key: cord-1056011-rkf971xn authors: Wang, Q.; Guo, H.; Li, Y.; Jian, X.; Hou, X.; Zhong, N.; Fei, J.; Su, D.; Bian, Z.; Zhang, Y.; Hu, Y.; Sun, Y.; Yu, X.; Jiang, B.; Qin, F.; Wu, Y.; Gao, Y.; Hu, Z. title: Efficacy and Safety of Leflunomide for Refractory COVID-19: An Open-label Controlled Study date: 2020-06-02 journal: nan DOI: 10.1101/2020.05.29.20114223 sha: a63eb2e3fb288e1b272d1dc5272fad7ee6b82f74 doc_id: 1056011 cord_uid: rkf971xn OBJECTIVE To evaluate the safety and efficacy of leflunomide for the treatment of refractory COVID-19 in adult patients. DESIGN Open-label controlled study SETTING A designated hospital for patients with refractory COVID-19 in Wuhan, China. PARTICIPANTS 27 hospitalized adult patients ([≥]18 years of age) with radiologically confirmed pneumonia and SARS-CoV-2 positive for more than 28 days despite standard care were assigned to receive standard of care (SOC, grp I) or leflunomide + SOC (grp 2). After 2 weeks, grp 1 and grp 2 patients who continued to be SARS-CoV-2-positive received leflunomide for 14 days while continuing SOC. MAIN OUTCOME MEASURES The primary outcomes were the rate of and time to SARS-CoV-2 clearance and the 14-day and 30-day hospital discharge rate. RESULTS Twelve patients enrolled in grp 1 and 15 patients were in grp 2. The 14 days SARS-CoV-2 viral clearance rate was 80.0% (12/15) for grp 2 patients receiving leflunomide versus 16.7% for grp 1 patients (2/12) (P=0.002). By day 14, the median time to SARS-CoV-2 clearance was 6.0 days (range 1-12; IQR 1-12) for grp 2 patients. In grp 1, two patients converted to viral negative on days 1 and 6 (P=0.002). The 14-day discharge rate was 73.3% (11/15) for the grp 2 versus 8.3% (1/12) for grp 1 (P=0.001). The 30-day discharge rate was 100% (15/15) for the grp 2 versus 66.7% (8/12) for grp 1. No severe adverse events or deaths were reported. CONCLUSION Leflunomide is effective in enhancing SARS-CoV-2 clearance and hospital discharge in refractory COVID-19 patients. The addition of leflunomide to SOC did not increase adverse events versus SOC. These preliminary observations underscore a need for a randomized clinical study of leflunomide in SARS-CoV-2 infection. 3 leflunomide versus 16.7% for grp 1 patients (2/12) (P=0.002). By day 14, the median time to SARS-CoV-2 clearance was 6.0 days (range 1-12， IQR 1-12) for grp 2 patients. In grp 1, two patients converted to viral negative on days 1 and 6 (P=0.002). The 14-day discharge rate was 73.3% (11/15) for the grp 2 versus 8.3% (1/12) for grp 1 (P=0.001). The 30-day discharge rate was 100% (15/15) for the grp 2 versus 66.7% (8/12) for grp 1. No severe adverse events or deaths were reported. Leflunomide is effective in enhancing SARS-CoV-2 clearance and hospital discharge in refractory COVID-19 patients. The addition of leflunomide to SOC did not increase adverse events versus SOC. These preliminary observations underscore a need for a randomized clinical study of leflunomide in SARS-CoV-2 infection. Based on the large numbers of global infected patients of SARS-CoV-2, there will be many patients on persist viral positive which is named refractory covid-19. Specific medication for the treatment of the refractory covid-19 has been approved. Leflunomide has been widely used in rheumatoid arthritis and psoriatic arthritis with good safety and tolerance. Recently, it is found an activity of anti-SARS-CoV-2 in vitro and the effective concentration of leflunomide is within the recognized therapeutic level for rheumatoid arthritis. Leflunomide is effective in enhancing SARS-CoV-2 clearance and hospital discharge . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint Coronavirus disease 2019 (COVID-19) is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 pandemic has so far spread to 210 countries and infected approximately three million people globally and caused more than 200,000 deaths 1 . Even though the majority of patients with COVID-19 clear SARS-CoV-2 within 14-20 days, our observations indicate that SARS-CoV-2 persists in 2-5% of infected hospitalized patients up to 4 weeks despite current best available care 2-6 . These patients are defined as having refractory or chronic COVID-19, and are not discharged from the hospital due to the risk of shedding the virus in communities. Given the current lack of effective treatment for COVID-19, repurposing existing drugs to treat COVID-19 offers a rational option to tackle this global public health emergency 7 . Leflunomide is an isoxazole derivate with immunosuppressive activities and has been used as disease-modifying anti-rheumatic drugs in rheumatoid arthritis and psoriatic arthritis [8] [9] [10] [11] [12] . The drug also possesses anti-viral activities against BK polyomavirus and cytomegalovirus [13] [14] [15] [16] [17] [18] 20 . Upon oral administration, over 80% of leflunomide is converted to teriflunomide via hepatic metabolism in the first pass through the liver 21, 22 . At standard doses for rheumatoid arthritis, teriflunomide . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 6 inhibits de novo pyrimidine synthesis via dihydroorotate dehydrogenase (DHODH), and at higher doses suppresses tyrosine and serine kinase activities 23 . Leflunomide is safe and well tolerated [9] [10] . A recent study suggested that leflunomide/teriflunomide could be repurposed for SARS-CoV-2 therapy 24 with a therapeutic range between 6-26 μ M of teriflunomide for SARS-CoV-2, which is within the recognized therapeutic level for rheumatoid arthritis 8 . This open-label study of leflunomide as adjunctive therapy to SOC was designed as a pilot study in anticipation for a randomized controlled trial (ChiCTR2000030058) and enrolled hospitalized adult patients (≥18 years of age) with radiologically confirmed COVID-19 pneumonia who were reverse transcriptase-polymerase chain reaction (RT-PCR)-positive for SARS-CoV-2 for more than 28 days despite standard care. Eligible patients had pneumonia confirmed by chest imaging and had an oxygen saturation (SaO2) of 94% or higher on room air PaO2/FiO2 ratio ≥ 300 mg Hg (ratio . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 7 of the partial pressure of oxygen (PaO2) to the fraction of inspired oxygen (FiO2)). Exclusion criteria were pregnancy, a history of liver disease, alanine aminotransferase level 5 times higher than the upper normal limit (50 U/L), and stage 4 chronic kidney disease. The study protocol adhered to the SPIRIT statement 25 and conducted under the International Conference on Harmonization Guidelines for Good Clinical Practice and the Declaration of Helsinki and the reporting of the study adhered to the CONSORT statement 26 . The corresponding author was responsible for the study design. All the authors contributed to the analysis of the data. All patients provided written informed consent to the study. Patients were assigned to the standard care group (grp 1) or the leflunomide group (grp 2) via patient choice. Standard care was provided to all patients according to the guideline 27 including supplemental oxygen and supportive care, as well as concurrent therapy with hydroxychloroquine, interferon-α, anti-human immunodeficiency virus drugs (lopinavir/ritonavir) or anti-influenza drugs (arbidol, oseltamivir) was allowed. Leflunomide (Airuohua, manufactured by Changzheng-Cinkate) was given at 30 mg/day to patients who were less than 64 years old, and 20 mg/day to patients who were ≥ 65 years old. The treatment lasted for 14 days as the first phase. All patients who continued to be RT-PCR-positive for SARS-CoV-2 by day 14 received leflunomide for 14 days as the second phase. Once RT-PCR for SARS-CoV-2 tests were negative for two consecutive assays over 24 hours apart, the physician could . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 8 terminate the treatment and observe for two days before discharge. Throat swap samples were obtained by skilled nurses, and sputum samples were collected after patients were trained by nurses one day before and daily after the start of leflunomide therapy until discharge from the hospital. RNA was extracted using the MagNA Pure 96 system and semiquantitative real-time RT-PCR was performed on The primary outcomes included the rate of and time to SARS-CoV-2 clearance, and the 14-day and 30-day hospital discharge rate. Secondary outcomes included the incidence of flares, defined as the event of conversion to being RT-PCR-positive for SARS-CoV-2 after turning negative for SARS-CoV-2 in the course of treatment, and adverse events. Patients were discharged when clinical symptoms and radiographic images of COVID-19 were significantly improved and RT-PCR-negative for SARS-CoV-2 with two consecutive assays over 24 hours apart. Adverse events (AEs) were graded and recorded according to NCI-CTCAE version 4.03. Safety events included AEs and severe adverse events (SAEs). SAEs included . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 9 any untoward medical occurrence that resulted in death, was life-threatening, required hospitalization or prolongation of hospitalization, or caused significant or persistent disability or incapacity, or birth defects. AEs were coded to a preferred term using the Medical Dictionary for Regulatory Activities (MedDRA) 22.0. Safety assessments were based mainly on the occurrence, frequency, and severity of AE and analyzed mainly using descriptive statistics. The analysis population included all patients. Analyses were descriptive in nature. Summary tabulations included the number of observations; mean, standard deviation, median, interquartile range (IQR), minimum and maximum for continuous variables; number and percentage per category for categorical data. The rate of SARS-CoV-2 clearance and discharge were analyzed by Fisher's Exact Test. Time to SARS-CoV-2 clearance and time to discharge from the hospital were described with Kaplan-Meier analysis. All analyses were conducted with SPSS software, version 25 (IBM Corp.). P<0.05 indicated a statistically significant difference. The study flowchart is shown in Figure 1 . 27 patients were enrolled in the study. Their median age was 62 (IQR 43-70), and 52% of the patients were men. Patient demographic and baseline characteristics are shown in Table 1 . The median duration of symptomatic onset or positive SARS-CoV-2 was 45 days (IQR 41-50). Twelve . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. Table S1 . The 14-day SARS-CoV-2 clearance rate was 80.0% (12/15) for patients in grp 2 (Figure 3 ). In order to further confirm the efficacy of leflunomide in enhancing the clearance of SARS-CoV-2, a sensitivity analysis was performed with the elimination of the patients whose viruses were cleared on day one after the enrollments. Based on this principle, four patients in the leflunomide group (Grp 2) and one patient in standard care (grp 1) were eliminated. Therefore, 11 patients left in each group for analysis. The results of the sensitivity analysis showed in Figures S1, S2 , and S3. The sensitivity analysis results are consistent with the overall primary endpoint results. Flares (SARS-CoV-2 recurrence) Flare occurred in one patient in group 2 ( Figure 4 ). The patient (Pt. 12) became viral . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. This open-label study demonstrated that leflunomide shortened the time to is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. To our knowledge, our study is the first clinical study evaluating a DHODH inhibitor, leflunomide, for the treatment of COVID-19. Leflunomide is the prodrug to the active drug teriflunomide. It has been approved for the treatment of rheumatoid arthritis 9 by the USA Food and Drug Administration (FDA), lupus nephritis 31 in China, and psoriatic arthritis 11 in the EU. Leflunomide is well tolerated 9-10 . Anti-viral activities have been described for leflunomide in CMV 16 and BK polyomavirus [13] [14] . Both the anti-viral and immunosuppressive activities of leflunomide have been attributed to its inhibition of de novo pyrimidine synthesis at concentrations active for rheumatoid arthritis 8,12, , and inhibition of tyrosine and serine kinase activities at higher teriflunomide concentrations 23 . Additionally, previous studies 14, 32 reported that leflunomide inhibits the PI3K-AKT-mTOR pathway, and the release of viral genetic . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 1 4 material from capsids depends on this signaling pathway. More recently, in vitro studies 24 indicated that teriflunomide has anti-SARS-CoV-2 activities at a range of μ M that falls within the recognized therapeutic levels for rheumatoid arthritis. Their data suggested that SARS-CoV-2 was sensitive to teriflunomide via inhibition of DHODH 24 , a rate-limiting enzyme in pyrimidine de novo synthesis. DHODH catalyzes the dehydrogenation of dihydroorotate to orotic acid to generate uridine and cytosine nucleotides. The authors further speculated that under normal conditions, nucleotides are supplied via both de novo biosynthesis and the salvage pathway that recycles pre-existing nucleotides from food or other nutrients. The salvage pathway is sufficient for supplying pyrimidines in non-proliferating quiescent cells, but in virus-infected cells, the de novo nucleotides biosynthesis is critical to supply the larger intracellular nucleotide pool required for viral replication. Finally, Xiong et al. DHODH-/-cells, even though cell growth is not affected 24 . In the current study, we investigated leflunomide as additional pharmacologic therapy for hospitalized patients who were RT-PCR-positive for SARS-CoV-2 for more than 28 days despite receiving standard of care. The study showed that leflunomide noticeably increased the 14 days SARS-CoV-2 clearance rate of refractory COVID-19 patients (80.0% versus 16.7% for standard care). In addition, leflunomide shortened the median time to SARS-CoV-2 clearance, which was 6.0 days for patients receiving leflunomide, whereas in the standard care group, only two of 12 patients converted to SARS-CoV-2 RT-PCR negative on day 1 and 6. Notably, 9 COVID-19 patients who . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 1 5 remained SARS-CoV-2 positive crossed over to leflunomide treatment after two weeks of standard care, 8 patients achieved SARS-CoV-2 clearance approximately 9 days from crossover. In addition, leflunomide significantly increased the 14-day discharge rate (73.3% versus 8.3% for standard care) and greatly shortened length of hospital stay. In this study, we applied leflunomide at 20-30 mg/day, matching the dose range for rheumatoid arthritis 9, 10 or systemic lupus erythematosus 30 . We found that leflunomide showed good tolerability and efficacy in treating COVID-19, with an acceptable safety profile of leflunomide, with no SAEs or deaths reported. Despite the demonstration of promising efficacy of leflunomiude, this study has several limitations. This pilot study was not randomized, the sample size of refractory COVID-19 patients was small, and all patients came from a single center. The RT-PCR assay utilized was semi-quantitative, and no plasma was saved to assay for serum teriflunomide levels. This non-randomized, open-label controlled trial has demonstrated that leflunomide has a favorable safety profile and is effective in enhancing SARS-CoV-2 clearance in refractory COVID-19 patients. The tolerability of the 14-28-day course of treatment with leflunomide is acceptable. Based on the efficacy and safety profiles of leflunomide on COVID-19 from this pilot study, a randomized controlled clinical study is warranted. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 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Inhibition of pyrimidine nucleotide synthesis and protein tyrosine phosphorylation is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 1 7 Contributors QW, HG, Yu-L, and XJ contributed equally to this paper and share joint first authorship. ZH was chief investigator of this study. QW, HG, Yu-L, and XJ were responsible for the design, analysing, and writing of the manuscript. XH, NZ, JF, DS, ZB, and YZ were responsible for recruitment and clinical care of the patients. YH, YS, XY, Yuan-L, BJ, and YG were responsible for the data analyses. Yan-L, FQ, and YWwere responsible for the sample collection and laboratory analysis. All authors reviewed and approved the manuscript. Project of Shandong University (Grant No. 2020XGA 01). All authors have completed the ICMJE uniform disclosure form at www.icmje.org/coi_disclosure.pdf and declare: no support from any organisation for the submitted work other than the listed above; no financial relationships with any organisations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work.Ethical approval: The study was approved by Qilu Hospital, Cheeloo College of Medicine, Shandong University (KYLL2020372). All patients gave written informed consent.Data sharing: After publication, study data will be available on reasonable request to the corresponding author.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 1 8 Dissemination to participants and related patient and public communities: We plan to submit this manuscript to a preprint.The corresponding author (ZH) affirms that the manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned have been explained.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 2 5 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 2 7 Patients 1 to 15 were in the leflunomide group and patients 16 to 27 were in the standard care group.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint 2 8 *ALT denotes alanine aminotransferase, and AST aspartate aminotransferase. Data are expressed as n (%) unless otherwise specified.. CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted June 2, 2020. . https://doi.org/10.1101/2020.05.29.20114223 doi: medRxiv preprint