In the present study, its actions on the central nervous system were investigated. Effects of Brotizolam, a New Thieno-Triazolo-Diazepine Derivative, on the Central Nervous System Kenjiro KIMISHIMA, Kyoko TANABE, Yukako KINOSHITA, Kooji TOKUYOSHI, Daisuke HOURI and Tatsuo KOBAYASHI Department of Pharmacology, Tottori University School of Medicine, Yonago 683, Japan Accepted August 24, 1984 Abstract-The effects of brotizolam, a new thieno-triazolo-diazepine derivative, on the central nervous system were analyzed in mice, rats and rabbits. Diazepam, estazolam and triazolam were used as control drugs. Brotizolam inhibited spon taneous motor activities; performances in the rotarod test, staircase test, and maximal electroshock seizure test; and pentetrazol or bemegride-induced convulsion. Moreover, catalepsy inducing action and potentiating effect on sleep elicited by pentobarbital or ethanol were observed. Following intraperitoneal or oral admin istration of brotizolam to rabbits with chronically implanted electrodes , the electro encephalographic profile in spontaneous EEG was characterized by slow waves with high amplitudes in the neocortex. The arousal responses by stimulation of the midbrain reticular formation and posterior hypothalamus were slightly inhibited, but the recruiting responses induced by stimulation of the diffuse thalamic projecting system were not inhibited, and seizure discharges induced by stimulation of the dorsal hippocampus were inhibited markedly. When motor activities and pente trazol-induced convulsions were observed as indices of tolerance for brotizolam, tolerance was not developed by repeated administration of brotizolam up to 14 days. These results suggested that brotizolam, a new thieno-triazolo-diazepine derivative, is judged to be a safer and stronger sleep inducer than diazepam and estazolam . Since the pioneering paper by Randall et et al. (11) and Nicholson et al. (12). al. (1) showed that chlordiazepoxide is a Brotizolam, 2-bromo-4-(2-chlorophenyl)-9 new type of minor tranquilizer with sedative, methyl-6H-thieno [3,2-f] [1, 2, 4]-triazolo muscle relaxant and anticonvulsive actions, [4, 3-a] [1, 4]-diazepine, has the chemical new derivatives of benzodiazepine such as structure shown in Fig. 1. It is a white, diazepam (2), nitrazepam (3), bromazepam odorless powder that is insoluble in water, (4) and medazepam (5) have been developed, soluble in chloroform, and slightly soluble in and they have been in wide clinical use as methyl alcohol. remedies for psychoneuroses such as anxiety and psychosomatic catatonia. Among them, diazepam, nitrazepam and flurazepam (4) are nowadays used as sleep inducers, and in Materials and Methods this field, estazolam (6) and triazolam (7), Animals: In this study ddY-strain mice, new triazolo-benzodiazepine compounds Wistar rats and matured rabbits were mainly having a trizol ring, have invited interest in used, and cats were used in part of the recent years. experiments. On the other hand, studies of benzodiaze General behavior: Each drug was adminis pine analogues led to research and develop tered orally or intraperitonea Ily, and general ment of thienodiazepine compounds (8-10). behavior was observed up to 8 hr after New thieno-triazolo-diazepines with a administration. triazol ring were recently developed by Weber Measurement of spontaneous locomotor time and duration of sleep were investigated. Anticonvulsive action: Male mice of each group consisting of 10-20 animals were orally administered the test or control drug, and at 2 hr after administration, the anti convulsive action was examined by both the maximal electroshock seizure test and drug induced convulsions method (pentetrazol and bemegride). The maximal electroshock (50 mA, for 0.2 sec) was given to mice using the apparatus described by Woodbury and Davenport (14) through corneal electrodes, and the disappearance of tonic extensor (TE) induced by the electroshock was taken as the determinant of the anti convulsive effect of drugs. As to chemo shock, the effects of these drugs were investigated on the minimal full (MF), tonic flexor (TF) and tonic extensor (TE) seizure induced by the subcutaneous in jection of pentetrazol and bemegride at doses of 95 and 38 mg/kg, respectively. Fig. 1. Chemical structures of brotizolam and other benzodiazepines. Staircase test: This is a simple test method devised by Thiebot et al. (15) for screening of anxiolytic drugs. The wooden device used activity: Spontaneous locomotor activity of in this test is a box, 30 cm high and 95 cm mice of each group consisting of 6 mice was long, that contains a 5 story staircase, with recorded up to 6 hr after administration with steps that are each 20 cm wide, 6 cm high an Animex activity meter (Farad Electronics, and 15 cm long. Rats weighing about 200 g Sweden) using the resonance circuit of the were placed quietly at the bottom of the induction coil. staircase with their back toward the staircase, Rotarod test: Mice of each group con and then the frequencies of rearing and sisting of 10 mice or more were put on a climbing the staircase were recorded for the rotarod having a diameter of 3 cm and subsequent 3 min. revolving at 16 r.p.m., and those which did EEG: Unanaesthetized rabbits were fixed not fall off for 3 min or more were regarded in a stereotaxic apparatus, and bipolar as normal. electrodes of insulated stainless steel of 0.25 Catalepsy test: According to Matsuda mm in diameter were implanted in 3 areas of method (13), mice were forced to lay their the cortex (the frontal, temporal and oc forelegs on a horizontal bar at 6 cm in cipital part) and 3-5 subcortical areas of height. Normal mice should try immediately the brain (the posterior hypothalamus, to free themselves from the forced posture. thalamic medial nucleus, midbrain reticular Those which had kept the posture for 30 sec formation, dorsal hippocampus and or more were judged to be catalepsy positive. amygdaloid nucleus). These rabbits were Sleep potentiating action: Male mice of subjected to the test at one week after the each group consisting of 8 or more, which above operation. These electrodes were con had been pre-treated with brotizolam or a nected to a pen-writing 8-lead electro control drug for comparison, were injected encephalograph for recording EEG at a rate with pentobarbital, intraperitoneally, at a of 1 .5 cm per sec. For the purpose of intra dose of 35 mg/kg or 20% ethanol, intra cerebral stimulation, the same electrodes venously, at a dose of 0.1 ml/10 g body were used, and after stimulation, these weight, and the drug effects on the onset electrodes were used as lead electrodes through turning of a switch. For stimulation, of them still showed frolicking behavior. At a rectangular pulse was used, parameters of 24 hr after the injection, some animals still which will be described in the related showed a slight ataxic gait. paragraph. 2. Spontaneous locomotor activity Tolerance test: In the tolerance-producing Mice of each group consisting of 6 animals liability test where motor activities and were placed in plastic observation cages. pentetrazol-induced convulsions were used After recording of their locomotor activity as indices of tolerance, brotizolam was for 2 hr, the test or the control drug was administered to mice for two consecutive administered, and the spontaneous locomotor weeks. activity of these animals was automatically Statistics: Statistical significance of the counted, up to the subsequent 6 hr. data was estimated using Student's t-test. Brotizolam administered orally at a dose The values of ED50 and the 95% confidence of 0.1 mg/kg did not exert any effect on the limit were calculated by the method of spontaneous locomotor activity of these Litchfield and Wilcoxon (16), animals. In the 0.5 mg/kg dose group, Drugs and administration route: As it is spontaneous locomotor activity was slightly insoluble in water, brotizolam was adminis inhibited from 30 min to 6 hr after adminis tered to animals orally or intraperitoneally tration. In the 1 mg/kg and 2 mg/kg dose as a 0.2% CIVIC suspension. As the active groups, a marked inhibition of spontaneous control, diazepam, estazolam or triazolam locomotor activity was observed from 30 min was used (Fig. 1). up to over 6 hr after administration as shown in Fig. 2. Results In both groups that were orally admin 1. General behavior istered diazepam and estazolam at a dose of a) Mice: Following the oral administration 2 mg/kg, moderate inhibition of spon of brotizolam at doses of 0.5 mg/kg or less, taneous locomotor activity was observed no change was observed in the general behavior of mice. At doses of 1-2 mg/kg, most of the mice kept a sedative crouching posture for 4-6 hr after administration. At dose of 5-20 mg/kg, mice were under sedation for longer periods, but recovered from the sedation by the morning of the following day. b) Rabbits: After intraperitoneal injection of brotizolam at doses of 0.5-1 mg/kg, no change was observed in the general behavior. However, at doses of 2-5 mg/kg, the spon taneous locomotor activity decreased; some rabbits kept crouching, motionless, and they somewhat dragged their hind legs when forced to move. Such symptoms lasted up to 3 hr after administration. c) Cats: In several minutes after intra peritoneal injection of brotizolam at a dose of 5 mg/kg, cats started to develop conspic uous muscular relaxation at the hind legs, showing a rather severe ataxic gait. With Fig. 2. Effect of brotizolam on spontaneous respect to consciousness, on the other hand, locomotor activities in mice. Ordinate: counts/min. they were rather excited, biting softly and Abscissa: time in hr. A: control, B: after adminis frolicking. In 30-40 min after the injection, tration of brotizolam, 1 mg/kg and C: brotizolam, the animals entered into sedation, but some 2 mg/kg, p.o.