Monolayers of cancer-derived cell lines are widely used in the modelling of the gastrointestinal (GI) absorption of drugs and in oral drug development. However, they do not generally predict drug absorption in vivo. Here, we report a robotically handled system that uses large porcine GI tissue explants that are functionally maintained for an extended period in culture for the high-throughput interrogation (several thousand samples per day) of whole segments of the GI tract. The automated culture system provided higher predictability of drug absorption in the human GI tract than a Caco-2 Transwell system (Spearman’s correlation coefficients of 0.906 and 0.302, respectively). By using the culture system to analyse the intestinal absorption of 2,930 formulations of the peptide drug oxytocin, we discovered an absorption enhancer that resulted in a 11.3-fold increase in the oral bioavailability of oxytocin in pigs in the absence of cellular disruption of the intestinal tissue. The robotically handled whole-tissue culture system should help advance the development of oral drug formulations and might also be useful for drug screening applications.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. The raw and analysed datasets generated during the study are too big to be publicly shared; however, they are available for research purposes from the corresponding authors upon reasonable request.
Goldberg, M. & Gomez-Orellana, I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug Discov. 2, 289–295 (2003).
Ensigna, L., Conea, R. & Hanes, J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. Drug Deliv. Rev. 64, 557–570 (2012).
Bjerknes, M. & Cheng, H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006).
Kaeffer, B. Mammalian intestinal ephithelial cells in primary culture: a mini review. In Vitro Cell. Dev. Biol. Anim. 38, 123–134 (2002).
Pageot, L. et al. Human cell models to study small intestinal functions: recapitulation of the crypt–villus axis. Microsc. Res. Tech. 49, 394–406 (2000).
Li, L. & Xie, T. Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol. 21, 605–631 (2005).
Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).
Meunier, V., Bourrie, M., Berger, Y. & Fabre, G. The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications. Cell Biol. Toxicol. 11, 187–194 (1995).
Artursson, P., Palm, K. & Luthman, K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46, 27–43 (2001).
Hubatsch, I., Ragnarsson, E. G. E. & Artursson, P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat. Protoc. 2, 2111–2119 (2007).
Fagerholm, U. Prediction of human pharmacokinetics—gastrointestinal absorption. J. Pharm. Pharmacol. 59, 905–916 (2007).
Sun, D. et al. Comparison of human duodenum and Caco-2 gene expression profiles for 12,000 gene sequences tags and correlation with permeability of 26 drugs. Pharm. Res. 19, 1400–1416 (2002).
Teksin, Z. S., Seo, P. R. & Polli, J. E. Comparison of drug permeabilities and BCS classification: three lipid-component PAMPA system method versus Caco-2 monolayers. AAPS J. 12, 238–241 (2010).
Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).
Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
Grabinger, T. et al. Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy. Cell Death Dis. 5, e1228 (2014).
Shamir, E. R. & Ewald, A. J. Three-dimensional organotypic culture: experimental models of mammalian biology and disease. Nat. Rev. Mol. Cell Biol. 15, 647–664 (2014).
Castellanos-Gonzalez, A., Cabada, M. M., Nichols, J., Gomez, G. & White, C. Human primary intestinal epithelial cells as an improved in vitro model for Cryptosporidium parvum infection. Infect. Immun. 81, 1996–2001 (2013).
Wang, Y. et al. Self-renewing monolayer of primary colonic or rectal epithelial cells. Cell. Mol. Gastroenterol. Hepatol. 4, 165–182 (2017).
Wang, Y. et al. Formation of human colonic crypt array by application of chemical gradients across a shaped epithelial monolayer. Cell. Mol. Gastroenterol. Hepatol. 5, 113–130 (2018).
Kasendra, M. et al. Development of a primary human small Intestine-on-a-Chip using biopsy-derived organoids. Sci. Rep. 8, 2871 (2018).
Medema, J. P. & Vermeulen, L. Microenvironmental regulation of stem cells in intestinal homeostasis and cancer. Nature 474, 318–326 (2011).
Kararli, T. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm. Drug Dispos. 16, 351–380 (1995).
Nejdfors, P., Ekelund, M., Jeppsson, B., Westro, B. R. & Nejdfors, P. Mucosal in vitro permeability in the intestinal tract of the pig, the rat, and man: species- and region-related differences. Scand. J. Gastroenterol. 5, 501–507 (2000).
Groenen, M. A. M. et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491, 393–398 (2012).
MacDonald, B. T., Tamai, K. & He, X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).
Söderholm, J. D. et al. Integrity and metabolism of human ileal mucosa in vitro in the ussing chamber. Acta Physiol. Scand. 162, 47–56 (1998).
Chowhan, Z. T. & Amaro, A. A. Everted rat intestinal sacs as an in vitro model for assessing absorptivity of new drugs. J. Pharm. Sci. 66, 1249–1253 (1977).
Barthe, L., Woodley, J. & Houin, G. Gastrointestinal absorption of drugs: methods and studies. Fundam. Clin. Pharm. 13, 154–168 (1999).
Westerhout, J. et al. A new approach to predict human intestinal absorption using porcine intestinal tissue and biorelevant matrices. Eur. J. Pharm. Sci. 63, 167–177 (2014).
Waiver of In Vivo Bioavailability and Bioequivalence Studies for Immediate-Release Solid Oral Dosage Forms Based on a Biopharmaceutics Classification System (US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, 2000).
Welling, P. G. Effects of food on drug absorption. Annu. Rev. Nutr. 16, 383–415 (1996).
Ingels, F. et al. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. Int. J. Pharm. 232, 183–192 (2002).
Lipinski, C. A., Lombardo, F., Dominy, B. W. & Feeney, P. J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 23, 3–25 (1997).
MacDonald, K. & Feifel, D. Helping oxytocin deliver: considerations in the development of oxytocin-based therapeutics for brain disorders. Front. Neurosci. 7, 35 (2013).
Kansy, M., Senner, F. & Gubernator, K. Physicochemical high throughput screening: parallel artificial membrane permeation assay in the description of passive absorption processes. J. Med. Chem. 41, 1007–1010 (1998).
Kerns, E. H. et al. Combined application of parallel artificial membrane permeability assay and Caco-2 permeability assays in drug discovery. J. Pharm. Sci. 93, 1440–1453 (2004).
Collett, A. et al. Influence of morphometric factors on quantitation of paracellular permeability of intestinal epithelia in vitro. Pharm. Res. 14, 767–773 (1997).
Artursson, P., Ungell, A. L. & Löfroth, J. E. Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments. Pharm. Res. 10, 1123–1129 (1993).
Hayeshi, R. et al. Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories. Eur. J. Pharm. Sci. 35, 383–396 (2008).
Thompson, S. L. & Compton, D. A. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–672 (2008).
Yamaura, Y., Chapron, B. D., Wang, Z., Himmelfarb, J. & Thummel, K. E. Functional comparison of human colonic carcinoma cell lines and primary small intestinal epithelial cells for investigations of intestinal drug permeability and first-pass metabolism. Drug Metab. Dispos. 44, 329–335 (2016).
Takenaka, T., Harada, N., Kuze, J., Chiba, M. & Iwao, T. Human small intestinal epithelial cells differentiated from adult intestinal stem cells as a novel system for predicting oral drug absorption in humans. Drug Metab. Dispos. 42, 1947–1954 (2014).
Bohets, H. et al. Strategies for absorption screening in drug discovery and development. Curr. Top. Med. Chem. 1, 367–383 (2001).
Gotoh, Y., Kamada, N. & Momose, D. The advantages of the Ussing chamber in drug absorption studies. J. Biomol. Screen. 10, 517–523 (2005).
Larregieu, C. A. & Benet, L. Z. Distinguishing between the permeability relationships with absorption and metabolism to improve BCS and BDDCS predictions in early drug discovery. Mol. Pharm. 11, 1335–1344 (2014).
Bergström, C. A. S. et al. Absorption classification of oral drugs based on molecular surface properties. J. Med. Chem. 46, 558–570 (2003).
Godbey, W. T., Wu, K. & Mikos, A. Poly(ethyenimine) and its role in gene delivery. J. Control. Release 60, 149–160 (1999).
Sato, T. & Clevers, H. Epithelial cell culture protocols. Methods Mol. Biol. 945, 319–328 (2013).
Zhang, S. et al. A pH-responsive supramolecular polymer gel as an enteric elastomer for use in gastric devices. Nat. Mater. 14, 1065–1071 (2015).
Traverso, G. et al. Physiologic status monitoring via the gastrointestinal tract. PLoS ONE 10, e0141666 (2015).
Schoellhammer, C. M. et al. Ultrasound-mediated gastrointestinal drug delivery. Sci. Transl. Med. 7, 310ra168 (2015).
Volpe, D. A. Variability in Caco-2 and MDCK cell-based intestinal permeability assays. J. Pharm. Sci. 97, 712–725 (2008).
Wishart, D. S. et al. DrugBank: a comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Res. 34, D668–D672 (2006).
We want to thank S. Kern, D. Hartman and S. Hershenson from the Bill and Melinda Gates Foundation for helpful discussions around the application and development of the GI-TRIS system. We thank J. Haupt and M. Jamiel for help with the in vivo porcine work. This work was funded in part by the National Institutes of Health (grant no. EB-000244) and the Bill and Melinda Gates Foundation (grant no. OPP1096734). T.v.E. and D.R. were funded by the Swiss National Foundation. We thank the Hope Babette Tang Histology Facility at the Koch Institute at MIT for the histology work and consultation. We would also like to thank the Microscopy Core Facility and the Swanson Biotechnology Center High Throughput Screening Facility. We are grateful for all members of the Langer and Traverso laboratories for helpful methodological suggestions.
The authors declare US Provisional Patent application no. 62/476,181 filed on 24 March 2017 covering the technologies described. T.v.E., G.T. and R.L. have a financial interest in Vivtex Corporation, a biotechnology company focused on the application of GI models for pharmaceutical applications. Complete details of all relationships for profit and not-for-profit for G.T. can be found at the following link: https://www.dropbox.com/sh/szi7vnr4a2ajb56/AABs5N5i0q9AfT1IqIJAE-T5a?dl=0. Complete details for R.L. can be found at the following link: https://www.dropbox.com/s/yc3xqb5s8s94v7x/Rev%20Langer%20COI.pdf?dl=0
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Supplementary figures and captions for the supplementary datasets.
Reported human absorption values of model drugs.
Overview of all publications that report Caco-2 permeability values for the specific drug listed.
Analysis of experimental parameters for selected Caco-2 permeability experiments reported in the literature.
Transporter–drug interactions based on published literature.
Absorption predictions for 39 model drugs.
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von Erlach, T., Saxton, S., Shi, Y. et al. Robotically handled whole-tissue culture system for the screening of oral drug formulations. Nat Biomed Eng 4, 544–559 (2020). https://doi.org/10.1038/s41551-020-0545-6
Nature Biomedical Engineering (2020)