RESUMO
Delivery of valproic acid (VPA) to the human brain is relatively inefficient as reflected by a low brain-to-unbound plasma concentration ratio (< or =0.5) at steady state. Previous pharmacokinetic studies suggested that the unfavorable brain-to-plasma gradient is maintained by coupled efflux transport processes at both the brain parenchymal cells and blood-brain barrier (BBB); one or both of the efflux transporters are inhibitable by probenecid. The present study in rabbits utilized microdialysis to measure drug concentration in the brain extracellular fluid (ECF) of the cerebral cortex during steady-state i.v. infusion with VPA alone or with VPA plus probenecid. Probenecid co-infusion elevated VPA concentration in the brain tissue surrounding the tip of the microdialysis probe to a greater extent than in the ECF (230% versus 47%). Brain intracellular compartment (ICC) concentration was estimated. In control rabbits, the ICC concentration was 2.8+/-0.28 times higher than the ECF concentration. Probenecid co-infusion elevated the ICC-to-ECF concentration ratio to 4.2+/-0.44, which confirms the existence of an efflux transport system in brain parenchymal cells. The ECF-to-unbound plasma concentration ratio was well below unity (0.029), indicating an uphill efflux transport of VPA across the BBB. Co-infusion of probenecid did not have a significant effect on VPA efflux at the BBB as evidenced by a minimal change in the ECF-to-unbound plasma concentration ratio. This study suggests the presence of distinctly different organic anion transporters for the efflux of VPA at the parenchymal cells and capillary endothelium in the brain.
Assuntos
Transporte Biológico Ativo/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Interações Medicamentosas/fisiologia , Espaço Extracelular/efeitos dos fármacos , Probenecid/farmacologia , Ácido Valproico/farmacocinética , Animais , Transporte Biológico Ativo/fisiologia , Barreira Hematoencefálica/fisiologia , Encéfalo/citologia , Encéfalo/metabolismo , Proteínas de Transporte/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Epilepsia/tratamento farmacológico , Espaço Extracelular/metabolismo , Líquido Intracelular/efeitos dos fármacos , Líquido Intracelular/metabolismo , Bombas de Íon/efeitos dos fármacos , Bombas de Íon/metabolismo , Masculino , Microdiálise/estatística & dados numéricos , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Coelhos , Ácido Valproico/sangueRESUMO
Cocultures of human umbilical vein endothelial cells (ECV304) and rat glioma cells (C6) from two commercial sources, American Type Culture Collection and European Collection of Animal Cell Cultures, were evaluated as an in vitro model for the blood-brain barrier. Monolayers of endothelial cells grown in the presence or absence of glial cells were examined for transendothelial electrical resistance, sucrose permeability, morphology, multidrug resistance-associated protein expression, and P-glycoprotein expression and function. Coculture of glial cells with endothelial cells increased electrical resistance and decreased sucrose permeability across European endothelial cell monolayers, but had no effect on American endothelial cells. Coculture of European glial cells with endothelial cells caused cell flattening and decreased cell stacking with both European and American endothelial cells. No P-glycoprotein or multidrug resistance-associated protein was immunodetected in endothelial cells grown in glial cell-conditioned medium. Functional P-glycoprotein was demonstrated in American endothelial cells selected in vinblastine-containing medium over eight passages, but these cells did not form a tight endothelium. In conclusion, while European glial cells confer blood-brain barrier-like morphology and barrier integrity to European endothelial cells in coculture, the European endothelial-glial cell coculture model does not express P-glycoprotein, normally found at the blood-brain barrier. Further, the response of endothelial cells to glial factors was dependent on cell source, implying heterogeneity among cell populations. On the basis of these observations, the umbilical vein endothelial cell-glial cell coculture model does not appear to be a viable model for predicting blood-brain barrier penetration of drug molecules.
Assuntos
Barreira Hematoencefálica , Endotélio Vascular/citologia , Glioma/patologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Técnicas de Cocultura , Endotélio Vascular/metabolismo , Estudos de Avaliação como Assunto , Glioma/metabolismo , Humanos , Camundongos , Microscopia Eletrônica , Modelos Biológicos , Ratos , Sacarose/metabolismo , Células Tumorais CultivadasRESUMO
The P-glycoprotein efflux system located on the apical membrane of brain capillary endothelial cells functions as part of the blood-brain barrier. In this study, primary cultures of bovine brain microvessel endothelial cells (BMECs) were investigated for the presence of a P-glycoprotein system and its contribution in regulating ivermectin distribution across the blood-brain barrier. Results of rhodamine 123 uptake studies with cyclosporin A and verapamil as substrates indicated that a functional efflux system was present on BMECs. Immunoblot analysis with the C219 monoclonal antibody to the product of the multidrug resistant member 1(MDR1) gene also confirmed the expression of MDR1 in the BMECs. Unbound ivermectin was shown to significantly increase the uptake of rhodamine 123 in BMECs, however, the drug only modestly enhanced the transcellular passage of rhodamine. The results of these studies affirmed that unbound ivermectin is an inhibitor of the MDR1 efflux system in BMECs.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Encéfalo/irrigação sanguínea , Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Ivermectina/metabolismo , Animais , Encéfalo/citologia , Bovinos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Corantes Fluorescentes/metabolismo , Immunoblotting , Ivermectina/farmacologia , Microcirculação/citologia , Microcirculação/metabolismo , Rodamina 123 , Rodaminas/metabolismoRESUMO
E-Delta 2-valproic acid (E-Delta 2-VPA), a major active metabolite of VPA, has been proposed as an alternative to VPA because it is less hepatotoxic and is nonteratogenic. In rodents, VPA and E-Delta 2-VPA have a brain tissue/free plasma concentration ratio less than unity, which suggests rapid removal of the alkanoate anticonvulsants from the central nervous system. This study in rabbits employed a simultaneous iv infusion-ventriculocisternal (VC) perfusion technique to investigate the steady-state kinetics of E-Delta 2-VPA transport at the blood-brain barrier, the blood-cerebrospinal fluid (CSF) barrier, and the neural cell membrane. Probenecid (PBD) was coadministered to probe the mediation of transport by organic anion transporter(s). Rabbits in the control group (N = 6) received an iv infusion of E-Delta 2-VPA to achieve a steady-state plasma concentration of 50 to 60 microg/ml. Blood and cisternal outflow of mock CSF perfusate were continuously sampled. Midway through the experiment, the VC perfusate was switched to one containing [3H]E-Delta 2-VPA. At 225 min, the rabbits were sacrificed, and each brain was removed and dissected into ten regions. Rabbits in the PBD group (N = 9) received an iv infusion and VC perfusion as in the control group as well as concomitant iv infusion of the inhibitor. The mean steady-state VC extraction ratio for [3H]E-Delta 2-VPA did not differ between the control and PBD groups (63.7 +/- 8.3% vs. 60. 6 +/- 9.6%), indicating the lack of a significant PBD-sensitive transport at the choroidal epithelium. Coadministration of PBD elevated brain concentration of cold E-Delta 2-VPA in the absence of a significant change in total or free steady-state plasma concentration. Mean E-Delta 2-VPA brain tissue/free plasma concentration ratios in the various brain regions were 3.5- to 5.2-fold higher in PBD-treated animals than in the controls. Significant increases (3.0- to 4.5-fold) in the mean brain tissue/cisternal perfusate concentration ratios were also observed. Compartmental modeling of the steady-state distribution data suggested that clearance of E-Delta 2-VPA from the brain parenchyma is governed jointly by efflux transporters at the neural cell membrane and brain capillary endothelium. Moreover, PBD-induced elevation of E-Delta 2-VPA tissue concentrations is attributed primarily to inhibition of E-Delta 2-VPA efflux transport at the neural cell membrane, resulting in both intracellular trapping and greater tissue retention of E-Delta 2-VPA.