BackgroundResearch into amisulpride use in Alzheimer’s disease (AD) implicates blood–brain barrier (BBB) dysfunction in antipsychotic sensitivity. Research into BBB transporters has been mainly directed towards the ABC superfamily, however, solute carrier (SLC) function in AD has not been widely studied. This study tests the hypothesis that transporters for organic cations contribute to the BBB delivery of the antipsychotics (amisulpride and haloperidol) and is disrupted in AD.MethodsThe accumulation of [3H]amisulpride (3.7–7.7 nM) and [3H]haloperidol (10 nM) in human (hCMEC/D3) and mouse (bEnd.3) brain endothelial cell lines was explored. Computational approaches examined molecular level interactions of both drugs with the SLC transporters [organic cation transporter 1 (OCT1), plasma membrane monoamine transporter (PMAT) and multi-drug and toxic compound extrusion proteins (MATE1)] and amisulpride with the ABC transporter (P-glycoprotein). The distribution of [3H]amisulpride in wildtype and 3×transgenic AD mice was examined using in situ brain perfusion experiments. Western blots determined transporter expression in mouse and human brain capillaries .ResultsIn vitro BBB and in silico transporter studies indicated that [3H]amisulpride and [3H]haloperidol were transported by the influx transporter, OCT1, and efflux transporters MATE1 and PMAT. Amisulpride did not have a strong interaction with OCTN1, OCTN2, P-gp, BCRP or MRP and could not be described as a substrate for these transporters. Amisulpride brain uptake was increased in AD mice compared to wildtype mice, but vascular space was unaffected. There were no measurable changes in the expression of MATE1, MATE2, PMAT OCT1, OCT2, OCT3, OCTN1, OCTN2 and P-gp in capillaries isolated from whole brain homogenates from the AD mice compared to wildtype mice. Although, PMAT and MATE1 expression was reduced in capillaries obtained from specific human brain regions (i.e. putamen and caudate) from AD cases (Braak stage V–VI) compared to age matched controls (Braak stage 0–II).ConclusionsTogether our research indicates that the increased sensitivity of individuals with Alzheimer’s to amisulpride is related to previously unreported changes in function and expression of SLC transporters at the BBB (in particular PMAT and MATE1). Dose adjustments may be required for drugs that are substrates of these transporters when prescribing for individuals with AD.

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates and inhibitors, and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride, and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT and 5xFAD mice, and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol), and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose accumulation in hCMEC/D3. 5xFAD mice showed increased brain [3H]amisulpride uptake, and no cerebrovascular space changes compared to WT. TEM revealed brain capillary degeneration in human AD. There was no significant effect of AD on mouse GLUT1 and P-gp BBB expression, and in human GLUT1 expression. In contrast, caudate P-glycoprotein expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1, and that BBB transporter expression is altered in AD. This suggests that antipsychotics exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.

Background Amisulpride is an atypical antipsychotic which antagonises dopamine (D2, D3) receptors in vitro and in vivo (Schoemaker et al., 1997). Older people, particularly those with dementia are more susceptible to antipsychotic side effects, including amisulpride (Reeves et al., 2017). Clinical and basic science research suggested that blood‐brain barrier (BBB) disruption underpins this heightened sensitivity by increasing central drug access (Sekhar et al., 2019; Harwood et al., 1994). The current study examines healthy and Alzheimer’s disease (AD) physiology to further understand this increased sensitivity. Method 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). 5xFAD mice express human amyloid precursor protein and presenilin 1 transgenes with five AD‐linked mutations, they develop Aβ plaques and cognitive impairments (Oakley et al., 2006). All experiments were performed according the Animal Scientific Procedures Act (1986) and Amendment Regulations 2012. Anaesthesia was applied via intraperitoneal injection of medetomidine hydrochloride and ketamine mixture. The mice were perfused with artificial plasma, containing [3H]amisulpride (6.5 nM) and [14C]sucrose (9.4 μM). Brain amyloid plaques were confirmed in 5xFAD mice by transmission electron microscopy. In silico molecular docking using AuDock Vina and GOLD software identified transporters of interest for our model substrate. Amisulpride was considered a substrate for a given transporter, if it had free energy binding lower than ‐5 kcal/mole and high chem score. Result Compared to WT (n=6), the 5xFAD (n=7) mice had increased striatal [3H]amisulpride uptake of 79% (t=1.975, df=11, p=0.0370). The [14C]sucrose (passive permeability measure) permeability was not significantly changed. Preliminary in silico analysis suggested that amisulpride is a substrate for glucose transporter 1 (GLUT1) and a very weak substrate for multidrug and toxin extrusion transporter (MATE2) (Table 1). Conclusion In silico studies suggested amisulpride interacts with BBB solute carrier (SLC) transporters: organic cation transporter (OCT1), plasma membrane monoamine transporter (PMAT), MATE1 (Sekhar et al., 2019), GLUT1 and MATE2, but not with the adenosine triphosphate binding cassette (ABC) transporter P‐glycoprotein. The increased brain permeability to amisulpride in 5xFAD mice suggests altered BBB transporter function, possibly due to SLC transporter expression changes associated with AD.

2 0 RUNNING TITLE: Brain delivery of drugs in Alzheimer's.2 1