Tomohiro Neichi

Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats.

The anemia associated with chronic renal failure is one of the best target diseases for erythropoietin (Epo) gene transfer. We previously reported a short-term (1 month) study of continuous rat Epo delivery by muscle-targeted gene transfer of plasmid DNA expressing rat Epo (pCAGGS-Epo) using in vivo electroporation in normal rats. Here, we performed a long-term pharmacokinetic study of continuous Epo delivery by this method in normal rats and uremic five-sixths nephrectomized rats. In normal rats, Epo gene expression and sufficient erythropoiesis occurred with Epo gene transfer in a dose-dependent manner, and persisted for at least 11 weeks. Repeated administration of the plasmid DNA effectively produced erythropoiesis. Similar erythropoiesis was observed in the uremic rats, and persisted for more than 15 weeks. Both normal and uremic rats showed a significant decrease in platelet count. Moreover, the uremic rats showed Epo-induced hypertension, which is the major side-effect of recombinant human Epo. These results demonstrate that muscle-targeted pCAGGS-Epo transfer by in vivo electroporation is a useful procedure for the long-term continuous delivery of Epo in both normal and uremic rats.

Selective inhibition of 5-lipoxygenase by natural compounds isolated from Chinese plants, Artemisia rubripes Nakai.

Three of four natural compounds, which are caffeic acid, eupatilin and 4′‐demethyleupatilin, isolated from Chinese plant, Artemisia rubripes Nakai selectively inhibited 5‐lipoxygenase of cultured mastocytoma cells. Half‐inhibition doses (ID 50) for caffeic acid, eupatilin and 4′‐demethyleupatilin were 3.7, 14 and 18 × 10−6 M, respectively. The inhibition by caffeic acid was non‐competitive types. Prostaglandin synthase activities were little inhibited by eupatilin and 4′‐demethyleupatilin, but rather stimulated by affeic acid. The formation of leukotriene C4 and D4 by mast tumor cells was almost completely suppressed by these compounds at 10−4 M.

Effects of Erythropoietin-Gene Electrotransfer in Rats with Adenine-Induced Renal Failure

Background: We previously demonstrated that erythropoietin (Epo) expression increases in five-sixths nephrectomized rats, after muscle-targeted gene transfer by in vivo electroporation, using plasmid DNA expressing rat Epo (pCAGGS-Epo). Here, we apply this method to a rat model with severe anemia associated with chronic renal failure; these rats have hematocrit levels in the 30–35% range, similar to those in humans with end-stage renal disease. Methods: Wistar rats were treated to produce adenine-induced uremia. The uremic rats were then treated with muscle-targeted gene transfer using pCAGGS-Epo. Some uremic rats died from chronic renal failure; one of these was dissected, and the kidneys were histologically examined. For the remaining rats, we measured body weight and blood pressure, and obtained blood samples regularly. Results: The uremic rats showed severe anemia, with hematocrit levels at 32.6 ± 3.3%. Epo-gene transfer increased Epo expression and serum Epo levels, and also increased the hematocrit levels to 64.5 ± 4.8%. The dose of pCAGGS-Epo used in this study did not induce severe hypertension. Conclusions: Continuous Epo-gene expression improves the anemia associated with chronic renal failure, and without severe side effects. Our results support the potential use of gene electrotransfer for human gene therapy applications.

Enhancement of PGI2 formation by a new vasodilator, 2-nicotinamidoethyl nitrate in the coupled system of platelets and aortic microsomes

Exogenous arachidonate addition to the coupled system of platelets and aortic microsomes resulted in production of TXA2 and PGI2 (detected as the stable degradation products, TXB2 and 6-keto PGF1 alpha, respectively). Imidazole, papaverine and dipyridamole increased PGI2 and decreased TXA2 in the coupled system. All of these agents inhibited TXA2 formation by platelets from arachidonate. Nitroglycerin did not show any effect on PGI2 and TXA2 formation in the coupled system and on TXA2 formation by platelets. In contrast with these compounds, in spite of showing no inhibitory effect on TXA2 formation by platelets alone, 2-nicotinamidoethyl nitrate (SG-75) increased PGI2 and decreased TXA2 in the coupled system. It is suggested that SG-75 accelerated the conversion of PGH2 to PGI2 so that smaller amounts of TXA2 was produced in the coupled system.

Thrombopoietic activity of human interleukin-6

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK‐01 cells cultured in the presence of human interleukin‐1. The N‐terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N‐terminal region of human interleukin‐6 (IL‐6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL‐6 by Western analysis using an anti‐IL‐6 antibody. Commercial recombinant human IL‐6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin‐3 and increased the number of peripheral platelets in mice in a dose‐dependent manner. From these results, it is concluded that human IL‐6 has thrombopoietic activity.

Post-secretion neutralization of transgene-derived effect: soluble erythropoietin receptor/IgG1Fc expressed in liver neutralizes erythropoietin produced in muscle

The regulation of transgene expression is a key issue for the development of safe gene therapy. Various strategies have been used to regulate protein production at the levels of transgene expression, transcription, translation, and secretion. Neutralization following secretion is another important backup system to prevent super‐therapeutic levels of a protein from being expressed by gene transfer.

The lipid-lowering profile in rodents AZ-1355, A New Dibenzoxazepine Derivative

The lipid-lowering profile of ethyl 10,11-dihydro-4-methoxydibenz[b,f]-(1,4)oxazepine-8-carboxylate (AZ-1355) has been evaluated using clofibrate as a reference compound. This compound is structurally unrelated to any other hypolipidemic agent. AZ-1355 was selected not only for its effect in reducing serum lipids, but also because it inhibits platelet aggregation in vivo and elevates the prostaglandin I2/thromboxane A2 ratio in vitro. It lowers serum total cholesterol in Triton-treated hyperlipidemic mice, and also lowers serum total cholesterol and triglyceride in dietary hyperlipidemic rats. In golden hamsters chosen for further evaluation, AZ-1355 reduced serum, liver and cardiac lipids, improved the beta/alpha-lipoprotein ratio and increased the HDL cholesterol. Thus, it is apparent that the lipid-lowering profile of AZ-1355 differs from that of clofibrate.