P-gp (Kd = 5.1 nM)[1]

在 CHrB30 细胞中，tariquidar (XR9576) 将 [3H]-长春花碱和 [3H]-紫杉醇的稳态积累提高至非 P-gp 表达 AuxB1 细胞中观察到的水平 (EC50=487±50 nM)，证明其作为 P-gp 介导的 [3H]-蛋氨酸转运调节剂的效力。 [3H]-Tariquidar 对 CHrB30 膜具有最强的亲和力（Kd=5.1±0.9 nM，n=7），最大结合容量（Bmax）为 275±15 pmol/mg 膜蛋白。与亲代细胞系不同，调节剂 Tariquidar (EC50=487±50 nM) 以剂量依赖性方式增加 [3H]-Vinblastine 的积累。 MDR 调节剂 Tariquidar 的 IC50 值为 43±9 nM，可抑制钒酸盐敏感性 ATP 酶 60-70% 的活性[1]。 Tariquidar (XR9576) 增强多种药物的细胞毒性，包括长春新碱、依托泊苷、紫杉醇和阿霉素； 25–80 nM XR9576 完全逆转耐药性。 [3H]叠氮平对 P-gp 的强光亲和标记被 tariquidar 抑制，表明与蛋白质有直接相互作用[2]。

此外，Tariquidar（6-12 mg/kg po）联合给药可完全恢复紫杉醇、依托泊苷和长春新碱对裸鼠体内两种高度耐药的 MDR 人类肿瘤异种移植物（2780AD、H69/LX4）的抗肿瘤活性。这些小鼠患有本质上耐药的 MC26 结肠肿瘤，与 Tariquidar (XR9576) 共同给药可增强多柔比星的抗肿瘤活性，而不会导致毒性显着增加。研究还发现，tariquidar 可显着提高阿霉素在体内对 sc MC26 肿瘤的抗癌功效[2]。

P-gp在CHrB30膜中的ATP水解活性[1]
先前描述的比色测定法用于测量ATP水解后无机磷酸盐的释放（Chifflet等人，1988）。膜（1 μg蛋白）与Na2ATP（2 mm），总测定体积为50 μl缓冲液，含（mm）：Tris pH 7.4 50，MgSO4 5，0.02%NaN3，NH4Cl 150用于20 最低37°C。ATP酶活性与40呈线性关系 最低37°C。调节剂（来自二甲基亚砜库存）和ATP酶抑制剂钒酸盐的添加浓度范围为10−9–10–5 m.最终DMSO浓度总是<1%，已知该水平不会改变ATP酶活性。药物对ATP酶活性的影响符合一般的剂量-反应关系（见上文）

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特异性药物与P-糖蛋白结合[1]
如前所述，使用快速过滤测定法测量[3H]-长春碱、[3H]-紫杉醇和[3H]-XR9576与CHrB30膜中P-gp的结合（Ferry等人，1992）。将膜与适当的放射性配体在总缓冲液体积为200的条件下孵育 μl（50 mm Tris pH 7.4），为期2-3年 h以达到平衡。洗涤缓冲液（3 ml）含有20 mm MgSO4，20 mm Tris（pH 7.4），并且样品在真空下通过过滤歧管中的单个GF/F过滤器过滤以分离结合的和游离的配体。进一步洗涤后（2×3 ml）通过液体闪烁计数测定结合配体的量。非特异性结合被定义为在存在至少100倍过量的竞争配体的情况下结合的[3H]-配体的量（如结果所示），并从所有值中减去
[3H]-配体结合的容量和亲和力的测定是通过饱和等温线分析实现的。将膜与增加浓度的标记药物和结合量（pmol mg−1）绘制为自由配体浓度的函数。

细胞培养[1]
中国仓鼠卵巢亲代（敏感）AuxB1和抗性CHrB30细胞如前所述在含有10%胎牛血清的α-最低必需培养基（α-MEM）中生长（Kartner等人，1983）。通过在秋水仙碱中逐步选择衍生自AuxB1细胞的CHrB30细胞（Kartner等人，1983）表达P-gp，并且通过在培养基中补充30 μg ml−1秋水仙碱

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质膜制备[1]
如前所述（Lever，1977），在使用氮空化和蔗糖密度离心收集破坏CHrB30细胞后制备质膜。最终的膜制剂储存在−70°C，蛋白质浓度为5-10 毫克 ml–1在含有0.25的缓冲液中 m蔗糖，10 mm Tris HCI（pH 7.5），并且包括蛋白酶抑制剂亮蛋白肽（0.1 毫克 ml−1），pepstatin A（0.1 毫克 ml−1）和苯甲脒（1 mm）

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稳态药物积累试验[1]
AuxB1和CHrB30细胞在12孔（24 mm）组织培养皿，并且如前所述测量[3H]-长春碱的稳态积累（Martin等人，1997）。通过添加0.1开始积累 μCi[3H]-长春碱和未标记的长春碱，最终浓度为100 nm。[3H]-紫杉醇的积累用0.1 μCi[3H]-紫杉醇和未标记药物，最终浓度为1 μm。将细胞在反应体积为1 60毫升 在37°C、5%CO2的条件下，以达到稳态。在10−9–10−6的浓度范围内，研究了调节剂XR9576和GF120918对[3H]-配体积累的影响 m.从DMSO原料中加入调节剂，得到0.2%（v v−1）的最终溶剂浓度。细胞采集后，通过液体闪烁计数测量积聚的药物，并对细胞蛋白质含量进行归一化。累积量作为调节剂浓度的函数的图符合一般的剂量反应方程（De Lean等人，1978）
使用几种浓度的放射性标记药物（1-300 nm）在1的存在和不存在下 μm GF120918，并在60 min周期，如上所述。

Dissolved in5% (w/v) D-( 1)-glucose (dextrose) solution; 8 mg/kg ; Coadministration of Tariquidar (p.o.) with doxorubicin (5 mg/kg, i.v.) Murine colon carcinoma xenografts MC26.

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Two different tariquidar formulations (A and B) were used, both at a dosage of 15 mg/kg, respectively. Formulation A was a solution and formulation B was a microemulsion which was previously shown to improve the oral bioavailability of the structurally related P-gp inhibitor elacridar in mice.[5]

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In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.[2]

[1]. The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein. Br J Pharmacol, 1999, 128(2), 403-411.

[2]. In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576. Cancer Res, 2001, 61(2), 749-758.

1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]-XR9576 with the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specific high-affinity binding to P-gp (Bmax = 275 pmol mg-1, Kd = 5.1 nM). The transport substrates [3H]-vinblastine and [3H]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action of XR9576 with P-gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. 2 The relative affinities of several modulators and transport substrates to interact with P-gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]-XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non-competitive interaction between XR9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [3H]-vinblastine and [3H]-paclitaxel transport as it increased the steady-state accumulation of these cytotoxics in CHrB30 cells to levels observed in non-P-gp-expressing AuxB1 cells (EC50 = 487+/-50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]-XR9576 accumulation was not influenced by P-gp expression or function. 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P-gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.[1]

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The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.[2]

C38H40CL2N4O6

719.653408050537

718.232

1992047-62-7

Tariquidar; 206873-63-4;625375-84-0 (mesylate);1992047-62-7 (2HCl); 625375-83-9 (methanesulfonate hydrate)

121513890

Solid powder

111Ų

4

8

11

50

1040

0

ZJCJRGRTDKIFJJ-UHFFFAOYSA-N

InChI=1S/C38H38N4O6.2ClH/c1-45-33-18-25-14-16-42(23-28(25)19-34(33)46-2)15-13-24-9-11-29(12-10-24)40-38(44)30-20-35(47-3)36(48-4)21-32(30)41-37(43)27-17-26-7-5-6-8-31(26)39-22-27;;/h5-12,17-22H,13-16,23H2,1-4H3,(H,40,44)(H,41,43);2*1H

N-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide;dihydrochloride

XR9576 dihydrochloride; D06008; XR 9576; D 06008; XR-9576;D-06008

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: > 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。