Albertus G. de Boer

The potential of mucoadhesive polymers in enhancing intestinal peptide drug absorption. III: Effects of chitosan-glutamate and carbomer on epithelial tight junctions in vitro

Two mucoadhesive polymers, chitosan-glutamate and carbomer, were studied in an in vitro model (Caco-2 cell monolayers) with respect to their ability to enhance intestinal peptide drug delivery. Preparations of the polymers at concentrations of 0.5, 1.0, and 1.5% w/v (chitosan), and of 0.5 and 1.0% w/v (carbomer) were applied to the apical side of Caco-2 cell monolayers. The effects on transepithelial electrical resistance (TEER), paracellular transport of a FITC-dextran of a molecular weight of 4400 (FD-4) and [14C]mannitol were measured. Paracellular transport of FD-4 was visualized by means of confocal laser scanning microscopy (CLSM). Furthermore, the impact of lowering the pH of the polymer solutions to pH 4 on the integrity of the cell layer was determined. The results show that both polymers were able to decrease TEER of Caco-2 cell layers significantly. In the case of carbomer, CLSM revealed a partial opening of epithelial tight junctions. Lowering of the pH in the control and polymer solutions to pH 4 resulted in every case in the irreversible damage of a large percentage of the cells, as shown by CLSM. Transport studies with [14C]mannitol and FD-4 showed only during co-application of carbomer significantly increased fluxes, whereas no difference from the control solution could be detected for chitosan-glutamate. A threshold value of about 50% of TEER reduction has been identified, which allows for transport of hydrophilic compounds across the cell monolayers of the Caco-2 cell model.

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.

The role of P-glycoprotein in blood-brain barrier transport of morphine: transcortical microdialysis studies in mdr1a (-/-) and mdr1a (+/+) mice.

The aim of this study was to investigate whether blood‐brain barrier transport of morphine was affected by the absence of mdr1a‐encoded P‐glycoprotein (Pgp), by comparing mdr1a (−/−) mice with mdr1a (+/+) mice. Mdr1a (−/−) and (+/+) mice received a constant infusion of morphine for 1, 2 or 4 h (9 nmol/min/mouse). Microdialysis was used to estimate morphine unbound concentrations in brain extracellular fluid during the 4 h infusion. Two methods of estimating in vivo recovery were used: retrodialysis with nalorphine as a calibrator, and the dynamic‐no‐net‐flux method. Retrodialysis loss of morphine and nalorphine was similar in vivo. Unbound brain extracellular fluid concentration ratios of (−/−)/(+/+) were 2.7 for retrodialysis and 3.6 for the dynamic‐no‐net‐flux at 4 h, with corresponding total brain concentration ratios of (−/−)/(+/+) being 2.3 for retrodialysis and 2.6 for the dynamic‐no‐net‐flux. The total concentration ratios of brain/plasma were 1.1 and 0.5 for mdr1a (−/−) and (+/+) mice, respectively. No significant differences in the pharmacokinetics of the metabolite morphine‐3‐glucoronide were observed between (−/−) and (+/+) mice. In conclusion, comparison between mdr1a (−/−) and (+/+) mice indicates that Pgp participates in regulating the amount of morphine transport across the blood‐brain barrier.

Targeted delivery across the blood–brain barrier

The safest and most effective way of targeting drugs to the entire brain is via delivery systems directed at endogenous receptor-mediated uptake mechanisms present at the cerebral capillaries. Such systems have been shown to be effective in animal models including primates, but no clinical trials have been performed so far. This review focuses on the well-characterised transferrin and insulin receptor-targeted systems, as well as on the more recently described systems that use the low-density lipoprotein-related protein 1 receptor, the low-density lipoprotein-related protein 2 receptor (also known as megalin and glycoprotein 330) or the diphtheria toxin receptor (which is the membrane-bound precursor of heparin-binding epidermal growth factor-like growth factor). The possibilities and limitations of these systems are compared and their future for human application is discussed.

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

ObjectiveTo 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. MethodsThe 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. ResultsAll 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. ConclusionsAmprenavir, 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.

Relationship between permeability status of the blood–brain barrier and in vitro permeability coefficient of a drug

OBJECTIVE The aim was to test the hypothesis that the assessment of basal and drug-induced changes in permeability of the blood-brain barrier (BBB) during in vitro drug transport assays is essential for an accurate estimation of the permeability coefficient of a drug. METHODS An in vitro BBB model was used, comprising of brain capillary endothelial cells (BCEC) and astrocytes co-cultured on semi-permeable filter inserts. Experiments were performed under control and challenged experimental circumstances, induced to simulate drug effects. The apparent BBB permeability coefficient for two markers for paracellular drug transport, sodium fluorescein (P(app,FLU), M(w) 376 Da) and FITC-labeled dextran (P(app,FD4), M(w) 4 kDa), was determined. Transendothelial electrical resistance (TEER) was used to quantify basal and (simulated) drug-induced changes in permeability of the in vitro BBB. The relationship between P(app) and TEER was determined. Drug effects were simulated by exposure to physiologically active endogenous and exogenous substances (i.e., histamine, deferroxamine mesylate, adrenaline, noradrenaline, bradykinin, vinblastine, sodium nitroprusside and lipopolysaccharide). RESULTS P(app,FLU) and P(app,FD4) in control experiments varied from 1.6 up to 17.6 (10(-6)cm/s) and 0.3 up to 7. 3 (10(-6)cm/s), respectively; while for individual filters P(app, FLU) was 4 times higher than P(app,FD4) (R(2)=0.97). As long as TEER remained above 131.Omega cm(2) for FLU or 122.Omega cm(2) for FD4 during the transport assay, P(app) remained independent from the basal permeability of the in vitro BBB. Below these TEER values, P(app) increased exponentially. This nonlinear relationship between basal BBB permeability and P(app) was described by a one-phase exponential decay model. From this model the BBB permeability status independent permeability coefficients for FLU and FD4 (P(FLU) and P(FD4)) were estimated to be 2.2+/-0.1 and 0.48+/-0.03 (10(-6)cm/s), respectively. In the experimentally challenged experiments, a reliable indication for P(FLU) and P(FD4) could be estimated only after the (simulated) drug-induced change in BBB permeability was taken into account. CONCLUSIONS The assessment of basal BBB permeability status during drug transport assays was essential for an accurate estimation of the in vitro permeability coefficient of a drug. To accurately extrapolate the in vitro permeability coefficient of a drug to the in vivo situation, it is essential that drug-induced changes in the in vitro BBB permeability during the drug transport assay are determined.

Astrocytes Increase the Functional Expression of P-Glycoprotein in an In Vitro Model of The Blood-Brain Barrier

AbstractPurpose. To investigate the influence of astrocytes on P-glycoprotein (Pgp) expression and intracellular accumulation of Pgp substrates, separate from their net transcellular transport across the blood-brain barrier (BBB). Methods. An in vitroBBB model was used, comprising of brain capillary endothelial cells (BCEC) monolayers or BCEC co-cultured with astrocytes. Results. BCEC+astrocyte co-cultures seemed to express a higher level of Pgp compared to BCEC monolayers. Inhibition of Pgp results in an increased intracellular accumulation of Pgp substrates in both BCEC monolayers and BCEC+astrocyte co-cultures, and increased the sensitivity for vinblastine mediated disruption of the in vitro BBB (called the vinblastine exclusion assay). BCEC monolayers were more sensitive to vinblastine mediated disruption compared to BCEC+astrocyte co-cultures. In the latter, but not in BCEC monolayers, an inhibitable polar transport of Pgp substrates was only found from the brain to the blood side of the filter. Conclusions. Astrocytes increase the functional expression of Pgp in our in vitroBBB model. These results also illustrate that an important role for Pgp on the BBB is to protect the barrier against intracellular accumulation of cytotoxic BBB disrupting compounds.

Effects of the Mucoadhesive Polymer Polycarbophil on the Intestinal Absorption of a Peptide Drug in the Rat

Abstract— The absorption across rat intestinal tissue of the model peptide drug 9‐desglycinamide, 8‐arginine vasopressin from bioadhesive formulations was studied in‐vitro, in a chronically isolated internal loop in‐situ and after intraduodenal administration in‐vivo. A controlled‐release bioadhesive drug delivery system was tested, consisting of microspheres of poly(2‐hydroxyethyl methacrylate) with a mucoadhesive Polycarbophil‐coating, as well as a fast‐release formulation consisting of an aqueous solution of the peptide in a suspension of Polycarbophil particles. Using the controlled‐release system, a slight improvement of peptide absorption was found in‐vitro in comparison with a non‐adhesive control system, but not in‐situ or in‐vivo. In contrast, bioavailability was significantly increased in all three models from the Polycarbophil suspension in comparison with a solution of the drug in saline. The effect appeared to be dose‐dependent, indicative of intrinsic penetration‐enhancing properties of the mucoadhesive polymer. A prolongation of the absorption phase in‐vitro and in the chronically isolated loop in‐situ suggested that the polymer was able to protect the peptide from proteolytic degradation. This could be confirmed by degradation studies in‐vitro. The duration of the penetration enhancing/enzyme inhibiting effect was diminished with increasing complexity of the test model, in the same way as was previously found for the bioadhesive effect. This interrelationship suggests that the observed improvement in peptide absorption and the mucoadhesive properties of this polymer are associated. The development of a fast‐release oral dosage form for peptide drugs on the basis of Polycarbophil appears to be possible.

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.

Critical factors of intracerebral microdialysis as a technique to determined the pharmacokinetics of drugs in rat brain

The purpose of this investigation was to determine the effect of experimental conditions on the concentrations of atenolol and acetaminophen in brain microdialysate, and to investigate the feasibility of performing repeated experiments within individual rats. Following intravenous bolus administration, reproducible concentration-time profiles were obtained in plasma and in brain dialysate. Based on corrections for in vitro recoveries of the intracerebral probe, the estimated ratio of the AUC in brain extracellular fluid (AUCbrain ECF) over the AUC in plasma (AUCplasma) +/- S.E.M. was 3.8 +/- 0.6% (n = 6) for atenolol and 18 +/- 2% (n = 6) for acetaminophen. Upon intracerebroventricular administration, interanimal differences in kinetics of acetaminophen in brain dialysate were observed while the concentrations of atenolol were below the detection limit of the assay. The influence of the use of isotonic versus hypotonic perfusate solutions on AUCbrain ECF values after intravenous bolus administration of both drugs was determined. Repeated experiments with the isotonic perfusate (24, 48 and 78 h post-surgery) resulted in AUCbrain ECF values with the ratio of 100: 98: 76% for acetaminophen and 100: 103: 98% for atenolol. Using a hypotonic perfusion solution the ratio of AUCbrain ECF values was 100: 154: 114% for acetaminophen and 100: 378: 427% for atenolol. A clear effect of the temperature of the hypotonic perfusate (24 vs 38 degrees C) on acetaminophen AUCbrain ECF values was revealed. The ratio of AUCbrain ECF values obtained at 24: 38 degrees C was 192: 100%.(ABSTRACT TRUNCATED AT 250 WORDS)