Rationale and design of the “Tocilizumab in patients with moderate to severe COVID-19: an open-label multicentre randomized controlled” trial (TOCIBRAS)

Introduction Pro-inflammatory markers play a significant role in the disease severity of patients with COVID-19. Thus, anti-inflammatory therapies are attractive agents for potentially combating the uncontrolled inflammatory cascade in these patients. We designed a trial testing tocilizumab versus standard of care intending to improve the outcomes by inhibiting interleukin-6, an important inflammatory mediator in COVID-19. Methods and analysis This open-label multicentre randomized controlled trial will compare clinical outcomes of tocilizumab plus standard of care versus standard of care alone in patients with moderate to severe COVID-19. Two of the following four criteria are required for protocol enrolment: D-dimer > 1,000ng/mL; C reactive protein > 5mg/dL, ferritin > 300mg/dL, and lactate dehydrogenase > upper limit of normal. The primary objective will be to compare the clinical status on day 15, as measured by a 7-point ordinal scale applied in COVID-19 trials worldwide. The primary endpoint will be assessed by an ordinal logistic regression assuming proportional odds ratios adjusted for stratification variables (age and sex). Ethics and dissemination The TOCIBRAS protocol was approved by local and central (national) ethical committees in Brazil following current national and international guidelines/directives. Each participating center had the study protocol approved by their institutional review boards before initiating protocol enrolment. The data derived from this trial will be published regardless of the results. If proven active, this strategy could alleviate the consequences of the inflammatory response in COVID-19 patients and improve their clinical outcomes.


Introduction:
Pro-inflammatory markers play a significant role in the disease severity of patients with COVID-19. Thus, anti-inflammatory therapies are attractive agents for potentially combating the uncontrolled inflammatory cascade in these patients. We designed a trial testing tocilizumab versus standard of care intending to improve the outcomes by inhibiting interleukin-6, an important inflammatory mediator in COVID- 19.

Methods and analysis:
This openlabel multicentre randomized controlled trial will compare clinical outcomes of tocilizumab plus standard of care versus standard of care alone in patients with moderate to severe COVID-19. Two of the following four criteria are required for protocol enrolment: D-dimer > 1,000ng/mL; C reactive protein > 5mg/dL, ferritin > 300mg/dL, and lactate dehydrogenase > upper limit of normal. The primary objective will be to compare the clinical status on day 15, as measured by a 7-point ordinal scale applied in COVID-19 trials worldwide. The primary endpoint will be assessed by an ordinal logistic regression assuming proportional odds ratios adjusted for stratification variables (age and sex).
Ethics and dissemination: The TOCIBRAS protocol was approved by local and central (national) ethical committees in Brazil following current national and international guidelines/ directives. Each participating center had the study protocol approved by their institutional review boards before initiating protocol enrolment. The data derived from this trial will be published regardless of the results. If proven active, this strategy could alleviate the consequences of the inflammatory response in COVID-19 patients and improve their clinical outcomes. February, and by June, the country accumulates more than one million cases and 50,000 deaths.
The development of pneumonia progressing to a systemic inflammatory response syndrome (SIRS) and multiorgan failure indicates that pro-inflammatory cytokines are a significant contributor to morbidity and mortality in SARS-CoV-2 infection. (5,6) The rapid rise in cytokines reminiscent of that seen in chimeric antigen receptor therapy (CAR-T) is proposed to be an essential element in the rapid clinical deterioration. (7)(8)(9) The COVID-19 SIRS is usually observed in the second week of infection, where a slew of pro-inflammatory signals are triggered, leading to respiratory compromise, and multiorgan failure. After SARS-CoV-2 initially binds to the angiotensin-converting enzyme 2 (ACE2) receptor, several cell types are infected thereafter from the respiratory, gastrointestinal, neurologic, endothelial, and reticuloendothelial systems, giving rise to a wide clinical presentation ranging from oligosymptomatic to multiorgan failure and death. (10)(11)(12)(13)(14)(15) Clinical and laboratory risk factors associated with worse outcomes are being recognized as the pandemic unfolds globally. (16)(17)(18) Macrophage infection and activation, initially in the lungs and then systemic, emerges as an essential source of pro-inflammatory cytokines such as interferon gamma (IFN-g), interleukin (IL)-6, IL-12, tumoral necrosis factor (TNF), IL-1RA, and C-X-C motif chemokine 10 (CXCL10) among others. (5) Other systems, such as the coagulation cascade, are activated by the crescent inflammatory response, leading to thrombotic and hemorrhagic events both in the lung and systemically. (19,20) This multisystem havoc triggered by SARS-CoV-2 is now recognized to be a critical element to its pathogenesis; this rationale has guided the proposal of therapeutic strategies that could possibly block its more lethal consequences. For example, an old drug, etoposide, and several more targeted approaches emerged in response to the macrophage activation, including anti-cytokine, anti-chemokine, anti-complement therapies, and Janus kinase (JAK) and Bruton tyrosine kinase (BTK) inhibitors. (8,21) It is likely that, if proven active, the benefit of these therapies will be incremental, and a combination strategy with antivirals, anticoagulants, or possibly convalescent plasma, will provide a better therapeutic approach. (22)(23)(24) Tocilizumab is an IL-6 inhibitor approved for rheumatoid arthritis, giant cell arteritis, and cytokine release syndrome during CAR-T. (25,26) Preliminary data have shown that tocilizumab and other IL-6 blockers may have a role in severe SIRS cases, but its role in COVID-19 patients has not yet been confirmed, and its use should be considered experimetal. (27,28) Given its mode of action, it is plausible that tocilizumab is active in patients with already established SIRS (that could worsen), as opposed to the early use during the infection cycle with no demonstrable inflammatory activity. Thus, to test this hypothesis, we devised a trial where tocilizumab will be tested to avert the more lethal consequences of COVID-19-related SIRS. To investigate the effects of blocking an ongoing inflammatory response, we designed a randomized controlled trial comparing the efficacy of tocilizumab plus standard of care to standard of care alone in patients with moderate to severe SARS-CoV-2 infection who require supplemental oxygen and have increased systemic inflammation markers levels. The requirement of having increased levels of systemic inflammatory markers, in our hypothesis, will better select those more likely to benefit from this strategy.

Study design
The TOCIBRAS (Tocilizumab in patients with moderate to severe COVID- 19: an open-label multicentre randomized controlled" trial) was developed according to SPIRIT guidelines (Appendix 1) and registered in ClinicalTrials.gov as NCT04403685. The TOCIBRAS trial is an open-label, parallel-group, superiority, multicenter, randomized controlled trial with the primary objective to show that tocilizumab, added to the standard of care, is superior to the standard of care alone in moderate to severe cases of COVID-19 ( Figure 1).  -Coagulation studies PT/PTT, fibrinogen, vWF, ristocetin cofactor, and factor 8. Details of the methodology for all these tests is provided in appendix 3.

Interventions
The concomitant use of hydroxychloroquine, azithromycin, corticosteroids, or other therapies are allowed in this trial as part of the standard of care if local institutional guidelines/protocols include these agents as part of the standard management for COVID-19 patients. Antibiotics are permitted at any time during the study at the discretion of the treating physician. The standard of care treatment for COVID- 19 is not yet defined thus investigators can apply what is considered their standard approach for these patients per local policies.
After providing written informed consent, the eligible patients will be randomized to receive either tocilizumab plus standard of care (n = 75) or standard of care alone (n = 75). In the experimental arm, tocilizumab will be given as a single intravenous infusion at 8 mg/kg dose. The maximum dose is 800 mg. To allow for homogenous dose rounding between centers, the following bodyweightbased scale will be applied: No dose adjustment for renal or hepatic impairment will be used. Infusion-related reactions will be treated with antihistamines and corticosteroids as needed and reported as AE. All infusions will be administered in hospitalized patients in a regular ward or intensive care unit.

Randomization
The participants will be randomly assigned to either the experimental (tocilizumab) or the control (standard of care) group with a 1:1 allocation considering blocks (2, 4, 6, and 8) with random variation and using age (< 60 and ≥ 60 years) and gender as strata, according to a computergenerated randomization schedule. The random sequence will be generated by a statistician not involved with patient's care using a Software R 3.6.3 algorithm. (29) Allocation concealment is ensured by a web-accessed system (REDCap) which only displays the assigned treatment after the participant is properly registered into the trial system and all eligibility criteria are met. (30,31) Blinding Patients, investigators, and caregivers will not be blinded to the assigned treatment. Clinical outcome assessors and statisticians performing the analysis are not involved with the patient care teams and are independent of the treating sites.

Data collection, management, analysis
Study data will be collected and managed using Research Electronic Data Capture (REDCap) electronic data capture tools hosted at BP -A Beneficência Portuguesa de São Paulo. (30,31) REDCap is a secure, web-based software platform designed to support data capture for research studies, providing an intuitive interface for validated data capture; audit trails for tracking data manipulation and export procedures; automated export procedures for seamless data downloads to common statistical packages and procedures for data integration and interoperability with external sources.
Data will be entered by the local study personnel who have a unique center designated access, which is not transferable. A limited number of staff members are provided access to REDCap for study-related interactions. The data will be kept on the institutional server, under the recommended REDCap Consortium security requirements. (31) This system's functionalities include patient registration, concealment of randomization, data entry, data cleaning, data export for statistical analysis, and event adjudication. Clinically relevant laboratory assessments will be conducted in real-time, while those included in the exploratory list will be analysed in a later time, from frozen serum or plasma. Data from the primary and secondary endpoints will be included in REDCap in the appropriate time points. The independent Data Monitoring Committee (DMC) will have full access to the study data .
The follow-up data for this trial are collected until day 29. A one-year follow-up study of the hospitalized patients enrolled in all Coalition trials will keep monitoring those patients enrolled in this study accepting to participate. Patients who die within this period will be censored at the time of death, and those still hospitalized will be followed on the study until discharge before enrolling in the long-term study. Contacts by telephone and other means will be used to ensure the highest possible retention rate during follow-up. We anticipate a high retention rate up to D29, considering that most of the patients will still be recovering from their recent viral pneumonia. Adherence will be monitored by reconciling protocol specified data collection and the data entry into REDCap.

Sample size
Considering an ordinal seven stages outcome with probabilities of 30%, 20%, 8%, 8%, 4%, 15%, 15%, respectively for stages 1 to 7, under the model of proportional odds ratios for the accumulated probabilities for the outcome levels, a sample of 75 cases per arm (150 cases) has an 80% power to detect an odds ratio of 0.44, with a 5% significance level.

Statistical methods
The main analysis will follow the intention-to-treat principle. The primary endpoint will be assessed by ordinal logistic regression assuming proportional odds ratios adjusted for the stratification variables (age and gender). Logistic regression models will assess binary cumulated outcomes. If odds proportionality does not hold in the final analysis, the primary outcome will be changed to a binomial endpoint collapsing categories 1 to 5 and 6 to 7 (alive versus dead or on mechanical ventilation). Secondary outcomes will be evaluated by generalized linear regressions using appropriate distributions. All models will be adjusted for age and the results will be presented with their 95% confidence intervals for the effect measures. Subgroup analyses will be presented in forest plots evaluated with interaction terms of group and the following variables: age (< or > 60 years), sex, types of comorbidities (cardiovascular, pulmonary, hepatic, renal, obesity, high blood pressure, cancer, diabetes), and inflammatory markers increased at the time of the study entry. Analyses will be performed with R software. (29)

Monitoring
An interim analysis will be performed when 50% of the planned accrual is reached (n = 75). The interim analysis will be performed by an independent DMC who will analyze efficacy and safety data, recruitment rate, adherence to the protocol, data quality, and follow-up losses. These data will be provided by the study coordinating site to the committee via a report. The Lan DeMets method and O'Brien Fleming thresholds will be applied as pre-defined criteria for study interruption. If the committee recommends continued accrual, the study will go onto completion (n = 150).

Adverse events
Adverse events are defined as any untoward medical occurrence, including an exacerbation of a pre-existing condition, in a subject during a clinical trial. The event does not necessarily have to have a causal relationship with the treatment.
A serious AE is defined as any AE that results in death, offers immediate risk to life, results in persistent or significant disability, requires or prolongs the patient's hospitalization, or if it is a major medical event that based on proper medical judgment could threaten the patient's life or could require medical or surgical intervention to prevent one of the other results listed above. Adverse events classified as serious will be notified within 24 hours to the coordinating site.
The AEs will be graded according to the most recent version of the Common Terminology Criteria for Adverse Events (CTCAE). Monthly monitoring will evaluate the integrity of the screening, inclusion/criteria, and data reporting from the sites for adequacy and consistency. Every 15 days, a statistical methodology will be applied to look at inconsistency and data errors.
Given the biologic characteristics of tocilizumab, the following AEs will be of specific interest and will be captured: -Secondary infections. -Anemia.

Ethics and dissemination
The TOCIBRAS study was approved by local and central (national) ethical committees following current national and international guidelines/directives. The National Committee in Research and Ethics (Comissão Nacional de Ética em Pesquisa -Conep) is the entity in Brazil regulating ethical standards for clinical research. TOCIBRAS was developed and will be conducted under the normative set forth by Conep, which adopts international ethical standards in clinical research. Each participating center had the study approved at their institutional review boards before initiating protocol enrolment. A first amendment was submitted to both central and local ethical committees, dealing with issues that were not clear in the original version of the protocol. This amendment did not change the primary or secondary endpoints, the study design, statistics, safety/efficacy monitoring, Informed Consent, exclusion criteria, or interim analysis. The inclusion criteria were not unchanged, except for the limit for the time of intubation up to 24 hours before the inclusion, as the benefit of an anti-inflammatory strategy, including tocilizumab, is more likely to be seen before a fully established SIRS.
Delegated investigators will obtain Informed Consent in this study. This trial only includes adult subjects; thus no rules for minor-aged patients' consent are considere d. Once the Informed Consent is signed, it is safeguarded by the center's research personnel. When a written Informed Consent cannot be provided due to the clinical status or other logistical impediments, a verifiable audio consent will be temporarily permitted for study inclusion. The process of audio recording is applied according to Conep guidelines for COVID-19 trials, given the eventual inability to timely obtain a written Informed Consent given the pace of the pandemic and the significant restrictions for visitations and companion stays in the regular wards or the intensive care units treating for COVID-19 patients. Once the clinical circumstances allow, written Informed Consent will be signed by the patient or next of kin and added to the patient's records. The physician will discuss the risks, benefits, and caveats in participating in the trial and answer all questions. This conversation will be documented in the medical record by the delegated physician, which will reflect the patient's knowledge about the study and consent.
The TOCIBRAS is an independent investigator-initiated trial funded by the COALITION COVID-19 Brazil. The exploratory laboratory analysis will be conducted and funded by Fleury Laboratory in São Paulo. A donation from Instituto Votorantim was kindly provided for the purchase of tocilizumab for this study.
The data derived from this trial will be published regardless of the results. The dataset will be analyzed independently by statisticians not involved with the teams involved with data entry and patient care in the participating centers. The publication policy will follow that of the Coalition members. The publication venues will include medical meetings/conferences and submission to peer review journals. Depending on the results, preliminary results could be disclosed via a press release if it is determined that it is in the public's best interest. The investigators in TOCIBRAS will write the manuscript, which will be approved by all authors before any submissions.

CONCLUSION
The result of this trial will shed light on the potential role of an interleukin blocking strategy in patients with moderate to severe COVID-19. Apart from the many other anti-cytokine approaches being investigated in COVID-19, our study selects patients who manifest an ongoing systemic inflammatory response, as evidenced by serum inflammatory biomarkers, which could make this strategy more effective. We hypothesize that tocilizumab could be more active in the earlier stages of the inflammatory response (which tends to occur in the second week of the infection) before it becomes fully established with multiorgan compromise. This approach differs, for example, from antiviral strategies where the more significant benefit is anticipated in the earlier stages of the infection (first week). Therefore the study was designed to detect a difference of tocilizumab plus standard of care versus standard of care in this more select 'window' in the disease course in moderate to severe COVID-19. As with most COVID-19 studies, assumptions had to be made in regards to anticipated treatment differences given the scarcity and inconsistency in the best available data available at the time of protocol development in regards to outcomes of patients with COVID-19. Nevertheless, we included a robust design and several secondary clinical and laboratory exploratory endpoints, which will allow us to detect if there is an activity of tocilizumab in this patient population. Our work will complement all the anti-inflammatory and other approaches being developed to mitigate the consequences of COVID-19.

Strengths and limitations of this study
-The randomized controlled study design permits a more definitive conclusion regarding the activity of tocilizumab in COVID-19. -The well-defined patient population with a higher propensity to develop inflammatory complications of COVID-19 may define the subset of patients in which a potent anti-inflammatory approach is warranted. -The 7-point ordinal scale is a robust endpoint for COVID-19 trials. -The study is not blinded which presents a weakness of the study design. -The total n is relatively small for a controlled study with more limited statistical power.

AUTHORS CONTRIBUTIONS
DLC Farias, J Prats, P Scheinberg, and VC Veiga conceptualized the study, wrote the protocol, recruited patients and drafted the manuscript; AB Cavalcanti, RG Rosa, FR Machado, FG Zampieri, O Berwanger, LCP Azevedo, RD Lopes, A Avezum, L Kawano-Dourado, CG Castro Júnior participated in the protocol development and approved its final version for the COALITION COVID-19 BRAZIL Group, recruited patients, participated in interim discussions and reviewed the manuscript; CZ Oliveira developed the REDCap database and attended to all data collection related issues; LP Damiani will be performing the statistical analysis; LEC Andrade, AF Sandez, MC Pintão coordinated exploratory sample collection and will be performing the exploratory analysis, participated in the protocol development an interim discussions and reviewed the manuscript.

FUNDING
This trial was funded by the COALITION COVID-19 Brazil. The exploratory laboratory analysis will be conducted and funded by Fleury Laboratory in São Paulo, Brazil. A donation from Instituto Votorantim has been kindly provided for the purchase of tocilizumab for this study. This research received no specific grant from any funding agency in the public, commercial, or not-forprofit sectors.

Methods: Assignment of interventions (for controlled trials)
Allocation: Sequence generation 16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions 7 Allocation concealment mechanism 16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned 7 Implementation 16c Who will generate the allocation sequence, who will enroll participants, and who will assign participants to interventions 7 Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how 7 17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant's allocated intervention during the trial 7

Appendix 1 -SPIRIT 2013 checklist
Methods: Data collection, management, and analysis Data collection methods 18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol 5,6 18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols NA Data management 19 Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol Statistical methods 20a Statistical methods for analyzing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the protocol 8 20b Methods for any additional analyses (eg, subgroup and adjusted analyses) NA 20c Definition of analysis population relating to protocol non-adherence (eg, as randomized analysis), and any statistical methods to handle missing data (eg, multiple imputations)

Methods: monitoring
Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent of the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed 21b Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial 9

Harms 22
Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events and other unintended effects of trial interventions or trial conduct 9 Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent of investigators and the sponsor 8

Ethics and dissemination
Research ethics approval 24 Plans for seeking research ethics committee/institutional review board (REC/IRB) approval 2,10 Protocol amendments 25 Plans for communicating important protocol modifications (eg, changes to eligibility criteria, outcomes, analyses) to relevant parties (eg, investigators, REC/IRBs, trial participants, trial registries, journals, regulators) 10 Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorized surrogates, and how (see item 32) 10 26b Additional consent provisions for collection and use of participant data and biological specimens in ancillary studies, if applicable NA

Confidentiality 27
How personal information about potential and enrolled participants will be collected, shared, and maintained to protect confidentiality before, during, and after the trial 10 Declaration of interests 28 Financial and other competing interests for principal investigators for the overall trial and each study site 11 Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that limit such access for investigators NA Ancillary and post-trial care 30 Provisions, if any, for ancillary and post-trial care, and for compensation to those who suffer harm from trial participation NA Dissemination policy 31a Plans for investigators and sponsor to communicate trial results to participants, healthcare professionals, the public, and other relevant groups (eg, via publication, reporting in results databases, or other data-sharing arrangements), including any publication restrictions NA 31b Authorship eligibility guidelines and any intended use of professional writers 11 31c Plans, if any, for granting public access to the full protocol, participant-level dataset, and statistical code NA

Appendices
Informed consent materials 32 Model consent form and other related documentation provided to participants and authorized surrogates NA Biological specimens 33 Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in the current trial and for future use in ancillary studies, if applicable NA NA -not applicable; DMC -data monitoring committee; REC/IRB -research ethics committee/institutional review board.