Influenza virus[1] Cap-dependent endonuclease (CEN); Deuterated form of Baloxavir

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.[5]

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

[2]. Antiviral Res. 2018 Dec;160:109-117.

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

[4].WO 2017104691 A1.

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

Baloxaviric acid (BXA), derived from the prodrug baloxavir ester (BXM), potently and selectively inhibits cap-dependent endonucleases in the PA subunit of influenza A and B virus polymerases. Clinical trials have shown that a single dose of BXM significantly reduces viral titers and alleviates influenza symptoms. This study aimed to elucidate the impact of variant viruses detected in post-treatment surveillance in clinical studies on the susceptibility and replication capacity of BXA. We found that the I38T mutation in the PA subunit is the main pathway leading to reduced viral susceptibility to BXA, with EC50 values of influenza A and B viruses decreasing by 30 to 50-fold and 7-fold, respectively. Viruses carrying the I38T mutation exhibited significantly impaired intracellular replication and correspondingly reduced in vitro endonuclease activity. Co-crystal structures of wild-type and I38T-mutant influenza A and B endonucleases bound to BXA indicate that this mutation reduces van der Waals contact with the inhibitor. The reduced stability of the BXA-bound endonuclease also supports the view that the I38T mutant has reduced affinity. These mechanistic insights provide biomarkers for future surveillance of treated populations. [1] Cap-dependent endonucleases (CENs), located in the PA subunit of influenza virus, mediate the key “cap capture” step in viral RNA transcription and are considered a promising anti-influenza target. This article describes the in vitro properties of a novel CEN inhibitor, baloxaviridine (BXA, the active form of baloxavir ester (BXM)). In enzyme activity assays, BXA inhibited viral RNA transcription by selectively inhibiting CEN activity; in cytopathic effect assays, BXA inhibited viral replication in infected cells without producing cytotoxicity. The antiviral activity of BXA was also confirmed by attenuation assays against seasonal influenza A and B viruses, including neuraminidase inhibitor-resistant strains. In addition, BXA exhibits broad-spectrum activity against multiple influenza A virus subtypes (H1N2, H5N1, H5N2, H5N6, H7N9, and H9N2). Furthermore, continuous passage of the virus in the presence of BXA can isolate PA/I38T variants with reduced sensitivity to BXA. Phenotypic and genotypic analyses by reverse genetics indicate that the mechanism of action of BXA is through the inhibition of CEN activity in infected cells. These results reveal the in vitro properties of BXA and support its use in the clinical treatment of influenza. [2] Baloxavir ester (Xofluza™) is an oral cap-dependent endonuclease inhibitor developed jointly by Roche and Shionogi. The drug blocks the proliferation of influenza virus by inhibiting the initiation of mRNA synthesis. In February 2018, baloxavir received the world's first approval in Japan for the treatment of influenza A or B virus infection. Currently, the drug is undergoing Phase III clinical trials for this indication in the United States, the European Union, and other countries. This article summarizes the key milestones in the development of baloxavir, which ultimately led to its first global approval for the treatment of influenza A or B virus infection. [3]

C24H19F2N3O4S

487.511818170547

487.131

2415027-80-2

Baloxavir marboxil;1985606-14-1;Baloxavir;1985605-59-1;Baloxavir-d5

169450565

Off-white to yellow solid powder

3.6

1

9

1

34

932

2

[2H]C1(C(OC[C@@H]2N1C(=O)C3=C(C(=O)C=CN3N2[C@H]4C5=C(CSC6=CC=CC=C46)C(=C(C=C5)F)F)O)([2H])[2H])[2H]

FIDLLEYNNRGVFR-KZRIHOBTSA-N

InChI=1S/C24H19F2N3O4S/c25-16-6-5-13-15(20(16)26)12-34-18-4-2-1-3-14(18)21(13)29-19-11-33-10-9-27(19)24(32)22-23(31)17(30)7-8-28(22)29/h1-8,19,21,31H,9-12H2/t19-,21+/m1/s1/i9D2,10D2

(3R)-6,6,7,7-tetradeuterio-2-[(11S)-7,8-difluoro-6,11-dihydrobenzo[c][1]benzothiepin-11-yl]-11-hydroxy-5-oxa-1,2,8-triazatricyclo[8.4.0.03,8]tetradeca-10,13-diene-9,12-dione

Baloxavir-d4; 2415027-80-2; HY-W751835; CS-0796197; (3R)-6,6,7,7-tetradeuterio-2-[(11S)-7,8-difluoro-6,11-dihydrobenzo[c][1]benzothiepin-11-yl]-11-hydroxy-5-oxa-1,2,8-triazatricyclo[8.4.0.03,8]tetradeca-10,13-diene-9,12-dione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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