A-803467

Nav1.8 sodium channel (IC50 = 8 nM)

A-803467 以选择性且显着的方式逆转 ABCG2 介导的多药耐药性。在用 ABCG2 转染的细胞系中，A-803467 (7.5 μM) 显着增强了米托蒽醌和拓扑替康的细胞毒性。 ABCG2 转染细胞中 MX 的积累。在不同的时间间隔，A-803467（7.5 μM；0~120 分钟）显着抑制 ABCG2 转染细胞的细胞内 [3H]-MX 流出。 A-803467 增加 ABCG2 的 ATP 酶活性[1]。

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在这项研究中，研究人员研究了河豚毒素抗性钠通道阻断剂A-803467对ABCG2过表达药物选择和转染细胞系的影响。我们发现，在无毒浓度下，A-803467可以显著增加过表达野生型或突变型ABCG2的耐药细胞对ABCG2底物的细胞敏感性。机制研究表明，A-803467（7.5μM）通过抑制ABCG2的转运活性显著增加了[（3）H]-米托蒽醌的细胞内积累，而不改变其表达水平。此外，A-803467刺激了ABCG2过表达的膜中的ATP酶活性。[1]

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在这项研究中，研究人员在这里报告了A-803467的发现，这是一种钠通道阻滞剂，可以有效地阻断河豚毒素抗性电流（IC（50）=140 nM），并在体外阻断大鼠背根神经节神经元自发和电诱发动作电位的产生。在重组细胞系中，A-803467有效地阻断了人Na（v）1.8（IC（50）=8 nM），并且与人Na（v）1.2、Na（v”1.3、Na（W）1.5和Na（v“1.7相比具有>100倍的选择性（IC（50中）值>或=1微M）。

在雄性 NCR 裸鼠中，A-803467（35 mg/kg；口服）没有表现出明显的毒性[1]。当 A-803467 和拓扑替康联合使用时，植入过表达 ABCG2 的 H460/MX20 细胞的小鼠体内肿瘤的生长大大减少。 -803467 可以恢复过度表达 ABCG2 转运蛋白的癌症对托泊替康的敏感性，但缺乏 ABCG2 表达的肿瘤不会受到显着影响[1]。

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在小鼠模型系统中，a-803467（35mg/kg）和拓扑替康（3mg/kg）的联合治疗显著抑制了移植有过表达ABCG2-的癌症细胞的小鼠的肿瘤生长。我们的研究结果表明，a-803467和ABCG2底物的组合可能是ABCG2阳性耐药性癌症的一种新的治疗方法。[1]

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A-803467（20mg/kg，静脉注射）阻断了大鼠脊髓背角中宽动态范围神经元的机械诱发放电。A-803467在多种大鼠疼痛模型中也剂量依赖性地减少了机械性异常性疼痛，包括：脊髓神经结扎（ED（50）=47mg/kg，i.p.）、坐骨神经损伤（ED（50中）=85mg/kg，i.p.，辣椒素诱导的继发性机械性异常疼痛（ED（50%）约为100mg/kg，i.p。A-803467对福尔马林诱导的伤害性疼痛和急性热痛和术后疼痛无效。这些数据表明，在神经性和炎性疼痛的动物模型中，体内对Na（v）1.8钠通道的急性和选择性药理学阻断会产生显著的镇痛作用[2]。

BCG2 ATP酶测定[1]
如前所述，测量了High Five昆虫细胞膜囊泡中ABCG2的Vi敏感性ATP酶活性。将膜囊泡（100μg蛋白质/ml）在37°C下在含或不含0.3 mM钒酸盐的ATP酶测定缓冲液中孵育5分钟，然后在37°℃下与不同浓度的0至80μM的A-803467、拓扑替康和MX（0-30μM）孵育3分钟。通过添加5 mM Mg ATP诱导ATP酶反应，总体积为0.1 ml。在37°C下孵育20分钟后，通过装载0.1 ml 5%SDS溶液停止反应。如前所述测量释放的无机磷酸盐（Pi）。

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药物选择性测定。[2]
在测定中评估了A-803467（10μM）的活性，以评估其相对于其他细胞表面受体、离子通道、转运位点和酶（包括阿片受体和环氧化酶1和2）的药理学选择性，使用所述的标准化测定方案（CEREP和内部测定）。

细胞活力测定[1]
如前所述，使用MTT比色法进行细胞毒性试验和逆转实验。对于HEK293/pcDNA3.1、HEK/ABCB1、HEK/AABCC10、HEK293/R482、HEK293/L482G和HEK293/R482T细胞，收获细胞并以6×103个细胞/孔的终浓度重新悬浮，对于H460和H460/MX20细胞，以4×103个电池/孔的最终浓度重新悬浮。将细胞均匀接种到96孔板中。为了测定A-803467的细胞毒性，在孵育24小时后，将不同浓度的药物加入每个孔中。为了确定A-803467的逆转能力，在与A-803467、FTC、维拉帕米或头孢菌素预孵育2小时后，将不同浓度的化疗药物加入指定孔中。药物孵育68小时后，加入MTT试剂（4mg/mL）。将平板再孵育4小时，丢弃上清液，加入100μl DMSO以溶解甲氮晶体。在570nm波长下测量细胞活力。所有实验重复至少3次，并计算平均值和标准偏差（SD）值。

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[3H]-MX积聚和流出试验[1]
如前所述，研究人员研究了A-803467对ABCG2过表达细胞中[3H]-MX的细胞内积累和外排的影响。简而言之，在37°C下，在有或没有A-803467（7.5μM）或FTC（5μM）的情况下，将细胞（5×106/细胞）重新悬浮并在RPMI 1640培养基中孵育2小时。然后将细胞与含有0.01μM[3H]-MX的培养基在37°℃下再孵育2个小时，有或没有A-503467（7.5µM）或FCC（5μM），随后用冰冷的PBS洗涤两次。对于累积试验，用10 mM裂解缓冲液（pH 7.4，含有1%Triton X-100和0.2%SDS）裂解细胞。然后放入闪烁液中。对于外排试验，然后将悬浮细胞在37°C的无[3H]-MX培养基中培养，培养基中有或没有A-803467（7.5μM）或FTC（5.0μM）。在指定时间（0、30、60和120分钟）收获细胞的等分试样，然后用冰冷的PBS洗涤并转移到相应的闪烁瓶中。使用Packard TRI-CARB1 190`A液体闪烁分析仪测量放射性。

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蛋白质印迹分析[1]
细胞裂解物如前所述制备。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）分离等量的总细胞裂解物（30μg蛋白质），并将其电泳转移到聚偏二氟乙烯（PVDF）膜上。在室温下在封闭溶液（5%牛奶）中孵育1小时后，用1:1000稀释的抗肌动蛋白一级单克隆抗体或1:500稀释的ABCG2在4°C下对膜进行免疫印迹过夜，然后在室温下用辣根过氧化物酶（HRP）偶联的二级抗体（1:1000稀释）进一步孵育2小时。通过增强化学发光检测系统检测蛋白质-抗体复合物。

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免疫荧光分析[1]
为了进行免疫荧光分析，将H460和H460/MX20细胞接种在24孔板中。细胞与或不与A-803467（7.5μM）一起孵育72小时。此后，用PBS洗涤细胞，在室温下用4%多聚甲醛固定15分钟，然后用PBS冲洗三次，然后在4°C下用1%曲拉通X-100渗透10分钟。再次用PBS洗涤细胞三次，然后在37°C下用2mg/ml BSA封闭1小时。将固定细胞与抗ABCG2单克隆抗体（BXP 21）（1:50）在4°C下孵育16小时，然后用PBS洗涤三次。然后将细胞与Alexa面粉488山羊抗小鼠IgG（1:60）在37°C下进一步孵育1小时。DAPI用于核复染。免疫荧光图像用尼康荧光显微镜拍摄。

Animal/Disease Models: Nude mice[1]
Doses: 35 mg/kg
Route of Administration: Po
Experimental Results: demonstrated no noticeable toxicity in the male NCR nude mice.

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Nude mouse MDR xenograft models [1]
The ABCG2-overexpressing NSCLC cell H460/MX20 xenograft mouse models were established as previously explained [41]. H460/MX20 cells (6 × 106) and H460 cells (4 × 106) were injected subcutaneously under the right and left armpit regions of the nude mice, respectively. We performed a pilot study using three different doses of A-803467 (17.5, 35 and 70 mg/kg) and we found that 35 mg/kg dose was effective in increasing the topotecan sensitivity in tumors without significantly increase toxicity, therefore 35 mg/kg dose was used throughout the following study. [1]
The mice were randomized into 4 groups (n = 6) when the tumors attained a mean diameter of 0.5 cm (day 0), and then received treatments as follows: (a) Vehicle (10% N-methyl pyrrolidine (NMP) in PEG-300, p.o., every 2nd and 3rd day; total 12 times), (b) A-803467 diluted in 10% NMP in PEG-300 (35 mg/kg, p.o., every 2nd and 3rd day; total 12 times), (c) Topotecan (3.0 mg/kg, i.p., every 3rd day; total 6 times), and (d) A-803467 (35 mg/kg, every 2nd and 3rd day; total 12 times, given 1 h before topotecan) + topotecan (3.0 mg/kg, i.p., every 3rd day: total 6 times). The body weights of the mice were monitored and the two perpendicular diameters of tumors (A and B) were recorded every 4th day, and tumor volumes (V) were calculated according to the following formula described previously

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A-803467 was dissolved in 5% DMSO/95% polyethylene glycol (PEG 400) for i.p. administration in a 2 ml/kg injection volume. The compound was injected 30 min before behavioral testing.
Spinal Dorsal Horn Neuronal Electrophysiology. [2]
Electrophysiological recording of spinal dorsal horn neurons was conducted as described. Briefly, neuropathic rats (L5–L6 spinal nerve ligation) were anesthetized with pentobarbital (50 mg/kg, i.p.), and catheters were placed in the left and right external jugular veins. A laminectomy was performed to remove vertebral segments T12-L3. The animals were then secured in a stereotaxic frame. Anesthesia was maintained for the duration of the experiment by a continuous infusion of propofol (8–12 mg/kg/hr, i.v.). Body temperature was kept at 37°C by placing the animals on a circulating water blanket. Platinum-plated stainless steel microelectrodes were used to record the activity of spinal wide dynamic range (WDR) neurons. Spike waveforms were monitored, digitized (32 points), and stored for offline analysis. Baseline spontaneous activity was recorded for 5 min before stimulation. Rats were stimulated three times (5 min apart) before drug administration with a von Frey hair (10 g) applied to the neuronal receptive field for 15 s located on the ipsilateral hindpaw. The mean of three stimulations represented baseline evoked activity. A-803467 (20 mg/kg, i.v.) or vehicle was infused over a 5- to 7-min period, and the von Frey hair was reapplied 35 min after this infusion. For comparison to baseline firing levels, statistical significance was established by using Wilcoxon's matched-pairs test. [2]
Pharmacological Selectivity Assays. [2]
The activity of A-803467 (10 μM) was evaluated in assays to assess pharmacological selectivity relative to other cell-surface receptors, ion channels, transport sites, and enzymes including the opioid receptors, and cycloxygenases 1 and 2, by use of standardized assay protocols (CEREP and in-house assays) as described.

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Analgesia and Side-Effect Assays. [2]
A-803467 was evaluated in well characterized in vivo models to assess acute, inflammatory, and neuropathic pain. The specific methodologies for these nociceptive assays, models of postoperative and visceral pain, and models of motor performance are described in the SI. Unless otherwise noted, all experimental and control groups contained at least six animals per group, and data are expressed as mean ± SEM. Data analysis was conducted by using analysis of variance and appropriate post hoc comparisons (P < 0.05). ED50 values were estimated by using least-squares linear regression.

[1]. A-803467, a tetrodotoxin-resistant sodium channel blocker, modulates ABCG2-mediated MDR in vitro and in vivo. Oncotarget. 2015;6(36):39276-39291.

[2]. A-803467, a potent and selective Nav1.8 sodium channel blocker, attenuates neuropathic and inflammatory pain in the rat. Proc Natl Acad Sci U S A. 2007;104(20):8520-8525.

To further understand the interaction of A-803467 with ABCG2, we performed an ATPase assay using ABCG2 overexpressed membranes. The majority of TKIs that interact with the ABC drug transporters stimulate ATP hydrolysis and the fact that A-803467 stimulates the ATP hydrolysis of ABCG2 in a concentration dependent manner (Fig. 3A) indicates that it behaves similar to other known substrates (Fig. 3B and 3C) of ABCG2 transporter, such as MX and topotecan. These results further prove that A-803467 not only interacts directly with the ABCG2 transporter, but may also be a competitive inhibitor of the transporter.
To identify the molecular interaction of A-803467 with the ABCG2 transporter, docking simulation was performed at various sites of the human ABCG2 homology model. The crystal structure of human ABCG2 transporter is not completely elucidated. Comparing the docking scores shown in Table 4, the most favorable binding site was identified as site-1. Molecular docking of topotecan, a well-known ABCG2 substrate, at the same site of ABCG2 was performed. The docking score of topotecan (−5.57 kcal/mol) is much higher than that of A-803467 (−8.07 kcal/mol). The lower docking score indicates stronger interaction between A-803467 to ABCG2 (Fig. 4). Moreover, molecular structure of A-803467 also exhibited the pharmacophoric features such as hydrophobic groups, aromatic ring centers (phenyl ring and furan ring) and hydrogen bond acceptors that have been reported as essential for ABCG2 inhibition. Overall, this molecular simulation will provide clues to optimize further derivatives of ABCG2 inhibitors. [1]

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Although lacking analgesic effects on acute thermal pain in normal rats, A-803467 significantly decreased acute mechanical nociception. This effect is in agreement with previous knockout and antisense data demonstrating that knockdown of Nav1.8 was associated with decreased acute mechanical nociception. More than 50% of C fibers and 10% of A fibers express Nav1.8 channels. The Nav1.8-positive C fibers are NGF- or GDNF-responsive, and many of these fibers also express TRPV1, supporting a role in acute thermal nociception. However, the expression of Nav1.8 channels on A-fibers and the data showing that disruption of Nav1.8 function reduces acute mechanical nociception provide evidence that activation of Nav1.8 channels contributes to normal sensitivity to noxious mechanical stimulation.
In summary, the present data demonstrate that A-803467 is a potent and highly selective blocker of Nav1.8 channels and that this compound effectively blocks sensory neuron excitability in vitro and in vivo. Evaluation of A-803467 in a wide range of animal pain models demonstrates that selective blockade of Nav1.8 channels in vivo results in a significant reduction in nociceptive sensitivity after nerve injury and inflammation. [2]

C19H16CLNO4

357.79

357.076

C, 63.78; H, 4.51; Cl, 9.91; N, 3.91; O, 17.89

944261-79-4

16038374

White to off-white solid powder

1.3±0.1 g/cm3

450.6±45.0 °C at 760 mmHg

128-130?C

226.3±28.7 °C

0.0±1.1 mmHg at 25°C

1.611

4.93

60.7

1

4

5

25

429

0

O=C(C1=CC=C(C2=CC=C(Cl)C=C2)O1)NC3=CC(OC)=CC(OC)=C3

VHKBTPQDHDSBSP-UHFFFAOYSA-N

InChI=1S/C19H16ClNO4/c1-23-15-9-14(10-16(11-15)24-2)21-19(22)18-8-7-17(25-18)12-3-5-13(20)6-4-12/h3-11H,1-2H3,(H,21,22)

5-(4-chlorophenyl)-N-(3,5-dimethoxyphenyl)furan-2-carboxamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.99 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 (6.99 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 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 3 中的溶解度: 30% PEG400+0.5% Tween80+5% Propylene glycol : 30 mg/mL

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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。