Sh-I-048A, an in vitro non-selective super-agonist at the benzodiazepine site of GABAA receptors: The approximated activation of receptor subtypes may explain behavioral effects

Abstract

Enormous progress in understanding the role of four populations of benzodiazepine-sensitive GABAA receptors was paralleled by the puzzling findings suggesting that substantial separation of behavioral effects may be accomplished by apparently non-selective modulators. We report on SH-I-048A, a newly-synthesized chiral positive modulator of GABAA receptors characterized by exceptional subnanomolar affinity, high efficacy and non-selectivity. Its influence on behavior was assessed in Wistar rats and contrasted to that obtained with 2 mg/kg diazepam. SH-I-048A reached micromolar concentrations in brain tissue, while the unbound fraction in brain homogenate was around 1.5%. The approximated electrophysiological responses, which estimated free concentrations of SH-I-048A or diazepam are able to elicit, suggested a similarity between the 10 mg/kg dose of the novel ligand and 2 mg/kg diazepam; however, SH-I-048A was relatively more active at α1- and α5-containing GABAA receptors. Behaviorally, SH-I-048A induced sedative, muscle relaxant and ataxic effects, reversed mechanical hyperalgesia 24 hours after injury, while it was devoid of clear anxiolytic actions and did not affect water-maze performance. While lack of clear anxiolytic actions may be connected with an enhanced potentiation at α1-containing GABAA receptors, the observed behavior in the rotarod, water maze and peripheral nerve injury tests was possibly affected by its prominent action at receptors containing the α5 subunit. The current results encourage further innovative approaches aimed at linking in vitro and in vivo data in order to help define fine-tuning mechanisms at four sensitive receptor populations that underlie subtle differences in behavioral profiles of benzodiazepine site ligands.

Novel Benzodiazepine-Like Ligands with Various Anxiolytic, Antidepressant, or Pro-Cognitive Profiles

“…A rapid equilibrium dialysis assay was used to determine the free fraction of the ligands in mouse plasma and brain tissue as described by Obradović et al [36]. Ligand-free brain concentrations were calculated by multiplying the total brain concentrations with the appropriate free fractions determined by rapid equilibrium dialysis.…”

“…To obtain pharmacokinetic profiles, mice were treated i.p. at 10 mg/kg and sacrificed at different time points (5 to 720 min post-injection) for brain and plasma quantification of ligands by ultraperformance LC-MS/MS [36]. The analytical lower limit of quantification for the ligands examined ranged from 0.5 to 1.0 ng/mL in plasma, and from 0.2 to 1.0 ng/g in brain tissue.…”

Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit

“…To determine the concentration of MP-III-022 and SH-053-2′F-R-CH3 in plasma and supernatants of brain tissue homogenates, MP-III-022 and SH-053-2′F-R-CH3 were extracted from these samples by solid phase extraction, using Oasis HLB cartridges (Waters Corporation, Milford, Massachusetts). The procedure of sample preparation and determination of MP-III-022 and SH-053-2′F-R-CH3 by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with Thermo Scientific Accela 600 UPLC system connected to a Thermo Scientific TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, California), equipped with electrospray ionization (ESI) source, has been already described in detail (Obradović et al, 2014). …”

Negative modulation of α5 GABAA receptors in rats may partially prevent memory impairment induced by MK-801, but not amphetamine- or MK-801-elicited hyperlocomotion

“…To determine the concentration of PWZ–029 in plasma and supernatants of brain tissue homogenates, PWZ–029 was extracted from these samples by solid phase extraction, using Oasis HLB cartridges (Waters Corporation, Milford, Massachusetts). The procedure of sample preparation and determination of PWZ–029 by ultraperformance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) with Thermo Scientific Accela 600 UPLC system connected to a Thermo Scientific TSQ Quantum Access MAX triple quadrupole mass spectrometer (Thermo Fisher Scientific, San Jose, California), equipped with electrospray ionization (ESI) source, was described in detail in Obradović et al, 2014.…”

“…These studies were aimed to assess if the in vivo concentrations attained at the time of behavioral testing enable a selective involvement of α 5 GABA A receptors (cf. Obradović et al, 2014; Redrobe et al, 2012), when compared with the in vitro findings of PWZ-029 (Savić et al, 2008). …”