Brain uptake of the drug of abuse γ-hydroxybutyric acid in rats

Abstract

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a substrate for the ubiquitous monocarboxylate transporter (MCT) family. GHB is also a drug of abuse due to its sedative/hypnotic and euphoric effects, with overdoses resulting in toxicity and death. The goal of this study was to characterize the distribution of GHB into the brain using in vivo microdialysis and in vitro uptake studies and to determine concentration-effect relationships for GHB in a rat animal model. GHB was administered to rats (400, 600, and 800 mg/kg i.v.), and blood, dialysate, and urine were collected for 6 h post-GHB administration. The GHB plasma and extracellular fluid (ECF) concentration-time profiles revealed that GHB concentrations in ECF closely followed plasma GHB concentrations. Sleep time increased in a dose-dependent manner (91 ± 18, 134 ± 11, and 168 ± 13 min, for GHB 400, 600, and 800 mg/kg, respectively). GHB partitioning into brain ECF was not significantly different at 400, 600, and 800 mg/kg. GHB uptake in rat and human brain endothelial cells exhibited concentration dependence. The concentration-dependent uptake of GHB at pH 7.4 was best-fit to a single-transporter model [K(m) = 18.1 mM (human), 23.3 mM (rat), V(max) = 248 and 258 pmol · mg(-1) · min(-1) for human and rat, respectively]. These findings indicate that although GHB distribution into the brain is mediated via MCT transporters, it is not capacity-limited over the range of doses studied in this investigation.

Fig. 1.

The microdialysis experimental protocol used to study GHB distribution into the frontal cortex and toxicodynamic effects in rats.

Fig. 2.

GHB concentration-time profiles in plasma (A) and brain extracellular fluid (B) after intravenous administration of GHB 400 mg/kg (□, dotted line), 600 mg/kg (▵, dashed line), or 800 mg/kg (○, solid line). The midpoint time represents the midpoint time for each 20-min fraction collection period. Data are plotted as the mean ± S.D., n = 4/dose.

Fig. 3.

GHB dose-normalized concentration-time profiles in plasma (A) and frontal cortex ECF (B) after 400, 600, or 800 mg/kg GHB i.v. GHB plasma and ECF concentrations were divided by their respective dose to assess linearity of distribution. The midpoint time represents the midpoint time for each 20-min fraction collection period. Data are plotted as the mean ± S.D., n = 4/dose.

Fig. 4.

Time course for GHB partitioning into brain (K_p,u,u) was determined from unbound plasma and brain ECF concentrations after GHB doses of 400, 600, and 800 mg/kg iv. GHB ECF concentrations were divided by GHB plasma concentrations for each plasma sample time point. No significant differences were detected. Data are plotted as the mean ± S.D., n = 4/dose.

Fig. 5.

GHB concentrations in plasma (A) or frontal cortex ECF (B) at RRR were calculated via noncompartmental analysis from the GHB concentration-time profiles. GHB concentrations at RRR were not significantly different across doses. Data are presented as mean ± S.D., n = 4/dose.

Fig. 6.

Time course for GHB uptake in RBE4 (A) and hCMEC/D3 (C) cells. Cells were incubated with 58 nM [³H]GHB up to 10 min at room temperature at pH 7.4 to characterize the time course for uptake. Concentration-dependent uptake of GHB in RBE4 (B) and hCMEC/D3 (D) cells. Uptake was done at pH 7.4 by incubating cells with up to 50 mM GHB for 15 s at room temperature. Data are displayed as the mean ± S.D. of three experiments, each experiment performed in triplicate, and GHB uptake data for each cell line were best fit to the simple Michaelis-Menten equation (eq. 2) as described under Materials and Methods.