Specific transcriptional inhibition of bone marrow-derived macrophage tumor necrosis factor-alpha gene expression and protein production using novel enantiomeric carbocyclic nucleoside analogues.

Tumor necrosis factor-alpha (TNF-alpha) is a powerful macrophage-derived proinflammatory cytokine, via both direct effects on host tissues as well as indirectly through the induction of other proinflammatory mediators, including interleukin- (IL) 1 beta and IL-6. Activation of murine bone marrow-derived macrophages (BMDM phi) with lipopolysaccharide (LPS) causes rapid expression of TNF-alpha, which as an autocrine factor enhances BMDM phi function through IL-1 beta and IL-6 production. In this study, we have examined the specific transcriptional inhibition of BMDM phi TNF-alpha using novel enantiomeric carbocyclic nucleoside analogues. BMDM phi were derived in vitro from murine bone marrow progenitors using colony stimulating factor-1 and treated with combinations of LPS (1-100 nG/ml) and the enantiomeric carbocyclic nucleoside (10-100 microM) analogues MDL 201, 112 (9-[(1S,3R)-cis-cyclopentan-3-ol]adenine); MDL 201,451 (9-[1R,3S)-cis-cyclopentan-3-ol]adenine); MDL 201,449 (9-[(1R,3R)-trans-cyclopentan-3-ol]adenine) and MDL 201,484 (9-[(1S,3S)-trans-cyclopentan-3-ol]adenine). Northern blot analysis showed that MDL 201,449 was the most effective agent in vitro at selectively inhibiting TNF-alpha. MDL 201,449 reduced TNF-alpha mRNA levels by nearly 50% for up to 4 hr after the simultaneous addition of LPS and the synthetic agent. In contrast, mRNA and secreted protein levels for IL-1 beta (measured by the D10.S bioassay) and mRNA for TNF-alpha receptor p60 and TNF-alpha receptor p80 were not significantly affected. Carbocyclic nucleoside analogues were effective when added to BMDM phi up-to 2 hr after LPS treatment and at concentrations as low as 10 microM. Regulation of BMDM phi IL-6 by carbocyclic nucleoside analogues in response to LPS appears to be both concentration and time dependent, because IL-6 mRNA and secreted protein levels were inhibited at only high drug concentrations (100 microM) and effective only at longer exposure times (+4 hr of incubation) to LPS. These data support the concept that M phi-derived proinflammatory cytokine gene expression is differentially, rather than coordinately, regulated by selective signal transduction and/or molecular pathways. Enantiomeric carbocyclic nucleoside analogues that specifically inhibit TNF-alpha may have therapeutic potential in inflammatory diseases, such as systemic inflammatory response syndrome, where TNF-alpha has been shown to have an important role in initiating the early stages of disease.

Effects of three irreversible inhibitors of ornithine decarboxylase on macrophage-mediated tumoricidal activity and antitumor activity in B16F1 tumor-bearing mice.

The objective of the present investigation was to compare the effects of three ornithine decarboxylase inhibitors on tumoricidal macrophage and antitumor activities in vivo. alpha-Difluoromethylornithine (DFMO), (2R,5R)-6-heptyne-2,5-diamine, and alpha-(fluoromethyl)dehydroornithine methyl ester (delta MFMOme) were administered continuously in drinking water starting on Day 1 to B16F1 tumor-bearing mice. DFMO, (2R,5R)-6-heptyne-2,5-diamine, and delta MFMOme reduced B16F1 tumor growth, measured on Day 18, up to 87, 79, and 95%, respectively. Similarly, all three ornithine decarboxylase inhibitors reduced B16F1 putrescine and spermidine levels. delta MFMOme was substantially more effective both as an antitumor agent and in reducing polyamines. Both DFMO and delta MFMOme augmented macrophage tumoricidal activity directed against B16F1 target cells. MAP had no effect on macrophage tumoricidal activity. Lipopolysaccharide-stimulated macrophages from delta MFMOme-treated mice also exhibited an increase in interleukin and tumor necrosis factor levels. Furthermore, treatment with a known macrophage activator, gamma-interferon, enhanced the antitumor activity of delta MFMOme. delta MFMOme did not alter natural killer cell activity; however, cytolytic T-lymphocyte induction was reduced by 40 to 50%. These results demonstrate that, in addition to their established antitumor activity, ornithine decarboxylase inhibitors may also potentiate specific tumoricidal effector cell generation in vivo.