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.

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.[1]

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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. [2]

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Baloxavir marboxil (Xofluza™; baloxavir) is an oral cap-dependent endonuclease inhibitor that has been developed by Roche and Shionogi. The drug blocks influenza virus proliferation by inhibiting the initiation of mRNA synthesis. 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. This article summarized the milestones in the development of baloxavir leading to this first global approval for influenza A or B virus infections.[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.)