Amisulpride is an atypical antipsychotic which antagonises dopamine (D2, D3) receptors in vitro and in vivo [1]. However, there is not enough data for its safe and effective dosage in older people or people with dementia, including Alzheimer’s disease (AD) [2]. Importantly, aged patients, and especially older patients with dementia show increased sensitivity to atypical antipsychotics, including amisulpride [2,3]. Studies have suggested that central pharmacokinetic alterations at the blood-brain barrier (BBB) might cause the increased sensitivity [3, 4]. Our present study examines healthy and Alzheimer’s disease physiology to further understand this increased sensitivity. In silico molecular docking studies were used to identify transporters of interest for our model substrate amisulpride. In vitro we studied the impact of the solute carrier transporters: plasma membrane monoamine transporter (PMAT), multi-antimicrobial extrusion proteins 1 and 2 (MATE1 and MATE2) on amisulpride transport in human cerebral microvessel endothelial cells/D3 (hCMEC/D3). We incubated the cells with [3H]amisulpride (3.8-7.7 nM) and [14C]sucrose (0.7-1.5 μM) with or without transporter inhibitors. In vivo, we investigated the BBB transport of amisulpride in 5xFamilial Alzheimer’s mouse model (5xFAD), and in age matched wild type mice (WT, C57/BL6) (12-15 months old). All experiments were performed in accordance with the Animal Scientific Procedures Act (1986) and Amendment Regulations 2012 and with consideration to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. All mice were anaesthetised via intraperitoneal injection of a mixture of medetomidine hydrochloride (2 mg/kg, Vetoquinol UK Limited) and ketamine (150 mg/kg, Pfizer, UK, and Chanelle, UK). Once the absence of surrogate indicators of consciousness was confirmed, the mice were perfused with artificial plasma, containing [3H]amisulpride (6.5 nM) and [14C]sucrose (9.4 μM). The presence of amyloid plaques was confirmed in 5 and 12 months old 5xFAD mice by transmission electron microscopy. The presence of PMAT in hCMEC/D3, and in WT and 5xFAD brain capillary samples was studied by Western blot. Preliminary in silico analysis and molecular docking suggested that amisulpride is a substrate of MATE1, PMAT, organic cation transporter 1 (OCT1), and glucose transporter 1 (GLUT1). In hCMEC/D3 cells PMAT inhibition led to a significant increase in [3H]amisulpride cell accumulation (F (1, 58) = 16.33, p=0.0002). The increase was observed at 20, 30, 60 and 120 minutes by 63.9% (p=0.0338); 83.3% (p=0.0237); 85.1% (p=0.0010); and 68.6% (p=0.0001), respectively. MATE1 and MATE2 inhibition did not cause an effect. Compared to WT (n=6), the 5xFAD (n=7) mice had increased [3H]amisulpride uptake in the striatum (t=1.975, df=11, p=0.0370), whereas [14C]sucrose (passive permeability measure) permeability was not significantly changed. Western blots confirmed PMAT expression in hCMEC/D3 cells. OCT1, MATE1 and MATE2 expression in hCMEC/D3 cells has previously been confirmed by our group [4, 5]. Also, we found that PMAT is expressed in WT and 5xFAD brain capillaries. PMAT, MATE1, OCT1 and GLUT1 are implicated in the transport of amisulpride at the BBB. The increased brain permeability to amisulpride in 5xFAD mice suggests altered BBB transporter function, possibly due to changes in brain capillary SLC transporter expression with AD [4].