Introduction

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Liposomes are well recognized drug delivery vehicles that have been shown to enhance the therapeutic activity of a number of anticancer drugs (Gabizon, 1992, 1994; Forssen et al., 1992; Perez-Soler et al., 1994; Mayer et al., 1990, 1993; Mayer and St. Onge, 1995). Appropriately designed liposomes have the ability to passively accumulate in tumor tissues that exhibit poorly defined or leaky vasculature. This process, termed "passive targeting", can result in the accumulation of significantly greater amounts of cytotoxic drug in tumor tissues than can be achieved by the administration of free drug (Huang et al., 1992, 1993; Bally et al., 1994). The increase in tumor-specific dose intensity achieved using liposomal delivery is frequently associated with increased antitumor activity (Webb et al., 1995).

The therapeutic activity of vincristine, a cell cycle-specific anticancer drug, is largely dictated by the duration of therapeutic vincristine concentrations at the tumor site (Horton et al., 1988). The therapeutic activity of conventional, aqueous, vincristine formulation (hereafter referred to as C-VINC) can be dramatically increased by increasing tumor drug exposure time (Boman et al., 1995; Mayer et al., 1995a). Furthermore, C-VINC administered to patients by long-term infusion has significant clinical activity, but is associated with severe toxicities (Jackson et al., 1981, 1984). Liposomal delivery may offer advantages based on maximizing tumor drug delivery while reducing accumulation of drug in most healthy tissues compared with conventional aqueous formulations. In support of this, preclinical studies have confirmed that liposomal vincristine formulations effect significant improvements in efficacy compared with C-VINC (Mayer et al., 1993; Boman et al., 1994; Webb et al., 1995, 1998) without increasing toxicities (Webb et al., 1998). A liposomal formulation of vincristine optimized for stability, pharmacokinetics, and efficacy (Webb et al., 1995, 1998) is currently in advanced clinical evaluation for the treatment of relapsed non-Hodgkin's lymphoma (NHL) (Sarris et al., 2000).

The development of therapeutically optimized lipid-based delivery systems requires tailoring the leakage rate of the drug from the liposomal carrier (Boman et al., 1997; Bally et al., 1998) such that drug is released from the carrier at a rate appropriate to achieve therapeutically active concentrations. However, drug released from liposomal carriers (hereafter referred to as nonliposomal drug) present in the plasma also has the potential to elicit toxicities in nontarget tissues. In clinical settings where the dose of liposomal vincristine is higher than that for the C-VINC (Sarris et al., 2000), potential toxicities arising from nonliposomal drug released from liposomes in the circulation may be a concern. Therefore, evaluations of the safety of liposomal systems for the delivery of cytotoxic drugs such as vincristine may benefit from characterization of the levels of nonliposomal drug associated with the administration of a liposomal drug delivery system.

The objectives for the present study were to 1) characterize the levels of nonliposomal drug associated with the administration of liposomal vincristine, and 2) evaluate and compare the pharmacokinetics of nonliposomal and liposome-encapsulated vincristine after the administration of liposomal vincristine formulations having different drug release properties and relating pharmacokinetic and biodistribution properties to historic data on toxicity and efficacy for liposomal vincristine and C-VINC. Specifically, vincristine encapsulated in sphingomyelin/cholesterol (SM/Chol) liposomes was compared with drug encapsulated in a more permeable distearoylphosphatidylcholine/cholesterol (DSPC/Chol) liposomal formulation. These evaluations were performed using the Lewis lung carcinoma (LLC) solid tumor model.

	Experimental Procedures

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Materials. Vincristine sulfate was purchased from David Bull Laboratories (Canada) Inc., Vaudreil, Quebec. [³H]Vincristine sulfate was purchased from Amersham Pharmacia Biotech (Bucks, UK). The lipids DSPC (>99% purity) and SM were obtained from Avanti Polar Lipids (Alabaster, AL), and Chol was obtained from Sigma Chemical Co. (St. Louis, MO). Saline (0.9% NaCl) was obtained from Baxter Corporation (Burnaby, BC, Canada). All other reagents were reagent grade and obtained from Sigma Chemical Co.

Animals and Tumor Models. Female BDF1 mice (Charles River, St. Constant, Quebec, Canada) used in these studies were between 20 and 23 g in weight. All animals were treated in accordance with the guidelines of the Canadian Council on Animal Care. LLC cells were obtained from the National Cancer Institute Repository (Bethesda, MD). Tumor cells (10⁶) were subcutaneously injected bilaterally into the flanks of these mice. Tumors were allowed to grow to 100 to 200 mg before the mice were administered with C-VINC or liposomal vincristine. Studies involving animals were performed in accordance with the standards of the Canadian Council on Animal Care.

Terminology. To facilitate the analysis of vincristine pharmacokinetic parameters, a set of terminology was selected to define the various formulations of vincristine that were administered as well as measured in plasma after drug administration. These definitions are as follows: 1) C-VINC, conventional, aqueous solution of vincristine sulfate in saline; 2) liposomal vincristine, vincristine encapsulated inside 100- to 120-nm-diameter unilamellar liposomes possessing a transmembrane pH gradient; 3) nonliposomal vincristine, vincristine present in the circulation that is not liposome encapsulated; and 4) free vincristine, vincristine that is neither protein-bound nor entrapped inside liposomes.

Liposome and Drug Preparation. Liposomes composed of DSPC/Chol, or SM/Chol (55/45; mol/mol) were prepared by dissolving the lipids in chloroform at the specified molar ratio, drying under vacuum, and then hydrating the dried lipid film with 300 mM citric acid (pH 4.0) at 100 mg of lipid/ml. The resulting multilamellar vesicles were subjected to five freeze-thaw cycles followed by 10 passes through two stacked polycarbonate filters containing pores with diameters of 100 nm (Nuclepore, Pleasanton, CA) using a Lipex Extruder (Lipex Biomembranes Inc., Vancouver, BC, Canada). The resulting large unilamellar vesicles had mean diameters ranging between 100 and 120 nm as determined using a Nicomp 370 submicron particle sizer.

Pharmacokinetics and Biodistribution Studies. Pharmacokinetic experiments were performed on female BDF1 mice bearing 100- to 200-mg LLC solid tumors. Mice were administered, via the tail vein, with a single bolus dose of either C-VINC (labeled with [³H]vincristine as described above) or liposomal [³H]vincristine (DSPC/Chol or SM/Chol formulations) at a dose of 2 mg of vincristine/kg (corresponding to 40 mg of lipid/kg). Select animals receiving saline alone were terminated at 24 h postadministration. At each time point after dosing, groups of four mice were anesthetized with 100 µl of 32 mg/ml i.p. ketamine and 4 mg/ml xylazine, in water, to achieve final doses of 160 and 20 mg/kg, respectively. Blood samples were collected by cardiac puncture and placed into EDTA-coated tubes (Microtainer; BD Biosciences, San Jose, CA). Plasma was obtained by centrifuging whole blood samples at 500g for 15 min at 4°C. After blood collection, animals were terminated and heart, spleen, lungs, lymph nodes, small intestine (approximately 3-4 inches, flushed with phosphate-buffered saline), muscle, kidneys, liver, and tumors were obtained from each animal. All tissues, with the exception of lymph nodes, were rinsed in phosphate-buffered saline, dried, and weighed, and then homogenized using a Polytron homogenizer. A 10% homogenate in distilled water prepared and 0.2-ml aliquot of the homogenate was digested with Solvable, decolorized with 30% v/v hydrogen peroxide, and mixed with 5 ml of Picofluor. Lymph nodes were placed in 7-ml scintillation vials, weighed, and digested as described for other tissues. The tissues obtained from control animals administered with saline were used as background samples for radioactivity determinations. All counts were converted to absolute radioactivity (dpm); samples that had dpm less than or equal to 2 times background values were considered as zero for all subsequent determinations. Tissue concentrations of vincristine were estimated by correcting for drug residing in the tissue vasculature as described previously (Mayer et al., 1995b).

Separation of Free and Liposomal Vincristine in Vivo. The concentrations of free vincristine present in the plasma after the administration of liposomal vincristine (SM/Chol versus DSPC/Chol) were determined. This procedure to separate free vincristine from protein-bound vincristine was also performed on mice administered with C-VINC. Mice were administered as described above then blood was collected by cardiac puncture and plasma obtained by centrifugation. After aliquots of the plasma were removed for assay of total vincristine concentrations in the plasma, the remaining plasma was used to separate free vincristine from liposomal and protein-bound vincristine using Microcon-30 ultrafiltration devices as described previously (Mayer and St. Onge, 1995). Specifically, plasma remaining from the pharmacokinetic sampling was transferred to Microcon-30 reservoirs and centrifuged at 10,000 rpm, 4°C, for 15 min in a microcentrifuge. Aliquots of 50 to 75 µl of the ultrafiltrate, containing free vincristine, were processed for liquid scintillation counting. Previous characterization of this method has demonstrated that protein-bound vincristine comprises approximately 40% of the total nonliposomal (free plus protein-bound) vincristine in the plasma over a drug concentration range of several orders of magnitude (Mayer and St. Onge, 1995). Therefore, the values reported here for free vincristine represent approximately 60% of the total nonliposomal vincristine present in the plasma.

Pharmacokinetics and Biodistribution Data Analyses. The plasma data were modeled using WinNonlin version 1.5 pharmacokinetic software (Pharsight Corporation, Mountain View, CA), to calculate pharmacokinetic parameters of free and liposomal vincristine according to standard equations. To determine appropriate models to fit the plasma data, the criteria used to evaluate the goodness of fit for each model included a visual assessment of distribution of residuals, rank, and Akaike's information criterion (Akaike, 1976). Calculated parameters included the maximum plasma concentration (C_max), the area under the concentration-time curve (AUC), half-life of elimination, volume of distribution (Vss), and the average time a particle remains in the plasma compartment, the mean residence time (MRT). Plasma AUC values for free vincristine, obtained using the ultrafiltration method, were calculated from the noncompartmental analysis of the concentration-time profiles. The linear trapezoidal summation was used to calculate the AUCs in tissue concentration-time profiles using a computer software AUC program provided by Dr. Wayne Riggs (Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada).

	Results

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Pharmacokinetics of Total Vincristine. The pharmacokinetics of vincristine after the administration of C-VINC or of either DSPC/Chol or SM/Chol formulations of liposomal vincristine are shown in Fig. 1. Key pharmacokinetic parameters were calculated from these data and are presented in Table 1. C-VINC was rapidly eliminated from the plasma and had AUC, half-life, MRT, and C_max values that were significantly lower, as well as Vss values that were significantly higher than those for both liposomal vincristine formulations (Table 1). After the administration of either liposomal vincristine formulation, elevated plasma concentrations of total vincristine were maintained up to 48 h postinjection. C_max values between 22.2 and 26.3 µg/ml were observed after i.v. administration of the liposomal formulations (Table 1). The elimination half-lives were 4.0 h for the DSPC/Chol liposomal vincristine formulation and 6.65 h for the SM/Chol liposomal vincristine formulation (Table 1). The higher C_max values, longer circulation half-lives, and longer mean residence times observed with the liposomal formulations, compared with C-VINC, were associated with significantly higher plasma AUC values for vincristine. Specifically, the AUC values for SM/Chol liposomal vincristine (213 µg · h/ml) and DSPC/Chol liposomal vincristine (153 µg · h/ml) were 361- and 259-fold greater than that for the C-VINC. Taken together, these data are similar to those described previously for both DSPC/Chol and SM/Chol liposomal formulations of vincristine (Webb et al., 1995, 1998).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Plasma concentrations of vincristine in mice bearing LLC tumors after i.v. administration of C-VINC or either the DSPC/Chol (A) or SM/Chol (B) liposomal formulations of vincristine. Insets provide additional detail on the comparison of free versus C-VINC in the first 4 h after administration. Data represent the means and standard deviations of C-VINC () or the total liposomal vincristine () and free vincristine (). Dose was 2 mg/kg of drug.

Comparison of Liposome-Encapsulated and Free Vincristine. It was anticipated that the majority of the total vincristine measured in plasma after the administration of liposomal drug was encapsulated in the liposomal carrier and was not bioavailable in a form able to cause toxicities. Furthermore, based on previous observations demonstrating reduced vincristine leakage from SM/Chol liposomes compared with DSPC/Chol systems (Webb et al., 1995), we hypothesized that this difference would be reflected by altered free drug concentrations in the plasma. To evaluate these parameters, the plasma concentrations of free vincristine were directly measured using a previously described ultrafiltration method (Mayer and St. Onge, 1995).

Biodistribution. The time course of vincristine distribution to various organs after the administration of the two liposomal drug formulations is given in Fig. 2. C_max and AUC values for C-VINC and the liposomal vincristine formulations are presented in Table 3. Vincristine accumulation in solid tumors was characterized to correlate tumor drug concentrations with efficacy properties characterized in previous studies (Webb et al., 1995) (Table 3; Fig. 2). Tumor vincristine levels following administration of C-VINC at 2 mg/kg resulted in relatively low accumulation of the drug with a C_max of 0.47 ± 0.1 µg/g and an AUC of 1.54 µg · h/g. Tumor vincristine concentrations following liposomal delivery were significantly higher than C-VINC at similar doses. For both liposomal formulations, the concentration-time plot (Fig. 2) indicated a relatively rapid distribution phase followed by sustained levels over the 72-h period. Specifically, there were 60- to 120-fold increases in AUC when liposomes were used as a carrier compared with C-VINC administered at 2 mg/kg. However, the AUC of vincristine following injection of SM/Chol liposomes (186.7 µg · h/g) was approximately 2-fold higher than that following DSPC/Chol liposomes (96.5 µg · h/g), consistent with previous observations (Webb et al., 1995).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Tissue distribution of vincristine in mice bearing LLC tumors after i.v. administration of either the DSPC/Chol (A) or SM/Chol (B) liposomal formulations of vincristine. Data represent the means and standard deviations of total vincristine in the liver (), kidney (), spleen (), heart (), lymph node (), and the tumor (). Tissues were collected and processed for the quantification of vincristine as described under Experimental Procedures.

	Discussion

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The toxicities associated with the conventional, aqueous formulation of vincristine are well characterized (Carter and Livingston, 1976; Sieber et al., 1976). Dose limiting toxicities associated with C-VINC include neurotoxicity, manifested primarily as peripheral neuropathy, and intestinal toxicity. In addition, the drug causes soft tissue necrosis and ulceration when accidentally extravasated during parenteral injections (Bellone, 1981).

One of the earliest studies evaluating the pharmacology and toxicology of C-VINC and a drug-permeable DSPC/Chol liposomal vincristine formulation in rats (Kanter et al., 1994) revealed that both C-VINC and liposomal vincristine resulted in dose-dependent weight loss where the liposomal formulation exhibited an elevated maximum tolerated dose compared with C-VINC. In dogs, toxicities included weight loss, gastrointestinal toxicity, myelosuppression, and serum chemistry alterations secondary to muscle, hepatic, gastrointestinal, and pancreatic toxicity for both C-VINC and liposomal vincristine. Interestingly, the dog studies revealed no patterns of toxicity that differentiated C-VINC from liposomal vincristine and maximum tolerated dose values for both formulations were comparable. The apparent discrepancy of toxicity properties in these two animal models is not readily resolved, partly because the contribution of different physical states of vincristine in plasma (e.g., liposome-entrapped, protein-bound, or free drug) to its toxicity profile is unknown. Furthermore, any propensity for vincristine to assume different physical states in different animal models is unknown.

The antitumor activity of liposomal anticancer drug formulations is dependent on drug release rates that can achieve therapeutic concentrations at the tumor site over relevant time frames in a manner that is minimally associated with toxicological consequences. We have shown that increasing vincristine retention in liposomal systems improves the therapeutic index by increasing the duration of drug exposure to tumor tissue (Boman et al., 1994, 1995, 1997; Webb et al., 1995; Bally et al., 1998). Liposomal encapsulation results in significant increases in plasma C_max, half-life, mean residence times, and AUC, and decreases in plasma clearance rates and volume of distribution compared with C-VINC. However, similar to toxicity considerations, it is unclear which vincristine states are responsible for providing antitumor activity. Presumably, vincristine must be released from the liposomes to access its target within tumor cells since liposomes themselves are not actively taken up intracellularly by tumor cells. Whether this bioavailable vincristine arises from drug released from liposomes in the central blood compartment or rather at the tumor site is not well understood.

To confirm that liposomal encapsulation of vincristine was not associated with detrimental levels of nonliposomal drug and better understand the relationship of vincristine pharmacokinetic parameters and toxicity/efficacy behavior, we developed an ultracentrifugation method that differentiated free from liposomal-associated and protein-bound drug in plasma under equilibrium conditions (Mayer and St. Onge, 1995). Using this method, we have demonstrated here that the plasma AUC values calculated for free vincristine represent only 2% of the total plasma vincristine for both the SM/Chol liposomal formulation and the leakier DSPC/Chol formulation. Importantly, the systemic exposure to free vincristine after administration of either liposomal formulation represented 52 to 56% of the exposure after the administration of the same dose of C-VINC. Similar relationships would be predicted for free plus protein-bound vincristine (i.e., nonliposomal vincristine) given that the partitioning of vincristine between free and protein-bound pools is approximately 1.5:1.0 over a very broad range of total plasma drug concentrations (Mayer and St. Onge, 1995). Therefore, increased toxicities would not be expected to arise from the administration of the liposomal formulations consistent with the decreased toxicity observed for liposomal vincristine in rodent models. Furthermore, the equivalent toxicity observed previously (Webb et al., 1995) between the DSPC/Chol and SM/Chol liposomal formulations studied here is consistent with the observation that the free vincristine plasma concentrations arising from these liposomal formulations (Fig. 1; Table 2) were very similar, even though the SM/Chol formulation displays approximately a 2-fold decreased drug leakage rate after i.v. injection compared with the DSPC/Chol vincristine formulation.

Alterations in vincristine biodistribution properties obtained with the liposome carriers studied here suggest that free drug concentrations in the plasma do not correlate with the extent of antitumor activity achieved with either C-VINC or liposomal vincristine formulations. These observations are consistent with a local drug infusion model where liposomal vincristine enhances therapeutic efficacy by providing an in situ drug infusion reservoir in the tumor, suggesting that activity is dictated by drug release properties at the site of action. In this model, presumably the extravasated liposomes in the tumor gradually release vincristine in the interstitial space whereby it is taken up by tumor cells over time. Consequently, drug released from liposomes in the central blood compartment may not contribute to the overall therapeutic activity of the anticancer drug. Although free drug concentrations appeared lower following liposomal vincristine injection than those observed following injection of C-VINC, it remains to be determined to what extent free drug concentrations may be predictive of toxicity behavior. Consequently, determining plasma drug-to-lipid ratios to establish drug release properties at the tumor site or noninvasive methodologies (e.g., microdialysis) determining free or extracellular levels of the drug in the peripheral tumor tissue compartment may be needed to provide additional insights into elucidating the relationships involving drug concentrations and antitumor efficacy. The latter technique may be particularly useful to understand pharmacokinetic/pharmacodynamic relationships given the complexity of interplay between factors such as tumor blood flow, hypoxia, and permeability differences that are characteristic of tumor heterogeneity.

In summary, the plasma concentrations of free vincristine following injection of liposomal drug were significantly lower than those observed following C-VINC administration at equivalent doses. This may explain the reduced toxicity observed for liposomal vincristine formulations compared with C-VINC in several previous rodent studies (Mayer et al., 1990, 1995b; Kanter et al., 1994). In contrast, increased efficacy correlates more closely with total vincristine concentrations regardless of the formulation used. These results appear consistent with early data being obtained in clinical studies with liposomal vincristine formulations, where significant plasma total vincristine concentrations have been observed (Mayer et al., 1995a) and promising signs of antitumor activity have been observed in relapsed NHL patients where the response rates were 41% with modest toxicities in a population that presented several adverse prognostic factors (Sarris et al., 2000). Definitive identification of relationships between free vincristine plasma concentrations and toxicity and/or efficacy in human cancer patients will require differentiation of free, protein-bound, and liposomal drug pools as was performed here.