Tuning Ciprofloxacin Release Profiles from Liposomally Encapsulated Nanocrystalline Drug.

PURPOSE: In order to attenuate the drug release rate, a single freeze-thaw step was previously shown to convert encapsulated drug into a single nanocrystal within each liposome vesicle. The goal of this study was to alter the nanocrystalline character, and thus the drug encapsulation state and release profile, by addition of surfactant prior to freeze-thaw. METHODS: A liposomal ciprofloxacin (CFI) formulation was modified by the addition of surfactant and frozen. After thawing, these formulations were characterized in terms of drug encapsulation by centrifugation-filtration, liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, and in vitro release (IVR) performance. RESULTS: The addition of increasing levels of polysorbate 20 (0.05 to 0.4%) or Brij 30 (0.05 to 0.3%) to the CFI preparations followed by subsequent freeze-thaw, resulted in a greater proportion of vesicles without drug nanocrystals and reduced the extent of growth of the nanocrystals thus leading to modified release rates including an increase in the ratio of non-encapsulated to sustained release of drug. CONCLUSIONS: This study provides another lever to achieve the desired release rate profile from a liposomal formulation by addition of surfactant and subsequent freeze-thaw, and thus may provide a personalized approach to treating patients.

Non-PEGylated liposomes for convection-enhanced delivery of topotecan and gadodiamide in malignant glioma: initial experience.

Convection-enhanced delivery (CED) of highly stable PEGylated liposomes encapsulating chemotherapeutic drugs has previously been effective against malignant glioma xenografts. We have developed a novel, convectable non-PEGylated liposomal formulation that can be used to encapsulate both the topoisomerase I inhibitor topotecan (topoCED) and paramagnetic gadodiamide (gadoCED), providing an ideal basis for real-time monitoring of drug distribution. Tissue retention of topoCED following single CED administration was significantly improved relative to free topotecan. At a dose of 10 microg (0.5 mg/ml), topoCED had a half-life in brain of approximately 1 day and increased the area under the concentration-time curve (AUC) by 28-fold over free topotecan (153.8 vs. 5.5 microg day/g). The combination of topoCED and gadoCED was found to co-convect well in both naïve rat brain and malignant glioma xenografts (correlation coefficients 0.97-0.99). In a U87MG cell assay, the 50% inhibitory concentration (IC(50)) of topoCED was approximately 0.8 microM at 48 and 72 h; its concentration-time curves were similar to free topotecan and unaffected by gadoCED. In a U87MG intracranial rat xenograft model, a two-dose CED regimen of topoCED co-infused with gadoCED greatly increased median overall survival at dose levels of 0.5 mg/ml (29.5 days) and 1.0 mg/ml (33.0 days) vs. control (20.0 days; P < 0.0001 for both comparisons). TopoCED at higher concentrations (1.6 mg/ml) co-infused with gadoCED showed no evidence of histopathological changes attributable to either agent. The positive results of tissue pharmacokinetics, co-convection, cytotoxicity, efficacy, and lack of toxicity of topoCED in a clinically meaningful dose range, combined with an ideal matched-liposome paramagnetic agent, gadoCED, implicates further clinical applications of this therapy in the treatment of malignant glioma.

Development of catheter-based procedures for transducing the isolated rabbit liver with plasmid DNA.

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.

DNA sequences in cationic lipid:pDNA-mediated systemic toxicities.

Systemic delivery of synthetic gene transfer vectors such as cationic lipid:plasmid DNA (pDNA) complexes elicits a range of acute physiologic responses, which in the context of therapeutic gene delivery represent dose-limiting toxicities. The most prominent responses are transient leukopenia, thrombocytopenia, serum transaminase elevations, and elevations of proinflammatory cytokines such as interferon-gamma (IFN-gamma), interleukin-12 (IL-12), and tumor necrosis factor-alpha (TNF-alpha). The unmethylated CpG sequences present in plasmid DNA have been implicated as a major cause of the robust cytokine response that follows systemic administration of cationic lipid:pDNA complexes. However, the factors causing the additional significant toxicities (leukopenia, thrombocytopenia, and serum transaminase elevations) recently shown to be associated with vector administration have not been defined. We show here that DNA sequences, such as immune stimulatory CpG sequences, play a significant role in inducing the additional acute toxicities associated with cationic lipid:pDNA complex administration. Importantly, while methylating these CpG sequences results in greatly reduced cytokine levels, this modification does not eliminate their ability to generate the other systemic toxicities. Examples of non-CpG DNA sequences that induce distinct toxicity profiles when administered systemically in the form of cationic lipid:DNA complexes are also identified. Taken together, these results imply that specific DNA sequences are responsible for a significant portion of the systemic toxicities observed after administration of cationic lipid:pDNA complexes.

Modifying the release properties of liposomes toward personalized medicine.

Surfactant-liposome interactions have historically been investigated as a simplified model of solubilization and breakdown of biological membranes by surfactants. In contrast, our goal was to utilize surfactants to modify the encapsulation and release properties of liposomes. The ability to manufacture one liposomal formulation, which could be modified by the addition of a surfactant to support a wide range of release profiles, would provide greater flexibility than manufacturing multiple batches of liposomes, each differing in composition and with its own specific release profile. A liposomal ciprofloxacin formulation was modified by the addition of various surfactants. These formulations were characterized in terms of liposome structure by cryo-TEM imaging, vesicle size by dynamic light scattering, drug encapsulation by centrifugation-filtration, and in vitro release (IVR) performance. The addition of polysorbate 20 or polysorbate 80 to liposomal ciprofloxacin, in a hypotonic environment, resulted in a concentration-dependent loss of encapsulated drug, and above 0.4% polysorbate 20, or 0.2% polysorbate 80, a modified IVR profile as well. This study demonstrates that the encapsulation and release properties of a liposomal formulation can be modified postmanufacture by the addition of judiciously chosen surfactants in combination with osmotic swelling of the liposomes and may support a personalized approach to treating patients.