HBsAg(IC50: 2.8 nM);HBeAg(IC50: 2.6 nM);HBV DNA(IC50: 3.2 nM); TENT; PAPD5 and PAPD7

感染 HBV 的人肝嵌合 uPA-SCID 小鼠表现出对 RG7834（4 mg/kg，每天两次，持续 21 天）的抗 HBV 功效[1]。
在小鼠中，RG7834（2、14.5 mg/kg、 po) 的半衰期为 4.9 小时，具有良好的口服生物利用度[1]。

感染 HBV 的人肝嵌合 uPA-SCID 小鼠表现出对 RG7834（4 mg/kg，每天两次，持续 21 天）的抗 HBV 功效[1]。
在小鼠中，RG7834（2、14.5 mg/kg、 po) 的半衰期为 4.9 小时，具有良好的口服生物利用度[1]。

微粒体稳定性测定[1]
为了测定微粒体的稳定性，将微粒体与试验化合物在37°C的磷酸钾缓冲液（100 mM，pH 7.4）中预孵育10分钟。最终孵育混合物由0.5mg微粒体蛋白/mL肝微粒体、1mM NADP、3mM葡萄糖6-磷酸、3mM MgCl2和0.05mg/mL葡萄糖6-磷酸脱氢酶组成，总体积为400μL磷酸钾缓冲液（100mM，pH 7.4）。反应是通过添加NADPH再生系统来引发的。在不同的时间点（0、3、6、9、15和30分钟），取等分试样（50μL），用150μL含内标乙腈骤冷。沉淀和离心后，通过LC-MS/MS分析上清液。

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血浆蛋白结合测定[1]
使用分子量截止膜为12–14 kDa的96孔微平衡透析装置测定未结合的化合物。地西泮作为阳性对照。汇集的小鼠和人血浆购自Biopredic。在2-5个试剂盒中测量化合物，初始总浓度为1μM，其中一个试剂盒化合物为阳性对照。通过测定阳性对照的未结合分数值来测试膜的完整性。将等体积的空白透析缓冲液（pH 7.4的Soerensen缓冲液）和含有物质的基质样品分别加载到受体和供体室中。然后密封HTD透析块，并在5%CO2环境下于37°C的培养箱中保存5小时。然后通过LC-MS/MS对药物浓度进行定量。

HBsAg检测[1]
将HepG2.2.15细胞以1.5×104个细胞/孔的速度一式两份接种到白色96孔板中。用DMSO中的化合物的3倍连续稀释系列处理细胞。所有孔中的最终DMSO浓度为1%，并且DMSO用作非药物对照。应用HBsAg化学发光免疫分析（CLIA）试剂盒半定量测定乙型肝炎病毒分泌抗原水平。对于检测，使用50μL/孔培养上清液，并按照制造商的说明进行程序。使用CellTiter Glo测定细胞毒性。使用E-WorkBook Suite生成剂量-反应曲线，并外推IC50和CC50值。IC50和CC50定义为化合物浓度（或条件培养基对数稀释度），在该浓度下，与无药物对照相比，HBsAg的诱导和细胞毒性分别降低50%。

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HBV DNA检测[1]
该检测采用实时qPCR直接测量细胞外HBV DNA拷贝数。将HepG2.2.15细胞接种在96孔微量滴定板中。只有内部孔用于减少细胞培养过程中观察到的“边缘效应”，外部孔填充完整的培养基以帮助最大限度地减少样品蒸发。第二天，洗涤HepG2.2.15细胞，并用含有不同浓度的试验化合物的完整培养基（一式三份）代替培养基。3TC被用作阳性对照，而单独的培养基被添加到细胞中作为阴性对照。三天后，用含有适当稀释的药物的新鲜培养基代替培养基。首次给药试验化合物后6天，收集细胞培养上清液，用Pronase处理，然后用于实时qPCR/TaqMan测定以测定HBV DNA拷贝数。抗病毒活性是根据HBV DNA水平（IC50）的降低来计算的。

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Caco-2含量测定[1]
该药物是用pH 7.4 HBSS中的10μM输入药物溶液制备的。对于顶端到基底外侧的方向，向Caco-2细胞的顶端侧添加200μL输入药物溶液，向基底外侧添加700μL pH 7.4 HBSS（1%DMSO）。对于从基底外侧到顶端侧的方向，向Caco-2细胞的基底外侧添加700μL输入药物溶液，向顶端侧添加200μL pH 7.4 HBSS（1%DMSO）。将平板在37°C、95%湿度条件下的5%CO2中孵育1小时。测定顶端侧和基外侧的药物量，并计算从A到B和从B到A方向的渗透性。

Animal Model: HBV-infected human liver chimeric uPA-SCID mice[1]
Dosage: 4 mg/kg
Administration: Twice daily for 21 days
Result:demonstrates good oral bioavailability[1]. reduced serum HBV DNA in mice by 0.6 log10 and lowered both HBsAg and HBeAg.

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Pharmacokinetic (PK) Analysis in Mice[1]
Compound was evaluated in mice at iv 1 mg/kg, po 2 mg/kg, and po 14.5 mg/kg. Compound solutions were prepared by dissolving the solid in 5% DMSO, 40% PEG400, and 55% saline for the iv dose and 1% RC-591 in water for the oral dose. Blood samples were collected at predetermined times into sodium heparin containing tubes, and plasma was separated via centrifugation (4 °C, 8000 rpm, 6 min) and stored frozen at −80 °C pending bioanalysis. Liver samples in the po group were harvested immediately after the collection of blood. The liver samples were then rinsed with saline, dried with filter paper, and stored at −80 °C until bioanalysis. Compound concentrations in the plasma and liver samples were determined by LC–MS/MS. The data were analyzed using a noncompartmental module of WinNonlin Professional 5.2. This PK study was approved by the Institutional Animal Care and Use Committee (IACUC) of Roche Pharma Research and Early Development China.

RG7834 was selected for further physicochemical and ADME characterization based on its favorable anti-HBV activity, cytotoxicity, solubility, and liver microsome stability. With favorable measured log D value of 1.28 and pKa value of 5.79 (acidic), RG7834 showed very good permeability with a Papp(A–B) of 12.8 × 10–6 cm s–1 and Papp ratio of 1.3 in Caco-2 assay. The unbound fractions of RG7834 in human and mouse plasma were determined to be 32.8% and 35.2%, respectively. The mouse single dose pharmacokinetics (SDPK) profile of RG7834 was evaluated in male BALB/C mice following intravenous (iv) and oral (po) administration. The results are summarized in Table 6. RG7834 had moderate plasma clearance (Cl) (41.9 mL min–1 kg–1) and good oral bioavailability (F) (62%) in mice. RG7834 also demonstrated satisfactory oral exposure and, particularly, good liver exposure, which was 4-fold higher than that in plasma. Although we did not have free liver drug concentration, the reasonably high total concentration of RG7834 in liver was considered as a desirable attribute for anti-HBV drugs because liver is the target organ in chronic HBV infections.
The cynomolgus monkey SDPK study of RG7834 was conducted subsequently, and it showed low plasma clearance (4.6 mL min–1 kg–1) following intravenous administration. RG7834 also showed good oral bioavailability (57%) with a half-life of 4.9 h following oral administration. [1]

Simultaneously, we also assessed the in vitro safety profile of RG7834. In the in vitro drug–drug interaction (DDI) assessment, RG7834 showed IC50 values of >50 μM against CYP3A4, CYP2D6, and CYP2C9. It had no time dependent inhibition (TDI) issue and no CYP induction issue. Moreover, RG7834 demonstrated an excellent in vitro toxicology profile, showing no inhibition of hERG channel, no flag for glutathione adduction (GSH), and clean in Ames assay (mutagenicity) and micronucleus test (MNT, clastogenicity). [1]

[1]. Discovery of RG7834: The First-in-Class Selective and Orally Available Small Molecule Hepatitis B Virus Expression Inhibitor with Novel Mechanism of Action. J Med Chem. 2018 Dec 13;61(23):10619-10634.

[2]. The Dihydroquinolizinone Compound RG7834 Inhibits the Polyadenylase Function of PAPD5 and PAPD7 and Accelerates the Degradation of Matured Hepatitis B Virus Surface Protein mRNA. Antimicrob Agents Chemother. 2020 Dec 16;65(1):e00640-20.

Chronic hepatitis B virus (HBV) infection is a serious public health burden, and current therapies cannot achieve satisfactory cure rate. There are high unmet medical needs of novel therapeutic agents with differentiated mechanism of action (MOA) from the current standard of care. RG7834, a compound from the dihydroquinolizinone (DHQ) chemical series, is a first-in-class highly selective and orally bioavailable HBV inhibitor which can reduce both viral antigens and viral DNA with a novel mechanism of action. Here we report the discovery of RG7834 from a phenotypic screening and the structure-activity relationship (SAR) of the DHQ chemical series. RG7834 can selectively inhibit HBV but not other DNA or RNA viruses in a virus panel screening. Both in vitro and in vivo profiles of RG7834 are described herein, and the data support further development of this compound as a chronic HBV therapy.[1]

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Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3' end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3' end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.[2]

C22H27NO6

401.452886819839

401.18

C, 65.82; H, 6.78; N, 3.49; O, 23.91

2072057-18-0

199482-36-3 (S-isomer hydrate);2072057-17-9 (S-isomer);2072057-18-0 (R-isomer);1802407-46-0 (racemic);

118261819

Solid powder

3.8

85.3Ų

1

7

8

29

685

1

CC(C)[C@H]1CC2=CC(=C(C=C2C3=CC(=O)C(=CN13)C(=O)O)OC)OCCCOC

KBXLMOYQNDMHQT-QGZVFWFLSA-N

InChI=1S/C22H27NO6/c1-13(2)17-8-14-9-21(29-7-5-6-27-3)20(28-4)10-15(14)18-11-19(24)16(22(25)26)12-23(17)18/h9-13,17H,5-8H2,1-4H3,(H,25,26)/t17-/m1/s1

(6R)-6-Isopropyl-10-methoxy-9-(3-methoxypropoxy)-2-oxo-6,7-dihydrobenzo[a]quinolizine-3-carboxylic acid

RG7834 R-isomer; RG 7834 R-isomer; RG-7834 R-isomer; RO7020322 R-isomer; RO 7020322 R-isomer; RO-7020322 R-isomer; RO0321; RO-0321; RO 0321;

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

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。