Abstract ID 15609

Poster Board 563

Purpose: Studies from our lab have shown that metformin attenuates post-ischemic brain injury through Nrf2 activation. Metformin is a small hydrophilic molecule that accumulates in the cells driven by membrane potential. Its potential interaction with transporters (uptake and efflux) is unknown. Four types of organic cationic transporters- OCT1, OCT2, OCT3, and PMAT have been identified at the luminal membrane of blood-brain barrier (BBB). MATE-1, expressed at the BBB, has also been shown to be responsible for the tubular and biliary secretion of metformin. Additionally, efflux transporters such as plasma glycoprotein (P-gp), expressed by the BBB, might influence brain access of metformin.

Method: A co-culture of bEnd3 cells and primary astrocytes for permeability experiments were used as an in-vitro BBB model. [14C] metformin treatment in presence of increasing amounts of unlabelled metformin was used to determine saturation of influx transporters. The permeability coefficient (PC) value of metformin was calculated as PC=(dQ/dt) *(1/(Co*A). For influx transporter-specific inhibition, unlabelled metformin (10 μM) treatment was performed in the presence of transporter-specific inhibitors. The amount of metformin in the samples was analyzed using LC-MS/MS method. For metformin’s interaction with the efflux transporter, MDR1 gene-transfected P-gp overexpressing cells were used. The PC of metformin in apical-to-basolateral (A>B) and basolateral-to-apical (B>A) in the presence and absence of P-gp potent inhibitor, Cyclosporine A (CsA) was calculated. Finally, the unidirectional flux ratio (UFR) and the efflux ratio (ER) were determined.

Result: It was shown that with inhibitory concentrations of 10 mM and 20 mM, there was a significant decrease in [14C] metformin transport (p<0.0001). This suggests that influx transporters for [14C] metformin start getting saturated at an inhibitory concentration of more than 1 mM. For the transporter-specific inhibition study, with mitoxantrone, metformin showed a significant reduction in permeability (p<0.005). With corticosterone, there is a further reduction in permeability (p<0.05), which suggests that, in addition to OCT-1, OCT-2 and/or OCT-3 are involved in metformin’s transport. However, there is no significant difference between corticosterone, desipramine, and MPP+, which rules out the possibility of other transporters like PMAT, SERT, or CHT involved in metformin’s transport. Next, for the interaction of metformin with p-gp, the UFR, and ER was found to be 1.24 and 0.64, respectively. Studies report that compounds with UFR and ER less than 2, are poor substrates for p-gp.

Conclusion: Our findings suggest that metformin uses a transporter-specific mechanism for its entry across the in vitro BBB model. OCT-1, 2, and 3 are involved in the influx of metformin into the brain, while it does not interact with efflux transporter such as p-gp. Our ongoing and future studies are related to metformin transport using in vitro ischemia model of oxygen-glucose derivation that is routinely utilized in our lab to more specifically model metformin transport into the ischemic brain.

This work was supported by NINDS R01 NS#117906⇓

Compounds and their transporter inhibition specificity.