A rapid solid-phase extraction and HPLC/DAD procedure for the simultaneous determination and quantification of different benzodiazepines in serum,blood and post-mortem blood

Summary A rapid and quantitative method for the determination of benzodiazepines using high-performance liquid chromatography (HPLC) with diode-array detection (DAD) is reported. The drugs were extracted from serum, blood or post-mortem blood using C₁₈ extraction columns. Brotizolam was used as internal standard. Experiments with spiked serum/blood samples resulted in recoveries between 75% and 94% for all investigated benzodiazepines. Excellent linearity was obtained over the concentration range 5–1500 ng benzodiazepine/ml. The limit of detection was approximately 2 ng/ml. The detection of low therapeutic serum levels of highly potent benzodiazepines is also possible.      

Quantification of naturally occurring benzodiazepine-like substances in human breast milk

The possible occurrence of benzodiazepine-like substances in human breast milk was investigated in 35 healthy, newly delivered women who were known not to be taking benzodiazepines. Maternal blood samples and a sample of breast milk were obtained on the fifth post partum day. A radioreceptor technique (lower limit of detection 1.5 ng/ml; difference between duplicates at various concentrations <7%) was used for measuring benzodiazepine-like substances in blood and breast milk (with and without prior extraction). No benzodiazepine-like substances could be demonstrated in any of the blood samples taken from the 35 women. Measurable concentrations of benzodiazepine-like substances were demonstrated in all but 1 of the 35 breast milk samples. The mean concentration of benzodiazepine-like substances for all 35 women was 4.3±2.3 ng/ml (range 0–9.3 ng/ml) expressed as lorazepam. The corresponding value for extracted breast milk was 2.6±1.5 ng/ml (range 0–7.0 ng/ml). There was no association between concentrations of benzodiazepine-like substances in breast milk and maternal age, weight, height and body mass or parity, or the sex of the infant and infant birth weight. We suggest that non-detectable amounts of benzodiazepine-like substances in serum are concentrated in the mammillary glands and excreted in a higher concentration in breast milk. It is less likely that the relevant benzodiazepines are produced in the mammillary glands.     

A comparison of the effects of scopolamine and diazepam on acquisition and retention of inhibitory avoidance in mice

Administration of either the muscarinic antagonist scopolamine or the benzodiazepine diazepam prior to training produced a dose-dependent impairment in the retention of one-trial inhibitory avoidance training in mice. To investigate the nature of this drug effect, the effects of scopolamine and diazepam were subsequently assessed on both acquisition and retention of inhibitory avoidance using a multiple-trial, training-to-criterion procedure. The training was conducted using either continuous trials in which the mouse was free to shuttle back and forth between shock and safe compartments or discrete trials in which the mouse was moved from the shock compartment of the safe compartment at the start of each trial. In either case, training continued until the mouse refrained from crossing into the shock compartment for a specified length of time on a single trial. Scopolamine (1.0 mg/kg) administered before training significantly increased the number of trials required to attain criterion, but did not affect retention when these mice were tested 2, 16, or 28 days later. In contrast, diazepam (1.0 mg/kg) did not significantly alter the number of trials necessary to reach criterion, but impaired retention of the inhibitory response in mice trained using discrete trials. The differences in the amnestic effects of scopolamine and diazepam revealed by this detailed analysis suggest that diazepam does not impair inhibitory avoidance performance through an effect on cholinergic function.     

An effect of triazolam on visual attention and information processing

This study explored whether benzodiazepines selectively affect aspects of attention and/or visual information processing, as they do memory. A cued visual-search paradigm was employed, using normal volunteers and a single dose of triazolam. This paradigm provided for a detailed examination of two aspects of visual attention and information processing: 1) controlled versus automatic attention allocation (via central and peripheral cues), and 2) the extent to which processing an item in a non-cued location affects performance (via cue-validity). Triazolam, compared to placebo, significantly increased response time, and Drug Condition interacted with Cue-Validity but not Cue-Type. Based on these data, we argue that triazolam doesnot affect attention allocation butdoes affect attentional disengagement and/or attention switching mechanisms.     

Disposition of oxazepam in patients on maintenance hemodialysis

Summary The pharmacokinetics of a single 30-mg oral dose of oxazepam was evaluated in seven patients with chronic renal failure on maintenance hemodialysis and in seven healthy controls matched for age and sex. Based on total (free plus bound) serum oxazepam concentrations, elimination half-life was prolonged in renal patients compared to controls (22 vs 8 h,p<0.001) and volume of distribution increased (3.0 vs 1.4 1/kg,p<0.02). However, total clearance was similar between groups (1.8 vs 1.9 ml/min per kilogram). These findings were confounded by the increased oxazepam free fraction in serum of renal failure patients (10.3%) as compared to healthy controls (4.3%). Correction for differences in binding indicates similar distribution of unbound oxazepam between groups, but reduced clearance of pharmacologically active unbound oxazepam in renal patients (18 vs 45 ml/min per kilogram). Oxazepam dosage, therefore, may require downward adjustment for renal failure patients on hemodialysis.Supported in part by grant OC 10/6-3 from the Deutsche Forschungsgemeinschaft, and by grant MH-34223 from the United States Public Health Service     

GABA[A] receptor level changes in female hamster forebrain following in vivo estrogen progesterone and benzodiazepine treatment: a quantitative autoradiography analysis

Summary The levels of gamma amino butyric acid (GABA_A) receptors (i.e. ³H-Muscimol binding sites) were determined by quantitative neurotransmitter receptors autoradiography in ovariectomized (OVX), OVX-estradiolo (E), OVX-progesterone (P), OVX-E+P, diazepam (DZ) and DZ + Ro15-1788 treated female hamsters.The various hormonal treatments altered ³H muscimol binding in many brain areas, whereas DZ and DZ + Ro15-1788 had little influence on GABA_A receptor levels at the onset of the blocked aggressive behavioral activity. For example, E, P and E+P all significantly increased ³H muscimol binding in medial preoptic area, ventromedial hypothalamic nuclei, and vertical diagonal bandmedial septal nucleus, whereas P treatment increased binding in the caudate-putamen and decreased it in reuniens nucleus of the thalamus. E and E+P treatments increased ³H muscimol binding in the corticomedial amygdala nucleus and hippocampus. Diazepam treatment decreased the GABA_A receptor binding in the caudate-putamen and basolateral amygdala, while having no effect in the other brain regions where hormone treatment was effective. In vitro incubation of brain sections with micromolar concentrations of E or P did not change muscimol binding. These results suggest that ovarian steroids, and benzodiazepines to a lesser extent, modulate ³H muscimol binding but do so in different brain regions. The hormone effects on ³H muscimol binding in the critical reproductive centres at a time period that coincides with the onset of its behavioral effect is consistent with a genomic controlled activity.     

Actions of theβ-carboline ZK 93426 in an animal test of anxiety and the holeboard: Interactions with Ro 15-1788

Summary The effects of the-carboline ZK 93426, a putative benzodiazepine receptor antagonist, were investigated in the social interaction test of anxiety and in the holeboard. Like the receptor antagonist Ro 15-1788, ZK 93426 (2.5–10 mg/kg) caused a specific reduction in social interaction (interpreted as an anxiogenic effect) and caused a significant elevation in exploratory head-dipping (5 mg/kg). When low (ineffective) doses of both compounds (1 mg/kg ZK 93426; 4 mg/kg Ro 15-1788) were administered together they significantly reduced social interaction. No further reductions in social interaction were observed when effective doses of both compounds (5 mg/kg ZK 93426; 10 mg/kg Ro 15-1788) were tested in combination; it is likely that this is due to almost total benzodiazepine receptor occupancy at effective doses of either compound. When doses of each compound (5 mg/kg ZK 93426; 10 mg/kg Ro 15-1788) that resulted in stimulation of head-dipping were examined in combination, the elevation in exploration was no longer observed. Since at higher doses of both compounds there is an attenuation of the elevation in head-dipping, it is again likely that the effects of the two compounds are additive.     

Duration of benzodiazepine clinical activity: Lack of direct relationship with plasma half-life

The anxiolytic activity and tolerance of two dosage schedules of prazepam, a long plasma half-life benzodiazepine, were compared under double-blind conditions in two groups of 10 inpatients each who met Research Diagnostic Criteria for Generalized Anxiety Disorder and presented chronic and severe symptomatology. Patients received prazepam 40 mg per day on one of two dosage schedules: 1) divided dosage (DD) - 10 mg in the morning and at noon and 20 mg in the evening; or 2) single dosage (SD) - 40 mg in the evening. The 3 weeks of therapy were preceded and followed by 1 week of wash-out for baseline and follow-up assessments, which were performed weekly with the Hamilton Anxiety Scale, Clinical Global Impression, rating of morning drowsiness and evening worsening of symptoms, and patient self-rating of anxiety by means of a visual analogue scale performed both in the morning and in the afternoon. The results showed a clear superiority of the DD over the SD schedule: better anxiolytic efficacy on the Hamilton Anxiety Scale (P<0.0005) and on both morning and afternoon visual analogue scales (P<0.01 andP<0.0002); less morning drowsiness (P<0.0001); and steadier anxiolytic effect during the daytime, as globally rated by the investigator (P<0.0001) or measured by morning-afternoon differences on the visual analogue scale (P<0.005). These results suggest that plasma pharmacokinetics alone may not be sufficient to predict the duration of benzodiazepine anxiolytic activity.