生物活性

Favipiravir is a RNA-directed RNA polymerase NS5B inhibitor which is useful for Antiviral agents. Favipiravir (T-705) is a new broad-spectrumantiviral drug with strong inhibitory activity on RNA-dependent RNA polymeraseof most RNA virus genome.

T-705inhibits IMPDH enzyme with an IC50 of ~600μM.[1]

inhibitoryeffects of T-705 against bunyaviruses in Vero 76 cells[2]

Antiviral activity of T-705

Anti-influenza virus activities IC50(ng/ml) of T-705

The cytotoxicity of favipiravir

Replication inhibition of T-705

体外研究

体内研究

激酶实验

Enzymatic assay with influenza virus polymerase[3]

Viral ribonucleoproteins (vRNPs) wereisolated from virions and purified by gradient ultracentrifugation. These vRNPscontain the native PA-PB1- PB2 polymerase enzyme and, as the template fortranscription, viral RNA complexed to nucleoprotein. To determine theirinhibitory effects on viral RNA elongation in competition with GTP, thecompounds were added to a reaction mixture (final volume: 25μl)containing 50mM Tris (pH 8.0), 100mMKCl, 1mM dithiothreitol, 5mM MgCl₂,0.4 U/μl of recombinant RNasin, 200μM ApG primer[adenyl(3’-5’)guanosine], 100μM UTP, 100μM CTP, 500μM ATP, 2.2μM GTP, and 1.5μM [8-³H]GTP. After addition of the isolated vRNPs, thesamples were incubated for 1 h at 30°C. The amount of radioactivity incorporatedinto viral RNA was quantified by a filter-based method.

细胞实验

Cell culture[1]

Vero cells were cultured in Dulbecco'sModified Eagle Medium (DMEM) supplemented with 2% fetal calf serum (FCS). QT-6cells persistently infected with PaBV-4 (strain 716) were cultured inDMEM/nutrient mixture F-12 (DMEM/F-12) supplemented with 10% FCS. OL cellspersistently infected with a wild-type BoDV-1 strain, He/80, (OB cells) werecultured in DMEM supplemented with 5% FCS. 293Tcells were cultured in DMEMsupplemented with 10% FCS.

Preparationof Vero-rBoDV-1-Gluc cells

Recombinant BoDV-1 carrying the Gaussialuciferase gene (rBoDV-1-Gluc) was produced using reverse genetics. Briefly,293T cells were transfected with a BoDV-1 cDNA-expressing plasmid and helperplasmids expressing the BoDV-1 nucleoprotein (N), phosphoprotein (P), and largeprotein (L) genes. Vero cells stably expressing a puromycin resistance genewere co-cultured with the transfected 293T cells and passaged every 3 days inthe presence of puromycin. After one month of culture, Vero cells persistentlyinfected with rBoDV-1-Gluc were obtained. Almost 100% of these cells wereinfected with rBoDV-1-Gluc as evaluated by immunofluorescence assay (IFA). Theactivity of Gaussia luciferase secreted in the culture medium was measuredusing the Gaussia Luciferase Assay kit according to the manufacturer'sinstructions in a single-well luminometer.

Cytotoxicityassay

Vero-rBoDV-1-Gluc cells were incubated at37 ℃ for 1h with PremixWST-1 (water-soluble tetrazolium) Assay. TheWST-1 reagent could be reduced to colorimetric formazan by cellulardehydrogenases, whose amount represents cell viability. The amount of formazanin the culture medium was measured at 440 nm, a maximum absorbance of formazan,in a microplate reader.

动物实验

Animal experiments[8]

IFNAR^-/- C57BL/6 mice were bredand maintained in an environmentally controlled specific-pathogen-free animalfacility of the NIID. Six- to 8-week-old female or male mice were used.

In infection experiments, each mouse wassubcutaneously inoculated with 100μl of virus solution (1.0 x 10⁸,1.0x10⁷, 1.0x10⁶, and 1.0x10⁵ TCID50/ml each).For mock infection, the same volume of DMEM (placebo) was used. Each groupconsisted of 5 to 10mice, which were given various doses of T-705, ribavirin,or placebo once a day by i.p. injection or per os (p.o.) using a stomach probe,just after subcutaneous inoculation of these mice with 1.0x10⁶ TCID50 of SFTSV (SPL010) in 100μl DMEM. Treatments were commenced 1h, 1day, 2days, 3days, 4days, or5days postinfection and continued for 5days.

Blood samples (20μl per animal)were obtained by tail vein puncture at intervals of 2 to 4days over a period of11 days (<4 blood drawings in total) for measurement of viral RNA levels.Body weight was recorded daily for 2 weeks, and each animal was monitored dailyfor the development of clinical signs, including hunched posture, ruffled fur,decreased activity, and response to stimuli, including neurological signs.

ViralRNA titration

The SFTSV genomic RNA was determined. TotalRNA was prepared from 20μl of blood samples using a High Pure viral RNA kit. Expression ofthe appropriate gene was estimated using a QuantiTect Probe RT-PCR kit accordingto the manufacturer’s protocol. Fluorescent signals were estimated using aLightCycler 96. Statistics were performed using GraphPad Prism6 Software. One-wayanalysis of variance with Bonferroni’s multiple-comparison test was used tocompare viral RNA copies between ribavirin-, T-705- and placebo-treated groups.

Histopathologyand immunohistochemistry

The mice treated with 300mg/kg/day T-705and placebo and with SFTSV were sacrificed using excess isoflurane at 12 and4days postinfection, respectively. The cervical lymph nodes, spleens, livers,and kidneys were collected for histopathological examination. The tissues wereroutinely processed and embedded in paraffin, sectioned, and stainedwithhematoxylin and eosin. Immunohistochemical (IHC) staining procedures were alsoperformed to detect the SFTSV antigens in the paraffin-embedded sections. Arabbit polyclonal antibody against SFTSV NP (number 75) was used as primaryantibody. Antigens were retrieved by hydrolytic autoclaving in citrate buffer(pH 6.0) for 10 min at 121°C. IHC staining was then performed using theEnVision/HRP immunodetection system.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Tokunaga T, Yamamoto Y, Sakai M, Tomonaga K, Honda T. Antiviral activity of favipiravir (T-705) against mammalian and avian bornaviruses. Antiviral Res. 2017;143:237-245.
[2] Gowen BB, Wong MH, Jung KH, et al. In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. Antimicrob Agents Chemother. 2007;51(9):3168-3176.
[3] Vanderlinden E, Vrancken B, Van Houdt J, et al. Distinct Effects of T-705 (Favipiravir) and Ribavirin on Influenza Virus Replication and Viral RNA Synthesis. Antimicrob Agents Chemother. 2016;60(11):6679-6691.
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体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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