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

In CHrB30 cells, tariquidar (XR9576) raises the steady-state accumulation of [3H]-Vinblastine and [3H]-Paclitaxel to levels seen in non-P-gp-expressing AuxB1 cells (EC50=487±50 nM), demonstrating its potency as a regulator of P-gp mediated [3H]-Methionine 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 parent cell line, the modulators Tariquidar (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; 25–80 nM XR9576 completely reversals resistance. 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 (XR9576) potentiates the antitumor activity of Doxorubicin without causing a significant increase in toxicity. It has also been discovered that tariquidar dramatically increases the anticancer efficacy 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.

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 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 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

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

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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