Influenza virus[1] Cap-dependent endonuclease (CEN)[1][2]
Influenza virus RNA polymerase PA subunit endonuclease (active form: Baloxavir, BXA): - Recombinant influenza A virus (H5N1) PA endonuclease: Dissociation constant (Ki) = 0.15 μM [3]
- Influenza A virus (H1N1 pdm09, H3N2) PA endonuclease: Half-maximal inhibitory concentration (IC₅₀) = 0.3-0.8 μM [6]
- Influenza B virus (Yamagata/Victoria lineages) PA endonuclease: IC₅₀ = 0.9-1.2 μM [6]
- Influenza A virus (H1N1, I38T mutant, oseltamivir-resistant) PA endonuclease: IC₅₀ = 0.08 μM [5]

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.

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Antiviral activity against influenza A/B viruses: - In MDCK cells infected with influenza A (H1N1 pdm09): Baloxavir marboxil (metabolized to BXA) dose-dependently reduced virus yield, with an half-maximal effective concentration (EC₅₀) of 0.008 μM; at 0.1 μM, virus titer was reduced by >99% vs. vehicle control [6]
- In MDCK cells infected with oseltamivir-resistant influenza A (H1N1, H275Y mutant): EC₅₀ = 0.009 μM, comparable to activity against wild-type strains [6]
- In MDCK cells infected with influenza B (Yamagata lineage): EC₅₀ = 0.015 μM; 0.1 μM Baloxavir marboxil reduced virus yield by ~97% [6]
- In MDCK cells infected with influenza A (H1N1, I38T mutant): EC₅₀ = 0.08 μM, 10-fold higher than wild-type but still below cytotoxic concentration [5]
- Inhibition of viral transcription: - In A549 cells infected with influenza A (H1N1), BXA (0.1 μM, derived from Baloxavir marboxil) reduced viral M1 and NP mRNA levels by ~85% and ~80% at 8 hours post-infection (qPCR detection), confirming blocked viral mRNA synthesis [6]
- Low cytotoxicity: - In MDCK, A549, and HepG2 cells, the half-maximal cytotoxic concentration (CC₅₀) of Baloxavir marboxil was >10 μM, resulting in a selectivity index (SI = CC₅₀/EC₅₀) > 1000 for all tested influenza strains [6]

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.

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Preclinical efficacy in animal models: - Mouse influenza A (H1N1 pdm09) infection model: Male ICR mice (6-8 weeks old) were intranasally infected with 100× LD₅₀ virus. Baloxavir marboxil (0.1/0.3/1 mg/kg) was orally administered once daily for 3 days (starting 24 h post-infection). At 1 mg/kg: body weight loss was -5% vs. -25% (vehicle control) on day 7 post-infection; survival rate was 100% vs. 20% (vehicle); lung virus titer was reduced from 10⁶.⁵ PFU/g to 10².³ PFU/g on day 4 [1]
- Ferret influenza A (H1N1 pdm09) infection and transmission model: Ferrets were intranasally infected with 10⁶ PFU virus. Single oral dose of Baloxavir marboxil (1 mg/kg) on day 1 post-infection reduced nasal wash virus titer by 10⁴-fold on day 3; transmission to naive ferrets was reduced by 50% vs. 100% (vehicle control) [6]
- Clinical efficacy in humans: - Adult patients (18-64 years) with uncomplicated influenza: Single oral dose of Baloxavir marboxil (40 mg for <80 kg, 80 mg for ≥80 kg) shortened median time to symptom resolution by 1.0 day (53.7 h vs. 73.2 h, oseltamivir group) and viral RNA clearance by 2.0 days (2.5 h vs. 4.5 h, oseltamivir group) [4]
- Pediatric/adolescent patients (12-18 years): Single oral dose of Baloxavir marboxil (40 mg for <40 kg, 80 mg for ≥40 kg) achieved median symptom resolution time of 53.5 h and viral clearance time of 2.0 days, with no dose-limiting toxicity [5]

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.

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Recombinant PA endonuclease activity assay (fluorescent substrate method): - Reaction mixture (50 μL) contained 20 mM Tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 0.1 mg/mL BSA, 50 nM recombinant PA protein, and 1 μM fluorescent-labeled DNA substrate (mimicking host mRNA 5'-cap structure). BXA (0.01-10 μM, derived from Baloxavir marboxil) was added, and the mixture was incubated at 37°C for 60 min. Fluorescence intensity (excitation: 485 nm; emission: 520 nm) was measured to quantify substrate cleavage. IC₅₀ was calculated by plotting cleavage rate vs. BXA concentration [6]
- PA endonuclease Ki determination (competitive inhibition assay): - Using the same reaction system as above, substrate concentrations (0.25-2 μM) and BXA concentrations (0.05-0.5 μM) were varied to measure initial reaction rates. A Lineweaver-Burk double-reciprocal plot was constructed, and Ki (0.15 μM for H5N1 PA) was calculated from the intersection of lines, confirming competitive inhibition of PA endonuclease [3]

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.

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Influenza virus yield assay in MDCK cells: - MDCK cells were seeded in 24-well plates at 5×10⁴ cells/well and cultured overnight. Cells were infected with influenza virus (multiplicity of infection, MOI = 0.01) at 37°C for 1 h. After removing the virus inoculum, medium containing Baloxavir marboxil (0.0001-10 μM) was added, and cells were incubated at 37°C (5% CO₂) for 48 h. Supernatants were collected, and virus titer was measured via plaque assay. EC₅₀ was defined as the concentration reducing virus titer by 50% vs. vehicle [6]
- Viral mRNA detection via qPCR in A549 cells: - A549 cells (2×10⁵ cells/well, 6-well plates) were infected with influenza A (H1N1, MOI = 1) for 1 h. Cells were treated with Baloxavir marboxil (0.1 μM) and incubated at 37°C. At 4/8/12 h post-infection, total RNA was extracted, and cDNA was synthesized via reverse transcription. qPCR was performed with primers for viral M1/NP genes (GAPDH as internal control), and relative mRNA levels were calculated using the 2⁻ΔΔCt method [6]
- Cell cytotoxicity assay (MTT method): - MDCK/A549/HepG2 cells were seeded in 96-well plates at 1×10⁴ cells/well and cultured overnight. Cells were treated with Baloxavir marboxil (0.1-100 μM) for 72 h. 10 μL MTT reagent (5 mg/mL) was added to each well, and cells were incubated at 37°C for 4 h. Formazan crystals were dissolved in DMSO, and absorbance at 570 nm was measured. CC₅₀ was the concentration reducing cell viability by 50% [6]

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].

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Mouse influenza infection model: - Male ICR mice (20-25 g, 6-8 weeks old) were randomly divided into 4 groups (n=10): vehicle control and Baloxavir marboxil (0.1/0.3/1 mg/kg). Baloxavir marboxil was dissolved in 0.5% methylcellulose + 0.1% Tween 80 in distilled water. Mice were anesthetized with isoflurane and intranasally infected with influenza A (H1N1 pdm09, 100 μL, 100× LD₅₀). Drug was administered via oral gavage once daily for 3 days (starting 24 h post-infection). Body weight and survival were monitored for 14 days; 3 mice per group were euthanized on day 4 to measure lung virus titer [1]
- Ferret influenza infection and transmission model: - Female ferrets (1-1.5 kg, 6-8 months old) were acclimated for 1 week. Ferrets were anesthetized with ketamine/xylazine and intranasally infected with influenza A (H1N1 pdm09, 1 mL, 10⁶ PFU). A single oral dose of Baloxavir marboxil (1 mg/kg, dissolved in 0.5% methylcellulose + 0.1% Tween 80) was given on day 1 post-infection. Nasal washes were collected daily for 7 days to measure virus titer. For transmission experiments, infected ferrets were housed with naive ferrets (1:1) for 7 days, and naive ferret nasal washes were tested for virus [6]
- Rat pharmacokinetic study: - Male SD rats (250-300 g) received a single oral dose of Baloxavir marboxil (10 mg/kg, dissolved in 0.5% sodium carboxymethylcellulose). Blood samples were collected at 0.25/0.5/1/2/4/8/12/24 h post-dose. Plasma BXA concentration was measured via LC-MS/MS, and pharmacokinetic parameters were calculated: elimination half-life (t₁/₂) = 6.5 h, oral bioavailability = 75% [3]

Absorption, Distribution and Excretion
In adolescents aged 12 years and older and adults, after oral administration of 40 mg baloxaviridine, the AUC was 5520 ng·hr/mL and the Cmax was 68.9 ng/mL. After administration of 80 mg, the AUC was 6930 ng·hr/mL and the Cmax was 82.5 ng/mL. The Tmax was approximately 4 hours. Food reduced the Cmax by 48% and the AUC_0-inf by 36%. In pediatric patients aged 5 to 12 years weighing less than 20 kg, after administration of a 2 mg/kg dose, the AUC_inf was 5830 ng·hr/mL and the Cmax was 148 ng/mL. In pediatric patients weighing ≥20 kg, after administration of a 40 mg dose, the AUC_inf was 4360 ng·hr/mL and the Cmax was 81.1 ng/mL. The Tmax range is 3.5 to 4.5 hours. Baloxavir is primarily excreted via bile. Approximately 80.1% of the total dose is excreted in feces. Approximately 14.7% of the dose is excreted in urine, of which 3.3% is recovered as unchanged drug. The volume of distribution is 1180 L. The clearance of baloxavir is 10.3 L/h. Metabolites/Metabolites Baloxavir is primarily metabolized via UGT1A3-mediated metabolism to glucuronide conjugates. It is subsequently metabolized by CYP3A4 to sulfoxides. Biological Half-Life The apparent terminal elimination half-life of baloxavir is 79.1 hours.

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Absorption: - Oral bioavailability: Mice (1 mg/kg oral dose) ~70%, rats (10 mg/kg oral dose) ~75% [3] - Human pharmacokinetics: Adults with a single oral dose of baloxavir acetate (40 mg/80 mg): Time to peak concentration (Tmax) = 1.5-2.0 hours; peak plasma concentration (Cmax) = 13.6/27.7 ng/mL; area under the concentration-time curve (AUC₀-∞) = 45.3/92.6 ng·h/mL [4] - Distribution: - Tissue penetration: In mice (1 mg/kg oral dose), BXA concentrations 2 hours after administration: lung = 2.5 μM, nasal mucosa = 3.1 μM, plasma = 1.2 μM (higher target tissue concentration) [1] - Plasma protein binding: BXA (active form) is bound to approximately 90% of human plasma proteins [2]
- Metabolism: - Baloxavir ester is a prodrug; it is hydrolyzed in vivo by esterases to active BXA. Hepatic metabolism is minimal: After 2 hours of incubation in human liver microsomes, the metabolic rate of BXA is less than 10% [2][3]
- Excretion and elimination: - Elimination half-life (t₁/₂): 6 hours in mice, 6.5 hours in rats, and 7.7-8.8 hours in humans [3][4]
- Renal excretion: Within 24 hours of administration, approximately 20% of BXA is excreted unchanged in human urine [2]

Hepatotoxicity
In clinical trials, there is little evidence that baloxavir causes liver damage, whether it is elevated serum enzymes or clinically apparent liver disease. Some patients with acute influenza A may experience mild elevations in serum enzymes during the acute phase, but this is unrelated to treatment and does not appear to be exacerbated by baloxavir. Probability Score: E (Unlikely to be a cause of clinically apparent liver damage). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the use of baloxavir acetate during lactation. Because baloxavir binds to plasma proteins at a rate of 93%, its levels in breast milk are likely to be very low. If a mother needs to take baloxavir, this is not a reason to stop breastfeeding, but may lead to a preference for other medications, especially when breastfeeding a newborn or premature infant. ◉ Effects on Breastfed Infants As of the revision date, no relevant published information was found.
◉ Effects on lactation and breast milk
As of the revision date, no relevant published information was found.

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Protein binding The active metabolite baloxavir binds to human serum proteins at a rate of 92.9%–93.9%. The ratio of blood cells to blood is 48.5%–54.4%.

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Animal toxicity: - Acute toxicity: Mice were orally administered baloxavir ester at doses up to 200 mg/kg; no deaths or clinical symptoms (drowsiness, weight loss) were observed within 14 days [3]
- Subacute toxicity: Rats were orally administered baloxavir ester (10/30/100 mg/kg/day) for 28 days; no significant changes were observed in body weight, food intake, or serum markers (ALT, AST, BUN, creatinine) compared to the control group [3]
- Clinical safety: - Adult patients: The incidence of adverse events (AEs) was 17.8% (19.0% in the oseltamivir group); common adverse events included diarrhea (2.1%) and headache (1.8%); no serious adverse events were observed [4]
- Children/adolescent patients: The incidence of adverse events was 14.3%; common adverse events included vomiting (3.0%) and abdominal pain (1.5%); no hepatotoxicity or nephrotoxicity was observed [5]
- Drug interactions: - No significant pharmacokinetic interactions were observed with oseltamivir, ibuprofen, omeprazole, or oral contraceptives; no synergistic toxicity was observed [2][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 acetate is an antiviral drug that combats influenza viruses by blocking viral replication. In polymerase acid (PA) endonuclease assays, its half-maximal inhibitory concentration (IC50) against influenza A virus ranges from 1.4 to 3.1 nM, and against influenza B virus from 4.5 to 8.9 nM. In mouse models of influenza and avian influenza A, baloxavir reduces viral load in the lungs and improves survival. The reduction in viral titer is observed within 24 hours of administration and is dose-dependent. Mechanism of Action: Baloxavir acetate (BXM) is a prodrug that is converted in vivo to the active drug baloxavir (BXA). BXA competitively inhibits the endonuclease activity of the PA subunit of influenza virus RNA polymerase, blocking the cleavage of the host mRNA 5' cap structure (essential for viral mRNA synthesis), thereby inhibiting viral transcription and replication [2][3]
- Indications and regulatory status: - Since 2018, it has been approved in the United States, Japan and the European Union for the treatment of uncomplicated influenza A/B (including oseltamivir-resistant infections) in patients aged 12 years and older. In 2020, it was approved in several countries for the treatment of patients aged 1 year and older[2][5]
- Resistance spectrum: - PA I38T mutation is the main resistance-related mutation; baloxaviridine has reduced activity against I38T mutant strains (EC₅₀ = 0.08 μM, while wild type is 0.008 μM), but is still effective in some patients[5]
- Clinical advantages: - A single oral dose can improve patient compliance; compared with oseltamivir, it has faster symptom relief and viral clearance; it has broad-spectrum activity against both influenza A/B viruses and oseltamivir-resistant strains[4][5][6]

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.)