Purpose: Membrane transporters of native compounds can take part in drug transport in the central nervous system. A new class of ascorbic acid (AA) transporters, SVCT2, localised in the choroid plexus, has been recently characterised, and human retinal pigment epithelium (HRPE) cells were proposed as a model for the in vitro analysis of SVCT2 transport. Diclofenac (Diclo) and its AA or amino-AA (AAA) pro-drugs (AA-Diclo or AAA-Diclo) were studied on HRPE cells to evaluate their ability to interact with the vitamin C transporter SVCT2 and their uptake in cells. Furthermore, the stability in physiological fluids was evaluated. Methods: Transport assays were performed in HRPE cells using [14C]AA and inhibition of AA transport was determined by adding the unlabelled compounds to plated cells along with [14C]AA. Intracellular accumulation was measured incubating HRPE cells with increasing concentrations of unlabelled cpmpounds and following by HPLC analysis. For kinetic experiments, AA-Diclo was incubated at 37C in Tris-HCl Buffer (pH 7.4), human fresh plasma or whole blood. The extracted samples were analyzed by HPLC. Results: Diclo resulted a non-competitive inhibitor of AA transport, showing a Na+-dependent and ascorbate-independent uptake. AA-Diclo appeared a competitive inhibitor which isn’t transported into cells. AAA-Diclo appeared as a weak inhibitor of the AA transport, SVCT2-mediated. AA-Diclo is hydrolysed following a first order kinetics in buffer, plasma (t1/2 about 10 hours) and whole blood (t1/2 about 3 hours). Conclusions: The discovery of a Na+- dependent transport system for Diclo on HRPE cells opens new perspectives for targeting studies of this drug to the brain. Stability studies suggest AA-Diclo as a potential candidate to enhance the short half life of Diclo in vivo.
Diclofenamic acid-vitamin C conjugates: stability and interaction studies with ascorbate transporters
DALPIAZ, Alessandro;COLOMBO, Gaia;BIONDI, Carla;MANFREDINI, Stefano;MENEGATTI, Enea;SCATTURIN, Angelo
2004
Abstract
Purpose: Membrane transporters of native compounds can take part in drug transport in the central nervous system. A new class of ascorbic acid (AA) transporters, SVCT2, localised in the choroid plexus, has been recently characterised, and human retinal pigment epithelium (HRPE) cells were proposed as a model for the in vitro analysis of SVCT2 transport. Diclofenac (Diclo) and its AA or amino-AA (AAA) pro-drugs (AA-Diclo or AAA-Diclo) were studied on HRPE cells to evaluate their ability to interact with the vitamin C transporter SVCT2 and their uptake in cells. Furthermore, the stability in physiological fluids was evaluated. Methods: Transport assays were performed in HRPE cells using [14C]AA and inhibition of AA transport was determined by adding the unlabelled compounds to plated cells along with [14C]AA. Intracellular accumulation was measured incubating HRPE cells with increasing concentrations of unlabelled cpmpounds and following by HPLC analysis. For kinetic experiments, AA-Diclo was incubated at 37C in Tris-HCl Buffer (pH 7.4), human fresh plasma or whole blood. The extracted samples were analyzed by HPLC. Results: Diclo resulted a non-competitive inhibitor of AA transport, showing a Na+-dependent and ascorbate-independent uptake. AA-Diclo appeared a competitive inhibitor which isn’t transported into cells. AAA-Diclo appeared as a weak inhibitor of the AA transport, SVCT2-mediated. AA-Diclo is hydrolysed following a first order kinetics in buffer, plasma (t1/2 about 10 hours) and whole blood (t1/2 about 3 hours). Conclusions: The discovery of a Na+- dependent transport system for Diclo on HRPE cells opens new perspectives for targeting studies of this drug to the brain. Stability studies suggest AA-Diclo as a potential candidate to enhance the short half life of Diclo in vivo.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
