In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats

Kupffer cells play an important role in the pathogenesis of liver diseases. During endotoxemia and alcohol-induced liver disease, tissue injury is pre ceded by an excessive release of cytokines by these macrophages. Tumor necr osis factor-alpha (TNF-alpha) is one of the key cytokines associated with l iver injury. Pre-exposure of animals to TNF-alpha antibodies has been shown to prevent macrophage-mediated liver injury in experimental animals. In th is article, we describe a method to encapsulate in pH-sensitive liposomes a nd to deliver an antisense phosphorothioate oligonucleotide (TJU-2755) agai nst TNF-alpha. We describe the efficacy of this formulation in inhibiting e ndotoxin-mediated production of TNF-alpha. The liposomes prepared were stab le for over 4 weeks at pH 7.4, but readily released their contents when exp osed to an acidic environment below pH 6, similar to the pH that exists in early endosomes. Male Sprague-Dawley rats were administered (i.v.) liposome -encapsulated TJU-2755 (1-2 mg/kg body wt.). Empty liposomes served as cont rols. Forty-eight hours postinjection, the animals were administered a sing le dose of lipopolysaccharide (50 mug/kg body wt.) and were sacrificed 90 m in later. The TNF-alpha produced by excised liver incubated ex vivo and the levels of plasma TNF-alpha were determined. After a single administration of liposome-encapsulated antisense TJU-2755, a 30% reduction in TNF-alpha p roduced by liver slices was observed. Two daily doses of the antisense olig onucleotide inhibited TNF-alpha production by 50%. This was associated with a 65 to 70% reduction in plasma levels of TNF-alpha, compared with control s. These results indicate that oligonucleotide TJU-2755 encapsulated in pH- sensitive liposomes can be used to effectively reduce endotoxin-mediated pr oduction of TNF-alpha in macrophages in vivo and thus may be of value in at tenuating or preventing macrophage-mediated liver injury.