Rational design of cationic lipids for siRNA delivery.

We adopted a rational approach to design cationic lipids for use in formulations to deliver small interfering RNA (siRNA). Starting with the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA), a key lipid component of stable nucleic acid lipid particles (SNALP) as a benchmark, we used the proposed in vivo mechanism of action of ionizable cationic lipids to guide the design of DLinDMA-based lipids with superior delivery capacity. The best-performing lipid recovered after screening (DLin-KC2-DMA) was formulated and characterized in SNALP and demonstrated to have in vivo activity at siRNA doses as low as 0.01 mg/kg in rodents and 0.1 mg/kg in nonhuman primates. To our knowledge, this represents a substantial improvement over previous reports of in vivo endogenous hepatic gene silencing.

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.

Optimization and characterization of a sphingomyelin/cholesterol liposome formulation of vinorelbine with promising antitumor activity.

Vinorelbine (VRL) is a particularly lipophilic member of the vinca alkaloids which, as a class of drugs, exhibit improved cytotoxicity and therapeutic activity through increased duration of exposure. Here, we describe and optimize a sphingomyelin/cholesterol (SM/Chol) liposome formulation of VRL to maximize in vivo drug retention, plasma circulation time, and therapeutic activity. VRL was efficiently encapsulated (>90%) into 100 nm liposomes using an ionophore-mediated loading method. VRL retention in SM/Chol liposomes after intravenous injection in mice was dependent on drug-to-lipid ratio (D/L), with higher D/L ratios exhibiting increased drug retention (0.3 > 0.2 > 0.1, wt/wt) and improved pharmacokinetics. Cryo-electron microscopic examination of a high D/L ratio formulation indicated that the intravesicular regions of these liposomes were electron dense compared with empty liposomes. The optimized, high D/L ratio SM/Chol VRL formulation showed promising activity against subcutaneous B16 melanoma tumors compared with VRL or SM/Chol formulations of vincristine or vinblastine. Finally, the stability of the formulation was excellent (<5% drug leakage, >99% intact VRL, no changes in liposome size after 1 year at 2-8 degrees C). The optimized drug retention properties of the SM/Chol formulation of VRL, combined with its promising antitumor activity and pharmaceutical stability, make this formulation an excellent candidate for future clinical development.

Immunogenicity and rapid blood clearance of liposomes containing polyethylene glycol-lipid conjugates and nucleic Acid.

Polyethylene glycol (PEG) is used widely in the pharmaceutical industry to improve the pharmacokinetics and reduce the immunogenicity of therapeutic and diagnostic agents. The incorporation of lipid-conjugated PEG into liposomal drug delivery systems greatly enhances the circulation times of liposomes by providing a protective, steric barrier against interactions with plasma proteins and cells. Here we report that liposome compositions containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide (ODN) or plasmid DNA elicit a strong immune response that results in the rapid blood clearance of subsequent doses in mice. The magnitude of this response is sufficient to induce significant morbidity and, in some instances, mortality. This effect has been observed in several strains of mice and was independent of sequence motifs, such as immunostimulatory CpG motifs. The ODN-to-lipid ratio and ODN dose was also determined to be important, with abrogation of the response occurring at a ratio between 0.04 and 0.08 (w/w). Rapid elimination of liposome-encapsulated ODN from blood depends on the presence of PEG-lipid in the membrane because the use of nonpegylated liposomes or liposomes containing rapidly exchangeable PEG-lipid also abrogated the response. These studies have important implications for the evaluation and therapeutic use of liposomal formulations of nucleic acid, as well as the potential development of liposomal vaccines.

Epicutaneous application of CpG oligodeoxynucleotides with peptide or protein antigen promotes the generation of CTL.

Immunostimulatory oligodeoxynucleotides (ODN) are effective adjuvants in the induction of humoral and cellular immune responses when administered parenterally with antigen. The skin has recently become a target organ for the design of non-invasive vaccine technologies. Using ovalbumin (OVA) as a model antigen, we demonstrate that the application of ODN sequences to tape-stripped skin promotes the induction of potent cytotoxic T lymphocyte (CTL) responses to co-administered peptide. Induction of peptide-specific CTL required the presence of CpG motifs within the ODN. CTL afforded tumor protection against a tumor expressing an immunodominant OVA CTL epitope. CTL could also be induced to whole protein administered onto the skin. Differential CpG sequence activity was noted with respect to the induction of CTL to epicutaneous protein with an ODN sequence containing a poly-G motif having an optimal effect. Peptide-specific CTL could be detected in the peripheral blood as early as 6 d after a single immunization. These results highlight the potential of the bare skin as a route for vaccine development and indicate an important role for immunostimulatory ODN as adjuvants to generate functional CTL with the help of the skin immune system.