Potent blockade of sodium channels and protection of brain tissue from ischemia by BIII 890 CL.

We have synthesized a new benzomorphan derivative, 2R-[2alpha,3(S*), 6alpha]-1,2,3,4,5,6-hexahydro-6,11, 11-trimethyl-3-[2-(phenylmethoxy)propyl]-2, 6-methano-3-benzazocin-10-ol hydrochloride (BIII 890 CL), which displaced [(3)H]batrachotoxinin A-20alpha-benzoate from neurotoxin receptor site 2 of the Na(+) channel in rat brain synaptosomes (IC(50) = 49 nM), but exhibited only low affinity for 65 other receptors and ion channels. BIII 890 CL inhibited Na(+) channels in cells transfected with type IIA Na(+) channel alpha subunits and shifted steady-state inactivation curves to more negative potentials. The IC(50) value for the inactivated Na(+) channel was much lower (77 nM) than for Na(+) channels in the resting state (18 microM). Point mutations F1764A and Y1771A in transmembrane segment S6 in domain IV of the alpha subunit reduced the voltage- and frequency-dependent block, findings which suggest that BIII 890 CL binds to the local anesthetic receptor site in the pore. BIII 890 CL inhibited veratridine-induced glutamate release in brain slices, as well as glutamate release and neurotoxicity in cultured cortical neurons. BIII 890 CL (3-30 mg/kg s.c.) reduced lesion size in mice and rats when administered 5 min after permanent focal cerebral ischemia at doses that did not impair motor coordination. In contrast to many other agents, BIII 890 CL was neuroprotective in both cortical and subcortical regions of the rat brain. Our results demonstrate that BIII 890 CL is a potent, selective, and highly use-dependent Na(+) channel blocker that protects brain tissue from the deleterious effects of focal cerebral ischemia in rodents.

BIIR 561 CL: a novel combined antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and voltage-dependent sodium channels with anticonvulsive and neuroprotective properties.

Antagonists of glutamate receptors of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype, as well as of voltage-gated sodium channels, exhibit anticonvulsive and neuroprotective properties in vivo. One can postulate that a compound that combines both principles might be useful for the treatment of disorders of the central nervous system, like focal or global ischemia. Here, we present data on the effects of dimethyl-(2-[2-(3-phenyl-[1,2, 4]oxadiazol-5-yl)-phenoxy]ethyl)-amine hydrochloride (BIIR 561 CL) on neuronal AMPA receptors and voltage-dependent sodium channels. BIIR 561 CL inhibited AMPA receptor-mediated membrane currents in cultured cortical neurons with an IC50 value of 8.5 microM. The inhibition was noncompetitive. In a cortical wedge preparation, BIIR 561 CL reduced AMPA-induced depolarizations with an IC50 value of 10.8 microM. In addition to the effects on the glutamatergic system, BIIR 561 CL inhibited binding of radiolabeled batrachotoxin to rat brain synaptosomal membranes with a Ki value of 1.2 microM. The compound reduced sodium currents in voltage-clamped cortical neurons with an IC50 value of 5.2 microM and inhibited the veratridine-induced release of glutamate from rat brain slices with an IC50 value of 2.3 microM. Thus, BIIR 561 CL inhibited AMPA receptors and voltage-gated sodium channels in a variety of preparations. BIIR 561 CL suppressed tonic seizures in a maximum electroshock model in mice with an ED50 value of 2.8 mg/kg after s.c. administration. In a model of focal ischemia in mice, i.p. administration of 6 or 60 mg/kg BIIR 561 CL reduced the area of the infarcted cortical surface. These data show that BIIR 561 CL is a combined antagonist of AMPA receptors and voltage-gated sodium channels with promising anticonvulsive and neuroprotective properties.

Synthesis and structure-activity relationships of 6,7-benzomorphan derivatives as antagonists of the NMDA receptor-channel complex.

We have synthesized a series of stereoisomeric 6,7-benzomorphan derivatives with modified N-substituents and determined their ability to antagonize the N-methyl-D-aspartate (NMDA) receptor-channel complex in vitro and in vivo. The ability of the compounds to displace [3H]-MK-801 from the channel site of the NMDA receptor in rat brain synaptosomal membranes and to inhibit NMDA-induced lethality in mice was compared with their ability to bind to the mu opioid receptor. Examination of structure-activity relationships showed that the absolute stereochemistry is critically important for differentiating these two effects. (-)-1R,9 beta,2"S-enantiomers exhibited a higher affinity for the NMDA receptor-channel complex than for the mu opioid receptor. The aromatic hydroxy function was also found to influence the specificity of the compounds. Shift of the hydroxy group from the 2'-position to the 3'-position significantly increased the affinity for the NMDA receptor-channel complex and considerably reduced the affinity for the mu opioid receptor. From this series of 6,7-benzomorphan derivatives, the compound 15cr.HCl [(2R)-[2 alpha, 3(R*),6 alpha]-1,2,3,4,5,6-hexahydro-3-(2-methoxypropyl)-6,11,11-trimethyl -2,6-methano-3-benzazocin-9-ol hydrochloride] was chosen as the optimum candidate for further pharmacological investigations.

BIII 277 CL is a potent and specific ion-channel blocker of the NMDA receptor-channel complex.

We determined the ability of a new benzomorphan derivative [2R-[2 alpha, 3(R*),6 alpha]]-1,2,3,4,5,6-hexahydro-3-(2-methoxypropyl)- 6,11,11-trimethyl-2,6-methano-3-benzazocin-9-ol hydrochloride (BIII 277 CL) to inhibit the N-methyl-D-aspartic acid (NMDA) receptor-channel complex in vitro and in vivo. BIII 277 CL potently displaced [3H]MK-801 binding from the NMDA receptor-channel complex in synaptosomal membrane preparations from rat brain cortex (Ki = 4.49 nmol/l). It was much less effective at displacing [3H]dihydromorphine, [3H]naloxone and [3H]ditolyguanidine binding in similar membrane preparations: the Ki values were 3323, 8031 and 1017 nmol/l, respectively. BIII 277 CL did not exhibit any marked affinities for a variety of other central neurotransmitter receptors. BIII 277 CL antagonized NMDA-induced [3H]noradrenaline release (EC50 = 1.7 mumol/l) and NMDA-induced inhibition of protein synthesis in rat hippocampal slices (EC50 = 3.0 mumol/l). In mice, BIII 277 CL prevented NMDA-induced lethality (ID50 = 0.54 mg/kg s.c.) and, as expected, also caused disturbances in motor coordination in the same dose range (ED50 = 0.47 mg/kg s.c.). The duration of BIII 277 CL was much shorter than than of (+)MK-801 in both tests. Finally, BIII 277 CL (0.3 mg/kg s.c. 5 times over 24 h) reduced the cortical infarct area in mice that had been subjected previously to focal cerebral ischemia by unilateral occlusion of the middle cerebral artery. In summary, these results indicate that BIII 277 CL is a potent and specific ion-channel blocker of the NMDA receptor-channel complex which could be used for the treatment of acute thromboembolic stroke in humans.

Therapeutic potential of CNS-active M2 antagonists: novel structures and pharmacology.

Clinical trials with muscarinic agonists or acetylcholine esterase inhibitors for the treatment of Alzheimer's dementia have shown disappointing or equivocal results. An alternative treatment of this disease is the development of drugs which enhance the release of acetylcholine. It is believed, that of the five muscarinic receptor subtypes so far identified in the brain, M2 receptors are located presynaptically in the cortex and hippocampus and upon stimulation inhibit the release of acetylcholine. Based on this hypothesis, we initiated a drug discovery program with the aim of identifying selective and centrally active M2 antagonists which are capable of enhancing cholinergic transmission. These efforts resulted in the successful design and synthesis of novel muscarinic antagonists able to cross the blood brain barrier. Moreover, these compounds show few peripheral effects and possess a superior M2 versus M1 selectivity. The prototype of this novel class of M2 selective compounds, BIBN 99, could be a valuable tool to test the hypothesis that lipophilic M2 antagonists show beneficial effects in the treatment of cognitive disorders.

Blood level, metabolism and excretion of [14C]-brotizolam in mice.

Blood level, metabolite pattern and excretion of [14C]-brotizolam, a hypnotic drug, were studied in mice following oral administration. [14C]-Brotizolam was rapidly absorbed which was indicated by a Tmax of the blood level of 0.5 h. Radioactive compounds were eliminated from the blood with a half-life of 5.6 h. Total excretion of radioactivity, the renal portion of which was 22.4%, was complete after 4 days. [14C]-Brotizolam was almost completely metabolized. Using TLC, HPLC and radioactivity measurement, the main metabolite in bile, urine and plasma was found to be brotizolam hydroxylated at the methyl group. Other major metabolites were brotizolam hydroxylated at the diazepine ring and a combination of both hydroxylations. In the bile, all metabolites were conjugated. The metabolism of brotizolam in mice is similar to that in dogs, monkeys and man but not in rats.

New tetracyclic guanidine derivatives with H1-antihistaminic properties. Chemistry of epinastine.

A series of new tetracyclic guanidines were synthesized by various methods. Specific binding of the described compounds to histamine-1 and histamine-2 receptors was determined. The compound 3-amino-9,13b-dihydro-1H-dibenz[c,flimidazo[1,5-a]azepine (epinastine, WAL 801, compound IIIa) combines high selectivity with high affinity for the H1 receptor and was selected from the compounds studied for further pharmacological and clinical investigations. Experimentally determined physicochemical parameters (pka-value, partition coefficient) and the hydrogen-bonding ability of epinastine are indications that this compound will not easily cross the blood-brain barrier. This explains the absence of CNS side-effects of epinastine in pharmacological and clinical studies.

Lack of induction of rat liver mixed-function oxidases after chronic administration of high brotizolam doses to rats.

Rats were treated with 10, 200 or 400 mg/kg brotizolam (Lendormin) for 4 weeks, then liver microsomes were prepared and the in vitro transformation of several model substances studied. Furthermore, after similar treatment of rats, the metabolite pattern in the plasma was studied using [14C]brotizolam as a marker. Finally the same investigations were performed after pretreating the rats with the enzyme inducers, phenobarbital or 3-methylchol-anthrene, for 3 days instead of brotizolam. The amount of microsomal protein in the rat liver was increased after all 3 doses of brotizolam, the liver weight after the highest dose only. Activity of the flavoenzyme NADPH cytochrome-c reductase was the only enzyme activity increased after 200 and 400 mg/kg brotizolam, whereas cytochrome P-450 content decreased after 400 mg/kg brotizolam. Activities of the mixed-function oxidases studied were not changed at all. Marked changes after brotizolam administration were seen in the metabolite pattern. The higher doses led to reduced amounts of both of the very polar metabolites. Simultaneously metabolite We 964 (= brotizolam hydroxylated at the methyl group) and the unchanged brotizolam increased several-fold. Treatment of rats with phenobarbital or 3-methylcholanthrene showed the typical but different changes in enzyme activities. The metabolite pattern of brotizolam, however, was not changed. From the results it is concluded that a 4-week treatment of rats with up to 400 mg/kg brotizolam causes no induction of mixed-function oxidases in the liver. The changes of the metabolite pattern described can be discussed as an effect of liver enzyme saturation.

Relative efficacies of 1,4-diazepines on GABA-stimulated chloride influx in rat brain vesicles.

The effects of 1,4-diazepines with two annelated heterocycles [brotizolam (WE 941), ciclotizolam (WE 973) and WE 1008] on gamma-aminobutyric acid (GABA)-stimulated chloride influx into rat brain membrane vesicles were examined. Brotizolam enhanced GABA (30 microM)-stimulated 36Cl- influx (146.1% of control), while ciclotizolam and WE 1008 showed only a small enhancement (119.3% and 119.1%, respectively) of GABA-stimulated 36Cl- uptake. Brotizolam resulted in a left shift of the GABA dose response curve at lower concentrations of GABA (10 microM), while at higher concentrations of GABA (1 mM), brotizolam caused a reduction of the maximal response. The enhancement of GABA-stimulated 36Cl- uptake by brotizolam (0.1 microM) was antagonized by Ro 15-1788. At higher concentration of GABA (300 microM), brotizolam inhibited GABA-stimulated 36Cl- uptake in a dose dependent manner and Ro15-1788 failed to antagonize this effect. These results suggest that 1) brotizolam produces an enhancement of GABA (30 microM)-stimulated chloride influx through the benzodiazepine receptor. 2) brotizolam inhibition of GABA (300 microM)-stimulated chloride influx involves an additional mechanism, and 3) the sedative-hypnotic action of brotizolam may be related to its high efficacy at the benzodiazepine/GABA-gated chloride channel.

In vitro and in vivo studies of the non-sedating antihistamine epinastine.

Epinastine (3-amino-9,13b-dihydro-1H-dibenz [c,f]imidazo[1,5-a]azepine hydrochloride, WAL 801 CL) was tested in vitro and in vivo in comparison with other H1-receptor antagonists. In the guinea pig ileum and in receptor binding studies the test substance showed a high affinity to H1-receptors. The following rank order was determined: WAL 801 CL greater than astemizole greater than terfenadine. These results were confirmed in vivo. The studies were carried out with oral and intravenous administration of WAL 801 CL to assess the inhibition of histamine-induced reactions in the skin or the lung of rats, dogs and guinea pigs. 10- to 100fold antihistaminic doses of WAL 801 CL showed no effect on the sleeping-waking behaviour of cats. From this and other results it is suggested that the compound does not penetrate in the central nervous system. The action pattern of WAL 801 CL as a non-sedating antihistamine corresponds more to that of terfenadine than that of ketotifen.

Blood level, distribution, metabolite pattern and excretion of [14C]alinidine in mice and rats.

Following oral and intravenous administration the absorption, distribution, metabolite pattern and excretion of [14C]alinidine, a drug with specific bradycardic efficacy, was studied in mice and rats. [14C]alinidine was rapidly and extensively absorbed. The distribution of radio-labelled drug over the entire animal body was rapid as indicated by blood level curves as well as by whole body autoradiography. In both species radioactive compounds were eliminated from blood with half-lives ranging from 5.6 h to 7.4 h. More than 50% of the renally excreted radioactivity was a uniform substance behaving in in TLC and HPLC experiments like the drug administered. From rat urine this compound could be identified as [14C]alinidine using mass spectrometry. In mice and rats no definite substance with clonidine-like chromatographic properties was found. Biliary excretion was demonstrated in both species. The renal portion of the total radioactivity elimination was 67.2-70.1% of the dose administered in mice and 68.1-85.1% in rats. Total excretion was 85.1-101.3% of radioactivity given and was complete 3-4 days after [14C]alinidine administration. No significant differences in pharmacokinetic behavior in mice and rats could be found.

Interactions of MEN 935 (adimolol), a long acting beta- and alpha-adrenolytic antihypertensive agent, with postsynaptic alpha-adrenoceptors in different isolated blood vessels--influence of angiotensin II.

MEN 935 [1-(3-[3-(1-naphthoxy)-2-hydroxypropyl) amino)-3,3-dimethylpropyl)-2-benzimidazolinone-hydrochloride monohydrate, adimolol] is a long acting antihypertensive agent with beta- and alpha-adrenolytic properties. Preliminary experiments in pithed rats had led to the suggestion that the alpha-adrenolytic activity was of the alpha 2-subtype. The alpha-adrenolytic properties of MEN 935 were now tested in isolated vascular preparations of rat aorta, rabbit vena ischiadica and rabbit vena cava inferior against the selective alpha 1-adrenergic agonist phenylephrine (PE) and the selective alpha 2-adrenergic agonist B-HT 920 [2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-(4,5-d)azepine]. The experiments were performed in absence and in presence of 5 X 10(-9) mol/l angiotensin II (A II). MEN 935 antagonized contractions to phenylephrine as well as those to B-HT 920 in each vessel. A twofold shift to the right of the concentration-response curves to both agonists was obtained with concentrations between 1.9 X 10(-8) and 1.4 X 10(-5) mol/l, depending on the vessel under investigation. A II modulated the adrenolytic properties of MEN 935 in each vessel. However, irrespective of the presence or absence of A II, no pharmacologically relevant difference between antagonism against PE or B-HT 920 could be seen. In isolated vessels, MEN 935 exerts a nonselective alpha-adrenergic antagonism. In receptor binding studies in rat cerebellar cortex, MEN 935 showed a Ki of 5.2 X 10(-7) mol/l at alpha 1-adrenoceptors and a Ki of 1.3 X 10(-5) mol/l at alpha 2-adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of brotizolam on mixed-function oxidases and glutathione metabolism in the rat.

Intra-gastric administration of brotizolam (0.1-200 mg/kg) daily for three days to rats resulted in no significant changes in the hepatic and intestinal cytochrome P-450-dependent or P-448-dependent mixed-function oxidases, or in the hepatic flavoprotein dimethylaniline N-oxidase. Liver microsomes from mouse, rat and man metabolized brotizolam by hydroxylation of the diazepine ring and of the methyl group at rates which were greater for mouse greater than rat greater than man. Brotizolam and its metabolites generated by rat-liver microsomes in vitro were not mutagenic in the Ames' test. Brotizolam, at 200 mg/kg per day for two to six weeks, depleted liver glutathione concentration and markedly increased liver gamma-glutamyl transpeptidase, glutathione reductase and glutathione transferase activities. Similar changes were not seen at the lower dose of 0.3 mg/kg. The observed increases in glutathione metabolism and the decreased tissue concentration of glutathione are indicative of high levels of glutathione conjugation, and provide a possible explanation for the equivocal increase in tumorigenicity seen in rats receiving brotizolam at high dosage.

Biochemical pharmacology of pirenzepine. Similarities with tricyclic antidepressants in antimuscarinic effects only.

5,11-Dihydro-11-[(4-methyl-1-piperazinyl) acetyl]-6H-pyrido[2,3-b][-1,4]-benzodiazepin-6-one dihydrochloride (pirenzepine, Gastrozepin) and some tricyclic antidepressant drugs which show a very close relationship concerning the chemical structure were investigated in numerous binding, uptake and enzymatic studies in vitro. With pirenzepine a high affinity binding could be demonstrated only to muscarinic receptors (Ki = 58 nmol/l). In all other studies pirenzepine had a very weak or no effect at all. In contrast, tricyclic antidepressants bound with high but different affinities to various receptors as known from numerous publications. The highest affinities were found with imipramine at the specific imipramine binding sites (Ki = 9.8 nmol/l) and at the alpha 1-receptor (Ki = 39 nmol/l), with desipramine at the muscarinic receptors (Ki = 88 nmol/l), with mianserin at the H1-(Ki = 3.4 nmol/l) and 5HT2-receptors (Ki = 7.3 nmol/l). Moreover, imipramine and desipramine showed their known substantial inhibition of noradrenaline and/or 5-hydroxytryptamine uptake. Thus, a homogeneous affinity or activity profile of the antidepressants studied does not exist. The only common property of pirenzepine and the tricyclic antidepressants was found to be the high affinity binding to the muscarinic receptors which might explain the common antisecretory action of these agents. Because of the unique specificity of pirenzepine lacking all other effects of the tricyclic antidepressants as demonstrated in this study, it is very unlikely that this drug exerts any antidepressant-like central action.

Biochemical studies with the new thienotriazolo-diazepine brotizolam.

Brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) is a newly developed thienotriazolo-diazepine with distinct hypnotic effects. It was biochemically characterized by performing various receptor binding studies, uptake and release studies in vitro and determining homovanillic acid brain concentrations in vivo. Using [3H]-flunitrazepam as a radioligand, brotizolam showed an extremely high affinity for specific benzodiazepine binding sites in the rat brain, demonstrated by an IC50 value of 1.0 nmol/l. Using both [3H]-flunitrazepam and [3H]-brotizolam as radioligands, a strong correlation between the IC50 values of brotizolam and various benzodiazepines could be shown. [3H]-Flunitrazepam as well as [3H]-brotizolam bound with similar affinities to cerebellar and hippocampal synaptosomal membranes. In contrast to [3H]-flunitrazepam binding, however, [3H]-brotizolam had twice the number of specific binding sites in the hippocampus. According to the maximum binding of [3H]-flunitrazepam, the inhibition curve of brotizolam also increased in the presence of 10(-4) mol/l gamma-aminobutyric acid and decreased remarkably in the presence of 10(-4) mol/l bicuculline. Using [3H]-muscimol as a ligand, a modulating effect of brotizolam could be shown by increasing the low affinity with no change in the number of binding sites. From various further receptor binding studies, as well as from in vitro uptake and release studies of [3H]-noradrenaline, [3H]-serotonin and [3H]-dopamine, no indication was found of another biochemical effect of brotizolam. There was also no change in the homovanillic acid concentration in the rat brain striata in vivo after brotizolam treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Pharmacological action of some known and possible metabolites of brotizolam.

The two major metabolites of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno [3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin) in man are the hydroxylation products We 964 (hydroxylation in the methyl group at 9-position) and We 1061 (hydroxylation in 6-position). A structural isomer originating from the latter, We 1064, was found in the urine of dogs. In the same species the demethylation product We 956 was identified. In line with this demethylation the oxidation product Web 1175 might also be formed but was not yet detected. The dihydroxylated product Web 1073 which could arise either from We 964 or from We 1061 was discovered in monkeys. All compounds were investigated with respect to their pharmacological and acute toxicological properties in various experimental situations in mice and rats, and exhibited a profile of action quite similar to brotizolam. Effects of other kinds did not occur. None of the examined metabolites had a longer duration of action than the parent substance. The strength of activity in the various tests was, with very minor exceptions, less than that of brotizolam. All findings favor the conclusion that the various actions of brotizolam are mainly caused by the latter itself and not by its active metabolites. Also, the duration of action of brotizolam is not substantially determined by the pharmacokinetics of its metabolites.

Blood level, distribution, excretion, and metabolite pattern of [14C]-brotizolam in the rat, dog, and rhesus monkey.

Following oral and intravenous administration the absorption, distribution, metabolite pattern and excretion of 14C-labeled brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3,2-f]-1,2,4-triazolo [4,3-a]-1,4-diazepine, We 941, Lendormin), a new hypnotic, was studied in rats, dogs, and rhesus monkeys. Given orally to these species, [14C]-brotizolam was rapidly and extensively absorbed. The distribution of the radiolabeled drug was very fast indicated by blood level curves as well as by rat whole body autoradiography. [14C]-Brotizolam and its metabolites crossed the placenta; they also occurred in the milk of rats. In all species studied radioactivity was eliminated from blood with very similar half-lives ranging from 14.8 to 20.8 h. The renal portion of the total radioactivity elimination increased from 5.5% of the dose given to rats to 33.5% in rhesus monkeys and to 51.4% in dogs. Total excretion was complete 3 to 4 days after [14C]-brotizolam administration. Biliary excretion also occurred. In the species studied thin layer chromatographic separation of the biliarily and renally excreted radioactivity indicated a similar but not the same pattern of 2 to 3 major metabolites which were mostly totally conjugated. Unchanged [14C]-brotizolam was only excreted in minor quantities, if at all.

Blood level, excretion, and metabolite pattern of [14C]-brotizolam in humans.

Following a single oral dose of 14C-labeled brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno[3, 2-f]-1,2,4-triazolo[4,3-a]-1,4-diazepine. We 941, Lendormin) the plasma level concentration and the total excretion of the radioactivity as well as the plasma level of the unchanged substance was measured in 4 healthy subjects. [14C]-Brotizolam was rapidly absorbed. The elimination half-life of the radioactivity from the plasma was 9.5 h, that of the unchanged [14C]-brotizolam 4.4 h. Using a two-compartment open model, the stimulation of giving 0.5 mg of the drug once daily showed no accumulation of brotizolam and its metabolites. Within the 4-day study 64.9% of the given radioactivity was eliminated renally. The total excretion was 86.5% of the dose. Extraction and thin layer chromatographic fractionation of the renally excreted radioactivity indicated two major metabolites one of which predominated. The metabolites were completely conjugated. Unchanged [14C]-brotizolam was excreted in minor amounts below 2-3% of the dose.

Metabolic fate of [14C]-brotizolam in the rat, dog, monkey and man.

The metabolism of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno [3,2-f]-1,2,4-triazolo[4,3-a]-1,4-diazepine, We 941, Lendormin) was studied in the bile of the rat and in the urine of the dog, rhesus monkey and man using 14C-labeled substance. Concentration, fractionation and purification of the metabolites were performed using thin layer chromatography, column chromatography or high pressure liquid chromatography. Metabolites were structurally characterized by thin layer chromatography, high pressure liquid chromatography, mass spectrometry and nuclear magnetic resonance spectrometry using reference compounds. Hydroxylation at different sites of the brotizolam molecule and subsequent conjugation were the metabolic pathways preferred by far in the species studied. Unchanged brotizolam was excreted in minute amounts only, if at all. In man and monkey We 964 (brotizolam hydroxylated in the methyl group) and We 1061 (brotizolam hydroxylated in the diazepine ring) represented the main metabolites. In the rat, the main metabolites were We 1061 and a brotizolam hydroxylated in the phenyl ring. The main metabolites found in the dog were We 964 and We 1064, an isomeric compound of We 1061. Since We 1061 is irreversibly transformed in an alkaline medium to We 1064, the latter could be formed due to the clean-up processes. Thus, in the dog also We 1061 was probably the metabolite which was actually excreted renally. The proposed structures of minor metabolites are presented.

Physical dependence capacity of brotizolam in rhesus monkeys. 1st communication: primary dependence studies.

Abstinence symptoms of doses acutely equi-effective on motocoordination of brotizolam (2-bromo-4-(2-chlorophenyl)-9-methyl-6H-thieno [3,2-f]-1,2,4-triazolo[4,3-a]-1,4-diazepine, We 941, Lendormin) and diazepam in rhesus monkeys were evaluated in a primary dependence study of 61 days. After termination of oral treatment with 3 X 5.4 mg/kg/d brotizolam and 3 X 13.5 mg/kg/d diazepam the duration of withdrawal symptoms varied. Most symptoms of brotizolam abstinence disappeared within 24 h of withdrawal, while the withdrawal symptoms following diazepam were more pronounced between the second and fifth day after termination of administration. Regularly during the whole study, determinations of the serum levels of brotizolam and diazepam were performed. Two days after termination of brotizolam treatment the substance could scarcely be detected in the serum. Diazepam serum levels, in contrast, declined more slowly. The physical dependence capacity of lower daily doses of brotizolam, 3 X 0.2, 3 X 0.6, and 3 X 1.8 mg/kg/d was tested in experiments with chronic administration for 4 weeks. 3 X 1.8 mg/kg/d was the lowest oral dose inducing physical dependence. Taking into consideration the great difference in human therapeutic single dosages, brotizolam is thought to have a very low physical dependence capacity in man, compared with diazepam.