key: cord-280005-i9fp5rys
authors: Wang, Mengmei; Zhao, Yang; Hu, Weihua; Zhao, Dong; Zhang, Yunting; Wang, Tao; Zheng, Zhishui; Li, Xiaochen; Zeng, Shaolin; Liu, Zhenlian; Lu, Li; Wan, Zhihui; Hu, Ke
title: Treatment of COVID-19 Patients with Prolonged Post-Symptomatic Viral Shedding with Leflunomide -- a Single-Center, Randomized, Controlled Clinical Trial
date: 2020-09-21
journal: Clin Infect Dis
DOI: 10.1093/cid/ciaa1417
sha: 
doc_id: 280005
cord_uid: i9fp5rys

OBJECTIVE: To evaluate the efficacy and safety of leflunomide, an approved dihydroorotate dehydrogenase inhibitor, to treat COVID-19 patients with prolonged post-symptomatic viral shedding. METHODS: We conducted a prospective, randomized, controlled, open-label trial involving hospitalized adult COVID-19 patients with prolonged PCR positivity. Patients were randomly assigned to receive either leflunomide (50 mg, q12h, three consecutive times, orally; then 20 mg, once daily for 8 days), in addition to nebulized interferon alpha 2a (IFN α-2a, 3 million IU each time, twice daily for 10 days), or nebulized IFN α-2a alone for 10 days. The primary end point was the duration of viral shedding. RESULTS: A total of 50 COVID-19 patients with prolonged PCR positivity were randomized into 2 groups; 26 were assigned to the leflunomide group, and 24 were assigned to the interferon alone group. Treatment with leflunomide was not associated with a difference from the interferon alone group in the duration of viral shedding (hazard ratio for negative RT-PCR, 0.70; 95% confidence interval, 0.391-1.256; P=0.186). In addition, the patients given leflunomide did not have a substantially shorter length of hospital stay than patients treated with interferon alone, with median (IQRs) durations of 29.0 (19.3-47.3) days and 33.0 (29.3-42.8) days, respectively, P=0.170. Two leflunomide recipients were unable to complete the full 10-day course of administration due to adverse events. CONCLUSIONS: In COVID-19 patients with prolonged PCR positivity, no benefit in terms of the duration of viral shedding was observed with the combined treatment of leflunomide and IFN α-2a beyond IFN α-2a alone.

Although clinical trials of compassionate or off-label uses of several drugs have been conducted, there is no specific and effective medication to treat patients with COVID-19 [1, 2, 3] . Partial clinical trial results of lopinavir-ritonavir, remdesivir, chloroquine and hydroxychloroquine have already been performed in different countries, but have shown only moderate and controversial effects [2, 4] . Therefore, it is still necessary to seek safe and solid strategies to treat COVID-19 when facing the increasing number of patients worldwide [5] .

The pandemic of COVID-19 has been under control in Wuhan, China since March, 2020, but some patients remained viral RNA-positive after their symptoms had resolved and their abnormal CT imaging had improved significantly [6, 7, 8] . Long-term COVID-19 positive patients cause many problems [9] , for example, they have to stay in the hospital for a long time and require more medical resources. In addition, they often had psychological disorders. Moreover, no specific therapeutic agents have been recommended for COVID-19 patients with prolonged post-symptomatic shedding [10] , which has become a great concern [11] .

Acute RNA virus replication, including SARS-CoV-2, largely depends on intracellular pyrimidine resources, and antagonists of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in the fourth step of the de novo pyrimidine biosynthesis pathway, can efficiently prohibit viral genome replication in infected cells [12] . Leflunomide, an approved DHODH inhibitor, has been widely used to treat patients with autoimmune diseases [13] , but whether leflunomide can be used to treat COVID-19 patients is unknown. As COVID-19 patients also suffer from excessive inflammations similar to autoimmune patients [14] , leflunomide may benefit COVID-19 patients through its antiviral and antiinflammation effects. A small-scale study of leflunomide treatment for confirmed patients with COVID-19 was conducted by our team, in which, leflunomide resulted in beneficial virologic clearance and length of hospital stay [15] . Based on that background, we conducted a prospective randomized, controlled, open-label trial, to evaluate the efficacy and safety of oral leflunomide to treat hospitalized COVID-19 patients with prolonged post-symptomatic viral shedding.

From March 10, 2020 to April 12, 2020, a total of 50 consecutive patients with confirmed COVID-19

with prolonged viral shedding were enrolled as study candidates. All patients were referred from other COVID- 19 The inclusion criteria were as follows: (1) aged 18 -70 years with a diagnosis of COVID-19 conforming to the Chinese Guidelines [16] ; (2) hospitalized for prolonged post-symptomatic viral shedding; (3) able to orally take medication; (4) non-pregnant women; (5) effective contraception for 7 days after taking the last medication. Candidates were excluded based on the following: (1) presence of any condition that would not allow the protocol to be followed, including known allergy to leflunomide, use of medications that are contraindicated with leflunomide and that could not be replaced or stopped during the trial period; (2) pregnant or breast-feeding; (3) known other serious comorbidities, such as liver, cardiovascular, cerebrovascular diseases, severe renal insufficiency or advanced cancer; (5) had received interferon before enrollment; (6) unwilling to participate in the study.

This clinical trial received approval from the Ethics Committee of the Renmin Hospital of Wuhan University (No.WDRY2020-K063) and written informed consent was obtained from each participant.

The study was registered at the Chinese Clinical Trial Registry (ChiCTR 2000030058).

Patients were assessed for eligibility on the basis of the inclusion and exclusion criteria (Figure 1 ). At the first interview, each candidate completed a comprehensive questionnaire including demographics, comorbidities, initial-episode syndromes and disease severity at the first admission, length of virus shedding from onset to enrollment, duration of post-symptomatic viral shedding, antiviral medication before enrollment,etc.However,the original protocol had been amended,which was for a multicenter, randomized, double-blind, controlled clinical trial. Due to few new COVID-19 patients in Wuhan, China since early March 2020, only convalescing patients with prolonged post-symptomatic viral shedding rather than those in the acute stage were enrolled in single center,with a small sample size.

Fifty eligible patients were randomly assigned to a combination treatment group that received leflunomide (50 mg, q12h, three consecutive times, orally; then 20 mg, once a day for 8 days; a total course of 10 days) plus nebulized IFN -2a (3 million IU each time, adding 2 ml of sterilized water, atomization inhalation twice daily for 10 days), or to a control group that received nebulized IFN -2a This was an open-label, prospective randomized, controlled trial, which was conducted at East Campus, Renmin Hospital of Wuhan University. The enrollment was initiated on March 10, 2020 and ended on April 12, 2020. The last patient studied was discharged on April 26, 2020 and was followedup until May 25, 2020. M a n u s c r i p t

Since there is no standard definition, we adopted the following definition of COVID-19 patients with prolonged post-symptomatic viral shedding, which refers to laboratory confirmed patients with COVID-19 who continued to have nasopharyngeal RT-PCR positivity at least two weeks after symptom resolution and after their abnormal CT imaging improved significantly.

After enrollment, serial nasopharyngeal swab specimens were obtained at the baseline (before leflunomide or IFN -2a was administered) and once every two days until nucleic acid tests were negative twice consecutively with an interval of ≥24 hours. RT-PCR for SARS-CoV-2 was performed using a commercial kit (GeneoDx Biotech Co., Ltd, Shanghai, China).

Clinical symptoms of patients were assessed once daily by trained nurses using diary cards, analysis of peripheral blood cells, biochemical indicators and chest imaging studies performed at the baseline, on day 3, one day after treatment and or one day before discharge for patients meeting discharge criteria within ten days of enrollment. Data were recorded on paper case record forms, then were entered into an electronic database and validated by the clinical trial staff.

Discharge criteria were as follows [16] : having a normal temperature for >3 days, significant improvements of respiratory symptoms and CT imaging, nucleic acid tests negative twice consecutively with an interval of ≥24 hours. After discharge, the patients were isolated at a designated place for 14 days as recommended [16] , which was arranged by community committees where the patients resided. They were followed-up by primary health-care facilities and were retested for viral nucleic acid on days 7 and 14. After that, they stayed in their homes for a second isolation period of 14 days, and were then retested for viral nucleic acid by the end of this quarantine period. We collected each patient's medical information during the isolation, which was shared with permission. In our study, enrolled patients with five consecutively negative nucleic acid tests were considered as having "true negative" results (two times during hospitalization, two times during the first isolation, and one time at the end of the second quarantine). If any patient at any time-point had a positive test for SARS-CoV-2, they were sent to a designated site for isolation and medical observation. 

The primary end point was the duration of viral shedding, which was defined as the time from randomization to the first negative nucleic acid test of five consecutive RT-PCR results. Other clinical outcomes included clinical status, i.e. progressive rate to severe illness, syndromes, peripheral blood cells and biochemical parameters, C-reactive protein and inflammatory cytokines, length of hospital stay, etc. Safety outcomes included adverse events that occurred during treatment, serious adverse events, and premature discontinuation of treatment.

Continuous variables are presented as medians (IQR). The normality of the distribution of variables was performed using the Kolmogorov-Smirnov test and statistical comparisons using a t-test.

Categorical variables are expressed as absolute numbers or percentages and are compared by the χ² test, Fisher's exact test or one-way ANOVA. The time to negative RT-PCR test was developed using the Kaplan-Meier method and was compared with a log-rank test. A P˂0.05 was considered statistically significant. All statistical analyses were performed using SAS 9.4 software (SAS Institute Inc, Cary, NC).

The characteristics of the patients in this study are summarized in Tables 1 and 2 . Of the 50 patients who underwent randomization and treatment assignment, 26 were assigned to the combination treatment group that orally received leflunomide plus nebulized IFN -2a, and 24 were assigned to the control group that received nebulized IFN -2a alone. In the combination treatment group, 24 patients (92.3%) received all treatments as assigned, but two patients did not complete the 10 day treatment regimen, one due to serious diarrhea 2 days after taking the drug, and the other due to impaired liver function. There were no significant differences in age (Table 1) .

There were no important between-group differences in baseline laboratory test results at enrollment, except for the level of creatine kinase in the control group, the level of tumor necrosis factor in the combination group was slightly higher, although both were within the normal range (Table 2) .

Twenty-four of the 26 patients in the combination treatment group and all 24 patients in the control group completed this study and were discharged. No deaths or severe illness occurred and the illness severity was not worse in either group. In terms of the duration of viral shedding after treatment, patients assigned to the combination treatment group had a time to negative RT-PCR results that was not different from patients assigned to the control group (Figure 2 ), the median time Laboratory examinations were conducted before and after treatment for all patients ( Table 2) . Of the post-treatment test results, there were no differences between the two groups except that the lymphocyte count in the control group was slightly higher than in the combination treatment group For safety, a total of 10 patients in the combination treatment group and 4 in the control group reported adverse events (Table 3) but that was not significantly different between the two groups (41.7% vs. 16.7%, P=0.057). There was one serious gastrointestinal adverse event that caused the discontinuation of treatment in the combination treatment group but none occurred in the control group, which was judged by the investigators to be related to the trial medication. For laboratory results, the absolute number of increased liver enzymes in the combination treatment group was higher than in the control group but was not statistically different (Table 2) Persistent viral shedding is a serious problem [17] . Cao and colleagues reported that SARS-CoV-2 RNA was detected in 40.7% of their patients on day 28 after a 14-day treatment regimen with lopinavir-ritonavir [18] . Another report showed that the median duration of viral shedding was 20 days in patients with COVID-19 and could be as long as 37 days [19] . An analysis of the transmission of COVID-19 revealed that 86% of subjects in China in January-February 2020 potentially contracted the virus from patients with no or minimal symptoms [20] . The prolonged existence of virus presents difficulties in attempts to control the community spread of SARS-CoV-2.

Partial in vitro studies or clinical trials have suggested the potential therapeutic activity of several compounds against coronaviruses [21] , however, there are no specific antiviral pharmaceutical treatments available for patients with COVID-19 [22] . The results of those studies did not show clinical improvement or the clinical trial results were controversial, including lopinavir-ritonavir [18] , remdesivir [23] , favipiravir [24] and chloroquine or hydroxychloroquine [25] .

We evaluated the efficacy and safety of leflunomide on SARS-CoV-2 infection in this study and compared it with the roles of interferon treatment alone. Interferon is recommended to be used for patients with COVID-19 by the Chinese guidelines [16] , for it has broad-spectrum antiviral activity [26] , has been widely used for the treatment of virus infections [26, 27, 28] , and is also effective for treating patients with COVID-19 [29, 30] . Leflunomide is capable of inhibiting viral RNA genome replication and rescues mice from advanced influenza infections [12] . Leflunomide directly targets DHODH, the host's de-novo pyrimidine synthesis enzyme, to cut off intercellular pyrimidine resources required as the starting step of building the viral RNA genome [12] . Like chloroquine and hydroxychloroquine, leflunomide has a dual mechanism of antiviral and immunoregulation and has been approved to treat arthritis for many years [31, 32]. Leflunomide has a clear-cut drug target of DHODH and has few off-target effects [33], whereas chloroquine and hydroxychloroquine are multitargeted and have more severe adverse effects [34] . Therefore, DHODH inhibitors may be attractive drugs for treating acute and severe virus infection diseases [35] . In a preliminary trial, we found that leflunomide resulted in beneficial virologic clearance and length of hospital stay for patients with COVID-19 [15] .

A c c e p t e d M a n u s c r i p t 9 In the present study, the baseline characteristics of the patients at enrollment were generally balanced across the two groups that did not differ with regard to duration, severity of illness and majority baseline laboratory results. However, differences in the negative conversion of virus nucleic acid between the combination treatment group and the control group were not observed. As compared to treatment with nebulized IFN -2a only, the combination of oral leflunomide and nebulized IFN - For safety, two leflunomide recipients discontinued treatment due to gastrointestinal adverse events or abnormal liver function, however, there was no statistical difference in the total number of adverse events between the two groups. The side-effect profile observed in the current trial arouses concern about the use of higher or more prolonged leflunomide dose regimens in efforts to improve outcomes. ＊ Length of virus shedding from onset to enrollment in patients with initial cough and expectoration.

※ Patients with initial cough and expectoration in patients with initial cough and expectoration.

# Lianhua Qingwen capsule is a kind of Chinese traditional medicine and is recommended for patients with COVID-19 [16] .

A c c e p t e d M a n u s c r i p t 17 

WHO launches global megatrial of the four most promising coronavirus treatments

Potential antiviral drugs for SARS-Cov-2 treatment: preclinical findings and ongoing clinical research

The epidemic of 2019-novel-coronavirus (2019-nCoV) pneumonia and insights for emerging infectious diseases in the future

Treatment options for COVID-19: the reality and challenges

Race to find COVID-19 treatments accelerates

Clinical features of COVID-19 convalescent patients with re-positive nucleic acid detection

Probable causes and risk factors for positive SARS-CoV-2 test in recovered patients: evidence from Brunei Darussalam

South Korea's COVID-19 Infection Status: From the perspective of re-positive test results after viral clearance evidenced by negative test results

Recurrence of positive SARS-CoV-2 in patients recovered from COVID-19

Prolonged viral RNA shedding duration in COVID-19

PCR assays turned positive in 25 discharged COVID-19 Patients

Novel and potent inhibitors targeting DHODH are broad-spectrum antiviral against RNA viruses including newly emerged coronavirus SARS-CoV-2

Leflunomide and teriflunomide: altering the metabolism of pyrimidines for the treatment of autoimmune diseases

Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

A small-scale medication of Leflunomide as a treatment of COVID-19 in an open-label blank-controlled clinical trial

Released by National Health Commission & National Administration of Traditional Chinese Medicine

Persistence and clearance of viral RNA in 2019 novel coronavirus disease rehabilitation patients

A trial of Lopinavir-Ritonavir in adults hospitalized with severe COVID-19

Clinical course and risk factors for mortality of adult in patients with COVID-19 in Wuhan, China: a retrospective cohort study

Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV-2)

Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro

Characteristics of and public health responses to the coronavirus disease 2019 outbreak in China

Remdesivir in adults with severe COVID-19: a randomised, double-blind, placebo-controlled, multicentre trial

Experimental treatment with favipiravir for COVID-19: an open-label control study. Engineering (Beijing) 2020

A rapid systematic review of clinical trials utilizing chloroquine and hydroxychloroquine as a treatment for COVID-19

Treatment with lopinavir/ritonavir or interferon-β1b improves outcome of MERS-CoV infection in a nonhuman primate model of common marmoset

BC Hepatitis Testers Cohort Team. Sustained virological response from interferon-based hepatitis C regimens is associated with reduced risk of extrahepatic manifestations

Combination group: leflunomide plus IFN -2a; Control group: IFN -2a alone

LDH = Lactate dehydrogenase; Ultra-TnI = Ultratroponin I; APTT = activated partial thromboplastin time

TNF = tumor necrosis factor. a: Comparison of baseline data between the two groups. b: Data comparison between after treatment the two groups

We respectfully thank all patients enrolled in this study. This work was supported by the National Key 

The funding agencies had no role in the study design and clinical medications; collection, analysis, and interpretation of the data; preparation, written, review, or approval of the manuscript.

Zhao Y and Hu WH contributed equally with Wang MM. The authors have no competing interest to declare for this study.

A c c e p t e d M a n u s c r i p t 19 A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 21 Figure 2