Mariska W. E. Langemeijer

In vitro and in vivo transport of zidovudine (AZT) across the blood-brain barrier and the effect of transport inhibitors

The transport of the antiviral nucleoside analogue zidovudine (3′-azido-3′-deoxythymidine; AZT) into the central nervous system (CNS) was characterized in vitro and in vivo. The in vitro model consisted of primary cultures of isolated bovine capillary endothelial cells. The transport rate of AZT across the monolayer, expressed as endothelial permeability P, was determined following luminal and abluminal administration. P did not differ between the two administration sites (luminal, 1.65 ± 0.44 cm/min/103; abluminal, 1.63 ± 0.28 cm/min/103). The transport of AZT across the endothelial cell monolayer was found to be concentration independent in the range between 0.4 and 50 µg/mL. AZT transport was not affected by pre-treatment of the cells with either metabolic inhibitors (DODG and DODG/NaN3) or probenecid. This suggests that AZT passes the monolayer mainly by passive diffusion. The in vivo transport of AZT across the blood–brain barrier and the blood–CSF barrier was studied in male Wistar rats after coadministration of potential inhibitors of active transport of AZT: probenecid (organic anion transport) and thymidine (nucleoside transport). Intracerebroventricular and intravenous coadministration of probenecid caused a significant (P < 0.001) increase in the CSF/plasma concentration ratio compared to the control phase, indicating that the organic anion carrier is involved in AZT transport from CSF to blood. Since there was no effect of probenecid on the transport of AZT in vitro, it is suggested that this carrier is located at the choroid plexus. Coadministration of thymidine did not affect the CSF/plasma concentration ratio, suggesting that a nucleoside carrier system is not involved in AZT transport into or out of the CNS.

Influence of Different Fat Emulsion-Based Intravenous Formulations on the Pharmacokinetics and Pharmacodynamics of Propofol

AbstractPurpose. The influence of different intravenous formulations on the pharmacokinetics and pharmacodynamics of propofol was investigated using the effect on the EEG (11.5-30 Hz) as pharmacodynamic endpoint. Methods. Propofol was administered as an intravenous bolus infusion (30 mg/kg in 5 min) or as a continuous infusion (150 mg/kg in 5 hours) in chronically instrumented male rats. Propofol was formulated as a 1% emulsion in an Intralipid 10%®-like fat emulsion (Diprivan-10®, D) or as a 1%- or 6% emulsion in Lipofundin® MCT/LCT-10% (Pl% and P6%, respectively). EEG was recorded continuously and arterial blood samples were collected serially for the determination of propofol concentrations using HPLC. Results. Following bolus infusion, the pharmacokinetics of the various propofol emulsions could adequately be described by a two-compart-mental pharmacokinetic model. The average values for clearance (Cl), volume of distribution at steady-state (Vd,ss) and terminal half-life (t1/2, λ2) were 107 ± 4 ml/min/kg, 1.38 ± 0.06 l/kg and 16 ± 1 min, respectively (mean ± S.E., n = 22). No significant differences were observed between the three propofol formulations. After continuous infusion these values were 112 ± 11 ml/min/kg, 5.19 ± 0.41 l/kg and 45 ± 3 min, respectively (mean±S.E., n = 20) with again no statistically significant differences between the three propofol formulations. Comparison between the bolus- and the continuous infusion revealed a statistically significant difference for both Vd,ss and t1/2, λ2 (p < 0.05), whereas Cl remained unchanged. In all treatment groups infusion of propofol resulted in a burst-suppression type of EEG. A profound hysteresis loop was observed between blood concentrations and EEG effect for all formulations. The hysteresis was minimized by a semi-parametric method and resulted in a biphasic concentration-effect relationship of propofol that was described non-parametrically. For P6% a larger rate constant onset of drug effect (t,1/2, keo) was observed compared to the other propofol formulations (p<0.05). Conclusions. The pharmacokinetics and pharmacodynamics of propofol are not affected by to a large extent the type of emulsion nor by the concentration of propofol in the intravenous formulation.

The Comparative Pharmacodynamics of Remifentanil and Its Metabolite, GR90291, in a Rat Electroencephalographic Model

BACKGROUND The purpose of this study was to investigate the in vivo pharmacodynamics and the pharmacodynamic interactions of remifentanil and its major metabolite, GR90291, in a rat electroencephalographic model. METHODS Remifentanil and GR90291 were administered according to a stepwise infusion scheme. The time course of the electroencephalographic effect (0.5-4.5 Hz) was determined in conjunction with concentrations of the parent drug and the metabolite in blood. RESULTS Administration of remifentanil resulted in concentrations of remifentanil and GR90291 in the ranges 0-120 ng/ml and 0-850 ng/ml, respectively. When the metabolite was administered, concentrations of the metabolite in the range 0-220 microg/ml and no measurable concentrations of remifentanil were observed. The mean +/- SE values of the pharmacokinetic parameters clearance and volume of distribution at steady state were 920+/-110 ml x min(-1) x kg(-1) and 1.00+/-0.93 l/kg for remifentanil and 15+/-2 ml x min(-1) x kg(-1) and 0.56+/-0.08 l/kg for GR90291. The relative free concentrations in the brain, as determined on the basis of the cerebrospinal fluid/total blood concentration ratio at steady state, were 25+/-5% and 0.30+/-0.11% for remifentanil and GR90291, respectively. Concentration-electroencephalographic effect relations were characterized on the basis of the sigmoidal Emax pharmacodynamic model. The mean +/- SE values for the maximal effect (Emax), the concentration at which 50% of the maximal effect is obtained (EC50), and Hill factor for remifentanil were 109+/-12 microV, 9.4+/-0.9 ng/ml, and 2.2+/-0.3, respectively (n = 8). For GR90291, the mean +/- SE values for EC50 and the Hill factor were 103,000+/-9,000 microg/ml and 2.5+/-0.4, respectively (n = 6). CONCLUSIONS Analysis of the data on the basis of a previously postulated, mechanism-based pharmacokinetic-pharmacodynamic model for synthetic opioids revealed that the low in vivo potency of GR90291 can be explained by a low affinity to the mu-opioid receptor in combination with a poor brain penetration.

Modelling of the pharmacodynamic interaction of an A1 adenosine receptor agonist and antagonist in vivo: N6-cyclopentyladenosine and 8-cyclopentyltheophylline

1 The purpose of this investigation was to develop a pharmacokinetic‐pharmacodynamic model for the interaction between an adenosine A1 receptor agonist and antagonist in vivo. The adenosine A1 receptor agonist, N6‐cyclopentyladenosine (CPA) and the antagonist, 8‐cyclopentyltheophylline (CPT) were used as model drugs. The CPA‐induced reduction in mean arterial pressure and heart rate were used as measurements of effect. 2 Four groups of eight rats each received 200 μg kg−1 of CPA i.v. in 5min during a steady‐state infusion of CPT at a rate of 0, 57, 114 or 228 μg kg−1 h−1. The haemodynamic parameters were continuously measured and frequent blood samples were taken to determine the pharmacokinetics of the drugs. 3 CPT had no influence on the pharmacokinetics of CPA and the baseline values of the haemodynamic variables. Furthermore, no clear antagonism by CPT was observed of the CPA‐induced reduction in mean arterial pressure. However, CPT antagonized the effect on heart rate, and with increasing CPT concentrations, a parallel shift of the CPA concentration‐effect relationship to the right was observed. 4 An agonist‐antagonist interaction model was used to characterize the interaction quantitatively. On the basis of this model, the pharmacodynamic parameters of both CPA and CPT could be estimated. For CPA the values were (mean±s.e.): Emax = 198 ± 11 b.p.m., EC50 = 2.1 ± 0.7ng ml−1, Hill factor = 2.3 ± 0.6 and for CPT: EC50 = 3.7 ± 0.3 ng ml−1 and Hill factor = 3.1 ± 0.1. 5 It is concluded that the competitive agonist‐antagonist interaction model may be of value to characterize quantitatively the pharmacodynamic interactions between adenosine A1 receptor ligands in vivo.

Pharmacokinetic‐pharmacodynamic modelling of the anti‐lipolytic and anti‐ketotic effects of the adenosine A1‐receptor agonist N6‐(p‐sulphophenyl)adenosine in rats

1 The purpose of this study was to develop and validate an integrated pharmacokinetic‐pharmacodynamic model for the anti‐lipolytic effects of the adenosine A1‐receptor agonist N6‐(p‐sulphophenyl)adenosine (SPA). Tissue selectivity of SPA was investigated by quantification of haemodynamic and anti‐lipolytic effects in individual animals. 2 After intravenous infusion of SPA to conscious normotensive Wistar rats, arterial blood samples were drawn for determination of blood SPA concentrations, plasma non‐esterified fatty acid (NEFA) and β‐hydroxybutyrate levels. Blood pressure and heart rate were monitored continuously. 3 The relationship between the SPA concentrations and the NEFA lowering effect was described by the indirect suppression model. Administration of SPA at different rates and doses (60 μg kg−1 in 5 min and 15 min, and 120 μg kg−1 in 60 min) led to uniform pharmacodynamic parameter estimates. The averaged parameters (mean±s.e., n=19) were Emax: −80±2% (% change from baseline), EC50: 22±2 ng ml−1, and Hill factor: 2.2±0.2. 4 In another group, given 400 μg kg−1 SPA in 15 min, pharmacodynamic parameters for both heart rate and anti‐lipolytic effect were derived within the same animal. The reduction in heart rate was directly related to blood concentration on the basis of the sigmoidal Emax model. SPA inhibited lipolysis at concentrations lower than those required for an effect on heart rate. The EC50 values (mean±s.e., n=6) were 131±31 ng ml−1 and 20±3 ng ml−1 for heart rate and NEFA lowering effect, respectively. 5 In conclusion, the relationship between blood SPA concentrations and anti‐lipolytic effect was adequately described by the indirect suppression model. For SPA a 6 fold difference in potency was observed between the effects on heart rate and NEFAs, indicating some degree of tissue selectivity in vivo.

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.

Pharmacodynamics of the anticonvulsant effect of oxazepam in aging BN/BiRij rats

1 The purpose of this investigation was to examine the influence of increasing age on the pharmacokinetics and the time course of the anticonvulsant response of oxazepam in BN/BiRij rats as an animal model of aging. 2 Oxazepam was administered intravenously in a dose of 12 mg kg−1 body weight and the anticonvulsant effect intensity was measured as elevation above baseline of a threshold for induction of localized seizure activity (TLS). Direct cortical stimulation with ramp shaped electrical pulse trains of increasing intensity was used to determine this threshold. 3 The pharmacological effect vs. time profile showed in young rats an anticonvulsant component followed by proconvulsant component which is suggestive for the occurrence of acute tolerance and/or withdrawal syndrome. With increasing age the proconvulsant component disappeared, resulting in a monophasic effect profile (anticonvulsant effect only) at the age of 35 months with significantly higher anticonvulsant effect intensity immediately following drug administration. No age‐related changes in the pharmacokinetic parameters of oxazepam were observed. 4 In five animals of each age group, benzodiazepine receptor binding characteristics were determined in vitro with [3H]‐flunitrazepam as a ligand. Both receptor density and affinity did not show age‐related changes. Available literature data on post‐receptor events do not indicate conclusive age‐related changes. 5 It is concluded, that the observed change in the pharmacodynamics of anticonvulsant effect of oxazepam can be explained by the disappearance of the tolerance/withdrawal phenomenon. This is compatible with a decreased efficiency of homeostatic control mechanisms in the elderly.

Pharmacokinetic-EEG effect relationship of midazolam in aging BN/BiRij rats

1 The purpose of the present investigations was to determine the influence of increasing age on the pharmacokinetics and pharmacodynamics of midazolam in male BN/BiRij rats as an animal model of aging. 2 Midazolam was administered intravenously at a dose of 2.5 mg kg−1 and its pharmacokinetics were determined on the basis of plasma concentrations as measured by high performance liquid chromatography (h.p.l.c.). Pharmacodynamics were studied using the midazolam‐induced changes in the electroencephalogram (EEG) as a measure of the pharmacological effect. Results were evaluated on the basis of stimultaneous pharmacokinetic‐pharmacodynamic modelling. In an attempt to differentiate between the effects of aging and of concurrent disease, an extensive clinical biochemical/pathological examination was conducted in individual rats by an independent pathologist. 3 The pharmacokinetics of midazolam were best characterized on the basis of a two exponential model. In the 4‐month‐old rats the values of the clearance, volume of distribution and elimination half‐life were 104 ± 13 ml min−1 kg−1 (mean ± s.e.mean), 3.4 ± 0.71 kg−1 and 30 ± 3 min, respectively. With increasing age, no changes in the pharmacokinetics of midazolam were observed. 4 The pharmacodynamics of midazolam were determined on the basis of the sigmoidal Emax model. In the 4‐month‐old rats the values of the parameters relative maximum effect, midazolam concentration at half maximum effect and Hill factor were 106 ± 10%, 50 ± 6 μg l−1 and 1.6 ± 0.3, respectively. In the group as a whole no significant changes in the pharmacodynamic parameters of midazolam were observed. However, when diseased animals were excluded from the evaluation, a tendency towards a decrease in the midazolam concentration at half maximum effect to 25 ± 14 μg l−1 was observed in the 36‐month‐old rats. 5 These findings suggest, that increasing age is associated with a tendency towards an increased brain sensitivity to midazolam, which is reflected in a parallel shift of the concentration vs. EEG effect relationship towards lower concentrations. However, it appears that factors other than age also contribute to interindividual variability in pharmacodynamics, considering the substantial interindividual variability within certain age groups.

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.

Pharmacokinetic-pharmacodynamic modelling of the analgesic effect of alfentanil in the rat using tooth pulp evoked potentials.

The purpose of the present study was to develop an animal model for the investigation of the concentration-effect relationship of alfentanil using tooth pulp evoked potentials (TPEP). Six chronically instrumented, freely moving rats received a computer-controlled intravenous infusion of alfentanil resulting in seven pseudo-steady-state blood concentration levels, each maintained for 30 min. At each concentration level, the tooth pulp of the rat upper incisor was electrically stimulated in a time-randomized order with different current intensities (400-800 microA, 1 msec duration) and the electroencephalogram (EEG) was recorded concomitantly. Arterial blood samples were collected serially and assayed for alfentanil using RIA. Repetitive evoked EEG responses were averaged per stimulus intensity and per session. The decrease of the area under the negative peak 15 msec after stimulation (% of preadministration value) was used as pharmacological endpoint. The concentration-TPEP effect of alfentanil was investigated by nonlinear mixed effect modeling (NONMEM). When the observed TPEP effect was plotted versus the alfentanil blood concentration no hysteresis or proteresis was observed, and the two could directly be related to each other on the basis of the sigmoidal Emax pharmacodynamic model. The (population) pharmacodynamic estimates were (+/-S.E.): Emax = 108 +/- 10%, EC50 = 24 +/- 17 ng/ml, Hill factor = 0.81 +/- 0.37. A large interindividual variability for EC50 (omegaEC50) of 164 +/- 107% was observed. The residual variability was 14 +/- 10%. It is concluded that the TPEP is a useful tool for the systematic investigation of the concentration-analgesic effect relationship of centrally acting analgesics in the freely moving rat.