In vitro P-glycoprotein efflux inhibition by atypical antipsychotics is in vivo nicely reflected by pharmacodynamic but less by pharmacokinetic changes.

BACKGROUND: P-glycoprotein (P-gp), an efflux transporter of the blood-brain barrier, limits the access of multiple xenobiotics to the central nervous system (CNS). Thus drug-dependent inhibition, induction or genetic variation of P-gp impacts drug therapy. METHODS: We investigated atypical antipsychotics and their interaction with P-gp. Amisulpride, clozapine, N-desmethylclozapine, olanzapine, and quetiapine were assessed in vitro on their inhibitory potential and in vivo on their disposition in mouse serum and brain, and behaviourally on the RotaRod test. In vivo wildtype (WT) and mdr1a/1b double knockout mice (mdr1a/1b (-/-, -/-); KO) were investigated. RESULTS: In rhodamine 123 efflux assay drugs inhibitory potency to P-gp could be ranked quetiapine>N-desmethylclozapine>clozapine>olanzapine. When treating WT and KO mice i.p. and assessing brain and serum levels by HPLC analysis, P-gp expression has the highest but a rather short effect on the distribution of amisulpride, whereas the others ranked N-desmethylclozapine>olanzapine>quetiapine>clozapine; contrasted by in vivo behavioral changes at various time points. Here quetiapine>clozapine>olanzapine impacts behavior most when P-gp is lacking. Present results indicate the relevance of P-gp expression for CNS-drug therapy. CONCLUSIONS: Combination of in vitro, and in vivo methods highlights that inhibitory potency did not reflect P-gp related drug disposition. But, when drugs were ranked for inhibitory potency, this order is reflected in pharmacodynamic changes or vice versa. Pharmacodynamic effects otherwise were at most correlated to drug brain levels, which however, were present only to a limited extent (by positron emission tomography) accessible in humans.

Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood-brain barrier.

In vitro models of the blood-brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol (P(app) = 1.30 x 10(-5) vs. 2.18 x 10(-5) cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.

Preserved pharmacological activity of hepatocytes-treated extracts of valerian and St. John's wort.

The two herbal extracts valerian (Valeriana officinalis L.) and St. John's wort (Hypericum perforatum L.) were studied for their metabolic changes upon incubation with freshly prepared rat hepatocytes and subsequently analysed phytochemically as well as pharmacologically in vitro. Quantitative HPLC analysis of valerian extracts revealed considerable metabolic activities with regard to sesquiterpenes and iridoids. The amount of acetoxyvalerenic acid decreased 9-fold, while that of hydroxyvalerenic acid correspondingly increased 9-fold due to O-deacetylation. The valepotriates didrovaltrate, isovaltrate and valtrate decreased 2-, 18- and 16-fold, respectively. However, the binding affinities of the incubated extracts to the benzodiazepine and picrotoxin binding site of the GABA (A) receptor were quite similar to those of the non-incubated extracts. Neither valerenic acids nor valepotriates exhibited any significant effect on the two binding sites when tested as single compounds. Therefore, either other constituents represent the active ones or multiple compounds are necessary for the observed inhibitory and allosteric effects at the GABA (A) receptor. Extracts of St. John's wort were less potently metabolised than valerian. The amount of pseudohypericin and the main flavonoids (hyperoside, rutin, isoquercitrin, quercitrin, quercetin and I3,II8-biapigenin) slightly decreased during the 4-h incubation period. Both the antagonist effect at the corticotropin-releasing factor (CRF) type 1 receptor and the binding inhibition at the 5-HT transporter were attenuated during the metabolic treatment. The reduced antagonist effect correlates with the decreasing amount of pseudohypericin known to be a CRF (1) receptor antagonist. In conclusion, the incubation of plant extracts with freshly prepared rat hepatocytes represents a useful approach to study the pharmacological action of metabolised plant extracts. The consistent pharmacological activity of both valerian and St. John's wort is concordant with the known clinical efficacy of pharmacological activities.