Influenza virus[1] Cap-dependent endonuclease (CEN)[1][2]

体外活性：Baloxavir marboxil（也称为 BXM 或 S-033188）是 Baloxavir（商品名 Xofluza；也称为 Baloxavir Acid、BXA 或 S-033447）的前药。它是由罗氏和盐野义开发的一种口服小分子帽依赖性核酸内切酶抑制剂。巴洛沙韦是基于多替拉韦 (DTG) 的双金属药效团概念，通过合理的分子设计发现的，多替拉韦 (DTG) 是一种人类免疫缺陷病毒 (HIV) 整合酶的链转移抑制剂。 Baloxavir 有效且选择性地抑制甲型和乙型流感病毒聚合酶 PA 亚基内的帽子依赖性核酸内切酶。 2018年2月，巴洛沙韦在日本获得全球首个批准，用于治疗甲型或乙型流感病毒感染。美国、欧盟和其他国家正在进行该适应症的 III 期开发。该药物通过抑制 mRNA 合成的起始来阻止流感病毒的增殖。在临床试验中，单剂量的巴洛沙韦可显着降低病毒滴度并减轻流感症状。 PA I38T 取代是降低 BXA 敏感性的主要途径，A 型病毒和 B 型病毒的 EC50 变化分别为 30 至 50 倍和 7 倍。带有 I38T 取代的病毒在细胞中表现出严重受损的复制适应性，并相应地降低了体外核酸内切酶活性。激酶测定：奥司他韦酸在 MES 测定缓冲液中连续稀释 [32.5 mmol/L MES 和 4 mmol/L CaCl2，溶于 DW（用 4 N NaOH 调节 pH 6.5）]。为了制备 NA 酶溶液，用 0.1% NP-40 灭活病毒原液，并用 MES 测定缓冲液稀释。 10 μL 奥司他韦酸溶液和 10 μL NA 酶溶液混合，37℃孵育 30 分钟，然后加入 30 μL 100 μmol/L 2'-(4-甲基伞形基)-α-DN -乙酰神经氨酸钠盐水合物(MUNANA；Sigma-Aldrich Co., Ltd.)。反应混合物在37℃下孵育60分钟，加入150μL终止液[0.1mol/L甘氨酸和25%乙醇（用4N NaOH调节pH 10.7）]终止反应。使用酶标仪EnVision 2103 (PerkinElmer Inc.)在激发波长355 nm和发射波长460 nm下测量荧光强度，然后使用XLfit软件计算IC50值。 FC是通过将每种测试病毒的IC50除以同源野生型病毒的IC50来计算的。细胞测定：犬肾MDCK细胞获自欧洲细胞培养物保藏中心。人准二倍体肿瘤RPMI2650和人胚胎肾293 T细胞由美国典型培养物保藏中心提供。 MDCK 和 RPMI2650 细胞维持在补充有 10% 胎牛血清 (FBS) 和 100 µg/mL 卡那霉素 (Thermo Fisher Scientific, Inc.) 的基本必需培养基 (MEM) 中。 293 T 细胞在含有 10% FBS 和 100 µg/mL 卡那霉素的 Dulbecco 改良 Eagle 培养基中培养。采用基于八个质粒的反向遗传学技术来产生所描述的重组病毒。 rgA/WSN/33 (H1N1) 质粒组和空载体 pHW2000 由 St. Jude 儿童研究医院的 Robert Webster 博士提供。用于生成 rgA/Victoria/3/75 和 rgB/Maryland 病毒的质粒是通过标准分子生物学技术用 pHW2000 构建的。所使用的引物序列可根据要求提供。 MDCK 和 293 T 细胞的共培养物用八种质粒转染并孵育 48 至 72 小时，然后在 MDCK 细胞中繁殖病毒。重组病毒的PA序列通过Sanger测序进行验证。通过MDCK细胞中的标准组织培养感染剂量(TCID)50测定或空斑形成单位(PFU)测定来测定病毒滴度。

病毒神经氨酸酶抑制剂对感染人分离的H7N9甲型流感病毒的小鼠疗效有限。尽管baloxavir marboxil/巴洛韦马博西可保护小鼠免受从人身上分离的低致病性H7N9禽流感病毒的致命攻击感染，但其对最近感染高致病性H7N9人分离株的小鼠的功效尚不清楚。本实验研究了baloxavir marboxil对感染高致病性人H7N9病毒a /Guangdong/17SF003/2016的小鼠的疗效。用单次1.5 mg/kg剂量的baloxavir marboxil治疗感染小鼠，保护小鼠免受高致病性人H7N9病毒感染的效果与奥司他韦50 mg/kg剂量的治疗相同，每天两次，连续5天。以15或50 mg/kg的剂量每日治疗5天，显示出卓越的治疗效果，在很大程度上阻止了病毒在呼吸器官中的复制。这些结果表明，baloxavir marboxil是人类高致病性H7N9病毒感染患者有价值的候选治疗药物。[6]

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在临床试验中，单剂量的巴洛沙韦可显着降低病毒滴度并减轻流感症状。

奥司他韦酸在 MES 测定缓冲液中连续稀释 [32.5 mmol/L MES 和 4 mmol/L CaCl2，溶于 DW（用 4 N NaOH 调节 pH 6.5）]。为了制备 NA 酶溶液，用 0.1% NP-40 灭活病毒原液，并用 MES 测定缓冲液稀释。 10 μL 奥司他韦酸溶液和 10 μL NA 酶溶液混合，37℃孵育 30 分钟，然后加入 30 μL 100 μmol/L 2'-(4-甲基伞形基)-α-DN -乙酰神经氨酸钠盐水合物(MUNANA；Sigma-Aldrich Co., Ltd.)。反应混合物在37℃下孵育60分钟，加入150μL终止液[0.1mol/L甘氨酸和25%乙醇（用4N NaOH调节pH 10.7）]终止反应。使用酶标仪EnVision 2103 (PerkinElmer Inc.)在激发波长355 nm和发射波长460 nm下测量荧光强度，然后使用XLfit软件计算IC50值。 FC是通过将每种测试病毒的IC50除以同源野生型病毒的IC50来计算的。

犬肾 MDCK 细胞获自欧洲细胞培养物保藏中心。人准二倍体肿瘤RPMI2650和人胚胎肾293 T细胞由美国典型培养物保藏中心提供。 MDCK 和 RPMI2650 细胞维持在补充有 10% 胎牛血清 (FBS) 和 100 µg/mL 卡那霉素 (Thermo Fisher Scientific, Inc.) 的基本必需培养基 (MEM) 中。 293 T 细胞在含有 10% FBS 和 100 µg/mL 卡那霉素的 Dulbecco 改良 Eagle 培养基中培养。采用基于八个质粒的反向遗传学技术来产生所描述的重组病毒。 rgA/WSN/33 (H1N1) 质粒组和空载体 pHW2000 由 St. Jude 儿童研究医院的 Robert Webster 博士提供。用于生成 rgA/Victoria/3/75 和 rgB/Maryland 病毒的质粒是通过标准分子生物学技术用 pHW2000 构建的。所使用的引物序列可根据要求提供。 MDCK 和 293 T 细胞的共培养物用八种质粒转染并孵育 48 至 72 小时，然后在 MDCK 细胞中繁殖病毒。重组病毒的PA序列通过Sanger测序进行验证。通过MDCK细胞中的标准组织培养感染剂量(TCID)50测定或空斑形成单位(PFU)测定来测定病毒滴度。

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].

Absorption, Distribution and Excretion
Following oral administration of 40 mg baloxavir marboxil in adolescents and adults aged 12 years and older, the AUC was 5520 ng x hr/mL and the Cmax was 68.9 ng/mL. Following a 80 mg dose, the the AUC was 6930 ng x hr/mL and the Cmax was 82.5 ng/mL. The Tmax is about four hours. Food decreased Cmax by 48% and AUC0-inf by 36%. In pediatric patients aged five to 12 years of age weighing less than 20 kg, the AUCinf was 5830 ng x hr/mL and the Cmax was 148 ng/mL following a 2 mg/kg dose. The AUCinf was 4360 ng x hr/mL and the Cmax was 81.1 ng/mL following a 40 mg dose in pediatric patients who weigh greater than or equal to 20 kg. The Tmax ranged from 3.5 to 4.5 hours.
Baloxavir is primarily eliminated by biliary excretion. About 80.1% of the total dose is excreted in feces. About 14.7% of the dose is excreted in urine, where 3.3% of the recovered dose is the unchanged parent drug.
The volume of distribution is 1180 L.
Clearance of baloxavir is 10.3 L/h.

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Metabolism / Metabolites
Baloxavir predominantly undergoes UGT1A3-mediated metabolism to form glucuronic acid conjugate. It is subsequently metabolized by CYP3A4 to form sulfoxide.

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Biological Half-Life
The apparent terminal elimination half-life of baloxavir is 79.1 hours.

Hepatotoxicity
In clinical trials, there was little evidence that baloxavir caused liver injury, either in the form of serum enzyme elevations or clinically apparent liver disease. A proportion of patients with acute influenza A may have minor serum enzyme elevations during the acute illness, but these are independent of therapy and do not appear to be exacerbated by baloxavir.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of baloxavir marboxil during breastfeeding. Because baloxavir is 93% bound to plasma proteins, the amount in milk is likely to be low. If baloxavir is required by the mother, it is not a reason to discontinue breastfeeding, but an alternate drug may be preferred, especially while nursing a newborn or preterm infant.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Baloxavir, the active metabolite, is 92.9–93.9% bound to human serum proteins. The ratio of blood cell to blood is 48.5–54.4%.

[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 marboxil is an antiviral drug that works against influenza virus to block viral replication. It has an 50% inhibitory concentration (IC₅₀) of 1.4 to 3.1 nM for influenza A viruses and 4.5 to 8.9 nM for influenza B viruses in a polymerase acidic (PA) endonuclease assay. In murine models of influenza and avian influenza A, baloxavir reduced pulmonary viral loads and increased survival rates of mice. The reduction of viral titer was observed within 24 hours of administration, in a dose-dependent manner.

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

123Ų

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.37 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900 μL 玉米油中，混合均匀。

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配方 2 中的溶解度: 10% DMSO+90% Corn Oil: ≥ 2.5 mg/mL (4.37 mM)

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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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。