LETERMOVIR   中文名称: 氯戊米特

Letermovir (formerly known as MK-8828 and AIC-246; trade name: Prevymis) is a new potent anticytomegalovirus drug in clinical development. On 11/8/2017, Letermovir was approved by FDA to prevent infection after bone marrow transplant. Despite modern prevention and treatment strategies, human cytomegalovirus (HCMV) remains a common opportunistic pathogen associated with serious morbidity and mortality in immunocompromised individuals, such as transplant recipients and AIDS patients. All drugs currently licensed for the treatment of HCMV infection target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance.

体外活性：莱特莫韦（原名MK-8828和AIC-246；商品名：Prevymis）是一种处于临床开发阶段的新型强效抗巨细胞病毒药物。 2017年11月8日，莱特莫韦被FDA批准用于预防骨髓移植后感染。尽管有现代的预防和治疗策略，人类巨细胞病毒（HCMV）仍然是一种常见的机会性病原体，与免疫功能低下个体（例如移植受者和艾滋病患者）的严重发病率和死亡率相关。目前获得许可用于治疗 HCMV 感染的所有药物均以病毒 DNA 聚合酶为靶标，并与严重的毒性问题和耐药性的出现有关。激酶检测：AIC246具有一致的抗病毒功效，并且AIC246对人巨细胞病毒具有显着的选择性。 AD169 突变株和指定的 rAIC246-1 和 rAIC246-2 对莱特莫韦 (AIC246) 高度耐药，EC50 分别为 5.6 nM、1.24 μM、0.37 μM。 Letermovir 通过涉及病毒基因产物 UL56 的特定抗病毒机制抑制 HCMV 复制。 Letermovir 通过干扰 HCMV 后代 DNA 的正确切割/包装来抑制细胞培养中的 HCMV 复制[2]。就 EC50 而言，莱特莫韦抑制当前金标准 GCV 超过 400 倍（平均值为 4.5 nM 与 2 μM），就 EC90 值而言抑制超过 2,000 倍（平均值为 6.1 nM 与 14.5 μM）[3] 。莱特莫韦与抗 HCMV 药物联合使用会产生附加的抗病毒作用，但莱特莫韦和抗 HIV 药物之间不存在相互作用。细胞测定：简而言之，将 5×103 AD169 感染的 NHDF 细胞/孔接种到 30 个 96 孔微量滴定板的孔中。允许感染在50 nM AIC246 (10×EC50)的暴露下进行，直到在一个或多个化合物处理的孔中出现CPE（表明抗性病毒突破）。未感染和未处理的细胞作为每个板上的对照。在存在 50 nM AIC246 的情况下，通过无细胞上清病毒的传代，培养物达到最大 CPE，从而完成突变病毒扩增。所得AIC246抗性子代病毒突变体通过在AIC246存在下有限稀释进行噬菌斑纯化3次。通过在没有选择压力的情况下连续传代空斑纯化的病毒（8至10次）来测试耐药性的稳定性。

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在斑块减少实验中，莱特莫韦对17种不同的临床HCMV分离株（WT1-WT17）表现出强大的抗病毒活性。其半数有效浓度（EC50）持续处于低纳摩尔范围（0.0008 µM 至 0.0031 µM），比更昔洛韦（GCV）强约1000倍。
莱特莫韦对HCMV实验室株（AD169，EC50 = 0.0051 µM）和一系列由AD169衍生的耐药变异株保持高活性。这些变异株在UL97（病毒激酶）和/或UL54（病毒DNA聚合酶）基因中带有已确认的对GCV和/或西多福韦（CDV）的耐药突变。莱特莫韦对这些耐药病毒的EC50值（0.0016 µM 至 0.0039 µM）与亲本AD169株相当或更低，表明不存在交叉耐药。
莱特莫韦对HCMV表现出显著的选择性。在基于细胞培养的复制实验中，它对其他人类疱疹病毒无显著活性（EC50 >10 µM），包括水痘-带状疱疹病毒（VZV）、1型和2型单纯疱疹病毒（HSV-1, HSV-2）、人类疱疹病毒6型（HHV-6）和爱泼斯坦-巴尔病毒（EBV）。对鼠巨细胞病毒（MCMV）的活性非常低（EC50 = 4.5 µM），对大鼠巨细胞病毒（RCMV）未检测到活性（EC50 >10 µM）。
莱特莫韦对来自其他病毒科的一系列重要人类致病病毒无抑制活性（EC50 >10 µM 至 >32 µM），包括人腺病毒2型（HAdV-2）、乙型肝炎病毒（HBV）、人类免疫缺陷病毒1型（HIV-1）、甲型流感病毒（H1N1）和丙型肝炎病毒（HCV）复制子系统。
平行进行的细胞毒性研究（使用alamarBlue和/或显微镜评估）表明，在抗病毒实验使用的最高药物浓度下（高达32 µM）未见毒性。[1]

使用小鼠异种移植模型，与安慰剂治疗的对照组相比，莱特莫韦（10-100 mg/kg/天，口服）导致移植细胞中 HCMV 滴度呈剂量依赖性降低

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来特莫韦 (AIC246) 在HCMV感染的小鼠异种移植模型中发挥强效的体内疗效。[2]
I期临床试验表明，来特莫韦总体耐受性良好，在人体内表现出高且持久的暴露量，支持每日一次给药。[2]
在IIa期试验及一名感染多重耐药HCMV株导致多器官疾病的患者中显示了概念验证。[2]

AIC246具有一致的抗病毒功效，并且AIC246对人巨细胞病毒具有显着的选择性。 AD169 突变株和指定的 rAIC246-1 和 rAIC246-2 对莱特莫韦 (AIC246) 高度耐药，EC50 分别为 5.6 nM、1.24 μM、0.37 μM。 Letermovir 通过涉及病毒基因产物 UL56 的特定抗病毒机制抑制 HCMV 复制。 Letermovir 通过干扰 HCMV 后代 DNA 的正确切割/包装来抑制细胞培养中的 HCMV 复制[2]。就 EC50 而言，莱特莫韦抑制当前金标准 GCV 超过 400 倍（平均值为 4.5 nM 与 2 μM），就 EC90 值而言抑制超过 2,000 倍（平均值为 6.1 nM 与 14.5 μM）[3] 。莱特莫韦与抗 HCMV 药物联合使用会产生附加的抗病毒作用，但莱特莫韦和抗 HIV 药物之间不存在相互作用。

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本文引用了莱特莫韦的作用模式，指出其通过靶向病毒terminase复合物干扰DNA多联体成熟。[1]
采用功能性病毒DNA切割实验评估来特莫韦对终止酶活性的影响。用HCMV感染细胞并用药物处理。孵育后，提取总DNA并用限制性内切酶（KpnI）消化。消化后的DNA通过凝胶电泳按大小分离，转移到膜上，并与针对HCMV基因组末端区域的地高辛标记探针进行杂交。约4 kb片段的存在表明终止酶正确切割并成熟为单位长度基因组，而其缺失则表明切割/包装过程被抑制。来特莫韦以浓度依赖的方式抑制~4 kb片段的形成，证实其干扰了终止酶介导的DNA加工过程。[2]

简而言之，将5×103 AD169感染的NHDF细胞/孔接种到30个96孔微量滴定板的孔中。允许感染在50 nM AIC246 (10×EC50)的暴露下进行，直到在一个或多个化合物处理的孔中出现CPE（表明抗性病毒突破）。未感染和未处理的细胞作为每个板上的对照。在存在 50 nM AIC246 的情况下，通过无细胞上清病毒的传代，培养物达到最大 CPE，从而完成突变病毒扩增。所得AIC246抗性子代病毒突变体通过在AIC246存在下有限稀释进行噬菌斑纯化3次。通过在没有选择压力的情况下连续传代空斑纯化的病毒（8至10次）来测试耐药性的稳定性。

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莱特莫韦的主要抗病毒活性使用标准的HCMV斑块减少实验进行评估。简而言之，在化合物系列稀释液存在下，用病毒分离株感染易感细胞单层。孵育一段时间后，对斑块进行显影（例如通过染色）并计数，以确定使斑块形成减少50%的药物浓度（EC50）。
对于一些HCMV变异株和其他病毒，采用了基于细胞病变效应（CPE）的实验或其他特定的基于细胞培养的复制实验。这些方法测量了化合物抑制病毒诱导的细胞死亡或其他病毒特异性复制标志物（例如，来自重组病毒的GFP表达、病毒抗原产生）的能力。
使用细胞活力测定（例如alamarBlue）和显微镜评估平行进行细胞毒性评估，以确保观察到的抗病毒效应并非由一般细胞毒性引起。[1]

10 mL/kg.; oral
Mice

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The in vivo antiviral activity was assessed using a mouse xenograft model. Immunodeficient mice were used. Gelfoam sponges were seeded with HCMV (strain Davis)-infected human fibroblasts and implanted subcutaneously in the dorsoscapular area.
Letermovir (AIC246) and the control drug valganciclovir (VGCV) were formulated in 2% dimethyl sulfoxide in 0.5% methylcellulose–99.5% phosphate-buffered saline.
Starting 4 hours after transplantation, mice were treated once daily via oral gavage for nine consecutive days. The administration volume was 10 ml/kg. Doses of Letermovir tested were 1, 3, 10, 30, and 100 mg/kg/day.
After 9 days of treatment, mice were sacrificed, implants were removed and digested with collagenase to recover human cells. Virus titers in the cell suspensions were determined by plaque assay. [3]

Absorption, Distribution and Excretion
In healthy subjects, the bioavailability of letermovir was 94% when not used in combination with cyclosporine; in hematopoietic stem cell transplantation (HSCT) recipients, the bioavailability was 35% when not used in combination with cyclosporine; and in HSCT recipients, the bioavailability was 85% when used in combination with cyclosporine. The time to peak concentration (Tmax) of letermovir ranged from 45 minutes to 2.25 hours. The time required to reach steady-state plasma concentrations was 9–10 days. Co-administration with food increased Cmax by a mean of 129.82% (range 104.35%–161.50%). No significant effect on AUC was observed. Letermovir is absorbed in the liver via the OATP1B1/3 transporter. 93% of the drug is excreted in the feces, of which 70% is the unchanged drug. <2% is excreted in the urine. The mean steady-state volume of distribution is 45.5 L.
The mean clearance rate in healthy subjects was 11.25 L/h.

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Metabolism/Metabolites
Letermovir is primarily metabolized in small amounts via UGT1A1/1A3.

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Biological Half-Life
After a once-daily intravenous injection of 480 mg of letermovir, a mean terminal half-life of 12 hours was observed.

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The paper mentions that letermovir showed favorable pharmacokinetic characteristics in a phase I clinical trial, but this in vitro study did not provide specific ADME/PK parameters (e.g., half-life, Cmax, AUC, bioavailability). [1]

Hepatotoxicity
In large pre-registration clinical trials, following hematopoietic stem cell transplantation, elevated ALT levels occurred in 18.5% of patients in the letermovir group and 21.9% in the placebo group. Of these, 3.5% had ALT levels exceeding five times the upper limit of normal, compared to only 1.6% in the placebo group. ALT elevations are typically transient, mild, and asymptomatic. There have been reports of recurrent elevated serum ALT levels after letermovir re-administration. In premarketing studies, jaundice and liver injury occurred in 0.5% of subjects; however, in the case of hematopoietic stem cell transplantation, all cases had other more likely causes of liver injury that could not be convincingly attributed to letermovir treatment. Since letermovir's approval, no clinically manifested cases of jaundice-related liver injury have been reported; however, overall clinical experience with letermovir treatment is limited. Probability Score: E (Unlikely to cause clinically significant liver injury).

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Effects during pregnancy and lactation
◉ Overview of use during lactation
There is currently no information on the use of letermovir during lactation. Because letermovir binds to plasma proteins at a rate of up to 99%, its concentration in breast milk may be very low. However, especially in breastfed newborns or preterm infants, other medications may be preferred.
◉ Effects on breastfed infants
No relevant published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
In vitro observations showed that letermovir binds to plasma proteins at a rate of up to 99% at concentrations of 0.2–50 mg/L.

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The paper states that letermovir showed good safety in a Phase I clinical trial. In the described in vitro experiments, cytotoxicity assessments showed no toxicity observed at the tested concentrations. [1]

[1]. Antimicrob Agents Chemother. 2012 Feb;56(2):1135-7.

[2]. J Virol. 2011 Oct;85(20):10884-93.

[3]. Antimicrob Agents Chemother. 2010 Mar;54(3):1290-7.

[4]. Antimicrob Agents Chemother. 2015;59(6):3140-8.

On November 8, 2017, letermovir was approved by the U.S. Food and Drug Administration (FDA) for the prevention of cytomegalovirus (CMV) infection in patients who have undergone allogeneic hematopoietic stem cell transplantation. It is the first novel CMV anti-infective drug classified as a DNA terminal transferase complex inhibitor. Letermovir has received FDA priority and orphan drug designation. Currently, it is marketed under the brand name Prevymis. Letermovir is a cytomegalovirus DNA terminal transferase complex inhibitor. Its mechanism of action is as an inhibitor of DNA terminal transferase complexes, cytochrome P450 3A, organic anion transport peptide 1B1, organic anion transport peptide 1B3, cytochrome P450 2C8, cytochrome P450 2C9, and cytochrome P450 2C19. Letermovir is an antiviral drug that targets the DNA terminal transferase complex of cytomegalovirus (CMV) to prevent CMV reactivation in immunocompromised patients. Mild to moderate elevations in serum transaminases may occur during literovir treatment, but no clinically significant cases of acute liver injury have been observed. Litermovir is a highly bioavailable, oral non-nucleoside analogue belonging to the 3,4-dihydroquinazoline acetate class. It is an inhibitor of the pUL56 subunit of the cytomegalovirus (CMV) viral terminal enzyme complex and possesses potential CMV-specific antiviral activity. After oral administration, literovir binds to the pUL56 subunit of the CMV viral terminal enzyme complex, preventing the tandem DNA from cleaving into monoclonal DNA of genome length. Because this drug interferes with viral DNA processing and subsequent viral DNA packaging into the procapsid, it blocks CMV replication, thereby preventing CMV infection. Drug Indications: Litermovir is indicated for the prevention of CMV infection and disease in CMV-seropositive adult allogeneic hematopoietic stem cell transplantation (HSCT) recipients. It is also indicated for the prevention of CMV disease in at-risk adult kidney transplant recipients (i.e., donor CMV seropositive/recipient CMV seronegative).
FDA Label
Previmix is indicated for the prevention of cytomegalovirus (CMV) reactivation and disease in CMV seropositive [R+] adult allogeneic hematopoietic stem cell transplant (HSCT) recipients. Antiviral medications should be used correctly according to official guidelines.
Prevention of Cytomegalovirus Infection

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Mechanism of Action
Cytomegalovirus (CMV) relies on a DNA terminal enzyme complex composed of multiple subunits (pUL51, pUL56, and pUL89) to process viral DNA. Viral DNA is produced as single-stranded repeat sequences, which are then cleaved by the DNA terminal enzyme complex into individual viral genomes, which can subsequently be packaged into mature viral particles. Letemovir inhibits the activity of this complex, thereby preventing the production of mature viral genomes and the formation of active viral particles. The exact mechanism by which letemovir binds to this complex is currently unknown. Initially, resistance mutations observed in pUL56 suggested that this subunit was the binding site for letemovir. However, resistance mutations have now been observed in pUL51, pUL56, and pUL89. A change in the amino acid sequence of one subunit could lead to a conformational change in the interacting subunit, thus affecting letemovir binding; or letemovir might interact with multiple subunits of the complex, but no evidence has yet been found to support either possibility. pUL89 is known to contain endonuclease activity of the complex, but because all members of the complex are essential for targeting and preventing proteasome degradation, it is difficult to determine whether letemovir directly inhibits the activity of pUL89.

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Pharmacodynamics
Letemovir inhibits the activity of the cytomegalovirus (CMV) DNA terminal enzyme complex, thereby preventing the viral DNA from being cleaved into a mature-length genome, thus preventing its packaging into viral particles. The EC50 value of letemovir for the DNA terminal enzyme complex is 2.1 nM. Human cytomegalovirus (HCMV) is a clinically significant herpesvirus that can cause complications in immunocompromised individuals. Current treatments (ganciclovir, valganciclovir, foscarnet, cidofovir) target viral DNA polymerases, but have limitations such as toxicity, low oral bioavailability, and drug resistance. Letermovir (AIC246) is a novel 3,4-dihydroquinazoline compound currently in clinical development with a unique mechanism of action that targets the viral terminal enzyme complex, for which there is no corresponding human counterpart. This unique mechanism enables it to combat HCMV strains resistant to existing standard treatments, as demonstrated in this study and in reported clinical cases of multidrug-resistant HCMV disease. The high selectivity of letermovir against HCMV relative to other herpesviruses and unrelated human pathogens suggests good safety and tolerability, and may address the limitations of existing therapies. [1]

C29H28F4N4O4

572.56

572.204

C, 60.84; H, 4.93; F, 13.27; N, 9.79; O, 11.18

917389-32-3

45138674

Solid powder

1.4±0.1 g/cm3

706.5±70.0 °C at 760 mmHg

381.1±35.7 °C

0.0±2.4 mmHg at 25°C

1.601

3.47

77.84

1

10

7

41

931

1

C([C@H]1C2C=CC=C(C=2N=C(N2CCN(C3C=CC=C(OC)C=3)CC2)N1C1C=C(C(F)(F)F)C=CC=1OC)F)C(=O)O

FWYSMLBETOMXAG-QHCPKHFHSA-N

InChI=1S/C29H28F4N4O4/c1-40-20-6-3-5-19(16-20)35-11-13-36(14-12-35)28-34-27-21(7-4-8-22(27)30)23(17-26(38)39)37(28)24-15-18(29(31,32)33)9-10-25(24)41-2/h3-10,15-16,23H,11-14,17H2,1-2H3,(H,38,39)/t23-/m0/s1

(S)-2-(8-fluoro-3-(2-methoxy-5-(trifluoromethyl)phenyl)-2-(4-(3-methoxyphenyl)piperazin-1-yl)-3,4-dihydroquinazolin-4-yl)acetic acid

MK-8828; MK 8828; MK8828; AIC-246; AIC 246; AIC246; Letermovir; Prevymis

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 ( ~174.65 mM )
Ethanol : ~100 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (4.37 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 25.0 mg/mL澄清DMSO储备液加入到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: ≥ 2.5 mg/mL (4.37 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 3 中的溶解度: ≥ 2.5 mg/mL (4.37 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 25.0 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: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (4.37 mM)

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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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