RESUMEN
The pharmacokinetics of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, were characterized within rodent whole liver, and cellular and subcellular compartments. Cross-species comparisons were performed using Sprague-Dawley rat and CD-1 mouse strains. Although whole liver oligonucleotide deposition and the proportion of drug found within parenchymal and nonparenchymal cells were similar between the two rodent species as a function of both time and dose, dramatic differences in subcellular pharmacokinetics were observed. Specifically, within murine hepatocyte nuclei, drug was observed at the 10 mg/kg dose, whereas in the rat nuclear-associated levels required the administration of 25 mg/kg. Under all experimental regimens, murine hepatic nuclear-associated drug concentrations were at least 2-fold higher than those found in rat liver cells. More detailed metabolic analysis was also performed using high performance liquid chromatography/electrospray-mass spectrometry (HPLC/ES-MS) and demonstrated that although the extent of metabolism was similar for rat and mouse, the pattern of n-1 metabolites varied as a function of both species and cell type. While rat and mouse hepatocytes and rat nonparenchymal cellular metabolites were predominantly products of 3'-exonuclease degradation, mouse nonparenchymal cells contained a majority of n-1 metabolites produced by 5'-exonucleolytic activity. Based upon these data, it would appear that subcellular oligonucleotide disposition and metabolism among rodent species are more divergent than whole organ pharmacokinetics might predict.
Asunto(s)
Hígado/metabolismo , Oligodesoxirribonucleótidos Antisentido/farmacocinética , Tionucleótidos/farmacocinética , Animales , Antivirales/metabolismo , Antivirales/farmacocinética , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Cinética , Hígado/efectos de los fármacos , Masculino , Oligodesoxirribonucleótidos Antisentido/metabolismo , Ratas , Ratas Sprague-Dawley , Fracciones Subcelulares/metabolismo , Tionucleótidos/metabolismo , Factores de Tiempo , Distribución TisularRESUMEN
It has been suggested that binding of phosphorothioate oligodeoxynucleotides (P=S ODNs) to macrophage scavenger receptors (SR-AI/II) is the primary mechanism of P=S ODN uptake into cells in vivo. To address the role of scavenger receptors in P=S ODN distribution in vivo, several pharmacokinetic and pharmacological parameters were compared in tissues from scavenger receptor knockout mice (SR-A-/-) and their wild-type counterparts after i.v. administration of 5- and 20-mg/kg doses of P=S ODN. With an antibody that recognizes P=S ODN, no differences in cellular distribution or staining intensity in livers, kidneys, lungs, or spleens taken from SR-A-/- versus wild-type mice could be detected at the histological level. There were no significant differences in P=S ODN concentrations in these organs as measured by capillary gel electrophoresis as well, although the concentration of P=S ODN in isolated Kupffer cells from livers of SR-A-/- mice was 25% lower than that in Kupffer cells from wild-type mice. Furthermore, a P=S ODN targeting murine A-raf reduced A-raf RNA levels to a similar extent in livers from SRA-/- (92.8%) and wild-type (88.3%) mice. Finally, in vitro P=S ODN uptake studies in peritoneal macrophages from SR-A-/- versus wild-type mice indicate that other high- and low-affinity uptake mechanisms predominate. Taken as a whole, our data suggest that, although there may be some contribution to P=S ODN uptake by the SR-AI/II receptor, this mechanism alone cannot account for the bulk of P=S ODN distribution into tissues and cells in vivo, including macrophages.
Asunto(s)
Proteínas de la Membrana , Oligodesoxirribonucleótidos Antisentido/metabolismo , Receptores Inmunológicos/clasificación , Receptores Inmunológicos/metabolismo , Receptores de Lipoproteína , Tionucleótidos/farmacocinética , Animales , Células Cultivadas , Electroforesis Capilar , Inmunohistoquímica , Técnicas In Vitro , Riñón/metabolismo , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , ARN/metabolismo , Receptores Depuradores , Receptores Depuradores de Clase A , Receptores Depuradores de Clase B , Bazo/metabolismoRESUMEN
Phosphorothioate antisense oligodeoxynucleotides are novel therapeutic agents designed to selectively and specifically inhibit production of various disease-related gene products. In vivo pharmacokinetic experiments indicate that these molecules are widely distributed in many species, with the majority of oligomers accumulating within liver and kidney. To better understand the metabolism of these agents, we studied the stability of several phosphorothioate oligodeoxynucleotides, their congeners, and second generation oligomer chemistries in rat liver homogenates. To examine metabolism, background nuclease activity was characterized in whole liver homogenates by using ISIS 1049, a 21-mer phosphodiester oligodeoxynucleotide. Nuclease activity could readily be detected in liver homogenates. Under optimized conditions, the predominant enzymatic activity was 3'-exonucleolytic and could be influenced by pH and ionic conditions. However, in addition to 3' exonucleases, 5' exo- and endonuclease activities were also observed. Our data indicate that metabolism of phosphorothioate oligodeoxynucleotides was more complex than that of phosphodiesters for many reasons, including phosphorothioate oligodeoxynucleotide inhibition of nucleases and the presence of R(p) and S(p) stereoisomers. The rate of phosphorothioate metabolism also appeared to be influenced by sequence, with pyrimidine-rich compounds being metabolized to a greater extent than purine-rich oligomers. Other factors affecting stability included oligomer chemistry and length. Concomitant experiments performed in rats dosed systemically with the same compounds mimic the activities seen in vitro and suggest that this liver homogenate system is a valuable model with which to study the mechanism of metabolism of antisense oligonucleotides.
Asunto(s)
Hígado/metabolismo , Oligodesoxirribonucleótidos/farmacología , Oligonucleótidos Antisentido/metabolismo , Animales , Estabilidad de Medicamentos , Endonucleasas/metabolismo , Exonucleasas/antagonistas & inhibidores , Exonucleasas/metabolismo , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Hígado/efectos de los fármacos , Masculino , Concentración Osmolar , Oxidación-Reducción , Perfusión , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Most drugs for cancer therapy are targeted to relative differences in the biological characteristics of cancer cells and normal cells. The therapeutic index of such drugs is theoretically limited by the magnitude of such differences, and most anticancer drugs have considerable toxicity to normal cells. Here we describe a new approach for developing anticancer drugs. This approach, termed variagenic targeting, exploits the absolute difference in the genotype of normal cells and cancer cells arising from normal gene sequence variation in essential genes and loss of heterozygosity (LOH) occurring during oncogenesis. The technology involves identifying genes that are: 1) essential for cell survival; 2) are expressed as multiple alleles in the normal population because of the presence of one or more nucleotide polymorphisms; and 3) are frequently subject to LOH in several common cancers. An allele-specific drug inhibiting the essential gene remaining in cancer cells would be lethal to the malignant cell and would have minimal toxicity to the normal heterozygous cell that retains the drug-insensitive allele. With antisense oligonucleotides designed to target two alternative alleles of replication protein A, 70-kDa subunit (RPA70) we demonstrate in vitro selective killing of cancer cells that contain only the sensitive allele of the target gene without killing cells expressing the alternative RPA70 allele. Additionally, we identify several other candidate genes for variagenic targeting. This technology represents a new approach for the discovery of agents with high therapeutics indices for treating cancer and other proliferative disorders.
Asunto(s)
Antineoplásicos/uso terapéutico , Pérdida de Heterocigocidad , Neoplasias/tratamiento farmacológico , Oligorribonucleótidos Antisentido/uso terapéutico , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Diseño de Fármacos , Estudios de Factibilidad , Marcación de Gen , Variación Genética , Genoma Humano , Células HeLa , Humanos , Neoplasias/genética , Oligorribonucleótidos Antisentido/farmacología , Proteína de Replicación A , Supresión Genética , Células Tumorales CultivadasRESUMEN
In the rat, the liver represents a major site of phosphorothioate oligodeoxynucleotide deposition after i.v. administration. For this reason, we examined the intracellular fate of ISIS 1082, a 21-base heterosequence phosphorothioate oligodeoxynucleotide, isolated from parenchymal and nonparenchymal cell types after systemic dosing using established perfusion and separation techniques followed by CGE. Isolated cells were further fractionated into nuclear, cytosolic and membrane constituents to assess the intracellular localization, distribution and metabolic profiles as a function of time and dose. After a 10-mg/kg i.v. bolus, intracellular drug levels where maximal after 8 hr and diminished significantly thereafter, suggesting an active efflux mechanism or metabolism. Nonparenchymal (i.e., Kupffer and endothelial) cells contained approximately 80% of the total organ cellular dose, and this was equivalently distributed between the two cell types, while the remaining 20% was associated with hepatocytes. Nonparenchymal cells contained abundant nuclear, cytosolic and membrane drug levels over a wide dose range. In contrast, at doses of less than 25 mg/kg, hepatocytes contained significantly less drug with no detectable nuclear-association. Doses at or above 25 mg/kg appeared to saturate nonparenchymal cell types, whereas hepatocytes continued to accumulate drug in all cellular compartments, including the nucleus. Our results suggest that although pharmacokinetic parameters vary as a function of hepatic cell type, significant intracellular delivery can be readily achieved in the liver after systemic administration.
Asunto(s)
Hígado/metabolismo , Oligodesoxirribonucleótidos Antisentido , Oligonucleótidos Antisentido/farmacocinética , Tionucleótidos/farmacocinética , Animales , Relación Dosis-Respuesta a Droga , Inyecciones Intravenosas , Hígado/ultraestructura , Masculino , Oligonucleótidos Antisentido/administración & dosificación , Ratas , Ratas Sprague-Dawley , Tionucleótidos/administración & dosificaciónRESUMEN
1. The effects of variations in substrates on the kinetic properties of Escherichia coli RNase H were studied using antisense oligonucleotides of various types hybridized to complementary oligoribonucleotides. The enzyme displayed minimal sequence preference, initiated cleavage through an endonucleolytic mechanism near the 3' terminus of the RNA in a DNA-RNA chimera and then was processively exonucleolytic. Phosphorothioate oligodeoxynucleotides hybridized to RNA supported cleavage more effectively than phosphodiester oligodeoxynucleotides. Oligonucleotides comprised of 2'-methoxy-, 2'-fluoro- or 2'-propoxy-nucleosides did not support RNase H1 activity. 2. The Km and Vmax. of cleavage of RNA duplexes with full phosphorothioate oligodeoxynucleotides were compared with methoxy-deoxy 'gapmers', i.e.; oligonucleotides with 2'-methoxy wings surrounding a deoxynucleotide centre. Such structural modifications resulted in substantial increases in affinity, but significant reductions in cleavage efficiency. The initial rates of cleavage increased as the deoxynucleotide gap size was increased. Multiple deoxynucleotide gaps increased the Vmax. but had little effect on Km. 3. The effects of several base modifications on the site of initial cleavage, processivity and initial rate of cleavage were also studied.