Flumazenil In Plasma Research Articles

Determination of flumazenil in human plasma by liquid chromatography–electrospray ionisation tandem mass spectrometry

A liquid chromatography–electrospray ionisation–tandem mass spectrometry (LC–ESI–MS/MS) method was developed to determine unlabelled flumazenil (Ro 15-1788) in human plasma in [ 11C]flumazenil positron emission tomography (PET) studies. N-Methyl tri-deuterated flumazenil was used as an internal standard. The analyte and internal standard were extracted from plasma samples using solid-phase extraction, with a recovery of 78%. This was determined through the convenience of radioactivity measurements of 11C-labelled flumazenil. The evaporated and reconstituted eluate was analysed by LC–ESI–MS/MS. The calibration curve was linear over the tested concentration range of 0.05–0.5 nM (15–150 pg/ml) with a correlation coefficient, R 2, of 0.998±0.001. A high precision was achieved, with mean intra-assay and inter-assay relative standard deviations of at most 6 and 7%, respectively. The accuracy of the method ranged from 95 to 104%. As a proof of concept, the validated method was applied in the determination of flumazenil in plasma from two healthy volunteers participating in a PET study with three repeated investigations. A bolus-infusion protocol was used to achieve a constant concentration level of flumazenil. The average plasma concentrations ranged from 0.11 and 0.19 nM and all measurements were within the calibration standard range. The flumazenil concentrations were relatively constant within each scan and the average intra-scan precision was 15%.

A rapid high-pressure liquid chromatographic procedure for determination of flumazenil in plasma

Introduction: Flumazenil antagonizes the effects of benzodiazepines at γ-aminobutyric acid (GABA) type A receptors in the central nervous system. Flumazenil has been reported to provoke panic attacks in patients with panic disorder (PD) but not in healthy controls. A rapid high-pressure liquid chromatographic (HPLC) method was developed for determination of flumazenil in plasma samples from PD patients receiving flumazenil and the results obtained with that assay are reported here. Methods: Samples from 37 PD subjects receiving 2 mg of flumazenil intravenously were analyzed. Extraction under basic conditions was followed by an HPLC assay with UV detection (250 nm). Lamotrigine was used as internal standard and a standard curve was constructed for each assay run. Flumazenil concentrations were measured in all the subjects in samples collected at 2 and 4 min after the drug administration and in some subjects, measurements were also done in samples collected at 7.5, 15, 30, 45, and 60 min. Results: The procedure was reproducible and linear (from 2.5 to 1000 ng/ml). At 2 and 4 min after flumazenil administration, the concentrations did not differ significantly between panicking and nonpanicking subjects, indicating that the pharmacokinetics of the drug is not the major determinant of the responses. There was a steep decline in the plasma concentration–time profile during the first 4 min, reflecting an extensive and rapid distribution after which the decline was slower. Discussion: The method described here is rapid, replicable, and convenient for the determination of flumazenil in plasma.

Benzodiazepine receptor quantification in Huntington's disease with [123I]iomazenil and SPECT

OBJECTIVESIncreasing evidence suggests that metabolic changes predate neuronal death in Huntington's disease and emission tomography methods (PET and SPECT) have shown changes in glucose consumption and receptor function in early...

Intrasubject correlation between static scan and distribution volume images for [11C]flumazenil PET.

Accumulation of [11C]flumazenil (FMZ) reflects central nervous system benzodiazepine receptor (BZR). We searched for the optimal time for a static PET scan with FMZ as semi-quantitative imaging of BZR distribution. In 10 normal subjects, a dynamic series of decay-corrected PET scans was performed for 60 minutes, and the arterial blood was sampled during the scan to measure radioactivity and labeled metabolites. We generated 13 kinds of "static scan" images from the dynamic scan in each subject, and analyzed the pixel correlation for these images versus distribution volume (DV) images. We also analyzed the time for the [11C]FMZ in plasma and tissue to reach the equilibrium. The intra-subject pixel correlation demonstrated that the "static scan" images for the period centering around 30 minutes post-injection had the strongest linear correlation with the DV image. The ratio of radioactivity in the cortex to that in the plasma reached a peak at 40 minutes after injection. Considering the physical decay and patient burden, we conclude that the decay corrected static scan for [11C]FMZ PET as semi-quantitative imaging of BZR distribution is to be optimally acquired from 20 to 40 minutes after injection.

Plasma concentration of flumazenil following intranasal administration in children.

A pharmacokinetic study in children to determine plasma flumazenil concentrations after the intranasal administration of 40 microg x kg(-1). Following institutional approval and informed written consent, 11 ASA physical status I-II patients, aged two to six years, undergoing general anesthesia for dental surgery were recruited. After induction, 40 microg x kg(-1) flumazenil Anexate, Roche, 0.1 mg x mL(-1) (0.4 mL x kg(-1))) were administered via a syringe as drops, prior to nasal intubation. Venous plasma samples were drawn prior to administration of flumazenil (t = 0), and then at 2, 4, 6, 8, 10, 15, 20, 30, 40, 60, and 120 min thereafter. The plasma samples were immediately processed by the on-site laboratory and then stored at -70 degrees C, before batch analysis via high performance liquid chromatography assay. Pharmacokinetic data calculations were performed using WinNonLin software (Scientific Consulting Inc.). Eleven patients were studied, but data for one patient were discarded due to insufficient sampling. The median age was 4.3 yr (range 3 to 6), with a median weight of 18.9 kg (range 14.9 to 22.2). There were seven boys and three girls. Mean Cmax was 67.8 ng x mL(-1) (SD 41.9), with Tmax at two minutes. The calculated half-life was 122 min (SD 99). The mean plasma concentrations of flumazenil attained were similar to those reported after intravenous administration, and may be sufficient to antagonize the side-effects of benzodiazepines. This route of administration may be useful when the intravenous route is not readily available.

Endotracheal flumazenil: A new route of administration for benzodiazepine antagonism

The purpose of this study was to determine if flumazenil is absorbed from broncho-pulmonary tissue after intratracheal administration and whether therapeutically significant plasma concentrations can be obtained. Six elective surgical patients received a dose of 1.0 mg flumazenil in 10 mL saline intratracheally during general anesthesia. Blood samples were drawn for 6 hours after administration and plasma concentrations were determined by gas chromatography-mass spectrometry (GC-MS). An average peak plasma flumazenil concentration of 65.9 ± 43.1 ng/mL was attained within 1 minute after administration. No patients reported chest discomfort or dyspnea upon awakening and there were no other side effects noted. Administration of flumazenil via an endotracheal tube results in rapid attainment of therapeutic blood levels.

Determination of flumazenil in plasma by gas chromatography-negative ion chemical ionization mass spectrometry.

A gas chromatographic-negative ion chemical ionization mass spectrometric (GC-NCI-MS) method for the determination of flumazenil in plasma is described. The GC of flumazenil (M(r) 303) is considered to be difficult as it is readily adsorbed in the GC column. Therefore, preconditioning the GC column with reconstituted extract from plasma and Silyl-8 was required to cover the active sites on the column. Monitoring the maximum mass peak (m/z 275) of the flumazenil resulted in a tenfold enhancement of sensitivity and signal-to-noise ratio (concentration = 1 ng/ml). Isotopically labeled flumazenil-d3 (M(r) 306, m/z 278) was used as the internal standard. The detection limit for flumazenil was found to be 0.1 ng/ml with an injection volume of 2 microliters. The signal-to-noise ratio was about 10. The routine quantification limit was set at 2 ng/ml for dog plasma and 1 ng/ml for human plasma. The sample volumes in both instances were 1 ml.

The Effects of Large-Dose Flumazenil on Midazolam-Induced Ventilatory Depression

Flumazenil, a benzodiazepine antagonist, clearly reverses midazolam-induced sedation; reversal of ventilatory depression has not been as well demonstrated. Thirty-two subjects completed this randomized, double-blind, placebo-controlled study investigating the dose-response relationship and duration of flumazenil's effects on ventilatory depression and hypnosis induced by a continuous midazolam infusion. A computer-controlled infusion of midazolam was used to titrate the predicted midazolam plasma concentration to a level at which subjects were unresponsive to verbal commands and then to maintain that concentration. Measurements of ventilation and hypnosis were repeated at predetermined intervals: before midazolam administration, before test drug (flumazenil [1, 3, or 10 mg] or placebo), and 5, 30, 60, 120, and 180 min after test drug administration. Ventilation and tidal volume were measured during an isocapnic hyperoxia clamp at a PETCO2 of 46 mm Hg (VE46 and VT46, respectively). A pseudo-rebreathing technique was used to measure the hypercapnic ventilatory response (HCVR) slope and ventilation intercept at a PETCO2 of 58 mm Hg (VE58). Midazolam reduced VE46, VT46, and VE58, as well as hypnosis scores, in all test drug groups. The reduction in HCVR slope, however, was significant only when all 32 subjects were considered in aggregate. All three doses of flumazenil reversed hypnosis and also reversed the reduction in VE46 and VT46 within 5 min. The reduction in VE58, however, was reversed less consistently. Flumazenil's effect on VE46 and VT46 lasted at least 30 min after 1 mg and at least 60 min after 3 mg, paralleling the effect of these doses on hypnosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Theophylline inhibits the elimination of flumazenil in rabbits

The effect of theophylline on the elimination of flumazenil during constant infusion of l mg/kg per h over 240 min was investigated in 10 rabbits. Theophylline was introduced as a single intravenous (i.v.) dose of 5 mg/kg (5 rabbits) and 15 mg/kg (5 rabbits), 100 min postinfusion. The addition of theophylline was associated with a new steady state which is approx. 32 and 46% higher for the 5 and 15mg/kg doses, respectively. The elimination half-life of the drug was also increased from 18 and 12 min to about 28 and 42 min, respectively, for the two doses. Despite the relatively wide margin of safety for flumazenil the changes observed in plasma concentration during concomitant administration of theophylline may be clinically important. The pharmacokinetic parameters obtained prior to theophylline injection in the 10 rabbits were 406 mg/ml and 15 min for the steady state and elimination half-life, respectively. The variations between subjects in these parameters were as much as 2-fold. A simple and sensitive HPLC procedure to measure flumazenil in plasma is also described.

Plasma concentrations of flumazenil during liver transplantation.

Six patients were given flumazenil (1 mg) during the anhepatic period of liver transplantation. We found higher mean plasma flumazenil concentrations during the anhepatic period than occurred in patients with hepatic cirrhosis. It was only possible to measure plasma flumazenil concentrations in four patients after revascularisation of the donor liver. Of these, elimination half-lives could be calculated in only three patients (normal in one and prolonged in two). In these two patients the elimination half-lives were prolonged to the same extent as in patients with fulminant hepatic failure. The changes during the anhepatic period occur because the liver contributes to the central volume of distribution. After revascularisation of the donor organ the prolonged half-lives indicate that transplanted livers may not recover normal metabolic functions immediately.

Flumazenil

Flumazenil is a specific benzodiazepine antagonist which is indicated when the central effects of a benzodiazepine need to be attenuated or terminated. Following intravenous administration of up to 1 mg, flumazenil effectively reverses sedation and improves psychomotor performance following administration of short and longer acting benzodiazepines used for sedation, or general anaesthesia supplemented with benzodiazepines. The duration of action is short at generally 30 to 60 minutes and supplemental doses of flumazenil may be needed to maintain the desired level of consciousness in some patients. After poisoning with high dosages of benzodiazepines alone or combined with other drugs, the initial single dose of flumazenil will require supplementing with repeated low intravenous doses or an infusion to maintain wakefulness. In such patients, flumazenil also facilitates differential diagnosis and reduces the necessity for interventions. Flumazenil thus enhances recovery and allows more rapid discharge of patients sedated with benzodiazepines for diagnostic procedures and facilitates management of patients during the initial recovery period following general anaesthesia supplemented with benzodiazepines, but does not preclude normal monitoring during the recovery period. Flumazenil is clearly very useful in treating drug poisoning when benzodiazepines are a major component. By virtue of its specific benzodiazepine antagonist effects, flumazenil provides an innovative and well tolerated approach in clinical situations requiring rapid reversal of benzodiazepine-induced central nervous system depressant effects.

Plasma Determination of Flumazenil, a Benzodiazepine Antagonist, by Immunotoxicology and by Capillary Gas Chromatography/Mass Spectrometry

The method presented describes a sensitive and specific quantitative assay for the determination of flumazenil in human plasma. Flumazenil and an internal standard, midazolam, are isolated by a basic extraction. The final extract is separated on a 25-m BP-1 capillary column and drugs are detected by selected ion monitoring at m/z 229 and m/z 310 for flumazenil and the internal standard, respectively. The minimum detectable quantity is 1.0 ng/mL, for flumazenil in plasma. Coefficients of variation for within-run data were less than 6%.

Pharmacokinetic-Pharmacodynamic Modelling of the Interaction between Flumazenil and Midazolam in Volunteers by Aperiodic EEG Analysis

The CNS effects resulting from the combined administration of midazolam and flumazenil were studied in 8 healthy volunteers to develop a model of the pharmacokinetic-pharmacodynamic interaction. Electroencephalograms (EEG) were recorded between Fp1-M1 and Fp2-M2. The EEG parameter total number of waves between 12 and 30 Hz (TNW12-30) derived by aperiodic analysis was used to quantify the effect. Following a 15 min baseline EEG recording, infusion of placebo or flumazenil was started. Infusion regimens for flumazenil were designed so that 'steady-state' concentrations of 10 and 20 micrograms/L were obtained. Doses of midazolam 15, 30 and 60 mg over 5 min were given 30 min after the start of placebo infusion (session A) or flumazenil infusion to 10 micrograms/L (session B) or 20 micrograms/L (session C), respectively. Venous blood samples were taken until 8 h after the start of the flumazenil or placebo infusion. A sigmoid maximum effect (Emax) model was used to characterise the relationship between the plasma concentration of midazolam which is in equilibrium with the effect compartment concentration (Cem) [Cem/Kp] and TNW12-30. Within 2 to 5 min of starting the midazolam infusion all subjects fell asleep, with loss of eyelid reflex. They awoke between 25 and 82 min later in all 3 sessions. The mean (+/- SD) plasma drug concentrations of midazolam corresponding to half the maximum increase in TNW12-30 (EC50) were 276 +/- 64, 624 +/- 187 and 1086 +/- 379 micrograms/L in sessions A, B and C, respectively. The half-lives reflecting equilibration between plasma concentration and effect (t1/2ke0), estimated by a nonparametric method, were 2.2 +/- 1.2, 3.3 +/- 3.3 and 2.9 +/- 1.2 min for the 3 different sessions. Emax and N were not affected by flumazenil. In each subject the plot of the average measured steady-state plasma flumazenil concentration versus the EC50 of midazolam showed a linear relationship. The plasma concentration of flumazenil that doubled the EC50 of midazolam (Cf,2) was 6.5 +/- 1.0 micrograms/L. The observed interaction is consistent with the competitive nature of the antagonism of midazolam by flumazenil.

Plasma pharmacokinetics and metabolism of the benzodiazepine antagonist [11C] Ro 15-1788 (flumazenil) in baboon and human during positron emission tomography studies.

Flumazenil is a specific antagonist of the central benzodiazepine receptor (CBZR). Labelled with 11C, this compound is the reference radioligand for positron emission tomography (PET) study of the CBZR in humans and primates. The time-course of [11C]-flumazenil radioactivity and its main acid metabolite [11C] Ro 15-3890 were reconstructed from the time-course of total radioactivity in plasma after administration with high or low SRA in primates and humans, applying an extraction procedure validated by TLC. The measured pharmacokinetics of [11C]-flumazenil (T1/2 beta = 45.1 +/- 12.3 min, T1/2 alpha = 1.5 +/- 1.5 min; K = 0.14 +/- 0.14 min-1; Vd area = 44.0 +/- 17.0 l; Clp = 40.0 +/- 8.5 l/h) exhibited a very rapid distribution phase followed by fast elimination, with a large volume of distribution; these results were confirmed by HPLC determinations and agree with previously published data on unlabelled flumazenil. Pharmacokinetics of [11C] Ro 15-3890 acid metabolite show that high drug concentrations in the blood are promptly achieved (kf = 0.13 +/- 0.004 min-1), with a very rapid elimination half-life (T1/2m = 4.47 +/- 1.31 min) comparable to that of [11C]-flumazenil. The percentage metabolization of parent compound to the acid [11C] Ro 15-3890 was constant from the 15th minute and was significantly higher in man compared to the monkey. This percentage was increased by prior eating. The other putative metabolites, i.e. labelled [11C] Ro 15-4965 and unlabelled Ro 15-5528, were never observed at detectable concentrations with TLC and HPLC in rabbit, baboon and human blood samples. This pharmacokinetic study of plasma flumazenil may be useful to implement a dynamic method of CBZR quantification using PET and for analysis of pharmacokinetics in brain tissue.