Tuesday, May 22, 2007

i-Fect and Nav1.8 Gene Silencing in vivo

Referenced in United States Patent 20070105806

Link to patent: Free Patents Online
[0241] The effect of siRNAs against Nav1.8, formulated with iFECT, on complete Freund's adjuvant-induced tactile hypersensitivity was evaluated in rats (FIG. 5). Adult male Sprague-Dawley rats received an injection of CFA (150 uL) into the hindpaw on day 0. siRNAs against Nav1.8 were then administered by intrathecal bolus to the lumbar region of the spinal cord on days 1, 2 and 3; specifically, for each bolus injection, 2 ug of siRNA was complexed with iFECT transfection reagent (Neuromics, Minneapolis Minn., USA) at a ratio of 1:4 (w:v) in a total volume of 10 uL. Five groups of rats (with 5 rats per group) were treated with either siRNA (AL-DP-6049, AL-DP-6209, AL-DP-6217 or AL-DP-6218; Table 1), or PBS, in the presence of iFECT. Tactile hypersensitivity was expressed as tactile withdrawal thresholds which were measured by probing the hindpaw with 8 calibrated von Frey filaments (Stoelting, Wood Dale Ill., USA) (0.41 g to 15 g). Each filament was applied to the plantar surface of the paw. Withdrawal threshold was determined by sequentially increasing and decreasing the stimulus strength and calculated with a Dixon non-parametric test (see Dixon, W. J. (1980) "Efficient analysis of experimental observations" Annu Rev Pharmacol Toxicol 20:441-462; Chaplan, S. R., F. W. Bach, et al.(1994) "Quantitative assessment of tactile allodynia in the rat paw" J Neurosci Methods 53:55-63). Tactile thresholds were measured before CFA injection to assess baseline thresholds, and then on day 4 after CFA and treatment with test articles. In rats treated with PBS, tactile hypersensitivity was pronounced on day 4, as evidenced by reduced paw withdrawal threshold, as expected. In rats treated with AL-DP-6209, tactile thresholds were nearly normalized on day 4, demonstrating that the Nav1.8 siRNA, AL-DP-6209, is efficacious in vivo against inflammation-induced hyperalgesia. Treatment with the Nav1.8 siRNA, AL-DP-6217, resulted in the average tactile threshold trending towards baseline, with one of five rats demonstrating a normal tactile response. AL-DP-6049 and AL-DP-6218 did not significantly alter tactile thresholds compared to PBS treatment, in this experimental paradigm.[0242] These results demonstrate that siRNAs targeting Nav1.8, formulated with transfection reagent and administered intrathecally, alleviate CFA-induced tactile hyperalgesia, and therefore represent a novel approach to providing effective treatment of clinical inflammatory pain.