RESUMO
Expression of multidrug resistance (MDR) features by acute myeloid leukemia (AML) cells predicts a poor response to many treatments. The MDR phenotype often correlates with expression of P-glycoprotein (Pgp), and Pgp antagonists such as cyclosporine (CSA) have been used as chemosensitizing agents in AML. Gemtuzumab ozogamicin, an immunoconjugate of an anti-CD33 antibody linked to calicheamicin, is effective monotherapy for CD33(+) relapsed AML. However, the contribution of Pgp to gemtuzumab ozogamicin resistance is poorly defined. In this study, blast cell samples from relapsed AML patients eligible for gemtuzumab ozogamicin clinical trials were assayed for Pgp surface expression and Pgp function using a dye efflux assay. In most cases, surface expression of Pgp correlated with Pgp function, as indicated by elevated dye efflux that was inhibited by CSA. Among samples from patients who either failed to clear marrow blasts or failed to achieve remission, 72% or 52%, respectively, exhibited CSA-sensitive dye efflux compared with 29% (P =.003) or 24% (P <.001) among samples from responders. In vitro gemtuzumab ozogamicin--induced apoptosis was also evaluated using an annexin V--based assay. Low levels of drug-induced apoptosis were associated with CSA-sensitive dye efflux, whereas higher levels correlated strongly with achievement of remission and marrow blast clearance. In vitro drug-induced apoptosis could be increased by CSA in 14 (29%) of 49 samples exhibiting low apoptosis in the absence of CSA. Together, these findings indicate that Pgp plays a role in clinical resistance to gemtuzumab ozogamicin and suggest that treatment trials combining gemtuzumab ozogamicin with MDR reversal agents are warranted. (Blood. 2001;98:988-994)
Assuntos
Aminoglicosídeos , Antibacterianos/farmacologia , Anticorpos Monoclonais/farmacologia , Ensaios Clínicos Fase II como Assunto , Resistência a Múltiplos Medicamentos/genética , Resistência a Múltiplos Medicamentos/imunologia , Leucemia Mieloide/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Doença Aguda , Anticorpos Monoclonais Humanizados , Apoptose/efeitos dos fármacos , Medula Óssea/patologia , Carbocianinas/farmacocinética , Ciclosporina/farmacologia , Sinergismo Farmacológico , Corantes Fluorescentes , Gemtuzumab , Humanos , Imunotoxinas/farmacologia , Leucemia Mieloide/patologia , Leucócitos Mononucleares/patologia , Fenótipo , Análise de Regressão , Indução de Remissão , Resultado do Tratamento , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
In this review, we summarize and update our data on the development of a multi-unit anti-BCR/ABL ribozyme. In vitro studies comparing several anti-BCR/ABL ribozymes demonstrated that a triple-unit ribozyme is the most efficient. Detailed kinetic analysis revealed this ribozyme to have a lower Kcat, most likely due to non homologous bases at restriction enzyme sites used in ribozyme construction. Delivery of this ribozyme to a BCR/ABL transformed cell line by a novel vehicle targeting the folate receptor resulted in a 3 log reduction in BCR/ABL mRNA when analyzed by RT-PCR. This delivery strategy reversed the IL-3 independence of this cell line. Retroviral vectors containing genes coding for the multi-unit ribozyme have been constructed and their use to effect BCR/ABL transformed cell biology is discussed.
Assuntos
Proteínas de Fusão bcr-abl/antagonistas & inibidores , Terapia Genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , RNA Catalítico/uso terapêutico , Animais , Sequência de Bases , Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Humanos , Cinética , Dados de Sequência Molecular , RNA Catalítico/química , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , Especificidade por Substrato , Transfecção , Células Tumorais CultivadasRESUMO
Chronic myelogenous leukemia is characterized by the Philadelphia chromosome, which at the molecular level results from the fusion of the bcr gene on chromosome 22 and the abl gene on chromosome 9. The bcr-abl fusion gene encodes a novel tyrosine kinase with transforming activity. In this study, we have synthesized a multi-unti ribozyme that targets bcr-abl mRNA. In vitro ribozyme cleavage reactions show increased cleavage efficiency of this multi-unit ribozyme compared with single or double ribozymes. The multiunit ribozyme was then transfected into murine myeloblasts transformed with the bcr-abl gene (32D cells). Ribozyme transfection was accomplished either by liposomes or using follic acid-polylysine as a carrier. Multi-unit ribozyme transfection reduced the level of bcr-abl mRNA 3 logs when transfected via folate receptor-mediated uptake into transformed 32D cells. These results suggest that a multi-unit ribozyme could be an effective therapeutic agent for the treatment of Philadelphia chromosome-positive chronic myelogenous leukemia.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , RNA Catalítico/metabolismo , RNA Mensageiro/metabolismo , RNA Neoplásico/metabolismo , Receptores de Superfície Celular , Animais , Sequência de Bases , Transporte Biológico , Purging da Medula Óssea , Proteínas de Transporte/metabolismo , Catálise , Linhagem Celular Transformada , Clonagem Molecular , DNA Complementar/síntese química , DNA Complementar/genética , Desenho de Fármacos , Receptores de Folato com Âncoras de GPI , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Vetores Genéticos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Antissenso/farmacologia , RNA Catalítico/farmacologia , RNA Catalítico/uso terapêutico , TransfecçãoAssuntos
Purging da Medula Óssea/métodos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , RNA Catalítico , Sequência de Bases , Transplante de Medula Óssea , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Dados de Sequência Molecular , RNA Catalítico/administração & dosagem , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Transfecção , Transplante Autólogo , Células Tumorais CultivadasRESUMO
Bactericidal levels of gentamicin were obtained in vitreous humor by iontophoresis of antibiotic directly through the sclera. A silicone rubber tube of small diameter filled with gentamicin sulfate formed the electrode. A two milliampere current applied for three minutes to each of four perilimbal sites introduced gentamicin sufficient to maintain therapeutic levels for more than 24 hours. The proportion of drug reaching vitreous versus aqueous humor was dependent on electrode position relative to retina and pars plana. An endogenous antibacterial agent was apparently released during iontophoretic stimulation and interfered with the bacterial growth inhibition assay. Ketoconazole, a water-insoluble antifungal agent, has also been introduced successfully into vitreous humor by anodal iontophoresis after protonation in weak acid.
