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A reliable prediction of the oral bioavailability in humans is crucial and of high interest for pharmaceutical and food industry. The predictive value of currently used in silico methods, in vitro cell lines, ex vivo intestinal tissue and/or in vivo animal studies for human intestinal absorption, however, is often insufficient, especially when food-drug interactions are evaluated. Ideally, for this purpose healthy human intestinal tissue is used, but due to its limited availability there is a need for alternatives. The aim of this study was to evaluate the applicability of healthy porcine intestinal tissue mounted in a newly developed InTESTine™ system to predict human intestinal absorption of compounds with different chemical characteristics, and within biorelevant matrices. To that end, first, a representative set of compounds was chosen of which the apparent permeability (Papp) data in both Caco-2 cells and human intestinal tissue mounted in the Ussing chamber system, and absolute human oral bioavailability were reported. Thereafter, Papp values of the subset were determined in both porcine jejunal tissue and our own Caco-2 cells. In addition, the feasibility of this new approach to study regional differences (duodenum, jejunum, and ileum) in permeability of compounds and to study the effects of luminal factors on permeability was also investigated. For the latter, a comparison was made between the compatibility of porcine intestinal tissue, Caco-2 cells, and Caco-2 cells co-cultured with the mucin producing HT29-MTX cells with biorelevant samples as collected from an in vitro dynamic gastrointestinal model (TIM). The results demonstrated that for the paracellularly transported compounds atenolol, cimetidine, mannitol and ranitidine porcine Papp values are within 3-fold difference of human Papp values, whereas the Caco-2 Papp values are beyond 3-fold difference. Overall, the porcine intestinal tissue Papp values are more comparable to human Papp values (9 out of 12 are within 3-fold difference), compared to Caco-2 Papp values (4 out of 12 are within 3-fold difference). In addition, for the selected hydrophilic compounds a significant increase in the permeability was observed from duodenum to ileum. Finally, this study indicated that porcine jejunal tissue segments can be used with undiluted luminal samples to predict human intestinal permeability and the effect of biorelevant matrices on this. In conclusion, viable porcine intestinal tissue mounted in the InTESTine™ system can be applied as a reliable tool for the assessment of intestinal permeability in the absence and presence of biorelevant samples. This would enable an accessible opportunity for a reliable prediction of human intestinal absorption, and the effect of luminal compounds such as digested foods, early in drug development. © 2014 Elsevier B.V. All rights reserved. Chemicals/CAS: acebutolol, 34381-68-5, 37517-30-9; atenolol, 29122-68-7, 93379-54-5; candesartan, 139481-59-7; cetirizine, 83881-51-0, 83881-52-1; cimetidine, 51481-61-9, 70059-30-2; ciprofloxacin, 85721-33-1; diazepam, 439-14-5; digoxin, 20830-75-5, 57285-89-9; famotidine, 76824-35-6; hydrocortisone, 50-23-7; ibuprofen, 15687-27-1, 79261-49-7, 31121-93-4, 527688-20-6; indometacin, 53-86-1, 74252-25-8, 7681-54-1; mannitol, 69-65-8, 87-78-5; melagatran, 159776-70-2; metoprolol, 37350-58-6; oxprenolol, 22972-97-0, 6452-71-7, 6452-73-9; phenazone, 60-80-0; pindolol, 13523-86-9, 21870-06-4; propranolol, 13013-17-7, 318-98-9, 3506-09-0, 4199-09-1, 525-66-6; ranitidine, 66357-35-5, 66357-59-3; salazosulfapyridine, 599-79-1; salicylic acid, 63-36-5, 69-72-7; testosterone, 58-22-0; verapamil, 152-11-4, 52-53-9 Manufacturers: Gibco, United Kingdom; Sigma Aldrich, Netherlands; Newport, United States

OrBiTo is a new European project within the IMI programme in the area of oral biopharmaceutics tools that includes world leading scientists from nine European universities, one regulatory agency, one non-profit research organization, four SMEs together with scientists from twelve pharmaceutical companies. The OrBiTo project will address key gaps in our knowledge of gastrointestinal (GI) drug absorption and deliver a framework for rational application of predictive biopharmaceutics tools for oral drug delivery. This will be achieved through novel prospective investigations to define new methodologies as well as refinement of existing tools. Extensive validation of novel and existing biopharmaceutics tools will be performed using active pharmaceutical ingredient (API), formulations and supporting datasets from industry partners. A combination of high quality in vitro or in silico characterizations of API and formulations will be integrated into physiologically based in silico biopharmaceutics models capturing the full complexity of GI drug absorption. This approach gives an unparalleled opportunity to initiate a transformational change in industrial research and development to achieve model-based pharmaceutical product development in accordance with the Quality by Design concept. Benefits include an accelerated and more efficient drug candidate selection, formulation development process, particularly for challenging projects such as low solubility molecules (BCS II and IV), enhanced and modified-release formulations, as well as allowing optimization of clinical product performance for patient benefit. In addition, the tools emerging from OrBiTo are expected to significantly reduce demand for animal experiments in the future as well as reducing the number of human bioequivalence studies required to bridge formulations after manufacturing or composition changes. © 2013 Elsevier B.V. All rights reserved.

Accurate prediction of the in vivo biopharmaceutical performance of oral drug formulations is critical to efficient drug development. Traditionally, in vitro evaluation of oral drug formulations has focused on disintegration and dissolution testing for quality control (QC) purposes. The connection with in vivo biopharmaceutical performance has often been ignored. More recently, the switch to assessing drug products in a more biorelevant and mechanistic manner has advanced the understanding of drug formulation behavior. Notwithstanding this evolution, predicting the in vivo biopharmaceutical performance of formulations that rely on complex intraluminal processes (e.g. solubilization, supersaturation, precipitation...) remains extremely challenging. Concomitantly, the increasing demand for complex formulations to overcome low drug solubility or to control drug release rates urges the development of new in vitro tools. Development and optimizing innovative, predictive Oral Biopharmaceutical Tools is the main target of the OrBiTo project within the Innovative Medicines Initiative (IMI) framework. A combination of physico-chemical measurements, in vitro tests, in vivo methods, and physiology-based pharmacokinetic modeling is expected to create a unique knowledge platform, enabling the bottlenecks in drug development to be removed and the whole process of drug development to become more efficient. As part of the basis for the OrBiTo project, this review summarizes the current status of predictive in vitro assessment tools for formulation behavior. Both pharmacopoeia-listed apparatus and more advanced tools are discussed. Special attention is paid to major issues limiting the predictive power of traditional tools, including the simulation of dynamic changes in gastrointestinal conditions, the adequate reproduction of gastrointestinal motility, the simulation of supersaturation and precipitation, and the implementation of the solubility-permeability interplay. It is anticipated that the innovative in vitro biopharmaceutical tools arising from the OrBiTo project will lead to improved predictions for in vivo behavior of drug formulations in the GI tract. © 2013 Elsevier B.V. All rights reserved.

In contrast to primary hepatocytes, estimating carrier-mediated hepatic disposition by using a panel of single transfected cell-lines provides direct information on the contribution of the individual transporters to the net disposition. The most direct way to correct for differences in transporter abundance between cell-lines and tissue is by using absolute protein quantification. In the present study, the performance of this strategy to predict human hepatic uptake transport was investigated and compared with traditional scaling from primary human hepatocytes. Rosuvastatin was used as a model compound. The uptake activity was measured in HEK293 cell-lines stably overexpressing OATP1B1⁄1a, OATP1B3 or OATP2B1, the major transporters involved in human hepatic uptake of rosuvastatin, or expressing OATP1B1⁄15, associated with reduced hepatic uptake of rosuvastatin. The abundance of these transporter proteins in the outer membranes of HEK293-cells, in human primary hepatocytes and in human liver tissue was determined by LC–MS/MS. The measured activity, corrected for protein abundance and scaled to the whole liver, gave a very accurate prediction of the hepatic intrinsic clearance observed in vivo. Embedded in a PBPK model describing the hepatic disposition and enterohepatic circulation, the collec- tive in vitro data resulted in a good explanation of the observed oral and intravenous pharmacokinetic profiles of rosuvastatin. The model allowed simulation of the effect of polymorphic variants of OATP1B1 on rosuvastatin pharmacokinetics. These results encourage a larger scale validation. This approach may facilitate prediction of drug–drug interactions, scaling of transporter processes across subpopulations (children, diseased patients), and may be extended to tissues for which primary cells may be more difficult to obtain. Chemicals/CAS: rosuvastatin, 147098-18-8, 147098-20-2 Manufacturers: Moravek, United States

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 Vmax for OATP1B1-mediated transport of E217b-G (estradiol 17b-D-glucuronide) was decreased

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Introduction: Reliable in vitro and in silico assays as alternatives for in vivo developmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivo developmental toxicity in in vitro assays are needed. Areas covered: The present review gives an overview of alternative assays, as described in literature, for in vivo developmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivo developmental toxicity, and how in vitro effect data can be translated to the in vivo situation using physiologically based kinetic (PBK) modeling. Expert opinion: Relevance of readouts in in vitro developmental toxicity assays as predictive biomarkers for in vivo developmental toxicity should be evaluated by comparing the obtained in vitro effect concentrations with in vivo internal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitro effect concentrations to in vivo dose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitro toxicity data in the safety assessment of compounds. Read More: http://informahealthcare.com/doi/abs/10.1517/17425255.2012.639762

INTRODUCTION: Reliable in vitro and in silico assays as alternatives for in vivo developmental toxicity studies are urgently needed, for the replacement, reduction and refinement (3Rs) of animal use in toxicological research. Therefore, relevant biomarkers for in vivo developmental toxicity in in vitro assays are needed. AREAS COVERED: The present review gives an overview of alternative assays, as described in literature, for in vivo developmental toxicity, including the effects (readouts) assessed in these assays. The authors discuss how these data may be used to obtain relevant biomarkers for in vivo developmental toxicity, and how in vitro effect data can be translated to the in vivo situation using physiologically based kinetic (PBK) modeling. EXPERT OPINION: Relevance of readouts in in vitro developmental toxicity assays as predictive biomarkers for in vivo developmental toxicity should be evaluated by comparing the obtained in vitro effect concentrations with in vivo internal concentrations at dose levels causing developmental toxicity. Extrapolation of the in vitro effect concentrations to in vivo dose levels using PBK modeling (i.e., reverse dosimetry) is promising in its use to derive points of departure for risk assessment, enabling the use of in vitro toxicity data in the safety assessment of compounds.

Embryotoxicity of glycol ethers is caused by their alkoxyacetic acid metabolites, but the mechanism underlying the embryotoxicity of these acid metabolites is so far not known. The present study investigates a possible mechanism underlying the embryotoxicity of glycol ether alkoxyacetic acid metabolites using the methoxyacetic acid (MAA) metabolite of ethylene glycol monomethyl ether as the model compound. The results obtained demonstrate an MAA-induced decrease of the intracellular pH (pHi) of embryonic BALB/c-3T3 cells as well as of embryonic stem (ES)-D3 cells, at concentrations that affect ES-D3 cell differentiation. These results suggest a mechanism for MAA-mediated embryotoxicity similar to the mechanism of embryotoxicity of the drugs valproic acid and acetazolamide (ACZ), known to decrease the pHi in vivo, and therefore used as positive controls. The embryotoxic alkoxyacetic acid metabolites ethoxyacetic acid, butoxyacetic acid and phenoxyacetic acid also caused an intracellular acidification of BALB/c-3T3 cells at concentrations that are known to inhibit ES-D3 cell differentiation. Two other embryotoxic compounds, all-trans-retinoic acid and 5-fluorouracil, did not decrease the pHi of embryonic cells at concentrations that affect ES-D3 cell differentiation, pointing at a different mechanism of embryotoxicity of these compounds. MAA and ACZ induced a concentration-dependent inhibition of ES-D3 cell differentiation, which was enhanced by amiloride, an inhibitor of the Na+/H+-antiporter, corroborating an important role of the pHi in the embryotoxic mechanism of both compounds. Together, the results presented indicate that a decrease of the pHi may be the mechanism of embryotoxicity of the alkoxyacetic acid metabolites of the glycol ethers. © 2010 Elsevier Inc.

At present, regulatory assessment of systemic toxicity is almost solely carried out using animal models. The European Commission's REACH legislation stimulates the use of animal-free approaches to obtain information on the toxicity of chemicals. In vitro toxicity tests provide in vitro concentration-response curves for specific target cells, whereas in vivo dose-response curves are regularly used for human risk assessment. The present study shows an approach to predict in vivo dose-response curves for developmental toxicity by combining in vitro toxicity data and in silico kinetic modeling. A physiologically based kinetic (PBK) model was developed, describing the kinetics of four glycol ethers and their embryotoxic alkoxyacetic acid metabolites in rat and man. In vitro toxicity data of these metabolites derived in the embryonic stem cell test were used as input in the PBK model to extrapolate in vitro concentration-response curves to predicted in vivo dose-response curves for developmental toxicity of the parent glycol ethers in rat and man. The predicted dose-response curves for rat were found to be in concordance with the embryotoxic dose levels measured in reported in vivo rat studies. Therefore, predicted dose-response curves for rat could be used to set a point of departure for deriving safe exposure limits in human risk assessment. Combining the in vitro toxicity data with a human PBK model allows the prediction of dose-response curves for human developmental toxicity. This approach could therefore provide a means to reduce the need for animal testing in human risk assessment practices. © The Author 2010. All rights reserved.

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds. © The Author 2009. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

The embryonic stem cell test (EST) has been proposed as an in vitro assay that might reduce animal experimentation in regulatory developmental toxicology. So far, evaluation of the EST was not performed using compounds within distinct chemical classes. Evaluation within a distinct class of chemically related compounds can define the usefulness of the assay for the chemical class tested. The aim of the present study was to evaluate the relative sensitivity of the EST for a selected series of homologous compounds and to compare the data to the relative developmental toxicity of the compounds in vivo. To this end a series of proximate developmentally toxic glycol ether alkoxy acid metabolites was tested in the EST. All glycol ether alkoxy acid metabolites tested showed a concentration-dependent inhibition of cardiomyocyte differentiation at noncytotoxic concentrations, with methoxyacetic acid as the most potent compound followed by ethoxyacetic acid, butoxyacetic acid, and phenoxyacetic acid, respectively. The potency ranking of the compounds in the EST corresponds with the available in vivo data. The relative differences between the potencies of the compounds appeared more pronounced in the in vivo studies than in the EST. A possible explanation for this discrepancy could be the difference in the kinetics of the compounds in vivo as compared with their in vitro kinetics. This study illustrates that the EST can be used to set priorities for developmental toxicity testing within classes of related compounds.

The new EU legislations for chemicals (Registration, Evaluation and Authorization of Chemicals, REACH) and cosmetics (Seventh Amendment) stimulate the acceptance of in vitro and in silico approaches to test chemicals for their potential to cause reproductive effects. In the current study seven compounds with known in vivo developmental effects were tested in the embryonic stem cell test (EST). The EST correctly classified 5-fluorouracil, methotrexate, retinoic acid, 2-ethoxyacetic acid and 2-methoxyacetic acid for their in vivo embryotoxic potential. The toxicity of 2-methoxyethanol and 2-ethoxyethanol was underestimated due to a lack of metabolic capacity in the EST. This study further investigated the possibility to use in silico techniques to extrapolate in vitro effect concentrations determined in the EST to in vivo exposure levels. This approach was evaluated by comparing in silico predicted in vivo effect levels with effect levels measured in rodents. The in vivo effect levels of 2-methoxyethanol, 2-ethoxyethanol, methotrexate and retinoic acid were correctly predicted with in silico modelling. Contrary, in vivo embryotoxicity of 5-fluorouracil was overestimated following this approach. It is concluded that a combination of in vitro and in silico techniques appears to be a promising alternative test method for risk assessment of embryotoxic compounds. © 2006 Elsevier Ireland Ltd. All rights reserved.

The nutritional quality of new functional or fortified food products depends on the bioavailability of the nutrient(s) in the human body. Bioavailability is often determined in human intervention studies by measurements of plasma or serum profiles over a certain time period. These studies are time and cost consuming and often appear to lack an optimal study design, leading to follow-up intervention trials. Therefore, an alternative approach is needed that will optimize the development of new products. This study describes an approach to predict human serum concentrations after the consumption of (fortified) food products. The concept is based on the integration of in vitro results with kinetic modeling. As a case study, human serum folate concentrations were predicted after the consumption of folate-fortified milk products for 4 wk. Oral bioavailability was investigated using a step-wise approach in which luminal bioaccessibility and intestinal absorption were independently evaluated. Subsequently, these in vitro data were integrated in a kinetic mathematical (in silico) model to predict serum folate concentrations after the intake of a single dose and during long-term consumption. This approach was evaluated in comparison to a human intervention study in which folic acid-fortified milk products were tested for their effect on serum folate concentrations. A high predictive quality of this alternative in vitro/in silico approach was demonstrated. Finally, this methodology was applied to predict serum folate concentrations after intake of different fortified milk products for 4 wk, showing its benefits for the development of new nutritional products. © 2006 American Society for Nutrition.

The gap between actual intake and recommended intake of folate could be bridged by the consumption of fortified food products. Milk is considered as a potential food matrix for folate fortification in countries (such as theNetherlands) witha highmilk consumption. The aim of the work described in this thesis was to study the bioavailability of folate from milk products to establish whether milk is a suitable matrix for fortification with folic acid or 5-CH 3 -H 4 folate. In addition, the role of folate-binding proteins (FBP) in the bioavailability of folate from milk was investigated.Studies with a dynamic in vitro gastrointestinal model showed that folic acid and 5-CH 3 -H 4 -folate are highly bioaccessible from fortified milk products. The bioaccessibility of folate from fortified milk products was lower in presence of additional FBP, with a more pronounced inhibitory effect for folic acid as compared with 5-CH 3 -H 4 folate. This was explained by the observed difference in extent of binding to FBP between folic acid and 5-CH 3 -H 4 -folate in the duodenal lumen. Before gastric passage, folic acid and 5-CH 3 -H 4 -folate were mainly bound to FBP (76-79%) while 7% was free. After gastric passage, folic acid remained bound to FBP to a similar extent (80-81%). For 5-CH 3 -H 4 -folate the FBP-bound fraction gradually decreased from 79% to 5% and the free fraction increased from 7% to 93%. So, while folic acid enters the proximal part of the small intestine bound to FBP, 5-CH 3 -H 4 -folate appears mainly to be present as free folate in the duodenal lumen. The intestinal absorption of folic acid and 5-CH 3 -H 4 folate was studied using monolayers of human colon carcinoma (Caco-2) cells. Only a small difference in transport, in rate and underlying transport mechanisms, across Caco-2 cells was found between folic acid and 5-CH 3 -H 4 -folate. In presence of FBP, the absorption of folic acid and 5-CH 3 -H 4 folate was found to be lower and dependent on the extent of binding to FBP at the luminal side of the intestinal cells.Results from a human intervention study showed that the consumption of 200mg of folic acid added to milk significantly increased folate concentrations in serum and red blood cells. Although only two fortified milk products were tested in a human study, several milk products fortified with folic acid or 5-CH 3 -H 4 -folate with or without additional FBP were tested in the in vitro studies with the gastrointestinal model. Finally, a kinetic model was used to integrate the in vitro results about the kinetics of folate bioaccessibility and intestinal absorption and to extrapolate the findings to the human situation. With this in silico approach, the blood folate levels in humans could be predicted accurately.In conclusion, the in vitro and in vivo studies described in this thesis show that milk is an appropriate food matrix for folate fortification. A dietary strategy with fortified milk products can be recommended to bridge the gap between actual and recommended folate intake to optimize the folate status of the population. Folic acid-fortified milk should, however, not be supplemented with additional FBP as this will lead to a lower bioavailability of folic acid.