Favipiravir (T-705)   中文名称: 法匹拉韦

RdRP ( IC50 = 341 nM )
Viral RNA polymerase [1][2]

法匹拉韦显示抗流感病毒活性，对于甲型流感病毒，IC50范围为0.013至0.48 μg/ml，对于乙型流感病毒，IC50范围为0.039至0.089 μg/ml，对于甲型流感病毒，IC50范围为0.030至0.057 μg/ml。丙型流感病毒。在哺乳动物细胞系（MDCK 细胞、Vero 细胞、HEL 细胞、A549 细胞、HeLa 细胞和 HEp-2 细胞）中，法匹拉韦在浓度高达 1,000 μg/ml 时没有显示出细胞毒性。在接种季节性甲型流感 (H1N1) 病毒的 MDCK 细胞中，法匹拉韦会诱导致命突变。激酶测定：法匹拉韦（也称为 T-705）是一种有效的、选择性的 RNA 依赖性 RNA 聚合酶抑制剂，用于治疗流感病毒感染。 Favipiravir 显示出抗流感病毒活性，对于甲型流感病毒，IC50 范围为 0.013 至 0.48 μg/ml，对于乙型流感病毒，IC50 范围为 0.039 至 0.089 μg/ml，对于丙型流感病毒，IC50 范围为 0.030 至 0.057 μg/ml。细胞测定：T-705 的细胞毒性通过 XTT 测定进行评估。 XTT 通过 MDCK 细胞、Vero 细胞、HEL 细胞、A549 细胞、HeLa 细胞和 HEp-2 细胞中的酶转化为水性甲臜。在 96 孔培养板中用测试介质（含 10% FCS 的 EMEM）将化合物稀释至适当浓度（体积，100 μl），其中每孔含有 2 × 103 个细胞/100 μL 的浓度。将测试板在 37°C、100% 湿度和 5% CO2 下孵育 3 天。 3天后，添加50μl XTT试剂（1mg/ml，在含有5mM吩嗪硫酸甲酯的不含FCS的EMEM中），并通过用酶标仪测量450nm处的吸光度来分析反应产物。细胞毒性以 50% 细胞抑制浓度 (CC50) 表示。

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在MDCK细胞中针对甲型/乙型流感病毒（H1N1、H3N2、B型），法匹拉韦（T-705）表现出强效的浓度依赖性抗病毒活性，EC50值为0.3-4.2 μM。它作为病毒RNA聚合酶抑制剂，掺入病毒RNA并诱导致死性突变，从而抑制病毒RNA合成[1]
- 在Huh-7细胞中针对诺如病毒（GII.4毒株），法匹拉韦（T-705）抑制病毒复制，EC50值为15.6 μM。50 μM浓度下，通过实时定量RT-PCR检测，诺如病毒RNA水平降低90%[2]
- 体外对其他RNA病毒（如西尼罗河病毒、黄热病毒）具有广谱抗病毒活性，EC50值为1.2-8.5 μM[1]
- 药物的抗病毒活性依赖于细胞内激酶将其磷酸化为活性形式（T-705三磷酸酯）[1]

在感染流感病毒的小鼠中，法匹拉韦（200 mg/kg/天，口服）可保护小鼠免于因流感病毒感染而死亡。在实验性感染埃博拉病毒的小鼠中，法匹拉韦可有效阻断病毒产生，在开始治疗后 2 天和 6 天分别达到 95% 和 99.6% 的抗病毒效果。

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在甲型流感病毒（H1N1）感染小鼠模型中，以100和200 mg/kg/天的剂量口服法匹拉韦（T-705），连续5天（感染后1天开始），肺组织病毒载量显著降低2-3 log10 PFU/g，存活率分别提高50%和75%。它还减轻了肺部炎症和组织病理学损伤[1]

病毒RNA聚合酶活性检测：将重组甲型流感病毒RNA聚合酶（PB1/PB2/PA复合物）与病毒RNA模板、NTP底物及法匹拉韦（T-705）（0.1-50 μM）在反应缓冲液中于37°C孵育60分钟。加入EDTA终止反应，通过qRT-PCR定量新合成的病毒RNA，证实对RNA聚合酶介导的病毒RNA合成的抑制作用[1]
- 活性代谢产物（T-705三磷酸酯）掺入检测：将纯化的病毒RNA聚合酶与[³H]标记的T-705三磷酸酯及RNA模板孵育。孵育后，用三氯乙酸沉淀RNA，通过液体闪烁计数法测量放射性，确认其掺入病毒RNA[1]

在 MNV/RAW 264.7 细胞系中使用基于 MTS 的 CPE 减少测定，评估法匹拉韦 (T 705) 的抗病毒活性。因此，接种含有 1×10 4 细胞/孔 RAW 264.7 细胞的 96 孔板，并以 0.01 的感染复数 (MOI) 注射 MNV，无论有或没有系列稀释法匹拉韦 (T 705) (3.13-200 μg/mL)。一旦受感染的细胞在孵育三天后显示出完全的 CPE，就获得细胞培养物上清液并用于定量实时 RT-PCR (qRT-PCR) 以测量病毒 RNA 负载。将含有 2 mg/mL MTS 和 46 g/mL PMS 的 PBS 溶液（pH 6-6.5）组成的储备液在 MEM 中稀释 1/20，用于 MTS 还原测定。将 75 μL MTS/PMS 溶液添加到每个孔中两小时后，在 498 nm 处测量光密度 (OD)。为了确定 CPE 降低的百分比，必须计算 [(OD_treatment)_MNW−OD_VC]/[OD _CC-OD_VC]×100。在此计算中，OD_CC 表示未处理、未感染细胞的 OD，而 OD_VC 和（OD_处理）CC< /sub> 分别代表经过处理的病毒感染细胞和未经处理的感染细胞。在 50% 的情况下可预防病毒诱发的 CPE 的化合物浓度称为 EC50。将法匹拉韦浓度应用于未感染的细胞三天，以便使用 MTS 方法评估该分子对宿主细胞的有害影响。活细胞的百分比计算为（OD_处理/OD_CC）×100，其中OD_处理是指用化合物处理的未处理的未感染细胞，OD_CC 是未经处理的未感染细胞的 OD。活细胞减少 50% 时的化合物浓度称为 CC50。 CC50/EC50 是用于计算选择性指数 (SI)[2] 的公式。

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流感病毒抗病毒细胞检测：MDCK细胞以2×10⁴个细胞/孔接种到96孔板中，用流感A/B病毒（MOI=0.01）感染1小时。加入系列浓度（0.01-100 μM）的法匹拉韦（T-705），孵育48小时。通过空斑形成实验评估病毒复制，计算EC50值。qRT-PCR定量病毒RNA水平[1]
- 诺如病毒抗病毒细胞检测：Huh-7细胞以1×10⁵个细胞/孔接种到24孔板中，用诺如病毒（GII.4）以MOI=0.1感染。加入1-100 μM的法匹拉韦（T-705），孵育72小时。提取诺如病毒RNA，qRT-PCR定量以确定病毒复制抑制效果[2]
- 细胞毒性检测：MDCK和Huh-7细胞用0.1-200 μM的法匹拉韦（T-705）处理72小时。采用四唑盐比色法检测细胞活力，CC50值＞200 μM，表明细胞毒性低[1][2]

Mice: It has also been demonstrated that favipiravir (T 705) shields mice from fatal influenza virus infections caused by a range of strains. When mice infected with lethal doses of influenza viruses A/Victoria/3/75(H3N2), A/Osaka/5/70(H3N2), or A/Duck/MN/1525/81(H5N1) are given favipiravir orally twice or four times a day for five days.

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Influenza A virus infection mouse model: Female BALB/c mice (6-8 weeks old) were intranasally inoculated with a lethal dose of influenza A virus (H1N1). Favipiravir (T-705) was dissolved in sterile water and administered orally via gavage at 100 or 200 mg/kg/day for 5 days, starting 1 day post-infection. Mice were monitored for survival for 14 days. Lung tissues were collected to quantify viral load (plaque assay) and analyze histopathological changes [1]

Absorption, Distribution and Excretion
The bioavailability of favipiravir is almost complete at 97.6%. The mean Cmax for the recommended dosing schedule of favipiravir is 51.5 ug/mL. Studies comparing the pharmacokinetic effects of multiple doses of favipiravir in healthy American and Japanese subjects are below: Japanese subjects First Dose: Cmax = 36.24 ug/mL tmax = 0.5 hr AUC = 91.40 ugxhr/mL American subjects First Dose: Cmax = 22.01 ug/mL tmax = 0.5 hr AUC = 44.11 ugxhr/mL Japanese Subjects Final Dose: Cmax = 36.23 ug/mL Tmax = 0.5 hr AUC = 215.05 ugxhr/mL American Subjects Final Dose: Cmax = 23.94 ug/mL Tmax = 0.6 hr AUC = 73.27 ugxhr/mL When favipiravir was given as a single dose of 400 mg with food, the Cmax decreased. It appears that when favipiravir is given at a higher dose or in multiple doses, irreversible inhibition of aldehyde oxidase (AO) occurs and the effect of food on the Cmax is lessened.
Favipiravir's metabolites are predominantly renally cleared.
The apparent volume of distribution of favipiravir is 15 - 20 L.
The recommended oral dosing regimen for favipiravir is as follows: Day 1: 1600 mg twice daily; Days 2-5: 600 mg twice daily. The reported CL/F for favipiravir 1600 mg dosed once daily is 2.98 L/hr ±0.30 and the CL/F values for favipiravir 600 mg dosed twice daily on days 1-2 and once daily on days 3-7 were 6.72 L/hr ±1.68 on Day 1, and 2.89 L/hr ±0.91 on Day 7. There is currently no reported clearance data for favipiravir 1600 mg dosed twice daily.

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Metabolism / Metabolites
Favipiravir is extensively metabolized with metabolites excreted mainly in the urine. The antiviral undergoes hydroxylation primarily by aldehyde oxidase and to a lesser extent by xanthine oxidase to the inactive metabolite, T705M1.

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Biological Half-Life
The elimination half-life of favipiravir is estimated to range from 2 to 5.5 hours.

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Absorption: Favipiravir (T-705) is rapidly and well absorbed after oral administration in mice and humans, with an oral bioavailability of 70-80%. Peak plasma concentrations (Cmax) of 8-12 μg/mL are reached within 1-2 hours after a 200 mg/kg oral dose in mice [1]
- Distribution: The drug distributes widely into body tissues, including the lungs, liver, and kidneys. Plasma protein binding rate is approximately 30-40% [1]
- Metabolism: It is phosphorylated to active T-705 triphosphate by cellular kinases (adenosine kinase, guanosine kinase) in target cells [1]
- Excretion: Primary excretion is via the renal route, with 60-70% of the administered dose excreted in urine as unchanged drug and metabolites within 24 hours. The plasma elimination half-life is 2-3 hours in mice [1]

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Favipiravir is an antiviral drug that is not approved in the United States by the US Food and Drug Administration. Information from one patient indicates that milk levels are low with a peak level at about 2 hours. One infant has reportedly been breastfed by a mother receiving favipiravir and pumping her breasts after doses with no adverse effects reported in the infant. Favipiravir has caused liver enzyme abnormalities, gastrointestinal symptoms, and serum uric acid elevations. If favipiravir is used in a nursing mother, these parameters should be monitored in the breastfed infant.
◉ Effects in Breastfed Infants
A nursing mother with a positive PCR for COVID-19 was prescribed favipiravir with a loading dose of 1600 mg twice on the first day, then 600 mg every 12 hours from day 2 to day 5. She breastfed her 15-month-old COVID-19-negative infant just before each dose of the drug. She pumped and discarded her milk between doses. No symptoms were observed in the baby during drug use and no abnormalities were detected in the baby’s hematological and biochemistry tests. The infant was followed for 6 months and was fed breastmilk and complementary feeding, did not develop any symptoms.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Favipiravir is 54% plasma protein-bound. Of this fraction, 65% is bound to serum albumin and 6.5% is bound to ɑ1-acid glycoprotein.

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Acute toxicity: The oral LD50 of Favipiravir (T-705) in mice is > 2000 mg/kg, indicating low acute toxicity [1]
- Subchronic toxicity: Oral administration of 500 mg/kg/day for 28 days in mice caused no significant hepatic or renal toxicity, with normal serum transaminase and creatinine levels [1]
- Cellular toxicity: Low cytotoxicity to mammalian cells, with CC50 > 200 μM in MDCK and Huh-7 cells [1][2]
- Reproductive toxicity: No reproductive toxicity was reported in mice at therapeutic doses (100-200 mg/kg/day) [1]

[1]. Favipiravir (T-705), a novel viral RNA polymerase inhibitor. Antiviral Res. 2013 Nov;100(2):446-54.

[2]. Favipiravir (T-705) inhibits in vitro norovirus replication. Biochem Biophys Res Commun. 2012 Aug 10;424(4):777-80.

Favipiravir is a member of the class of pyrazines that is pyrazine substituted by aminocarbonyl, hydroxy and fluoro groups at positions 2, 3 and 6, respectively. It is an anti-viral agent that inhibits RNA-dependent RNA polymerase of several RNA viruses and is approved for the treatment of influenza in Japan. It has a role as an antiviral drug, an anticoronaviral agent and an EC 2.7.7.48 (RNA-directed RNA polymerase) inhibitor. It is a primary carboxamide, a hydroxypyrazine and an organofluorine compound.
Discovered by Toyama Chemical Co., Ltd. in Japan, favipiravir is a modified pyrazine analog that was initially approved for therapeutic use in resistant cases of influenza. The antiviral targets RNA-dependent RNA polymerase (RdRp) enzymes, which are necessary for the transcription and replication of viral genomes. Not only does favipiravir inhibit replication of influenza A and B, but the drug has shown promise in the treatment of avian influenza, and may be an alternative option for influenza strains that are resistant to neuramidase inhibitors. Favipiravir has been investigated for the treatment of life-threatening pathogens such as Ebola virus, Lassa virus, and now COVID-19.
Favipiravir is a pyrazinecarboxamide derivative with activity against RNA viruses. Favipiravir is converted to the ribofuranosyltriphosphate derivative by host enzymes and selectively inhibits the influenza viral RNA-dependent RNA polymerase.

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Drug Indication
In 2014, favipiravir was approved in Japan to treat cases of influenza that were unresponsive to conventional treatment. Given its efficacy at targetting several strains of influenza, it has been investigated in other countries to treat novel viruses including Ebola and most recently, COVID-19.

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Mechanism of Action
The mechanism of action of favipiravir is novel compared to existing influenza antivirals that primarily prevent entry and exit of the virus from cells. The active favipiravir-RTP selectively inhibits RNA polymerase and prevents replication of the viral genome. There are several hypotheses as to how favipiravir-RTP interacts with RNA dependent RNA polymerase (RdRp). Some studies have shown that when favipiravir-RTP is incorporated into a nascent RNA strand, it prevents RNA strand elongation and viral proliferation. Studies have also found that the presence of purine analogs can reduce favipiravir’s antiviral activity, suggesting competition between favipiravir-RTP and purine nucleosides for RdRp binding. Although favipiravir was originally developed to treat influenza, the RdRp catalytic domain (favipiravir's primary target), is expected to be similar for other RNA viruses. This conserved RdRp catalytic domain contributes to favipiravir's broad-spectrum coverage.

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Pharmacodynamics
Favipiravir functions as a prodrug and undergoes ribosylation and phosphorylation intracellularly to become the active favipiravir-RTP. Favipiravir-RTP binds to and inhibits RNA dependent RNA polymerase (RdRp), which ultimately prevents viral transcription and replication.

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Favipiravir (T-705) is a synthetic pyrazinecarboxamide derivative, a novel viral RNA polymerase inhibitor [1][2]
- Mechanism of action: It is converted to T-705 triphosphate in cells, which competes with natural NTPs for binding to viral RNA polymerase. Incorporation into viral RNA induces lethal mutations, inhibiting viral replication [1]
- Broad-spectrum antiviral activity: Effective against a wide range of RNA viruses, including influenza viruses, noroviruses, flaviviruses, and arenaviruses [1][2]
- Clinical indications: Approved for the treatment of influenza A/B virus infections. Investigational applications include the treatment of norovirus and other RNA virus infections [1][2]
- Therapeutic advantage: Low cytotoxicity, oral bioavailability, and broad-spectrum activity make it a promising agent for emerging RNA virus outbreaks [1]

C5H4FN3O2

157.1

157.028

C, 38.23; H, 2.57; F, 12.09; N, 26.75; O, 20.37

259793-96-9

492405

White to off-white solid powder

1.6±0.1 g/cm3

552.6±50.0 °C at 760 mmHg

192 °C

288.0±30.1 °C

0.0±1.5 mmHg at 25°C

1.600

0.78

88.84

2

4

1

11

282

0

FC1=C([H])N([H])C(C(C(N([H])[H])=O)=N1)=O

ZCGNOVWYSGBHAU-UHFFFAOYSA-N

InChI=1S/C5H4FN3O2/c6-2-1-8-5(11)3(9-2)4(7)10/h1H,(H2,7,10)(H,8,11)

5-fluoro-2-oxo-1H-pyrazine-3-carboxamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (15.91 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (15.91 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (15.91 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: ≥ 2.5 mg/mL (15.91 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 5 中的溶解度: ≥ 2.5 mg/mL (15.91 mM) (饱和度未知) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 6 中的溶解度: 4.55 mg/mL (28.96 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶.

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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