Therapeutically optimized rates of drug release can be achieved by varying the drug-to-lipid ratio in liposomal vincristine formulations

The anti-tumor efficacy of liposomal formulations of cell cycle dependent anticancer drugs is critically dependent on the rates at which the drugs are released from the liposomes. Previous work on liposomal formulations of vincristine have shown increasing efficacy for formulations with progressively slower release rates. Recent work has also shown that liposomal formulations of vincristine with higher drug-to-lipid (D/L) ratios exhibit reduced release rates. In this work, the effects of very high D/L ratios on vincristine release rates are investigated, and the antitumor efficacy of these formulations characterized in human xenograft tumor models. It is shown that the half-times (T(1/2)) for vincristine release from egg sphingomyelin/cholesterol liposomes in vivo can be adjusted from T(1/2) = 6.1 h for a formulation with a D/L of 0.025 (wt/wt) to T(1/2) = 117 h (extrapolated) for a formulation with a D/L ratio of 0.6 (wt/wt). The increase in drug retention at the higher D/L ratios appears to be related to the presence of drug precipitates in the liposomes. Variations in the D/L ratio did not affect the circulation lifetimes of the liposomal vincristine formulations. The relationship between drug release rates and anti-tumor efficacy was evaluated using a MX-1 human mammary tumor model. It was found that the antitumor activity of the liposomal vincristine formulations increased as D/L ratio increased from 0.025 to 0.1 (wt/wt) (T(1/2) = 6.1-15.6 h respectively) but decreased at higher D/L ratios (D/L = 0.6, wt/wt) (T(1/2) = 117 h). Free vincristine exhibited the lowest activity of all formulations examined. These results demonstrate that varying the D/L ratio provides a powerful method for regulating drug release and allows the generation of liposomal formulations of vincristine with therapeutically optimized drug release rates.     

Qualitative and Quantitative One-step Analysis of Lipids and Encapsulated Bioactive Molecules in Liposome Preparations by HPTLC/FID (IATROSCAN)

Liposomes are widely used vehicles for the delivery of bioactive molecules. They are composed mainly from acyl-phosphatidylcholines, cholesterol, and charged lipids (e.g., stearylamine, dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine).

The incorporation efficiencies of the bioactive molecule and the drug to lipid molar ratio are important factors for the assessment of the liposomal formulation. In order to successfully characterize a liposomal formulation, it is necessary to be able to accurately measure the lipids and the encapsulated molecule, using the smallest possible sample.

The present work describes an analytical methodology on qualitative and quantitative determination of all the lipid ingredients that are involved in the liposome formulation, as well as the drug incorporation and the drug–lipid ratio, by a simultaneous measurement of all the liposomal ingredients using thin-layer chromatography coupled with a flame ionization detector (HPTLC/FID).

The procedure requires only one measurement per sample, and it can be applied even in very small or much diluted samples.

The proposed analytical method can be applied in general on all steps of the development of liposomal formulations. The purity and stability of the raw materials can also be easily evaluated. In addition the preparation procedure can be tracked in order to locate possible losses of raw material and errors of the preparation method resulting in the amelioration of the method.     

Qualitative and quantitative one-step analysis of lipids and encapsulated bioactive molecules in liposome preparations by HPTLC/FID (IATROSCAN)

Liposomes are widely used vehicles for the delivery of bioactive molecules. They are composed mainly from acyl-phosphatidylcholines, cholesterol, and charged lipids (e.g., stearylamine, dipalmitoylphosphatidylglycerol (DPPG), phosphatidylethanolamine).The incorporation efficiencies of the bioactive molecule and the drug to lipid molar ratio are important factors for the assessment of the liposomal formulation. In order to successfully characterize a liposomal formulation, it is necessary to be able to accurately measure the lipids and the encapsulated molecule, using the smallest possible sample.The present work describes an analytical methodology on qualitative and quantitative determination of all the lipid ingredients that are involved in the liposome formulation, as well as the drug incorporation and the drug-lipid ratio, by a simultaneous measurement of all the liposomal ingredients using thin-layer chromatography coupled with a flame ionization detector (HPTLC/FID).The procedure requires only one measurement per sample, and it can be applied even in very small or much diluted samples.The proposed analytical method can be applied in general on all steps of the development of liposomal formulations. The purity and stability of the raw materials can also be easily evaluated. In addition the preparation procedure can be tracked in order to locate possible losses of raw material and errors of the preparation method resulting in the amelioration of the method.     

Optimization of prednisolone-loaded long-circulating liposomes via application of Quality by Design (QbD) approach

Quality by design principles (QbD) were used to assist the formulation of prednisolone-loaded long-circulating liposomes (LCL-PLP) in order to gain a more comprehensive understanding of the preparation process. This approach enables us to improve the final product quality in terms of liposomal drug concentration, encapsulation efficiency and size, and to minimize preparation variability. A 19-run D-optimal experimental design was used to study the impact of the highest risk factors on PLP liposomal concentration (Y₁- μg/ml), encapsulation efficiency (Y₂-%) and size (Y₃-nm). Out of six investigated factors, four of them were identified as critical parameters affecting the studied responses. PLP molar concentration and the molar ratio of DPPC to MPEG-2000-DSPE had a positive impact on both Y₁ and Y₂, while the rotation speed at the formation of the lipid film had a negative impact. Y₃ was highly influenced by prednisolone molar concentration and extrusion temperature. The accuracy and robustness of the model was further on confirmed. The developed model was used to optimize the formulation of LCL-PLP for efficient accumulation of the drug to tumor tissue. The cytotoxicity of the optimized LCL-PLP on C26 murine colon carcinoma cells was assessed. LCL-PLP exerted significant anti-angiogenic and anti-inflammatory effects on M2 macrophages, affecting indirectly the C26 colon carcinoma cell proliferation and development.     

Fast high-throughput screening of temoporfin-loaded liposomal formulations prepared by ethanol injection method

A new strategy for fast, convenient high-throughput screening of liposomal formulations was developed, utilizing the automation of the so-called ethanol-injection method. This strategy was illustrated by the preparation and screening of the liposomal formulation library of a potent second-generation photosensitizer, temoporfin. Numerous liposomal formulations were efficiently prepared using a pipetting robot, followed by automated size characterization, using a dynamic light scattering plate reader. Incorporation efficiency of temoporfin and zeta potential were also detected in selected cases. To optimize the formulation, different parameters were investigated, including lipid types, lipid concentration in injected ethanol, ratio of ethanol to aqueous solution, ratio of drug to lipid, and the addition of functional phospholipid. Step-by-step small liposomes were prepared with high incorporation efficiency. At last, an optimized formulation was obtained for each lipid in the following condition: 36.4 mg·mL(-1) lipid, 13.1 mg·mL(-1) mPEG(2000)-DSPE, and 1:4 ethanol:buffer ratio. These liposomes were unilamellar spheres, with a diameter of approximately 50?nm, and were very stable for over 20 weeks. The results illustrate this approach to be promising for fast high-throughput screening of liposomal formulations.     

Liposomal amphotericin-B in the control of experimental aspergillosis in mice: Part I--Relative therapeutic efficacy of free and liposomal amphotericin-B

An animal model system for aspergillosis in BALB/c mice has been developed to evaluate the therapeutic efficacy of liposomal Amphotericin-B (Amp-B) and commercial Amp-B (Fungizone). Amp-B was intercalated into liposomes composed of egg phosphatidylcholine, phosphatidylethanolamine and cholesterol in a molar ratio of 6:1:3. A single dose (0.5 mg/kg body wt) of Amp-B both alone as well as in liposomal preparation was injected (i.v.) into animals infected with Aspergillus fumigatus. An increase in survival rate of animals and decrease in fungal count in lung, the most affected organ, were observed with liposomal formulation. Inclusion of Amp-B into liposomes also reduced the toxicity of the drug. Tissue distribution analysis of Amp-B by HPLC showed an increase in concentration of the drug in lung for both free and liposomal Amp-B in infected animals as compared to normal. Use of liposomal Amp-B increased the concentration of the drug in the disease affected organs such as lung, spleen. A longer persistence of the drug in the infected organs was also observed. The results suggest that inclusion of Amp-B in liposomes decreases its toxicity and improves therapeutic efficacy which at least in part could be due to more deposition and longer persistence of the drug in infected tissues.     

Advanced protein formulations

It is well recognized that protein product development is far more challenging than that for small‐molecule drugs. The major challenges include inherent sensitivity to different types of stresses during the drug product manufacturing process, high rate of physical and chemical degradation during long‐term storage, and enhanced aggregation and/or viscosity at high protein concentrations. In the past decade, many novel formulation concepts and technologies have been or are being developed to address these product development challenges for proteins. These concepts and technologies include use of uncommon/combination of formulation stabilizers, conjugation or fusion with potential stabilizers, site‐specific mutagenesis, and preparation of nontraditional types of dosage forms—semiaqueous solutions, nonfreeze‐dried solid formulations, suspensions, and other emerging concepts. No one technology appears to be mature, ideal, and/or adequate to address all the challenges. These gaps will likely remain in the foreseeable future and need significant efforts for ultimate resolution.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

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.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

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 degrees 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.     

Design of liposomes to improve delivery of amphotericin-B in the treatment of aspergillosis

The efficacy and toxicity of free and liposome intercalated amphotericin-B (Amp-B) in controlling Aspergillosis, caused byAspergillus fumigatus in BALB/c mice were studied. Liposomal Amp-B had higher LD₅₀ (8.1 mg/kg) as compared to that of the free drug (1.2 mg/kg). An improvement in the therapeutic index of the drug was observed with liposomal formulation of the drug. We also focussed on the effect of lipid composition and surface sugar in modulating the therapeutic potency of Amp-B. The most effective liposomal preparation was composed of egg phosphatidylcholine (EPC) : L--phosphatidylethanolamine, dipalmitoyl (DPPE): cholesterol (Chol) in the molar ratio of 6:1:3. Amp-B intercalated into mannose grafted liposomes (LD₅₀ = 9.3 mg/kg) was more effective as compared to the other formulation tested.     

Effect of Lipid Composition on Topical Delivery of Cyclosporin-A from Nonionic Ldtosomal Formulations: An in Vitro Study with Hairless Mouse Skin

Abstract

Previous studies in our laboratories showed that a novel nonionic liposome formulation composed of glyceryl dilaurate (GDL), cholesterol (CH), and polyoxyethylene-10-stearyl ether (POE-10) at a weight ratio composition of 57:15:28 delivered more cyclosporin-A (CsA) into and through the skin than phospholipid-based liposomal formulations and more conventional formulations that were tested. Since only a single GDL:CH:POE-10 composition was tested, we initiated studies to determine if it would be possible to control the rate and extent of drug uptake by varying the ratios of the liposome-forming components of the formulation. This report describes how the GDL to POE-10 ratio (CH being held constant at 15 wt%) influences the rate and extent of uptake of CsA following topical application of nonionic liposomal formulations to hairless mouse skin mounted on Franz diffusion cells. The results indicate that the rate and extent of CsA uptake is highest between GDL/POE-10 ratios of about 1 to 1.5 and decreases steadily at ratios above and below this range. The effect of liposomal composition on CsA deposition is probably the result of a number of complex and interrelated factors including partitioning of CsA from the formulation into the skin and permeation enhancer effects.     

Simultaneous Determination of Polyethylene Glycol-Conjugated Liposome Components by Using Reversed-Phase High-Performance Liquid Chromatography with UV and Evaporative Light Scattering Detection

Liposomes incorporating polyethylene glycol (PEG)-conjugated lipids (PEGylated liposomes) have attracted attention as drug delivery carriers because they show good in vivo stability. The lipid component of PEGylated liposomal formulations needs to be quantified for quality control. In this study, a simple reversed-phase high-performance liquid chromatography (HPLC) method with an evaporative light-scattering detector (ELSD) was established for simultaneous determination of hydrogenated soy phosphatidylcholine, cholesterol, PEG-conjugated lipid, and hydrolysis products of phospholipid in PEGylated liposomal formulations. These lipids were separated using a C18 column with a gradient mobile phase consisting of ammonium acetate buffer and ammonium acetate in methanol at a flow rate of 1.0 ml/min. This method provided sufficient repeatability, linearity, and recovery rate for all lipids. However, the linearity and recovery rates of cholesterol achieved using a ultraviolet (UV) detector were better than those achieved using an ELSD. This validated method can be applied to assess the composition change during the preparation process of liposomes and to quantify lipid components and hydrolysis products contained in a commercially available liposomal formulation DOXIL®. Taken together, this reversed-phase HPLC-UV/ELSD method may be useful for the rapid or routine analysis of liposomal lipid components in process development and quality control.     

Sulfosalicylate mediates improved vinorelbine loading into LUVs and antineoplastic effects

Liposomal vinorelbine formulation is desirable, as it might improve the therapeutic activity of vinorelbine. However, because of its lipophilic and membrane-permeable properties, vinorelbine is hard to be formulated into liposomes using conventional drug-loading technologies. To improve vinorelbine retention, ammonium salts of several anionic agents were employed to prepare liposomal vinorelbine formulations. It was found that 5-sulfosalicylate (5ssa) could form stable complexes with vinorelbine and stabilize entrapped vinorelbine. The resultant vesicles had an in vitro release t(1/2) of ~12.49 hours in NH(3)-containing media, which is longer than those of sulfate and phytate vesicles (~0.57 hours). The circulation half-life of vinorelbine after the injection of 5ssa vesicles into normal mice was ~13.01 hours, accounting for ~2-fold increase relative to that of sulfate vesicles. Improved drug retention correlated with enhanced antitumor efficacy. In the RM-1/c57 model, 5ssa vesicles were more efficacious than sulfate vesicles (P?相似文献   

Development of stealth liposome coencapsulating doxorubicin and fluoxetine

Stealth liposomes form an important subset of liposomes, demonstrating prolonged circulation half-life and improved safety in vivo. Caelyx? (liposomal doxorubicin; Merck & Co., Whitehouse Station, New Jersey, USA) is a successful example of the application of stealth liposomes in anticancer treatment. However, multidrug resistance (MDR) to chemotherapy still remains a critical problem, accounting for more than 90% of treatment failure in patients with advanced cancer. To circumvent MDR, fluoxetine and doxorubicin were tested in combination for synergistic activity in MCF-7 (human breast carcinoma) and MCF-7/adr (doxorubicin-resistant human breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cell-viability assay. Coencapsulation of doxorubicin and fluoxetine, using an ammonium sulphate gradient, was investigated, and a factorial experiment was designed to determine the optimal drug-to-lipid (D/L) ratio for coencapsulation. Drug release from Dox-Flu-SL (stealth liposome coencapsulating doxorubicin and fluoxetine) under both in vitro and in vivo conditions was determined. In MCF-7 cells, synergism was demonstrated at specific doxorubicin:fluoxetine ratios of between 0.09 and 0.5 (molar ratio), while MCF/7/adr cells demonstrated synergism across all drug ratios. Coencapsulation of doxorubicin and fluoxetine (Dox-Flu-SL) was successfully achieved (optimal doxorubicin:fluoxetine:lipid molar ratio of 0.02:0.05:1), obtaining a mean concentration of 257 ± 12.1 and 513 ± 29.3 μM for doxorubicin and fluoxetine, respectively. Most important, Dox-Flu-SL demonstrated drug release in synergistic ratios in cell-culture media, accounting for the improved cytotoxicity of Dox-Flu-SL over liposomal doxorubicin (LD) in both MCF-7 and MCF-7/adr cells. Pharmacokinetic studies also revealed that Dox-Flu-SL effectively prolonged drug-circulation time and reduced tissue biodistribution. Dox-Flu-SL presents a promising anticancer formulation, capable of effective reversal of drug resistance, and may constitute a novel approach for cancer therapy.     

Separation and determination of liposomal and non-liposomal daunorubicin from the plasma of patients treated with Daunoxome

Several liposomal formulations of anthracyclines have been developed recently and are currently used in the clinical setting. We describe a technique of separation and quantification of the liposomal and non-liposomal forms of daunorubicin in the plasma of patients treated with DaunoXome, a liposomal formulation of daunorubicin. The method we propose is based upon the property of liposomes to cross reversed-phase C₁₈ silicagel cartridges without being retained, while non-liposomal drug is retained on the stationary phase and is eluted with methanol. Extraction of liposomal and non-liposomal daunorubicin from plasma, therefore, is performed in two steps. This technique is rapid, can be automated in order to handle large series of samples, and the plasma can be frozen after sampling by addition of glycerol. The recovery of liposomal daunorubicin as well as the precision, linearity and accuracy of the technique appear satisfactory for pharmacokinetic purposes.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Factors affecting the pharmacokinetics and pharmacodynamics of liposomal drugs

Various attempts to increase the therapeutic index of the drug while minimizing side effects have been made in drug delivery systems. Among several promising strategies, liposomes represent an advanced technology to target active molecules to the site of action. Rapid clearance of circulating liposomal drugs administered intravenously has been a critical issue because circulation time in the blood affects drug exposure at the target site. The clinical use of liposomal drugs is complicated by large intra- and interindividual variability in their pharmacokinetics (PK) and pharmacodynamics (PD). Thus, it is important to understand the factors affecting the PK/PD of the liposomal formulation of drugs and to elucidate the mechanisms underlying the variability in the PK/PD of liposomal drugs. In this review article, we describe the characteristics of liposome formulations and discuss the effects of various factors, including liposome-associated factors, host-associated factors, and treatment on the PK/PD of liposomal agents.     

Strategies for Optimizing Liposomal Doxorubicin

Abstract

Liposome encapsulation of doxorubicin can dramatically alter its biological activity, resulting in decreased toxicity and equivalent or increased antitumor potency. Since the physical characteristics of the liposome carrier system (size, lipid composition, and lipid dose) can have profound effects on the pharmacologic properties of vesicles administered intravenously, it may be expected that the therapeutic activity of liposomal doxorubicin will be sensitive to these properties. To determine the influence of these variables on the toxicity and efficacy properties of liposomal doxorubicin, transmembrane pH gradient-dependent active encapsulation techniques have been utilized to generate liposomal doxorubicin preparations in which the vesicle size, lipid composition, and drug to lipid ratio can be independently varied. these studies indicate that the toxicity of liposomal doxorubicin is related to the stability of the preparation in the circulation. This property is dictated primarily by vesicle lipid composition, although the drug to lipid ratio can also exert an influence. In contrast, the antitumor activity of liposomal doxorubicin appears most sensitive to the size of the vesicle system. Specifically, antitumor drug potency increases as the vesicle size is decreased. these studies demonstrate that manipulating the physical characteristics of liposomal anticancer pharmaceuticals can lead to preparations with optimized therapeutic activity.     

Sulfosalicylate mediates improved vinorelbine loading into LUVs and antineoplastic effects

Liposomal vinorelbine formulation is desirable, as it might improve the therapeutic activity of vinorelbine. However, because of its lipophilic and membrane-permeable properties, vinorelbine is hard to be formulated into liposomes using conventional drug-loading technologies. To improve vinorelbine retention, ammonium salts of several anionic agents were employed to prepare liposomal vinorelbine formulations. It was found that 5-sulfosalicylate (5ssa) could form stable complexes with vinorelbine and stabilize entrapped vinorelbine. The resultant vesicles had an in vitro release t_1/2 of ~12.49 hours in NH₃-containing media, which is longer than those of sulfate and phytate vesicles (~0.57 hours). The circulation half-life of vinorelbine after the injection of 5ssa vesicles into normal mice was ~13.01 hours, accounting for ~2-fold increase relative to that of sulfate vesicles. Improved drug retention correlated with enhanced antitumor efficacy. In the RM-1/c57 model, 5ssa vesicles were more efficacious than sulfate vesicles (P?<?0.05). In RM-1/BDF1 and Lewis lung cancer/c57 models, antitumor efficacy was also considerably improved after vinorelbine encapsulation into 5ssa vesicles. For instance, in the RM/BDF1 model, liposomal vinorelbine was at least 4-fold more therapeutically active than free vinorelbine. Our results demonstrated that 5ssa could stabilize vinorelbine relative to other anions, resulting in the formulation with improved drug retention and efficacy. Improved vinorelbine retention might be associated with the formation of insoluble precipitate, which could be proved by precipitation study and decreased drug-release rate at a high D/L ratio.     

Liposomal Anticancer Drugs as Agents to be used in Combination with other Anticancer Agents: Studies on a Liposomal Formulation with two Encapsulated Drugs

Abstract

When considering the use of combination therapies with liposomal anticancer agents several approaches can be defined. One approach could rely on administration of one liposomal formulation with more than one entrapped cytotoxic drug. This study focuses on an assessment of a liposomal formulation containing vincristine and mitoxantrone. Distearoyl phosphatidylcholine (DSPC)/Cholesterol (Choi) (55:45 molar ratio) liposomes were loaded with vincristine using transmembrane pH gradients. These systems were subsequently incubated with mitoxantrone to effect uptake of the second drug. Retention of both drugs was determined in vitro and in vivo. In vitro drug release indicated >95% retention of mitoxantrone and approximately 75% retention of vincristine when liposomes were prepared with an initial interior pH of 2.0. In vivo results however, demonstrated that greater than 80% of the encapsulated vincristine was released within 1 hour following i.v. administration. The instability of a liposomal formulation containing two anticancer drugs following i.v. administration may be a consequence of a combination of factors including drug-loading induced collapse of the transmembrane pH gradient, loss due to osmotic effects and an associated insertion of serum proteins into the bilayer, as well as the presence of a large biological “sink” which can alter the transbilayer drug gradient in favor of drug release.