The purpose of this study was to evaluate differentiated human colonoid-derived monolayers (hCDMs) as an in vitro platform for investigating small molecule accumulation, toxicity, and pharmacodynamics (PD), in comparison to Caco-2 monolayers. Differentiated hCDMs formed polarized monolayers with a physiological barrier function (332.1 ± 52.02 Ω × cm2). In toxicity assays for drug-induced diarrhea, hCDMs demonstrated enhanced sensitivity to the epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) erlotinib and gefitinib, evidenced by lower concentrations inducing 20% inhibition (IC20) of cell viability and IC20/30× maximal plasma concentration (Cmax) ratios, and decreased sensitivity to SN-38, indicated by higher IC20 and IC20/30× Cmax, compared to Caco-2 monolayers. Baseline eicosanome profiling showed that hCDMs provided a colon-like representation of the prostaglandin (PG) pathway, whereas Caco-2 monolayers displayed cancer-associated profiles, such as elevated PGF2α and 15k PGF2α. Treatment with nonsteroidal anti-inflammatory drugs (NSAIDs) revealed that celecoxib elicited a dose-dependent reduction in the abundance of a subset of PGs within hCDMs, an effect not observed in the Caco-2 system. In conclusion, hCDMs present a more physiologically relevant alternative to conventional Caco-2 monolayers for integrated studies of cellular accumulation, toxicity, and PD in the colon. Incorporating additional donors and a broader compound panel will further enhance understanding of the model's clinical relevance.

BACKGROUND:The gut-kidney axis is crucial in chronic kidney disease (CKD). As kidney function declines, uremic toxins accumulate in plasma, and gut dysbiosis, oxidative stress, and inflammation arise, potentially affecting the expression of proteins involved in absorption, distribution, metabolism, and excretion (ADME). We aimed to determine the colonic expression of ADME proteins in CKD and explore modulatory mechanisms. METHODS:RNA and protein expression of key gut transporters, enzymes and receptors were determined in colon biopsies from 17 patients with CKD and 12 healthy volunteers using RNA sequencing and targeted mass spectrometry. In parallel, we induced CKD in rats by 5/6th nephrectomy, and we administered an antibiotic cocktail to assess differences when depleting the gut microbiome. Caco-2 cells exposed to human serum or fecal water derived from CKD patients were used to elucidate modulatory mechanisms. RESULTS:CKD downregulated the colon efflux transporter proteins P-gp (apical), MRP3 (basolateral) and BCRP (apical) (BCRP only in patients not undergoing hemodialysis). P-gp downregulation was mediated by aromatic gut microbiome-derived uremic toxins in patients, consistently with in vitro observation that P-gp was downregulated in Caco-2 cells exposed to CKD serum. In CKD rats' colon, Mdr1a was downregulated, and Bcrp was upregulated only when antibiotics were administered, indicating the gut microbiome influences P-gp and BCRP in CKD. CONCLUSIONS:We confirmed kidney-gut crosstalk, highlighting how uremic environment and gut dysbiosis consequent to CKD impact gut transport physiology. P-gp and BCRP can be downregulated via gut microbiome metabolites, thus shedding light on the importance of therapies targeting microbial activity.

Cystinosis is a rare, lysosomal storage disorder caused by mutations in the CTNS gene encoding the lysosomal cystine transporter, resulting in lysosomal cystine accumulation, the phenotypic hallmark of cystinosis, and progressive cellular dysfunction. Accurate quantification of cystine levels is therefore essential for assessing lysosomal transport deficiency and treatment response. In vitro cell models provide a controlled platform to investigate disease mechanisms and to evaluate emerging therapeutic strategies. To determine intracellular cystine concentrations in these models, adequate sample preparation, storage, and highly sensitive analytical methods are essential. In this work, a rapid hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed for the direct determination of cystine in cellular extracts. Use of a PEEK-lined HILIC-Z column proved essential to minimize metal-induced peak tailing and improve chromatographic performance. The total run time of 5 min enabled high-throughput analysis, facilitating efficient screening of novel therapeutic approaches in cellular systems. Validation demonstrated excellent linearity (R2 ≥ 0.9986) and a lower limit of quantification (LLOQ) of 12.5 nM, representing a > 8-fold improvement over reported reversed-phase LC methods. Addition of N-ethylmaleimide (NEM) prior to cell lysis effectively limited cysteine oxidation and maintained sample stability at 4 °C. Applicability was demonstrated in an isogenic laboratory HEK293T cell model, where CTNS-knockout (KO) cells exhibited significantly elevated intracellular cystine levels compared to wild-type (WT) cells. As additional controls, elevated levels were successfully restored following cysteamine treatment or lentiviral-vector (LV)-mediated CTNS protein re-expression. The developed method hence provides a sensitive and reliable analytical platform for in vitro evaluation of novel therapeutic strategies in cystinosis research.

This study examines the influence of aging on the protein abundance of key drug transport proteins (DTPs) and drug-metabolizing enzymes (DMEs). Duodenal and colonic biopsies were collected from 58 volunteers aged 19-85 years who underwent routine endoscopic procedures. Targeted mass spectrometry-based proteomics was used to quantify the abundance of major ATP-binding cassette (ABC) and solute carrier (SLC) transporters, as well as cytochrome P450 (CYP) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes involved in drug disposition. Multiple regression analysis was employed to assess the effects of age, sex, and sex-specific age effects on DTP and DME expression, providing insight into potential age-related variability in intestinal drug metabolism and transport. The analysis revealed significant age-related changes in duodenal protein abundance, with age coefficients ranging from -0.36 to 0.51. Specifically, a decrease in duodenal abundance with age was observed for carboxylesterase 2 (CES2), peptide transporter 1 (PEPT1), and villin-1. Additionally, a significant age-dependent increase in the duodenal abundance of breast cancer resistance protein (BCRP), multidrug resistance-associated proteins 1, 3, 4 (MRP1, MRP3, MRP4) and permeability-glycoprotein (P-gp) was observed. No significant impact of sex or sex-specific age effects was detected. These findings provide novel insights into the aging human proteome and may inform future research in this area.

The pH-mediated effect of drug ionization on solubility is well-described. However, pH can also indirectly influence solubility by altering the colloidal structures in human intestinal fluids. This study investigates the indirect pH effect on the apparent solubility of 13 uncharged drugs across a pH range of 4.5 to 7.5 in fed-state simulated intestinal fluids (SIF) composed of taurocholate and lecithin, with or without added lipids (monoolein and/or sodium oleate). A pronounced indirect pH effect on drug solubility was observed when oleate was present in the SIF, whereas monoolein had only a minor effect. Below pH 6.5, sodium oleate was converted to oleic acid, resulting in lipid droplet formation that enhanced lipophilic compound solubility in the total sample (lipid phase + micellar phase), while the micellar solubility remained similar to the reference SIF (without oleate). This resulted in an up to 50-fold increase of the ratio total/micellar drug solubility, which correlated well with drug lipophilicity or its combination with total polar surface area (R2 ≈ 0.8). At higher pH, a lipid phase was not formed because the ionized sodium oleate partitioned in the micellar phase, where it significantly increased drug solubilization. These findings highlight the importance of considering indirect pH effects in solubility assessments by tuning simulated intestinal fluids composition to better reflect in vivo reality.

Food effects on intestinal drug solubility and dissolution are a critical consideration in drug development, commonly investigated using simulated intestinal fluids (SIF) such as FaSSIF and FeSSIF. While these media represent average fasted- and fed-state conditions, their lack of a lipid fraction and disregard for compositional variability limit their physiological relevance and predictive power. To address these limitations, this proof-of-concept study introduces a novel set of fed-state SIF, termed digestion-induced SIF (DiSIF), featuring two key enhancements: (i) inclusion of a physiologically relevant lipid fraction generated through in vitro digestion of a liquid meal, and (ii) incorporation of variability via modulation of bile salt concentration and stage of digestion. Nine DiSIF media were developed to reflect both the average composition and the variability observed in fed-state human intestinal fluids (HIF). Overall, DiSIF media adequately predicted the micellar solubility of seven poorly water-soluble model compounds. Unlike commonly used SIF, they also enabled estimation of drug solubilization in total samples containing both micellar and lipid fractions, as observed in fed-state HIF. Furthermore, the media’s compositional variability allowed for the prediction of compound sensitivity to physiological variability with relative accuracy. These findings support further refinement and validation of DiSIF media as a versatile tool for formulation development and food-effect prediction in oral drug delivery.

Most available data on the composition and solubilizing properties of postprandial human intestinal fluid (HIF) are derived from studies involving liquid meals. These data inform the development of simulated intestinal fluids, widely used in in vitro assays for predicting intestinal drug behavior. However, the typical human diet primarily consists of solid meals, and the physical form of food has been shown to influence gastrointestinal transit and digestion, thereby affecting drug disposition and bioavailability. This study compares the characteristics of fed-state HIF collected after solid meal ingestion (SM-HIF) with previously published data on pooled liquid meal-derived HIF (LM-HIF) and newly generated data from individual LM-HIF samples. Time-dependent samples were analyzed over 180- and 90 min postprandial sampling periods to assess compositional changes following the administration of a solid and liquid meal, respectively. In addition, pooled samples were used to evaluate the solubilizing capacity for seven lipophilic model compounds. After intake of the solid meal, duodenal concentrations of exogenous (lipids, cholesterol, proteins) and endogenous (bile salts, phospholipids) components gradually increased to peak levels reached after 45-75 min. After 180 min, lipid and protein concentrations were still elevated compared to fasted state levels. In comparison to the liquid meal, the ingestion of the solid meal resulted in reduced concentrations of exogenous components, while endogenous components (bile salts and phospholipids) were relatively similar. For most compounds, the reduction in lipid content led to diminished solubilizing capacity of SM-HIF compared to LM-HIF when considering the combined micellar and lipid fractions. In contrast, the solubilizing capacity of the micellar fraction as such was largely independent of the meal type. Both the composition (particularly the micellar lipid concentration) and the solubilizing capacity of SM-HIF were highly variable between pools, albeit to a lesser extent than in LM-HIF. The findings of this study highlight that the physical form of the meal influences the composition and solubilizing capacity of HIF. These insights should be taken into account when refining biorelevant media for in vitro models to better predict food effects during drug product development.

Modelling and simulation of hepatic bile acids (BA) kinetics is instrumental to understand mechanisms underlying drug-induced cholestasis (DiCho). A recent study has shown that the loss of tankyrase1/2 (TNKS1/2) matured the hepatic phenotype in vitro in terms of cellular respiration rate and metabolism. However, whether this phenotype was accompanied with more in vivo relevant hepatic BA handling was not investigated. The present study explored whether tankyrase1/2 loss improved hepatic BA handling through an integrated in vitro-in silico approach. To do so, double knockout (DKO) TNKS1/2 HepG2 cells were exposed to a 10 µM BA mixture containing chenodeoxycholic acid (CDCA), cholic acid, deoxycholic acid, and lithocholic acid. BA levels and their metabolites were subsequently quantified in medium and cell extracts using liquid chromatography-tandem mass spectrometry (LC-MSMS). The in vitro data were then used as input in an ordinary differentially equation (ODE)-based kinetics model that was solved in R, using CDCA and its metabolites as index. The analyses revealed that glycine and taurine conjugation were enhanced by 1.5- and 2.2-fold, respectively, in the HepG2-DKO cells compared to the control. Further, the mechanistic model unveiled that efflux of taurochenodeoxycholic acid was elevated. In conclusion, HepG2-DKO cells provide a robust foundation for building a sensitive in vitro model for DiCho studies. Furthermore, this study discovered that tankyrase1/2 loss improved BA metabolism and kinetics, promoting the utility of tankyrase1/2 inhibitors, like XAV-939, in future pre-clinical BA disposition interaction studies.

The purpose of this study was to evaluate EpiColon, a novel human organotypic 3D colon microtissue prototype, developed to assess colonic drug disposition, with a particular focus on permeability ranking, and compare its performance to Caco-2 monolayers. EpiColon was characterized for barrier function using transepithelial electrical resistance (TEER), morphology via histology and immunohistochemistry, and functionality through drug transport studies measuring apparent permeability (Papp). Cutoff thresholds for the permeability of FITC-dextran 4 kDa (FD4), FITC-dextran 10 kDa (FD10S), and [14C]mannitol were established to monitor microtissue integrity. Permeability of EpiColon for 20 benchmark drugs was compared with Caco-2 data, and the activity of pivotal efflux transporters, including multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp), along with multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP), was evaluated using selective substrates. EpiColon exhibited a physiological barrier function (272.0 ± 53.05 Ω x cm2) and effectively discriminated between high (e.g., budesonide and [3H]metoprolol) and low permeable compounds (e.g., [3H]atenolol and [14C]mannitol). The model demonstrated functional activity for key efflux transporters, with efflux ratios of 2.32 for [3H]digoxin (MDR1/P-gp) and 3.34 for sulfasalazine (MRP2 and BCRP). Notably, EpiColon showed an enhanced dynamic range in the low permeability range, differentiating Papp between FD4 and FD10S, in contrast to Caco-2 monolayers. Significant positive correlations were observed between human fraction absorbed (fabs) and logarithmically transformed Papp [AP-BL] values for both EpiColon (rs = 0.68) and Caco-2 (rs = 0.68). Furthermore, EpiColon recapitulates some essential phenotypic and cellular features of the human colon, including the expression of critical marker genes (Pan-Cytokeratin+: epithelial/colonocytes, Vimentin+: mesenchymal/fibroblast, and Alcian Blue+: goblet cell/mucus). In conclusion, EpiColon is a promising platform that offers a valuable complement to conventional Caco-2 monolayers for studying colonic drug disposition. However, the presence of flat and some cuboidal cells, along with low throughput, must be addressed to improve its applicability in both academic research and pharmaceutical industry.

BACKGROUND:Bariatric surgery alters gastrointestinal anatomy and physiology, complicating functional assessments such as gastric emptying. The 13C-octanoic acid breath test is a simple, non-invasive alternative to scintigraphy, though its validity in bariatric populations requires further validation. METHODS:For this proof-of-concept analysis, gastric emptying data were derived from a cross-sectional study including individuals with obesity, sleeve gastrectomy, and Roux-en-Y gastric bypass (RYGB). Gastric emptying was measured simultaneously using the 13C-octanoic breath test and the reference method, scintigraphy. Gastric emptying half-times (GET1/2) were compared between the two methods using Wilcoxon signed-rank tests in each group. Concordance between both methods was assessed using Kendall's tau correlation coefficients, and Bland-Altman plots. RESULTS:No significant inter-method differences were observed for GET1/2 in any group. Mean differences were -26.7 min (95 % CI: -72.3; 18.6) for obesity, -3.92 min (95 % CI: -30.8; 23.0) for sleeve gastrectomy, and -8.55 min (95 % CI: -21.3; 4.18) for RYGB. Kendall's tau coefficients indicated positive rank associations within each group, but were non-significant (Obesity: 0.8, P = 0.16; sleeve gastrectomy: 0.90, P = 0.13; RYGB: 0.57, P = 0.33). Bland-Altman plots demonstrated acceptable agreement between the measurements across all groups. CONCLUSION:This proof-of-concept analysis suggests that the 13C-octanoic acid breath test has potential as a valid, non-invasive method for assessing gastric emptying in post-bariatric surgery patients. However, larger validation studies are warranted to confirm these preliminary findings.

Physiologically based biopharmaceutics modeling (PBBM) is crucial for drug absorption prediction due to its ability to integrate drug-specific, physiological, and biopharmaceutic properties. Recent reports highlight the need to improve prediction accuracy for regional, colonic absorption, and extended-release (ER) formulations, often using average physiological data due to rare concurrent individual measurements (e.g., luminal pH and transit time). This investigation evaluated if in-vivo measured individual pH and transit time data could enhance absorption predictions. Metoprolol (BCS I), with low colon absorption risk, and AZ3 (BCS III), with high risk, were chosen based on IntelliCap study data. Metoprolol had 6-hour and 14-hour release profiles, and AZ3 had a 12-hour profile. Individual PBBMs used GastroPlus and GI-Sim, comparing default and individual gastrointestinal models. Model accuracy was evaluated using Cmax, AUC0-t, and relative bioavailability (Frel) with absolute average fold error (AAFE). For metoprolol's 6-hour profile, individual models' AAFE ranged 1.18-1.23 (GastroPlus) and 1.23-1.45 (GI-Sim), similar to defaults (1.15-1.27, 1.22-1.32). For the 14-hour profile, AAFE was 1.56-1.68 (GastroPlus), 1.35-1.37 (GI-Sim) against defaults (1.61-1.66, 1.37-1.40). AZ3's individual AAFE was 1.23-1.56 and 1.35-1.72 versus defaults (1.29-1.58, 1.29-1.56). The results indicate that using individual data did not significantly improve prediction accuracy compared to default models, highlighting the effectiveness of current small intestine models but also emphasizing the need for better models in predicting colonic absorption.

This manuscript compiles a set of temporal pH profiles of the upper gastrointestinal (GI) tract under varying physiological and pharmacological conditions, including water and food intake as well as proton pump inhibitor (PPI) co-medication. A pooled analysis was conducted using data from 19 clinical studies previously performed in our laboratory, all of which employed standardised protocols and measurement techniques. Intraluminal pH was recorded in aspirated samples from the gastric antrum, duodenum, and proximal jejunum. Test conditions involved administering 240 to 250 mL of water in either the fasted state, the fed state after intake of the nutritional supplement Ensure Plus, or the fasted state with co-medication of esomeprazole. Trends in time-dependent pH were visualised using generalised additive models for location and shape (GAMLSS). Fed state profiles showed different temporal pH developments compared to the fasted state post water ingestion in the stomach and duodenum. An exploratory analysis using an FDA-standard high-fat breakfast yielded pH profiles similar in shape to those with Ensure Plus, though with a trend towards lower median pH values. Co-administration of esomeprazole resulted in pH increases not only in the stomach but also in the duodenum and jejunum, compared to the fasted state. These findings provide detailed insight into the dynamic luminal pH environment of the upper GI tract, potentially relevant to the design, optimisation, and evaluation of oral dosage forms. Furthermore, the provided data may support the development and refinement of in silico tools used to predict oral drug behaviour.

Understanding the kinetics of hepatic processes, such as bile acid (BA) handling and cellular aerobic metabolism, is crucial for advancing our knowledge of liver toxicity, particularly drug-induced cholestasis (DiCho). This article aimed to construct interpretable models with parameter estimations serving as reference values when investigating these cell metrics. Longitudinal datasets on BA disposition and oxygen consumption rates were collected using sandwich-cultured human hepatocytes. Chenodeoxycholic acid (CDCA), lithocholic acid (LCA), as well as their amidated and sulfate-conjugated metabolites were quantified with liquid chromatography-mass spectrometry. The bile salt export pump (BSEP) abundance was monitored with targeted proteomics and modelled for activity assessment. Oxygen consumption was measured using Seahorse XFp analyser. Ordinary differential equation-based models were solved in R. The basolateral uptake and efflux clearance of glycine-conjugated CDCA (GCDCA) were estimated at 1.22 µL/min/106 cells (RSE 14

Fluctuating severity of symptoms is a common hallmark of many inflammatory disorders, including inflammatory bowel disease (IBD). Addressing the pH changes during active and resting phases in IBD-affected tissue, a disease-adaptive nanocarrier system is designed for oral administration, enabling pH-dependent local drug release. The hybrid carrier combines poly(lactic-co-glycolic acid) and an amphiphilic cyclodextrin derivative, with physicochemical properties and drug release kinetics controlled by adjusting polymer ratios. The systems exhibited baseline drug release at pH 5 with increased rates at pH 2, which is characteristic of actively inflamed IBD tissue. Assessing the impact of biomolecule adhesion, biocorona formation was studied using ex vivo human intestinal fluids. Corona composition highly depended on the patient's prandial state and the nanocarrier matrix, with proteins predominating in the fasted state and lipids in the fed state. Notably, differences in the attachment of proteins and free fatty acids are detected in the latter. Transport studies using human in vitro models of the inflamed intestine revealed mucosal accumulation, facilitating localized drug delivery and effectively reducing cytokine levels to basal concentrations. This hybrid system highlights the potential of disease-adaptive drug release for inflammatory disease treatment and underscores the impact of biocorona formation on therapeutic performance in the gastrointestinal tract.

The tiny-TIM system offers an in vitro platform for the simulation of physiological processes occurring in human stomach and small intestine aiding in drug product development by predicting the bioperformance of oral formulations under fasted and fed state intake conditions. To assess this in vitro system in terms of its physiological relevance, we performed a detailed analysis of the composition as well as the solubilizing capacity of tiny-TIM intestinal fluids (TIF), and compared this to previously collected and analysed human intestinal fluids (HIF). Moreover, the impact of meal type on TIF composition and solubilising capacity was investigated by using either a liquid meal or a solid meal. In the fasted state, TIF exhibited lower lipid concentrations with a TIF/HIF ratio of 0.27, and elevated bile salt levels (TIF/HIF ratio of 1.8). Fasted state TIF generally overpredicted the solubilizing capacity of HIF, likely due to its higher bile salt concentrations. In the fed state, TIF contained biorelevant lipid concentrations but remained monophasic without phase separation, unlike HIF. This was likely due to higher bile salt levels (5.3 times that of HIF), which solubilized all lipids into the micellar phase. This resulted on average in a 3.3-fold increase in solubility of the poorly water-soluble model compounds in the micellar fraction of TIF as compared to HIF. Shifting from a liquid to a solid meal had minimal impact on TIF composition and solubilizing capacity.

Mesalamine is a standard first-line therapy for managing chronic inflammatory bowel diseases, such as Crohn's disease and ulcerative colitis. Despite its established efficacy, the precise mechanism of action within enterocytes remains unclear. This study aimed to develop and validate Physiologically Based Biopharmaceutics Models (PBBM) for mesalamine (5-ASA) and its metabolite, acetyl mesalamine (Ac-5-ASA), to predict drug concentrations in plasma, colonic lumen, and colonic tissue of healthy subjects and compare the results to measured concentrations. Using the Simcyp Simulator (V22), the models accurately predicted plasma concentrations for various formulations, including intravenous, oral immediate-release and controlled release formulations within a two-fold range. Results also captured the intestinal and hepatic metabolism converting mesalamine to acetyl mesalamine. However, significant discrepancies were observed in predicting luminal and tissue concentrations, with underpredictions for Claversal and Pentasa formulations reaching factors of up to 506 and 55 for 5-ASA and Ac-5-ASA in colonic tissue, respectively. These discrepancies highlight limitations in current modeling approaches, particularly in simulating drug accumulation within enterocytes. Despite these challenges, this investigation highlights both the potential benefits and the complexities of using PBBMs. Future work should focus on generating definitive N-acetyl transferase (NAT1) abundance data with an in-vitro in-vivo extrapolation link, improving approaches to better explore local drug concentrations in the gastrointestinal tract, and addressing the gap in accurately predicting luminal and tissue concentrations in the colon.

Colonic drug absorption is a prerequisite for a drug's suitability for colon-targeted and extended-release formulations. Since drug solubility is a key factor for uptake in the gastrointestinal tract, reliably estimating solubility in the human colon is essential for determining the feasibility of such formulation approaches. To date, our understanding of colonic drug solubility, how it compares to the proximal small intestine, and how it is linked to luminal fluid composition is limited by the scarcity of reference data available. Therefore, this study aimed to measure and compare the apparent solubility of eight drugs with varying physicochemical properties (apixaban, danoprevir, dexloxiglumide, febuxostat, fenofibrate, rofleponide, ticagrelor and tofacitinib) in pooled aspirates from the proximal human colon and small intestine, along with simulated media and buffers commonly used in solubility assessment. Additionally, the composition of the pooled luminal fluids was characterized. Whereas solubility in colonic and small intestinal fluids was comparable for most drugs, the small intestine's solubilizing capacity clearly exceeded that of the colon for the lipophilic drugs fenofibrate and ticagrelor. Extensive degradation of danoprevir was observed in both luminal fluids. Prediction of small intestinal solubility of the lipophilic compounds fenofibrate and ticagrelor was improved in fasted state simulated intestinal fluid compared to blank buffer, although the solubilizing capacity of the human fluids was only captured partially. Fasted state simulated colonic fluid solely improved the colonic solubility prediction of fenofibrate, while the prediction of ticagrelor remained outside the 2-fold prediction error threshold. Solubilities of all other drugs were predicted reasonably well in blank buffers and simulated media. The results generated in this study may serve as reference data for the validation of improved in vitro and in silico tools for colonic drug solubility prediction.

The widespread prevalence of colorectal cancer and its high mortality rate emphasize the urgent need for more effective therapies. When developing new drug products, a key aspect is ensuring that sufficiently high concentrations of the active drug are reached at the site of action. Drug transporters and drug-metabolizing enzymes can significantly influence the absorption and local accumulation of drugs in intestinal tissue. To understand how their presence may affect local drug disposition, the protein abundance of multiple drug transporters and drug-metabolizing enzymes was quantified in paired healthy colonic mucosa and colorectal adenocarcinoma tissue from colorectal cancer patients, utilizing mass spectrometry-based targeted proteomics. Statistically significant changes in protein expression were observed for two transporters (MRP1 and BCRP) and three of the studied enzymes (CES1, CES2 and UGT2B17). MRP1 displayed higher levels in cancerous tissue compared to healthy mucosa samples, while BCRP, CES1, CES2 and UGT2B17 showed the opposite. Other proteins of interest which could be quantified in colonic samples were the drug transporters P-gp, MRP3, MRP4, OATP2B1, MCT1 and enzymes CYP4F2, CYP2J2 and UGT1A1. The insights from this study enhance our understanding of the extent to which drug disposition in tumor tissue of colorectal cancer patients could be impacted by drug transporters and drug-metabolizing enzymes and may facilitate a more accurate prediction of local drug concentrations.

The role of intraluminal enzymes for the hydrolysis of active pharmaceutical ingredients (API), prodrugs and pharmaceutical excipients will be reviewed. Carboxylesterases may hydrolyze ester-based API, prodrugs and ester-bond containing polymer excipients, whereas lipases digest lipid formulation excipients, such as mono-, di- and triglycerides. To clarify the conditions that should be mimicked when designing in vitro studies, we briefly review the upper gastrointestinal physiology and provide new data on the inter-individual variability of enzyme activities in human intestinal fluids. Afterwards, the methodology for studying enzymatic hydrolysis of API, prodrugs, lipid and polymeric excipients, as well as the main results that have been obtained, are summarized. In vitro digestion models used to characterize lipid formulations are well described, but data about the hydrolysis of lipid excipients (including surfactants) has been scarce and contradictory. Data on API and prodrug hydrolysis by esterases is available; however, inconsistent use of enzyme types and concentrations limits structure-stability relationships. Hydrolysis of polymer excipients in the lumen has not been significantly explored, with only qualitative data available for cellulose derivates, polyesters, starches, etc. Harmonization of the methodology is required in order to curate larger enzymatic hydrolysis datasets, which will enable mechanistic understanding and theoretical prediction.