Expression and functional relevance of UGT1A4 in a cohort of human drug‐resistant epileptic brains

Abstract

Summary Purpose Brain drug bioavailability is regulated by the blood–brain barrier (BBB). It was recently suggested that cytochrome P450 (CYP) enzymes could act in concert with multidrug transporter proteins to regulate drug penetration and distribution into the diseased brain. The possibility that phase II metabolic enzymes could be expressed in the epileptic brain has been not evaluated. Phase II enzymes are involved in the metabolism of common antiepileptic drugs (AEDs). Methods Phase II enzyme UGT1A4 brain expression was evaluated in temporal lobe resections from patients with epilepsy. UGT1A4 expression was determined by western blot and immunocytochemistry in primary cultures of human drug-resistant brain endothelial human brain epileptic endothelial cells (EPI-EC)s and commercially available control cells human brain microvascular endothelial cells (HBMECs). Lack of DNA condensation measured by 4′,6-diamidino-2-phenylindole (DAPI) was used as a surrogate marker of cell viability and was correlated to UGT1A4 expression high performance liquid chromatography ultraviolet detection (HPLC-UV) was used to quantify lamotrigine metabolism by EPI-EC and HBMEC. The appearance of the specific lamotrigine metabolite, 2-n glucuronide (MET-1), was also evaluated. Lamotrigine and MET-1 levels were measured in selected surgical brain and matched blood samples. Key Findings UGT1A4 expression was observed in BBB endothelial cells and neurons. Our quantification study revealed variable levels of UGT1A4 expression across the brain specimens analyzed. Neurons devoid of UGT1A4 expression displayed nuclear DAPI condensation, a sign of cellular distress. UGT1A4 overexpression in EPI-EC, as compared to HBMEC, was reflected by a proportional increase in lamotrigine metabolism. The lamotrigine metabolite, MET-1, was formed in vitro by EPI-EC and, to a lesser extent, by HBMEC. HPLC-UV measurements of brain and blood samples obtained from patients receiving lamotrigine prior to surgery revealed the presence of lamotrigine and its metabolites in the brain. Significance These initial results suggest the presence of a phase II enzyme in the epileptic brain. Further studies are required to fully describe the pattern of brain UGT1A4 expression in relation to clinical variables and drug resistance.

Modulation of glucocorticoid receptor in human epileptic endothelial cells impacts drug biotransformation in an in vitro blood–brain barrier model

“…Pharmacoresistance in epilepsy has been supported by several theories . The role of drug‐metabolizing enzymes and the formation of reactive metabolites due to enzymatic conversion by local ECs affecting drug bioavailability to the brain in DRE is one such theory . In the current study, CYP involvement was therefore explored in the DIV‐BBB and was found to be regulated by GR modulation in EPI‐ECs (Figures and ).…”

“…36,40,41 The role of drug-metabolizing enzymes and the formation of reactive metabolites due to enzymatic conversion by local ECs affecting drug bioavailability to the brain in DRE is one such theory. 7,10,11,16,42 In the current study, CYP involvement was therefore explored in the DIV-BBB and was found to be regulated by GR Figures 5 and 6). The in vitro BBB model, which mimics the drug-resistant phenotype, could be an ideal screening platform for pharmacologic compounds, similar to microfluidic liver devices used to evaluate CYP activity and function.…”

Blood–brain barrier, bulk flow, and interstitial clearance in epilepsy

“…In addition, data from several groups support that the epileptic BBB presents a variety of molecular signatures that are in one way or another involved in the disease. These span from expression of multiple drug resistance-related transporters (Dombrowski et al, 2001) and enzymes (Boussadia et al, 2014; Ghosh et al, 2013; Ghosh et al, 2011), to abnormal levels of GLUT-1, a glucose transporters (Cornford et al, 1998). Most of human data derive from analysis of resected tissue, where expression of an array of drug extrusion molecules has been reported (Dombrowski et al, 2001) and leakage of capillaries or vessels reported by several groups after the original observation by Cornford (Cornford and Hyman, 1999; Cornford et al, 1998).…”

Frequencies of UGT1A4*2 (P24T) and *3 (L48V) and their effects on serum concentrations of lamotrigine

“…This phenomenon, called auto-induction, is subject to great interindividual variation (Brodie et al 2013). In a recent study on endothelial epileptic brain cells collected surgically, individual expression of UGT1A4 protein and metabolism of LTG was highly variable (Ghosh et al 2013). Thus, a variety of genetic polymorphisms as well as a number of epigenetic factors may explain interindividual and interethnic variation in UGT1A4 protein expression and respective changes in metabolic activity.…”