Metabolism of two new benzodiazepine-type anti-leishmanial agents in rat hepatocytes and hepatic microsomes and their interaction with glutathione in macrophages.

OBJECTIVES: To measure the metabolism and toxicity of 7-chloro-4-(cyclohexylmethyl)-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-1) and 4-cyclohexylmethyl-1-methyl-3,4-dihydro-1H-1,4-benzodiazepine-2,5-dione (BNZ-2), two new benzodiazepine analogues found to be effective against Leishmania amastigotes in vitro. METHODS: The metabolism of BNZ-1 and -2 was investigated in isolated rat hepatocytes and rat liver microsomes. The toxicity of the compounds was assessed in a murine macrophage cell line by determining cell viability and reduced glutathione (GSH) content. The metabolism and toxicity of flurazepam was assessed for comparison. KEY FINDINGS: BNZ-1 and BNZ-2 underwent similar metabolic transformations by the liver systems, forming N-demethylated and hydroxylated metabolites, with subsequent O-glucuronidation. Flurazepam and both analogue compounds depleted macrophage GSH levels without affecting cell viability at the concentrations used (up to 100 microM), but only flurazepam inhibited glutathione reductase activity, indicating that it is acting by a different mechanism. CONCLUSIONS: The exact mechanism responsible for GSH depletion is unknown at present. Further experiments are needed to fully understand the effects of BNZs on the parasite GSH analogue, trypanothione, which may be a direct or indirect target for these agents. Pharmacokinetic evaluation of these compounds is required to further progress their development as potential new treatments for leishmaniasis.

Characterization and in vitro phase I microsomal metabolism of designer benzodiazepines: An update comprising flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864).

The number of newly appearing benzodiazepine derivatives on the new psychoactive substances (NPS) drug market has increased over the last couple of years totaling 23 'designer benzodiazepines' monitored at the end of 2017 by the European Monitoring Centre for Drugs and Drug Addiction. In the present study, three benzodiazepines [flunitrazolam, norflurazepam, and 4'-chlorodiazepam (Ro5-4864)] offered as 'research chemicals' on the Internet were characterized and their main in vitro phase I metabolites tentatively identified after incubation with pooled human liver microsomes. For all compounds, the structural formula declared by the vendor was confirmed by gas chromatography-mass spectrometry (GC-MS), liquid chromatography-tandem mass spectrometry (LC MS/MS), liquid chromatography-quadrupole time of flight-mass spectrometry (LC-QTOF-MS) analysis and nuclear magnetic resonance (NMR) spectroscopy. The metabolic steps of flunitrazolam were monohydroxylation, dihydroxylation, and reduction of the nitro function. The detected in vitro phase I metabolites of norflurazepam were hydroxynorflurazepam and dihydroxynorflurazepam. 4'-Chlorodiazepam biotransformation consisted of N-dealkylation and hydroxylation. It has to be noted that 4'-chlorodiazepam and its metabolites show almost identical LC-MS/MS fragmentation patterns to diclazepam and its metabolites (delorazepam, lormetazepam, and lorazepam), making a sufficient chromatographic separation inevitable. Sale of norflurazepam, the metabolite of the prescribed benzodiazepines flurazepam and fludiazepam, presents the risk of incorrect interpretation of analytical findings.

Metabolism of flurazepam by the small intestine.

The metabolism of flurazepam-5-14C has been studied in man following catheterization of the portal and hepatic veins. Flurazepam was administered through a tube into the stomach in one patient and into the duodenum in two patients. Thin-layer chromatographs of portal vein blood showed that there was a rapid and early appearance of metabolites of flurazepam consistent with the metabolism of the flurazepam by the intestinal mucosa and at times when the concentrations in the hepatic vein and peripheral blood were very much lower than those in the portal vein. The major metabolites identified in portal vein blood were the mono- and didesetyl metabolies of flurazepam. Considerable hepatic uptake of flurazepam and its metabolites occurred, as evidenced by the lower concentrations of the parent compound and metabolites in the hepatic vein. Thus, "first-pass" metabolism of flurazepam following oral administration occurs in the small bowel mucosa of man as well as in the liver.

Comparative efficacy of estazolam, flurazepam, and placebo in outpatients with insomnia.

The efficacy and safety of estazolam, an investigational triazolobenzodiazepine, and flurazepam were compared in 65 insomniac outpatients. Patients completed sleep questionnaires each morning. Global evaluations demonstrated that both treatments were significantly superior to placebo. However, estazolam was preferred over flurazepam in a global rating that reflected how well rested and refreshed the subjects felt on arising. Improvement in complaints of difficulty in going to sleep showed only a trend toward significance favoring estazolam and flurazepam over placebo. Residual daytime drowsiness and fatigue accounted for approximately 70% of all side effects with both active treatments. Significantly more side effects occurred with flurazepam than with estazolam. Flurazepam-treated patients had a significantly more severe rating of adverse reactions than did placebo-treated patients.

Quazepam and flurazepam: differential pharmacokinetic and pharmacodynamic characteristics.

Quazepam and flurazepam share pharmacokinetic properties that result in prevention of early-morning insomnia, daytime rebound anxiety, and withdrawal rebound insomnia. Yet sleep laboratory and performance studies demonstrated that during a 1- to 4-week administration period quazepam had a low potential for causing daytime drowsiness or impairment. This profile may be related to several factors, such as differences in quazepam's metabolic pathways; plasma pharmacokinetics; rate of brain uptake, redistribution, and clearance; as well as differences in receptor binding and kinetics.

Objective measurements of daytime sleepiness and performance comparing quazepam with flurazepam in two adult populations using the Multiple Sleep Latency Test.

Daytime residual drowsiness and psychomotor performance were assessed for two long half-life benzodiazepines, quazepam and flurazepam, in two randomized, parallel, and double-blind studies in insomniacs. Seventeen middle-aged patients took quazepam 15 mg or 30 mg, or flurazepam 30 mg; the 47-night study included 4 placebo baseline nights, 28 consecutive treatment nights, and 15 posttreatment nights. Forty-eight geriatric patients took either flurazepam 15 mg, quazepam 15 mg, or placebo; the 15-night study included 1 placebo baseline night, 7 treatment nights, and 7 posttreatment nights. The Multiple Sleep Latency Test (MSLT), an objective test for measuring daytime sleepiness, and performance tests were administered. In the first study, flurazepam patients were significantly (p less than .05) sleepier after the 7th and 14th treatment nights when compared to baseline, whereas quazepam patients were not. In the second study, flurazepam patients were sleepier at midday (p less than .10) and late afternoon (p less than .05) after 1 treatment week than were quazepam and placebo patients. Performance test results suggested quazepam has a relatively low potential for daytime impairment. Thus, quazepam 15 mg produces less daytime somnolence and fewer psychomotor performance decrements than does flurazepam.

Kinetics, brain uptake, and receptor binding characteristics of flurazepam and its metabolites.

The benzodiazepine derivative flurazepam (FLZ) is widely used as a hypnotic, but the relative contributions of FLZ and its metabolites desalkylflurazepam (DA-FLZ), hydroxyethylflurazepam (ETOH-FLZ), and flurazepam aldehyde (CHO-FLZ) to overall clinical activity remain uncertain. A single 20 mg/kg dose of FLZ.HCl was administered to mice, with plasma and brain concentrations of FLZ and metabolites determined during 5 h after dosage. Brain and plasma concentrations of FLZ were maximal at 0.5 h after dosage, then declined rapidly in parallel, whereas those of DAFLZ were maximal at 2 h, then declined slowly. Concentrations of ETOH-FLZ, the most polar metabolite, were maximal at 0.5 h, and were undetectable after 3 h. Little CHO-FLZ was detected in either brain or plasma. A single 30-mg oral dose of FLZ.HCl was given to 18 human volunteers, with plasma levels determined over 9 days. FLZ was detected in plasma at low concentrations for no more than 3 h after dosage. ETOH-FLZ concentrations were higher and persisted for 8 h after dosage. CHO-FLZ reached intermediate peak levels and was present longer than FLZ or ETOH-FLZ. In contrast, DA-FLZ achieved the greatest peak concentrations, occurring at 10 h after dosage. Levels declined very slowly, with a mean half-life of 71.4 h, and were still detectable 9 days after FLZ dosage. Plasma free fractions (percent unbound) in mice were 40.3, 51.4, and 25.0% for FLZ, ETOH-FLZ and DA-FLZ, respectively; in humans, values were 17.2, 35.2, and 3.5%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Excretion of quazepam into human breast milk.

Previous metabolic studies have established that two major metabolites, 2-oxoquazepam and N-desalkyl-2-oxoquazepam, are present in plasma after dosing with quazepam, a new benzodiazepine hypnotic. The excretion of quazepam, 2-oxoquazepam, and N-desalkyl-2-oxoquazepam into human breast milk was studied in four lactating nonpregnant volunteers. Each volunteer received one 15-mg quazepam tablet following an overnight fast. Nursing of offspring was discontinued after drug administration. Milk and blood samples were collected prior to and at specified times (up to 48 hours) after dosing. Plasma and milk levels of quazepam, 2-oxoquazepam, and N-desalkyl-2-oxoquazepam were determined by specific GLC methods. The concentrations of the three compounds found in milk appeared to depend on their relative lipophilicities, which were determined by log P values. The mean milk/plasma AUC ratios of quazepam, 2-oxoquazepam, and N-desalkyl-2-oxoquazepam were 4.19, 2.02, and 0.091, respectively. Levels of quazepam and 2-oxoquazepam declined at about the same rate in plasma and in milk. The total amount of the administered quazepam dose found in the milk as quazepam, 2-oxoquazepam, and N-desalkyl-2-oxoquazepam through 48 hours was only 0.11 per cent.

Interaction of cimetidine with oxazepam, lorazepam, and flurazepam.

The influence of cimetidine coadministration, 300 mg every 6 hours, on the kinetics of single oral doses of oxazepam (30 mg), lorazepam (2 mg), and flurazepam (30 mg) was evaluated in healthy volunteers. Cimetidine had no significant effect on the peak plasma concentration or the time of peak concentration for either oxazepam, lorazepam, or desalkylflurazepam (formed from flurazepam). Cimetidine likewise did not alter the elimination half-life of oxazepam (9.4 hours) or lorazepam (11.6 hours), and did not change total AUC for lorazepam. Oxazepam AUC was increased an average of 10 per cent by cimetidine (P less than 0.02). In contrast, cimetidine prolonged desalkylflurazepam elimination half-life (141 vs. 94 hours, P less than 0.1) and increased AUC an average of 65 per cent (P less than 0.05). Thus, cimetidine has little or no influence on the absorption or disposition of oxazepam and lorazepam, two benzodiazepines biotransformed by glucuronide conjugation. However, cimetidine slows the elimination of flurazepam's metabolite, desalkylflurazepam, which is biotransformed by oxidation.

Rebound insomnia and elimination half-life: assessment of individual subject response.

Following abrupt withdrawal of five benzodiazepine hypnotics, the presence of rebound insomnia on individual subject nights was evaluated in comparison to a placebo group. During the first three nights of withdrawal, the frequency of occurrence of rebound insomnia for drugs with relatively rapid rates of elimination (triazolam, midazolam, and lormetazepam) was significantly higher than that for the placebo control group. In contrast, the frequency of withdrawal sleep difficulty for two slowly eliminated hypnotics (flurazepam and quazepam) was similar to that of the placebo control group during each of five successive three-night segments of a 15-night withdrawal period. These findings, based on individual subject-night data, confirm and extend previous reports using group mean values that demonstrate a frequent, immediate, and intense degree of rebound insomnia following abrupt withdrawal of relatively rapidly eliminated hypnotic drugs and an infrequent, delayed, and milder degree of sleep difficulty following withdrawal of slowly eliminated drugs.

Behavioral measurement of benzodiazepine tolerance and GABAergic subsensitivity in the substantia nigra pars reticulata.

Rotational behavior was elicited by unilateral microinjection of the benzodiazepine flurazepam, and the gamma-aminobutyric acid (GABA) agonist, muscimol, into the substantia nigra pars reticulata (SNpr). This response was used to quantitate benzodiazepine tolerance and GABAergic subsensitivity after chronic benzodiazepine treatment. Studies in naive rats established the dose requirements for inducing contralateral circling and demonstrated the reproducibility of the behavioral response as a measure of SNpr function. There was a large difference in potency between the two drugs for causing dose-related rotation. The response to microinjected flurazepam could be blocked by 16 mg/kg of the benzodiazepine antagonist, Ro15-1788. Tolerance to intranigral flurazepam (50 micrograms) was measured by a reduction in the turning response after a 1- or 4-week chronic flurazepam treatment. The time course for the reversal of tolerance after a 4-week benzodiazepine treatment correlates with the time course of the reversal of benzodiazepine receptor down-regulation in the SNpr. Subsensitivity of the GABAergic system was demonstrated by the decreased rotational response to muscimol (10 ng), confirming the idea that the GABAergic system is also functionally altered by chronic benzodiazepine treatment. The time course of the decreased sensitivity to muscimol does not coincide with the development and reversal of tolerance to the turning produced by flurazepam or with benzodiazepine receptor down-regulation. These data suggest differential regulation of SNpr sensitivity to benzodiazepine and GABA agonists following chronic benzodiazepine treatment and may provide a basis for differential tolerance; the development of tolerance to some but not other benzodiazepine actions.

Radioreceptor assay of benzodiazepines in cerebrospinal fluid during chronic flurazepam treatment in cats.

A radioreceptor assay was used to measure benzodiazepine-like activity in cerebrospinal fluid (CSF) of cats during chronic flurazepam treatment. Benzodiazepine-like activity was measured by the displacement of [3H]flunitrazepam from benzodiazepine receptors in homogenates of rat cerebral cortex. Cats were given 5 mg/kg flurazepam daily, and tolerance was measured by rating several indicators of neurological impairment. CSF was sampled 1 h after flurazepam administration, when drug actions were greatest. Ataxia and muscle relaxation were greatest on the first treatment day, then decreased despite increasing CSF benzodiazepine-like activity. By day 11, CSF activity was 3 times that measured 1 h after the first chronic dose. CSF benzodiazepine-like activity declined slowly after treatment and approached zero by the 10th day after treatment. This residual activity, probably due to active flurazepam metabolites, correlates with the observation that physical dependence is present in these cats up to 7 days after the end of chronic treatment. The rapid loss of drug effect despite increasing active drug levels shows that the nervous system is capable of a rapid and profound adaptation to the presence of benzodiazepines.

Benzodiazepine agonist-type activity of raubasine, a rauwolfia serpentina alkaloid.

Raubasine produced a dose-related inhibition of specific [3H]flunitrazepam binding to rat brain membranes. Scatchard analyses revealed a significant increase in the affinity constant but no change in the number of binding sites, suggesting that raubasine acts as a competitive inhibitor. Raubasine also inhibited the in vivo binding of [3H]flunitrazepam to mouse brain sites. Behavioral studies showed raubasine to possess anticonvulsant properties against pentylenetetrazol- and bicuculline-induced convulsions in mice. These effects were inhibited by the benzodiazepine antagonist, Ro 15-1788. The results suggest that raubasine interacts directly at benzodiazepine sites with a benzodiazepine agonist-type activity.

Several new benzodiazepines selectively interact with a benzodiazepine receptor subtype.

The potency of several new benzodiazepines as inhibitors of [3H]flunitrazepam binding was investigated in membranes from rat cerebellum or hippocampus. It was found that quazepam and two of its metabolites have a higher affinity for benzodiazepine receptors in cerebellum than for those in hippocampus, indicating a preferential interaction of these compounds with BZ1-receptors. Other experiments indicated that these compounds have benzodiazepines agonistic properties similar to diazepam or flunitrazepam. Thus for the first time, benzodiazepines have been identified with differentially interact with different benzodiazepine receptors.

Pharmacokinetics of benzodiazepines: metabolic pathways and plasma level profiles.

Large differences exist among the various benzodiazepines with regard to their pharmacokinetic properties and metabolism in man. Some are eliminated from the body at a relatively slow rate, e.g. desmethyldiazepam, and others are metabolized rapidly, e.g. midazolam, triazolam. Several benzodiazepines have major active metabolites that are slowly eliminated, e.g. medazepam, halazepam , quazepam and, consequently, should be considered as potentially long-acting. Such differences may be very important clinically because pharmacokinetic data will help to optimize drug therapy with respect to the choice of the proper drug and drug preparation, as well as with the choice of a proper dose and dosage regimen. The therapeutic objectives of drug therapy differ quite considerably for the various clinical indications of benzodiazepines. In anti-anxiety and anti-epileptic therapy, prolonged or continuous treatment is pursued, so that compounds with relatively long or intermediate elimination half-lives of parent drug or active metabolites are of advantage. In hypnotic treatment, on the other hand, the duration of drug action should be restricted to the duration of the night, hence a compound with a short elimination half-life may be preferred. An overview is given of the pharmacokinetics of the major benzodiazepines currently available and of some interesting new ones that are still in the development stage.

Bidirectional effects of chronic treatment with agonists and inverse agonists at the benzodiazepine receptor.

We have studied in rodents the effects of beta-carboline inverse agonists on chronic treatment and after repeated administration of benzodiazepine agonists. Chronically, the inverse agonist FG 7142 caused chemical kindling, i.e., a decrease in the threshold to the convulsive effects of the drug. This change was accompanied by decreases in the effects of beta-carboline but not benzodiazepine agonists. In addition the effects of GABA receptor agonists were decreased and the effects of GABA antagonists marginally increased. The GABA stimulated benzodiazepine binding was lower after FG 7142 kindling. Some evidence was found in mice to suggest that these changes were accompanied by behavioural alterations, but studies in rats did not show any changes. Repeated administration of benzodiazepine agonists, sufficient to cause tolerance to their pharmacological actions and to those of beta-carboline agonists, increased all of the effects of the partial inverse agonists and some of the actions of the full inverse agonists. We suggest that this is due not to precipitation of withdrawal but to a "withdrawal shift" in the coupling at the receptor inophore. This would increase the intrinsic properties of inverse agonists and decrease those of agonists. Evidence for this hypothesis is summarised.

Quantitative estimation of potentiation and antagonism by dose ratios corrected for slopes of dose-response curves deviating from one.

A shift of dose-response curves of a receptor agonist A by a receptor antagonist B to the right is frequently expressed or quantitated by calculating the dose ratio (DR) from the ED50 values obtained in the absence and presence of B. A comparison of ED50 values or a DR is also used in a more general way to express the effects of other antagonists or of potentiators. For this situation, where B is not competing with A for a binding site, slope-values may often deviate from one. Because the slope of shifted dose-response curves (deviating from one) affects the magnitude of enhancement or diminution at a given DR, we have to take it into account. For example, the same changes in effects are associated with DR = 10 at curves with slope = 1, but with DR = 2.15 in case of slope = 3. Enhancement and diminution expressed by dose ratios is more or less underestimated in case of curves with slope > 1. We therefore propose to quantitate potentiation and antagonism by a corrected DR (DRcorr), which can simply be calculated from the uncorrected DR at a given slope. Consequently, a DRcorr reflects a true measure of enhancement or diminution for curves with slope = 1, equivalent to that which would have been observed for curves with slope = 1. The practical value of this modification is exemplified and illustrated by analysis of experimental data.

Relationships of brain and plasma levels of quazepam, flurazepam, and their metabolites with pharmacological activity in mice.

The relationships between the pharmacological activities of quazepam and flurazepam and the concentrations of each drug and its major active metabolites in brain and plasma following single oral doses of either drug to mice were investigated. At various time points after either quazepam or flurazepam administration, pharmacological activity was measured by the inhibition of electroconvulsive shock (ECS)-induced seizures. After quazepam, the plasma and brain samples obtained at the same time points were assayed for concentrations of quazepam, 2-oxoquazepam and N-desalkyl-2-oxoquazepam by specific GLC methods. After flurazepam, the plasma and brain samples were assayed for flurazepam, hydroxyethyl-flurazepam, and N-desalkyl-2-oxoquazepam, also by specific GLC methods. The results showed that both quazepam and flurazepam were rapidly metabolized and that parent drugs and metabolites were rapidly distributed to the brain. The brain levels of all the benzodiazepines analyzed in this study paralleled plasma levels. After quazepam, pharmacological activity most closely paralleled the combined brain concentrations of quazepam and 2-oxoquazepam rather than N-desalkyl-2-oxoquazepam levels. In contrast, following the flurazepam dose, activity most closely paralleled N-desalkyl-flurazepam concentrations. From these data, it can be concluded quazepam is distinctly different from flurazepam, and that, in the presence of quazepam and 2-oxoquazepam, N-desalkyl-2-oxoquazepam does not contribute extensively to the observed pharmacological activity.

Swim stress selectively alters the specific binding of a benzodiazepine antagonist in mice.

The ability of flurazepam to antagonize the electrical precipitation of tonic hindlimb extension is reduced 24 h after mice are forced to swim for 10 min in cold water (6 degrees C). Presumably, this reduction in flurazepam's antiseizure efficacy reflects an environmental stress-induced modification of the GABAA receptor complex. The current study employed a variety of complementary in vitro approaches to characterize the delayed effects of cold-water swim stress on binding parameters of the GABAA receptor complex that may be associated with flurazepam's reduced antiseizure efficacy. The specific binding of [3H]flunitrazepam and the potentiation of this binding by chloride ions did not change after stress in the cerebral cortex, hippocampus, and cerebellum. Moreover, swim stress did not alter the ability of GABA to inhibit the binding of [35S]t-butylbicyclophosphorothionate (TBPS), a ligand that is a specific biochemical marker of the GABA-associated chloride ionophore, to crude membranes prepared from the cerebral cortex and cerebellum. Swim stress was associated with alterations of the specific binding of [3H]Ro 15-1788, a benzodiazepine receptor antagonist, to crude hippocampal and cerebellar membranes. The results are considered in the context of new insights derived from molecular cloning studies of the GABAA receptor complex.

The inhibitory effect of endogenous convulsants quinolinic acid and kynurenine on the pentobarbital stimulation of [3H]flunitrazepam binding.

The metabolites of tryptophan-kynurenines with convulsant action quinolinic acid (QA) and l-kynurenine (l-KYN) antagonized the enhancing effect of pentobarbital (1 mM) on [3H]Flunitrazepam binding. IC50 for l-KYN were 35.9 +/- 14.8 microM and for QA 31.2 +/- 7.2 microM respectively. The inhibitory effect of KYN was stereoselective: IC50 of l-isomer was about two fold lower than IC50 of racemic form, d,l-KYN. Scatchard analysis revealed that inhibitory effect of QA and l-KYN on [3H]Flunitrazepam binding enhanced by pentobarbital is due to the decrease in affinity of benzodiazepine receptors. On the basis of these data it is proposed that QA and l-KYN possess their convulsant action interacting with barbiturate/picrotoxin sensitive sites of GABA-benzodiazepine-barbiturate complex.