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Accepted Manuscript A Systematic Review of therapeutic agents for the treatment of the Middle East Respiratory Syndrome Coronavirus MERS CoV Hisham Momattin Anfal Y Al Ali Jaffar A Al Tawfiq Tawfiq PII S1477 8939 19 30109 7 DOI https doi org 10 1016 j tmaid 2019 06 012 Reference TMAID 1441 To appear in Travel Medicine and Infectious Disease Received Date 3 March 2019 Revised Date 23 June 2019 Accepted Date 24 June 2019 Please cite this article as Momattin H Al Ali AY Al Tawfiq Tawfiq JA A Systematic Review of therapeutic agents for the treatment of the Middle East Respiratory Syndrome Coronavirus MERS CoV Travel Medicine and Infectious Disease 2019 doi https doi org 10 1016 j tmaid 2019 06 012 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting typesetting and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content and all legal disclaimers that apply to the journal pertain M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 1 A Systematic Review of Therapeutic Agents for the Treatment of the Middle East Respiratory Syndrome Coronavirus MERS CoV Hisham Momattin1 Anfal Y Al Ali2 and Jaffar A Al Tawfiq Tawfiq3 4 5 1Deparment of Pharmacy Services King Khalid Hospital Najran Saudi Arabia 2 Department of Pharmacy Services Dhahran Eye Specialist Hospital Dhahran Saudi Arabia 3 Infectious Disease Unit Specialty Internal Medicine Department Johns Hopkins Aramco Healthcare Dhahran Saudi Arabia 4 Department of Medicine Indiana University School of Medicine Indianapolis IN USA 5 Department of Medicine Johns Hopkins University School of Medicine Baltimore MD USA Corresponding author Dr Jaffar A Al Tawfiq P O Box 76 Room A 428 2 Building 61 Dhahran Health Center Dhahran 31311 Saudi Arabia Email address jaffar tawfiq jaltawfi Tel 966 13 870 9748 Fax 966 13 870 3790 Key words MERS therapy Middle East Respiratory Syndrome Coronavirus Financial support None Conflicts of Interest None M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 2 Abstract Background The Middle East Respiratory Syndrome Coronavirus MERS CoV was first described in 2012 and attracted a great international attention due to multiple healthcare associated outbreaks The disease carries a high case fatality rate of 34 5 and there is no internationally or nationally recommended therapy Method We searched MEDLINE Science direct Embase and Scopus databases for relevant papers published till March 2019 describing in vitro in vivo or human therapy of MERS Results Initial search identified 62 articles 52 articles were from Medline 6 from Embase and 4 from science direct Based on the inclusions and exclusions criteria 30 articles were included in the final review and comprised 22 in vitro studies 8 studies utilizing animal models 13 studies in humans and one study included both in vitro and animal model There are few promising therapeutic agents in the horizon The combination of lopinavir ritonavir and interferon beta 1b showed excellent results in common marmosets and currently is in a randomized control trial Ribavirin and interferon were the most widely used combination and experience comes from a number of observational studies Although the data are heterogenous this combination might be of potential benefit and deserve further investigation There were no randomized clinical trials to recommend specific therapy for the treatment of MERS CoV infection Only one such study is planned for randomization and is pending completion The study is based on a combination of lopinavir ritonavir and interferon beta 1b A fully human polyclonal IgG antibody SAB 301 was safe and well tolerated in healthy individuals and this agent may deserve further testing for efficacy M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 3 Conclusion Despite multiple studies in humans there is no consensus on the optimal therapy for MERS CoV Randomized clinical trials are needed and potential therapies should be evaluated only in such clinical trials In order to further enhance the therapeutic aroma for MERS CoV infection repurposing old drugs against MERS CoV is an interesting strategy and deserves further consideration and use in clinical settings M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 4 Introduction Middle East Respiratory Syndrome Coronavirus MERS CoV was first identified in 2012 and since then the disease attracted an increased international interest to resolve issues related to the epidemiology clinical features and therapy This interest is further enhanced by the fact that MERS CoV infection resulted in 2428 cases in 27 countries around the world as of June 23 2019 1 and most of the cases are linked to the Middle East 2 So far there had been three patterns of the transmission of MERS CoV virus mainly sporadic cases 3 intra familial transmissions 4 6 and healthcare associated transmission 3 7 26 The disease carries a high case fatality rate of 34 5 1 and so far there had been no proven effective therapy and no approved therapies for MERS CoV infection by international or national societies Few therapeutic agents were reported in the literature but all were based on retrospective analysis In this study we review available literature on the current therapeutic options for the disease including in vitro animal studies and studies in human Search strategy We searched four electronic databases MEDLINE Science direct Embase and Scopus for articles in accordance with the referred Reporting Items for Systematic Reviews and Meta Analyses PRISMA guidelines 27 We used the following terms 1 Middle East Respiratory Syndrome Coronavirus OR MERS virus OR MERS Viruses OR MERS CoV OR Novel Coronavirus AND 2 Drug effect OR Drug Therapy OR Combination drug therapy OR Drug Ther OR Combination drug ther M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 5 In addition we reviewed the references of retrieved articles in order to identify additional studies or reports not retrieved by the initial search The included studies were arranged as in vitro studies animal studies and human studies We included studies conducted in the vitro animal or humans that measured the impact of drug therapy against MERS CoV We excluded studies that examined the impact of drug therapy against Coronaviruses other than MERS CoV any study that focused on drug synthesis and extractions review articles studies of supplemental therapy and articles focused on the mechanism of action of medications Results Initial search identified 62 articles 52 articles were from Medline 6 articles from Embase and 4 articles from science direct Of those 32 studies were excluded review studies n 16 drug synthesis and extraction n 3 supplemental therapy n 1 drug therapy in Coronavirus in general n 4 and site of action of different drugs modalities n 8 Based on the inclusions and exclusions criteria only 30 articles were included in the final review 13 studies were conducted in vitro 8 studies were done in animal models 8 studies were done in humans and one study included both in vitro and animal model Figure 1 In Vitro Studies There were many in vitro studies evaluating various agents against MERS CoV such as interferon INF ribavirin and HIV protease inhibitors nelfinavir ritonavir and lopinavir as summarized in table 1 In vitro studies showed that IFN has a lower 50 inhibitory concentration IC50 for MERS CoV compared with IFN a2b 28 In addition IFN has a superior anti MERS CoV activity in the magnitude of 16 41 83 and 117 fold higher compared to IFN 2b IFN IFN universal type 1 and IFN 2a respectively 28 Pegylated M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 6 Interferon PEG IFN inhibited the effect of MERS CoV at a dose of 1 ng ml with complete inhibition of cytopathic effect CPE at doses of 3 1000 ng ml in MERS CoV infected Vero cells 29 Ribavirin a nucleoside analog requiring activation by host kinases to a nucleotide required high in vitro doses to inhibit MERS CoV replications and these doses are too high to be achieved in vivo 30 31 The combination of interferon alfa 2b INF 2b and ribavirin in Vero cells resulted in a an 8 fold reduction of the IFN 2b dose and a 16 fold reduction in ribavirin dose 30 The HIV protease inhibitors Nelfinavir and lopinavir were thoughts to inhibit MERS CoV based on results from SARS 32 Nelfinavir mesylate hydrate and lopinavir showed suboptimal 50 effective concentration EC50 in the initial CPE inhibition assay and were not evaluated further 31 In another study the mean EC50 of lopinavir using Vero E6 and Huh7 cells was 8 0 M 33 MERS CoV requires fusion to the host cells to replicate thus MERS CoV fusion inhibitors such as camostat and the Heptad Repeat 2 Peptide HR2P were evaluated in vitro 34 35 Camostat inhibited viral entry into human bronchial submucosal gland derived Calu 3 cells but not immature lung tissue 34 HR2P was shown to inhibit MERS CoV replication and the spike protein mediated cell cell fusion 35 Camostat was effective in reducing viral entry by 15 folds in the Vero TMPRSS2 cells infected with MERS CoV 36 Nitazoxanide a broad spectrum antiviral agent and teicoplanin an inhibitor of Cathepsin L in the Late Endosome Lysosome cycle and a blocker of the entry of MERS CoV showed inhibitory effects of MERS CoV in vitro 37 38 M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 7 The ability of recombinant receptor binding domain RBD Fd to inhibit MERS CoV has been studied in DPP 4 expressing Huh 7 infected cells The 50 inhibition dose ID50 for RBD Fd was 1 5 g ml compared with no inhibitory activity in untreated cells even at highest dose 39 Cyclosporin affects the function of many cyclophilins that act as chaperones and facilitate protein folding 29 40 In vitro cyclosporine inhibited MERS CoV replication 29 40 Three days post infection cytopathic effects CPE of MERS CoV was inhibited by Cyclosporine Vero cells and mock infected Huh7 cells 29 Toremifene Chlorpromazine and Chloroquine were evaluated using Vero cells human monocyte derived macrophages MDMs and immature dendritic cells MDDCs 41 These drugs were transferred to cells one hour prior to infection with MERS CoV After 48 hours viral replication was inhibited by Toremifene with 50 effective concentration EC50 of 12 9 M but the MDMs dose was too low to have a calculated EC50 Chlorpromazine inhibited MERS CoV in Vero cells with an EC50 of 9 5 M and no cytotoxicity In MDMs cells the EC50 was 13 58 M with high 50 cytotoxicity concentration CC50 of 25 64 M Chloroquine showed no antiviral activity in the MDMs Toremifene reduced virus by 1 1 5 log10 at a dose more than 20 M Chlorpromazine reduced MERS CoV by 2 log10 and had a narrow therapeutic window and a high toxicity 41 Chloroquine Chloropromazine and loperamide were tested on Huh7 cells 43 The cells were treated 1 hour prior to infection Antiviral activity of chloroquine was dose dependent Chlorpomazine showed activity against MERS CoV with EC50 of 4 9 1 2 M and CC50 of 21 3 1 0 M Loperamide an antidiarrheal drug inhibited MERS CoV and induced CPE Two kinase signaling ABL1 pathway inhibitors Imatinib mesylate and Dasatinib were active M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 8 against MERS CoV in vitro 42 In Vero E6 and MRC5 cells imatinib had a dose dependent killing 43 Saracatinib has a broad spectrum antiviral activity against different strain of MERS CoV After 72 hours of infection of Huh 7 cells Saracatinib exhibited an EC50 of 2 9 M and CC50 of more than 50 M 44 Whereas gemcitabine was shown to be effective against MERS CoV infected Huh 7 cells with an EC50 of 1 2 M and a complete viral depletion at a dose of 1 M 44 Inhibitory effect of resveratrol against MERS CoV was tested using infected Vero E6 cells After 48 hours cell death was significantly reduced in the treatment group with resveratrol The study showed that resveratrol inhibited MERS CoV after entry in the cells and when resveratrol was added at same time of MERS CoV there was no difference in cell proliferations and viral titers compared with cells treated after infections 45 The antiviral activity of GS 441524 and its pro drug GS 5734 Remdesivir were tested on MERS CoV infected human airway epithelial cell HAE 46 GS 441524 has a mean EC50 of 0 86 M and GS 5734 has a mean EC50 of 0 074 M with more reduction in viral titer if the drug was added 24 72 hours post infection 46 Utilizing HAE cells infected with MERS CoV there was a significant reduction in viral replication and dsRNA level when cells were treated with K22 compound 47 A novel peptide P9 showed an in vitro activity against MERS CoV at an IC50 of 5 g ml and more than 95 infection reduction at concentration higher than 25 g ml 48 The two neurotransmitter antagonists Chlorpromazine hydrochloride and triflupromazine hydrochloride inhibit MERS CoV infected Vero E6 cells 42 The DNA synthesis and repair inhibitor Gemcitabine Hydrochloride and an Estrogen receptor I antagonist Toremifene citrate had antiviral activity M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 9 against MERS CoV 42 An Estrogen receptor I antagonist Toremifene citrate had activity against MERS CoV 42 In addition MERS CoV is inactivated by amotosalen and ultraviolet light in fresh frozen plasma 49 Animal Studies Monoclonal antibodies against MERS CoV had been tested in animal models of MERS CoV infection The monoclonal antibodies 3B11 N and 4E10 N were compared with no treatment in Rhesus Monkey model 50 Antibodies 3B11 N were administered as a prophylaxis one day prior to animal inoculation and showed significant reduction in lung disease radiographically However there was no significant diffrence when 3B11 N and 4E10 N were compared in term of lung pathology P 0 1122 50 Interferon alfa 2a in conjunction with ribavirin were tested in rhesus macaques model of MERS CoV infection The animals were randomly assigned to either treatment or control groups and therapy was started eight hours post infection Necropsy showed a normal appearance of the lung in the treatment group compared with the control group Virus replication was significantly reduced in the lung of treated animal Serum interferon alfa was 37 times the level in untreated group by day 2 In addition the treated group showed reduced systemic and local levels of pro inflammatory markers such as interleukin 2 monocyte chemotactic protein 1 interleukin 2 receptor antagonist interleukin 6 interleukin 15 and interferon gamma 51 Another study was conducted utilizing 12 healthy common marmosets inoculated with MERS Cov and then assigned to four groups control group Mycophenolate mofetil intraperitoneally 8 hours after inoculation Lopinavir with Ritonavir at 6 30 and 54 hours after inoculation or Interferon Beta 1b subcutaneous at 8 and 56 hours post inoculation 52 Lopinavir Ritonavir M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT 10 and Interferon beta 1b treated groups had better clinical scores less weight reduction less pulmonary infiltrate and lower viral load than the untreated group The Mycophenolate group had a higher viral load with severe disease compared with the control group The fatality rate was higher in untreated and Mycophenolate treated groups 67 than Lopinavir Ritonavir treated and Interferon Beta 1 b treated groups 0 33 after 36 hours of inoculation 52 The human dipeptyl peptidase 4 hDPP4 is a receptor for cell binding and entry of MERS CoV A transgenic mouse model with hDPP4 was utilized to test the effects of humanized mAb hMS 1 In the model a single dose of hMS 1 protected the transgenic mouse from MERS CoV infection and all control mice died ten days post infection 53 The Humanized antibodies mAb 4C2h are mouse derived neutralizing spike receptor binding domain of MERS CoV MERS RBD that were further humanized 54 A single intravenous dose was injected one day pre and post MERS CoV inoculation and showed that h mAb 4C2h significantly decreased viral titer in the lungs in the mouse model p 15 M Chloroquine No virus reduction MDMs Toremifene Dose treated too low to determine EC50 with high cytotoxicity Virus reduction by 1 1 5 log10 if dose 20 M with increased in the toxicity Chlorpromazine EC50 13 58 M with high cytotoxicity CC50 25 64 M SI was 1 9 Virus reduction by 2 log10 with narrow therapeutic window and high toxicity Chloroquine No antiviral activity and no cytotoxicity MDDCs Toremifene Virus reduction by 1 1 5 log10 if dose 20 M with increased in the toxicity Chlorpromazine Virus reduction by 2 log10 with narrow therapeutic window and M A N U S C R I P T A C C E P T E D A CCEPTED MANUSCRIPT 32 high toxicity Chloroquine No antiviral activity and no cytotoxicity 33 In vitro Comparator study Huh7 cells Chloroquine Chlorpromazine Loperamide Lopinavir Pre infection Chloroquine dose dependent EC50 3 0 1 1 M and CC50 58 1 1 1 M SI was 19 4 Chlorpromazine Complete inhibition at 12 M EC50 4 9 1 2 M and CC50 21 3 1 0 M SI was 4 3 Loperamide Complete inhibition at 8 M EC50 4 8 1 5 M and CC50 15 5 1 0 M SI was 3 2 Lopinavir Complete inhibition at 12 M EC50 8 1 5 M and CC50 24 4 1 0 M SI was 3 1 43 In vitro Comparator study Vero E6 MRC5 Imatinib in the first 4hrs of infection versus 5 hrs post infection Iamtinib at time of infection is dose dependent Viral level higher at post infection compared to before infection P 0 05 Genomic RNA inhibited if drug added before infection P 0 05 but no effect if added post infection M A N U S C R I P T A C C E P T E D A CCEPTED MANUSCRIPT 33 CCF2 cleavage reduced by 80 P 50 M SI 17 Dose 1 M viral titer reduced by 50 P 0 05 with no effect on viral N protein after 24 hrs Dose 10 M reduced by 90 P 0 05 with complete depletion on the viral N protein after 24 hrs Complete inhibition of viral genomic RNA and mRNA synthesis P 0 0001 Viral titer Pretreatment no difference At time of infection marked reduction with significant a decrease M A N U S C R I P T A C C E P T E D A CCEPTED MANUSCRIPT 34 of viral genomic RNA and mRNA synthesis Post treatment within 2 hrs complete inhibition P 0 0001 Post treatment after 4hrs less effect P 0 05 Huh 7 cells infected with rMERS Cov Saracatinib rMERS CoV infected cells EC50 9 3 M Huh 7 cells infected with rMERS Cov S2 Saracatinib rMERS CoV S2 infected cells EC50 9 0 M Huh 7 cells infected with MERS CoV Gemcitabine EC50 1 2 M with complete viral depletion at dose 1 M Saracatinib Gemcitabine Synergistic effect at combination index of 0 529 Cytotoxicity no difference compared with Saracatinib and less compared with Gemcitabine 45 In vitro Comparator study Vero E6 Resveratrol Reduced cell death at 125 250 M MTS assay P 0 05 neutral red uptake assay P 0 005 Less cytotoxicity even at higher concentration Viral RNA level At concentration 31 25 250 M after 48hr lower than after 24 hrs M A N U S C R I P T A C C E P T E D A CCEPTED MANUSCRIPT 35 After 48 hr at concentration 150 M lower P 0 05 at concentration 200 M P 0 01 at concentration 250 M P 0 001 If the drug added at time of infection no difference in the cell proliferations and viral titers After 24hr the inhibition of N protein is dose dependent manner At concentration 150 M limited decrease in the N protein At concentration 250 M elimination of N protein Inhibited Caspase 3 cleavage dose dependent manner If drug administered consecutively at lower dose Ever 24 hrs dose 62 5 M the cell proliferation and cells viability were higher compared with untreated group P 0 001 The cytotoxicity and viral titer were lower P 95 reduction at concentration 25 g ml 36 In vitro Comparator study Vero TMPRSS2 infected cells Camostat At dose 10 M decreased viral entry by 15 fold Vero TMPRSS2 negative infected cells Camostat At dose 10 M no effect on the viral entry Calu 3 cells Camostat At dose 10 M decreased viral ent