No abstract

This study investigates 1) the anticancer efficacy of a new squalenoyl prodrug of gemcitabine (SQgem) in nanoassembly form compared with gemcitabine at equitoxic doses and 2) the subacute and acute preclinical toxicity of these compounds. The toxicity studies revealed that SQgem nanoassemblies, like gemcitabine, were toxic, and they led to dose-dependent mortality after daily i.v. injections for 1 week, irrespective of the route of administration. However, a 4-to 5-day spaced dosing schedule (injections on day 0, 4, 8, and 13) was proved to be safer in terms of weight loss and hematological and other toxicity. Using this spaced dosing schedule, SQgem nanoassemblies exhibited impressive anticancer activity in mice bearing L1210 leukemia because this treatment led to 75% long-term survivors. In contrast, at equitoxic doses, neither free gemcitabine nor cytarabine led to longterm survivors and all the mice of these groups died of the disease. Further toxicity studies performed at lethal doses by blood and serum analysis and organ weight determinations revealed that the hematological toxicity was the dose-limiting toxicity in both SQgem nanoassemblies and gemcitabine, whereas probable gastrointestinal toxicity was also associated with free gemcitabine. The SQgem nanoassemblies did not display hepatotoxicity, which is one of the clinically encountered toxicities of gemcitabine. To summarize, these preclinical studies demonstrated that the toxicological profile of new squalenoyl gemcitabine nanomedicine was not distinct from that of the parent gemcitabine, whereas it was much more potent than gemcitabine at equitoxic doses and cytarabine at clinically relevant doses. These data support the candidature of SQgem for clinical trials.Gemcitabine is an anticancer nucleoside analog indicated in the clinic for the treatment of various solid tumors. Although the anticancer activity of gemcitabine against leukemia has been demonstrated in preclinical models in which the leukemia cells were introduced intraperitoneally (Hertel et al., 1990), it is not indicated in the clinic for this purpose. Furthermore, these preclinical models do not mimic the real clinical situations. In addition, gemcitabine was recently shown to be insufficiently active against leukemia in phase II clinical trials (Angiolillo et al., 2006, Wagner-Bohn et al., 2006. We have recently developed a new prodrug of gemcitabine by conjugation with squalene (squalenoylation) (Couvreur et al., 2006a). The linkage of squalene was performed at the level of the 4-amino group of gemcitabine, rendering the molecule more amphiphilic, and resulting in their spontaneous aggregation as nanoassemblies of approximately 130 nm in diameter. These squalenoyl gemcitabine (SQgem) nanoassemblies were demonstrated to prevent the deamination of gemcitabine in vitro, thus overcoming one of the main drawbacks to its use, its short biological half-life (Couvreur et al., 2006b). In addition, in vitro, the SQgem nanoassemblies were demonstrated to be more cytotoxic than gemcitabin...

In an earlier report, we demonstrated the superior anticancer efficacy of orally administered squalenoyl gemcitabine (SQdFdC) nanomedicine over its parent drug gemcitabine on rats bearing RNK-16 large granular lymphocytic (LGL) leukemia. In the present communication, we investigated the mechanisms behind this observation both at the cell and tissue level. The mechanisms were investigated by performing cytotoxicity, cell uptake, and biodistribution experiments. In the presence of cytidine deaminase, SQdFdC nanoassemblies resisted deamination and exerted significant anticancer activity in vitro against RNK-16 LGL leukemia cells, whereas the cytotoxicity of free gemcitabine decreased by approximately 83-fold, indicating its degradation due to deamination. Additionally, the SQdFdC showed considerably higher intracellular accumulation and retention compared with gemcitabine (P<0.05). Unlike gemcitabine, the cellular access to SQdFdC was not influenced by nucleoside transporters. When administered orally to rats, unlike H-gemcitabine, the H-SQdFdC absorbed slowly, but exhibited an improved pharmacokinetics and tissue distribution profile, particularly in the lymphoid organs (the major organs of metastasis). The resistance to deamination, followed by the improved pharmacokinetic and tissue distribution, and greater accumulation and retention at the level of cancer cells, are the key factors for the superiority of SQdFdC nanoassemblies over free gemcitabine against RNK-16 LGL leukemia in rats.