 Chemical Structure.png)
Physicochemical Properties
| Molecular Formula | C38H40CL2N4O6 |
| Molecular Weight | 719.653408050537 |
| Exact Mass | 718.232 |
| CAS # | 1992047-62-7 |
| Related CAS # | Tariquidar; 206873-63-4;625375-84-0 (mesylate);1992047-62-7 (2HCl); 625375-83-9 (methanesulfonate hydrate) |
| PubChem CID | 121513890 |
| Appearance | Solid powder |
| Hydrogen Bond Donor Count | 4 |
| Hydrogen Bond Acceptor Count | 8 |
| Rotatable Bond Count | 11 |
| Heavy Atom Count | 50 |
| Complexity | 1040 |
| Defined Atom Stereocenter Count | 0 |
| InChi Key | ZJCJRGRTDKIFJJ-UHFFFAOYSA-N |
| InChi Code | 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 |
| Chemical Name | N-[2-[[4-[2-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)ethyl]phenyl]carbamoyl]-4,5-dimethoxyphenyl]quinoline-3-carboxamide;dihydrochloride |
| Synonyms | XR9576 dihydrochloride; D06008; XR 9576; D 06008; XR-9576;D-06008 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | P-gp (Kd = 5.1 nM)[1] |
| ln Vitro | 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]. |
| ln Vivo | 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]. |
| Enzyme Assay |
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). 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 Assay |
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. 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). 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. |
| Animal Protocol |
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] 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] |
| References |
[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. |
| Additional Infomation |
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] 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] |
Solubility Data
| Solubility (In Vitro) | DMSO: > 10 mM |
| Solubility (In Vivo) |
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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.3896 mL | 6.9478 mL | 13.8956 mL | |
| 5 mM | 0.2779 mL | 1.3896 mL | 2.7791 mL | |
| 10 mM | 0.1390 mL | 0.6948 mL | 1.3896 mL |