Jasper Dingemanse

Determination of the threshold for convulsions by direct cortical stimulation

In this study we investigated whether determination of the convulsion threshold by electrical stimulation of the cortex could be used as a simple test for measuring anticonvulsant drug activity in unrestrained, unanaesthetized rats. Pulse trains delivered to electrodes implanted in the frontoparietal cortex elicited convulsions, similar to those seen in the classical electroshock tests. The threshold could be determined rapidly with pulse trains which increased in strength in a ramp-shaped fashion (bipolar pulses of 2 msec, 50 pulses/sec, increment 1.3 microA/pulse). The threshold was defined as the current needed to elicit forelimb clonus. Upon repeated stimulation the threshold declined from a value of about 600 microA to about 350 microA in 20 sessions. Thereafter, continued testing did not result in considerable changes in threshold. After stabilization, the convulsion threshold could be determined repeatedly with intervals as short as 5 min. Following i.p. injection of 5 mg/kg of diazepam an elevation of the threshold of 30% was observed 0.5 h after injection. After 5 daily injections, evidence for the development of complete tolerance was obtained. After i.v. injection of 8 mg/kg oxazepam, the threshold increase reached a peak level of 75% after 20 min. The changes in threshold followed arterial blood concentration of oxazepam, which was maximally 4.8 micrograms/ml immediately after injection. The threshold returned to baseline in approximately 6 h. The results of the present study show that with our procedure anticonvulsant drug activity can be accurately, rapidly and repeatedly determined in individual animals, both in acute and chronic experiments.

Pharmacokinetic-pharmacodynamic modeling of midazolam effects on the human central nervous system

The effect of midazolam on α‐activity of the EEG and latency of the P‐100 of the visual evoked response (VER) was studied in six healthy subjects. Drug concentration was related to effect with the Emax model that was used with either a threshold drug concentration or a sigmoid exponent. An effect compartment was included in the pharmacokinetic‐pharmacodynamic model. Four subjects showed hysteresis, and mean values of half‐lives‐keo ranged from 0.26 to 0.60 hour. Mean values of EC50 ranged from 42.0 to 48.1 ng/ml. Goodness of fit did not differ significantly between the sigmoid Emax model and the threshold Emax model. The sigmoid exponent estimated was 3.7 ± 1.8 (EEG, mean ± SD) and 2.9 ± 1.4 (VER); the threshold concentration was estimated at 15.7 ± 11.1 ng/ml (EEG) and 11.3 ± 7.0 ng/ml (VER). We conclude that the Emax model adequately describes the relationship between midazolam concentration and effect and that the sigmoid exponent can be substituted by a threshold drug concentration, with a comparable fit of the model to the data.

Pharmacokinetic modeling of the anticonvulsant response of oxazepam in rats using the pentylenetetrazol threshold concentration as pharmacodynamic measure

This investigation developed strategies along which the anticonvulsant effect of oxazepam in the rat could be pharmacokinetically modeled. After determination of the pharmacokinetics of oxazepam, which could be described with a two-compartment model (halflives of distribution and elimination 6 and 52 min, respectively), the drug was administered iv to groups of animals to achieve a serum concentration range of 0.1–2.5 mg/L at 10, 45, and 120 min after administration. At these time points pentylenetetrazol (PTZ) was infused slowly until the first myoclonic jerk occurred. The anticonvulsant response, expressed as the elevation of the serum or brain threshold concentration of PTZ, was modeled versus the serum (both total and free) and brain oxazepam concentration, according to the sigmoid Emax model. The total serum and brain oxazepam EC50values are about 0.5 mg/L and 1.1 mg/kg, respectively, and Emax 120 mg/L PTZ. No marked differences in pharmacodynamic parameters between the three time groups were found, which indicates that serum and brain are pharmacokinetically indistinguishable from the effect compartment, that there is no (inter) activity of oxazepam metabolites and absence of development of acute tolerance during the investigated time frame. An interfering role of metabolites was also excluded by a direct radioreceptor assay of oxazepam, yielding very similar results as the specific Chromatographic assay. It is concluded that the concentration-anticonvulsant effect relationship of oxazepam can satisfactorily be described by the sigmoid Emax model, when utilizing the employed experimental strategies.

The influence of dosage time of midazolam on its pharmacokinetics and effects in humans

The influence of dosage time of midazolam on its pharmacokinetics and effects on the central nervous system were investigated in six healthy volunteers, with pharmacokinetic‐pharmacodynamic modeling. Each volunteer received single oral doses of 15 mg midazolam on four separate occasions: 8 AM, 2 PM, 8 PM, and 2 AM. An almost significant circadian variation was found in elimination half‐life, shortest at 2 PM (1.26 ± 0.47 hours, mean ± SD) and longest at 2 AM (1.57 ± 0.44 hours) (p = 0.05). Drug effects measured were α activity of the electroencepalograph and P100 latency of the visual‐evoked response. The maximum drug effect (Emax) model described the concentration‐effect relationship, extended with either a threshold drug concentration or a sigmoidicity parameter. A significant circadian variation was found in baseline α activity: highest at 8 AM (109% ± 19% of the 24‐hour mean) and lowest at 2 AM (80% ± 12%). For α activity the drug concentration at half‐maximum effect of both threshold Emax model and sigmoid Emax model showed lower values at 8 AM and 2 AM and higher values at 2 PM and 8 PM. However, these differences were either not significant (p = 0.10, threshold model) or on the verge of statistical significance (p = 0.05, sigmoid model). No circadian variation was found in the parameters describing the effect on the visual‐evoked response. We conclude that the sensitivity of the central nervous system to midazolam, as reflected in α activity, possibly shows a circadian variation.

Pharmacokinetics and bioavailability of intravenous, oral, and rectal nitrazepam in humans

The pharmacokinetics and bioavailability of nitrazepam following intravenous, oral (tablet), and rectal (solution) administration were studied in seven healthy, young male volunteers. Nitrazepam plasma concentrations were determined by electron-capture GLC; pharmacokinetic evaluations were made by compartmental analysis (NONLIN) and compared with the results obtained by a less stringent modelling of the data. The plasma concentration-time profile was similar for all three routes of administration. Mean kinetic parameters as obtained by compartmental analysis of i.v. nitrazepam were: distribution half-life 17 min; volume of distribution after equilibrium 2.14 liters/kg; total plasma clearance 61.6 ml/min; elimination half-life 29.0 h. The mean protein unbound fraction of nitrazepam in plasma was 12.3% and the clearance of the unbound fraction was 506 ml/min. Absorption of oral nitrazepam started after the elapse of a lag time (mean value 12 min) and occurred as an apparent first-order process in all but one subject, with a mean absorption half-life of 16 min. Distribution and elimination half-lives were comparable with those following i.v. administration. Following rectal administration of the nitrazepam solution, rapid first-order absorption occurred with a mean lag time of 4 min and a mean absorption half-life of 9 min. Peak times (median 18 min) were significantly shorter than following oral administration (median 38 min), but there was little difference in peak concentrations. The distribution half-life was similar to i.v. and oral administration, but the elimination half-lives were longer with a mean value of 33.1 h. Following i.v. administration a good agreement was found between the results obtained by compartmental analysis using NONLIN and those obtained by a less stringent modelling of the data. Following oral and rectal administration, a good agreement between the two procedures was found for the elimination half-life; estimation of bioavailability, however, was higher by compartmental analysis. The mean bioavailability data showed that absorption is complete when nitrazepam is given orally and almost 20% lower when it is given rectally, but considerable interindividual differences were observed.

Phannacokinetic‐pharmacodynamic modelling of the anticonvulsant effect of oxazepam in individual rats

1 The purpose of this investigation was to examine in vivo drug‐concentration anticonvulsant effect relationships of oxazepam in individual rats following administration of a single dose. 2 Whole blood concentration vs time profiles of oxazepam were determined following administration of doses of 4, 8 and 12 mg kg−1. The pharmacokinetics could be described by an open 2‐compartment pharmacokinetic model. Following 12 mg kg−1 the values (mean ± s.e., n = 11) of clearance and volume of distribution were 28 ± 2 ml min−1 kg−1 and 2.6 ± 0.31 kg−1, respectively, and were not significantly different from the values obtained at the other doses. 3 The anticonvulsant effect was quantitated by a new technique which allows repetitive determination of the convulsive threshold by direct cortical stimulation within one rat. Significant dose‐dependent elevations of the seizure threshold were observed. 4 By pharmacokinetic‐pharmacodynamic modelling, a log‐linear relationship was found between concentration and anticonvulsant effect. Following 12 mg kg−1 the values (mean ± s.e., n = 11) of the pharmacodynamic parameters slope and minimal effective concentration (Cmin) were 243 ± 27 μA and 0.11 ± 0.02 mg l−1, respectively and not significantly different from the values obtained at the other doses. 5 In a repeatability study the pharmacodynamic parameters were determined twice on two different occasions with an interval of two weeks in the same group of 11 rats. The inter‐animal variability in the pharmacodynamic parameter slope was 46%, whereas the intra‐animal variability was 24 ± 18%. The value of the minimal effective concentration was in each animal and on each occasion close to zero within the relatively narrow range of 0.01–0.30 mgl−1. 6 The results of this study showed that it is possible to determine in vivo concentration‐anticonvulsant effect relationships of oxazepam under non‐steady‐state conditions in individual rats. The anti‐convulsant effect of oxazepam appeared to be a rapidly reversible direct effect and acute tolerance did not develop within the time frame of the experiments.

Strategy to assess the role of (inter)active metabolites in pharmacodynamic studies in-vivo: a model study with heptabarbital

Abstract— The purpose of this investigation was to develop a universal experimental strategy by which the role of (inter)active metabolites in in‐vivo pharmacodynamic studies can be examined. Heptabarbital was chosen as a model drug and several pharmacokinetic variables which may affect in‐vivo concentration‐pharmacological response relationships were examined. Adult female rats received an i.v. infusion of the drug at one of three different rates (0.225–1.50 mg min−1) until the animals lost their righting reflex (after 11 ± 1 to 88 ± 8 min of infusion). The serum concentration of the drug at onset of loss of righting reflex (LRR) increased slightly with increasing infusion rate. The drug concentrations in brain tissue and cerebrospinal fluid (CSF), (mean ± s.d.: 67 ± 5 mg kg−1 and 24 ± 4 mg L−1, respectively, for the lowest infusion rate) were not affected by the infusion rate. The possible contribution of (inter)active metabolites to the pharmacological response of heptabarbital was determined by administration of different i.v. bolus doses (14.1–22.5 mg) resulting in widely differing sleeping‐times (7 ± 3 to 119 ± 20 min). The concentrations of heptabarbital in serum, brain tissue and CSF at offset of LRR (mean ± s.d.: 77 ± 8 mg L−1, 76 ± 7 mg kg−1 and 29 ± 5 mg L−1, respectively, for the highest dose) were not affected by the administered dose. Kinetic analysis of the relationship between dose and the duration of the pharmacological response revealed an elimination half‐life of heptabarbital of 2.8 ± 0.2 h, which is in close agreement with the value determined on the basis of the plasma concentration vs time profile following administration of 22.5 mg i.v. (2.8 ± 0.4 h). In a separate investigation no statistically significant differences were observed in heptabarbital concentrations at onset of LRR during an i.v. infusion (0.563 mg min−1) and at offset of LRR following an i.v. bolus dose (22.5 mg; sleeping time: 100 ± 20 min). These results show that (a) there is a rapid equilibration between the concentrations of heptabarbital (heptabarbitone) in CSF and those at the site of action (i.e. the CSF compartment is pharmacokinetically indistinguishable from the site of action), (b) metabolites do not interfere with the pharmacological effect of heptabarbital and (c) within the time‐frame of the experiments there is no development of ‘acute’ tolerance to the anaesthetic effect of heptabarbital. It is concluded that a combination of determination of the concentrations at offset of a certain pharmacological effect following administration of different drug doses, and evaluation of the dose vs duration of pharmacological response relationship, can be a powerful tool in examining the role of unknown (inter)active metabolites.

Application of serial sampling of cerebrospinal fluid in pharmacodynamic studies with a drug active in the CNS: Heptabarbital concentrations at onset and offset of loss of righting reflex in rats

As cerebrospinal fluid (CSF) possesses unique characteristics in order to explore concentration-pharmacological response relationships of drugs active in the CNS, the practicability of serial sampling of CSF was tested in a study with heptabarbital. Concentrations in CSF and plasma were measured simultaneously in individual rats during and after an intravenous infusion for 30 min. At the end of the infusion, the distribution equilibrium was attained with a CSF/plasma concentration ratio of 0.38, roughly equal to the fraction unbound to protein. When concentrations in blood and CSF were determined at the onset and offset of loss of righting reflex concentrations in blood were significantly greater at onset (146 +/- 19 mg/l) than at offset (108 +/- 16 mg/l, n = 6), whereas concentrations in CSF were identical (39 +/- 5 and 38 +/- 5 mg/l, respectively). This confirmed the earlier observation that the CSF is pharmacokinetically indistinguishable from the site of action. When the duration of the loss of righting reflex was varied, concentrations of heptabarbital in CSF at onset and offset were similar, independent of the duration of the loss of righting reflex (1-5 hr). These findings demonstrate the absence of the development of acute tolerance and confirmed that no (inter)active metabolites interfered with the pharmacological response. In a total number of 26 rats the concentrations in CSF at onset and offset of loss of the righting reflex were compared. The interindividual variation was 13-15% and the intra-individual variation was only 4-6%. The results demonstrate the usefulness of serial sampling of CSF in pharmacodynamic studies with centrally acting drugs.

Determination of Benzodiazepines: The Present-Day Scene

Methods that have been used for determining benzodiazepines in biological fluids include GC, HPLC, direct DPP, RIA and RRA methods*, of which GC, HPLC and RRA have proved the most valuable in clinical research on benzodiazepines. For low concentrations GC is particularly suitable, with ECB and possibly a SCOT column and a solids injector. Some hydroxylated benzodiazepines have to be derivatized. For thermally unstable compounds such as these, HPLC-UV is advantageous, although less sensitive than GC-ECD. HPLC with fluorescence detection is feasible but requires derivatization. HPLC with EC detection has so far been unpromising. HPLC could be useful for drug enantiomers.

The Influence of Dosage Time of Midazolam on Its Pharmacokinetics and Effects in Humans

The influence of dosage time of midazolam on its pharmacokinetics and effects on the central nervous system were investigated in six healthy volunteers, with pharmacokinetic-pharmacodynamic modeling. Each volunteer received single oral doses of 15 mg midazolam on four separate occasions: 8 AM, 2 PM, 8 PM, and 2 AM. An almost significant circadian variation was found in elimination half-life, shortest at 2 PM (1.26 +/- 0.47 hours, mean +/- SD) and longest at 2 AM (1.57 +/- 0.44 hours) (p = 0.05). Drug effects measured were alpha activity of the electroencepalograph and P100 latency of the visual-evoked response. The maximum drug effect (Emax) model described the concentration-effect relationship, extended with either a threshold drug concentration or a sigmoidicity parameter. A significant circadian variation was found in baseline alpha activity: highest at 8 AM (109% +/- 19% of the 24-hour mean) and lowest at 2 AM (80% +/- 12%). For alpha activity the drug concentration at half-maximum effect of both threshold Emax model and sigmoid Emax model showed lower values at 8 AM and 2 AM and higher values at 2 PM and 8 PM. However, these differences were either not significant (p = 0.10, threshold model) or on the verge of statistical significance (p = 0.05, sigmoid model). No circadian variation was found in the parameters describing the effect on the visual-evoked response. We conclude that the sensitivity of the central nervous system to midazolam, as reflected in alpha activity, possibly shows a circadian variation.