key: cord-0981085-5b7kq08x
authors: Kaur, Hardeep; Sarma, Phulen; Bhattacharyya, Anusuya; Sharma, Saurabh; Chhimpa, Neeraj; Prajapat, Manisha; Prakash, Ajay; Kumar, Subodh; Singh, Ashutosh; Singh, Rahul; Avti, Pramod; Thota, Prasad; Medhi, Bikash
title: Efficacy and safety of Dihydroorotate dehydrogenase (DHODH) inhibitors “Leflunomide” and “Teriflunomide” in Covid-19: A narrative review.
date: 2021-06-07
journal: Eur J Pharmacol
DOI: 10.1016/j.ejphar.2021.174233
sha: c53bd3a12bc7e5f30839cda388961467bc6e4796
doc_id: 981085
cord_uid: 5b7kq08x

Dihydroorotate dehydrogenase (DHODH) is rate-limiting enzyme in biosynthesis of pyrimidone which catalyzes the oxidation of dihydro-orotate to orotate. Orotate is utilized in the biosynthesis of uridine-monophosphate. DHODH inhibitors have shown promise as antiviral agent against Cytomegalovirus, Ebola, Influenza, Epstein Barr and Picornavirus. Anti-SARS-CoV-2 action of DHODH inhibitors are also coming up. In this review, we have reviewed the safety and efficacy of approved DHODH inhibitors (leflunomide and teriflunomide) against COVID-19. In target-centered in silico studies, leflunomide showed favorable binding to active site of MPro and spike: ACE2 interface. In artificial-intelligence/machine-learning based studies, leflunomide was among the top 50 ligands targeting spike: ACE2 interaction. Leflunomide is also found to interact with differentially regulated pathways [identified by KEGG (Kyoto Encyclopedia of Genes and Genomes) and reactome pathway analysis of host transcriptome data] in cogena based drug-repurposing studies. Based on GSEA (gene set enrichment analysis), leflunomide was found to target pathways enriched in COVID-19. In vitro, both leflunomide (EC50 41.49±8.8μmol/L) and teriflunomide (EC50 26μmol/L) showed SARS-CoV-2 inhibition. In clinical studies, leflunomide showed significant benefit in terms of decreasing the duration of viral shredding, duration of hospital stay and severity of infection. However, no advantage was seen while combining leflunomide and IFN alpha-2a among patients with prolonged post symptomatic viral shredding. Common adverse effects of leflunomide were hyperlipidemia, leucopenia, neutropenia and liver-function alteration. Leflunomide/teriflunomide may serve as an agent of importance to achieve faster virological clearance in COVID-19, however, findings needs to be validated in bigger sized placebo controlled studies.

COVID-19 pandemic has become a major cause of mortality and morbidity at this current point 39 of time across the world Vm et al., 2020) . The coronavirus invades the 40 host via binding of S1-domain of the spike protein of SARS-CoV-2 with the host receptor 41 angiotensin Converting enzyme-2 (ACE-2) leading to conformational changes in spike protein compounds. After oral administration, it is rapidly metabolized to the active-metabolite 114 teriflunomide (A77 1726) and hepatic cytosolic & microsomal fractions are implicated in its 115 metabolism. In kinetic studies, the metabolite teriflunomide is primarily evaluated for PK-PD 116 correlations (Rozman, 2002) . These agents are approved as immunomodulators for the treatment 117 of rheumatoid arthritis and multiple sclerosis . These 118 agents are also reported to have antiviral effect against different viruses e.g. cytomegalovirus 119 (Gokarn et al., 2019; Silva et al., 2018) , BK viremia (Chen et al., 2013; Nesselhauf et al., 2016) , 120 HIV-1 (Read et al., 2010) , Junín virus (Sepúlveda et al., 2018) and Epstein-Barr virus (Zivadinov 121 et al., 2019).

124 DHODH inhibitors are reported to have anti-SARS-CoV-2 effect (Xiong et al., 2020) and 125 clinical case reports and studies are increasingly coming up on the same (Maghzi et al., 2020, p. 126 1). In this context, we have reviewed the safety and efficacy of FDA approved DHODH 127 inhibitors (leflunomide and its metabolite teriflunomide) against "SARS-CoV-2" and in the 128 evidence generation process; we reviewed data from in silico, in vitro, preclinical in-vivo and 129 clinical studies.

-5.7 kcal/mol). In case of binding of leflunomide to spike protein: ACE2 interface, Smith et al. 137 found leflunomide to be among the top posers among the 8000 screened candidates for binding AI/ML based approach is increasingly being used to identify novel drugs or to repurpose existing 142 drugs against COVID-19. Batra R et al, 2020 (Batra et al., 2020 used a sequential machine 143 learning based combined methodology to screen the ligand databases against spike protein of 144 SARS-CoV-2 or the S:ACE2 interaction surface, which was followed by validation using 145 Autodock. For training of the ML-model and validation, they used dataset from Smith et al, 2020 146 .Testing the trained model against "FDA approved CureFFI" dataset, 147 common active ingredient from "DrugCentral" and "BindingDB dataset" of small molecules 148 revealed a total of 187 ligands with good binding scores. Biological networks provide an excellent platform for understanding and characterizing complex 154 multilevel biological interactions i.e. interactomes and is increasingly being used in the new drug 155 discovery and drug repurposing process (Zhang et al., 2014) . Jha Z et al, 2020, (Jia et al., 2020) 156 used transcriptomic data of broncho-alveolar lavage fluid from healthy controls and COVID-19 "reactome pathway analysis" and "computational repositioning" was then performed for the co-159 expressed genes. For the pathway analysis they used "KEGG" and "reactome gene sets" and the 160 drug-induced transcriptome was used for cogena-based drug-repositioning. Leflunomide was 161 identified as one of the top 20 enriched drugs (leflunomide ranked 15 th ).

In another transcriptional response of host-cell to SARS-CoV2 based drug re-purposing, Li et al, 163 2020, (Li et al., n.d., p. 2) , used RNA sequence data of normal human bronchial cells, lung 164 cancer cell line without ACE2 expression (A549), lung cancer cells with ACE2 expression 165 (CALU-3) and SARS-CoV-2 infected cells and signaling network analysis was done.

Identification of potential gene candidates was identified through signaling network analysis. On 167 the basis of gene ontology (GO) analysis, genes that were upregulated within each super GO 168 were used as signatures to identify potential drugs that can inhibit activation of these super GOs. In another multi-omics based study, disease-atlas of drug-targets for COVID-19 were 177 constructed and using this 726 target disease associations were prioritized (on the basis of 178 mendelian randomization and co-localization evidence). The whole study resulted in 179 identification of three potential genes as targets of COVID-19 therapy, which included DHODH and leflunomide and teriflunomide are known inhibitors of DHODH . Data 181 from all these studies is showed in Table 1 . 

Efficacy of leflunomide and teriflunomide was evaluated in vitro settings (Xiong et al., 2020) 185 and both Leflunomide and teriflunomide showed inhibitory action against "SARS-CoV-2" in 186 "vero E6 cells" (MOI 0.05) with EC50 of 41.49 ± 8.84 µmol/L. However, teriflunomide was 187 found to be more potent with EC50 of 26 µmol/L (at MOI 0.05) and 6 µmol/L (at MOI 0.03).

Notably, EC50 of leflunomide was found to be higher than fevipiravir, but lower than remdesivir 189 and chloroquine. At MOI 0.05, leflunomide had higher selectivity index compared to 190 teriflunomide, whereas at MOI 0.03, teriflunomide showed a higher selectivity index. Data 191 showed in Table 1 . After exhaustive search of different databases, we couldn't find a single study which evaluated 195 the "safety and efficacy" of leflunomide/teriflunomide in animal models of "COVID-19". 198 We could find two comparative clinical studies, where leflunomide was compared against 199 control/standard of care Wang et al., 2020) , and one dedicated case series on 200 teriflunomide in COVID-19, which evaluated the "safety and efficacy" of 201 J o u r n a l P r e -p r o o f leflunomide/teriflunomide in "COVID-19" (Mantero et al., 2020a) [Details shown in table 2]. 202 Among these studies, the study by Wang Q et al, 2020 is a pre-print . . Another study by Hu et al, 2020 , all the 3 patients (100%) showed 225 virological clearance in the leflunomide arm, whereas, in the control arm none (0%) showed 226 virological clearance on day 8 post initiation of therapy . (Dhingra et al., 2007) . CRP also serves an important prognostic 230 biomarker for COVID-19 with higher level correlating well with disease severity (Ali, 2020, p. 231 19). In the study by Hu et al, 2020 , compared to baseline (median 37.4, IQR: 232 7.8-120.6) a significant decrease in CRP level was seen in the leflunomide treatment group post 233 treatment (median 5, IQR 5-5). (Baek et al., 2018) .

In a single study , "median duration of hospital stay" was 11 days 238 in the leflunomide group and it was median 24 days in the SOC group (P<0.05) showing lower 239 duration of hospital stay in leflunomide group. 240 2.4.5. 14 day and 30 day hospital discharge-rate: 241 In a single study Although we don't have data from dedicated controlled studies, two case series on teriflunomide 247 addressed this issue. In a case series of five "COVID-19" infected cases among teriflunomide 248 treated patients with multiple sclerosis (Maghzi et al., 2020) , All the patients had mild disease 249 (100%, 5/5) and 4 patients recovered with medications and no hospital admission was required, 250 while the 5 th patient developed the disease while in hospital for other disease condition, however 251 after restarting teriflunomide, his fever didn't exceed 37.5 degree (Maghzi et al., 2020) . In 252 another case series of 6 multiple sclerosis patients on teriflunomide therapy (Mantero et al., 253 2020a) with average duration of teriflunomide use of 2.1 ± 1.6 years (no additional co- In s single study, 73% in the leflunomide group showed adverse events, however, more adverse 258 events were seen in the control group (83.3%) . In a single study, 40% patients in the leflunomide group showed treatment emergent adverse 261 event, whereas only 25% in the SOC group showed TEAE . Alterations in hematological profile reported in the leflunomide group (single study) were 267 leucopenia (20%) and Neutropenia (13.3%) . Other less common TEAEs 268 were lymphopenia, thrombocytopenia each of which occurred in the leflunomide Group (6.7% 269 for both). However, no such adverse effects were noted in the control group . One study reported higher level of liver enzymes (AST and ALT) in the leflunomide group 272 (p<0.05 for each) when compared to the control/SOC group . In the study by 273 Wang et al, 2020 elevation of ALT was seen in 6.7% of case (1/15) in the 274 leflunomide and 0% cases in the SOC group. These changes were commonly reversible after 275 stopping of leflunomide and standard clearance protocols were sufficient for dealing with such 276 an increase . difference was seen between the interferon alpha-2a + leflunomide arm compared to interferon 282 alpha-2a alone arm with regards to "duration of viral shredding (confirmed by RT-PCR)" and 283 "duration of hospital stay". It is to be noted that viral shredding was not confirmed by culture (Wang et al., n.d.) . Interestingly, two patients in the leflunomide arm couldn't complete owing to 285 occurrence of adverse events. Table 3 ]. To summarize the evidence of efficacy, in vitro studies, both leflunomide and teriflunomide 301 showed inhibitory action against "SARS-CoV-2" in vero E6 cells [multiplicity of infection 302 (MOI) 0.05] with EC50 of 41.49 ± 8.84 µmol/L. However, teriflunomide was found to be more 303 potent with EC50 of 26 µmol/L (at MOI 0.05) and 6 µmol/L (at MOI 0.03). Notably, EC50 of 304 leflunomide was found to be higher than Favipiravir, but lower than remdesivir and chloroquine.

In clinical studies, compared to control/standard of care, leflunomide showed significant benefit 306 in terms of decreasing the duration of viral shredding (2 studies), decreasing CRP level (1 study, 307 pre post comparison), duration of hospital stay (1 study), 14-day and 30-day hospital discharge 308 rate (1 study) and severity of infection (2 case series on teriflunomide). However, among the four 309 clinical studies included, 1 was a small sample size RCT (n=5 in each arm), one was a non-310 randomized controlled trial, and rest two were case series (in both teriflunomide was used, n≥5 311 for both the cases). However, no add on advantage was seen with leflunomide treatment when it Table 1 : Details of studies based upon target centered in silico approach, AI/ML based screening 347 approach, host transcriptional response and network biology based approach and in vitro studies. Incidence of hospitalization.

Elevated level of C-reactive protein may be an early marker to predict risk for severity of COVID-19

Analysis of length of hospital stay using electronic health records: A statistical and data mining approach

Screening of Therapeutic Agents for COVID-19 Using Machine Learning and Ensemble Docking Studies

Safety and efficacy of lopinavir/ritonavir combination in COVID-19: A systematic review, meta-analysis, and metaregression analysis

Hydroxychloroquine in Nonhospitalized Adults With Early COVID-19

Interferon Regulatory Factor-1 (IRF-1) Shapes Both Innate and CD8+ T Cell Immune Responses against West Nile Virus Infection

Efficacy and safety of leflunomide for the treatment of BK virus-associated hemorrhagic cystitis in allogeneic hematopoietic stem cell transplantation recipients

Broadspectrum inhibition of common respiratory RNA viruses by a pyrimidine synthesis inhibitor with involvement of the host antiviral response

Clinical expression of leflunomide-induced pneumonitis

Dihydroorotate dehydrogenase inhibitors in SARS-CoV-2 infection

C -Reactive Protein, Inflammatory Conditions and Cardiovascular Disease Risk

Identification of FDA Approved Drugs Targeting COVID-19 Virus by Structure-Based Drug Repositioning

Use of Leflunomide for Treatment of Cytomegalovirus Infection in Recipients of Allogeneic Stem Cell Transplant

Association Between Early Treatment With Tocilizumab and Mortality Among Critically Ill Patients With COVID-19

Structure and regulation of the human interferon regulatory factor 1 (IRF-1) and IRF-2 genes: implications for a gene network in the interferon system

A Small-Scale Medication of Leflunomide as a Treatment of COVID-19 in an Open-Label Blank-Controlled Clinical Trial

Transcriptome-based drug repositioning for coronavirus disease 2019 (COVID-19)

Clinical efficacy of antivirals against novel coronavirus (COVID-19): A review

Folic acid as placebo in controlled clinical trials of hydroxychloroquine prophylaxis in COVID-19: Is it scientifically justifiable? Med Hypotheses 149, 110539

Ivermectin as a potential drug for treatment of COVID-19: an in-sync review with clinical and computational attributes

Repurposing 0.5% povidone iodine solution in otorhinolaryngology practice in Covid 19 pandemic

Repurposing drugs for COVID-19 based on transcriptional response of host cells to SARS-CoV-2

Inhibition of Pyrimidine Biosynthesis Pathway Suppresses Viral Growth through Innate Immunity

Inhibiting pyrimidine biosynthesis impairs Ebola virus replication through depletion of nucleoside pools and activation of innate immune responses

COVID-19 in teriflunomide-treated patients with multiple sclerosis

Mild COVID-19 infection in a group of teriflunomide-treated patients with multiple sclerosis

Mild COVID-19 infection in a group of teriflunomide-treated patients with multiple sclerosis

Antiviral effects of selected IMPDH and DHODH inhibitors against foot and mouth disease virus

Evaluation of leflunomide for the treatment of BK viremia and biopsy proven BK nephropathy; a single center experience

COVID-19: Current understanding of its pathophysiology, clinical presentation and treatment

Update on the target structures of SARS-CoV-2: A systematic review

Virtual screening and molecular dynamics study of approved drugs as inhibitors of spike protein S1 domain and ACE2 interaction in SARS-CoV-2

The effect of leflunomide on cycling and activation of T-cells in HIV-1-infected participants

Remdesivir for treatment of COVID-19; an updated systematic review and meta-analysis

Clinical Pharmacokinetics of Leflunomide

Efficacy and safety of steroid therapy in COVID-19: A rapid systematic review and Metaanalysis

Virological and clinical cure in COVID-19 patients treated with hydroxychloroquine: A systematic review and meta-analysis

Possible prophylactic or preventive role of topical povidone iodine during accidental ocular exposure to 2019-nCoV

Letter to the editor: Possible role of topical povidone iodine in case of accidental ocular exposure to SARS-CoV-2

Leflunomide-induced interstitial lung disease: prevalence and risk factors in Japanese patients with rheumatoid arthritis

Drug Repurposing for Candidate SARS-CoV-2 Main Protease Inhibitors by a Novel In Silico Method

Antiviral activity of A771726, the active metabolite of leflunomide, against Junín virus

In Silico Structure-Based Repositioning of Approved Drugs for Spike Glycoprotein S2 Domain Fusion Peptide of SARS-CoV-2: Rationale from Molecular Dynamics and Binding Free Energy Calculations

Experience with leflunomide as treatment and as secondary prophylaxis for cytomegalovirus infection in lung transplant recipients: A case series and review of the literature

Repurposing Therapeutics for COVID-19: Supercomputer-Based Docking to the SARS-CoV-2 Viral Spike Protein and Viral Spike Protein-Human ACE2 Interface

COVID-19 pandemic: A review based on current evidence

Treatment of COVID-19 Patients with Prolonged Post-Symptomatic Viral Shedding with Leflunomide --a Single-Center, Randomized, Controlled Clinical Trial. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America

Efficacy and Safety of Leflunomide for Refractory COVID-19: An Open-label Controlled Study

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

Potential treatment of COVID-19 by inhibitors of human dihydroorotate dehydrogenase

The IRF family of transcription factors

Network Biology in Medicine and Beyond

Multiomics study revealing tissue-dependent putative mechanisms of SARS-CoV-2 drug targets on viral infections and complex diseases

Teriflunomide's effect on humoral response to Epstein-Barr virus and development of cortical gray matter pathology in multiple sclerosis