Publications

@article{sharma_developing_2024,

title = {Developing an adult stem cell derived microphysiological intestinal system for predicting oral prodrug bioconversion and permeability in humans},

author = {Abhinav Sharma and Liang Jin and Xue Wang and Yue-Ting Wang and David M. Stresser},

url = {https://pubs.rsc.org/en/content/articlelanding/2024/lc/d3lc00843f},

doi = {10.1039/D3LC00843F},

issn = {1473-0189},

year  = {2024},

date = {2024-01-17},

urldate = {2024-01-17},

journal = {Lab Chip},

volume = {24},

number = {2},

pages = {339–355},

abstract = {Microphysiological systems (MPS) incorporating human intestinal organoids have shown the potential to faithfully model intestinal biology with the promise to accelerate development of oral prodrugs. We hypothesized that an MPS model incorporating flow, shear stress, and vasculature could provide more reliable measures of prodrug bioconversion and permeability. Following construction of jejunal and duodenal organoid MPS derived from 3 donors, we determined the area under the concentration–time (AUC) curve for the active drug in the vascular channel and characterized the enzymology of prodrug bioconversion. Fosamprenavir underwent phosphatase mediated hydrolysis to amprenavir while dabigatran etexilate (DABE) exhibited proper CES2- and, as anticipated, not CES1-mediated de-esterification, followed by permeation of amprenavir to the vascular channel. When experiments were conducted in the presence of bio-converting enzyme inhibitors (orthovanadate for alkaline phosphatase; bis(p-nitrophenyl)phosphate for carboxylesterase), the AUC of the active drug decreased accordingly in the vascular channel. In addition to functional analysis, the MPS was characterized through imaging and proteomic analysis. Imaging revealed proper expression and localization of epithelial, endothelial, tight junction and catalytic enzyme markers. Global proteomic analysis was used to analyze the MPS model and 3 comparator sources: an organoid-based transwell model (which was also evaluated for function), Matrigel embedded organoids and finally jejunal and duodenal cadaver tissues collected from 3 donors. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) of global proteomic data demonstrated that all organoid-based models exhibited strong similarity and were distinct from tissues. Intestinal organoids in the MPS model exhibited strong similarity to human tissue for key epithelial markers via HCA. Quantitative proteomic analysis showed higher expression of key prodrug converting and drug metabolizing enzymes in MPS-derived organoids compared to tissues, organoids in Matrigel, and organoids on transwells. When comparing organoids from MPS and transwells, expression of intestinal alkaline phosphatase (ALPI), carboxylesterase (CES)2, cytochrome P450 3A4 (CYP3A4) and sucrase isomaltase (SI) was 2.97-, 1.2-, 11.3-, and 27.7-fold higher for duodenum and 7.7-, 4.6-, 18.1-, and 112.2-fold higher for jejunum organoids in MPS, respectively. The MPS approach can provide a more physiological system than enzymes, organoids, and organoids on transwells for pharmacokinetic analysis of prodrugs that account for 10% of all commercial medicines.},

note = {Publisher: The Royal Society of Chemistry},

keywords = {absorption, Bioavailability, Biological Transport, Caco-2 Cells, drug absorption, drug metabolising enzymes ({DME}), drug permeability, intestinal barrier, oral bioavailability, permeability},

pubstate = {published},

tppubtype = {article}

}

@article{gomez-martinez_planar_2022,

title = {A Planar Culture Model of Human Absorptive Enterocytes Reveals Metformin Increases Fatty Acid Oxidation and Export},

author = {Ismael Gomez-Martinez and R. Jarrett Bliton and Keith A. Breau and Michael J. Czerwinski and Ian A. Williamson and Jia Wen and John F. Rawls and Scott T. Magness},

doi = {10.1016/j.jcmgh.2022.04.009},

issn = {2352-345X},

year  = {2022},

date = {2022-01-01},

urldate = {2022-01-01},

journal = {Cellular and Molecular Gastroenterology and Hepatology},

volume = {14},

number = {2},

pages = {409–434},

abstract = {BACKGROUND & AIMS: Fatty acid oxidation by absorptive enterocytes has been linked to the pathophysiology of type 2 diabetes, obesity, and dyslipidemia. Caco-2 and organoids have been used to study dietary lipid-handling processes including fatty acid oxidation, but are limited in physiological relevance or preclude simultaneous apical and basal access. Here, we developed a high-throughput planar human absorptive enterocyte monolayer system for investigating lipid handling, and then evaluated the role of fatty acid oxidation in fatty acid export, using etomoxir, C75, and the antidiabetic drug metformin. 

METHODS: Single-cell RNA-sequencing, transcriptomics, and lineage trajectory was performed on primary human jejunum. In vivo absorptive enterocyte maturational states informed conditions used to differentiate human intestinal stem cells (ISCs) that mimic in vivo absorptive enterocyte maturation. The system was scaled for high-throughput drug screening. Fatty acid oxidation was modulated pharmacologically and BODIPY (Thermo Fisher Scientific, Waltham, MA) (B)-labeled fatty acids were used to evaluate fatty acid handling via fluorescence and thin-layer chromatography. 

RESULTS: Single-cell RNA-sequencing shows increasing expression of lipid-handling genes as absorptive enterocytes mature. Culture conditions promote ISC differentiation into confluent absorptive enterocyte monolayers. Fatty acid-handling gene expression mimics in vivo maturational states. The fatty acid oxidation inhibitor etomoxir decreased apical-to-basolateral export of medium-chain B-C12 and long-chain B-C16 fatty acids, whereas the CPT1 agonist C75 and the antidiabetic drug metformin increased apical-to-basolateral export. Short-chain B-C5 was unaffected by fatty acid oxidation inhibition and diffused through absorptive enterocytes. 

CONCLUSIONS: Primary human ISCs in culture undergo programmed maturation. Absorptive enterocyte monolayers show in vivo maturational states and lipid-handling gene expression profiles. Absorptive enterocytes create strong epithelial barriers in 96-Transwell format. Fatty acid export is proportional to fatty acid oxidation. Metformin enhances fatty acid oxidation and increases basolateral fatty acid export, supporting an intestine-specific role.},

keywords = {Absorptive Enterocyte Monolayers, Caco-2 Cells, Diabetes Mellitus, Drug Screening, Enterocytes, Fatty Acid Oxidation, Fatty Acids, Humans, Hypoglycemic Agents, intestinal stem cells, Metformin, Type 2},

pubstate = {published},

tppubtype = {article}

}

@article{gunasekara_monolayer_2018,

title = {A monolayer of primary colonic epithelium generated on a scaffold with a gradient of stiffness for drug transport studies},

author = {Dulan B. Gunasekara and Jennifer Speer and Yuli Wang and Daniel L. Nguyen and Mark I. Reed and Nicole M. Smiddy and Joel S. Parker and John K. Fallon and Philip C. Smith and Christopher E. Sims and Scott T. Magness and Nancy L. Allbritton},

url = {https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6339567/},

doi = {10.1021/acs.analchem.8b02845},

year  = {2018},

date = {2018-11-11},

urldate = {2018-11-11},

volume = {90},

number = {22},

pages = {13331},

abstract = {Animal models are frequently used for in vitro physiologic and drug transport studies of the colon, but there exists significant pressure to improve assay throughput as well as achieve tighter control of experimental variables than can be achieved with ...},

note = {Publisher: NIH Public Access},

keywords = {Atenolol, Biological Transport, Caco-2 Cells, Colon, Epithelium, Humans, Propranolol, Riboflavin, Tissue Engineering},

pubstate = {published},

tppubtype = {article}

}