra™ was approved by the European Medicines Agency (EMA) and the FDA as an infliximab biosimilar for all indications of the reference infliximab, including RA, AS, PsA, PsO, CD, and UC. In the case of etanercept, the biosimilar HD203 was recently approved in South Korea. In addition, Samsung Bioepis's SB4, known as BRENZYS™ (Samsung Bioepis Co., Ltd., Korea) received regulatory approval from the Korean Ministry of Food and Drug Safety (MFDS), the European Commission (EC), and the Australian Therapeutic Goods Administration (TGA) to be used as a biosimilar alternative to etanercept [18,28].
In this randomized, double-blind, parallel-group, noninferiority study, the efficacy and safety of CinnoRA® were compared with those of adalimumab in the The percentage of our patients achieving moderate and good EULAR responses increased significantly in both the CinnoRA® and the Humira® arms, and the difference between the two arms was not statistically significant. Further, the percentage of patients reaching ACR20, ACR50, and ACR70 increased significantly during the 6-month period. In the retrospective study of Takeuchi et al. investigating the ability of adalimumab to reduce disease activity in 167 patients with RA, the mean DAS28-ESR score decreased from 5.3 ± 1.3 at baseline to 3.5 ± 1.5 at week 52 (p < 0.0001), which is consistent with our findings [32]. Furst et al. conducted a double-blind, placebocontrolled study and assigned patients into groups receiving either adalimumab 40 mg subcutaneously every other week or placebo. The study aimed to evaluate the efficacy of adalimumab when given with standard antirheumatic therapy over 24 weeks in patients with active Fig. 2 Evaluation of non-inferiority of test-adalimumab to reference adalimumab in terms of the proportion of patients who met good and moderate disease activity score in 28 joints based on erythrocyte sedimentation rate (DAS28-ESR) European League Against Rheumatism (EULAR) response in the per-protocol (PP) population at 12 weeks, 95% CI for the difference −0.009 to 0.16 (a); the intention-to-treat (ITT) population at 12 weeks, 95% CI for the difference −0.02 to 0.17 (b); the PP population at 24 weeks, 95% CI for the difference −0.04 to 0.04 (c); and the ITT population at 24 weeks, 95% CI for the difference −0.09 to 0.09 (d) RA, who were not adequately responding to such therapies. Similarly, a 24-week follow-up period was considered to evaluate the efficacy and safety of test-adalimumab in rheumatic patients [33].
Aletaha et al. chose the 3-month time point as a critical decision point in the treatment of patients with RA. It seems that patients who have significantly improved by 3 months are more likely to reach their treatment target by 6 months. In fact, achieving responses at 3 months is a good indicator of remission at 12 months, whereas patients with poor responses at 3 months will probably benefit from changing the treatment [34]. In agreement with previous studies, patients in our study who had noticeable improvements at 3 months also had better remission status. Patients in the CinnoRA® arm responded to treatment within a shorter period of time; however, this difference was not statistically significant. In the study of Burmester et al. in rheumatic patients receiving adalimumab for approximately 5 years, the mean HAQdisability index score decreased in the first 6 months and then remained steady till the end of the study [35]. Similarly, in our study the mean HAQ score decreased significantly in both treatment arms and the difference between the groups was not statistically significant. In fact, as an important therapeutic goal, adalimumab improved social and physical functions in rheumatic patients.
In the safety analyses of the study of Takeuchi et al., the most frequently noted adverse events during one year of treatment with adalimumab were reactions at the drug administration site, with a frequency of 11.40/100 patient-years [32]. Safety was evaluated based on the adverse events reported by patients. All the adverse events were summarized according to the Medical Dictionary for Regulatory Activities system organ class (MedDRA SOC). We did not notice any significant difference in the incidence of injection site reactions as the most prevalent adverse events between treatment arms, and our findings were consistent with previous studies. Despite having a negative PPD test at the beginning of the study, one of the patients had a positive PPD test 8 weeks later that was probably due to close contact with a patient infected with TB close to the time of study enrollment.

Conclusion
Based on our findings, CinnoRA®, as a biosimilar adalimumab, was shown to be non-inferior to Humira® in the treatment of adult patients with active RA.

Consent for publication
Informed consent forms were obtained from all the patients before conducting the screening procedure. Prior to collecting the signed informed consent forms from the patients, all of the contents were explained thoroughly to the patients by the coordinating investigators and they signed the forms with full awareness about the clinical trial conditions. These forms were signed and dated by the coordinating investigators as well. As specific data that would identify the participants are included in the consent forms, we are unable to provide them; hence, this is not applicable.

Competing interests
The authors of the study declare no competing interests.

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