Purification of a benzodiazepine from bovine brain and detection of benzodiazepine-like immunoreactivity in human brain.

Abstract

An endogenous brain substance that binds to the central-type benzodiazepine receptors with agonist properties is present in both rat and bovine brains. This substance has been purified to homogeneity from bovine brain by immunoafflmity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reversed-phase HPLC steps. The purified substance was characterized as the benzodiazepine N-desmethyldiazepam (nordiazepam The anti-benzodiazepine mAb 21-7F9 was obtained after immunizing BALB/c mice with 3-hemisuccinyloxyclonazepam coupled to bovine serum albumin. The production, purification, and characterization of mAb 21-7F9 have been reported (2).Immunoaffinity Chromatography. Fifty grams of bovine cerebellum and cerebral cortex were homogenized (Waring blender, 6 min) in 60 ml of 5 mM Tris-HCl, pH 8.1/0.3 mM phenylmethylsulfonyl fluoride/1 mM EDTA containing soybean trypsin inhibitor (20 pug/ml) and pepstatin (1 pug/ml). The preparation of the soluble extract and the procedure for immunoaffinity chromatography have been described (1). The sample was loaded on a 10-ml Affi-Gel 10 (Bio-Rad) column containing 50 mg ofimmobilized anti-benzodiazepine mAb 21-7F9, and 100 ml of each washing solution was passed through the column at a flow rate of 10 ml/hr. The retained material was eluted with 100 ml of 0.2 M acetic acid at a flow rate of 50 ml/hr.Gel Filtration on Sephadex G-25. The immunoaffinity eluate was dried under reduced pressure, dissolved in 0.9 ml of 40% formic acid, and layered on a Sephadex G-25 column (51.5 cm x 1.2 cm i.d.) that had been equilibrated with 40% formic acid. Fractions (2 ml) were collected at a flow rate of 5 ml/hr. The active fractions were pooled, concentrated under reduced pressure, and resuspended in 600 Al of 0.1% trifluoroacetic acid. The benzodiazepine receptor binding activity of the fractions was assayed as indicated below.Reversed-Phase HPLC.Step I. A 200-Al sample of the Sephadex G-25-purified substance was subjected to reversed-phase HPLC on a C8 column [length, 25 cm; particle size, 10 /Lm; pore size, 300 A; Brownlee Laboratory (Santa Clara, CA)] and eluted with a linear gradient (5-60%) of acetonitrile in 0.1% trifluoroacetic acid.Step II. tTo whom all reprint requests should be addressed. 9236The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy.

“…In contrast, the concentrations of diazepam and N-desmethyldiazepam were increased 2-to 9-fold in brain extracts from rats with HE. The variability in the concentrations of these substances in HE may reflect, in part, the wide range of BZ concentrations in control brains, a phenomenon that has been observed previously (18,20). In addition to the elevated concentrations of diazepam and N-desmethyldiazepam present in brain extracts of rats with HE, the overall concentration of HPLC-purified substances inhibiting radioligand binding to BZ receptors (expressed in diazepam equivalents) was elevated 4-to 6-fold compared to controls ( Table 1, Fig.…”

“…Nonetheless, substances comigrating with the N-desmethyldiazepam and diazepam standards were consistently present. Mass spectroscopic analysis confirmed the identity of two of these unknown substances as N- (18,19). In contrast, the concentrations of diazepam and N-desmethyldiazepam were increased 2-to 9-fold in brain extracts from rats with HE.…”

Mechanisms of Action and Persistent Neuroplasticity by Drugs of Abuse

“…Its intrinsic efficacy has been explained by antagonism of endogenous positive or negative modulators. Indeed, there is evidence for endogenous production of compounds with binding and functional activity at BZ sites ("endozepines"), such as diazepam and its metabolite desmethyldiazepam (Sangameswaran et al, 1986;Klotz, 1991), and molecules with other structures, including heme derivates (Ruscito and Harrison, 2003) and the diazepam binding inhibitor (DBI) and its proteolytic peptide fragments, such as octadecaneuropeptide (Alho et al, 1985;Costa and Guidotti, 1991). Recently, DBI and octadecaneuropeptide have been shown to increase neurogenesis in postnatal mouse subventricular zone by negative Drug-Induced Neuroplasticity modulation of GABA A receptors (Alfonso et al, 2012), and, surprisingly, DBI was described to positively modulate reticular thalamic a3 subunit-containing GABA A receptors, an effect that appears to be needed for antiabsence seizure effect in a mouse model (Christian et al, 2013).…”

Identification of Inosine as an Endogenous Modulator for the Benzodiazepine Binding Site of the GABA_A Receptors

“…The search for naturally occurring ligands for BZ binding site is, therefore, conceptually analogous to the work involving opiate receptors in which the discovery of a functional receptor for the non-physiologic opiates have led to the isolation and identification of endorphins and enkephalins as being endogenous substrates for these receptors [8,26]. Indeed, several substances have been isolated and proposed as endogenous ligands for the BZ binding site, referred to as EBZs [1,4,6,19,25,28,30]. Similarly, several endogenous substances which inhibit the binding of [ 3 H]muscimol to the GABA binding site have been isolated and proposed to be involved in the modulation of the GABA receptor [4,12,13,15,24,29,32,[35][36][37][38].…”