A Randomized, Controlled Study on the Safety and Ecacy of Maraviroc and/or Favipiravir vs Currently Used Therapy in Severe COVID-19 Adults. “COMVIVIR” Trial.

Multiple studies have established that hyperinammatory response induced by SARS CoV-2 is a main cause of complications and death in infected subjects. Such dysfunctional immune response has been described as a dysregulated and exacerbated production of cytokines and chemokines that attracts and activates inammatory cells, which start and sustain pulmonary and systemic damage, thus causing complications that lead to multi organ failure and death. Therefore, we suggest that blocking key inammation receptors could help to reduce migration and activation of T cells, monocytes/macrophages and neutrophils, thus mitigating the cytokine dysregulation and averting severe complications and death. Importantly, the optimum treatment for COVID-19 severe patients should combine a modulator of the immune response plus a direct antiviral drug against SARS-CoV-2, in order to address both the hyperinammatory effects of the immune dysregulation and the viral load. Methods: Maraviroc (MVC), a CCR5 antagonist, and Favipiravir (FPV), an antiviral, will be evaluated single and combined, added to the treatment currently used at the Hospital General de México Dr. Eduardo Liceaga for severe COVID-19 patients. One hundred patients will be allocated in four arms [Current treatment only (CT), CT+MVC, CT+FPV, CT+MVC+FPV]. Percentage of patients free of mechanical ventilation or death at day 28, immunophenotyping and viral load will be compared between groups. Discussion: New immune focused therapies are targeting strong inammation mediators such as IL-6 and IL1-β; nevertheless, to our best knowledge, only one study explores chemotaxis control. The use of a drug therapy that addresses both the regulation of the immune response and the inhibition of viral replication could at the same time, help to alleviate the hyperinammatory condition and reduce the time of the viral clearance process, therefore improving treatment outcomes.


Introduction
The COVID-19 (Coronavirus Disease 2019) pandemic caused by infection of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) has caused a global mortality surrounding 3% (1). Multiple studies have found that the hyperin ammatory response induced by SARS-CoV-2 is one of the main causes of severity and death. In severe COVID-19 patients, an association was found between pneumonitis and/or ARDS (Acute Respiratory Distress Syndrome), high serum levels of proin ammatory cytokines, extensive lung damage and microthrombosis (2). The late stage of the disease is di cult to manage and many patients die (3,4). Based on the reports of the Chinese Disease Control Center, Cascella and cols. (5) classi ed the patients according to clinical severity in three groups (Table 1).
It has been proposed that the critical course of the disease is caused by an exacerbated and poorly understood immune response, linked to the phenomenon known as "cytokine storm" or cytokine release syndrome (6). Albeit it is not completely clear what initiates and propagates the cytokine storm, the severity of COVID-19, combined with rapid pandemic spread, has placed unprecedented pressure on the global healthcare system, and therapeutic strategies are urgently needed.
Pathological studies of patients lethally infected by COVID-19 had reported acute pulmonary edema, abundant in ltration of in ammatory cells, multiple organ failure, thromboembolic complications and septicemia (7,8). One of the mechanisms that could precede functional and tissue damage is the in ltration of in ammatory cells, triggered by the release of chemokines (9, 10).
Gene studies in lung samples identi ed overexpression of CCL2 and CCL3 chemokines (11). Furthermore, Huang and cols. (12) reported that besides leukopenia and lymphopenia, hospitalized patients had higher plasma concentrations of CCL3 and CCL4 upon admission than healthy subjects. They also mention that SARS and MERS physiopathology is outlined by an increase of proin ammatory cytokines and chemokines in serum (IL-Iβ, IL-6, IL-12, IFN-γ and CCL2). Previously, serum CXCL10 and CCL7 were identi ed as predictors of progression (13). Hence, it is of notice that the cytokine storm is accompanied by chemokine-induced migration of white cells, particularly CCL2, CCL3, CCL7 and CXCL10.
In respiratory diseases, it has been shown that CCR5 is involved in neutrophil recruitment to the lungs (15). In that sense, in human subjects with chronic pulmonary in ammatory diseases, in ltrated neutrophils overexpress CCR5, induced by activation of TLRs and NOD2 (16). Neutrophils' in ltration in pulmonary capillaries, alveolar extravasation and neutrophilic mucositis have already been observed in . Despite the precise mechanism that drives such in ltration remains unknown, it is feasible that CCR5 may play a critical role in the immunopathology of COVID-19 (18).
Along with phagocytosis and oxidative burst, neutrophils have another resource to eliminate pathogens: NETosis, a distinct form of programmed, necrotic cell death characterized by the neutrophilic release of network organized protein and DNA structures known as "neutrophil extracellular traps" (NETs), which are able to capture and entangle such pathogens (19). Though bene cial against pathogens, NETs could stimulate certain disease processes (20). Excessive formation of NETs could trigger a chain of in ammatory reactions that destroys surrounding tissue and facilitates micro thrombosis (21). Previous reports associate aberrant formation of NETs to pulmonary disorders, namely ARDS (22). The increase in D dimer described as a severity marker in COVID-19 severe patients, could be related to NETosis, since it has an essential role in the start and progression of thrombosis in veins and arteries (23). Hence, all these neutrophil functions could be part of both the tissue damage and microthrombosis in COVID-19.
As previously mentioned, the chemokines increased in COVID-19 severe patients are CCL3, CCL5 and CCL7. All these are CCR5 ligands; thus, our group hypothesizes that a CCR5 blockade could prevent leukocyte migration to the lung and attenuate the cytokine storm, and can be considered a therapeutic target (24). Moreover, a monoclonal antibody targeted against CCR5 (Leronlimab, also known as PRO140) was able to restore lymphocyte levels and decrease IL-6 in 10 COVID-19 patients (18).
One of these drugs is Maraviroc (MVC) (25), an oral CCR5 antagonist, mainly used as an anti-retro viral that impedes binding of the gp120 protein to CCR5, thus avoiding viral internalization. (26). MVC has not been widely studied in the context of reduction of hyperin ammatory conditions. However, some reports have found interesting effects in modulation or resolving of general in ammatory conditions, such as reduction of cytokine expression by in vitro human adipocytes (27), and as an alleviating agent of hemorrhage-induced hepatic injury in rats by a PPAR-γ depending pathway (28). Furthermore, it was used in a phase II study to minimize the graft vs. host disease in bone marrow stem cell transplant in pediatric patients (29). It has also been observed that MVC decreases mucosal in ammation (30), VCAM-1 (31), T cell in ltration, neuroin ammation (32) and endothelial dysfunction (33). Regarding the lung, a model of induced hemorrhagic shock in rats reported that MVC has a protective role against pulmonary damage (34). All the aforementioned, along with the broad safety range of MVC, good tolerance an low incidence of adverse effects (35) makes it an excellent candidate to be used as a modulator of the dysregulated immune response in COVID-19.
On the other hand, in silico studies (36,37), aimed to nd possible candidates for the treatment of SARS-CoV-2, and found that MVC could have a direct antiviral activity by binding to the main protease of the virus (Mpro). Other in silico studies propose that MVC could have an antiviral effect based on structural similarity to inhibitors of viral proteins (38), and on its binding capacity to the hinge site receptor binding motif (RBM), and fusion peptide (FP) (39). Moreover, there is also a report of MVC inhibiting SARS-CoV2 multiplication in cell culture (40). Altogether, this body of evidence suggests that MVC could not only block the CCR5-dependent migration to the lung, but also reduce the viral load.
We hypothesized that an effective treatment for COVID-19 severe patients should combine a modulator of the immune response with a direct antiviral drug against SARS-CoV-2, in order to address both the hyperin ammatory effects of the immune dysregulation and the viral load, thus yielding best results. Favipiravir (FPV), directly inhibits viral replication and transcription by selective inhibition of the viral enzyme RNA-dependent RNA polymerase (RdRP) (41)(42)(43)(44).
FPV has been used successfully against A H1N1 in uenza (45,46). Regarding COVID- 19, an open randomized study in 80 mild patients found that FPV reduced the time of viral clearance by 50% compared to Lopinavir/Ritonavir with less adverse effects (47). Another open randomized study in moderate patients reported FPV to be more effective in clinical recovery compared to Arbidol (48). An in vitro study found that FPV is capable to suppress the SARS-CoV-2 infection at high concentrations (49). A clinical study combining FPV with methylprednisolone for severe COVID-19 pneumonia reported good results (50). Finally, to date there are more than 30 studies registered in the U.S. National Library of Inclusion criteria These will con rm the selected subjects for enrollment.
Normal hepatic function, de ned as a maximum of a vefold increase of transaminases.
Signed informed consent.
Women in fertile capability must accept the use of a contraceptive method for 90 days after treatment completion.  Therefore, 25 patients will be allocated by group: 100 total.

Randomization
Subjects will be randomized using EPIDAT v. 4.2 considering strati cation by sex.

Statistical analysis
For the calculation of the e cacy, all those patients that completed the study with no major deviations from the foreseen results (improvement, progression to critical or death) will be considered as the Population per Protocol (PP). In order to avoid bias by the effect of termination of treatment due to causes different from the foreseen ones, the modi ed intention to treat (mITT) will be calculated, based on the criterion of at least one dose taken and at least one measurement after basal of the endpoints.
The primary endpoint (PFS for mechanical ventilation or death) [time frame: day 28] will be analyzed using a Kaplan-Meier Log Rank model to compare the 4 arms.
The secondary endpoints will be compared between arms as follows: Change rate in the patterns of activation, tra cking and exhaustion in peripheral blood lymphocytes, monocytes and neutrophils [Time frame: day 10-1]: Data will be analyzed for subpopulations, frequencies (%) and mean uorescence intensities (MFI) for each molecule and their interactions.
By subsets: Gating using Flowing Software By clusters: PCA using RStudio Data will be controlled by confounding factors (BMI, sex, hypertension, and diabetes).
IBM SPSS version 21 and RStudio v. 3.6.3 will be used for the statistical analysis. A con dence interval of 95% and a signi cance level of p ≤ 0.05 will be considered.

Procedure
Patients eligible by screening criteria are invited to participate in the protocol. Upon acceptance, a detailed explanation is given, and signature of a written informed consent form is requested. One copy of the signed informed consent is kept in the patient's le, and one more is given to the patient.
If the con rmatory tests for inclusion criteria show that not all are met, or any exclusion criterion is found, the subject is informed and is not recruited.
Once participation is con rmed, patients are registered in the data collection sheet and undergo a general clinical and medical record assessment. Samples for blood count, blood chemistry and blood gases are collected, as well as saliva for viral load and blood for evaluation of immune status. A general approach to the study is presented in Table 2 NPE will be used to measure SARS-CoV-2. Blood samples will consist of 25ml: for cytokines and chemokines measurement on serum and activation and tra cking markers on leukocytes. Saliva, plasma, and leukocytes samples will be transported to the BSL3 laboratory of CIENI-INER (Research Center for Infectious Diseases at the National Respiratory Diseases Institute) for these analysis.
Participation of the subjects will end upon discharge by improvement or death. If patients progress to critical condition, tablets will be crushed and administered using a nasogastric tube.
Even in the occurrence of anticipated discharge or death, the patient's information is kept and processed for data analysis, as long as the patient or their relatives do not retire their consent. Likewise, participation of the subjects is terminated before term if a complication or adverse effect occurs that can be related to the drug under study and is ruled so by the responsible physician. If that is the case, the necessary medical support is given to correct the adverse effect, and the patient is kept in observation.
Reporting of AE/SAE is done by trial physicians on a daily basis in compliance with the current procedure of the Hospital, which demands reporting to the Pharmacovigilance, Ethics, and Biosafety Committees of the Hospital within the rst 24 hours. In turn, the Pharmacovigilance Committee informs the Uppsala Monitoring Centre.

Follow up
Discharged patients are contacted by telephone on a weekly basis until day 28. Questions are addressed for symptoms and possible adverse effects.
The study will be considered nished when all the enrolled subjects calculated in the sample size complete their participation.

Discussion
In this study, we propose the use of MVC and/or FPV as a therapeutic resource to prevent or ameliorate the immune dysregulation, decrease the viral load and reduce complications and death in severe patients in risk of progressing to critical. The cells accountable for such dysregulation express receptors like CCR5, which mediate their tra cking to the lungs. Therefore, it is considered a therapeutic target.
In recent studies with other CCR5 inhibitors such as Leronlimab, a signi cant decrease in peripheral blood levels of CCL5, IL-6 and IL-1β, as well as in ammation and viral load has been observed. This could be related with a reduction of in ammation markers like ferritin, D-dimer and CRP (54,55). Therefore, MVC could, as mentioned before, prevent the tra cking of pro-in ammatory leukocytes to the lungs through CCR5 blockade in a similar manner. Nevertheless, MVC has a technical advantage, because unlike Leronlimab, it is an orally administered tablet that does not require special conditions for transport, handling and administration.
MVC is a CCR5 antagonist that could also have an antiviral effect, albeit it has not been evaluated in the context of COVID-19. Alongside, the results could be potentiated by the direct effect of an antiviral such as FPV. It has been reported that FPV decreases the viral load by day 4-7 (47), which leads us to think that an early intervention would bring better results for the arms where FPV is going to be evaluated . Moreover, it has been observed that there exists a relationship between viral load and severity (56).
Despite newly developed SARS-CoV-2 vaccines are being massively applied, current evidence suggests that COVID-19 is a disease that will coexist with mankind for a very long period of time. Moreover, pharmacologic strategies used for treatment have not proven completely effective.
Thence, more studies of different therapeutic targets are needed to search for resources that help to improve the patients' prognosis. The use of a drug therapy that addresses both the regulation of the immune response and the inhibition of viral replication could at the same time, help to alleviate the hyperin ammatory condition and reduce the time of the viral clearance process. This study is intended to compare the therapy currently used at Hospital General de Mexico "Dr. Eduardo Liceaga" to treat COVID-19 on severe non-critical patients, which does not comprise either an in ammation modulator or an antiviral, to a strategic therapy focused on the aforementioned mechanisms of pathogenicity.   Figure 1 Overall design of the study and description of the arms, main events and sampling days. CT: Currently used treatment. MVC: Maraviroc. FPV: Favipiravir. NPE: Nasopharyngeal exudate