Benzodiazepines in the brain

Great progress has been made in the last 5 yr in demonstrating the presence of benzodiazepines (BDZs) in mammalian tissues, in beginning studies on the origin of these natural compounds, and in elucidating their possible biological roles. Many unanswered questions remain regarding the sources and biosynthetic pathways responsible for the presence of BDZs in brain and their different physiological and/or biochemical actions. This essay will focus on recent findings supporting that: (1) BDZs are of natural origin; (2) mammalian brain contains BDZs in concentrations ranging between 5.10⁻¹⁰–10⁻⁸ M; (3) dietary source of BDZs might be a plausible explanation for their occurrence in animal tissues, including man; (4) the formation of BDZ-like molecules in brain is a possibility, experimentally supported; (5) BDZ-like molecules including diazepam andN-desmethyldiazepam are elevated in hepatic encephalopathy; and (6) natural BDZs in the brain are involved in the modulation of memory processes. Future studies using the full range of biochemical, physiological, behavioral, and molecular biological techniques available to the neuroscientist will hopefully continue to yield exciting and new information concerning the biological roles that BDZs might play in the normal and pathological functioning of the brain.     

USE OF A PETHIDINE AND MIDAZOLAM COMBINATION FOR THE REVERSIBLE SEDATION OF CRABEATER SEALS (LOBODON CARCINOPHAGUS)

Between October and December of 1996–1999, off eastern Antarctica (60°-150°E), we darted 31 crabeater seals with midazolam and pethidine at estimated dose rates of 0.15–0.4 mg/kg and 1–3 mg/kg, respectively. Maximum sedation was reached at 23 ± 9 min (n = 18) and first signs of recovery were noted at 54 ± 24 min (n = 4). Seals greater than 250 kg body-mass were sedated by administration of approximately 90–100 mg midazolam and 600 mg pethidine, but the degree of sedation was unpredictable and did not permit invasive procedures in some cases. Behavior of the seal and adjacent conspecifics affected the success of procedures and our ability to monitor vital signs. Naloxone and flumazenil reversed sedation, making this combination attractive for use in animals adjacent to water. Additional ketamine was administered to two seals, resulting in improved restraint.     

Effect of flumazenil on GABAA receptors in isolated rat hippocampal neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

Changes in mACh, NMDA and GABA(A) receptor binding after lateral fluid-percussion injury: in vitro autoradiography of rat brain frozen sections

Adult rats were subjected to a moderate lateral fluid percussion injury (FPI), followed by survival periods of 2 and 12 h. Regional NMDA subtype glutamate, muscarinic acetylcholine and GABA(A) receptor binding in various brain regions was analysed by quantitative in vitro autoradiography and short-lived positron emission tomography tracers [11C]cyano-dizocilpine, 4-N-[11C]methylpiperidylbenzilate (4-N-[11C]MPB), and [11C]flumazenil, respectively. The binding potential (BP, Bmax/KD) was calculated. The data with [11C]cyano-dizocilpine showed a significant decrease in BP bilaterally for the frontoparietal cortex and hippocampus at both time points, in comparison with that of the sham-operated controls. At 12 h the decrease was significantly more prominent for the ipsilateral cortex and hippocampus than for the contralateral side. The BP of 4-N-[11C]MPB was significantly decreased after 2 h for the trauma-side hippocampus, and after 12 h it had decreased for the trauma-site cortex and the bilateral hippocampus. The [11C]flumazenil exhibited a significant decrease in BP for the trauma-site cortex and the underlying hippocampus by 2 h after the traumatic brain injury. After 12 h a significantly decreased BP was observed only for the trauma-site cortex. The finding of a decreased BP demonstrates the involvement of these receptor systems in the development of cellular dysfunction, which is widespread and not limited to the site of lateral FPI.     

Isoliquiritigenin, a chalcone compound, is a positive allosteric modulator of GABAA receptors and shows hypnotic effects

Isoliquiritigenin (ILTG) is a chalcone compound and has valuable pharmacological properties such as antioxidant, anti-inflammatory, anticancer, and antiallergic activities. Recently, the anxiolytic effect of ILTG has been reported; however, its action mechanism and hypnotic activity have not yet been demonstrated. Therefore, we investigated the hypnotic effect and action mechanism of ILTG. ILTG significantly potentiated the pentobarbital-induced sleep in mice at doses of 25 and 50 mg/kg. The hypnotic activity of ILTG was fully inhibited by flumazenil (FLU), a specific gamma-aminobutyric acid type A (GABA_A)–benzodiazepine (BZD) receptor antagonist. The binding affinity of ILTG was 0.453 μM and was found to be higher than that of the reference compound, diazepam (DZP, 0.012 μM). ILTG (10⁻⁵ M) potentiated GABA-evoked currents to 151% of the control level on isolated dorsal raphe neurons. ILTG has 65 times higher affinity for GABA_A–BZD receptors than DZP, and the dissociation constant for ILTG was 4.0 × 10⁻¹⁰ M. The effect of ILTG on GABA currents was blocked by 10⁻⁷ M FLU and ZK-93426. These results suggest that ILTG produces hypnotic effects by positive allosteric modulation of GABA_A–BZD receptors.     

JM-1232(−) and propofol,a new combination of hypnotics with short-acting and non-cumulative preferable properties

Drug interactions are significant in anesthesiology because drug combinations can potentially possess novel properties. The pharmacological advantages of a new combination of the benzodiazepine receptor agonist JM-1232(−) and propofol were investigated in mice. Male adult mice were administered JM-1232(−) or propofol or combinations of the two drugs intravenously. Loss of the righting reflex was evaluated as achieving hypnosis, and the time until recovery of the reflex was measured as hypnosis time. After determining the ED₅₀, doses double and triple the ED₅₀ of propofol were injected with JM-1232(−) to compare hypnosis time. The injections were repeated four times, and the hypnosis times were compared. Flumazenil was administered separately immediately after the last dose was injected. The ED₅₀ values ([95% confidence interval]) for hypnosis were 3.76 [3.36–4.10] for JM-1232(−) and 9.88 [8.03–11.58] mg kg⁻¹ for propofol. Co-administration of 0.5 and 1 mg kg⁻¹ JM-1232(−) reduced the ED₅₀ values of propofol to 1.76 [1.21–2.51] and 1.00 [0.46–1.86] mg kg⁻¹, respectively. The drug combination for hypnosis produced a supra-additive interaction. Hypnosis time was significantly shorter in the groups given the mixtures compared to each hypnotic administered alone. After repeated injections, hypnosis time with the mixtures showed smaller prolongation than that with the hypnotic alone. Flumazenil completely restored the recovery time after anesthesia. The combination of JM-1232(−) and propofol showed a supra-additive interaction, and the reduced hypnotic dose contributed to a faster recovery even after multiple injections.