Identification of Dihydromyricetin and Metabolites in Serum and Brain Associated with Acute Anti-Ethanol Intoxicating Effects in Mice

Abstract

Dihydromyricetin is a natural bioactive flavonoid with unique GABA_A receptor activity with a putative mechanism of action to reduce the intoxication effects of ethanol. Although dihydromyricetin's poor oral bioavailability limits clinical utility, the promise of this mechanism for the treatment of alcohol use disorder warrants further investigation into its specificity and druggable potential. These experiments investigated the bioavailability of dihydromyricetin in the brain and serum associated with acute anti-intoxicating effects in C57BL/6J mice. Dihydromyricetin (50 mg/kg IP) administered 0 or 15-min prior to ethanol (PO 5 g/kg) significantly reduced ethanol-induced loss of righting reflex. Total serum exposures (AUC0_→24) of dihydromyricetin (PO 50 mg/kg) via oral (PO) administration were determined to be 2.5 µM × h (male) and 0.7 µM × h (female), while intraperitoneal (IP) administration led to 23.8-fold and 7.2- increases in AUC0_→24 in male and female mice, respectively. Electrophysiology studies in α5β3γ2 GABA_A receptors expressed in Xenopus oocytes suggest dihydromyricetin (10 µM) potentiates GABAergic activity (+43.2%), and the metabolite 4-O-methyl-dihydromyricetin (10 µM) negatively modulates GABAergic activity (-12.6%). Our results indicate that administration route and sex significantly impact DHM bioavailability in mice, which is limited by poor absorption and rapid clearance. This correlates with the observed short duration of DHM's anti-intoxicating properties and highlights the need for further investigation into mechanism of DHM's potential anti-intoxicating properties.

Keywords: GABAA receptors; acute alcohol intoxication; alcohol use disorder; bioavailability; dihydromyricetin; loss of righting reflex; metabolism.

Figure 1

Dihydromyricetin (DHM) time course for attenuating the loss of righting reflex (LORR) following acute oral gavage of ethanol (5 g/kg) in male and female 8-week-old C57BL/6J mice. Each mouse received an IP injection of DHM or vehicle (10% DMSO) at a time prior to an oral gavage of ethanol (ETOH). Each time point (0, 15, 45, 90, 180 min) or vehicle was 1 week apart for a total of 6 weeks. (A) Males (n = 8), (B) Female (n = 8). To determine whether the observed DHM effects were a consequence of repeated oral ETOH tolerance, mice were tested for LORR responsivity prior to (Baseline) and after (Post) the 6-week repeated IP dosing schedule. For this, mice received an oral ETOH without any IP injections. Mice were divided by the significant difference in baseline and post LORR. (C) Mice demonstrating a significantly reduced LORR displayed tolerance (n = 8; 3 males/5 females), whereas mice that retained the LORR were responsive (n = 8; 5 males/3 females). (D): Time course of DHM on LORR in Responsive only mice. * indicates p < 0.05 from vehicle treated condition. ** indicates p < 0.005 from baseline.

Figure 2

Serum AUC0_→24 of DHM 50 mg/kg with oral (PO) and intraperitoneal (IP) administration in male and female mice. *^s Statistically significant (p < 0.05) difference between male and female mice. ***^a Statistically significant (p < 0.0001) between administration routes of same sex.

Figure 3

Proposed metabolic pathway of dihydromyricetin (DHM) based on detection of metabolites in feces and/or urine samples of rats [31,32]. Boxed compounds are those that were detected in this study.

Figure 4

Synthesis route of 3′-Me-DHM, 4′-Me-DHM, 4′-DeOH-DHM (a) NaOH, EtOH/H₂O (b) H₂O_2, NaOH, MeOH/Dioxane (c) 1. HCl, MeOH, THF 2. (4′-Me-DHM only) Pd/C, H₂, MeOH.