Liposome encapsulation enhances the anti-inflammatory activity of an ICAM-1 antisense oligonucleotide

Autor: Klimuk, Sandra K.
Jazyk: angličtina
Rok vydání: 2000
Druh dokumentu: Text
Popis: Antisense oligonucleotides are designed to specifically bind to a variety of DNA and RNA targets to inhibit the translation of the genetic code into protein. By targeting disease-associated proteins this technology offers the potential for rapid design of highly specific drugs. However, the development of antisense molecules as a novel class of therapeutics has been slowed because of the undesirable pharmacokinetic and pharmacodynamic characteristics of these molecules. For example, antisense oligonucleotides cannot readily penetrate cells, they bind non-specifically to proteins, and are cleared rapidly from the circulation. Liposomal encapsulation has been used to overcome similar issues for other drugs. The objective of this research program was to investigate the effect of encapsulation on the activity of a phosphorothioate (PS) antisense oligodeoxynucleotide (ODN) targeted against murine intercellular adhesion molecule-1 (ICAM-1). This work has been divided into three distinct areas: (1) Establishing and characterizing an animal model of acute inflammation and assessing the pharmacokinetic and biodistribution of empty liposomes in the model. (2) A description of the formulation process and characterization of the encapsulated ODN, using both in vitro and in vivo techniques. (3) Measuring the effects of lipid encapsulation on the antiinflammatory activity of an antisense ODN specific for murine ICAM-1. To achieve my objectives, it was necessary to characterize an animal model in which the biological activity of an antisense molecule could be readily and reproducibly measured. The model chosen was of murine contact hypersensitivity (CHS) induced by the chemical 2,4- dinitrofluorobenzene (DNFB), in which an inflammatory response was generated in one ear while the other ear acted as a control. Inflammation was monitored by measuring ear thickness and inflammatory cell infiltration, both of which peaked 24 h after applying DNFB to the ear. Large unilamellar vesicles (LUV) accumulated in the inflammation site to a level more then 20-fold higher than that measured in the untreated control ear. This accumulation occurred only during the first 24 h of inflammation and is attributed to increased vascular permeability. Maximal extravasation of lipid was obtained using vesicles 120 nm in diameter and accumulation at the inflammation site was found to be proportional to the concentration of vesicles in the circulation. The next series of experiments focused on formulating the antisense ODN in conventional liposomes and measuring the effects of encapsulation on various in vitro and in vivo properties exhibited by ODN. For the majority of these studies radiolabeled phosphorothioate (PS) ODN was passively encapsulated into neutral 120 nm LUV composed of egg phosphatidylcholine (EPC) and cholesterol (CH) (55:45, molar ratio). Encapsulation reduced ODN induced activation of the human complement cascade by at least 1000-fold and protected the antisense molecule from degradation by plasma nucleases. Free ODN was cleared from the circulation rapidly, exhibiting a half-life of only a few minutes with concomitant accumulation of radioactive tracer primarily in the liver and kidneys. In contrast the circulation half-life of an equivalent dose of encapsulated ODN was increased to several hours with clearance occurring mostly via the liver and spleen. Furthermore, the encapsulated ODN was not released from the vesicles during circulation and remained intact for at least 24 h. Disease-site targeting was also demonstrated for encapsulated ODN as both the lipid vesicle and drug accumulated in the inflamed ear. The final experiments were designed to measure the biological activity of a free and encapsulated antisense ODN targeted against ICAM-1 in the murine CHS model. Anti-inflammatory activity for the free ODN was not detected at doses as high as 50 mg/kg. Mice receiving encapsulated ODN exhibited normal ear thickness and minimal cellular infiltration, indicating a greatly reduced inflammatory response. Comparable anti-inflammatory activity was observed in mice treated with topical corticosteroids. The ODN activity was sequence specific, as both a lipid encapsulated ODN specific for human ICAM-1 and a scrambled version of the murine ICAM-1 ODN failed to inhibit inflammation. A dose response was also observed for the encapsulated murine sequence. Immunohistochemical analysis showed a reduction in ICAM-1 protein expression on endothelial cells in ear tissue following treatment with the encapsulated active compound. These data indicate that liposomes may represent an effective vehicle for delivering antisense ODN to sites of inflammation.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate
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