CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Biochimica et Biophysica Acta 1768 (2007) 678–687 www.elsevier.com/locate/bbamem Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo ⁎ Paul G. Tardi a, , Ryan C. Gallagher a, Sharon Johnstone a, Natashia Harasym a, Murray Webb b, Marcel B. Bally b, Lawrence D. Mayer a a Celator Pharmaceuticals Corp., 1779 West 75th Avenue, Vancouver, BC, Canada V6P 6P2 b Department of Advanced Therapeutics, British Columbia Cancer Agency, 600 West 10th Avenue, Vancouver, BC, Canada V5Z 4E6 Received 7 September 2006; received in revised form 23 November 2006; accepted 30 November 2006 Available online 6 December 2006 Abstract A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/ distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 °C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled. © 2006 Elsevier B.V. All rights reserved. Keywords: Liposomes; Irinotecan; Floxuridine; Coordinated release; Metal loading 1. Introduction effects associated with solid tumors [3–6]. This in turn has often resulted in improvements of the overall therapeutic activity of Liposomes have been used extensively to improve the the drug and has led to the regulatory approval of several therapeutic index of a variety of drugs by ameliorating toxicity liposome-based anticancer products [7,8]. Interestingly, very and/or increasing the therapeutic potency of the encapsulated little work has been undertaken to deliver drug combinations in agent [1,2]. This is perhaps best exemplified in the delivery of liposomes. This is likely the result of difficulties associated with anticancer drugs where it has been well documented both the efficient and stable encapsulation of two chemotherapeutics preclinically and clinically that small (approximately 100 nm) inside a single liposome as well as challenges in controlling the liposomes reduce exposure of entrapped drugs to susceptible release of chemically disparate drugs with one liposome healthy tissues while preferentially accumulating in sites of composition. tumor growth due to enhanced permeability and retention (EPR) Our interest in developing liposome formulations containing co-encapsulated anticancer drug combinations stems from the fact that virtually all curative cancer treatment regimens utilize ⁎ Corresponding author. Tel.: +1 604 675 2105; fax: +1 604 708 5883. drug combinations. We hypothesized that enhanced antitumor E-mail address: [email protected] (P.G. Tardi). activity may be achieved by simultaneously delivering and 0005-2736/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2006.11.014 P.G. Tardi et al. / Biochimica et Biophysica Acta 1768 (2007) 678–687 679 exposing anticancer drug combinations to tumor cells in vivo, 2.2. Preparation of liposomes since tumor cells would be less able to develop compensatory resistance mechanisms compared to single or sequentially Based on the appropriate molar composition, DSPC and DSPG were dissolved in chloroform/methanol/water (5/1/.1) at 50 mg/ml and combined with administered agents. This approach became of even greater cholesterol dissolved in chloroform. Where appropriate, cholesteryl hexadecyl importance when we recently observed that the antitumor ether [14C]CHE or [3H]CHE was added as a non-exchangeable, non- activity of drug combinations can be dramatically dependent on metabolizable lipid marker [18]. The solvent was removed under a stream of the molecular ratio of the combined drugs [9]. Specifically, nitrogen gas and the lipid films placed under high vacuum overnight. Typically, some ratios of a drug combination can be synergistic whereas the lipid films were rehydrated at 70 °C with 100 mM copper gluconate, 220 mM triethanolamine (TEA), pH 7.4. The pH of the copper gluconate other ratios of the same drugs can be additive or even solution was adjusted by varying the TEA content and not through the addition antagonistic [10]. This highlighted the need to control drug of sodium hydroxide. In experiments involving the sequential loading of ratios being exposed to tumor cells after systemic administration floxuridine and irinotecan, the floxuridine was passively trapped in the since the uncontrolled and dissimilar pharmacokinetics of liposomes by adding 122 mM floxuridine plus [3H]floxuridine as a tracer to individual drugs utilized in conventional drug “cocktails” no the copper gluconate buffer. The newly formed multilamellar vesicles (MLVs) were passed 10 times through an extruding apparatus (Northern Lipids) doubt results in exposure to sub-optimal drug ratios with a containing two stacked 100 nm polycarbonate filters. The mean diameter and concomitant loss in therapeutic activity. Further, although an size distribution of each liposome preparation, analyzed by a NICOMP Model increasing number of individual liposomal anticancer drugs are 270 Submicron particle sizer (Pacific Scientific, Santa Barbara, CA, USA) being developed, the pharmacokinetics of currently available operating at 632.8 nm, was typically 110±20 nm. Following extrusion the formulations vary greatly which would hinder attempts to external liposomal buffer was exchanged for 300 mM sucrose, 20 mM HEPES, 30 mM EDTA, pH 7.4 (SHE) using tangential flow chromatography. coordinate the exposure of different drugs using existing lipo- some formulations [11,12]. In addition, combining multiple liposome-based anticancer drugs poses potential difficulties due 2.3. Time course of irinotecan encapsulation to high lipid doses which have been shown in humans to lead to The following conditions were used to examine irinotecan encapsulation infusion-related adverse events [13,14]. into liposomes containing various metal salts, copper gluconate at different pH In view of the above considerations, we undertook a series of and irinotecan to lipid ratios. Irinotecan (Camptosar) and liposomes were heated studies aimed at developing single liposome formulations that separately at 50 °C for 1 min and then combined at t=0 by adding the lipid to the are able to stably co-encapsulate drug combinations and drug while vortexing. The final lipid concentration was diluted to 30 mM using SHE buffer. Aliquots of 100 μl were removed at various time points and applied coordinate the release of the two agents after intravenous (i.v.) to 1 ml Sephadex G-50 spin columns. The columns were prepared by adding injection. In this report we describe the co-encapsulation of glass wool to a 1 ml syringe and Sephadex G-50 beads hydrated in HEPES irinotecan and floxuridine. This drug combination was selected buffered saline (HBS; 20 mM HEPES, 150 mM NaCl, pH 7.4). The columns based on the fact that irinotecan and fluoropyrimidine (typically were packed by spinning at 500×g for 2 min. Following addition of the sample 5-fluorouracil, 5-FU) combination treatment is standard of care to the column, the liposome fraction was collected in the void volume by centrifuging at 650×g for 1 min. Aliquots of the spin column eluant and the pre- for metastatic colorectal cancer. The focus on floxuridine rather column solution were taken and analyzed by liquid scintillation counting to than 5-FU was based on the superior encapsulation and determine the lipid concentration at each time point. The irinotecan retention properties of floxuridine compared to 5-FU [15] and concentration in each liposomal fraction was determined using a UV based the fact that trials conducted in the 1960s established the clinical assay. Briefly, a 100 μl aliquot of each liposomal sample (or smaller volume μ μ equivalency of these two fluoropyrimidines [16,17]. We have adjusted to 100 l with distilled water) was solubilized in 100 l of 10% Triton X-100 plus 800 μl of 50 mM citrate/trisodium citrate, 15 mM EDTA, pH 5.5 and shown in previous studies that a 1:1 molar