Cellular Uptake of the Atypical Antipsychotic Clozapine Is a Carrier-Mediated Process

Abstract

The weak base antipsychotic clozapine is the most effective medication for treating refractory schizophrenia. The brain-to-plasma concentration of unbound clozapine is greater than unity, indicating transporter-mediated uptake, which has been insufficiently studied. This is important, because it could have a significant impact on clozapine's efficacy, drug-drug interaction, and safety profile. A major limitation of clozapine's use is the risk of clozapine-induced agranulocytosis/granulocytopenia (CIAG), which is a rare but severe hematological adverse drug reaction. We first studied the uptake of clozapine into human brain endothelial cells (hCMEC/D3). Clozapine uptake into cells was consistent with a carrier-mediated process, which was time-dependent and saturable ( V = 3299 pmol/million cells/min, K = 35.9 μM). The chemical inhibitors lamotrigine, quetiapine, olanzapine, prazosin, verapamil, indatraline, and chlorpromazine reduced the uptake of clozapine by up to 95%. This could in part explain the in vivo interactions observed in rodents or humans for these compounds. An extensive set of studies utilizing transporter-overexpressing cell lines and siRNA-mediated transporter knockdown in hCMEC/D3 cells showed that clozapine was not a substrate of OCT1 (SLC22A1), OCT3 (SLC22A3), OCTN1 (SLC22A4), OCTN2 (SLC22A5), ENT1 (SLC29A1), ENT2 (SLC29A2), and ENT4/PMAT (SLC29A4). In a recent genome-wide analysis, the hepatic uptake transporters SLCO1B1 (OATP1B1) and SLCO1B3 (OATP1B3) were identified as additional candidate transporters. We therefore also investigated clozapine transport into OATP1B-transfected cells and found that clozapine was neither a substrate nor an inhibitor of OATP1B1 and OATP1B3. In summary, we have identified a carrier-mediated process for clozapine uptake into brain, which may be partly responsible for clozapine's high unbound accumulation in the brain and its drug-drug interaction profile. Cellular clozapine uptake is independent from currently known drug transporters, and thus, molecular identification of the clozapine transporter will help to understand clozapine's efficacy and safety profile.

IPEC-J2 rMdr1a, a New Cell Line with Functional Expression of Rat P-glycoprotein Encoded by Rat Mdr1a for Drug Screening Purposes

“…K p,uu values larger than unity could indicate active uptake transport of olanzapine across the BBB in the rodent animal models. This is corroborated by a report by Dickens et al, who found that clozapine, a second-generation antipsychotic structurally similar to olanzapine, was actively taken up by the human brain endothelial cell line hCMEC/D3 [32]. It is therefore not unlikely that the discrepancy between in vitro observation of efflux transport of olanzapine and K p,uu values larger than unity in animal models can be explained by active uptake processes outcompeting potential weak efflux transport, rather than species differences.…”

The biology of ergothioneine, an antioxidant nutraceutical

“…Although this view remains controversial, even hydrophobic molecules do not normally 'float across' whatever phospholipid bilayer portion of cells may be untrammelled by proteins. Xenobiotics in particular need to 'hitchhike' on protein transporters that have presumably evolved for 'natural' substrates but that are capable of their uptake (142)(143)(144)(145)(146)(147)(148)(149)(150)(151)(152) . While transporters seem to have remained somewhat understudied (153) , those transporters involved in uptake and encoded by the human genome are now catalogued formally as SLC for solute carriers (154,155) , with efflux transporters mainly being classed as ABC families (156) .…”

Golgi localized β1-adrenergic receptors stimulate Golgi PI4P hydrolysis by PLCε to regulate cardiac hypertrophy

“…5B) and lamotrigine (10 µM, Fig. 5C)(25) to determine if OCT3 is required for NE-stimulated intracellular PI4P hydrolysis.Preincubation of NRVMs with any of these inhibitors prevented NE from inducing PI4P hydrolysis, suggesting that OCT3-mediated transport is required for PI4P hydrolysis by this catecholamine. In addition, we sought to determine if receptor internalization is required for NE-mediated PI4P hydrolysis and bAR activation at the Golgi.…”