An ex Vivo Model for Evaluating Blood–Brain Barrier Permeability, Efflux, and Drug Metabolism

Abstract

The metabolism of drugs in the brain is difficult to study in most species because of enzymatic instability in vitro and interference from peripheral metabolism in vivo. A locust ex vivo model that combines brain barrier penetration, efflux, metabolism, and analysis of the unbound fraction in intact brains was evaluated using known drugs. Clozapine was analyzed, and its major metabolites, clozapine N-oxide (CNO) and N-desmethylclozapine (NDMC), were identified and quantified. The back-transformation of CNO into clozapine observed in humans was also observed in locusts. In addition, risperidone, citalopram, fluoxetine, and haloperidol were studied, and one preselected metabolite for each drug was analyzed, identified, and quantified. Metabolite identification studies of clozapine and midazolam showed that the locust brain was highly metabolically active, and 18 and 14 metabolites, respectively, were identified. The unbound drug fraction of clozapine, NDMC, carbamazepine, and risperidone was analyzed. In addition, coadministration of drugs with verapamil or fluvoxamine was performed to evaluate drug-drug interactions in all setups. All findings correlated well with the data in the literature for mammals except for the stated fact that CNO is a highly blood-brain barrier permeant compound. Overall, the experiments indicated that invertebrates might be useful for screening of blood-brain barrier permeation, efflux, metabolism, and analysis of the unbound fraction of drugs in the brain in early drug discovery.

Metabolism and Distribution of Clozapine-N-oxide: Implications for Nonhuman Primate Chemogenetics

“…Collectively, these data indicate that CNO is a substrate for Pgp-mediated efflux at the BBB in vitro . Our results are in agreement with previous studies demonstrating low blood–brain permeability of CNO 19 and may account for its limited CNS distribution (i.e., exposure) in vivo in NHP.…”

Pharmacosynthetic Deconstruction of Sleep-Wake Circuits in the Brain

“…The major argument resides in the fact that the radiolabeled CNO signal could have been due to converted radiolabeled clozapine. Indeed, contrasting results from clozapine/CNO plasma measurements were reported in rodents and in monkeys detecting clozapine after CNO systemic injections (Hellman et al 2016;Gomez et al 2017;Raper et al 2017) or not (Alexander et al 2009;Guettier et al 2009;Nagai et al 2016). It still remains elusive to explain or reconcile these differences.…”

“…From many reports, CNO appeared to be pharmacologically and behaviorally inert in rodents (Bender et al 1994;Armbruster et al 2007;Alexander et al 2009;Guettier et al 2009). However, recent studies performed in mice, rats, and monkeys further investigated this possibility by measuring the presence of clozapine following CNO administration through various assays (Hellman et al 2016;Gomez et al 2017;Raper et al 2017). Additionally, the blood-brain permeability of CNO has been reexamined (Hellman et al 2016;Ji et al 2016;Nagai et al 2016;Gomez et al 2017;Raper et al 2017).…”

“…However, recent studies performed in mice, rats, and monkeys further investigated this possibility by measuring the presence of clozapine following CNO administration through various assays (Hellman et al 2016;Gomez et al 2017;Raper et al 2017). Additionally, the blood-brain permeability of CNO has been reexamined (Hellman et al 2016;Ji et al 2016;Nagai et al 2016;Gomez et al 2017;Raper et al 2017). These recent papers emphasize three major points: (1) the affinity of clozapine to bind DREADDs is higher than CNO (Gomez et al 2017); (2) CNO may not cross the blood-brain barrier (Hellman et al 2016;Gomez et al 2017;Raper et al 2017); and (3) CNO can be converted to clozapine under certain doses in monkeys and rodents (Hellman et al 2016;Gomez et al 2017;Raper et al 2017).…”

Blood‐brain barrier development: Systems modeling and predictive toxicology

“…To assess the metabolic capacity of the BBB, recent studies have shifted attention to investigating phase I and phase II metabolism at the site of the barrier. To assess cytochrome P450 metabolism, Hellman et al () utilized an ex vivo locust model to investigate local phase I metabolic rates of selected pharmaceuticals at the site of the BBB (Hellman, Aadal Nielsen, Ek, & Olsson, ). While this invertebrate model does not have an endothelial cell based physical barrier, the authors demonstrated CYP activity through xenobiotic metabolism.…”