Fluoxetine pharmacokinetics and tissue distribution quantitatively supports a therapeutic role in COVID-19 at a minimum dose of 20 mg per day

Background. Various in vitro studies have shown fluoxetine inhibits multiple variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogen causing the coronavirus disease 2019 (COVID-19) worldwide pandemic and multiple observational clinical studies have shown that patients receiving fluoxetine experienced clinical benefit by lowering the risk of intubation and death. The aim of this study is to conduct population pharmacokinetic dosing simulations to quantify the percentage of patients achieving a trough level for the effective concentration resulting in 50% (EC50) and 90% (EC90) inhibition of SARS-CoV-2 as reported in Calu-3 human lung cells. Methods. Pharmacometric parameter estimates used in this study were obtained from the U.S. FDA website from a new drug application for fluoxetine hydrochloride. A population of 1,000 individuals were simulated at standard fluoxetine antidepressant doses (20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, and 60 mg/day) to estimate the percentage of the patients achieving a trough plasma level for the EC50 and EC90 SARS-CoV-2 inhibition. All analyses were conducted in R. Results. By day-10 at 20 mg/day, 93.2% and 47% of the population will achieve the trough target plasma EC50 and EC90 concentrations, respectively, which translates to a lung tissue distribution coefficient of 60-times higher EC50 (283.6 ng/ml [0.82 mM]) and EC90 (1390.1 ng/ml [4.02 mM]). Further, by day-10 at an ideal dose of 40 mg/day, 99% and 93% of patients will reach the trough EC50 and EC90 concentrations, respectfully. Lastly, only a dose of 60 mg/day will reach the SARS-CoV-2 EC90 inhibitory concentration in the brain at pharmacokinetic steady-state. Conclusion. Overall, with a minimum treatment period of 10-days and a minimum dose of 20 mg/day, this study corroborates in vitro studies reporting fluoxetine inhibiting SARS-CoV-2 titers and also multiple observational clinical studies showing therapeutic benefit of fluoxetine in COVID-19 patients.


Introduction
Fluoxetine is a selective serotonin reuptake inhibitor (SSRI) that is a racemic mixture of two stereoisomers, R-fluoxetine and S-fluoxetine, and maintains regulatory approvals for a wide-array of clinical indications in the medical specialty of psychiatry.Several recent in vitro studies have shown that fluoxetine inhibits replication of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pathogen causing the worldwide novel coronavirus disease 2019 (COVID-19) pandemic (Schloer et al., 2020;Zimniak et al., 2020Zimniak et al., , 2021;;Dechaumes et al., 2021;Fred et al., 2021).The potential underlying mechanisms showing clinically protective factors in the SARS-CoV-2 infection is at least in-part due to fluoxetine being a functional inhibitor of acid sphingomyelinase (FIASMA)the ceramide systemwhich is reported to play a critical central role in COVID-19, as shown in both preclinical (Carpinteiro et al., 2020a(Carpinteiro et al., , 2021) ) and observational clinical studies (Darquennes et al., 2021;Hoertel et al., 2021a,c).Further, another important mechanism is that fluoxetine (sigma-1 receptor, Ki=191.2nM) and as fluvoxamine (sigma-1 receptor, Ki=17.0 nM), are endoplasmic reticulumderived sigma-1 receptor agonists, which is known to inhibit cytokine production; thereby mitigating inflammation by decreasing tumor necrosis factor (TNF-α) and is protective against clinical deterioration in sepsis (Roumestan et al., 2007;Hashimoto, 2015;Köhler et al., 2018;Rosen et al., 2019;Sukhatme et al., 2021).Similarly, Marín-Corral et al. evaluated 22 metabolomics biomarkers from plasma samples in hospitalized COVID-19 patients (n=49) in an effort to guide COVID-19 clinical decisions by disease severity and found that ceramide metabolism, tryptophan degradation, as well as reduction in nicotinamide adenine nucleotide reactions were significantly associated with respiratory severity and inflammation patients in COVID-19 patients (Marín-Corral et al., 2021).These known facts not only point to further understanding of the pathogenesis and efficacy of fluoxetine in depression, but also is suggestive of the compound being repurposed in infectious diseases.

REVISED Amendments from Version 2
In this revised version of this manuscript the following were made: 1) For all graphs, there is an additional y-axis scaled showing concentrations in micromolar.
2) In the Limitations subsection of the discussion, references are added to provide clarity the time to achieving the simulated concentrations in the organs will likely be more accurate at pharmacokinetic steady-state; more specifically at 10-days to 21-days.3) An extra figure was added to show the fluoxetine organ tissue distribution results in micromolar concentrations with horizontal dashed lines depicting the EC50 and EC90 thresholds inhibiting SARS-CoV-2 in human lung cells.4) In the discussion section, sub-headings are added for clarity for the reader.
Any further responses from the reviewers can be found at the end of the article Hoertel et al. investigated compounds classified as FIASMAs, due to the ceramide/acid sphingomyelinase system being related to SARS-CoV-2 infection, found that out of a total of 2,846 hospitalized cases (277, 9.7% taking a FIASMAbased compounds), patients with FIASMA medications were significantly associated with lower likelihood of intubation or death (HR=0.71;95% CI=0.58-0.87,p<0.001) (Hoertel et al., 2021c).Another study (n=545) by Hoertel et al. which also investigated FIASMA psychotropics against COVID-19, reported that in 164 (30.1%) patients who were treated with FIASMA-based compounds at baseline, had a significant lower risk of intubation or death (HR=0.42;95%CI=0.31-0.57;p<0.01) (Hoertel et al., 2021a).Recently, Oskotsky and colleagues reported that among SSRIs, fluoxetine alone had a statistically significant lower relative risk of mortality in comparison to COVID-19 patients who were not prescribed the SSRI (Oskotsky et al., 2021).Nemeth et al. reported in a retrospective case-control study conducted at the Uzsoki Street Teaching Hospital at the Semmelweis University found that, when compared to patients not taking fluoxetine, patients taking fluoxetine (20 mg/day) and diagnosed with COVID-19 pneumonia was associated with a 70% decrease of mortality (odds-ratio = 0. Considering the COVID-19 clinical symptoms affecting the lungs, fluoxetine lung concentrations is a critically important factor to consider when interpreting study results of SARS-CoV-2 inhibition.Johnson et al. reported human-tissue concentrations of fluoxetine in airline pilots in whole-blood ranged from 0.021-1.4μg/ml and lung concentrations ranged from 1.56 μg/ml to 51.9 μg/ml, leading to a fluoxetine distribution coefficient of 60, and is clinically relevant when investigating the pharmacokinetics of fluoxetine (Johnson, Lewis & Angier, 2007).In this context, the aim of this study is to conduct in silico population pharmacokinetic dosing simulations, which serve as the pharmacometrics standard in clinical trials and regulatory drug application submissions, to quantify the percentage of patients expected to achieve the trough effective concentration resulting in 90% inhibition of SARS-CoV-2 as found in Calu-3 human lung cells.

Pharmacokinetic model
The pharmacometrics model that incorporates differential equation variables and the respective variances for a structural one-compartment pharmacokinetic model with first-order absorption was referenced from the United State Food and Drug Administration's (FDA) Center for Drug Evaluation and Research Clinical Pharmacology and Biopharmaceutics Review(s) for a New Drug Application (NDA) 19-936 SE5-064 for Prozac (Fluoxetine Hydrochloride) that was submitted by Eli Lilly (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/18936S064_Prozac%20Pulvules_biopharmr.pdf)(Center for Drug Evaluation and Research, 2002).For this study, the pharmacokinetic parameters referenced from the NDA were calculated by the FDA after combining 3 pharmacokinetic study datasets resulting in the following pharmacometric parameters: volume of distribution (Vd) value of

Dosing simulations
To estimate the percentage of patients from a population of one thousand simulated patients who would achieve the trough target EC90 concentration, pharmacokinetic dosing of fluoxetine consisted of three dosing trials of fluoxetine: 20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, and lastly 60 mg/day.

Software and statistics
All pharmacokinetic dosing simulations are conducted with a population of 1,000 patients using mrgsolve and pharmacokinetic parameter estimates using PKNCA in R version 3.6.3(R Core Team, 2015).The overall R script has been adapted from a study published in Clinical Pharmacology and Therapeutics using hydroxychloroquine

Results
The fraction of fluoxetine bound in human plasma is 94%, which leaves only 6% of the compound being unbound in human plasma (Sommi, Crismon & Bowden, 1987).Despite fluoxetine being highly protein bound, a study by Mantinieks et al. reported in paired antemortem and postmortem cases (n=18), fluoxetine concentrations had a human whole-blood to plasma ratio of 0.8-1.0,meaning that the fluoxetine whole-blood concentrations are actually less than plasma or, at most, up to a 1:1 ratio and would not require to be scaled from plasma to whole-blood (Mantinieks et al., 2020).Moreover, Mantinieks et al. found the postmortem (range: 0.031-1.4mg/L) to antemortem (range: 0.018-0.51mg/L) fluoxetine drug concentration ratio as 1.8, but was not statistically significant as the p-value > 0.05 and thus the 1.8 ratio is not applicable to this study (Mantinieks et al., 2020).Therefore, this study directly translates the simulated fluoxetine plasma concentrations and directly applied the tissue distribution coefficients (60 for lung, 15 for brain, 10 for heart, 38 for liver, 20 for spleen, and 9 for kidneys At the peak fluoxetine concentration (Cmax), the corresponding median time Tmax is 220 hours (range, 49-220) and has a half-life (t ½)expressed as arithmetic mean (standard deviation, SD)of 46.6 hours (SD=40.8)for the study population.Further, in a population of 1,000 patients during a fluoxetine treatment period of once daily dosing for 10-days, the following are the maximum lung concentrations (Cmax-lungs): 20 mg/day (Cmax-lungs = 1950 ng/mL [5.6 μM]), 30 mg/day (Cmax-lungs=2922 ng/mL [8.4 μM]), 40 mg/day (Cmax-lungs=3900 ng/mL [11.3 μM]), 50 mg/day (Cmax-lungs=4872 ng/mL [14.1 μM], 60 mg/day (Cmax=5844 ng/mL [16.9 μM]).At all doses ranging from 20 mg/day to 60 mg/day, oral fluoxetine doses were able to exceed the target SARS-CoV-2 EC50 [0.82 μM] and EC90 [4.02 μM] inhibitory values.Detailed results of all of the maximum concentrations in the plasma, whole-blood, lungs, heart, liver, spleen, and kidney are found in Table 1.
The difference between the Cmax and Cmin in this study is approximately 20 ng/ml and to calculate the equivalent micromolar concentration, the ng/ml value is divided by the molecular weight of 345.8 g/mol.With a t ½ of 46.6 hours and summing the t ½ with the standard deviation of 40.8 hours, a portion of the population will take 21-days to reach steady-state.Further, assuming all patients are treated with fluoxetine for either 10-days or 21-days and with the last dose being on the morning of either day-10 or day-21, the amount of time it would take for the plasma concentration to fall beneath the threshold plasma EC50 (4.7 ng/ml [0.014 uM]) and EC90 (23.2 ng/ml [0.067 μM]) values that results in 60-times higher lungs (EC50=283.6ng/ml [0.82 μM]) and EC90=1390.1 ng/ml [4.02 μM]), are as follows: 20 mg/day (3.4 days to EC50; 0 days to EC90), 30 mg/day (4.2 days to EC50; 0.8 days to EC90), 40 mg/day (4.8 days to EC50; 1.5 days to EC90), 50 mg/day (5.3 days to EC50; 1.9 days to EC90), and 60 mg/day (5.7 days to EC50; 2.3 days to EC90). Figure 6 illustrates the fluoxetine pharmacokinetics over 10-days and Figure 7 shows fluoxetine pharmacokinetics throughout 21-days for once daily dosing in the morning with EC50 and EC90 threshold values.Figure 8 shows fluoxetine median organ concentrations (μM) at pharmacokinetic steady-state with EC50 (0.82 μM) and EC90 (4.02 μM) thresholds known to inhibit SARS-CoV-2.

Brain EC50 and EC90 concentrations
Extrapolating from in vitro to in vivo concentrations are dependent on intracellular versus extracellular concentrations, as well as the methodology of quantifying either whole-blood versus plasma concentrations in human pharmacokinetic studies.The EC50 and EC90 target concentrations represent the extracellular fluoxetine concentrations in the

Fluoxetine adverse drug reactions and drug-drug-interactions
According to the United States Food and Drug Administration Adverse Events Reporting System (FAERS) during the window period of 1982 to June 30, 2022, fluoxetine was reported to have a total of 85,407 cases, 67,924 serious cases, and 11,046 end of life cases (https://www.fda.gov/drugs/questions-and-answers-fdas-adverse-event-reporting-system-faers/fda-adverse-event-reporting-system-faers-public-dashboard).Females (n=49,467) represented 58% of the adverse drug reaction (ADR) cases, males (n=23,240) represented 27% of the ADRs cases, and 15% of the ADR cases did not specify (n=12,700) a gender.The most common adverse drug event reported for fluoxetine is Drug Interaction and amounts to 4,347 cases (5.1% of total).Given this information, drug interactions associated with fluoxetine are due to inhibition of the cytochrome P450 (CYP) system.Specifically, CYP2C19 and CYP2D6 may have interactions such as in patients taking tamoxifen for breast cancer by inhibiting conversion to the active endoxifen metabolite via CYP2D6 or in cases of clopidogrel in cardiology by inhibiting the conversion of clopidogrel to the active 2-oxo-clopidogrel metabolite (Spina, Trifirò & Caraci, 2012; Eugene, 2019).Overall, from a drug-safety perspective, prior to administering fluoxetine, a careful review of all current medications and clinical status by a physician clinical pharmacologist to avoid drug interactions due to fluoxetine's ability to strongly inhibit CYP2C19 and CYP2D6 (Hefner, 2018).Compounds that are sensitive and moderate CYP2C19 substrates (e.g.omeprazole, diazepam, lansoprazole, rabeprazole, voriconazole) and CYP2D6 substrates (e.g.dextromethorphan, eliglustat, nebivolol, tolterodine, encainide, metoprolol, propranolol, tramadol) will have an increased total area under the concentration-time curve of ≥ 5-fold drug exposure when treated with fluoxetine (https://www.fda.gov/drugs/druginteractions-labeling/drug-development-and-drug-interactions-table-substrates-inhibitors-and-inducers).Lastly, patients who have a pharmacogenomic profile of being a CYP2D6 Ultra-rapid Metabolizers may have sub-therapeutic fluoxetine concentrations; while, CYP2D6 Poor Metabolizer or Intermediate Metabolizers may have supratherapeutic concentrations and should be monitored for potential fluoxetine side-effects; but may also have a higher rate of achieving the target trough EC90 concentration at a 20 mg/day fluoxetine dose relative to CYP2D6 Normal (Extensive) Metabolizers.Further, of note, the over-the-counter compound famotidine (PEPCID ® ), a histamine type 2 H 2 receptor antagonists, which has been shown to inhibit the human immunodeficiency virus (HIV) (Bourinbaiar & Fruhstorfer, 1996), has also been shown to provide clinical benefit in patients with COVID-19particularly COVID-19 pneumoniaand future    suggests of the lack of knowledge of the etiologies of depression and clinical pharmacology of fluoxetine beyond serotonin transporters to inhibiting inflammatory cytokines and more that will be idientied (Kirsch & Sapirstein, 2004).

Fluoxetine dose for COVID-19 clinical trials
Optimal dose selection for clinical trials are of importance to ensure the maximum number of patients achieve the effective concentration resulting in 90% inhibition of the SARS-CoV-2 pathogen similarly to that reported in Calu-3 human lung cells.Based on the study findings showing the percentage of the population achieving the EC50 and EC90 inhibitory concentrations in the lungs, a dose of 40 mg/day would be recommended to achieve therapeutic potential inhibiting SARS-CoV-2 titers.Overall, given the abundance of clinical trial data and published clinical studies investigating various dosing approaches (e.g.loading dose) for fluoxetine, this author defers all final dosing recommendations for fluoxetine to the FDA approved package-insert for adults and for the pediatrics population (Eli Lilly / Dista Products Company, 2021).For an application in pediatrics, fluoxetine doses in children are recommended to start at 10 mg/day and then titrate to 20 mg/day, whereas in adults with depression, a 20 mg/day is recommended as a starting dose (Eli Lilly/Dista Products Company, 2021).Lastly, given pharmacogenomic (drug-gene), DDIs, and drug-drug-gene interactions that are likely in patients taking multiple medications, therapeutic drug monitoring of fluoxetine and other co-administered medications are recommended.

Study limitations
A limitation of this study is in respect to how fast the trough concentrations are achieved which assumes that fluoxetine immediately distributes to lung tissue at the estimated 60-fold concentration compared to plasma levels.There is some evidence that suggests fluoxetine may accumulate into tissues over time as is reported by Erb et al. who found that certain antidepressants accumulate in lipid rafts in vitro over several days of exposure (Erb, Schappi & Rasenick, 2016).In reference to human brain fluoxetine concentrations, Karson et al. used spectroscopy and determined that fluoxetine and the norfluoxetine metabolite are not detectable in human brain at about 1 week, but actually detectable at about 2-to 3weeks of treatment the concentrations were able to be assayed indicating that fluoxetine accumulates in brain over time (Karson et al., 1993).Therefore, with this evidence, it is likely that the fluoxetine lung concentrations would not be evident as quickly as reported in the simulations and the data should be interpreted assuming the target EC50 and EC90 concentrations inhibiting SARS-CoV-2 at fluoxetine's pharmacokinetic stead-state concentrationsthat is between 10-days and 21-days of treatmentand patients should in-turn be treated for 21-days with fluoxetine in order to have a valid clinical trial with a dose of at least 20 mg/day and ideally at 40 mg/day.
Another limitation of this study is that the results are purely quantitative using pharmacometrics simulations based on differential equations and not a randomized controlled clinical trial.Despite this limitation, it is important for the reader to note that the methodology used in this study is standard practice for all drugs undergoing Phase-1 Clinical Trials in drug development by clinical pharmacologists for later regulatory approval for human use.Moreover, the pharmacometric parameters used were estimated from an NDA submitted to the United States Food and Drug Administration with parameter estimates actually calculated by the FDA clinical pharmacologists as reported in the Clinical Pharmacology and Biopharmaceutics Review after combining three human study datasets provided by the study sponsor (https://www.accessdata.fda.gov/drugsatfda_docs/nda/2003/18936S064_Prozac%20Pulvules_biopharmr.pdf).

Conclusions
This study investigated fluoxetine pharmacokinetics and human organ distribution which confirmed that previously published median effective concentrations and specifically, the EC90 fluoxetine value inhibiting SARS-CoV-2 in Calu-3 human lung cells are achievable using standard fluoxetine antidepressant doses (20 mg/day, 30 mg/day, 40 mg/day, 50 mg/day, and 60 mg/day) and also corroborates findings from multiple retrospective clinical studies showing patients who were exposed to fluoxetine during COVID-19 were associated with reduced risk of clinical deterioration and death.Overall, assuming patients are not treated with medications that result in significant DDIs or have the clinically relevant pharmacogenomic concerns, a minimum dose of 20 mg/day for at least 10-days inhibits SARS-CoV-2 viral titers, but a dose of 40 mg/day would be ideal for clinical trials evaluating fluoxetine in COVID-19 due to efficient lung distribution at a 60-times higher concentration.
This project contains the following underlying data: • Data File 1: v2_fluoxetine_20mg_PO_QAM.csv

Eero Castren
Neuroscience Center, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki, Finland The author has only partially responded to the comments I gave to the previous version.It is also very difficult to see what changes have actually been made to the version 1.

Eero Castren
Neuroscience Center, Helsinki Institute of Life Science HiLIFE, University of Helsinki, Helsinki, Finland Andy Eugene has written an interesting paper on a very timely topic.Preclinical as well as limited clinical data support the idea that some antidepressants and related compounds may protect cells from SARS-CoV2 infection, but it has been unclear whether concentrations that are found in cell culture studies to be inhibitory are really achieved in clinical treatments.It is unfortunate that there is only limited amount of high-quality data on the tissue concentrations and distribution of fluoxetine in human tissues, but Eugene has taken advantage of the data that is available to model fluoxetine pharmacokinetics in human lung tissue.His results suggest that sufficiently high lung tissue concentrations are achieved within a 10 day treatment period in majority of patients with a standard 20 mg/day dosing, and with higher doses, the trough concentration is achieved within the first days of treatment.The simulations appear properly conducted and the manuscript is well written.I have a few issues that might improve the manuscript further.
The conclusions about how fast trough concentrations are achieved is based on the assumption that fluoxetine immediately distributes to lung tissue at the estimated 60 fold concentration compared to plasma levels.However, there is some (although clearly incomplete) information that fluoxetine may accumulate into tissues over time.Rasenick and colleagues have found that certain antidepressants accumulate in lipid rafts in vitro over several days of exposure (Erb et al. 2016 1 ) For human brain, Karson et al. have found that, using spectroscopy, fluoxetine/norfluoxetine is not detectable in human brain at about 1 week, but at about 2-3 weeks of treatment, concentrations of several micromolar can be assayed (Karson et al. (1993 2 )) indicating that fluoxetine accumulates in brain over time.If the same is true for lung tissue, the 60-fold concentrations may not be reached in lung tissues as quickly as estimated here.Although paucity of information prevents any proper estimations, I think it would be important to discuss this issue in the discussion section. 1.
The author should be complemented for indicating in many places in the manuscript fluoxetine concentrations in both ng/ml and in molar units, however, this is not done systematically.It would be very helpful to, wherever possible, indicate both units.In the figures, it may not be feasible to have double set of units in the Y-axis, I would prefer molar units, but if the author prefers ng/ml, that is not a major problem.

2.
The author may want to mention that a clinical phase 3 trial evaluating the effect of fluvoxamine vs. placebo is ongoing and should be completed by September this year.
3.  On a more general, and potentially more innovative front, it is one of a small (but quickly increasing) number of studies contributing to this topic and the first study to the reviewer's knowledge addressing this specific pharmacokinetic issue, which is an important area of inquiry with major implications in this context of pandemic.There is much to like about this manuscript.

Yes
The results are presented clearly, the methods are sound, the discussion follows well from it, and the manuscript is very well written.Below are several points that would strengthen the submission:

Title:
A minor suggestion would be a modification of the title to increase the interest to this important study, such as "Fluoxetine pharmacokinetics and tissue distribution suggest a possible therapeutic role in COVID-19 at usual antidepressant dose".

Introduction:
The introduction section should be updated and include a description of results from recent studies focused on the association between antidepressant use and the course of COVID-19, which would be very helpful to readers to understand the clinical context of this study and why this study (and its results) are important.In addition to the medRxiv one, the reference to the published article of Moreover, a recent study (Marín-Corral et al. (2021 16 )) showed that ceramide plasma concentration is associated with inflammatory markers and clinical outcomes in COVID-19 patients.I think that results from all those studies should also be recognized and shortly summarized in the Introduction.

○
The abstract might include some of these points listed above.

Results:
Importantly, results from that study can help guide the choice of the right dose to use in COVID-19 clinical trials.I would strongly recommend the author to present in the result section as well as in the abstract (for main findings) for each following dose: 20 mg/d, 30mg/d (which is also interesting for clinical tolerability purpose), 40 mg/d and 60 mg/d the percentage of patients reaching a) EC90 and b) EC50 at (i) 1 day, (ii) 2 days, and (ii) 3 days.In addition, given that 40 mg/d might lead to increased risk of mild side effects among older adults and that at the same time reaching the efficient dose as fast as possible is desirable given the potential antiviral properties of fluoxetine, would a loading dose of 30 mg or 40 mg the first or two first days, followed by a dose of 20 mg/day, perform better than only taking 20 mg/d?Finally, is there a way to ensure that at no point of the 10 day treatment course, no patient (typically older adults) will reach a toxic plasma dose with the different scenarios, as I think 40 mg/d is the maximum recommended dose in the elderly population?Even if the reviewer recognizes that it may represent a substantial additional amount of work, these detailed results will certainly be of great help to clinicians to balance the risks associated for each dose and the potential benefits, and help them design ideal clinical trial.

○
With the different scenarios, at the end of the 10-day treatment, how long would it take to have a substantial decrease in the fluoxetine plasma concentration (and its metabolite norfluoxetine)?This information would give an idea of the duration of potential protection of the treatment against SARS-CoV-2 once stopped and of the required duration of medical follow-up.

Conclusion:
Finally, with these different dose scenarios, what recommendation(s) of dose would do the author for a clinical trial including fluoxetine for COVID-19?Would it be desirable to do a loading dose then decrease to the standard 20 mg/d dose which is known to be very well tolerated in older adults?Or keeping 30 mg/d or 40 mg/d throughout the trial would be the best choice?
I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com 33 [95% CI, 0.16-0.68,p=0.002]) (Németh et al., 2021).Most recently, in a large state psychiatric hospital operated by the New York State Office of Mental Health, Clelland et al. showed patients treated with fluoxetine (p=0.023) or trazodone (p = 0.001) had statistically significant lower risk of infection with COVID-19, while there was a trend of higher risk of infections with patients treated with the atypical antipsychotic olanzapine (p=0.084)(Clelland et al., 2022).Two clinical trials showed another SSRI antidepressant fluvoxaminealso a FIASMA-based compound with sigma-1 receptor agonist propertieshad clinical benefit against COVID-19 (Lenze et al., 2020; Seftel & Boulware, 2021).Lenze et al. reported in a double-blind randomized clinical trial (n=152) between April 10, 2020 and August 5, 2020, that none (0 of 80) of the patients who received fluvoxamine (n=80) at 100 mg/day compared to 8.3% (6 of 72) of patients receiving placebo (n = 72) three-times per day for 15 days experienced clinical deterioration (absolute difference=8.7%,95% CI, 1.8%-16.4%,p=0.009) (Lenze et al., 2020).Seftel et al. found in a prospective cohort study, none (0 of 65) of the patients who received fluvoxamine 50 mg twice daily (100 mg/day) versus 12.5% (6 of 48) of patients who were observed alone were hospitalized and by day-14, residual COVID-19 symptoms were evident in none (0 of 65) of patients treated with fluvoxamine versus 60% (29 of 48) of patients who observed alone (Seftel & Boulware, 2021).
(Al-Kofahi et al., 2020).Statistical results providing percentage estimates are calculated from trough concentrations of patients achieving the effective concentrations and is referenced from the Schloer et al. study reporting the EC50 and EC90 values in human-lung Calu-3 cells (Schloer et al., 2020).
) from the Johnson et al. study and the original preprint version of this manuscript is updated to account for the findings from Mantinieks et al. (Johnson, Lewis & Angier, 2007; Eugene, 2020, 2021; Mantinieks et al., 2020).The target EC90 endpoint lung concentration for fluoxetine is 1390.1 ng/ml[4.02μM] and 1/60 of this concentration is the new EC90-plasma concentration of 23.2 ng/ml [0.067 μM].Similarly, the fluoxetine SARS-CoV-2 lung EC50 is 283.6 ng/ml [0.82 μM] and 1/60 of the EC50 at the target lung concentration in the plasma results in 4.7 ng/mL.The percentage of the 1,000 simulated patients are illustrated in the following figures: Figure1(20 mg/day), Figure2(30 mg/day), Figure3(40 mg/day), Figure4(50 mg/day), and Figure5(60 mg/day) with a horizontal dashed-line throughout the pharmacokinetic dosing figures showing the required trough EC50 and EC90-plasma levels that translates to the 60-times higher lung concentrations.

Figure 1 .
Figure 1.Fluoxetine population (n=1,000) dosing simulation results for an oral dose of 20 mg/day for 10 days.The shaded regions illustrate the 10th (lower) and 90th (upper) percentiles with the solid line within the shaded region representing the median fluoxetine concentration.The dashed horizontal line depicts the effective concentration resulting (A) 50% and (B) 90% inhibition (EC90) of SARS-Cov-2 that will result in 60-times higher level in the lungs.

Figure 2 .
Figure 2. Fluoxetine population (n=1,000) dosing simulation results for an oral dose of 30 mg/day for 10 days.The shaded regions illustrate the 10th (lower) and 90th (upper) percentiles with the solid line within the shaded region representing the median fluoxetine concentration.The dashed horizontal line depicts the effective concentration resulting (A) 50% and (B) 90% inhibition (EC90) of SARS-CoV-2 that will result in 60-times higher level in the lungs.

Figure 3 .
Figure 3. Fluoxetine population (n=1,000) dosing simulation results for an oral dose of 40 mg/day for 10 days.The shaded regions illustrate the 10th (lower) and 90th (upper) percentiles with the solid line within the shaded region representing the median fluoxetine concentration.The dashed horizontal line depicts the effective concentration resulting (A) 50% and (B) 90% inhibition (EC90) of SARS-CoV-2 that will result in 60-times higher level in the lungs.

Figure 4 .
Figure 4. Fluoxetine population (n=1,000) dosing simulation results for an oral dose of 50 mg/day for 10 days.The shaded regions illustrate the 10th (lower) and 90th (upper) percentiles with the solid line within the shaded region representing the median fluoxetine concentration.The dashed horizontal line depicts the effective concentration resulting (A) 50% and (B) 90% inhibition (EC90) of SARS-CoV-2 that will result in 60-times higher level in the lungs.

Figure 5 .
Figure 5. Fluoxetine population (n=1,000) dosing simulation results for an oral dose of 60 mg/day for 10 days.The shaded regions illustrate the 10th (lower) and 90th (upper) percentiles with the solid line within the shaded region representing the median fluoxetine concentration.The dashed horizontal line depicts the effective concentration resulting (A) 50% and (B) 90% inhibition (EC90) of SARS-CoV-2 that will result in 60-times higher level in the lungs.

Figure 6 .
Figure 6.Fluoxetine concentration-time profile in human plasma at standard daily antidepressant doses (20 mg/day, 30 mg/day, 40mg/day, 50 mg/day, 60 mg/day) throughout 10 days.The vertical line illustrates the morning dose on day-10, the highest (EC90) and lowest (EC50) horizontal lines illustrates the threshold for the plasma effective concentrations inhibiting 50% and 90% of SARS-CoV-2 titers that distributes to 60-times higher concentrations within the lungs, respectively.

Figure 7 .
Figure 7. Fluoxetine concentration-time profile in human plasma at standard daily antidepressant doses (20 mg/day, 30 mg/day, 40mg/day, 50 mg/day, 60 mg/day) throughout 21 days.The two vertical line illustrates the morning doses on day-10 and on day-21.The highest (EC90) and lowest (EC50) horizontal lines illustrates the threshold for the plasma effective concentrations inhibiting 50% and 90% of SARS-CoV-2 titers that distributes to 60-times higher concentrations within the lungs, respectively.

© 2021
Hoertel N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Nicolas Hoertel 1 Hôpital Corentin-Celton, Service de Psychiatrie, Assistance Publique-Hôpitaux de Paris (AP-HP), Issy-les-Moulineaux, France 2 Paris University, Paris, France This is a review of the manuscript "Fluoxetine pharmacokinetics and tissue distribution suggest a possible role in reducing SARS-CoV-2 titers" submitted for publication in F1000Research.This is a very interesting and important study, which addresses a timely and important question -in a context of a growing number of preclinical and clinical studies showing a potential efficacy of certain antidepressants, and particularly fluoxetine, in COVID-19, what dose of fluoxetine should be optimally prescribed in clinical trials to ensure a maximum of patients achieve the effective concentration resulting in 90% inhibition (EC90) of SARS-CoV-2 as reported in Calu-3 human lung cells.
trough target plasma concentrations will be referenced from the Schloer et al. study who reported after a 48-hour incubation period in Calu-3 lung cells (Schloer et al., 2020) which are significantly higher than the EC90 in Vero E6 cell results from Zimniak et al. study (Schloer et al., 2020; Zimniak et al., 2020, 2021).
(Hoertel et al., 2020(Hoertel et al., , 2021b)) are capable of achieving the aforementioned EC50s for all of the aforementioned microbes and fluoxetine may be combined with antipsychotics (e.g.olanzapine) to treat bipolar depression, treatment-resistant depression, schizophrenia in general and these antipsychotics may have notable associations with sedation and somnolence (Eugene et al., 2021).Direct clinical translation of this current pharmacokinetic study supports the findings from a retrospective multicenter observational study by Hoertel et al., who found a median fluoxetine dose of 20 mg/day resulted in a significantly lower risk of intubation and death in a population composed of 63% women and 37% men(Hoertel et al., 2020(Hoertel et al., , 2021b)).
Comparing the Hoertel et al. and Zimniak et al. publications, Hoertel et al. found that in addition to fluoxetine, venlafaxine (median dose of 75 mg/day) and escitalopram (median dose of 10 mg) were also associated with a lower risk of intubation and death; however, Zimniak et al. showed that neither escitalopram nor paroxetine inhibited SARS-CoV-2 in vitro (Hoertel et al., 2020, 2021b; Zimniak et al., 2020, 2021).There have been authors who have suggested that the effect of fluoxetine in treating depression is due to a placebo effect; however, with all due respect, it The R programming language pharmacokinetic script that produces the general results of this study are available for download at: https://doi.org/10.17605/OSF.IO/RVYPZ (Eugene, 2021).

Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Partly Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Reviewer Report 21 June 2021 https://doi.org/10.5256/f1000research.56641.r87881