A new nanomedicine of gemcitabine displays enhanced anticancer activity in sensitive and resistant leukemia types

Abstract

Gemcitabine is an anticancer nucleoside analogue active against various solid tumors. However, it possesses important drawbacks like a poor biological half-life and the induction of resistance. With the objective of overcoming the above drawbacks, we designed a new nanomedicine of gemcitabine and studied its anticancer efficacy against leukemia at preclinic. Gemcitabine has been covalently coupled with 1,1′,2-tris-nor-squalenic acid to obtain the new anticancer nanomedicine 4-(N)-Tris-nor-squalenoyl-gemcitabine (SQdFdC NA). The SQdFdC NA exhibited, in comparison to gemcitabine, 3.26- and 3.22-folds higher cytotoxicity respectively, in murine resistant leukemia L1210 10K cells and in human leukemia resistant cell line CEM/ARAC8C. Following intravenous treatment of murine aggressive metastatic leukemia L1210 wt bearing mice, the SQdFdC NA caused significant increase in survival time compared to gemcitabine and also led to long-term survivals, which was not the case after gemcitabine treatment. This was attributed to significantly higher deposition of SQdFdC NA in spleen and liver (P < 0.05), the major metastatic organs. In comparison to gemcitabine, SQdFdC NA displayed greater ability to induce S-phase arrest of the cancer cells followed by increased apoptotic induction. Interestingly, like gemcitabine, SQdFdC NA didn't induce appreciable differences in blood parameters even at doses higher than those used for anticancer evaluation. The preclinical data obtained in vitro and in vivo with SQdFdC NA demonstrate that this nanomedicine represents a new therapeutic system for the effective treatment of leukemia.

Introduction

Squalene (Fig. 1A) is an isoprenoid compound, precursor in the biosynthesis of cholesterol; it is widely distributed in nature. In humans, about 60% of the dietary squalene is absorbed [1]. It has been previously used as an excipient vehicle for various categories of drugs [2], [3] and may enhance the therapeutic efficacy of drugs such as cholesterol-lowering agents (e.g. Pravastatin), when co-administered [4]. Apart from its other properties such as antioxidant for skin and eye, an interesting therapeutic application of squalene is its use as an adjunct in the treatment of variety of cancers, even though the therapeutic improvement in vivo, when combined with anticancer drug was very low [5]. Squalene was also found to be able to inhibit the effect of the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate in mouse-skin carcinogenesis [6]. On the other hand, prolonged administration of squalene did not demonstrate appreciable side-effects and toxic signs in animals [7]. Very recently, we have developed the concept of “squalenoylation” involving the chemical linkage of squalene with various nucleosides analogues which surprisingly, self-organized in water as nanoassemblies of 100–300 nm, irrespective of the nucleoside analogue used [8].

In the group of anticancer nucleoside analogues, gemcitabine is a potent compound demonstrated to be effective in the treatment of a variety of solid tumors including colon, lung, pacreatic, breast, bladder and ovarian cancers [9], [10]. However, gemcitabine undergoes rapid deamination into the inactive uracil derivative, hence resulting in a short half-life. Another concern is the observed induction of resistance in preclinical models both in vitro and in vivo which would be important in future clinical situations.

Since gemcitabine is a hydrophilic compound, it requires membrane proteins called nucleoside transporters for entering the cells. The main transporter types include hENT1, hENT2, hCNT1 and hCNT3 [11]. Intracellularly, a part of gemcitabine molecules may undergo deamination into the inactive uracil derivative (dFdU) [12]. In fact, gemcitabine needs to be phosphorylated into its active form, primarily into monophosphate mainly by deoxycytidine kinase and, to a lower extent, by other intracellular kinases [13], [14]. The monophosphate is further phosphorylated into di- and triphosphate forms. The triphosphate form (dFdCTP) of gemcitabine is the only active form which inhibits DNA synthesis. However, any modification in the above sequence of events would lead to resistance. For instance, in the case of leukemia, the cellular resistance to gemcitabine due to nucleoside transporter and dCK deficiency has been well documented [11], [15], [16]. Clearly, the drug resistance is an important clinical concern in the treatment with gemcitabine.

Control over the spread of cancer cells and metastasis is obviously another challenge in chemotherapy, since it accounts for 90% of lethality in cancer patients [17]. In many clinical situations, gemcitabine like other anticancer agents is not efficient in inhibiting metastasis, because it doesn't reach the metastasized tissues in sufficient concentrations. In leukemia, the abnormal cells generally spread to the tissues such as bone marrow, spleen and liver [18].

In this view, we investigated in the present communication, the anticancer activity of the 4-(N)-Tris-nor-squalenoyl-gemcitabine nanoassemblies (SQdFdC NA) in vitro on resistant murine and human leukemia cells as well as in vivo on murine aggressive metastatic leukemia L1210 wt. Although the main indication of gemcitabine in the clinic is solid tumors, the initial preclinical evaluation of gemcitabine was done on L1210 murine leukemia [19]. However, in vivo, high doses of gemcitabine were required probably due to the deamination process and subsequently the shorter half-life, contributing to its poor activity in controlling the extensive leukemic metastasis. It was, therefore, of interest to investigate whether the squalenoyl conjugation improved or not the anticancer activity of gemcitabine on leukemia in vivo. Tissue distribution studies have been carried out using similar treatment schedule. The cell cycle parameters such as S-phase and apoptosis have also been evaluated in vivo in L1210 wt leukemia ascitic bearing mice. Finally, the blood parameters have been evaluated after treating the animals with gemcitabine or SQdFdC NA at doses higher than those used for the treatment of the L1210 wt leukemia bearing mice.