PCIS are discussed and highlighted as a promising method for drug ADME-tox studies in the intestine to be used as alternative to the in vitro cell culturing and in vivo animal modeling which are conventionally used in academia and industry. They show relatively good viability and functionality up to 8 - 24 hours of incubation.
Due to the physiological levels of DTs and DMEs in PCIS, at least during the first hours of culturing, they can provide physiological relevant results, including metabolic profiles, induction and inhibition potency, and local drug/metabolite exposure, and can represent the functional differences between duodenum, jejunum, ileum and colon. Therefore, PCIS are expected to serve as a translational model and a bridge between animal and human. The development of substrates and inhibitors with higher specificity for the enzymes and transporters could be instrumental to identify the influence of the proteins involved in the disposition of a certain substrate. In addition, PCIS made from the intestine of genetic deficient animals such as the Mrp2-/- rat, or genetically modified animals (i.e. Mdr1a knockout mouse) can be helpful for to identify transporters involved in the transport of a drug under study [76].
The sensitive nature of the intestine necessitates to monitor the quality of the intestine and the viability of PCIS using viability markers, e.g. the intracellular ATP and morphology, in each experiment and for every compound of interest. Moreover, a further optimization of culture medium would be very useful for maintenance not only of viability, but also of DME and DT activity. Addition of different supplementary compounds, such as ligands for the nuclear factors involved in regulation of the DME and DT, into the medium to optimize the culture conditions of PCIS should be investigated for each species separately, since rat PCIS seem more vulnerable in the currently used medium (Williams medium E) compared to mouse and human PCIS.
Extension of the viability is also instrumental for studies on DIGI, which were up to now limited to the short term effects of NSAIDs and bile acids. Future applications may lead to the discovery of mechanisms of damage using transcriptomics technologies and of new protecting
7
drugs for ischemia-reperfusion injury, which would be useful also for transplantation purposes [53].
The very efficient use of human tissue makes the PCIS the preferred model for translational studies. However as human intestinal tissue is scarce and not widely available, PCIS may be also applied to identify other animal species that in some aspects may be more similar to human than rat or mouse, for instance monkey or (mini)pig.
The wider application of PCIS would certainly benefit from development of better cold- and cryopreservation techniques. Development of long-term preservation technologies for PCIS hopefully will benefit from the fast growing knowledge of cryopreservat ion techniques. When cryopreserved viable PCIS, especially human PCIS, become available at any time and location, a larger application in academia and industry for the ADME-tox tests during drug development will emerge.
The co-culture of PCIS with intestinal bacteria will be a promising new area, since the microbiome plays an important role in the intestinal metabolism and toxicity. The gut flora can contribute to the generation of toxic metabolites that may injure the intestine [91]. The gut microenvironment was also incorporated in the „human gut-on-a-chip‟ microdevice [92, 93] in which Caco-2 cells formed four different types of differentiated epithelial cells and recapitulated the structure of intestinal villi. In this system the gut microenvironment was recreated with flow of fluid and peristaltic motions, together with the co-culture of normal intestinal bacteria on the luminal surface.
Selection of slices with and without Peyer‟s patches, aggregations of lymphoid cells that are usually found in the ileum, may allow investigating the influence of the immunological function of the Peyer‟s patches on the intestinal metabolism and transport function and on DIGI.
Article highlights box
After the introduction tissue slicer and agarose filling and embedding, PCIS has been established as ex vivo model for the intestine, which can be easily applied to the human intestine and that of various animals.
As an alternative model, PCIS have an important potential for ADME-tox studies due to sufficient maintenance of tissue viability, activity and functionality to perform toxicity and induction studies, maintenance of cell polarity and cell- cell and cell- matrix contacts, relatively easy and fast preparation, efficient use of the scarce tissue resulting in 100 - 200
slices per experiment, applicability to the human situation and convenience in studying regional and species differences, in compliance with 3Rs. However, its limitations should also be taken into account.
PCIS were applied to study drug transport related to efflux transporters (P-gp, MRPs) and influx transporters (ASBT). The ex vivo assessment of the transporters involved and the evaluation of the inhibitory potencies of their inhibitors are expected to make more accurate predictions for potential DDIs in vivo.
Applications of PCIS on drug toxicity were focused on DIGI by NSAIDs and toxic bile acids. PCIS are also promising in evaluating the toxicity of anticancer compounds.
PCIS can be applied to predict DDIs by studying the induction and inhibition of drugs on the activity of DTs and DMEs. Furthermore, the transport- metabolism interplay, e.g.
P-gp/CYP3A interplay, and interorgan interactions can be studied in PCIS.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. The work was funded by the China Scholarship Council (CSC) and the University of Groningen.
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References
1. Otto W. Versuche und uberledbeudem carcino mgewebe (methoden). Bioche mische Zeitschr 1923;142:317-33.
2. HA K. Untersuchungen über den Stoffwechsel der Aminosäuren im Tierkörper. Hoppe -Seyl Z 1933;217:191.
3. Kru mdiec k CL, dos Santos J, Ho K-J. A new instrument for the rapid preparation of t issue slices. Analytical Biochemistry 1980;104(1):118-23. the cyclosporin derivative SDZ IMM 125 using human liver and kidney slices and intestine. Co mparison with rat liver slices and cyclosporin A metabolism. Drug Metabolism and Disposition 1995;23(3):327-33.
7. Smith PF, Gandolfi AJ, Kru mdiec k CL, Putnam CW, Zu koski CF, 3rd, Davis WM, et al. Dyna mic organ culture of precision liver slices for in vitro toxicology. Life Sci 1985;36(14):1367 -75.
8. Ruegg CE, Gandolfi AJ, Nag le RB, Kru mdiec k CL, Brendel K. Preparat ion of positional renal slices for study of cell-specific toxicity. Journal of Pharmacological Methods 1987;17(2):111-23.
9. van de Bovenka mp M , Groothuis GMM, Draa isma AL, Mere ma MT, Be zuijen JI, van Gils MJ, et a l.
Precision-Cut Liver Slices as a New Model to Study To xicity -Induced Hepatic Stellate Ce ll Activation in a Physiologic Milieu. Toxicological Sciences 2005;85(1):632-38.
10. Martignoni M, Groothuis G, de Kanter R. Co mparison of mouse an d rat cytochrome P450-med iated metabolism in liver and intestine. Drug Metabolism and Disposition 2006;34(6):1047 -54.
11. Groothuis GM, de Graa f IA. Prec ision-cut intestinal slices as in vitro tool for studies on drug metabolis m.
Current drug metabolism 2012;14(1):112-19.
12. Westra IM, Oosterhuis D, Groothuis GMM , Olinga P. The Effect of Antifibrotic Drugs in Rat Prec ision -Cut Fibrotic Liver Slices. PloS one 2014;9(4):e95462.
13. Westra IM, Oosterhuis D, Groothuis GMM, Olinga P. Prec ision -cut liver slices as a model for the early onset of liver fibrosis to test antifibrotic drugs. Toxicology and Applied Pharmacology 2014;274(2):328 -38.
14. Van de Bovenka mp M, Groothuis GMM, Meijer DKF, Olinga P. Liver fibrosis in v itro: Cell culture mode ls and precision-cut liver slices. Toxicology in Vitro 2007;21(4):545-57.
15. Oene ma TA, Maarsingh H, Smit M, Groothuis GMM, Meurs H, Gosens R. Bronchoconstriction Induces TGF-β Release and Airway Remodelling in Guinea Pig Lung Slices. PloS one 2013;8(6):e 65580.
16. Schlepütz M, Rieg AD, Seehase S, Sp illner J, Pe re z-Bouza A, Braunschweig T, et al. Neura lly Mediated Airway Constriction in Hu man and Other Species: A Co mparat ive Study Using Prec ision -Cut Lung Slices (PCLS). PloS one 2012;7(10):e47344.
17. Lavoie TL, Krishnan R, Siegel HR, Maston ED, Fredberg JJ, Solway J, et al. Dilatation of the Constricted Hu man Airway by Tidal Expansion of Lung Parenchyma. A me rican Journal of Respiratory and Critica l Care Medicine 2012;186(3):225-32.
18. Groothuis GMM, Casin i A, Meurs H, Olinga P. Chapter 3 Translational Research in Pharmacology and
To xico logy Using Precision-Cut Tissue Slices. Human-based Systems for Translational Research: The Royal Society of Chemistry 2015:38-65.
19. Fisher RL, Vic kers AEM. Preparat ion and culture of precis ion-cut organ slices fro m human and anima l.
Xenobiotica 2013;43(1):8-14.
20. de Kanter R, Tuin A, van de Ke rkhof E, Mart ignoni M, Draa isma A L, de Jager MH, et a l. A ne w technique for preparing precision-cut slices fro m sma ll intestine and colon for drug biotransformation studies. Journal of Pharmacological and Toxicological Methods 2005;51(1):65 -72.
21. Vic kers A EM, Zollinger M , Dannecker R, Tynes R, He itz F, Fischer V. In Vitro Metabolis m of Tegaserod in Hu man Liver and Intestine: Assessment of Drug Interactions. Drug Metabolis m and Disposition 2001;29(10):1269-76.
22. Malfatti MA, Connors MS, Mauthe RJ, Fe lton JS. The Capability of Rat Co lon Tissue Slices to Metabolize the Cooked-Food Ca rcinogen 2-A mino-1-methyl-6-phenylimidazo[4,5-b]pyrid ine. Cancer Research 1996;56(11):2550-55.
23. De Kanter R, De Jager M H, Draais ma A L, Ju rva JU, Olinga P, Meijer DKF, et al. Drug -metabolizing activity of human and rat liver, lung, kidney and intestine slices. Xenobiotica 2002;32(5):349-62.
24. Le rche-Langrand C, Toutain HJ. Precision-cut liver slices: characteristics and use for in vitro pharmaco-toxicology. Toxicology 2000;153(1–3):221-53.
25. de Kanter R, Monshouwer M, Meijer DK, Groothuis GM. Precision -cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special re ference to non-hepatic tissues. Current drug metabolis m 2002 Feb;3(1):39-59.
26. van de Kerkhof EG, Ungell A-LB, Sjöberg ÅK, de Jager MH, Hilgendorf C, de Graaf IAM, et a l.
Innovative Methods to Study Human Intestinal Drug Metabolism in Vit ro: Precision-Cut Slices Co mpa red with Ussing Chamber Preparations. Drug Metabolism and Disposition 2006;34(11):1893 -902.
27. van de Kerkhof EG, de Graa f IAM, Ungell A -LB, Groothuis GMM. Induction of metabolis m and transport in human intestine: validation of precision-cut slices as a tool to study induction of drug metabolis m in human intestine in vitro. Drug Metabolism and Disposition 2008;36(3):604 -13.
28. Punyadarsaniya D, Winter C, Mork A-K, A miri M , Naim HY, Rautenschlein S, et a l. Prec ision-cut intestinal slices as a culture system to analy ze the infection of differentiated intestinal epithelia l ce lls by avian influenza viruses. Journal of Virological Methods 2015;212:71-75.
29. de Graaf IAM, Olinga P, de Jager MH, Mere ma MT, de Kanter R, van de Ke rkhof EG, et a l. Preparat ion and incubation of precision-cut liver and intestinal slices for applicat ion in d rug metabolis m and to xic ity studies.
Nat Protocols 2010;5(9):1540-51.
30. Brouwe r KLR, Kepple r D, Hoffmaster KA, Bow DAJ, Cheng Y, La i Y, et a l. In Vit ro Methods to Support Transporter Evaluation in Drug Discovery and Development. Clinica l Pharmaco logy & Therapeutics 2013;94(1):95-112.
31. Kis O, Zastre J, Hoque MT, Walms ley S, Bendayan R. Role of drug efflu x and uptake transporters in atazanavir intestinal permeability and drug-drug interactions. Pharm Res 2013;30(4):1050-64.
32. Rozehnal V, Naka i D, Hoepner U, Fischer T, Ka miya ma E, Takahashi M, et al. Hu man s ma ll intestinal and colonic tissue mounted in the Ussing chamber as a tool for characteri zing the intestinal absorption of drugs.
European Journal of Pharmaceutical Sciences 2012;46(5):367-73.
33. Ellis LCJ, Ha wksworth GM, Weaver RJ. ATP-dependent transport of statins by human and rat MRP2/Mrp2.
Toxicology and Applied Pharmacology 2013;269(2):187-94.
34. Li M, Si L, Pan H, Rabba AK, Yan F, Qiu J, et a l. Exc ipients enhance intestinal absorption of ganciclovir by P-gp inhib ition: Assessed in vitro by everted gut sac and in situ by improved intestinal perfusion. International
7
Journal of Pharmaceutics 2011;403(1– 2):37-45.
35. The International Transporter Consortium. Me mbrane transporters in drug development. Nature Revie ws Drug Discovery 2010;9(3):215-36.
36. Hu M, Ling J, Lin H, Chen J. Use of Caco-2 Cell Monolayers to Study Drug Absorption and Metabolism.
In: Yan Z, Caldwell G, eds. Optimization in Drug Discovery: Humana Press 2004:19-35.
37. Siissalo S, Zhang H, St ilgenbauer E, Kau konen AM, Hirvonen J, Finel M. The Exp ression of Most UDP-Glucuronosyltransferases (UGTs) Is Increased Significantly during Caco-2 Ce ll Differentiation, whereas UGT1A6 Is Highly Expressed Also in Undifferentiated Ce lls. Drug Metabolis m and Disposition 2008 Nove mber 1, 2008;36(11):2331-36.
38. Ka miya ma E, Sugiya ma D, Na kai D, M iura S -i, Oka za ki O. Culture period-dependent change of function and expression of ATP-Binding Cassette transporters in caco-2 Ce lls. Drug Metabolism and Disposition 2009 September 1, 2009;37(9):1956-62.
39. Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, et a l. Sing le Lgr5 ste m cells build crypt–villus structures in vitro without a mesenchymal niche. Nature 2009;459(7244):262 -65.
40. Pham BT, van Haaften WT, Oosterhuis D, Nieken J, de Graaf IAM, Olinga P. Precision -cut rat, mouse, and human intestinal slices as novel models for the early-onset of intestinal fibrosis. Physiological Reports 2015;3(4):e 12323.
41. W.M.S. Russell, Burch RL. The princ iples of hu mane e xperimental technique. England Unive rsities Federation for Animal Welfare Wheathampstead, 1959.
42. van der Logt EM J, Blokzijl T, van der Meer R, Faber KN, Dijkstra G. Westernized high -fat diet acce lerates weight loss in de xtran sulfate sodiu m-induced colitis in mice, which is further aggravated by supplementation of heme. The Journal of Nutritional Biochemistry 2013;24(6) :1159-65.
43. Wang WP, Guo X, Koo MWL, Wong BCY, La m SK, Ye YN, et al. Protective ro le of he me o xygenase -1 on trinitrobenzene sulfonic acid-induced colitis in rats, 2001.
44. Bertrand B, Ste fan L, Pirrotta M, Monchaud D, Bodio E, Richard P, et a l. Caffeine-Based Gold(I) N-Heterocyclic Ca rbenes as Possible Anticancer Agents: Synthesis and Biologica l Properties. Inorganic Chemistry 2014;53(4):2296-303.
45. De Kanter R, Monshouwer M, Draa isma A L, De Jager MH, De Graaf IAM, Proost JH, et al. Predict ion of whole-body metabolic clea rance of drugs through the combined use of slices from rat liver, lung, kidney, s mall intestine and colon. Xenobiotica 2004;34(3):229-41.
49. Li M, Si LQ, Pan HP, Rabba AK, Yan F, Qiu J, et a l. Excip ients enhance intestinal absorption of ganciclovir by P-gp inhibit ion: Assessed in vit ro by everted gut sac and in situ by improved intestinal perfusion.
International Journal of Pharmaceutics 2011 Jan;403(1-2):37-45.
50. Parsa A, Saadati R, Abbasian Z, Azad Arama ki S, Dadashzadeh S. Enhanced Permeability of Etoposide across Everted Sacs of Rat Sma ll Intestine by Vita min E-TPGS. Iranian Journal of Pharmaceutica l Research :
IJPR 2013 Winter;12(Suppl):37-46.
51. van de Kerkhof EG, de Graa f IAM, de Jager M H, Groothuis GMM. Induction of phase I and II d rug metabolism in rat small intestine and colon in vitro. Drug Metabolism and Disposition 2007;35(6):898-907.
52. van de Kerkhof EG, de Graaf IAM, de Jager MH, Me ijer DKF, Groothuis GMM. Characterization of rat small intestinal and colon precision-cut slices as an in vitro system for drug metabolis m and induction studies.
Drug Metabolism and Disposition 2005;33(11):1613-20.
53. Roskott AM, Nieuwenhuijs VB, Leuvenink HG, Dijkstra G, Ottens P, de Jager MH, et al. Reduced ischemia -reo xygenation injury in rat intestine after lu minal preservation with a ta ilored solution. Transplantation 2010;90(6):622-9.
54. Li M, de Graaf IAM, de Jager MH, Groothuis GMM. Rat p rec ision-cut intestinal slices to study P-gp activity and the potency of its inhibitors ex vivo. Toxicology in Vitro 2015;29(5):1070-78.
55. Possidente M, Dragoni S, Franco G, Gori M, Berte lli E, Teodori E, et al. Rat intestinal precision -cut slices as an in v itro mode l to study xenobiotic interaction with t ransporters. European Journal of Pharmaceutics and Biopharmaceutics 2011;79(2):343-48.
56. Khan AA, Chow ECY, Porte RJ, Pang KS, Groothuis GMM . Expression and regulation of the bile acid transporter, OSTα-OSTβ in rat and human intestine and liver. Biopharmaceutics & Drug Disposition 2009;30(5):241-58.
57. Khan AA, Chow ECY, van Loenen-Weemaes A-mMA, Porte RJ, Pang KS, Groothuis GMM. Co mparison of effects of VDR versus PXR, FXR and GR ligands on the regula tion of CYP3A isozymes in rat and human intestine and liver. European Journal of Pharmaceutical Sciences 2009;37(2):115 -25.
58. Khan AA, Dragt BS, Porte RJ, Groothuis GMM. Regulation of VDR e xp ression in rat and human intestine and liver – Consequences for CYP3A expression. Toxicology in Vitro 2010;24(3):822-29.
59. Khan AA, Chow ECY, Porte RJ, Pang KS, Groothuis GMM. The role of lithocholic acid in the regulation of bile ac id deto xication, synthesis, and transport proteins in rat and hu man intestine and liver slices. To xicology in Vitro 2011;25(1):80-90.
60. Niu X. Prec ision-cut intestinal slices as an ex vivo model to study NSAID-induced intestinal toxic ity (chapter 6). Groningen: University of Groningen; 2014.
61. Takano M, Yu moto R, Muraka mi T. Expression and function of efflu x drug transporters in the intestine.
Pharmacology & Therapeutics 2006;109(1–2):137-61.
62. MacLean C, Moenning U, Re ichel A, Fricke r G. Closing the Gaps: A full scan of the intestinal exp ression of P-g lycoprotein, breast cancer resistance protein, and mult idrug resistance-associated protein 2 in ma le and female rats. Drug Metabolism and Disposition 2008;36(7):1249 -54.
63. Sekine S, Ito K, Sae ki J, Ho rie T. Interaction of M rp2 with radixin causes reversible canalicula r M rp2 localization induced by intracellula r redo x status. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 2011;1812(11):1427-34.
64. Knutson T, Fridblo m P, Ahlström H, Magnusson A, Tannergren C, Lennernäs H. Increased Understanding of Intestinal Drug Pe rmeability Determined by the LOC-I-GUT Approach Using Multislice Co mputed Tomography. Molecular Pharmaceutics 2009;6(1):2-10.
65. Fisher M B, Lab issiere G. The role of the intestine in drug metabolis m and pharmacokinetics: an industry perspective. Current drug metabolism 2007 Oct;8(7):694-9.
66. Laffont CM, Toutain P-L, Alv inerie M , Bousquet-Mélou A. Intestinal secretion is a major route for parent ivermectin elimination in the rat. Drug Metabolism and Disposition 2002 June 1, 2002;30(6):626-30.
67. Chosidow O, Delch ier J-C, Chaumette M-T, Wechsler J, Wolkenstein P, Bourgault I, et al. Intestinal
7
involvement in drug-induced toxic epidermal necrolysis. The Lancet 1991;337(8746):928.
68. Niu X, de Graaf IAM, Groothuis GMM. Evaluation of the intestinal to xicity and transport of xenobiotics utilizing precision-cut slices. Xenobiotica 2013;43(1):73-83.
69. Li M, Vokral I, Evers B, de Graa f IAM, de Jager MH, Groothuis GMM. Rat and human prec ision -cut intestinal slices as ex vivo models to study the bile acid uptake by the apical sodium-dependent bile acid transporter. to be submitted.
70. Li M, de Graaf IAM, de Jager MH, Groothuis GMM. P-gp Activ ity and Inhib ition in the Different Regions of Human Intestine Ex Vivo. to be submitted.
71. Dro zdzik M, Gröer C, Penski J, Lapczuk J, Ostrowski M, La i Y, et al. Protein Abundance of Clinica lly Re levant Multidrug Transporters along the Entire Length of the Human Intestine. Molecular Pharmaceutics 2014;11(10):3547-55.
72. Sugimoto H, Hirabayashi H, Kimura Y, Furuta A, A mano N, Moriwaki T. Quantitative investigation of the impact of P-glycoprotein inhibit ion on drug transport across blood -brain barrie r in rats. Drug Metabolis m and Disposition 2011;39(1):8-14.
73. Li M, de Graaf IAM, Siissalo S, de Jager MH, van Da m A, Groothuis GMM. The Consequence of Drug-Drug Interactions Influencing the Interplay Between P-gp and Cyp3a: An Ex Vivo Study with Rat Precision-Cut Intestinal Slices. submitted.
74. Li M, de Graaf I, de Jager M, Groothuis G. Hu man PCIS as e x vivo model to study the in terplay between P-gp and CYP3A4 in the intestine. ISSX Online Abstracts 2014;Supplement 9(2).
75. Niu X, de Graa f IAM, van der Bij HA, Groothuis GMM. Prec ision cut intestinal slices are an appropriate ex vivo model to study NSAID-induced intestinal toxicity in rats. Toxicology in Vitro 2014;28(7):1296-305.
76. Niu X, de Graaf IAM, van de Vegte D, Langelaar-Ma kkin je M, Se kine S, Groothuis GMM. Consequences of Mrp2 deficiency for diclofenac toxicity in the rat intestine ex vivo. Toxicology in Vitro 2015;29(1) :168-75.
77. Niu X, de Graaf IM, Langelaar-Makkin je M, Horvatovich P, Groothuis GM . Dic lofenac toxic ity in human intestine ex vivo is not related to the formation of intestinal metabolites. Arch Toxicol 2015;89(1):107-19.
77. Niu X, de Graaf IM, Langelaar-Makkin je M, Horvatovich P, Groothuis GM . Dic lofenac toxic ity in human intestine ex vivo is not related to the formation of intestinal metabolites. Arch Toxicol 2015;89(1):107-19.