Anxiolytic-like activity of pregabalin in the Vogel conflict test in α2δ-1 (R217A) and α2δ-2 (R279A) mouse mutants.

The α(2)δ auxiliary subunits (α(2)δ-1 and α(2)δ-2) of voltage-sensitive calcium channels are thought to be the site of action of pregabalin (Lyrica), a drug that has been shown to be anxiolytic in clinical trials for generalized anxiety disorder. Pregabalin and the chemically related drug gabapentin have similar binding and pharmacology profiles, demonstrating high-affinity, in vitro binding to both α(2)δ-1 and α(2)δ-2 subunits. Two independent point mutant mouse strains were generated in which either the α(2)δ-1 subunit (arginine-to-alanine mutation at amino acid 217; R217A) or the α(2)δ-2 subunit (arginine-to-alanine mutation at amino acid 279; R279A) were rendered insensitive to gabapentin or pregabalin binding. These strains were used to characterize the activity of pregabalin in the Vogel conflict test, a measure of anxiolytic-like activity. Pregabalin showed robust anticonflict activity in wild-type littermates from each strain at a dose of 10 mg/kg but was inactive in the α(2)δ-1 (R217A) mutants up to a dose of 320 mg/kg. In contrast, pregabalin was active in the α(2)δ-2 (R279A) point mutants at 10 and 32 mg/kg. The positive control phenobarbital was active in mice carrying either mutation. These data suggest that the anxiolytic-like effects of pregabalin are mediated by binding of the drug to the α(2)δ-1 subunit.

Effect of chitosan glutamate, carbomer 974P, and EDTA on the in vitro Caco-2 permeability and oral pharmacokinetic profile of acyclovir in rats.

BACKGROUND: Chitosan glutamate and polyacrylic acid (e.g., carbomer 974P) are known to modulate the tight junctions in the intestinal wall and increase permeability and blood exposure of drugs absorbed orally by the paracellular route. AIM: To assess the impact of chitosan glutamate and carbomer 974P on the absorption of paracellularly absorbed model drug, acyclovir, in vitro and in rat in vivo. METHODS: The influence of chitosan glutamate and carbomer 974P (alone and in combination with EDTA-Na2) on the in vitro Caco-2 permeability and oral pharmacokinetic profile in the rat of acyclovir was investigated. RESULTS: In the presence of chitosan glutamate, the apparent permeability of acyclovir across Caco2 monolayer increased 4.1 times relative to control. This increase was accompanied by a significant ( approximately 60%) decrease in transepithelial electrical resistance values indicating opening of the tight junctions in the cell monolayer. In rat, chitosan glutamate doubled oral bioavailability of acyclovir and tripled the amount of acyclovir excreted unchanged into urine. In contrast, the effect of carbomer 974P was not statistically significant at 5% level. CONCLUSIONS: In conclusion, chitosan glutamate (1-3%) and chitosan glutamate (1%)/EDTA-Na2 (0.01%) are effective excipients to increase permeability of acyclovir across Caco-2 cell monolayers and the oral absorption in the rat in vivo.

The use of 1-aminobenzotriazole in differentiating the role of CYP-mediated first pass metabolism and absorption in limiting drug oral bioavailability: a case study.

Preliminary studies in our laboratory demonstrated low oral bioavailability of Drug X in male Sprague Dawley rats. However, the factors responsible for the observed poor bioavailability were not well understood. The objective of this study was to investigate the contribution of cytochrome P450(s) metabolism to the observed poor oral bioavailability of Drug X in male Sprague-Dawley rats in the presence of 1-aminobenzotriazole, a non-specific irreversible inhibitor of cytochrome P450s. Male Sprague-Dawley rats were pre-treated with or without oral 1-aminobenzotriazole (50 mg/kg) two hours prior to receiving a single intravenous or oral dose of Drug X (3 mg/kg). Blood samples were collected from animals at different time points over six hours following Drug X dosing. Plasma concentrations of Drug X were determined using LC/MS/MS. Pharmacokinetic data obtained from an intravenous dose study in rats suggested that Drug X exhibited a high clearance (55 mL/min/kg) and moderate volume of distribution (1.3 L/kg) with short half-life in rats (0.7 hr). Oral dosing of Drug X to rats resulted in low oral bioavailability (19%). 1-aminobenzotriazole pre-treatment of male Sprague Dawley rats followed by an intravenous dose of Drug X resulted in a decrease in plasma clearance by 71% and an increase in half-life by 100%, without affecting the volume of distribution. Furthermore, the oral bioavailability of Drug X increased markedly with 1-aminobenzotriazole pre-treatment. However, the fraction absorbed of Drug X did not significantly change with 1-aminobenzotriazole pre-treatment. The results of this study indicated that CYP-mediated metabolism played a major role in limiting the oral bioavailability of Drug X in rats. The data suggests that 1-aminobenzotriazole can be used as an effective tool in assessing the factors contributing to the poor oral bioavailability of drugs.

Porcine brain microvessel endothelial cells as an in vitro model to predict in vivo blood-brain barrier permeability.

The objective of the study was to establish primary cultured porcine brain microvessel endothelial cells (PBMECs) as an in vitro model to predict the blood-brain barrier (BBB) permeability in vivo. The intercellular tight junction formation of PBMECs was examined by electron microscopy and measured by transendothelial electrical resistance (TEER). The mRNA expression of several BBB transporters in PBMECs was determined by reverse transcriptionpolymerase chain reaction analysis. The in vitro permeability of 16 structurally diverse compounds, representing a range of passive diffusion and transporter-mediated mechanisms of brain penetration, was determined in PBMECs. Except for the perfusion flow rate marker diazepam, the BBB permeability of these compounds was determined either in our laboratory or as reported in literature using in situ brain perfusion technique in rats. Results in the present study showed that PBMECs had a high endothelium homogeneity, an mRNA expression of several BBB transporters, and high TEER values. Culturing with rat astrocyte-conditioned medium increased the TEER of PBMECs, but had no effect on the permeability of sucrose, a paracellular diffusion marker. The PBMEC permeability of lipophilic compounds measured under stirred conditions was greatly increased compared with that measured under unstirred conditions. The PBMEC permeability of the 15 test compounds, determined under the optimized study conditions, correlated with the in situ BBB permeability with an r2 of 0.60. Removal of the three system L substrates increased the r2 to 0.89. In conclusion, the present PBMEC model may be used to predict or rank the in vivo BBB permeability of new chemical entities in a drug discovery setting.