RESUMO
Influenza remains a worldwide health concern. Antiviral drugs are considered as one of the useful options for its prevention as a complementary measure to vaccination. Baloxavir acid selectively inhibits the cap-dependent endonuclease of influenza viruses and exhibits marked viral titre reduction in patients. Here, we describe the prophylactic potency of baloxavir acid against lethal infection with influenza A and B viruses in mice. BALB/c mice were subcutaneously administered once with baloxavir acid suspension, or orally administered once daily for 10 days with oseltamivir phosphate solution at human relevant doses. Next, the mice were intranasally inoculated with A/PR/8/34 (H1N1) or B/Hong Kong/5/72 strain at 24 to 96 h after the initial dosing. Prophylactic treatment with the antiviral drugs significantly reduced the lung viral titres and prolonged survival time. In particular, baloxavir acid showed a greater suppressive effect on lung viral titres compared to oseltamivir phosphate. In this model, baloxavir acid maintained significant prophylactic effects against influenza A and B virus infections when the plasma concentration at the time of infection was at least 0.88 and 3.58 ng/mL, respectively. The significant prophylactic efficacy observed in our mouse model suggests the potential utility of baloxavir marboxil for prophylaxis against influenza in humans.
Assuntos
Herpesvirus Cercopitecino 1 , Vírus da Influenza A Subtipo H1N1 , Influenza Humana , Tiepinas , Humanos , Animais , Camundongos , Influenza Humana/tratamento farmacológico , Influenza Humana/prevenção & controle , Oseltamivir/farmacologia , Oseltamivir/uso terapêutico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/farmacologia , Tiepinas/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Camundongos Endogâmicos BALB C , FosfatosRESUMO
Baloxavir marboxil (BXM) is an FDA-approved antiviral prodrug for the treatment of influenza A and B infection and postexposure prophylaxis. The active form, baloxavir acid (BXA), targets the cap-snatching endonuclease (PA) of the influenza virus polymerase complex. The nuclease activity delivers the primer for transcription, and previous reports have shown that BXA blocks the nuclease activity with high potency. However, biochemical studies on the mechanism of action are lacking. Structural data have shown that BXA chelates the two divalent metal ions at the active site, like inhibitors of the human immunodeficiency virus type 1 (HIV-1) integrase or ribonuclease (RNase) H. Here we studied the mechanisms underlying the high potency of BXA and how the I38T mutation confers resistance to the drug. Enzyme kinetics with the recombinant heterotrimeric enzyme (FluB-ht) revealed characteristics of a tight binding inhibitor. The apparent inhibitor constant (Kiapp) is 12 nM, while the I38T mutation increased Kiapp by â¼18-fold. Order-of-addition experiments show that a preformed complex of FluB-ht, Mg2+ ions and BXA is required to observe inhibition, which is consistent with active site binding. Conversely, a preformed complex of FluB-ht and RNA substrate prevents BXA from accessing the active site. Unlike integrase inhibitors that interact with the DNA substrate, BXA behaves like RNase H inhibitors that compete with the nucleic acid at the active site. The collective data support the conclusion that BXA is a tight binding inhibitor and the I38T mutation diminishes these properties.
Assuntos
Dibenzotiepinas/farmacologia , Endonucleases/antagonistas & inibidores , Vírus da Influenza B/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/virologia , Morfolinas/farmacologia , Piridonas/farmacologia , RNA Polimerase Dependente de RNA/antagonistas & inibidores , Triazinas/farmacologia , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Domínio Catalítico , Endonucleases/metabolismo , Humanos , Vírus da Influenza B/enzimologia , Vírus da Influenza B/isolamento & purificação , Influenza Humana/enzimologia , RNA Polimerase Dependente de RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Virais/antagonistas & inibidores , Proteínas Virais/metabolismoRESUMO
Baloxavir marboxil is a new drug developed in Japan by Shionogi to treat seasonal flu infection. This cap-dependent endonuclease inhibitor is a prodrug that releases the biologically active baloxavir acid. This new medicine has been marketed in Japan, the USA and Europe. It is well tolerated (more than 1% of the patients experienced diarrhea, bronchitis, nausea, nasopharyngitis, and headache), and both influenza A and B viruses are sensitive, although the B strain is more resistant due to variations in the amino acid residues in the binding site. The drug is now in post-marketing pharmacovigilance phase, and its interest will be especially re-evaluated in the future during the annual flu outbreaks. It has been also introduced in a recent clinical trial against COVID-19 with favipiravir.
RESUMO
Neuraminidase inhibitors (NAIs), that currently include oseltamivir (Tamiflu® ), zanamivir (Relenza® ), peramivir (Rapivab® ) and laninamivir (Inavir® ), constitute an important class of antivirals recommended against seasonal influenza A and B infections. NAIs target the surface NA protein whose sialidase activity is responsible for virion release from infected cells. Because of their pivotal role in the transcription/translation process, the polymerase acidic (PA) and polymerase basic 1 and 2 (PB1 and PB2, respectively) internal proteins also constitute targets of interest for the development of additional anti-influenza agents. Baloxavir marboxil (BXM), an inhibitor of the cap-dependent endonuclease activity of the influenza PA protein, was approved in the United States and Japan in 2018. Baloxavir acid (BXA), the active compound of BXM, demonstrated a potent in vitro activity against different types/subtypes of influenza viruses including seasonal influenza A/B strains as well as avian influenza A viruses with a pandemic potential. A single oral dose of BXM provided virological and clinical benefits that were respectively superior or equal to those displayed by the standard (5 days, twice daily) oseltamivir regimen. Nevertheless, BXM-resistant variants have emerged at relatively high rates in BXM-treated children and adults. Consequently, there is a need to study the fitness (virulence and transmissibility) characteristics of mutants with a high potential to emerge as such variants can compromise the clinical usefulness of BXM. The purpose of this manuscript is to review the fitness properties of influenza A and B isolates harbouring mutations of reduced susceptibility to BXA.
Assuntos
Antivirais/farmacologia , Dibenzotiepinas/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza B/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Morfolinas/farmacologia , Piridonas/farmacologia , Triazinas/farmacologia , Farmacorresistência Viral , Humanos , Vírus da Influenza A/isolamento & purificaçãoRESUMO
Baloxavir marboxil (BXM) demonstrated a rapid and profound decline in infectious viral titer 1 day after BXM administration. Rapid reduction in virus titer is a characteristic of BXM. There may be a possibility that drug carryover effects have impacts on the observed antiviral effects due to the poor correlation that was observed between viral titer reduction and alleviation of influenza symptoms. Here, we report possible carryover effects of baloxavir acid (BXA), an active form of BXM, on infectious titer testing. Our findings indicate that there is little impact of BXA carryover on the infectious titer testing.
Assuntos
Antivirais/administração & dosagem , Dibenzotiepinas/administração & dosagem , Influenza Humana/tratamento farmacológico , Morfolinas/administração & dosagem , Nasofaringe/virologia , Orthomyxoviridae/efeitos dos fármacos , Faringe/virologia , Piridonas/administração & dosagem , Triazinas/administração & dosagem , Humanos , Influenza Humana/diagnóstico , Influenza Humana/virologia , Orthomyxoviridae/genética , Orthomyxoviridae/crescimento & desenvolvimento , Orthomyxoviridae/fisiologiaRESUMO
Susceptibility of influenza A viruses to baloxavir can be affected by changes at amino acid residue 38 in the polymerase acidic (PA) protein. Information on replicative fitness of PA-I38-substituted viruses remains sparse. We demonstrated that substitutions I38L/M/S/T not only had a differential effect on baloxavir susceptibility (9- to 116-fold) but also on in vitro replicative fitness. Although I38L conferred undiminished growth, other substitutions led to mild attenuation. In a ferret model, control viruses outcompeted those carrying I38M or I38T substitutions, although their advantage was limited. These findings offer insights into the attributes of baloxavir-resistant viruses needed for informed risk assessment.
Assuntos
Antivirais/uso terapêutico , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H3N2/genética , Infecções por Orthomyxoviridae/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Replicação Viral/genética , Substituição de Aminoácidos , Animais , Dibenzotiepinas , Modelos Animais de Doenças , Cães , Furões , Sequenciamento de Nucleotídeos em Larga Escala , Células Madin Darby de Rim Canino , Masculino , Testes de Sensibilidade Microbiana , Morfolinas , Infecções por Orthomyxoviridae/virologia , Piridonas , RNA Polimerase Dependente de RNA/genética , Estações do Ano , Resultado do Tratamento , Proteínas Virais/genéticaRESUMO
In 2019, influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic gene, which confers reduced susceptibility to baloxavir, were detected in Japan in an infant without baloxavir exposure and a baloxavir-treated sibling. These viruses' whole-genome sequences were identical, indicating human-to-human transmission. Influenza virus isolates should be monitored for baloxavir susceptibility.
Assuntos
Antivirais/farmacologia , Suscetibilidade a Doenças , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Influenza Humana/transmissão , Influenza Humana/virologia , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , Adolescente , Adulto , Antivirais/uso terapêutico , Criança , Dibenzotiepinas , Humanos , Lactente , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/epidemiologia , Japão/epidemiologia , Pessoa de Meia-Idade , Morfolinas , Mutação , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Piridonas , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Adulto JovemRESUMO
In January 2019, two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA), which confers reduced susceptibility to baloxavir, were detected from epidemiologically unrelated hospitalised children in Japan. The viruses exhibited reduced susceptibility to baloxavir but were susceptible to neuraminidase inhibitors. Only one of the two children had been treated with baloxavir. An epidemiological analysis suggests possible transmission of the PA I38T mutant A(H3N2) virus among humans.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/genética , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/farmacologia , Piridinas/farmacologia , Tiepinas/farmacologia , Triazinas/farmacologia , Adolescente , Adulto , Antivirais/uso terapêutico , Criança , Dibenzotiepinas , Inibidores Enzimáticos/farmacologia , Humanos , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Pacientes Internados , Japão , Pessoa de Meia-Idade , Morfolinas , Oxazinas/uso terapêutico , Reação em Cadeia da Polimerase , Piridinas/uso terapêutico , Piridonas , Tiepinas/uso terapêutico , Resultado do Tratamento , Triazinas/uso terapêutico , Adulto JovemRESUMO
The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza virus infection in Japan in February 2018. Two influenza A(H3N2) viruses carrying an I38T substitution in the polymerase acidic subunit (PA) were detected in baloxavir-treated children in December 2018. This mutation is known to confer reduced susceptibility to baloxavir, and the two mutant viruses exhibited 76- and 120-fold reduced susceptibility to baloxavir.
Assuntos
Antivirais/uso terapêutico , Endonucleases/antagonistas & inibidores , Vírus da Influenza A Subtipo H3N2/efeitos dos fármacos , Vírus da Influenza A Subtipo H3N2/genética , Influenza Humana/tratamento farmacológico , Oxazinas/uso terapêutico , Piridinas/uso terapêutico , Tiepinas/uso terapêutico , Triazinas/uso terapêutico , Substituição de Aminoácidos/genética , Antivirais/administração & dosagem , Dibenzotiepinas , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Endonucleases/genética , Humanos , Vírus da Influenza A Subtipo H3N2/isolamento & purificação , Influenza Humana/diagnóstico , Japão , Testes de Sensibilidade Microbiana , Morfolinas , Piridonas , Resultado do TratamentoRESUMO
The novel cap-dependent endonuclease inhibitor baloxavir marboxil was approved for the treatment of influenza A and B virus infections in February 2018 in Japan. Because of the need to monitor influenza viruses for reduced susceptibility to this drug, we used two cell-based screening systems - a conventional plaque reduction assay and a focus reduction assay - to evaluate the susceptibility of influenza viruses to baloxavir. First, we generated a reference virus possessing an I38T substitution in the polymerase acidic subunit (PA), which is known to be associated with reduced susceptibility to baloxavir, and demonstrated the validity of our systems using this reference virus. We then determined the susceptibility of a panel of neuraminidase (NA) inhibitor-resistant viruses and their sensitive counterparts to baloxavir. No significant differences in baloxavir susceptibilities were found between the NA inhibitor-resistant and -sensitive viruses. We also examined seasonal influenza viruses isolated during the 2017-2018 influenza season in Japan and found that no currently circulating A(H1N1)pdm09, A(H3N2), or B viruses had significantly reduced susceptibility to baloxavir and none of the viruses possessed an amino acid substitution at PA residue 38. Use of a combination of methods to analyze antiviral susceptibility and detect amino acid substitutions is valuable for monitoring the emergence of baloxavir-resistant viruses.
RESUMO
Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.