Vascular effects of midazolam, flumazenil, and a novel imidazobenzodiazepine MP-III-058 on isolated rat aorta.

Hypotensive influences of benzodiazepines and other GABAA receptor ligands, recognized in clinical practice, seem to stem from the existence of "vascular" GABAA receptors in peripheral blood vessels, besides any mechanisms in the central and peripheral nervous systems. We aimed to further elucidate the vasodilatatory effects of ligands acting through GABAA receptors. Using immunohistochemistry, the rat aortic smooth muscle layer was found to express GABAA γ2 and α1-5 subunit proteins. To confirm the role of "vascular" GABAA receptors, we investigated the vascular effects of standard benzodiazepines, midazolam, and flumazenil, as well as the novel compound MP-III-058. Using two-electrode voltage clamp electrophysiology and radioligand binding assays, MP-III-058 was found to have modest binding but substantial functional selectivity for α5ß3γ2 over other αxß3γ2 GABAA receptors. Tissue bath assays revealed comparable vasodilatory effects of MP-III-058 and midazolam, both of which at 100 µmol/L concentrations had efficacy similar to prazosin. Flumazenil exhibited weak vasoactivity per se, but significantly prevented the relaxant effects of midazolam and MP-III-058. These studies indicate the existence of functional GABAA receptors in the rat aorta, where ligands exert vasodilatory effects by positive modulation of the benzodiazepine binding site, suggesting the potential for further quest for leads with optimized pharmacokinetic properties as prospective adjuvant vasodilators.

High amount of lecithin facilitates oral delivery of a poorly soluble pyrazoloquinolinone ligand formulated in lipid nanoparticles: Physicochemical, structural and pharmacokinetic performances.

Preclinical development of deuterated pyrazoloquinolinone ligands, promising drug candidates for various neuropsychiatric disorders, was hindered by unusually low solubility in water and oils. DK-I-60-3 (7-methoxy-d3-2-(4-methoxy-d3-phenyl)-2,5-dihydro-3Hpyrazolo[4,3-c]quinolin-3-one) is one of such pyrazoloquinolinones, and we recently reported about increased oral bioavailability of its nanocrystal formulation (NC). Lipid nanoparticles (LNP) with a high concentration of lecithin, which enhances loading capacity of the lipid matrix, may give rise to further improvement. After preformulation studies by differential scanning calorimetry and polarized light microscopy, LNP were prepared by the hot high pressure homogenization, and characterized in terms of particle size, morphology, and encapsulation efficacy. The layered structure visible on atomic force micrographs was confirmed by nuclear magnetic resonance. Obtained formulations were desirably stable, with small particle size (<100 nm), and high encapsulation efficacy (>99 %). Lecithin was partially fluid and most probably located in the outer shell of the particle, together with DK-I-60-3. While the hydrophobic part of polysorbate 80 was completely immobilized, its hydrophilic part was free in the aqueous phase. In oral neuropharmacokinetic study in rats, an around 1.5-fold increase of area under the curve with LNP compared to NC was noticed both in brain and plasma. In bioavailability study, F value of LNP (34.7 ± 12.4 %) was 1.4-fold higher than of NC (24.5 ± 7.8 %); however, this difference did not reach statistical significance. Therefore, employment of LNP platform in preclinical formulation of DK-I-60-3 imparted an incremental improvement of its physicochemical as well as pharmacokinetic behavior.

Parenteral nanoemulsions of risperidone for enhanced brain delivery in acute psychosis: Physicochemical and in vivo performances.

This work aimed to deepen the lately acquired knowledge about parenteral nanoemulsions as carriers for brain delivery of risperidone, a poorly water-soluble antipsychotic drug, through establishing the prospective relationship between their physicochemical, pharmacokinetic, biodistribution, and behavioral performances. For this purpose, two optimized risperidone-loaded nanoemulsions, stabilized by lecithin or lecithin/polysorbate 80 mixture, and costabilized by sodium oleate, were produced by high-pressure homogenization. The characterization revealed the favorable droplet size, narrow size distribution, high surface charge, with proven stability to autoclaving and long-term stability for at least one year at 25±2°C. Pharmacokinetic and tissue distribution results demonstrated improved plasma, liver, and brain pharmacokinetic parameters, resulting in 1.2-1.5-fold increased relative bioavailability, 1.1-1.8-fold decreased liver distribution, and about 1.3-fold improved brain uptake of risperidone active moiety following intraperitoneal administration of nanoemulsions relative to solution in rats. In behavioral study, investigated nanoemulsions showed pronounced reduction in basal and, more pertinently, amphetamine-induced locomotor activity in rats, with an early onset of antipsychotic action, and this effect lasted at least 90min after drug injection. Together, these findings corroborate the applicability of parenteral nanoemulsions as carriers for enhanced brain delivery of risperidone, further suggesting their promise in acute psychosis treatment or other emergency situations.

Parenteral nanoemulsions as promising carriers for brain delivery of risperidone: Design, characterization and in vivo pharmacokinetic evaluation.

This paper describes design and evaluation of parenteral lecithin-based nanoemulsions intended for brain delivery of risperidone, a poorly water-soluble psychopharmacological drug. The nanoemulsions were prepared through cold/hot high pressure homogenization and characterized regarding droplet size, polydispersity, surface charge, morphology, drug-vehicle interactions, and physical stability. To estimate the simultaneous influence of nanoemulsion formulation and preparation parameters--co-emulsifier type, aqueous phase type, homogenization temperature--on the critical quality attributes of developed nanoemulsions, a general factorial experimental design was applied. From the established design space and stability data, promising risperidone-loaded nanoemulsions (mean size about 160 nm, size distribution <0.15, zeta potential around -50 mV), containing sodium oleate in the aqueous phase and polysorbate 80, poloxamer 188 or Solutol(®) HS15 as co-emulsifier, were produced by hot homogenization and their ability to improve risperidone delivery to the brain was assessed in rats. Pharmacokinetic study demonstrated erratic brain profiles of risperidone following intraperitoneal administration in selected nanoemulsions, most probably due to their different droplet surface properties (different composition of the stabilizing layer). Namely, polysorbate 80-costabilized nanoemulsion showed increased (1.4-7.4-fold higher) risperidone brain availability compared to other nanoemulsions and drug solution, suggesting this nanoemulsion as a promising carrier worth exploring further for brain targeting.

Experimental design in formulation of diazepam nanoemulsions: physicochemical and pharmacokinetic performances.

With the aid of experimental design, we developed and characterized nanoemulsions for parenteral drug delivery. Formulations containing a mixture of medium-chain triglycerides and soybean oil as oil phase, lecithin (soybean/egg) and polysorbate 80 as emulsifiers, and 0.1 M phosphate buffer solution (pH 8) as aqueous phase were prepared by cold high-pressure homogenization. To study the effects of the oil content, lecithin type, and the presence of diazepam as a model drug and their interactions on physicochemical characteristics of nanoemulsions, a three factor two-level full factorial design was applied. The nanoemulsions were evaluated concerning droplet size and size distribution, surface charge, viscosity, morphology, drug-excipient interactions, and physical stability. The characterization revealed the small spherical droplets in the range 195 -220 nm with polydispersity index below 0.15 and zeta potential between -30 and - 60 mV. Interactions among the investigated factors, rather than factors alone, were shown to more profoundly affect nanoemulsion characteristics. In vivo pharmacokinetic study of selected diazepam nanoemulsions with different oil content (20%, 30%, and 40%, w/w) demonstrated fast and intense initial distribution into rat brain of diazepam from nanoemulsions with 20% and 30% (w/w) oil content, suggesting their applicability in urgent situations.

Behavioural characterization of four endemic Stachys taxa.

We performed a basic behavioral characterization of methanol extracts of four Balkan endemic Stachys taxa: S. anisochila (SA), S. beckeana (SB), S. plumosa (SP) and S. alpina subsp. dinarica (SAD). The behavioral activity of extracts dosed intraperitoneally in the range 100-400 mg/kg was examined in adult male Wistar rats, in the elevated plus maze, spontaneous locomotor activity, and grip strength tests, mainly predictive of anxiolytic, sedative and myorelaxant actions, respectively. All investigated Stachys extracts lacked anxiolytic or myorelaxant activities, while SB at 400 mg/kg exerted an anxiogenic-like effect. The study with the selective antagonist beta-CCt showed that the sedative effect of SAD was only partially mediated by GABAA receptors containing the alpha1-subunit. While discernible, the behavioral effects of SA and SP were not distinct. In all extracts, chlorogenic acid and verbascoside were identified. In SA, SB, and SAD the flavonoid fraction was constituted of isoscutellarein and hypolaetine glycosides, while in SP chrysoeriol and apigenin glycosides were present. The results reveal the psychotropic potential of four endemic Stachys taxa, of which SAD appeared most promising as a natural sedative.

Gaba-benzodiazepine receptor complex in brain oxidative metabolism regulation.

This study investigated the impact of modulating the gamma-aminobutyric acid(A) (GABA)(A)-benzodiazepine receptor complex activity on the rat frontal cortex slices oxygen consumption (QO(2)), polarographically determined using the biological oxygen monitor. Throughout the study, diazepam, flumazenil and picrotoxin were administered i.p. 30 min before sacrificing animals and obtaining slice preparations, while GABA was added directly into the medium in the reaction chamber. GABA decreased QO(2) in concentrations of 5 x 10(-4), 10(-2) and 5 x 10(-2)mol l(-1), while 10(-5) and 10(-6)mol l(-1) GABA had no effect, as well as diazepam, flumazenil and picrotoxin. All diazepam doses (1, 2.5 and 5 mg kg(-1)) increased action of 5 x 10(-4)mol l(-1) GABA, whereas 2.5 mg kg(-1) dose amplified the effect of 10(-6)mol l(-1) GABA. Flumazenil and picrotoxin (5 mg kg(-1) both) blocked diazepam's effects. Flumazenil augmented 10(-6)mol l(-1) GABA effects, while picrotoxin and flumazenil abolished the effects of 5 x 10(-4)mol l(-1) GABA. To our knowledge, this is the first study to examine the influence of modulation of GABA(A)-benzodiazepine receptor function on cerebral metabolism of oxygen in in vitro settings. The results are in accordance with those obtained in numerous in vivo studies, pointing to the moderate level of influence of GABA(A)-benzodiazepine receptor complex on QO(2) regulation.