Mice: For this study, HBV-transgenic mice are employed. The compounds (BAY 41-4109) are given to mice twice a day for 28 days in a suspension solution formulated with 0.5% tylose. The placebo is the 0.5% tylose. The animals are killed six hours after the last treatment, and the livers are taken out and frozen right away for further examination. The anesthetized animals' hearts are punctured to obtain blood[1].

小鼠：本研究使用 HBV 转基因小鼠。将化合物(BAY 41-4109)以0.5％tylose配制的悬浮液形式给予小鼠，每天两次，持续28天。安慰剂是 0.5% tylose。最后一次治疗后六小时将动物处死，并立即取出肝脏并冷冻以供进一步检查。刺穿麻醉动物的心脏以获取血液[1]。

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在HepG2.2.15细胞中，BAY 41-4109抑制HBV复制的半数抑制浓度（IC50）估计为53 nM。其对HepG2.2.15细胞的半数细胞毒性浓度（CC50）为7 µM。[1]
基于细胞实验，该化合物被鉴定为杂芳基嘧啶类化合物中一种有效的人乙型肝炎病毒（HBV）复制抑制剂。[1]

BAY 41-4109 可剂量依赖性地减少肝脏和血浆中的病毒 DNA，其功效与 3TC 相似。当添加 BAY 41 -4109 时，HBV 转基因小鼠肝脏的乙型肝炎病毒核心抗原 (HBcAg) 水平较低。根据对小鼠、大鼠和狗进行的药代动力学研究，其吸收速度快，与剂量成比例的血浆浓度约为 60%，生物利用度为 30% [1]。 BAY41-4109 通过专注于病毒衣壳，防止病毒在体内生长 [2]。

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在HBV转基因小鼠（Tg [HBV1.3 fsX−3′5′]）中，每日两次口服给予BAY 41-4109（剂量为15和30 mg/kg），持续28天，能剂量依赖性地显著降低肝脏和血浆中的HBV特异性DNA。在血浆中的抗病毒效果比在肝脏中更显著。在降低肝脏病毒DNA方面，其效力与测试剂量的3TC（拉米夫定）相当或更强。[1]
对经BAY 41-4109处理（30 mg/kg，每日三次，持续20天）的转基因小鼠肝组织进行免疫组化分析显示，与3TC处理不同，雄性和雌性小鼠肝细胞中的胞质乙肝核心抗原（HBeAg）均显著减少或完全消失。核内HBeAg染色未受BAY 41-4109处理的显著影响。[1]

使用HepG2.2.15细胞评估BAY 41-4109的抗HBV复制活性。将细胞接种于96孔板中，并在37°C、5% CO2条件下与系列稀释的化合物共孵育。在第4天更换培养基。暴露于化合物8天后，收集细胞培养上清，并裂解细胞。使用斑点杂交法检测上清和/或裂解液中的HBV-DNA，随后进行化学发光检测并用成像系统分析。IC50值根据此数据计算得出。[1]

Rodents: HBV-transgenic mice are used in this study. For a duration of 28 days, mice are administered the compounds (BAY 41-4109) twice daily in a suspension solution containing 0.5% tylose. 0.5% tylose serves as the placebo. After the animals are killed six hours after the last treatment, the livers are immediately removed and frozen for additional analysis. To get blood, punctures are made in the hearts of the unconscious animals[1].

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HBV-transgenic mice (Tg [HBV1.3 fsX−3′5′]), approximately 10 weeks old, producing high levels of serum HBV-DNA, were used. Animals were pre-selected based on serum HBV-DNA levels. Compounds were formulated as a suspension in 0.5% Tylose (serving as a placebo/vehicle control).
For the main efficacy study, groups of mice (5 males and 5 females per group) were treated orally (per os) with BAY 41-4109 or 3TC at doses of 7.5, 15, or 30 mg/kg, administered twice daily (b.i.d.) for 28 consecutive days. Animals were sacrificed 6 hours after the last dose. Livers were removed, frozen, and subsequently analyzed for HBV-DNA by dot-blot hybridization. Blood was collected via cardiac puncture, and plasma was analyzed for HBV-DNA by quantitative real-time PCR. [1]
For immunohistochemical analysis of HBeAg, a separate experiment was conducted where mice were treated with 30 mg/kg of BAY 41-4109 or 3TC three times daily (t.i.d.) for 20 consecutive days. Livers were then fixed, paraffin-embedded, sectioned, and stained using an immunohistochemistry protocol involving a polyclonal rabbit antibody against HBeAg, followed by detection using an avidin-biotin-peroxidase system and 3,3'-diaminobenzidine as the chromogen. [1]

In mice, BAY 41-4109 showed rapid absorption after oral administration, with a time to maximum concentration (tmax) of 0.17 hours.
The oral bioavailability of BAY 41-4109 was approximately 30% in mice, about 60% in rats and dogs.
The compound exhibited a high clearance rate in mice, which decreased with increasing animal size. The half-life in mice was approximately 2 hours.
Based on allometric scaling from animal data (mice, rats, dogs), the predicted steady-state volume of distribution (Vss) in humans was 2.9 L/kg. Human clearance and bioavailability were also predicted using scaling approaches, assuming complete absorption. [1]

[1]. Inhibition of human hepatitis B virus (HBV) by a novel non-nucleosidic compound in a transgenic mouse model. Antiviral Res. 2002 May;54(2):69-78.

[2]. BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly. J Mol Recognit. 2006 Nov-Dec;19(6):542-8.

[3]. Inhibition of hepatitis B virus replication by Bay 41-4109 and its association with nucleocapsid disassembly. J Chemother. 2008 Aug;20(4):458-67.

BAY 41-4109 (methyl (R)-4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoro-2-pyridinyl)-6-methyl-1,4-dihydro-pyrimidine-5-carboxylate) is a novel non-nucleosidic compound belonging to the class of heteroaryl-pyrimidines. [1]
The reduction of cytoplasmic HBeAg by BAY 41-4109, unlike nucleoside analogue 3TC, indicates a distinct mode of antiviral action, potentially involving direct inhibition of the viral replication machinery and/or inhibition of the viral assembly process. [1]
The study concludes that BAY 41-4109 represents a promising anti-HBV drug candidate based on its potent antiviral activity in a transgenic mouse model, favorable pharmacokinetic profile in animals, and lack of observed toxicity in the conducted experiments. [1]

C18H13CLF3N3O2

395.7629

395.064

476617-51-3

Bay 41-4109;298708-81-3;Bay 41-4109 racemate;298708-79-9

10188768

Light yellow to yellow solid powder

3.1

63.6

1

7

4

27

645

1

ClC1C([H])=C(C([H])=C([H])C=1[C@]1([H])C(C(=O)OC([H])([H])[H])=C(C([H])([H])[H])N([H])C(C2=C(C([H])=C(C([H])=N2)F)F)=N1)F

FVNJBPMQWSIGJK-UHFFFAOYSA-N

InChI=1S/C18H13ClF3N3O2/c1-8-14(18(26)27-2)15(11-4-3-9(20)5-12(11)19)25-17(24-8)16-13(22)6-10(21)7-23-16/h3-7,15H,1-2H3,(H,24,25)

(S)-Methyl 4-(2-chloro-4-fluorophenyl)-2-(3,5-difluoropyridin-2-yl)-6-methyl-1,4-dihydropyrimidine-5-carboxylate

BAY-414109 S-isomer; BAY414109 S-isomer; BAY 414109; BAY-41-4109; BAY41-4109; BAY 41-4109

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)

DMSO : ≥ 37 mg/mL (~93.49 mM)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。