Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles i n cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucle otide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mR NA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposo mal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 mu g/ml and 1 3.5 mu g/ml for LE-5132 and 5132, respectively. The decrease in plasma conc entration of LE-5132 and 5132 followed a biexponential pattern, with initia l distribution half-lives (t(1/2 alpha)) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t(1/2 beta)) with LE-5132 and 5132 w ere 14.5 hours and 4.3 hours, respectively. The area under the plasma conce ntration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs wi thin 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-f old, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did n ot affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encap sulated mismatched 5132 (0.5 mu M LE-5132, 71.3% +/- 22.5%; 1.0 mu M LE-513 2, 79.6% +/- 16.7%). In addition, LE-5132 appeared to be a more potent anti tumor compound than 5132 (p < 0.001). These data established the suitabilit y of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous adm inistration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf -1 expression in tumor tissue compared with blank liposome-treated or untre ated control groups. LE-5132 or ionizing radiation (IR) treatment alone cau sed significant but transient inhibition of SQ-20B tumor growth but not tum or regression. Remarkably, a combination of LE-5132 and IR treatments led t o significant and sustained tumor regression for at least 27 days after the last treatment (p < 0.001). Histopathologic examination of tumor samples r evealed a significant proportion of cells containing fragmented chromatin i n the LE-5132 + IR treatment group as compared with single agent and untrea ted control groups. These in vivo data support the notion that Raf-1 has pr oliferative and survival functions and advance the scientific and technolog ic bases for the use of antisense raf ODN in the management of radioresista nt malignancies.