Broad-spectrum antiviral

严重急性呼吸综合征冠状病毒2的RNA依赖性RNA聚合酶是当前治疗2019冠状病毒病药物开发工作的重要靶点。莫努匹拉韦是一种广谱抗病毒药物，是核苷类似物β-D-N4-羟基胞苷（NHC）的口服生物可利用前药。Molnupiravir或NHC可以增加复制冠状病毒的G到A和C到U过渡突变。突变频率的增加可能与抗病毒作用的增加有关；然而，莫努匹拉韦诱导突变的生化数据尚未报道。在这里，我们研究了活性化合物NHC5'-三磷酸（NHC-TP）对纯化的严重急性呼吸综合征冠状病毒2 RNA依赖性RNA聚合酶复合物的作用。天然核苷酸的掺入效率高于NHC-TP掺入模型RNA底物的效率，其顺序为GTP（12841）>ATP（424）>UTP（171）>CTP（30），表明NHC-TP主要与CTP竞争掺入。由于在RNA引物链中掺入一磷酸，没有观察到对RNA合成的显著抑制。当嵌入模板链中时，一磷酸NHC以相似的效率支持NHC:G和NHC:A碱基对的形成。NHC:G产物的延伸受到适度抑制，但较高的核苷酸浓度可以克服这种阻碍。相反，NHC:A碱基对导致观察到的G到A（G:NHC:A）或C到U（C:G:NHC:A:U）突变。总之，这些生物化学数据支持莫努匹拉韦的作用机制，该机制主要基于通过模板链介导的RNA诱变[3]。

莫努匹拉韦是一种强效抗病毒药物，以50–500 mg/kg的剂量每12小时口服一次，持续三天，可以阻止严重急性呼吸系统综合征冠状病毒的复制和疾病[1]
莫努匹拉韦（7 mg/kg；口服；每天两次，持续3.5天）显著降低了病毒的脱落量，缩短了发烧的持续时间[2]。冠状病毒（CoV）在物种之间频繁传播，导致新的疾病爆发，最近的例子是新出现的SARS-CoV-2，新冠肺炎的病原体。在这里，我们发现核糖核苷类似物β-d-N4-羟基胞苷（NHC；EIDD-1931）对严重急性呼吸系统综合征冠状病毒2型、MERS-CoV、严重急性呼吸综合征冠状病毒和相关人畜共患2b或2c组蝙蝠冠状病毒具有广谱抗病毒活性，并对携带核苷类似物抑制剂瑞德西韦耐药性突变的冠状病毒具有更高的效力。在感染严重急性呼吸系统综合征冠状病毒或MERS-CoV的小鼠中，预防和治疗性给予EIDD-2801，一种口服生物可利用的NHC前药（β-d-N4-羟基胞苷-5’-异丙基酯），可以改善肺功能，降低病毒滴度和体重减轻。体外和体内MERS-CoV产量的降低与病毒中过渡突变频率的增加有关，但与宿主细胞RNA无关，这支持了CoV致命突变的机制。NHC/EIDD-2801对抗多种冠状病毒的效力和口服生物利用度突出了其作为对抗严重急性呼吸系统综合征冠状病毒2型和其他未来人畜共患冠状病毒的有效抗病毒药物的潜在效用[1]。

蛋白质的表达和纯化[3]
如上所述，通过使用杆状病毒表达系统将nsp-5、-7、-8和-12表达为多蛋白，并通过在nsp-8 N-末端组氨酸标签上的Ni-NTA亲和层析纯化nsp-7-8-12复合物，产生了严重急性呼吸系统综合征冠状病毒2型RdRp复合物

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NTP掺入和包埋引物或模板的NHC-MP对病毒RNA合成的影响[3]
根据我们的报告，通过严重急性呼吸系统综合征冠状病毒2型RdRp掺入NTP，并进行数据采集和定量。单核苷酸和多核苷酸掺入测定的酶浓度分别为100或200 nM。RNA合成孵育时间为10分钟。使用来自单核苷酸掺入测定的数据来确定天然核苷酸相对于NHC-TP的偏好。选择性值计算为天然核苷酸与核苷酸类似物的结合效率之比。核苷酸掺入的效率由Michaelis–Menten常数Vmax与Km的比值决定。核苷酸掺入底物是通过将[α-32P]NTP掺入4-nt引物而产生的5-nt引物。5-nt引物的形成在给定的时间点是最大的；然而，它的确切浓度是未知的。因此，通过量化对应于6-nt引物产物的信号并将其除以反应中的总信号（5-nt引物和6-nt引物）来测量反应中产生的产物。这定义了产品分数。产物分数通常乘以总底物浓度，以确定Vmax的摩尔单位，这在这里是不可能的，如上所述。因此，Vmax的单位被报告为随时间变化的乘积分数。选择性值是无单位的，因为它是具有相同单位的两个Vmax/Km测量的比率。如我们所述，制备了具有嵌入NHC-MP的RNA模板。图S1中解释了与NHC相关的方案修改。

Madin-Darby犬肾（MDCK）细胞（ATCC CCL-34）在补充有7.5%胎牛血清（FBS）的Dulbecco改良Eagle培养基（DMEM）中于37°C和5%CO2下生长。来自30岁健康女性供体的正常原代人支气管气管上皮细胞（HBTECs）在支气管生命细胞培养基中生长。这些细胞由供应商在知情同意的情况下获得，并符合赫尔辛基宣言、《人体组织法》（英国）、CFR第21篇和HIPAA法规。所有监管审批均由供应商负责。本研究中使用的永生细胞系定期检查微生物污染（间隔约6个月）。2017年7月25日，LifeLine Cell Technology对HBTEC进行了微生物污染测试。本研究仅使用了通道编号为1-4的重型作战电子计算机[2]。

Animal Model: C57BL/6 mice (intranasal infection with SARS-CoV)[1]
Dosage: 50, 150, 500 mg/kg
Administration: Oral; every 12 hours for 3 days
Result: Body weight loss is significantly diminished or prevented.

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PK in cynomolgus macaques[2]
Non-naïve cynomolgus macaques (4 males, 4 females; 2 to 6 years of age) retrieved from the colony stock at Concord Biosciences and originally received from Chares River Laboratories were each dosed orally with 100 mg/kg of NHC dissolved in 240 mM citrate buffer, followed by blood collection from the femoral vein at the specified time points. After a 7-day washout period, each animal was dosed orally with 130 mg/kg of EIDD-2801 dissolved in 80% (v/v) PEG-400, 20% (v/v) N,N-Dimethylacetamide, followed by blood collection from the femoral vein at the specified time points. In a separate study, non-naïve cynomolgus macaques (3 males, 3 females; 2 to 6 years of age) retrieved from the colony stock at MPI, Inc and originally received from Chares River Laboratories were each dosed intravenously with 10 mg/kg of NHC dissolved in 0.9% sterile sodium chloride, followed by blood collection from the femoral vein at the specified time points. For all samples, plasma was separated from heparinized blood and stored at −80°C before analysis as described in NHC. For calibration, standard curves were prepared in blank plasma (concentrations range 10 to 10,000 ng/ml). Quality-control samples of 30, 500, and 5000 ng/ml in blank plasma were analyzed at the beginning of each sample set. Calibration showed linearity with R2 values >0.99.

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PK and PD in ferrets[2]
Female ferrets (6 to 8 months of age) received from Marshall BioRescoursces were rested for one week, assigned randomly to study groups and dosed orally with EIDD-2801 dissolved in 1% methylcellulose, followed by blood collection from the anterior vena cava and tissue sampling at the specified time points. Three animals per groups were sampled for PK analyses and 2-3 animals for PD testing. Plasma was separated from heparinized blood, and tissue samples snap-frozen and stored at −80°C before analysis as described in NHC. For calibration, standard curves were prepared in blank plasma (concentrations range 10 to 100,000 ng/ml) and blank tissue lysate (concentration range 1.56 to 3,130 ng/ml), respectively. Quality-control samples of 30, 500 and 5,000 ng/ml in blank plasma were analyzed at the beginning of each sample set. Calibration in each matrix showed linearity with R2 values >0.99.

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Influenza infection studies in ferrets[2]
Female ferrets (6 to 8 months of age) were received from Marshall BioRescoursces and housed in an ABSL-2 (animal biosafety level) facility. Ferrets were rested for 1 week, weighed, assigned randomly to groups, anesthetized with dexmedetomidine/ketamine, and infected intranasally with 1 × 105 (A/California/07/2009 (H1N1)) or 1 × 106 pfu (A/Wisconsin/67/2005 (H3N2)); infection volume 200 μl. Treatment with EIDD-2801 was initiated 3 hours before infection (prophylactic regimen), 12 hours post-infection (post-exposure prophylactic regimen), or 24 hours post-infection (therapeutic regimen), and continued for 3.5 days b.i.d. Compound was administered orally in 3.5 ml doses in 1% methylcellulose formulation and chased with 3.5 ml high-calorie liquid dietary supplement. Control groups received vehicle (1% methylcellulose in water) volume equivalents. Body temperature was monitored continuously (readings every 2-15 minutes) using implanted telemetric sensors. Bodyweight of animals was measured at start and end of each experiment, and for some experiments daily; no changes in body weight were detected. Additional monitoring of phenotypically appreciable adverse effects included assessment of animals for changes in overall composure, activity level or vocation and occurrence of diarrhea, vomiting or reduced food uptake. Viral load was measured from nasal lavages (collected in 12-hour intervals) and nasal turbinates (upper respiratory tract), harvested 3.5 days after infection if not specified otherwise.

[1]. An orally bioavailable broad-spectrum antiviral inhibits\nSARS-CoV-2 in human airway epithelial cell cultures and multiple\ncoronaviruses in mice. Sci Transl Med. 2020 Apr 6. pii: eabb5883.

[2]. Characterization of orally efficacious influenza drug with\nhigh resistance barrier in ferrets and human airway epithelia. Sci\nTransl Med. 2019 Oct 23;11(515). pii: eaax5866.

[3]. Molnupiravir promotes SARS-CoV-2 mutagenesis via the RNA template. Biol Chem. 2021 Jul; 297(1): 100770.

C13H19N3O7

329.3059

329.12

C, 47.42; H, 5.82; N, 12.76; O, 34.01

2349386-89-4

3258-02-4 (EIDD-1931);2492423-29-5

White to off-white solid powder

-0.8

141Ų

O[C@@H]([C@H]([C@H](N1C(N/C(C=C1)=N/O)=O)O2)O)[C@H]2COC(C(C)C)=O

HTNPEHXGEKVIHG-QCNRFFRDSA-N

InChI=1S/C13H19N3O7/c1-6(2)12(19)22-5-7-9(17)10(18)11(23-7)16-4-3-8(15-21)14-13(16)20/h3-4,6-7,9-11,17-18,21H,5H2,1-2H3,(H,14,15,20)/t7-,9-,10-,11-/m1/s1

((2R,3S,4R,5R)-3,4-dihydroxy-5-((E)-4-(hydroxyimino)-2-oxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydrofuran-2-yl)methyl isobutyrate

Molnupiravir; MK-4482; MK4482; MK 4482; EIDD-2801; EIDD 2801; EIDD2801; prodrug-EIDD-1931; prodrug-EIDD 1931; prodrug-EIDD1931.

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;
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