RESUMEN
Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins--the 170-kDa P-glycoprotein (P-gp) and the 190-kDa multidrug resistance-associated protein (MRP1)--that pump drugs out of MDR cells. The intracellular level of a drug, which influences the drug's cytotoxic effect, is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). One possible pharmacological approach to overcoming drug resistance is the use of specific inhibitors that enhance the cytotoxicity of known antineoplastic agents. Many compounds have been proven to be very efficient in inhibiting P-gp activity, but only some of them can inhibit MRP1. However, the clinical results obtained so far by this approach have been rather disappointing. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs whose physicochemical properties favor the uptake of such drug by resistant cells. Our recent studies have shown that whereas the P-gp- and MRP1-mediated efflux of different anthracycline-based drugs may not differ considerably, their kinetics of uptake do. Thus, the high uptake of drug by cells may lead to concentrations at the cellular target site high enough to achieve the needed cytotoxicity against MDR cells. Therefore, increased drug lipophilicity might be one factor in improving drug cytotoxicity in MDR cells. In vitro studies have shown that idarubicin, an analogue of daunorub cin, is more effective than daunorubicin and doxorubicin against MDR tumor cell lines and that this increased effectiveness is related in part to the increased lipophilicity of idarubicin. Other studies have also confirmed the strong impact of lipophilicity on the uptake and retention of anthracyclines in MDR cells.
Asunto(s)
Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Resistencia a Múltiples Medicamentos/genética , Resistencia a Antineoplásicos/genética , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Animales , Antibióticos Antineoplásicos/metabolismo , Antibióticos Antineoplásicos/farmacología , Transporte Biológico Activo/efectos de los fármacos , Transporte Biológico Activo/genética , Humanos , Células Tumorales CultivadasRESUMEN
A major problem in the treatment of cancer is cellular resistance to cytotoxic drugs. In tumor cells in vitro, the development of multidrug resistance is usually accompanied by increased expression of drug transporters, either P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP(1)). Both proteins belong to the superfamily of ATP-binding cassette (ABC) transporter proteins and mediate the transport of a broad range of drugs. Altenberg et al. (Proc Natl Acad Sci USA90: 9735-9738, 1993) have shown that changes in intra- or extracellular pH do not mediate P-gp-dependent multidrug resistance. Therefore, we similarly studied whether changes in intra- or extracellular pH could mediate MRP(1)-dependent multidrug resistance. In particular, we measured the MRP(1)-mediated efflux of hydroxyrubicin from GLC4/ADR cells. Since hydroxyrubicin is a fully neutral anthracycline derivative that has no deprotonable function at pH lower than 10 and so cannot accumulate in non-nuclear compartments under the influence of pH or transmembrane gradients, we hypothesized that any modifications of its kinetics of efflux as a function of pH can be assigned to a modification of the transporter efficiency. However, as our data show, modifications of extra- and/or intracellular pH yielded no modification of the MRP(1)-mediated efflux of hydroxyrubicin.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Proteínas de Unión al ADN/fisiología , Epirrubicina/análogos & derivados , Epirrubicina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Transporte Biológico , División Celular/efectos de los fármacos , Citosol/metabolismo , Resistencia a Múltiples Medicamentos/fisiología , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Epirrubicina/farmacología , Humanos , Concentración de Iones de Hidrógeno , Proteína 3 Homóloga de MutS , Células Tumorales CultivadasRESUMEN
Multidrug resistance phenotype in mammalian cells is often correlated with overexpression of P-glycoprotein or Multidrug Resistance-Associated protein (MRP(1)). Both proteins are energy-dependent drug efflux pumps that efficiently reduce the intracellular accumulation and hence the cytotoxicity of many natural cytotoxins. Overexpression of these transporters by tumor cells is thought to be a significant factor in both intrinsic and acquired resistance to anticancer drugs. Consequently a great deal of interest is focused on identifying chemical agents that can either antagonise drug transport by these proteins or that can inhibit the proliferation of tumors cells despite the expression of these transporters. P-glycoprotein-mediated multidrug resistance is reversed by a variety of compounds, but surprisingly, few agents reverse the MRP(1)-mediated multidrug resistance. However, it has recently been shown that 2-[4-(diphenylmethyl)-1-piperazinyl]ethyl-5-(trans-4,6-dimethyl-1, 3, 2-dioxaphosphorinan-2-yl)-2, 6-dimethyl-4-(3-nitrophenyl)-3-pyridinecarboxylate P oxide (PAK-104P) was able to inhibit the P-glycoprotein and MRP(1)-mediated efflux of several compounds. Understanding of the interactions between transporters and multidrug resistance reversing agents is important in the design of more effective multidrug resistance modulators. We now examined the effect of PAK-104P on Pgp-and MRP1-mediated efflux of three anthracyclines, daunorubicin, pirarubicin, hydroxydoxorubicin and of calcein acetoxymethyl ester and calcein. Our data show that PAK-104P non-competitively inhibits the P-glycoprotein-mediated efflux of anthracycline derivatives and calcein acetoxymethyl ester with an inhibitory constant K(I)=0. 25+/-0.05 microM. PAK-104P also non-competitively inhibits the MRP(1)-mediated efflux of daunorubicin, pirarubicin, hydroxyrubicin, calcein acetoxymethyl ester and calcein. However, surprisingly, in this case the K(I) values obtained were very different ranging from 0.06 for hydroxyrubicin to 10 microM for calcein. These data strongly suggested the existence of two different mechanisms for the inhibition by PAK-104P of the MRP(1)-mediated efflux of molecules: a first mechanism, involving a low-affinity site for PAK-104P, and which would concern molecules such as calcein, cysteinyl leukotriene LCT(4) etc. whose efflux do not depend on glutathione. A second mechanism involving a high-affinity site for PAK-104P and which would concern molecules such as anthracyclines, calcein acetoxymethyl ester whose efflux depends on the presence of glutathione.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Transportadoras de Casetes de Unión a ATP/fisiología , Antibióticos Antineoplásicos/metabolismo , Óxidos P-Cíclicos/farmacología , Fluoresceínas/metabolismo , Proteínas de Neoplasias/fisiología , Ácidos Nicotínicos/farmacología , Daunorrubicina/metabolismo , Depresión Química , Doxorrubicina/análogos & derivados , Doxorrubicina/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Epirrubicina/análogos & derivados , Epirrubicina/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Células Tumorales CultivadasRESUMEN
Multidrug resistance (MDR) in model systems is known to be conferred by two different integral proteins, the 170-kDa P-glycoprotein (Pgp) and the 190-kDa multidrug resistance-associated protein (MRP1). One possible pharmacological approach to overcome drug resistance is the use of specific inhibitors, which enhance the cytotoxicity of known antineoplastic agents. However, while many compounds have been proven to be very efficient in inhibiting Pgp activity only some of them are able to inhibit MRP1. The other likely approach is based on the design and synthesis of new non-cross-resistant drugs with physicochemical properties favoring the uptake of the drug by the resistant cells. The intracellular drug retention influences its cytotoxic effect. The level of the intracellular drug content is a function of the amount of drug transported inside the cell (influx) and the amount of drug expelled from the cell (efflux). In this work, the kinetics of drug uptake and the kinetics of active efflux of several anthracycline derivatives in both Pgp expressing K562/Adr cells and MRP1 expressing GLC4/Adr cells was determined. Our data have shown that in both cell lines there is no correlation between the resistance factor and the kinetics of drug efflux by these pumping systems. However, a very good correlation between the resistance factor and the kinetics of drug uptake has been established in both cell lines: the resistance factor decreases when the kinetics of drug uptake increases. This work has clearly shown that when the rate of transmembrane transport of anthracycline is high enough, the efflux mediated by the protein transporter is not able to pace with it. The protein transporter essentially operates in a futile cycle and the resistance factor is tending to one. It does not mean, however, that when the resistance factor is close to one the anthracycline is not transported by the pump.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antraciclinas/metabolismo , Membrana Celular/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Antraciclinas/química , Antraciclinas/farmacología , División Celular/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Daunorrubicina/farmacología , Resistencia a Medicamentos , Humanos , Cinética , Estructura Molecular , Proteína 3 Homóloga de MutS , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The multidrug resistance protein (MRP) has been shown to mediate ATP-dependent efflux of anticancer agents of diverse structure, such as daunorubicin (DNR), vincristine and etoposide. Thus, this protein does confer a multidrug resistant phenotype to cancer cells, similar to P-glycoprotein (Pgp). The substrate specificity of both transporter proteins is partly overlapping but is otherwise very distinct; because MRP is a multiple organic anion transporter, it transports certain glutathione conjugates and may be partly dependent on intracellular glutathione levels for the transport of anthracyclines. We have studied the transport kinetics of a series of anthracyclines in MRP and Pgp that overexpress tumor cell lines to obtain information on the substrate specificity of these proteins. The anthracyclines have modifications in the sugar moiety. The mean active efflux coefficient Ka, used to characterize the efficiency of the active efflux, was very similar for DNR and one of its 4'-deoxy-derivatives (eso-DNR) for MRP and Pgp [10-20 x 10(-10)/sec/(cells/ml)]. The permanently neutral derivatives 3'-deamino-3'-hydroxy-doxorubicin (OH-DOX) and 3'-deamino-3'-hydroxy-daunorubicin (OH-DNR) were effluxed by both proteins but had a lower Ka [2 x 10(-10) and 6 x 10(-10)/sec/(cells/ml) (OH-DOX)] and 2 x 10(-10) and 5 x 10(-10)/sec/(cells/ml) (OH-DNR)] for MRP and Pgp. Two anthracyclines, the doxorubicin derivative pirarubicin and 2'-bromo-4'-epi-DNR seemed to have a slightly higher Ka value for Pgp than for MRP. The apparent Michaelis-Menten constants (K(m)) and maximal efflux rates (VM) for the active transport were within a narrow range for both transporters, except for OH-DOX and OH-DNR, which had a lower VM in the case of MRP-mediated transport, suggesting a role of the amino group in the interaction with glutathione. Determination of the Hill coefficient (nH) of the MRP-mediated efflux gave most values close to 2, which suggests cooperativity of the transport of anthracyclines as reported before for Pgp. In conclusion, the transport kinetics of anthracyclines by MRP and Pgp are very similar.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Carcinoma de Células Pequeñas/metabolismo , Daunorrubicina/análogos & derivados , Daunorrubicina/farmacocinética , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacocinética , Humanos , Cinética , Leucemia Eritroblástica Aguda/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas Asociadas a Resistencia a Múltiples MedicamentosRESUMEN
OBJECTIVE: The purpose of this study was to compare breath-hold three-dimensional (3D) rapid gradient-echo (GRE) MR imaging obtained before and after gadolinium chelate injection with T2-weighted fast spin-echo and T2-weighted breath-hold fast spin-echo (BHFSE) MR imaging in the detection of focal hepatic masses. SUBJECTS AND METHODS: Fifty-three patients with 108 focal hepatic masses had, prospectively, MR of the liver at 1.5 T. T2-weighted fast spin-echo (6000/117 [TR/effective TE]; echo train length=16; acquisition time = 3 min 12 sec) images obtained with and without fat suppression, T2-weighted BHFSE (2700/105; echo train length = 20; acquisition time = 22 sec), and 3D rapid GRE images (10.1/1.9/30 degrees [TR/TE/alpha]) obtained during one breath-hold (12 scan locations in 21 sec or 20 scan locations in 32 sec) before and after injection of gadolinium chelate were blindly and independently analyzed in consensus by three readers. RESULTS: Gadolinium chelate-enhanced 3D rapid GRE images allowed depiction of more focal hepatic masses (90 of 108, sensitivity = 83%) than did T2-weighted fast spin-echo with fat suppression images (76 of 108, sensitivity = 70%), T2-weighted fast spin-echo without fat suppression images (74 of 108, sensitivity = 69%), T2-weighted BHFSE images (73 of 108, sensitivity = 68%), and unenhanced 3D rapid GRE images (54 of 108, sensitivity = 50%) (p < .01). No difference in sensitivity was found between the three T2-weighted sequences. CONCLUSION: Gadolinium chelate-enhanced 3D rapid GRE imaging is superior to T2-weighted fast spin-echo images obtained with or without fat suppression for the detection of focal hepatic masses. T2-weighted BHFSE is similar to T2-weighted fast spin-echo images in detecting focal hepatic lesions.
Asunto(s)
Medios de Contraste , Compuestos Heterocíclicos , Hepatopatías/diagnóstico , Hígado/patología , Imagen por Resonancia Magnética/métodos , Compuestos Organometálicos , Adulto , Anciano , Diagnóstico Diferencial , Femenino , Humanos , Imagen por Resonancia Magnética/instrumentación , Imagen por Resonancia Magnética/normas , Imagen por Resonancia Magnética/estadística & datos numéricos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Respiración , Sensibilidad y Especificidad , Estadísticas no Paramétricas , Factores de TiempoRESUMEN
OBJECTIVE: The goal of our study was to compare a T2-weighted breath-hold fast spin-echo (BHSE) technique with T2-weighted non-breath-hold fast spin-echo techniques for imaging the liver. SUBJECTS AND METHODS: Thirty-three patients with hepatic lesions had T2-weighted BHSE images obtained in 22 sec and conventional T2-weighted non-breath-hold fast spin-echo images obtained in 3 min 12 sec with and without fat suppression. Images were analyzed quantitatively by measuring the lesion-liver contrast, spleen-liver contrast, and signal-to-noise ratios of lesions and qualitatively by evaluating the sharpness of hepatic contours, visibility of intrahepatic vessels and other segmental landmarks, and presence of artifacts. RESULTS: Quantitatively, lesion-liver contrast, spleen-liver contrast, and signal-to-noise ratios obtained with the BHSE technique were inferior to those obtained with fast spin-echo techniques with and without fat suppression (11.2 +/- 7.1 versus 15.4 +/- 10.6 and 14.5 +/- 9.8, p < .001; 5.3 +/- 3.7 versus 8.7 +/- 3.5 and 7.0 +/- 3.8, p < .001; 16.2 +/- 8.2 versus 20.1 +/- 10.9 and 19.7 +/- 9.5, p < .01, respectively; Student's t test). Qualitatively, image artifacts and intrahepatic vessel depiction on BHSE images were similar to those obtained with the fast spin-echo techniques. The BHSE technique was superior to fat-suppressed fast spin-echo technique for showing hepatic contours (p < .01; Wilcoxon signed-rank test). CONCLUSION: The BHSE technique is quantitatively inferior to non-breath-hold fast spin-echo techniques. However, further studies with a surgical standard of reference are needed to compare the three techniques in terms of sensitivity.
Asunto(s)
Hepatopatías/diagnóstico , Hígado/patología , Imagen por Resonancia Magnética/métodos , Tejido Adiposo/patología , Adulto , Anciano , Femenino , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Persona de Mediana EdadRESUMEN
OBJECTIVE: Hepatic metastases from neuroendocrine tumors are often markedly hyperintense on unenhanced T2-weighted MR images, making their appearance similar to that of cavernous hemangiomas. In contrast, cavernous hemangiomas show characteristic enhancement on dynamic gadolinium chelate-enhanced gradient-recalled echo MR images. The purpose of this study was to determine the relative merits of dynamic gadolinium chelate-enhanced gradient-recalled echo MR imaging versus MR imaging with unenhanced spin-echo pulse sequences for distinguishing between hepatic metastases from neuroendocrine tumors and cavernous hemangiomas. MATERIALS AND METHODS: The unenhanced spin-echo and dynamic gradient-recalled echo MR images obtained after IV administration of a gadolinium chelate in 28 patients (14 patients with pathologically proven hepatic metastases from neuroendocrine tumors and 14 patients with hepatic cavernous hemangiomas) were reviewed blindly and independently by three interpreters. Unenhanced spin-echo and dynamic gadolinium chelate-enhanced gradient-recalled echo MR images were compared for accuracy in characterizing liver lesions. RESULTS: The most intense enhancement of hepatic metastases from neuroendocrine tumors was observed on early dynamic gadolinium chelate-enhanced gradient-recalled echo MR images; enhancement was peripheral in four patients, global and heterogeneous in seven patients, and global and homogeneous in three patients. On late dynamic gadolinium chelate-enhanced gradient-recalled echo MR images, enhancement of hepatic metastases from neuroendocrine tumors was predominantly peripheral in five patients, global and heterogeneous in five patients, and global and homogeneous in four patients. Differentiation between cavernous hemangiomas and hepatic metastases from neuroendocrine tumors was impossible in five cases with unenhanced spin-echo MR imaging alone, in five cases with dynamic gadolinium chelate-enhanced gradient-recalled echo MR imaging alone, and in no case with the combination of unenhanced spin-echo MR imaging and dynamic gadolinium chelate-enhanced gradient-recalled echo MR imaging. In comparison with unenhanced spin-echo MR imaging alone or dynamic gadolinium chelate-enhanced gradient-recalled echo MR imaging alone, the combination of unenhanced spin-echo MR imaging and dynamic gadolinium chelate-enhanced gradient-recalled echo MR imaging allowed significantly (p < .001) clearer differentiation between hepatic metastases from neuroendocrine tumors and cavernous hemangiomas. CONCLUSIONS: Early enhancement and heterogeneity on dynamic gadolinium chelate-enhanced gradient-recalled echo MR images are the most common features of hepatic metastases from neuroendocrine tumors. The combination of unenhanced spin-echo and dynamic gadolinium chelate-enhanced gradient-recalled echo MR images allows more accurate characterization of hepatic metastases from neuroendocrine tumors and clearer differentiation from cavernous hemangiomas.