Interaction of beta-blockers with the renal uptake transporter OCT2.

AIM: The uptake of drugs from the blood into the renal tubular cells is a key determinant for renal secretion and may influence their systemic plasma concentrations and extrarenal effects. Metformin, used for treatment of type 2 diabetes, is taken up into renal tubular cells by the organic cation transporter 2 (OCT2). Because many patients with type 2 diabetes receiving metformin are concomitantly treated with beta-blockers, we tested whether beta-blockers can inhibit OCT2-mediated drug transport. METHOD: Using Madin-Darby canine kidney II cells stably expressing the uptake transporter OCT2, we analysed whether the beta-blockers bisoprolol, carvedilol, metoprolol and propranolol inhibit the transport of OCT2 substrates 1-methyl-4-phenylpyridinium (MPP(+)) and metformin. RESULTS: Neither bisoprolol nor metoprolol significantly inhibited the uptake of MPP(+), whereas a significant inhibition was observed for carvedilol und propranolol (half maximal inhibitory concentration IC(50): 26.3 and 67.5 microM) respectively. Moreover, all beta-blockers significantly inhibited OCT2-mediated metformin uptake (IC(50) for bisoprolol: 2.4 microM, IC(50) for carvedilol: 2.3 microM, IC(50) for metoprolol: 50.2 microM and IC(50) for propranolol: 8.3 microM). CONCLUSION: These in vitro results demonstrate that alterations of uptake transporter function by beta-blockers have to be considered as potential mechanisms underlying drug-drug interactions in the kidney.

Induction of intestinal P-glycoprotein by St John's wort reduces the oral bioavailability of talinolol.

St John's wort (SJW) is known to induce cytochrome P450 (CYP) 3A4 and P-glycoprotein through pregnane X-receptor activation. Our study evaluated the effects of long-term SJW administration on oral and intravenous pharmacokinetics of the nonmetabolized in vivo probe of P-glycoprotein, talinolol, in relation to intestinal P-glycoprotein expression. In a controlled, randomized study (N=9), the pharmacokinetics of oral (50 mg) and intravenous talinolol (30 mg) was determined before and after 12 days SJW (900 mg daily, Jarsin 300). Duodenal biopsies were taken and MDR1 genotypes assessed. SJW reduced the oral talinolol bioavailability by 25% (P=0.049) compared with water control. A 93% increase in oral clearance (P=0.177) and a 31% reduction in area under the serum concentration time curve (AUC; P=0.030) were observed. Renal and nonrenal clearance (CLNR), elimination half-life, peak serum drug concentration (Cmax), and time to reach Cmax were not significantly altered. After intravenous talinolol, SJW affected only CLNR (35% increase compared with water, P=0.006). SJW increased MDR1 messenger ribonucleic acid (mRNA) as well as P-glycoprotein levels in the duodenal mucosa. Subjects with the combined MDR1 genotype comprising 1236C>T, 2677G>T/A, and 3435C>T polymorphisms had lower intestinal MDR1 mRNA levels and displayed an attenuated inductive response to SJW as assessed by talinolol disposition. Long-term SJW decreased talinolol AUC with a corresponding increase in intestinal MDR1 expression, suggesting that SJW has a major inductive effect on intestinal P-glycoprotein. Interestingly, the magnitude of induction appeared to be affected by MDR1 genotype.

Intestinal drug transporter expression and the impact of grapefruit juice in humans.

The goals of this study were to assess the extent of human intestinal drug transporter expression, determine the subcellular localization of the drug uptake transporter OATP1A2, and then to assess the effect of grapefruit juice consumption on OATP1A2 expression relative to cytochrome P450 3A4 and MDR1. Expression of drug uptake and efflux transporters was assessed using human duodenal biopsy samples. Fexofenadine uptake by different transporters was measured in a transporter-transfected cell line. We investigated the influence of grapefruit juice on pharmacokinetics of orally administered fexofenadine. The effect of grapefruit juice on the expression of intestinal transporters was determined using real-time polymerase chain reaction and Western blot analysis. In the duodenum of healthy volunteers, an array of CYP enzymes as well as uptake and efflux transporters was expressed. Importantly, uptake transporters thought to be liver-specific, such as OATP1B1 and 1B3, as well as OATP2B1 and 1A2 were expressed in the intestine. However, among OATP transporters, only OATP1A2 was capable of fexofenadine uptake when assessed in vitro. OATP1A2 colocalized with MDR1 to the brush border domain of enterocytes. Consumption of grapefruit juice concomitantly or 2 h before fexofenadine administration was associated with reduced oral fexofenadine plasma exposure, whereas intestinal expression of either OATP1A2 or MDR1 remained unaffected. In conclusion, an array of drug uptake and efflux transporters are expressed in the human intestine. OATP1A2 is likely the key intestinal uptake transporter for fexofenadine absorption whose inhibition results in the grapefruit juice effect. Although short-term grapefruit juice ingestion was associated with reduced fexofenadine availability, OATP1A2 or MDR1 expression was unaffected.

Detection of pathogenic micro-organisms--a contribution to discussion.

When applying methods for the detection of pathogenic micro-organisms in foodstuffs, information on the distribution of the target organism in the foodstuff submitted to a test should be available. The sampling plan used should allow to detect the presence of low levels of the target organism with a high probability. The individual steps of a detection procedure (pre-enrichment, selective enrichment, isolation and confirmation) need careful examination. It is important that inoculation of low levels of the target organism leads to successful enrichment even in the presence of relatively large numbers of competing organisms. In cases where competing micro-organisms form suspect colonies, there is a risk that false-negative results are obtained, because colonies of the target organism may not be isolated. Collaborative trials have to be carried out to assess the performance of presence/absence tests. Meaningful results are obtained only, if the test samples contain low levels of the target organism and if the effect of competing micro-organisms is checked. While it can hardly be disputed that the determination of the sensitivity and specificity of the test method provides valuable information, this cannot be said for "accordance" and "concordance", two recently proposed parameters which correspond to repeatability and reproducibility in quantitative tests. A better alternative may be to specify the probability to obtain two positive results, when analysing samples containing the target organism under repeatability or reproducibility conditions. In an analogous way, the probability to obtain two negative results with samples containing competing micro-organisms, but not the target organism, could be specified.

Green tea ingestion greatly reduces plasma concentrations of nadolol in healthy subjects.

This study aimed to evaluate the effects of green tea on the pharmacokinetics and pharmacodynamics of the ß-blocker nadolol. Ten healthy volunteers received a single oral dose of 30 mg nadolol with green tea or water after repeated consumption of green tea (700 ml/day) or water for 14 days. Catechin concentrations in green tea and plasma were determined. Green tea markedly decreased the maximum plasma concentration (C(max)) and area under the plasma concentration-time curve (AUC(0-48)) of nadolol by 85.3% and 85.0%, respectively (P < 0.01), without altering renal clearance of nadolol. The effects of nadolol on systolic blood pressure were significantly reduced by green tea. [(3)H]-Nadolol uptake assays in human embryonic kidney 293 cells stably expressing the organic anion-transporting polypeptides OATP1A2 and OATP2B1 revealed that nadolol is a substrate of OATP1A2 (Michaelis constant (K(m)) = 84.3 µmol/l) but not of OATP2B1. Moreover, green tea significantly inhibited OATP1A2-mediated nadolol uptake (half-maximal inhibitory concentration, IC(50) = 1.36%). These results suggest that green tea reduces plasma concentrations of nadolol possibly in part by inhibition of OATP1A2-mediated uptake of nadolol in the intestine.

Interaction of innovative small molecule drugs used for cancer therapy with drug transporters.

Multiple new small molecules such as tyrosine kinase, mammalian target of rapamycin (mTOR) and proteasome inhibitors have been approved in the last decade and are a considerable progress for cancer therapy. Drug transporters are important determinants of drug concentrations in the systemic circulation. Moreover, expression of drug transporters in blood-tissue barriers (e.g. blood-brain barrier) can limit access of small molecules to the tumour (e.g. brain tumour). Finally, transporter expression and (up)regulation in the tumour itself is known to affect local drug concentrations in the tumour tissue contributing to multidrug resistance observed for multiple anticancer agents. This review summarizes the current knowledge on: (i) small molecules as substrates of uptake and efflux transporters; (ii) the impact of transporter deficiency in knockout mouse models on plasma and tissue concentrations; (iii) small molecules as inhibitors of uptake and efflux transporters with possible consequences for drug-drug interactions and the reversal of multidrug resistance; and (iv) on clinical studies investigating the association of polymorphisms in genes encoding drug transporters with pharmacokinetics, outcome and toxicity during treatment with the small molecules.

Relevance of conserved lysine and arginine residues in transmembrane helices for the transport activity of organic anion transporting polypeptide 1B3.

BACKGROUND AND PURPOSE: Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin. EXPERIMENTAL APPROACH: Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated. KEY RESULTS: Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V(max) normalized to cell surface protein expression for Lys41>Ala (wild type: 190 +/- 8, Lys41>Ala:16 +/- 4 pmol (mg protein)(-1) min(-1), P < 0.001), whereas V(max) of Lys41>Arg and Arg580>Lys (103 +/- 8 and 123 +/- 14 pmol (mg protein)(-1) min(-1), P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala. CONCLUSIONS AND IMPLICATIONS: We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.

Variability in human hepatic MRP4 expression: influence of cholestasis and genotype.

The multidrug resistance protein 4 (MRP4) is an efflux transporter involved in the transport of endogenous substrates and xenobiotics. We measured MRP4 mRNA and protein expression in human livers and found a 38- and 45-fold variability, respectively. We sequenced 2 kb of the 5'-flanking region, all exons and intron/exon boundaries of the MRP4 gene in 95 patients and identified 74 genetic variants including 10 non-synonymous variations, seven of them being located in highly conserved regions. None of the detected polymorphisms was significantly associated with changes in the MRP4 mRNA or protein expression. Immunofluorescence microscopy indicated that none of the non-synonymous variations affected the cellular localization of MRP4. However, in cholestatic patients the MRP4 mRNA and protein expression both were significantly upregulated compared to non-cholestatic livers (protein: 299+/-138 vs 100+/-60a.u., P<0.001). Taken together, human hepatic MRP4 expression is highly variable. Genetic variations were not sufficient to explain this variability. In contrast, cholestasis is one major determinant of human hepatic MRP4 expression.

Influence of rifampicin on the expression and function of human intestinal cytochrome P450 enzymes.

AIMS: To investigate the potential induction by rifampicin of intestinal CYP2C8, CYP2C9, CYP2D6 and CYP3A4 using preparations of human enterocytes. METHODS: Using a multilumen perfusion catheter shed human enterocytes were collected from 6 healthy subjects before and after 10 days of 600 mg day(-1) oral rifampicin administration. The protein expression of CYP2C8, CYP2C9, CYP2D6 and CYP3A4 as well as that of CYP3A4 mRNA was determined using Western blotting and RT-PCR, respectively. RESULTS: CYP3A4 mRNA expression in shed enterocytes increased from 74.6 +/- 44.2 to 143.2 +/- 68.4 a.u. (P < 0.05, 95% CI: 21.8-115.3). Expression of CYP2C8 and CYP2C9 increased from 5.1 +/- 0.9 to 10.4 +/- 2.3 pmol mg(-1) protein (P < 0.01, 95% CI: 2.8-7.7) and from 4.2 +/- 1.4 to 5.7 +/- 1.1 pmol mg(-1) protein (P < 0.01, 95% CI: 0.6-2.4), respectively. No significant difference in CYP2D6 expression before and during rifampicin intake was observed. Rifampicin administration also resulted in a significant induction of CYP3A4 protein (34.1 +/- 10.7 vs. 113.9 +/- 31.1 pmol mg(-1) protein (P < 0.001, 95% CI: 51.8-107.6)). Ex vivo incubation of enterocyte homogenates with verapamil resulted in a significantly increased production of the metabolites formed via CYP3A4 (D-617: 125.9 +/- 118.8 vs. 277.2 +/- 145.5 pmol min(-1) mg(-1) protein (P < 0.05, 95% CI: 30.1-272.5); norverapamil: 113.0 +/- 57.9 vs. 398.4 +/- 148.2 pmol min(-1) mg(-1) protein (P < 0.05, 95% CI: 47.2-523.6)). CONCLUSION: Our findings indicate that shed enterocytes are a useful tool to study the expression, regulation and function of drug metabolizing enzymes. Induction of intestinal CYP2C8 and CYP2C9 might contribute in part to rifampicin - mediated drug interactions, in addition to their hepatic counterparts and intestinal and hepatic CYP3A4.

[Adenosine-dependent death of Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16 (author's transl)]. / Durch Adenosin hervorgerufene Abtötung von Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16

Heterotrophic growth with fructose and autotrophic growth with hydrogen and carbon dioxide was entirely inhibited by adenosine at a concentration of 0.6 mg/ml in Hydrogenomonas eutropha (Alcaligenes eutrophus) H 16. Growth inhibition was not accompanied by a detectable uptake of the nucleoside. Adenosine caused a rapid inhibition of protein synthesis, followed by a decrease in nucleic acid formation. Enzyme synthesis was also impared, whilst cell respiration remained uneffected. Adenosin also caused a drastic but temporary decrease in viable cell counts. Cells incubated in presence of adenosine and fructose for several days, however, were no longer susceptable to this nucleoside. Adenosine-dependent growth inhibition turned out to be contingent upon the nature of the organic substrate. Cells growing with succinate, glutamate or peptone were less effected than cells, growing autotrophically or with fructose. No inhibition was observed in fructose-growing cells, if amino acids were also present in the medium. Several other nucleosides tested, did not show such growth inhibition, except desoxyadenosine, which, however, did not effect viable cell counts.