Planta Med 2016; 82(13): 1192-1201
DOI: 10.1055/s-0042-110323
Pharmacokinetic Investigations
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Caco-2 Permeability Studies and In Vitro hERG Liability Assessment of Tryptanthrin and Indolinone

Evelyn A. Jähne
1   Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
,
Daniela E. Eigenmann
1   Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
,
Fahimeh Moradi-Afrapoli
1   Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
,
Sheela Verjee
2   Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland
,
Veronika Butterweck
2   Institute for Pharma Technology, School of Life Sciences, University of Applied Sciences Northwestern Switzerland, Muttenz, Switzerland
,
Simon Hebeisen
3   BʼSYS GmbH, The Ionchannel Company, Witterswil, Switzerland
,
Timm Hettich
4   Institute for Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences, Northwestern Switzerland, Muttenz, Switzerland
,
Götz Schlotterbeck
4   Institute for Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences, Northwestern Switzerland, Muttenz, Switzerland
,
Martin Smieško
5   Molecular Modeling, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
,
Matthias Hamburger
1   Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
,
Mouhssin Oufir
1   Pharmaceutical Biology, Department of Pharmaceutical Sciences, University of Basel, Basel, Switzerland
› Author Affiliations
Further Information

Publication History

received 23 March 2016
revised 27 May 2016

accepted 07 June 2016

Publication Date:
15 July 2016 (online)

Abstract

Tryptanthrin and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolinone (indolinone) were recently isolated from Isatis tinctoria as potent anti-inflammatory and antiallergic alkaloids, and shown to inhibit COX-2, 5-LOX catalyzed leukotriene synthesis, and mast cell degranulation at low µM to nM concentrations. To assess their suitability for oral administration, we screened the compounds in an in vitro intestinal permeability assay using human colonic adenocarcinoma cells. For exact quantification of the compounds, validated UPLC-MS/MS methods were used. Tryptanthrin displayed high permeability (apparent permeability coefficient > 32.0 × 10−6 cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and unchanged apparent permeability coefficient values in the presence of the P-glycoprotein inhibitor verapamil (50 µM) indicated that tryptanthrin was not involved in P-glycoprotein interactions. For indolinone, a low recovery was found in the human colon adenocarcinoma cell assay. High-resolution mass spectrometry pointed to extensive phase II metabolism of indolinone (sulfation and glucuronidation). Possible cardiotoxic liability of the compounds was assessed in vitro by measurement of an inhibitory effect on human ether-a-go-go-related gene tail currents in stably transfected HEK 293 cells using the patch clamp technique. Low human ether-a-go-go-related gene inhibition was found for tryptanthrin (IC50 > 10 µM) and indolinone (IC50 of 24.96 µM). The analysis of compounds using various in silico methods confirmed favorable pharmacokinetic properties, as well as a slight inhibition of the human ether-a-go-go-related gene potassium channel at micromolar concentrations.

Supporting Information

 
  • References

  • 1 Hamburger M. Isatis tinctoria–From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical. Phytochem Rev 2002; 1: 333-344
  • 2 Danz H, Stoyanova S, Thomet OA, Simon HU, Dannhardt G, Ulbrich H, Hamburger M. Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis. Planta Med 2002; 68: 875-880
  • 3 Pergola C, Jazzar B, Rossi A, Northoff H, Hamburger M, Sautebin L, Werz O. On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo . Br J Pharmacol 2012; 165: 765-776
  • 4 Recio MC, Cerda-Nicolas M, Potterat O, Hamburger M, Rios L. Anti-inflammatory and antiallergic activity in vivo of lipophilic Isatis tinctoria extracts and tryptanthrin. Planta Med 2006; 72: 539-546
  • 5 Ishihara T, Kohno K, Ushio S, Iwaki K, Ikeda M, Kurimoto M. Tryptanthrin inhibits nitric oxide and prostaglandin E2 synthesis by murine macrophages. Eur J Pharmacol 2000; 407: 197-204
  • 6 Kiefer S, Mertz AC, Koryakina A, Hamburger M, Küenzi P. (E,Z)-3-(3′,5′-Dimethoxy-4′-hydroxy-benzylidene)-2-indolinone blocks mast cell degranulation. Eur J Pharm Sci 2010; 40: 143-147
  • 7 Oufir M, Sampath C, Butterweck V, Hamburger M. Development and full validation of an UPLC-MS/MS method for the determination of an anti-allergic indolinone derivative in rat plasma, and application to a preliminary pharmacokinetic study. J Chromatogr B 2012; 902: 27-34
  • 8 Jähne EA, Eigenmann DE, Culot M, Cecchelli R, Walter FR, Deli MA, Tremmel R, Fricker G, Smiesko M, Hamburger M, Oufir M. Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models. J Pharm Biomed Anal 2014; 98: 235-246
  • 9 Jähne EA, Eigenmann DE, Sampath C, Butterweck V, Culot M, Cecchelli R, Gosselet F, Walter FR, Deli MA, Smieško M, Hamburger M, Oufir M. Pharmacokinetics and in vitro blood-brain barrier screening of the plant-derived alkaloid tryptanthrin. Planta Med 2016;
  • 10 Hubatsch I, Ragnarsson EG, Artursson P. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nat Protoc 2007; 2: 2111-2119
  • 11 Laverty HG, Benson C, Cartwright EJ, Cross MJ, Garland C, Hammond T, Holloway C, McMahon N, Milligan J, Park BK, Primohamed M, Pollard C, Radford J, Roome N, Sager P, Singh S, Suter T, Suter W, Trafford A, Volders PGA, Wallis R, Weaver R, York M, Valentin JP. How can we improve our understanding of cardiovascular safety liabilities to develop safer medicines?. Br J Pharmacol 2011; 163: 675-693
  • 12 FDA. Guidance for industry: bioanalytical method validation. US Department of Health and Human Services, Food and Drug Administration (CDER and CVM), May 2001. Available at. http://www.fda.gov/downloads/Drugs/Guidances/ucm070107.pdf Accessed February 15, 2016
  • 13 European Medicines Agency E. Guideline on bioanalytical method validation, EMEA/CHMP/EWP/192217/2009, Committee for Medicinal Products for Human Use (CHMP), London, 21 July 2011. Available at. http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf Accessed February 15, 2016
  • 14 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: 71-76
  • 15 Zhu X, Zhang X, Ma G, Yan J, Wang H, Yang Q. Transport characteristics of tryptanthrin and its inhibitory effect on P-gp and MRP2 in Caco-2 cells. J Pharm Pharm Sci 2011; 14: 325-335
  • 16 Hayeshi R, Hilgendorf C, Artursson P, Augustijns P, Brodin B, Dehertogh P, Fisher K, Fossati L, Hovenkamp E, Korjamo T, Masungi C, Maubon N, Mols R, Müllertz A, Mönkkönen J, OʼDriscoll C, Oppers-Tiemissen HM, Ragnarsson EGE, Rooseboom M, Ungell AL. Comparison of drug transporter gene expression and functionality in Caco-2 cells from 10 different laboratories. Eur J Pharm Sci 2008; 35: 383-396
  • 17 Broeders JJ, Van Eijkeren JC, Blaauboer BJ, Hermens JL. Transport of chlorpromazine in the Caco-2 cell permeability assay: a kinetic study. Chem Res Toxicol 2012; 25: 1442-1451
  • 18 Heikkinen AT, Mönkkönen J, Korjamo T. Kinetics of cellular retention during Caco-2 permeation experiments: role of lysosomal sequestration and impact on permeability estimates. J Pharmacol Exp Ther 2009; 328: 882-892
  • 19 Palmgren JJ, Monkkonen J, Korjamo T, Hassinen A, Auriola S. Drug adsorption to plastic containers and retention of drugs in cultured cells under in vitro conditions. Eur J Pharm Biopharm 2006; 64: 369-378
  • 20 Engman HA, Lennernas H, Taipalensuu J, Otter C, Leidvik B, Artursson P. CYP3A4, CYP3A5, and MDR1 in human small and large intestinal cell lines suitable for drug transport studies. J Pharm Sci 2001; 90: 1736-1751
  • 21 Korjamo T, Mönkkönen J, Uusitalo J, Turpeinen M, Pelkonen O, Honkakoski P. Metabolic and efflux properties of Caco-2 cells stably transfected with nuclear receptors. Pharm Res 2006; 23: 1991-2001
  • 22 Meunier V, Bourrie M, Berger Y, Fabre G. The human intestinal epithelial cell line Caco-2; pharmacological and pharmacokinetic applications. Cell Biol Toxicol 1995; 11: 187-194
  • 23 Chen J, Lin H, Hu M. Metabolism of flavonoids via enteric recycling: role of intestinal disposition. J Pharmacol Exp Ther 2003; 304: 1228-1235
  • 24 Usta M, Wortelboer HM, Vervoort J, Boersma MG, Rietjens IM, Van Bladeren PJ, Cnubben NH. Human glutathione S-transferase-mediated glutathione conjugation of curcumin and efflux of these conjugates in Caco-2 cells. Chem Res Toxicol 2007; 20: 1895-1902
  • 25 Liu W, Feng Q, Li Y, Ye L, Hu M, Liu Z. Coupling of UDP-glucuronosyltransferases and multidrug resistance-associated proteins is responsible for the intestinal disposition and poor bioavailability of emodin. Toxicol Appl Pharmacol 2012; 265: 316-324
  • 26 Jancova P, Anzenbacher P, Anzenbacherova E. Phase II drug metabolizing enzymes. Biomed Pap Med Fac Univ Palacký Olomouc Czechoslov 2010; 154: 103-116
  • 27 Tukey RH, Strassburg CP. Human UDP-glucuronosyltransferases: metabolism, expression, and disease. Annu Rev Pharmacol Toxicol 2000; 40: 581-616
  • 28 Rüster GU, Hoffmann B, Hamburger M. Inhibitory activity of indolin-2-one derivatives on compound 48/80-induced histamine release from mast cells. Pharmazie 2004; 59: 236-237
  • 29 Lee JK, Abe K, Bridges AS, Patel NJ, Raub TJ, Pollack GM, Brouwer KL. Sex-dependent disposition of acetaminophen sulfate and glucuronide in the in situ perfused mouse liver. Drug Metab Dispos 2009; 37: 1916-1921
  • 30 Jiang W, Xu B, Wu B, Yu R, Hu M. UDP-glucuronosyltransferase (UGT) 1A9-overexpressing HeLa cells is an appropriate tool to delineate the kinetic interplay between breast cancer resistance protein (BRCP) and UGT and to rapidly identify the glucuronide substrates of BCRP. Drug Metab Dispos 2012; 40: 336-345
  • 31 Shawahna R, Uchida Y, Declèves X, Ohtsuki S, Yousif S, Dauchy S, Jacob A, Chassoux F, Daumas-Duport F, Couraud PO, Terasaki T, Scherrmann JM. Transcriptomic and quantitative proteomic analysis of transporters and drug metabolizing enzymes in freshly isolated human brain microvessels. Mol Pharm 2011; 8: 1332-1341
  • 32 Shawahna R, Declèves X, Scherrmann JM. Hurdles with using in vitro models to predict human blood-brain barrier drug permeability: a special focus on transporters and metabolizing enzymes. Curr Drug Metab 2013; 14: 120-136
  • 33 Zhou Z, Gong Q, Ye B, Fan Z, Makielski JC, Robertson GA, January CT. Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature. Biophys J 1998; 74: 230-241
  • 34 Redfern WS, Carlsson L, Davis AS, Lynch WG, MacKenzie I, Palethorpe S, Siegl PKS, Strang I, Sullivan AT, Wallis R. Relationships between preclinical cardiac electrophysiology, clinical QT interval prolongation and torsade de pointes for a broad range of drugs: evidence for a provisional safety margin in drug development. Cardiovasc Res 2003; 58: 32-45
  • 35 Food and Drug Administration. Guidance for industry: S7B nonclinical evaluation of the potential for delayed ventricular repolarization (QT interval prolongation) by human pharmaceuticals, 2005. Available at. http://www.fda.gov/ Accessed February 15, 2016
  • 36 Schramm A, Saxena P, Chlebek J, Cahlíková L, Baburin I, Hering S, Hamburger M. Natural products as potential human ether-a-go-go-related gene channel inhibitors-screening of plant-derived alkaloids. Planta Med 2014; 80: 740-746
  • 37 Witchel HJ, Milnes JT, Mitcheson JS, Hancox JC. Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K+ channels expressed in mammalian cell lines and Xenopus oocytes. J Pharmacol Toxicol Methods 2002; 48: 65-80
  • 38 Po SS, Wang DW, Yang ICH, Johnson JP, Nie L, Bennett PB. Modulation of HERG potassium channels by extracellular magnesium and quinidine. J Cardiovasc Pharmacol 1999; 33: 181-185
  • 39 Sun L, Tran N, Tang F, App H, Hirth P, McMahon G, Tang C. Synthesis and biological evaluations of 3-substituted indolin-2-ones: a novel class of tyrosine kinase inhibitors that exhibit selectivity toward particular receptor tyrosine kinases. J Med Chem 1998; 41: 2588-2603
  • 40 Friedländer P, Roschdestwenski N. Über ein Oxidationsprodukt des Indigoblaus. Ber Dtsch Chem Ges 1915; 48: 1841-1847
  • 41 Mohn T, Potterat O, Hamburger M. Quantification of active principles and pigments in leaf extracts of Isatis tinctoria by HPLC/UV/MS. Planta Med 2007; 73: 151-156
  • 42 Gerlier D, Thomasset N. Use of MTT colorimetric assay to measure cell activation. J Immunol Methods 1986; 94: 57-63
  • 43 Tofighi Z, Asgharian P, Goodarzi S, Hadjiakhoondi A, Ostad SN, Yassa N. Potent cytotoxic flavonoids from Iranian Securigera securidaca . Med Chem Res 2014; 23: 1718-1724
  • 44 Youdim KA, Avdeef A, Abbott NJ. In vitro trans-monolayer permeability calculations: often forgotten assumptions. Drug Discov Today 2003; 8: 997-1003
  • 45 Verjee S, Brügger D, Abdel-Aziz H, Butterweck V. Permeation characteristics of hypericin across Caco-2 monolayers in the absence or presence of quercitrin – a mass balance study. Planta Med 2015; 81: 1111-1120
  • 46 Hebeisen S, Pires N, Loureiro AI, Bonifácio MJ, Palma N, Whyment A, Spanswick D, Soares-da-Silva P. Eslicarbazepine and the enhancement of slow inactivation of voltage-gated sodium channels: a comparison with carbamazepine, oxcarbazepine and lacosamide. Neuropharmacology 2015; 89: 122-135
  • 47 Vedani A, Dobler M, Hu Z, Smieško M. OpenVirtualToxLab – a platform for generating and exchanging in silico toxicity data. Toxicol Lett 2015; 232: 519-532