The role of ABC transporters and inflammation in drug-resistant epilepsy

Abstract

This thesis explores two pathologies thought to be related to drug resistance in epilepsy that may themselves be causally related: 1) overexpression of drug transporters in capillaries and 2) inflammation. With regard to the first hypothesis, overexpression of the ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), at the blood-brain barrier are thought to contribute to drug resistance in epilepsy. To measure ABC transporter activity in vivo, positron emission tomography (PET) imaging can be used, which requires a radiolabeled substrate and non-radiolabeled inhibitor. The second hypothesis suggests that inflammation increases P-gp expression and, conversely, that inhibiting inflammation decreases P-gp expression. Questions remain however, as to the interactions between ABC transporters and specific inflammatory proteins, as well as the cellular expression of the same inflammatory proteins in the human brain.

In Paper I, we characterized the BCRP inhibitor Ko143 to determine if it was a candidate for use in PET imaging. P-gp activity has been measured using PET imaging with tracers such as [11C]Ndesmethyl-loperamide (a P-gp substrate) along with a P-gp inhibitor such as tariquidar, but a similar imaging paradigm has not yet been developed for BCRP. Therefore, we performed multiple in vitro assays to characterize Ko143 and to measure its interaction with P-gp, BCRP, and the multidrug resistance transporter 1 (MRP1). Data from the in vitro assays indicated that while Ko143 was a potent BCRP inhibitor (IC50 = 9.7 nM), at higher concentrations it was a substrate for P-gp (IC50 = 2.7 µM) and MRP1.

In Paper II, because of reports questioning whether tariquidar is a P-gp inhibitor, we investigated tariquidar to determine the mechanism by which it interacts with P-gp. Using similar methods as outlined in Paper I, we found that tariquidar was a potent P-gp inhibitor at low concentrations (IC50 = 100 nM), but at higher micromolar concentrations it was a substrate and competitive inhibitor of BCRP.

In Paper III, we sought to determine whether a relationship exists between ABC transporter expression and expression of the inflammatory enzymes cyclooxygenase (COX)-1 and -2, as well as the inflammation biomarker, translocator protein 18 kDa (TSPO). We used multiplex immunofluorescence to measure the expression of P-gp and BCRP as well as COX-1, COX-2, and TSPO in brain tissue samples from people with drug-resistant epilepsy. These tissue samples were classified as either having mesial temporal sclerosis (MTS) or not (non-MTS), in which the non-MTS samples acted as control tissue for MTS samples. When investigating the relationship between ABC transporters and the inflammatory proteins, the only correlation we observed was between BCRP and TSPO, in which increased BCRP density correlated linearly with increased TSPO density (P = 0.0003, r = 0.72131). No significant differences were found in the expression of any protein measured between MTS and nonMTS tissue samples.

In Paper IV, we investigated the cellular expression of three inflammatory proteins COX-1, COX-2, and TSPO in brain tissue samples from people with drug-resistant epilepsy. To do so, we used multiplex immunofluorescence microscopy to measure the expression of these proteins in microglia, astrocytes, and neurons. We found that that COX-1 was predominately expressed in microglia, while COX-2 and TSPO were expressed in microglia and neurons.

In summary, this thesis explored the mechanisms underlying drug resistance in epilepsy. We studied overexpression of ABC transporters and inflammation, two pathologies hypothesized to be involved in drug-resistant epilepsy that may themselves possibly be related. While Ko143 is specific for BCRP at nanomolar concentrations (similar to tariquidar for P-gp), its potential utility as a radiolabeled inhibitor was diminished by the fact that PET requires picomolar affinity—rather than the 9.7 nM we measured— to measure the low density of BCRP in the brain. With regard to inflammation, we found that COX-1 is primarily expressed in microglia, a trait that makes it, rather than COX-2, a better radioligand for studying neuroinflammation in patients with drug-resistant epilepsy, given that microglia produce the majority of pro-inflammatory cytokines in the brain.