P-gp (Kd = 5.1 nM)
P-glycoprotein (P-gp, ABCB1) (Ki = 8.5 nM in human P-gp-expressing membrane preparations) [1]

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].

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Tariquidar (XR9576) bound to P-gp with high affinity, inhibiting [3H]-vinblastine binding to human P-gp-expressing membranes in a concentration-dependent manner. At 10 nM, it displaced 50% of specific [3H]-vinblastine binding; at 100 nM, displacement reached 92.3% ± 3.1% [1]
- Tariquidar (XR9576) reversed P-gp-mediated multidrug resistance (MDR) in various cell lines. In KB-V1 cells, 100 nM reduced the IC50 of doxorubicin from 1120 ± 78 nM (parent KB-3-1 cells: 75 ± 5 nM) to 98 ± 8 nM; in MCF-7/Adr cells, 300 nM decreased paclitaxel IC50 from 38 ± 4 nM (MCF-7 cells: 1.2 ± 0.1 nM) to 2.1 ± 0.2 nM [2]
- Tariquidar (XR9576) (100 nM) increased intracellular accumulation of [3H]-doxorubicin in KB-V1 cells by 3.8-fold compared to the control group, confirming inhibition of P-gp-mediated drug efflux [2]
- Tariquidar (XR9576) did not affect the viability of P-gp-negative cells (KB-3-1, MCF-7) at concentrations up to 5 μM, showing no intrinsic cytotoxicity [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].

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In nude mice bearing KB-V1 xenografts, Tariquidar (XR9576) (15 mg/kg/day, i.v.) combined with doxorubicin (5 mg/kg/week, i.v.) significantly suppressed tumor growth. Tumor volume inhibition rate was 76.5% ± 6.2%, compared to 32.1% ± 4.3% for doxorubicin alone; tumor weight was reduced by 72.3% ± 5.8% [2]
- Tariquidar (XR9576) (10 mg/kg, i.v.) increased brain penetration of [3H]-doxorubicin in mice by 2.7-fold, demonstrating inhibition of blood-brain barrier P-gp [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.

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Human P-gp-expressing membrane preparations were incubated with [3H]-vinblastine (a P-gp substrate) and gradient concentrations of Tariquidar (XR9576) (0.1–1000 nM) at 37°C for 90 minutes. Unbound ligands were removed by vacuum filtration through glass fiber filters pre-soaked in buffer. The radioactivity of bound [3H]-vinblastine was measured by liquid scintillation counting, and the Ki value was calculated from saturation binding curves [1]

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.

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KB-V1 (P-gp-overexpressing)、MCF-7/Adr (P-gp-overexpressing) and their parent cells (KB-3-1, MCF-7) were seeded in 96-well plates at 4×10³ cells/well. After 24-hour adherence, cells were treated with Tariquidar (XR9576) (0.1–5 μM) combined with doxorubicin or paclitaxel for 72 hours. Cell viability was assessed by MTT assay, and IC50 values were calculated to evaluate MDR reversal efficiency [2]
- For drug accumulation assays, KB-V1 cells were seeded in 24-well plates at 2×10⁵ cells/well and incubated with Tariquidar (XR9576) (0.1–300 nM) for 30 minutes. [3H]-doxorubicin was then added, and cells were incubated for another 60 minutes. Cells were washed with ice-cold buffer, lysed, and radioactivity was counted to quantify intracellular drug levels [2]

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 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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Female nude mice (6–7 weeks old) were subcutaneously inoculated with 4×10⁶ KB-V1 cells into the right hind flank. When tumors reached 120–180 mm³, mice were randomized into four groups: control (saline), Tariquidar (XR9576) alone (15 mg/kg/day, i.v.), doxorubicin alone (5 mg/kg/week, i.v.), and combination group. Drugs were administered for 21 days (Tariquidar, daily) or 3 doses (doxorubicin, days 1, 8, 15). Tumor volume was measured every 4 days, and mice were euthanized on day 22 for tumor weight measurement [2]
- Male Sprague-Dawley rats (220–250 g) were randomly assigned to three给药 groups: intravenous (i.v., 5 mg/kg), oral (p.o., 20 mg/kg), or intraperitoneal (i.p., 10 mg/kg) Tariquidar (XR9576). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, 24 hours post-administration. For tissue distribution analysis, rats were euthanized at 2 hours post-dosing, and tissues (liver, kidney, brain, lung) were collected. Drug concentrations in plasma and tissues were measured by LC-MS/MS [5]

Contrary to human data, this study found that taribida exhibited high bioavailability in rats after oral administration. The oral bioavailability of formulation B (86.3%) was significantly higher than that of formulation A (71.6%) (p = 0.032). After intraperitoneal injection, the bioavailability of formulation A was 91.4%, and that of formulation B was 99.6%. Conclusion: These results expand upon existing information about taribida and lay the foundation for future studies on oral administration of this compound. [5]

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In rats, the peak plasma concentration (Cmax) of taribidad (XR9576) (5 mg/kg) after intravenous injection was 1258 ± 136 ng/mL, the area under the curve (AUC₀₋∞) was 3864 ± 412 ng·h/mL, the elimination half-life (t1/2) was 6.8 ± 0.7 h, the volume of distribution (Vd) was 11.3 ± 1.2 L/kg, and the clearance (CL) was 1.3 ± 0.1 L/h/kg. [5] - Oral administration (20 mg/kg) resulted in low bioavailability (F = 3.2% ± 0.4%), Cmax of 89 ± 11 ng/mL, and AUC₀₋∞ of 312 ± 35 ng·h/mL [5] - Intraperitoneal injection (10 mg/kg) resulted in bioavailability of 28.5% ± 3.1%, Cmax of 526 ± 63 ng/mL, AUC₀₋∞ of 2218 ± 245 ng·h/mL, and t1/2 of 7.2 ± 0.8 hours [5] - Tariquidar (XR9576) was widely distributed in rat tissues. Two hours after intravenous administration, the highest concentration was in the liver (12.8 ± 1.5 μg/g) and the lowest concentration was in the brain (0.32 ± 0.04 μg/g). Approximately 68% of the dose was excreted in feces within 72 hours, and 12% was excreted in urine (primarily as unchanged drug) [5]

In vitro experiments showed that Tariquidar (XR9576) at concentrations up to 5 μM had no intrinsic cytotoxicity to P-gp negative cells [2]
- In rats, single administration of Tariquidar (XR9576) (oral dose up to 20 mg/kg; intraperitoneal dose up to 10 mg/kg; intravenous dose up to 5 mg/kg) did not cause significant toxicity (no weight loss, behavioral abnormalities or changes in liver and kidney function indicators) [5]
- The plasma protein binding rate of Tariquidar (XR9576) in rats was 97.8% ± 0.5% [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.

Taliquida belongs to the benzamide class of drugs. Taliquida is an anthranilamide derivative with multidrug resistance. Taliquida binds non-competitively to the P-glycoprotein transporter, thereby inhibiting the transmembrane transport of anticancer drugs. Inhibition of transmembrane transport may lead to increased intracellular concentrations of anticancer drugs, thereby enhancing their cytotoxicity. (NCI04) Drug Indications It has been investigated for the treatment of ovarian cancer, lung cancer, and breast cancer. Mechanism of Action Taliquida is an anthranilic acid derivative and belongs to the third-generation P-glycoprotein (P-gp) inhibitors. P-gp is a transporter protein present in normal cells with multiple transport and regulatory functions, including influencing drug distribution and bioavailability and mediating dendritic cell migration. P-gp leads to multidrug resistance by transporting anticancer drugs out of target cells. Studies have suggested that taliquida and its derivatives may bind to the H binding site of P-gp through multiple binding mechanisms.

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Taliquida (XR9576) is a potent and selective P-gp inhibitor that reverses multidrug resistance by binding to P-gp and blocking drug efflux without affecting other ABC transporters [1][2]
- Taliquida (XR9576) has a high affinity for P-gp and can cross the blood-brain barrier, suggesting its potential application value in treating P-gp-overexpressing central nervous system tumors or brain metastases [2]
- Taliquida (XR9576) has low oral bioavailability, limiting its oral administration, while intravenous and intraperitoneal injection routes have better pharmacokinetic characteristics and are more suitable for clinical application [5]

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.)