EIDD-1931 (Beta-d-N4-hydroxycytidine; NHC)   中文名称: EIDD-1931（β-d-N4-羟基胞苷；NHC）

P2Y12 Receptor

抗 VEEV（委内瑞拉马脑炎病毒）剂 β-d-N4-hydroxycytidine (NHC)的 EC50、EC90 和 EC99 值分别为 0.426、1.036 和 2.5 μM[1]。
在 Huh-7–CHIKV 中复制子细胞系，β-d-N4-羟基胞苷抑制CHIKV复制子活性，50%有效浓度（EC50）为0.8 μM。关于 BHK-21 细胞中的复制子，已报告了类似的结果 (EC50=1.8 μM)。根据 MTT 测定，NHC 在 Huh-7 细胞培养系统中浓度高达 100 μM 时不会引起任何细胞毒性。对于外周血单核 (PBM)、Vero 和 CEM 细胞，NHC 的 50% 细胞毒性浓度 (CCsub>50) 值分别为 30.6 μM、7.7 μM 和 2.5 μM。NHC 充当嘧啶类似物；外源核苷，例如嘧啶 C 和 U，可以逆转 NHC 介导的 CHIKV 复制子抑制；然而，复制子不受 dA、dC、dG、dU 或 T 的影响。无论有或没有 NHC，嘧啶 A 和 G 都在复制子抑制中发挥作用 [2]。

在小鼠模型中对VEEV抑制剂核糖核苷类似物EIDD-1931（β-D-N4-羟基胞苷）进行了表征。[4]
• EIDD-1931可口服，成功递送至脑组织并转化为活性5′-三磷酸形式。[4]
• EIDD-1931在长达1000mg/kg/天的7天剂量范围毒理学研究中表现出良好的耐受性。[4]
• EIDD-1931在预防性或治疗性给药时保护小鼠免受VEEV的致命鼻内攻击。[4]

细胞内NHC-TP t1/2s的测定。[2]
Huh-7细胞（六孔板每孔2.5×106）与10μM[3H]NHC（β-d-N4-羟基胞苷，500 dpm/pmol）在37°C、5%CO2气氛中孵育24小时。然后用无药物培养基洗涤细胞三次以去除细胞外NHC，并用常规培养基孵育特定时间段（0、1、2、4、8和24小时）。如下所述提取细胞内代谢物。

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细胞内代谢物的测定。[2]
在NHC-TP积累或NHC-TP t1/2研究时间点测定的选定时间，去除细胞外培养基，用冷磷酸缓冲盐水洗涤细胞层。用60%甲醇（1ml）刮除细胞后，在-20°C下孵育过夜提取NHC及其各自的代谢物，然后以14000 rpm离心样品5分钟，收集上清液。第二天，在冰上提取1小时（200μl，含60%甲醇），然后再次以14000 rpm的速度离心样品（Eppendorf 5415C型离心机）5分钟。将提取物合并，在温和的过滤气流下干燥，然后储存在-20°C下，直至通过HPLC分析。将残留物重新悬浮在200μl水中，并将等分试样注入HPLC柱中。

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猴和人全血中NHC的稳定性研究。[2]
将10微摩尔[3H]NHC（1000 dpm/pmol）在猴子或人类血液中孵育不同时间段（0、0.08、0.16、1、2、4和24小时）。在选定的时间点，取200μl的等分试样，以14000 rpm离心5分钟。收集上清液，加入500μl乙腈并混合。将样品在14000rpm下重新离心5分钟，并使用DNA快速真空干燥上清液。将残留物重新悬浮在200μl水中，并将等分试样注入HPLC柱中。

病毒复制分析。将总共5×10~5个Vero细胞接种到6孔Costar平板中，并以图例中所示的MOI感染。在指定时间将β-d-N4-hydroxycytidine (NHC)添加到细胞中，收获培养基，并通过Vero细胞上的空斑试验测定样品中的病毒滴度，如别处所述[1]。

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发现β-d-N4-hydroxycytidine (NHC)在HCV复制子系统（克隆A）中具有选择性抗丙型肝炎病毒（HCV）活性。在HCV复制子系统、Huh-7细胞、HepG2细胞和原代人肝细胞中研究了氚化NHC的细胞内代谢。用10微M放射性标记的NHC孵育细胞表明，所有肝细胞中都存在广泛而快速的磷酸化。除了NHC的5'-单磷酸、-二磷酸和-三磷酸代谢物外，还对其他代谢物进行了表征。这些包括胞苷和尿苷单磷酸盐、二磷酸盐和三磷酸盐。UTP是Huh-7细胞和原代人肝细胞中主要的早期代谢产物，表明NHC的脱氨基是主要的分解代谢途径。经计算，放射性标记的NHC三磷酸盐和来源于Huh-7细胞中NHC孵育的CTP和UTP的细胞内半衰期分别为3.0+/-1.3、10.4+/-3.3和13.2+/-3.5小时（平均值+/-标准差）。使用猴子和人类全血的研究表明，猴子细胞的脱氨基和氧化速度比人类细胞快，这表明NHC在血浆中的持续时间可能不足以输送到肝细胞。

Pharmacokinetics and tissue distribution in mice[4]
Female ICR (CD-1®) mice, 6–8 weeks of age, were used in the studies (to match mice used in efficacy studies). EIDD-1931 was administered by oral gavage (PO) in 240 mM citrate buffer pH 3 ± 0.3 or intraperitoneally (IP) in saline. The oral doses tested were 50, 150 and 500 mg/kg of body weight, and the IP doses were 10 and 50 mg/kg of body weight. Blood samples were collected at 0.08, 0.25, 0.5, 1, 2, 4, 8, and 24 h post IP administration, and at 0.25, 0.5, 1, 2, 3, 4, 8, and 24 h post oral administration. Plasmas were prepared within 30 min after collection by centrifugation at 2000g for 10 min at 4 °C and stored at −80 °C before processing for analysis by LC-MS/MS. Mouse organs (lung, spleen liver, kidney, heart and brain) were collected from all mice immediately following blood collection starting from 0.5 h post dose. The tissues were immediately snap-frozen in liquid nitrogen and stored at −80 °C before processing for analysis by LC-MS/MS.

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Dose range finding (DRF) toxicology and toxicokinetic study[4]
This study was conducted in two phases: Phase A (single dose, acute toxicity) and Phase B (multiple doses). During Phase A, two groups of 6 mice (3 males and 3 females each) were administered EIDD-1931 once via oral gavage at 500 and 1000 mg/kg dose levels, and following a four-day washout period the same animals were administered 1500 and 2000 mg/kg doses. The compound was delivered at 10 ml/kg volumes in sodium citrate vehicle (0.24M sodium citrate, pH 3 ± 0.3). After dosing, the animal's weight, food consumption, general physical appearance and behavior were monitored twice daily for four days. During Phase B, EIDD-1931 was administered once daily for 7 consecutive days. Ten male and ten female mice per dose (80 mice total) were tested at dose levels of 200, 500, and 1000 mg/kg/day administered at a dose volume of 10 mL/kg. [4]

Animals in the toxicokinetic (TK) arm of the study received EIDD-1931 at the same doses and dose volumes and in the same manner as the main study groups at doses of 200, 500, and 1000 mg/kg/day. Thirty six male and 36 female mice per dose level were used in the TK arm. Blood samples were collected from TK animals for determination of the plasma concentrations of EIDD-1931. Samples were collected from cohorts of 3 TK animals/sex/group/timepoint at 1, 2, 4, 6, 8, and 24 h postdose on Day 1 and at predose and 1, 2, 4, 6, and 8 h postdose on Day 7. Samples were collected in tubes containing lithium heparin as an anticoagulant and kept on ice.

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Murine models of intranasal VEEV infection[4]
Seven to eight-week-old ICR (Crl:CD1) female mice were used in all studies. The dose dependency of EIDD-1931 was determined in a prophylaxis study. Four groups of mice were dosed via gavage with 150, 300 or 500 mg/kg EIDD-1931 in 240 mM sodium citrate buffer pH 3 ± 0.3 or mock-treated with vehicle only, all at 10 ml/kg dose volume, starting at 2 h before infection. The second treatment was delivered at +2 h post-infection (PI), and then the treatment was continued twice daily (b.i.d.) for 6 days. In a second (therapeutic) study, treatment with 500 mg/kg EIDD-1931 was initiated starting at 6, 12, 24 or 48 h post-infection and the treatment was compared to a vehicle (mock) treated group. For the +6 h group, the second treatment was performed at 12 h post-infection and then, for all groups, the treatment was continued every 12 h (b.i.d.) for 6 days.

Absorption, Distribution and Excretion
N4-hydroxycytidine is orally bioavailable in mice but poorly bioavailable in non-human primates.

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Metabolism / Metabolites
N4-hydroxycytidine distributes into tissues where it is is phosphorylated to the 5'-triphosphate form.

[1]. β-d-N4-Hydroxycytidine Is a Potent Anti-alphavirus Compound That Induces a High Level of Mutations in the Viral Genome. J Virol. 2018 Jan 17;92(3). pii: e01965-17.

[2]. Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells. Antimicrob Agents Chemother. 2004 Dec;48(12):4636-42

[3]. Characterization of β-d- N4-Hydroxycytidine as a Novel Inhibitor of Chikungunya Virus. Antimicrob Agents Chemother. 2017 Mar 24;61(4):e02395-16.

[4]. The prophylactic and therapeutic activity of a broadly active ribonucleoside analog in a murine model of intranasal venezuelan equine encephalitis virus infection. Antiviral Res . 2019 Nov:171:104597.

N(4)-hydroxycytidine is a nucleoside analogue that is cytidine which carries a hydroxy group at the N(4)-positon. It has broad-spectrum antiviral activity against influenza, SARS-CoV , SARS-CoV-2 and MERS-CoV. It has a role as a drug metabolite, a human xenobiotic metabolite, an anticoronaviral agent and an antiviral agent. It is a nucleoside analogue and a ketoxime. It is functionally related to a cytidine.
N4-Hydroxyctidine, or EIDD-1931, is a ribonucleoside analog which induces mutations in RNA virions. N4-hydroxycytidine was first described in the literature in 1980 as a potent mutagen of bacteria and phage. It has shown antiviral activity against Venezuelan equine encephalitis virus, and the human coronavirus HCoV-NL63 in vitro. N4-hydroxycytodine has been shown to inhibit SARS-CoV-2 as well as other human and bat coronaviruses in mice and human airway epithelial cells. It is orally bioavailable in mice and distributes into tissue before becoming the active 5’-triphosphate form, which is incorporated into the genome of new virions, resulting in the accumulation of inactivating mutations. In non-human primates, N4-hydroxycytidine was poorly orally bioavailable. A [remdesivir] resistant mutant mouse hepatitis virus has also been shown to have increased sensitivity to N4-hydroxycytidine. The prodrug of N4-hydroxycytidine, [EIDD-2801], is also being investigated for its broad spectrum activity against the coronavirus family of viruses.

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Drug Indication
N4-hydroxycytidine and its prodrug [EIDD-2801] is being studied for its activity against a number of viral infections including influenza, MERS-CoV, and SARS-CoV-2.

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Mechanism of Action
N4-hydroxycytidine is phosphorylated in tissue to the active 5’-triphosphate form, which is incorporated into the genome of new virions, resulting in the accumulation of inactivating mutations, known as viral error catastrophe. A [remdesivir] resistant mutant mouse hepatitis virus has also been shown to have increased sensitivity to N4-hydroxycytidine.

C9H13N3O6

259.21602

259.08

C, 41.70; H, 5.06; N, 16.21; O, 37.03

3258-02-4

197020

White to off-white solid powder

-2.2

137.07

5

6

3

18

398

4

C1(N2C=C/C(=N\O)/NC2=O)OC(CO)C(O)C1O

XCUAIINAJCDIPM-XVFCMESISA-N

InChI=1S/C9H13N3O6/c13-3-4-6(14)7(15)8(18-4)12-2-1-5(11-17)10-9(12)16/h1-2,4,6-8,13-15,17H,3H2,(H,10,11,16)/t4-,6-,7-,8-/m1/s1

N4-Hydroxycytidine

EIDD-1931; EIDD 1931; EIDD1931; N4-Hydroxycytidine; β-D-N4-hydroxycytidine; Uridine, 4-oxime; N(4)-Hydroxycytidine; 3258-02-4; EIDD-1931; Beta-D-N4-hydroxycytidine; Uridine, 4-oxime; N-hydroxycytidine; 4-N-Hydroxycytidine; NHC; EIDD-2801-metabolite; Molnupiravir-,etabolite

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 该产品在溶液状态不稳定，请现配现用。

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 (~385.77 mM)
H2O : ≥ 25 mg/mL (~96.44 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (8.02 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 20.8 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 2 中的溶解度: ≥ 2.08 mg/mL (8.02 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 20.8 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 3 中的溶解度: ≥ 2.08 mg/mL (8.02 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 20.8 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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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：
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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；
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