In Vitro Simulation of the Environment in the Upper Gastrointestinal Lumen After Drug Administration in the Fed State Using the TIM-1 System and Comparison With Luminal Data in Adults.

We evaluated the environment in TIM-1 luminal compartments using paracetamol and danazol solutions and suspensions and the fed state configuration. Data were compared with recently published data in healthy adults. TIM-1 experiments were performed with a 3-fold downscale. Volumes of secretions in gastric and duodenal compartments adequately reflected the luminal data in adults up to 3 h post drug dosing. pH values in duodenal and jejunal compartments adequately reflected average pH values in adults. In gastric compartment pH values where initially higher than average values in adults and reached baseline levels earlier than in adults. The environment in the TIM-1 gastric compartment and jejunal compartment adequately reflected the average total paracetamol and danazol amounts per volume of contents in the adult stomach and upper small intestine, respectively. Total bile acids concentrations in the micellar phase of contents in duodenal and jejunal compartments overestimated micellar concentrations in the upper small intestine of adults. Adjustments in gastric emptying/acid secretion rates and bile acids identities in the duodenal and jejunal compartments, and application of dynamic bile acids secretion rates are expected to further improve the relevance of luminal conditions in TIM-1 compartments with those in adults.

A standardised semi-dynamic in vitro digestion method suitable for food - an international consensus.

The link between food and human health is increasingly a topic of interest. One avenue of study has been to assess food disintegration and interactions within the gastrointestinal tract. In vitro digestion models have been widely used to overcome the constrictions associated with in vivo methodology. The COST Action INFOGEST developed an international, harmonised protocol for static simulation of digestion in the upper gastrointestinal tract of adults. This protocol is widely used; however, it is restricted to providing end-point assessment without considering the possible structural changes. On the other hand, there are dynamic models that provide more physiologically relevant data but are expensive and difficult to access. There is a gap between these models. The method outlined in this article provides an intermediate model; it builds upon the harmonised static model and now includes crucial kinetic aspects associated with the gastric phase of digestion, including gradual acidification, fluid and enzyme secretion and emptying. This paper provides guidance and standardised recommendations of a physiologically relevant semi-dynamic in vitro simulation of upper gastrointestinal tract digestion, with particular focus on the gastric phase. Adaptations of this model have already been used to provide kinetic data on nutrient digestion and structural changes during the gastric phase that impact on nutrient absorption. Moreover, it provides a simple tool that can be used in a wide range of laboratories.

Microbial communities in a dynamic in vitro model for the human ileum resemble the human ileal microbiota.

The important role for the human small intestinal microbiota in health and disease has been widely acknowledged. However, the difficulties encountered in accessing the small intestine in a non-invasive way in healthy subjects have limited the possibilities to study its microbiota. In this study, a dynamic in vitro model that simulates the human ileum was developed, including its microbiota. Ileostomy effluent and fecal inocula were employed to cultivate microbial communities within the in vitro model. Microbial stability was repetitively achieved after 10 days of model operation with bacterial concentrations reaching on average 107 to 108 16S rRNA copy numbers/ml. High diversities similar to those observed in in vivo ileum samples were achieved at steady state using both fecal and ileostomy effluent inocula. Functional stability based on Short Chain Fatty Acid concentrations was reached after 10 days of operation using fecal inocula, but was not reached with ileostomy effluent as inoculum. Principal Components and cluster analysis of the phylogenetic profiles revealed that in vitro samples at steady state clustered closest to two samples obtained from the terminal ileum of healthy individuals, independent of the inoculum used, demonstrating that the in vitro microbiota at steady state resembles that of the human ileum.

Appetite ratings of foods are predictable with an in vitro advanced gastrointestinal model in combination with an in silico artificial neural network.

The expected increase of global obesity prevalence makes it necessary to have information about the effects of meal intakes on the feeling of appetite. Because human clinical studies are time and cost intensive, there is a need for a reliable alternative. The aim of this study was to develop and evaluate an in vitro-in silico technology to predict the feelings of fullness and hunger after consumption of different types of meals. In this technology the results from an in vitro gastrointestinal model (tiny-TIMagc) on gastric viscosity and intestinal digestion of different meals were used as input data for an in silico artificial neural network (ANN). The predictions of the feeling of fullness and hunger were compared with actual human scores for these parameters after intake of the same type of meals. From these first series of experiments, with a relatively small number of in vitro digestive parameters as input for in silico modeling, a reasonable prediction of appetite rating for foods can be realized at a time- and cost-effective way.

INFOGEST static in vitro simulation of gastrointestinal food digestion.

Developing a mechanistic understanding of the impact of food structure and composition on human health has increasingly involved simulating digestion in the upper gastrointestinal tract. These simulations have used a wide range of different conditions that often have very little physiological relevance, and this impedes the meaningful comparison of results. The standardized protocol presented here is based on an international consensus developed by the COST INFOGEST network. The method is designed to be used with standard laboratory equipment and requires limited experience to encourage a wide range of researchers to adopt it. It is a static digestion method that uses constant ratios of meal to digestive fluids and a constant pH for each step of digestion. This makes the method simple to use but not suitable for simulating digestion kinetics. Using this method, food samples are subjected to sequential oral, gastric and intestinal digestion while parameters such as electrolytes, enzymes, bile, dilution, pH and time of digestion are based on available physiological data. This amended and improved digestion method (INFOGEST 2.0) avoids challenges associated with the original method, such as the inclusion of the oral phase and the use of gastric lipase. The method can be used to assess the endpoints resulting from digestion of foods by analyzing the digestion products (e.g., peptides/amino acids, fatty acids, simple sugars) and evaluating the release of micronutrients from the food matrix. The whole protocol can be completed in ~7 d, including ~5 d required for the determination of enzyme activities.

In vitro models for the prediction of in vivo performance of oral dosage forms: Recent progress from partnership through the IMI OrBiTo collaboration.

The availability of in vitro tools that are constructed on the basis of a detailed knowledge of key aspects of gastrointestinal (GI) physiology and their impact on formulation performance and subsequent drug release behaviour is fundamental to the success and efficiency of oral drug product development. Over the last six years, the development and optimization of improved, biorelevant in vitro tools has been a cornerstone of the IMI OrBiTo (Oral Biopharmaceutics Tools) project. By bringing together key industry and academic partners, and by linking tool development and optimization to human studies to understand behaviour at the formulation/GI tract interface, the collaboration has enabled innovation, optimization and implementation of the requisite biorelevant in vitro tools. In this paper, we present an overview of the in vitro tools investigated during the collaboration and offer a perspective on their future use in enhancing the development of new oral drug products.

Subjective feelings of appetite of wholegrain breakfasts evaluated under controlled, laboratory and 'at home' conditions.

BACKGROUND: Appetite regulating properties of foods are usually investigated under laboratory conditions, whereas in real life, foods are consumed under at home conditions. The objective of this study was to compare the acute effects of breakfasts when tested in a laboratory condition and in an at home condition. Appetite regulating properties of two bread breakfasts and two cereal breakfasts were also compared. SUBJECTS AND METHODS: In this randomized cross-over trial balanced for laboratory and at home test conditions, thirty-two women consumed five breakfasts, i.e. two bread breakfasts, two cereal breakfasts and one fried-egg breakfast. Visual analogue scales for measuring appetite were captured via an on-line scoring system and were analyzed as incremental area under the curve, as satiation phase and as satiety phase. RESULTS: Location effects were limited to two small effects only. An overall location effect in hunger feelings was observed (p = 0.040), which occurred specifically during the short satiation period (p = 0.0002) where hunger feelings scored higher under laboratory conditions. Similarly, a location effect was observed for desire to eat (p = 0.001); this was again higher under laboratory conditions. No other location effects were observed. Bread breakfasts did not differ in their appetite regulating properties. The Steel Cut oatmeal breakfast was reported to be more satiating (p = 0.001) as compared to the ready-to-eat cereal. CONCLUSIONS: Whereas the five breakfasts varied somewhat in their appetite regulating properties, evaluation under laboratory conditions overall did not result in different appetite scores compared to the at home conditions. This suggests that at home testing may be a useful alternative to laboratory test conditions for nutrition research.

Gastrointestinal and Systemic Disposition of Diclofenac under Fasted and Fed State Conditions Supporting the Evaluation of in Vitro Predictive Tools.

This study aimed to gain further insight into the gastrointestinal disposition of the weakly acidic BCS class II drug diclofenac and the implications for systemic drug exposure in humans under fasted and fed state conditions. For this purpose, gastrointestinal and blood samples were collected from healthy volunteers after oral intake of a commercially available tablet of the potassium salt of diclofenac (i.e., Cataflam) in different prandial states. Subsequently, these in vivo data served as a reference for the evaluation of in vitro tools with different levels of complexity, i.e., a conventional USP II dissolution apparatus, a modified version of the dynamic open flow through test apparatus, and the TNO gastrointestinal model equipped with the recently developed advanced gastric compartment (TIMagc). In vivo data suggested impaired drug dissolution and/or immediate precipitation in the fasted stomach, linked to the acidity of the gastric environment. Similarly, a vast presence of solid drug material in the stomach was observed under fed state conditions, which could be attributed to a marked delay in intragastric tablet disintegration after drug intake with a meal. Emptying of solid drug from the stomach into the duodenum generally resulted in rapid intestinal drug (re)dissolution in both test conditions, explaining the absence of a food effect on the extent of overall systemic exposure for diclofenac. In vitro tools were found to be capable of predicting in vivo intraluminal (and systemic) disposition of this compound, the extent of which depended on the degree to which the dynamic nature of the gastrointestinal process(es) to be investigated was simulated.

Effects of inhomogeneity on triglyceride digestion of emulsions using an in vitro digestion model (Tiny TIM).

The colloidal behaviour and extent of lipolysis of various emulsions stabilized by whey protein and Tween were studied using the TNO Intestinal Model (TIM) extended with a purposely designed gastric compartment. The in vitro results suggest that creaming of a fatty layer in the gastric region causes a delay in fat entering the small intestinal region, delays and reduces the free fatty acid content in the small intestinal lumen and delays fat absorption. It was shown that controlling the pH with pig gastric juice instead of simulated gastric juice delayed creaming of the emulsions significantly, which resulted in faster gastric lipolysis. However, because the digestive conditions are not adjusted by physiological regulation mechanisms such as the regulation of gastric emptying by the detection of nutrients in the small intestine, care must be taken to translate these results to the in vivo reality. It is expected that the differences between the systems will be tempered by the physiological feedback regulation mechanisms of digestion.

Evaluation of two dynamic in vitro models simulating fasted and fed state conditions in the upper gastrointestinal tract (TIM-1 and tiny-TIM) for investigating the bioaccessibility of pharmaceutical compounds from oral dosage forms.

Pharmaceutical research needs predictive in vitro tools for API bioavailability in humans. We evaluated two dynamic in vitro gastrointestinal models: TIM-1 and tiny-TIM. Four low-soluble APIs in various formulations were investigated in the TIM systems under fasted and fed conditions. API small-intestinal bioaccessibility profiles were evaluated between the two systems and in comparison with human data. Both TIM systems showed a higher bioaccessibility of ciprofloxacin and nifedipine during 3-4h after dosing immediate release (IR) compared to modified release (MR) formulations. Higher bioaccessibility levels from IR formulations were observed under fasted state in the first 30-90 min in tiny-TIM as compared to TIM-1, resulting in a tmax similar to clinical data. Absence (ciprofloxacin) or presence (posaconazole) of a food effect on bioaccessibility was observed in both TIM systems in line with human data. A higher bioaccessibility of fenofibrate from nano- vs micro-particle formulation was found in both TIM systems. This dataset shows the predictive quality of the TIM systems for clinical data on API small-intestinal bioaccessibility from IR and MR formulations and food effects. Tiny-TIM provides higher throughput and better prediction for IR formulations. TIM-1 provides detailed information on site-specific release of APIs, relevant for MR formulations and food effects.

Food matrix effects on bioaccessibility of ß-carotene can be measured in an in vitro gastrointestinal model.

Since the food matrix determines ß-carotene availability for intestinal absorption, food matrix effects on the bioaccessibility of ß-carotene from two diets were investigated in vitro and compared with in vivo data. The "mixed diet" consisted of ß-carotene-rich vegetables, and the "oil diet" contained ß-carotene-low vegetables with supplemental ß-carotene. The application of extrinsically labeled ß-carotene was also investigated. The bioaccessibility of ß-carotene was 28 µg/100 µg ß-carotene from the mixed diet and 53 µg/100 µg ß-carotene from the oil diet. This ratio of 1.9:1 was consistent with in vivo data, where the apparent absorption was 1.9-fold higher in the oil diet than in the mixed diet. The labeled ß-carotene was not equally distributed over time. In conclusion, the food matrix effects on bioaccessibility of ß-carotene could be measured using an in vitro model and were consistent with in vivo data. The application of extrinsically labeled ß-carotene was not confirmed.

In vitro models for the prediction of in vivo performance of oral dosage forms.

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.

In vitro gastrointestinal model (TIM) with predictive power, even for infants and children?

There is a need for information on the bioavailability in pediatric patients of drugs from manipulated dosage forms when applied in combination with food and/or co-medication under realistic daily practice circumstances. We describe the development, validation and application of a dynamic, computer-controlled in vitro system mimicking the conditions in the upper gastrointestinal tract of neonates, infants and toddlers: TIMpediatric. Paracetamol and diclofenac in age-related food matrices and with esomeprazole co-medication were tested. The experiments showed relevant results on the impact of drug manipulation and co-medication on the availability for absorption of active compounds. Without ethical constraints, alternative approaches for oral dosing and new pediatric formulations can be studied in TIMpediatric with a high predictive value.

Development of the Digestive System-Experimental Challenges and Approaches of Infant Lipid Digestion.

At least during the first 6 months after birth, the nutrition of infants should ideally consist of human milk which provides 40-60 % of energy from lipids. Beyond energy, human milk also delivers lipids with a specific functionality, such as essential fatty acids (FA), phospholipids, and cholesterol. Healthy development, especially of the nervous and digestive systems, depends fundamentally on these. Epidemiological data suggest that human milk provides unique health benefits during early infancy that extend to long-lasting benefits. Preclinical findings show that qualitative changes in dietary lipids, i.e., lipid structure and FA composition, during early life may contribute to the reported long-term effects. Little is known in this respect about the development of digestive function and the digestion and absorption of lipids by the newborn. This review gives a detailed overview of the distinct functionalities that dietary lipids from human milk and infant formula provide and the profound differences in the physiology and biochemistry of lipid digestion between infants and adults. Fundamental mechanisms of infant lipid digestion can, however, almost exclusively be elucidated in vitro. Experimental approaches and their challenges are reviewed in depth.

Comparison of five in vitro digestion models to in vivo experimental results: lead bioaccessibility in the human gastrointestinal tract.

This paper presents a multi-laboratory comparison study of in vitro models assessing bioaccessibility of soil-bound lead in the human gastrointestinal tract under simulated fasted and fed conditions. Oral bioavailability data from a previous human in vivo study on the same soil served as a reference point. In general, the bioaccessible lead fraction was significantly (P<0.05) different between the in vitro methods and ranged for the fasted models from 2% to 33% and for the fed models from 7% to 29%. The in vivo bioavailability data from literature were 26.2+/-8.1% for fasted conditions, compared to 2.5+/-1.7% for fed conditions. Under fed conditions, all models returned higher bioaccessibility values than the in vivo bioavailability; whereas three models returned a lower bioaccessibility than bioavailability under fasted conditions. These differences are often due to the method's digestion parameters that need further optimization. An important outcome of this study was the determination that the method for separating the bioaccessible lead from the non-bioaccessible fraction (centrifugation, filtration, ultrafiltration) is crucial for the interpretation of the results. Bioaccessibility values from models that use more stringent separation methods better approximate in vivo bioavailability results, yet at the expense of the level of conservancy. We conclude from this study that more optimization of in vitro digestion models is needed for use in risk assessment. Moreover, attention should be paid to the laboratory separation method since it largely influences what fraction of the contaminant is considered bioaccessible.

Effect of partially hydrolyzed guar gum (PHGG) on the bioaccessibility of fat and cholesterol.

The addition of a compound that lowers the intestinal uptake of fat and cholesterol might be an interesting strategy to reduce the risk of vascular disease. Partially hydrolyzed guar gum (PHGG) has been shown to have this effect in healthy volunteers after intake of a yogurt drink with 3 to 6% PHGG. In the present study a yogurt drink with 3% sunflower oil and 4% egg yolk was tested with 3% and 6% PHGG, and compared to a control without PHGG. Experiments were performed in a multi-compartmental model of the gastrointestinal tract, equipped to study the digestion and availability for absorption (bioaccessibility) of lipids. The results show that PHGG decreases the bioaccessibility of both fat and cholesterol in a dose-dependent manner. The bioaccessibility of fat was 79.4+/-1.7%, 70.8+/-2.5% and 60.1+/-1.1% for the control experiments and the experiments with 3% and 6% PHGG respectively. The bioaccessibility of cholesterol was 82.2+/-2.0%, 75.4+/-1.2% and 64.0+/-4.3% for the control and the experiments with 3% and 6% PHGG respectively. Additional experiments indicated that PHGG reduces bioaccessibility through the depletion flocculation mechanism. Depletion flocculation antagonizes the emulsification by bile salts and thus decreases lipolytic activity, resulting in a lower bioaccessibility of fat and cholesterol. Depletion flocculation with polymers might be an interesting mechanism, not described before, to reduce fat and cholesterol absorption.

Adaptive function of soil consumption: an in vitro study modeling the human stomach and small intestine.

Despite occurring in a wide variety of taxa, deliberate soil consumption (geophagy) is a poorly understood behavior. In humans, geophagy is sometimes considered aberrant or a sign of metabolic dysfunction. However, geophagy is normally assigned an adaptive function in nonhuman primates and various other organisms. One hypothesis submits that clay-rich soil adsorbs intestinal insults, namely plant metabolites or diarrhoea-causing enterotoxins. Here we test the capacity of kaolin, a commonly ingested clay, to adsorb quinine (an alkaloid) and two types of tannin (digestion-inhibitors). Trials were conducted in vitro using the TNO Intestinal Model, a device that closely simulates digestion by the human stomach and small intestine. Kaolin reduced the bioavailability of each compound by < or =30%. However, because we could not replicate clay-epithelial adhesion and reduced motility, these results may underestimate adsorption in vivo. We also show that kaolin fails to render calcium oxalate soluble. We conclude that gastrointestinal adsorption is the most plausible function of human geophagy. Adaptive advantages include greater exploitation of marginal plant foods and reduced energetic costs of diarrhoea, factors that could account for the high frequency of geophagy in children and pregnant women across the tropics.

Comparison of five in vitro digestion models to study the bioaccessibility of soil contaminants.

Soil ingestion can be a major exposure route for humans to many immobile soil contaminants. Exposure to soil contaminants can be overestimated if oral bioavailability is not taken into account. Several in vitro digestion models simulating the human gastrointestinal tract have been developed to assess mobilization of contaminants from soil during digestion, i.e., bioaccessibility. Bioaccessibility is a crucial step in controlling the oral bioavailability for soil contaminants. To what extent in vitro determination of bioaccessibility is method dependent has, until now, not been studied. This paper describes a multi-laboratory comparison and evaluation of five in vitro digestion models. Their experimental design and the results of a round robin evaluation of three soils, each contaminated with arsenic, cadmium, and lead, are presented and discussed. A wide range of bioaccessibility values were found for the three soils: for As 6-95%, 1-19%, and 10-59%; for Cd 7-92%, 5-92%, and 6-99%; and for Pb 4-91%, 1-56%, and 3-90%. Bioaccessibility in many cases is less than 50%, indicating that a reduction of bioavailability can have implications for health risk assessment. Although the experimental designs of the different digestion systems are distinct, the main differences in test results of bioaccessibility can be explained on the basis of the applied gastric pH. High values are typically observed for a simple gastric method, which measures bioaccessibility in the gastric compartment at low pHs of 1.5. Other methods that also apply a low gastric pH, and include intestinal conditions, produce lower bioaccessibility values. The lowest bioaccessibility values are observed for a gastrointestinal method which employs a high gastric pH of 4.0.