Proxalutamide (GT0918) Reduces the Rate of Hospitalization and Death in COVID-19 Male Patients: A Randomized Double-Blinded Placebo- Controlled Trial

John McCoy Applied Biology Inc., Irvine, CA, USA Andy Goren Applied Biology Inc., Irvine, CA, USA Flavio Adsuara Cadegiani (  avio.cadegiani@unifesp.br ) Federal University of São Paulo, São Paulo, Brazil https://orcid.org/0000-0002-2699-4344 Sergio Vaño-Galván Dermatology Department, Ramón y Cajal Hospital, Madrid, Spain. Maja Kovacevic Department of Dermatology and Venereology, University Hospital Center “Sestre milosrdnice”, Zagreb, Croatia. Daniel Fonseca Samel Hospital, Manaus, Brazil Edinete Dorner Samel Hospital, Manaus, Brazil Dirce Costa Onety Samel Hospital, Manaus, Brazil Ricardo Ariel Zimerman Hospital da Brigada Militar, Porto Alegre, Brazil Carlos Gustavo Wambier Department of Dermatology, Alpert Medical School of Brown University


Introduction
Androgen signaling plays a central role in SARS-CoV-2 infectivity. SARS-CoV-2 cell entry is mediated by priming of viral spike proteins by the androgen-promoted enzyme, transmembrane protease, serine 2 (TMPRSS2). Structural modi cation of viral spike proteins enhances binding of SARS-CoV-2 to angiotensin converting enzyme 2 (ACE2) and facilitates viral entry into cells. Thus, targeting the transcription of TMPRSS2 with antiandrogens can affect viral cell entry. 3,4 It has been demonstrated that ACE2 and TMPRSS2 levels in lung and cardiac cells are reduced by antiandrogens. 1,2 Conversely, patients with androgen sensitive phenotypes have been shown to have increased COVID-19 disease burden. The androgen-mediated phenotype androgenetic alopecia (AGA) has been associated with greater COVID-19 disease severity [5][6][7] . Further, the progression of AGA in hospitalized men showed a direct correlated with COVID-19 disease severity; men with more advanced AGA were more likely to require intensive care or die. 5,8 In a large population based study of COVID-19 patients in northern Italy, it was reported that prostate cancer patients undergoing androgen depravation therapy (ADT) had lower SARS-CoV-2 infection rates compared to prostate cancer patients not receiving ADT (OR 4.05; 95% CI 1.55-10.59). 9 Similarly, we have reported the protective effect of 5-alpha-reductase inhibitors (5ARi) for men with COVID-19 in two observational studies. 10,11 5ARis such as nasteride and dutasteride block the conversion of testosterone to a more potent intracellular androgen, dihydrotestosterone. 2 In a study of 77 men hospitalized with COVID-19 we found that among chronic antiandrogen users (primarily dutasteride) 8% were admitted to the ICU, compared to 58% of men not taking antiandrogens (p=0.0015). 10 Moreover, the frequency of COVID-19 symptoms among men with AGA was drastically reduced in users of 5ARis in an outpatient setting. A statistically signi cant reduction in the frequency of 20 of 29 clinical symptoms was observed in 5ARi users compared to non-users. For example, 38% and 2% of men presented with low-grade fever, 60% and 6% with dry cough, and 88% and 15% reported anosmia in the non-5ARi and 5ARi groups, respectively. 11 In a randomized, placebo-controlled trial, early antiandrogen therapy with dutasteride resulted in faster recovery from symptoms, improvement in in ammatory markers, and reduced detection rates of SARS-CoV-2 in nasopharyngeal swabs after 7 days compared to placebo (35.7% versus 88.2%, p=0.0094). 12 Similarly, in a randomized clinical trial that enrolled 80 hospitalized men, the use of nasteride (a 5ARi with a shorter half-life than dutasteride) was associated with improvement in oxygen saturation by day 5, and a 75% lower mortality rate. 13 Proxalutamide is a potent second-generation nonsteroidal androgen receptor (AR) antagonist. 14 AR antagonist competitively inhibit androgen hormones from binding to the AR, which prevents AR dimerization and binding of AR dimers to DNA. 15 We have previously reported our preliminary analysis of the effects of proxalutamide 200mg per day in COVID-19 patients, which showed an overall 74% reduction in nasopharyngeal detection of SARS-CoV-2 on the 7 th day of treatment. 16 The objective of this study was to evaluate the e cacy of proxalutamide in preventing hospitalization in males diagnosed with COVID-19 in an outpatient setting.

Study Design and Oversight
This was double-blinded, randomized, placebo-controlled clinical trial of proxalutamide for the treatment of male outpatients with COVID-19 (NCT04446429). The study was approved by the Brazilian National

Study Population and Covariates
Patients were recruited through social media or by referral from primary care physicians. Potential study patients presented one of two outpatient centers (Centro Clínico Advance and Exame Imagem e Laboratorio, Brasilia, Brazil). Self-referred patients that contacted the study sites for participation underwent a medical screening visit prior to randomization. Nasopharyngeal swabs were collected from all potential patients by trained medical personal. SARS-CoV-2 status was con rmed by real-time reverse transcription polymerase reaction (rtPCR) testing following the Cobas rtPCR kit protocol (Automatized Platform, Roche, USA).
The inclusion criteria were: males with mild COVID-19 disease (COVID-19 8-point ordinal scale <3), positive rtPCR for SARS-CoV2 within seven days of randomization, initiation of symptoms seven days prior to randomization, creatinine below 1.5 times the upper limit of normalily (ULN), alanine transferase (ALT) below 3.0 times the ULN, total bilirubin below 2.0 times the ULN, and International Normalized Ratio of Prothrombin Time below 1.5 times the ULN, and age 18 years or older. The exclusion criteria included: signs of severe COVID-19 including respiratory rate >30 breaths/min, tachycardia >120 beats per minute, systolic and diastolic arterial blood pressure <90mmHg and <60mmHg, respectively, criteria for hospitalization at the time of randomization, uncontrolled types 1 or 2 diabetes mellitus or hypertension, congestive heart failure (CHF) grades 3 or 4 (New York Heart Association), or left ventricle ejection rate below 50%, rate corrected Q-T interval above 450mm in the electrocardiogram, HIV, active hepatitis B or C, active syphilis, cancer diagnosed in the past ve years (except for skin and thyroid cancer), and refusal to use condom in the 90 days following treatment.
Baseline characteristics, comorbidities, test results, and medications used were recorded. For each patient, the age, frequency and duration of medication used and the following pre-existing conditions were recorded: type 2 diabetes, hypertension, obesity (de ned as body mass index >30 kg/cm 2 ), and chronic obstructive pulmonary disease. Pre-existing conditions were chosen based on previous drug trials in COVID-19.

Procedures
Patients were randomized to receive either proxalutamide 200 mg/day for up to 7 days or placebo. Proxalutamide 200mg and placebo were manufactured to appear identical (Kintor Pharmaceuticals Ltd., Suzhou, China). A blocked randomization list was created with block size of 10 and the sequence of dispensing was followed throughout the trial. The group assignment was known only to the study monitor. The patients and doctors directly managing patient care were blinded to the study group allocation until the close of the study. Patients were instructed to take one tablet once a day. The telephone contact and clinical follow-up were independently performed by study assistants that were blinded to group assignments. No changes in the randomization process occurred during the trial.
Previous prescriptions from referring medical doctors, initiated upon the diagnosis of COVID-19, were continued. Usual care consisted of various combinations of medications and was individualized to each patient's needs. Azithromycin 500mg q.d. (5 days) and nitazoxanide 500mg b.i.d. (6 days) were offered to all patients, while the following medications were prescribed as needed: dipyrone, paracetamol, ondansetron, dexamethasone, rivaroxaban, and enoxaparin. In cases of hospitalization, additional drugs were prescribed according to the clinical judgement of the hospital staff. Treatment compliance was not evaluated during the trial.

Protocol Modi cations
The initial protocol approved by the IRB proposed the use proxalutamide 200 mg per day for 3 days only (approval #4.282.108). However, after the rst safety analysis conducted from Oct 30 to Nov 01 (35 patients randomized), the safety committee observed that 3 patients allocated in the same group experienced unexpected relapse of previous symptoms after a few days of interruption of the investigational product. The study director agreed to unblind the 3 patients, which were observed to be from the proxalutamide group. The protocol was amended to change the posology from one tablet per day for 3 days to 1 tablet per day for 7 days (IRB approval #4.513.428). Upon study closing, a total of 75 patients were randomized and given bottles with 3 tablets (38 received proxalutamide), and a total of 193 patients were randomized and given bottles with 7 tablets (96 received proxalutamide).

Study Outcomes
The primary outcome for the study was the percentage of patients hospitalized due to COVID-19. Results 268 men were randomized to the study. 134 men were randomized to the proxalutamide arm and 134 men were assigned to placebo. Six patients in the placebo arm discontinued treatment and withdrew consent. A total of 134 patients from the proxalutamide group and 128 patients from the placebo group were followed throughout the study period. The six patients from the placebo arm that were not followed were assumed to be non-hospitalized (COVID-19 8-point ordinal scale 1) and were included in the intention-to-treat analysis. The ow chart of patients recruited into the study is shown in Figure 1.
Baseline characteristics of the two study groups are displayed in Table 1. Concomitant treatments were di cult to assess owing to the outpatient nature of the sampled population and were not monitored.
A summary of the distribution of COVID-19 8-point ordinal scale scores at day 15 and day 30 postrandomization is included in Table 2. At day 30, 12 patients were still hospitalized in the placebo group, compared to zero in the proxalutamide arm. Figure 2 shows the cumulative hospitalization rates over the follow-up period of 30-days post-randomization. The 3 patients that were hospitalized in the proxalutamide group had later hospitalizations compared to the placebo group, which showed a higher rate of hospitalizations in the rst 10 days. After 10 days, the rate of hospitalization was similar between groups.
A summary of treatment emergent adverse events (TEAEs) is presented in Table 3. Gastrointestinal TEAEs were more frequent in the proxalutamide arm: diarrhea (21% proxalutamide versus 9% placebo), nausea (14% proxalutamide versus 6% placebo), abdominal pain (12% proxalutamide versus 5% placebo). Death within 30 days of enrollment in the study was reported for 2 patients (1.6%) in the placebo arm. No deaths were reported in the proxalutamide arm (Table 3). In the placebo arm, the primary cause of death was reported as acute respiratory distress syndrome (ARDS) resulting from progressive COVID-19 disease. The percentage of patients reported to have serious adverse events (SAEs) was 0% in proxalutamide arm and 29% in the placebo arm. The primary cause of SAEs in was due to progressive COVID-19.

Discussion
These results show the improvement of clinical outcomes in men with COVID-19 after the use of proxalutamide 200 mg per day. We have previously reported the protective effect of the 5-alpha-reduactase inhibitor, dutasteride, in men with COVID-19, both in hospitalized patients and in an outpatient setting. 11,12,18 As dutasteride has a half-life of 5 weeks and carries a labeled contraindication for the use in women of childbearing age, 19 we were keen to explore an antiandrogen that could be applied more broadly. Proxalutamide has been evaluated for 28 days in Phase 1 safety studies for both men and women; furthermore, the pharmacokinetics clearance rate for proxalutamide is 22 hours. 20 As such, proxalutamide would be more suitable for application in men and women. We are currently completing a similar outpatient trial focused on female patients (NCT04853134). Additionally, we have completed a randomized, placebo-controlled trial of proxalutamide for male and female hospitalized COVID-19 patients (NCT04728802). Future trials should also explore other non-steroidal antiandrogens (NSAA) with proven safety data in the treatment of COVID-19, e.g., apalutamide, enzalutamide, darolutamide, bicalutamide, or utamide.
The dosage used in this trial, 200mg for up to 7 days, was effective in reducing the hospitalization rates by 91%. It is still unknown if different posology could improve e cacy in the outpatient setting. However, higher dosages, higher initial loading dose, or a longer treatment course could be explored in future trials. For example, 300 mg per day has been used in hospitalized patients (NCT04728802). Additionally, a loading dosage 600 mg followed by 200 mg per day for 7 days has been reported in a case report. 21 SARS-CoV-2 cell entry into type II pneumocytes is blocked by TMPRSS2 inhibitors 27,28 and TMPRSS2 transcription inhibitors (antiandrogens). 1, 3 We expect an antiandrogen-class effect on protection from COVID-19, which may be explained by mechanisms distinct from the androgen-mediated SARS-COV-2 cell entry mechanism. 22 A non-immunosuppressive anti-in ammatory effect may occur, as the activation of the androgen receptor by dihydrotestosterone leads to increased IL-6 production in macrophages, delaying repair of injury. 24 Furthermore, NSAA use was associated with a reduction in plasma IL-6 and TNF post-trauma, 25 and reduction in in ammatory response induced by radiation. 26 Of note, the androgen receptor may be activated though the IL-6 STAT3 activation pathway. 1,23 More reseach is needed to elucidate the mechanism of NSAA on COVID-19.
We acknowledge that there are important limitations in the present study. Primarily, no direct monitoring of treatment compliance was conducted; this combined with the protocol change from a 3-day to a 7-day intervention schedule could have masked differences between treatment with placebo and proxalutamide, i.e., treatment with 3 days of proxalutamide may have been ineffectual. Nevertheless, proxalutamide retained its e cacy in intention-to-treat analysis. Second, owing to the open recruitment, data for the usual care medications was not analyzed. Finally, the six patients that withdrew from the study were all from the placebo arm. To account for this we assumed the greatest bias against the tested drug, and included all six patients as non-hospitalized in the intention-to-treat analysis.
Here we demonstrate that proxalutamide was a bene t to men with mild to moderate COVID-19. Male outpatients with SARS-CoV-2 were 91% less likely to be hospitalized versus those treated with placebo.
We hope the results of this study encourage more trials of proxalutamide and other antiandrogens for the treatment of COVID-19.

Conclusion
In COVID-19 male outpatients, treatment with proxalutamide reduced the rate of hospitalization by 91%.
The results of the present study further elucidate the protective impact of early antiandrogen treatment for COVID-19 in the outpatient setting. Further studies of antiandrogen therapies in COVID-19 patients are needed.    Figure 1 Enrollment and analyzed population.