Favipiravir pharmacokinetics in immunocompromised infants and children with chronic RNA viral infections

IntroductionFavipiravir selectively inhibits RNA polymerase responsible for single-stranded viral replication. It is licensed for treating influenza and repurposed to treat other diseases such as Ebola and COVID-19. It is metabolised by hepatic aldehyde oxidase (AO) and is an AO inhibitor with complex pharmacokinetics. We have used favipiravir, in combination with other antivirals, in severely immunocompromised children with life-threatening RNA virus infections. As an unlicensed indication, favipiravir pharmacokinetics were routinely monitored at our institution. Population pharmacokinetic model is used to describe the favipiravir pharmacokinetic properties, drug exposure and sources of variability in these children.MethodsRoutine favipiravir plasma levels of 9 patients (0.8–11yrs, mean age=5.3yrs;median weight=15kg) were analysed retrospectively (62 samples). All patients received favipiravir 200mg or 400mg tds and had at least one plasma level 45min (peak), 3h and 8h (trough) post-dose. Parameter estimation and model simulation properties (visual predictive check) were assessed using R language (v 4.1.2) and RStudio (2022.02.0+443).ResultsA one-compartment model with weight as covariate best describes the data, with (1) elimination clearance=1L/h and volume of distribution=7.54L, both allometric scaled centring at median weight, and (2) estimated t1/2=5.17h with Cmax = 24µg/mL at 200mg and 41µg/mL at 400mg.ConclusionsTo our knowledge this is the first report of favipiravir pharmacokinetic parameters in infants and young children. Weight significantly improves the model fit as a covariate. Reported EC50 for norovirus in vitro was 19–39µg/mL and enterovirus 71 was 23µg/mL, indicating higher favipiravir doses or combination with other antivirals are required.

Attacking the SARS-CoV-2 Replication Machinery with the Pathogen Box's Molecules

Introduction: The world is currently facing a pandemic initiated by the new coronavirus disease 2019 (COVID-19), caused by the SARS-CoV-2 virus. Viral transcription and replication are the fundamental processes of any virus. They allow the synthesis of genetic material and the consequent multiplication of the virus to infect other cells or organisms.Methods: The most important protein in SARS-CoV-2 is the RNA polymerase (RdRp or nsp12), responsible for both processes. The structure of this protein (PDB ID: 6M71) was used as a target in the application of computational strategies for a drug search, like virtual screening and molecular docking. Here, the Pathogen Box database of chemical compounds was used together with Remdesivir, Beclabuvir, and Sofosbuvir drugs as potential inhibitors of nsp12.Results: The results showed Top10 potential target inhibitors with binding energy (ΔG) higher than those of the positive controls, of which TCMDC-134153 and TCMDC-135052, both with ΔG = −7.53 kcal/mol, present interactions with three important residues of the nsp12 catalytic site.Conclusion: These proposed ligands would be used for subsequent validation by molecular dynamics, where they can be considered as drugs for the development of effective treatments against this new pandemic.

Safety Profile of Molnupiravir with Significant Effect on COVID-19: A Review

Background: As the COVID era unfolds, researchers reveal that rapid changes in viral genetic material allow viruses to circumvent challenges triggered by the host immune system and resist anti-viral drugs, potentially leading to persistent viral manifestations in host cells. Molnupiravir (RNA-dependent RNA polymerase inhibitor) is a novel anti-viral medicine promising a vital role in coming setbacks.Objectives: This review aims to clarify the safety and efficacy of the molnupiravir molecule in light of existing case studies. As a result, it is intended to explore and discuss the molecular structure, mechanism of action, discovery and development process, preclinical research, clinical investigations, and other subtopics.Methods: A total of 75 publications were searched using multiple engines, such as Google Scholar, PubMed, Web of Science, Embase, Cochrane Library, ClinicalTrials.gov, and others, with a constraint applied to exclude publications published over 11 years ago. Molnupiravir, safety, efficacy, COVID- 19, RdRp, PK-PD, and clinical study were utilized as keywords.Results: Clinical results on molnupiravir are supported by investigations that were recently disclosed in a study on both sex volunteers (male and female) with an age restriction of 19 to 60 years, followed by a Phase-3 Clinical Trial (NCT04575584) with 775 randomly assigned participants and no fatalities reported due to treatment.Conclusion: Molnupiravir proved a high level of safety, allowing it to be tested further. This review supports the safety and efficacy of this molecule based on the established evidence, which claims the most anticipated employment of molnupiravir in COVID protocol.

In-silico studies of active phytochemicals from siddha medicinal herbs of karisalai chooranam against SARS-CoV-2 main protease (3 CLpro), RNA dependent RNA polymerase and angiotensin-converting enzyme II receptor

Aim: The contagious disease COVID 19 is a recently out-broken pandemic situation which threatens humankind all over the world. Siddha system of medicine is one of the traditional medical systems of India, which has provided a novel remedy for many epidemics like Dengue, Chicken guinea earlier. On evaluating the literature evidence and considering the mortality and severity of the disease, we have attempted to identify the possible inhibition of viral replication by "Karisalai Chooranam" - a polyherbal Siddha formulation which contains herbs like Karisalai (Wedelia chinensis), Thoodhuvelai (Solanum trilobatum), Musumusukai (Melothria maderaspatana) and Seeragam (Cuminum cyminum). The aim of this study was to identify the bioactive components present in Karisalai chooranam and pin down the components that inhibit COVID 19 protease by In Silico molecular docking analysis. Material and methods: The study was performed for the active compounds present in the herbs (Wedelia chinensis - Benzoic acid, Solanum trilobatum- Disogenin, Melothria maderaspatana- beta-sitosterol, Cuminum cyminum L- Coumaric acid and Limonene) with three potential targets, PDB id: 6LU7 3-chymotrypsin-like protease (3CLpro), PDB id: 6-NUR RNA dependent RNA polymerase and PDB id: 2AJF Angiotensin-converting enzyme II (ACE2) receptor using Autodock Vina. Key findings: The active phytocomponents present in "Karisalai chooranam" was found to inhibit the target 3CL proenzyme and hereby halt the formation of 16 non-structural proteins (nsp1-nsp16) that are highly essential for viral replication and there by prevents viral survival in the host environment. The phytocomponents also inhibited the target RNA dependent RNA polymerase (PDB)-6NUR RdRp which possess versatile action in mediating nonstructural protein (nsp 12) essential for viral replication. A significant binding against the target Angiotensin-converting enzyme II (ACE2) receptors - PDB- 2AJF was found which was recognized as a binding site for novel coronavirus to cause its pathogenesis. Among the five active components present in the herb, the binding ability of Disogenin and beta-sitosterol with COVID19 protease suggests a possible mechanism of protease inhibition and thus preventing viral replication. Significance: The results strongly suggest that phytocomponents of "Karisalai chooranam" may act as a potential therapeutic agent for the management of COVID-19 and related symptoms. Further, the efficacy of the active compounds should be tested in vitro before being recommended as a drug.

Investigation of coronavirus and hepatitis E virus in rodents in Dali City, Yunnan Province

An investigation of coronavirus (CoV) and hepatitis E virus (HEV) in rodents was performed to understand CoV and HEV infection of rodents in Dali City, Yunnan Province. Rodent samples were obtained in the four towns of Dali city through traps from August 2020 to August 2021. A total of 76 rodents belonging to six species and five genera were captured: Rattus tanezumi, Rattus norvegicus, Apodemus chevrieri, Eothenomys miletus, Niviventer fulvescens, and Mus Pahari. Detection of CoV and HEV was performed by nested-PCR. The infection rate of CoV was 40.74% (11/27) and 2.38% (1/42) in R. norvegicus and R. tanezumi, respectively. The infection rate of HEV was 14.81% (4/27) and 2.38% (1/42) in R. norvegicus and R. tanezumi, respectively. Co-infection with CoV and HEV was detected in two R. norvegicus, with a co-infection rate of 7.41% (2/27). A Basic Local Alignment Search Tool (BLAST) search was performed on partial RNA-dependent RNA polymerase (RdRp) sequences of CoV and HEV. Eleven strains from R. norvegicus were a-CoV, and matched best to strain KY370050 from Rattus losea (Fujian, China), with 99.73% to 99.74% nucleotide (nt) sequency identity. One strain was ss-CoV from R. tanezumi, which displayed 98.21% nt sequence identity with strain MT820632 from Bandicota indica (Yunnan, China). Five strains from R. norvegicus were all HEV-C, and showed 95.87% to 96.21% sequence similarity to strain MN450853 from a patient in Hong Kong, China. In conclusion, CoV and HEV infections are present in rodents in Dali City. Because the host animals of the two viruses are closely related to humans, surveillance and investigations of related viruses should be strengthened.

Scleroderma Renal Crisis and COVID—Is There an Association?

Scleroderma renal crisis (SRC) is a rare but potentially devastating complication of systemic sclerosis as it is associated with significant morbidity and mortality. We present an interesting case of a patient who developed SRC following infection with COVID-19. A 37-year-old female presented with new-onset hypertension, AKI, anemia and thrombocytopenia. She had a history of diffuse cutaneous systemic sclerosis diagnosed 8 years ago, that had been well controlled with immunosuppression. The patient had contracted COVID-19 infection about 2 weeks ago but had remained largely asymptomatic except for a sore throat. Urinalysis revealed sub-nephrotic proteinuria but was otherwise bland. Peripheral blood smear was notable for 12-15 schistocytes per HPF. ADAMTS13 and complement levels were normal. Serologies for ANA, ANCA, anti-Scl70, anti-Jo1, anti-Sm, lupus anticoagulant, anti-beta2-glycoprotein I, anti-RNA polymerase III, RF, cryoglobulin, RPR, hepatitis and HIV, all returned negative. Renal biopsy revealed an arterial predominant thrombotic microangiopathy (TMA) (Figure) consistent with a diagnosis of SRC. The patient was treated with anti-hypertensives including an ACE-inhibitor, but her AKI continued to worsen, ultimately leading to dialysis dependence. SRC classically develops in patients with early or progressive diffuse cutaneous disease or positivity for anti-RNA polymerase III antibodies. Our patient did not have any such risk factors and rather developed SRC following infection with COVID-19. COVID-19 has been reported to cause TMA by inducing immune dysregulation via an overactive complement system. It is plausible that infection with COVID-19 triggered an exaggerated immune response, in turn leading to the development of SRC in our patient. COVID-19 may trigger SRC in patients with systemic sclerosis in the absence of other risk factors. (Figure Presented)

GETTING TO THE HEART OF THE MATTER

CASE: 45-year-old woman with PMHx systemic sclerosis presents with fever, weight loss, chest tightness, weakness and altered mental status for 2 weeks. Home meds are prednisone, mycophenolic acid, lasix. On presentation she is febrile to 38.9C, HR 110, BP 97/64, SpO2 96% on RA. Exam shows telangiectasis, normal cardiopulmonary exam, mild sclerodactyly. Oriented only to self, has bilateral LE 3/5 weakness. Labs with WBC 2.6K, Hgb 7.1, plts 126K. Cr normal. Liver enzymes mildly elevated. BNP 3900. Trop 251. Lactate 4.9 Blood cultures negative, CMV/EBV negative, COVID-19 negative, Ferritin > 15,000, Triglycerides 274 LDH 495, Fibrinogen 274, D-Dimer 755, ANA 1:1280, + dsDNA, low titer Smith, + RNP, + SSA, + RNA Pol III. TTE with EF 27% and diffuse hypokinesis. Cardiac MRI with myocardial fibrosis no active myocarditis, suggestive of scleroderma. Lumbar puncture with high protein, borderline increased oligoclonal bands, elevated IgG index but elevated synthesis rate, suggestive of CNS inflammation. Patient is in cardiogenic shock secondary to hemophagocytic lymphohistiocytosis/macrophage activating syndrome (HLH/MAS) related to systemic sclerosis/scleroderma with SLE overlap requiring inotropes and aggressive diuresis. She develops severe pain and bright red purpura on bilateral legs. Hypercoagulable w/u showed low protein C/S, low complement, negative cryoglobulin. Skin biopsy showed vaso-occlusive process c/w HLH/MAS. Receives IV methylprednisolone for empiric treatment of HLH/MAS and IV cyclophosphamide for possible lupus cerebritis. Patient improves and is discharged on long-term milrinone, Plaquenil, and steroids. IMPACT/DISCUSSION: Secondary HLH or MAS is a life-threatening condition of extreme inflammation that can occur in autoimmune conditions, infection, or malignancy Diagnosing HLH requires high clinical suspicion - >10K ferritin level is highly sensitive and specific for diagnosis of HLH This patient has multisystem involvement of autoimmune disease given history of scleroderma The LP studies raise concern for lupus cerebritis, specifically the IgG index and IgG synthesis rate are helpful for this diagnosis Underline subtype of systemic sclerosis-overlap syndromes and here particularly scleroderma lupus overlap Highlight the utility of cardiac MRI in characterizing myocarditis / fibrosis Discuss need for high alert for necrotizing fasciitis with painful palpable purpura Overview treatment of HLH/MAS with high dose steroids Reflection on high mortality of HLH/MAS and question of recovered heart function CONCLUSION: Teaching Point 1: Secondary HLH is a syndrome of extreme inflammation caused by underlying malignancy, autoimmune condition, or infection. Teaching Point 2: HLH and MAS have a great deal of symptom/clinical presentation overlap. Ferritin level > 10,000 is highly sensitive and specific for diagnosis of HLH Teaching Point 3: Systemic sclerosis can present in a variety of ways including cardiac, lung, skin involvement.

Potential of Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) as a Simple Rapid Molecular Diagnostic Tool to Detect SARS-CoV-2 Nucleocapsid Gene

Introduction: The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), identified in December of 2019, is the cause of the coronavirus disease 2019 (COVID-19). Due to the high reproductive rate of the virus, the best way to slow down the spread is to identify and isolate patients at the early stage of infections. The current diagnostic methods are either too expensive, slow or have low accuracy. Variants of SARS-CoV-2 with mutations at the primer binding sites may cause evasion of polymerase chain reaction (PCR) detection using current primers. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) has potential as a rapid molecular test that is easy to conduct. Methods: LAMP primers were design based on the highly conserved regions of the SARS-CoV-2 Nucleocapsid (N) gene. RT-LAMP assays were conducted using an optimized Bst 3.0 polymerase protocol on T7 RNA polymerase synthesized RNA template. The LAMP sensitivity assay was tested on 1:10 serial diluted pJET1.2 vector with SARSCoV2 N gene inserts. A specificity test was conducted by running the test on plasmids containing SARS-CoV and MERS-CoV N genes. The results were visualised via gel electrophoresis, SYBR Green staining and Lateral Flow Dipstick (LFD). Results: The optimized protocol is sensitive enough to detect SARS-CoV-2 genetic material within 10 minutes but is most sensitive at 30 minutes. Additionally, it is specific to only the genetic materials of SARS-CoV-2. Furthermore, an LFD with multiple test lines was successful for multiplexed LAMP reactions with different genic regions of the virus. Conclusion: The multiplexed LFD-LAMP is potentially a simple yet specific and sensitive method of rapid molecular diagnostics of COVID-19.

Molecular docking and dynamics of phytochemicals from Chinese herbs with SARS-CoV-2 RdRp

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is causing coronavirus disease 2019 (COVID-19) pandemic. Ancient Chinese herbal formulas are effective for diseases caused by viral infection, and their effects on COVID-19 are currently being examined. To directly evaluate the role of Chinese herbs in inhibiting replication of SARS-CoV-2, we investigated how the phytochemicals from Chinese herbs interact with the viral RNA-dependent RNA polymerase (RdRP). Total 1025 compounds were screened, and then 181compounds were selected for molecular docking analysis. Four phytochemicals licorice glycoside E, diisooctyl phthalate, (-)-medicocarpin, and glycyroside showed good binding affinity with RdRp. The best complex licorice glycoside E/RdRp forms 3 hydrogen bonds, 4 hydrophobic interactions, 1 pair of Pi-cation/stacking, and 4 salt bridges. Furthermore, docking complexes licorice glycoside E/RdRp and diisooctyl phthalate/RdRp were optimized by molecular dynamics simulation to obtain the stable conformation. These studies indicate that they are promising as antivirals against SARS-CoV-2.

Terpenoids from Centella asiatica, a novel inhibitor against RNA-dependent-RNA polymerase activity of NSP12 of the SARS CoV-2 (COVID-19)

Covid-19, the first case of which was reported in Wuhan (China) in December 2019 was found to be a strain of coronaviruses like SARS and MERS reported earlier. These viruses are positive strain RNA viruses composed of both structural as well as non-structural proteins. The enzyme RdRp (RNA dependent RNA polymerase) stands responsible for catalyzing the replication of this virus within the host cell. A disruption in the core catalytic subunit composed of nsp12, nsp7, and nsp8 may inhibit the replication of the same. Different drugs targeting different sites on the virus have been developed. In this context, some of the natural products of the plant Centella asiatica was lead for further drug development against the target proteins of RdRp protein (PDB ID: 6NUR) through molecular docking. These compounds are 2,3-dihydroxyurs-12-en-28-oic acid, corosolic acid and pomolic acid which are triterpenoids and have highest binding affinity against RdRp protein thereby arresting the viral replication. Several previous studies showed triterpenoids as pertinent mediators implicated in the in vitro immune response.

In silico study identifies RO 28-2653 as a novel drug against SARS-CoV2 mutant strains

Introduction: Concerning the current pandemic situation, the world is facing due to the highly infectious coronavirus (SARS-CoV2), we aim to gain some insight into the pre-existing drugs and compounds for curing the disease. Method: Here, we have studied the interaction of 10 drug molecules by in silico study against three targets, Angiotensin Convertase Enzyme-2 receptor (ACE-2), main protease (Mpro) and RNA dependent RNA polymerase (RDRP) and further analysed the interaction of the best docked compound against spike mutants. Results: By analysing the protein-ligand interactions by docking, and molecular dynamics simulation, it proves that RO 28-2653 can be a potent candidate drug for future COVID treatment even against the mutant strains. Conclusion: The used drugs have been implicated in asthma, hypertension, etc., so repurposing these drugs can have a beneficial role on COVID-19, keeping in mind that any drug should be used in a certain prescribed dosage.

Susceptibility of spike glycoprotein and RNA-dependent RNA polymerase of SARS-CoV-2 to mutation: in silico structural dynamics study

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a microorganism that causes coronavirus disease 2019 (COVID-19). Mutations affect evolutionary conservation of microorganisms. The fast pace evolutionary changes are currently affecting pathogenicity of SARS-CoV-2. In this study, the structural fluctuations of the amino acid residues in the spike glycoprotein and RNA-dependent RNA polymerase (nsp12) of SARS-CoV-2 were investigated by in silico approach using structural flexibility dynamics to decipher susceptibility to mutation. The result of this study implicated key amino acid residues (with rmsf) which could be very susceptible to mutation, which include residues 50 (3.79 Å), 119 (4.56 Å), 120 (3.53 Å), 220 (3.84 Å), 265 (4.31 Å) of RNA-dependent RNA polymerase (nsp12), as well as residues 477 (4.21 Å), 478 (4.82 Å), 479 (5.40 Å), 481 (5.94 Å), 560 (4.63 Å), 704 (4.02 Å), 848 (4.58 Å), 1144 (4.56 Å) and 1147 (4.61 Å) of spike glycoprotein. The SARS-CoV-2 mutations destabilized the overall protein structure in multiples of amino acid residues which could interfere with active site leading to insensitivity or resistance to the inhibitors. Mutation T478K of Spike glycoprotein showed the highest deviation in the structure. Overall, spike glycoprotein has the highest number of mutations, and these variants could increase the risk to human health if not mitigated in the population.

Progress in drug development for treatment of coronavirus disease 2019 (COVID-19)

Since the outbreak of the novel coronavirus (SARS-CoV-2), the number of people infected worldwide has been increasing, and the medical situation is very severe. In emergency situations, the development of innovative drugs and the treatment of new coronavirus pneumonia (COVID-19) new adaptations on the market The development of the certificate has become the only way to find specific therapeutic drugs and the best treatment plan for COVID-19. The mechanism of angiotensin-converting enzyme 2 (ACE2) that mediates the invasion of host cells by SARS-CoV-2 has been discovered and is based on SARS-CoV- 2. Potential therapeutic targets of host and host, mainly including RNA-dependent RNA polymerase, 3CL protease, papain-like protease, Janus kinase, interleukin 6 and immunomodulators, etc. According to the above-mentioned pharmacological mechanism of action, the treatment of marketed drugs Great progress has been made in the development of new indications for COVID-19 and the clinical research and development of innovative drugs, but no specific drugs have been found. Some traditional Chinese medicines in China can block the SARS-CoV-2 replication cycle, regulate the body's immune response, and treat COVID-19. Biopharmaceuticals are currently undergoing phase I clinical studies in the world for the treatment of COVID-19. Biopharmaceuticals are progressing rapidly, accounting for 67%. At present, the research and development of drugs for the treatment of COVID-19 in China is facing severe challenges and biosafety The number of protection laboratories is small, the research on the mechanism of SARS-CoV-2 infection and the body's response mechanism is not in-depth, the resources of non-clinical cells and animal models are scarce, and the professional quantitative pharmacology research platform and professional talent training system are not perfect to treat COVID-19 The informatization of drug clinical trials and sample testing capabilities are in urgent need of improvement. If China can use this to improve its ability to develop new drugs in emergency situations, it will be able to better protect people's health.