The potential of Sutherlandia frutescens for herb-drug interaction.

In Africa, Sutherlandia frutescens is a popular medicinal herb widely consumed by people living with human immunodeficiency virus/AIDS. Concomitant use with antiretroviral drugs has generated concerns of herb-drug interaction (HDI). This study investigated the inhibitory effects of the crude extracts of S. frutescens on the major cytochrome P450 isozymes with the use of pooled human liver microsomes. Its effect on the metabolic clearance of midazolam using cryopreserved hepatocytes was also monitored. The potential of S. frutescens to inhibit human ATP-binding cassette transporters (P-gp and BCRP) and the human organic anion transporting polypeptide (OATP1B1 and OATP1B3) activity was assessed using cell lines overexpressing the transporter proteins. S. frutescens showed inhibitory potency for CYP1A2 (IC(50) = 41.0 µg/ml), CYP2A6 (IC(50) = 160 µg/ml), CYP2B6 (IC(50) = 20.0 µg/ml), CYP2C8 (IC(50) = 22.4 µg/ml), CYP2C9 (IC(50) = 23.0 µg/ml), CYP2C19 (IC(50) = 35.9 µg/ml), and CYP3A4/5 (IC(50) = 17.5 µg/ml [with midazolam1'-hydroxylation]; IC(50) = 28.3 µg/ml [with testosterone 6ß-hydroxylation]). Time-dependent (irreversible) inhibition by S. frutescens was observed for CYP3A4/5 (K(I) = 296 µg/ml, k(inact) = 0.063 min(-1)) under the conditions of this study. S. frutescens also delays the production of midazolam metabolites in the hepatocytes, decreasing its clearance by 40%. Furthermore, S. frutescens inhibited P-gp (IC(50) = 324.8 µg/ml), OATP1B1 (IC(50) = 10.4 µg/ml), and OATP1B3 (IC(50) = 6.6 µg/ml). The result indicates the potential for HDI between S. frutescens and the substrates of the affected enzymes, if sufficient in vivo concentration of the extract is attained.

The potential of Hypoxis hemerocallidea for herb-drug interaction.

CONTEXT: Aqueous decoction of Hypoxis hemerocallidea Fisch. & C.A. Mey. (Hypoxidaceae) (Hypoxis) is widely consumed in Southern Africa by people living with HIV/AIDS, some of whom are on ARV and other medications. OBJECTIVE: The aim of this study was to investigate the potential of the crude aqueous extracts of Hypoxis to inhibit major forms of CYP450 and transport proteins. MATERIALS AND METHODS: Corms of Hypoxis were water-extracted and incubated (in graded concentrations: 1-100 µg/mL) with human liver microsomes (20 min) to monitor the effects on phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, paclitaxel 6α-hydroxylation, diclofenac 4'-hydroxylation, S-mephenytoin 4'-hydroxylation, bufuralol 1'-hydroxylation, chlorzoxazone 6-hydroxylation, midazolam 1'-hydroxylation and testosterone 6ß-hydroxylation as markers for the metabolic activities of CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4/5, respectively. The generation of metabolites were monitored and quantified with the aid of LC-MS/MS. The potential of the extracts to inhibit human ATP-binding cassette transporter activity was assessed using recombinant MDCKII and LLC-PK1 cells over-expressing human breast cancer resistant protein and human P-glycoprotein , respectively (with Ko143 and cyclosporin A as positive controls). Similar assessment was performed with human organic anion transporting polypeptide (OATP1B1 and OATP1B3) using recombinant HEK293 cells over-expressing OATP1B1 and OATP1B3, respectively (with rifamycin and 10 µM atorvastatin as positive controls). RESULTS: Extracts of Hypoxis inhibited the production of the metabolites of the substrates of the following enzymes (as compared to controls) with the indicated IC50 values (µg/mL): CYP1A2 (120.6), CYP2A6 (210.8), CYP2B6 (98.5), CYP2C8 (195.2), CYP2C9 (156) and CYP3A4/5 (185.4). The inhibition of the uptake activity of OATP1B1 and OATP1B3 were also observed with IC50 values of 93.4 and 244.8 µg/mL, respectively. DISCUSSION: Extract concentrations higher than the estimated IC50 values are achievable in the gastrointestinal tract when traditional doses of Hypoxis are considered. This may have profound effects on presystemic metabolism of the drug substrates. If absorbed, systemic inhibition of metabolic enzymes/transporters by Hypoxis may be expected. CONCLUSION: The result suggests that there is the potential for HDI between Hypoxis and the substrates of the affected enzymes/transporters, if sufficient in vivo concentration of Hypoxis extracts is attained.

Regulation of BCRP (ABCG2) and P-glycoprotein (ABCB1) by cytokines in a model of the human blood-brain barrier.

Brain capillary endothelial cells form the blood-brain barrier (BBB), a highly selective permeability membrane between the blood and the brain. Besides tight junctions that prevent small hydrophilic compounds from passive diffusion into the brain tissue, the endothelial cells express different families of drug efflux transport proteins that limit the amount of substances penetrating the brain. Two prominent efflux transporters are the breast cancer resistance protein and P-glycoprotein (P-gp). During inflammatory reactions, which can be associated with an altered BBB, pro-inflammatory cytokines are present in the systemic circulation. We, therefore, investigated the effect of the pro-inflammatory cytokines interleukin-1beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) on the expression and activity of BCRP and P-gp in the human hCMEC/D3 cell line. BCRP mRNA levels were significantly reduced by IL-1beta, IL-6 and TNF-alpha. The strongest BCRP suppression at the protein level was observed after IL-1beta treatment. IL-1beta, IL-6 and TNF-alpha also significantly reduced the BCRP activity as assessed by mitoxantrone uptake experiments. P-gp mRNA levels were slightly reduced by IL-6, but significantly increased after TNF-alpha treatment. TNF-alpha also increased protein expression of P-gp but the uptake of the P-gp substrate rhodamine 123 was not affected by any of the cytokines. This in vitro study indicates that expression levels and activity of BCRP, and P-gp at the BBB may be altered by acute inflammation, possibly affecting the penetration of their substrates into the brain.

A new intestinal cell culture model to discriminate the relative contribution of P-gp and BCRP on transport of substrates such as imatinib.

P-glycoprotein (P-gp/MDR1/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) play an important role in transport of a wide variety of endogenous compounds, drugs and toxins. Transport of some drugs, for example the tyrosine kinase inhibitor imatinib, is influenced by both P-gp and BCRP. Establishing an intestinal Caco-2 cell culture model with specific knock-downs of P-gp and BCRP and double knock-down of both proteins, we aimed to elucidate the impact of each transporter on transport of imatinib. Stable single and double knock-downs of P-gp and BCRP were obtained by RNA interference (RNAi). Transporter expression was measured on RNA and protein level using real-time RT-PCR and Western blot, respectively. Functional activity was quantified by transport of specific substrates across Caco-2 cells. MDR1 and BCRP mRNA expression was reduced to 75% and 90% compared to wild-type control in single MDR1- and BCRP-knock-down clones, respectively. In double knock-down clones, MDR1 expression decreased to 95% and BCRP expression to 80%. Functional activity of P-gp and BCRP was diminished as transport of the P-gp-specific substrate (3)H-digoxin and the BCRP-specific substrate (14)C-PhIP was augmented in the opposite direction, when the respective transporter was knocked down. Similar effects were observed by chemical inhibition of the respective transporter. Bidirectional transport studies with (14)C-imatinib revealed an abrogation of asymmetric transport when P-gp was knocked down, either in single or double knock-down clones compared to wild-type cells. This was not observed in single BCRP-knock-down clones. In conclusion, this newly established cell system with single and concomitant knock-down of P-gp and BCRP can be used to quantify the specific partial impact of the transporters on transport of substrates that are transported by both proteins. For imatinib transport, the contribution of P-gp seems to be more important compared to BCRP in this Caco-2 cell system.

Effects of Curcuma extracts and curcuminoids on expression of P-glycoprotein and cytochrome P450 3A4 in the intestinal cell culture model LS180.

Curcuma longa L. is a widely used spice. Its main ingredients, the curcuminoids, are used in the treatment of inflammatory diseases and cancer. Bioavailability of curcuminoids is low, and huge amounts remain in the intestine. We therefore aimed to investigate their interaction potential with the ABC-transporter P-glycoprotein (P-gp, product of the MDR1/ABCB1 gene) and cytochrome P450 3A4 (CYP3A4) in an intestinal cell line (LS180). Intestinal P-gp and CYP3A4 play a major role in drug absorption, and consequently changes in their expression level could lead to interactions. The intestinal LS180 cell line was incubated with different Curcuma extracts, the single curcuminoids (curcumin, demethoxycurcumin, and bisdemethoxycurcumin), as well as a curcuminoid mixture. Changes in mRNA expression of MDR1 and the cytochrome CYP3A4 were measured by real-time RT-PCR. MDR1 mRNA expression was significantly but not relevantly downregulated by the curcuminoids, whereas the extracts had no significant effect on it. CYP3A4 mRNA expression did not alter significantly after treatment. Curcuma extracts, the single curcuminoids, and a curcuminoid mixture had no relevant effect on MDR1 and CYP3A4 mRNA expression in our intestinal cell system. Further studies are required to evaluate their effects in vivo.

Development of decision tree models for substrates, inhibitors, and inducers of p-glycoprotein.

In silico classification of new compounds for certain properties is a useful tool to guide further experiments or compound selection. Interaction of new compounds with the efflux pump P-glycoprotein (P-gp) is an important drug property determining tissue distribution and the potential for drug-drug interactions. We present three datasets on substrate, inhibitor, and inducer activities for P-gp (n = 471) obtained from a literature search which we compared to an existing evaluation of the Prestwick Chemical Library with the calcein-AM assay (retrieved from PubMed). Additionally, we present decision tree models of these activities with predictive accuracies of 77.7 % (substrates), 86.9 % (inhibitors), and 90.3 % (inducers) using three algorithms (CHAID, CART, and C4.5). We also present decision tree models of the calcein-AM assay (79.9 %). Apart from a comprehensive dataset of P-gp interacting compounds, our study provides evidence of the efficacy of logD descriptors and of two algorithms not commonly used in pharmacological QSAR studies (CART and CHAID).

Changes in mRNA expression levels of solute carrier transporters in inflammatory bowel disease patients.

Inflammatory bowel disease (IBD) is an inflammatory condition that affects the gastrointestinal tract. The solute carrier (SLC) superfamily of transporters comprise proteins involved in the uptake of drugs, hormones, and other biologically active compounds. The purpose of this study was to determine the mRNA expression levels of 15 solute carrier transporters in two regions of the intestine in IBD patients. Endoscopic biopsy specimens were taken from two locations (terminal ileum and colon) for histological examination and RNA extraction. We quantitatively measured the mRNA expression of 15 SLC transporters in 107 IBD patients (53 with Crohn's disease and 54 with ulcerative colitis) and 23 control subjects. mRNA expression was evaluated using the quantitative reverse transcription-polymerase chain reaction technique. We observed that in the ileum of IBD patients, mRNA levels for serotonin transporter, equilibrative nucleoside transporter (ENT) 1, ENT2, and organic anion-transporting polypeptide (OATP) 2B1 were significantly elevated, whereas levels for apical sodium-dependent bile acid transporter (ASBT) and organic zwitterion/cation transporter (OCTN) 2 were significantly lower. In colon, mRNA levels for ENT1, ENT2, concentrative nucleoside transporter (CNT) 2, OATP2B1, and OATP4A1 were significantly higher, whereas mRNA levels for OCTN2 were significantly decreased. In inflamed colon of IBD patients the mRNA expression levels of ENT1, ENT2, CNT2, OATP2B1, OATP4A1, and peptide transporter 1 were significantly higher. We conclude that intestinal SLC mRNA levels are dysregulated in IBD patients, which may be linked to the inflammation of the tissue and provides an indication about the role of inflammatory signaling in regulation of SLC expression.

Classification of cytochrome p(450) activities using machine learning methods.

The cytochrome P(450) (CYP) system plays an integral part in the metabolism of drugs and other xenobiotics. Knowledge of the structural features required for interaction with any of the different isoforms of the CYP system is therefore immensely valuable in early drug discovery. In this paper, we focus on three major isoforms (CYP 1A2, CYP 2D6, and CYP 3A4) and present a data set of 335 structurally diverse drug compounds classified for their interaction (as substrate, inhibitor, or any interaction) with these isoforms. We also present machine learning models using a variety of commonly used methods (k-nearest neighbors, decision tree induction using the CHAID and CRT algorithms, random forests, artificial neural networks, and support vector machines using the radial basis function (RBF) and homogeneous polynomials as kernel functions). We discuss the physicochemical features relevant for each end point and compare it to similar studies. Many of these models perform exceptionally well, even with 10-fold cross-validation, yielding corrected classification rates of 81.7 to 91.9% for CYP 1A2, 89.2 to 92.9% for CYP 2D6, and 87.4 to 89.9% for CYP3A4. Our models help in understanding the structural requirements for CYP interactions and can serve as sensitive tools in virtual screenings and lead optimization for toxicological profiles in drug discovery.

The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies.

The human brain endothelial capillary cell line hCMEC/D3 has been developed recently as a model for the human blood-brain barrier. In this study a further characterization of this model was performed with special emphasis on permeability properties and active drug transport. Para- or transcellular permeabilities (P(e)) of inulin (0.74 x 10(-3) cm/min), sucrose (1.60 x 10(-3) cm/min), lucifer yellow (1.33 x 10(-3) cm/min), morphine (5.36 x 10(-3) cm/min), propranolol (4.49 x 10(-3) cm/min) and midazolam (5.13 x 10(-3) cm/min) were measured. By addition of human serum the passive permeability of sucrose could be reduced significantly by up to 39%. Furthermore, the expression of a variety of drug transporters (ABCB1, ABCG2, ABCC1-5) as well as the human transferrin receptor was demonstrated on the mRNA level. ABCB1, ABCG2 and transferrin receptor proteins were detected and functional activity of ABCB1, ABCG2 and the ABCC family was quantified in efflux experiments. Furthermore, ABCB1-mediated bidirectional transport of rhodamine 123 was studied. The transport rate from the apical to the basolateral compartment was significantly lower than that in the inverse direction, indicating directed p-glycoprotein transport. The results of this study demonstrate the usefulness of the hCMEC/D3 cell line as an in vitro model to study drug transport at the level of the human blood-brain barrier.

Breast cancer resistance protein and P-glycoprotein expression in patients with newly diagnosed and therapy-refractory ulcerative colitis compared with healthy controls.

AIMS: Efflux transporters such as breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (Pgp; MDR1/ABCB1) are protecting the enterocytes from potentially toxic compounds. Both transporters have been reported to be downregulated in patients with active ulcerative colitis (UC). The aim of this study was to evaluate transporter expression in both unaffected and inflamed mucosa of patients with active UC, in drug-naïve and treated patients with UC and compare the results with transporter expression in healthy subjects. METHODS: Transporter expression was determined with real-time RT-PCR (TaqMan) in inflamed and unaffected mucosa of newly diagnosed (n = 12) and therapy-refractory (n = 11) patients with UC. Expression levels were compared with UC patients in remission (n = 11) and control subjects (n = 26). BCRP and Pgp expression was evaluated by immunohistochemistry. RESULTS: Compared with unaffected mucosa, BCRP expression was significantly reduced in inflamed mucosa of newly diagnosed drug-naïve patients with UC (expression reduced to 30%) as well as in patients not responding to treatment (reduced to 25%) with either 5-aminosalicylates (n = 7) or prednisone (n = 4). Unaffected mucosa of UC patients showed comparable transporter expression to unaffected mucosa of control subjects. MDR1 expression depicts a similar pattern. Protein staining for Pgp and BCRP was significantly reduced in the inflamed mucosa of patients with active UC. CONCLUSIONS: Expression of both efflux transporters BCRP and MDR1 is reduced, but only in inflamed tissue of patients with active UC. Transporter expression in unaffected mucosa of patients with active UC is comparable to healthy controls. The data suggest that the inflammatory process is responsible for the reduced levels. A major role in the pathogenesis of UC is unlikely.

Induction of cytochrome P450 3A4 and P-glycoprotein by the isoxazolyl-penicillin antibiotic flucloxacillin.

Clinical findings indicate that co-administration of the isoxazolyl-penicillin flucloxacillin with cyclosporine may reduce the plasma concentrations of cyclosporine. We have explored in the present study if induction of cytochrome P450 3A4 or P-glycoprotein may offer a mechanistic explanation of the observed effects. Flucloxacillin is neither an inhibitor nor a substrate of drug metabolizing cytochrome P450 isoenzymes (CYP3A4, 1A2, 2C9, 2C19 and 2D6) or P-glycoprotein as shown by an in vitro assay for CYP inhibition, a fluorescent indicator assay for P-glycoprotein inhibition and a functional P-glycoprotein ATPase assay. However, incubation of human LS 180 colorectal adenocarcinoma cells with flucloxacillin led to a dose-dependent induction of MDR1 as well as of CYP3A4 mRNA, which was also confirmed in primary human hepatocytes. At high concentrations, flucloxacillin activated the human Pregnane-X-Receptor, PXR, a ligand-dependent transcription factor that is the target of many drugs that induce CYP3A4, with consequences for the metabolism of other drugs. Liver microsomes from control rats or rats, which received for 3 consecutive days 100 mg/kg of oral flucloxacillin, were used to study the metabolism and metabolite pattern of midazolam, a model substrate of CYP 3A4. There was a trend towards a higher intrinsic microsomal clearance of midazolam using microsomes from flucloxacillin treated rats. In addition, there was a significant increase in the formation of the principal midazolam metabolites 1-hydroxy midazolam, 4-hydroxy midazolam and 1,4-dihydroxy midazolam as compared to controls. These findings indicate that flucloxacillin has the potential to induce expression of both CYP3A4 as well as P-glycoprotein, most likely through activation of the nuclear hormone receptor PXR. This would offer an explanation for the observed clinical drug-drug interactions between the antibiotic and cyclosporine.

Thalidomide does not interact with P-glycoprotein.

BACKGROUND: There is growing clinical interest in thalidomide for the treatment of various disorders due to its anti-inflammatory, immunomodulatory, and anti-angiogenic properties. In numerous clinical trials thalidomide is used as an adjunct to standard therapy. Therefore, clinicians should be aware of all possible drug-drug interactions that might occur with this drug. P-glycoprotein (P-gp), a drug efflux transporter that is expressed in many tissues, is the cause of several drug-drug interactions. P-gp induction or inhibition can lead to ineffective therapy or side-effects. In this study, we investigated thalidomide's potential to cause drug-drug interactions on the level of P-gp. METHODS: LS180 cells were incubated with thalidomide for 72 h in order to determine P-gp induction using real-time RT-PCR. A human leukaemia cell line over-expressing MDR1 (CCRF-CEM/MDR1) was used to measure uptake of rhodamine 123, a P-gp substrate, in the presence of thalidomide. Dose-dependent and bi-directional transport of thalidomide through Caco-2 cell monolayers was performed to assess site-directed permeability. Transport rates were determined using HPLC including chiral separation of the thalidomide enantiomers. RESULTS: Thalidomide did not induce P-gp expression in LS180 cells. The uptake of rhodamine 123 in CCRF cells over-expressing MDR1 was not influenced by co-incubation with thalidomide. The transport through Caco-2 monolayers was linear and the permeability was similar for both directions. No differences between the thalidomide enantiomers were observed. CONCLUSIONS: Our study indicates that thalidomide is neither a substrate, nor an inhibitor or an inducer of P-gp. Therefore, P-gp-related drug-drug interactions with thalidomide are not likely.

Primary porcine proximal tubular cells as a model for transepithelial drug transport in human kidney.

BACKGROUND: Kidney proximal tubular cells play a major role in the transport of endogenous and exogenous compounds. A multitude of different transporters are expressed starting with multidrug ABC transporters (e.g. abcb1, abcc1-6), slc22a6-8 (organic anion transporters) and slc22a1-3 (organic cation transporters). For transport studies of renal drug transport, cell lines like MDCK and LLC-PK1 are often used to overexpress and study one or two transporters, such as abcb1 or abcc1-6. However, the use is limited since under physiological conditions xenobiotics are transported through different transporters at the same time. Therefore, a primary in vitro model expressing functionally different transporters simultaneously, as it is the case in vivo, would be of great benefit. METHODS: Primary proximal tubular cells were isolated from porcine kidney. Cells were cultured under selective culturing conditions leading to specific growth of primary proximal tubular cells. Expression of important proximal transporters was checked at mRNA level with RT-PCR, at protein level with immunocytochemistry and functionally by transport and uptake assays. RESULTS: A model of primary proximal tubular cells was established expressing the most important transporters: abcb1, abcc1, abcc2, slc22a8, slco1a2, slc15a1, slc5a2 and slc4a4. In freshly isolated cells, slc22a1 and slc22a6 were expressed, but were down-regulated in culture. Abcb1, abcc1, abcc2 and slc4a4 were detected at protein level with immunostaining. Functional activity was confirmed for abcb1, abcc1/2, slc22a8, slc15a1/2 and slc5a1/2. The tightness of the monolayers of this model was better than in previously established in vitro models. CONCLUSION: This primary cell culture model might be an interesting tool to investigate proximal tubular transport and to predict toxicity and drug interactions since it expresses functionally several transporters simultaneously.

P-glycoprotein and surfactants: effect on intestinal talinolol absorption.

BACKGROUND AND OBJECTIVE: Surfactants used in pharmaceutical formulations can modulate drug absorption by multiple mechanisms including inhibition of intestinal P-glycoprotein (P-gp). Our objective was to analyze the effect of 2 surfactants with different affinity for P-gp in vitro on the intestinal absorption and bioavailability of the P-gp substrate talinolol in humans. METHODS: In vitro, the influence of surfactants on talinolol permeability was studied in Caco-2 cells. In vivo, an open-label 3-way crossover study with 9 healthy male volunteers was performed. Subjects were intubated with a 1-lumen nasogastrointestinal tube. The study solution, containing either talinolol (50 mg), talinolol and D-alpha-tocopheryl polyethylene glycol 1000 succinate (TPGS) (0.04%), or talinolol and Poloxamer 188 (0.8%), was administered through the tube. RESULTS: TPGS, but not Poloxamer 188, inhibited the P-gp-mediated talinolol transport in Caco-2 cells. In healthy volunteers TPGS increased the area under the plasma concentration-time curve with extrapolation to infinity (AUC 0-infinity ) of talinolol by 39% (90% confidence interval, 1.10-1.75) and the maximum plasma concentration (C max) by 100% (90% confidence interval, 1.39-2.88). Poloxamer 188 did not significantly alter the AUC 0-infinity or C max of talinolol. CONCLUSIONS: This in vivo intraduodenal perfusion study showed that low concentrations of TPGS, close to the concentrations that showed P-gp inhibition in vitro, significantly increased the bioavailability of talinolol. The study design excluded modulation of solubility by TPGS and unspecific surfactant-related effects. The latter was supported by the absence of modulation of the talinolol pharmacokinetics by Poloxamer 188, which does not modulate P-gp. Therefore we consider intestinal P-gp inhibition by TPGS as the major underlying mechanism for the increase in talinolol bioavailability.

Distribution of breast cancer resistance protein (BCRP/ABCG2) mRNA expression along the human GI tract.

Human breast cancer resistance protein (BCRP/ABCG2) is an ABC-transporter that is present on the luminal membrane of intestinal epithelial cells and restricts absorption of anticancer drugs such as methotrexate, topotecan, mitoxantrone, and doxorubicin. The exact anatomic distribution of BCRP along the gastrointestinal (GI) tract, however, has not been determined before. The aim of this study was, therefore to investigate BCRP mRNA expression pattern along the GI tract in 14 healthy subjects. Furthermore, BCRP duodenal mRNA expression was compared with MDR1/ABCB1 mRNA. Additionally, BCRP mRNA expression was investigated in two human intestinal cell lines (Caco-2 and LS180). Since previous animal studies have suggested sex specific differences in BCRP expression, we analyzed intestinal BCRP expression with respect to sex. Biopsies were taken from different gut segments (duodenum, terminal ileum and ascending, transverse, descending and sigmoid colon). Gene expression was assessed by quantitative real-time PCR (Taqman). BCRP mRNA expression was maximal in the duodenum and decreased continuously down to the rectum (terminal ileum 93.7%, ascending colon 75.8%, transverse colon 66.6%, descending colon 62.8%, and sigmoid colon 50.1% compared to duodenum, respectively). BCRP expression in the duodenum was comparable to MDR1/ABCB1 gene expression. Caco-2 cells showed a comparable expression of BCRP as human duodenal tissue. Gender specific differences in BCRP expression were not observed. These findings represent the first systematic site-specific analysis of BCRP expression along the GI tract. This information might be helpful to develop target strategies for orally administered anticancer drugs.

Evaluation of the immortalized human brain capillary endothelial cell line BB19 as a human cell culture model for the blood-brain barrier.

In vitro models of the blood-brain barrier (BBB) play a major role in the study of BBB permeability of drug candidates. However, most established in vitro models use cells of non-human origin, which is not optimal for the prediction of brain permeability in humans. The aim of this study was to assess the human brain capillary endothelial cell line BB19 for its usefulness as an in vitro model of the human BBB. Restrictive tight junctions are a prerequisite for drug transport studies. Sucrose permeability of BB19 cells on different filters was compared to porcine brain capillary endothelial cells (BCEC). Tightness of BB19 cell monolayers still needs further optimization. Hardly any discrimination between Sucrose and Propranolol (P(app) = 1.30 x 10(-5) vs. 2.18 x 10(-5) cm/s) was seen. Cells showed an improvement towards a more primary BCEC morphology with C6 conditioned medium, dexamethasone, and 1,25-dihydroxyvitamin D3. The presence of P-glycoprotein (P-gp), MRP4 and BCRP, ABC-transporters located in the BBB, has been shown on mRNA level, by immunostaining, and western blot. MRP1, MRP2, MRP5, OAT3, and OAT4 were also detected by RT-PCR. Functional properties of the BBB were shown with uptake of propranolol, morphine, and sucrose. Uptake studies with daunomycin and the P-gp inhibitor verapamil showed functional activity of P-gp. We conclude that BB19 cells might be feasible as a human in vitro model of the BBB for drug uptake studies. However, for the assessment of transport studies, further improvements of this model are necessary.

Glucagon-like peptide 1 induces natriuresis in healthy subjects and in insulin-resistant obese men.

Glucagon-like peptide-1-(7-36)-amide (GLP-1) is involved in satiety control and glucose homeostasis. Animal studies suggest a physiological role for GLP-1 in water and salt homeostasis. This study's aim was to define the effects of GLP-1 on water and sodium excretion in both healthy and obese men. Fifteen healthy subjects and 16 obese men (mean body mass index, 36 kg/m2) were examined in a double-blind, placebo-controlled, crossover study to demonstrate the effects of a 3-h infusion of GLP-1 on urinary sodium excretion, urinary output, and the glomerular filtration rate after an i.v. 9.9-g salt load. Infusion of GLP-1 evoked a dose-dependent increase in urinary sodium excretion in healthy subjects (from 74 +/- 8 to 143 +/- 18 mmol/180 min, P = 0.0013). In obese men, there was a significant increase in urinary sodium excretion (from 59 to 96 mmol/180 min, P = 0.015), a decrease in urinary H+ secretion (from 1.1 to 0.3 pmol/180 min, P = 0.013), and a 6% decrease in the glomerular filtration rate (from 151 +/- 8 to 142 +/- 8 ml/min, P = 0.022). Intravenous infusions of GLP-1 enhance sodium excretion, reduce H+ secretion, and reduce glomerular hyperfiltration in obese men. These findings suggest an action at the proximal renal tubule and a potential renoprotective effect.

Vasospastic persons exhibit differential expression of ABC-transport proteins.

PURPOSE: To quantify the gene expression levels of the ABC-proteins MDR1 (P-glycoprotein) and MRP (multidrug resistance-associated protein) isoforms in isolated mononuclear cells of vasospastic persons with increased Endothelin-1 plasma levels. METHODS: Quantitative real-time RT-PCR was performed to determine the expression levels of the MDR1 (P-glycoprotein) gene and MRP1 to MRP5 genes as well as the expression of the ETA and ETB receptor in mononuclear cells derived from 11 vasospastic subjects compared to 10 healthy controls. RESULTS: Mononuclear cells of vasospastic subjects showed a significant decrease in the expression of MDR1 (P-glycoprotein) gene (p=0.029), MRP2 gene (p=0.003), and MRP5 gene (p=0.013) when compared to healthy controls. These effects were poorly correlated with ET-1 plasma levels. No significant ETA and ETB receptor expression was observed in both groups. CONCLUSIONS: Vasospastic persons differ in their expression pattern of MDR proteins from healthy controls. This might be an indirect effect of elevated ET-1 levels.

Gene expression of CYP3A4, ABC-transporters (MDR1 and MRP1-MRP5) and hPXR in three different human colon carcinoma cell lines.

Colon carcinoma cell lines are used widely as screening models for intestinal absorption of drugs. However, the expression of important transport systems and of metabolic enzymes is not completely characterized yet. The expression and inducibility of multidrug resistance gene 1 (MDR1) and cytochrome P450 isoform 3A4 (CYP3A4) was investigated in Caco-2 parental, Caco-2 TC-7 (TC-7) and LS180 cell lines. In the same three cell lines, we investigated the expression of isoforms of the multidrug resistance associated protein family (MRP1-MRP5) and the human pregnane X receptor (hPXR), which may be important for MDR1 and CYP3A4 induction. Cells were treated with rifampicin or 1alpha,25-dihydroxycholecalciferol (1,25(OH)(2)D(3)) for 72 h and the total RNA was extracted. Afterwards reverse transcription real-time polymerase chain reaction (TaqMan) assay was performed to determine the mRNA expression level. We have shown that in LS180 cells, MDR1 and CYP3A4 were inducible with both inducers. In Caco-2 parental and TC-7 cells, CYP3A4 was only inducible with 1,25(OH)(2)D(3). Furthermore, differences were shown in gene expression of several transport proteins (MDR1 and MRP1-MRP5) and CYP3A4 in different human colon carcinoma derived cell lines. hPXR mRNA was expressed in all three cell lines but the amount of mRNA detected was significantly higher in LS180 cells than in Caco-2 and TC-7 cells. We concluded that LS180 cells were a suitable model to study MDR1 and CYP3A4 induction, but for drug transport studies Caco-2 parental and TC-7 cells would be preferred as the more physiological model.

In vitro-in vivo extrapolation method to predict human renal clearance of drugs.

Renal clearance is a key determinant of the elimination of drugs. To date, only few in vitro-in vivo extrapolation (IVIVE) approaches have been described to predict the renal organ clearance as the net result of glomerular filtration, tubular secretion, and tubular reabsorption. In this study, we measured in LLC-PK1 cells the transport of 20 compounds that cover all four classes of the Biopharmaceutical Drug Disposition System. These data were incorporated into a novel kidney model to predict all renal clearance processes in human. We showed that filtration and secretion were main contributors to the renal organ clearance for all compounds, whereas reabsorption was predominant for compounds assigned to classes 1 and 2. Our results suggest that anionic drugs were not significantly secreted in LLC-PK1 cells, resulting in under-predicted clearances. When all study compounds were included a high overall correlation between the reported and predicted renal organ clearances was obtained (R² = 0.83). The prediction accuracy in terms of percentage within twofold and threefold error was 70% and 95%, respectively. In conclusion, our novel IVIVE method allowed to predict the human renal organ clearance and the contribution of each underlying process.