Environmental concentrations of a delorazepam-based drug impact on embryonic development of non-target Xenopus laevis.

Benzodiazepines, psychotropics drugs used for treating sleep disorders, anxiety and epilepsy, represent a major class of emerging water pollutants. As occurs for other pharmaceutical residues, they are not efficiently degraded during sewage treatment and persist in effluent waters. Bioaccumulation is already reported in fish and small crustaceans, but the impact and consequences on other "non-target" aquatic species are still unclear and nowadays of great interest. In this study, we investigated the effects of a pharmaceutical preparation containing the benzodiazepine delorazepam on the embryogenesis of Xenopus laevis, amphibian model species, taxa at high risk of exposure to water contaminants. Environmental (1 µg/L) and two higher (5 and 10 µg/L) concentrations were tested on tadpoles up to stage 45/46. Results demonstrate that delorazepam interferes with embryo development and that the effects are prevalently dose-dependent. Delorazepam reduces vitality by decreasing heart rate and motility, induces marked cephalic and abdominal edema, as well as intestinal and retinal defects. At the molecular level, delorazepam increases ROS production, modifies the expression of some master developmental genes and pro-inflammatory cytokines. The resulting stress condition significantly affects embryos' development and threatens their survival. Similar effects should be expected as well in embryos belonging to other aquatic species that have not been yet considered targets for these pharmaceutical residues.

A case report on fatal intoxication by tapentadol: Study of distribution and metabolism.

We report a case in which a tapentadol acute intoxication was suspected as the cause of death of a 39-year-old man: approximately two days after death, cardiac and femoral blood, as well as urine, bile, gastric content and chest hair, were collected during the autopsy. Tapentadol was detected before and after hydrolysis in femoral (530 ng/mL unconjugated and 1570 ng/mL conjugated) and cardiac (680 ng/mL unconjugated and 3440 ng/mL conjugated) blood, and additionally in bile (3200 ng/mL), urine (9300 ng/mL), chest hair (2850 pg/mg) and gastric content. LC-QTOF screening analysis confirmed the presence of five different tapentadol metabolites (tapentadol-O-glucuronide, tapentadol-O-sulfate, N-desmethyltapentadol, N-desmethyltapentadol-glucuronide and N-desmethyltapentadol-O-sulfate), in urine, bile, cardiac and femoral blood. Positivity of body hairs allowed us to conclude that the man had used tapentadol in the last weeks/months. Autopsy and toxicological results (also positive for clotiapine, diazepam and chlordesmethyldiazepam) suggested that tapentadol could have caused, even at low concentrations, a severe respiratory depression, which contributed to the death of the subject. This is one of the few cases in literature where tapentadol was detected in blood, together with its metabolites, and the only one in which the parent drug was identified in hairs.

Organ distribution of diclazepam, pyrazolam and 3-fluorophenmetrazine.

The organ distribution of 3-fluorophenmetrazine (3-FPM), pyrazolam, diclazepam as well as its main metabolites delorazepam, lormetazepam and lorazepam, was investigated. A solid phase extraction (SPE) and a QuEChERS (acronym for quick, easy, cheap, effective, rugged and safe) - approach were used for the extraction of the analytes from human tissues, body fluids and stomach contents. The detection was performed on a liquid chromatography-tandem mass spectrometry system (LCMS/MS). The analytes of interest were detected in all body fluids and tissues. Results showed femoral blood concentrations of 10 µg/L for 3-FPM, 28 µg/L for pyrazolam, 1 µg/L for diclazepam, 100 µg/L for delorazepam, 6 µg/L for lormetazepam, and 22 µg/L for lorazepam. Tissues (muscle, kidney and liver) and bile exhibited higher concentrations of the mentioned analytes than in blood. Additional positive findings in femoral blood were for 2-fluoroamphetamine (2-FA, approx. 89 µg/L), 2-flourometamphetamine (2-FMA, hint), methiopropamine (approx. 2.2 µg/L), amphetamine (approx. 21 µg/L) and caffeine (positive). Delorazepam showed the highest ratio of heart (C) and femoral blood (P) concentration (C/P ratio = 2.5), supported by the concentrations detected in psoas muscle (430 µg/kg) and stomach content (approx. 210 µg/L, absolute 84 µg). The C/P ratio indicates that delorazepam displays susceptibility for post-mortem redistribution (PMR), supported by the findings in muscle tissue. 3-FPM, pyrazolam, diclazepam, lorazepam and lormetazepam did apparently not exhibit any PMR. The cause of death, in conjunction with autopsy findings was concluded as a positional asphyxia promoted by poly-drug intoxication by arising from designer benzodiazepines and the presence of synthetic stimulants.

Validation of an LC-MS/MS Method for the Quantification of 13 Designer Benzodiazepines in Blood.

The misuse of designer benzodiazepines, as an alternative to prescription benzodiazepines and for drug-facilitated sexual assaults, has emerged as a growing threat, due in part to the ease of purchasing these drugs on the internet at low prices. Causing concern for safety is the lack of dosage information resulting in users self-medicating, often leading to unintended overdoses, coma or death at higher doses. With limited published data regarding the quantification of designer benzodiazepines in forensic cases, a method was validated for the determination of 13 designer benzodiazepines in postmortem blood, to add to the in-house method that already included a limited number of common designer benzodiazepines. The developed method included 3-hydroxyphenazepam, clobazam, clonazolam, delorazepam, deschloroetizolam, diclazepam, flualprazolam, flubromazepam, flubromazolam, flunitrazolam, meclonazepam, nifoxipam and pyrazolam in 0.5 mL postmortem blood using liquid chromatography-tandem mass spectrometry. The analytes were treated with solid phase extraction before undergoing separation on a C18 column and analyzed on the mass spectrometer in electrospray positive mode using multiple reaction monitoring. The linear range of the calibration curve was 1-200 ng/mL and up to 500 ng/mL for 3-hydroxyphenazepam, clobazam, flubromazepam and pyrazolam. The limits of detection and quantitation were 0.5 ng/mL (signal-to-noise ratio >3) and 1 ng/mL, respectively. The calculated bias, intra-day imprecision, relative standard deviation (RSD) and inter-day imprecision RSD were ±12%, 3-20% and 4-21%. Matrix effects ranged from -52% to 33% with RSD values ranging from 3-20%, indicating consistent effects throughout multiple sources. Recovery ranged from 35 to 90%, where only two compounds were <50%. Other parameters tested included carryover, stability, interference and dilution integrity, which all yielded acceptable results. With the application of this method to blood specimens from the New York City Office of Chief Medical Examiner, this validated method proved to be simple, reproducible, sensitive and robust.

The unmasking of hidden severe hyponatremia after long-term combination therapy in exacerbated bipolar patients: a case series.

Hyponatremia is occasionally unmasked in psychiatric patients during hospitalization after routine blood and urinary tests, and correlates in most cases with an inappropriate secretion of antidiuretic hormone, mainly due to iatrogenic factors. Only a few studies have regarded the combination of psychotropic drugs as triggers of chronic, asymptomatic hyponatremia in bipolar patients, who require to be hospitalized because of the exacerbation of their mental illness. We presented three clinical cases of patients affected by a long-term psychiatric disorder and under polypharmacotherapy for several months. After excluding other potential factors, we hypothesized that pharmacological treatment with a mood stabilizer (oxcarbazepine) associated with a benzodiazepine (delorazepam), a second-generation antipsychotic (olanzapine) or an antidepressant (fluvoxamine), triggered severe hyponatremia ([Na+] ≤125 mEq/L), serum hypo-osmolarity, and elevated inappropriate urine osmolarity added to more diluted sodium concentration. When we discontinued the treatment, clinical conditions of our patients improved, despite the previous administration of hypertonic saline jointly with water restriction. Psychiatrists should consider that bipolar patients on long-term polypharmacotherapy may present a higher risk of severe hyponatremia not clinically detectable. Consequently, routine laboratory tests should be periodically repeated as they represent the only available tool to unmask such electrolyte imbalances.

Combined occlusal and pharmacological therapy in the treatment of temporo-mandibular disorders.

OBJECTIVE: Aim of the present work is to assess the effectiveness of a scientific protocol built up to relieve pain in chronic temporo-mandibular disorders (TMD) using Michigan splint together with a pharmacological therapy compared to the traditional occlusal therapy by Michigan splint alone. PATIENTS: 35 adult patients, with signs and symptoms of TMD lasting more than 6 months, were enrolled into this study and divided into two groups: the first receiving occlusal therapy by Michigan splint and pharmacological therapy with Delorazepam and Thiocolchicoside, while the second receiving occlusal therapy by Michigan splint and "placebo" administration. The comparisons between the two experimental groups were assessed using a 5 steps visual-analogue scale (V.A.S.). RESULTS AND CONCLUSIONS: The outcomes from the experimental groups were statistically compared resulting significantly different with an improvement or disappearance of signs and symptoms in the treated group with respect to the placebo group at 12 and 18 months from the beginning of the experiment (p < 0.001).

Comparison by means of bispectral index score, between anxiolysis induced by diazepam and sedation induced by midazolam.

AIM: Bispectral Index Score (BIS) is an objective tool to assess sedation depth. Benzodiazepines have different pharmacological profiles and diazepam may be safer than midazolam in this setting. The aim of this study was to compare BIS values observed during anxiolysis after diazepam versus sedation after midazolam. METHODS: Thirty-six patients were randomly assigned to 3 groups: group 1 was treated with i.v. diazepam, groups 2 and 3 with iv midazolam 1 and 3 mg, respectively. Sedation was monitored clinically and by means of BIS. BIS values were evaluated as area under the curve (AUC) and compared by variance analysis. The statistical comparison of other data was performed by variance analysis or, alternatively, the χ2 according to Yates. The statistical significance was indicated by P values <0.05. RESULTS: AUC values were significantly lower after midazolam when compared to AUC values registered in diazepam treated patients; 22.6% of the group 3 patients showed BIS values <80, versus 0.4% of group 1 patients. CONCLUSION: Diazepam has a safer profile, with BIS values and clinical conditions according to the definition of minimal and/or moderate sedation. Diazepam represents the safer drug for anxiety management in dentistry, because regularly produces a state of sedation during which verbal contact with the patient is maintained and carry a margin of safety wide enough to render loss of consciousness unlikely.

Reduction of temazepam to diazepam and lorazepam to delorazepam during enzymatic hydrolysis.

It has been previously reported that treatment of urinary oxazepam by commercial ß-glucuronidase enzyme preparations, from Escherichia coli, Helix pomatia and Patella vulgata, results in production of nordiazepam (desmethyldiazepam) artefact. In this study, we report that this unusual reductive transformation also occurs in other benzodiazepines with a hydroxyl group at the C3 position such as temazepam and lorazepam. As determined by liquid chromatography-mass spectrometry analysis, all three enzyme preparations were found capable of converting urinary temazepam into diazepam following enzymatic incubation and subsequent liquid-liquid extraction procedures. For example, when H. pomatia enzymes were used with incubation conditions of 18 h and 50 °C, the percentage conversion, although small, was significant--approximately 1% (0.59-1.54%) in both patient and spiked blank urines. Similarly, using H. pomatia enzyme under these incubation conditions, a reductive transformation of urinary lorazepam into delorazepam (chlordesmethyldiazepam) occurred. These findings have both clinical and forensic implications. Detection of diazepam or delorazepam in biological samples following enzyme treatment should be interpreted with care.

Quantification of chlordesmethyldiazepam by liquid chromatography-tandem mass spectrometry: application to a cloxazolam bioequivalence study.

A rapid, sensitive and specific LC-MS/MS method was developed and validated for quantifying chlordesmethyldiazepam (CDDZ or delorazepam), the active metabolite of cloxazolam, in human plasma. In the analytical assay, bromazepam (internal standard) and CDDZ were extracted using a liquid-liquid extraction (diethyl-ether/hexane, 80/20, v/v) procedure. The LC-MS/MS method on a RP-C18 column had an overall run time of 5.0 min and was linear (1/x weighted) over the range 0.5-50 ng/mL (R > 0.999). The between-run precision was 8.0% (1.5 ng/mL), 7.6% (9 ng/mL), 7.4% (40 ng/mL), and 10.9% at the low limit of quantification-LLOQ (0.500 ng/mL). The between-run accuracies were 0.1, -1.5, -2.7 and 8.7% for the above mentioned concentrations, respectively. All current bioanalytical method validation requirements (FDA and ANVISA) were achieved and it was applied to the bioequivalence study (Cloxazolam -- test, Eurofarma Lab. Ltda and Olcadil -- reference, Novartis Biociências S/A). The relative bioavailability between both formulations was assessed by calculating individual test/reference ratios for Cmax, AUClast and AUC0-inf. The pharmacokinetic profiles indicated bioequivalence since all ratios were as proposed by FDA and ANVISA.

Combined sedation with oral chlordemethyldiazepam and midazolam by nasal route in third molar surgery.

AIM: This study was performed to evaluate the effects on the cardiocirculatory system, on perioperative anxiety and compliance of sedation with 2 benzodiazepines, chlordemethyldiazepam (CDDZ) a long acting oral drug for presedation and midazolam, a short acting drug, administered by nasal route to induce intraoperative sedation. METHODS: Fifty randomized patients undergoing third molar extraction at the Dental Clinic, University of Padua, were preoperatively evaluated. Anxiety was evaluated through a visual, analogue, scale (VAS) of 10 cm, a questionnaire of adjectives called interval scale of anxiety response (ISAR) and the Newman test was applied to evaluate the changes in psychomotor functions. All patients were treated with 1 ml of oral CDDZ for presedation and midazolam by the nasal route for intraoperative sedation at doses of 1 mg in Group 1 (25 patients) and 2 mg in Group 2 (25 patients). In all patients preoperative cardiocirculatory parameters were evaluated and in the first 20 min after the beginning of intervention. At the end of intervention the Newman test was reapplied, anxiety and postoperative cardiocirculatory data were reevaluated and the quality of the intervention judged in an interview made 1 week after the intervention (quality of the sedation technique, perioperative pain intensity, assumption of analgesic drugs, swelling, amnesia etc. after intervention). RESULTS: The treatment with 1 mg CDDZ + 2 mg midazolam by nasal route is the best association to slightly attenuate intra- and postoperative cardiocirculatory response, anxiety and to improve the quality of the treatment without interfering on the psychomotor response of patients at the time of the discharge. CONCLUSION: To conclude, the sedative technique employed is easily applied by the dentist, and is safe, efficacious and well tolerated by patients.

SPME-LC with UV detection to study delorazepam-serum albumin interactions.

Solid phase microextraction coupled to high performance liquid chromatography with UV detection (SPME/LC-UV) has been employed to study the binding of delorazepam to human serum albumin (HSA) and bovine serum albumin (BSA). The procedure could also be potentially extended to the measurement of partition coefficients between a wide variety of semi- or non-volatile compounds and matrices. The method is solvent free, simple, fast, and drawbacks of the conventional analytical techniques are avoided. Moreover, the matrix did not interfere with the measurement by binding to the fibre and the amount extracted by the fibre was negligibly small; thus it did not disturb the delorazepam-protein binding.

Determination of delorazepam in urine by solid-phase microextraction coupled to high performance liquid chromatography.

An SPME-HPLC-UV method for the determination of delorazepam, a representative benzodiazepine, in spiked human urine samples was developed for the first time. The performances of two commercially available fibers, a carbowax/templated resin (Carbowax/TPR-100) and a polydimethylsiloxane/divinylbenzene (PDMS/DVB), were compared, indicating the latter as the most suitable for urine samples analysis. All the aspects influencing adsorption (extraction time, pH, temperature, salt addition) and desorption (desorption and injection time, desorption solvent mixture composition) of the analyte on the fiber have been investigated. In particular, short extraction times were necessary to reach the equilibrium and very short desorption times were employed. The procedure required simple sample pre-treatment and was able to detect 5 ng/ml in spiked urine, regardless of the complexity of the matrix.

Binding of [3H]CB 34, a selective ligand for peripheral benzodiazepine receptors, to rat brain membranes.

The 2-phenyl-imidazo[1,2-a]pyridine derivative CB 34 is a ligand for peripheral benzodiazepine receptors. The binding of [3H]CB 34 to rat cerebrocortical membranes was characterized. Specific binding was rapid, reversible, saturable and of high affinity. Kinetic analysis yielded association and dissociation rate constants of 0.2x10(8) M(-1) min(-1) and 0.29 min(-1), respectively. Saturation binding experiments revealed a single class of binding sites with a total binding capacity of 188+/-8 fmol/mg protein and an apparent dissociation constant of 0.19+/-0.02 nM. Specific [3H]CB 34 binding was inhibited by ligands selective for peripheral benzodiazepine receptors, whereas, with the exception of flunitrazepam and diazepam, ligands for central benzodiazepine receptors were inactive. Of the brain regions examined, the density of the [3H]CB 34-binding sites was greatest in the hypothalamus and lowest in the cerebral cortex. [3H]CB 34 is thus a potent and selective ligand for peripheral benzodiazepine receptors and should be proven useful for studies of the roles of these receptors.

Peripheral benzodiazepine receptor messenger RNA is decreased in lymphocytes of generalized anxiety disorder patients.

BACKGROUND: The aim of this study was to assess whether the decrease of peripheral benzodiazepine receptor (pBR) number in peripheral blood mononuclear cells (PBMC), previously observed in patients with generalized anxiety disorder, is paralleled by changes in the relative content of messenger RNA (mRNA) encoding pBR. METHODS: Eight patients with a DSM-III-R diagnosis of generalized anxiety disorder were examined before, during, and after 2'-chloro-N-desmethyl-diazepam treatment. Eight healthy subjects were analyzed in parallel. The relative content of pBR mRNA was determined by reverse-transcriptase-polymerase chain reaction, using beta-actin as internal standard. Kinetic binding properties of pBR were measured using 3H-PK11195 as a ligand. RESULTS: pBR and pBR mRNA were significantly decreased in untreated generalized anxiety disorder patients as compared to controls (by 45% and 70%, respectively). Both pBR density and mRNA levels returned to control values during treatment or after withdrawal, which also coincided with recovery from anxiety. CONCLUSIONS: These results suggest that the turnover rate of pBR is reduced in PBMC of generalized anxiety disorder patients, and that this change occurs at the transcriptional level.

Paroxetine efficacy in the treatment of generalized anxiety disorder.

Recently, there has been a renewed interest in alternatives to the benzodiazepines for the treatment of generalized anxiety disorder (GAD). The aim of the present study was to compare the efficacy of paroxetine vs. imipramine and 2'-chlordesmethyldiazepam in 81 patients with a DSM-IV diagnosis of GAD. Approximately two-thirds of the patients who completed the study improved greatly or moderately on all three active drugs. During the first 2 weeks of treatment, 2'-chlordesmethyldiazepam treatment resulted in the greatest improvement in anxiety ratings. Both paroxetine and imipramine treatment resulted in more improvement than 2'-chlordesmethyldiazepam by the fourth week of treatment. Paroxetine and imipramine affect predominantly psychic symptoms, whereas 2'-chlordesmethyldiazepam affects predominantly somatic symptoms. Our results suggest that paroxetine is effective for the treatment of GAD.

Abnormal ventricular repolarization mimicking myocardial infarction after heterocyclic antidepressant overdose.

In 2 young adult women who experienced acute heterocyclic antidepressant intoxication, we found a quite unusual electrocardiographic pattern characterized by abnormal ST-tract elevation in the right precordial leads associated with a marked QRS widening (right bundle branch block and left anterior fascicular block type). Because serum electrolyte imbalance and acute myocardial ischemic events were excluded, the mechanism by which antidepressant overdose may produce such elevation of the ST tract remains unclear.

Effects of liver disease on the pharmacokinetics of intravenous and oral chlordesmethyldiazepam.

We studied the pharmacokinetics of a single 0.5-mg i.v. dose of chlordesmethyldiazepam in 8 patients with liver disease and in 12 age-matched healthy controls. The kinetics were also studied of a single 1-mg oral dose in the patients with liver disease. After i.v. administration the kinetics of total chlordesmethyldiazepam in patients with liver disease differed from those in controls: elimination half-life was almost twice that in controls (395 and 204 h), as a consequence of a marked reduction in total clearance (0.13 and 0.25 ng.ml-1.h-1), whereas the apparent volume of distribution was similar in patients and controls (4.7 and 3.9 l/kg-1). The free fraction of the drug in patients was higher (5.5%) than in controls (2.9%). Correction for differences in protein binding revealed clearance in the patients was one-fifth (1.8 and 10.5 ng ml-1.kg-1) and volume of distribution one-half (65.0 and 118.4 l.kg-1) that in controls. The systemic availability of oral chlordesmethyldiazepam was high (110%) in spite of a relatively slow absorption rate. These results indicate a need for caution in the administration of chlordesmethyldiazepam to patients with liver disease.