murine hepatitis virus, delayed brain tumor cell ( EC50 = 30 nM ); SARS-CoV, HAE cell ( EC50 = 74 nM ); MERS-CoV, HAE cell ( EC50 = 74 nM ); SARS-CoV-2 ( IC50 = 3.3 μM ); SARS-CoV-2 alpha ( IC50 = 4.7 μM ); SARS-CoV-2 beta ( IC50 = 32 μM ); SARS-CoV-2 gamma ( IC50 = 3.7 μM ); SARS-CoV-2 delta ( IC50 = 9.2 μM )
GS-5734 demonstrates broad-spectrum antiviral activity against other pathogenic RNA viruses in vitro and antiviral activity against multiple EBOV variants in cell-based assays (EC50=0.06-0.14 μM).[1] With an EC50 of 0.03 μM for the murine hepatitis virus in delayed brain tumor cells and 0.074 μM for SARS-CoV and MERS-CoV in HAE cells, GS-5734 functions as a broad-spectrum therapeutic to protect against CoVs.[2]

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• Antiviral activity against coronaviruses: Inhibited SARS-CoV replication in primary human airway epithelial (HAE) cells with EC₅₀ = 0.069 μM; reduced viral titers by >4 log₁₀ units at 1 μM after 48 hours. Similarly inhibited MERS-CoV in HAE cells (EC₅₀ = 0.074 μM) [1].
• Activity against Ebola virus: Suppressed Ebola virus (EBOV) replication in HeLa cells (EC₅₀ = 0.086 μM) and primary human macrophages (EC₅₀ = 0.17 μM) [1].
• Mechanism of action: Remdesivir triphosphate (active metabolite) acts as an adenosine triphosphate (ATP) analog, causing delayed chain termination during viral RNA synthesis. Incorporation into nascent RNA strands inhibited subsequent nucleotide additions after 3-5 bases, terminating replication [1].
• Activity against Nipah virus: Inhibited Nipah virus replication in primary human lung microvascular endothelial cells (EC₅₀ = 0.03 μM) [2].
• Cytotoxicity: CC₅₀ > 10 μM in Vero E6 cells and >20 μM in Huh7 cells, indicating high selectivity index [1]

GS-5734 在体外表现出针对其他致病性 RNA 病毒的广谱抗病毒活性，并在基于细胞的检测中表现出针对多种 EBOV 变体的抗病毒活性 (EC50=0.06-0.14 μM)。 [1] GS-5734 对迟发性脑肿瘤细胞中的鼠肝炎病毒的 EC50 为 0.03 μM，对 HAE 细胞中的 SARS-CoV 和 MERS-CoV 的 EC50 为 0.074 μM，可作为一种广谱治疗剂来预防 CoV。 [2]

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• 抗冠状病毒活性： 在原代人呼吸道上皮（HAE）细胞中抑制SARS-CoV复制，EC₅₀ = 0.069 μM；1 μM浓度处理48小时后病毒滴度降低>4 log₁₀单位。在HAE细胞中同样抑制MERS-CoV（EC₅₀ = 0.074 μM）[1]。
• 抗埃博拉病毒活性： 在HeLa细胞（EC₅₀ = 0.086 μM）和原代人巨噬细胞（EC₅₀ = 0.17 μM）中抑制埃博拉病毒（EBOV）复制[1]。
• 作用机制： Remdesivir三磷酸盐（活性代谢物）作为三磷酸腺苷（ATP）类似物，在病毒RNA合成中引起延迟链终止。掺入新生RNA链后，在3-5个核苷酸下游阻止后续核苷酸添加，终止复制[1]。
• 抗尼帕病毒活性： 在原代人肺微血管内皮细胞中抑制尼帕病毒复制（EC₅₀ = 0.03 μM）[2]。
• 细胞毒性： 在Vero E6细胞中CC₅₀ > 10 μM，在Huh7细胞中CC₅₀ > 20 μM，表明高选择性指数[1]

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Remdesivir (GS-5734) 在 HeLa 细胞中对多种丝状病毒（埃博拉病毒 Makona 株、Kikwit 株、马尔堡病毒、本迪布焦病毒、苏丹病毒）具有广谱抗病毒活性，EC₅₀ 值在低纳摩尔至亚微摩尔水平。[1]

在 HEP-2 细胞中，对呼吸道合胞病毒（RSV）的 EC₅₀ 为 0.019 μM，CC₅₀ 为 6.0 μM。[1]

对胡宁病毒（EC₅₀ = 0.47 μM）、拉沙热病毒（EC₅₀ = 1.48 μM）和中东呼吸综合征冠状病毒（MERS-CoV，EC₅₀ = 0.34 μM）也有抑制活性，但对基孔肯雅病毒、委内瑞拉马脑炎病毒和 HIV-1 无活性（EC₅₀ > 20 μM）。[1]

在人单核细胞来源的巨噬细胞中，与 1 μM GS-5734 孵育可快速形成并持续维持细胞内 NTP，半衰期为 16 ± 1 小时。[1]

无论何时开始治疗，施用 3 mg/kg GS-5734 均可提高生存率。病毒暴露三天后，所有接受 10 mg/kg GS-5734 治疗的动物均到达其生命阶段的末期。然而，重复给予 10 mg/kg GS-5734 剂量的动物始终表现出更强的抗病毒作用。当使用 10 mg/kg D3 方案（病毒暴露后 3 天开始）治疗时，与 EVD 相关的临床疾病体征和凝血病标志物以及终末器官病理生理学与改善相关。[1]

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• 埃博拉病毒小鼠模型： 感染后1小时开始腹腔注射（25 mg/kg，每日1次，持续6天）可100%预防小鼠适应性埃博拉病毒（maEBOV）感染小鼠的死亡，而载体对照组存活率为0%。治疗后第6天血清中病毒RNA检测不到[1]。
• SARS-CoV小鼠模型： 感染后24小时开始皮下渗透泵给药（负荷剂量50 mg/kg + 维持剂量25 mg/kg/天，持续12天），使SARS-CoV感染小鼠的肺部病毒滴度降低约2 log₁₀单位，并改善肺部组织病理学[1]。
• 尼帕病毒雪貂模型： 感染前1天开始预防性给药（10 mg/kg，每日1次，持续3天），可预防体重减轻，并使咽拭子病毒RNA减少>99%[2]

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在埃博拉病毒感染的猕猴模型中，感染后第3天开始，每天一次静脉注射 10 mg/kg Remdesivir (GS-5734)，连续12天，可实现100%存活率，显著抑制病毒复制，并改善临床症状和病理生理指标。[1]

较低剂量（3 mg/kg）在感染后第0天或第2天开始给药，提供部分保护（存活率33–66%）。[1]

体外RSV RNA合成试验[1]
使用纯化的RSV L/P复合物和RNA寡核苷酸模板（Dharmacon）在体外重建RSV聚合酶的RNA合成，该模板代表RSV前导启动子31,32,33（3′-UGCCUUUUACG-5′）的核苷酸1-14。RNA合成反应如前所述进行，不同之处在于反应混合物含有250μM三磷酸鸟苷（GTP）、10μM三磷酸尿苷（UTP）、10µM三磷酸胞苷（CTP），补充了10μCi[α-32P]CTP，并且含有10μM三磷酸腺苷（ATP）或不含ATP。在这些条件下，聚合酶能够从启动子的3位位点开始合成，但不能从1位位点开始。GS-5734的NTP代谢物在DMSO中连续稀释，并以10、30或100μM的浓度包含在每种反应混合物中，如图1f所示。在含有7M尿素的25%聚丙烯酰胺凝胶上，在Tris-牛磺酸-EDTA缓冲液中通过电泳分析RNA产物，并通过放射自显影检测放射性标记的RNA产物。

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呼吸道合胞病毒A2聚合酶抑制试验[1]
转录反应在30μL反应缓冲液（50 mM三乙酸盐（pH 8.0）、120 mM乙酸钾、5%甘油、4.5 mM MgCl2、3 mM DTT、2 mM EGTA、50μg ml−1 BSA、2.5 U RNasin、20μM ATP、100μM GTP、100μM UTP、100μMCTP和1.5μCi[α-32P]ATP（3000 Ci mmol−1））中含有25μg粗制RSV-RNP复合物。选择转录测定中使用的放射性标记的核苷酸，以匹配正在评估的RSV RNP转录抑制的核苷酸类似物。
为了确定核苷酸类似物是否抑制RSV RNP转录，使用六步连续稀释法以五倍增量添加化合物。在30°C下孵育90分钟后，用350μl Qiagen RLT裂解缓冲液停止RNP反应，并使用Qiagen RNeasy 96试剂盒纯化RNA。纯化的RNA在65°C的RNA样品加载缓冲液中变性10分钟，并在含有2 M甲醛的1.2%琼脂糖/MOPS凝胶上运行。将琼脂糖凝胶干燥，暴露于Storm磷光屏，并使用Storm磷光仪显影。

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抑制人RNA聚合酶II[1]
对于25μl的反应混合物，将7.5μl 1×转录缓冲液（20 mM HEPES（pH 7.2-7.5）、100 mM KCl、0.2 mM EDTA、0.5 mM DTT、20%甘油）、3 mM MgCl2、100 ng CMV阳性或阴性对照DNA以及ATP、GTP、CTP和UTP的混合物与不同浓度（0-500μM）的抑制剂在30°C下预孵育5分钟。混合物含有5-25μM（等于Km）的竞争性33P标记ATP和400μM的GTP、UTP和CTP。通过加入3.5μl HeLa和提取物开始反应。在30°C下孵育1小时后，通过加入10.6μl蛋白酶K混合物停止聚合酶反应，该混合物含有终浓度为2.5μgμl−1蛋白酶K、5%SDS和25 mM EDTA。在37°C下孵育3-12小时后，将10μl反应混合物与10μl负载染料（98%甲酰胺、0.1%二甲苯氰基和0.1%溴酚蓝）混合，在75°C下加热5分钟，然后负载到6%聚丙烯酰胺凝胶（8M尿素）上。将凝胶在70°C下干燥45分钟，并暴露于磷光屏。使用Typhoon Trio Imager和Image Quant TL软件对全长产物363个核苷酸径流RNA进行定量。

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抑制人线粒体RNA聚合酶[1]
在含有10 mM HEPES（pH 7.5）、20 mM NaCl、10 mM DTT、0.1 mg ml-1 BSA和10 mM MgCl234的缓冲液中，将20纳摩尔的POLRMT与含有POLRMT轻链启动子区和线粒体（mt）转录因子TFA（100 nM）和mtTFB2（20 nM）的20 nM模板质粒（pUC18 LSP）一起孵育。将反应混合物预温至32°C，通过加入2.5μM的天然NTPs和1.5μCi的[32P]GTP来引发反应。在32°C下孵育30分钟后，在DE81纸上发现反应并定量。

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• RdRp抑制实验： 重组SARS-CoV RdRp复合物（nsp7/nsp8/nsp12）与RNA模板/引物及核苷三磷酸（NTPs）孵育。加入梯度浓度的Remdesivir三磷酸盐（RTP）。通过放射性标记核苷酸掺入定量RNA合成。RTP竞争性抑制ATP掺入，IC₅₀ = 3.65 μM [1]。
• RNA链终止实验： 使用纯化的RdRp复合物进行引物延伸反应。通过凝胶电泳分析RTP掺入RNA模板的情况。结果显示RTP在掺入位点下游3-5个核苷酸处引起终止[1]

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通过体外 RNA 合成实验评估活性代谢物 NTP 对 RSV RNA 聚合酶的抑制作用。使用纯化的 RSV 核糖核蛋白复合物和 RNA 寡核苷酸模板，在含放射性标记 CTP 的反应体系中加入系列稀释的 NTP。RNA 产物通过尿素-聚丙烯酰胺凝胶电泳和放射自显影分析。NTP 通过引起链提前终止来抑制 RSV 聚合酶催化的 RNA 合成。[1]

在转录实验中测试了 NTP 对人 RNA 聚合酶 II 和线粒体 RNA 聚合酶的抑制作用，使用 HeLa 核提取物或重组 POLRMT 与转录因子。NTP 在浓度高达 500 μM 时不抑制人源聚合酶。[1]

HeLa和HFF-1细胞中的EBOV检测[1]
抗病毒检测在USAMRIID的BSL-4中进行。HeLa或HFF-1细胞以每孔2000个细胞的速度接种在384孔板上。在感染前2小时，使用HP D300数字分配器以两倍稀释增量从10μM开始，将化合物的十个连续稀释液（一式三份）直接添加到细胞培养物中。使用HP D300数字分配器将每个孔中的DMSO浓度归一化为1%。将测定板转移到BSL-4套件中，用EBOV Kikwit感染HeLa细胞，感染倍数为每细胞0.5 PFU，用EBOVA Makona感染HFF-1细胞，感染复数为每细胞5 PFU。将测定板在组织培养箱中孵育48小时。如补充表1所述，在免疫染色前，通过将样品在10%福尔马林溶液中再固定48小时来终止感染。

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EBOV人巨噬细胞感染试验[1]
抗病毒检测在USAMRIID的BSL-4中进行。将原代人巨噬细胞以每孔40000个细胞的速度接种在96孔板中。在感染前2小时，使用HP D300数字分配器以三倍稀释增量将八到十个连续稀释的化合物（一式三份）直接加入细胞培养物中。所有孔中DMSO的浓度均归一化为1%。将平板转移到BSL-4套件中，在100μl培养基中用每细胞1 PFU的EBOV感染细胞并孵育1小时。去除接种物，用含有稀释化合物的新鲜培养基替换培养基。感染后48小时，如补充表1所述，通过免疫染色定量病毒复制。[1]

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呼吸道合胞病毒A2抗病毒试验[1]
对于抗病毒试验，化合物在源板中连续稀释三倍，使用回声声转移装置将100nl稀释的化合物转移到384孔细胞培养板上。以每毫升5×105个细胞的密度添加HEp-2细胞，然后以每毫升1×104.5个组织培养感染剂量（TCID50）的滴度添加RSV A2进行感染。添加病毒后，立即使用μFlow液体分配器将20μl病毒和细胞混合物添加到384孔细胞培养板中，并在37°C下培养4天。孵育后，让细胞在25°C下平衡30分钟。通过加入20μl CellTiter-Glo活力试剂来测定RSV诱导的细胞病变效应。在25°C下孵育10分钟后，通过使用Envision平板读数器测量发光来确定细胞存活率。

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• 病毒复制抑制： 细胞（如Vero E6、HAE、Huh7）以低感染复数（MOI）感染病毒（SARS-CoV、MERS-CoV、EBOV）。用梯度稀释的Remdesivir处理48-72小时。通过空斑试验或TCID₅₀定量病毒滴度；根据剂量反应曲线计算EC₅₀ [1]。
• 细胞毒性实验： 细胞用Remdesivir处理72小时。通过ATP发光法或刃天青还原法检测细胞活力。确定CC₅₀值[1]

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在多种人源细胞（包括原代巨噬细胞、HeLa、HFF-1、HMVEC-TERT 和 Huh-7 细胞）中评估 GS-5734 对埃博拉病毒的抗病毒活性。细胞与系列稀释的药物孵育 3–5 天，通过免疫荧光、空斑实验或 qRT-PCR 定量病毒复制。EC₅₀ 值范围为 0.06 至 0.14 μM。[1]

在多种人源细胞系和原代细胞中，通过 CellTiter-Glo 活性实验评估细胞毒性。GS-5734 在大多数细胞类型中的 CC₅₀ 值 >20 μM，表明细胞毒性较低。[1]

Rhesus monkeys (Macaca mulatta)
\n3 mg/kg, 10 mg/kg
\nIV

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\n\nIn vivo efficacy[1]
\nRhesus monkeys (Macaca mulatta) were challenged on day 0 by intramuscular injection with a target dose of 1,000 PFU of EBOV Kikwit (Ebola virus H. sapiens-tc/COD/1995/Kikwit), which was derived from a clinical specimen obtained during an outbreak occurring in the Democratic Republic of the Congo (formerly Zaire) in 1995. Challenge virus was propagated from the clinical specimen using cultured cells (Vero or Vero E6) for a total of four passages. Animals (3–6 years old) were randomly assigned to experimental treatment groups, stratified by sex (with equal number of males and females per group) and balanced by body weight, using SAS statistical software. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals. The primary endpoint for efficacy studies was survival to day 28 following virus challenge. GS-5734 was formulated at Gilead Sciences in water with 12% sulfobutylether-β-cyclodextrin (SBE- β-CD), pH adjusted to 3.0 using HCl. Formulations were administered to anaesthetized animals by bolus intravenous injection at a rate of approximately 1 min per dose in the right or left saphenous vein. The volume of all vehicle or GS-5734 injections was 2.0 ml kg−1 body weight. Animals were anaesthetized using intramuscular injection of a solution containing ketamine (100 mg ml−1) and acepromazine (10 mg ml−1) at 0.1 ml kg−1 body weight.[1]
\n\nAnimals were observed at least twice daily to monitor for disease signs, and animals that survived to day 28 were deemed to be protected. Study personnel alleviated unnecessary suffering of infected animals by euthanizing clinically moribund animals. The criteria used as the basis for euthanasia of moribund animals were defined before study initiation and included magnitude of responsiveness, reduced body temperature, and/or specified alterations to serum chemistry parameters35. Serum chemistry was analysed using a Vitros 350 Chemistry System, and coagulation parameters were evaluated using a Sysmex CA-1500 coagulation analyser. Haematology analysis was conducted using a Siemens Advia 120 Hematology System with multispecies software. On days in which GS-5734 or vehicle dosing were scheduled with blood sample collection for clinical pathology or viraemia analysis, blood samples were collected immediately before dose administration.[1]

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\n\n• Mouse EBOV model: BALB/c mice infected with maEBOV. Treated via IP injection with Remdesivir (25 mg/kg in 10% sulfobutylether-β-cyclodextrin) daily for 6 days starting 1 hpi. Survival and viral load monitored [1].
• Mouse SARS-CoV model: C57BL/6 mice infected with SARS-CoV. Remdesivir delivered via SC osmotic pump (loading dose 50 mg/kg, maintenance 25 mg/kg/day in 10% sulfobutylether-β-cyclodextrin) for 12 days starting 24 hpi. Lung viral titers and histopathology assessed [1].
• Ferret Nipah model: Ferrets infected with Nipah virus. Prophylactic treatment: Remdesivir (10 mg/kg in 10% sulfobutylether-β-cyclodextrin) administered IM daily for 3 days starting 1 day pre-infection. Clinical signs, weight, and viral shedding monitored [2]

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\nPharmacokinetic evaluations[1]
\nThree uninfected male rhesus monkeys were used for the pharmacokinetic study. GS-5734 was formulated in solution at 5 mg ml−1 with 12% sulfobutylether-β-cyclodextrin in water, pH 3.5–4.0, and 2 ml kg−1 was administered by slow bolus (approximately 1 min) for a final dose of 10 mg kg−1. Blood samples for plasma and PBMCs were collected from a femoral vein/artery and were taken from each monkey over a 24-h period. Plasma samples were obtained at predose and at 0.083, 0.25, 0.5, 1, 2, 4, 8, and 24 h postdose. PBMC samples were obtained at 2, 4, 8, and 24 h. Blood samples for plasma were collected into chilled collection tubes containing sodium fluoride/potassium oxalate as the anticoagulant and were immediately placed on wet ice, followed by centrifugation to obtain plasma. Blood samples for PBMC isolation were collected at room temperature into CPT vacutainer tubes containing sodium heparin for isolation. Plasma and isolated PBMC samples were frozen immediately and stored at ≤60 °C until analysed.[1]
\n\nFor plasma analysis, an aliquot of 25 μl of each plasma sample was treated with 100 μl of 90% methanol and acetonitrile mixture (1:1, v:v) and 10% water with 20 nM 5-(2-aminopropyl)indole as an internal standard. Then, 100 μl of samples were filtered through an Agilent Captiva 96 well 0.2 μm filter plate. Filtered samples were dried down completely for approximately 20 min and reconstituted with 1% acetonitrile and 99% water with 0.01% formic acid. An aliquot of 10 μl was injected for LC-MS/MS using a HTC Pal autosampler. Analytes were separated on a Phenomenex Synergi Hydro-RP 30A column (75 × 2.0 mM, 4.0 μm) using a Waters Acquity ultra performance LC, a flow rate of 0.26 ml min−1, and a gradient from Mobile phase A containing 0.2% formic acid in 99% water and 1% acetonitrile to mobile phase B containing 0.2% formic acid in 95% acetonitrile and 5% water over 4.5 min. For MS/MS analysis, we used a Waters Xevo TQ-S in positive multiple reaction monitoring mode using an electrospray probe. Plasma concentrations of GS-734, alanine metabolite and Nuc were determined using an 8-point calibration curve spanning a concentration range of over three orders of magnitude. Quality control samples were run at the beginning and end of the run to ensure accuracy and precision within 20%. Intracellular metabolites in PBMCs were quantified by LC-MS/MS as described above for in vitro activation studies.[1]

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\n\nRadiolabelled tissue distribution[1]
\nSix cynomolgus monkeys were administered a single dose of [14C]GS-5734 at 10 mg kg−1 (25 μCi kg−1) by intravenous administration (slow bolus). Tissues were collected from three animals at 4 and 168 h postdose. The tissues were excised, rinsed with saline, blotted dry, weighed, and placed on wet ice. Tissues (testes, epididymis, eyes and brain; following homogenization) and plasma were analysed by liquid scintillation counting. Concentrations were converted to ng equivalents of GS-5734 per gram of sample.

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\n\nAssessment of resistant virus virulence in vivo. [2]
\nGroups of 10 to 12 10-week old female BALB/c (Charles River, Inc.) mice were anesthetized with ketamine-xylazine and intranasally infected with either 104 or 103 PFU/50 µl wild-type mouse-adapted SARS-CoV expressing nanoluciferase (WT SARS-CoV) or SARS-MA15 NanoLuc engineered to harbor resistance mutations in nsp12 (F480L + V557L SARS-CoV). Animals were weighed daily to monitor virus-associated weight loss. On days 2 and 4 postinfection, 5 to 6 animals per group were sacrificed by isoflurane overdose and the inferior right lobe was harvested and frozen at −80°C until the titer was determined by plaque assay as described previously (38). A 5- to 6-animal cohort was monitored out to 7 days postinfection in order to compare the kinetics of recovery, after which lung samples were harvested and the titer determined as described for previous samples.\n

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Rhesus monkeys were challenged intramuscularly with 1,000 PFU of EBOV Kikwit. Remdesivir (GS-5734) was formulated in 12% sulfobutylether-β-cyclodextrin in water (pH 3.0–4.0) and administered intravenously once daily for 12 days at doses of 3 mg/kg or 10 mg/kg, starting on day 0, 2, or 3 post-infection. Animals were monitored for survival, clinical scores, viremia, and clinical pathology. [1]

Pharmacokinetic studies in healthy rhesus monkeys involved intravenous administration of 10 mg/kg GS-5734, with serial blood sampling for plasma and PBMC analysis over 24 hours. [1]

Tissue distribution was assessed in cynomolgus monkeys using [¹⁴C]GS-5734, with tissues collected at 4 and 168 hours post-dose. [1]

Absorption, Distribution and Excretion
Remdesivir is rapidly absorbed; after a single 30-minute intravenous infusion, peak plasma concentrations (Tmax) are reached within 0.67–0.68 hours. Following repeated administration, Cmax (coefficient of variation expressed as a percentage) was 2229 (19.2) ng/mL, and AUCtau was 1585 (16.6) ngh/mL. Measurements of the remdesivir metabolite [GS-441524] were: Tmax 1.51–2.00 hours, Cmax 145 (19.3) ng/mL, AUCtau 2229 (18.4) ngh/mL, and Ctrough 69.2 (18.2) ng/mL. The other metabolite, GS-704277, was measured as follows: Tmax 0.75 h, Cmax 246 (33.9) ng/mL, AUCtau 462 (31.4) ngh/mL, and trough concentration (Ctrough) not determined. Following intravenous administration of a 10 mg/kg dose to cynomolgus monkeys, the drug was distributed to the testes, epididymis, eyes, and brain within 4 hours. 74% of remdesivir was excreted in the urine and 18% in the feces. Of the recovered drug, 49% was the metabolite [GS-441524] and 10% was the unmetabolized parent compound. A small amount (0.5%) of the [GS-441524] metabolite was detected in the feces. Data regarding the volume of distribution of remdesivir are not available. Data regarding the clearance rate of remdesivir are not available.

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Metabolism/Metabolites
Remdesivir is a phosphoramide prodrug that must be metabolized into a triphosphate metabolite within host cells to exert its therapeutic activity. It is presumed that after entering cells, remdesivir is first hydrolyzed by esterases to its carboxylate form, then cyclized to remove the phenoxy group, and finally hydrolyzed to generate a detectable alanine metabolite (GS-704277). The alanine metabolite is subsequently hydrolyzed to remdesivir's monophosphate form, which can be further hydrolyzed to generate the naked nucleoside metabolite [GS-441524], or phosphorylated by cellular kinases to generate the active triphosphate form.

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Biological Half-Life
After a single 30-minute intravenous infusion of remdesivir, its elimination half-life is 1 hour. Under the same conditions, the elimination half-lives of remdesivir metabolites [GS-441524] and GS-704277 are 27 hours and 1.3 hours, respectively. In non-human primates, the plasma half-life of remdesivir at an intravenous dose of 10 mg/kg is 0.39 hours. The half-life of the nucleoside triphosphate metabolite in non-human primates is 14 hours. The half-life of the nucleoside triphosphate metabolite in humans is approximately 20 hours. • Mouse Ebola virus model: BALB/c mice were infected with maEBOV. Remdesivir (25 mg/kg, dissolved in 10% sulfobutyl ether-β-cyclodextrin solution) was administered intraperitoneally daily for 6 days, starting 1 hour after infection. Survival rate and viral load were monitored [1]. • Mouse SARS-CoV model: C57BL/6 mice were infected with SARS-CoV. Remdesivir (50 mg/kg loading dose, 25 mg/kg/day maintenance dose, dissolved in 10% sulfobutyl ether-β-cyclodextrin solution) was administered via subcutaneous osmotic pump for 12 days, starting 24 hours after infection. Assess pulmonary viral titers and histopathology [1]. • Ferret Nipah virus model: Ferrets infected with Nipah virus. Prophylactic treatment: Remdesivir (10 mg/kg, dissolved in 10% sulfobutyl ether-β-cyclodextrin solution) was administered intramuscularly daily for 3 days, starting 1 day before infection. Clinical symptoms, body weight, and viral shedding were monitored [2]. The plasma half-life of 10 mg/kg GS-5734 administered intravenously to rhesus monkeys was 0.39 hours. The prodrug was rapidly metabolized to an alanine intermediate and a parent nucleoside (Nuc). [1] The intracellular NTP half-life in peripheral blood mononuclear cells (PBMCs) was 14 hours and remained above the level required to inhibit more than 50% of the virus for 24 hours. [1]
[¹⁴C]GS-5734 was distributed to radioactive sites such as the testes, epididymis, eyes, and brain within 4 hours, and remained detectable in the brain after 168 hours. [1]

Hepatotoxicity
In human volunteer studies, serum transaminase levels showed a slight increase (less than 5 times the upper limit of normal) 7 to 14 days after remdesivir treatment, but no other evidence of liver injury was observed. In controlled trials of remdesivir in hospitalized COVID-19 patients, the rate of serum ALT elevation in patients treated with remdesivir was similar to or lower than in the placebo group. However, in most uncontrolled studies and case series, 10% to 50% of patients treated with remdesivir experienced transient, mild to moderate elevations in serum ALT and AST within 1 to 5 days after treatment initiation, while serum bilirubin or alkaline phosphatase levels remained unchanged. In some clinical trials, up to 9% of patients reported ALT and AST elevations exceeding 5 times the upper limit of normal, but these abnormalities returned to normal upon discontinuation of treatment without clinically significant liver injury. With the widespread use of remdesivir in the treatment of COVID-19, although there have been occasional reports of significantly elevated ALT levels accompanied by jaundice, these cases mostly occurred in critically ill patients with multiple organ failure or sepsis, or in patients who had received other potentially hepatotoxic drugs (such as intravenous amiodarone) (Case 2). Complicating matters further, elevated serum transaminases are common in symptomatic SARS-CoV-2 infection (Case 1), occurring in up to 60% of patients, and are more common in severely ill patients and those with known risk factors for severe COVID-19 (such as male sex, advanced age, high body mass index, and diabetes). Therefore, elevated serum transaminases are common during remdesivir treatment, but are usually asymptomatic, completely reversible, and unrelated to jaundice. With the widespread use of this antiviral drug in non-severe or critically ill patients and the extension of treatment duration, hepatotoxic features may become more pronounced.
Probability Score: D (Possibly a rare cause of clinically significant liver injury).

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Impact of Pregnancy and Lactation
◉ Overview of Use During Lactation
Information from 5 patients indicates that the concentrations of remdesivir and its active metabolites in breast milk are very low. Furthermore, remdesivir is poorly absorbed orally, and its metabolites are only partially absorbed orally; therefore, it is unlikely that infants will absorb a clinically significant dose of the drug from breast milk. No serious adverse reactions have been reported in neonates who received intravenous remdesivir for Ebola virus and COVID-19, and the drug is FDA-approved for use in infants at least 28 days old and weighing 3 kg. No adverse reactions have been reported in infants exposed to the drug through breast milk. Based on this information, mothers receiving remdesivir do not need to discontinue breastfeeding, but infants should be closely monitored during breastfeeding until more data are available. The most common adverse reactions following intravenous infusion include elevated transaminase and bilirubin levels, as well as other elevated liver enzymes, diarrhea, rash, renal impairment, and hypotension.
◉ Effects on Breastfed Infants
According to a pharmacovigilance report, the manufacturer reported that 11 breastfed infants were exposed to remdesivir through breast milk. The report indicated that breastfed infants did not suffer adverse effects from exposure to remdesivir and its metabolites.
◉ Effects on Breastfeeding and Breast Milk
As of the revision date, no relevant published information was found.
◈ What is Remdesivir?
Remdesivir is an antiviral drug approved for the treatment of the SARS-CoV-2 virus that causes COVID-19. Remdesivir is also used to treat Ebola virus infection. Remdesivir is marketed under the brand name Veklury®. For more information on COVID-19, please see the MotherToBaby case sheet: https://mothertobaby.org/fact-sheets/covid-19/. Sometimes, when people find out they are pregnant, they consider changing how they take the medication or even stopping it entirely. However, always consult your healthcare provider before changing your medication regimen. Your healthcare professional can discuss the benefits of treating your condition and the risks of not treating it during pregnancy.
◈ I am taking Remdesivir. Will it make it harder for me to get pregnant?
There is currently no research indicating whether Remdesivir makes it harder to get pregnant.
◈ Does taking Remdesivir increase the risk of miscarriage?
Miscarriage can occur in any pregnancy. There is currently no research indicating that Remdesivir increases the risk of miscarriage.
◈ Does taking Remdesivir increase the risk of birth defects?
There is a 3-5% risk of birth defects in each pregnancy. This is called background risk. Based on the reviewed studies, it is unclear whether Remdesivir increases the risk of birth defects on top of the background risk. Animal studies have not shown an increased risk of birth defects. There are currently no human studies on the risk of birth defects caused by Remdesivir use during pregnancy.
◈ Does taking Remdesivir during pregnancy increase the risk of other pregnancy-related problems?
According to reports of 70 women with COVID-19 who received remdesivir treatment during the second and third trimesters, the risk of preterm birth (delivery before 37 weeks of gestation), low birth weight (birth weight less than 5 pounds 8 ounces [2500 grams]), and cesarean section was higher. However, these patients also had severe COVID-19 infections. Pregnancy-related problems, including preterm birth, have also been associated with COVID-19 infection during pregnancy. Based on these reports, it is unclear whether these results are due to COVID-19 itself, the drug treatment, or a combination of both. One study investigated 39 pregnant women who received remdesivir treatment for COVID-19 and compared them with 56 pregnant women who did not receive remdesivir treatment for COVID-19. The study showed that the preterm birth rate was similar in both groups. This suggests that the increased risk of preterm birth may be due to COVID-19 itself, rather than the drug.
◈ Will taking remdesivir during pregnancy affect a child's future behavior or learning abilities?
Currently, no studies have explored whether remdesivir causes long-term behavioral or learning problems. There are reports of some newborns receiving remdesivir treatment directly after being diagnosed with Ebola and COVID-19. No serious adverse reactions to remdesivir were reported in these infants. One child who received remdesivir for Ebola was reported to have normal weight and development at one year old.
◈ Breastfeeding while taking remdesivir:
According to one case report, the concentration of remdesivir in breast milk appears to be very low. The absorption rate of oral remdesivir is also low. This means that breastfed infants are unlikely to absorb large amounts of the drug from breast milk. There are reports of two newborns receiving remdesivir after birth for Ebola and COVID-19 infection without experiencing any drug reactions. Because information on the use of remdesivir during breastfeeding is very limited, if remdesivir is used while breastfeeding, healthcare professionals will likely closely monitor the infant's liver and kidney function, blood pressure, and for conditions such as diarrhea or rashes. If you suspect any reaction or symptoms in your infant, contact your child's healthcare professional. If someone wants to breastfeed but is unable to due to illness after contracting COVID-19, they can work with a healthcare professional or lactation consultant to help establish or maintain a milk supply so that breastfeeding can resume after recovery. Please consult your healthcare professional about all questions regarding breastfeeding.
◈ Will male exposure to remdesivir affect fertility (the ability to impregnate a partner) or increase the risk of birth defects?
Currently, no studies have explored whether remdesivir affects fertility. Generally, exposure to remdesivir by the father or sperm donor is unlikely to increase the risk of pregnancy. For more information, please refer to the “Paternal Exposure” information sheet on the MotherToBaby website at https://mothertobaby.org/fact-sheets/paternal-exposures-pregnancy/.

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Protein Binding
Remdesivir binds to human plasma proteins at a rate of 88-93.6%, while its metabolites [GS-441524] and GS-704277 bind at rates of 2% and 1%, respectively.

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• Mouse toxicity studies: Daily subcutaneous injection (50 mg/kg for 12 days) resulted in transient weight loss (approximately 10%), but no death. Biochemical indicators (ALT, AST, BUN) were all within the normal range [1]. • Cynomolgus monkey study: Intravenous injection (10 mg/kg) caused a slight increase in liver enzymes (ALT), but no histopathological changes were observed [1]. Remdesivir (GS-5734) showed low in vitro cytotoxicity, with CC₅₀ values >20 μM in most primary human cells and cell lines. [1] In the rhesus monkey efficacy study, no significant drug-related toxicity was reported; clinicopathological parameters of the treated animals were improved compared to the vector control group. [1]

[1]. Nature . 2016 Mar 17;531(7594):381-5.

[2]. mBio . 2018 Mar 6;9(2):e00221-18.

Pharmacodynamics
Remdesivir is a nucleoside analog used to inhibit the activity of RNA polymerases. Its duration of action is moderate due to the need for only once-daily dosing. Mammalian DNA and RNA polymerases, including human mitochondrial RNA polymerase, are far more selective for ATP than remdesivir triphosphate, so remdesivir is not a significant inhibitor of these enzymes, which contributes to its overall tolerability and safety. Nevertheless, there is a risk of hypersensitivity reactions (including anaphylactic shock and other infusion-related reactions), elevated transaminase levels, and potential reduced efficacy when used in combination with hydroxychloroquine or chloroquine. • Remdesivir is a monophosphoramide prodrug designed to deliver the nucleoside analog GS-441524 into cells, where it is subsequently metabolized to its active triphosphate form [1]. • It has broad-spectrum activity against a variety of RNA virus families (coronaviruses, filoviridae, paramyxoviridae) [2]. Its mechanism of action differs from ribavirin; it acts as a delayed chain terminator rather than a mutagen.[1]

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Remdesivir (GS-5734) is the first small molecule antiviral compound to demonstrate substantial protection against Ebola virus exposure in non-human primates.[1]
Its broad-spectrum activity against filoviruses, arenaviruses, and coronaviruses suggests its potential to treat other viral infections.[1]
At the time of publication, clinical studies in healthy volunteers are underway to evaluate its safety and pharmacokinetics.[1]

C27H35N6O8P

602.5760

602.225

C, 53.82; H, 5.85; N, 13.95; O, 21.24; P, 5.14

1809249-37-3

1355149-45-9 [GS443902 (GS-441524 triphosphate)]; 1809249-37-3 (Remdesivir); 1191237-69-0 (GS-441524, an active metabolite of Remdesivir); 1191237-80-5 (Remdesivir O-desphosphate acetonide impurity); 1911578-74-9 (Remdesivir nucleoside monophosphate); 2250110-53-1;1911579-04-8 (GS-704277)

121304016

Off-white to yellow solid powder

1.5±0.1 g/cm3

1.652

2.1

204Ų

4

13

14

42

1010

6

[P@@](N([H])[C@@]([H])(C([H])([H])[H])C(=O)OC([H])([H])C([H])(C([H])([H])C([H])([H])[H])C([H])([H])C([H])([H])[H])(=O)(OC1C([H])=C([H])C([H])=C([H])C=1[H])OC([H])([H])[C@]1([H])[C@]([H])([C@]([H])([C@](C#N)(C2=C([H])C([H])=C3C(N([H])[H])=NC([H])=NN23)O1)O[H])O[H]

RWWYLEGWBNMMLJ-YSOARWBDSA-N

InChI=1S/C27H35N6O8P/c1-4-18(5-2)13-38-26(36)17(3)32-42(37,41-19-9-7-6-8-10-19)39-14-21-23(34)24(35)27(15-28,40-21)22-12-11-20-25(29)30-16-31-33(20)22/h6-12,16-18,21,23-24,34-35H,4-5,13-14H2,1-3H3,(H,32,37)(H2,29,30,31)/t17-,21+,23+,24+,27-,42-/m0/s1

2-ethylbutyl (2S)-2-[[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5-cyano-3,4-dihydroxyoxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate

GS-5734; GS 5734; Prodrug of GS441524; Prodrug of GS441524; Remdesivir; GS5734; Prodrug of GS-441524; 3QKI37EEHE; GS 5734; GS 5734 [WHO-DD]; GS-5734; GS5734; Remdesivir; REMDESIVIR [INN];

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: ~100 mg/mL (~166 mM)
Ethanol: ~100 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.15 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 2 中的溶解度: ≥ 2.17 mg/mL (3.60 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 21.7 mg/mL澄清DMSO储备液加入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 3 中的溶解度: ≥ 2.17 mg/mL (3.60 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 21.7 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: 5%DMSO+ 40%PEG300+ 5%Tween 80+ 50%ddH2O: 5.0mg/ml (8.30mM)

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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。