Mechanism of brain targeting by dexibuprofen prodrugs modified with ethanolamine-related structures

Abstract

The first molecular insights into how prodrugs modified with ethanolamine-related structures target the brain were generated using an in vitro BBB model and in situ perfusion technique. Prodrugs were delivered safely and efficiently to the brain through tight interaction with the anionic membrane of brain capillary endothelial cells, observed as a shift in zeta potential, followed by uptake into the cells. Prodrugs III and IV carrying primary and secondary amine modifications appeared to enter the brain via energy-independent passive diffusion. In contrast, besides the passive diffusion, prodrugs I and II carrying tertiary amine modifications also appeared to enter via an active process that was energy and pH dependent but was independent of sodium or membrane potential. This active process involved, at least in part, the pyrilamine-sensitive H(+)/OC antiporter, for which the N,N-diethyl-based compound II showed a much lower affinity than the N,N-dimethyl-based compound I, likely due to steric hindrance. These new insights into brain-targeting mechanisms may help guide efforts to design new prodrugs.

Figure 1

Chemical structures of dexibuprofen and prodrugs I, II, III, and IV.

Figure 2

Morphologic analysis of in vitro models of blood–brain barrier. Scanning electron micrographs of (A) blood to brain by capillary endothelial cells (BCECs) and (B) ACs; (C) Light micrographs of BCECs. (D) Transmission electron micrograph of the in vitro BCEC-AC coculture model.

Figure 3

Effects of prodrugs on the tight junctions of the in vitro model of blood–brain barrier (BBB). (A) Cytotoxicity of dexibuprofen (DIBU) and of prodrug I, II, III, or IV on blood to brain by capillary endothelial cells (BCECs) after 4-hour incubation. Results were presented as average cell viability %±s.d. (n=5). (B) Transendothelial electrical resisitance (TEER) changes in the BBB model before and after exposure to DIBU or one of the four prodrugs. Data represent mean±s.d. (n=3). (C) F-actin filaments in a BCEC monolayer were incubated for 30 minutes with DIBU or prodrug, then observed by laser confocal scanning microscopy. Cells without any drug treatment served as controls. Scale bar=20 μm. (D) Effects of DIBU and prodrugs on the zeta potential of BCECs. Cultures were treated for 30 minutes at 37°C with 80 μmol/L DIBU or prodrug. Cultures were treated with TMA-DPH served as a positive control, while cultures incubated with phosphate-buffered saline (PBS) served as a negative control. Data represent mean±s.d. (n=3). **P<0.01 versus control group. TMA-DPH, 1-(4-(trimethylamino) phenyl)-6-phenyl-1,3,5-hexatriene.

Figure 4

Uptake of dexibuprofen (DIBU) and prodrugs I, II, III, and IV by blood to brain by capillary endothelial cell (BCEC) monolayers under various conditions. (A) Cultures were exposed for 1 hour at 37°C to various DIBU or prodrug doses (20, 40, 60, 80, and 120 μmol/L). (B) Cultures were exposed to 80 μmol/L DIBU or prodrug and analyzed at 10, 20, 30, 60, and 120 minutes. (C) Cultures were exposed for 1 hour to 80 μmol/L DIBU or prodrug at either 37 °C or 4 °C. (D) Cultures were exposed for 1 hour at 37 °C to 80 μmol/L prodrug in the presence of metabolic energy inhibitors. (E) Cultures of either BCECs or L929 cells were exposed at 37°C for 1 hour to 80 μmol/L DIBU or prodrug. Data represent mean±s.d. (n=3). **P<0.01.

Figure 5

Mechanistic studies of the uptake of prodrugs I and II by blood to brain by capillary endothelial cell (BCEC) monolayers. (A) Effect of extracellular pH on uptake of 80 μmol/L prodrug after 1-hour incubation at 37°C. (B) Effect of intracellular pH on uptake of 80 μmol/L prodrug after 1-hour incubation at 37°C. (C) Effects of replacing sodium ion and altering membrane potential on uptake of 80 μmol/L prodrug after 1-hour incubation at 37°C. Cultures were incubated in sodium-containing buffer (control) or sodium-free buffer in which sodium had been replaced with N-methylglucamine or potassium. Cultures were also pretreated with 10 μmol/L valinomycin, and then uptake was measured in buffer containing sodium and valinomycin. Potential competitive inhibitors (pyrilamine, propranolol, imipramine, hemicholinium-3, choline, l-carnitine, tetraethylammonium chloride (TEA), l-lysine, l-arginine, and spermine) were incubated with cultures for 1 hour at 37°C, and uptake of (D) prodrug I and (E) prodrug II was examined. Lineweaver-Burk plot of the inhibitory effect of pyrilamine on the uptake of prodrug I (F) and prodrug II (G). Cellular uptake of I and II (20 to 120 μmol/L, for 20 minutes) was measured in the absence (−) or presence of 200 μmol/L pyrilamine (•). Data represent mean±s.d. (n=3). *P<0.05; **P<0.01.

Figure 6

Mechanism of transendothelial transport of DIBU and prodrugs in an in vitro BCEC-AC coculture model of the blood–brain barrier. (A) Accumulated cleared volume (μL) of DIBU and prodrugs across the barrier. (B) P_app value of DIBU and prodrugs at 37°C or 4°C or in the presence of NaN₃. *P<0.05; **P<0.01, compared with P_app values obtained at 37°C. Transport efficiency of (C) prodrugs I and (D) II in the presence of the inhibitors pyrilamine, propranolol, or imipramine. The amount of I or II transported at 37°C served as the control. *P<0.05; **P<0.01, compared with the control group. Data represent mean±s.d. (n=3). BCEC, blood to brain by capillary endothelial cell.

Figure 7

In vivo prodrug transport from blood to brain in rats, measured using the in situ brain perfusion method. (A) Brain uptake of DIBU or prodrug after 30-second perfusion at 37°C or 4°C or in the presence of NaN₃. *P<0.05 versus 37°C; **P<0.01 versus 37°C. (B) Uptake of I and II by the brain after perfusion with or without pyrilamine (1 mmol/L). *P<0.05 versus the control; **P<0.01 versus the control. Data represent mean±s.d. (n=5).