Characterization of theIn VitroHydrolysis of Areca (Betel) Nut Xenobiotics, Arecoline and Guvacoline

Abstract ID 129510 Poster Board 239 Background/Hypothesis: Areca nut (AN), often called betel nut, is widely consumed for its intoxicating effects. Users experience euphoria, warming sensations of the body, and increased mental stamina. In fact, AN is the fourth-most common self-administered psychoactive substance worldwide. Previous studies indicate psychoactivity is due to alkaloids, mainly arecoline and to some extent guvacoline. The arecoline and guvacoline structures, interestingly, both have a labile methyl ester moiety, suggesting clearance by a Phase I hydrolysis mechanism. However, kinetics describing their hydrolysis in human liver remains unknown. We hypothesized that human liver microsomes (HLM) predominantly hydrolyze arecoline and guvacoline to their corresponding carboxylate metabolites, arecaidine and guvacine, respectively. Methods: A HPLC-PDA assay utilizing ion exchange chromatography was developed to quantitate arecoline, arecaidine, guvacoline and guvacine. Traditional microscale metabolic reactions were conducted in buffered microsome suspensions kept under simulated physiological conditions in a shaking water bath. Using a range of alkaloid concs (10-8000 μM; n = 8-9), formation of hydrolytic metabolites was subjected to enzyme kinetic curve fitting. Kinetic parameters were subsequently extrapolated to the in vivo condition using human scaling factors. Finally, arecoline hydrolase activity was compared between control HLM and HLM pre-exposed to chemical inhibitors of carboxylesterase (CES): loperamide (CES2 inhibitor), digitonin (CES1 inhibitor), and potent non-specific CES inhibitors disulfiram and simvastatin. Results: Validation of the bioanalytical assay demonstrated its capability to measure alkaloids and their carboxylate metabolites with good sensitivity, accuracy, and precision. Arecoline hydrolase activity in HLM was 4903%, 166%, and 100% greater than in human intestinal microsomes, liver cytosol and liver S9, respectively (p<0.0001). Arecoline depletion in HLM best followed a monoexponential decay model with elimination half-life of 16 (± 0.4) min. In samples with thermally inactivated HLM, arecoline hydrolysis was negligible. As assessed by Akaike Information Criterion (AIC), arecaidine formation was best fitted to the Michaelis-Menten equation. The resultant Km, Vmax and Clint were >8000 μM, 783 nmol/min/mg, and 90 μl/min/mg, respectively. The apparent in vivo Clint was 110 ml/min/kg. Only minor guvacoline depletion was observed in HLM; the Vmax, Clint (in vitro), and Clint (in vivo) for guvacine formation were 35 nmol/min/mg, 1.02 μl/min/mg, and 1.25 ml/min/kg, respectively. In the presence of CES inhibitors, arecoline hydrolase activity was unimpeded by loperamide, whereas digitonin, disulfiram and simvastatin significantly reduced activity by 37% (p < 0.01), 86% (p < 0.0001) and 98% (p < 0.0001), respectively. Conclusions: For the first time, we report the enzyme kinetics for hydrolysis of areca nut methyl ester xenobiotics (arecoline and guvacoline) in HLM. HLM rapidly hydrolyzes arecoline to arecaidine, but with minimal capacity to hydrolyze guvacoline, an unexpected finding. Combined, structural characteristics of the molecule and experimental data reported herein suggests arecoline is a substrate for CES1. Finally, arecoline hydrolysis was almost completely abolished by simvastatin and significantly impeded by disulfiram, a largely unknown human CES inhibitor at this time. NIH/NIDA 1R01DA056544-01A1