The aim of this study was not to prove the efficacy of INDO for the treatment of SARS-CoV-2 but to show that INDO could be considered as a promising candidate for the treatment of patients infected with the SARS-COV-2 virus based on the evidence derived from studies conducted in-vitro, in animals, and on the model-based simulations.
The Food and Drug administration (FDA) approved INDO in 1965 under the brand name Indocin ®. INDO is approved to treat moderate to severe osteoarthritis, rheumatoid arthritis, and ankylosing spondylitis. Today, several drug manufacturers make generic versions of the drug. INDO was also found to have significant anticancer activity against a wide variety of cancer cell types, in vitro and in vivo [29, 30]. INDO performs its anticancer activity in different fashions, inhibits proliferation via induction of apoptotic death of tumor cells [28, 30], reduces tumorigenesis by enhancing the immune response [31, 32] and inhibiting the angiogenesis  as well. Recently, extensive studies on various cancer cell types including colorectal carcinoma justified the efficacy of INDO to reduce the levels of anti-apoptotic proteins and progressive cell proliferation [34, 35].
Coronavirus is an envelope virus with four structural proteins: spike (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein . S protein is responsible for the virus attachment and entry to the target cells, which initiate the infection process. S protein plays key roles in the induction of protective humoral and cellular immunity during SARS-CoV. This is the reason why the pseudovirus model that contains the SARS-CoV-2 spike was considered as the most attractive target for SARS-CoV vaccine and therapeutic development [36,38].
In-vitro, INDO was found to be active against several viruses, including SARS-CoV-1 virus, and SARS-CoV-2 pseudovirus [11, 16]. This antiviral activity was also shown in CCoV and in several other RNA-viruses suggesting a cellular rather than a viral target for the drug [11, 16].
The in-vitro results presented in this paper indicated that INDO is active in the human SARS-CoV-2 pseudovirus at low micromolar range, that a good correlation exists between viral load and rate of response in CCoV infected dogs, that INDO has a similar in-vitro inhibitory effect on SARS-CoV-2 pseudovirus and CCov, and that a good expectation exists for the human performance of INDO in the treatment of patients infected with SARS-CoV-2 .
A meta-analysis recently conducted on 9 studies including laboratory-confirmed 1426 patients affected by SARS-CoV-2 suggests that there were mild or severe cytokine storm in severe patients, which was an important cause of death. Therefore, the treatment of cytokine storm has become an important part of rescuing severe patients. Interleukin-6 (IL-6) plays an important role in cytokine release syndrome. If a drug can block the signal transduction pathway of IL-6, it is expected to become a new treatment of severe patients .
To explore the role of IL-6 in the SARS-CoV-2 infection, the CORIMUNO-TOCI trial was conducted using tocilizumab (clinicaltrials.gov reference NCT04331808). Tocilizumab is a blocker of IL-6R, which can effectively block IL-6 signal transduction pathway [40, 41]. The results of this study have yet to be published, but the outcomes were reported in a press release. It included patients who were hospitalized with SARS-COV-2 moderate to severe pneumonia in intensive care or at high risk of requiring intensive care but did not need resuscitation upon admission. The study included 129 patients who were randomized to either usual treatment plus tocilizumab (n=65) or usual treatment alone (n=64).
One of the interesting pharmacological effects of INDO is its modulation of cytokine production  and its robustly effect on the reduction of proinflammatory IL-6 as shown in a study conducted in mice where a decrease of 75% to 80% of IL-6 has been observed . This feature in combination with the antiviral properties of INDO on human SARS-CoV-1, canine CCoV, and SARS-CoV-2 [11, 16], further highlight how INDO could be used to potentially aid the fight against the coronavirus.
Different simulations were conducted to evaluate the expected performances of different dosage regimens using the time during which the INDO exposure remain above the effective concentration as a criterion for assessing efficacy.
Two thresholds for the effective INDO concentration were considered: the first one (best case scenario) was associated with the concentration at which pseudovirus replication is inhibited by 50 percent (i.e. 0.358 mg/L) and the second one (worst case scenario) associated with the concentration at which virus replication is inhibited by 95 percent (i.e. 1.074 mg/L).
The results of the analysis indicated that the 75 mg SR BID dosage regimen was expected to deliver an improved clinical benefit given the larger time/day during which the exposure was expected to remain above the effective concentration following this dosage regimen.
The doses used in the simulations were selected based on the recommended dosage regimen of INDO reported in the label for the IR  and the SR formulations . The recommended dosage for the IR formulation is 25 mg BID or TID. If this is well tolerated, the daily dosage can be increased by 25 mg or by 50 mg, but the total daily dose should not exceed 200 mg. INDO SR, 75 mg once a day can be substituted for INDO IR capsules, 25 mg three times a day; and INDO SR, 75 mg BID can be substituted for INDO IR 50 mg three times a day.
Among the limitations that could affect the assessment of the INDO effect:
1) The in-vitro test showed that INDO had a directly and potently antiviral activity against the SARS CoV-2 pseudovirus and not on the SARS Cov-2 virus. The pseudovirus model that contains the SARS-CoV-2 spike was used to study the S/receptor interactions and this test is not necessarily indicative of an effect on virus replication;
2) The assumption that the broad range effects on coronaviruses (e.g., SARS-CoV-1, CCoV, and SARS-CoV-2) can be used to predict the INDO efficacy in the treatment of SARS-CoV-2;
3) The assumption that INDO inhibits viral replication of human SARS-CoV-2 at the same concentration that inhibits CCoV;
4) There are no deep pools of the virus and what is in the blood reflects what is in the target;
5) The assumption that the time above the effective concentration is the driver of the efficacy;
6) The limited number of dogs used to assess viral load and efficacy in animal studies;
7) The use of the proportionality criterion in the translational model for estimating the clinical response in humans.
The limitations of the present analyses mainly concern the reliability and the predictive performances of the outcomes of the animal and in-vitro studies. The relevance of these studies mainly concerns the proof of mechanism of new candidate drugs. These studies provide only indicative information on the potential performances of new treatments in controlled clinical trials as shown by the outcomes of recent clinical trials on HCQ and remdesivir [44, 45, 46]. Two drugs with an in-vitro potency very closed to the in-vitro potency of INDO .