Pharmaceutical Research

, 35:72 | Cite as

Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption

  • Seyoum Ayehunie
  • Tim Landry
  • Zachary Stevens
  • Alex Armento
  • Patrick Hayden
  • Mitchell Klausner
Research Paper

Abstract

Purpose

The study evaluates the use of new in vitro primary human cell-based organotypic small intestinal (SMI) microtissues for predicting intestinal drug absorption and drug-drug interaction.

Methods

The SMI microtissues were reconstructed using human intestinal fibroblasts and enterocytes cultured on a permeable support. To evaluate the suitability of the intestinal microtissues to model drug absorption, the permeability coefficients across the microtissues were determined for a panel of 11 benchmark drugs with known human absorption and Caco-2 permeability data. Drug-drug interactions were examined using efflux transporter substrates and inhibitors.

Results

The 3D–intestinal microtissues recapitulate the structural features and physiological barrier properties of the human small intestine. The microtissues also expressed drug transporters and metabolizing enzymes found on the intestinal wall. Functionally, the SMI microtissues were able to discriminate between low and high permeability drugs and correlated better with human absorption data (r2 = 0.91) compared to Caco-2 cells (r2 = 0.71). Finally, the functionality of efflux transporters was confirmed using efflux substrates and inhibitors which resulted in efflux ratios of >2.0 fold and by a decrease in efflux ratios following the addition of inhibitors.

Conclusion

The SMI microtissues appear to be a useful pre-clinical tool for predicting drug bioavailability of orally administered drugs.

KEY WORDS

caco-2 drug-drug interaction drug metabolizing enzymes drug permeation drug transporters organotypic small intestinal microtissues 

ABBREVIATIONS

Ω

Ohm

2D

Two dimensional

3D

Three dimensional

Ǻ

Angstrom

(A)

Apical

ABCB1

ATP binding cassette subfamily B member 1

ABCC1

ATP Binding Cassette Subfamily C Member 1

ABCC2

ATP Binding Cassette Subfamily C Member 2

ABCG2

ATP-binding cassette sub-family G member 2

ADR

Adverse drug reaction

ALI

Air–liquid interface

(B)

Basolateral

BCRP

Breast cancer resistance protein

BCS

Biopharmaceutical Classification System

CDCF

5(6)-carboxy-2′,7′-dichlorofluorescein (CDCF)

cDNA

Complementary DNA

CK

Cytokeratin

Cq

PCR cycles

Ct

Threshold cycle

CYP450

Cytochrome P450

DDI

Drug-drug interaction

FDA

Food and Drug Administration

FT

Full-thickness

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GI

Gastrointestinal

HPLC

High performance liquid chromatography

H

Hour

IIAM

International Institute for the Advancement of Medicine

LC/MS

Liquid chromatography–mass spectrometry

LY

Lucifer Yellow

MDCK

Madin-Darby canine kidney

MDR1

Multi-drug resistance gene (MDR)-1

Met

Metabolite

Min

Minutes

MRP-1

Multidrug-resistance associated protein-1

MRP-2

Multidrug-resistance associated protein-2

MS MRM

Mass spectroscopy multiple reaction monitoring

MTT

3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide

OPO

Organ Procurement Organization

Papp

Apparent permeability coefficient

P-gp

p-Glycoprotein

PR

Parental

PT

Partial thickness

qPCR

Quantitative polymerase chain reaction

RFU

Relative fluorescence unit

RNA

Ribonucleic acid

RT

Room temperature

RT-PCR

Reverse Transcription Polymerase Chain Reaction

SEM

Scanning electron microscopy

SMI

Small intestine

TEER

Transepithelial electrical resistance

TEM

Transmission electron microscopy

TTT

TEER of treated tissues

TUT

TEER of untreated tissues

References

  1. 1.
    Pretorius E, Bouic PJD. Permeation of four oral drugs through the human intestinal mucosa. AAPS PharmSciTech. 2009;10:270–5.CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Brendon M, Baker BM, Christopher S, Chen CS. Deconstructing the third dimension – how 3D culture microenvironments alter cellular cues. J Cell Sci. 2012;125:3015–24.CrossRefGoogle Scholar
  3. 3.
    Loriot Y, Perlemuter G, Malka D, Penault-Llorca F, Boige V, Deutsch E, et al. Drug insight: gastrointestinal and hepatic adverse effects of molecular-targeted agents in cancer therapy. Nat Clin Pract Oncol. 2008;5:268–78.CrossRefPubMedGoogle Scholar
  4. 4.
    Hubatsch I, Ragnarsson EGE, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc. 2007;2:2111–9.CrossRefPubMedGoogle Scholar
  5. 5.
    Lee MKK, Dilq. Drug development in cell culture: crosstalk from the industrial prospects. J Bioequivalence Bioavailab. 2014;6:O96–O114.Google Scholar
  6. 6.
    Volpe DA. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines. Future Med Chem. 2011;3:2063–77.CrossRefPubMedGoogle Scholar
  7. 7.
    Gupta V, Doshi N, Mitragotri S. Permeation of insulin, calcitonin, and exenatide across Caco-2 monolayers: measurement using rapid 3-day system. PLoS One. 2013;8:e77136.CrossRefGoogle Scholar
  8. 8.
    Tavelin S, Taipalensuu J, Söderberg L, Morrison R, Chong S, Artursson P. Prediction of the oral absorption of low permeability drugs using small intestine-like 2/4/A1 cell monolayers. Pharm Res. 2003;20:397–405.CrossRefPubMedGoogle Scholar
  9. 9.
    Ferrec EL, Chesne C, Artusson P, Brayden D, Fabre G, Gires P, et al. In vitro models of the intestinal barrier. The report and recommendations of ECVAM workshop 46. ATLA. 2001;29:649–68.PubMedGoogle Scholar
  10. 10.
    Huang Y, Adams MC. An in vitro model for investigating intestinal adhesion of potential dairy propionibacteria probiotic strains using cell lineC2BBe1. Lett Appl Microbiol. 2003;36:213e216.CrossRefGoogle Scholar
  11. 11.
    Fang Y, Eglen RM. Three-dimensional cell cultures in drug discovery and development. SLAS Disc. 2017;22:456–72.Google Scholar
  12. 12.
    Mathur A, Loskill P, Shao K, Huebsch N, Hong SG, Marcus MG, et al. Human iPSC-based cardiac microphysiological system for drug screening applications. Sci Rep. 2017;5:8883.CrossRefGoogle Scholar
  13. 13.
    Li AP. Preclinical in vitro screening assays for drug-like properties. Drug Discov Today. 2005;2:179–85.CrossRefGoogle Scholar
  14. 14.
    Amdion GL, Lennerlas H, Shah VP, Crison JR. A theoretical basis for biopharmaceutics drug classification: the correlation of in vitro drug product dissolution and in vivo drug bioavailability. Pharm Res. 1995;12:413–20.CrossRefGoogle Scholar
  15. 15.
  16. 16.
    Maschmeyer I, Hasenberg T, Jaenicke A, Lindner M, Lorenz AK, Zech J, et al. Chip-based human liver-intestine and liver-skin co-cultures - a first step toward systemic repeated dose substance testing in vitro. Eur J Pharm Biopharm. 2015;95:77–87.CrossRefPubMedGoogle Scholar
  17. 17.
    Wang Z, Hop CE, Leung KH, Pang J. Determination of in vitro permeability of drug candidates through a caco-2 cell monolayer by liquid chromatography/tandem mass spectrometry. J Mass Spectrom. 2000;35:71–6.CrossRefPubMedGoogle Scholar
  18. 18.
    Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.CrossRefPubMedGoogle Scholar
  19. 19.
    Ito S, Karnovsky MJ. Formaldehyde-glutaraldehyde fixative containing trinitro compounds. J Cell Biol. 1968;39:168–169-A.Google Scholar
  20. 20.
    Zimmermann C. Expression of drug transporters in intestine and blood. 2005. Doctoral thesis. http://edoc.unibas.ch/213/1/DissB_7139.pdf.
  21. 21.
    Rubas W, Jezyk N, Grass GM. Comparison of the permeability characteristics of a human colonic epithelial (Caco-2) cell line to colon of rabbit, monkey, and dog intestine and human drug absorption. Pharm Res. 1993;10:113–8.CrossRefPubMedGoogle Scholar
  22. 22.
    Shugarts S, Benet L. The role of transporters in the pharmacokinetics of orally administered drugs. Pharm Res. 2009;26:2039–54.CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Christians U. Transport proteins and intestinal metabolism; P-glycoprotein and cytochrome P450 (CYP)-3A. Ther Drug Monit. 2004;26:104–6.CrossRefPubMedGoogle Scholar
  24. 24.
    Szarka CE, Pleiffer GR, Hum ST, Everley LC, Baishem AM, Moore DF, et al. Glutathione S-transferase activity and glutathione s-transferase μ expression in subjects with risk for colorectal cancer. Cancer Res. 1995;55:2789–93.PubMedGoogle Scholar
  25. 25.
  26. 26.
    Zhao Y, Le J, Abraham MH, Hersey A, Eddershaw PJ, Luscombe CN, et al. Evaluation of human intestinal absorption data and subsequent derivation of a quantitative structure activity-relationship (QSAR) with the Abraham descriptors. J Pharm Sci. 2001;90:749–83.CrossRefPubMedGoogle Scholar
  27. 27.
    Shirasaka Y, Kuraoka E, Spahn-Langguth H, Nakanishi T, Langguth P, Tamai I. Species difference in the effect of grapefruit juice on intestinal absorption of Talinolol between human and rat. J Pharmacol Exp Ther. 2010;332:181–9.CrossRefPubMedGoogle Scholar
  28. 28.
    Bock U, Flototto T, Haltner E. Validation of cell culture models for the intestine and the blood-brain barrier and comparison of drug permeation. ALTEX. 2004;21(Suppl 3):57–64.PubMedGoogle Scholar
  29. 29.
    Zaki NM, Artursson P, Bergström C. A modified physiological BCS for prediction of intestinal absorption in drug discovery. Mol Pharm. 2010;7:1478–87.CrossRefPubMedGoogle Scholar
  30. 30.
    Kiela PR, Ghishan FK. Physiology of intestinal absorption and secretion. Best Pract Res Clin Gastroenterol. 2016;30:145–59.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Crawley SW, Mooseker MS, Tyska MJ. Shaping the intestinal brush border destruction by enterohemorrhagic Escherchia coli (EHEC): new insights from organoid culture. J Cell Biol. 2014;207:441–51.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Holmes R, Lobley RW. Intestinal brush border revisited. Gut. 1998;30:1667–78.CrossRefGoogle Scholar
  33. 33.
    Tyska MJ. Brush border destruction by enterohemorrhagic Escherchia coli (EHEC): new insights from organoid culture. Cell Mol Gastroenterol Hepatol. 2016;2:7–8.CrossRefPubMedGoogle Scholar
  34. 34.
    Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–89.CrossRefPubMedGoogle Scholar
  35. 35.
    Gilbert PM, Havenstrite KL, Magnusson KEG, Sacco A, Leonardi NA, Kraft P, et al. Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science. 2010;329:1078–81.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickma JJ. TEER measurement techniques for in vitro barrier model systems. J Lab Autom. 2015;20:107–26.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Balimane PV, Han Y-H, Chong S. Current industrial practices of assessing permeability and p-glycoprotein interaction. AAPS J. 2006;8:E1–E13.CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Hersman EM, Bumpus NN. A targeted proteomics approach for profiling murine cytochrome P450 expressions. J Pharmacol Exp Ther. 2014;349:221–8.CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    US FDA. Guidance for industry drug interaction studies — study design, data analysis, implications for dosing, and labeling recommendations. 2006. https://www.fda.gov/OHRMS/DOCKETS/98fr/06d-0344-gdl0001.pdf.
  40. 40.
    DiMarco RL, Hunt DR, Dewi RE, Heilshorn SC. Improvement of paracellular transport in the Caco-2 drug screening model using protein-engineered substrates. Biomaterials. 2017;129:152–62.CrossRefPubMedGoogle Scholar
  41. 41.
    Cummins CL, Mangravite LM, Benet LZ. Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodium butyrate and the phorbol ester 12-O-tetradecanoylphorbol-13 acetate. Pharm Res. 2001;18:1102–9.CrossRefPubMedGoogle Scholar
  42. 42.
    Maschmeyer I, Lorenz AK, Schimek K, Hasenberg T, Ramme AP, Hubner J, et al. A four-organ-chip for interconnected long-term co-culture of human intestine, liver, skin and kidney equivalents. Lab Chip. 2015;15:2688–99.CrossRefPubMedGoogle Scholar
  43. 43.
    Car D, Ayehunie S, Davies A, Duckworth C, French S, Hall N, et al. Towards better modeling and mechanistic biomarkers for drug-induced gastrointestinal injury. Pharmacol Ther. 2017;172:181–94.CrossRefGoogle Scholar
  44. 44.
    Eaton AD, Zimmermann Delaney CB, Hurley BP. Primary human polarized small intestinal epithelial barriers respond differently to a hazardous and an innocuous protein. Food Chem Toxicol. 2017;106:70–7.CrossRefPubMedGoogle Scholar
  45. 45.
    Maldonado-Conteras A, Birtley JR, Boll E, Zhao Y, Mumy KL, Toscano J, et al. Shigella depends on SepA to destabilize the intestinal epithelial integrity via cofilin activation. Gut Microbes. 2017;8:544–60.CrossRefGoogle Scholar
  46. 46.
    Anselmo AC, McHugh KJ, Webster J, Langer R, Jaklenec A. Layer-by-layer encapsulation of probiotics for delivery to the microbiome. Adv Mater. 2016;28:9486–90.CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.MatTek CorporationAshlandUSA

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