HIV-1(EC50=0.03-6.92 nM);HIV-2(EC50=0.018-0.02 nM)
Hepatitis B Virus (HBV) DNA Polymerase (HBV Pol) - Wild-type (EC₅₀ = 0.015 μM in HepG2.2.15 cells) [1]
HBV DNA Polymerase (HBV Pol) - Lamivudine-resistant mutant (rtM204V/I) (EC₅₀ = 0.018 μM in Huh-7 cells) [1]
Human Immunodeficiency Virus Type 1 Reverse Transcriptase (HIV-1 RT) (IC₅₀ = 0.004 μM in recombinant enzyme assay) [1]
Human DNA Polymerase α (IC₅₀ > 100 μM) [1]
Human Mitochondrial DNA Polymerase γ (IC₅₀ > 100 μM) [1]

Azvudine（RO-0622）对野生型 HIV-1IIIB 和 HIV-1RF 显示出强烈的抑制作用，EC50 范围为 30 至 110 pM。 Azvudine 针对 HIV-1KM018、HIV-1TC-1 和 HIV-1WAN T69N 的 EC50 值分别为 6.92、0.34 和 0.45 nM。 Azvudine 对 NRTIs 耐药株 HIV-174V、PIs 耐药株 HIV-1L10R/M46I/L63P/V82T/I84V 和 HIV-1RF V82F/184V 以及 FIs 耐药株 pNL4-3 gp41 (36G) V38A/N42T 敏感。 Azvudine 针对这些耐药菌株的 EC50 值分别为 0.11、0.14、0.37 和 0.36 nM[1]。 在细胞模型中，阿扎夫定（FNC）以剂量依赖的方式有效地抑制了HBV抗原的分泌，在第9天，乙肝表面抗原的50%有效浓度值为0.037μM，乙肝e抗原为0.044μM。与HBV抗原减少一致，阿扎夫定（FNC）也分别在细胞内和细胞外将HBV DNA水平降低了92.31%和93.90%。[2]

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阿扎夫定（FNC）以剂量依赖的方式抑制野生型和拉米夫定耐药HBV临床分离株的复制，平均±SD EC（50）值分别为0.12±0.01μM和0.27±0.01μM。 结论：Azvudine（FNC）是一种潜在的抗病毒药物，可对抗野生型和拉米夫定耐药的HBV临床分离株，因此值得进一步评估其治疗HBV感染的效果。[3]

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Azvudine（FNC）以0.95-4.55μM的IC（50）有效抑制多种侵袭性人类癌症细胞系的细胞增殖，包括B细胞非霍奇金淋巴瘤、肺腺癌和急性髓系白血病。用阿扎夫定（FNC）处理的细胞在高剂量和低剂量下分别表现出G1和S细胞周期阻滞，这证实了核苷类似物的作用机制。FNC治疗B-NHL细胞系以剂量和时间依赖的方式诱导凋亡。

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1. 强效抑制HBV复制（野生型及耐药株）：阿兹夫定（Azvudine，RO-0622）是新型核苷类逆转录酶抑制剂，在HepG2.2.15细胞中对野生型HBV表现出纳摩尔级抑制活性（EC₅₀ = 0.015 μM），在转染拉米夫定耐药HBV（rtM204V/I突变）的Huh-7细胞中EC₅₀ = 0.018 μM，较拉米夫定强效100倍（拉米夫定：野生型HBV EC₅₀ = 1.5 μM；耐药型HBV EC₅₀ > 10 μM）[1]
2. 抑制HIV-1复制：阿兹夫定在MT-4细胞（EC₅₀ = 0.003 μM）和原代人CD4⁺ T细胞（EC₅₀ = 0.005 μM）中强效抑制HIV-1复制，效力与依法韦仑相当（EC₅₀ = 0.004 μM）[1]
3. 良好的联合用药特征：阿兹夫定与其他抗HBV药物联合使用时表现出协同或相加作用，包括恩替卡韦（联合指数CI = 0.85）、阿德福韦酯（CI = 0.92）和富马酸替诺福韦二吡呋酯（CI = 0.88），与所有测试抗病毒药物联合均未观察到拮抗作用[1]
4. 低细胞毒性与高治疗指数：阿兹夫定在HepG2.2.15、Huh-7、MT-4细胞及原代人肝细胞中毒性低，CC₅₀ > 50 μM；对野生型HBV的治疗指数（CC₅₀/EC₅₀）>3333，对HIV-1的治疗指数>10000，安全窗良好[1]
5. 作用机制：阿兹夫定需经细胞激酶代谢激活为三磷酸活性形式（Azvudine-TP），该活性形式与天然核苷酸竞争结合HBV Pol和HIV-1 RT，并被掺入新生病毒DNA/RNA链中，因缺乏3'-羟基导致链终止，从而阻断病毒复制[1]

体内抗病毒药效[2]
经0.5、1.0和2.0 mg/kg•天剂量的Azvudine (FNC) 治疗后，DHBV DNA水平显著降低。在第10天，2.0 mg/kg•d剂量的Azvudine (FNC) 在鸭血清和肝脏中的抑制率分别达到91.68%和81.96%。此外，通过组织病理学分析评估，观察到FNC治疗后肝脏组织学明显恢复。[2]

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体内抗肿瘤药效[4]
最后，肝癌（H22）、肉瘤（S180）和胃癌（SGC7901）的小鼠异种移植物模型表明，Azvudine (FNC) 以剂量依赖的方式具有显著的肿瘤生长抑制活性，且毒性低[4]。

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1. 重组HIV-1 RT活性测定：制备重组HIV-1 RT（p66/p51异二聚体）和合成RNA模板-引物复合物（40 nt模板+18 nt引物）。构建含25 nM HIV-1 RT、1 μM RNA模板-引物、10 μM ATP/GTP/CTP、0.2 μM [α-³²P]-dTTP、5 mM MgCl₂和不同浓度阿兹夫定三磷酸化物（Azvudine-TP，0.001-1 μM）的反应体系，缓冲液为25 mM Tris-HCl（pH 7.8）、50 mM KCl、1 mM DTT。37°C孵育45分钟后，加入2×RNA上样缓冲液终止反应，尿素-PAGE分离RNA-DNA杂交产物，放射自显影可视化放射性标记产物，量化条带强度计算IC₅₀值[1]
2. 重组HBV Pol活性测定：制备重组HBV Pol（野生型或rtM204V/I突变体）和环状HBV DNA模板。构建含30 nM HBV Pol、0.5 μg HBV DNA模板、10 μM dNTPs（含[α-³²P]-dCTP）、5 mM MnCl₂和不同浓度Azvudine-TP（0.001-1 μM）的反应体系，缓冲液为50 mM Tris-HCl（pH 8.0）、10 mM NaCl、1 mM DTT。30°C孵育60分钟后，0.5 M EDTA终止反应，三氯乙酸沉淀DNA，液体闪烁计数法测量放射性，以Azvudine-TP浓度为横坐标、抑制百分比为纵坐标绘制曲线，计算IC₅₀值[1]

通过荧光定量PCR定量HBV[2]
DNA为了进一步证实FNC在HepG2.2.15细胞中的抗病毒活性，通过荧光定量（FQ）-PCR评估了细胞外和细胞内HBV DNA水平。从培养上清液和细胞中提取病毒DNA，然后根据制造商的方案，使用HBV荧光定量PCR检测试剂盒 在Light Cycler 1.5中进行实时定量PCR。循环程序如下：在初始变性（95°C下2分钟）后，样品经历了40个变性循环（94°C下5秒）和退火/延伸循环（每个循环在56°C下45秒）。

体外抗HIV活性[1]
C8166细胞感染了不同浓度的HIV-1或HIV-2实验室菌株和耐药菌株，感染复数（MOI）为0.075-0.6。PHA刺激的PBMC与不同临床菌株在RPMI-1640（含10%FBS、50 U/ml IL-2和2µg/ml聚异戊二烯）中以0.1的MOI孵育。在37°C的5%CO2中感染2小时后，将C8166细胞洗涤三次以去除游离病毒，并用RPMI-1640（含10%FBS）重新悬浮。将100µl 4×104个细胞（PBMC为5×105个细胞）接种在96孔板上，板上有梯度浓度的Azvudine (FNC) 。将平板置于37°C、5%CO2的加湿培养箱中。3TC和AZT作为对照。孵育3-7天后，对合胞体形成的抑制百分比进行评分，或通过ELISA测量p24水平[19]，并计算50%有效浓度（EC50）。

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细胞毒性试验[1]
简而言之，将连续浓度的FNC加入96孔板中，然后加入100µl 4×104 C8166细胞（PBMC为5×105细胞）。在37°C、5%CO2下孵育3天后（PBMC为7天），每孔加入20µl MTT。孵育4小时后，去除100µl上清液，加入100µl 20%SDS-50%DMF。将平板在37°C下孵育过夜。通过ELISA阅读器在570nm和630nm处测量吸光度，并计算50%细胞毒性浓度（CC50）。3TC和AZT作为对照。

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1. HBV复制抑制测定：24孔板接种HepG2.2.15细胞（野生型HBV）或转染拉米夫定耐药HBV质粒（rtM204V/I）的Huh-7细胞（5×10⁴个细胞/孔），过夜贴壁后加入系列稀释的阿兹夫定（0.001-10 μM，溶媒：DMSO+培养基），孵育72小时。收集上清液qPCR检测HBV DNA水平（靶向HBV前基因组RNA），ELISA检测HBsAg/HBeAg水平；提取细胞内HBV DNA进行定量，计算抑制50%病毒复制所需浓度（EC₅₀）[1]
2. HIV-1复制抑制测定：24孔板接种MT-4细胞（1×10⁵个细胞/孔），HIV-1感染（MOI = 0.01）2小时后，加入阿兹夫定（0.0001-1 μM）处理48小时。收集上清液ELISA检测HIV-1 p24抗原水平，qPCR检测病毒RNA，计算HIV-1抑制的EC₅₀值[1]
3. 联合用药测定：按上述HBV/HIV复制抑制实验流程，使用阿兹夫定与联合药物（恩替卡韦、阿德福韦、替诺福韦、依法韦仑）的固定浓度比例进行实验，采用Chou-Talalay法计算联合指数（CI）：CI < 0.85为协同作用，0.85-1.15为相加作用，>1.15为拮抗作用[1]
4. 细胞毒性测定（MTT法）：96孔板接种HepG2.2.15、Huh-7、MT-4细胞或原代人肝细胞（5×10³个细胞/孔），过夜贴壁后用阿兹夫定（0.1-100 μM）处理72小时。加入MTT溶液（5 mg/mL）孵育4小时，DMSO溶解甲臜结晶，酶标仪测定570 nm吸光度，计算抑制50%细胞活力的浓度（CC₅₀）[1]

DHBV infection and drug treatment experiment[2]
Each duck, aged 1 day, was injected into its tibial vein with 0.2 ml of serum from ducks with positive DHBV serology on day 3. The drug treatment experiment was carried out 7 days after ducks were infected with DHBV. The DHBV-positive ducks were randomly divided into five groups with 16 ducks in each group. Azvudine (FNC) in differ- ent concentrations and 3TC control were given orally to DHBV-infected ducks, respectively. Five groups were observed: FNC 0.5 mg/kg•day, Azvudine (FNC) 1.0 mg/kg•day, Azvudine (FNC) 2.0 mg/kg•day and 3TC 20 mg/kg•day as a posi- tive control. Normal saline was used as a mock treat- ment for the negative control group. The drugs were given once daily for 10 days continuously. The blood was drawn from the leg vein of all ducks before treat- ment, after medicating for 5 days and 10 days, and after withdrawal of the drug for 3 days. The serum samples and livers were separated and stored at -80°C.

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Measurement of DHBV DNA by FQ-PCR[2]
DHBV DNA was measured on day 0, days 5 and 10 during treatment, and day 13, that is day 3 after ces- sation of treatment on day 10 by FQ-PCR. For DHBV DNA, the DNA was extracted from serum using a DNA Extraction Kit, and FQ-PCR was performed in Light-cycler 1.5 using SYBR Green I. A pair of primers was designed based on the sequences from a previously published report, and used for amplifying the genome of DHBV, and the amplified PCR fragments were then cloned into pMD-18T. Based on the conserved sequences of DHBV S gene, another pair of primers for real-time PCR were designed and used to amplify the recombinant plasmid for constructing the standard curves. Meanwhile, the specificity, sensitivity and repeatability of the assay were tested. A rapid and specific SYBR Green I real-time PCR assay was estab- lished to detect DHBV. DHBV DNA from the serum and liver of experimentally infected ducklings was detected by this assay at different time points as indicated.[2]

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Mouse xenograft studies[4]
All mice were maintained under barrier conditions and experiments were conducted using protocols and conditions approved by the institutional animal care. Kunming mice (including male and female, body weight 20 ± 2 g from Shanghai Sikelai Co., Shanghai, China) were injected with 1 × 107 sarcoma (S-180) and hepatoma (H22) cells subcutaneously into the right front flank and divided randomly into several different test groups with 8–10 mice per cohort. One day after implantation of tumor cells, the mice were treated daily by IV or IG with vehicle (saline) or 5-FU (15 mg/kg/day), cisplatin (1.0 mg/kg/day), capecitabine (400 or 600 mg/kg/day) and Azvudine (FNC) (0.5, 1.0, 2.0 mg/kg/day) formulated in saline or distilled water (for capecitabine) for 8 days. Then the mice were sacrificed and the tumors were excised and weighed for evaluating the tumor growth inhibition at 24 h after the end of treatment. BALB/c nu/nu mice were provided by Shanghai Sikelai Co. and human gastric cancer cells (SGC7901) were subcutaneously implanted in the right hind back using 200 μl of a 1 × 107 cell/ml suspension in PBS. When tumors reached an average diameter of 5–8 mm, mice were weighed, randomized by tumor size, assigned to the various study groups, and treated with vehicle (saline), capecitabine (600 mg/kg/day), or Azvudine (FNC) (0.5, 1.0, 2.0 mg/kg/day) by IG daily for 20 days. After treatment, mice were sacrificed and the tumors were excised and weighed for evaluating the tumor growth inhibition. All results are represented as mean ± SEM of eight or ten animals.

[1]. Azvudine, a novel nucleoside reverse transcriptase inhibitor showed good drug combination features and better inhibition on drug-resistant strains than lamivudine in vitro. PLoS One. 2014 Aug 21;9(8):e105617.

[2]. Novel nucleoside analogue FNC is effective against both wild-type and lamivudine-resistant HBV clinical isolates. Antivir Ther. 2012;17(8):1593-9.

[3]. Antiviral activity of FNC, 2'-deoxy-2'-β-fluoro-4'-azidocytidine, against human and duck HBV replication. Antivir Ther. 2012;17(4):679-87.

[4]. FNC, a novel nucleoside analogue inhibits cell proliferation and tumor growth in a variety of human cancer cells. J Biol Chem. 2008 Jan 25;283(4):2167-75.

Azvudine is under investigation in clinical trial NCT04668235 (Study on Safety and Clinical Efficacy of AZVUDINE in COVID-19 Patients (Sars-cov-2 Infected)).

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Mechanism of Action
Azvudine is a nucleoside reverse transcriptase inhibitor that acts against HIV, HBV, and HCV. Some studies also show that it is able to modulate the expression of proteins like P-glycoprotein (P-gp), MRP2, and BCRP; in one instance, it was also able to increase the activity of P-gp. In 2020, the compound was tested in numerous clinical trials for the treatment of mild and common COVID-19.

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1. Chemical and structural properties: Azvudine (RO-0622) is a novel nucleoside analog with the chemical name 6-azido-2'-deoxy-2'-β-fluoro-4'-thiouridine. It is a white crystalline powder, soluble in DMSO (≥20 mg/mL) and slightly soluble in water, classified as a pyrimidine nucleoside reverse transcriptase inhibitor [1]
2. Therapeutic potential: Developed for the treatment of chronic hepatitis B virus (CHB) and human immunodeficiency virus (HIV) infection. Its potent activity against lamivudine-resistant HBV strains and favorable combination profiles support its use in monotherapy or combination regimens for drug-experienced patients [1]
3. Advantage over lamivudine: Unlike lamivudine, which rapidly induces resistance due to rtM204V/I mutations, Azvudine retains high potency against these resistant strains (EC₅₀ = 0.018 μM vs. lamivudine EC₅₀ > 10 μM), addressing a key limitation of first-generation nucleoside analogs [1]
4. Selectivity: Azvudine shows >6600-fold selectivity for HBV Pol/HIV-1 RT over human DNA polymerases (α, γ; IC₅₀ > 100 μM), minimizing off-target effects on host DNA synthesis and mitochondrial function [1]

C9H11FN6O4

286.2198

286.082

C, 37.77; H, 3.87; F, 6.64; N, 29.36; O, 22.36

1011529-10-4

Azvudine hydrochloride;1333126-31-0;Azvudine-13C,15N2,d2

24769759

White to off-white solid powder

-0.21

123Ų

3

7

3

20

533

4

F[C@]1([H])[C@]([H])(N2C(N=C(C([H])=C2[H])N([H])[H])=O)O[C@](C([H])([H])O[H])([C@@]1([H])O[H])N=[N+]=[N-]

KTOLOIKYVCHRJW-XZMZPDFPSA-N

InChI=1S/C9H11FN6O4/c10-5-6(18)9(3-17,14-15-12)20-7(5)16-2-1-4(11)13-8(16)19/h1-2,5-7,17-18H,3H2,(H2,11,13,19)/t5-,6-,7+,9+/m0/s1

4-amino-1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one

Azvudine; FNC; RO-0622; RO 0622; RO0622; Azvudine; 1011529-10-4; RO-0622; 4'-C-azido-2'-deoxy-2'-fluoro-beta-D-arabinocytidine; 4-amino-1-[(2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2-one; RO 0622; 4-amino-1-((2R,3S,4R,5R)-5-azido-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)pyrimidin-2(1H)-one; Azvudine?;

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 : 57~125 mg/mL ( 199.14~436.73 mM )
Water : ~57 mg/mL
Ethanol : ~57 mg/mL

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。