RESUMEN

OBJECTIVE: Our objective was to explore the relationships between imatinib pharmacokinetics and 9 allelic variants in 7 genes coding for adenosine triphosphate-binding cassette transporters (ABCB1 and ABCG2) and enzymes (cytochrome P450 [CYP] 2C9, CYP2C19, CYP2D6, CYP3A4, and CYP3A5) of putative relevance for imatinib. METHODS: Imatinib transport in vitro was studied by use of human embryonic kidney 293 cells transfected with wild-type ABCG2 and an ABCG2 Q141K clone. Steady-state pharmacokinetics of imatinib was obtained in 82 patients with gastrointestinal stromal tumors treated with oral imatinib at doses ranging from 100 to 1000 mg/d. Genotyping was carried out via direct sequencing or restriction fragment length polymorphism-based techniques. RESULTS: Human embryonic kidney 293 cells transfected with ABCG2 Q141K exhibited greater drug accumulation in vitro in comparison with cells expressing wild-type ABCG2 (P = .028). However, pharmacokinetic parameters of imatinib in vivo were not statistically significantly different in 16 patients who were heterozygous for ABCG2 421C>A compared with 66 patients carrying the wild-type sequence (P = .479). The apparent oral clearance of imatinib was potentially reduced in individuals with at least 1 CYP2D6*4 allele (median, 7.78 versus 10.6 L/h; P = .0695). Pharmacokinetic parameters were not related to any of the other multiple-variant genotypes (P >or= .230), possibly because of the low allele frequencies. CONCLUSIONS: This study indicates that common genetic variants in the evaluated genes have only a limited impact on the pharmacokinetics of imatinib. Further investigation is required to quantitatively assess the clinical significance of homozygous variant ABCG2 and CYP2D6 genotypes in patients treated with imatinib.

Asunto(s)

Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Preparaciones Farmacéuticas/metabolismo , Piperazinas/farmacocinética , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Pirimidinas/farmacocinética , Transportadoras de Casetes de Unión a ATP/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Benzamidas , Transporte Biológico Activo , Línea Celular Tumoral , Estudios de Cohortes , Sistema Enzimático del Citocromo P-450/genética , Femenino , Neoplasias Gastrointestinales/genética , Neoplasias Gastrointestinales/metabolismo , Frecuencia de los Genes , Genotipo , Humanos , Mesilato de Imatinib , Isoenzimas/genética , Masculino , Persona de Mediana Edad , Fenotipo , Proteínas Proto-Oncogénicas c-kit/genética , Células del Estroma/metabolismo

RESUMEN

Antiestrogens, such as tamoxifen, are widely used for endocrine treatment of estrogen receptor-positive breast cancer. However, as breast cancer progresses, development of tamoxifen resistance is inevitable. The mechanisms underlying this resistance are not well understood. To identify genes involved in tamoxifen resistance, we have developed a rapid screening method. To alter the tamoxifen-sensitive phenotype of human ZR-75-1 breast cancer cells into a tamoxifen-resistant phenotype, the cells were infected with retroviral cDNA libraries derived from human placenta, human brain, and mouse embryo. Subsequently, the cells were selected for proliferation in the presence of 4-hydroxy-tamoxifen (OH-TAM) and integrated cDNAs were identified by sequence similarity searches. From 155 OH-TAM-resistant cell colonies, a total of 25 candidate genes were isolated. Seven of these genes were identified in multiple cell colonies and thus cause antiestrogen resistance. The epidermal growth factor receptor, platelet-derived growth factor receptor-alpha, platelet-derived growth factor receptor-beta, colony-stimulating factor 1 receptor, neuregulin1, and fibroblast growth factor 17 that we have identified have been described as key regulators in the mitogen-activated protein kinase pathway. Therefore, this pathway could be a valuable target in the treatment of patients with breast cancer resistant to endocrine treatment. In addition, the putative gene LOC400500, predicted by in silico analysis, was identified. We showed that ectopic expression of this gene, designated as breast cancer antiestrogen resistance 4 (BCAR4), caused OH-TAM resistance and anchorage-independent cell growth in ZR-75-1 cells and that the intact open reading frame was required for its function. We conclude that retroviral transfer of cDNA libraries into human breast cancer cells is an efficient method for identifying genes involved in tamoxifen resistance.

Asunto(s)

Neoplasias de la Mama/genética , Resistencia a Antineoplásicos/genética , Antagonistas de Estrógenos/farmacología , Pruebas Genéticas , Tamoxifeno/análogos & derivados , Animales , Química Encefálica , División Celular , Línea Celular Tumoral , Embrión de Mamíferos , Mutación del Sistema de Lectura , Biblioteca de Genes , Genómica/métodos , Humanos , Ratones , Placenta/química , Retroviridae/genética , Tamoxifeno/farmacología , Transducción Genética

RESUMEN

The effectiveness of platinum drugs in the treatment of cancer is hindered by intrinsic and acquired resistance. The cause of clinical resistance to platinum compounds is still unknown. In an attempt to identify new cellular mechanisms of cisplatin resistance, a one-step cisplatin-selection procedure was used to generate resistant sublines of the platinum sensitive A2780 ovarian cancer cell line. In the present study we selected an A2780 subline, A2780-Pt, that has a significantly reduced ability to accumulate cisplatin (36% of the parent A2780 cell line) and consequently shows a clear cisplatin-resistant phenotype (resistance factor, i.e., RF: 8.6). The A2780-Pt cell line was specifically cross-resistant to carboplatin (RF: 12.0), tetraplatin (RF: 8.1) and oxaliplatin (RF: 6.1) which was associated with a reduced cellular platinum accumulation (50%, 54% and 58% of A2780, respectively). No cross-resistance was found for a variety of other anticancer agents. Further experiments to determine the cause of the platinum resistance of the A2780-Pt cell line revealed that: (1) impaired cellular platinum accumulation could not be attributed to aberrant expression of MRP2 (ABCC2), CTR1 (SLC31A1), ATP7A or ATP7B, (2) resistance was not associated with platinum inactivation by metallothionein and glutathione, (3) the platinum efflux rate was similar to that of A2780, (4) the defect in cellular accumulation and the resistance could be overcome by treatment with cisplatin nanocapsules, consistent with impaired influx, and (5) the defect in accumulation is specific for platinum compounds in the cis-configuration, since A2780-Pt cells did not show reduced accumulation of transplatin. This specificity suggests that not passive diffusion but an inward transporter is impaired in A2780-Pt. In conclusion, we generated an A2780 subline that showed a uniquely stable platinum resistance phenotype, which could theoretically be caused by an impaired inward transporter specific for cis-configurated platinum compounds.

Asunto(s)

Antineoplásicos/farmacocinética , Proteínas de Transporte de Catión/fisiología , Cisplatino/farmacocinética , Resistencia a Antineoplásicos , Neoplasias Ováricas/tratamiento farmacológico , Carboplatino/farmacocinética , Femenino , Glutatión/metabolismo , Humanos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Nanotecnología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Compuestos Organoplatinos/farmacocinética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Oxaliplatino , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo

RESUMEN

PURPOSE: The majority of cytotoxic drugs for adults are dosed based on body surface area (BSA), aiming to reduce interpatient variability in drug exposure. We prospectively studied the usefulness of BSA-based dosing of cisplatin in patients at extremes of BSA values. PATIENTS AND METHODS: Patients were randomly assigned to receive a fixed dose of cisplatin in course 1, and a BSA-adjusted dose in course 2, or vice versa. The fixed dose was based on the average BSA for males and females, while extremes were set at BSA values exceeding the average +/- 1 standard deviation. Subsequently, we retrospectively analyzed data from a normal population. RESULTS: In 25 patients assessable for both courses, the use of a fixed dose of cisplatin resulted in reduced exposure to unbound platinum in patients at the upper extremes of BSA (P = .003) and higher exposures in patients at the lower extremes (P = .009), as compared with exposures following the BSA-adjusted dose. Although clearance was related to BSA (R2 = 0.44; P < .001), only a small reduction in interpatient variability in clearance after correction for BSA was achieved (20.8% v 17.1%). In the retrospective analysis, compared with the average patient, the clearance of unbound platinum in patients with a BSA value < or = 1.65 m2 was 16% slower (P < .001), while an 18% faster clearance (P < .001) was observed in patients with a BSA value > or = 2.05 m2. CONCLUSION Unless better predictors for platinum clearance are identified, fixed-dose regimens per BSA cluster (< or = 1.65 m2; 1.66 m2 to 2.04 m2; > or = 2.05 m2) are recommended.

Asunto(s)

Antineoplásicos/administración & dosificación , Superficie Corporal , Cisplatino/administración & dosificación , Adulto , Anciano , Cisplatino/farmacocinética , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Platino (Metal)/farmacocinética

RESUMEN

The aim of this study is to discover a gene set that can predict resistance to platinum-based chemotherapy in ovarian cancer. The study was performed on 96 primary ovarian adenocarcinoma specimens from 2 hospitals all treated with platinum-based chemotherapy. In our search for genes, 24 specimens of the discovery set (5 nonresponders and 19 responders) were profiled in duplicate with 18K cDNA microarrays. Confirmation was done using quantitative RT-PCR on 72 independent specimens (9 nonresponders and 63 responders). Sixty-nine genes were differentially expressed between the nonresponders (n=5) and the responders (n=19) in the discovery phase. An algorithm was constructed to identify predictive genes in this discovery set. This resulted in 9 genes (FN1, TOP2A, LBR, ASS, COL3A1, STK6, SGPP1, ITGAE, PCNA), which were confirmed with qRT-PCR. This gene set predicted platinum resistance in an independent validation set of 72 tumours with a sensitivity of 89% (95% CI: 0.68-1.09) and a specificity of 59% (95% CI: 0.47-0.71)(OR=0.09, p=0.026). Multivariable analysis including patient and tumour characteristics demonstrated that this set of 9 genes is independent for the prediction of resistance (p<0.01). The findings of this study are the discovery of a gene signature that classifies the tumours, according to their response, and a 9-gene set that determines resistance in an independent validation set that outperforms patient and tumour characteristics. A larger independent multicentre study should further confirm whether this 9-gene set can identify the patients who will not respond to platinum-based chemotherapy and could benefit from other therapies.

Asunto(s)

Antineoplásicos/farmacología , Cisplatino/farmacología , Perfilación de la Expresión Génica , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/genética , Adulto , Anciano , Antineoplásicos/uso terapéutico , Cisplatino/uso terapéutico , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Genes Relacionados con las Neoplasias , Humanos , Persona de Mediana Edad , Neoplasias Ováricas/patología , Valor Predictivo de las Pruebas , Pronóstico , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Resultado del Tratamiento

RESUMEN

Previous studies have shown that Imatinib mesylate (Gleevec), a selective tyrosine kinase inhibitor of c-KIT and platelet-derived growth factor receptors (PDGFR), is highly effective in c-KIT/CD117-positive gastrointestinal stromal tumors (GIST), especially in those having activating mutations in c-kit exon 11. In addition, gain-of-function mutations in the juxtamembrane domain (exon 12) and the kinase activation loop (exon 18) of PDGFRalpha were found in GISTs. Importantly, the presence and type of these mutually exclusive c-KIT or PDGFRalpha mutations were found to be associated with the response to imatinib. Here, we examined the prevalence of c-kit exon 11 and PDGFRalpha exons 12 and 18 mutations in other tumor types known to express these tyrosine kinase receptors in order to explore which other cancer types may potentially benefit from imatinib treatment. We determined the mutational status of these commonly mutated exons by direct sequencing in 11 different tumor types (in total: 215 unrelated cases), including GIST, chordoma, and various distinct tumors of lung, brain and its coverings, and skin cancer. Of the 579 exons examined (211 c-kit exon 11, 192 PDGFRalpha exon 12, 142 PDGFRalpha exon18, 17 PDGFRbeta exon 12 and 17 PDGFRbeta exon 18), only 12 (all GIST) harbored mutations (10 c-kit exon 11 and 2 PDGFRalpha exon18). From these data we conclude that activating c-KIT and PDGFR mutations are sporadic in human cancers known to overexpress these tyrosine kinase receptor genes and suggest that, except in GIST, this overexpression is not correlated with activating mutations. The latter may imply that these wild-type c-KIT and PDGFR tumor types will probably not benefit from imatinib treatment.

Asunto(s)

Antineoplásicos/farmacología , Tumores del Estroma Gastrointestinal/genética , Mutación , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-kit/genética , Pirimidinas/farmacología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Benzamidas , Análisis Mutacional de ADN , Activación Enzimática/genética , Tumores del Estroma Gastrointestinal/patología , Humanos , Mesilato de Imatinib , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo

RESUMEN

Imatinib mesylate is a selective tyrosine kinase inhibitor that is successfully used in the treatment of Philadelphia-positive chronic and acute leukaemia's, and gastrointestinal stromal tumors. We investigated whether the intended chronic oral administration of imatinib might lead to the induction of the intestinal ABC transport proteins ABCB1, ABCC1 (MRP1), ABCC2 (MRP2) and ABCG2. Using Caco2 cells as an in vitro model for intestinal drug transport, we found that continuous exposure (up to 100 days) with imatinib (10 microM) specifically upregulates the expression of ABCG2 (maximal approximately 17-fold) and ABCB1 (maximal approximately 5-fold). The induction of gene expression appeared to be biphasic in time, with a significant increase in ABCG2 and ABCB1 at day 3 and day 25, respectively, and was not mediated through activation of the human orphan nuclear receptor SXR/NR1I2. Importantly, chronic imatinib exposure of Caco2 cells resulted in a approximately 50% decrease in intracellular accumulation of imatinib, probably by enhanced ABCG2- and ABCB1-mediated efflux, as a result of upregulated expression of these drug pumps. Both ABCG2 and ABCB1 are normally expressed in the gastrointestinal tract and it might be anticipated that drug-induced upregulation of these intestinal pumps could reduce the oral bioavailability of imatinib, representing a novel mechanism of acquired pharmacokinetic drug resistance in cancer patients that are chronically treated with imatinib.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/biosíntesis , Antineoplásicos/administración & dosificación , Transporte Biológico , Expresión Génica/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas de Neoplasias/biosíntesis , Piperazinas/administración & dosificación , Pirimidinas/administración & dosificación , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Animales , Benzamidas , Células COS , Chlorocebus aethiops , Receptor de Androstano Constitutivo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Humanos , Mesilato de Imatinib , Proteínas de Transporte de Membrana , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias/metabolismo , Receptor X de Pregnano , Proteínas Tirosina Quinasas/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales Cultivadas

RESUMEN

ABCG2 (BCRP/MXR/ABCP) functions as an efflux transporter for many agents, including topotecan, and the protein is expressed at high levels in the human intestine. Some individuals possess a nonsynonymous variant in the ABCG2 gene at nucleotide 421, substituting lysine for glutamine on position 141 at exon 5. The present pilot study indicates that this genotype results in a 30% reduced efflux transport of topotecan in vitro compared to the wild-type. In a preliminary fashion, the heterozygous CA allele observed in two patients was associated with a 1.34-fold increased oral bioavailability of topotecan compared to the bioavailability in ten patients with the wild-type allele (42.0% versus 31.4%; p = 0.037). It is suggested that the high frequency of the A allele in certain ethnic groups may have therapeutic implications for individuals treated with topotecan or other ABCG2 substrates.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/genética , Antineoplásicos/farmacocinética , Carcinoma de Células Pequeñas/genética , Proteínas de Neoplasias/genética , Neoplasias Ováricas/genética , Topotecan/farmacocinética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Administración Oral , Adulto , Anciano , Sustitución de Aminoácidos , Antineoplásicos/administración & dosificación , Disponibilidad Biológica , ADN de Neoplasias/genética , Femenino , Frecuencia de los Genes , Genotipo , Heterocigoto , Humanos , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Farmacogenética , Proyectos Piloto , Polimorfismo de Nucleótido Simple , Estudios Prospectivos , Topotecan/administración & dosificación , Células Tumorales Cultivadas

RESUMEN

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.

Asunto(s)

Antineoplásicos/farmacocinética , Resistencia a Antineoplásicos/genética , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Antineoplásicos/farmacología , Área Bajo la Curva , Transporte Biológico/fisiología , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/fisiología , Polimorfismo Genético

RESUMEN

Imatinib mesylate is a selective tyrosine kinase inhibitor that is successfully used in the treatment of chronic myeloid leukaemias and gastrointestinal stromal tumours. The drug is taken orally on a daily basis in order to suppress tumour growth. Unfortunately, the vast majority of patients will eventually progress while on therapy. It is generally thought that this acquired unresponsiveness is due to gene amplification or somatic mutations in the drug's target genes. However, we have now evidence, based on several in vitro and in vivo observations suggesting that pharmacokinetic resistance may also play a definitive role in the ultimate resistance of patients on chronic imatinib. Our findings may have serious implications for the chronic imatinib treatment of cancer patients.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/metabolismo , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Piperazinas/metabolismo , Piperazinas/farmacocinética , Pirimidinas/metabolismo , Pirimidinas/farmacocinética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Administración Oral , Benzamidas , Disponibilidad Biológica , Humanos , Mesilato de Imatinib , Proteínas de Neoplasias/genética

RESUMEN

Imatinib mesylate (STI571), a potent tyrosine kinase inhibitor, is successfully used in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. However, the intended chronic oral administration of imatinib may lead to development of cellular resistance and subsequent treatment failure. Indeed, several molecular mechanisms leading to imatinib resistance have already been reported, including overexpression of the MDR1/ABCB1 drug pump. We examined whether imatinib is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump that is frequently overexpressed in human tumors. Using a panel of well-defined BCRP-overexpressing cell lines, we provide the first evidence that imatinib is a substrate for BCRP, that it competes with mitoxantrone for drug export, and that BCRP-mediated efflux can be reversed by the fumitremorgin C analog Ko-143. Since BCRP is highly expressed in the gastrointestinal tract, BCRP might not only play a role in cellular resistance of tumor cells but also influence the gastrointestinal absorption of imatinib.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Neoplasias de la Mama/patología , Resistencia a Antineoplásicos , Proteínas de Neoplasias/metabolismo , Piperazinas/metabolismo , Pirimidinas/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Benzamidas , Unión Competitiva , Neoplasias de la Mama/tratamiento farmacológico , Radioisótopos de Carbono , Línea Celular Tumoral , Doxorrubicina/metabolismo , Humanos , Mesilato de Imatinib , Mitoxantrona/metabolismo , Micotoxinas/análogos & derivados , Micotoxinas/farmacología , Especificidad por Sustrato

RESUMEN

Male germ cell tumors (GCTs) are extremely sensitive to platinum-containing chemotherapy, with only 10% of patients showing therapy resistance. However, the biological basis of the high curability of disseminated GCTs by chemotherapy is still unknown. Recently, we demonstrated that the mammalian serine/arginine-rich protein-specific kinase 1 (SRPK1) is a cisplatin-sensitive gene, inactivation of which leads to cisplatin resistance. Because, in mammalians, the expression of SRPK1 is preferentially high in testicular tissues, cisplatin responsiveness of male GCTs might be associated with SRPK1 levels. In the present study, we monitored SRPK1 protein expression in a unique series of nonseminomatous GCTs by immunohistochemistry. Randomly selected GCTs (n = 70) and tumors from patients responding to standard chemotherapy (n = 20) generally showed strong SRPK1 staining. In contrast, expression in refractory GCTs (n = 20) as well as in GCTs from poor-prognosis patients responding to high-dose chemotherapy only (n = 11) was significantly lower (two-sided Wilcoxon rank sum test: P < .001). In conclusion, our data suggest that SRPK1 expression might be an important prognostic indicator for the chemoresponsiveness of nonseminomatous GCTs.

Asunto(s)

Germinoma/enzimología , Compuestos de Platino/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Neoplasias Testiculares/enzimología , Adolescente , Adulto , Resistencia a Antineoplásicos , Regulación Neoplásica de la Expresión Génica , Germinoma/tratamiento farmacológico , Germinoma/mortalidad , Germinoma/patología , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/metabolismo , Empalme del ARN , Análisis de Supervivencia , Neoplasias Testiculares/tratamiento farmacológico , Neoplasias Testiculares/mortalidad , Neoplasias Testiculares/patología

RESUMEN

To improve the curative success of chemotherapy, it will be essential to understand the molecular basis of drug resistance (DR) and sensitivity. We have developed a cell culture system that enables the functional cloning of mammalian DR genes based on phenotypic selection after overexpression of mammalian retroviral cDNA libraries and validated our system using the anticancer drug cisplatin. ERCC1-deficient and therefore cisplatin-hypersensitive mouse embryonic fibroblast target cells were transduced with a human placenta retroviral cDNA library. Subsequent cisplatin selection yielded 20 DR clones, each containing a recurring human ERCC1 gene. Surprisingly, nine of these clones contained 5'-truncated ERCC1 sequences that required alternative splicing of the vector sequence to encode a functional ERCC1 protein. The usage of cryptic splice sites in the vector sequence should be taken into consideration when interpreting results from retroviral gene expression applications, and might have consequences for the safe application of retroviral constructs in gene therapy.

Asunto(s)

Clonación Molecular/métodos , Proteínas de Unión al ADN , Resistencia a Medicamentos/genética , Endonucleasas , Biblioteca de Genes , Retroviridae/genética , Empalme Alternativo , Animales , Antineoplásicos/farmacología , Línea Celular , Supervivencia Celular , Cisplatino/farmacología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Genes , Vectores Genéticos , Humanos , Ratones , Proteínas/genética , Proteínas/metabolismo , Eliminación de Secuencia

RESUMEN

The therapeutic potential of antitumor drugs is seriously limited by the manifestation of cellular drug resistance. We used the budding yeast Saccharomyces cerevisiae as a model system to identify novel mechanisms of resistance to one of the most active anticancer agents, cisplatin. We pinpointed NPR2 (nitrogen permease regulator 2) as a gene whose disruption conferred resistance to cisplatin. In addition, we observed a 4-fold cross-resistance of yeast npr2Delta cells (i.e., cells from which the NPR2 gene had been disrupted) to the anticancer drug doxorubicin, in combination with hypersensitivity to cadmium chloride. Furthermore, npr2Delta cells displayed unaltered cellular cisplatin and doxorubicin accumulation and showed an enhanced rate of spontaneous mutation compared with the isogenic parent. These data indicate that the npr2Delta phenotype overlaps that of the sky1Delta cells that we characterized previously (Mol Pharmacol 61:659-666, 2002). Therefore, we generated yeast npr2Delta sky1Delta double-knockout cells and performed clonogenic survival assays for cisplatin and doxorubicin, which revealed that NPR2 and SKY1 (SR-protein-specific kinase from budding yeast) are epistatic. The double-knockout strain was just as resistant to cisplatin and doxorubicin as the single-knockout strain that was most resistant to either drug. In conclusion, we identified NPR2 as a novel component involved in cell kill provoked by cisplatin and doxorubicin, and our data support the hypothesis that NPR2 and SKY1 may use mutual regulatory routes to mediate the cytotoxicity of these anticancer drugs.

Asunto(s)

Antineoplásicos/farmacología , Cisplatino/farmacología , Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/efectos de los fármacos , Resistencia a Medicamentos/fisiología , Resistencia a Antineoplásicos , Péptidos y Proteínas de Señalización Intracelular , Fenotipo , Platino (Metal)/farmacocinética , Saccharomyces cerevisiae/fisiología , Proteínas de Saccharomyces cerevisiae/genética

RESUMEN

Previously, a gender dependency of topotecan was found in the pharmacokinetics in the plasma compartment. Here, we prospectively studied the red blood cell (RBC) partitioning of topotecan and evaluated its consequences for overall drug disposition. Blood samples were obtained from 12 patients receiving cisplatin followed by i.v. topotecan. Topotecan pharmacokinetic analysis was performed in whole blood, plasma and RBCs. Significantly slower clearance was noted in females (n=7) compared to males (n=5) for lactone and total topotecan in plasma (p<0.0001), and for total drug in RBCs (p=0.027), but not in whole blood. In addition, no gender-dependent differences were observed in the terminal half-lives of topotecan in any of the compartments. The area under the curve ratios for RBC total to plasma lactone were 2.53+/-0.0640 and 2.13+/-0.442 in males and females, respectively. Hence, topotecan displays preferential affinity for RBCs compared to plasma, although these cells do not act as a depot in which drug accumulates over time. RBCs thus play a principal role in the distribution kinetics of topotecan and have a major impact on its plasma pharmacokinetics. The data warrant a change from current practice in pharmacokinetic studies with this agent and provide further evidence that, in general, the choice of the appropriate assay matrix should be rationally based.

Asunto(s)

Antineoplásicos/farmacocinética , Eritrocitos/metabolismo , Topotecan/farmacocinética , Adulto , Anciano , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Área Bajo la Curva , Cromatografía Líquida de Alta Presión/métodos , Cisplatino/administración & dosificación , Femenino , Semivida , Humanos , Infusiones Intravenosas , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Estudios Prospectivos , Factores Sexuales , Topotecan/administración & dosificación , Topotecan/sangre

RESUMEN

PURPOSE: The aim of this study was to investigate whether expression of particular drug resistance genes in primary operable breast cancer correlates with response to first-line chemotherapy in advanced disease. EXPERIMENTAL DESIGN: We determined mRNA levels of BCRP, LRP, MRP1, MRP2, and MDR1 in 59 primary breast tumor specimens of patients who received chemotherapy as first-line systemic treatment after diagnosis of advanced disease. The relative expression levels were measured by quantitative real-time reverse transcription-PCR and subsequently analyzed in relation to the type of response to chemotherapy, the length of progression-free survival (PFS), and post-relapse overall survival. RESULTS: For each of these drug resistance genes, a large variation in expression level was observed among the tumors of the different patients. When analyzing mRNA expression in relation to overall response, it was found that the median expression level of these five drug resistance genes in the responding tumors, as compared with nonresponding tumors, was markedly lower. Classification of tumors as high versus low with respect to the expression level of these genes showed that the overall response in the MDR1-high subset (17%), as compared with the MDR1-low subset (68%), was significantly lower (P = 0.005). Although similar differences in response rate were found for subsets of tumors stratified by the expression level of the other drug resistance genes, none of the observed differences were statistically significant. However, in the subgroup of patients treated with anthracycline-based chemotherapy (5-fluorouracil, Adriamycin/epirubicin, and cyclophosphamide), a correlation between response and the expression of BCRP and MRP1 (only PFS) was found, whereas such an association was not present in the cyclophosphamide, methotrexate, and 5-fluorouracil-treated group of patients. Furthermore, high expression of LRP as well as MDR1 was found to be significantly associated with a poor PFS (P = 0.04 and P < 0.001, respectively). For lung resistance-related protein, this association was limited to 5-fluorouracil, Adriamycin/epirubicin, and cyclophosphamide. Expression levels of BCRP, MRP1, or MRP2 were not related with the length of PFS. Furthermore, no correlation between the expression level of these drug resistance genes and post-relapse overall survival was found. CONCLUSIONS: In this pilot study, MDR1 expression in primary breast tumors was inversely related with the efficacy of first-line chemotherapy, and high expression level was a significant predictor of poor prognosis for patients with advanced disease. Apart from MDR1, the expression levels of BCRP, LRP, and MRP1 might have some additional predictive value for clinical outcome.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Proteínas de Transporte de Membrana , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/genética , Partículas Ribonucleoproteicas en Bóveda/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Adulto , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Secuencia de Bases , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Neoplasias de la Mama/cirugía , Cartilla de ADN , Resistencia a Múltiples Medicamentos/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Metástasis Linfática , Persona de Mediana Edad , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Valor Predictivo de las Pruebas , ARN Mensajero/genética , ARN Neoplásico/genética , Recurrencia , Estudios Retrospectivos , Análisis de Supervivencia , Factores de Tiempo , Transcripción Genética/genética

RESUMEN

PURPOSE: Despite dose calculation using body-surface area (BSA), pharmacokinetics of most anticancer drugs show wide interindividual variability. In this study, we evaluated the role of BSA in paclitaxel disposition. PATIENTS AND METHODS: Paclitaxel pharmacokinetics were prospectively studied in 12 patients that were treated in a randomized cross-over design with paclitaxel (3-hour infusion at a 3-week interval) at 175 mg/m2 in cycle 1 (A) and a flat-fixed dose of 300 mg in cycle 2 (B), or vice versa. Blood samples were collected up to 24 hours after dosing and analyzed for total and unbound paclitaxel. RESULTS: The area under the curves (AUC) of unbound paclitaxel were similar in both dosing groups, with mean values +/- SD (A v B) of 1.34 +/- 0.158 versus 1.30 +/- 0.329 microM x h, indicating that BSA-based dosing reduced the coefficient of variation by 53.3%. Unbound and total paclitaxel clearance was also significantly related to various body-size measures, including BSA (R > or = 0.617; P < or =.033), weight (R >or = 0.621; P < or =.031), and lean-body mass (r > or = 0.630; P < or = .028). We hypothesize that this is caused by the association of paclitaxel in the circulation with Cremophor EL, the distribution of which is linked to total blood volume, and thus to BSA. CONCLUSION: This study indicates that paclitaxel disposition is significantly related to BSA. This provides a pharmacokinetic rationale for BSA-based dosing of this drug.

Asunto(s)

Antineoplásicos Fitogénicos/farmacocinética , Superficie Corporal , Neoplasias/metabolismo , Paclitaxel/farmacocinética , Adulto , Anciano , Animales , Antineoplásicos Fitogénicos/administración & dosificación , Área Bajo la Curva , Estudios Cruzados , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Tasa de Depuración Metabólica , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Paclitaxel/administración & dosificación , Estudios Prospectivos

RESUMEN

New solubilizers, including Sorporol 230, Sorporol 120Ex, Aceporol 345-T, Aceporol 460 and Riciporol 335, as potential new delivery vehicles for paclitaxel were investigated, since recent studies have shown that the paclitaxel delivery vehicle Cremophor EL significantly alters the pharmacokinetics of paclitaxel. Cremophor EL and Tween 80 were used as a reference. As in the case of Cremophor EL, alteration of blood distribution of paclitaxel occurred in the presence of all tested vehicles. Also, no differences in the affinity of paclitaxel for the tested solubilizers was found during equilibrium dialysis experiments. The different vehicles could be distinguished by a different rate of esterase-mediated breakdown, which was correlated with the fatty acid content of the solubilizers. The activation of the complement cascade was less pronounced for all solubilizers, except Riciporol 335, compared to Cremophor EL. The strategies presented here provide the possibility to rapidly screen future candidate delivery vehicles with optimal characteristics for use as a solubilizer in clinical formulations of paclitaxel or other poorly water-soluble drugs.

Asunto(s)

Antineoplásicos Fitogénicos/administración & dosificación , Paclitaxel/administración & dosificación , Vehículos Farmacéuticos/química , Antineoplásicos Fitogénicos/sangre , Antineoplásicos Fitogénicos/química , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Humanos , Técnicas In Vitro , Indicadores y Reactivos , Paclitaxel/sangre , Paclitaxel/química , Soluciones Farmacéuticas , Estándares de Referencia , Solubilidad

RESUMEN

It has been hypothesized that the paclitaxel vehicle Cremophor EL (CrEL) is responsible for nonlinear drug disposition by micellar entrapment. To gain further insight into the role of CrEL in taxane pharmacology, we studied the pharmacokinetics of paclitaxel in the presence and absence of CrEL after i.p. and i.v. dosing. Patients received an i.p. tracer dose of [G-(3)H]paclitaxel in ethanol without CrEL (100 microCi diluted further in isotonic saline) on day 1, i.p. paclitaxel formulated in CrEL (Taxol; 125 mg/m(2)) on day 4, an i.v. tracer of [G-(3)H]paclitaxel on day 22, and i.v. Taxol (175 mg/m(2)) on day 24. Four patients (age range, 54-74 years) were studied, and serial plasma samples up to 72 h were obtained and analyzed for total radioactivity, paclitaxel, and CrEL. In the presence of CrEL, i.v. paclitaxel clearance was 10.2 +/- 3.76 liters/h/m(2) (mean +/- SD), consistent with previous findings. The terminal disposition half-life was substantially prolonged after i.p. dosing (17.0 +/- 11.3 versus 28.7 +/- 8.72 h), as was the mean residence time (7.28 +/- 2.76 versus 40.7 +/- 13.8 h). The bioavailability of paclitaxel was 31.4 +/- 5.18%, indicating insignificant systemic concentrations after i.p. treatment. CrEL levels were undetectable after i.p. dosing (<0.05 microl/ml), whereas after i.v. dosing, the mean clearance was 159 +/- 58.4 ml/h/m(2), in line with earlier observations. In the absence of CrEL, the bioavailability and systemic concentrations of i.p. paclitaxel were significantly increased. This finding is consistent with the postulated concept that CrEL is largely responsible for the pharmacokinetic advantage for peritoneal cavity exposure to total paclitaxel compared with systemic delivery.

Asunto(s)

Antineoplásicos Fitogénicos/farmacocinética , Glicerol/análogos & derivados , Glicerol/farmacología , Mesotelioma/tratamiento farmacológico , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacocinética , Anciano , Antineoplásicos Fitogénicos/efectos adversos , Antineoplásicos Fitogénicos/sangre , Líquido Ascítico/metabolismo , Disponibilidad Biológica , Femenino , Enfermedades Gastrointestinales/inducido químicamente , Enfermedades Hematológicas/inducido químicamente , Humanos , Inyecciones Intraperitoneales , Inyecciones Intravenosas , Mesotelioma/metabolismo , Persona de Mediana Edad , Neoplasias Ováricas/metabolismo , Paclitaxel/efectos adversos , Paclitaxel/sangre

RESUMEN

An additional chromatographic peak was observed in plasma samples of patients receiving NX 211, a liposomal formulation of the topoisomerase I inhibitor lurtotecan. We have isolated and purified this product by sequential solid-phase extractions, and we report its structure and cytotoxicity relative to lurtotecan and related agents. Nuclear magnetic resonance data indicate that cleavage of the piperazino moiety occurred at the N-C bond of the B-ring, yielding 7-methyl-10,11-ethylenedioxy-20(S)-camptothecin (MEC). Tests of the growth inhibition potential of MEC in seven human tumor cell lines showed that the compound was approximately 2-18-fold more cytotoxic than lurtotecan, topotecan, and 7-ethyl-10-hydroxy-20(S)-camptothecin (SN-38). Subsequently, we found that MEC was the product of rapid photolysis of lurtotecan, with the rate of degradation inversely proportional to NX 211 concentrations, and greatly depends on light intensity. Furthermore, MEC concentrations were found to increase significantly in plasma samples exposed to laboratory light but not in blood. MEC was not produced from NX 211 in the presence of human liver microsomes, suggesting that it is not a product of cytochrome P-450 metabolism. Using a validated analytical method, trace levels of MEC were quantitated in blood samples of two patients. These observations confirm that the precautions for protection from light currently specified for preparation and administration of NX 211 dose solutions are critical. Procedures to minimize formation of MEC, by the use of amber vials for NX 211 and by preparation of dilutions immediately before clinical use in a fashion completely protected from light, are now being routinely implemented.

Asunto(s)

Antineoplásicos/efectos de la radiación , Camptotecina/química , Camptotecina/farmacología , Camptotecina/efectos de la radiación , Adulto , Camptotecina/análogos & derivados , Camptotecina/aislamiento & purificación , División Celular/efectos de los fármacos , Cromatografía Líquida de Alta Presión , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Luz , Masculino , Persona de Mediana Edad , Estructura Molecular , Células Tumorales Cultivadas/efectos de los fármacos