Elsevier

Journal of Controlled Release

Volume 100, Issue 3, 10 December 2004, Pages 331-346
Journal of Controlled Release

Preparation, characterization, cytotoxicity and pharmacokinetics of liposomes containing lipophilic gemcitabine prodrugs

https://doi.org/10.1016/j.jconrel.2004.09.001Get rights and content

Abstract

Gemcitabine is a known anticancer agent rapidly deaminated to the inactive metabolite 2′,2′-difluorodeoxyuridine; it must therefore be administered at very high dose. Many different approaches have been tried to improve the metabolic stability; we synthesized a series of increasingly lipophilic prodrugs of gemcitabine by linking the 4-amino group with valeroyl, heptanoyl, lauroyl and stearoyl linear acyl derivatives. We studied their stability at storage, in plasma and with the lysosomal intracellular enzyme cathepsins. We studied incorporation of these lipophilic prodrugs in liposomes, where their encapsulation efficiency (EE) closely depends on the length of the saturated 4-(N)-acyl chain, the phospholipids chosen and the presence of cholesterol. A maximum EE of 98% was determined for 4-(N)-stearoyl-gemcitabine incorporated in DSPC/DSPG 9:1. This formulation was correlated with the highest stability in vitro and in vivo. Cytotoxicity of gemcitabine prodrugs, free or encapsulated in liposomes, was between two- and sevenfold that of free gemcitabine. Encapsulation of long-chain lipophilic prodrugs of gemcitabine in liposomes protected the drug from degradation in plasma, assuring a long plasma half-time and intracellular release of the free drug.

Introduction

Gemcitabine (2′,2′-difluorodeoxycytidine) is a pyrimidine antimetabolite structurally and pharmacologically similar to cytarabine (1-β-d-arabinofuranosylcytosine, ara-C). It differs from ara-C only for substitution of geminal fluorines for the hydroxyl group at the 2′ position and was specifically developed to extend the activity of ara-C to non-hematological malignancies. Like ara-C, gemcitabine requires intracellular phosphorylation to the active 5′-triphosphate; this conversion is mediated by deoxycytidine kinase, the rate-limiting enzyme in activation of gemcitabine [1]. The gemcitabine action mechanism entails gemcitabine triphosphate being incorporated on the end of elongating DNA strand, followed by addition of an additional deoxynucleotide that stops DNA synthesis and inhibition of ribonucleoside reductase [2], [3], [4]. Gemcitabine is rapidly and extensively deaminated by cytidine deaminase in blood, liver, kidney and other tissues [5] to the inactive metabolite 2′,2′-difluorodeoxyuridine (dFdU), and excreted in the urine. Gemcitabine (<10%) and main metabolite dFdU accounted for 99% of the excreted dose. For this reason, gemcitabine has a very short plasma half-life (8–17 min). In order to achieve therapeutic drug levels, gemcitabine is currently administered at 1000 mg/m2 as 30-min intravenous infusion. When used in monotherapy, gemcitabine is indicated for pancreatic cancer and non-small cell lung cancer [5], but it has also demonstrated activity against many solid tumours [6], [7]. Many different approaches have been tried to improve gemcitabine metabolic stability and, consequently, its in vivo cytotoxic activity. Eli Lilly patented the synthesis of saturated and monounsaturated C18 and C20 long-chain 4-(N)-acylderivatives and 5′-esters of gemcitabine. Among these, the 5′ C20 ester and 4-amide derivatives showed better cytotoxic activity than the parent compound [8]. It has been shown that liposomes provide protection against rapid metabolic inactivation of drugs [9]. Nevertheless, low-molecular-weight water-soluble molecules, such as ara-C, a drug strictly related to gemcitabine, or 5-fluorouridine, diffuse rapidly through liposome bilayers [10], [11] thus limiting the shelf life and clinical utility of these liposomes. To overcome this problem, a large number of lipophilic prodrugs of 5-fluorouridine or ara-C have been synthesized [11], [12], [13] and encapsulated in liposomes, with high efficiency. The in vivo antitumor activity of these liposomal prodrugs against a variety of tumours is generally superior to that of the pure drug. Many alkyl derivatives of ara-C, considered less susceptible to hydrolysis than the corresponding acyl derivatives, have also been synthesised: impressive antitumor activity against various solid tumours has been shown by some alkyl derivative of ara-C, such as 4-(N)-hexadecyl (NHAC) and 4-(N)-octadecyl (NOAC) 1-β-d-arabinofuranosylcytosine [14]. NHAC has also been entrapped within liposomes, and has shown stronger antitumor activity than ara-C, probably due to a mechanism that overcomes the ara-C resistance in ara-C-resistant HL-60 cell line [15]. After incubation with human blood, NHAC rapidly transferred from vesicles to plasma proteins, erythrocytes and leucocytes; the addition of PEG-modified phospholipids in the liposomes did not significantly prevent this transfer [16]. It is probable that, like ara-C, gemcitabine also offers some resistance against being entrapped as free drug in conventional liposomes or PEG liposomes: in a recent study, gemcitabine was successfully entrapped in vesicular phospholipid gels (VPG) composed of very densely packed liposomes prepared by high-pressure homogenization [17]. Another patent describes stable incorporation of gemcitabine in liposomes obtained through electrostatic interaction with a selected negatively charged phospholipid, such as cardiolipin, within the liposomes [18].

We here present the synthesis and encapsulation in liposome of a series of 4-(N)-acyl derivatives prodrugs of gemcitabine. The technological properties of these formulations are discussed, together with a preliminary examination of their biological properties, including their antitumor activity and pharmacokinetics.

Section snippets

Chemicals

Gemcitabine was synthesized in our laboratory as described in [19]. The 1H nuclear magnetic resonance (NMR) spectra were recorded on a Bruker 300 Ultrashild (Karlsruhe, Germany) in CD3COCD3 or pyridine-d5 solutions at room temperature, with SiMe4 as internal standard. Mass spectra were obtained on a Finnigan-MAT TSQ 700 spectrometer (San Jose, CA). The reactions were monitored by thin-layer chromatography (TLC) on F254 silica gel pre-coated sheets (Merck, Milan, Italy); after development, the

Synthesis of 4-(N)-acyl-gemcitabine derivatives

In order to obtain a progressive lipophilicity in the gemcitabine prodrugs for liposome encapsulation, we synthesized 4-(N)-valeroyl- (C5) 1, 4-(N)-heptanoyl- (C7) 2, 4-(N)-lauroyl- (C12) 3 and 4-(N)-stearoyl- (C18) 4 gemcitabine derivatives using different procedures (Scheme 1). The best synthetic method was the well known mixed-anhydride techniques [23], which affords linkage between a molecule bearing a carboxylic group and an amino group of another molecule in two steps: (1) by reacting

Discussion

Liposomes, because of their biphasic character, can act as carriers for both lipophilic and hydrophilic drugs. Highly hydrophilic drugs, such as ara-C or gemcitabine, are located exclusively in the aqueous compartment of liposomes. These molecules, which are uncharged at physiological pH, rapidly diffuse through the liposome bilayer. This limits their stability and therefore their possible applications. On the contrary, highly lipophilic drugs are entrapped almost completely within the

Conclusions

In conclusion, we successfully prepared a formulation providing dual protection of gemcitabine from plasma catabolism, first by covalently linking the amino group of gemcitabine to a long fatty chain, as in 4, and then by encapsulating this lipophilic prodrug in the best of a number of liposomal formulations we had prepared, which is characterized by a rigid vesicle. Jointly, the cytotoxicity, pharmacokinetic and plasma stability studies clearly show that our liposomal gemcitabine formulation

Acknowledgements

Mr. Daniele Zonari's excellent technical assistance is appreciated. We wish to thank Prof. R. Cavalli (Università degli Studi di Torino, Dipartimento di Scienza e Tecnologia del Farmaco, Italy) for DSC analysis. This work was supported by MIUR 40–60%.

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