The therapeutic efficacy and tumor accumulation of a liposome formulation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), an effective agent used in the treatment of malignant brain tumors, was examined in an animal tumor model. Pharmacokinetic studies in normal and tumor-bearing rats indicated that a 2-fold greater plasma exposure was achieved with liposome-formulated CCNU compared with the free drug. In Fisher rats bearing s.c. tumors 36B-10, tumor growth was delayed substantially when liposomal CCNU was delivered compared with free-drug treatment. In single-dose treatments of 20, 35, and 50 mg/kg, tumor progression after each dose was reduced ∼2-fold with liposomal compared with free CCNU (four animals in each treatment group). Multiple-dose treatments (given as three weekly doses with eight animals in each treatment group) with cumulative doses of 80 and 100 mg/kg of free and liposomal CCNU also resulted in a 2-fold reduction in tumor progression when compared with free-drug treatment. When drug levels in tumors relative to plasma were examined, it was observed that tumor drug concentrations did not exceed those found in plasma after administration of free CCNU; after administration of liposomal CCNU, however, tumor concentrations exceeded those in plasma by nearly 10-fold. These results suggest that the increased efficacy of liposome-formulated CCNU may be attributable to enhanced drug accumulation in tumor tissues.

Liposome encapsulation of anticancer drugs has been studied extensively both in the laboratory and in the clinic; increased plasma drug concentrations, improved tumor delivery, decreased toxicity, and increased efficacy for a variety of cytotoxic agents have been reported (1, 2, 3). The benefits associated with anticancer drug delivery using liposome formulations have been primarily the reduced toxicity of the encapsulated form of the drug. However, variable improvements in antitumor potency have been reported. This may be the case in part because most efforts to date have focused on anthracyclines, which exhibit only a modest degree of cytotoxic potency relative to the duration of tumor drug exposure (4).

The combined properties of liposome formulations, including the increase of circulation times and tumor drug accumulation, and decreased toxicity are particularly well suited for delivery of highly active alkylating agents such as CCNU3. It is an extremely potent nitrosourea, a class of drugs that are some of the most widely used chemotherapeutic compounds in the treatment of CNS tumors (5). The intermediate lipophilicity of these agents allows high levels to partition into the CNS after systemic administration. However, the clinical use of these drugs is restricted by dose-related toxicities, including neurotoxicity and delayed and cumulative myelosuppression (6, 7). Moreover, the nitrosoureas do not produce durable long-term responses and, even in combination with radiation therapy, do not increase survival time for most patients (8). The development of novel agents with increased antitumor activity and decreased toxicity is imperative to improving the treatment of CNS tumors.

Toward this end, we developed and optimized a liposome-formulated CCNU that enhanced the antitumor activity of the drug without exacerbating toxic side effects. Incorporating CCNU into negatively charged, SUVs resulted in profound increases in antitumor potency against malignant medulloblastoma cells but decreased toxicity to normal fetal brain cells in vitro compared with free drug (1). Additionally, we found that systemic administration of liposomal CCNU in normal rats significantly increased plasma drug exposure without concomitant increases in behavioral neurotoxicity (1).

In light of the improvements in in vitro antitumor potency and plasma exposure of CCNU accompanied by a decrease in toxicity in normal rats, in this report, we further characterize the efficacy of liposome-formulated CCNU to suppress the growth of brain tumors in a rat model. In addition, we have sought to elucidate the possible mechanisms underlying the improved therapeutic effects of liposomal CCNU in this model by examining tumor-to-plasma drug accumulation and plasma free-fraction drug levels.

Free and Liposomal CCNU Formulations.

CCNU was kindly provided by the Drug Synthesis and Chemistry Branch of the Developmental Therapeutics Division of Cancer Treatment from the NCI. The phospholipids, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) were purchased from Sygena, Inc. (Cambridge, MA). Routinely, lipid-encapsulated CCNU (drug:lipid molar ratio 1:10) was prepared by first dissolving 100 mg of DMPC and DMPG (1:1, mol/mol) with CCNU in 1 ml of chloroform in a test tube and evaporating off the solvent with a stream of N2 gas to form a dry film. Where needed, [3H]cholesteryl ether (specific activity, 8.5 × 103 cpm/μg lipid; NEN, Wilmington, DE) a nonmetabolizable lipid marker, was added to the organic phase. The final specific activity of [3H]liposome suspension was 8.5 × 103 cpm/μg lipid. The dry film was then vacuum desiccated for at least 30 min. To prepare desired lipid concentrations, a 1-ml volume of sterile PBS (pH 7.4), composed of 8 g/liter NaCl, 0.2 g/liter KCl and KH2PO4, and 0.16 g/liter Na2HPO4 was then added to create a 100-mg/ml suspension. The mixture was then sonicated at 27°C in a (water) bath type sonicator (Laboratory Supplies, Inc., Hicksville, NY) until a uniform translucent suspension of SUVs was obtained. The diameters of liposomes were determined by photon correlation spectroscopy (Malvern Zetasizer 5000, Southborough, MA) to be 80 ± 8 nm in size. Under these conditions, we found that almost (∼96%) all of the CCNU in the suspension was lipid associated. Details of these results have been published (1). Free CCNU dosages were prepared just prior to drug administration and consisted of dissolving CCNU in a carrier solution of sterile 0.9% NaCl with 10% ethanol and 2% Tween 80.

Animal Model and Tumor Cells.

The 36B-10 cells, an ethylnitrosourea-induced Fisher F-344 rat malignant astrocytoma, were generated in the Department of Neurological Surgery, University of Washington (Seattle, WA) as has been described in detail elsewhere (9). The glioma cells were cultured in tissue culture flasks (Becton Dickinson Labware, Franklin Lake, NJ) in Waymouth‘s medium containing 5% antibiotic/antimycotic, 2 mm glutamine, and 10% fetal bovine serum in a humidified atmosphere of 5% CO2 at 37°C. In vitro growth inhibition attributable to CCNU exposure was performed with a [3H]thymidine uptake assay, which has been described previously (1).

Female Fisher F-344 rats, weighing ∼180 g and obtained from Charles River Laboratories (Wilmington, MA), were used in these experiments. The rats were maintained in a specific pathogen-free environment and fed sterile laboratory pellets and water ad libitum. Cultured 36B-10 cells were harvested and centrifuged at 900 × g for 5 min; the resulting supernatant was removed, and the cells were resuspended with plain Waymouth‘s media and kept on ice until the suspension was drawn into 1-ml syringes for s.c. injection. For tumor inoculation, 0.2 ml of a cell suspension containing 5 × 105 cells was administered s.c. to each flank (4 sites total) of rats using 22-gauge needles. Animals were monitored daily for weight and tumor development. There was 100% tumor take in all of the animals, and tumors were discernable 14 days after inoculation. All of the animals were evaluated for their tumor size at day 14 and randomly assigned into treatment groups. The tumor size distribution between control and treatment groups were similar. Tumor measurements were performed with Vernier calipers, and volume was determined with the following equation:

\[Tumor\ volume\ (mm^{3})\ {=}\ \frac{a^{2}{\times}b}{2}\]

where a = shorter length and b = longer length. The fold increase was calculated by comparing the size of each tumor at day 14 to that of subsequent measurements after treatments.

Chemotherapy.

Rats bearing s.c. gliomas received free and lipid-encapsulated CCNU, as well as control carrier solutions, i.p. on 14 days after tumor inoculation. Animals inoculated with tumors and receiving free-drug carrier solution or blank liposomes served as controls. Single- and multiple-dosing schedules were examined for the effect on tumor volume and hematological toxicity. Single-dose response studies were done with treatments of 0, 20, 35, and 50 mg/kg free and liposomal CCNU (n = 4 animals each treatment). Multiple-dosing schedules consisted of (a) 40 mg/kg on day 14 after tumor inoculation followed by 20 mg/kg 7 and 14 days later and (b) 20 mg/kg 14 days after tumor inoculation followed by 40 mg/kg 7 and 14 days later (n = 8 animals for each treatment schedule). Tumor volume data, reported 21 days after treatment was initiated, were normalized by dividing these final values with pretreatment tumor volumes.

Toxicity.

Before and during the course of chemotherapy, blood was collected from tail veins using the Unopette Microcollection System (Becton Dickinson, Rutherford, NJ); WBCs and platelets were counted, and complete blood cell analysis was done by differential microscopic analysis of the glass slide prepared according to the manufacturer‘s recommendation. Because significant changes were detectable only in WBCs and platelets, these two values were presented in this report. In addition, animal weight was monitored daily throughout each experiment.

Pharmacokinetic Studies.

Free or liposome-encapsulated CCNU (50 mg/kg) was administered i.p. to tumor-bearing rats; blood (by cardiac puncture) and tumorous tissues were collected 5, 10, 15, 20, 30, 120, 240, and 360 min after dosing. Plasma was quickly separated from the blood samples after collection and placed with the tumors in dry ice. This procedure prevents CCNU decomposition during sample preparation for drug analysis. Plasma and tumor tissue concentrations were determined by first diluting and homogenizing the samples with chilled homogenizing buffer [50 mm Tris-HCl and 0.1 mm EDTA (pH 2 with HCl)]. Sample homogenates were extracted (three times) with five volumes of ethyl acetate and centrifuged at 800 × g to separate the organic phase from the aqueous layer. The organic supernatants were combined and evaporated to dryness under a stream of nitrogen. As described previously, dried extracts were reconstituted with high-performance liquid chromatography mobile phase [acetonitrile:acetate, 60:40, v/v (pH 4)] and analyzed by isocratic reverse-phase chromatography (1). A small fraction of tissue homogenate and plasma were used to determine lipid concentration by counting the [3H]cholesteryl ether activity in Protosol (NEN, Wilmington, DE) after bleaching according to the Protosol manufacturer‘s recommendations before scintillation counting. From these data, lipid:drug molar ratios were determined.

Separation of free from liposomal and protein-bound drug was performed using Centrifree (1.0-ml capacity) ultrafiltration devices (Amicon, Inc., a division of Millipore, Bedford, MA) with a Mr cutoff of 30,000. As previously characterized for liposome-associated doxorubicin and vincristine, these devices separate free from bound drug under equilibrium conditions. Centrifree devices hold filtration membranes that are anisotropic and hydrophilic, exhibit low adsorption properties, and have a sample hold-up volume of ≤10 μl (10). After i.p. administration of free CCNU, plasma samples (n = 2) were collected at 10, 15, 20, and 30 min. After dosing with liposomal CCNU, plasma samples were collected at 30, 60, 120, 240, and 360 min. Centrifree devices containing rat plasma were spun at 2000 × g for 20 min in a refrigerated (4°C) Beckman J21-C centrifuge equipped with a fixed-angle rotor (JA-20). The ultrafiltrate and bound fractions were then frozen on dry ice and stored at −80°C until drug concentrations were analyzed by high-performance liquid chromatography analysis. The ultrafiltrate and bound concentrations of CCNU were compared with CCNU concentrations in total plasma to determine percentage of free and percentage of bound fractions of drug.

Statisical Analysis.

Data were expressed as mean values ± SD or SE. Student‘s t test (two-tailed) and two way ANOVA were performed comparing the differences between two experimental groups and among three groups of animals, respectively. Differences were considered significant when Ps were less than 0.05.

The sensitivity of 36B-10 cells to both free and liposomal CCNU was verified. The growth inhibition curves for the free and liposomal formulations of CCNU [after accounting for the nearly 2-fold lower degradation rate of CCNU in liposomes (1)], appeared to be identical (Fig. 1). The time course of plasma CCNU concentrations in tumor-bearing rats receiving the same dose of free or liposome-formulated CCNU was then determined. For the same dose, plasma CCNU exposure increased by at least 2-fold when it was administered to rats in liposomal as compared with free-drug formulation. Between 10 and 30 min after dose administration, ∼5-fold higher plasma CCNU concentrations were detected in rats administered liposome-formulated compared with free drug (Fig. 2). Plasma CCNU concentrations at 10 min were 10.2 ± 1.7 versus 47.0 ± 9.3 μg/ml, and 2.2 ± 0.3 versus 14.4 ± 0.6 μg/ml 30 min after administration of free and liposome-formulated drug, respectively. At extended times (2 and 4 h), plasma CCNU was detectable only in animals treated with liposome-formulated CCNU.

To determine whether increased plasma exposure of liposomal CCNU may also lead to increased tumor exposure and, therefore, to increased efficacy, we compared the effects of free and liposome-encapsulated CCNU on tumor progression in rats implanted with 36B-10 tumors. The first study was a single-dose drug administration study after establishment of the 36B-10 tumor (14 days after tumor implantation). Tumor progression was substantially delayed after a single bolus dose of 0, 20, 35, or 50 mg/kg CCNU when the drug was delivered as a liposomal (Fig. 3,B) compared with free-drug formulation (Fig. 3,A). There was a clear dose-response effect on tumor volumes measured 21 days after treatment up to 35 mg/kg for both free and liposomal CCNU (Fig. 3,C). Increasing the dose to 50 mg/kg did not provide further tumor growth reduction. For each dose examined, tumor volumes observed after liposomal CCNU treatment were found to be ∼2-fold lower than that observed for free-CCNU therapy (Table 1). Compared with control, (untreated animals with tumors) liposome-formulated CCNU reduced the tumor volume by ∼3.5-fold by the end of 3 weeks at the highest dose (50 mg/kg; Fig. 3).

In addition to evaluating tumor response, systemic toxicity between free and liposome-encapsulated CCNU was evaluated by monitoring peripheral WBCs and platelets as well as body weight. In these studies, the lowest WBC and platelet counts were found at 4 days after drug administration (Table 1). Although there was a CCNU-dependent decrease in WBC and platelet count in all of the drug-treated animals, no significant differences in toxicity were found between animals treated with free and with liposomal CCNU even at the highest dose of 50 mg/kg. Also, single-dose treatments with both free and liposomal CCNU did not inhibit tumor progression beyond 12 days after drug administration.

To optimize tumor response and reduce systemic toxicity with a more aggressive dosing regimen, two multiple-dosing regimens with free and liposomal CCNU were performed. The first regimen consisted of single weekly doses of 40, 20, and 20 mg/kg (80 mg/kg total dose) and was designed with the intent to suppress early tumor progression with an initial dose of 40 mg/kg, followed weekly by 20 mg/kg maintenance doses. The second regimen, with weekly doses of 20, 40, and 40 mg/kg (100 mg/kg total) was chosen with the intent to control the exponential tumor growth found at the later stage of therapy. As with the single-dose treatments (Table 1), end-point tumor volumes from multiple doses of liposome-encapsulated CCNU treatments were ∼2-fold lower than those observed after free-drug treatments (Table 2), and the time course of tumor progression was significantly altered (data not shown) from that in single-dose studies. Hematological toxicity 7 days after a cumulative dose of 80 mg/kg liposome-encapsulated CCNU was significantly lower than that after the same dosing regimen of free drug (Table 2). However, with a cumulative dosage of 100 mg/kg, both free and liposomal CCNU showed equivalent decreases in WBC and platelet counts, and body weight measures.

To elucidate the mechanisms of increased antitumor activity provided by liposome-formulated CCNU, we monitored tumor concentrations of CCNU after free and liposome-encapsulated drug administration. We hypothesized that liposomal CCNU may accumulate in tumors as a liposome-associated complex. If CCNU levels in tumors exceeded those in plasma after delivery of the liposome formulation, it would suggest retention of the drug with liposomes in plasma and subsequent passive uptake of liposomal drug by the tumor. If drug levels in the tumors were the same as those found in plasma, it might indicate rapid dissociation of CCNU from the liposomes in plasma and the tumor:plasma ratio would be similar to that found after free CCNU administration. Initial time points indicate that, after administration of free CCNU, there is rapid drug accumulation in the tumor, such that the ratio between tumor and plasma concentrations reached unity by 30 min (Fig. 4). Two h after free-drug administration, there was no CCNU detectable in plasma or tumors. In contrast, tumor accumulation from plasma took considerably longer after liposome-encapsulated drug delivery and tumor concentrations exceeded plasma levels by nearly 10-fold from 2 to 6 h after liposomal CCNU administration. At 2–6 h, about 3–6 μg/ml CCNU in tumor tissue was detected in animals treated with liposomal CCNU (compared with the initial blood concentrations of ∼10 μg/ml in these animals). Analysis of tumors isolated from animals treated with radiolabeled liposomal CCNU exhibited lipid:drug ratios in the range of 8.6–9 (m/m), values similar to the initial formulation of liposomal CCNU.

Free and protein-bound fractions of CCNU in rat plasma were examined to explain the observations of lower hematotoxicity and neurotoxicity (1) for rats treated with liposomal CCNU. After a 50-mg/kg dose, plasma free-fractions were 5.0 ± 1.5% and 13.2 ± 1.4% after liposomal and free CCNU administration, respectively (Table 3). These results indicate that, compared with free-drug formulations, plasma free-fractions were reduced by about one-half with liposome-formulated CCNU.

The antitumor activity of liposome-encapsulated CCNU was significantly greater than that of free drug in the s.c.-inoculated 36B-10 rat glioma model. Previous studies with this rat-tumor model revealed that increased survival times could not be achieved with escalating doses of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU), a more hydrophilic analogue of CCNU (11). These investigators suggested that the effectiveness of the nitrosourea is in part limited by drug delivery problems. We have substantiated these speculations. Whereas we found liposomal CCNU to inhibit 36B-10 cell growth to the same extent as free CCNU in vitro, treatments to tumor-bearing rats indicated a significant 2-fold decrease in tumor progression with liposomal CCNU therapy compared with free drug.

In addition to the 2- to 4-fold increase in stability of CCNU bound to lipids, one of the likely mechanisms leading to enhanced therapeutic effect is probably the increased circulation time found with lipid-associated CCNU, which allows increased tumor exposure. When formulated in small unilamellar liposomes (SUVs) of ∼80-nm diameter, we found substantially greater and prolonged plasma concentrations of liposomal CCNU compared with those of free drug. Many studies have shown that the size of liposomes is one of the most important determinants of their longevity in the bloodstream (12, 13). With decreased recognition by phagocytic cells of the reticuloendothelial systems, SUVs can remain significantly longer in the circulation than larger liposomes (12, 14, 15). The observation that long-circulating liposomes of small size (<100 nm) accumulate in the interstitial fluid of transplanted tumors at levels comparable with those organs enriched with cells of the reticuloendothelial systems, such as the liver, is the basis for renewed momentum in the search for liposomal drug formulations with potential applications in cancer therapy (11, 13, 16, 17). The enhanced drug accumulation in tumors is apparently related to prolonged circulation times of liposomes in vivo. When we compared CCNU concentrations in 36B-10 tumors to those in plasma after free and liposome-encapsulated drug administration, we found >10-fold higher CCNU concentrations in the tumor than plasma when the liposomal drug was administered. The results of tumor drug concentrations reaching, but not exceeding, the concentrations in plasma after free CCNU delivery is consistent with the knowledge that all of the clinically available nitrosoureas enter tumor cells via passive diffusion (18).

There are two likely mechanisms of liposome-encapsulated CCNU delivery to tumors: (a) release of drug from circulating liposomes followed by free distribution into all of the body compartments; and/or (b) extravasation of liposomes into the tumor interstitial fluid followed by in situ release of drug. Pharmacologically, the latter mechanism is the most interesting because it represents first-order targeting. Many types of tumors are well vascularized, increasing the opportunity for liposome delivery. In general, vessels within tumors have wide endothelial junctions, a large number of fenestrae, and discontinuous or absent basement membranes (19). Also, tumor angiogenesis includes the formation of capillary sprouts that are highly permeable because of gaps between adjacent endothelial cells and openings at the vessel termini (20). The 36B-10 glioma, when implanted s.c., is well vascularized and grows rapidly. The ability of liposome-encapsulated CCNU to extravasate into s.c. 36B-10 tumors was supported in this study by the observed slow increases in tumor drug concentrations that exceeded those in plasma at extended time points. This is consistent with the hypothesis that enhanced tumor drug accumulation by liposome-associated drug is attributable to extravasation of small liposomes to tumor and is similar to that described in other preclinical studies (12, 21). We found that tumor:plasma ratios were much higher than unity after liposomal CCNU administration. This can only be achieved if a significant fraction of the liposome-CCNU complex accumulates intact in the tumor. However, we cannot rule out concomitant drug release from a small fraction of liposomes in circulation and an eventual partitioning into tumor tissues. It is possible that sustained drug-release of CCNU from liposome may provide some therapeutic enhancement also. Additional studies are needed to examine whether CCNU is released exclusively in the plasma compartment and/or after transport to tumor sites.

Whereas liposome-formulated CCNU was found to increase therapeutic effects by reducing tumor volumes, we did not observe a concomitant increase in adverse effects. Similar results of increased efficacy and decreased toxicity have also been observed with liposomal formulations of doxorubicin (22, 23, 24). The relationship between plasma exposure to free-drug and hematological toxicity has been demonstrated for a number of commonly used anticancer agents, including CCNU (25). For liposomal drug delivery, bone marrow is considered a major site of interest for liposome localization because of possible drug toxicity and attendant hematotoxicity. In a detailed study of liposome biodistribution, Huang et al.(12) observed SUVs residing exclusively in the resident macrophages and not in the hematopoietic cells, in the bone marrow of mice after i.v. injection. This selective uptake in bone marrow may also be present with liposome-formulated CCNU and may explain the equal or decreased hematological toxicity compared with free CCNU administration. Additional studies are needed to elucidate these mechanisms.

To investigate potential mechanisms for the equal or decreased toxicity observed with liposomal CCNU, we quantitated the percentage of free and percentage of protein-bound drug after i.p. dosing. In this study, we found plasma free-fractions of CCNU to be approximately one-half of that after liposomal drug delivery compared with free CCNU delivery. These findings may provide one of the mechanisms that partially explains the reduced hematotoxicity observed with liposomal CCNU in this tumor model. It is likely that both altered cellular distribution and decreased plasma free-fractions of CCNU lead to the reduced side effects.

The plasma free-fraction results reported here also have relevance on clinical applications of systemic delivery of liposome-formulated CCNU to treat brain tumors. Whether a decrease in plasma free-fractions found after liposome delivery of CCNU may decrease the availability of CCNU to the brain remains to be determined. However, a number of studies indicate that plasma-protein-bound agents, such as tryptophan, oxicams, and methylprednisolone, are as available to cross the blood-brain barrier and enter the CNS as free pools of drug (29–28). We found that the decrease in plasma free-fractions, observed after liposomal CCNU treatment, did not result in a decreased tumoricidal effect in s.c. brain tumors. Therefore, the degree of transfer of CCNU to the brain after systemic delivery of liposomal CCNU is likely to be strongly influenced by the relative affinity of CCNU for the liposomes compared with its affinity for the cellular membranes of brain capillary endothelium. To elucidate whether lipid association alters CNS availability of CCNU, additional studies investigating the brain uptake of CCNU in normal and tumor-bearing animals after systemic administration of liposome-formulated CCNU should be performed. Additionally, direct administration of liposomal CCNU to tumors, possibly through Ommaya reservoirs, often implanted after surgical removal of brain tumor, may be used to evaluate whether CCNU that is formulated in liposomes can provide localized and sustained release of CCNU locally at or near brain tumors.

Liposome-formulated CCNU provided increased plasma exposure and tumor selectivity in tumor-bearing rats without exacerbating the systemic toxicity seen with free-drug treatment. The evidence presented here provides possible mechanisms for the increased therapeutic efficacy of CCNU found in a rat tumor model. Liposomal CCNU can be formulated with high efficiency and results in an improved margin of safety and activity of the drug to reduce tumor volumes compared with the effects of free-drug delivery.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

                  
3

The abbreviations used are: CCNU, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CNS, central nervous system; SUV, small unilamellar vesicle.

Fig. 1.

Free (▴) and liposomal CCNU (▪) inhibition of rat glioma cell growth in vitro. Cell growth inhibition was determined by [3H]thymidine incorporation assay as described previously (1). Data are expressed as means ± SD of a typical experiment of four repeated curves.

Fig. 1.

Free (▴) and liposomal CCNU (▪) inhibition of rat glioma cell growth in vitro. Cell growth inhibition was determined by [3H]thymidine incorporation assay as described previously (1). Data are expressed as means ± SD of a typical experiment of four repeated curves.

Close modal
Fig. 2.

Comparison of CCNU plasma concentrations in rats that were given free (solid bars) and liposomal (open bars) CCNU at indicated time points. Tumor-bearing rats were given 50 mg/kg by i.p. route. Date represent mean values from three animals at each time point.

Fig. 2.

Comparison of CCNU plasma concentrations in rats that were given free (solid bars) and liposomal (open bars) CCNU at indicated time points. Tumor-bearing rats were given 50 mg/kg by i.p. route. Date represent mean values from three animals at each time point.

Close modal
Fig. 3.

The time course of tumor progression after a single-dose treatment of 0 (▪), 20 (▴), 35 (○), and 50 (♦) mg/kg free (A) and liposomal (B) CCNU. Data points represent mean values from 3 animals at each time point. C, the effect of dose escalation on tumor progression after a single dose of free (▴) or liposomal CCNU (▪). Data represent mean ± SE for tumor volumes measured 21 days after treatment; n = 4 for each treatment; *, P < 0.05.

Fig. 3.

The time course of tumor progression after a single-dose treatment of 0 (▪), 20 (▴), 35 (○), and 50 (♦) mg/kg free (A) and liposomal (B) CCNU. Data points represent mean values from 3 animals at each time point. C, the effect of dose escalation on tumor progression after a single dose of free (▴) or liposomal CCNU (▪). Data represent mean ± SE for tumor volumes measured 21 days after treatment; n = 4 for each treatment; *, P < 0.05.

Close modal
Fig. 4.

The relative tumor:plasma concentration ratio at indicated time points for free (A) and liposomal (B) CCNU in tumor-bearing rats receiving 50 mg/kg CCNU i.p. ( The scales differ on the Y axes.) Data represent means ± SD.

Fig. 4.

The relative tumor:plasma concentration ratio at indicated time points for free (A) and liposomal (B) CCNU in tumor-bearing rats receiving 50 mg/kg CCNU i.p. ( The scales differ on the Y axes.) Data represent means ± SD.

Close modal
Table 1

Effect of single-dose treatment with free and liposome-encapsulated CCNU on tumor response and systemic toxicity

TreatmentaDose (mg/kg)Tumor volumeb (fold increase)WBCcPlateletcBody weightd (% change)
Control 89 ± 20 11 ± 1 
Free CCNU 20 54 ± 2 17 ± 7 23 ± 5 13 ± 3 
 35 39 ± 8 29 ± 6 19 ± 4 7 ± 5 
 50 45 ± 6 28 ± 8 14 ± 6 14 ± 3 
Liposomal CCNU 20 30 ± 8e 31 ± 5 16 ± 4 4 ± 7 
 35 23 ± 4e 20 ± 4 18 ± 4 0 ± 2 
 50 20 ± 3e 35 ± 4 7 ± 3 2 ± 1 
TreatmentaDose (mg/kg)Tumor volumeb (fold increase)WBCcPlateletcBody weightd (% change)
Control 89 ± 20 11 ± 1 
Free CCNU 20 54 ± 2 17 ± 7 23 ± 5 13 ± 3 
 35 39 ± 8 29 ± 6 19 ± 4 7 ± 5 
 50 45 ± 6 28 ± 8 14 ± 6 14 ± 3 
Liposomal CCNU 20 30 ± 8e 31 ± 5 16 ± 4 4 ± 7 
 35 23 ± 4e 20 ± 4 18 ± 4 0 ± 2 
 50 20 ± 3e 35 ± 4 7 ± 3 2 ± 1 
a

n = 4 animals for each treatment.

b

Tumor volume is expressed as the fold increase over the 21-day study period. Data are expressed as mean ± SD.

c

Mean % decrease ± SD change from untreated control group 4 days after treatment.

d

Mean % ± SD change from pretreatment value to 21 days after treatment.

e

P < 0.05 compared with free CCNU treatment.

Table 2

Effect of multiple-dose treatment with free and liposomal CCNU on tumor response and systemic toxicity

TreatmentDosea (mg/kg)Tumor volumeb (fold increase)WBCcPlateletcBody weightd (% decrease)
Control 82 ± 13 
Free CCNU 80 20 ± 9 73 ± 12 49 ± 10 15 ± 2 
 Ae (40, 20, 20)     
 100 18 ± 5 60 ± 8 63 ± 7 36 ± 2 
 Be (20, 40, 40)     
Liposomal CCNU 80 11 ± 2f 49 ± 3f 18 ± 5f 12 ± 3 
 Ae (40, 20, 20)     
 100 6 ± 4f 87 ± 6 87 ± 5 39 ± 4 
 Be (20, 40, 40)     
TreatmentDosea (mg/kg)Tumor volumeb (fold increase)WBCcPlateletcBody weightd (% decrease)
Control 82 ± 13 
Free CCNU 80 20 ± 9 73 ± 12 49 ± 10 15 ± 2 
 Ae (40, 20, 20)     
 100 18 ± 5 60 ± 8 63 ± 7 36 ± 2 
 Be (20, 40, 40)     
Liposomal CCNU 80 11 ± 2f 49 ± 3f 18 ± 5f 12 ± 3 
 Ae (40, 20, 20)     
 100 6 ± 4f 87 ± 6 87 ± 5 39 ± 4 
 Be (20, 40, 40)     
a

Total dose administered (the numbers in parentheses indicate the dose and sequence of CCNU given on a weekly basis; n = 8 animals for each treatment).

b

Tumor volume is expressed as the fold increase over the 21-day study period. Data are expressed as mean ± SD.

c

Mean % decrease ± SD change from control group 7 days after last dose.

d

Mean % ± SD change from pretreatment value to 22 days after treatment.

e

Dosing regimens of both lipid-encapsulated and free drug.

f

P < 0.05 compared with free CCNU treatment.

Table 3

Effect of liposome association on free and protein-bound fractions of CCNU in rat plasma

CCNU formulationFree fraction (%)Bound fraction (%)
Free 13.2 ± 1.4a 86.8 ± 1.4 
Liposomal 5.0 ± 1.5b,c 95.0 ± 1.5c 
CCNU formulationFree fraction (%)Bound fraction (%)
Free 13.2 ± 1.4a 86.8 ± 1.4 
Liposomal 5.0 ± 1.5b,c 95.0 ± 1.5c 
a

Mean ± SD from animals (n = 2) measured at 10, 15, 20, and 30 min after dosing.

b

Mean ± SD from animals (n = 2) measured at 30, 60, 120, and 360 min after dosing.

c

P < 0.05 between free and liposome-encapsulated CCNU; n = 8 (four samples collected at indicated time points above from two animals) for each formulation.

1
Bethune C., Blum A., Geyer J., Silber J., Ho R. Lipid association increases the potency against primary medulloblastoma cells and systemic exposure of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) in rats.
Pharm. Res. (NY).
,
16
:
896
-903,  
1999
.
2
Mayer L., Cullis P., Bally M. Designing therapeutically optimized liposomal anticancer delivery systems: lessons from conventional liposomes Lasic D. D. Papahadjopoulos D. eds. .
Medical Applications of Liposomes
,
:
231
-258, Elsevier Science B.V. New York  
1998
.
3
Gabizon A., Catane R., Uziely B. Prolonged circulation time and enhanced accumulation in malignant exudates of doxorubicin encapsulated in polyethylene-glycol coated liposomes.
Cancer Res.
,
54
:
987
-992,  
1994
.
4
Boman N. L., Bally B. B., Cullis P. R. Encapsulation of vincristine in liposomes reduces its toxicity and improves its antitumor efficacy.
J. Liposome Res.
,
5
:
523
-541,  
1995
.
5
Leserman L. D., Machy P., Barbet J. Cell-specific drug transfer from liposomes bearing monoclonal antibodies.
Nature (Lond.).
,
293
:
226
-228,  
1981
.
6
Nagahiro S., Yamamoto Y., Diksic M., Mitsuka S., Sugimoto S., Feindel W. Neurotoxicity after intracarotid nitrosourea administration: hemodynamic changes studied by double-tracer autoradiography.
Neurosurgery (Baltimore)
,
29
:
19
-26,  
1991
.
7
Carter K. S., Schabel T. M., Jr., Broler L. E., Jonston T. P. 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and other nitrosoureas in cancer treatment: a review.
Adv. Cancer Res.
,
16
:
273
-332,  
1972
.
8
Walker M. D., Green S. B., Byar D. P., Alexander E., Jr., Batzdorf M. J., Owens G., Ransohoff J., Roberston J. T., Shapiro W. R., Smith K. R., Jr., Wilson C. B., Strike T. A. Randomized comparisons of radiation and nitrosoureas for the treatment of malignant glioma after surgery.
N. Engl. J. Med.
,
303
:
1323
-1329,  
1980
.
9
Spence A. M., Coates P. W. Scanning electron microscopy of cloned astrocytic lines derived from ethylnitrosourea-induced rat glioma.
Virchows Arch. B Cell Pathol.
,
28
:
77
-85,  
1978
.
10
Mayer L., St.-Onge G. Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques.
Anal. Biochem.
,
232
:
149
-157,  
1995
.
11
Spence A. M., Geraci J. P. Combined cyclotron fast-neutron and BCNU therapy in a rat brain-tumor model.
J. Neurosurg.
,
54
:
461
-467,  
1981
.
12
Huang S. K., Lee K.-D., Hong K., Friend D. S., Papahadjopoulos D. Microscopic localization of sterically stabilized liposomes in colon carcinoma-bearing mice.
Cancer Res.
,
52
:
5135
-5143,  
1992
.
13
Proffitt R. T., Williams L. E., Presant C. A., Tin G. W., Uliana J. A., Gamble R. C., Baldeschwieler J. D. Liposomal blockade of the reticuloendothelial system: improved tumor imaging with small unilamellar vesicles.
Science (Wash. DC)
,
220
:
502
-505,  
1983
.
14
Allen T., Everest J. The effect of particle size and drug release properties on pharmacokinetics of encapsulated drug in rats.
J. Pharmacol. Exp. Ther.
,
266
:
539
-554,  
1983
.
15
Juliano R., Stamp D. The effect of particle size and charge on the clearance rates of liposomes and liposome encapsulated drugs.
Biochem. Biophys. Res. Commun.
,
63
:
651
-658,  
1975
.
16
Ogihara-Umeda I., Kojima S. Increased delivery of gallium-67 to tumors using serum-stable liposomes.
J. Nucl. Med.
,
29
:
516
-523,  
1988
.
17
Gabizon A., Price D., Huberty J., Bresalier R. S., Papahadjopoulos D. Effect of liposome composition and other factors on the targeting of liposomes to experimental tumors: biodistribution and imaging studies.
Cancer Res.
,
50
:
6371
-6378,  
1990
.
18
Begleiter A., Lam H-Y. P., Goldenberg G. J. Mechanism of uptake of nitrosourea by L5178Y lymphoblasts in vitro.
Cancer Res.
,
37
:
1022
-1027,  
1977
.
19
Jain R. Transport of molecules across tumor vasculature.
Cancer Metastasis Rev.
,
6
:
559
-593,  
1987
.
20
Folkman J., Haudenschild C. Induction of capillary growth in vitro Dingle J. Gordon J. eds. .
Cellular Interactions
,
:
119
-136, Elsevier Amsterdam  
1981
.
21
Gabizon A. Selective tumor localization and improved therapeutic index of anthracyclines encapsulated in long-circulating liposomes.
Cancer Res.
,
52
:
891
-896,  
1992
.
22
Gabizon A., Dagan A., Goren D., Barenholz Y., Fuks Z. Liposomes as in vivo carriers of Adriamycin: reduced cardiac uptake and preserved antitumor activity in mice.
Cancer Res.
,
42
:
4734
-4739,  
1982
.
23
Gabizon A., Shiota R., Papahadjopoulos D. Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times.
J. Natl. Cancer Inst. (Bethesda)
,
81
:
1484
-1488,  
1989
.
24
Mayer L. D., Bally M. B., Cullis P. R., Wilson S. L., Emerman J. T. Comparison of free and liposome encapsulated doxorubicin tumor drug uptake and antitumor efficacy in the SC115 murine mammary tumor.
Cancer Lett.
,
53
:
183
-190,  
1990
.
25
Ratain M. J., Schilsky R. L. R., Conley B. A. Pharmacodynamics in cancer therapy.
J. Clin. Oncol.
,
8
:
1739
-1753,  
1990
.
26
Pardridge W. New directions in blood-brain barrier research. Studies with isolated human brain capillaries.
Ann. NY Acad. Sci.
,
529
:
50
-60,  
1988
.
27
Jolliet P., Simon N., Bree F., Urien S., Pagliara A., Carrupt P. A., Testa B., Tillement J. P. Blood-to-brain transfer of various oxicams: effects of plasma binding on their brain delivery.
Pharm. Res. (NY)
,
14
:
650
-656,  
1997
.
28
Chen T., Mackic J., McComb J., Giannotta S., Weiss M., Zlokovic B. Cellular uptake and transport of methylprednisolone at the blood-brain barrier.
Neurosurgery (Baltimore)
,
38
:
348
-354,  
1996
.