Dynamic high performance liquid chromatography on chiral stationary phases. Low temperature separation of the interconverting enantiomers of diazepam, flunitrazepam, prazepam and tetrazepam.

Diazepam and the structurally related 1,4-benzodiazepin-2-ones tetrazepam, prazepam and flunitrazepam are chiral molecules because they adopt a ground state conformation featuring a non-planar seven membered ring devoid of any reflection-symmetry element. The two conformational enantiomers of this class of benzodiazepines interconvert rapidly at room temperature by a simple ring flipping process. Low temperature HPLC on the Whelk-O1 chiral stationary phase allowed us to separate the conformational enantiomers of diazepam and of the related 1,4-benzodiazepin-2-ones, under conditions where the interconversion rate is sufficiently low, compared to the chromatographic separation rate. Diazepam, tetrazepam and prazepam showed temperature dependent dynamic HPLC profiles with interconversion plateaus indicative of on-column enantiomer interconversion (enantiomerization) in the temperature range between -10 °C and -35 °C, whereas for flunitrazepam on-column interconversion was observed at temperatures between -40 °C and -66 °C. Simulation of exchange-deformed HPLC profiles using a computer program based on the stochastic model yielded the apparent rate constants for the on-column enantiomerization and the corresponding free energy activation barriers. At -20 °C the enantiomerization barriers, ΔG(≠), for diazepam, prazepam and tetrazepam were determined to be in the range 17.6-18.7 kcal/mol. At -55 °C ΔG(≠) for flunitrazepam was determined to be in the 15.6-15.7 kcal/mol range. The experimental dynamic chromatograms and the corresponding interconversion barriers reported in this paper call for a reinterpretation of previously published results on the HPLC behavior of diazepam on chiral stationary phases.

Oral lichen planus and neurogenic inflammation: new observations and therapeutic implications from four clinical cases.

Oral lichen planus (OLP) is a usually chronic and relapsing mucocutaneous disease with unknown etiology. Immunosuppressive treatment is sometimes unsatisfactory. We describe four cases of reticular OLP localized on the internal side of cheek, in the territory innervated by sensory free endings of the buccinator nerve, poorly responding to immunosuppressive treatment (topical/systemic corticosteroids, topical cyclosporin). Addition of prazepam 10 mg/day to standard therapy achieved significant improvement and clinical healing in 30-40 days. Because of the complex interplay between the nervous and immune systems, neuroinflammation, acting through conventional axon reflex and/or indirect reflex mechanism involving localized efferent vasodilatory parasympathetic fibers, could have an important pathogenic role in OLP. Such hypothesis could explain, at least partly, the spreading of lesions in OLP primarily triggered (and possibly sustained) by infections, irritants, or autoimmunity. Moreover, neuroinflammation could have a relevant role in OLP related to psychosomatic diseases, where the nervous component is the primary trigger and the main pathogen responsible for the lesions observed. Benzodiazepines modulate neuroinflammation through central, and, possibly, peripheral action. In OLP patients with mild/subclinical psychological conditions, low doses may effectively modulate neuroinflammatory pathways that are not always completely inhibited by immunosuppressive treatment and can contribute to the persistence of inflammation.

General unknown screening procedure for the characterization of human drug metabolites in forensic toxicology: applications and constraints.

LC coupled to single (LC-MS) and tandem (LC-MS/MS) mass spectrometry is recognized as the most powerful analytical tools for metabolic studies in drug discovery. In this article, we describe five cases illustrating the utility of screening xenobiotic metabolites in routine analysis of forensic samples using LC-MS/MS. Analyses were performed using a previously published LC-MS/MS general unknown screening (GUS) procedure developed using a hybrid linear IT-tandem mass spectrometer. In each of the cases presented, the presence of metabolites of xenobiotics was suspected after analyzing urine samples. In two cases, the parent drug was also detected and the metabolites were merely useful to confirm drug intake, but in three other cases, metabolite detection was of actual forensic interest. The presented results indicate that: (i) the GUS procedure developed is useful to detect a large variety of drug metabolites, which would have been hardly detected using targeted methods in the context of clinical or forensic toxicology; (ii) metabolite structure can generally be inferred from their "enhanced" product ion scan spectra; and (iii) structure confirmation can be achieved through in vitro metabolic experiments or through the analysis of urine samples from individuals taking the parent drug.

Noradrenergic regulation of GABAergic inhibition of main olfactory bulb mitral cells varies as a function of concentration and receptor subtype.

The main olfactory bulb (MOB) receives a rich noradrenergic innervation from the pontine nucleus locus coeruleus (LC). Previous studies indicate that norepinephrine (NE) modulates the strength of GABAergic inhibition in MOB. However, the nature of this modulation and the NE receptors involved remain controversial. The goal of this study was to investigate the role of NE receptor subtypes in modulating the GABAergic inhibition of mitral cells using patch-clamp electrophysiology in rat MOB slices. NE concentration dependently and bi-directionally modulated GABA(A) receptor-mediated spontaneous and miniature inhibitory postsynaptic currents (sIPSCs/mIPSCs) recorded in mitral cells. Low doses of NE suppressed sIPSCs and mIPSCs because of activation of alpha2 receptors. Intermediate concentrations of NE increased sIPSCs and mIPSCs primarily because of activation of alpha1 receptors. In contrast, activation of beta receptors increased sIPSCs but not mIPSCs. These results indicate that NE release regulates the strength of GABAergic inhibition of mitral cells depending on the NE receptor subtype activated. Functionally, the differing affinity of noradrenergic receptor subtypes seems to allow for dynamic modulation of GABAergic inhibition in MOB as function of the extracellular NE concentration, which in turn, is regulated by behavioral state.

Contribution of neuroinflammation in burning mouth syndrome: indications from benzodiazepine use.

Characterized by burning and painful oral sensations in absence of clinically significant mucosal abnormalities, the burning mouth syndrome is, despite numerous researches made, basically idiopathic and, consequently, difficult to treat effectively. Therapy with tricyclic antidepressants and benzodiazepines has been proposed, although the exact pathomechanism is not clear. The objective of this study is to define the possible reasons for the efficacy of benzodiazepines in the treatment of the burning mouth syndrome. Starting from the report of eight cases successfully treated with prazepam, the present authors examined the clinical features and the evidence from literature that support the possibility of a role of neuroinflammation in the pathogenesis of the burning mouth syndrome. Available data suggest that the nervous system could be crucial in the pathogenesis of the syndrome (altered perception of pain, disturbance of neural transmission, increased excitability, negative involvement of trigeminal-vascular system), and the present authors' experience lets them suppose a role for neuroinflammation. This hypothesis could also explain the positive response to benzodiazepines in some patients. The important role of neuroinflammation in dermatologic and oral diseases has been only recently investigated and acknowledged. Further studies on the connection between neuroinflammation and burning mouth syndrome could open interesting perspectives in the understanding and management of this difficult clinical condition.

Induction of a mixed depressive episode during rTMS treatment in a patient with refractory major depression.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive experimental technique which has mostly been investigated in the treatment of mood disorders with possible efficacy in depression. Among its potential side effects, there have been some reports of rTMS-induced (hypo)mania in the literature but none for rTMS-induced mixed episodes. We report the case of a 39-year-old woman suffering from refractory chronic major depression who developed a depressive mixed episode associated with a mild serotonin syndrome during her second week of rTMS treatment. She was receiving a combination of antidepressants, the doses of which were kept unchanged during rTMS treatment. Mixed as well as manic episodes may be induced by transcranial magnetic stimulation, complications already observed with antidepressants and electroconvulsive therapy. Therefore, caution should be exercised among clinicians using this experimental procedure, particularly in the treatment of bipolar depressed patients.

Benzodiazepines prescription in Dakar: a study about prescribing habits and knowledge in general practitioners, neurologists and psychiatrists.

Benzodiazepines are relatively well-tolerated medicines but can induce serious problems of addiction and that is why their use is regulated. However, in developing countries like Senegal, these products are used without clear indications on their prescription, their dispensation or their use. This work focuses on the prescription of these medicines with a view to make recommendations for their rational use. Benzodiazepine prescription was studied with psychiatrists or neurologists and generalists in 2003. Specialist doctors work in two Dakar university hospitals and generalists in the 11 health centres in Dakar. We did a survey by direct interview with 29 of 35 specialists and 23 of 25 generalists. All doctors were interviewed in their office. The questionnaire focused on benzodiazepine indications, their pharmacological properties, benzodiazepines prescribed in first intention against a given disease and the level of training in benzodiazepines by doctors. Comparisons between specialists and generalists were made by chi-square test. Benzodiazepines were essentially used for anxiety, insomnia and epilepsy. With these diseases, the most benzodiazepines prescribed are prazepam against anxiety and insomnia and diazepam against epilepsy. About 10% of doctors do not know that there is a limitation for the period of benzodiazepine use. The principal reasons of drugs choice are knowledge of the drugs, habit and low side effects of drugs. All generalists (100%) said that their training on benzodiazepines is poor vs. 62.1% of specialists, and doctors suggest seminars, journals adhesions and conferences to complete their training in this field. There are not many differences between specialists and generalists except the fact that specialists prefer prazepam in first intention in the insomnia treatment where generalists choose bromazepam. In addition, our survey showed that specialists' training in benzodiazepines is better than that of generalists. Overall, benzodiazepine prescription poses problems particularly in training, and national authorities must take urgent measures for rational use of these drugs.

Naltrexone plus benzodiazepine aids abstinence in opioid-dependent patients.

Naltrexone (NTX) is widely used to prevent relapse of opioid-dependent patients but its association with insomnia and "hyperexcitability" can result in treatment withdrawal. We evaluated whether NTX combined with the benzodiazepine prazepam was more effective than NTX in keeping patients opioid-free. We determined the relapse rate over 6 months in 56 opioid-dependent subjects, divided into 4 equal groups. All groups received psychological support and underwent urine tests for drug metabolites twice weekly. Group 1 did not receive pharmacological treatment (controls). Group 2 received NTX alone (one 50-mg tablet daily); group 3 received NTX (one 50-mg tablet daily) plus placebo (one tablet twice daily); and group 4 received NTX (one 50-mg tablet daily) plus prazepam (one 10-mg tablet twice daily). Ten patients of group 1 relapsed within 3 months, one after 6 months and three remained opioid-free. Six patients of group 2 relapsed within three months, two after 6 months, and six remained opioid-free. Seven patients of group 3 relapsed three months, one after 6 months and six patients remained opioid-free. In group 4, one patient relapsed within 3 months and one patient after 6 months; 12 patients of this group remained opioid-free. At urine tests, a significantly higher percent patients of group 4 remained free of Delta(9)-tetrahydrocannabinol versus patients of groups 2 and 3. In conclusion, many patients remained opioid-free on NTX alone or combined with prazepam, with a significant advantage for the NTX plus prazepam group.

Development and validation of a liquid chromatographic/electrospray ionization mass spectrometric method for the quantitation of prazepam and its main metabolites in human plasma.

A method was developed and fully validated for the quantitation of prazepam and its major metabolites, oxazepam and nordiazepam, in human plasma. Sample pretreatment was achieved by solid-phase extraction using Oasis HLB cartridges. The extracts were analysed by high-performance liquid chromatography (HPLC) coupled with single-quadrupole mass spectrometry (MS) with an electrospray ionization interface. The MS system was operated in the selected ion monitoring mode. HPLC was performed isocratically on a reversed-phase XTerra MS C18 analytical column (150 x 3.0 mm i.d., particle size 5 microm). Diazepam was used as the internal standard for quantitation. The assay was linear over a concentration range of 5.0-1000 ng ml(-1) for all compounds analyzed. The limit of quantitation was 5 ng ml(-1) for all compounds. Quality control samples (5, 10, 300 and 1000 ng ml(-1)) in five replicates from three different runs of analysis demonstrated an intra-assay precision (CV) of < or = 9.1%, an inter-assay precision of < or = 6.0% and an overall accuracy (relative error) of < 4.6%. The method can be used to quantify prazepam and its metabolites in human plasma covering a variety of pharmacokinetic or bioequivalence studies.

Electrospray ionization gas-phase electrophoresis under ambient conditions and it's potential or high-speed separations.

A moderately high resolution nanoelectrospray ionization gas-phase electrophoresis instrument was constructed and evaluated for simple high-speed separations of several groups of compounds. The insertion of a plate containing a 1.6 cm diameter exit orifice, 2.5 cm from the location of electrospray, allowed ions to be created and desolvated under ambient conditions with minimal solvent contamination to the drift tube. Ion separation selectivity is discussed and shown to be slightly altered by changing the drift gas flow rate. Issues of using gas-phase electrophoresis as a high-speed separation technique are discussed. Gas-phase electrophoresis-spectra of selected benzodiazepines, triazine herbicides, and simple combinatorial chemistry libraries are demonstrated.

Radiopacity of clomipramine conglomerations and unsuccessful endoscopy: report of 4 cases.

BACKGROUND: The radiopacity of ingested substances may serve as a clue to the presence of particular compounds, as this characteristic varies considerably among medications and household products. Tablet conglomerations are also variably radiopaque. We report 4 cases of clomipramine poisoning associated with formation of radiopaque masses, believed to be clomipramine, in the area of the stomach. CASE REPORTS: Four patients were admitted to the Toxicological Intensive Care Unit after ingestions of, respectively, 8.5 g (180 tablets of mixed strength), 7.5 g (100 tablets), 10.5 g (140 tablets), and 4.5 g (60 tablets) of clomipramine, along with other sedatives and antipsychotics. In each case, a rounded density was observed in the gastric area on plain chest radiograph. The hospital courses of each patient were marked by tachycardia, hypotension, QRS and QT prolongation, seizures, and decreased mental status. Three of 4 patients underwent unsuccessful endoscopy to remove tablet fragments and subsequently suffered gastrointestinal hemorrhage requiring transfusion. All patients were discharged recovered from the hospital. DISCUSSION: Clomipramine, a potent tricyclic antidepressant, has been previously reported to be nonradiopaque, and has not been reported to induce formation of concretions. These cases suggest that massive ingestions of clomipramine may form bezoars which are radiopaque and may be associated with serious toxicity. Careful consideration should be given prior to the use of gastric endoscopy for the retrieval of tablet fragments since significant hemorrhage, attributed to the procedure itself rather than to clomipramine toxicity, may ensue.

Kinetic study on the degradation of prazepam in acidic aqueous solutions by high-performance liquid chromatography and fourth-order derivative ultraviolet spectrophotometry.

A reversed-phase HPLC method was developed for the kinetic investigation of the acidic hydrolysis of prazepam which was carried out in hydrochloric acid solutions of 0.01, 0.1 and 1.0 M. In addition, a fourth-order derivative method for monitoring the parent compound itself was proposed and evaluated. One intermediate was observed by HPLC, which should be formed from breakage of the azomethine linkage. Further slow hydrolysis of the amide bond led to the benzophenone product that was isolated and identified. The mechanism of hydrolysis was biphasic, showing a consecutive reaction with a reversible step. Relative standard deviation was less than 2% for HPLC and less than 5% for the derivative method. Detection limits were 1.2 x 10(-7) M for the former method and 6.7 x 10(-7)M for the latter. Accelerated studies at higher temperatures were employed. Results of HPLC and fourth-order derivative methods were statistically the same.

Development of a supercritical fluid extraction method for the determination of temazepam in whole blood.

A supercritical fluid extraction (SFE) procedure for the analysis of temazepam from whole blood was developed. Quantitative recoveries were obtained by high-performance liquid chromatography using prazepam as an internal standard and carefully monitoring the extraction temperature and pressure. The results were found to compare well with those obtained by solid-phase extraction techniques, but they also had the advantages of reduced solvent consumption and minimal sample handling. The application of this method to authentic forensic blood specimens showed the SFE method to be useful as an alternative procedure for the extraction of temazepam in the toxicology laboratory.

Simultaneous high-performance liquid chromatographic analysis of flunitrazepam and four metabolites in serum.

A high-performance liquid chromatographic method for the simultaneous determination of flunitrazepam and four metabolites, desmethylflunitrazepam (DMF), 7-aminodesmethylflunitrazepam (7-NH2DMF), 7-aminoflunitrazepam (7-NH2F) and 3-hydroxyflunitrazepam (3-OHF), in serum is described. The method involves a simple extraction from alkalinized plasma (pH 9.5) into diethyl ether-chloroform (80:20, v/v). Prazepam was used as an internal standard for the quantification of the five compounds. Separation was achieved with a 10 microns RSil CN column (300 x 3.9 mn I.D.). The detection wavelength was set at 242 nm. The limits of detection ranged from 2.5 to 5 micrograms/l with a limit of quantification of 10 micrograms/l for all analytes.

Molecular determinants of recognition and activation at the cerebellar benzodiazepine receptor site.

Semiempirical quantum mechanical and molecular mechanics calculations were carried out to identify and characterize the steric and electronic properties that modulate ligand recognition and activation of the cerebellar GABAA/benzodiazepine (BDZ) receptor. For this hypothesis development, thirteen compounds belonging to structurally diverse chemical families were selected for study. Among the compounds selected were nine that bind and four that do not bind with appreciable affinity to this receptor and some that are known agonists, antagonists and inverse agonists, as measured by their modulation of GABA (gamma-aminobutyric acid) enhanced chloride ion flux in cerebellum. The stereoelectronic requirements for recognition deduced from commonalities among the ligands are the presence of at least two of three hydrogen bonding centers, and a lipophilic aromatic ring, in a specific spatial relationship. The results suggest that the selectivity for the cerebellar or Type I subtype, demonstrated by some of these ligands, could be failure to meet the requirements for binding at other receptors because of the absence of one of the proton accepting centers or the larger surface area and volume of these ligands. The requirement for activation, deduced from comparisons of agonist, antagonist, and inverse agonist properties is the presence of an electron accepting aromatic ring in a specific geometric arrangement with respect to the components of recognition. The validity of the '3D-Pharmacophore' developed was probed by using it for predictions of the behavior of 11 additional compounds not used for its development.

[Exacerbation of an affective psychosis after terminating a decade of benzodiazepine treatment. Which therapeutic procedure is sensible?]. / Exazerbation einer affektiven Psychose nach Beendigung einer jahrzehntelangen Benzodiazepinbehandlung. Welches therapeutische Vorgehen ist sinnvoll?

We report the case of a 61-year old patient with an affective disorder who had been treated with benzodiazepines in low dosages over a 16 year period. This treatment had been prescribed by his physician following several depressive episodes. During this time, the patient remained able to work and exhibited little psychopathological symptomatology. Following discontinuation of medication, however, the depressive phases resumed and were particularly intractable. The clinical implications of this case and the therapeutic strategies for approaching patients with long-term benzodiazepine treatment are discussed.

[Comparative double-blind study of bromazepam versus prazepam in non-psychotic anxiety]. / Etude comparative en double insu du bromazépam versus prazépam dans l'anxiété non psychotique.

The efficacy of bromazepam and prazepam for the different components of anxiety: inhibition, asthenia and somatisation is evaluated in a multi-centric, comparative and randomised study, conducted as double blind and in parallel groups in 159 adult patients showing a manifest anxiety according to the F.D.A. criteria. After a 7 day wash-out period, the patients receive either bromazepam in a 12 mg/d dose or prazepam in a 40 mg/d dose, over 4 weeks (D0-D28), then in a decreasing dose from D28 to D43; follow-up is carried out using the anxious inhibition scale W.P.2, auto-questionnaire A.D.A., the Hamilton anxiety scale and the Tyrer questionnaire (benzodiazepine withdrawal symptoms questionnaire). Patients are evaluated seven times during the study: at day 7 for inclusion, day 0 for randomisation, then day 7 and day 14 for following visits, at day 28 for efficacy and tolerance evaluation, and at day 50 for utilisation and withdrawal evaluation. The major efficacy criteria are the evolution of inhibition, asthenia and somatisation as compounds of anxiety respectively evaluated by W.P.2 scale, asthenic partial score of autoquestionnaire A.D.A. and somatic partial score of Hamilton anxiety scale. The analysis of results don't show any significant difference between the two groups on the evolution of the components asthenia and inhibition. However the evolution of the somatic component clearly makes a significant difference in favour of bromazepam. There is also a significant difference in terms of global anxiolytic action efficacy, in favour of bromazepam.(ABSTRACT TRUNCATED AT 250 WORDS)