Benzodiazepine Concentrations Research Articles

A Methodological Comparison of Two Formulations of Temazepam in Pharmacokinetic and Pharmacodynamic Aspects

In a double-blind and cross-over study 12 healthy subjects took temazepam 20 mg in two different formulations (soft gelatine capsule or uncoated tablet) and matched placebo at one-week intervals. Plasma temazepam concentrations at 0.5, 1, 2, 3, 8, 12 and 24 hours after treatment were analyzed by gas chromatography. Psychomotor performance was measured objectively (digit symbol substitution, letter cancellation, Maddox wing test) and subjectively (visual analogue scales) before the drug intake and 1, 2 and 3 hours later and the plasma benzodiazepine concentrations were analyzed also by radioreceptor bioassay. The two different formulations were compared in pharmacokinetic and pharmacodynamic terms, and the gas chromatographic and radioreceptor assays were compared. The soft gelatine capsule produced higher peak plasma concentrations than the uncoated tablet. The computed AUCs and elimination half-lives proved to be similar after either formulation. A satisfactory correlation between the bioassayed benzodiazepine concentrations and chemically assayed temazepam was shown. In pharmacodynamic terms the results suggest a shorter and somewhat smaller subjective response for the capsule than for the tablet form.

Teratogenic effects of benzodiazepine use during pregnancy

Eight children exposed in utero to benzodiazepines had characteristic dysmorphic features, growth aberrations, and central nervous system abnormalities from birth. Their dysmorphic characteristics resembled those of the fetal alcohol syndrome, although they had greater focal involvement of cranial nerves, with a sullen and expressionless face, and they more often had impairment of vitality at birth. One infant died and at autopsy had varying degrees of distortion of neuronal migration, with concomitant heterotopias. Five of the eight mothers had regularly consumed benzodiazepines, and the three remaining mothers had blood samples during pregnancy revealing benzodiazepine concentrations indicative of regular use. Our findings indicate that maternal consumption of benzodiazepines may be teratogenic in humans.

Protracted Tinnitus after Discontinuation of Long-Term Therapeutic Use of Benzodiazepines

The presence of protracted tinnitus after discontinuation of long-term therapeutic doses of diazepam (less than or equal to 30 mg/day) is described in three patients. In one of these patients, the association of the tinnitus appearance with the drug discontinuation was documented in a double-blind, randomized, crossover single case study. Objective confirmation of drug use or of abstinence was performed by obtaining plasma benzodiazepine concentrations. The findings provide further documentation that sensory disturbances of short- and long-term duration are among the most distinctive clinical features of the benzodiazepine withdrawal syndrome.

Benzodiazepines Modulate Voltage-Sensitive Calcium Channels in GH3Pituitary Cells at Sites Distinct from Thyrotropin-Releasing Hormone Receptors

Benzodiazepines (BZs) have been shown to modulate voltage-sensitive Ca2+ channels in a number of neuronal and nonneuronal cell types and to competitively antagonize TRH binding to receptors on cells of the nervous system and anterior pituitary gland. Because interaction of TRH with its receptor is known to cause enhanced influx of Ca2+ through voltage-sensitive channels in rat pituitary GH3 cells, it was determined whether BZs and TRH were interacting with the same binding site on these cells. The potencies of three BZs, Ro5-4864, diazepam (DZP), and chlordiazepoxide (CDE), were compared as modulators of Ca2+ channels and as inhibitors of TRH binding in GH3 cells. Modulation of Ca2+ channel activity was measured as the inhibition by BZs of K+ depolarization-induced Ca2+ influx using intracellularly trapped quin 2 or 45Ca2+ uptake. The three BZs caused dose-dependent inhibition of Ca2+ influx with an order of potency of Ro 5-4864 greater than DZP greater than CDE. In contrast, the order of potency of the three BZs to inhibit [3H]TRH binding was CDE greater than DZP much greater than Ro 5-4864. The concentrations of BZs needed to inhibit Ca2+ influx and TRH binding were in the micromolar range. These data show that BZs can modulate Ca2+ channel activity in endocrine cells and that these sites are distinct from those that modulate TRH binding on pituitary cells.

Mouse lines selected for genetic differences in diazepam sensitivity.

Selective breeding techniques were used to alter allelic frequencies responsible for diazepam sensitivity and resistance. We used the rotarod test to determine the duration of diazepam-induced neurologic deficit in genetically heterogeneous mice. Males were more sensitive than females in the initial population. We then selectively bred for diazepam resistance and sensitivity. A significant difference between the lines was apparent in both sexes after two generations, and divergence has continued over seven generations. Brain benzodiazepine assays indicated that absorption and distribution of diazepam do not differ in the two lines. Differences in brain benzodiazepine concentrations at recovery from ataxia indicated that the two lines differ in central nervous system sensitivity. We found diazepam-induced rotarod impairment to be blocked in a dose-dependent manner by the specific benzodiazepine antagonist Ro 15-1788, indicating that this effect is mediated through BZ receptors. A dose-response curve obtained from generations 6 and 7 indicates a 9- to 14-fold difference in dose required to obtain similar effects in the two lines. These mice are expected to be useful experimental subjects in studies of benzodiazepine mechanisms.

The role of benzodiazepine receptors in the induction of differentiation of HL‐60 leukemia cells by benzodiazepines and purines

A series of benzodiazepines was evaluated for their capacity to induce the differentiation of HL-60 acute promyelocytic leukemia cells. Benzodiazepines were effective initiators of maturation in the concentration range of 50 to 150 microM. The possible involvement of benzodiazepine receptors in mediating the differentiation induced by these agents was investigated. The presence of high affinity, peripheral type benzodiazepine binding sites (KD = 7.3 nM, TB = 14.5 pmol/mg protein with Ro5-4864) was demonstrated in HL-60 membranes. The occupancy of peripheral type high affinity benzodiazepine receptors by various benzodiazepines showed some correlation (r = 0.76) with their differentiation-inducing capabilities, but binding potencies were 1,000-fold higher than the concentrations required to produce differentiation. A class of benzodiazepine receptors with lower binding affinity was also detected in HL-60 membranes (KD = 28.6 microM; TB = 199 pmol/mg protein with diazepam). A higher level of correlation (r = 0.88) was demonstrated between benzodiazepine occupancy of these lower affinity receptors and the capacity to induce maturation. Significantly, benzodiazepine concentrations needed for low affinity binding and induction of differentiation were the same (25-200 microM), suggesting that low affinity benzodiazepine receptors may be involved in the induction process. We have shown that the molecular form responsible for the induction of the differentiation of HL-60 cells to mature forms by 6-thioguanine (TGua) is the free base, TGua, itself [Ishiguro, Schwartz, and Sartorelli (1984) J. Cell. Physiol., 121:383-390]. Since hypoxanthine (Hyp) and inosine (Ino) have been identified as putative endogenous ligands for high affinity benzodiazepine receptors in brain tissue, the potential involvement of benzodiazepine receptors in the differentiation of HL-60 cells by the purines was investigated. Physiological purines such as Hyp and Ino were inactive in displacing the benzodiazepines from their high and low affinity binding sites in HL-60 membranes. In contrast, TGua caused inhibition of benzodiazepine binding to high and low affinity sites. The inhibition of Ro5-4864 binding to high affinity binding sites by TGua appeared to be due to the binding of TGua to membranes through the formation of a mixed disulfide between the 6-thiopurine and protein thiols, since the inhibition was reversed by the presence of 2-mercaptoethanol. The findings suggest a possible relationship between the occupancy of benzodiazepine receptors by TGua and the induction of leukemic cell differentiation.

Withdrawal reaction after long-term therapeutic use of benzodiazepines

We conducted a double-blind, placebo-controlled trail in which 40 patients who had undergone long-term therapy with benzodiazepines were switched to placebo or to diazepam in a dose approximately equivalent to their usual dose of the benzodiazepine; the dose of diazepam was then tapered during an eight-week period. Patients were assessed clinically and psychologically and had weekly sessions of behavioral therapy. The subjects who received placebo had more symptoms, assessed their symptoms as more severe, and stopped taking the study drug at a higher rate than those receiving the tapering doses of diazepam. The subjects in the placebo group also had symptoms shortly after being switched to placebo, whereas those in the diazepam group had symptoms much later. Some withdrawal symptoms were distinct from those of anxiety (e.g., tinnitus, involuntary movement, and perceptual changes). Withdrawal symptoms occurred earlier in patients who had received short-acting benzodiazepines than in those who had received long-acting benzodiazepines. Symptoms gradually disappeared over a four-week period in both the placebo and the diazepam groups. Serial determination of plasma benzodiazepine concentrations was a useful way to assess compliance, treatment outcome, and relapse during withdrawal. We conclude that a clinically important, mild, but distinct withdrawal syndrome occurs after discontinuation of long-term therapeutic use of benzodiazepines.

Actual versus reported benzodiazepine usage by medical outpatients.

Benzodiazepines are widely prescribed, and in 1979 almost 10% of the adult population was taking them. Prior studies of outpatient usage of benzodiazepines have relied on survey or prescription data, which may be confounded by noncompliance. To determine the actual use of benzodiazepines, plasma benzodiazepine concentrations were measured in 225 consecutive outpatients from a university cardiology outpatient service. Self reports indicated that the great majority of the patients (191) were taking at least one medicine, and 70 reported being on a psychotropic drug. Seventy-seven patients reported taking benzodiazepines, the majority being on bromazepam (20), diazepam (26) or oxazepam (19). In 25 of those 77 patients, the reported drug could not be detected in plasma. Conversely, in 10 of the 225 patients, benzodiazepines which were not reported were detected (diazepam or flurazepam). Of those taking benzodiazepines, many had a low concentration, suggesting intermittent rather than regular use. Thus, many patients for whom benzodiazepines are prescribed take them irregularly, and a small group uses them without reporting their prescription. These findings have implications for the clinical presentation of illness and for the possibility of drug interactions.

Reinterpretation of the pharmacokinetic mechanism of oral benzodiazepine ethanol interaction.

Previously published studies investigating the oral benzodiazepine ethanol interaction have utilized a single dose of ethanol, a single dose of oral benzodiazepine, and measured plasma benzodiazepine concentration over varying time periods. Most studies reported an increase or no change in benzodiazepine plasma concentrations following ethanol administration, which the investigators usually ascribed to an ethanol-induced increase in the benzodiazepine absorption rate constant. However, ethanol decreases the hepatic clearance of benzodiazepines that are biotransformed via the P450 enzyme system and this effect was not taken into account in evaluation of the results of these studies. Computer simulations have been used to investigate possible mechanisms of the oral benzodiazepine-ethanol interaction. The effects of a constant or transient decrease in clearance and an increase in absorption rate constant upon maximum concentration, time of maximum concentration, and area under the benzodiazepine plasma concentration curve (AUC) have been examined. A transient 75% decrease in benzodiazepine clearance resulted in a 13.6% increase in AUC (0-36 hr), a 3.4% increase in Cmax and a 5.7% increase in tmax. These changes are qualitatively consistent with, but quantitatively shy, of those observed in most studies. Consequently, an effect of ethanol upon benzodiazepine absorption must still be considered.

Midazolam as an Intravenous Induction Agent in the Elderly

Midazolam, the first benzodiazepine derivative with water-soluble salts, was studied as an induction agent in general anesthesia for the elderly. In group 1 (n = 14), 5 or 10 mg oral diazepam was used as premedication, but in group 2 (n = 9), both oral (10 mg dixyrazin as a night-time sedative) and intramuscular (0.01 mg/kg atropine + 1 mg/kg meperidine) premedicants were used. Serum concentrations of benzodiazepines were determined using both gas-liquid chromatographic (unchanged midazolam) and radioreceptor assays (binding equivalents of benzodiazepines plus their active metabolites). In general, midazolam, 0.15 mg/kg, intravenously resulted in smooth induction of anesthesia, although the time required for induction was rather long and a sudden but transient decrease in blood pressure was found in a significant number of patients. The course of anesthesia was otherwise satisfactory. A marked amnesic effect was observed, especially when diazepam was used as premedication. The pharmacokinetic parameters based on gas-liquid chromatographic measurements were quite comparable with those in young, healthy persons published earlier. In both groups, the binding equivalents measured with radioreceptor assay (reflecting total benzodiazepine activity) were higher than the levels of unchanged midazolam determined with gas-liquid chromatography. The relatively low dose of 0.15 mg/kg of midazolam needed for anesthetic induction in the elderly indicates not pharmacokinetic, but pharmacodynamic, alterations in older patients. We conclude that midazolam is a new intravenous induction agent for use in the elderly, but careful titration of the dosage according to the response of the patient is required. Diazepam premedication prior to midazolam causes a marked anterograde amnesic effect.

The Frequency in the Danish Population of Benzodiazepines in Blood Samples Received for Blood Ethanol Determination for the Period June 1978 to June 1979

The Institute of Forensic Chemistry in Copenhagen receives about 26 000 blood samples per year for ethanol determination from drivers suspected of driving under the influence of ethanol. The frequency of benzodiazepines in these was estimated by analyzing a sample of 1382, for the period June 1978–June 1979. Detection was by radioreceptor assay based upon the presence of specific binding sites for benzodiazepines in mammalian brain membranes and prepared for analytical purposes in a stable and dry form by the Ferrosan Company, Denmark. A benzodiazepine concentration higher than a therapeutic lower level of 0. 20 μmol/kg (calculated as diazepam) was found in 80 blood samples, but for only 15 of these had a specific request for benzodiazepine analysis been made by the police.

Imaging of [ 11C]-labelled RO 15-1788 binding to benzodiazepine receptors in the human brain by positron emission tomography

The benzodiazepine antagonist Ro 15-1788 was labelled with [ 11C] and examined for possible use as ligand for PET scan studies on benzodiazepine receptors in the brain of cynomolgus monkeys and human subjects. [ 11C] Ro 15-1788 allowed the in vivo visualization of benzodiazepine receptor binding in cerebral and cerebellar cortical areas as well as in basal brain nuclei in PET scan images. [ 11C] Ro 15-1788 exhibited a high ratio of specific benzodiazepine receptor binding (cerebral cortex) to non-specific binding (pons) and the kinetics of binding should be satisfactory for quantitative clinical PET scan studies using [ 11C]. The in vivo binding of [ 11C] Ro 15-1788 in the cerebral cortex of cynomolgus monkeys and healthy human subjects was reduced by approximately 90% within 10 min after the intravenous injection of a high dose of unlabelled Ro 15-1788 (0.5 mg/kg i.v.). Different areas of the healthy human brain showed an approximately 10-fold variation in maximal [ 11C] Ro 15-1788 binding that corresponded to the previously known distribution of benzodiazepine receptors in these regions. The highest degree of binding was obtained in the medial occipital cerebral cortex followed by frontal cortex, cerebellum, thalamus, striatum and pons. Two psychiatric patients with anxiety syndromes who had been treated for a long time with high doses of benzodiazepines had roughly the same degree of maximal [ 11C] Ro 15-1788 binding in brain regions as the healthy subjects but the rate of decline of [ 11C] Ro 15-1788 in the brain was higher. This indicates that there is measurable competition between [ 11C] Ro 15-1788 binding and clinical benzodiazepine concentrations in the body fluids of psychiatric patients. The results demonstrate that [ 11C] Ro 15-1788 should be a valuable tool for quantitative analyses of benzodiazepine receptor characteristics and receptor occupancy in the brain of patients with neuropsychiatric disorders.

Diazepam-stimulated increases in the synaptosomal uptake of 45ca 2+: Reversal by dihydropyridine calcium channel antagonists

Pharmacologically relevant concentrations of benzodiazepines have previously been reported to increase 45Ca 2+ uptake into synaptosomes. This observation, coupled with the recent report that nifedipine may block the hypnotic effect of flurazepam, led us to study the effects of nifedipine and nitrendipine on 45Ca 2+ uptake into synaptosomes. Diazepam (1 μM) significantly increased the uptake of 45Ca 2+ to a crude synaptosomal fraction (P 2) prepared from rat cerebral corted and depolarized with 55 mM K +. Nifedipine (1 μM) did not alter the uptake of Ca 2+, while nitrendipine (1 μM) reduced uptake by 37%. Both nifedipine and nitrendipine completely antagonized the ability of diazepam to increase 45Ca 2+ uptake following K + depolarization. These observations support the notion that the pharmacologic actions of benzodiazepines may be mediated through effects on a calcium channel.

Plasma concentrations of benzodiazepines — A review of clinical findings and implications

1. Methods for benzodiazepine analysis are briefly discussed and the pharmacokinetics of the benzodiazepines reviewed. 2. Studies of the relationship between plasma concentration and anxiolytic response have produced conflicting results. Routine monitoring in anxiety states is not warranted. 3. Some specific indications for monitoring are in elderly patients; in cases of suspected non-compliance; in patients with renal and hepatic disease. 4. The relationship between plasma concentration and ECG changes following benzodiazepine overdose showed that monitoring was of little value in these cases.

Behavioural pharmacology of benzodiazepines

1. Acute injections of benzodiazepines produce sedative effects in the rat that can be detected by decreases in spontaneous motor activity and exploration. The effects are found with low doses, are dose-related and correlate well with plasma concentrations. 2. With repeated daily doses there is tolerance to the sedative effects within 3–5 days even with benzodiazepines with short half-lives and no active metabolites. 3. In the rat there may be some pharmacokinetic tolerance accounting for some of the behavioural tolerance, but pharmacokinetic changes cannot explain the tolerance to low doses of chlordiazepoxide. 4. With chronically treated rats there is no relationship between plasma benzodiazepine concentrations and sedation. 5. There is rapid recovery from tolerance to the sedative effects and if the animals are tested undrugged there is no detectable effect 24 h after the last dose. 6. Anxiolytic effects emerge after a few days of benzodiazepine treatment, but after 10–20 days of treatment there is tolerance to these effects in the social interaction test and in the corticosterone-stress response.

Benzodiazepine drug-drug interactions commonly occurring in clinical practice.

Pharmacokinetic drug-drug interactions which involve currently available benzodiazepines may be classified into two major categories: interactions which affect benzodiazepine rate of absorption, and interactions which affect clearance and, therefore, elimination half-life. Ethanol is the prototype for absorptive interactions. Concurrent ethanol use and oral ingestion of benzodiazepine derivatives uniformly slows the rate but does not change the extent of benzodiazepine absorption. Interactions which affect benzodiazepine clearance affect only those derivatives which are oxidatively metabolized or cleared as a function of hepatic blood flow (high first-pass clearance). Conjugated benzodiazepines are not implicated in such interactions. Rifampin and chronic ethanol use induce benzodiazepine oxidation, while cimetidine, oral contraceptives, ethanol (acute ingestion), disulfiram, isoniazid, and propranolol inhibit benzodiazepine oxidation. In addition, ethanol, cimetidine and isoniazid decrease first-pass hepatic extraction of triazolam, enhancing its systemic availability and decreasing oral clearance. The pharmacokinetic consequence of induction of benzodiazepine oxidation is higher clearance and decreased steady-state concentrations during chronic dosing. Conversely, inhibition of benzodiazepine oxidation decreases clearance and increases steady-state benzodiazepine concentrations during chronic dosing. Because a correlation of benzodiazepine plasma concentration and pharmacological effect is not established, the pharmacodynamic consequences of these interactions are not currently well characterized.

Potentiating action of midazolam on GABA-mediated responses and its antagonism by Ro 14-7437 in the frog spinal cord

The effect of midazolam, a new water-soluble benzodiazepine, on an in vitro slice preparation of the frog spinal cord was investigated using electrophysiological recordings. Midazolam potently (ED 50 = 1 nM) enhanced the depolarizing action of GABA on primary afferent fibres while leaving the depolarizing effect of glutamate, glycine or high K + solutions unchanged. Concentrations of midazolam higher than 100 nM had an antagonistic effect on GABA responses. Ro 14-7437 was a powerful and selective antagonist of the midazolam potentiation without affecting control responses to GABA, glutamate or high K +. The antagonism of GABA responses induced by high doses of midazolam was not sensitive to Ro 14-7437. Our data suggest that midazolam is a very potent and selective modulator of GABA responses: this finding illustrates that electrophysiological techniques can detect specific effects of very low concentrations of benzodiazepines on a CNS since preparation with well preserved architectural organization.

Benzodiazepine Dependence A Review of the Evidence

Classical pharmacological dependence accompanied by euphoria, clinical evidence of tolerance and escalation of dosage are very rare with the benzodiazepines. However, there is now abundant evidence that prolonged administration of doses within the therapeutic range leads to true dependence in a significant minority of patients. This dependence is characterised by withdrawal symptoms on stopping treatment; these include perceptual disturbances, epileptic seizures, weight loss, insomnia and autonomic symptoms. As some of the symptoms are qualitatively different from those of anxiety neurosis, and as the withdrawal syndrome commonly lasts between 5 and 15 days, the possibility that the symptoms represent a return of pre-existing anxiety can be discounted. The withdrawal syndrome is more likely if: (a) the benzodiazepine has been taken in regular dosage for more than 4 months; (b) higher dosages have been used; (c) the drug is stopped suddenly; and (d) a short acting benzodiazepine has been taken. The withdrawal syndrome is most likely to occur when there is a rapid fall in blood benzodiazepine concentrations; this may be associated with altered sensitivity of benzodiazepine receptors. The syndrome can best be avoided by gradual reduction of dosage. The temporary prescription of other drugs, particularly beta-adrenoceptor blocking drugs, may attenuate withdrawal symptoms, but antipsychotic drugs in low dosage are of no benefit.

Micromolar affinity benzodiazepine receptors: identification and characterization in central nervous system.

Receptors that selectively bind micromolar concentrations of benzodiazepines are present in rat brain membrane. These micromolar receptors exhibit saturable, stereospecific binding, and the potency of benzodiazepine binding to these receptors is correlated with the ability of the benzodiazepines to inhibit maximum electric shock-induced convulsions. Benzodiazepine receptors with nanomolar affinity differ from the micromolar receptors in their binding, kinetic, and pharmacologic characteristics. The micromolar receptors also bind phenytoin, a non-benzodiazepine anticonvulsant. These results provide evidence for a distinct class of clinically relevant benzodiazepine receptors that may regulate neuronal excitability and anticonvulsant activity.

The relationship between peak velocity of saccadic eye movements and serum benzodiazepine concentration.

1 Six healthy male volunteers received single oral doses of 10 mg diazepam, 20 mg temazepam, 15 mg flurazepam, 5 mg nitrazepam, 10 mg desmethyl-diazepam and placebo in a double-blind randomized fashion. 2 Peak velocity of saccadic eye movements, serum benzodiazepine concentration, and subjective ratings of wakefulness and co-ordination were measured at intervals up to 12 h after drug administration. 3 All active treatments produced a statistically significant decrease in peak saccadic velocity. The effect of temazepam and diazepam was generally more pronounced than that of flurazepam, nitrazepam and desmethyl-diazepam. 4 There were log-linear correlations between peak saccadic velocity and serum benzodiazepine concentration after ingestion of temazepam, diazepam and nitrazepam. 5 These results demonstrate a clear relationship between serum benzodiazepine concentration and its effect on a convenient measure of brainstem reticular formation function.