Comparative genomics and characterization of SARS-CoV-2 P.1 (Gamma) Variant of Concern (VOC) from Amazonas, Brazil (preprint)

Background P.1 lineage (Gamma) was first described in the State of Amazonas, northern Brazil, in the end of 2020, and has emerged as a very important variant of concern (VOC) of SARS-CoV-2 worldwide. P.1 has been linked to increased infectivity, higher mortality and immune evasion, leading to reinfections and potentially reduced efficacy of vaccines and neutralizing antibodies. Methods The samples of 276 patients from the State of Amazonas were sent to a central referral laboratory for sequencing by gold standard techniques, through Illumina MiSeq platform. Both global and regional phylogenetic analyses of the successfully sequenced genomes were conducted through maximum likelihood method. Multiple alignments were obtained including previously obtained unique human SARS-CoV-2 sequences. The evolutionary histories of spike and non-structural proteins from ORF1a of northern genomes were described and their molecular evolution was analyzed for detection of positive (FUBAR, FEL, and MEME) and negative (FEL and SLAC) selective pressures. To further evaluate the possible pathways of evolution leading to the emergence of P.1, we performed specific analysis for copy-choice recombination events. A global phylogenomic analysis with subsampled P.1 and B.1.1.28 genomes was applied to evaluate the relationship among samples. Results Forty-four samples from the State of Amazonas were successfully sequenced and confirmed as P.1 (Gamma) lineage. In addition to previously described P.1 characteristic mutations, we find evidence of continuous diversification of SARS-CoV-2, as rare and previously unseen P.1 mutations were detected in spike and non-structural protein from ORF1a. No evidence of recombination was found. Several sites were demonstrated to be under positive and negative selection, with various mutations identified mostly in P.1 lineage. According to the Pango assignment, phylogenomic analyses indicate all samples as belonging to the P.1 lineage. Conclusion P.1 has shown continuous evolution after its emergence. The lack of clear evidence for recombination and the positive selection demonstrated for several sites suggest that this lineage emergence resulted mainly from strong evolutionary forces and progressive accumulation of a favorable signature set of mutations.

Proxalutamide Improves Inflammatory, Immunologic, and Thrombogenic Markers in Mild-to-Moderate COVID-19 Males and Females: an Exploratory Analysis of a Randomized, Double-Blinded, Placebo-Controlled Trial Early Antiandrogen Therapy (EAT) with Proxalutamide (The EAT-Proxa Biochemical AndroCoV-Trial) (preprint)

Background: The androgen theory on COVID-19 is based on the fact that males, in particular when affected by androgenetic alopecia, and females with hyperandrogenic states are more severely affected by COVID-19, while chronic users of antiandrogens experiment lower rates of COVID-19 complications. The theory finds plausibility on the androgen-mediated transmembrane protease serine 2 (TMPRSS-2), a key protein for SARS-CoV-2 cell entry. We demonstrated reduction of hospitalization rate using a potent non-steroidal antiandrogen (NSAA), proxalutamide, in both females and males COVID-19 outpatients. In this joint exploratory analysis, we aimed to demonstrate whether the efficacy of proxalutamide on mild-to-moderate COVID-19 could be justified by improvements in inflammatory, immunologic, and thrombogenic responses. Materials and methods: This is a joint post-hoc analysis of two double-blind, placebo-controlled two-arm randomized clinical trials (RCTs) on proxalutamide 200mg/day for seven days for female and male COVID-19 outpatients, respectively, compared to standard of care (SOC), of hematocrit, neutrophils lymphocytes, eosinophils, platelets, neutrophil-to-lymphocyte (N/L) ratio, ferritin, fibrinogen, D-dimer, ultrasensitive C-reactive protein (usCRP) lactate 1-hour erythrocyte sedimentation rate (1hESR), total testosterone, estradiol, sex hormone binding globulin (SHBG), oxygen saturation and heart rate measured on days 0, 1 and 7. Results: A total of 445 subjects were enrolled (268 males and 177 females) between October 21th 2020 and February 28th 2021, with similar baseline characteristics. Neutrophils were lower in proxalutamide group in Day 1 (p = 0.005) and Day 7 (p < 0.0001). Lymphocytes were higher in the proxalutamide group in Day 7 (p = 0.0001). Eosinophils were higher in the proxalutamide arm in Day 1 (p = 0.04) and Day 7 (p < 0.00010. In Day 7, platelets were higher in proxalutamide arm (p = 0.03). Ferritin levels were lower in proxalutamide arm in Day 7 (p = 0.03) Fibrinogen levels were lower in proxalutamide group in Days 1 and 7 (p < 0.0001 for both days). D-dimer levels were lower in proxalutamide group in Days 1 and 7 (p < 0.0001 for both days). UsCRP levels were reduced in proxalutamide group in Day 7 (p < 0.0001). 1hESR) was reduced in proxalutamide arm in Day 1 (p = 0.0009) and Day 7 (p < 0.0001). In males, testosterone levels were higher in proxalutamide group in Day 1 (p = 0.048) and Day 7 (p = 0.0001). In females, testosterone levels were higher in proxalutamide group in Day 7 (p = 0.018), and estradiol levels were higher in proxalutamide arm in Day 1 (p = 0.044). Oxygen saturation was higher in proxalutamide in Day 1 (p = 0.0006) and Day 7 (p < 0.0001). Conclusions: The substantial improvements observed in immunologic, inflammatory, thrombotic and oxygen markers with proxalutamide may support the reduction of hospitalization rate observed in both females and males with COVID-19 using proxalutamide, compared to standard of care.

Proxalutamide (GT0918) Reduces the Rate of Hospitalization in mild-to-moderate COVID-19 Female Patients: A Randomized Double-Blinded Placebo-Controlled Two-Arm Parallel Trial. (preprint)

Background: Antiandrogens were shown to be effective in mild-tomoderate COVID-19 male patients, supported by the SARS-CoV-2 dependency on transmembrane protease, serine 2 (TMPRSS2), which is solely modulated by androgens, for cell entry. While women with hyperandrogenism experiment more symptoms in COVID-19 compared to women without hyperandrogenism, and the chronic use of an antiandrogen seemed to mitigate these symptoms, whether the benefits would be observed in overall females is unknown. The objective of this study is to evaluate the efficacy of proxalutamide as a treatment for mild-to-moderate COVID-19 in females. Methods: This was a randomized, double-blind, placebo-controlled, two-arm, parallel study on COVID-19 female outpatients, that compared the use of proxalutamide versus placebo. The primary outcome was hospitalization rates throughout 30 days after randomization. Patients with laboratory confirmed COVID-19 not hospitalized were recruited in two sites in Brasilia, Brazil, between January 4 and February 28, 2021, were randomized on a 2:3 ratio between proxalutamide and placebo, and were administered proxalutamide 200mg/day or placebo for seven days, in addition to usual care. Results: A total of 177 women were randomized, including 75 in the proxalutamide arm and 102 patients in the placebo arm. None of the patients lost follow-up or discontinued treatment. The 30-day hospitalization rate was 2.7% in the proxalutamide arm and 18.6% in the placebo arm (p<0.001), with a hospitalization risk ratio (RR) of 0.14 [95% confidence interval (CI), 0.03-0.59]. Conclusions: These findings suggest that treatment of COVID-19 patients with proxalutamide in combination with standard of care was reduced hospitalization rate by 86% (p < 0.001), with no safety concerns.

Proxalutamide (GT0918) Improves Lung Injury in Hospitalized COVID-19 Patients - an Analysis of the Radiological Findings of the Proxa-Rescue AndroCoV Trial (preprint)

Introduction: Antiandrogen are good candidates against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease (COVID-19) due to the inhibition of its entry into host cells by the suppression of TMPRSS2, an enzyme that primes the SARS-CoV-2 spike (S) protein and is key for its cell entry. Proxalutamide is a second-generation nonsteroidal anti-androgen (NSAA) with strong activities on androgen receptor (AR) antagonism, suppression of AR nuclear expression, and downregulation of the membrane-attached angiotensin converting enzyme-2 (ACE2). The efficacy of proxalutamide was previously demonstrated for early COVID-19 patients, and has now demonstrated efficacy to reduce deaths in hospitalized COVID-19 patient in a double-blind, placebo-controlled randomized clinical trial (RCT). Whether radiological changes would follow the improvement in clinical outcomes with proxalutamide is not established. The present post-hoc analysis aims to evaluate whether proxalutamide improves lung injury observed through chest computed tomography (CT) scans, in addition to the clinical improvement, thus providing further objective evidence of the drug response in COVID-19. Methods: This is a post-hoc analysis of the radiological findings of a double-blinded, placebo-controlled, prospective, two-arm RCT (The Proxa-Rescue AndroCoV Trial) with all enrolled patients from the three participating institutions of the city of Manaus, Amazonas, Brazil, that had at least two chest CT scans during hospitalization. The quantification of lung parenchyma involvement was performed by independent board-certified radiologists with expertise in analysis of COVID-19 images, that were blind to the assigned intervention in the RCT. A first chest CT scan was performed upon randomization and a second CT scan was performed approximately five days later, whenever patient transportation was feasible. Results: Of the 395 patients initially evaluated, 72 and 179 patients from the proxalutamide and placebo arms, respectively, were included (n=251). Baseline and clinical characteristics, interval between first and second chest CT scans, and percentage of lung parenchyma affected in the baseline chest CT scan were similar between groups. In the second chest CT scan, the percentage of lungs affected (Median - IQR) was 35.0% (25.0-57.5%) in the proxalutamide group versus 67.5% (50.0-80.0%) in the placebo group (p < 0.001). The absolute and relative change between the second and first chest CT scans (Median - IQR) were -15.0 percent points (p.p.) (-30.0 - 0.0p.p.) and -25.0% (-50.0 - 0.0%) in the proxalutamide group, respectively, and +15.0p.p. (0.0 - +30.0p.p.) and +32.7% (0.0 - +80.0%) in the placebo group, respectively (p < 0.001 for both absolute and relative changes). Conclusion: Proxalutamide improves lung opacities in hospitalized COVID-19 patients when compared to placebo. (NCT04728802)

Proxalutamide (GT0918) Reduction of Mortality Rate in Hospitalized COVID-19 Patients Depends on Treatment Duration - an Exploratory Analysis of the Proxa-Rescue AndroCoV Trial (preprint)

Introduction: Proxalutamide, a second generation non-steroidal antiandrogen (NSAA), primarily developed for castration-resistant prostate cancer, demonstrated reduction in 28-day mortality rate of 77.7% in hospitalized COVID-19 patients in a double-blind, placebo-controlled, two-arm randomized clinical trial (RCT), through intention-to-treat (ITT) analysis. However, the intriguingly high 28-day mortality rate of patients that did not complete the 14-day treatment with proxalutamide, compared non-completers of the placebo arm and overall placebo arm, raised the hypotheses of the existence of non-neglectable differences between ITT and on-treatment (OT) analysis in terms of drug efficacy. Despite the inherent limitations of OT analysis, we aimed to respond to unanswered questions regarding the drug efficacy when the 14-day treatment with proxalutamide was complete, and secondarily understand the causality relationship between treatment interruption and mortality rate. Methods: This is a post-hoc exploratory analysis of a double-blinded, randomized, placebo-controlled, prospective, multicentric, two-arm RCT of 300mg-daily 14-day proxalutamide therapy for hospitalized COVID-19 patients not requiring mechanical ventilation, using OT population, i.e., excluding patients that did not complete treatment or interrupted at least 24 hours before death. Patients above 18 years old with confirmed COVID-19 not presenting kidney, liver, or heart failure were eligible. The primary outcome was 8-point COVD-19 ordinal scale at day 14. Secondary outcomes included 28-day 8-point COVID-19 ordinary scale, 14-day and 28-day all-cause mortality rate, and median hospital length. Results: In total, 580 patients completed the 14-day treatment or died during treatment, including 288 patients in the proxalutamide arm and 292 patients in the placebo arm, with similar baseline characteristics between groups. The 28-day all-cause mortality rate was 4.2% in the proxalutamide group and 49.0% in the placebo group. The mortality risk ratio (RR) was 0.08 (95% CI, 0.05-0.15), with a number needed to treat (NNT) of 2.2 to prevent death. The median hospital length stay after randomization was 05 days (interquartile range - IQR = 3 to 7.2 days) in the proxalutamide group and 09 days (IQR = 6 to 15 days) in the placebo group (p < 0.001). The 28-day all-cause mortality rate of patients that received proxalutamide but interrupted treatment before 14 days was 79.3%, while those that received placebo and interrupted before 14 days was 52.8% (p = 0.054 between groups). Conclusion: The reduction in 28-day all-cause mortality rate with 14-day proxalutamide treatment for hospitalized COVID-19 patients was more significant treatment completers (92%), compared to the reduction when all patients enrolled in the proxalutamide arm were considered (77.7%). However, the magnitude of statistical significance of the reduction in all-cause mortality and the NNT were similar between the OT and ITT analysis. The apparent high mortality risk rate with early interruption of proxalutamide treatments suggests that strategies for treatment compliance should be reinforced for future RCTs with proxalutamide. (NCT04728802)

Efficacy of Proxalutamide in Hospitalized COVID-19 Patients: A Randomized, Double-Blind, Placebo-Controlled, Parallel-Design Clinical Trial (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is mediated by the androgen-promoted protease, transmembrane protease, serine 2 (TMPRSS2). Previously, we have shown that treatment with proxalutamide, a non-steroidal androgen receptor antagonist, accelerates viral clearance and clinical remission in outpatients with coronavirus disease 2019 (COVID-19) compared to placebo. The effects in hospitalized COVID-19 patients were unknown. Methods: Men and women hospitalized but not requiring mechanical ventilation were randomized (1:1 ratio) to receive 300 mg of proxalutamide per day or placebo for 14 days. The study was conducted at eight sites in the state of Amazonas, Brazil. The primary outcome measure was the clinical status (8-point ordinal scale) at 14-days post-randomization. The primary efficacy endpoint was the 14-day recovery ratio (alive hospital discharge [scores 1, 2]). Findings: A total of 645 patients were randomized (317 received proxalutamide, 328 placebo) and underwent intention-to-treat analysis. The 14-day median ordinal scale score in the proxalutamide group was 1 (interquartile range [IQR]=1-2) versus 7 (IQR=2-8) for placebo, P<0.001. The 14-day recovery rate was 81.4% for proxalutamide and 35.7% for placebo (recovery ratio, 2.28; 95% CI 1.95-2.66 [P<0.001]). The 28-day all-cause mortality rate was 11.0% for proxalutamide versus 49.4% for placebo (hazard ratio, 0.16; 95% CI 0.11-0.24). The median post-randomization time to recovery was 5 days (IQR=3-8) for proxalutamide versus 10 days (IQR=6-15) for placebo. Interpretation: Hospitalized COVID-19 patients not requiring mechanical ventilation receiving proxalutamide had a 128% higher recovery rate than those treated with placebo. All-cause mortality was reduced by 77.7% over 28 days. (ClinicalTrials.gov number, NCT04728802).

Efficacy of Proxalutamide (GT0918) in Hospitalized COVID-19 Patients (preprint)

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is mediated by the androgen-promoted protease, transmembrane protease, serine 2 (TMPRSS2). Previously, we have shown that treatment with proxalutamide, a non-steroidal androgen receptor antagonist, accelerates viral clearance and clinical remission in outpatients with coronavirus disease 2019 (COVID-19) compared to placebo. The effects in hospitalized COVID-19 patients were unknown. Methods: Men and women hospitalized but not requiring mechanical ventilation were randomized (1:1 ratio) to receive 300 mg of proxalutamide per day or placebo for 14 days. The study was conducted at eight sites in the state of Amazonas, Brazil. The primary outcome measure was the clinical status (8-point ordinal scale) at 14-days post-randomization. The primary efficacy endpoint was the 14-day recovery ratio (alive hospital discharge [scores 1, 2]). Findings: A total of 645 patients were randomized (317 received proxalutamide, 328 placebo) and underwent intention-to-treat analysis. The 14-day median ordinal scale score in the proxalutamide group was 1 (interquartile range [IQR]=1–2) versus 7 (IQR=2–8) for placebo, P<0.001. The 14-day recovery rate was 81.4% for proxalutamide and 35.7% for placebo (recovery ratio, 2.28; 95% CI 1.95–2.66 [P<0.001]). The 28-day all-cause mortality rate was 11.0% for proxalutamide versus 49.4% for placebo (hazard ratio, 0.16; 95% CI 0.11–0.24). The median post-randomization time to recovery was 5 days (IQR=3–8) for proxalutamide versus 10 days (IQR=6–15) for placebo.Interpretation: Hospitalized COVID-19 patients not requiring mechanical ventilation receiving proxalutamide had a 128% higher recovery rate than those treated with placebo. Clinical Trial Registration Details: ClinicalTrials.gov number, NCT04728802Funding Information: Kintor Pharmaceuticals, Ltd.Declaration of Interests: Kintor Pharmaceuticals, Ltd. manufactures and plans to market proxalutamide, and has an investigational new drug (IND) application under United States Food and Drugs Administration to conduct a Phase 3 study for proxalutamide for COVID19. Applied Biology, Inc. has patents pending regarding antiandrogen therapy for COVID19. Dr. Goren, Dr. McCoy, and Dr. Li are employees of Applied Biology, Inc. Dr. Cadegiani has served as a clinical director for Applied Biology, Inc. Dr. Wambier has served as an advisor to Applied Biology, Inc. The other authors have no conflict of interest to declare.Ethics Approval Statement: The study was approved by an ethics committee and registered in clinicaltrials.gov (NCT04728802), and also approved by Brazilian National Ethics Committee, approval number 4.513.425; CAAE 41909121.0.0000.5553; Comitê de Ética em Pesquisa (CEP) of the Comitê Nacional de Ética em Pesquisa (CONEP) of the Ministry of Health (MS). (CEP/CONEP/MS).

Clinical diagnosis of COVID-19: a prompt, feasible, and sensitive diagnostic tool for COVID-19 based on a 1,757-patient cohort (The AndroCoV Clinical Scoring for COVID-19 diagnosis) (preprint)

Importance In the COVID-19 pandemic, a limiting barrier for more successful approaches to COVID-19 is the lack of appropriate timing for its diagnosis, during the viral replication stage, when antiviral approaches could demonstrate efficacy, precluding progression to severe stages. Three major reasons that hamper the diagnosis earlier in the disease are the unspecific and mild symptoms in the first stage, the cost- and time-limitations of the rtPCR-SARS-CoV-2, and the insufficient sensitivity of this test as desired for screening purposes during the pandemic. More sensitive and earlier methods of COVID-19 detection should be considered as key for breakthrough changes in the disease course and response to specific therapeutic strategies. Our objective was to propose a clinical scoring for the diagnosis of COVID-19 (The AndroCoV Clinical Scoring for COVID-19 Diagnosis) that has been validated in a large population sample, aiming to encourage the management of patients with high pre-clinical likelihood of presenting COVID-19, at least during the pandemics, independent of a rtPCR-SARS-COV-2 test. Materials and methods This is a compounded retrospective and prospective analysis of clinical data prospectively collected from the Pre-AndroCoV and AndroCov Trials that resulted in a clinical scoring for COVID-19 diagnosis based on likelihood of presenting COVID-19 according to the number of symptoms, presence of anosmia, and known positive household contact, in a variety of combinations of scoring criteria, aiming to the detect scorings that provided the highest pre-test probability and accuracy. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and accuracy were calculated for subjects screened in two different periods and altogether, for females, males, and both, in a total of nine different scenarios, for combinations between one, two, or three or more symptoms, or presence of anosmia in subjects without known positive household contacts, and no symptoms, one, two, or three or more symptoms, or presence of anosmia or ageusia in subjects with known positive household contacts. Results 1,757 patients were screened for COVID-19. Among the multiple combinations, requiring two or more symptoms with or without anosmia or ageusia for subjects without known contact and one or more symptoms with or without anosmia or ageusia with known positive contacts presented the highest accuracy (80.4%), and higher pretest probability and accuracy than virtually all rtPCR-SARS-CoV-2 commercially available kit tests. Conclusion The AndroCoV Clinical Scoring for COVID-19 Diagnosis was demonstrated to be a feasible, quick, inexpensive and sensitive diagnostic tool for clinical diagnosis of COVID-19. A clinical diagnosis of COVID-19 should avoid delays and missed diagnosis, and reduce costs, and should therefore be recommended as a first-line option for COVID-19 diagnosis for public health policies, at least while SARS-CoV-2 is the prevailing circulating virus. Key Points Question Is a clinical diagnosis of COVID-19 sensitive, accurate, and feasible? Findings The present analysis of a 1,757-subject cohort of the AndroCoV trials demonstrated that clinical scoring for COVID-19 diagnosis can deliver a more sensitive and prompter diagnosis than the current gold-standard diagnostic method, rtPCR-SARS-CoV-2, with an accuracy above 80%. Meaning A clinical diagnosis of COVID-19 avoids missed diagnosis due to insufficient sensitivity or incorrect timing of the performance of rtPCR-SARS-CoV-2, reduces costs, avoid delays on specific managements, and allows the testing of potentially effective antiviral therapeutic approaches that should work if administered in the early stage of COVID-19

Proxalutamide (GT0918) Reduces the Rate of Hospitalization for COVID-19 Male Outpatients: A Randomized Double-Blinded Placebo-Controlled Trial. (preprint)

Background: Antiandrogens have demonstrated a protective effect for COVOD-19 patients in observational and interventional studies. The goal of this study was to determine if proxalutamide, an androgen receptor antagonist, could be an effective treatment for men with COVID-19 in an outpatient setting. Study design and methods: A randomized, double-blinded, placebo-controlled clinical trial was conducted at two outpatient centers (Brasilia, Brazil). Patients were recruited from October 21 to December 24, 2020 (ClinicalTrials.gov number, NCT04446429). Male patients with confirmed COVID-19 but not requiring hospitalization (COVID-19 8-point ordinal scale <3) were administered proxalutamide 200mg/day or placebo for up to 7 days.  The primary endpoint was hospitalization rate at 30 days post-randomization.Results: A total of 268 men were randomized in a 1:1 ratio.  134 patients receiving proxalutamide and 134 receiving placebo were included in the intention-to-treat analysis.  The 30-day hospitalization rate was 2.2% in men taking proxalutamide compared to 26% in placebo, P<0.001. The 30-day hospitalization risk ratio was 0.09; 95% confidence interval (CI) 0.03 to 0.27.  Patients in the proxalutamide arm more frequently reported gastrointestinal adverse events, however, no patient discontinued treatment.  In placebo group, 6 patients were lost to follow-up, and 2 patients died from acute respiratory distress syndrome.Conclusion: The hospitalization rate in proxalutamide treated men was reduced by 91% compared to usual care. Trial Registration: NCT04446429

Dutasteride Reduces Viral Shedding, Inflammatory Responses and Time-to-Remission in COVID-19: Biochemical Findings of a Randomized Double-Blind Placebo Controlled Interventional Trial (DUTA AndroCoV-Trial - Biochemical). (preprint)

Importance: SARS-CoV-2 cell entry and infectivity is indirectly dependent on androgenic status and phenotype through the regulation of transmembrane protease serine 2 (TMPRSS2), an androgen-mediated proteolytic enzyme that facilitates SARS-CoV-2 entry.  Males, particularly those affected by androgenetic alopecia (AGA) are overrepresented in severe COVID-19, while the use of 5-alpha-reductase inhibitors (5ARis), an antiandrogenic drug class, may reduce COVID-19 severity. Objective: Our objective was to determine if dutasteride, a wide and potent 5ARi, would bring biochemical and virological benefits in early COVID-19.Design, Setting, and Participants: A double-blinded, randomized, prospective, investigational study of dutasteride for the treatment of COVID-19, as add-on therapy to the local standard of care, for mild or moderate, non-hospitalized subjects confirmed for SARS-CoV-2 (The Duta AndroCoV Trial).Interventions:  Dutasteride 0.5mg/day or placebo for 30 days or until full COVID-19 remission. Nitazoxanide was given 500mg twice a day for six days and azithromycin was given 500mg/day for five days for all subjects.Main Outcome(s) and Measure(s): Remission times for fatigue, ageusia, anosmia, and overall disease, oxygen saturation (%), real-time polymerase chain reaction (rtPCR-SARS-CoV-2), ultrasensitive C-reactive protein (usCRP), D-dimer, lactate, dehydrogenase lactate (DHL), erythrocyte sedimentation rate (ESR), ultrasensitive troponin and ferritin.Results:  Compared to placebo group (n=44) with similar baseline characteristics, dutasteride (n=43) presented reduced fatigue, anosmia and overall disease duration (46.6%, 49.6% and 43.2% lower duration, respectively; p<.0001 for all), and in Day 7 presented higher rates of virologic cure (64.3% versus 11.8% cure; p=.0094), , increased recovery rate (84.7% versus 57.5%; p=.03), higher mean [SD] oxygen saturation (97.0% [1.4%] versus 95.7% [2.0%]; p=.02), lower median [IQR] usCRP (0.34mg/L [0.23mg/L -0.66mg/L] versus 1.47mg/L [0.70mg/L-3.37mg/L]; p<.0001),  lower median [IQR] lactate (2.01mmol/L [1.12mmol/L-2.43mmol/L] versus 2.66mmol/L [2.05mmol/L-3.55mmol/L]; p=.0049), lower median [IQR] ESR (5.0mm/1h [3.0mm/1h-11.0mm/1h] versus 14.0mm/1h [7.25mm/1h-18.5mm/1h]; p=.0007), lower median [IQR] LDH (165U/L [144U/L -198U/L] versus 210U/L [179U/L-249U/L]; p=.0013 and lower median [IQR] troponin levels (0.005ng/mL [0.003ng/mL-0.009ng/mL] versus 0.007ng/mL [0.006ng/mL-0.010ng/mL]; p=.048).Conclusions and Relevance: These findings suggest that dutasteride reduces clinical and virologic disease duration and inflammatory markers in males with mild-to-moderate, early-stage COVID-19, and should be considered as a therapeutic option in the current context of the COVID-19 pandemic.Trial Registration: NCT04446429

Clinical Diagnosis of COVID-19: a Prompt, Feasible, Costless, and Highly Sensitive Diagnostic Tool for COVID-19 Based on a 1,757-Patient Cohort (The AndroCoV Clinical Scoring for COVID-19 Diagnosis). (preprint)

Importance: A major barrier for successful therapeutic approaches to COVID-19 is the inability during the viral replication stage, when drugs with potential antiviral activity could demonstrate efficacy and preclude progression to more severe stages. Reasons that hamper an earlier diagnosis of COVID-19 include the unspecific and mild symptoms during the first stage, the delay in the diagnosis and specific management caused by the requirement of a rtPCR-SARS-CoV-2 for the diagnosis of COVID-19, and the insufficient sensitivity of the a rtPCR-SARS-CoV-2, oppositely to what is recommended for a screening test during an outbreak. More sensitive and earlier diagnostic tools for COVID-19 should be unraveled as a key strategy for an breakthrough change in the disease course and response to specific therapies, particularly those that target the blockage of viral shedding. We aimed to create an accurate, sensitive, easy-to-perform and intuitive clinical scoring for the diagnosis of COVID-19 without the need of a rtPCR-SARS-CoV-2 (termed as The AndroCoV Clinical Scoring for COVID-19 Diagnosis), resulted from a 1,757 population cohort, eo eventually encourage the management of patients with high pre-clinical likelihood of presenting COVID-19, independent of a rtPCR-SARS-COV-2 test, to avoid delays and loss of appropriate timing for potential therapies.Materials and methods: This is a post-hoc analysis of clinical data prospectively collected of the Pre-AndroCoV and AndroCov Trials, that resulted in scorings for clinical diagnosis of COVID-19, based on the likelihood of presenting actual COVID-19 according to the number of symptoms, presence of anosmia, and known positive household contact. Sensitivity, specificity, positive predictive value, negative predictive value, positive likelihood ratio, and accuracy were calculated for subjects screened in two different periods and both periods together, for females, males and both, in a total of nine different scenarios, according to combinations between one, two, or three or more symptoms, or presence of anosmia in subjects without known positive household contacts, and no symptoms, one, two, or three or more symptoms, or presence of anosmia or ageusia in subjects with known positive household contacts. Scorings that yielded the highest pre-test probability, sensitivity and accuracy were selected.Results: Of the 1,757 patients screened, 1,284 were diagnosed for COVID-19. The scoring that required: 1. Two or more symptoms, or anosmia or ageusia alone, for subjects without known contact; or 2. One or more symptoms, including anosmia or ageusia alone, when with known positive contacts presented the highest accuracy (80.4%) among all combinations attempted, and higher sensitivity (85.7%) than rtPCRSARS-CoV-2 commercially available kit tests.Conclusion: The AndroCoV Clinical Scoring for COVID-19 Diagnosis demonstrated to be a feasible, easy, costless, and sensitive diagnostic tool for clinical diagnosis of COVID-19. Because clinical diagnosis of COVID-19 avoids delays in specific treatments, particularly for high-risk populations, prevents false-negative diagnosis, and reduces diagnostic costs, this diagnostic tool should be considered as an option for COVID-19 diagnosis, at least while SARS-CoV-2 is the prevailing circulating virus and vaccination rate is below the required for herd immunity.

5-Alpha-Reductase Inhibitors Reduce Remission Time of COVID-19: Results From a Randomized Double Blind Placebo Controlled Interventional Trial in 130 SARS-CoV-2 Positive Men (preprint)

BackgroundSARS-CoV-2 entry into type II pneumocytes is depended on the TMPRSS2 proteolytic enzyme. The only known promoter of TMPRSS2 in humans is an androgen response element. As such, androgen sensitivity may be a risk factor for COVID-19. Previously, we have reported a retrospective cohort analysis demonstrating the protective effect of 5-alpha-reductase inhibitors (5ARis) in COVID-19. Men using 5ARis were less likely to be admitted to the ICU than men not taking 5ARis. Additionally, men using 5ARis had drastically reduced frequency of symptoms compared to men not using 5ARis in an outpatient setting. Here we aim to determine if 5ARis will be a beneficial treatment if given after SARS-CoV-2 infection. MethodsA double-blinded, randomized, prospective, investigational study of dutasteride for the treatment of COVID-19 (NCT04446429). Subjects confirmed positive for SARS-CoV-2 were treated in an outpatient setting. Endpoints for the study were remission times for a predetermined set of symptoms: fever or feeling feverish, cough, shortness of breath, sore throat, body pain or muscle pain/ache, fatigue, headache, nasal congestion, nasal discharge (runny nose), nausea or vomiting, diarrhea, loss of appetite, and loss of taste or smell (Ageusia or Anosmia). ResultsA total of 130 SARS-CoV-2 positive men were included in the study, 64 subjects in the dutasteride arm and 66 subjects in the placebo-controlled group. The differences in the average remission times for fatigue and anosmia or ageusia was statistically different between the groups (5.8 versus 10.1 days for fatigue and 7.3 versus 13.4 days for anosmia or ageusia, in dutasteride and placebo groups, respectively), however, the total remission time was significantly reduced for the men given dutasteride; 9.0 days versus 15.6 days in the placebo group (p < 0.001). Excluding loss of taste and smell the average total remission time was 7.3 days in the dutasteride group versus 11.7 in the placebo arm (p < 0.001). ConclusionThe total remission time for men using 5ARis was significantly reduced compared to men not taking 5ARis.

Early COVID-19 Therapy with Azithromycin Plus Nitazoxanide, Ivermectin or Hydroxychloroquine in Outpatient Settings Significantly Reduced Symptoms Compared to Known Outcomes in Untreated Patients. (preprint)

Background: While there was a lack of pharmacological interventions proven to be effective in early, outpatient settings for COVID-19, in a prospective, open-label observational study (pre-AndroCoV Trial) the use of nitazoxanide, ivermectin and hydroxychloroquine demonstrated similar effects, and apparent improvement of outcomes compared to untreated patients. The unexpected apparent positive results led to ethical questions on the employment of further full placebo-control studies in early stage COVID-19. The objective of the present study was to elucidate whether the conduction of a full placebo-control RCT was still ethically viable, through a comparative analysis with two control-groups. Materials and methods: Active group (AG) consisted of mild-to-moderate early stage COVID-19 patients enrolled in the Pre AndroCoV-Trial, treated with nitazoxanide ivermectin, or hydroxychloroquine in selected cases, in association with azithromycin. Vitamin D, vitamin C, zinc, glucocorticoids and anticoagulants, when clinically recommended. Control Group 1 (CG1) consisted of a retrospectively obtained group of untreated patients from the same population as those from the Pre-AndroCoV Trial, and Control Group 2 (CG2) resulted from a precise prediction of clinical outcomes, based on a thorough and structured review of articles indexed in PubMed and MEDLINE and statements by official government agencies and specific medical societies. For both CGs, patients were matched for proportion between sex, age, obesity and other comorbidities. Results: Compared to CG1 and CG2, AG showed a reduction of 31.5 to 36.5% in viral shedding (p < 0.0001), 70 to 85% and 70 to 73% in duration of COVID-19 clinical symptoms when including and not including anosmia and ageusia, respectively ((p < 0.0001 for both), and 100% in respiratory complications through the parameters of the Brescia COVID-19 Respiratory Scale (p < 0.0001). For every 1,000 confirmed cases for COVID-19, a minimum of 140 patients were prevented from hospitalization (p < 0.0001), 50 from mechanical ventilation, and five deaths, when comparing to age-, sex- and comorbidity-matched non-treated patients with similar initial disease severity at the moment of diagnosis. Conclusion: Apparent benefits of the combination between early detection and early pharmacological approaches for COVID-19 demonstrated to be consistent when when compared to different control groups of untreated patients. The potential benefits could allow a large number of patients prevented from hospitalizations, deaths and persistent symptoms after COVID-19 remission. The potential impact on COVID-19 disease course and numbers of negative outcomes and the well-established safety profile of the drugs proposed by the Pre-AndroCoV Trial led to ethical questions regarding the conduction of further placebo control randomized clinical trials (RCTs) for early COVID-19. Early pharmacological approaches including azithromycin in combination with any of the options between nitazoxanide, ivermectin or optionally hydroxychloroquine should be considered for those diagnosed with COVID-19 presenting less than seven days of symptoms. Of the three drugs, we opted for nitazoxanide, due to more extensive demonstration of in vitro and in vivo antiviral activity, proven efficacy against other viruses in humans, and steadier safety profile.

Early COVID-19 Therapy with Azithromycin Plus Nitazoxanide, Ivermectin or Hydroxychloroquine in Outpatient Settings Significantly Reduced Symptoms Compared to Known Outcomes in Untreated Patients. (preprint)

Background: While there was a lack of pharmacological interventions proven to be effective in early, outpatient settings for COVID-19, in a prospective, open-label observational study (pre-AndroCoV Trial) the use of nitazoxanide, ivermectin and hydroxychloroquine demonstrated similar effects, and apparent improvement of outcomes compared to untreated patients. The unexpected apparent positive results led to ethical questions on the employment of further full placebo-control studies in early stage COVID-19. The objective of the present study was to elucidate whether the conduction of a full placebo-control RCT was still ethically viable, through a comparative analysis with two control-groups.Materials and methods: Active group (AG) consisted of mild-to-moderate early stage COVID-19 patients enrolled in the Pre AndroCoV-Trial, treated with nitazoxanide ivermectin, or hydroxychloroquine in selected cases, in association with azithromycin. Vitamin D, vitamin C, zinc, glucocorticoids and anticoagulants, when clinically recommended. Control Group 1 (CG1) consisted of a retrospectively obtained group of untreated patients from the same population as those from the Pre-AndroCoV Trial, and Control Group 2 (CG2) resulted from a precise prediction of clinical outcomes, based on a thorough and structured review of articles indexed in PubMed and MEDLINE and statements by official government agencies and specific medical societies. For both CGs, patients were matched for proportion between sex, age, obesity and other comorbidities. Results: Compared to CG1 and CG2, AG showed a reduction of 31.5 to 36.5% in viral shedding (p < 0.0001), 70 to 85% and 70 to 73% in duration of COVID-19 clinical symptoms when including and not including anosmia and ageusia, respectively ((p < 0.0001 for both), and 100% in respiratory complications through the parameters of the Brescia COVID-19 Respiratory Scale (p < 0.0001). For every 1,000 confirmed cases for COVID-19, a minimum of 140 patients were prevented from hospitalization (p < 0.0001), 50 from mechanical ventilation, and five deaths, when comparing to age-, sex- and comorbidity-matched non-treated patients with similar initial disease severity at the moment of diagnosis.Conclusion: Apparent benefits of the combination between early detection and early pharmacological approaches for COVID-19 demonstrated to be consistent when when compared to different control groups of untreated patients. The potential benefits could allow a large number of patients prevented from hospitalizations, deaths and persistent symptoms after COVID-19 remission. The potential impact on COVID-19 disease course and numbers of negative outcomes and the well-established safety profile of the drugs proposed by the Pre-AndroCoV Trial led to ethical questions regarding the conduction of further placebo control randomized clinical trials (RCTs) for early COVID-19. Early pharmacological approaches including azithromycin in combination with any of the options between nitazoxanide, ivermectin or optionally hydroxychloroquine should be considered for those diagnosed with COVID-19 presenting less than seven days of symptoms. Of the three drugs, we opted for nitazoxanide, due to more extensive demonstration of in vitro and in vivoantiviral activity, proven efficacy against other viruses in humans, and steadier safety profile.

Hydroxychloroquine, nitazoxanide and ivermectin have similar effects in early COVID-19: a head-to-head comparison of the Pre-AndroCoV Trial. (preprint)

Background: COVID-19 pandemic requires urgent responses in terms of identification of effective and safe therapies to reduce hospitalization, death, and post-COVID symptoms, while vaccines are not extensively available. Repurposing already existing medications for COVID-19 should be preferred over the development of new drugs due to their inherent advantages of well-established safety profile, familiarity, and cost. Although antiandrogens have strong plausibility to be effective against COVID-19, hydroxychloroquine, nitazoxanide and ivermectin gained unquestionable popularity due to their in vitro and in vivo direct or indirect antiviral activity, and preliminary observations of efficacy against COVID-19. The objective of the present open-label prospective observational study (the pre-AndroCoV trial) was to make a head-to-head comparative analysis between hydroxychloroquine, nitazoxanide and ivermectin, in terms of potential efficacy for COVID-19, combined with early COVID-19 detection, aiming to choose one of these three drugs to include in the AndroCoV randomized clinical trial (RCT).Materials and methods: Participants were recruited from social media and referred from other medical centers. Patients confirmed for COVID-19 with positive rtPCR-SARS-CoV-2 with fewer than seven days of symptoms and four days of treatment were included. Patients were actively questioned for age, sex, body mass index (BMI), presence of approximately 40 existing diseases and regular use of 30 drug classes, and COVID-19 symptomatology. Hydroxychloroquine 400mg/day for five days, nitazoxanide 500mg twice daily for six days, or ivermectin 0.2mg/kg/day for three consecutive days was given in a quasi-random manner, in association with azithromycin 500mg/day for five days, and optional addition of vitamin C, vitamin D and zinc, and glucocorticoids and anticoagulants in case of signs of lung injury or higher risk for thrombosis, respectively. Patients were followed up for 60 days, including active questions on disease course and symptoms on Days 0, 1, 2, 3, 7, 14 and 30, and virtual medical visits on Days 0 and 14, and whenever symptoms got worse on in the presence of severe adverse effects. Results: In total, 585 participants, including 270 females and 305 males, were included. Of these, 159, 357, and 110 patients received hydroxychloroquine, nitazoxanide, and ivermectin, respectively, with similar baseline characteristics and time-to-treat between them. The three groups had similar duration of positive rtPCR-SARS-CoV-2, clinical disease duration and recovery speed. Of the 585 patients, none was hospitalized, needed mechanical ventilation, or died, and 1.5% persisted with symptoms after recovery.Conclusion: Hydroxychloroquine, nitazoxanide and ivermectin seem to be similarly effective for overall clinical outcomes in COVID-19 when used before seven days of symptoms, and overwhelmingly superior compared to untreated COVID-19 population, even for those outcomes not influenced by placebo effect, at least when combined with azithromycin, and vitamin C, D and zinc in the majority of the cases. Between these drugs, nitazoxanide demonstrated the strongest broad spectrum antiviral activity, plausibility to act as an anti-COVID agent, and safety profile, at least at the time of the choice of the drug for the AndroCoV Trial.

Azithromycin with nitazoxanide, hydroxychloroquine or ivermectin, with or without dutasteride, for early stage COVID-19: an open-label prospective observational study in males with mild-to-moderate COVID-19 (The Pre-AndroCoV Male Trial). (preprint)

Background: COVID-19 is a multisystemic disorder caused by SARS-CoV-2 that has led to more than 1,000,000 deaths until the end of September 2020. Besides aging, obesity, and metabolic diseases, males, in particular those affected by androgenetic alopecia (AGA), are at higher risk to develop complications. While policies for diagnosis of COVID-19 still focus on the presence of fever or shortness of breath, these symptoms tend to appear only in later and more severe stages of the disease, when viral infectivity is already, hampering potential antiviral approaches. In addition, clinical characterization of early COVID-19 stages still lacks. The objective of the present observational study was to characterize prospectively clinical features and predictors in males during early COVID-19, and to evaluate whether the combination of more sensitive case-detection, early diagnosis and early pharmacological approaches would lead to improved clinical outcomes.Material and methods: Males confirmed for COVID-19 through positive real-time polymerase chain reaction (rtPCR) for SARS-CoV-2 with less than seven days of symptoms and three days of COVID-19 confirmation were divided into non-AGA, AGA not using dutasteride (AGA no-5ARi), and AGA using dutasteride (AGA-5ARi) groups. Patients were actively characterized for baseline and lifestyle characteristics, 22 different diseases, 42 drug classes and vaccines, 26 different symptoms, and 10 different parameters to measure COVID-19 related clinical outcomes. Azithromycin plus hydroxychloroquine, nitazoxanide 500mg or ivermectin, with or without dutasteride or spironolactone were used. Patients were then evaluated for COVID-19 clinical course, duration, and progression.Results:  A total of 305 males were enrolled, including 192 non-AGA, 71 AGA non-ARi and 52 AGA-5ARi. The prevailing symptoms were anosmia (68.9%), ageusia (61.2%), headache (37.5%), hyporexia (37.5%), fatigue (35.2%), dry cough (35.2%), fever or “feverish” (33.9%), thoracic pain (32.4%), conjunctival hyperemia (29.5%), weakness (29.5%), nasal congestion or rhinorrhea (28.6% and myalgia (26.3%). ARi users remained asymptomatic throughout COVID-19 treatment in 82.7% (43 of 52 males), and the only symptoms present in more than two AGA-5ARi patients were anosmia and ageusia. Thoracic, upper back, lower back pain, arthralgia affected a higher percentage of AGA no-5ARi than non-AGA males (all p < 0.01), but had similar durations (p = n/s). Anosmia, ageusia, headache, fatigue, myalgia and conjunctival hyperemia were more commonly present and lasted for longer periods in AGA no 5ARi patients (all p < 0.01). Self-reported perception of “sinusitis” and “sore throat”, dry cough and weakness were equally present (p = n/s) but had longer duration in AGA no-5ARi males (all p < 0.01).The different drug combinations were equally distributed (p > 0.05). AGA males were more severely affected than non-AGA in terms of duration of clinical manifestations (9.4 ± 6.0 vs 14.2 ± 7.3 days, p < 0.0001) and viral shedding (14.0 ± 5.2 vs 17.8 ± 6.2 days; p < 0.0001), which has been fully mitigated by the chronic use of dutasteride (p < 0.0001 and < 0.0001 vs non-AGA and AGA no-5ARi, respectively, for both clinical manifestations and viral shedding duration. Non-AGA, AGA no-5ARi and AGA-5ARi achieved 95% clinical recovery in seven, 14, and two days, respectively. In regards functional capacity, AGA no -5ARi males at Days 30, 14, 7, and 3 after treatment initiation were similar than non-AGA at Days 14, 7, 3, and 0, respectively (all p > 0.9). None of the patients required hospitalization and mechanical ventilation, or progressed to more severe states.Conclusion: The combination of more sensitive and earlier diagnosis of COVID-19 with  a variety of drug combinations with preliminary demonstration of direct or indirect antiviral activity against SARS-CoV-2 demonstrated indisputable improved COVID-19 related clinical outcomes compared to the extensively described COVID-19 clinical course, and avoided the progression to more severe state in all patients included in the present analysis, independently of risk factors, demonstrating that any additional risk factor can be completely mitigated by the combination of more sensitive clinical suspect with early pharmacological approaches.The overwhelming differences indicate that full placebo control RCTs for early COVID-19 may be ethically questionable. Instead, double blind therapies with different options, or mixed open label placebo control for COVID-19 should be considered.

An open-label prospective observational study of antiandrogen and non-antiandrogen early pharmacological approaches in females with mild-to-moderate COVID-19. The Pre-AndroCoV Female Trial. (preprint)

Background: While COVID-19 remains largely unclear and mortality continues to raise, early effective approaches prior to complications lack, as well as researches for characterization and therapeutical potential options in actual early COVID-19. Although females seem to be less affected than females, hyperandrogenic (HA) phenotype, like polycystic ovary syndrome (PCOS), idiopathic hirsutism, congenital adrenal hyperplasia (CAH) female androgenetic alopecia (AGA), or idiopathic HA may be at higher risk due to its inherent enhanced androgenic activity. The present study aimed to evaluate the effects of any early pharmacological approach to females diagnosed with COVID-19 before seven days of symptoms, as well as investigate whether HA is an additional risk factor in this population. Materials and methods: Females with symptoms for less than seven days confirmed for COVID-19 through positive real-time polymerase chain reaction (rtPCR-SARS-CoV-2) were classified and divided as non-HA, HA, and HA using spironolactone (HA-spiro) groups. Patients were questioned for baseline characteristics, 23 different diseases, 44 drug classes and vaccines, 28 different symptoms, and eight different parameters to measure COVID-19 related clinical outcomes. Treatment was then provided, including azithromycin 500mg/day for five days in all cases, associated with hydroxychloroquine 400mg/day for five days, nitazoxanide 500mg twice a day for six days, or ivermectin 0.2mg/kg/day por three days, and optionally spironolactone 100mg twice a day until cure. Patients were assessed for COVID-19 clinical course, clinical and viral duration, and disease progression. Results: In total, 270 females were enrolled, including 195, 67, and eight in non-HA, HA, and HA-spiro groups, respectively. Prevailing symptoms were anosmia (71.1%), ageusia (67.0%), headache (48.1%), myalgia (37.4%), dry cough (36.3%), nasal congestion or rhinorrhea (34.1%), fatigue (33.3%), weakness (29.5%), hyporexia (27.8%), thoracic pain (24.8%), diarrhea (24.1%) and dizziness (21.5%). Earliest symptoms (days) were dizziness (1.0 +- 0.2 day), abdominal pain (1.1 +- 0.3); conjunctival hyperemia (1.1 +- 0.5), nasal congestion or rhinorrhea (1.2 +- 0.5), headache (1.2 +- 0.5), dry cough (1.2 +- 61617; 0.5), myalgia (1.2 +- 0.4), nauseas (1.3 +- 0.5) and weakness (1.3 +- 0.5). Time-to-treat, positive rtPCR, and duration of symptoms with and without anosmia and ageusia were significantly lower in HA-spiro than non-HA, HA, and overall non-users. Time-to-treat was similar while all duration of symptoms and positive rtPCR-SARS-CoV-2 were significantly shorter in non-HA than HA. Spironolactone users were more likely to be asymptomatic than non-users during COVID-19. Fewer non-HA than HA females were affected by anosmia, ageusia, dry cough, fatigue, weakness and hyporexia. Ageusia, weakness and myalgia lasted shorter in non-HA than HA. None of the patients needed hospitalization or any other COVID-19 complication. Conclusions: A sensitive, early detection of COVID-19 followed by a pharmaceutical approach with different drug combinations yielded irrefutable differences compared to sex-, age-, body mass index (BMI)-, and disease-matched non-treated controls in terms of clinical outcomes, ethically disallowing placebo-control randomized clinical trials in the early stage of COVID-19 due to the marked improvements. HA females presented more severe and prolonged clinical manifestations, although none progressed to worse outcomes. Spironolactone mitigated the additional risks due to HA.