Alternative to LRRK2-IN-1 for Pharmacological Studies of Parkinson's Disease.

BACKGROUND/AIMS: LRRK2 (leucine-rich repeat protein kinase 2) is one of the most commonly accepted genes associated with Parkinson's disease (PD). The overexpression of disease-associated mutations in LRRK2 is toxic to the cells, while reduction or elimination of LRRK2 expression promotes cell health and growth. Thus, the identification of an LRRK2 inhibitor with good physiochemical and pharmacokinetic properties is of great interest for the treatment of PD. METHODS: In this study, we have investigated LRRK2 compounds, LRRK2-IN-1 and Compound 1, in vitro and in vivo to determine how suitable they are as a selective LRRK2 tool compound. RESULTS: We report that Compound 1, patented by GSK, is a potent and selective LRRK2 inhibitor with good blood-brain barrier permeability as reflected by its high brain to plasma ratio in rats. In addition, Compound 1 can significantly promote neurite outgrowth in a primary cortical culture, indicating an optimistic cellular function of this compound in a biological system. In contrast, LRRK2-IN-1 is a less selective LRRK2 inhibitor and has low brain penetration. Furthermore, LRRK2-IN-1 is cyto- and genotoxic, while Compound 1 does not exhibit any toxicity. CONCLUSIONS: These results suggest that Compound 1 may be a superior tool compound than LRRK2-IN-1 to advance future pharmacological research on LRRK2.

Utilizing Internal Standard Responses to Assess Risk on Reporting Bioanalytical Results from Hemolyzed Samples.

Bioanalytical analysis of toxicokinetic and pharmacokinetic samples is an integral part of small molecule drugs development and liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the technique of choice. One important consideration is the matrix effect, in which ionization of the analytes of interest is affected by the presence of co-eluting interfering components present in the sample matrix. Hemolysis, which results in additional endogenous components being released from the lysed red blood cells, may cause additional matrix interferences. The effects of the degree of hemolysis on the accuracy and precision of the method and the reported sample concentrations from hemolyzed study samples have drawn increasing attention in recent years, especially in cases where the sample concentrations are critical for pharmacokinetic calculation. Currently, there is no established procedure to objectively assess the risk of reporting potentially inaccurate bioanalytical results from hemolyzed study samples. In this work, we evaluated the effect of different degrees of hemolysis on the internal standard peak area, accuracy, and precision of the analyses of BMS-906024 and its metabolite, BMS-911557, in human plasma by LC-MS/MS. In addition, we proposed the strategy of using the peak area of the stable isotope-labeled internal standard (SIL-IS) from the LC-MS/MS measurement as the surrogate marker for risk assessment. Samples with peak areas outside of the pre-defined acceptance criteria, e.g., less than 50% or more than 150% of the average IS response in study samples, plasma standards, and QC samples when SIL-IS is used, are flagged out for further investigation.

Effect of netazepide, a gastrin/CCK2 receptor antagonist, on gastric acid secretion and rabeprazole-induced hypergastrinaemia in healthy subjects.

AIMS: To compare gastric acid suppression by netazepide, a gastrin/CCK2 receptor antagonist, with that by a proton pump inhibitor (PPI), and to determine if netazepide can prevent the trophic effects of PPI-induced hypergastrinaemia. METHODS: Thirty healthy subjects completed a double-blind, randomized, parallel group trial of oral netazepide and rabeprazole, alone and combined, once daily for 6 weeks. Primary end points were: basal and pentagastrin-stimulated gastric acid and 24 h circulating gastrin and chromogranin A (CgA) at baseline, start and end of treatment, gastric biopsies at baseline and end of treatment and basal and pentagastrin-stimulated gastric acid and dyspepsia questionnaire after treatment withdrawal. RESULTS: All treatments similarly inhibited pentagastrin-stimulated gastric acid secretion. All treatments increased serum gastrin, but the combination and rabeprazole did so more than netazepide alone. The combination also reduced basal acid secretion. Rabeprazole increased plasma CgA, whereas netazepide and the combination reduced it. None of the biopsies showed enterochromaffin-like (ECL) cell hyperplasia. Withdrawal of treatments led neither to rebound hyperacidity nor dyspepsia. CONCLUSIONS: Netazepide suppressed pentagastrin-stimulated gastric acid secretion as effectively as did rabeprazole. The reduction in basal acid secretion and greater increase in serum gastrin by the combination is consistent with more effective acid suppression. Despite our failure to show rabeprazole-induced ECL cell hyperplasia and rebound hyperacidity, the increase in plasma CgA after rabeprazole is consistent with a trophic effect on ECL cells, which netazepide prevented. Thus, netazepide is a potential treatment for the trophic effects of hypergastrinaemia and, with or without a PPI, is a potential treatment for acid-related conditions.

A phase Ib/IIa trial to evaluate the CCK2 receptor antagonist Z-360 in combination with gemcitabine in patients with advanced pancreatic cancer.

AIM: To evaluate the combination of the gastrin antagonist Z-360 and gemcitabine for advanced pancreatic cancer. METHODS: Previously untreated patients with PC were randomly allocated to Z-360 120 mg, 240 mg or placebo. Z-360/placebo was given on day -3 and gemcitabine 1000 mg/m(2) commenced on day 1 followed by Z-360 on day 2. Thereafter Z-360/placebo was given twice daily concurrently with standard dose of gemcitabine. Pharmacokinetics for both drugs was measured alone and in combination. Toxicity, response and quality of life were also recorded. RESULTS: Thirty-three patients with a median age of 62 years were randomised of which six had locally advanced disease and 26 had metastatic disease. Analysis of the area under the plasma concentration versus time curve (AUC), the maximum observed concentration (Cmax(obs)) and the time of the maximum observed concentration (Tmax(obs)) for Z-360, gemcitabine and 2,2-difluorodeoxyuridine (dFdU), could not exclude an effect on the systemic exposure to Z-360, gemcitabine and dFdU when co-administration of Z-360 and gemcitabine was compared with single agent administration. The most commonly reported adverse events were nausea, abdominal pain, vomiting and fatigue. At the end of the study, 62.5%, 25% and 60% had stable disease in the 120 mg, 240 mg and placebo group, respectively. A higher proportion of patients in Z-360 groups reported improvement in pain. CONCLUSIONS: Z-360 is safe and well tolerated when combined with gemcitabine. A Phase III trial is needed to determine whether the combination of Z-360 and gemcitabine is superior to gemcitabine alone in advanced PC.

Simplified kinetic calibration of solid-phase microextraction for in vivo pharmacokinetics.

Solid-phase microextraction (SPME) has been demonstrated to be useful for in vivo sampling in pharmacokinetic studies. In this study, a single time-point kinetic calibration for in vivo dynamic monitoring was developed by simplification of the laborious multiple time-point kinetic calibration, based on the independent desorption kinetics of the preloaded standards from SPME fibers with the changing analyte concentrations. The theoretical foundation and practical application conditions, such as the replicate numbers, the optimal time-point for desorption, and the sampling time, were systematically investigated. Furthermore, the feasibility of using regular standards rather than deuterated ones for the kinetic calibration was justified by comparing to the data obtained using the deuterated standards. All the methods were verified by in vitro and in vivo experiments. The results from in vivo SPME were validated by the blood drawing and chemical assay. These simplified calibration methods improved the quantitative applications of SPME for dynamic monitoring and in vivo sampling, enhance the multiplexing capability and automatic potentials for high throughput analysis, and decrease expenses on reagents and instruments.

Determination of chemically reduced pyrrolobenzodiazepine SJG-136 in human plasma by HPLC-MS/MS: application to an anticancer phase I dose escalation study.

SJG-136 1,1'-[[(propane-1,3-diyl)dioxy]bis[(11aS)-7-methoxy-2-methylidene-1,2,3,11a-tetrahydro-5H-pyr- rolo[2,1-c][1,4]benzodiazepin-5-one]] (NSC 694501), is a bifunctional pyrrolobenzodiazepine (PBD) dimer that forms selective, irreversible, interstrand DNA cross-links via exocyclic N2 atoms of two guanine bases, with a preference for 5'PuGATCPy binding sites. SJG-136 is highly cytotoxic in human tumor cells in vitro and in human tumor xenograft models in vivo at subnanomolar concentrations and is currently in anticancer phase I clinical trials in the United Kingdom and United States. To support correlative pharmacokinetics studies, a highly sensitive HPLC-MS/MS assay was developed and validated for the reliable quantitation of SJG-136 in human plasma, using the structurally similar PBD dimer DSB-120 as an internal standard. Chemical reduction of SJG-136 to its corresponding amine (SJG-136-H(4), [M + H](+)m/z 561) improved HPLC peak resolution and sensitivity by minimizing complications that arose from the reactivity of the labile imine moieties. Plasma samples were processed by protein precipitation and centrifugal membrane dialysis; components were separated by HPLC using an Agilent Rapid Resolution HT 1.8 mm (2.1 mm x 50 mm) analytical column. The total analysis time from injection to injection was 11 min. Electrospray MS/MS detection of SJG-136-H(4) was based on the selected reaction monitoring (SRM) transition [M + H](+)m/z 561 --> 301. The analytical response ratio was linearly proportional to the plasma concentration of SJG-136 over the nominal concentration range of 25 pg/ml to 250 ng/ml, with a coefficient of determination of r > or = 0.999. The intrarun absolute %RE was < or =19.6, 14.2, and 14.0% at 0.056, 2.83, and 56.3 ng/ml, respectively. The corresponding %RSD was < or =14.9%, 9.01, and 4.59%. The interday %RSD was < or =2.72, 3.46, and 5.20%. The lower and upper limits of quantitation were 0.056 and 56 ng/ml, respectively; recovery of SJG-136 from plasma was > or = 62% across the validated concentration range. The sensitivity of the validated assay was sufficient to detect SJG-136 in human subjects for up to 6 h after intravenous administration of 6 microg/m(2), the starting dose of an NCI-sponsored dose escalation study.

Preliminary pharmacokinetic and bioanalytical studies of SJG-136 (NSC 694501), a sequence-selective pyrrolobenzodiazepine dimer DNA-cross-linking agent.

SJG-136 is a synthetic pyrrolobenzodiazepine (PBD) dimer in which two DNA-alkylating subunits are linked through an inert propanedioxy tether. Biophysical and biochemical studies of SJG-136 have shown a remarkable affinity for DNA and potent cytotoxicity in vitro. On this basis, together with its unique sequence selectivity and interstrand DNA cross-linking activity, SJG-136 has been selected for clinical trials. This study examines the pharmacological characteristics of SJG-136 and provides the first report of pharmacokinetic properties for this agent. A sensitive, selective and reproducible reversed-phase gradient LC/MS assay has been developed for detection and analysis, where a molecular ion ( m / z 557.2) is detectable for the SJG-136 parent imine. Fluorescence detection (260 nm excitation, 420 nm emission) gives a limit of sensitivity of 5 nM (2.5 ng ml(-1)) for analysis of SJG-136 in mouse plasma. Extraction efficiencies from plasma were >65% across a range of concentrations (5-1000 nM). Following administration to mice at the MTD (i.p., 0.2 mg kg(-1)), high peak plasma concentrations of SJG-136 were seen ( C (max) = 336 nM) at 30 min after dosing. A calculated terminal t (1/2) of 0.98 h and AUC of 0.34 microM.h resulted in a clearance rate of 17.7 ml min(-1) kg(-1). The PBD dimer binds only moderately to proteins (65-75%), and in vitro cytotoxicity studies confirmed IC(50) values of 4-30 nM with a panel of human cell lines. This finding demonstrates that plasma concentrations achieved in the mouse are substantially higher than those required to elicit an anti tumour response in vitro. This report forms an important phase in the pre-clinical characterization of the compound.

Validation of a liquid chromatographic-tandem mass spectrometric method for the measurement of (R)-1-[2,3-dihydro-2-oxo-1-pivaloylmethyl-5-(2-pyridyl)-1H-1,4-benzodiazepin-3-yl]-3-(3-methylaminophenyl)urea (YF476) in human plasma.

A sensitive and specific liquid chromatographic-tandem mass spectrometric (LC-MS-MS) method has been validated for the measurement of YF476 in human plasma. The method involves a simple liquid-liquid extraction procedure, chromatography of the extracts on a C(18) column, atmospheric pressure chemical ionisation and detection in the multiple reaction monitoring mode. The calibration line was linear over the concentration range 0.1 ng/ml (the limit of quantification) to 25.0 ng/ml. Intra- and inter-batch precision was <14% and intra- and inter-batch accuracy was <11% over the entire calibration range. The bioanalytical method is robust and has been used for the analysis of many samples from human subjects involved in early clinical studies (Phase I).

Carbon-11 labelled cholecystokininB antagonists: radiosynthesis and evaluation in rats.

Three cholecystokinin type B (CCKB) receptor antagonists were labelled with 11C and evaluated ex vivo in rat biodistribution studies. The CCKB antagonists were YF 476 and two other compounds of the basic 3-ureido-1,4-benzodiazepine class. Following tail-vein administration of [11C]-YF 476 exceedingly low levels of radioactivity were found in all brain regions from 5 to 60 min post-injection. Similar results were obtained using the other two 11C-labelled CCKB antagonists. In light of the very poor brain penetration of these compounds, reports on the central nervous system activity of this class of CCKB antagonists should be viewed with caution.

Long-lasting cholecystokinin(2) receptor blockade after a single subcutaneous injection of YF476 or YM022.

Histamine-forming ECL cells in the rat stomach operate under the control of gastrin. They represent a convenient target for studying cholecystokinin-B/gastrin (CCK(2)) receptor antagonists in vivo. We examined the effectiveness and duration of action of two CCK(2) antagonists, YM022 and YF476, with respect to their effect on ECL-cell histidine decarboxylase (HDC) activity in the rat. Oral administration of subcutaneous deposition of YF476 or YM022 reduced the HDC activity. The maximum/near-maximum dose for both drugs and for both modes of administration was 300 micromol kg(-1) (effects measured 24 h after dose). At this dose and time the serum concentration of YF476 was 20 - 40 nmol l(-1). The dose 300 micromol kg(-1) was used in all subsequent studies. A single subcutaneous injection of YF476 inhibited the HDC activity for 8 weeks. The circulating concentration of YF476 remained high for the same period of time (>/=15 nmol l(-1)). Subcutaneous YM022 suppressed the HDC activity for 4 weeks. A single oral dose of YF476 or YM022 inhibited the HDC activity for 2 - 3 days. Chronic gastric fistula rats were used to study the effect of subcutaneous YF476 on gastrin-stimulated acid secretion. A single injection of YF476 prevented gastrin from causing an acid response for at least 4 weeks (the longest time studied). We conclude that a single subcutaneous injection of 300 micromol kg(-1) YF476 causes blockade of CCK(2) receptors in the stomach of the rat for 8 weeks thus providing a convenient method for studies of the consequences of long-term CCK(2) receptor inhibition.

The development of a sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A receptor antagonist, in human plasma.

A sensitive and specific enzyme immunoassay for FK480, a novel cholecystokinin type-A (CCK-A) receptor antagonist, was developed to study the pharmacokinetics of the drug at low-dose administration using a specific monoclonal antibody. The high performance liquid chromatography (HPLC) method had been used for studying toxicokinetics, but its determination limit (2.5 ng ml-1) was too high for use in clinical studies. Subsequently we developed an enzyme immunoassay (EIA) using rabbit anti-FK480 serum (polyclonal antibody). It had higher sensitivity (0.1 ng ml-1) when 0.5 ml of plasma was used but its specificity was low because of the cross-reactivity of the metabolites of FK480. Therefore we produced several monoclonal antibodies for FK480 by cell fusion, and selected the antibody which was least cross-reactive for the isolated metabolites of FK480. Finally we developed a sensitive and specific EIA using this monoclonal antibody. The lower limit of quantification of this method was 0.2 ng ml-1 when 0.2 ml of human plasma was used. The coefficient of variation over the calibration range (0.2-10 ng ml-1) was less than 15%. We used this method for clinical studies, and it showed a good correlation to the HPLC method when plasma concentration was 2.5 ng ml-1 or more.

Carbamazepine intoxication with negative myoclonus after the addition of clobazam.

We report a case a carbamazepine (CBZ) intoxication with negative myoclonus that occurred 4 weeks after clobazam (CLB) had been added to a stable regimen of CBZ and topiramate (TPM). Both CBZ and CBZ-epoxide (CBZ-E) blood levels were elevated, and the symptoms resolved quickly when CBZ dosage was reduced and CLB discontinued. CLB was reintroduced a year later with the patient's consent, and the time course of the interaction was studied: CBZ and CBZ-E levels increased slowly over 12 days. The interaction is thus probably related to the progressive increase in Nor-CLB.

Micro-extraction of chlordiazepoxide and its primary metabolites, desmethylchlordiazepoxide and demoxepam, from plasma and their measurement by liquid chromatography.

We have developed a micro-extraction procedure for the analysis of chlordiazepoxide and its two unique metabolites, demoxepam and desmethylchlordiazepoxide, in plasma, using liquid chromatography. The method is both reliable and sensitive for the quantitation of low plasma concentrations of these three compounds. The extraction procedure allows rapid sample processing, which, together with the small sample volume (100 microL), makes it ideal for routine sample handling. The limit of detection for the three compounds ranged from 0.075 to 0.125 mg/L and recovery of the three different benzodiazepines ranged from 87 to 94%. Within- and between-assay coefficients of variation ranged from 3.1-4.5% and from 4.7 to 7.6%, respectively.

[Death in the bathtub--rectal drug administration]. / Tod in der Badewanne--Rektale Medikamentenbeibringung.

A young nurse was found dead in a bathtub. An autopsy revealed the following results: pulmonary emphysema, severe edema of both lungs, transudation in both pleural cavities. Conspicuous were skin sticks of a white wax material. In chemical-toxicological analysis diazepam, tetrazepam and phenobarbital were detected in this material. After anal-rectal and additionally oral ingestion the following blood concentrations were determined: BAC 0.03/1000; diazepam 500 ng/ml; nordiazepam 65 ng/ml; tetrazepam 180 ng/ml; phenobarbital 9.4 mg/l. In connection with this drug effects an acute, multifocal, suppurating bronchopneumonia in both lungs was revealed as the cause of death.

Interactions of clobazam with conventional antiepileptics in children.

Clobazam is a 1,5-benzodiazepine effective in antiepileptic therapy of children and adults. Presently it is mainly used as adjuvant therapy for intractable seizures. Our objective was to evaluate the effect of clobazam on the apparent clearance of other antiepileptic drugs at steady state, and to determine the factors that determine the plasma levels of clobazam and its active metabolite N-desmethylclobazam. Patients were 74 children with intractable seizures who received treatment with clobazam at our institution as part of the Canadian Cooperative Clobazam Study Group during the years 1987 to 1991. Serum concentrations of clobazam, N-desmethylclobazam, and of concomitant antiepileptic drugs were monitored and prospectively collected. The effect of clobazam treatment on the apparent clearance steady state of the other antiepileptic drugs was determined by statistical comparison of the clearances of each drug before and after initiation of clobazam treatment using Wilcoxon's signed rank test. The effects of dosage, age, and concomitant antiepileptic therapy on the levels of clobazam and N-desmethylclobazam was assessed by multivariate analysis. Response to treatment and incidence of adverse effects were evaluated for each conventional antiepileptic drug to possibly identify favorable or unfavorable combinations with clobazam. Whereas the clearances of most conventional antiepileptics are not affected by cotherapy with clobazam, the apparent clearances of valproic acid and primidone are significantly reduced in the presence of clobazam. Serum concentrations of clobazam increased with dosage and age, and decreased with phenobarbital cotherapy. Serum concentrations of N-desmethylclobazam significantly correlated with clobazam serum levels, age, or clobazam dosage and were significantly increased by cotherapy with phenytoin or carbamazepine. None of the concomitantly used drugs were associated with increased or decreased rate of seizure control. Twelve patients experienced mild adverse drug effects that were not associated with particular cotherapy, clobazam dose, or plasma concentrations. When clobazam is added to a therapy regimen that includes valproic acid, the patient should be closely followed for possible adverse drug reactions caused by elevated valproic acid serum concentrations, and monitoring of valproate serum levels should be considered. When clobazam doses are gradually increased to achieve an optimal clinical effect, the interactions with phenobarbital, carbamazepine, and phenytoin do not necessitate therapeutic drug monitoring of clobazam or N-desmethylclobazam, because there is a large therapeutic window and a poor correlation between plasma concentrations and therapeutic efficacy.

Effects of high-fat diet and cholecystokinin receptor blockade on promotion of pancreatic ductal cell tumors in the hamster.

The mechanism by which high-fat diets potentiate pancreatic cancer is not known, but pancreaticotrophic hormones such as cholecystokinin (CCK) may be involved. The effect of CCK receptor blockade on carcinogenesis during the entire promotion period was investigated in Syrian Golden hamsters fed a high- or low-fat diet and treated with N-nitrosobis(2-oxopropyl)amine (3 x 10 mg/kg at weekly intervals). One-half of the hamsters fed a high-fat diet received the CCK-A receptor antagonist devazepide (25 nmol/kg/hr) for the duration of the experiment. At 39 weeks the incidence of pancreatic malignancies was significantly higher in hamsters fed the high-fat diet than in those fed the low-fat diet (p < 0.05). Tumor incidence was not changed by CCK receptor blockade. Potentiation of pancreatic cancer by a high-fat diet in hamsters does not appear to be influenced by endogenous CCK during the tumor promotion period.

Analysis of clobazam and its active metabolite norclobazam in plasma and serum using HPLC/DAD.

This report describes a simple reversed-phase high-performance liquid chromatographic (HPLC) method with automated solid-phase extraction (SPE) for analysing clobazam and norclobazam concentrations in human serum or plasma. For the HPLC analysis the samples and standards are prepared with an ASPEC automatic sample preparer using 100-mg Bond-Elut C-18 solid-phase extraction columns. The HPLC method is an isocratic method with a mobile phase of acetonitrile:methanol:10 mmol l-1 dipotassium hydrogen phosphate, pH 3.7 (30:2:100), at a flow rate of 1.5 ml min-1. The benzodiazepines are detected with a diode array detector (DAD) at 240 nm and the peak purity analyses are performed at 210-365 nm. The recovery is over 97% for both analytes, and it is independent of the drug concentration. The intra-assay CVs vary between 0.7 and 2.2% and inter-assay CVs between 3.8 and 4.6% at therapeutic drug concentrations. The detection limit is 15 nmol l-1. The assay is linear from 30 to 20,000 nmol l-1 (clobazam) and from 170 to 105,000 nmol l-1 (norclobazam). This method leads to a very good separation of norclobazam from carbamazepine and phenytoin. None of the anti-epileptic or antidepressant drugs tested interfere with the assay.

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.

Simultaneous determination of twelve benzodiazepines in human serum using a new reversed-phase chromatographic column on a 2-microns porous microspherical silica gel.

A high-performance liquid chromatographic method has been developed for the simultaneous analysis of twelve frequently used benzodiazepines (BZPs) (bromazepam, clonazepam, chlordiazepoxide, estazolam, etizolam, flutazoram, haloxazolam, lorazepam, nitrazepam, oxazolam, triazolam and diazepam, internal standard) by using commercially available 2 or 5 microns particle size reversed-phase columns and a microflow cell-equipped ultraviolet detector. The separation was achieved using a C18 reversed-phase column (condition 1: 100 x 4.6 mm I.D., particle size 2 microns, TSK gel Super-ODS: conditon 2: 100 x 4.6 mm I.D., particle size 5 microns, Hypersil ODS-C18). The mobile phase was composed of methanol-5 mM NaH2PO4 (pH 6) (45:55, v/v), and the flow-rate was 0.65 ml/min (condition 1 and 2). The absorbance of the eluent was monitored at 254 nm. Retention times under condition 1 were shorter than those of condition 2. When the twelve benzodiazepines were determined, sensitivity and limits of quantification were about four to ten times better under condition 1 than under condition 2. The rate of recovery and linearity in condition 1 were approximately the same as those in condition 2. These results show that a new ODS filler with a particle size of 2 microns was more sensitive, provided better separation and was more rapid than that with conventional ODS filler.

Pharmacokinetic and pharmacodynamic interactions of bretazenil and diazepam with alcohol.

1. Interaction between alcohol and bretazenil (a benzodiazepine partial agonist in animals) was studied with diazepam as a comparator in a randomized, double-blind, placebo controlled six-way cross over experiment in 12 healthy volunteers, aged 19-26 years. 2. Bretazenil (0.5 mg), diazepam (10 mg) and matching placebos were given as single oral doses after intravenous infusion of alcohol to a steady target-blood concentration of 0.5 g l-1 or a control infusion of 5% w/v glucose at 1 week intervals. 3. CNS effects were evaluated between 0 and 3.5 h after drug administration by smooth pursuit and saccadic eye movements, adaptive tracking, body sway, digit symbol substitution test and visual analogue scales. 4. Compared with placebo all treatments caused significant decrements in performance. Overall, the following sequence was found for the magnitude of treatment effects: bretazenil+alcohol > diazepam+alcohol > or = bretazenil > diazepam > alcohol > placebo. 5. There were no consistent indications for synergistic, supra-additive pharmacodynamic interactions between alcohol and bretazenil or diazepam. 6. Bretazenil with or without alcohol, and diazepam+alcohol had marked effects. Because subjects were often too sedated to perform the adaptive tracking test and the eye movement tests adequately, ceiling effects may have affected the outcome of these tests. 7. No significant pharmacokinetic interactions were found. 8. Contrary to the results in animals, there were no indications for a dissociation of the sedative and anxiolytic effects of bretazenil in man.