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

Tariquidar methanesulfonate, hydrate (XR9576 methanesulfonate, hydrate) raises the steady-state accumulation of these cytotoxics in CHrB30 cells to levels seen in non-P-gp-expressing AuxB1 cells (EC50=487±50 nM), making it a potent modulator of P-gp mediated [3H]-Vinblastine and [3H]-Paclitaxel transport. [3H]-Tariquidar has the strongest affinity (Kd=5.1±0.9 nM, n=7) and the maximum binding capacity (Bmax) of 275±15 pmol/mg membrane protein when it comes to CHrB30 membranes. Unlike the ancestral cell line, the modulators XR9576 (EC50=487±50 nM) increase the accumulation of [3H]-Vinblastine in a dose-dependent manner. With a strong IC50 value of 43±9 nM, the MDR modulator Tariquidar can inhibit 60–70% of the activity of the vanadate-sensitive ATPase[1]. Tariquidar (XR9576) enhances the cytotoxicity of several medications, including as Vincristine, Etoposide, Paclitaxel, and Doxorubicin; in the presence of 25–80 nM Tariquidar, resistance is completely reversed. Strong photoaffinity labeling of P-gp by [3H]Azidopine is inhibited by tariquidar, suggesting a direct interaction with the protein[2].

Additionally, coadministration of Tariquidar (6–12 mg/kg po) fully restores the antitumor activity of Paclitaxel, Etoposide, and Vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. These mice bear the intrinsically resistant MC26 colon tumors, and coadministration of Tariquidar methanesulfonate, hydrate (XR9576 methanesulfonate, hydrate) potentiates the antitumor activity of Doxorubicin without a significant increase in toxicity. It has also been discovered that tariquidar dramatically increases the antitumor activity of doxorubicin against sc MC26 tumors in vivo[2].

ATP hydrolytic activity of P-gp in CHrB30 membranes[1]
A previously described colorimetric assay was used to measure inorganic phosphate liberation following ATP hydrolysis (Chifflet et al., 1988). Membranes (1 μg protein) were incubated with Na2ATP (2 mm) in a total assay volume of 50 μl in buffer containing (mm): Tris pH 7.4 50, MgSO4 5, 0.02% NaN3, NH4Cl 150 for 20 min at 37°C. The ATPase activity was linear to 40 min at 37°C. Modulators (from DMSO stocks) and the ATPase inhibitor vanadate, were added in the concentration range 10−9–10–5 m. The final DMSO concentration was always <1%, a level known not to alter ATPase activity. The effect of drugs on the ATPase activity was fitted by the general dose-response relationship (see above).

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Specific drug binding to P-glycoprotein[1]
A rapid filtration assay was used to measure the binding of [3H]-vinblastine, [3H]-paclitaxel and [3H]-XR9576 to P-gp in CHrB30 membranes as previously described (Ferry et al., 1992). Membranes were incubated with appropriate radioligand in a total buffer volume of 200 μl (50 mm Tris pH 7.4) for a period of 2–3 h to reach equilibrium. Washing buffer (3 ml) containing 20 mm MgSO4, 20 mm Tris (pH 7.4) was then added and the samples filtered under vacuum through a single GF/F filter in a filtration manifold to separate bound and free ligand. After further washing (2×3 ml) the amount of bound ligand was determined by liquid scintillation counting. Non-specific binding was defined as the amount of [3H]-ligand bound in the presence of at least a 100 fold excess of competing ligand (indicated in Results) and was subtracted from all values.
Determination of the capacity and affinity of [3H]-ligand binding was achieved by saturation isotherm analysis. Membranes were incubated with increasing concentration of labelled drug and the amount bound (pmol mg−1) plotted as a function of free ligand concentration.

Cell culture[1]
The Chinese hamster ovary parental (sensitive) AuxB1 and the resistant CHrB30 cells were grown as previously described in α-minimum essential medium (α-MEM) containing 10% foetal calf serum (Kartner et al., 1983). The CHrB30 cells, derived from AuxB1 cells by step wise selection in colchicine (Kartner et al., 1983), express P-gp and selection pressure was maintained by supplementing media with 30 μg ml−1 colchicine.

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Plasma membrane preparation[1]
Plasma membranes were prepared following disruption of CHrB30 cells using nitrogen cavitation and collection with sucrose density centrifugation as previously described (Lever, 1977). The final membrane preparation was stored at −70°C, at protein concentrations of 5–10 mg ml–1 in buffer containing 0.25 m sucrose, 10 mm Tris HCI (pH 7.5) and including the protease inhibitors leupeptin (0.1 mg ml−1), pepstatin A (0.1 mg ml−1) and benzamidine (1 mm).

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Steady-state drug accumulation assay[1]
AuxB1 and CHrB30 cells were grown to confluency in 12-well (24 mm) tissue culture dishes and the steady-state accumulation of [3H]-vinblastine was measured as previously described (Martin et al., 1997). Accumulation was initiated by the addition of 0.1 μCi [3H]-vinblastine and unlabelled vinblastine to a final concentration of 100 nm. The accumulation of [3H]-paclitaxel was measured using 0.1 μCi [3H]-paclitaxel and unlabelled drug to a final concentration of 1 μm. Cells were incubated in a reaction volume of 1 ml for 60 min at 37°C under 5% CO2 in order to reach steady-state. The effect of the modulators XR9576 and GF120918 on [3H]-ligand accumulation was investigated in the concentration range 10−9–10−6 m. Modulators were added from a DMSO stock giving a final solvent concentration of 0.2% (v v−1). Following cell harvesting, accumulated drug was measured by liquid scintillation counting and normalized for cell protein content. Plots of amount accumulated as a function of modulator concentration were fitted with the general dose-response equation (De Lean et al., 1978).
The accumulation of [3H]-XR9576 was also measured in AuxB1 and CHrB30 cells using several concentrations of radiolabelled drug (1–300 nm) in the presence and absence of 1 μm GF120918 and followed over a 60 min period, as described above.

[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.

[3]. A new method measuring the interaction of radiotracers with the human P-glycoprotein (P-gp) transporter. Nucl Med Biol. 2018 Feb 14;60:29-36.

1. The molecular interaction between the anthranilic acid derivative [3H]-XR9576 and multidrug-resistant P-glycoprotein (P-gp) was characterized using equilibrium binding kinetics and properties. XR9576 exhibited specific high affinity binding to P-gp (Bmax = 275 pmol mg⁻¹, Kd = 5.1 nM). The transport substrates [3H]-vincaline and [3H]-paclitaxel showed affinity for P-gp that was 4-fold and 20-fold lower, respectively, than XR9576. Compared to vincaline, XR9576 exhibited a longer duration of interaction with P-gp, while vincaline showed a slower binding rate and a faster dissociation rate. 2. The relative affinity of several modulators and transport substrates for P-gp interactions was determined using a drug-displacement equilibrium binding experiment. Vincristine and paclitaxel could only partially displace the binding of [3H]-XR9576, with Ki values significantly different from the measured Kd values. This indicates a non-competitive interaction between XR9576 and the P-gp substrates vincristine and paclitaxel. XR9576 was confirmed as a potent regulator of P-gp-mediated transport of [3H]-vincristine and [3H]-paclitaxel, as it increased the steady-state accumulation of these cytotoxic drugs in CHrB30 cells to levels observed in P-gp-non-expressing AuxB1 cells (EC50 = 487 ± 50 nM). This drug transport inhibition was not mediated through competitive transport, as the accumulation of [3H]-XR9576 was unaffected by P-gp expression or function. These results demonstrate that the P-gp regulator XR9576 exhibits higher selectivity, more durable inhibitory activity, and stronger interaction potency with this transporter than any other reported regulator. Multiple pieces of evidence suggest that XR9576 inhibits P-gp function by binding to a site different from the transport substrate interaction site. These two sites may have regulatory or transport functions, respectively. [1]

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Overexpression of P-glycoprotein (P-gp) on the surface of tumor cells leads to multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump, reducing the intracellular concentration of structurally unrelated drugs. P-gp function modulators can restore the sensitivity of MDR cells to these drugs. XR9576 is a novel anthranilic acid derivative that has been developed as a highly potent and specific P-gp inhibitor. In this study, we evaluated the in vitro and in vivo regulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a range of human (H69/LX4, 2780AD) and mouse (EMT6 AR1.0, MC26) multidrug-resistant (MDR) cell lines. XR9576 enhanced the cytotoxicity of several drugs, including doxorubicin, paclitaxel, etoposide, and vincristine; resistance was completely reversed in the presence of 25–80 nM XR9576. Direct comparative studies with other modulators showed that XR9576 is one of the most potent modulators reported to date. Accumulation and efflux studies using the P-gp substrates [3H]daunorubicin and rhodamine 123 showed that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but this inhibition persisted for more than 22 hours after removal of the modulator from the culture medium. This contrasts sharply with P-gp substrates such as cyclosporine A and verapamil, which lose activity within 60 minutes, indicating that XR9576 is not transported via P-gp. Furthermore, XR9576 is a potent inhibitor of [3H]azidopine on P-gp photoaffinity labeling, suggesting a direct interaction with this protein. In mice carrying inherently resistant MC26 colon tumors, co-administration of XR9576 with doxorubicin enhanced the antitumor activity of doxorubicin without significantly increasing toxicity. Maximum synergistic effects were observed at intravenous or oral doses of 2.5–4.0 mg/kg. Furthermore, in nude mice, co-administration with XR9576 (6–12 mg/kg, orally) completely restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenograft models (2780AD and H69/LX4). Importantly, all effective co-administration regimens were well-tolerated. Moreover, intravenous co-administration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results indicate that XR9576 is a potent, selective, and long-lasting modulator. It possesses strong intravenous and oral activity without significantly enhancing the plasma pharmacokinetics of paclitaxel or increasing the toxicity of co-administration. Therefore, XR9576 has great potential in treating P-gp-mediated multidrug resistance (MDR) cancers. [2]

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In drug development, the success rate of biomarkers for brain applications is lower than that of cardiovascular or oncology drugs. One reason is that tracers cannot penetrate the blood-brain barrier due to their interaction with efflux transporters such as P-gp (MDR1 or ABCB1). This study aimed to develop a reliable model for parallel measurement of the interaction between radiotracers and human efflux transporters P-gp during radiolabeling. This study used LigandTracer® technology with the wild-type cell line MDCKII and an equivalent cell line overexpressing human P-gp (MDCKII-hMDR1). The method was based on PET tracers known to interact with human P-gp transporters and evaluated at nanomolar concentrations (15 nM). [11C]SNAP-7941 and [18F]FE@SNAP were used as P-gp substrates and compared with uptake experiments and μPET images using a real-time model. [11C]DASB, [11C]Harmine, [18F]FMeNER, [18F]FE@SUPPY, and [11C]Me@HAPTHI were used as tracers, and no interaction with P-gp was observed in vitro. However, the SUV value of [11C]Me@HAPTHI increased significantly after being blocked by Tariquidar. The established real-time kinetic model directly used PET tracers at compound concentrations, reflecting the in vivo situation. This method can be used in the early stages of radiopharmaceutical development to measure their interaction with P-gp before conducting animal experiments. [3]

C40H52N4O12S2

892.99

892.287

625375-83-9

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

71576652

Light yellow to yellow solid

7.746

7

17

11

61

1130

0

S(C([H])([H])[H])(=O)(=O)O[H].S(C([H])([H])[H])(=O)(=O)O[H].O(C([H])([H])[H])C1=C(C([H])=C2C(=C1[H])C([H])([H])N(C([H])([H])C([H])([H])C1C([H])=C([H])C(=C([H])C=1[H])N([H])C(C1=C([H])C(=C(C([H])=C1N([H])C(C1C([H])=NC3=C([H])C([H])=C([H])C([H])=C3C=1[H])=O)OC([H])([H])[H])OC([H])([H])[H])=O)C([H])([H])C2([H])[H])OC([H])([H])[H].O([H])[H].O([H])[H].O([H])[H]

MNKRYFJEUQPYTI-UHFFFAOYSA-N

InChI=1S/C38H38N4O6.2CH4O3S.3H2O/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;2*1-5(2,3)4;;;/h5-12,17-22H,13-16,23H2,1-4H3,(H,40,44)(H,41,43);2*1H3,(H,2,3,4);3*1H2

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)