Skip to main content

Advertisement

SpringerLink
Log in

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

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

Similar content being viewed by others

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. Pretorius E, Bouic PJD. Permeation of four oral drugs through the human intestinal mucosa. AAPS PharmSciTech. 2009;10:270–5.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  5. Lee MKK, Dilq. Drug development in cell culture: crosstalk from the industrial prospects. J Bioequivalence Bioavailab. 2014;6:O96–O114.

    Google Scholar 

  6. Volpe DA. Drug-permeability and transporter assays in Caco-2 and MDCK cell lines. Future Med Chem. 2011;3:2063–77.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  11. Fang Y, Eglen RM. Three-dimensional cell cultures in drug discovery and development. SLAS Disc. 2017;22:456–72.

    CAS  Google Scholar 

  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.

    Article  Google Scholar 

  13. Li AP. Preclinical in vitro screening assays for drug-like properties. Drug Discov Today. 2005;2:179–85.

    Article  CAS  Google Scholar 

  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.

    Article  Google Scholar 

  15. http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm072101.pdf.

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  18. Mosmann T. Rapid calorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983;65:55–63.

    Article  CAS  PubMed  Google Scholar 

  19. Ito S, Karnovsky MJ. Formaldehyde-glutaraldehyde fixative containing trinitro compounds. J Cell Biol. 1968;39:168–169-A.

    Google Scholar 

  20. Zimmermann C. Expression of drug transporters in intestine and blood. 2005. Doctoral thesis. http://edoc.unibas.ch/213/1/DissB_7139.pdf.

  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.

    Article  CAS  PubMed  Google Scholar 

  22. Shugarts S, Benet L. The role of transporters in the pharmacokinetics of orally administered drugs. Pharm Res. 2009;26:2039–54.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Christians U. Transport proteins and intestinal metabolism; P-glycoprotein and cytochrome P450 (CYP)-3A. Ther Drug Monit. 2004;26:104–6.

    Article  CAS  PubMed  Google Scholar 

  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.

    CAS  PubMed  Google Scholar 

  25. https://www.fda.gov/downloads/Drugs/Guidances/ucm070246.pdf.

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  30. Kiela PR, Ghishan FK. Physiology of intestinal absorption and secretion. Best Pract Res Clin Gastroenterol. 2016;30:145–59.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Holmes R, Lobley RW. Intestinal brush border revisited. Gut. 1998;30:1667–78.

    Article  Google Scholar 

  33. Tyska MJ. Brush border destruction by enterohemorrhagic Escherchia coli (EHEC): new insights from organoid culture. Cell Mol Gastroenterol Hepatol. 2016;2:7–8.

    Article  PubMed  Google Scholar 

  34. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126:677–89.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Balimane PV, Han Y-H, Chong S. Current industrial practices of assessing permeability and p-glycoprotein interaction. AAPS J. 2006;8:E1–E13.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hersman EM, Bumpus NN. A targeted proteomics approach for profiling murine cytochrome P450 expressions. J Pharmacol Exp Ther. 2014;349:221–8.

    Article  PubMed  PubMed Central  Google Scholar 

  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. 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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  Google Scholar 

  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.

    Article  Google Scholar 

  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.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. MatTek Corporation, 200 Homer Avenue, Ashland, Massachusetts, USA

    Seyoum Ayehunie, Tim Landry, Zachary Stevens, Alex Armento, Patrick Hayden & Mitchell Klausner

Authors
  1. Seyoum Ayehunie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tim Landry
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zachary Stevens
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alex Armento
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Patrick Hayden
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mitchell Klausner
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Seyoum Ayehunie.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ayehunie, S., Landry, T., Stevens, Z. et al. Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption. Pharm Res 35, 72 (2018). https://doi.org/10.1007/s11095-018-2362-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-018-2362-0

KEY WORDS

Search

Navigation