Abstract
l-Type Amino Acid Transporter 1 (LAT1/Lat1) is responsible for carrying large, neutral l-amino acids as well as several drugs and prodrugs across the blood-brain barrier (BBB). However, the BBB is not the only barrier that hinders drugs acting effectively within the brain; the brain parenchymal cell membranes represent a secondary barrier for the drugs with intracellular target sites. In this study, expression and function of Lat1 was quantified in mouse primary neuron, astrocyte and immortalized microglia (BV2) cultures. Moreover, ability of Lat1 to carry prodrugs inside these brain cells was evaluated. The results showed that Lat1 was localized at the similar level in all studied cells (3.07 ± 0.92–3.77 ± 0.91 fmol/µg protein). The transporter was also functional in all three cell types, astrocytes having the highest transport capacity and affinity for the LAT1/Lat1-substrate, [14C]-l-leucine, followed by neurons and microglia. The designed prodrugs (1-6) were able to utilize Lat1 for their cellular uptake and it was mainly much higher than the one of their parent drugs. Interestingly, improved cellular uptake was also achieved in cells representing Alzheimer’s Disease phenotype. Therefore, improved delivery and intra-brain targeting of drugs can be attained by utilizing LAT1/Lat1 and prodrug approach.
Highlights
Johanna Huttunen[1], Soile Peltokangas[1], Mikko Gynther[1], Teemu Natunen[2], Mikko Hiltunen[2], Seppo Auriola[1], Marika Ruponen[1], Kati-Sisko Vellonen1 & Kristiina M
The expression of Glut[1] varied between the cell types, being highest in primary astrocytes (26.93 ± 1.16 fmol/μg protein), followed by immortalized microglia (17.65 ± 0.07 fmol/μg protein) and lowest in primary neurons (1.13 ± 0.22 fmol/μg protein) (Fig. 1b), which was in accordance to the literature
Instead, do not express nearly at all any form of Glut[1], since glucose is transported into the neurons mainly via Glut[3]
Summary
Johanna Huttunen[1], Soile Peltokangas[1], Mikko Gynther[1], Teemu Natunen[2], Mikko Hiltunen[2], Seppo Auriola[1], Marika Ruponen[1], Kati-Sisko Vellonen1 & Kristiina M. It has been reported that murine primary neurons, astrocytes and microglia express Lat[1] mRNA16, this does not reflect the expression or functionality of the protein on the plasma membrane This is important to understand, in order to develop intra-brain targeted drugs, i.e., drugs that can in addition of crossing the BBB, penetrate the secondary barrier of brain parenchymal cell membranes. We have recently reported that neither Alzheimer’s disease (AD) induced alterations of transgenic mice nor lipopolysaccharide (LPS)-induced neuroinflammation changed the expression or function of Lat[1] at the BBB or primary astrocytes[27,28] This signifies that LAT1/Lat[1] can be utilized for targeted drug delivery into the healthy brain and brain predisposed to pathological changes of brain diseases. The results reported here will increase the current understanding of CNS-drug disposition and highlight the importance of transporters’ role in intra-brain targeted drug delivery that can aid in future CNS-drug development
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