European Journal of Pharmaceutical Sciences 24 (2005) 539–543 Solubilization of poorly soluble lichen metabolites for biological testing on cell lines Th´ ord´ ıs Kristmundsd ´ ottir a,∗ , Elsa J ´ onsd´ ottir a , Helga M. ¨ Ogmundsd´ ottir b,c , Krist´ ın Ing ´ olfsd´ ottir a a Faculty of Pharmacy, University of Iceland, Hagi, Hofsvallagata 53, Reykjavik IS-107, Iceland b Faculty of Medicine, University of Iceland, Reykjavik, Iceland c Molecular and Cell Biology Research Laboratory, Icelandic Cancer Society, Reykjavik, Iceland Received 8 July 2004; received in revised form 11 January 2005; accepted 14 January 2005 Abstract The depside atranorin and depsidone fumarprotocetraric acid, isolated from the lichens Stereocaulon alpinum and Cetraria islandica, respectively, were chosen as prototypes for poorly soluble natural compounds in an effort to facilitate testing in pharmacological models. Solubilizing agents previously identified as being non-toxic towards a malignant leukemic (K-562) cell line and suitable for testing of anti- proliferative activity of the dibenzofuran lichen metabolite (+)-usnic acid were used in solubilization studies of the depside and depsidone. Cyclodextrin derivatives were found to be most suitable for solubilizing the lichen compounds, the greatest rise in solubility being witnessed for fumarprotocetraric acid, increasing almost 300-fold from 0.03 mg/ml in water to 8.98 mg/ml in 10% 2-hydroxypropyl--cyclodextrin (HPCD). Subsequently, the lichen compounds, including (+)-usnic acid, were solubilized in 10% HPCD and tested for effects on three malignant human cell lines; T-47D (breast), Panc-1 (pancreas) and PC-3 (prostate) in a standard proliferation assay. Atranorin and fumarpro- tocetraric acid did not exhibit anti-proliferative effects but usnic acid was active against all test cell lines with EC 50 values of 4.3–8.2 g/ml. The non-toxic solubilizing agents used in this study could prove useful for pharmacological testing of other poorly soluble natural products. © 2005 Elsevier B.V. All rights reserved. Keywords: Lichen metabolites; Solubility; Cell lines; Fumarprotocetraric acid; Atranorin; Usnic acid 1. Introduction Some secondary metabolites found in Icelandic lichens have shown promising anti-proliferative results in in vitro tests on malignant human cell lines ( ¨ Ogmundsd´ ottir et al., 1998; Haraldsd ´ ottir et al., 2004). Further testing of other can- didates of the same origin has however often been hampered by the poor solubility that many of these metabolites show in non-toxic solvents. Numerous ways are possible when try- ing to increase the solubility of poorly soluble substances, for example the use of co-solvents, surfacants and complex forming agents (Tinwalla et al., 1993; Jonkman-de Vries et al., 1996; Li et al., 1999a, b). These methods must however ∗ Corresponding author. Tel.: +354 525 4370; fax: +354 525 4071. E-mail address: thordisk@hi.is (T. Kristmundsd´ ottir). produce solvent systems that are non-toxic for the cells in culture. In a previous study the lichen metabolite (+)-usnic acid (Fig. 1), a dibenzofuran derivative, was used as a pro- totype for a water-insoluble natural product with the aim to find a solvent that was both capable of solubilizing usnic acid and was free of direct activity against a test cell line. The di- rect effects of various solvents and complexants were tested on the human leukemia cell line K-562 in a standard prolif- eration assay. Most of the compounds proved toxic with the exception of the solvents propylene glycol and polyethylene glycol 400 (PEG 400) and the complexant 2-hydroxypropyl- -cyclodextrin (HPCD). Anti-proliferative activity of usnic acid could be demonstrated with an EC 50 of 4.7 g/ml us- ing PEG 400 and 2-hydroxypropyl--cyclodextrin but only the latter gave satisfactory solubility. 2-Hydroxypropyl-- cyclodextrin was thus identified as a solubilizing agent that 0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.01.011