Several specific experimental investigations have been performed during the COVID-19 pandemic that underlie the possibility that fluoxetine might be beneficial in the fight against the development of severe outcomes of COVID-19 infection.
2.1. Experimental Data from the Ursula Rensher Group
In a paper accepted in September 2020, before vaccines became available, the group of Ursula Rensher from Münster (Germany) reported a series of experiments in which they had investigated molecules that interfered with cholesterol accumulation in late endosomes 
The model cell lines were Vero E6 cells and polarized bronchial Calu-3 cells. The line of Vero cells was established in 1962 from the kidneys of a normal African green monkey. They are non-tumorigenic cells, widely used in vaccine production in standardized conditions 
. VeroE6 cells are widely used in experiments that study the pathomechanisms of SARS-CoV-2 infection as they can be easily infected with the virus and the released virus particles from these cells can be easily and precisely quantitated by real-time QPCR measurements 
. While Vero E6 cells are excellent for assessing viral replication, the Calu-3 cells established in 1975 from a pleural effusion of lung adenocarcinoma 
are good models for epithelial lung cell modelling.
The authors used fluoxetine, imipramine, amiodarone, and the NPC1 inhibitor U18666A. NPC1 is an intracellular cholesterol transport protein that transports low-density lipoproteins into the late endosomes, mutation of NPC1 can cause Niemann Pick type C disease. As they could show that fluoxetine was active against viral replication for influenza virus strains (EC50 = 1 µM and EC90 = 5–6 µM), they tested its effect on SARS-CoV-2 cells and found that EC90 for SARS-CoV-2 was in the range of 2–4 µM depending on the used cell lines. U18666A could reduce viral titers by 99% at the concentration of 10 µM-s. Imipramine and amiodarone were also effective on viral replication in different cell lines.
The authors investigated cholesterol accumulation in late endosomes using microscopic methods. As a positive control, they used U18666A treated cells. They could show a significant accumulation of cholesterol at a very high dose of fluoxetine of 20 µM-s, but at a lower dose of 5 µM-s the results were not significant. The authors tested the changes in the pH of the endosomes by microscopic methods. In this assay, both 5 µM and 20 µM-s of fluoxetine produced significant changes in lysosomal pH, comparable to those caused by 2–10 µM-s of U18666A.
Finally, they compared the infectivity of Vero E6 cells pre-treated with 5 µM fluoxetine or 10 µM of U18666A. Both pre-treatments produced a significant reduction of infectivity, as assessed by intracellular nucleocapsid detection with microscopic methods.
In their next study, accepted for publication in February 2021, at the time when global vaccination campaigns had just been launched, the same group published a manuscript where they argued that remdesivir, itraconazole, and fluoxetine might have synergistic effects on SARS-CoV-2 infection in vitro 
. They used the same cell lines as in their previous report. The concentration of fluoxetine used in their study was between 0.5 µM and 2.5 µM. At the highest dose of 2.5 µM fluoxetine concentration, without remdesivir co-treatment, the virus titer reduction was negligible. Although they could show synergies for both the combination of remdesivir with Itraconazole and the combination of remdesivir with fluoxetine, a potential limitation of their experimental work was that remdesivir had a very short half-life in serum. In mice experiments, remdesivir was detectable in serum only half an hour after dosing. It is to be noted that the potentially active metabolites of remdesivir could be detected in serum up to 24 h post-dosing 
. At the same time, an observation that might underlie the potential synergy between remdesivir and fluoxetine in the lung is, that in the same investigation, the concentration of remdesivir in lung tissue could be detected much longer, up to 24 h with the highest concentration at two hours. The reported concentration of remdesivir in the lung tissue was 0.35 µM. At a similar concentration of remdesivir (0.25 µM) in the Renscher study, 2.5 µM of fluoxetine produced only a modest 10% reduction in the virus titer.
In September 2021 the Reschler group published a study 
wherein they investigated the synergy of fluoxetine with the GS-441524 nucleoside analogue. GS-441524 is the main plasma metabolite of remdesivir with a plasma half-life of 24 h. Human data on GS-441524 are scarce, the current applications are mainly experimental and veterinary 
. In this paper, the Rescher group investigated the synergy between various doses of fluoxetine and GS-441524. The same Vero E6 and Calu-3 cells were used. Similar synergies were observed as for remdesivir. The highest used concentration was 2.5 µM of fluoxetine, which had marginal inhibitory effects as seen in their previous report. A combination of 2.5 µM fluoxetine with 1 µM of GS-441524 produced a 99.9% inhibition of virus production on polarized Calu-3 cells.
2.2. Experimental Data from the Jochen Bodem Group
In March 2021, the group of Jochen Bodem published a short but important paper 
. Their results confirmed the findings of the Rescher group, namely that fluoxetine dramatically reduced the viral replication of SARS-CoV-2 in the Vero E6 cell line that originates from the kidneys of a normal African green monkey. Similarly to the results of the Rensher group, in the experiments of the Bodem group, the concentration of 2.5 µM of fluoxetine resulted in the reduction of virus titer by one order of magnitude, approx. 90% inhibition, moreover, 5 µM of fluoxetine had a dramatic effect of more than three orders of magnitude reduction of the viral titer. At these concentrations of fluoxetine, no significant inhibition of cell growth was seen in Vero6 cells. Escitalopram or Paroxetine had marginal effects. The results were quantified by virus-specific QPCR and confirmed by microscopic staining. Fluoxetine did not affect other tested viruses such as RSV, Rabies, HSV-1, and HHV8.
The same paper by Bodem 
reports a very important experiment assessing the effect of fluoxetine on viral replication in normal human lung tissue preparations. Human, disease-free lung tissue slices of 300 µm width with intact peripheral airways were prepared and cultivated from samples originating from lobe resection due to cancer. The tissue slices were treated with 5 µM of fluoxetine and then infected with SARS-CoV-2. After 3 days, supernatants were harvested, and infectivity was assessed in Vero E6 cells. The resulting virus titers were quantified by QPCR. Fluoxetine treatment of the lung slices at 5 µM-s resulted in a more than two orders of magnitude reduction of viral output in the developed assay, which corresponds to more than 99% of inhibition. These fluoxetine concentrations are in line with the concentrations measured in postmortem human lung samples as described later in point 5.2 of this review.
2.3. Enantiomer Indifference of the Antiviral effect of Fluoxetine
A third very important observation is reported in the same paper of Bodem 
that addresses the stereoselectivity of fluoxetine effects. The currently used fluoxetine is the racemic mixture of both S and R enantiomers. In their report, the authors investigated the viral replication inhibitory effect of both the racemic mixture and the two enantiomers separately. In their experiments they found that there was no difference in the inhibitory effects on the virus between the two enantiomers. This observation is of extreme importance if we take into account our previous knowledge about the specifically psychiatric effects of the two fluoxetine enantiomers, which was accumulated in the 1990s. Although the enantiomers of fluoxetine were not studied extensively, the enantiomers of norfluoxetine, the metabolic product generated by demethylation in the liver and which are also active serotonin reuptake inhibitors, have been well investigated. The topic was examined in several studies assessing rat brains and complemented with studies on human platelets 
. These investigations showed that the S enantiomer of norfluoxetine was over 20 fold more potent than the R enantiomer regarding the SSRI effect of the enantiomers. Interestingly, the less effective R enantiomer, if administered orally at 80 or 120 mg/day, resulted in QT elongations on ECG measurements. These QT elongations were statistically significant, underlying the possibility that the cardiac effects of fluoxetine observed in other studies 
might involve other mechanisms beyond SSRI activity. It is to be noted that the QT elongation effect of fluoxetine, although reported, is still less pronounced compared to the similar effect of citalopram 
, where these cardiac side effects led to the development of escitalopram, which contains the S enantiomer of citalopram to reduce potential side effects. The observations on the similar inhibitory effect on SARS-CoV-2 replication of both S and R fluoxetine enantiomers, together with the 20 fold higher SSRI effect of the S enantiomer, raise the possibility of the development of an antiviral formulation based on the R enantiomer that might have fewer CNS effects. As this is the first report on the enantiomer indifference of fluoxetine antiviral effects, further studies are needed to confirm these observations.