RESUMO
We constructed a series of adenoviral (Ad) vectors that express the Candida albicans cytosine deaminase (CD) suicide gene under the transcriptional control of either the human alpha-lactalbumin (ALA) or ovine beta-lactoglobulin (BLG) promoter (Ad.ALA.CD and Ad.BLG.CD, respectively). The Ad.ALA.CD and the Ad.BLG.CD vectors converted the prodrug 5-fluorocytosine (5-FC) to the toxic nucleotide analog 5-fluorouracil in a breast cancer cell-specific manner, with a conversion rate of 40% and 52% in T47D cells and 50% and 41% in MCF7 cells, respectively. No significant conversion (< or =3%) was observed in an immortalized nontumorigenic breast epithelial cell line (MCF10A) and a human osteosarcoma cell line (U2OS). Adenovirus vector-based prodrug conversion of the 5-FC in T47D and MCF7 in the presence of 1 mg/mL of 5-FC led to cytotoxicity that resulted in a nearly complete cell death (> or =90%) after 5 days, whereas MCF10A and U2OS cells remained resistant (< or =10%). Nude mice harboring T47D-derived breast tumors that were injected intratumorally (i.t.) with therapeutic adenovirus vectors at a dose of 2 x 10(8) plaque-forming units and treated systemically with 5-FC at a concentration of 500 mg/kg/day showed a marked reduction in tumor mass within 30 days when compared with animals that received vector alone. Animal survival was significantly prolonged after 72 days in mice treated with therapeutic vectors in conjunction with prodrug when compared with control animals. These preclinical data are sufficiently promising to warrant further studies of this transcriptional targeting approach to breast cancer treatment.
Assuntos
Neoplasias da Mama/enzimologia , Candida albicans/enzimologia , Flucitosina/metabolismo , Fluoruracila/metabolismo , Terapia Genética/métodos , Nucleosídeo Desaminases/genética , Receptores de Estrogênio , Ativação Transcricional , Adenoviridae/enzimologia , Adenoviridae/genética , Animais , Neoplasias da Mama/patologia , Proteínas de Transporte/metabolismo , Sobrevivência Celular , Citosina Desaminase , Feminino , Expressão Gênica , Marcação de Genes , Vetores Genéticos , Humanos , Lactalbumina/genética , Lactalbumina/metabolismo , Lactoglobulinas/genética , Lactoglobulinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nucleosídeo Desaminases/biossíntese , Pró-Fármacos/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
Direct measurements of cutaneous drug levels and kinetics have long been hampered by lack of appropriate methods. Recently, studies have indicated that microdialysis, a method of continuous in vivo sampling of extracellular fluid, may also be performed in human skin. The present study was designed to evaluate this technique for kinetic analyses of cutaneous drug levels. Using a transdermal nicotine delivery system with 35 mg of nicotine as a model, nicotine levels were determined in the dialysate of human skin by means of high performance liquid chromatography. In vitro studies demonstrated that nicotine levels in the dialysate strictly correlated with nicotine concentrations in the dialyzed medium. In nine healthy male volunteers receiving nicotine by transdermal delivery, nicotine was detectable within 90-180 min, and peak levels of approximately 1000 ng/ml were detected within 240-360 min of patch application. Correlation analyses of the individual data from our subjects revealed that nicotine kinetics were independent of skin barrier function, as assessed by transepidermal water loss, but indicated that the detectable maximum nicotine levels may depend on the location of the probe. In summary, the present study demonstrates that microdialysis may be a novel, powerful tool to study cutaneous pharmacology in vivo.
