The effect of vesnarinone on TNFα production in human peripheral blood mononuclear cells and microglia: a preclinical study for the treatment of multiple sclerosis

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Abstract

Vesnarinone (OPC-8212) is a synthetic quinolinone derivative with inotropic and immunomodulatory effects. Vesnarinone has been shown to inhibit tumor necrosis factor-alpha (TNFα) produced by mitogen stimulated macrophages, and to inhibit phosphodiesterase (PDE) type III in cardiac muscle. TNFα and interferon-gamma (IFNγ) have been implicated in the pathogenesis of autoimmune diseases, and both cytokines are targets for therapeutic intervention. IFNγ can enhance autoimmune disease through direct effects, and indirectly by priming macrophages to produce TNFα. In this study, we demonstrate that while vesnarinone enhances basal TNFα levels, it inhibits TNFα production in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy donors stimulated with lipopolysaccharide (LPS) or primed with IFNγ and stimulated with suboptimal doses of LPS. In addition, vesnarinone inhibited TNFα production in primary adult human microglial cultures. However, in contrast to rolipram, another TNFα inhibiting agent, vesnarinone failed to inhibit TNFα production by myelin basic protein specific T-cell lines. As oral TNF inhibitors are currently being considered in the USA for clinical application in MS, the implications of our findings on the development of vesnarinone for treatment of MS are discussed.

Introduction

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-,3,4-dimethoxybenzoyl) 1-piperazinyl]-2(1H)-quinolinone) is a synthetic quinolinone derivative with positive inotropic as well as immunomodulatory effects. The inotropic effect is believed to be related to a decrease in potassium currents, prolonged opening of the sodium channels that result in increased intracellular sodium, and an increase in inward calcium currents (Feldman et al., 1993). Vesnarinone has been used extensively in therapeutic trials for congestive heart failure both in Japan and in the United States. Vesnarinone administration to human patients was complicated by neutropenia in some cases suggesting that the drug has immunosupressive effects (Feldman et al., 1993). A number of studies demonstrated that vesnarinone can inhibit lipopolysaccharide (LPS)-induced cytokines in peripheral blood lymphocytes including tumor necrosis factor alpha (TNFα) (Maruyama et al., 1993; Matsumori et al., 1994; Shioi et al., 1994; Kambayashi et al., 1996; Oyaizu et al., 1996; Sasayama and Matsumori, 1996). Although vesnarinone is a phosphodiesterase (PDE) III inhibitor, and multiple immune cells express PDE III and IV isoforms, the mechanisms responsible for the immunological effects of vesnarinone are not understood.

The proinflammatory cytokines, TNFα and interferon-gamma (IFNγ) produced by macrophages and T cells respectively have been implicated in the pathogenesis of a number of autoimmune diseases including rheumatoid arthritis and multiple sclerosis (MS) (Panitch et al., 1987; Hofman et al., 1989; Powell et al., 1990; Kroemer and Martinez-A, 1992). Therefore inhibitors of TNFα production could have potential therapeutic effects in such diseases. IFNγ by itself does not induce TNFα in macrophages but can prime TNFα production in these cells (Gifford and Lohmann-Matthes, 1987; Hayes et al., 1995; de Wit et al., 1996), and therefore could potentially enhance autoimmune diseases. This study examined the effects of vesnarinone on TNFα production by human peripheral blood mononuclear cells (PBMC) and myelin basic protein (MBP) specific T-cell lines from healthy donors and MS patients, and in primary cultures of adult human microglia. In addition, the effect of vesnarinone on PBMC primed with IFNγ and exposed to a suboptimal dose of LPS (defined as a dose that by itself does not result in substantial TNFα induction) was examined.

Section snippets

Cells

PBMC were obtained from nine normal volunteers by leukapheresis performed either at the Red Cross Center in Baltimore, MD, or at the National Institutes of Health Clinical Center, Bethesda, MD. Blood was processed for PBMC isolation by ficoll hypaque gradients. PBMC from MS patients were obtained either by venipuncture from patients diagnosed at the University of Maryland MS center, or by leukapheresis from MS patients diagnosed at the National Institutes of Health. The purified lymphocytes

Effect of vesnarinone on TNFa production in PBMC from MS patients and controls

We have examined the effect of venarinone on uninduced as well as LPS induced TNFα production by PBMC from eight patients with definite MS (Table 1 and Fig. 1) and four healthy controls (Fig. 2). In initial experiments we observed that vesnarinone by itself was able to induce TNFα in PBMC from two of three MS patients (MD, BU, but not GA; Table 1). Therefore, we subsequently examined the effect of serial doses of vesnarinone on basal TNFα production by PBMC from five additional MS patients (

Discussion

This study demonstrates that vesnarinone inhibits TNFα production in human PBMC stimulated with LPS. In addition, we demonstrate an inhibitory effect of vesnarinone on IFNγ priming of PBMC to produce TNFα which may be relevant to the pathogenesis of autoimmune diseases. Furthermore, vesnarinone inhibited TNFα production in LPS-stimulated microglia.

The effect of vesnarinone on basal TNFα levels produced by PBMC varied among the eight MS patients and four controls tested (Table 1). In initial

Acknowledgements

We would like to thank Mrs. Nayereh Dehghan for typing the manuscript. Supported by a grant from Otsuka Pharmaceutical, Japan.

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