Baloxavir Acid Research Articles

An Improved Enzyme-Linked Focus Formation Assay Revealed Baloxavir Acid as a Potential Antiviral Therapeutic Against Hantavirus Infection.

Hantaviruses, etiologic pathogens responsible for two severe human diseases, exist in areas ranging from Eurasia to America and remain global public health concerns. Conventionally, plaque formation assays have been used for hantavirus titering. However, hantaviruses replicate slowly within cells and produce minimal cytopathic effects, making this technique difficult to master. The improved enzyme-linked immunosorbent assay-based antigen detection method is easier to perform but is still time consuming. Here, we established an enzyme-linked focus formation assay (FFA) for Hantaan virus titering that is twice as fast as traditional assays. Moreover, using this method, we evaluated the effects of favipiravir (T-705) and another influenza virus drug, baloxavir acid (BXA), on hantavirus replication. We found that the endonuclease inhibitor BXA exerted similar anti-hantavirus effects as T-705. Overall, we developed a time-saving method for hantavirus titering and suggest BXA as a potential treatment choice for hantavirus-exposed individuals.

Population Pharmacokinetics of Baloxavir Marboxil in Japanese Pediatric Influenza Patients

Baloxavir marboxil is a prodrug of baloxavir acid, an inhibitor of cap-dependent endonuclease, and suppresses the replication of influenza virus. The aim of this study was to investigate its pharmacokinetic characteristics in Japanese pediatrics. Population pharmacokinetic analysis was conducted for baloxavir acid with 328 plasma concentration data points in a clinical study of 107 Japanese pediatric influenza patients. The plasma baloxavir acid concentration profiles were well captured by a 2-compartment model including first-order absorption and lag time. Body weight was considered to be the most crucial covariate, which affects clearance and volume of distribution. The body weight–based dose regimen (10 mg for 10 kg to less than 20 kg pediatrics, 20 mg for 20 kg to less than 40 kg pediatrics, and 40 mg for at least 40 kg pediatrics) for Japanese pediatrics can provide comparable exposure to baloxavir acid to that for adults. In conclusion, the population pharmacokinetic model would be useful to comprehend the characteristics of baloxavir acid pharmacokinetics in pediatric patients.

Inhibition of avian-origin influenza A(H7N9) virus by the novel cap-dependent endonuclease inhibitor baloxavir marboxil

Human infections with avian-origin influenza A(H7N9) virus represent a serious threat to global health; however, treatment options are limited. Here, we show the inhibitory effects of baloxavir acid (BXA) and its prodrug baloxavir marboxil (BXM), a first-in-class cap-dependent endonuclease inhibitor, against A(H7N9), in vitro and in vivo. In cell culture, BXA at four nanomolar concentration achieved a 1.5–2.8 log reduction in virus titers of A(H7N9), including the NA-R292K mutant virus and highly pathogenic avian influenza viruses, whereas NA inhibitors or favipiravir required approximately 20-fold or higher concentrations to achieve the same levels of reduction. A(H7N9)-specific amino acid polymorphism at position 37, implicated in BXA binding to the PA endonuclease domain, did not impact on BXA susceptibility. In mice, oral administration of BXM at 5 and 50 mg/kg twice a day for 5 days completely protected from a lethal A/Anhui/1/2013 (H7N9) challenge, and reduced virus titers more than 2–3 log in the lungs. Furthermore, the potent therapeutic effects of BXM in mice were still observed when a higher virus dose was administered or treatment was delayed up to 48 hours post infection. These findings support further investigation of BXM for A(H7N9) treatment in humans.

Baloxavir marboxil susceptibility of influenza viruses from the Asia-Pacific, 2012–2018

Baloxavir Marboxil (BXM) is an influenza polymerase inhibitor antiviral that binds to the endonuclease region in the PA subunit of influenza A and B viruses. To establish the baseline susceptibility of viruses circulating prior to licensure of BXM and to monitor for susceptibility post-BXM use, a cell culture-based focus reduction assay was developed to determine the susceptibility of 286 circulating seasonal influenza viruses, A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, including neuraminidase inhibitor (NAI) resistant viruses, to Baloxavir Acid (BXA), the active metabolic form of BXM. BXA was effective against all influenza subtypes tested with mean EC50 values (minimum-maximum) of 0.7 ± 0.5 nM (0.1–2.1 nM), 1.2 ± 0.6 nM (0.1–2.4), 7.2 ± 3.5 nM (0.7–14.8), and 5.8 ± 4.5 nM (1.8–15.5) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses, respectively. Using reverse genetics, amino acid substitutions known to alter BXA susceptibility were introduced into the PA protein resulting in EC50 fold change increases that ranged from 2 to 65. Our study demonstrates that currently circulating viruses are susceptible to BXA and that the newly developed focus reduction assay is well suited to susceptibility monitoring in reference laboratories.

Population Pharmacokinetic and Exposure-Response Analyses of Baloxavir Marboxil in Adults and Adolescents Including Patients With Influenza

Baloxavir marboxil, a prodrug that is metabolized to baloxavir acid, suppresses viral replication by inhibiting cap-dependent endonuclease. Our aim is to characterize its pharmacokinetics and exposure-response relationships. Population pharmacokinetic analysis of the baloxavir acid was performed using 8310 plasma concentration data points from 1109 subjects. Exposure-response analyses were performed regarding the time to alleviation of symptoms and the reduction in the influenza virus titer. A 2-compartment model with first-order absorption and lag time well described the plasma concentration data for baloxavir acid, and body weight and race were found to be the most important factors influencing the clearance and distribution volume. A dose regimen based on the body weight (40 mg for patients weighing <80 kg and 80 mg for patients weighing ≥80 kg) could provide sufficient exposures for expecting efficacy irrespective of body weight or race; however, the exposures were dependent on the body weight and race. Exposure-response analyses suggested that the reduction in the influenza virus titer was greater in any exposure-based groups in baloxavir marboxil treatment than in the oseltamivir phosphate treatment and placebo groups. In conclusion, the population pharmacokinetic model and exposure-response relationships would be useful for understanding the pharmacokinetic and pharmacodynamic characteristics of baloxavir acid.

1350. Therapeutic Effects of Baloxavir Marboxil against Influenza A Virus Infection in Ferrets

BackgroundBaloxavir marboxil (BXM) is a novel small molecule inhibitor of cap-dependent endonuclease that is essential for influenza virus transcription and replication. In this study, pharmacokinetic profiles of BXM and baloxavir acid (BXA), an active form of BXM, were first examined in ferrets, and then the therapeutic effects of BXM against influenza A virus infection were compared with that of oseltamivir phosphate in ferrets.MethodsThe plasma exposure of BXA and BXM was examined after a single oral administration of BXM at doses of 10 and 30 mg/kg. The concentrations in plasma were determined by liquid chromatography-tandem mass spectrometry(LC/MS/MS). For efficacy study, ferrets infected intranasally with A/Kadoma/2006 (H1N1) were administrated 10 or 30 mg/kg of BXM orally twice daily for 1 day, starting at 1 day post-infection (p.i.) or administrated 10 mg/kg of BXM orally twice daily for 1 day, starting at 2 days p.i.. Oseltamivir phosphate was administered at doses of 5 mg/kg orally twice daily for 2 days as a comparison. The virus titer in the nasal washes and body temperature change were monitored during infection.ResultsBXA was detected in ferret plasma after a single oral administration of BXM at 10 and 30 mg/kg, in more than a dose-proportional manner. When the treatment was initiated at 1 day p.i., BXM at 10 and 30 mg/kg showed reduction of virus titer to an undetectable level on day 2 p.i. and statistically significant reduction in virus titer over time from day 2 to 3 p.i. compared with vehicle and oseltamivir phosphate. Moreover, the change of body temperature over time from 8 hours after the first administration to 3 days p.i. was significantly lower in BXM at 10 and 30 mg/kg than vehicle and oseltamivir phosphate. These effects were also observed in ferrets treated with BXM at 10 mg/kg even when administered at 2 day p.i. where ferret exhibit fever that is more than 1 degree higher than on 1 day p.i..ConclusionSingle-day oral administration of BXM had beneficial effects on viral titer and symptoms in ferrets infected with influenza A virus, which were superior to those observed with oseltamivir phosphate and vehicle.Disclosures M. Kitano, Shionogi & Co., Ltd.: Employee, Salary. T. Matsuzaki, Shionogi & Co., Ltd.: Employee, Salary. R. Oka, Shionogi & Co., Ltd.: Employee, Salary. K. Baba, Shionogi TechnoAdvance Research & Co., Ltd.: Employee, Salary. T. Noda, Shionogi TechnoAdvance Research & Co., Ltd.: Employee, Salary. Y. Yoshida, Shionogi & Co., Ltd.: Employee, Salary. K. Sato, Shionogi & Co., Ltd.: Employee, Salary. R. Yoshida, Shionogi & Co., Ltd.: Employee, Salary. A. Sato, Shionogi & Co., Ltd.: Employee, Salary. H. Kamimori, Shionogi & Co., Ltd.: Employee, Salary. T. Shishido, Shionogi & Co., Ltd.: Employee, Salary. A. Naito, Shionogi & Co., Ltd.: Employee, Salary.

Combination treatment with the cap-dependent endonuclease inhibitor baloxavir marboxil and a neuraminidase inhibitor in a mouse model of influenza A virus infection.

ObjectivesBaloxavir marboxil (formerly S-033188) is a first-in-class, orally available, cap-dependent endonuclease inhibitor licensed in Japan and the USA for the treatment of influenza virus infection. We evaluated the efficacy of delayed oral treatment with baloxavir marboxil in combination with a neuraminidase inhibitor in a mouse model of lethal influenza virus infection.MethodsThe inhibitory potency of baloxavir acid (the active form of baloxavir marboxil) in combination with neuraminidase inhibitors was tested in vitro. The therapeutic effects of baloxavir marboxil and oseltamivir phosphate, or combinations thereof, were evaluated in mice lethally infected with influenza virus A/PR/8/34; treatments started 96 h post-infection.ResultsCombinations of baloxavir acid and neuraminidase inhibitor exhibited synergistic potency against viral replication by means of inhibition of cytopathic effects in vitro. In mice, baloxavir marboxil monotherapy (15 or 50 mg/kg twice daily) significantly and dose-dependently reduced virus titre 24 h after administration and completely prevented mortality, whereas oseltamivir phosphate treatments were not as effective. In this model, a suboptimal dose of baloxavir marboxil (0.5 mg/kg twice daily) in combination with oseltamivir phosphate provided additional efficacy compared with monotherapy in terms of virus-induced mortality, elevation of cytokine/chemokine levels and pathological changes in the lung.ConclusionsBaloxavir marboxil monotherapy with 96 h-delayed oral dosing achieved drastic reductions in virus titre, inflammatory response and mortality in a mouse model. Combination treatment with baloxavir acid and oseltamivir acid in vitro and baloxavir marboxil and oseltamivir phosphate in mice produced synergistic responses against influenza virus infections, suggesting that treating humans with the combination may be beneficial.

In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit

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.

Characterization of influenza virus variants induced by treatment with the endonuclease inhibitor baloxavir marboxil

Baloxavir acid (BXA), derived from the prodrug baloxavir marboxil (BXM), potently and selectively inhibits the cap-dependent endonuclease within the polymerase PA subunit of influenza A and B viruses. In clinical trials, single doses of BXM profoundly decrease viral titers as well as alleviating influenza symptoms. Here, we characterize the impact on BXA susceptibility and replicative capacity of variant viruses detected in the post-treatment monitoring of the clinical studies. We find that the PA I38T substitution is a major pathway for reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC50 changes in A and B viruses, respectively. The viruses harboring the I38T substitution show severely impaired replicative fitness in cells, and correspondingly reduced endonuclease activity in vitro. Co-crystal structures of wild-type and I38T influenza A and B endonucleases bound to BXA show that the mutation reduces van der Waals contacts with the inhibitor. A reduced affinity to the I38T mutant is supported by the lower stability of the BXA-bound endonuclease. These mechanistic insights provide markers for future surveillance of treated populations.