Development of a new sense oligonucleotide drug targeting inducible nitric oxide synthase for sepsis model rats

<p>Backgrounds and aim: During the inflammation, a variety of inflammatory cytokines are produced in the liver, and nitric oxide (NO), which is synthesized by inducible nitric oxide synthase (iNOS), is produced as well. Excess NO is assumed to be one of the factors involved in hepatic injuries, and it is required to suppress the iNOS induction to reduce the injuries. We have been pursuing the compounds with anti-inflammatory effects by monitoring NO production in primary cultured rat hepatocytes and in acute liver failure of rats in vivo. Because the operation of hepatocellular carcinoma (HCC) is highly invasive, and adverse effects of chemotherapy for unresectable HCC are intense, the treatment of HCC is difficult in many cases. When we use a new anti-inflammatory compound for the therapy, it is possible to decrease the operative invasiveness and the adverse effects. Previously, we reported that introduction of a short DNA that corresponds to iNOS mRNA sequence (i.e., iNOS sense oligonucleotide) into hepatocytes specifically decreased expression level of iNOS mRNA as well as production of NO in vitro. In this study, we investigated effects of the iNOS sense oligonucleotides in sepsis model rats in vivo.</p><p>Methods: Each iNOS sense oligonucleotide (SO1 or its derivatives) was introduced into primary cultured rat hepatocytes, and the expression levels of iNOS mRNA were measured and compared to the cells transfected with control DNA. A sepsis model was prepared by the administration of D-galactosamine (GalN) and bacterial lipopolysaccharide (LPS) with or without injection of an iNOS sense oligonucleotide to rats. Survival of the treated rats was recorded by 72 hours after GalN/LPS injection, and the expression of iNOS and cytokine mRNAs in liver were analyzed. Liver tissues of the treated rats were pathologically examined.</p><p>Results: Among prepared iNOS sense oligonucleotides, which include chemically modified derivatives, SO1 was the most effective oligonucleotide for decreasing expression level of iNOS mRNA in rat hepatocytes. When SO1 was simultaneously administered with GalN and LPS to rats (GalN/LPS+SO1 group), their survival rate was significantly increased (58%) compared to the rats administered with GalN and LPS alone (GalN/LPS group). The expression of mRNAs for iNOS and TNF-α was decreased, and the number of apoptotic hepatocytes was also markedly suppressed in the livers of GalN/LPS+SO1 group compared to that of GalN/LPS group.</p><p>Conclusion: Administration of an iNOS sense oligonucleotide inhibited the induction of iNOS and TNF-α in sepsis model rats and showed hepatoprotective effects by the improvement of the pathological findings in the liver. These data imply that natural antisense transcript-targeted regulation (NATRE) technology using the iNOS sense oligonucleotide may be used to treat human sepsis with hepatic failure.</p>