Induction of human P-glycoprotein in Caco-2 cells: development of a highly sensitive assay system for P-glycoprotein-mediated drug transport.

The aim of this work is to develop a highly sensitive assay system for P-gp-mediated transport by using two methods, induction of P-gp and short-term culture of Caco-2 cells. To induce P-gp in Caco-2 cells, cells were cultured in vinblastine-containing medium. The mRNA level of P-gp was approximately 7-fold higher in Caco-2 cells cultured with vinblastine (P-gp-induced Caco-2 cells) than in control cells.

Bidirectional membrane transport: simulations of transport inhibition in uptake studies explain data obtained with flavonoids.

The purpose of the simulations was to obtain an estimate of concentration-dependent uptake curves when two counteracting transporters are present. On the basis of this experimental data obtained with a pair of ovarian carcinoma cell lines, one of which was not expressing the exsorptive transporter P-glycoprotein and one of which was an MDR1-transfected, P-glycoprotein expressing variant, the kinetics of cellular uptake of the radiolabel (3)H-talinolol were calculated and the inhibitory constants at P-gp were determined for different flavonoids. With respect to the inhibition of P-gp function, among others, naringenin and isoquercitrin were identified as inhibitors, yet estimation of the inhibitory constant was only possible for uptake values corrected for non-P-glycoprotein-mediated processes.

In silico modeling of non-linear drug absorption for the P-gp substrate talinolol and of consequences for the resulting pharmacodynamic effect.

Purpose: The aim of the present work was to demonstrate P-glycoprotein's involvement in the non-linear talinolol pharmacokinetics using an advanced compartment and transit model (ACAT) and to compare the results predicted from the model to the finding of a phase I dose escalation study with oral talinolol doses increasing from 25 to 400 mg.

Materials And Methods: Besides minimum input parameters for the compound (pKa(s), solubility at one or more pH's, Peff, doses, formulation, diffusivity), physiological and pharmacokinetic properties, transporter data are included in these predictions. The simulations assumed higher expression levels in lower gastrointestinal regions, in particular in the colon, which is in accordance with the results of intestinal rat perfusion studies and intestinal distribution data from rats, catfishes, micropigs and humans reported in the literature.

Effects of controlled-release on the pharmacokinetics and absorption characteristics of a compound undergoing intestinal efflux in humans.

Objective: The number of active pharmaceutical ingredients (API) undergoing inhibitable and saturable intestinal efflux is considerable. As a consequence, absorption and bioavailability may depend on the intestinal concentration profile of the drug and may vary as a function of dose and release rate of the drug from the dosage form. The impact of controlled versus immediate-release on the absorption of P-glycoprotein substrates is currently unknown.

High-performance liquid chromatography of lactose with evaporative light scattering detection, applied to determine fine particle dose of carrier in dry powder inhalation products.

A method for quantification of the fine particle dose of lactose is described, using a hydrophilic interaction chromatography (HILIC) method and evaporative light scattering detection. The HILIC method used an aminopropyl column and a mobile phase consisting of acetonitril/water (80/20, v/v) for isocratic elution. Sensitive chromatography was obtained using a low concentration of water in the extraction solvent.

Cyclosporine A (CsA) affects the pharmacodynamics and pharmacokinetics of the atypical antipsychotic amisulpride probably via inhibition of P-glycoprotein (P-gp).

The importance of P-glycoprotein (P-gp) in the pharmacokinetics of amisulpride and the effects of a P-gp inhibitor cyclosporine A (CsA) was investigated both, in vitro and in vivo. In vitro and in vivo results indicated amisulpride as a substrate of P-gp. Amisulpride was not metabolized by rat liver microsomes.

Modulation of drug transport by selected flavonoids: Involvement of P-gp and OCT?

Flavonoids, as a common component of daily nutrition, are a possible source of interference with absorption processes, due to modulation of transporting proteins. In this study, the influence of selected flavonoids (quercetin, isoquercitrin, spiraeoside, rutin, kaempferol, naringenin, naringin, and kaempferol) on the transport of the P-gp substrate [3H]talinolol across Caco-2 cell monolayers was investigated. To elucidate the mechanism behind the interaction observed in this system the potency of the flavonoids to replace [3H]talinolol from its P-gp binding site as well as their activity to inhibit OCT2-mediated [14C]TEA uptake into LLC-PK(1) cells were measured, as P-gp and OCT have been shown to be present in Caco-2 cells.

Nanosuspension formulations for low-soluble drugs: pharmacokinetic evaluation using spironolactone as model compound.

Various particle sizes of spironolactone as a model low solubility drug were formulated to yield micro-and nanosuspensions of the type solid lipid nanoparticles and DissoCubes. Seven oral and one i.v.

Selective downregulation of the MDR1 gene product in Caco-2 cells by stable transfection to prove its relevance in secretory drug transport.

Considerable interest is focused on overcoming multidrug resistance (MDR) in cancer chemotherapy. The in vitro experiments to characterize P-glycoprotein's (P-gp) function and to decrease its effects have led to a variety of strategies such as addition of competitors or supplementation of the medium with oligonucleotides complementary to the 5'-end of the MDR1-mRNA. For the Caco-2 cell line, an in vitro model for absorption screening, expressing multiple transporters including P-gp, which pumps substances back into the apical solution, P-gp activity might mask other relevant transport proteins' activity.

Identification of P-glycoprotein substrates and inhibitors among psychoactive compounds--implications for pharmacokinetics of selected substrates.

The pharmacokinetics of antipsychotic drugs has become an integral part in understanding their pharmacodynamic activity and clinical effects. In addition to metabolism aspects, carrier-mediated transport, particularly secretion by ABC transporters, has been discussed as potentially relevant for this group of therapeutics. In this study, the psychoactive compounds perphenazine, flupentixol, domperidone, desmethyl clozapine, haloperidol, fluphenazine, fluvoxamine, olanzapine, levomepromazine, perazine, desmethyl perazine, clozapine, quetiapine and amisulpride were characterized in terms of P-glycoprotein (P-gp) affinity and transport.

In vivo evaluation of a novel pH- and time-based multiunit colonic drug delivery system.

Background: Targeted drug delivery to the colon is important for topical treatment of inflammatory bowel diseases. Established targeting systems predominantly focus on either pH- or time-dependent release, or bacterial degradation.

Aim: To perform a three-phase, crossover design trial evaluating a novel combined pH- and time-based multiunit delivery system.

Development of a sterilizing in-place application for a production machine using Vaporized Hydrogen Peroxide.

The use of steam in sterilization processes is limited by the implementation of heat-sensitive components inside the machines to be sterilized. Alternative low-temperature sterilization methods need to be found and their suitability evaluated. Vaporized Hydrogen Peroxide (VHP) technology was adapted for a production machine consisting of highly sensitive pressure sensors and thermo-labile air tube systems.

Conditioning following powder micronization: influence on particle growth of salbutamol sulfate.

Micronization is a high-energy process that induces changes in the crystallinity of materials. As a result, the crystalline structures on the particles' surface are being destroyed and amorphous areas are formed. After micronization of salbutamol sulfate to be used in dry powder inhalers, only small amounts of amorphous material are produced.

Influence of mechanical activation on the physical stability of salbutamol sulphate.

In order to obtain the optimal particle size distribution for pharmaceutical powders in dry powder inhalers the particles have to be micronised. In most cases the process of micronisation is connected with a high input of energy which induces disorder and defects on the surface of the drug particles and as a result changes in the crystallinity. Consequently, changes in the physical stability of the powders may occur.

Different dissolution media lead to different crystal structures of talinolol with impact on its dissolution and solubility.

During the performance of dissolution tests with immediate and controlled-release talinolol tablets it was detected that the type of the buffer used as dissolution medium had a strong influence on the solubility and the dissolution behavior of the drug. It was proven that talinolol appeared in different crystal structures with strongly differing solubilities when pure water, acetate, or phosphate buffers were employed as dissolution media. The resulting crystal structures were characterized by means of light microscopy, differential scanning calorimetry, and X-ray powder diffraction.

Presystemic metabolism and intestinal absorption of antipsoriatic fumaric acid esters.

Psoriasis is a chronic inflammatory skin disease. Its treatment is based on the inhibition of proliferation of epidermal cells and interference in the inflammatory process. A new systemic antipsoriasis drug, which consists of dimethylfumarate and ethylhydrogenfumarate in the form of their calcium, magnesium and zinc salts has been introduced in Europe with successful results.

How does the benzamide antipsychotic amisulpride get into the brain?--An in vitro approach comparing amisulpride with clozapine.

This study evaluated the disposition of the two atypical antipsychotics, amisulpride (AMS) and clozapine (CLZ), and its main metabolite N-desmethylclozapine (DCLZ), to their target structures in the central nervous system by applying an in vitro blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier based on monolayers of porcine brain microvessel endothelial cells (PMEC) or porcine choroid plexus epithelial cells (PCEC). Permeation studies through PMEC- and PCEC-monolayers were conducted for 60 min at drug concentrations of 1, 5, 10, and 30 muM applied to the donor compartment. PMEC were almost impermeable for AMS (permeation coefficient, P<1 x 10(-7) cm/s) in the resorptive direction, whereas transport in the secretory direction was observed with a P (+/-SD) of 5.

Lipophilicities of baclofen ester prodrugs correlate with affinities to the ATP-dependent efflux pump P-glycoprotein: relevance for their permeation across the blood-brain barrier?

Purpose: Distribution to the effect site is a prerequisite for the therapeutic effect and determined by physicochemical properties and affinities to inside- and outside-directed membrane transporters. Based on the hypothesis that lipophilic esters of the GABA-derivative baclofen have a higher affinity to brain tissue, baclofen esters (methyl, ethyl, 1-propyl, 2-propyl, butyl) were studied regarding their penetration through the blood-brain barrier and their affinities to P-glycoprotein (P-gp).

Methods: Octanol-water distribution coefficients (D) served as lipophilicity parameters.

Transfer of lipophilic markers from PLGA and polystyrene nanoparticles to caco-2 monolayers mimics particle uptake.

Purpose: The objective of this study was to evaluate nanoparticle uptake by the Caco-2 monolayer model in vitro. Special emphasis was placed on the localization and the quantification of the uptake of fluorescently labeled polystyrene and poly(lactic-co-glycolic acid) (PLGA) nanoparticles.

Methods: Intracellular fluorescence was localized by fluorescence and confocal laser scanning microscopy.

Compromised integrity of excised porcine intestinal epithelium obtained from the abattoir affects the outcome of in vitro particle uptake studies.

Excised porcine intestinal tissue obtained from the local abattoir was studied for its suitability to examine the uptake and transport of poly(lactic-co-glycolic acid) (PLGA) nanoparticles in Peyer's (PP) and non-Peyer's patch (NPP) tissue in vitro. Incubation of such tissue with fluorescent PLGA and polystyrene particles revealed negligible uptake into the intercellular space with no noticeable difference between PP and NPP tissue. Similarly, yeast cells, which were used as a positive control for selective uptake into PP tissue, were found in the subepithelial area of both PP and NPP tissue.

In vitro assessment of intestinal IGF-I stability.

Insulin-like growth factor (IGF-I) is a 7648-Da polypeptide consisting of 70 amino acids. Clinically, IGF-I might be used in type II diabetes, which requires a life-long treatment. Therefore, delivery routes other than parenteral injections are highly desirable.

Intestinal drug efflux: formulation and food effects.

The intestine, primarily regarded as an absorptive organ, is also prepared for the elimination of certain organic acids, bases and neutral compounds depending on their affinity to intestinal carrier systems. Several of the transport systems known to mediate efflux in the major clearing organs--liver and kidney--are also expressed in the intestine. Examples of secretory transporters in the intestine are P-glycoprotein, members of the multidrug resistance associated protein family, breast cancer resistance protein, organic cation transporters and members of the organic anion polypeptide family.

Compound mixtures in Caco-2 cell permeability screens as a means to increase screening capacity.

The purpose of this investigation was a study of simultaneous permeability measurement using compound mixtures (cassette dosing) as an alternative to single compound evaluation in order to increase the capacity of screens for intestinal drug permeability. Drug transport across Caco-2 monolayers was studied, both in the apical to basolateral and the basolateral to apical direction. The apparent permeability coefficients for ten compounds displaying different intestinal transport mechanisms were determined, first as single compounds and then as components of a mixture.

Pretreatment with potent P-glycoprotein ligands may increase intestinal secretion in rats.

The expression of P-glycoprotein is induced in cell cultures upon exposure to various inducers. Therefore, the aim of the present study was to evaluate the in-vivo relevance of this observation, i.e.

Grapefruit juice enhances intestinal absorption of the P-glycoprotein substrate talinolol.

Grapefruit juice (GFJ) is known to affect the pharmacokinetics of various drugs, presumably mainly via inhibition of oxidative metabolism. In order to evaluate the effect of GFJ on P-glycoprotein-related transport processes, measurements of transport characteristics through Caco-2 monolayers and in vivo drug absorption studies were performed with the transported, yet not metabolized model compound talinolol. Apical-to-basolateral talinolol transport in the Caco-2 model at 1 mM racemate concentration was increased almost 3-fold when GFJ was present (S-talinolol P(eff): 0.