Structural Requirements for Uptake of Diphenhydramine Analogs into hCMEC/D3 Cells Via the Proton-Coupled Organic Cation Antiporter

There is increasing evidence that a proton-coupled organic cation (H⁺/OC) antiporter facilitates uptake of various central nervous system-active drugs, such as the histamine H1 receptor antagonist diphenhydramine, into the brain. The purpose of this study was to clarify the structural requirements for H⁺/OC antiporter-mediated uptake into hCMEC/D3 cells, an established in vitro model of the human blood-brain barrier, by using a series of diphenhydramine analogs. For this purpose, we synthesized seven tertiary amine analogs and three amide analogs. Uptake of all the amines was facilitated by an outwardly directed H⁺ gradient and inhibited by pyrilamine, a typical substrate and a strong inhibitor of the H⁺/OC antiporter. Further, uptake of most of the amines was trans-stimulated by pyrilamine. Uptake of the amines was 21 times faster than that of the amides on average, even though the lipophilicity (log D_7.4) of the amines is lower than that of the amides. Amines containing a pyrrolidine or piperidine ring showed the highest uptake rates. Our results suggest that an amine moiety, especially a heterocyclic amine moiety, is important for recognition and transport by the H⁺/OC antiporter.