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Robotically handled whole-tissue culture system for the screening of oral drug formulations

Abstract

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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Fig. 1: Characterization of ex vivo cultured intestinal tissue.
Fig. 2: GI-TRIS interface device development.
Fig. 3: GI-TRIS transport variability validation.
Fig. 4: In vivo absorption predictability analysis using model drugs.
Fig. 5: In vivo absorption predictability comparison to the Caco-2 Transwell system.
Fig. 6: GI-TRIS intestinal transport screening of Alexa-488–oxytocin formulations.
Fig. 7: Validation experiments and in vivo pharmacokinetic and histological analysis of oxytocin formulations.

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Data availability

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.

References

  1. Goldberg, M. & Gomez-Orellana, I. Challenges for the oral delivery of macromolecules. Nat. Rev. Drug Discov. 2, 289–295 (2003).

    Article  CAS  PubMed  Google Scholar 

  2. Ensigna, L., Conea, R. & Hanes, J. Oral drug delivery with polymeric nanoparticles: the gastrointestinal mucus barriers. Adv. Drug Deliv. Rev. 64, 557–570 (2012).

    Article  CAS  Google Scholar 

  3. Bjerknes, M. & Cheng, H. Intestinal epithelial stem cells and progenitors. Methods Enzymol. 419, 337–383 (2006).

    Article  CAS  PubMed  Google Scholar 

  4. Kaeffer, B. Mammalian intestinal ephithelial cells in primary culture: a mini review. In Vitro Cell. Dev. Biol. Anim. 38, 123–134 (2002).

    Article  PubMed  Google Scholar 

  5. 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).

    Article  CAS  PubMed  Google Scholar 

  6. Li, L. & Xie, T. Stem cell niche: structure and function. Annu. Rev. Cell Dev. Biol. 21, 605–631 (2005).

    Article  CAS  PubMed  Google Scholar 

  7. Ootani, A. et al. Sustained in vitro intestinal epithelial culture within a Wnt-dependent stem cell niche. Nat. Med. 15, 701–706 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. 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).

    Article  CAS  PubMed  Google Scholar 

  9. 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).

    Article  CAS  PubMed  Google Scholar 

  10. 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).

    Article  CAS  PubMed  Google Scholar 

  11. Fagerholm, U. Prediction of human pharmacokinetics—gastrointestinal absorption. J. Pharm. Pharmacol. 59, 905–916 (2007).

    Article  CAS  PubMed  Google Scholar 

  12. 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).

    Article  CAS  PubMed  Google Scholar 

  13. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).

    Article  CAS  PubMed  Google Scholar 

  15. Sato, T. et al. Single Lgr5 stem cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Wang, Y. et al. Self-renewing monolayer of primary colonic or rectal epithelial cells. Cell. Mol. Gastroenterol. Hepatol. 4, 165–182 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  20. 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).

    Article  PubMed  Google Scholar 

  21. Kasendra, M. et al. Development of a primary human small Intestine-on-a-Chip using biopsy-derived organoids. Sci. Rep. 8, 2871 (2018).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Medema, J. P. & Vermeulen, L. Microenvironmental regulation of stem cells in intestinal homeostasis and cancer. Nature 474, 318–326 (2011).

    Article  CAS  PubMed  Google Scholar 

  23. Kararli, T. Comparison of the gastrointestinal anatomy, physiology, and biochemistry of humans and commonly used laboratory animals. Biopharm. Drug Dispos. 16, 351–380 (1995).

    Article  CAS  PubMed  Google Scholar 

  24. 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).

    Google Scholar 

  25. Groenen, M. A. M. et al. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491, 393–398 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. MacDonald, B. T., Tamai, K. & He, X. Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev. Cell 17, 9–26 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    Article  PubMed  Google Scholar 

  28. 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).

    Article  CAS  PubMed  Google Scholar 

  29. Barthe, L., Woodley, J. & Houin, G. Gastrointestinal absorption of drugs: methods and studies. Fundam. Clin. Pharm. 13, 154–168 (1999).

    Article  CAS  Google Scholar 

  30. 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).

    Article  CAS  PubMed  Google Scholar 

  31. 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).

  32. Welling, P. G. Effects of food on drug absorption. Annu. Rev. Nutr. 16, 383–415 (1996).

    Article  CAS  PubMed  Google Scholar 

  33. Ingels, F. et al. Simulated intestinal fluid as transport medium in the Caco-2 cell culture model. Int. J. Pharm. 232, 183–192 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. MacDonald, K. & Feifel, D. Helping oxytocin deliver: considerations in the development of oxytocin-based therapeutics for brain disorders. Front. Neurosci. 7, 35 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    Article  CAS  PubMed  Google Scholar 

  37. 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).

    Article  CAS  PubMed  Google Scholar 

  38. Collett, A. et al. Influence of morphometric factors on quantitation of paracellular permeability of intestinal epithelia in vitro. Pharm. Res. 14, 767–773 (1997).

    Article  CAS  PubMed  Google Scholar 

  39. 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).

    Article  CAS  PubMed  Google Scholar 

  40. 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).

    Article  CAS  PubMed  Google Scholar 

  41. Thompson, S. L. & Compton, D. A. Examining the link between chromosomal instability and aneuploidy in human cells. J. Cell Biol. 180, 665–672 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Article  PubMed  CAS  Google Scholar 

  44. Bohets, H. et al. Strategies for absorption screening in drug discovery and development. Curr. Top. Med. Chem. 1, 367–383 (2001).

    Article  CAS  PubMed  Google Scholar 

  45. Gotoh, Y., Kamada, N. & Momose, D. The advantages of the Ussing chamber in drug absorption studies. J. Biomol. Screen. 10, 517–523 (2005).

    Article  CAS  PubMed  Google Scholar 

  46. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bergström, C. A. S. et al. Absorption classification of oral drugs based on molecular surface properties. J. Med. Chem. 46, 558–570 (2003).

    Article  PubMed  CAS  Google Scholar 

  48. Godbey, W. T., Wu, K. & Mikos, A. Poly(ethyenimine) and its role in gene delivery. J. Control. Release 60, 149–160 (1999).

    Article  CAS  PubMed  Google Scholar 

  49. Sato, T. & Clevers, H. Epithelial cell culture protocols. Methods Mol. Biol. 945, 319–328 (2013).

    Article  PubMed  CAS  Google Scholar 

  50. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Traverso, G. et al. Physiologic status monitoring via the gastrointestinal tract. PLoS ONE 10, e0141666 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Schoellhammer, C. M. et al. Ultrasound-mediated gastrointestinal drug delivery. Sci. Transl. Med. 7, 310ra168 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  53. Volpe, D. A. Variability in Caco-2 and MDCK cell-based intestinal permeability assays. J. Pharm. Sci. 97, 712–725 (2008).

    Article  CAS  PubMed  Google Scholar 

  54. Wishart, D. S. et al. DrugBank: a comprehensive resource for in silico drug discovery and exploration. Nucleic Acids Res. 34, D668–D672 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Contributions

T.v.E., R.L. and G.T. conceived the study and designed experiments. T.v.E. performed experiments and data analyses. S.S. performed experiments and assisted in data analyses. D.R. performed computational modelling and helped with data analyses. D.M. manufactured the device and helped in its design. F.J. performed finite element analysis. Y.S. performed PCR analysis and helped with western blotting. Y.-A.L.L. performed SEM analysis. C.S. provided help and guidance regarding ultrasound-mediated transfection experiments. T.E. performed transport experiments and helped with Caco-2 meta-analysis. J.L. and H.L. assisted in oxytocin screening and mechanistic experiments. S.B. helped with Caco-2 meta-analysis. C.C., L.B. and A.H. performed pharmacokinetics experiments in pigs. T.v.E. and G.T. performed data interpretation and wrote the manuscript. R.L. and G.T. supervised the research.

Corresponding authors

Correspondence to Robert Langer or Giovanni Traverso.

Ethics declarations

Competing interests

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 information

Supplementary Information

Supplementary figures and captions for the supplementary datasets.

Reporting Summary

Supplementary Dataset 1

Reported human absorption values of model drugs.

Supplementary Dataset 2

Overview of all publications that report Caco-2 permeability values for the specific drug listed.

Supplementary Dataset 3

Analysis of experimental parameters for selected Caco-2 permeability experiments reported in the literature.

Supplementary Dataset 4

Transporter–drug interactions based on published literature.

Supplementary Dataset 5

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

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