The pharmacokinetics of racemic (rac) felodipine, rac-nitrendipine and nifedipine (all given as an oral dose of 20 mg in solution) have been investigated in a randomised cross-over study in 12 healthy male subjects using stereoselective assays. Both felodipine and nitrendipine exhibited stereoselective pharmacokinetics. On average, the AUCs of the active (S)-enantiomers of felodipine and nitrendipine were 139% and 104% higher than those of their optical antipodes, but the elimination half-lives of the enantiomers of each racemate were not different. The AUCs of nifedipine, rac-felodipine, rac-nitrendipine and of their enantiomers were highly correlated (all r > 0.83), suggesting closely related rate limiting steps in the in vivo first-pass metabolism of these high-clearance drugs. Stereoselectivity was only a minor contributor to inter-individual variability in the oral pharmacokinetics of these compounds in healthy subjects.

The markets for the traditional output of schools of pharmacy, namely education, research and graduates, are changing. The main private client in these markets, the pharmaceutical industry, is moving fast to become more efficient, under pressure from overly costly drug development. The challenges to the industry that emanate from the fantastic rate of advances in the biomedical sciences and pharmaceutical development are considerable. The many agents that were unheard of 10 years ago, such as gene-regulators, together with new technologies, all require new approaches to fundamental pharmaceutical issues. The concept of disciplines in graduate education may have to be reconsidered in the light of the multidisciplinary problems to be tackled. In addition, graduates will need to acquire a range of non-disciplinary skills, such as better communication or team working, in order to be effective in the commercial market place. The concept of 'research schools' following either a local or network model may provide the way forward to help academia meet the graduate education needs of industry. The objectives and mission of such institutions must be clearly defined to ensure that the current scientific environment is embraced fully.

Objective: The aim was to establish a flexible, abundantly available, reproducible and functionally characterized in vitro model of the blood-brain barrier (BBB). Methods: In a first step, bovine brain capillaries and newborn rat astrocytes were isolated. Subsequently, a co-culture of primary brain capillary endothelial cells (BCEC) on semi-permeable filter inserts, with astrocytes on the bottom of the filter was established. The cell material was characterized on the basis of specific cell-type properties and (functional expression of) specific BBB properties. Results: BCEC displayed: (1) characteristic endothelial cell morphology; (2) expression of endothelial cell markers (i.e., CD51, CD62P, CD71 and cadherin 5); (3) marginal F-actin localization; (4) tight junction formation between the cells; (5) expression of gamma -glutamyl-transpeptidase (gamma -GTP); (6) expression of P-glycoprotein (Pgp); (7) functional transendothelial transferrin transport and uptake; (8) restriction of paracellular transport; and (9) high transendothelial electrical resistance (TEER). Astrocytes displayed characteristic astrocyte morphology and expressed glial fibrillary acidic protein (GFAP). Co-culture with astrocytes increased TEER and decreased paracellular transport. In addition, expression of the glucocorticoid receptor (GR) was demonstrated in the endothelial cells of the BBB, while no expression of the mineralocorticoid receptor (MR) was found. Conclusions: A high quality and mass-production in vitro BBB model was established in which experiments with physiological (e.g., regulation of BBB permeability), pharmacological (e.g., pharmacokinetics and pharmacodynamics) and pathophysiological (e.g., disease influence on BBB permeability) objectives can be reproducibly performed. (C) 2001 Elsevier Science B.V. All rights reserved.

Microdialysis is an in vivo technique that permits monitoring of local concentrations of drugs and metabolites at specific sites in the body. Microdialysis has several characteristics, which makes it an attractive tool for pharmacokinetic research. About a decade ago the microdialysis technique entered the field of pharmacokinetic research, in the brain, and later also in peripheral tissues and blood. Within this period much has been learned on the proper use of this technique. Today, it has outgrown its child diseases and its potentials and limitations have become more or less well defined. As microdialysis is a delicate technique for which experimental factors appear to be critical with respect to the validity of the experimental outcomes, several factors should be considered. These include the probe; the perfusion solution; post-surgery interval in relation to surgical trauma, tissue integrity and repeated experiments; the analysis of microdialysate samples; and the quantification of microdialysate data. Provided that experimental conditions are optimized to give valid and quantitative results, microdialysis can provide numerous data points from a relatively small number of individual animals to determine detailed pharmacokinetic information. An example of one of the added values of this technique compared with other in vivo pharmacokinetic techniques, is that microdialysis reflects free concentrations in tissues and plasma. This gives the opportunity to assess information on drug transport equilibration across membranes such as the blood–brain barrier, which already has provided new insights. With the progress of analytical methodology, especially with respect to low volume/low concentration measurements and simultaneous measurement of multiple compounds, the applications and importance of the microdialysis technique in pharmacokinetic research will continue to increase.

We describe a new simple, selective and sensitive micromethod based on HPLC and fluorescence detection to measure debrisoquine (D) and 4-hydroxydebrisoquine (4-OHD) in urine for the investigation of xenobiotic metabolism by debrisoquine hydroxylase (CYP2D6). Four hundred microl of urine was required for the analysis of D and 4-OHD. Peaks were eluted at 8.3 min (4-OHD), 14.0 min (D) and 16.6 min for the internal standard, metoprolol (20 microg/ml). The 5-microm CN-reverse-phase column (Shimpack, 250 x 4.6 mm) was eluted with a mobile phase consisting of 0.25 M acetate buffer, pH 5.0, and acetonitrile (9:1, v/v) at 0.7 ml/min with detection at lambdaexcitation = 210 nm and lambdaemission = 290 nm. The method, validated on the basis of measurements of spiked urine, presented 3 ng/ml (D) and 6 ng/ml (4-OHD) sensitivity, 390-6240 ng/ml (D) and 750-12000 ng/ml (4-OHD) linearity, and 5.7/8.2% (D) and 5.3/8.2% (4-OHD) intra/interassay precision. The method was validated using urine of a healthy Caucasian volunteer who received one 10-mg tablet of Declinax(R), po, in the morning after an overnight fast. Urine samples (diuresis of 4 or 6 h) were collected from zero to 24 h. The urinary excretion of D and 4-OHD, Fel (0-24 h), i.e., fraction of dose administered and excreted into urine, was 6.4% and 31.9%, respectively. The hydroxylation capacity index reported as metabolic ratio was 0.18 (D/4-OHD) for the person investigated and can be compared to reference limits of >12.5 for poor metabolizers (PM) and <12.5 for extensive metabolizers (EM). In parallel, the recovery ratio (RR), another hydroxylation capacity index, was 0.85 (4-OHD: SigmaD + 4-OHD) versus reference limits of RR <0.12 for PM and RR >0. 12 for EM. The healthy volunteer was considered to be an extensive metabolizer on the basis of the debrisoquine test.

Non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists show antiparkinsonian-like activity in animal models, and possess neuroprotective properties. However they also induce a number of behavioral side effects in rodents at higher doses; these include learning impairment, hyperlocomotion, and ataxia. The present study focused on the possible development of tolerance, or sensitization, to any of these effects after sustained administration, either by repeated injection or continuous infusion. When memantine or (+)MK-801 (20 and 0.31 mg/kg/day respectively) were either infused or repeatedly injected for 14 days, tolerance was observed to their learning impairing effect at high doses, in a passive avoidance test. Tolerance to their ataxic effect developed after repeated administration ((+)MK-801 and memantine), or after infusion (memantine). Sensitization to the locomotor stimulation was seen following repetitive injections of memantine for 14 days, but not seen with (+)MK-801. In animals with an unilateral 6-OHDA lesion of the nigrostriatal system, acute administration of memantine caused ipsilateral rotations, which were augmented following 14 days of infusion. The potency of amantadine to antagonize neuroleptic-induced catalepsy was unchanged following either infusion or repeated injections. The various acute effects of non-competitive NMDA receptor antagonists were modified differently by sustained treatment (i.e. tolerance to learning impairment and ataxia; sensitization to memantine's locomotor stimulation). The anti-cataleptic activity of amantadine remained unaltered. However, differences between drugs and the two treatment regimens (i.e. repetitive versus continuous treatments) were apparent.

Behavioral changes have previously been reported following administrations of uncompetitive NMDA receptor antagonists memantine, amantadine and MK-801 for 14 days, at the doses that produce plasma levels comparable to those seen in patients (20, 100 and 0.31 mg/kg/day respectively). Using the same doses, the effect on receptor binding (autoradiography) was studied in rats. [3H]MK-801 binding was increased in the dentate gyrus and CA3 region of the hippocampus (35.2 and 24.3% respectively) following 3 days S.C. infusion of memantine by ALZET minipumps. One daily injection of memantine for 14 days, increased [3H]MK-801 binding in the frontal cortex by 40.3%. The same treatment with amantadine did increase [3H]raclopride binding to dopamine D2 receptors by 13.5%. None of these treatments changed the expression of muscarinic receptors. It is concluded that subchronic blockade of the NMDA receptor by uncompetitive antagonists at moderate (therapeutically-relevant) doses induced only minor changes in NMDA and dopamine D2 receptor expression.

The intracerebral microdialysis technique represents an important tool for monitoring free drug concentrations in brain extracellular fluid (brain(EcF)) as a function of time. With knowledge of associated free plasma concentrations, it provides information on blood-brain barrier (BBB) drug transport. However, as the implantation of the microdialysis probe evokes tissue reactions, it should be established if the BBB characteristics are maintained under particular microdialysis experimental conditions. Several studies have been performed to evaluate the use of intracerebral microdialysis as a technique to measure drug transport across the BBB and to measure regional pharmacokinetics of drugs in the brain. Under carefully controlled conditions, the intracerebral microdialysis data did reflect passive BBB transport under normal conditions, as well as changes induced by hyperosmolar opening or by the presence of a tumor in the brain. Studies on active BBB transport by the mdr1a-encoded P-glycoprotein (Pgp) were performed, comparing mdr1a(-/-) with wild-type mice. Microdialysis surgery and experimental procedures did not affect Pgp functionality, but the latter did influence in vivo concentration recovery, which was in line with theoretical predictions. It is concluded that intracerebral microdialysis provides meaningful data on drug transport to the brain, only if appropriate methods are applied to determine in vivo concentration recovery.

Memantine is an uncompetitive N-methyl-D-aspartate receptor antagonist which blocks the NMDA receptor with moderate-affinity in a use- and voltage dependent manner. In clinical practice it is used chronically in the treatment of dementia and does not induce psychotomimetic effects as, high affinity, uncompetitive antagonists. Thus, it was of interest to determine dopamine (DA) and metabolite (DOPAC - dihydroxyphenylacetic acid and HVA - homovanillic acid) concentrations in the prefrontal cortex (PFC) in response to 14 days administration of memantine (20 mg/kg/day). It was previously determined that in rats this treatment induces sensitization to the locomotor effect and tolerance to the learning impairing properties of high doses of memantine. Acute administration of memantine (20 mg/kg, ip) did not affect dopamine levels in the PFC. It did however increase DA metabolite (DOPAC and HVA) concentrations. Administration of memantine (20 mg/kg/day) for 14 days before the acute challenge only slightly changed memantine's effect on PFC neurochemistry even though pharmacokinetic tolerance was observed. When memantine was administered to the sham group, which had been repeatedly treated with Hypnorm (including neuroleptic), an increase in PFC dopamine and metabolite content was seen. In accordance with the fact that memantine does not possess psychotomimetic activity at therapeutically relevant doses, these experiments showed that it does not affect the prefrontal cortex dopamine levels.

Various methods are used to quantify sedative drug effects, but it is unknown how these surrogate measures relate to clinically relevant sleepiness. This study assessed the sensivity of different surrogates of sedation to clinically relevant sleepiness induced by sleep deprivation. Nine healthy volunteers completed a balanced three-way cross-over study with 1-week wash-out periods. Adaptive tracking, smooth-pursuit and saccadic eye movements, body sway, digit symbol substitution (DSST), visual analogue scales (VAS) and electroencephalograms (EEG) were evaluated on three occasions: (1) during the day after normal sleep, (2) during wakefulness at night; and (3) during the day after a night of sleep deprivation.VAS of alertness showed a gradual decline at night and a constant average reduction of 38 percent [95% Confidence intervals (CI), 28–47%] during the day after sleep deprivation. Average mood scores diminished by 14 percent (95%, CI 2–24%) during the day after sleep deprivation. Adaptive tracking, saccadic eye movements and body sway tended to deteriorate at night, but overall this was not statistically significant. After a night of sleep deprivation, adaptive tracking decreased by 21 percent (95% CI, 11–30%), saccadic eye movements decreased by 9–10 percent (95% CI, 5–13%/6–15%) and body sway increased by 37 percent (95% CI, 5–79%). In contrast, EEG b2-amplitudes declined significantly at night by 18 percent (95% CI, 6–29%), without changes during the day after sleep deprivation. Smooth pursuit, DSST and other EEG-amplitudes remained unchanged. These resultsemphasize that reductions in adaptive tracking, saccadic peak velocity and body sway caused by sedative drugs really reflect sedation. They also provide a level of clinical significance for these surrogates of sedation. EEG parameters and smooth pursuit were unaffected by sleep deprivation, so drug-induced changes in these measures may notreflect sedation in a stricter sense. The motivation and alertnessnecessary for DSST may overcome mild sedation.