Alpha-hydroxy Metabolite Research Articles

Flubromazolam-Derived Designer Benzodiazepines: Toxicokinetics and Analytical Toxicology of Clobromazolam and Bromazolam.

Flubromazolam is widely known as highly potent designer benzodiazepine (DBZD). Recently, the two flubromazolam-derived new psychoactive substances (NPS) clobromazolam and bromazolam appeared on the drugs of abuse market. Since no information concerning their toxicokinetics in humans is available, the aims of the current study were to elucidate their metabolic profile and to identify the isozymes involved in their phase I and phase II metabolism. In vitro incubations with pooled human liver S9 fraction were performed and analyzed by liquid chromatography coupled to orbitrap-based high-resolution tandem mass spectrometry (LC-HRMS-MS). Biosamples after the ingestion of bromazolam allowed the identification of metabolites in human plasma and urine as well as the determination of bromazolam plasma concentrations by LC-HRMS-MS using the standard addition method. In total, eight clobromazolam metabolites were identified in vitro as well as eight bromazolam metabolites in vitro and in vivo. Predominant metabolic steps were hydroxylation, glucuronidation and combinations thereof. Alpha-hydroxy bromazolam glucuronide and bromazolam N-glucuronide are recommended as screening targets in urine. Bromazolam and its alpha-hydroxy metabolite are recommended if conjugate cleavage is part of the sample preparation procedure. The bromazolam plasma concentrations were determined to be 6 and 29 μg/L, respectively. Several cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) isozymes were shown to catalyze their metabolic transformations. CYP3A4 was involved in the formation of all phase I metabolites of both NPS, while UGT1A4 and UGT2B10 catalyzed their N-glucuronidation. Several UGT isoforms catalyzed the glucuronidation of the hydroxy metabolites. In conclusion, the determined bromazolam plasma concentrations in the low micrograms per liter range underlined the need for sensitive analytical methods and the importance of suitable urine screening procedures including DBZD metabolites as targets. Such an analytical strategy should be also applicable for clobromazolam.

Application of pharmacokinetic studies to a novel antidepressant, E2011.

1. The original drug tested here, (5R)-3-[2-(3-cyanopropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxaz olidinone (ER-4539), exhibited strong MAO-A inhibitory activity in vitro, but its bioavailability in rat was very low. After ER-4539 was administered orally to dog, a metabolite was found in plasma. 2. The metabolite was isolated by hplc after incubation with dog liver microsomal preparations. Its structure, determined by ms and nmr analysis, was alpha-hydroxy-ER-4539. The configuration of the alpha-hydroxy metabolite was (S), determined in comparison with the authentic sample of (R) and (S) by hplc. The isolated metabolite had potent MAO-A inhibitory action in vitro, indicating that it would have antidepressant action. 3. (5R)-3-[2-((1S)-3-Cyano-1-hydroxypropyl)benzothiazol-6-yl]-5- methoxymethyl-2-oxazolidinone (E2011), the synthesized metabolite, has been improved in regard to biopharmaceutical characteristics in rat and dog.

Determination of midazolam and its alpha-hydroxy metabolite in human plasma and urine by high-performance liquid chromatography.

We describe a new high-performance liquid chromatography (HPLC) method for measurement of midazolam and its major metabolite, alpha-hydroxymidazolam, in clinical samples. Plasma or urine was mixed with 100 ng internal standard Ro 05-6669 and borate buffer, 0.1 M, pH 9. Midazolam and its related compounds were extracted into diethylether. The organic phase was evaporated to dryness. The residue was dissolved in HPLC mobile phase [methanol-isopropyl alcohol-perchloric acid, 0.5 microM (57:25:18)] and injected into the chromatograph. The separation of substances was performed on an Spherisorb S5CN 250 x 4.6 mm HPLC column maintained at 45 degrees C. The detection was performed by absorption measurement at 245 nm. At a flow rate of 1.7 ml/min, the retention times of Ro 05-6669, 1,4 dihydroxymidazolam, alpha-hydroxymidazolam, 4-hydroxymidazolam and midazolam were 4.0, 6.7, 7.8, 9.6, and 10.8 min, respectively. In the concentration range of 5-1,000 ng/ml, the calibration graphs for both compounds were linear. The coefficients of variation of the between-day and within-day assay were < 14% for the concentration range 5-10 and < 7% for the range 10-600 ng/ml. The limits of detection for midazolam and alpha-hydroxymidazolam were 2 and 4 ng/ml, respectively. This assay is more sensitive than earlier methods; it is simple and rapid, and it enables the quantification of midazolam and its alpha-hydroxy metabolite with very good precision and accuracy in human plasma and urine.

Influence of nitrendipine and verapamil on plasma levels, urinary excretion, and beta-blocking effect of metoprolol

Following randomized allocation eight healthy volunteers were treated for 1 week each with metoprolol alone (100 mg twice daily), verapamil 80 mg three times a day plus metoprolol 100 mg twice daily, and with nitrendipine 20 mg twice daily. Plasma levels and urinary recovery of the beta-blocker, antipyrine clearance, and heart rate on exercise were measured. Verapamil and nitrendipine slightly prolonged elimination half-life of metoprolol. The urinary recovery of the parent beta-blocker and of its alpha-hydroxy metabolite was elevated by both calcium antagonists (verapamil and nitrendipine). Exercise tachycardia (150 beats/min without drugs) was inhibited more pronounced on the combination therapies than under metoprolol administration alone. Results of the present study indicate that calcium antagonists enhance inhibition of exercise tachycardia caused by metoprolol, possibly due to their binding to myocardial beta-adrenergic receptors which is known from the literature. As both calcium antagonists did not increase plasma levels of metoprolol, in the present study a kinetic interaction between the beta-blocker and the calcium channel blockers investigated does not appear to be responsible for the pharmacodynamic effects observed.

Polymorphic drug oxidation: pharmacokinetic basis and comparison of experimental indices.

The pharmacokinetic basis for using various experimental indices, (urinary drug: metabolite and metabolite:drug + metabolite ratios, urinary metabolite recovery and AUC values), for detecting polymorphic oxidative drug metabolism was examined. Pharmacokinetic determinants in addition to partial metabolic clearance down the polymorphic route were identified in each index. The ability of the various indices to discriminate bimodality in population data was assessed using a computer simulation. With the exception of the AUC data, bimodality was apparent to varying extents in all of the frequency distributions and, in general, logarithmic transformation allowed clearer visualisation of the two phenotypic groups. Simulated distributions were compared with those observed experimentally for metoprolol and its alpha-hydroxy metabolite. Detailed pharmacokinetic data from controlled studies in small numbers of volunteers can form the basis of the input to the simulation programme. Inspection of the output may help in the design of further studies in larger numbers of subjects in whom only limited data collection is possible.

Determination of metoprolol in human plasma by column switching high-performance liquid chromatography

Retention characteristics of metoprolol have been studied in reversed phase mode on RP2, RP8 and CN columns. The plots of retention time as a function of the acetonitrile content and of the ionic strength of the mobile phase permitted the choice of the best conditions to separate metoprolol from plasma components by switching of these three types of columns. Human plasma (0.5–1 ml) diluted with water is first injected on a RP2 column (25–40 μm particle diameter, prepared by dry packing) and rinsed with water. The sample is then back eluted with acetonitrile-0.022 M acetate buffer (75∶25, v:v) and switched to a CN column (10 cm long, 5 μm particle diameter). The heart cut of the eluate is selected and loaded on a RP8 analytical column (25 cm long, 5 μm particle diameter) with acetonitrile-0.088 M acetate buffer (75∶25, v:v) as mobile phase. Auto-sampler and switching valves are actuated automatically by a computing integrator based on a fixed time schedule. The duration of one cycle is about 30 min, but the last analytical step is about 15 min and represents the time interval between two injections. Metoprolol, its alpha-hydroxy metabolite and the internal standard are detected by fluorescence (λex= 225 nm; λem > 320 nm).

ChemInform Abstract: PHARMACOLOGY OF SOME METABOLITES OF TRIAZOLAM, ALPRAZOLAM, AND DIAZEPAM PREPARED BY A SIMPLE, ONE‐STEP OXIDATION OF BENZODIAZEPINES

AbstractDas Imidazobenzodiazepin (Ia) und Triazolam (Ib) werden mit einem geringem Überschuß Kalium‐tert.‐ butylat in einem Gemisch aus tert.‐Butylalkohol, Dimethylformamid, Tetrahydrofuran, Dimethoxyethan und Triethylphosphit in ihre Anionen übergeführt, deren Oxidation bei ‐20°C mit Sauerstoff zu den Hydroxy‐Analoga (II) führt.

Pharmacology of some metabolites of triazolam, alprazolam, and diazepam prepared by a simple, one-step oxidation of benzodiazepines.

A simple, one-step chemical oxidation of triazolam (7) to its 4-hydroxy analogue, 7a, has been developed and applied to other triazolo- and imidazobenzodiazepines. The reaction may be used to convert diazepam to temazepam. 4-Hydroxytriazolo[4,3-a][1,4]benzodiazepines have low central nervous system sedative and anticonvulsant activity in sharp contrast to metabolites of diazepam which remain active. While 10, an alpha-hydroxy metabolite of triazolam, retains much of the activity of 7, 10a, and alpha,4-dihydroxy metabolite of triazolam, is 250 times less potent than 7 on the nicotine-antagonism assay and over 300 times less potent on the traction assay.