Brincidofovir (CMX001, HDP CDV, HDPCDV)   中文名称: 布罗福韦酯

Antiviral; DNA polymerase

CMX001是西多福韦的一种口服活性脂质缀合物，在体外对抗单纯疱疹病毒（HSV）复制的活性是阿昔洛韦或西多福韦的50倍。[2]
在21世纪，我们面临着天然或重组VARV和MPXV作为生物武器的潜在用途，以及人类MPXV的出现。这种情况需要抗病毒药物的治疗和预防干预。Cidovovir是一种抗病毒药物，被批准用于治疗艾滋病患者的巨细胞病毒视网膜炎，对痘病毒有活性，但必须通过静脉注射给药，并与肾毒性有关。CDV的醚脂类似物CMX001（HDP-CDV）对包括痘病毒在内的一系列DNA病毒具有强大的抗病毒活性，具有良好的口服生物利用度和最小的肾毒性。[1]

CMX001和CDV在通过鼻内途径或小颗粒气雾剂引入高剂量（10000×LD（50））后保护小鼠免于死亡方面同样有效。使用10mg/kg剂量的CMX001，然后每隔一天使用2.5mg/kg剂量，持续14天，提供了坚实的保护，防止在（100-200）x LD（50）的鼻疽病毒鼻腔内激发后死亡和体重减轻。此外，当延迟给药至感染后5天，即未经治疗的对照组死亡前3-4天时，实现了对死亡率的完全保护。这个治疗窗口相当于在普通天花的皮疹阶段进行干预。[1]
这些研究比较了CMX001与阿昔洛韦在鼻内接种1型或2型HSV的BALB/c小鼠中的疗效。CMX001使用每天一次口服5至1.25 mg/kg的剂量有效降低死亡率，即使在病毒接种后48-72小时延迟治疗时也是如此。从用CMX001处理的小鼠获得的器官样品的滴度为每克组织3-5 log（10）斑块形成单位，低于从用阿昔洛韦处理的小鼠中获得的样品，包括大脑的5个不同区域。口服（14）C-CMX001后，在小鼠的中枢神经系统中记录了与药物相关的放射性的可检测浓度。这些研究表明，CMX001可穿透血脑屏障，是传播性感染和中枢神经系统感染中HSV复制的有效抑制剂，并且优于阿昔洛韦[2]。

使用的HSV-1菌株是E-377，HSV-2菌株是MS。这些病毒的起源以前已经报道过。从新鲜获得的新生儿人类包皮制备HFF细胞作为原代培养物。在体外和体内使用的原代兔肾细胞中制备并定量病毒库。两种细胞系的培养基均为含有10%FBS和2μM L-谷氨酰胺、100单位/ml青霉素和25 mM庆大霉素的厄尔盐的MEM[2]。

BSC-1细胞（ATCC CCL 26）在含有10%胎儿克隆III、2mM l-谷氨酰胺、100U/ml青霉素和100μg/ml链霉素的Eagle最低必需培养基（MEM）中生长。在非洲绿猴肾细胞系BSC-1（Chen et al.，1992）中繁殖被命名为MOS-3P2的ECTV的MOS菌株（ATCC VR-1374）的斑块纯化分离物。如其他地方所述，通过蔗糖缓冲液纯化病毒（Moss和Earl，1998）。如前所述估计了病毒的传染性（Wallace和Buller，1985）。简言之，将病毒悬浮液在PBS+1%血清中连续稀释，在37°C下吸收到单层中1小时，并用DMEM+5%胎儿克隆III中的1%羧甲基纤维素悬浮液覆盖。在37°C下4天后，通过向每个孔中加入0.5 ml 0.3%结晶紫/10%福尔马林溶液，可见病毒斑块并灭活病毒[1]。

Antiviral compounds[1]
Solutions of CDV and CMX001 were prepared fresh prior to each experiment by dissolving the compounds in sterile, distilled water, and stored at 4 °C over the course of the experiment.
Aerosol challenge[1]
Mice were exposed to aerosolized ECTV suspended in MEM using a nose-only inhalation exposure system (NOIES) equipped with a 1-jet BioAerosol Nebulizing Generator, and operated within a class 2 biological safety cabinet. The NOIES was operated with a primary air pressure of 20 psi giving 1.5 l/min flow rate to the aerosol chamber (without secondary air), a virus suspension flow rate of 0.5 ml/min, and a system operating pressure of ∼−0.5 in vacuum relative to the outside atmospheric pressure. The quantity of virus delivered to the mice over the course of exposure was estimated by multiplying the concentration of virus in the aerosol (C A expressed in PFUs) by the total volume (V M) of air respired by a mouse of given body weight over the exposure time using Guyton's formula for minute volumes administered to rodents (Guyton, 1947). This presented virus dose is likely an upper limit as it assumes that the entire virus challenge was optimally aerosolized and completely taken up on inhalation.
Intra-nasal challenge[1]
Mice were anesthetised with 0.1 ml/10 g body weight of ketamine HCl (9 mg/ml) and xylazine (1 mg/ml) by intraperitoneal injections. Anesthetised mice were laid on their dorsal side with their bodies angled so that the anterior end was raised 45° from the surface, a plastic mouse holder was used to ensure conformity. ECTV was diluted in PBS to the required concentration and slowly loaded into each nare (5 μl/nare). Mice were subsequently left in situ for 2–3 min before being returned to their cages.
At indicated times following exposure to ECTV, groups of mice were treated by gavage with 0.1 ml sterile, distilled water (placebo) or water containing the desired concentration of CMX001. CDV was delivered by an intraperitoneal injection at the desired dose. This treatment was repeated as described throughout the results. To determine infectious viral titres, mice were sacrificed at 4, 6, and 8 days post-challenge, and lung, spleen, and liver tissues and nasal wash were isolated. Tissue was ground in PBS (10%, w/v), frozen and thawed three times, and sonicated for 20 s. Virus infectivity (PFU/ml) in tissue homogenates was estimated by titration on BSC-1 monolayers. Arithmetic means were calculated for PFU/ml values above the limit of detection (102 PFU/ml). Remaining mice were observed for clinical signs of disease (morbidity) and mortality. Moribund mice were euthanized.

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Antiviral compounds[2]
CMX001 was suspended in 0.4% carboxymethylcellulose (CMC) for oral delivery to mice. ACV was weighed and suspended in sterile water for oral treatment of mice. Compounds were prepared in a 0.2 ml volume which was administered once daily (CMX001) or twice daily (ACV). Treatments for efficacy evaluations were administered to mice for 7 consecutive days beginning 24-72 h post viral inoculation by oral gavage using doses of 5, 2.5 and 1.25 mg/kg of CMX001 administered once daily or doses of 120, 60 or 30 mg/kg of ACV given twice daily at approximately 12 h intervals. For pathogenesis experiments, treatments began 24 h post viral inoculation and were administered for 7 consecutive days using 5 mg/kg of CMX001 or 100 mg/kg of ACV. These doses were selected based upon the results obtained in the mortality experiments.
Experimental infections[2]
Mice were manually restrained for intranasal inoculations using a total volume of 0.04 ml/mouse containing an approximate LD90 of either HSV-1, E-377 or HSV-2, strain MS. For these studies, the inoculum contained 4.4 × 104 pfu/mouse for HSV-1 or 1.1 × 105 pfu/mouse for HSV-2. For mortality experiments, animals were evaluated at least once daily for 21 days and four times daily during peak occurrence of clinical neurological signs so that mice could be humanely euthanized prior to death. Pathogenesis studies were performed for both HSV-1 and HSV-2 in order to compare the effect of CMX001 and ACV on viral replication of both viral types in target organs of mice. Three mice each from vehicle- and drug-treated groups were euthanized on days 1, 3, 5, 7 or 10 post inoculation for collection of lung, liver, spleen, kidney, olfactory bulbs, cerebral cortex, pons/medulla, diencephalon, cerebellum and trigeminal ganglia. Organ samples were pooled by tissue type and homogenized in a 10% w/v suspension, and frozen until assayed for virus. Virus titers were determined by plating of tissue homogenates on HFF cells and plaques were enumerated after three days incubation. The trigeminal ganglia were collected individually and co-cultured directly on primary rabbit kidney (RK) cells with N’ N’ dimethylbisacetamide for detection of latent virus as described previously. Briefly, the ganglia were minced, placed onto tissue culture cell monolayers and monitored for viral cytopathic effects for 3 weeks. Ganglia were transferred weekly onto fresh RK cells with new media.

Absorption, Distribution and Excretion
The oral bioavailability of brincidofovir is 13.4% in its tablet formulation and 16.8% in its suspension formulation. Following oral administration, the Cmax and AUCtau of brincidofovir were 480 ng/mL and 3400 ng·hr/mL, respectively. The Cmax and AUCtau of the active metabolite, cidofovir diphosphate, were 9.7 pg/106 cells and 1200 pg·hr/106 cells, respectively. Maximum plasma concentrations (Tmax) of brincidofovir are reached at approximately 3 hours post-administration, while maximal plasma concentrations for cidofovir diphosphate are reached at approximately 47 hours post-administration.
Brincidofovir is eliminated as metabolites in both the urine (~51%) and feces (~40%).
The apparent volume of distribution of brincidofovir is 1230 L.
The apparent clearance of brincidofovir in healthy adult patients is 44.1 L/h.

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Metabolism / Metabolites
Brincidofovir is a pro-drug of [cidofovir] and as such must undergo some basic metabolic reactions to become pharmacologically active. Upon entering the target cell, the phosphodiester bond of brincidofovir is hydrolyzed to generate cidofovir, which is then phosphorylated to generate the active agent: cidofovir diphosphate. The specific enzyme(s) responsible for this reaction have not been elucidated, but _in vitro_ findings suggest sphingomyelin phosphodiesterase plays a major role in the initial hydrolysis of brincidofovir. There are two major inactive metabolites of brincidofovir, CMX103 and CMX064, which are generated via carboxylation of the terminal carbon followed by several cycles of CYP-mediated oxidative reactions and fatty acid oxidation. These reactions are mediated, at least in part, by CYP4F2.

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Biological Half-Life
The mean terminal half-lives of brincidofovir and its pharmacologically active metabolite, cidofovir diphosphate, are 19.3 hours and 113 hours, respectively.

Hepatotoxicity
In preregistration clinical trials of brincidofovir as prevention of cytomegalovirus and adenovirus infection in adults and children after hematopoietic stem cell transplantation (HSCT), serum aminotransferase elevations were more frequent with brincidofovir (22%) than placebo (16%) treatment, the usual rate of hepatic enzyme elevations being high after HSCT. ALT elevations above 5 times the upper limit of normal (ULN) arose in 1.8% vs 1.4% , and one of 269 brincidofovir recipients discontinued therapy because of ALT elevations above 10 times ULN. The ALT and AST elevations arose early as brincidofovir was given only 2 or 3 times, on days 1 and 8 and in some trials day 21 of treatment. In the same trials mild-to-moderate elevations in serum bilirubin arose in 6.5% of those on brincidofovir vs 5.9% of placebo recipients, again reflecting the high rate of bilirubin elevations in patients after HSCT. In small studies in healthy human volunteers abnormalities of liver tests were not observed. Since approval of brincidofovir, there have been no instances of smallpox infection that qualified for its use. However, the emergence of mpox infection shortly after its approval has led to its use for this non-approved indication. Aminotransferase elevations were frequent in the mpox virus infected subjects treated with brincidofovir but were invariably asymptomatic, transient and without symptoms or concurrent bilirubin elevations. Thus, the total clinical experience with use of brincidofovir is limited. While transient serum aminotransferase elevations have been reported with its use, there have been no reports of clinically apparent liver injury linked to brincidofovir therapy.
Likelihood score: D (possible cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the use of brincidofovir during breastfeeding. Individuals with smallpox are recommended not to breastfeed their infant because of the risk of passing variola virus to the infant through direct contact. This precaution probably applies to monkeypox, also. Providing pumped milk to the infant may be possible if no lesions are near the breast and adequate precautions are taken with respect to cleaning hands, breasts, breast pumps and any other apparatuses used to provide milk to the infant. Until more safety data become available, an alternate drug may be preferred.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Brincidofovir is >99% protein-bound in plasma, although the specific protein(s) to which it binds have not been elucidated.

[1].Efficacy of therapeutic intervention with an oral ether-lipid\nanalogue of cidofovir (CMX001) in a lethal mousepox model. Antiviral\nRes. 2008 Jan;77(1):39-49.

[2]. Efficacy of CMX001 against herpes simplex virus infections\nin mice and correlations with drug distributionstudies. J Infect Dis.\n2010 Nov 15;202(10):1492-9.

[3]. CMX001 (1-O-hexadecyloxypropyl-cidofovir) inhibits\npolyomavirus JC replication in human brain progenitor-derived\nastrocytes. Antimicrob Agents Chemother. 2011 May;55(5):2129-36.

[4]. CMX001 to prevent cytomegalovirus disease in\nhematopoietic-cell transplantation. N Engl J Med. 2013 Sep\n26;369(13):1227-36

[5].The lipid moiety of brincidofovir is required for in vitro antiviral activity against Ebola virus. Antiviral Res. 2016 Jan;125:71-8.

Pharmacodynamics
The pharmacologically active agent resulting from brincidofovir metabolism, cidofovir diphosphate, has an exceedingly long duration of action that allows for it to be dosed once weekly. The entirety of a brincidofovir smallpox treatment consists of only two doses, on days 1 and 8, which seemingly reduces the risk of adverse reactions. Regimens involving a longer duration of administration (i.e. more than a single dose on days 1 and 8) have been shown to increase mortality compared to placebo and should therefore be avoided. Brincidofovir is considered a potential human carcinogen and has demonstrated the potential to cause infertility - as such, its use should be restricted to situations in which it is absolutely necessary.

C27H52N3O7P

561.69148

561.354

C, 57.73; H, 9.33; N, 7.48; O, 19.94; P, 5.51

444805-28-1

483477

Solid powder

5.592

156.93

3

7

26

38

721

1

NC1=NC(N(C[C@@H](CO)OCP(O)(OCCCOCCCCCCCCCCCCCCCC)=O)C=C1)=O

WXJFKKQWPMNTIM-VWLOTQADSA-N

InChI=1S/C27H52N3O7P/c1-2-3-4-5-6-7-8-9-10-11-12-13-14-15-19-35-20-16-21-37-38(33,34)24-36-25(23-31)22-30-18-17-26(28)29-27(30)32/h17-18,25,31H,2-16,19-24H2,1H3,(H,33,34)(H2,28,29,32)/t25-/m0/s1

3-(hexadecyloxy)propyl hydrogen ((((S)-1-(4-amino-2-oxopyrimidin-1(2H)-yl)-3-hydroxypropan-2-yl)oxy)methyl)phosphonate

CMX001; BCV; CMX 001; HDP-CDV; CMX-001; HDPCDV

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)

Typically soluble in DMSO (e.g. > 10 mM)

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。