УДК 577.114.7:577.352.2:616-006
LIPOPHILIC PRODRUGS OF A TRIAZOLE-CONTAINING COLCHICINE ANALOGUE IN LIPOSOMES: BIOLOGICAL EFFECTS ON HUMAN TUMOR CELLS
© 2013 г. N. R. Kuznetsova", E. V. Svirshchevskaya", N. S. Sitnikov4, L. Abodoc, H. Sutoriusc, J. Zapked, J. Velder®, P. Thomopoulou®, H. Oschkinaf, A. Prokopc, H.-G. Schmalz®, A. Yu. Fedorov4, E. L. Vodovozova"- #
aShemyakin—Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, Moscow, 117997Russia bDepartment of Organic Chemistry, Chemical Faculty, N.I. Lobachevsky Nizhni Novgorod State University,
Nizhni Novgorod, Russia cDepartment of Oncology/Hematology, Childrens Hospitel Cologne, Köln, Germany dLeibniz-Institute of Molecular Pharmacology (FMP), Berlin, Germany eDepartment für Chemie, Universität zu Köln, Köln, Germany
Colchicine site binders — blockers of tubulin polymerization — are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable formulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine — yeast phosphatidylinositol — palmitic or oleic prodrug, 8 : 1 : 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.
Keywords: liposomes, colchicinoids, lipophilic prodrugs, fatty acid esters, anti-proliferative activity, proapoptotic activity
DOI: 10.7868/S0132342313050102
INTRODUCTION
Among cytotoxic anticancer drugs, antimitotic agents have generated considerable interest due to the tremendous success of taxanes and the widespread use of the Vinca alkaloids in clinical oncology [1]. A search for new substances that inhibit tubulin polymerization via interaction at the sites distinct from those of taxanes and Vinca alkaloids, is focused mostly
Abbreviations: EPR, enhanced permeability and retention; PEG, polyethylene glycol; LDH, lactate dehydrogenase; PC, phosphatidylcholine; PI, phosphatidylinositol; PBS, phosphate buffered saline; DAPI, 4',6-diamidino-2-phenylindol; EGTA, ethylene glycol tetraacetic acid; PIPES, piperazine-N,N-bis(2-ethanesulfonic acid).
# Corresponding author (phone: +7 (495) 330-66-10; fax: +7 (495) 330-66-01; e-mail: elvod@ibch.ru).
on small molecules such as those binding the colchicine site [1, 2]. Among them, combretastatin A4 [3] and allocolchicine [4] are less toxic than colchicines, yet reveal high antitubulin activity and therefore are promising agents for antitumor therapy.
An effective approach to reduce general toxicity and improve biodistribution upon systemic administration is the delivery of drugs in liposomes [5]. Accumulation of vehicles of about 100 nm in diameter in tumors results from increased permeability and disordered architecture of the neovasculature (effect of enhanced permeability and retention, EPR) [6]. Meanwhile, it is often difficult to incorporate therapeutic concentrations of medicines in the hydrophilic core of nanosized liposomes by the practically feasible remote loading technique, which demands that the
Fig. 1. Molecular structures of colchicine and allocolchicine analogues 1 and 2, respectively, in comparison with colchicine and corresponding oleic (3, 5) and palmitic (4, 6) esters.
drug molecule should be week amphiphilic base or acid [7]. As for colchicine as such, it is a neutral lipophilic molecule, which localizes mainly in the lipid-ic environment, however is prone to be expelled to the aqueous phase and to form stacked aggregates [8]. Recently, an elegant strategy to overcome leakage of colchicine from long-circulating liposomes was reported [9]: the authors developed biodegradable polyethylene glycol (PEG) conjugates of colchicine for reliable retention in the aqueous core of liposomes. An alternative approach may be anchoring colchicine deeply in the bilayer by the additional modification with lipid moiety, i.e. employing a lipophilic prodrug. Preparation of liposomes loaded with an applicable compound in the bilayer is simplified, as compared to encapsulation ofwater soluble-drug in the inner volume. A number oflipophilic prodrugs, including those ofAra-C [10], 5-fluorouridine [11], gemcitabine [12], and mitomycin C [13] applied in liposome formulations have been shown to surpass the initial drugs in pharmacokinetics and antitumor activity. Previously, we reported that li-
posomes loaded with octadecyl ester of sarcolysine (DL-melphalan) caused significant increase in survival of mice with transplanted mammary cancer as compared to the intact alkylating agent [14].
Recently, triazole-containing colchicine and allocolchicine analogues 1 and 2, respectively, along with their fatty acid esters 3—6 have been synthesized [15, 16] (Fig. 1) as potential colchicine site binders. Indeed, the lauric ester of colchicinoid 1 showed a threefold increase in antimitotic activity in the whole-cell assay as compared to colchicine. At the same time, its affinity to the colchicine-binding site of tubulin decreased, suggesting the importance of secondary effects such as transport across membrane [16]. On the other hand, the use of esters as prodrugs is known to benefit from facile enzymatic hydrolysis in the target cell, since typically esterases are present throughout different tissues of the body and are characterized by wide substrate specificity (as compared to amidases) [17]. Our previous results [16] correlate well with rather fast intracellular cleavage of the colchicinoid
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Fig. 2. Inhibition of proliferation (A) and apoptosis induction (B) in Burkitt-like lymphoma BJAB cells induced by colchicine and allocolchicine analogues 1 and 2 and lipid derivatives 3—6. A. Numbers of BJAB cells were calculated after 24-h incubation, using CASY® Cell Counter. B. Apoptosis rate was estimated after 72 h incubation by flow cytometry using propidium iodide staining. Experiments were carried out in quadruplicate. Standard deviation was within 5% (not shown).
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laurate. Palmitic and oleic esters seem to be of even more interest not only owing to their propensity for facilitated transport across cell membranes, but also due to the evident high affinity to the lipid bilayer, which should provide reliable anchoring in liposomes, as well as high loading capacity of the nanosized vehicle. The goal of this study was (1) to test the anti-proliferative properties and apoptosis-inducing potential of colchicine and allocolchicine analogues 1—6, (2) prepare liposome formulations of lipophilic prodrugs of the most potent compounds, and (3) assess biological activity of the liposomes on a panel of human tumor cell lines.
RESULTS AND DISCUSSION
First of all, compounds 1—6 were tested for antiproliferative activity using Burkitt-like lymphoma cells (BJAB tumor cells). Proliferation inhibition (IC50) was determined by cell counting after 24 h treatment with different concentrations of the agents. The IC50 values are presented in Table 1, the corresponding plots of cell viability are shown in Fig. 2A. According to these results, proliferation inhibition potency of colchicine analogue 1 is similar to that of colchicine itself. Colchicinoid esters 3 and 4 exhibited even higher activity than colchicinoid 1 (IC50 4 and 5 nM against 25 nM); this may be explained by the facilitat-
ed transfer of the amphiphilic compounds across the plasma membrane followed by fast intracellular hydrolysis and transportation to the tubulin molecule. Indeed, both compounds displayed significant inhibition of tubulin polymerization in an in vitro cell-free polymerization assay at a concentration of 40 ^M, however initial colchicinoid 1 showed almost complete inhibition under the same conditions (Fig. 3A). These data correlate well with our previous results obtained for lauric ester of colchicinoid 1 [16].
In contrast to the colchicine derivatives, allocolch-icine analogue 2 exhibited significantly lower anti-proliferative activity with a 15-fold IC50 value as compared to colchicine (Table 1 and Fig. 2A). Surprisingly, fatty acid esters 5 and 6 showed further decrease in the activity. In support of this unexpected outcome, in tu-bulin polymerization inhibition assay, compounds 5 and 6 produced no effect in cell-free medium, while compound 2 fully inhibited tubulin polymerization (Fig. 3B). Altogether, these results imply some hindrances to delivery of allocolchicinoid 2 to the target tubulin molecule in the cell and slowdown of intracellular hydrolysis of esters 5 and 6.
The potency to induce apoptosis (AC50) was determined by means of a DNA fragmentation assay [18]. Activity of all tested compounds correlated well with
Table 1. Cytotoxicity (IC50) and proapopto
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