Anna-Lena Ungell

Expression of Thirty-six Drug Transporter Genes in Human Intestine, Liver, Kidney, and Organotypic Cell Lines

This study was designed to quantitatively assess the mRNA expression of 36 important drug transporters in human jejunum, colon, liver, and kidney. Expression of these transporters in human organs was compared with expression in commonly used cell lines (Caco-2, HepG2, and Caki-1) originating from these organs to assess their value as in vitro transporter system models, and was also compared with data obtained from the literature on expression in rat tissues to assess species differences. Transporters that were highly expressed in the intestine included HPT1, PEPT1, BCRP, MRP2, and MDR1, whereas, in the liver, OCT1, MRP2, OATP-C, NTCP and BSEP were the main transporters. In the kidney, OAT1 was expressed at the highest levels, followed by OAT3, OAT4, MCT5, MDR1, MRP2, OCT2, and OCTN2. The best agreement between human tissue and the representative cell line was observed for human jejunum and Caco-2 cells. Expression in liver and kidney ortholog cell lines was not correlated with that in the associated tissue. Comparisons with rat transporter gene expression revealed significant species differences. Our results allowed a comprehensive quantitative comparison of drug transporter expression in human intestine, liver, and kidney. We suggest that it would be beneficial for predictive pharmacokinetic research to focus on the most highly expressed transporters. We hope that our comparison of rat and human tissue will help to explain the observed species differences in in vivo models, increase understanding of the impact of active transport processes on pharmacokinetics and distribution, and improve the quality of predictions from animal studies to humans.

Characterization of fluids from the stomach and proximal jejunum in men and women

AbstractPurpose. To chemically characterize the fluids available for drug dissolution in the upper gastrointestinal tract during the fasted state in humans, and to examine variations and potential gender differences regarding the physico-chemical properties of these fluids. Methods. Twenty-four healthy volunteers, 12 females and 12 males, were intubated, and fluids from the stomach and upper jejunum were collected separately. Bulk pH, osmolality, electrolytes and total concentrations of bile acids and proteins were assessed. To study intraindividual variations, eleven of the individuals were studied on more than one occasion. Results. The stomach and upper jejunal fluids varied significantly in all the measured entities, except the total concentration of proteins. The intraindividual variability was pronounced in some of the individuals, both in the stomach and the upper jejunum. We did not, however, observe any gender differences. Conclusions. This study demonstrates the complex nature of the fluids available for drug dissolution in the stomach and the upper small intestine in humans. The results can be used when designing a more physiological in vitro dissolution media representative for the fasted state. When designing such a medium, we suggest that gender differences not be taken into account.

Selective paracellular permeability in two models of intestinal absorption : cultured monolayers of human intestinal epithelial cells and rat intestinal segments

New data on the permeabilities of hydrophilic markers in two commonly used in vitro models, i.e., excised intestinal segments from the rat and monolayers of Caco-2 cells, are presented. The results are compared to human in vivo data. Two groups of hydrophilic marker molecules were tested: (1) monodisperse polyethylene glycols of molecular weights ranging from 194 to 502 g/mol and (2) a heterogeneous group of molecules consisting of urea, creatinine, erythritol, and mannitol (60–182 g/mol). The permeabilities of the marker molecules showed a nonlinear dependence on the molecular weight and decreased in the order rat ileum > rat colon > Caco-2 cells. Surprisingly, the polyethylene glycols permeated more easily than the other marker molecules, indicating that characteristics other than molecular weight, e.g., the flexibility of the structure, may also be important for permeation through the membrane. Comparisons with the published permeability profiles of polyethylene glycols in human intestinal segments in vivo (i.e., calculated permeability coefficients as a function of molecular weight) indicate that the human intestine is more permeable than the in vitro models. However, the permeability profiles of the corresponding segments in the human intestine and the in vitro models were comparable. Thus, good correlations were established between permeabilities of the human ileum and rat ileum and between those of human colon, rat colon, and the Caco-2 cells. We conclude that the paracellular absorption in humans can be studied mechanistically in these in vitro models.

Evaluation of viability of excised rat intestinal segments in the Ussing chamber: investigation of morphology, electrical parameters, and permeability characteristics.

AbstractPurpose. To clarify relations between alterations in electrical and permeability data with time and to elaborate accompanying structural changes of intestinal segments in Ussing chamber experiments. Methods. Excised intestinal segments from the rat were studied in a modified Ussing chamber. Experiments were run up to 180 minutes during which the electrical parameters, PD, SCC, and R, were measured and the permeability coefficients (Papp) of mannitol and propranolol were determined. Each segment was observed under the light microscope for morphological evaluation. Results. PD and SCC values showed a decrease for most segments while the R values remained steady throughout the experiment. The Papp for propranolol increased aborally to the small intestine. For mannitol, the reversed was observed. In some cases, there was a time-dependent change in permeability for these marker molecules. The main morphological changes observed were a decreased nucleo-apical distance, decreased villi amplification factor, initial edema, cell sloughing, and epithelial restitution. Conclusions. The time-dependent changes in permeability coefficients of mannitol and propranolol are suggested to be related to changes in electrical parameters and morphological alterations. Presented data illustrates the importance of information regarding time-dependent structural changes for correct interpretation of permeability data.

Innovative methods to study human intestinal drug metabolism in vitro: precision-cut slices compared with ussing chamber preparations.

Predictive in vitro methods to investigate drug metabolism in the human intestine using intact tissue are of high importance. Therefore, we studied the metabolic activity of human small intestinal and colon slices and compared it with the metabolic activity of the same human intestinal segments using the Ussing chamber technique. The metabolic activity was evaluated using substrates to both phase I and phase II reactions: testosterone, 7-hydroxycoumarin (7HC), and a mixture of cytochrome P450 (P450) substrates (midazolam, diclofenac, coumarin, and bufuralol). In slices of human proximal jejunum, the metabolic activity of several P450-mediated and conjugation reactions remained constant up to4hof incubation. In the colon slices, conjugation rates were virtually equal to those in small intestine, whereas P450-mediated conversions occurred much slower. In both organs, morphological evaluation and ATP content implied tissue integrity within this period. P450 conversions using the Ussing chamber technique showed that the metabolic rate (sum of metabolites measured in apical, basolateral, and tissue compartments) was constant up to 3 h. For 7HC conjugations, the metabolic rate remained constant up to 4 h. The distribution of the metabolites in the compartments differed between the substrates. Overall, metabolic rates were surprisingly similar in both techniques and appear similar to or even higher than in liver. In conclusion, this study shows that both human intestinal precision-cut slices and Ussing chamber preparations provide useful tools for in vitro biotransformation studies.

Jejunal permeability and hepatic extraction of fluvastatin in humans

The primary objective was to investigate the effective permeability and the hepatic extraction of fluvastatin, a new 3‐hydroxy‐3‐methylglutaryl‐coenzyme A (HMG‐CoA) reductase inhibitor, during a jejunal perfusion in humans. The secondary objective was to investigate the relationship between human jejunal effective permeability values and physicochemical properties for four different drugs.

Influence of Hydrodynamics and Particle Size on the Absorption of Felodipine in Labradors

AbstractPurpose. To study the influence of GI hydrodynamics and drug particle size on felodipine absorption in the dog. Methods. Labradors fistulated at midjejunum were used to selectively study the influence of hydrodynamics and particle size on the in vivodissolution and absorption of the poorly soluble, lipophilic drug felodipine. A combination of infusion and oral administration of either normal saline or a 5% glucose solution was used to maintain “fasted” and establish “fed” state motility patterns, respectively. The absorption characteristics of both a micronized (8 μm) and a coarse fraction (125 μm) of felodipine were subsequently studied under these two motility patterns. Results. A reduction in particle size led up to an approximate 22-fold increase in maximum plasma concentration and up to an approximate 14-fold increase in area under the curve, with a commensurate decrease in the time at which the maximum plasma concentration occurred. Although the absorption of felodipine from the solution and micronized suspension was not influenced by a change in the hydrodynamics, felodipine was absorbed from the coarse suspension almost twice as well in the “fed” state as under “fasted” conditions. Conclusions. Absorption from coarse suspensions of felodipine was sensitive to luminal hydrodynamics, whereas micronized suspensions were not. However, the particle size seems to have a much more important influence on the bioavailability of felodipine than the hydrodynamics per se.

Concentration- and region-dependent intestinal permeability of fluvastatin in the rat.

The purpose of this study was to investigate the mechanisms of transport of fluvastatin across the intestinal mucosa in various regions of the intestine in the rat. In‐situ single‐pass perfusions of the jejunum, ileum and colon were performed and the effective permeability (Peff) of fluvastatin, antipyrine and D‐glucose were assessed in each region, at three different perfusate fluvastatin concentrations (1.6, 16 and 160 μM). The effect of lovastatin acid on the bi‐directional transport of fluvastatin across the ileal mucosa was also studied.

In Vitro and in Silico Identification and Characterization of Thiabendazole as a Mechanism-Based Inhibitor of CYP1A2 and Simulation of Possible Pharmacokinetic Drug-Drug Interactions

Thiabendazole (TBZ) and its major metabolite 5-hydroxythiabendazole (5OH-TBZ) were screened for potential time-dependent inhibition (TDI) against CYP1A2. Screen assays were carried out in the absence and presence of NADPH. TDI was observed with both compounds, with kinact and KI values of 0.08 and 0.02 min–1 and 1.4 and 63.3 μM for TBZ and 5OH-TBZ, respectively. Enzyme inactivation was time-, concentration-, and NADPH-dependent. Inactivation by TBZ was irreversible by dialysis and oxidation by potassium ferricyanide, and there was no protection by glutathione. 5OH-TBZ was a weak TDI of CYP1A2, and enzyme activity was recovered by dialysis. IC50 determination of TBZ and 5OH-TBZ showed both compounds to be potent inhibitors, with IC50 values of 0.83 and 13.05 μM, respectively. IC50 shift studies also demonstrated that TBZ was a TDI of CYP1A2. In silico methods identified the thiazole group as a TDI fragment and predicted it as the site of metabolism. The observation pointed to epoxidation of the thiazole and the benzyl rings of TBZ as possible routes of metabolism and mechanisms of TDI. Drug-drug interaction (DDI) simulation studies using SimCyp showed good predictions for competitive inhibition. However, predictions for mechanism-based inhibition (MBI)-based DDI were not in agreement with clinical observations. There was no TBZ accumulation upon chronic administration of the drug. The in vitro MBI findings might therefore not be capturing the in vivo situation in which the proposed bioactivation route is minor. This might be the case for TBZ in which, in vivo, UDP glucuronosyltransferases and sulfanotransferase metabolize and eliminate the 5OH-TBZ.

Surface activity and concentration dependent intestinal permeability in the rat

AbstractPurpose. To investigate the relation between intestinal effective permeability (Peff) and surface activity of fluvastatin and verapamil. Methods. Peff values were determined for fluvastatin, antipyrine and D-glucose following colon perfusions in the rat in situ. The perfusion solutions differed regarding concentrations of fluvastatin (0-2500 μM) and surface tension (58.9-43.7 mN/m). A cellulose derivative, ethyl-(hydroxyethyl) cellulose (EHEC), was added to lower the surface tension of one of the perfusion solutions. The surface tension of perfusion solutions containing R/S-verapamil (8-814 μM) and R/S-verapamil + chlorpromazine (814 μM + 10 mM) were related to the corresponding Peff values from the literature. Results. The Peff of fluvastatin correlated inversely (r2 = 0.985, p < 0.05) with the surface tension of the perfusion solutions below the critical micelle concentration (CMC, 1 mM). Decreasing the surface tension with EHEC increased the Peff of fluvastatin by 36% (p < 0.001), but not to the extent anticipated from the correlation between the Peff and the surface tension. EHEC also increased the Peff of antipyrine by 49% (p < 0.01) but not for D-glucose. The Peff of R/S-verapamil correlated inversely with the surface tension (r2 = 0.980, p < 0.001). Conclusions. The ability of fluvastatin to decrease the surface tension at the membrane surface can partly explain the concentration dependent colonic Peff of fluvastatin. This study shows that the surface activity of the drug molecule itself is an important physicochemical factor that should be taken into consideration when evaluating drug absorption studies performed in vitro or in situ.