Assessment of active transport of HIV protease inhibitors in various cell lines and the in vitro blood--brain barrier.

OBJECTIVE: To investigate the involvement of P-glycoprotein (Pgp) and the multidrug resistance-associated protein (MRP) on the active transport of the HIV protease inhibitors amprenavir, ritonavir and indinavir. METHODS: The transport behaviour of ritonavir, indinavir and amprenavir in the presence and absence of Pgp modulators and probenecid was investigated in an in vitro blood--brain barrier (BBB) co-culture model and in monolayers of LLC-PK1, LLC-PK1:MDR1, LLC-PK1:MRP1 and Caco-2 cells. RESULTS: All three HIV protease inhibitors showed polarized transport in the BBB model, LLC-PK1:MDR1 and Caco-2 cell line. The Pgp modulators SDZ-PSC 833, verapamil and LY 335979 inhibited polarized transport, although their potency was dependent on both the cell model and the HIV protease inhibitor used. Ritonavir and indinavir also showed polarized transport in the LLC-PK1 and LLC-PK1:MRP1 cell line, which could be inhibited by probenecid. HIV protease inhibitors were not able to inhibit competitively polarized transport of other HIV protease inhibitors in the LLC-PK1:MDR1 cell line. CONCLUSIONS: Amprenavir, ritonavir and indinavir are mainly actively transported by Pgp, while MRP also plays a role in the transport of ritonavir and indinavir. This indicates that inhibition of Pgp could be useful therapeutically to increase HIV protease inhibitor concentrations in the brain and in other tissues and cells expressing Pgp. The HIV protease inhibitors were not able to inhibit Pgp-mediated efflux when given simultaneously, suggesting that simultaneous administration of these drugs will not increase the concentration of antiretroviral drugs in the brain.

Specificity of doxorubicin versus rhodamine-123 in assessing P-glycoprotein functionality in the LLC-PK1, LLC-PK1:MDR1 and Caco-2 cell lines.

The LLC-PK1:MDR1, LLC-PK1 and Caco-2 cell lines were used to investigate whether rhodamine-123 or doxorubicin would be the preferred substrate to study P-glycoprotein (P-gp) functionality in vitro. Both rhodamine-123 and doxorubicin showed highly polarised transport in the Caco-2 cell line and the LLC-PK1:MDR1 cell line, indicating that P-gp is actively transporting these drugs. However, for rhodamine-123 polarised transport was also seen in the monolayers of the wild-type LLC-PK1 cell line, indicating the presence of another active transporter for this compound. Polarised transport of doxorubicin in the Caco-2 and the LLC-PK1:MDR1 cell lines could be inhibited by the P-gp inhibitors SDZ-PSC 833 (PSC 833), cyclosporin A (CsA), verapamil and quinine, but not by the inhibitors for the organic cation carrier systems cimetidine and tetraethylammonium (TEA). Polarised transport of rhodamine-123 in the Caco-2 cell line could only be inhibited by P-gp inhibitors. In the LLC-PK1:MDR1 and LLC-PK1 cell lines transport was also inhibited by inhibitors for the organic cation transport systems. In conclusion, rhodamine-123 is a substrate for both P-gp and the organic cation carrier systems in the kidney cell line. This indicates that rhodamine-123 is not selective enough to study P-gp functionality in cell systems were organic cation carrier systems are also present. Doxorubicin appears to be a more selective P-gp substrate and therefore more useful in studying P-gp functionality in vitro.

Effect of endotoxin on permeability of bovine cerebral endothelial cell layers in vitro.

The effect of lipopolysaccharide (LPS) on cultured cerebral endothelial cells was investigated to assess the changes in the trans endothelial electrical resistance (TEER) across the blood-brain barrier that may occur during inflammatory diseases of the central nervous system. Primary cultures of bovine cerebral endothelial cells were cultured to tight monolayers with a TEER of 250 to 300 omega.cm2 on polycarbonate Transwell filters. LPS induced a time- and dose-dependent decline in TEER. Transport of the hydrophilic model compounds sodium fluorescein and fluorescein dextran (MR, 4 kDa) across monolayers of bovine cerebral endothelial cells increased more than 3-fold after treatment of the cells with LPS (50 ng/ml). Treatment of the monolayers with various concentrations of LPS caused a 3-to 4-fold increase in the permeability of bovine cerebral endothelial cells for [125I]bovine serum albumin, which was also preceded by a decrease in TEER. The reduction of TEER by LPS could be inhibited completely by indomethacin (10(-6)M for 30 min), a cyclooxygenase inhibitor, but not by dexamethasone, a glucocorticoid (10(-7) M for 16 hr). In conclusion, LPS administration to blood-brain barrier endothelial cells causes a decrease in TEER which leads to enhanced transport of low and high molecular weight molecules. During this process the production of eicosanoids by the endothelial cells seem to play a key role.

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.

Absorption enhancement of a hydrophilic model compound by verapamil after rectal administration to rats.

The use of verapamil as an absorption enhancer for the paracellular route in-vivo was studied using FITC-labelled dextran (molecular weight 4000) (FD-4) as a hydrophilic model compound for transport enhancement. The kinetics of FD-4 after intravenous doses of 1 or 10 mg could be described by a two-compartment model with a systemic clearance of approximately 2 mL min-1 and a terminal plasma half-life of approximately 36 min. Rectal administration to rats, performed as a rectal infusion of 10 mg FD-4 together with 7 mM verapamil, resulted in a 10-fold increase in the percentage of the dose absorbed over a 5-h period compared with the control and a 6-fold increase compared with a bolus administration, although the total amount absorbed remained relatively low (approx. 3% maximum). Large inter-animal variation in effect values were noted. The data indicate that although verapamil is able to enhance the absorption of hydrophilic compounds in-vivo, practical application of verapamil for this purpose doses not seem feasible.

Absorption enhancement of hydrophilic compounds by verapamil in Caco-2 cell monolayers.

Caco-2 monolayers were used to determine whether verapamil enhanced the transport of hydrophilic compounds across epithelial cells. Transepithelial electrical resistance (TEER) measurements, as an indicator of the opening of tight junctions, and transport experiments with fluorescein-Na (Flu) and FITC-dextran Mw 4000 (FD-4) were used to assess the effect. (+/-) Verapamil concentrations up to 3 x 10(-4) M increased TEER dose-dependently, whereas from concentrations of 7 x 10(-4) M onwards a dose-dependent drop was found. After removal of verapamil (< 10(-3) M) the effects on TEER were reversible within 30 min. A second administration of verapamil after different time intervals produced a much larger effect on TEER than the first administration. The separate R- and S-enantiomers did not reveal a difference in enantiomer effect. (+/-) Verapamil at 7 x 10(-4) M increased Flu transport about 13-fold and 26-fold after the first and second treatment in the same monolayers, respectively. Transport of FD-4 increased approximately 4-fold and 6-fold after the first and second treatment, respectively. Potential damaging effects were assessed by trypan blue exclusion (cell death) and cell detachment. No cell death occurred at verapamil concentrations of 8.5 x 10(-4) M or lower, whereas cell detachment did not occur within 1 hr at all concentrations used in these experiments. At later times detachment was observed at concentrations of 7 x 10(-4) M and higher. Confocal laser scanning microscopy showed that verapamil opens the paracellular route, thereby enhancing the permeability of hydrophilic compounds. However, relatively high concentrations are needed to achieve this effect and only a narrow concentration range can be used without cytotoxic effects, which limits the potential application of verapamil as an absorption enhancing agent.

Effect of anisotonic conditions on the transport of hydrophilic model compounds across monolayers of human colonic cell lines.

The effect of anisotonic solutions on the enhancement of the transport of hydrophilic model compounds across monolayers of Caco-2 and HT-29.cl19A intestinal epithelial cells was studied. In filter-grown monolayers of the highly differentiated villus-like Caco-2 cell line, a profound and dose-dependent drop in the transepithelial electrical resistance was found after apical treatment with a 30 or a 50% hypotonic solution (200 and 150 mOsmol, respectively). This drop was not observed after basolateral and two-sided application of a 50% hypotonic solution. During apical hypotonic treatment a 12- and 8-fold increase also was observed in transepithelial transport of two hydrophilic model compounds, i.e., fluorescein-Na and fluorescein-isothiocyanate-labeled dextran, MW 4000, respectively. Through confocal laser scanning microscopy, it was revealed that this enhanced transport was predominantly via the paracellular route. Moreover, morphological changes in the cell layers indicating cell swelling were observed after apical hypotonic, but not after basolateral or bilateral treatment, probably resulting from an incomplete regulatory volume decrease response. This swelling, and slight lateral retraction of the cells, allowed the hydrophilic compounds to pass between the cells. The effects of hypotonic challenge also were studied in monolayers of the more crypt cell-like HT-29.cl19A cell line. After apical hypotonic shock, these cells showed no effect on transepithelial electrical resistance, whereas an increase was observed after basolateral and bilateral treatment. Hypotonic shock failed to increase the transport of the hydrophilic model compounds in this cell line.(ABSTRACT TRUNCATED AT 250 WORDS)

Lymphocyte adhesion to brain capillary endothelial cells in vitro.

The presence and upregulation of adhesion molecules on bovine brain endothelial cells (BBEC) were investigated. Monolayers of BBEC were incubated with lipopolysaccharide (LPS), interleukin-1 beta (rhIL-1 beta), and interleukin-6 (rhIL-6) to simulate in vitro an inflammatory site in the cerebral capillaries. Adhesion of lymphocytes to BBEC increased 4.1-fold after stimulation of the endothelial cells for 4 h with 5 or 10 ng/ml LPS. Lymphocyte adhesion increased after incubation of the BBEC for 4 h with IL-1 and was increased 3.7-fold using 100 ng/ml IL-1. BBEC pre-incubated with IL-6 for 4 h also showed an increase in adhesion of lymphocytes, and cells pretreated with 100 ng/ml IL-6 showed a 3-fold increase in lymphocyte adherence. Specific monoclonal antibodies directed against CD11a, CD18, and VLA-4 were able to block adherence of lymphocytes to stimulated BBEC. These results indicate that the in vitro activation of BBEC may serve as a model for the study of inflammation of the blood-brain barrier.

Permeability enhancement in Caco-2 cell monolayers by sodium salicylate and sodium taurodihydrofusidate: assessment of effect-reversibility and imaging of transepithelial transport routes by confocal laser scanning microscopy.

The effects of sodium salicylate and sodium tauro-24,25-dihydrofusidate (STDHF) on the aqueous permeability of confluent monolayers of Caco-2 cells were studied. Measurements of transepithelial electrical resistance (TEER) showed a concentration-dependent effect of both compounds after apical incubation for 1 hr. Reductions in TEER resulting from EC50 concentrations (2.8 mM for STDHF; 173 mM for salicylate) were reversible within 5.75 hr. The transpithelial fluxes of two hydrophilic model compounds, sodium fluorescein F (molecular weight 376) and a fluorescein isothiocyanate-labeled dextran (mean molecular weight 4000) was significantly increased by STDHF (2.8 mM). Sodium salicylate (173 mM) only enhanced the transport of sodium fluorescein significantly. At the EC50 concentrations, confocal laser scanning microscopy (CLSM) visualized both fluorescent tracers mainly in the paracellular route. With higher enhancer concentrations (373 mM sodium salicylate and 8 mM STDHF), both transport markers appeared intracellularly as a result of cell death. STDHF rapidly extracted an exogenous lipophilic membrane probe, 5-(N-hexadecanoyl)aminofluorescein (HEDAF), from the apical part of Caco-2 plasma membranes, indicating qualitatively that STDHF interacts with the lipid portion of cell membranes. These results suggest that both sodium salicylate and STDHF can be used to reversibly increase paracellular permeability of Caco-2 cell monolayers, whereby STDHF appears to be advantageous compared to sodium salicylate. By adapting the Costar cell culture system to CLSM, we have shown that this technique is suitable to study membrane interactions qualitatively and for visualizing transport routes of hydrophilic tracers through nonfixed, filter-grown monolayers.

Grapefruit juice and cimetidine inhibit stereoselective metabolism of nitrendipine in humans.

The effects of grapefruit juice (150 ml at -15, -10, -1/4, +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.

High-performance liquid chromatographic determination of the polar metabolites of nifedipine in plasma, blood and urine.

A relatively simple reversed-phase high-performance liquid chromatographic method for the determination of the polar metabolites of nifedipine in biological fluids is described. After conversion of 2-hydroxymethyl-6-methyl-4-(2-nitrophenyl)pyridine-3,5-dicarboxylic acid 5-methyl ester (IV) into 5,7-dihydro-2-methyl-4-(2-nitrophenyl)-5-oxofuro[3,4-b] pyridine-3-carboxylic acid methyl ester (V) by heating under acidic conditions, V was extracted with n-pentane-dichloromethane (7:3) and analysed on a C18 column with ultraviolet detection. Subsequently, 2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic acid monomethyl ester (III) was extracted with chloroform and analysed on the same system. Limits of determination in blood were 0.1 microgram/ml for III and 0.05 microgram/ml for IV and V; these limits were two to ten times higher for urine. This inter-assay variability was always less than 7.5%.

Analysis of nifedipine and its pyridine metabolite dehydronifedipine in blood and plasma: review and improved high-performance liquid chromatographic methodology.

A reversed-phase HPLC method is described for the simultaneous determination of nifedipine and its primary pyridine metabolite dehydronifedipine in blood and plasma, that involves UV detection and neutral (blood) or alkaline (plasma) extraction. The limit of reliable determination is found to be 3 ng ml-1 with an inter-assay RSD of below 11%. In the presence of haemoglobin, nifedipine is unstable at pH greater than 10, necessitating neutral extraction for the measurement of nifedipine in haemolysed blood. Published methods for analysis of nifedipine are reviewed, emphasizing the lack of specificity and sensitivity which render many of them unsuitable for the investigation of nifedipine disposition in man.

Depression of force by phosphate in skinned skeletal muscle fibers of the frog.

The relation between isometric force and phosphate concentration in skinned skeletal muscle fibers of the frog is found to depend on fiber size. Force decreased with increasing phosphate concentration, but depression of force in thick fibers was smaller than in thin segments. When the external phosphate concentration was abruptly altered during a sustained contracture, force changed. The half-time of the force change was proportional to the cross-sectional area of the preparation. From this relation, a value for the diffusion constant of phosphate in skinned fibers of 0.9 x 10(-10) m2/s was derived. The rate of phosphate production was determined photometrically via the enzymatic coupling of the resynthesis of ATP to the oxidation of nicotinamide adenine dinucleotide. The average value (+/- SE) of the rate of ATP hydrolysis (at 4 degrees C) was 2.7 +/- 0.3 mumol.s-1.g dry wt-1, which corresponds to 0.34 mmol.l-1.s-1. From a calculation based on the diffusion constant and the rate of phosphate production determined, it follows that the dependency of the force-phosphate relation on fiber diameter is due to phosphate accumulation inside the fiber.

Enantioselective determination of felodipine and other chiral dihydropyridine calcium entry blockers in human plasma.

A sensitive method for the enantioselective determination of felodipine in human plasma is described. Following alkaline extraction with dichloromethane-pentane, racemic felodipine and its primary pyridine metabolite are simultaneously assayed using capillary gas chromatography on a DB-1 column, with electron-capture detection. The enantiomers of felodipine are quantitatively separated by high-performance liquid chromatography on a Chiralcel OJ column, containing tris(4-methylbenzoate)-modified cellulose coated on silica, and off-line detection using the same gas chromatographic system is applied. The limits of determination in plasma (and the inter-assay coefficient of variation (C.V.) at levels below 1 ng/ml) were 0.1 ng/ml (C.V. 13%) for felodipine, 0.1 ng/ml (C.V. 15%) for the enantiomers of felodipine and 0.3 ng/ml (C.V. 7%) for its pyridine metabolite. The method has proved to be applicable to several other chiral dihydropyridine calcium entry blockers, including nitrendipine, with comparable sensitivities.