An in vitro study of dual drug combinations of anti-viral agents, antibiotics, and/or hydroxychloroquine against the SARS-CoV-2 virus isolated from hospitalized patients in Surabaya, Indonesia
Abstract
A potent therapy for the infectious coronavirus disease COVID-19 is urgently required with, at the time of writing, research in this area still ongoing. This study aims to evaluate the in vitro anti-viral activities of combinations of certain commercially available drugs that have recently formed part of COVID-19 therapy. Dual combinatory drugs, namely; Lopinavir-Ritonavir (LOPIRITO)-Clarithromycin (CLA), LOPIRITO-Azithromycin (AZI), LOPIRITO-Doxycycline (DOXY), Hydroxychloroquine (HCQ)-AZI, HCQ-DOXY, Favipiravir (FAVI)-AZI, HCQ-FAVI, and HCQ-LOPIRITO, were prepared. These drugs were mixed at specific ratios and evaluated for their safe use based on the cytotoxicity concentration (CC50) values of human umbilical cord mesenchymal stem cells. The anti-viral efficacy of these combinations in relation to Vero cells infected with SARS-CoV-2 virus isolated from a patient in Universitas Airlangga hospital, Surabaya, Indonesia and evaluated for IC50 24, 48, and 72 hours after viral inoculation was subsequently determined. Observation of the viral load in qRT-PCR was undertaken, the results of which indicated the absence of high levels of cytotoxicity in any samples and that dual combinatory drugs produced lower cytotoxicity than single drugs. In addition, these combinations demonstrated considerable effectiveness in reducing the copy number of the virus at 48 and 72 hours, while even at 24 hours, post-drug incubation resulted in low IC50 values. Most combination drugs reduced pro-inflammatory markers, i.e. IL-6 and TNF-α, while increasing the anti-inflammatory response of IL-10. According to these results, the descending order of effective dual combinatory drugs is one of LOPIRITO-AZI>LOPIRITO-DOXY>HCQ-AZI>HCQ-FAVI>LOPIRITO-CLA>HCQ-DOX. It can be suggested that dual combinatory drugs, e.g. LOPIRITO-AZI, can potentially be used in the treatment of COVID-19 infectious diseases.
Introduction
At the end of 2019, a case of pneumonia was diagnosed on the basis of a viral infection in Wuhan, China . The pathogen was identified as a novel enveloped RNA betacoronavirus2, currently referred to as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which has a phylogenetic similar to SARS-CoV. Since that time, it has developed into a global pandemic due to Coronavirus SARS-CoV-2, also referred to as COVID-19 On March 2nd 2020, the Indonesian Ministry of Health reported the first confirmed domestic positive case of SARS-CoV-2. By September 2020, more than 262,000 individuals had been infected with 10,105 cases culminating in death
COVID-19 infection causes severe pneumonia with symptoms such as fever, a persistent cough, and progressive breathing failure associated with respiratory complications. The high hospitalization rate, risk of mortality and lack of a specific established treatment rendered urgent the need for an effective therapy for COVID-19 to be developed. The main viral proteinase has recently been considered positively as a suitable target for drug design against COVID-19 infection due to its vital role in the poly-protein processing necessary for coronavirus reproduction
The term ‘antiviral agents’ refers to the medications prescribed to combat Middle East Respiratory Syndrome (MERS) and SARS pandemics. Interferon α (IFN-α), lopinavir-ritonavir, chloroquine phosphate, ribavirin, and Arbidol have been highlighted in the latest version of the Guidelines for the Prevention, Diagnosis, and Treatment of Novel Coronavirus-induced Pneumonia issued by the Republic of China’s National Health Commission (NHC) as potential treatments for COVID-19. In addition to antiviral agents, antibiotics such as amoxicillin, azithromycin or fluoroquinolones are also being employed in an attempt to eradicate the SARS-CoV-2 virus. However, given the continuing lack of data regarding their efficacy as a form of COVID-19 therapy, this study aims to evaluate the use of dual combinatory drugs as an antiviral therapy against the SARS-CoV-2 virus, specifically COVID-19, within the Indonesian context.
During the present research, the respective in vitro antiviral activities of Lopinavir-Ritonavir (LOPIRITO), Favipiravir (FAVI), Azithromycin (AZI), Clarithromycin (CLA), Doxycycline (DOXY), and Hydroxychloroquine (HCQ) as dual combinatory drugs at determined ratios were analyzed. These ratios were established based on the plasma concentration of drugs administered at the usual dose during clinical therapy, (see Table 1). However, in many cases, there were limited or even no reports regarding the pharmacokinetic profiles in dual drug combination
Lopinavir, Ritonavir, and Favipiravir have all been used as antiviral agents which act as virus protease inhibitors . Azithromycin is classified as a macrolide antibiotic which has been used extensively in the treatment of severe respiratory lower tract infections such as pneumonia. It can be employed for preventing secondary infection often resulting from viral infection, thereby avoiding a severe prognosis. Azithromycin has been reported to be an immune modulator and anti-inflammatory agent [ while also inhibiting virus replication and the cytopathic effect mediated by the Zika virus in Glial cell lines and astrocytes. Moreover, the use of clarithromycin has been regarded in the same manner as that of Azithromycin. Clarithromycin demonstrates a high affinity with the protein target of HIV-1 protease in the molecular docking study which is superior to that of doxycycline due to high hydrophobicity and partition co-efficiency . The combined application of Clarithromycin and antiviral agents, i.e. Oseltamivir or Zanamivir, increased systemic immunity while reducing rates of infection-related relapse in children infected with the influenza virus [. Doxycyline, a tetracycline-derived drug, has an inhibitory effect on dengue fever viral replication and reduces the proinflammatory marker IL-6 during viral infections Consequently, it may prove effective as a form of COVID-19 therapy [. Hydroxychloroquine is an aminoquinoline-derivate compound producing fewer severe side effects than chloroquine. It has been employed as an antiviral agent which impedes the viral pre-entry stage, inhibits both viral replication mediated by acidic endocytosis and viral replication through modification of post-translation virus protein, hinders virus maturation via pH modulation, and produces anti-inflammatory effects by reducing IL-6 levels in serum In this present work, the efficacy of these drugs as a form of COVID-19 therapy was evaluated on Vero cells as viral hosts cultured with SARS-CoV-2 virus isolated from hospitalized patients in Universitas Airlangga Hospital, Surabaya, Indonesia. Furthermore, an analysis of the structure-based computational modelling of ligand-receptor interactions evaluated their potential use as the main protease of SARS-CoV-2 inhibitor [24].
Material and methods Materials Lopinavir-Ritonavir (LOPIRITO) was produced by Abbott Laboratories (Aluvia®, Chicago, USA); Favipiravir (FAVI) by Toyama Chemical (Fujifilm Group) (Avigan®, Japan); Azithromycin (AZI) tablets by Gentec Pharmaceutical Group (Spain); Clarithromycin (CLA) by Ind Swift Laboratories Limited (India); Doxycycline (DOXY) by Genero Pharmaceuticals (Doxicor®, Indonesia); Hydroxychloroquine (HCQ) by Imedco Djaja (Hyloquin®, Indonesia); and dimethyl sulfoxide by Sigma Aldrich (Singapore). All other reagents and solvents employed in this study were of the highest quality available. Milli-Q water was used in all experiments. Virus and cell collection Vero cells were used for virus inoculation against SARS-CoV-2 isolates in Indonesia. Cells were seeded in a 12-well microplate at a cell density of 5x104 cells/well cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, USA) containing 10% foetal bovine serum (Gibco, USA), 1% penicillin-streptomycin (Gibco, USA) and 1% amphotericin-B (Gibco, USA). Cells were incubated in a CO2 incubator at 37°C in a humidified atmosphere of 5% CO2 for 24 hours and cultured to reach 80–90% confluence. SARS-CoV-2 virus isolates were collected from PCR-positive confirmed patients in Universitas Airlangga Hospital, Surabaya. Patient sputum sampling and clinical procedures were performed in accordance with the ethical clearance issued by The Ethics Commission of Universitas Airlangga Hospital (Certificate number 136/KEP/2020 dated April 20, 2020). The sputum of conscious patients was collected in viral transport medium (VTM) containing Gentamycin sulphate (100μg/ml) and Amphotericin B (0.5μg/ml). Further experiments were conducted in the Biosafety Level (BSL)-3 Laboratory at The Institute of Tropical Disease, Universitas Airlangga, Surabaya, Indonesia. In order to isolate the virus, the sputum samples were inserted into a new conical tube, subsequently vortexed for five minutes, and centrifuged at 13,000 rpm for ten minutes. After centrifugation, the supernatant of each sample was extracted for the purposes of further experiments. Preparation of drugs solution Each tablet containing drugs was triturated and mixed until homogenous. Approximately 50 mg equivalent mass of drugs were weighed and added to dimethyl sulfoxide in order to solubilize the drugs. The suspension was sonicated in a water bath for 15 minutes before being added to Rosewell Park Memorial Institute (RPMI) media, sonicated again and vortexed to mix it until homogenous. The suspension was then filtered through a polycarbonate membrane with a pore size of 0.45 μm and then a pore size of 0.22 μm under aseptic conditions. The filtrate was mixed with 10% foetal bovine serum and penicillin streptomycin before being vortexed to produce a homogenous mixture to be used as a stock solution. The samples were prepared by diluting the stock solution of each drug with RPMI complete media at an appropriate level of dilution to produce a determined concentration. The dual combinatory drugs mixtures were prepared by mixing appropriate amounts of two drug stock solutions in order to produce a final concentration at the required level. The combinatory drugs were evaluated at both constant and non-constant ratios to evaluate their effects on the cytotoxicity, including; antagonistic, synergistic, or additive. A constant ratio of the mixture was achieved by adding drug solutions at the same ratio, thereby increasing each drug concentration, to produce dose escalation. In contrast, at a non-constant ratio, a fixed determined concentration of drug was added to increased doses of other drug solution in order to produce different levels of drug concentration. Cytotoxicity assay for dual combinatory drugs The cytotoxic concentration (CC50) of drugs was performed by means of MTT assay at the Stem Cell Research and Development Center, Universitas Airlangga using human umbilical cord mesenchymal stem cells which had been obtained from human placenta tissue as approved by the Ethical Committee of Universitas Airlangga Hospital (Certificate number 101/KEH/2019 dated January 10, 2019). The cells were prepared as the primary cell culture and used for the cytotoxicity assay because of their sensitivity to chemicals. Cells were seeded into 96-well microplates at a concentration of 1x103 cells/well in 100 μL Alpha Minimum Essentials Medium (α-MEM, Gibco, USA) supplemented with 10% foetal bovine serum, 1% penicillin-streptomycin and 1% amphotericin-B. The plates were then incubated in a CO2 incubator at 37°C with 5% CO2 for 24 hours, at which point, the supernatant was replaced with α-MEM containing drugs at each concentration and incubated for a further 48 hours. Approximately 25μL of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium-bromide (MTT) reagent at a concentration of 5mg/mL was subsequently added to each well and incubated for four hours at 37°C with 5% CO2. Purple formazan crystals were formed and observed under an inverted microscope. Dimethyl sulfoxide was added to each well with the complete solubilisation of formazan crystals subsequently being observed. The greater the number of formazan crystals formed, the lower the toxicity of the samples which were read for optical density of formazan using a multi reader at a measurement wavelength of 595 nm (Promega Glomax, USA). The CC50 value was analyzed by CompuSyn software (the ComboSyn Inc., accessed from www.combosyn.com). Virus inoculation and antiviral assay for dual combinatory drugs Vero cells obtained from Elabscience® (Catalog No. EP-CL-0242, USA) were seeded in a 12-well plate and confirmed as reaching 80–90% confluence on the day of virus inoculation. The culture medium was removed and the cells were then added to RPMI media containing SARS-CoV-2 isolates, previously diluted with RPMI media at a ratio of 1:2. In this study, about 2,000 virus copies were added to 50,000 cells of Vero cells, with a multiplicity of infection (MoI) degree of 0.04. The plate was gently agitated for 30 minutes and incubated at 37°C, 5% CO2 for 24 hours. About 3 mL of complete culture medium were subsequently added to the plate and incubated at 5% CO2 37°C for 24 hours, at which point 3 mL of RPMI media containing a drug combination were introduced and incubated at 5% CO2 37°C for 24, 48, and 72 hours. The drug mixtures were prepared at appropriate weight constant ratios selected on the basis of the optimum safety profiles in the cytotoxicity study. The Vero cells were observed post-treatment to observe the cytopathic effects, including; the rounding and detachment of cells. Moreover, the IC50 values were determined in order to quantify antiviral activity by measuring the proviral load in each well. The determination of the proviral load was performed by means of a Seegene COVID-19 detection Kit (Beijing, China) which detected three target genes, i.e. N-gene, E-gene and RdRP-gene. Amplification and data acquisition were carried out using the ABI Prism 7500 Sequence detector system (Applied Biosystems, USA). The IC50 value was further analyzed using CompuSyn software (The ComboSyn Inc., accessed from www.combosyn.com). Measurement of IL-6, IL-10 and TNF-α levels of virus-infected Vero cells incubated with dual combinatory drugs To enable measurement of IL-6, IL-10 and TNF-α levels, the culture medium of the treated cells was collected in sterile micro-tubes and centrifuged at 3,500 rpm for 20 minutes. The supernatants were carefully collected and diluted with aquadest at a 1:5 volume ratio and vortexed until homogenous. The samples were deposited onto a well-plate, added to ELISA reagents (Bioassay Technology Laboratory, Shanghai, China), and incubated at 37°C for 60 minutes. Reagent substrate solution was then added to the well and incubated for ten minutes at 37°C. The samples were measured for antigen concentration using the optical density (OD) plotted into the standard curves of IL-6, IL-10, and TNF-α. Molecular docking study of drugs against main protease of SARS-CoV-2 virus The molecular docking study was carried out by using Schrodinger Maestro 2019–2 Maestro software including protein preparation, ligand preparation, grid generation and receptor-ligand docking. The Linux operating system was used for the computational study. Ligands (Lopinavir, Ritonavir, Favipiravir, Azithromycin, Clarithromycin, Doxycycline, and Hydroxychloroquine) were downloaded from the NCBI (http://www.ncbi.nlm.nih.gov/pccompound). The crystal Structure of SARS-CoV-2 main protease, PDB ID: ALU6 was retrieved from the Protein Data Bank (PDB) (https://www.rcsb.org/). The main protease protein was prepared for a docking study by using in Schrodinger 2019–2 Maestro software. All ligand compounds were prepared using LigPrep, which can produce low energy isomer of the ligand in optimization by using the OPLS_2005 force field. The OPLS_2005 force field was used for generating Grid on protein receptors. Schrodinger 2019–2 version was used to predict the binding affinity, ligand competence, and inhibitory candidate to the protein by performing rigid, flexible docking. The ligands were docked with generated Grid of receptor protein PDB ID: ALU6 The optimal ligand selection for the receptor was done based on the docking score. Preparation of ligands and receptors. Ligand-receptor complex. The complex in the form of a crystal structure consisting of native ligands and receptors was downloaded from the Protein Data Bank (PDB) server at the web address https://www.rcsb.org with I protein structure consists of two chains (A and C). The Main protease (Mpro) is in the A chain (shown in brown), while the native ligand appears as blue in the C chain, as presented in Fig 1.
Credited to PLOSE ONE
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