title: Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.
abstract: Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in 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 oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-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 within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, 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 not 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-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.
authors.affiliation: Department of Radiation Medicine, Georgetown University Medical Center, Washington, DC 20007, USA.
authors.forename: P C
authors.initials: PC
authors.lastname: Gokhale
authors.forename: D
authors.initials: D
authors.lastname: McRae
authors.forename: B P
authors.initials: BP
authors.lastname: Monia
authors.forename: A
authors.initials: A
authors.lastname: Bagg
authors.forename: A
authors.initials: A
authors.lastname: Rahman
authors.forename: A
authors.initials: A
authors.lastname: Dritschilo
authors.forename: U
authors.initials: U
authors.lastname: Kasid
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D000818:Animals; D001777:Blood Coagulation / Q000187:drug effects; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000532:radiotherapy*; D004339:Drug Compounding; D006801:Humans; D007822:Laryngeal Neoplasms / Q000188:drug therapy / Q000532:radiotherapy*; D008081:Liposomes; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020319:Oligodeoxyribonucleotides, Antisense / Q000493:pharmacokinetics / Q000627:therapeutic use*; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000627:therapeutic use*; D013873:Thionucleotides / Q000493:pharmacokinetics / Q000627:therapeutic use*
chemical_list: D020413:3' Untranslated Regions; D008081:Liposomes; D020319:Oligodeoxyribonucleotides, Antisense; D011838:Radiation-Sensitizing Agents; D013873:Thionucleotides; C099587:ISIS 5132; D019908:Proto-Oncogene Proteins c-raf