A novel liposomal irinotecan formulation with significant anti-tumour activity: Use of the divalent cation ionophore A23187 and copper-containing liposomes to improve drug retention

Abstract

We determined whether the method used to encapsulate irinotecan into 1,2-distearoyl-sn-glycero-phosphocholine/cholesterol (DSPC/Chol; 55:45 mol%) liposomes influenced: (i) irinotecan release rate and (ii) therapeutic efficacy. DSPC/Chol (55:45 mol%) liposomes were prepared with: (i) unbuffered CuSO₄; (ii) buffered (pH 7.5) CuSO₄; (iii) unbuffered MnSO₄ and the ionophore A23187 (exchanges internal metal²⁺ with external 2H⁺ to establish and maintain a transmembrane pH gradient); and (iv) unbuffered CuSO₄ and ionophore A23187. All formulations exhibited >98% irinotecan encapsulation (0.2 drug-to-lipid molar ratio; 10 min incubation at 50 °C). Following a single intravenous injection (100 mg/kg irinotecan) into Balb/c mice, the unbuffered CuSO₄ plus A23187 formulation mediated a half-life of irinotecan release of 44.4 h; a ⩾4-fold increase compared to the other liposome formulations. This surprising observation demonstrated that the CuSO₄ plus A23187 formulation enhanced irinotecan retention compared to the MnSO₄ plus A23187 formulation, indicating the importance of the divalent metal. A single dose of the CuSO₄ plus A23187 formulation (50 mg/kg irinotecan) mediated an 18-fold increase in median T − C (the difference in days for treated and control subcutaneous human LS 180 adenocarcinoma xenografts to increase their initial volume by 400%) when compared to a comparable dose of Camptosar^®. Improved irinotecan retention was associated with increased therapeutic activity.

Introduction

Irinotecan (CPT-11) is a water-soluble camptothecin derivative that has demonstrated clinical activity against colorectal [1], [2] and small cell lung cancers [3], as well as showing promising activity in other cancer indications [4], [5]. Camptothecins promote apoptosis by stabilising the cleavable complex formed between topoisomerase I (topo I) and DNA [6]. This mechanism is dependent on the integrity of the lactone ring common to all camptothecins; however, the lactone ring undergoes a pH-dependent reversible hydrolysis that favours the carboxy derivative at physiological pH [7]. Consequently, drug delivery technologies, including liposomes, have been investigated as a means to stabilise the lactone ring. Liposome formulations of topotecan and irinotecan, the two approved water-soluble camptothecins, which trap the drug in the acidic aqueous core of the liposome, have been reported [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22]. Further, liposomes have been used to solubilise hydrophobic camptothecins such as SN-38 (the active metabolite of CPT-11) [23], [24], [25] and 9-nitrocamptothecin (9-NC) [13], [26]. Parental formulations of liposomal lurotecan (NX211/OSI-211) [27] and an aerosol preparation of liposomal 9-NC have been tested clinically [28], [29].

Weakly basic drugs such as irinotecan can be loaded into preformed liposomes that exhibit a transmembrane pH gradient (acidic inside) [30]. This approach traps the drug in an acidic environment as well as achieving efficient (>98%) loading [9] using a method that is suitable for pharmaceutical development [31]. Transmembrane pH gradients can be created directly by preparing liposomes in a well-buffered acidic solution [32]. Alternatively, “self-generating/self-maintaining” systems can generate a pH gradient by the presence of a transmembrane ammonium sulfate gradient [33], or by the intravesicular entrapment of a monovalent, or divalent, metal ion coupled with an appropriately selected ionophore [34]. As an alternative to these methods, we have recently described an encapsulation method that relies on formation of a copper ion/camptothecin complex, which can facilitate encapsulation of irinotecan or topotecan in the presence or absence of a transmembrane pH gradient [15], [35].

The ideal drug delivery system would retain its therapeutic payload until it reaches the target site whereupon the drug would be released. Resultantly, research has focused on strategies to engineer drug retention and controlled release [36]. This study investigated the influence of transition metal-based drug encapsulation technology on irinotecan retention by a generic liposomal formulation (DSPC/Chol (55:45 mol%)). The therapeutic activity of simple liposomal anti-cancer drug formulations is dependent on the rate of release of the encapsulated drug from the liposomes following intravenous administration [37], [38], [39]. For example, increased drug retention of the vinca alkaloid vincristine was associated with increased therapeutic effects [40]; however, if vincristine retention exceeded a certain limit, a decrease in therapeutic effect was noted [41]. Similarly, liposomal formulations of the anthraquinone mitoxantrone that achieved optimal drug delivery to sites of tumour growth (e.g., formulations that retain the drug well) exhibited less therapeutic activity when compared to formulations that released the drug more rapidly and were less efficient in mediating tumour-specific drug delivery [42], [43].

The studies reported herein demonstrated that the method of irinotecan encapsulation by DSPC/Chol liposomes can have surprising effects on drug retention following in vivo administration and that better irinotecan retention was associated with significant improvements in therapeutic efficacy.