Pharmacologic Evaluations of Novel LAT1-Targeting Cannabidiol Prodrugs for CNS Delivery Open Access

Abstract

Epilepsy is a chronic neurological condition characterized by recurrent seizures, significantly decreasing quality of life and increasing the risk of premature mortality for patients. Refractory epilepsies represent a particularly severe, treatment-resistant class wherein patients fail to achieve durable seizure control from traditional antiepileptic drugs. Epidiolex, a purified form of cannabidiol (CBD), was recently FDA approved as an adjunct to the standard of care for refractory epilepsy treatment, exerting unique and multimodal mechanisms of action. However, its clinical utility is constrained by poor oral bioavailability, high exposure to peripheral tissues due to non-specific CNS penetration mechanisms, and extensive Cytochrome P450 (CYP450)-mediated first‑pass metabolism into pharmacologically inactive forms. To address these limitations, our group designed a series of novel LAT1‑targeting CBD prodrugs that append aromatic amino acid–based promoieties to the 6‑phenolic hydroxyl of CBD to enable carrier‑mediated blood–brain barrier transport via the L‑type amino acid transporter 1 (LAT1), highly expressed by brain-resident cell types. We hypothesize that transporter-mediated uptake could enhance brain selectivity and regional targeting, delivering more CBD to the brain at a given plasma concentration and thus improving its peripheral exposure and distribution profile. Here, we evaluated numerous pharmacological aspects of this prodrug class. We first endeavored to elucidate LAT1 utilization in-vitro through Transwell permeability and competitive cellular uptake assays against a known LAT1 inhibitor, complemented by in-silico affinity predictions against LAT1 and a preliminary subcellular sequestration experiment. Prodrugs displayed robust cellular accumulation but minimal sensitivity to LAT1-inhibited conditions, suggesting passive diffusion attributed to high lipophilicity could dominate transport under the tested conditions, given that in-silico models predict nanomolar affinity comparable to literature prodrugs. We next tested P-gp efflux liability using an MDCK-MDR1 permeability assay, as MDR1 overexpression is highly correlated with refractory epilepsy. Our data suggested high levels of P-gp-mediated efflux for two prodrugs, which may additionally counteract LAT1-mediated influx since hCMEC/D3 cells express functional P-gp levels. Lastly, CYP450 inhibition at the CYP3A4 and CYP2D6 isoforms of highest clinical relevance was tested. The majority of prodrugs exhibited weaker inhibition than CBD, indicating an improved drug-drug interaction profile favorable for the continued development of this proprietary compound class.

Table of Contents

Introduction 1

Materials and Methods 15

Results and Discussion 24

Conclusions and Future Directions 44

Supplementary Information 48

References 51 

About this Honors Thesis

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School	Emory College
Department	Chemistry
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Chemistry, Biochemistry
Keyword	hCMEC/D3 cells Cannabidiol (CBD) Refractory epilepsy LAT1 transporter Prodrug design P-glycoprotein (P-gp) Cytochrome P450 (CYP450) Blood-brain barrier (BBB)
Committee Chair / Thesis Advisor	Liotta, Dennis, Emory University
Committee Members	Lynn, David, Emory University Miller, Eric, Emory University

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