Influenza virus[1] Cap-dependent endonuclease (CEN)[1][2]

Baloxavir marboxil (also known as BXM or S-033188) is the prodrug of Baloxavir (trade name Xofluza; also known as Baloxavir acid, BXA,or S-033447). It is an orally available small molecule inhibitor of the cap-dependent endonuclease developed by Roche and Shionogi. Baloxavir was discovered by rational molecular design based on the two-metal pharmacophore concept for dolutegravir (DTG), a strand transfer inhibitor of human immunodeficiency virus (HIV) integrase. Baloxavir potently and selectively inhibits the cap-dependent endonuclease within the polymerase PA subunit of influenza A and B viruses. In February 2018, baloxavir received its first global approval in Japan for the treatment of influenza A or B virus infections. Phase III development is underway in the USA, EU and other countries for this indication.The drug blocks influenza virus proliferation by inhibiting the initiation of mRNA synthesis. In clinical trials, single doses of Baloxavir profoundly decrease viral titers as well as alleviating influenza symptoms. PA I38T substitution is a major pathway for reduced susceptibility to BXA, with 30- to 50-fold and 7-fold EC50changes 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.

Viral neuraminidase inhibitors show limited efficacy in mice infected with H7N9 influenza A viruses isolated from humans. Although baloxavir marboxil protected mice from lethal challenge infection with a low pathogenic avian influenza H7N9 virus isolated from a human, its efficacy in mice infected with a recent highly pathogenic version of H7N9 human isolates is unknown. Here, we examined the efficacy of baloxavir marboxil in mice infected with a highly pathogenic human H7N9 virus, A/Guangdong/17SF003/2016. Treatment of infected mice with a single 1.5 mg/kg dose of baloxavir marboxil protected mice from the highly pathogenic human H7N9 virus infection as effectively as oseltamivir treatment at 50 mg/kg twice a day for five days. Daily treatment for five days at 15 or 50 mg/kg of baloxavir marboxil showed superior therapeutic efficacy, largely preventing virus replication in respiratory organs. These results indicate that baloxavir marboxil is a valuable candidate treatment for human patients suffering from highly pathogenic H7N9 virus infection.[6]

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In clinical trials, single doses of Baloxavir profoundly decrease viral titers as well as alleviating influenza symptoms.

Oseltamivir acid was serially diluted in MES assay buffer [32.5 mmol/L MES and 4 mmol/L CaCl2 in DW (pH 6.5 adjusted with 4 N NaOH)]. To prepare NA enzyme solution, virus stocks were inactivated by 0.1% NP-40, and diluted with MES assay buffer. Ten μL of the oseltamivir acid solution and 10 μL of the NA enzyme solution were mixed and incubated at 37 °C for 30 minutes, followed by addition of 30 μL of 100 μmol/L 2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid sodium salt hydrate (MUNANA; Sigma-Aldrich Co., Ltd.). The reaction mixtures were incubated at 37 °C for 60 minutes, and the reaction was stopped by addition of 150 μL of stop solution [0.1 mol/L glycine and 25% ethanol (pH 10.7 adjusted with 4 N NaOH)]. The fluorescence intensity was measured with a microplate reader EnVision 2103 (PerkinElmer Inc.) at excitation wavelength of 355 nm and an emission wavelength of 460 nm, followed by calculation of IC50 values with XLfit software. FC was calculated by dividing IC50 of each tested virus to IC50 of the cognate wild-type virus.

Canine kidney MDCK cells were obtained from European Collection of Cell Cultures. Human quasi-diploid tumor RPMI2650 and human embryonic kidney 293 T cells were provided by American Type Culture Collection. MDCK and RPMI2650 cells were maintained in minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and 100 µg/mL kanamycin (Thermo Fisher Scientific, Inc.). 293 T cells were cultured in Dulbecco’s modified Eagle’s medium with 10% FBS and 100 µg/mL kanamycin. Eight plasmids-based reverse genetics technique was employed to generate recombinant viruses as described. The plasmid set of rgA/WSN/33 (H1N1) and empty vector pHW2000 were provided by Dr. Robert Webster at St. Jude Children’s Research Hospital. The plasmids for the generation of rgA/Victoria/3/75 and rgB/Maryland viruses were constructed with the pHW2000 by standard molecular biology techniques. The primer sequences used are available upon request. Co-culture of MDCK and 293 T cells were transfected with the eight plasmids and incubated 48 to 72 hours, followed by propagation of the viruses in MDCK cells. The PA sequences of the recombinant viruses were verified by Sanger sequencing. Viral titers were determined by standard tissue culture infectious dose (TCID)50 assay or plaque-forming unit (PFU) assay in MDCK cells.

To evaluate the efficacy of baloxavir marboxil in vivo infection with the H7N9 virus, baloxavir marboxil was orally administered to mice at 5 and 50 mg/kg twice a day for five days and was shown to have completely protected them from lethal challenge infection with a low pathogenic avian H7N9 human isolate, A/Anhui/1/2013. Highly pathogenic A/Guangdong/17SF003/2016 virus, which possesses enhanced polymerase activity in mammals due to PB2-482R, PB2-588V, and PA-497R is more pathogenic than A/Anhui/1/2013 because it causes a systemic infection in mice, ferrets, and macaques; this greater pathogenicity may affect the efficacy of baloxavir marboxil. Although A/Guangdong/17SF003/2016 showed reduced growth in human bronchial epithelial cells, this virus possesses the A100V, R262K, V387I, N394D, I465V, and K497R mutations in PA that may affect sensitivity to baloxavir marboxil compared with A/Anhui/1/2013. Accordingly, here, we assessed the efficacy of baloxavir marboxil against this highly pathogenic human H7N9 virus in vitro and in vivo.[6]
Next, we assessed the efficacy of baloxavir marboxil in mice infected with the highly pathogenic human H7N9 virus. Six-week-old female mice (BALB/c, Japan SLC Inc.) were anesthetized with isoflurane and intranasally infected with 10 mouse lethal dose 50 (MLD50; 104.3 PFU) of highly pathogenic A/Guangdong/17SF003/2016 (H7N9) possessing NA-294R (arginine at position 294 of NA indicates sensitive to NA inhibitors). Five infected mice per group were orally treated with oseltamivir phosphate at 5 or 50 mg/kg twice a day for 5 days or with baloxavir marboxil at 1.5, 15, or 50 mg/kg once or twice a day for 5 days. The negative control mice received 0.5% methylcellulose because this reagent was used as a solvent. Body weight changes of these mice were monitored for 14 days and mice that lost 25% or more of their initial body weight were scored as dead and euthanized according to institutional guidelines. All animal experiments were conducted in accordance with the University of Tokyo’s Regulations for Animal Care and Use, which were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval no. PA15-12). Mice given methylcellulose exhibited immediate body weight loss and died up to 8 days after infection. Oseltamivir phosphate treatment at 5 mg/kg for 5 days slightly improved the survival time of the infected mice (p = 0.009, log-rank test) but failed to protect them from the lethal challenge infection. Oseltamivir phosphate treatment at 50 mg/kg for 5 days showed 80% protection with severe body weight loss. In contrast, 60% of mice treated once with baloxavir marboxil at 1.5 mg/kg survived for 14 days, whereas all of the mice in the other baloxavir-treated groups survived without any body weight loss (p = 0.0016, log-rank test). These results show that a single dose of baloxavir marboxil with 15 mg/kg is sufficient to protect mice from infection with a highly pathogenic human H7N9 virus[6].

Absorption, Distribution and Excretion
Following oral administration of 40 mg baloxavir marboxil in adolescents and adults aged 12 years and older, the AUC was 5520 ng x hr/mL and the Cmax was 68.9 ng/mL. Following a 80 mg dose, the the AUC was 6930 ng x hr/mL and the Cmax was 82.5 ng/mL. The Tmax is about four hours. Food decreased Cmax by 48% and AUC0-inf by 36%. In pediatric patients aged five to 12 years of age weighing less than 20 kg, the AUCinf was 5830 ng x hr/mL and the Cmax was 148 ng/mL following a 2 mg/kg dose. The AUCinf was 4360 ng x hr/mL and the Cmax was 81.1 ng/mL following a 40 mg dose in pediatric patients who weigh greater than or equal to 20 kg. The Tmax ranged from 3.5 to 4.5 hours.
Baloxavir is primarily eliminated by biliary excretion. About 80.1% of the total dose is excreted in feces. About 14.7% of the dose is excreted in urine, where 3.3% of the recovered dose is the unchanged parent drug.
The volume of distribution is 1180 L.
Clearance of baloxavir is 10.3 L/h.

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Metabolism / Metabolites
Baloxavir predominantly undergoes UGT1A3-mediated metabolism to form glucuronic acid conjugate. It is subsequently metabolized by CYP3A4 to form sulfoxide.

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Biological Half-Life
The apparent terminal elimination half-life of baloxavir is 79.1 hours.

Hepatotoxicity
In clinical trials, there was little evidence that baloxavir caused liver injury, either in the form of serum enzyme elevations or clinically apparent liver disease. A proportion of patients with acute influenza A may have minor serum enzyme elevations during the acute illness, but these are independent of therapy and do not appear to be exacerbated by baloxavir.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of baloxavir marboxil during breastfeeding. Because baloxavir is 93% bound to plasma proteins, the amount in milk is likely to be low. If baloxavir is required by the mother, it is not a reason to discontinue breastfeeding, but an alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Baloxavir, the active metabolite, is 92.9–93.9% bound to human serum proteins. The ratio of blood cell to blood is 48.5–54.4%.

[1]. Sci Rep.2018 Jun 25;8(1):9633

[2]. Drugs.2018 Apr;78(6):693-697

[3]. WO 2017104691 A1.

[4]. N Engl J Med. 2018 Sep 6;379(10):913-923.

[5]. N Engl J Med. 2020 Jul 23;383(4):309-320.

[6]. Viruses. 2019 Nov; 11(11): 1066.

Pharmacodynamics
Baloxavir marboxil is an antiviral drug that works against influenza virus to block viral replication. It has an 50% inhibitory concentration (IC₅₀) of 1.4 to 3.1 nM for influenza A viruses and 4.5 to 8.9 nM for influenza B viruses in a polymerase acidic (PA) endonuclease assay. In murine models of influenza and avian influenza A, baloxavir reduced pulmonary viral loads and increased survival rates of mice. The reduction of viral titer was observed within 24 hours of administration, in a dose-dependent manner.

C27H23F2N3O7S

571.55

571.122

C, 56.74; H, 4.06; F, 6.65; N, 7.35; O, 19.59; S, 5.61

1985606-14-1

Baloxavir;1985605-59-1;Baloxavir-d5;Baloxavir-d4;2415027-80-2

124081896

White to yellow solid powder

1.6±0.1 g/cm3

712.8±70.0 °C at 760 mmHg

384.9±35.7 °C

0.0±2.3 mmHg at 25°C

1.696

2.24

0

12

6

40

1090

2

S1CC2C(=C(C=CC=2[C@@H](C2C=CC=CC1=2)N1[C@@H]2COCCN2C(C2=C(C(C=CN12)=O)OCOC(=O)OC)=O)F)F

RZVPBGBYGMDSBG-GGAORHGYSA-N

InChI=1S/C27H23F2N3O7S/c1-36-27(35)39-14-38-25-19(33)8-9-31-24(25)26(34)30-10-11-37-12-21(30)32(31)23-15-6-7-18(28)22(29)17(15)13-40-20-5-3-2-4-16(20)23/h2-9,21,23H,10-14H2,1H3/t21-,23+/m1/s1

({(12aR)-12-[(11S)-7,8-difluoro-6,11-dihydrodibenzo[b,e]thiepin-11-yl]-6,8-dioxo-3,4,6,8,12,12ahexahydro-1H-[1,4]oxazino[3,4-c]pyrido[2,1-f][1,2,4]triazin-7-yl}oxy)

Trade name Xofluza; Baloxavir acid; 1985606-14-1; Xofluza; S-033188; baloxavir-marboxil; 505CXM6OHG; Baloxavir marboxil [INN]; UNII-505CXM6OHG; BXA; Baloxavir marboxil; S-033188; S 033188; S033188

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 33~100 mg/mL ( 58.32~174.96 mM )
Ethanol : 7 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (4.37 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 10% DMSO+90% Corn Oil: ≥ 2.5 mg/mL (4.37 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)