title: In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats.
abstract: Kupffer cells play an important role in the pathogenesis of liver diseases. During endotoxemia and alcohol-induced liver disease, tissue injury is preceded by an excessive release of cytokines by these macrophages. Tumor necrosis factor-alpha (TNF-alpha) is one of the key cytokines associated with liver injury. Pre-exposure of animals to TNF-alpha antibodies has been shown to prevent macrophage-mediated liver injury in experimental animals. In this article, we describe a method to encapsulate in pH-sensitive liposomes and to deliver an antisense phosphorothioate oligonucleotide (TJU-2755) against TNF-alpha. We describe the efficacy of this formulation in inhibiting endotoxin-mediated production of TNF-alpha. The liposomes prepared were stable for over 4 weeks at pH 7.4, but readily released their contents when exposed 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 controls. Forty-eight hours postinjection, the animals were administered a single dose of lipopolysaccharide (50 microg/kg body wt.) and were sacrificed 90 min 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 produced by liver slices was observed. Two daily doses of the antisense oligonucleotide inhibited TNF-alpha production by 50%. This was associated with a 65 to 70% reduction in plasma levels of TNF-alpha, compared with controls. These results indicate that oligonucleotide TJU-2755 encapsulated in pH-sensitive liposomes can be used to effectively reduce endotoxin-mediated production of TNF-alpha in macrophages in vivo and thus may be of value in attenuating or preventing macrophage-mediated liver injury.
authors.affiliation: Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA. biddanda.ponnappa@mail.tju.edu
authors.forename: B C
authors.initials: BC
authors.lastname: Ponnappa
authors.forename: I
authors.initials: I
authors.lastname: Dey
authors.forename: G C
authors.initials: GC
authors.lastname: Tu
authors.forename: F
authors.initials: F
authors.lastname: Zhou
authors.forename: M
authors.initials: M
authors.lastname: Aini
authors.forename: Q N
authors.initials: QN
authors.lastname: Cao
authors.forename: Y
authors.initials: Y
authors.lastname: Israel
mesh_terms: D000818:Animals; D056486:Chemical and Drug Induced Liver Injury; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D004355:Drug Stability; D011992:Endosomes; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007275:Injections, Intravenous; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008081:Liposomes; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008107:Liver Diseases / Q000378:metabolism / Q000517:prevention & control; D008297:Male; D016376:Oligonucleotides, Antisense / Q000008:administration & dosage* / Q000378:metabolism; D009946:Organothiophosphorus Compounds / Q000008:administration & dosage* / Q000378:metabolism; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013154:Spleen / Q000378:metabolism; D014018:Tissue Distribution; D016896:Treatment Outcome; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D008070:Lipopolysaccharides; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D009946:Organothiophosphorus Compounds; D054735:Phosphorothioate Oligonucleotides; D012333:RNA, Messenger; C428116:TJU 2755; D014409:Tumor Necrosis Factor-alpha