Pharmacological resistance to brain drugs is a common clinical event affecting patient management; it is also a common cause for neuro-surgical interventions. In particular, the number of drug resistant subjects is significant among those suffering from epilepsy. The International League against Epilepsy estimates that 20-25% of epileptic subjects are resistant to available anti-epileptic drugs (AED). Incomplete understanding of the pattern of brain AED biotransformation in the diseased brain represents a major hindrance to the development of new drugs. Consensus is gathering on the fact that modeling of the drug resistant phenotype requires a multi-modal experimental approach, including the use of human brain tissue (and of their in vitro manipulation) paired with animal models of disease. We now propose to 1) Test the hypothesis that, in DRE, the brain bioavailability of AEDs is affected by BBB P450 enzymes; 2) Test the hypothesis that BBB P450 produce metabolites with neurotoxic properties. We also propose the corollary hypothesis that a concerted metabolic-transport mechanism determines AED bioavailability in the DRE brain. Our recent published data and preliminary results show that: a) Transcripts of P450 enzymes are elevated in primary endothelial cells (EC) isolated from drug resistant epileptic patients (DRE); these include AED- metabolizers such as CYP3A4, CYP2C9, etc. Data were compared to available, control brain EC (non-DRE); b) Transcripts for PHASE II metabolic enzymes are present in DRE EC; these enzymes are responsible for the metabolism of 1st and 2nd generation AEDs; c) CYP3A4 and MDR1 co-localize at the BBB (and neurons) in human DRE brain; d) Overexpression of CYP3A4 in DRE EC is associated with exaggerated carbamazepine (CBZ) metabolism. This new metabolic pathway produces the toxic CBZ metabolite quinolic acid (QA). The parent (14C CBZ) origin of QA was evaluated using HPLC-Accelerated Mass Spectrometry (AMS) in vitro and ex vivo (DRE brain specimens). AMS results were corroborated by mass spectroscopy (MS) and by two HPLC protocols optimized for QA detection; e) DRE endothelial cells metabolize lamotrigine (LMT) and levetiracetam (LEV). In our proposal we will approach the issue of human control brain tissues by using brain samples resected to treat diseases other than drug resistant seizures, autoptic brain, and internal controls consisting of non-spiking regions in resected DRE brains. To dissect the role of EC, we will use primary BBB cell cultures derived from resected brain specimens. The BBB is recapitulated in vitro by a flow-based device. A combination of AMS and MS is used to determine the molecular nature of new metabolites in the DRE brain. Finally, two models of epilepsy are used to study the temporal and topographic pattern of brain expression and function of P450 enzymes. To our knowledge, these studies represent the first multimodal attempt to elucidate the expression and the role of brain P450 enzymes in DRE. Our ultimate goal is improved clinical management of DRE. The proposed studies will provide new insight into the mechanisms contributing to human DRE, improving the understanding of the pathophysiological significance of BBB P450. Modeling of the DRE BBB may serve as a tool for personalized medicine and specific disease modeling (e.g., type of drug resistant epilepsy and underlying pathology) allowing for the screening of new AED.
Failure of generating new brain therapeutics stems from an insufficient knowledge of the mechanisms regulating drug brain distribution under pathological conditions. As a result, a significant percentage of subjects affected by brain disorders are, to various extents, resistant to available drug treatment. For example, 20-25% of subjects with epilepsy do not respond to two or more anti-epileptic drugs (AED), administered alone or in combination. In these cases, brain surgery remains the only medical intervention. We wish to test the hypothesis that, in drug resistant forms of epilepsy, cerebrovascular P450 enzymes metabolize AED reducing their pharmacological efficacy. We will test the hypothesis that cerebrovascular P450 enzymes produce metabolites which may have neurotoxic effect. We also propose that P450 enzymes act in concert with drug transporter proteins, affecting the pattern of AED brain availability. Our proposal has the potential to elucidate mechanisms of brain biotransformation implied in brain drug resistance and to establish methods modeling human drug resistance, allowing for the screening of new AED for the improved clinical management of refractory forms of epilepsy.
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