Danny Riethorst

Characterization of Human Duodenal Fluids in Fasted and Fed State Conditions

This work provides an elaborate characterization of human intestinal fluids (HIF) collected in fasted- and fed-state conditions. HIF from 20 healthy volunteers (10 M/F) were aspirated by intubation near the ligament of Treitz in a time-dependent manner (10-min intervals) and characterized for pH, bile salts, phospholipids, cholesterol, triacylglycerides (TAG), diacylglycerides (DAG), monoacylglycerides (MAG), free fatty acids (FFA), pancreatic lipase, phospholipase A2, and nonspecific esterase activity. For almost all parameters, a food-induced effect was observed. Results were characterized by a high variability, as illustrated by the broad ranges observed for each parameter: pH (fasted: 3.4-8.3; fed: 4.7-7.1), bile salts (fasted: 0.03-36.18 mM; fed: 0.74-86.14 mM), phospholipids (fasted: 0.01-6.33 mM; fed: 0.16-14.39 mM), cholesterol (fasted: 0-0.48 mM; fed: 0-3.29 mM), TAG (fed: 0-6.76 mg/mL), DAG (fed: 0-3.64 mg/mL), MAG (fasted: 0-1.09 mg/mL; fed: 0-11.36 mg/mL), FFA (fasted: 0-3.86 mg/mL; fed: 0.53-15.0 mg/mL), pancreatic lipase (fasted: 26-86 g/mL; fed: 146-415 g/mL), phospholipase A2 (fasted: 3-6 ng/mL; fed: 4.3-27.7 ng/mL), and nonspecific esterase activity (fasted: 270-4900 U/mL; fed: 430-4655 U/mL). This comprehensive overview may serve as reference data for physiologically based pharmacokinetic modeling and the optimization of biorelevant simulated intestinal fluids for the use in in vitro dissolution, solubility, and permeability profiling.

Evaluation of fasted state human intestinal fluid as apical solvent system in the Caco-2 absorption model and comparison with FaSSIF

To date, the Caco-2 model is considered as the gold standard to predict intestinal drug absorption. Often, aqueous phosphate buffers are used as apical medium. The purpose of this study was to use fasted state human intestinal fluid (FaHIF) as apical solvent system to generate biorelevant permeability values for a series of 16 model drugs that can be used as reference data to critically evaluate fasted state simulated intestinal fluid (FaSSIF) as possible substitute medium. Caco-2 compatibility with FaHIF was achieved when 50mg/ml mucus was applied on top of the cells before adding the apical medium. The use of FaHIF as solvent system generated a broad range of apparent permeability values (Papp) for the series of model compounds. When Papp values obtained with FaHIF were compared to those obtained with FaSSIF, a strong correlation was observed (R=0.951). The use of FaSSIF in the absence of mucus did not significantly alter this correlation. For FaHIF, FaSSIF and reference phosphate buffer blank FaSSIF, a strong sigmoidal relationship was found between Papp and fahuman, illustrated by correlation coefficients of 0.961, 0.893 and 0.868, respectively. In terms of inter-subject variability, the use of FaHIF from different volunteers originating from two distinct age groups (18-25 years; 65-72 years) exhibited an average coefficient of variance (CV) of 30%. However, no age dependency in permeability could be observed. In conclusion, the data generated in this article justify the use of FaSSIF as biorelevant apical medium in the Caco-2 assay to accurately predict in vivo drug absorption. Also, the optimized mucus-containing Caco-2 model can be used in combination with intestinal fluid samples aspirated after drug administration to further investigate intraluminal drug and formulation behavior.

Evaluation of fasted and fed state simulated and human intestinal fluids as solvent system in the Ussing chambers model to explore food effects on intestinal permeability

The Ussing chambers model is almost exclusively used in the presence of plain aqueous phosphate buffers as solvent system. In an attempt to further elucidate the effect of luminal ingredients and postprandial conditions on intestinal permeability, pooled fasted and fed state human intestinal fluids (FaHIFpool, FeHIFpool) were used. In addition, simulated intestinal fluids of both nutritional states (FaSSIF, FeSSIF) were evaluated as possible surrogate media for HIF. The use of FaHIFpool generated a broad range of Papp values for a series of 16 model drugs, ranging from 0.03×10(-6)cm/s (carvedilol) to 33.8×10(-6)cm/s (naproxen). A linear correlation was observed between Papp values using FaSSIF and FaHIFpool as solvent system (R=0.990), justifying the use of FaSSIF as surrogate medium for FaHIF in the Ussing chambers. In exclusion of the outlier carvedilol, a strong sigmoidal relationship was found between Papp and fahuman of 15 model drugs, illustrated by correlation coefficients of 0.961 and 0.936 for FaHIFpool and FaSSIF, respectively. When addressing food effects on intestinal permeability, the use of FeHIFpool resulted in a significantly lower Papp value for nine out of sixteen compounds compared to fasting conditions. FeSSIF as solvent system significantly overestimated Papp values in FeHIFpool. To conclude, the optimized Ussing chambers model using biorelevant media as apical solvent system holds great potential to investigate food effects in a more integrative approach, taking into account drug solubilisation, supersaturation and formulation effects.

Altered duodenal bile salt concentration and receptor expression in functional dyspepsia

Background Functional dyspepsia is a common functional gastrointestinal disorder in which a variety of pathophysiological mechanisms such as increased intestinal permeability and low-grade inflammation are involved. The factor causing these alterations, however, has not been identified. Objective We aimed to evaluate the luminal bile salt content and receptor expression in patients with functional dyspepsia and healthy volunteers. Methods Gastroduodenoscopy was performed to obtain duodenal biopsies from 25 healthy volunteers and 25 patients with functional dyspepsia (Rome III) to measure duodenal bile salt receptor expression with Western blot. Duodenal fluid aspirates were collected at fixed time points during fasted and fed state conditions and bile salt composition analysis was performed by liquid chromatography-mass spectrometry/mass spectrometry. Results Patients (N = 17) displayed decreased fasted bile salt concentrations compared to healthy volunteers (N = 20) over time (1.8 ± 0.3 mM vs 3.6 ± 0.5 mM; p = 0.03). In addition, an increased expression of duodenal bile salt sensor vitamin D receptor was found in patients (3.7 ± 1.0-fold; p < 0.0005; N = 24 for both groups). Conclusion Patients with functional dyspepsia are characterized by a decreased duodenal bile salt concentration in fasted state and an increased duodenal vitamin D receptor expression.

Human intestinal fluid layer separation: The effect on colloidal structures & solubility of lipophilic compounds

Graphical abstract Figure. No caption available. &NA; In addition to individual intestinal fluid components, colloidal structures are responsible for enhancing the solubility of lipophilic compounds. The present study investigated the link between the ultrastructure of fed state human intestinal fluids (FeHIF) and their solubilizing capacity for lipophilic compounds, taking into account interindividual variability. For this purpose, FeHIF samples from 10 healthy volunteers with known composition and ultrastructure were used to determine the solubility of four lipophilic compounds. In light of the focus on solubility and ultrastructure, the study carefully considered the methodology of solubility determination in relation to colloid composition and solubilizing capacity of FeHIF. To determine the solubilizing capacity of human and simulated intestinal fluids, the samples were saturated with the compound of interest, shaken for 24 h, and centrifuged. When using FeHIF, solubilities were determined in the micellar layer of FeHIF, i.e. after removing the upper (lipid) layer (standard procedure), as well as in total FeHIF (without removal of the upper layer). Compound concentrations were determined using HPLC‐UV/fluorescence. To link the solubilizing capacity with the ultrastructure, all human and simulated fluids were imaged using transmission electron microscopy (TEM) before and after centrifugation and top layer (lipid) removal. Comparing the ultrastructure and solubilizing capacity of individual FeHIF samples demonstrated a high intersubject variability in postprandial intestinal conditions. Imaging of FeHIF after removal of the upper layer clearly showed that only micellar structures remain in the lower layer. This observation suggests that larger colloids such as vesicles and lipid droplets are contained in the upper, lipid layer. The solubilizing capacity of most FeHIF samples substantially increased with inclusion of this lipid layer. The relative increase in solubilizing capacity upon inclusion of the lipid layer was most pronounced in samples that contained mainly vesicles alongside the micelles. Current fed state simulated intestinal fluids do not contain the larger colloids observed in the lipid layer of FeHIF and can only simulate the solubilizing capacity of the micellar layer of FeHIF. While the importance of drug molecules solubilized in the micellar layer of postprandial intestinal fluids for absorption has been extensively demonstrated previously, the in‐vivo relevance of drug solubilization in the lipid layer is currently unclear. In the dynamic environment of the human gastrointestinal tract, drug initially entrapped in larger postprandial colloids may become available for absorption upon lipid digestion and uptake. The current study, demonstrating the substantial solubilization of lipophilic compounds in the larger colloids of postprandial intestinal fluids, warrants further research in this field.

Human intestinal fluid factors affecting intestinal drug permeation in vitro

&NA; Intestinal permeability assessment is an important aspect of drug development, which strongly depends on the solvent system used in the intestinal donor compartment. For this purpose, human intestinal fluids (HIF) can be considered as the golden standard. A recent study demonstrated a reduced apparent permeation across rat intestinal tissue from fed versus fasted state HIF for 9 out of 16 compounds tested. Commercially available fed and fasted state simulated fluids (FeSSIF and FaSSIF) reproduced this food effect for only 3 out of 16 compounds. To elucidate this observed difference, the current study assessed the impact of relevant intestinal fluid factors (bile salt, phospholipids, cholesterol, free fatty acids (FFA), monoacylglycerides (MAG)) and 2‐factor interactions at a fixed pH of 6.5 on drug permeation across both rat tissue (Ussing chambers setup) and an artificial membrane (dialysis setup). Four representative compounds were selected for the permeation experiments: for propranolol and indomethacin, a food‐induced permeation reduction was previously seen in both HIF and SIF; for metoprolol and darunavir, a reduction was only seen in HIF. Using a fractional 25–1 design of experiments (DoE) approach, 16 SIF combinations were defined as donor media for permeation studies. In the Ussing chambers (rat tissue), FFA and MAG reduced the permeation for all 4 compounds. Only for propranolol and indomethacin, permeation was further reduced by bile salts and phospholipids. This explains why the use of FeSSIF vs FaSSIF, lacking FFA and MAG, predicted a negative food effect for propranolol and indomethacin but not for metoprolol and darunavir. In the dialysis set‐up using an artificial membrane, significantly higher permeation rates compared to the Ussing chambers were observed. Under those conditions, FFA and MAG no longer reduced permeation, while bile salts and phospholipids still did. This may indicate that lipidic structures can provide depot release in the case of a dynamic equilibrium between free and entrapped drug. Graphical abstract Figure. No caption available.

Gastric and Duodenal Ethanol Concentrations after Intake of Alcoholic Beverages in Postprandial Conditions

This study determined intraluminal ethanol concentrations (stomach and duodenum) in fed healthy volunteers after the consumption of common alcoholic beverages (beer, wine, and whisky). The results of this study were compared with a previous study in fasted volunteers. Five healthy volunteers were recruited in a crossover study. The fed state was simulated by ingestion of 250 mL of Nutridrink Compact Neutral. Volunteers subsequently consumed two standard units of beer (Stella Artois, 500 mL, 5.2% ethanol), wine (Blanc du Blanc, 200 mL, 11% ethanol), or whisky (Gallantry Whisky, 80 mL, 40% ethanol). Gastric and duodenal fluids were aspirated through two catheters over time and analyzed for ethanol content by head space gas chromatography. The capability of ethanol to permeate gastric and duodenal rat mucosa was examined in an Ussing chambers setup. A similar average gastric Cmax was observed in the beer and the wine conditions: 3.3% and 3.7% ethanol, respectively. The gastric Cmax in the whisky condition amounted to 8.5% ethanol. Lower ethanol concentrations were observed in the duodenum compared to the stomach. The duodenal Cmax was similar in all three conditions: 1.3%, 1.2%, and 1.6% ethanol for beer, wine, and whisky, respectively. Compared to the fasted state (reported in a previous study), higher gastric ethanol concentrations were observed during a longer time period. In the beer and wine conditions, similar concentrations were observed in the intestine regardless of the prandial state. After intake of whisky, however, the ethanol concentration was lower in the fed intestine. Alcohol was observed to permeate both gastric and duodenal rat mucosa. Higher intragastric ethanol concentrations were maintained for a longer period of time in fed compared to fasted state conditions. However, the observed concentration profiles were not in line with current FDA guidelines for alcohol resistance testing of formulations, stating that in vitro tests should investigate the impact of up to 40% ethanol for 2 h. The presented intraluminal ethanol concentrations may serve as reference data for the further development of relevant in vitro models to assess ethanol effects on formulation performance.