Permeation Characteristics of Hypericin across Caco-2 Monolayers in the Absence or Presence of Quercitrin – A Mass Balance Study^[*]

Abstract

Hypericin is a natural polycyclic quinone found in Hypericum perforatum. Although hypericin reportedly has numerous pharmacological activities, only a limited number of studies have been performed on the absorption and transport characteristics of this compound, presumably because hypericin is a highly lipophilic compound that is poorly soluble in a physiological medium. The major aim of this study was to get a detailed understanding of the exposure and fate of hypericin in the Caco-2 cell system under different experimental conditions. The permeation characteristics of hypericin (5 µM) in the absence or presence of the model flavonoid quercitrin (20 µM) were studied in the absorptive direction, without or with the addition of 10 % FBS to the transport buffer apically. Following the application of hypericin to the apical side of the monolayer, only negligible amounts of the compound were found in the basolateral compartment when the experiment was performed with a transport buffer. The amount of hypericin in the basolateral compartment increased in the presence of quercitrin (from 0 to 4 %). The majority of hypericin was found after cell extraction (44 % in the absence and 64 % in the presence of quercitrin). When 10 % FBS was added to the transport buffer in the apical compartment to improve the solubility of hypericin in the aqueous solution, around 68 % of hypericin was bound to the serum proteins. Under these experimental conditions, the amount of hypericin in the cells/cell membrane was only 13 % in the absence and 18 % in the presence of quercitrin. The low recovery and significant amounts of hypericin found after cell extraction and bound to the surface of the culture dish made a correct estimation of permeability constants impossible. Fluorescence microscopy and imaging analysis revealed that hypericin is mainly accumulated in the cell membrane. The precise mechanism through which hypericin might overcome the hydrophobic barrier of cell membranes remains to be elucidated. However, our experiments demonstrated that regardless of the experimental conditions, the permeation characteristics of hypericin improved in the presence of the model flavonoid quercitrin.

Key words

hypericin - quercitrin - Caco-2 - uptake - absorption

^* Dedicated to Professor Dr. Dr. h. c. mult. Adolf Nahrstedt on the occasion of his 75th birthday.

• References

• 1 Karioti A, Bilia AR. Hypericins as potential leads for new therapeutics. Int J Mol Sci 2010; 11: 562-594
• 2 Falk H. From the Photosensitizer Hypericin to the Photoreceptor Stentorin – The Chemistry of Phenanthroperylene Quinones. Angew Chem Int Ed Engl 1999; 38: 3116-3136
• 3 Chaloupka R, Obsil T, Plasek J, Sureau F. The effect of hypericin and hypocrellin-A on lipid membranes and membrane potential of 3T3 fibroblasts. Biochim Biophys Acta 1999; 1418: 39-47
• 4 Miskovsky P, Sureau F, Chinsky L, Turpin PY. Subcellular distribution of hypericin in human cancer cells. Photochem Photobiol 1995; 62: 546-549
• 5 Siboni G, Weitman H, Freeman D, Mazur Y, Malik Z, Ehrenberg B. The correlation between hydrophilicity of hypericins and helianthrone: internalization mechanisms, subcellular distribution and photodynamic action in colon carcinoma cells. Photochem Photobiol Sci 2002; 1: 483-491
• 6 Ho YF, Wu MH, Cheng BH, Chen YW, Shih MC. Lipid-mediated preferential localization of hypericin in lipid membranes. Biochim Biophys Acta 2009; 1788: 1287-1295
• 7 Ali SM, Olivo M. Bio-distribution and subcellular localization of hypericin and its role in PDT induced apoptosis in cancer cells. Int J Oncol 2002; 21: 531-540
• 8 Theodossiou TA, Noronha-Dutra A, Hothersall JS. Mitochondria are a primary target of hypericin phototoxicity: synergy of intracellular calcium mobilisation in cell killing. Int J Biochem Cell Biol 2006; 38: 1946-1956
• 9 Vandenbogaerde AL, Cuveele JF, Proot P, Himpens BE, Merlevede WJ, deWitte PA. Differential cytotoxic effects induced after photosensitization by hypericin. J Photochem Photobiol B 1997; 38: 136-142
• 10 Kamuhabwa AR, Augustijns P, de Witte PA. In vitro transport and uptake of protohypericin and hypericin in the Caco-2 model. Int J Pharm 1999; 188: 81-86
• 11 Sattler S, Schaefer U, Schneider W, Hoelzl J, Lehr CM. Binding, uptake, and transport of hypericin by Caco-2 cell monolayers. J Pharm Sci 1997; 86: 1120-1126
• 12 Butterweck V, Petereit F, Winterhoff H, Nahrstedt A. Solubilized hypericin and pseudohypericin from Hypericum perforatum exert antidepressant activity in the forced swimming test. Planta Med 1998; 64: 291-294
• 13 Juergenliemk G, Nahrstedt A. Dissolution, solubility and cooperativity of phenolic compounds from Hypericum perforatum L. in aqueous systems. Pharmazie 2003; 58: 200-203
• 14 Butterweck V, Lieflaender-Wulf U, Winterhoff H, Nahrstedt A. Plasma levels of hypericin in presence of procyanidin B2 and hyperoside: a pharmacokinetic study in rats. Planta Med 2003; 69: 189-192
• 15 Sieger R, Nahrstedt A. Effects of phenolic compounds from Hypericum perforatum L. on the solubility and permeation of Hypericin in vitro . Planta Med 2006; 72: 1073 (P_310)
• 16 Artursson P, Palm K, Luthman K. Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv Drug Deliv Rev 1996; 22: 67-84
• 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 Brügger D. Die Veränderung der Wasserlöslichkeit von Hypericin beeinflusst durch verschiedene Flavonoide aus dem Johanniskraut (Masterthesis). Basel: University of Basel; 2013
• 19 Lipinski CA, Lombardo F, Dominy BW, Feeney PJ. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv Drug Deliv Rev 1997; 23: 3-25
• 20 Bruggisser R, von Daeniken K, Jundt G, Schaffner W, Tullberg-Reinert H. Interference of plant extracts, phytoestrogens and antioxidants with the MTT tetrazolium assay. Planta Med 2002; 68: 445-448
• 21 Peng L, Wang B, Ren P. Reduction of MTT by flavonoids in the absence of cells. Colloids Surf B Biointerfaces 2005; 45: 108-111
• 22 Shoemaker M, Cohen I, Campbell M. Reduction of MTT by aqueous herbal extracts in the absence of cells. J Ethnopharmacol 2004; 93: 381-384
• 23 Wang P, Henning SM, Heber D. Limitations of MTT and MTS-based assays for measurement of antiproliferative activity of green tea polyphenols. PLoS One 2010; 5: e10202
• 24 Wisman KN, Perkins AA, Jeffers MD, Hagerman AE. Accurate assessment of the bioactivities of redox-active polyphenolics in cell culture. J Agric Food Chem 2008; 56: 7831-7837
• 25 Maioli E, Torricelli C, Fortino V, Carlucci F, Tommassini V, Pacini A. Critical appraisal of the MTT assay in the presence of rottlerin and uncouplers. Biol Proced Online 2009; 11: 227-240
• 26 Sugawara K, Fujikawa M, Yoshida M. Screening of protein kinase inhibitors and knockdown experiments identified four kinases that affect mitochondrial ATP synthesis activity. Febs Lett 2013; 587: 3843-3847
• 27 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
• 28 Heikkinen AT, Monkkonen 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
• 29 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
• 30 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
• 31 Korjamo T, Monkkonen 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
• 32 Kocisova E, Chinsky L, Miskovsky P. Sequence specific interaction of the antiretrovirally active drug hypericin with 5′ATGGCAGGATAT3′ oligonucleotide: a resonance Raman spectroscopy study. J Biomol Struct Dyn 1998; 15: 1147-1154
• 33 English DS, Doyle RT, Petrich JW, Haydon PG. Subcellular distributions and excited-state processes of hypericin in neurons. Photochem Photobiol 1999; 69: 301-305
• 34 Ho YF, Wu MH, Cheng BH, Chen YW, Shih MC. Lipid-mediated preferential localization of hypericin in lipid membranes. Biochim Biophys Acta 2009; 1788: 1287-1295
• 35 Strejckova A, Stanicova J, Jancura D, Miskovsky P, Bano G. Spatial orientation and electric-field-driven transport of hypericin inside of bilayer lipid membranes. J Phys Chem B 2013; 117: 1280-1286
• 36 Uzdensky AB, Ma LW, Iani V, Hjortland GO, Steen HB, Moan J. Intracellular localisation of hypericin in human glioblastoma and carcinoma cell lines. Lasers Med Sci 2001; 16: 276-283
• 37 Tarahovsky YS, Kim YA, Yagolnik EA, Muzafarov EN. Flavonoid-membrane interactions: involvement of flavonoid-metal complexes in raft signaling. Biochim Biophys Acta 2014; 1838: 1235-1246
• 38 Schinazi RF, Chu CK, Babu JR, Oswald BJ, Saalmann V, Cannon DL, Eriksson BF, Nasr M. Anthraquinones as a new class of antiviral agents against human immunodeficiency virus. Antiviral Res 1990; 13: 265-272
• 39 Kapitza SB, Michel BR, van Hoogevest P, Leigh MLS, Imanidis G. Absorption of poorly water soluble drugs subject to apical efflux using phospholipids as solubilizers in the Caco-2 cell model. Eur J Pharm Biopharm 2007; 66: 146-158