ABSTRACT
Cyclosporine A (CsA) produces oxidative stress and apoptosis in rat hepatocytes, but it is not known whether membrane lipid peroxidation plays a role in CsA toxicity. The objective of the present study was to determine whether iron-catalyzed hydroxyl or membrane alkoxyl radical formation is causally involved in the prooxidative cell injury and apoptosis. As previously reported, cultured rat hepatocytes exposed to CsA exhibited concentration-dependent signs of apoptotic cell injury, including chromatin condensation and fragmentation, increased caspase-3 activity, and release of cytosolic lactate dehydrogenase. Addition of the ferric iron chelator desferrioxamine or the novel potent lipophilic iron chelator CGP72670 did not protect against CsA-induced cell death, indicating that iron-catalyzed lipid peroxidation is unlikely to be involved in CsA-mediated apoptosis. In contrast, the classical chain-breaking lipid peroxidation inhibitor alpha-tocopherol was able to rescue cells from CsA-induced cytotoxicity and apoptosis. alpha-Tocopherol not only effectively inhibited the production of thiobarbituric acid-reactive substances and the formation of reactive oxygen species, but it also prevented proteins from being oxidized and forming mixed disulfides. Furthermore, alpha-tocopherol inhibited the cellular uptake of extracellular calcium (45 Ca 2+) into cells, similar to the reducing agent dithiothreitol. By decreasing the extracellular calcium concentrations or by adding calcium channel blockers (diltiazem, nifedipine) or a cell-permeable calcium chelator Bis-(o-aminophenoxy)-ethane-N,N,N',N'-tertraacetic acid (BAPTA), both CsA-induced caspase-3 activation and apoptosis were inhibited, indicating a pivotal role of Ca 2+ in mediating CsA-induced cell injury. These results suggest that alpha-tocopherol protects from CsA-mediated apoptosis and cytotoxictiy by preventing the oxidation of redox-sensitive Ca 2+ -ATPase. Thus, it is the attenuation of increased calcium influx and, hence, the inhibition of caspase-3 activation, rather than the downstream inhibition of lipid peroxidation, that is a key mechanism of the protection provided by alpha-tocopherol against CsA-induced cell injury.
ABSTRACT
The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.
Subject(s)
Antioxidants/pharmacology , Apoptosis , Cyclosporins/pharmacology , Hepatocytes/drug effects , Animals , Antimetabolites/pharmacology , Buthionine Sulfoximine/pharmacology , Calcium/metabolism , Caspase 3 , Caspases/metabolism , Chromatin/drug effects , Cyclosporine/pharmacology , DNA Fragmentation/drug effects , Drug Interactions , Hepatocytes/pathology , L-Lactate Dehydrogenase/metabolism , Male , Rats , Rats, WistarSubject(s)
HIV Infections/drug therapy , HIV Protease Inhibitors/pharmacokinetics , Nelfinavir/pharmacokinetics , Saquinavir/administration & dosage , Saquinavir/pharmacokinetics , Biological Availability , Child , Child, Preschool , Dose-Response Relationship, Drug , Drug Administration Schedule , Drug Therapy, Combination , Female , Follow-Up Studies , HIV Infections/diagnosis , HIV Protease Inhibitors/administration & dosage , Humans , Long-Term Care , Male , Nelfinavir/administration & dosage , Prospective Studies , Treatment OutcomeABSTRACT
OBJECTIVE: Our objective was to investigate the clinical pharmacologic characteristics of saquinavir given as a soft gelatin capsule, either alone or in combination with nelfinavir, to children and adolescents with human immunodeficiency virus infection. METHODS: The pharmacokinetics of 50 mg/kg saquinavir 3 times a day (tid) alone versus 33 mg/kg saquinavir tid plus 30 mg/kg nelfinavir tid was assessed after single-dose administration and after short- and long-term administration. The single-dose pharmacokinetics of fixed (1200 mg) versus unrestricted weight-adjusted dosing (50 mg/kg) was also investigated. RESULTS: Saquinavir as the sole protease inhibitor resulted in lower saquinavir exposure in children (steady-state geometric mean area under the concentration-time curve from time zero to 24 hours [AUC (0-24 h)], 5790 ng x h/ml; steady-state concentration 8 hours after drug administration [C(8h,SS)], 65 ng/ml) and adolescents [steady-state geometric mean AUC(0-24 h), 5914 ng x h/ml] than that reported in adults treated with 1200 mg tid [steady-state geometric mean AUC(0-24 h), 21,700 ng x h/ml; C(8h,SS), 223 ng/ml]. This finding appeared to be attributable to markedly higher apparent oral clearance, potentially as a result of increased systemic clearance and reduced oral bioavailability. Nelfinavir combined with saquinavir reduced apparent oral clearance, increasing saquinavir exposure in children [steady-state geometric mean AUC(0-24 h), 11,070 ng x h/ml; C(8h,SS), 380 ng/ml] to levels that approach those observed in adults. A significant correlation between average trough concentration and sustained viral load suppression was observed in children. The apparent threshold for maintaining viral load suppression was a mean trough saquinavir concentration above 200 ng/ml. CONCLUSIONS: The pharmacokinetics of saquinavir in children is different from that of adults, and administration of saquinavir alone will not give consistently efficacious plasma levels. The best way of improving saquinavir exposure in children is through combination therapy with other protease inhibitors that inhibit saquinavir metabolism.
Subject(s)
HIV Infections , HIV Protease Inhibitors , Nelfinavir , Saquinavir , Adolescent , Area Under Curve , Blood Proteins/metabolism , Child , Child, Preschool , Drug Administration Schedule , Drug Therapy, Combination , HIV Infections/drug therapy , HIV Infections/metabolism , HIV Protease Inhibitors/pharmacokinetics , HIV Protease Inhibitors/pharmacology , HIV Protease Inhibitors/therapeutic use , Humans , Nelfinavir/pharmacokinetics , Nelfinavir/therapeutic use , Randomized Controlled Trials as Topic , Saquinavir/pharmacokinetics , Saquinavir/pharmacology , Saquinavir/therapeutic useABSTRACT
La cefalosporina A (CsA) se utiliza frecuentemente en hepatocitos de rata y fracciones mitocondriales hepáticas aisladas como inhibidor específico de la liberación de Ca2+ y como bloqueador selectivo de la permeabilidad y del potencial de membrana mitocondriales, procesos implicados en la inhibición de la apoptosis. Sin embargo, hasta ahora no se ha descrito ni la inhibición ni la inducción de apoptosis por CsA en cultivos primarios de hepatocitos de rata tras su incubación por un periodo de 4 a 20 horas. El propósito de este estudio ha sido examinar con criterios morfológicos y bioquímicos los efectos de la CsA sobre la apoptosis y esclarecer las características de los mecanismos subyacentes. Los hepatocitos de rata se cultivaron durante 4-20 horas con CsA a concentraciones de 0, 10, 25 y 50 µM. Se investigaron los fenómenos de condensación y fragmentación de la cromatina, fragmentación de ADN (TUNEL), distribución de fosfatidilcolina en la membrana (Anexina V), así como la actividad enzimática de caspasas 1, -3 y -6, el potencial de membrana mitocondrial (Rhodamina 123) y la liberación de citocromo C en el citosol. Tras cuatro horas de incubación con CsA, la condensación y fragmentación de la cromatina y el número de células TUNEL y Anexina V positivas aumentaron en función de la dosis sin que se observara pérdida enzimática, lo que indica la integridad de la membrana celular externa. Después de 20 horas de incubación con CsA, experimentaron unmayor incremento acompañado del aumento de las actividad de cistein-proteasa, caspasa-3 (CPP32) y de un ligero incremento de caspasa-6 (Mch 2), pero no de caspasa-1 (ICE). El inhibidor de caspasa-6, Ac-VEID-CHO, únicamente inhibió la apoptosis inducida por CsA. El descenso de potencial de membrana mitocondrial y la liberación de citocromo C fueron paralelos a los cambios de ultraestructuras mitocondriales y pudieran considerarse reacciones tempranas que desencadenan la cascada de fenómenos apoptóticos. La microscopía de transmisión electrónica (TEM) confirmó el incremento del número de células necróticas al cabo de 20 horas, pero no tras 4 horas de incubación, en comparación con los controles. (AU)
