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. from 1 up 6 hr after administration (Fig. 3), hr after administration of each drug. In both but the inhibition was found to be weaker tests, the number of animals which fell off than in the 1 mg/kg brotizolam group. the rotarod increased at doses of 0.1 mg/kg 3. Rotarod test brotizolam or more. The ED50 of brotizolam The test was performed with mice which 1 hr after administration was 0.26 mg/kg could stay on the rotarod for 3 min or more (Table 1). in each preliminary test, performed thrice The ED50 of estazolam was calculated to on the day before the start of this test. The be 0.53 mg/kg and that of triazolam, 0.66 test was conducted twice at 30 min and 1 mg/kg. 4. Catalepsy test The test was performed with 10 mice in each group. As shown in Table 2, brotizolam caused catalepsy at an oral dose of 0.05 mg/ kg, but the incidence of catalepsy was not observed to be dose-dependent. In estazolam groups, catalepsy also appeared in a dose independent manner in one case each at 0.05 mg/kg and 0.5 mg/kg. 5. Sleep potentiating action a) Pentobarbital-induced sleep: One hr after the oral administration of brotizolam at different doses to mice of each group consisting of 9-10 animals, pentobarbital was intraperitoneally injected at a dose of 35 mg/kg, and the onset and duration of sleep was measured. Fig. 3. Effects of diazepam and estazolam on As shown in Table 3, brotizolam slightly spontaneous locomotor activities in mice. Ordinate: shortened the onset time of sleep at doses of counts/min. Abscissa: time in hr. A: after adminis tration of diazepam, 2 mg/kg and B: estazolam, 0.1-1 mg/kg, whereas the duration of sleep 2 mg/kg, p.o. was prolonged by more than two times that

Table 1. Effects of brotizolam and estazolam on the rotarod test in mice Table 2. Effects of brotizolam and estazolam on the catalepsy test in mice

Table 3. Effects of brotizolam and estazolam on sleeping time induced by pentobarbital (35 mg/kg, i.p.)

observed with the control at a dose of 0.01 brotizolam, ethanol (20 V/V %) was injected mg/kg, by about three times at 0.5 mg/kg, and intravenously into male mice of each group 4 times at 1 mg/kg. In estazolam-adminis consisting of 8-12 at a dose of 0.1 ml/10 g. tered mice, the onset time of sleep tended to As shown in Table 4, sleeping time was delay, while the duration of sleep was markedly prolonged in a dose-dependent prolonged significantly at doses of 0.05-1 manner by brotizolam, though the values mg/kg. showed considerable variation. The ED50 of brotizolam and estazolam 6. Anticonvulsive action calculated in animals that kept sleeping for a) Maximal electroshock seizure: Two hr periods at least two times as long as the after the oral administration of the drugs, an animals of the control group to which electric shock was given to mice of each pentobarbital alone was administered (29 group composed of at least 1 5 mice, and the min) were 0.042 mg/kg and 0.068 mg/kg, anticonvulsive effects of these drugs were respectively. examined. b) Ethanol-induced sleep: One hr after As indicated in Table 5, 75 mg/kg Table 4. Effect of brotizolam on sleep induced by ethanol (2 g/kg, i.v.)

Table 5. Effects of brotizolam and estazolam on MES in mice

brotizolam inhibited the appearance of TE in The effects of the drugs on the pentetrazol half of the mice, but the rate of inhibition did induced convulsions at 2 hr after the adminis not increase further even when the dose was tration were examined. increased to 100 mg/kg, the ED50 of As shown in Table 6, the appearance of brotizolam for inhibition of TE being more pentetrazol-induced convulsions were than 75 mg/kg. markedly inhibited after administration of Estazolam also inhibited TE dose brotizolam at a dose of 0.2 mg/kg or more dependently, the ED50 of estazolam being and estazolam at 0.5 mg/kg or more. The 41.0 mg/kg. ED50 of brotizolam, estazolam, diazepam and b) Pentetrazol-induced convulsions: Fol triazolam were 0.27 mg/kg, 0.66 mg/kg, lowing the subcutaneous injection of pen 0.48 mg/kg and 0.012 mg/kg, respectively. tetrazol at a dose of 95 mg/kg, mice began to c) Bemegride-induced convulsions: When develop MF at 5-15 min after injection and bemegride was subcutaneously injected at a showed slight excitation, jumping repeatedly dose of 38 mg/kg (100% convulsive dose), and moving around in the cage, and some mice developed a series of convulsions in a mice developed transient clonic con process almost similar to that described vulsions (CL). At 7-50 min after injection, above for pentetrazol-induced convulsions. however, almost all of the mice developed TF At this dose, all mice suffered from con and subsequent TE and died. Judgement on vulsions such as M F, TF and TE seizure and anticonvulsive action was made on the about 95% of them died immediately after basis of the disappearance of M F, TF or TE TE. seizure. At 2 hr after oral administration of the test Table 6. Effects of drugs on convulsion induced by pentetrazol (95 mg/kg, s.c.)

Table 7. Effects of drugs on convulsion induced by bemegride (38 mg/kg, s.c.)

and control drugs, bemegride was injected or more and estazolam at 1 mg/kg or more, subcutaneously. As shown in Table 7, the the calculated ED50 values being 0.21 mg/ appearance of bemegride-induced con kg and 0.71 mg/kg, respectively. The ED50 of vulsions were markedly inhibited after treat diazepam and triazolam were 1.2 mg/kg ment with brotizolam at a dose of 0.2 mg/kg and 0.014 mg/kg, respectively. 7. Staircase test and thereafter, the patterns recovered to the The effects of brotizolam and benzodiaze normal condition (Fig. 5). pines on the staircase test which is generally b) Effects on arousal responses induced used for screening of anxiolytic drugs were by stimulation to the posterior hypothalamus: examined. Each drug was administered When the posterior hypothalamus was orally 2 hr before the test. stimulated with 200 Hz, 1 msec, 1-4 V, for As shown in Fig. 4, brotizolam showed 5-7 sec, the desynchronization with low almost the same tendency as that of other voltage and fast waves appeared at the benzodiazepines. Compared with the results cortical EEG. obtained with untreated rats, brotizolam Brotizolam was intraperitoneally injected clearly augmented the frequency of climbing at doses of 2-5 mg/kg. At a dose of 5 mg/kg, the staircase at doses of 0.1-2 mg/kg, but 2 out of 3 rabbits showed moderately reduced the frequency at 4-8 mg/kg, while inhibited arousal response from 10 to 40 min the drug reduced the frequency of rearing after administration (Fig. 6). Whereas at a dose-dependently within the range of 1-4 dose of 2 mg/kg, no inhibitory action was mg/kg. After administration of estazolam clearly detected. also, the frequency of climbing was aug c) Effects on arousal responses induced mented at doses of 0.1-4 mg/kg, but reduced by stimulation to the midbrain reticular clearly at a dose of 8 mg/kg, while the formation: When the ascending activating frequency of rearing was reduced dose system of the midbrain reticular formation dependently within the range of 1-8 mg/kg. was stimulated with 100 Hz, 0.75-2 V, for 8. Effects on EEG 5-7 sec, the cortical EEG showed arousal The effects of brotizolam on EEG were responses with low-voltage and fast waves investigated by administering the drug during stimulation. orally or intraperitoneally to rabbits with Brotizolam was intraperitoneally injected chronically implanted electrodes. at dose of 2-5 mg/kg. At 10-70 min after a) Spontaneous EEG: At 5-10 min after injection, arousal responses were inhibited administration of brotizolam at doses of clearly in 2 out of 3 rabbits in the 2 mg/kg 2-5 mg/kg, the fast-wave components in group and in all 3 rabbits in the 5 mg/kg the cortex began to decrease and high group. All rabbits became normal at 90-120 voltage waves appeared sometimes. The 0) min after injection (Fig. 7). wave at the hippocampus became rather d) Effects on recruiting responses irregular when the drug was administered at induced by stimulation to the thalamic a dose of 5 mg/kg. Such EEG patterns medial nucleus: The thalamic medial nucleus continued till 60-80 min after administration, was stimulated with 6-8 Hz, for 5-7 sec, to

Fig. 4. Effects of brotizolam and other benzodiazepines on the staircase test. Ordinate: rate of climbing and rearing frequency to the control. Abscissa: dose (mg/kg, i.p.). A: brotizolam, B: estazolam and C: triazolam. ------: No. of climbing, - : No. of rearing. Fig. 5. Effect of brotizolam on spontaneous EEG. A: control, B: 5 min after intraperitoneal injection of brotizolam 5 mg/kg, C: 20 min and D: 90 min. Abbreviations (Figs. 5-8): I: left, r: right, Fr: frontal, Pa: parietal and Oc: occipital cortex. Hy: hypothalamus, Th: thalamus, Hipp: hippocampus, RF: midbrain reticular formation and Am: amygdala. investigate the effects of brotizolam on the e) Hippocampal after-discharges: When diffuse thalamic projecting system. The the dorsal hippocampus of rabbits was cortical EEG of untreated rabbits showed stimulated with 100 Hz, 1-5 V, for 5-7 sec, recruiting responses. In all 5 rabbits injected typical hippocampal after-discharges with intraperitoneally with brotizalam at doses of spikes and waves appeared in the hip 2-5 mg/kg, no effect on the system was pocampus, and the after-discharges extended observed, even up to 180 min after injection. to other areas. Fig. 6. Effect of brotizo!am on arousal response induced by stimulation of the posterior hypothalamus. A: control (scale line, 1.0 V), B: 10 min after intraperitoneal iniection of 5 mg/kg brotizolam, C: 25 min and D: 40 min.

At 10-50 min after intraperitoneal in Thus, no development of tolerance producing jection of brotizolam, the durations of the liability to the spontaneous locomotor after-discharges were shortened in 2 out of activity was observed. Neither was tolerance 3 rabbits at a dose of 2 mg/kg, while the developed when estazolam was administered after-discharges disappeared or were in repeatedly at a dose of 3.3 mg/kg. hibited markedly in all 3 rabbits at a dose of b) Pentetrazol-induced convulsions: On 5 mg/kg (Fig. 8). the 1st, 7th and 14th day of brotizolam 9. Tolerance test administration, 95 mg/kg pentetrazol was On the basis of the spontaneous locomotor subcutaneously injected into the mice, and activity and pentetrazol-induced convulsions, the appearance of convulsions was in the tolerance producing liability of brotizolam vestigated. As shown in Table 8, brotizolam was tested in mice. Brotizolam at 1.35 mg/ continued to show the anticonvulsive effect kg, p.o., which was five times the ED50 of up to the day 14, without producing tolerance. brotizolam for inhibition of pentetrazol Also, when estazolam was repeatedly admin induced convulsions, was given consecu istered at a dose of 3.3 mg/kg, which was tively to mice and the effects of the drug on five times the ED50 of estazolam for inhibition these items were examined on the 1st, 7th of pentetrazol-induced convulsions, no and 14th day of administration. tolerance was observed to develop. a) Spontaneous locomotor activity: As shown in Fig. 9, the effect of brotizolam on Discussion the spontaneous locomotor activity recorded Brotizolam, a new thieno-triazolo by the Animex activity meter was found to diazepine derivative, has a chemical structure be almost the same on days 1, 7 and 14. different from those of benzodiazepine Fig. 7. Effect of brotizolam on arousal responses nduced by stimulation of the midbrain reticular formation . A: control (scale line, 1.25 V), B: 10 min after intraperitoneal injection of 5 mg/kg brotizolam, C: 30 min and D: 90 min.

Table 8. Effects of consecutive administration of brotizolam and estazolam on pentetrazol-induced convulsion

compounds, but its pharmacological pro actions on the central nervous system such perties are similar to those of triazolam, a as sedative, anti-anxiety and anticonvulsive benzodiazepine derivative with a triazol effects; production of anti-aggressive ring. In 1979, Koch (17) reported that the behavior; and analgesic, anti-amphetamine drug has a variety of pharmacological and anti-apomorphine actions. For this Fig. 8. Effect of brotizolam on hippocampal after-discharge induced by stimulation of dorsal hip pocampus. A: control (scale line, 2.25 V), B: 15 min after intraperitoneal injection of 5 mg/kg bro tizolam, C: 30 min and D: 50 min. reason, it is generally considered that regard to sedation, decrease in spontaneous brotizolam has a neuroleptic action as well locomotor activity, inhibitory effect in the as actions specific to benzodiazepine rotarod test, potentiation of pentobarbital compounds. induced sleep, marked inhibition of pente In the present study, we obtained results trazol and bemegride-induced convulsions similar to those obtained already in a series and findings from the staircase test. The of benzodiazepine compounds (18-21) in action of brotizolam in inducing catalepsy Fig. 9. Effect of consecutive administration of brotizolam and estazolam on spontaneous locomotor activity. Ordinate: counts/min. Abscissa: time in hr. left: brotizolam, 1.35 mg/kg, p.o.; right: estazolam, 3.3 mg/kg, p.o. A: 1st day, B: 7th day and C: 14th day. was also similar to those of prazepam, appropriate to use brotizolam as a sleep triazolam and diazepam observed already by inducer in clinical therapy, considering its Kimishima et al. (19, 20) and Matsuda (13), strong hypnogenic properties (26), its pos and brotizolam did not show any neuroleptic session of fewer characteristics of anxiolytic action in our study. drugs, and speedy metabolism (27, 28). Regarding EEG findings, brotizolam tended Toxicity of brotizolam is known to be to cause a change to slow waves in the extremely low. The drug caused acute spontaneous EEG and inhibited arousal toxicity in oral administration to mice or rats responses induced by stimulation to the at doses of 3,000 mg/kg or more (29). In posterior hypothalamus and midbrain our study on the actions of the drug on the reticular formation, but all of these actions spontaneous locomotor activity and drug were not different from those of benzodiaze induced convulsions, it exerted these actions pine compounds (18, 19, 21). without development of tolerance, even if it As to the potency of this drug, brotizolam was administered to animals at a dose five was proved to be clearly more potent than times that of the ED50 for anticonvulsive diazepam in a series of our experiments. In effects, once daily for 2 consecutive weeks. view of the anticonvulsive action on pen For these reasons, brotizolam, a new tetrazol and bemegride-induced con thieno-triazolo-diazepine compound, is vulsions, brotizolam was more potent than judged to be a safer and stronger sleep estazolam, but less potent than triazolam. inducer than diazepam and estazolam. 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