Simultaneous Determination of Tramadol and its Metabolite in Human Plasma by GC/MS.

A simple and sensitive GC/MS method for the determination of tramadol and its metabolite (O-desmethyltramadol) in human plasma was developed and validated. Medazepam was used as an internal standard. The calibration curves were linear (r=0.999) over tramadol and O-desmethyltramadol concentrations ranging from 10 to 200 ng/mL and 7.5 to 300 ng/mL, respectively. The method had an accuracy of >95% and intra- and interday precision (RSD%) of ≤4.83% and ≤4.68% for tramadol and O-desmethyltramadol, respectively. The extraction recoveries were 97.6±1.21% and 96.3±1.66% for tramadol and O-desmethyltramadol, respectively. The LOQ using 0.5 mL human plasma was 10 ng/mL for tramadol and 7.5 ng/mL for O-desmethyltramadol. Stability studies showed that tramadol and O-desmethyltramadol were stable in human plasma after 8 h incubation at room temperature or after 1 week storage at -20°C with three freeze-thaw cycles. Also, this method was successfully applied to six patients who had been given an intravenous formulation of 100 mg tramadol with Cmax results of 2018.1±687.8 and 96.1±22.7 ng/mL for tramadol and O-desmethyltramadol, respectively.

Is long-term use of benzodiazepine a risk for cancer?

The carcinogenicity of benzodiazepines (BZDs) is still unclear. We aimed to assess whether long-term benzodiazepines use is risk for cancer.We conducted a longitudinal population-based case-control study by using 12 years from Taiwan National Health Insurance database and investigated the association between BZDs use and cancer risk of people aged over 20 years. During the study period, 42,500 cases diagnosed with cancer were identified and analyzed for BZDs use. For each case, six eligible controls matched for age, sex, and the index date (ie, free of any cancer in the date of case diagnosis) by using propensity score. For appropriate risk estimation, we observed the outcomes according to their length of exposure (LOE) and defined daily dose (DDD). To mimic bias, we adjusted with potential confounding factors such as medications and comorbid diseases which could influence for cancer risk during the study period. The data was analyzed by using Cox proportional hazard regression and conditional logistic regression.The finding unveils benzodiazepines use into safe and unsafe groups for their carcinogenicity. The use of diazepam (HR, 0.96; 95%CI, 0.92-1.00), chlorodizepoxide (HR, 0.98; 95%CI, 0.92-1.04), medazepam (HR, 1.01; 95%CI, 0.84-1.21), nitrazepam (HR, 1.06; 95%CI, 0.98-1.14), oxazepam (HR, 1.05; 95%CI, 0.94-1.17) found safer among BZDs. However, clonazepam (HR, 1.15; 95%CI, 1.09-1.22) were associated with a higher risk for cancers. Moreover, specific cancer risk among BZDs use was observed significantly increased 98% for brain, 25% for colorectal, and 10% for lung, as compared with non-BZDs use.Diazepam, chlordiazepoxide, medazepam, nitrazepam, and oxazepam are safe among BZDs use for cancer risk. Our findings could help physicians to select safer BZDs and provide an evidence on the carcinogenic effect of benzodiazepines use by considering the LOE and DDD for further research.

Acute kidney injury with medazepam-hyoscine buthylbromide.

An 84-year-old female patient was admitted to our internal medicine outpatient clinic complaining of stomach ache, nausea, and vomiting. She had hepatitis C infection for 10 years that was managed with antiviral treatment. On the second day of admission, she developed anxiety and complained about dysuria. Medazepam and hyoscine butylbromide combined tablet was administered. The day after medazepam and hyoscine butylbromide administration, patient's creatinine level increased to 2.3 mg/dL (0.57-1.11 mg/dL). Medazepam and hyoscine butylbromide administration was stopped on the fourth day. After 10 days of follow-up, her creatinine levels were normalized. In this article, we present an elderly patient with acute kidney injury induced by medazepam and hyoscine butylbromide that was managed with best supportive care.

Electronic properties of neuroleptics: ionization energies of benzodiazepines.

Vertical ionization energies (VIEs) of medazepam, nordazepam and their molecular subunits have been calculated using the electron propagator method in the P3/CEP-31G* approximation. Vertical electron affinities (VEAs) have been obtained with a ∆SCF procedure at the DFT-B3LYP/6-31+G* level of theory. Excellent correlations have been achieved between IE(calc) and IE(exp), allowing reliable assignment of the ionization processes. Our proposed assignment differs in many instances from that previously reported in the literature. The electronic structure of the frontier Dyson orbitals shows that the IE and EA values of the benzodiazepines can be modulated by substitution at the benzene rings. Hardness values, evaluated as (IE - EA)/2, follow the trend of the experimental singlet transition energies. Medazepam is a less hard (i.e., less stable) compound than nordazepam.

Benzodiazepines medazepam and midazolam are activators of pregnane X receptor and weak inducers of CYP3A4: investigation in primary cultures of human hepatocytes and hepatocarcinoma cell lines.

Benzodiazepines have wide-spread used in pharmacotherapy for their anxiolytic, myorelaxant, hypnotic, amnesic and anticonvulsive properties. Despite benzodiazepines are used in clinics over 50 years, they have not been surprisingly tested for capability to induce major drug-metabolizing cytochromes P450. In the current study, we have examined the potency of Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Medazepam, Midazolam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam to induce CYP1A2 and CYP3A4 in primary cultures of human hepatocytes. Benzodiazepines were tested in therapeutic concentrations and in concentrations corresponding to their plasma levels in intoxicated patients. We found weak but significant induction of CYP3A4 mRNA by Midazolam and Medazepam, while other benzodiazepines did not induce CYP3A4 expression. None of the tested compounds induced CYP1A2 mRNA in three independent human hepatocytes cultures. In addition, employing gene reporter assays with transiently transfected hepatocarcinoma cells, we found that tested benzodiazepines did not activate aryl hydrocarbon receptor (AhR), whereas Midazolam and Medazepam slightly activated pregnane X receptor (PXR). Consistently, two-hybrid mammalian assay using hybrid fusion plasmids GAL4-PXR ligand-binding domain (LBD) and VP16-SRC-1-receptor-interacting domain (RID) confirmed PXR activation by Midazolam and Medazepam. In conclusion, Alprazolam, Bromazepam, Chlordiazepoxide, Clonazepam, Diazepam, Lorazepam, Nitrazepam, Oxazepam, Tetrazepam and Triazolam can be considered as safe drugs in term of their inability to induce PXR- and AhR-dependent cytochrome P450 enzymes CYP1A2 and CYP3A4. Medazepam and Midazolam slightly activated pregnane X receptor and displayed weak potency to induce CYP3A4 mRNA in human hepatocytes.

A study of the effects of large doses of medazepam used for self-poisoning in 10 pregnant women on fetal development.

The purpose of this article is to report an evaluation of the teratogenic and fetotoxic potential of medezepam in humans based on pregnant women who used very large doses of medazepam for a suicide attempt. All self-poisoned patients were cared for at the Department of Toxicology Internal Medicine, Koranyi Hospital, a toxicological inpatients clinic in Budapest, Hungary, between 1960 and 1993. Pregnant women were identified from self-poisoned subjects admitted from a population base of three million people of Budapest and the surrounding region. The rates of congenital abnormalities (CAs), intrauterine fetal development, cognitive and behavioral status in children born to mothers who attempted suicide with medazepam alone or in combination with other drugs during pregnancy was compared in their sib controls. Between 1980 and 1993, 835 pregnant women in our study attempted suicide during pregnancy with drugs. Of these, 314 delivered live-born infants and 283 were examined and/or evaluated. Thirty-two (3.8%) of these 835 pregnant women used medazepam with or without other drugs for self-poisoning; 10 of these women delivered live-born babies. The dose of medazepam used for the suicide attempt ranged between 60 and 500 mg, with a mean of 276 mg. Eight of the 32 suicide attempts involving medazepam occurred between the 4th and 12th postconceptional weeks. Of the 10 live-born exposed children, one was affected with congenital inguinal hernia; one of the 13 sib controls had a lethal hydronephrosis. No adverse effects were observed on intrauterine growth, cognitive status, or behavioral deviations in the 10 children born to mothers who attempted suicide with medazepam during pregnancy. Very large doses of medazepam were used for self-poisoning during pregnancy. These doses did not increase the rate of CAs even though eight mothers attempted suicide during the most critical period for production of CAs. No fetotoxic, including neurotoxic, effects of exposure of live-born children to a very large dose of medazepam were observed. Our experiences show the feasibility and benefits of use of the self-poisoning model in estimating human teratogenic and fetotoxic risks of drugs.

[Efficacy and possible mechanisms of action of a new peptide anxiolytic selank in the therapy of generalized anxiety disorders and neurasthenia].

Sixty-two patients with generalized anxiety disorder (GAD) and neurasthenia were studied. The effect of selank (30 patients) was compared to that of medazepam (32 patients). Patient's state was assessed with psychometric scales (Hamilton, Zung, CGI). Enkephalin activity in the blood serum was measured as well. The anxiolytic effects of both drugs were similar but selank had also antiasthenic and psychostimulant effects. The clinical-biological study revealed that patients with GAD and neurasthenia had the decreased level of tau(1/2) leu-enkephalin which was correlated with disease duration, severity of symptoms related to anxiety and asthenia and autonomic disorders. The increase of this parameter and stronger positive correlations with anxiety level were observed during the treatment with selank mostly in patients with GAD.

Determination of diazepam in cream biscuits by liquid chromatography.

An analytical procedure was developed for the detection and quantitation of diazepam in cream biscuits, which were used to commit crime. The method involves the extraction of diazepam with ethanol at room temperature, and the extract is filtered, evaporated to dryness, and redissolved in the mobile phase, methanol-acetonitrile-tetrahydrofuran-water (15 + 55 + 4 + 26, v/v). The separation is achieved on a C18 reversed-phase column with the mobile phase and diode array detection (lambda(max)) at 230 nm. Medazepam is used as the internal standard is for quantification. The calibration plot for the determination of diazepam is based on linear regression analysis (y = 0.6687x + 0.0372; r2 = 0.995). The limit of detection for diazepam in the biscuit samples was estimated as 600 ng/mL. The limit of quantitation for diazepam was estimated as 1.75 microg/mL. The diazepam detected per piece of biscuit was found to be in the range of 0.27-0.45 mg. Pure diazepam was added to biscuit samples at 3 levels (100 and 500 microg/g, and 1 mg/g), and the recoveries were found to be 95%. The mean retention time of diazepam was 2.7 min and that of medazepam (IS) was 4 min. The relative standard deviations of the diazepam level in the biscuit samples were estimated to be 0.4% for retention time and 1.02% for peak area in intraday analysis, whereas the corresponding values were and 0.61 and 2.34% in interday analysis. The method is rapid and reliable for qualitative and quantitative analysis of cream biscuits laced with diazepam, and it can be used by law enforcement laboratories for routine analysis.

[Determination of diazepine derivatives: alprazolam, medazepam, chlordiazepoxid mixture by high performance liquid chromatography]. / Diazepino dariniu: alprazolamo, medazepamo, chlordiazepoksido misinio tyrimas didelio slegio skysciu chromatografijos metodu.

The aim of this research was to determine possibility of qualitative analysis of diazepine derivatives: alprazolam, medazepam, chlordiazepoxid in the mixture. The high performance liquid chromatography was used for the investigation, the reagents--tablets of alprazolam, medazepam, chlordiazepoxid. It was found out that the most acceptable eluent for the analysis of the mixture by high pressure liquid chromatography is phosphate buffer, containing 0.02 M penthanesulfonic acid:etannitrile (55:45) (pH=3.5). The optimal wavelength for identification of the drugs in the mixture is 254 nm.

Pharmacological activity of phenazepam and flunitrazepam in ultralow doses.

Experiments on male outbred albino rats showed that benzodiazepine tranquilizers phenazepam and flunitrazepam in ultralow doses (10(-9)-10(-15) mol/kg) produced an anxiolytic effect in the conflict situation test. This effect was not accompanied by myorelaxing and sedative side effects typical of standard doses of tranquilizers.

[Analysis of benzodiazepine derivative mixture by gas-liquid chromatography]. / Benzodiazepino dariniu misinio analize duju ir skysciu chromatografijos metodu.

The analysis of mixture of benzodiazepine derivates (chlordiazepoxide, flunitrazepam, medazepam, nitrazepam, oxazepam and tetrazepam) by gas--liquid chromatography (GLC) in purpose to separate and identify these psychotropic drugs in mixture is presented in this article. The experiment was carried out in vitro, accommodating this method for identification and separation of drugs, isolated from biological objects (blood and urine). Referring to data of annual reports of chemical investigations (1) above-mentioned psychotropic drugs are very frequent among drug intoxication. In most cases they are detected in the mixture of the same or different pharmacological group, and this causes difficulty for separation and identification. The analysis of the mixture was carried out by GLC, which is widely used in practice of forensic-chemical examination. Adsorbents and stationery phases were changed; the conditions and parameters of chromatography were modified, in purpose totally separate preparations in the mixture. For the separation and identification of all three preparation the column packed with Inerton Super with stationary phase 3% OV-17 is suitable. The column temperature-290 degrees C. The mixture of these drugs was excreted from body fluids (blood and urine) in vitro and investigated by GLC under these conditions. The results of investigation were similar.

A population-based case-control teratologic study of nitrazepam, medazepam, tofisopam, alprazolum and clonazepam treatment during pregnancy.

OBJECTIVE: To study the association between nitrazepam, medazepam, tofisopam, alprazolum and clonazepam treatments during pregnancy and prevalence of different congenital abnormalities (CAs). MATERIALS AND METHODS: A matched case-control study using cases with CAs and population controls from the dataset of the nationwide Hungarian Case-Control Surveillance of Congenital Abnormalities (HCCSCA), 1980-1996. RESULTS: Of 38,151 pregnant women who had babies without any defects (population control group), 75 (0.20%) were treated with these five benzodiazepines during pregnancy. Of 22,865 pregnant women who delivered offspring with CAs, 57 (0.25%) had benzodiazepine treatment. The occurrence of five benzodiazepine treatments during the second and third months of gestation, i.e. in the critical period for most major CAs did not show significant differences in matched case-control pairs. CONCLUSION: Treatment with five benzodiazepines studied during pregnancy did not present detectable teratogenic risk to the fetus in humans but the amount of information was limited for different CAs.

Agorafobia simple y agorafobia compleja: estudio de tres casos / Simple agoraphobia and complex agoraphobia: study of three cases

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[Effect of the activation of GABA A, benzodiazepine, and D2 dopamine receptors on the passive avoidance extinction in submissive and aggressive mice]. / Vliianie aktivatsii GAMK A benzodiazepinovykh i D2 dofaminergicheskikh retseptorov na ugashenie uslovnoi reaktsii passivnogo izbeganiia u submissivnykh i agressivnykh myshei.

The effect of activation of GABAA, benzodiazepine, and D2 dopamine receptors on extinction of passive avoidance and their dependence on the initial state of aggressive and submissive C57BL/6J mice were studied. It was found that in mice with the submissive stereotype of behavior produced by experience of defeats in daily agonistic confrontations, extinction of the conditioned reaction occurred faster than in control mice. The activation of D2 receptors by quinpirole and of benzodiazepine receptors by medasepam before training restored the retrieval of the memory trace. A prolongation of extinction was observed in aggressive mice in comparison with control and submissive animals, and activation of GABAA by muscimol and benzodiazepine receptors by medazepam led to acceleration of extinction. Activation of D2 receptors was ineffective. Thus, the difference in initial behavioral strategy determined both the development of extinction of the passive avoidance and variability of participation of D2, GABAA, and benzodiazepine receptors in the maintenance of availability of the memory trace to retrieval.

Effect of benzodiazepines on the epithelial and neuronal high-affinity glutamate transporter EAAC1.

EAAC1-mediated glutamate transport concentrates glutamate across plasma membranes of brain neurons and epithelia. In brain, EAAC1 provides a presynaptic uptake mechanism to terminate the excitatory action of released glutamate and to keep its extracellular concentration below toxic levels. Here we report the effect of well known anxiolytic compounds, benzodiazepines, on glutamate transport in EAAC1-stably transfected Chinese hamster ovary (CHO) cells and in EAAC1-expressing Xenopus laevis oocytes. Functional properties of EAAC1 agreed well with already reported characteristics of the neuronal high-affinity glutamate transporter (Km D-Asp,CHO cells: 2.23+/-0.15 microM; Km D-Asp,oocytes: 17.01+/-3.42 microM). In both expression systems, low drug concentrations (10-100 microM) activated substrate uptake (up to 200% of control), whereas concentrations in the millimolar range inhibited (up to 50%). Furthermore, the activation was more pronounced at low substrate concentrations (1 microM), and the inhibition was attenuated. The activity of other sodium cotransporters such as the sodium/D-glucose cotransporter SGLT1, stably transfected in CHO cells, was not affected by benzodiazepines. In electrophysiological studies, these drugs also failed to change the membrane potential of EAAC1-expressing Xenopus laevis oocytes. These results suggest a direct action on the glutamate transporter itself without modifying the general driving forces. Thus, in vivo low concentrations of benzodiazepines may reduce synaptic glutamate concentrations by increased uptake, providing an additional mechanism to modulate neuronal excitability.

[The effect of rudotel on the synaptic activity of dopaminergic neurons]. / Vliianie rudotelia na sinapticheskuiu aktivnost' dofaminergicheskikh neironov.

The work discusses the neurochemical correlates of rat brain dopamin-synaptic activity after injection of 0.5 mg/kg rudotel (medazepam) as compared with intact animals. The parameters of functioning of D2-receptors in [3H]-dopamine binding, the content of catecholamines and their metabolites (high-performance liquid chromatography with electrochemical detection) were studied in the dopaminergic system in different brain areas (the striatum, frontal cortex, amygdala, hippocampus, hypothalamus, and accumbence).

[The effects of medazepam on memory trace reproduction during learning and amnesia in aggressive and submissive C57Bl/6J mice]. / Effekty medazepama na vosproizvedenie sleda pamiati pri obuchenii i amnezii u agressivnykh i submissivnykh myshei linii C57Bl/6J.

Experiments were conducted on C57B1 mice to study the role of the social status on the reproduction of a conditioned reaction of passive avoidance and the effect of medazepam on the processes of reproduction. Aggressive and submissive animals were selected according to the test for agonistic confrontations. No effect of the animals' social status on the formation of a conditioned habit was revealed, but a significant increase in the level of defecation was recorded in the aggressive mice. Medazepam administration before the test reduced the reproduction of the conditioned reaction only in the control mice. An amnestic effect blocked reproduction in control and submissive mice but did not change it in the aggressors. Medazepam restored the amnestic memory trace only in submissive individuals. In aggressive mice it reduced the emotional response but did not change reproduction. The data obtained suggest that variations of the social status determine both the behavioral responsiveness to training and the changes in reproduction in response to medazepam.

Automated on-line dialysis for sample preparation for gas chromatography: determination of benzodiazepines in human plasma.

An on-line dialysis-solid-phase extraction-gas chromatographic (GC) approach has been developed for the determination of drugs in plasma, using some benzodiazepines as model compounds. Clean-up is based on performing the dialysis of 100 microliters samples for 7 min using water as acceptor phase and trapping the diffused analytes on a PLRP-S copolymer precolumn. After drying of the precolumn with nitrogen for 15 min, the analytes are desorbed with ethyl acetate (275 microliters) and injected on-line into the GC system via a loop-type interface. The system provides a very efficient clean-up, and offers the possibility of adding chemical agents which can help to reduce drug-protein binding and, thus, increase sensitivity. To demonstrate the potential of the described approach, the determination of benzodiazepines in plasma at their therapeutical levels is used as an example with flame ionization, thermionic and mass-selective detection.