P-gp (Kd = 5.1 nM)

Tariquidar (XR9576) increases the steady-state accumulation of P-gp, making it a potent modulator of P-gp-mediated transport of [3H]-Vinblastine and [3H]-Paclitaxel. The levels of P-gp observed in AuxB1 cells (EC50=487±50 nM) are not required for the cytotoxic effects observed in CHrB30 cells. [3H]-Tariquidar has the strongest affinity (Kd=5.1±0.9 nM, n=7) and a binding capacity (Bmax) of 275±15 pmol/mg membrane protein when it comes to CHrB30 membranes. The modulator Tariquidar (EC50=487±50 nm) increased the accumulation of [3H]-vinblastine in a dose-dependent manner when compared to the parental cell line. With an effective IC50 value of 43±9 nM, the MDR modulator Tariquidar can inhibit 60–70% of vanadate-sensitive ATPase activity[1]. Several medications, such as doxorubicin, paclitaxel, etoposide, and vincristine, are made more cytotoxic by tiriquidar (XR9576); in the presence of 25–80 nM XR9576, 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].

In mice with intrinsically resistant MC26 colon cancers, coadministration of Tariquidar (XR9576) at a dose of 2.5–4.0 mg/kg maximally increased the anticancer efficacy of doxorubicin without appreciably increasing toxicity was noted. Moreover, in nude mouse xenografts of two highly resistant MDR human cancers (2780AD, H69/LX4), coadministration with Tariquidar (6–12 mg/kg po) completely restored the efficacy of paclitaxel, etoposide, and vincristine Antitumor activity. Additionally, when doxorubicin is subcutaneously injected in vivo against MC26 tumors, tartiquidar greatly increases its anti-tumor effectiveness [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.

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]

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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

In contrast to human data, the present study found a high bioavailability of tariquidar in rats after oral dosing. Oral bioavailability was significantly higher (p = 0.032) for formulation B (86.3%) than for formulation A (71.6%). After intraperitoneal dosing bioavailability was 91.4% for formulation A and 99.6% for formulation B. Conclusion: The present findings extend the available information on tariquidar and provide a basis for future studies involving oral administration of this compound.[5]

[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]. Simultaneous Semimechanistic Population Analyses of Levofloxacin in Plasma, Lung, and Prostate To Describe the Influence of Efflux Transporters on Drug Distribution following Intravenous and Intratracheal Administration. Antimicrob Agents Chemother. 2015 Nov 30;60(2):946-54.

[4]. Regulation of P-glycoprotein expression in brain capillaries in Huntington's disease and its impact on brain availability of antipsychotic agents risperidone and paliperidone. J Cereb Blood Flow Metab. 2016 Aug;36(8):1412-23.

[5]. Pharmacokinetics of the P-gp Inhibitor Tariquidar in Rats After Intravenous, Oral, and Intraperitoneal Administration. Eur J Drug Metab Pharmacokinet. 2018 Apr 3.

Tariquidar is a member of benzamides.
Tariquidar is an anthranilamide derivative with multidrug resistance properties. Tariquidar non-competitively binds to the p-glycoprotein transporter, thereby inhibiting transmembrane transport of anticancer drugs. Inhibition of transmembrane transport may result in increased intracellular concentrations of an anticancer drug, thereby augmenting its cytotoxicity. (NCI04)

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Drug Indication
Investigated for use/treatment in ovarian cancer, lung cancer, and breast cancer.

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Mechanism of Action
Tariquidar is an anthranilic acid derivative third generation P-glycoprotein (P-gp) inhibitors. P-gp is a transport protein found in normal cells that has many trasport and modulatory functions, from affecting the distribution and bioavailability of drugs to mediating the migration of dendritic cells. P-gp is responsible for multidrug resistance through transport of anti-cancer drugs out of their target cells. It is proposed that tariquidar's and its derivatives may bind to the H-binding site of P-gp through multiple mechanisms of binding.

C38H38N4O6

646.73

646.279

C, 70.57; H, 5.92; N, 8.66; O, 14.84

206873-63-4

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

148201

Off-white to yellow solid

1.3±0.1 g/cm3

716.0±60.0 °C at 760 mmHg

386.8±32.9 °C

0.0±2.3 mmHg at 25°C

1.662

6.38

2

8

11

48

1040

0

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]

LGGHDPFKSSRQNS-UHFFFAOYSA-N

InChI=1S/C38H38N4O6/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)

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 30% propylene glycol, 5% Tween 80, 65% D5W: 30 mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)