METHOXYETHYL MODIFICATION IMPROVES INHIBITORY EFFECT OF C-RAF ANTISENSE OLIGODEOXYNUCLEOTIDES IN VITRO AND IN VIVO : Transplantation

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Abstracts of the 18th Annual Meeting of the American Society of Transplantation (AST), May 15-19, 1999, Chicago, Illinois

METHOXYETHYL MODIFICATION IMPROVES INHIBITORY EFFECT OF C-RAF ANTISENSE OLIGODEOXYNUCLEOTIDES IN VITRO AND IN VIVO

Stepkowski, S; Qu, X; Wang, M-E; Tian, L; Chen, W; Wancewicz, E; Johnston, J; Bennett, F C; Kahan, B D; Monia, B

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Abstract 826

C-raf is a well-characterized serine/threonine (Ser/Thr) protein kinase involved in the multiple transduction signals in T cells. We postulated that inhibition of C-raf mRNA expression prevents heart allograft rejection. Methods: Two 20-mer C-raf antisense oligodeoxynucleotides (oligo)-each with identical molecular sequence-were synthesized with different modifications: IP-11061, phosphorothioate (PS) backbone alone; and IP15770, PS backbone with methoxyethyl (ME) substitutions at the 2′-sugar positions in the first five and last five nucleotides. ME IP-15770 was more effective than PS IP-11061 (25-400 nM) to inhibit C-raf mRNA expression in rat A10 smooth muscle cells: 25 nM IP-15770 inhibited C-raf mRNA expression by 70%, compared with 25 nM IP-11061 by only 10%. The effect was sequence-specific; 200 nM IP-11061 failed to affect the expression of a housekeeping G3PDH mRNA. Untreated ACI (RT1a) recipients rejected Lewis (LEW; RT11) heart allografts at a mean survival time (MST) of 8 ± 0.6 days. A 14-day intravenous (i.v.) infusion by osmotic pump of 5.0 mg/kg/day IP-11061 prolonged heart allograft survival to 11.0 ± 1.0 days (p<0.05); 10.0 mg/kg/day IP-11061 to 14.8 ± 2.6 days (p<0.01); and, 20.0 mg/kg/day IP-11061 to 15.0 ± 2.6 days (p<0.01). However, ME-modification improved in vivo results: 5.0 mg/kg/day IP-15770 extended survivals to 14.8 ± 1.8 days (p<0.05); 10.0 mg/kg/day IP-15770 to 17.3 ± 2.6 days (p<0.01); and, 20.0 mg/kg/day to 19.0 ± 3.3 days (p<0.01). In contrast, 5.0 (8.7 ± 1.2 days; NS), 10.0 (9.3 ± 0.6 days; NS) or 20.0 (10.0 ± 0.8 days; NS) mg/kg/day scrambled control IP-11483 was ineffective, documenting sequence-specificity of C-raf oligo. A 3-day oral gavage with 2.5, 5.0, or 10.0 mg/kg/day cyclosporine (CsA) produced MSTs of 8.7 ± 1.2 days, 10.3 ± 1.2 days, and 12.2 ± 0.8 days, respectively. Addition to 10 mg/kg/day CsA therapy of 14-day 10.0 mg/kg/day IP-11061 extended survival to 21.3 ± 7.8 days or of IP1570 to 17.0 ± 2.0 days (p<0.01). Combination index (CI) values of 1.0-2.7 indicated no synergistic interaction (CI<1 shows synergistic, and CI>1 shows antagonistic, interaction). In contrast, 7-day therapy with 1.0, 2.0, or 3.0 mg/kg/day sirolimus (SRL) resulted in MSTs of 7.0 ± 0.7 days, 10.4 ± 1.5 days, and 18.0 ± 2.7 days, respectively. Combination of 2.0 mg/kg/day SRL with 10.0 mg/kg/day IP-11061 prolonged survivals to 32.6 ± 4.8 days (CI=0.2), and with 10.0 mg/kg/day IP-15770 to 62.7 ± 35.2 days (CI=0.02), with 3 out of 6 hearts surviving more than 100 days. Conclusions: The C-raf inhibition blocks allograft rejection, and the effect is synergistic with SRL. The ME modification improves C-raf inhibitory effects both in vitro and in vivo.

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