Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3

Abstract

R( )-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson's disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R( )-apomorphine and its S-enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R( )-or S( )-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy-and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R( )-and S( )-apomorphine increased with time. The initial uptake velocities of R( )-and S( )-apomorphine were concentration-dependent, with similar K m and V max values. The cell-to-medium (C/M) ratio of R( )-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R( )-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R( )-apomorphine transport in human blood-brain barrier (BBB) model cells is similar to S( )-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium-or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.Key words apomorphine; blood-brain barrier; proton-coupled organic cation antiporter; hCMEC/D3 cell R(−)-Apomorphine, a dopamine agonist, has been approved as a subcutaneous injection formulation for rescue management of motor function impairment associated with levodopa therapy in patients with Parkinson's disease. It has been reported to have a 12 times higher unbound concentration in the brain than the blood (brain-to-blood unbound concentration ratio, K puu =12, 1) whereas S(+)-apomorphine, the dopamine receptor-inactive enantiomer, has a K puu of 5.1) These results suggest that both enantiomers are actively transported into the brain across the blood-brain barrier (BBB) in rats. However, the mechanism of blood-to-brain transport of this drug across the BBB remains unknown.The immortalized human brain capillary endothelial cell line, hCMEC/D3 has been extensively validated as a BBB model by means of pharmacological, toxicological, immunological and infection studies in numerous laboratories worldwide, and it is established that hCMEC/D3 cells retain many of the morphologi...

Involvement of a proton‐coupled organic cation antiporter in the blood–brain barrier transport of amantadine

“…A proton‐coupled organic cation transporter has been shown to be functionally expressed in hCMEC/D3 cells and in TR‐BBB13 cells and is known to be responsible for the in vitro uptake of the ionized forms of other organic cationic drugs such as tramadol , diphenhydramine , pyrilamine , oxycodone , memantine , nicotine and apomorphine . Higuchi et al have recently suggested that the predicted steady‐state brain interstitial concentration of memantine in human produced by the proton‐coupled organic cation antiporter could be sufficient for therapeutic efficacy .…”

“…Higuchi et al have recently suggested that the predicted steady‐state brain interstitial concentration of memantine in human produced by the proton‐coupled organic cation antiporter could be sufficient for therapeutic efficacy . Furthermore, amantadine has been demonstrated to have extensive potency for modulating the proton‐coupled organic cation transporter . Interestingly, since the adamantane moiety is a very small molecule, it has been conjugated to compounds that are poorly absorbed into the CNS in order to create a brain‐directed lipid carrier with improved BBB permeability .…”

Development of a Rat Plasma and Brain Extracellular Fluid Pharmacokinetic Model for Bupropion and Hydroxybupropion Based on Microdialysis Sampling, and Application to Predict Human Brain Concentrations

“…The net uptake asymmetry observed is similar to oxycodone BBB transport in rats, (Boström et al, 2006), which showed a net 3-fold asymmetry favoring rat brain ECF relative to unbound plasma concentrations. Subsequent in vitro studies with oxycodone (Okura et al, 2008)-as well as more recent in vitro transport or in situ brain perfusion studies with several other low molecular weight weakly basic drugs, such as apomorphine (Okura et al, 2014), clonidine (André et al, 2009), codeine (Fischer et al, 2010), diphenhydramine (Shimomura et al, 2013), and nicotine (Cisternino et al, 2013)-implicate a pH-dependent proton-coupled antiporter at the BBB (Gharavi et al, 2015). It is possible that this transporter may also apply to bupropion and hydroxybupropion.…”

Involvement of Proton-Coupled Organic Cation Antiporter in Varenicline Transport at Blood-Brain Barrier of Rats and in Human Brain Capillary Endothelial Cells

“…9,10 In addition to these transporters, it has been reported that proton-coupled organic cation (H þ /OC) antiporter functions at human and rodent BBB, 10,11 although the molecular entity involved has not been identified yet. This antiporter contributes to BBB transport of various weakly basic drugs, including oxycodone, 11 pyrilamine, 12 diphenhydramine, 13 apomorphine, 14 tramadol, 15 memantine, 16 and clonidine. 17 Thus, it appears to recognize bulky and lipophilic type II cations with amine structures.…”