Douwe D. Breimer

The influence of cytokines on the integrity of the blood-brain barrier in vitro

The effects of the cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on the permeability of monolayers of rat cerebral endothelial cells (RCEC) were investigated to assess potential changes in the integrity of the blood-brain barrier (BBB). RCEC were cultured to tight monolayers with a trans endothelial electrical resistance (TEER) of 100-150 ohm . cm2 on polycarbonate filters. Exposure of the RCEC to TNF-alpha, IL-1 beta and IL-6 induced a decline in the TEER, which could be completely abolished by 1 muM of indomethacin, a cyclooxygenase inhibitor. In addition, the effect of IL-1 beta on TEER across monolayers of RCEC could be completely inhibited by IL-1 receptor antagonist. In conclusion, cytokines induce a disruption of the BBB in vitro. In this process, cyclooxygenase activation within the endothelial cells seems to play a key role.

Multidrug resistance protein 1 protects the choroid plexus epithelium and contributes to the blood-cerebrospinal fluid barrier

Multidrug resistance protein 1 (MRP1) is a transporter protein that helps to protect normal cells and tumor cells against the influx of certain xenobiotics. We previously showed that Mrp1 protects against cytotoxic drugs at the testis-blood barrier, the oral epithelium, and the kidney urinary collecting duct tubules. Here, we generated Mrp1/Mdr1a/Mdr1b triple-knockout (TKO) mice, and used them together with Mdr1a/Mdr1b double-knockout (DKO) mice to study the contribution of Mrp1 to the tissue distribution and pharmacokinetics of etoposide. We observed increased toxicity in the TKO mice, which accumulated etoposide in brown adipose tissue, colon, salivary gland, heart, and the female urogenital system. Immunohistochemical staining revealed the presence of Mrp1 in the oviduct, uterus, salivary gland, and choroid plexus (CP) epithelium. To explore the transport function of Mrp1 in the CP epithelium, we used TKO and DKO mice cannulated for cerebrospinal fluid (CSF). We show here that the lack of Mrp1 protein causes etoposide levels to increase about 10-fold in the CSF after intravenous administration of the drug. Our results indicate that Mrp1 helps to limit tissue distribution of certain drugs and contributes to the blood-CSF drug-permeability barrier.

Establishment and functional characterization of an in vitro model of the blood-brain barrier, comprising a co-culture of brain capillary endothelial cells and astrocytes

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.

Methodological issues in microdialysis sampling for pharmacokinetic studies

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.

Pharmacokinetic-pharmacodynamic modeling of the central nervous system effects of midazolam and its main metabolite α-hydroxymidazolam in healthy volunteers

The pharmacodynamics of midazolam and its main metabolite α‐hydroxymidazolam were characterized in individual subjects by use of saccadic eye movement and electroencephalographic (EEG) effect measurements. Eight healthy volunteers received 0.1 mg/kg midazolam intravenously in 15 minutes, 0.15 mg/kg α‐hydroxymidazolam intravenously in 15 minutes, 7.5 mg midazolam orally and placebo in a randomized, double‐blind, four‐way crossover experiment. Plasma concentrations of midazolam, α‐hydroxymidazolam and 4‐hydroxymidazolam were measured by gas chromatography. The amplitudes in the 11.5 to 30 Hz (beta) frequency band were used as EEG effect measure. The concentration‐effect relationships were quantified by the sigmoid maximum effect model. The median effective concentrations of midazolam and α‐hydroxymidazolam were (mean ± SE) 77 ± 15 and 98 ± 17 ng/ml, respectively, for the EEG effect measure. For peak saccadic velocity the values were 40 ± 7 ng/ml for midazolam and 49 ± 10 ng/ml for α‐hydroxymidazolam. The maximum effect values were similar for both compounds. The effects observed after oral administration of midazolam could not be predicted accurately by an additive and competitive interaction model. It seems that α‐hydroxymidazolam is highly potent with respect to the measured effects and contributes significantly to those effects of midazolam after oral administration.

RECTAL DRUG ADMINISTRATION - CLINICAL PHARMACOKINETIC CONSIDERATIONS

SummaryThe human rectum represents a body cavity in which drugs can be easily introduced and retained and from which absorption is well possible. There are important therapeutic reasons why it is sometimes preferable to give a drug rectally rather than orally, e.g. in cases of nausea and vomiting. Drawbacks of rectal drug administration include the interruption of absorption by defaecation and lack of patient acceptability. The mechanism of drug absorption from the rectum is probably no different to that in the upper part of the gastrointestinal tract, despite the fact that the physiological circumstances (e.g. pH, fluid content) differ substantially. Absorption from aqueous and alcoholic solutions may occur very rapidly, which has proved to be of considerable therapeutic value in the rapid suppression of acute convulsive attacks by diazepam (e.g. in children), but absorption from suppositories is generally slower and very much dependent on the nature of the suppository base, the use of surfactants or other additives, particle size of the active ingredient, etc. There is some evidence that hepatic first-pass elimination of high clearance drugs is partially avoided after rectal administration, e.g. lignocaine. This can be explained by the rectal venous blood supply: the upper part is connected with the portal system, whereas the lower part is directly connected with the systemic circulation.Plasma concentration data following rectal administration of representatives of several classes of drugs are reviewed: anticonvulsants, non-narcotic analgesics and non-steroidal anti-inflammatory agents, hypnosedatives and anaesthetics, strong analgesics, theophylline and derivatives, corticosteroids, antibacterial agents, thiazinamium, promethazine, hyoscine-N-butyl-bromide, streptokinase, progesterone, ergotamine tartrate and levodopa. Only in a limited number of cases has it been adequately shown that the rectal route of administration gives plasma concentrations which are comparable to the oral route. Potentially the rectal route offers the same possibilities as the oral route, but the influence of the formulation seems to be very critical. It is also likely that in the future novel drug delivery systems with zero order release characteristics will be applied rectally. Interesting preliminary results have already been obtained with theophylline administered by 2ml osmotic pumps.

Assay of Antipyrine and Its Primary Metabolites in Plasma, Saliva and Urine by High-Performance Liquid Chromatography and Some Preliminary Results in Man

A reversed phase system for the HPLC separation of antipyrine and its primary metabolites is described. Based on this system an assay procedure for antipyrine in plasma and saliva was developed with a lowest measurable concentration of 25 ng/ml and precision of +/- 3.6 and +/- 4.5%, respectively. Furthermore, assays for the parent compound, 3-hydroxymethyl-antipyrine, norantipyrine and 4-hydroxy-antipyrine in urine were developed. The lowest measurable concentration for these compounds is about 100 ng/ml except for 3-hydroxymethyl-antipyrine with a lowest measurable concentration of about 200 ng/ml. The precision was established at +/- 3.6 and +/- 5.0% for 3-hydroxymethyl-antipyrine, and antipyrine, respectively, and +/- 7.0 and +/- 3.6% for norantipyrine and 4-hydroxy-antipyrine, respectively. The method was applied to studies on antipyrine metabolism in humans. Following administration of a single dose of 500 mg antipyrine to 5 healthy volunteers, 3.3 +/- 1.2% of the dose was recovered from 48-hour hydrolyzed urine as unchanged drug, 39.7+/- 8.7% as 3-hydroxymethyl-antipyrine, 14.5 +/- 6.8% as norantipyrine and 28.5 +/- 2.2% as 4-hydroxy-antipyrine.

The role of glutathione conjugation in the mutagenicity of 1,2-dibromoethane

Abstract Two mechanisms for the toxic actions of 1,2-dibromoethane have been postulated, both of which involve biotransformation. The first is oxidation to 2-bromoacetaldehyde, a highly reactive substance, the second a possible direct conjugation to glutathione, giving rise to a reactive half-mustard. It was the purpose of this investigation to determine to what extent these two reactive species are responsible for the mutagenicity of 1,2-dibromoethane. To assess quantitatively the importance of the conjugation to glutathione in vivo, rats were administered single doses of 1,2-dibromoethane; 30–55 per cent of the dose was excreted as mercapturic acid. The conjugation of 1,2-dibromoethane to glutathione was also studied in vitro. Specific activities of the metabolizing systems used in the mutagenicity experiments were determined. The mutagenicity of 1,2-dibromoethane towards Salmonella typhimurium TA100 was considerably enhanced by the addition of 100,000 g supernatant fraction, whereas the addition of microsomes had no effect, indicating that the primary glutathione adduct is responsible for the mutagenic effect. As a model for the mutagenic intermediate, S-2-bromoethyl-N-acetyl-cysteine methyl ester was synthesized. This proved to be a very reactive and highly mutagenic compound, which can be further metabolized and thereby detoxified by glutathione conjugation. A similar phenomenon is likely to occur in the mutagenicity test with 1,2-dibromoethane, where after an initial rise in the number of mutants with increasing amounts of glutathione, the number of mutations decreases again. These results clearly indicate that glutathione conjugation plays an important role in the mutagenicity of 1,2-dibromoethane.

The influence of phenobarbital, 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin on glutathione S-transferase activity of rat liver cytosol.

Abstract Specific activities and apparent Michaelis-Menten kinetic parameters were determined for glutathione (GSH) S-transferase activity (E.C. 2.5.1.18) in rat liver cytosol, towards styrene oxide (STOX), 1,2-butylene oxide (BOX) and 1-chloro-2,4-dinitrobenzene (CDNB) as electrophilic substrates, before and after pretreatment with the drug-metabolizing enzyme inducers phenobarbital (PB), 3-methylcholanthrene (MC) and 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD). The measured GSH S-transferase activities appear to obey Michaelis-Menten kinetics. In non-induced animals the apparent K m values of the transferase activities were equal for STOX vs GSH, but they differed by a factor of 2 for CDNB vs GSH and by a factor of 14 for BOX vs GSH. The apparent V max values in each combination of GSH and electrophilic substrate were equal, but differed by one order of magnitude for the mutual substrate combinations. Pretreatment of the rats with MC resulted in enhancement of all measured activities expressed in terms of cytosol protein, while TCDD only enhanced the activities expressed as per gram body wt. PB enhanced both activities when STOX was employed as substrate, but when CDNB was used as the substrate, only the activity per gram body wt increased. All pretreatments increased the V max values using CDNB as the substrate, while PB and MC had an enhancing effect using STOX; the V max using BOX was enhanced after TCDD administration only. The K m values using BOX as the substrate was lowered after MC pretreatment; TCDD pretreatment decreased the K m using STOX, while it increased the K m using CDNB. It is concluded that the GSH S-transferase system is inducible, but in contrast to the induction of the mixed function oxidase system, qualitative differences between the inducing effects of PB and MC were not observed. Use of TCDD as inducing agent, however. resulted in a different induction pattern, which may indicate that during induction with this agent different types of GSH S-transferases are involved.

Grapefruit juice and cimetidine inhibit stereoselective metabolism of nitrendipine in humans

The effects of grapefruit juice (150 ml at −15, −10, ¼, +5, and +10 hours) and cimetidine (200 mg at the same times) on the stereoselective pharmacokinetics and effects of 20 mg oral racemic nitrendipine were investigated in a placebo‐controlled crossover study in nine healthy men. In all subjects the AUC of racemic nitrendipine was increased by grapefruit juice (mean increase 106%; 95% confidence interval 64% to 158%) and cimetidine treatment (+154%; 95% confidence interval 77% to 265%). Comparable results were obtained for the peak plasma drug concentration and for both parameters of (S)‐ and (R)‐nitrendipine. There were highly significant differences in the area under the concentration‐time curve and peak plasma drug concentration between enantiomers within all treatments. Grapefruit juice had no effect on this stereoselectivity, but cimetidine increased the mean S/R ratio of areas under the curve (2.25) by 20% (95% confidence interval 12% to 29%) compared with placebo treatment (1.89). Half‐lives and time to reach peak concentration of the enantiomers were not different within and between treatments. There were no consistent effects on blood pressure with all treatments, but in most subjects there was a small temporary increase in heart rate after intake of nitrendipine. Grapefruit juice and cimetidine did not affect these hemodynamic parameters and did not cause additional adverse effects.