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The gastrointestinal mucus is a hydrogel lining the luminal side of the gastrointestinal epithelium. Mucus is vital for gut homeostasis because it protects the epithelium from the noxious external environment. However, from a drug delivery perspective, drugs have to permeate through the mucus to reach the epithelium; therefore, mucus might pose a barrier to drug absorption. Most of the information about mucus derives from fundamental studies performed on rodents. However, information from larger preclinical animal species is highly warranted for improving study designs and guiding better interpretation of data from preclinical assessments. Furthermore, improved understanding of the nature of the gastrointestinal mucus would enable the development of in vitro mucus models with increased biorelevance. These could then be implemented in drug absorption assays to improve the (bio)predictability. Well-informed in vitro mucus models would enable quick and less variable screening of drug candidates in the early drug development stages. Finally, these models would contribute to reduction, refinement, and replacement (the 3Rs) of animal usage in the drug development process. 

This thesis aims to improve our understanding of the nature of gastrointestinal mucus and its impact on drug absorption. For this purpose, mucus from the complete gastrointestinal tract of pigs and dogs was characterized and the diffusion of physicochemically diverse FITC-dextrans through colonic mucus was studied, both ex vivo and in vitro. The characterization of the gastrointestinal mucus focused on properties relevant for drug absorption and revealed the physiological characteristics, composition, and structural profiles from the various gastrointestinal regions. The findings pointed towards substantial differences between small intestinal and colonic mucus in both species and served as the basis for developing artificial colonic mucus models for drug permeation assessments. Porcine and canine artificial mucus models were developed and validated against the respective native secretions in terms of structural properties and demonstrated their potential to capture the key diffusion patterns of FITC-dextrans observed in native colonic mucus. Overall, this work provided insights into key properties of mucus from large preclinical species and validated tools for the assessment of the impact of mucus on drug absorption.

Colonic diseases affect more than 10 million people in Europe, and by 2045, more than 1% of the global population is expected to be affected by inflammatory bowel diseases. Drug development targeting colonic diseases is urgently needed. However, translating in vitro research into in vivo clinical relevance remains a challenge, with significant time and effort required in drug discovery and pre-clinical stages. Therefore, the development of efficient drug study platforms is necessary, in accordance with the ethical 3Rs principles and the UN SDGs 2030. In addition, the FDA Modernization Act 2.0 encourages the improvement of in vitro models with in vivo relevance.

Colonic mucus is the first interface in contact with drugs targeting colonic diseases. Mucus is a hydrogel composed of complex macromolecular crosslinks and acts as a structural barrier. In healthy conditions, colonic mucus consists of a stratified layer, with the outer layer hosting bacteria. Previous studies have reported changes in mucus in 85% of patients with various colonic diseases. However, characterization of the viscoelastic and barrier properties of colonic mucus in diseased states is still underexplored.

This PhD project focuses on studying the characteristics of the mucus barrier that may influence drug diffusion. Properties of native colonic mucus from human patients are characterized. Combined with studies of particle diffusion and viscoelastic properties in colonic mucus, general parameters influencing drug diffusion in the mucus are identified. Characteristics dependent on pH, surface charge, viscosity, and macromolecular composition of mucus have been investigated. Studies of drug interaction with mucus models from pig, dog, and artificial colonic mucus were performed to observe drug diffusion, drug binding, and drug permeability. Various methods to improve experimental (3D printing) and analytical (machine learning classifiers and physiology-based pharmacokinetic models) approaches were incorporated to enhance reproducibility and provide in-depth data analysis.

From this study, the macrorheology and microrheology profiles of the mucus were compared, and artificial colonic mucus was shown to capture the properties of native colonic mucus. By formulating a biosimilar artificial colonic mucus based on the native form, computational studies connecting to in vivo settings allow for better prediction and improved clinical relevance.