The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects

Abstract

The functional characterization of hispidulin (4',5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABA(A)/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nm and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (alpha(1-3,5,6)beta(2)gamma(2)S) indicating positive allosteric properties. Maximal stimulation at alpha(1)beta(2)gamma(2)S was observed with 10 microm hispidulin. In contrast to diazepam, hispidulin modulated the alpha(6)beta(2)gamma(2)S-GABA(A) receptor subtype. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg(-1) body weight (BW) per day) and diazepam (2 mg kg(-1) BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood-brain barrier for the chemically synthesized, (14)C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K(in)) of 1.14 ml min(-1) g(-1), measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 microm, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites.

Figure 1

Chemical syntheses of ¹⁴C-labelled hispidulin 11 (c) and its building blocks 5 (a) and 7 (b) and the structure of apigenin (d).

Figure 2

Functional properties of hispidulin and apigenin. (a) Hispidulin at 10 μM by itself did not elicit any current but stimulated currents elicited by 4 μM GABA at recombinant α1β2γ2S GABA_A receptors expressed by X. leavis oocytes. (b) Concentration dependence of allosteric stimulation by hispidulin. (c) Stimulation by hispidulin (10 μM) at α1β2γ2S GABA_A receptors is partially inhibited by co-application of the BZD receptor antagonist Ro15-1788 (1 μM). α1β2 GABA_A receptors are not stimulated by 10 μM hispidulin. (d) Allosteric stimulation by hispidulin at α6β2γ2S GABA_A receptors. (e) Inhibition by apigenin of GABA-induced currents at recombinant α1β2γ2S GABA_A receptors. The points indicate the mean±s.e.m. (n=3–5).

Figure 3

Effects of vehicle, hispidulin and diazepam on the occurrence of epileptic seizures in the Mongolian gerbil model (M. unguiculatus) before and after the application of test substances. Paired proportion tests (http://www.ubmail.ubalt.edu/~harsham/Business-stat/otherapplets/PairedProp.htm) were used to examine the null hypothesis that 7 days' pharmacological treatment had no modulatory effect on seizure propensity. ^*P<0.04.

Figure 4

Volume of distribution (V_d) in the rat brain (right hemisphere) for [¹⁴C]hispidulin plotted against time (n=3), as assessed using the Takasato in situ perfusion method. R²=0.9929.

Figure 5

Relationship between log K_in (experimental) and log P_oct (calculated) of passively permeating compounds (diamonds), other flavonoids (naringenin, quercetin), substrates for known uptake (glucose) and efflux (colchicine) transport. Log K_in values with the exception of hispidulin are taken from Qaiser et al. (in preparation; see also Youdim et al., 2004). Log P_oct was calculated using the KowWin (LogKow) software at http://www.esc.syrres.com/interkow/logkow.htm. This program uses fragmental analysis of the compound structure for its prediction (values show high correlation with quoted experimental values, P=0.98).

Figure 6

HPLC traces at 320 nm obtained from receiver wells of the Caco-2 cells showing permeability of hispidulin (a) and apigenin (b) in their intact aglycone forms.