Ex vivo gut-hepato-biliary organ perfusion model to characterize oral absorption, gut-wall metabolism, pre-systemic hepatic metabolism and biliary excretion; application to midazolam.

To date, characterization of the first-pass effect of orally administered drugs consisting of local intestinal absorption and metabolism, portal vein transport and hepatobiliary processes remains challenging. Aim of this study was to explore the applicability of a porcine ex-vivo perfusion model to study oral absorption, gut-hepatobiliary metabolism and biliary excretion of midazolam. Slaughterhouse procured porcine en bloc organs (n = 4), were perfused via the aorta and portal vein. After 120 min of perfusion, midazolam, atenolol, antipyrine and FD4 were dosed via the duodenum and samples were taken from the systemic- and portal vein perfusate, intestinal faecal effluent and bile to determine drug and metabolite concentrations. Stable arterial and portal vein flow was obtained and viability of the perfused organs was confirmed. After intraduodenal administration, midazolam was rapidly detected in the portal vein together with 1-OH midazolam (EG-pv of 0.16±0.1) resulting from gut wall metabolism through oxidation. In the intestinal faecal effluent, 1-OH midazolam and 1-OH midazolam glucuronide (EG-intestine 0.051±0.03) was observed resulting from local gut glucuronidation. Biliary elimination of midazolam (0.04±0.01 %) and its glucuronide (0.01±0.01 %) only minimally contributed to the enterohepatic circulation. More extensive hepatic metabolism (FH 0.35±0.07) over intestinal metabolism (FG 0.78±0.11) was shown, resulting in oral bioavailability of 0.27±0.05. Ex vivo perfusion demonstrated to be a novel approach to characterize pre-systemic extraction of midazolam by measuring intestinal as well as hepatic extraction. The model can generate valuable insights into the absorption and metabolism of new drugs.

Evaluation of Normothermic Machine Perfusion of Porcine Livers as a Novel Preclinical Model to Predict Biliary Clearance and Transporter-Mediated Drug-Drug Interactions Using Statins.

There is a lack of translational preclinical models that can predict hepatic handling of drugs. In this study, we aimed to evaluate the applicability of normothermic machine perfusion (NMP) of porcine livers as a novel ex vivo model to predict hepatic clearance, biliary excretion, and plasma exposure of drugs. For this evaluation, we dosed atorvastatin, pitavastatin, and rosuvastatin as model drugs to porcine livers and studied the effect of common drug-drug interactions (DDIs) on these processes. After 120 minutes of perfusion, 0.104 mg atorvastatin (n = 3), 0.140 mg pitavastatin (n = 5), or 1.4 mg rosuvastatin (n = 4) was administered to the portal vein, which was followed 120 minutes later by a second bolus of the statin coadministered with OATP perpetrator drug rifampicin (67.7 mg). After the first dose, all statins were rapidly cleared from the circulation (hepatic extraction ratio > 0.7) and excreted into the bile. Presence of human-specific atorvastatin metabolites confirmed the metabolic capacity of porcine livers. The predicted biliary clearance of rosuvastatin was found to be closer to the observed biliary clearance. A rank order of the DDI between the various systems upon coadministration with rifampicin could be observed: atorvastatin (AUC ratio 7.2) > rosuvastatin (AUC ratio 3.1) > pitavastatin (AUC ratio 2.6), which is in good agreement with the clinical DDI data. The results from this study demonstrated the applicability of using NMP of porcine livers as a novel preclinical model to study OATP-mediated DDI and its effect on hepatic clearance, biliary excretion, and plasma profile of drugs. SIGNIFICANCE STATEMENT: This study evaluated the use of normothermic machine perfusion (NMP) of porcine livers as a novel preclinical model to study hepatic clearance, biliary excretion, plasma (metabolite) profile of statins, and OATP-mediated DDI. Results showed that NMP of porcine livers is a reliable model to study OATP-mediated DDI. Overall, the rank order of DDI severity indicated in these experiments is in good agreement with clinical data, indicating the potential importance of this new ex vivo model in early drug discovery.

Mechanisms and immunomodulatory properties of pre- and probiotics.

The human body is exposed to many xenobiotic, potentially harmful compounds. The intestinal immune system is crucial in protecting the human body from these substances. Moreover, many microorganisms, residing in the gastrointestinal tract, play an important role in modulating immune responses. Pre- and probiotics may have beneficial effects on the microbial composition and activity within the human gut, subsequently affecting the immune system. Prebiotics can exert their effects via different mechanisms, like selectively stimulating the growth of bacteria by providing substrates or via direct immune stimulation. Probiotics may have beneficial health effects via competition with pathogens for substrates and binding intestinal sites, bioconversions of for example sugars into fermentation products with inhibitory properties, production of growth substrates like vitamins for the host, direct antagonism of pathogens via antimicrobial peptide production, reduction of inflammation and stimulation of immune cells. This review focuses on the different mechanisms via which the pre- and probiotics exert their beneficial effects on the host, addressing their immunomodulatory properties in particular.

An Ex Vivo Fermentation Screening Platform to Study Drug Metabolism by Human Gut Microbiota.

Colon microbiota-based drug metabolism has received little attention thus far in the process of drug development, whereas the role of gut microbiota in clinical safety and efficacy of drugs has become more clear. Many of these studies have been performed using animal studies, but the translational value of these data with respect to drug pharmacokinetics, efficacy, and safety is largely unknown. To investigate human colon microbiota-mediated drug metabolism, we applied a recently developed ex vivo fermentation screening platform, in which human colonic microbiota conditions are simulated. A set of 12 drugs (omeprazole, simvastatin, metronidazole, risperidone, sulfinpyrazone, sulindac, levodopa, dapsone, nizatidine, sulfasalazine, zonisamide, and acetaminophen) was incubated with human colon microbiota under strictly anaerobic conditions, and samples were analyzed using high-performance liquid chromatograph-UV-high-resolution mass spectrometry analysis. The human microbiota in the fermentation assay consisted of bacterial genera regularly encountered in human colon and fecal samples and could be reproducibly cultured in independent experiments over time. In addition, fully anaerobic culture conditions could be maintained for 24 hours of incubation. Five out of the 12 included drugs (sulfasalazine, sulfinpyrazone, sulindac, nizatidine, and risperidone) showed microbiota-based biotransformation after 24 hours of incubation in the ex vivo fermentation assay. We demonstrated that drug metabolites formed by microbial metabolism can be detected in a qualitative manner and that the data are in accordance with those reported earlier for in vivo metabolism. In conclusion, we present a research tool to investigate human colon microbiota-based drug metabolism that may be applied to enable translatability of microbiota-based drug metabolism.

Silver iodide nanowires grown within tubular J-aggregates.

Silver iodide nanowires have been grown within tubular J-aggregates of the cyanine dye 3,3'-bis(2-sulfopropyl)-5,5',6,6'-tetrachloro-1,1'-dioctylbenzimida-carbo-cyanine (C8S3) from aqueous AgNO3 solutions. Crystal structure analysis by selected area electron diffraction (SAED), high resolution transmission electron microscopy (HRTEM) and energy-dispersive X-ray spectroscopy (EDXS) of single nanowires revealed that they are of silver iodide (AgI), while previously they were presumed to be of metallic silver. Iodine has not been added intentionally, but it is a remnant from the chemical synthesis of the dye and present in a dye:iodine ratio of almost 2:1, as revealed by inductively coupled plasma mass spectrometry (ICP-MS). The AgI wires grow as single crystals with lengths of several 10-100 nm and width of 6.5 ±â€¯0.5 nm. The width and the orientation of the crystal relative to the aggregate axis are defined by the tubular structure of the templating dye aggregate. Caused by the nucleation at the tube wall the main growth is not along the usually preferred [0 0 0 1] direction but along the extension of the basal plane, which is furthermore tilted by an angle of 6°â€¯±â€¯2° against the main axis of the aggregate. This self-assembled system represents an organic-inorganic hybrid system with a well-defined semiconductor nanowire, AgI, that is strictly oriented with respect to the aggregated phase of conjugated molecules.

Generation of Bayesian prediction models for OATP-mediated drug-drug interactions based on inhibition screen of OATP1B1, OATP1B1∗15 and OATP1B3.

Human organic anion-transporting polypeptide 1B1 (OATP1B1) and OATP1B3 are important hepatic uptake transporters. Early assessment of OATP1B1/1B3-mediated drug-drug interactions (DDIs) is therefore important for successful drug development. A promising approach for early screening and prediction of DDIs is computational modeling. In this study we aimed to generate a rapid, single Bayesian prediction model for OATP1B1, OATP1B1∗15 and OATP1B3 inhibition. Besides our previously generated HEK-OATP1B1 and HEK-OATP1B1∗15 cells, we now generated and characterized HEK-OATP1B3 cells. Using these cell lines we investigated the inhibitory potential of 640 FDA-approved drugs from a commercial library (10µM) on the uptake of [(3)H]-estradiol-17ß-d-glucuronide (1µM) by OATP1B1, OATP1B1∗15, and OATP1B3. Using a cut-off of ⩾60% inhibition, 8% and 7% of the 640 drugs were potent OATP1B1 and OATP1B1∗15 inhibitors, respectively. Only 1% of the tested drugs significantly inhibited OATP1B3, which was not sufficient for Bayesian modeling. Modeling of OATP1B1 and OATP1B1∗15 inhibition revealed that presence of conjugated systems and (hetero)cycles with acceptor/donor atoms in- or outside the ring enhance the probability of a molecule binding these transporters. The overall performance of the model for OATP1B1 and OATP1B1∗15 was ⩾80%, including evaluation with a true external test set. Our Bayesian classification model thus represents a fast, inexpensive and robust means of assessing potential binding of new chemical entities to OATP1B1 and OATP1B1∗15. As such, this model may be used to rank compounds early in the drug development process, helping to avoid adverse effects in a later stage due to inhibition of OATP1B1 and/or OATP1B1∗15.

Combined analysis of pharmacokinetic and efficacy data of preclinical studies with statins markedly improves translation of drug efficacy to human trials.

Correct prediction of human pharmacokinetics (PK) and the safety and efficacy of novel compounds based on preclinical data, is essential but often fails. In the current study, we aimed to improve the predictive value of ApoE*3Leiden (E3L) transgenic mice regarding the cholesterol-lowering efficacy of various statins in humans by combining pharmacokinetic with efficacy data. The efficacy of five currently marketed statins (atorvastatin, simvastatin, lovastatin, pravastatin, and rosuvastatin) in hypercholesterolemic patients (low-density lipoprotein ≥ 160 mg/dl) was ranked based on meta-analysis of published human trials. Additionally, a preclinical combined PK efficacy data set for these five statins was established in E3L mice that were fed a high-cholesterol diet for 4 weeks, followed by 6 weeks of drug intervention in which statins were supplemented to the diet. Plasma and tissue levels of the statins were determined on administration of (radiolabeled) drugs (10 mg/kg p.o.). As expected, all statins reduced plasma cholesterol in the preclinical model, but a direct correlation between cholesterol lowering efficacy of the different statins in mice and in humans did not reach statistical significance (R(2) = 0.11, P < 0.57). It is noteworthy that, when murine data were corrected for effective liver uptake of the different statins, the correlation markedly increased (R(2) = 0.89, P < 0.05). Here we show for the first time that hepatic uptake of statins is related to their cholesterol-lowering efficacy and provide evidence that combined PK and efficacy studies can substantially improve the translational value of the E3L mouse model in the case of statin treatment. This strategy may also be applicable for other classes of drugs and other preclinical models.

Drug-drug interactions between rosuvastatin and oral antidiabetic drugs occurring at the level of OATP1B1.

Organic anion-transporting polypeptide 1B1 (OATP1B1) is an important hepatic uptake transporter, of which the polymorphic variant OATP1B1*15 (Asn130Asp and Val174Ala) has been associated with decreased transport activity. Rosuvastatin is an OATP1B1 substrate and often concomitantly prescribed with oral antidiabetics in the clinic. The aim of this study was to investigate possible drug-drug interactions between these drugs at the level of OATP1B1 and OATP1B1*15. We generated human embryonic kidney (HEK)293 cells stably overexpressing OATP1B1 or OATP1B1*15 that showed similar protein expression levels of OATP1B1 and OATP1B1*15 at the cell membrane as measured by liquid chromatography-tandem mass spectrometry. In HEK-OATP1B1*15 cells, the V(max) for OATP1B1-mediated transport of E(2)17ß-G (estradiol 17ß-d-glucuronide) was decreased >60%, whereas K(m) values (Michaelis constant) were comparable. Uptake of rosuvastatin in HEK-OATP1B1 cells (K(m) 13.1 ± 0.43 µM) was nearly absent in HEK-OATP1B1*15 cells. Interestingly, several oral antidiabetics (glyburide, glimepiride, troglitazone, pioglitazone, glipizide, gliclazide, and tolbutamide), but not metformin, were identified as significant inhibitors of the OATP1B1-mediated transport of rosuvastatin. The IC(50) values for inhibition of E(2)17ß-G uptake were similar between OATP1B1 and OATP1B1*15. In conclusion, these studies indicate that several oral antidiabetic drugs affect the OATP1B1-mediated uptake of rosuvastatin in vitro. The next step will be to translate these data to the clinical situation, as it remains to be established whether the studied oral antidiabetics indeed affect the clinical pharmacokinetic profile of rosuvastatin in patients.

Dioxins and dioxin-like PCBs in different fish from the river Elbe and its tributaries, Germany.

In a long-term program polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/Fs) as well as dioxin-like polychlorinated biphenyls (DL-PCBs) were analyzed in the muscle tissue of eels (Anguilla anguilla), bream (Abramis brama), European chub (Leuciscus cephalus) and ide (Leuciscus idus) from the river Elbe and its tributaries Mulde and Saale. The variation of the PCDD/F and DL-PCB concentrations in all fish samples is very large, whereby the DL-PCBs predominate in comparison to the PCDD/Fs. In the eels, the concentrations (pg WHO-TEQ/g ww) for the PCDD/Fs lie in the range of 0.48-22 and for the DL-PCBs between 8.5 and 59. In the whitefish, the concentration range is 0.48-12 for the PCDD/Fs and 1.2-14 for the DL-PCBs. Statistical analysis using relative congener patterns for PCDD/Fs allow spatial correlations to be examined for sub-populations of eels and whitefish. The results are compared to the maximum levels laid down in the European Commission Regulation (EC) No. 466/2001 and the action levels of the European Commission Recommendation 2006/88/EC. Eels caught directly after the major flood in August 2002 as well as eels near Hamburg (years 1996 and 1998) show high concentration peaks. Compared to the eels whitefish is less contaminated with PCDD/Fs and DL-PCBs.

Contamination of the alluvial plain, feeding-stuffs and foodstuffs with polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (PCDD/Fs), dioxin-like polychlorinated biphenyls (DL-PCBs) and mercury from the River Elbe in the light of the flood event in August 2002.

Meadow soils, feeding-stuffs and foodstuffs from the alluvial plain of the river Elbe were analyzed in respect of PCDD/Fs, DL-PCBs and mercury with a view to assessing the consequences of the extreme flood of August 2002. The PCDD/F concentrations in the soils range from 3 to 2100 ng WHO-TEQ/kg dm, and for the DL-PCBs the range was 0.32 to 28 ng WHO-TEQ/kg dm. On the basis of established threshold values >40% of the areas are only fit for restricted usage. Mercury concentrations range from 0.11 to 17 mg/kg dm, whereby the action value of 2 mg/kg dm is exceeded in about 50% of the soil samples. A cumulative memory effect from past floods rather than a recent contamination from August 2002 is documented. Soils taken from behind broken dykes showed significantly lower concentrations. Grass, hay and grass silage originating from pasture land in Lower Saxony were taken before and immediately after the flooding. PCDD/Fs range from 0.29 to 16 ng WHO-TEQ/kg, the maximum permitted value of 0.75 ng WHO-TEQ/kg was exceeded in about 50% of the samples. Muscle-tissue from cattle, sheep, lamb and a roe deer as well as untreated milk from individual cows returned values ranging from 0.76 to 5.9 pg WHO-PCDD/F-TEQ/g fat, and 10% of the samples returned values higher than the permitted maximum of 3 pg WHO-PCDD/F-TEQ/g fat. The action value of 2 pg WHO-PCDD/F-TEQ/g fat was exceeded in 33% of the samples. No direct connection between these results and the effects of the flood could be established. A major input path for PCDD/Fs is the tributary Mulde, which discharges contaminated sediments from its catchment area into the Elbe.

The Elbe flood in August 2002--occurrence of polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans (PCDD/F) and dioxin-like PCB in suspended particulate matter (SPM), sediment and fish.

As a result of extreme precipitation in August 2002 major flooding occurred in the catchment area of the rivers Elbe, Vltava (Moldau) and Mulde. Pollutants from industrial sites and from municipal sewage treatment works (STW) entered the Elbe and led to a serious pollution problem in the river. PCDD/F concentrations (in pg WHO-TEQ/g dw) in SPM ranged from 7-150, in sediments from 3-140; the "safe sediment value" of 20 was exceeded in 46% of the samples. 24 eels showed a wide concentration variation for these contaminants. The WHO-PCDD/F+PCB-TEQ values lay in the range from 11-56 pg/g ww, whereby the WHO-PCB-TEQ values were several times higher than the WHO-PCDD/F-TEQ values. The maximum permitted value of 4 pg WHO-PCDD/F/g ww (EU Directive No. 2375/2001) was reached or exceeded in 54% of the individuals. A statistical analysis using data from SPM and sediment samples showed that in the Czech river section the flooding activated a contamination source in the vicinity of the Spolana works. The influence of the tributary Mulde could be clearly demonstrated. Only a major clean-up of the contaminated sites in Bitterfeld can lead to a mid to long term improvement in respect of PCDD/F and dioxin-like PCB input into the Elbe.

Application of a novel and fast information-theoretic method to the discovery of higher-order correlations in protein databases.

We present a fast, discrete data-mining approach to the problem of finding kappa-tuples of correlated amino acid residues in protein sequence data. When sets of sequence-distant sites display high mutual information, they may bespeak important structural or functional features. Our novel methodology overcomes the limitations of previous methods which examined only single-residue features or pairwise interactions.

Protein model representation and construction.

Crystallographic studies play a major role in current efforts towards protein structure determination. However, despite recent advances in computational tools for molecular modeling and graphics, the task of constructing a protein model from crystallographic data remains complex and time-consuming, requiring extensive expert intervention. This paper describes an approach to automating the process of model construction, where a model is represented as an annotated trace (or partial trace) of the three-dimensional backbone of the structure. Potential models are generated using an evolutionary algorithm, which incorporates multiple fitness functions tailored to different structural levels in the protein. Preliminary experimental results, which demonstrate the viability of the approach, are reported.

From electron density and sequence to structure: integrating protein image analysis and threading for structure determination.

This paper presents a computational methodology for integrating techniques from protein image interpretation and protein sequence threading, applied to the problem of structure determination from experimental X-ray crystallographic electron density maps. In the proposed architecture, image interpretation of an electron density map produces candidate structural segments; threading is applied to evaluate these hypothesized segments and thus to constrain the set of possible image interpretations. We present the results of experiments designed to test ability of the threading module to discriminate between correct and incorrect alignments of protein sequences onto structural models derived from protein image interpretation. The long-term goal of this research is to improve our ability to determine protein structures from crystallographic data, and to further our understanding of the underlying relationship between sequence and structure.

Comments on ;parallel algorithms for finding a near-maximum independent set of a circle graph' [with reply].

The authors refers to the work of Y. Takefuji et al. (see ibid., vol.1, pp. 263-267, Sept. (1990)), which is concerned with the problem of RNA secondary structure prediction, and draws the reader's attention to his own model and experiments in training the neural networks on small tRNA subsequences. The author admits that Takefuji et al. outline an elegant way to map the problem onto neural architectures, but suggests that such mappings can be augmented with empirical knowledge (e.g., free energy values of base pairs and substructures) and the ability to learn. In their reply, Y. Takefuji and K.-C. Lee hold that the necessity of the learning capability for the RNA secondary structure prediction is questionable. They believe that the task is to build a robust parallel algorithm considering more thermodynamic properties in the model.

[The analysis of polycyclics in vegetable samples]. / Zur Analytik von Polycyclen in Gemüseproben.

PAH analyses in vegetables are often difficult to carry out; therefore two different clean-up methods, which allow elimination of interfering matrix components such as essential oils, waxes, carotinoids and chlorophylls were developed. Clean-up of 32 different vegetables samples consisted of either Bio-Beads S-X3 chromatography or semi-preparative HPLC on silica gel Si-60. PAH were determined by capillary GC-MS, which allowed the application of deuterium-labeled internal standards. The results are presented; higher PAH contamination was detected in kale and parsley.

Update for users of the Cornell sequence analysis package.

This paper reports some additions and improvements to the Cornell sequence analysis package for microcomputers (Fristensky, Lis, and Wu, 1982, Nucleic Acids Res. 10, 6451-6463).