Effects of Two Anti-TNF-α Compounds: Etanercept and 5-Ethyl-1-Phenyl-2-(1H)-Pyridone on Secreted and Cell-Associated TNF-α In Vitro
Ken J. Grattendick, James M. Nakashima, Shri N. Giri
DOI: 10.4236/pp.2011.24031   PDF   HTML     4,678 Downloads   9,410 Views   Citations


Tumor necrosis factor-alpha (TNF-α) is a potent inflammatory cytokine and its exaggerated production has been implicated in acute, chronic and autoimmune inflammatory diseases. Proteinaceous and non-proteinaceous anti-TNF-α agents have been developed to reduce its circulating levels either by neutralizing, binding or inhibiting the de novo synthesis with the aim of achieving desirable therapeutic effects. In the present study, we compared the effects of a protein-based anti-TNF-α drug, etanercept, and a non-protein-based anti-TNF-α small molecule, 5-ethyl-1-phenyl-2-(1H) pyridone (5-EPP), on the LPS-stimulated secretion of TNF-α in the medium and TNF-α associated with the THP-1 cells in vitro. Both drugs had marked concentration-dependent inhibitory effects on the LPS-stimulated secretion of TNF-α. However, their effects on the LPS-stimulated cell-associated TNF-α were diametrically opposed to each other. For instance, etanercept further increased the level by up to 12-fold, whereas 5-EPP inhibited the level in a dose dependent manner. In addition, 5-EPP caused a significant reduction in the elevated level of cell associated TNF-α caused by LPS + etanercept. The differences in the levels of cell-associated TNF-α as reported in the present study may partly explain the adverse effects of some protein-based anti-TNF-α drugs including etanercept as opposed to a non-protein-based anti-TNF-α drug such as pirfenidone, a structural analogue of 5-EPP, for treatment of some TNF-α mediated diseases. It was concluded from the findings of this study that drugs which elevate the levels of cell associated-TNF-α will potentially have more adverse events even after reducing the secreted levels of TNF-α than the drugs which reduce both the secreted and cell-associated TNF-α levels.

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K. Grattendick, J. Nakashima and S. Giri, "Effects of Two Anti-TNF-α Compounds: Etanercept and 5-Ethyl-1-Phenyl-2-(1H)-Pyridone on Secreted and Cell-Associated TNF-α In Vitro," Pharmacology & Pharmacy, Vol. 2 No. 4, 2011, pp. 238-247. doi: 10.4236/pp.2011.24031.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] R. A. Black, C. T. Rauch, C. J. Kozlosky, J. J. Peschon, J. L. Slack, M. F. Wolfson, et al., “A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells,” Nature, Vol. 385, 1997, pp. 729-733.
[2] E. Decoster, B. Vanhaesebroeck, P. Vandenabeele, J. Grooten and W. Fiers, “Generation and biological characterization of membrane-bound, uncleavable murine tumor necrosis factor,” The Journal of Biological Chemistry, Vol. 270, 1995, pp. 18473-18478.
[3] M. Grell, E. Douni, H. Wajant, M. Lohden, M. Clauss, B. Maxeiner, et al., “The transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kDa tumor necrosis factor receptor,” Cell, Vol. 83, 1995, pp. 793-802.
[4] M. Grell, H. Wajant, G. Zimmermann and P. Scheurich, “The type 1 receptor (CD120a) is the high-affinity receptor for soluble tumor necrosis factor,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 95, 1998, pp. 570-575.
[5] D. Wallach, E. E. Varfolomeev, N. L. Malinin, Y. V. Goltsev, A. V. Kovalenko and M. P. Boldin, “Tumor necrosis factor receptor and Fas signaling mechanisms,” Annual Review of Immunology, Vol. 17, 1999, pp. 331- 367.
[6] L. J. Old, “Tumor necrosis factor (TNF),” Science, Vol. 230, 1985, pp. 630-632.
[7] P. Vandenabeele, W. Declercq, R. Beyaert and W. Fiers, “Two tumour necrosis factor receptors: structure and function,” Trends in Cell Biology, Vol. 5, 1995, pp. 392- 399.
[8] P. Vassalli, “The pathophysiology of tumor necrosis factors,” Annual Review of Immunology, Vol. 10, 1992, pp. 411-452.
[9] J. M. Bathon, R. W. Martin, R. M. Fleischmann, J. R. Tesser, M. H. Schiff, E. C. Keystone, et al., “A comparison of etanercept and methotrexate in patients with early rheumatoid arthritis,” The New England Journal of Medicine, Vol. 343, 2000, pp. 1586-1593.
[10] P. E. Lipsky, D. M. van der Heijde, E. W. St Clair, D. E. Furst, F. C. Breedveld, J. R. Kalden, et al., “Infliximab and methotrexate in the treatment of rheumatoid arthritis. Anti-Tumor Necrosis Factor Trial in Rheumatoid Arthritis with Concomitant Therapy Study Group,” The New England Journal of Medicine, Vol. 343, 2000, pp. 1594- 1602.
[11] S. R. Targan, S. B. Hanauer, S. J. van Deventer, L. Mayer, D. H. Present, T. Braakman, et al., “A short-term study of chimeric monoclonal antibody cA2 to tumor necrosis factor alpha for Crohn’s disease. Crohn’s Disease cA2 Study Group,” The New England Journal of Medicine, Vol. 337, 1997, pp. 1029-1035.
[12] P. J. Mease, B. S. Goffe, J. Metz, A. VanderStoep, B. Finck and D. J. Burge, “Etanercept in the treatment of psoriatic arthritis and psoriasis: a randomised trial,” Lancet, Vol. 356, 2000, pp. 385-390.
[13] J. Braun, J. Brandt, J. Listing, A. Zink, R. Alten, W. Golder, et al., “Treatment of active ankylosing spondylitis with infliximab: a randomised controlled multicentre trial,” Lancet, Vol. 359, 2002, pp. 1187-1193.
[14] J. D. Gorman, K. E. Sack and J. C. Davis, Jr., “Treatment of ankylosing spondylitis by inhibition of tumor necrosis factor alpha,” The New England Journal of Medicine, Vol. 346, 2002, pp. 1349-1356.
[15] S. Munoz-Fernandez, V. Hidalgo, J. Fernandez-Melon, A. Schlincker and E. Martin-Mola, “Effect of infliximab on threatening panuveitis in Behcet’s disease,” Lancet, Vol. 358, 2001, pp. 1644.
[16] G. Caron, Y. Delneste, J. P. Aubry, G. Magistrelli, N. Herbault, A. Blaecke, et al., “Human NK cells constitutively express membrane TNF-alpha (mTNFalpha) and present mTNFalpha-dependent cytotoxic activity,” European Journal of Immunology, Vol. 29, 1999, pp. 3588- 3595.
[17] T. Decker, M. L. Lohmann-Matthes and G. E. Gifford, “Cell-associated tumor necrosis factor (TNF) as a killing mechanism of activated cytotoxic macrophages,” Journal of Immunology, Vol. 138, 1987, pp. 957-962.
[18] M. Higuchi, K. Nagasawa, T. Horiuchi, M. Oike, Y. Ito, M. Yasukawa, et al., “Membrane tumor necrosis factor- alpha (TNF-alpha) expressed on HTLV-I-infected T cells mediates a costimulatory signal for B cell activation- characterization of membrane TNF-alpha,” Clinical Immunology and Immunopathology, Vol. 82, 1997, pp. 133- 140.
[19] A. D. Watts, N. H. Hunt, Y. Wanigasekara, G. Bloomfield, D. Wallach, B. D. Roufogalis, et al., “A casein kinase I motif present in the cytoplasmic domain of members of the tumour necrosis factor ligand family is implicated in ‘reverse signalling’,” The EMBO Journal, Vol. 18, 1999, pp. 2119-2126.
[20] C. Ferran, F. Dautry, S. Merite, K. Sheehan, R. Schreiber, G. Grau, et al., “Anti-tumor necrosis factor modulates anti-CD3-triggered T cell cytokine gene expression in vivo,” The Journal of Clinical Investigation, Vol. 93, 1994, pp. 2189-2196.
[21] G. H. Waetzig, D. Seegert, P. Rosenstiel, S. Nikolaus and S. Schreiber, “p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease,” Journal of Immunology, Vol. 168, 2002, pp. 5342-5351.
[22] S. Harashima, T. Horiuchi, N. Hatta, C. Morita, M. Higuchi, T. Sawabe, et al., “Outside-to-inside signal through the membrane TNF-alpha induces E-selectin (CD62E) expression on activated human CD4+ T cells,” Journal of Immunology, Vol. 166, 2001, pp. 130-136.
[23] G. Eissner, S. Kirchner, H. Lindner, W. Kolch, P. Janosch, M. Grell, et al., “Reverse signaling through transmembrane TNF confers resistance to lipopolysaccharide in human monocytes and macrophages,” Journal of Immunology, Vol. 164, 2000, pp. 6193-6198.
[24] W. C. Cain, R. W. Stuart, D. L. Lefkowitz, J. D. Starnes, S. Margolin and S. S. Lefkowitz, “Inhibition of tumor necrosis factor and subsequent endotoxin shock by pirfenidone,” International Journal of Immunopharmacology, Vol. 20, 1998, pp. 685-695.
[25] M. L. Hale, S. B. Margolin, T. Krakauer, C. J. Roy and B. G. Stiles, “Pirfenidone blocks the in vitro and in vivo effects of staphylococcal enterotoxin B,” Infection and Immunity, Vol. 70, 2002, pp. 2989-2994.
[26] H. Nakazato, H. Oku, S. Yamane, Y. Tsuruta and R. Suzuki, “A novel anti-fibrotic agent pirfenidone suppresses tumor necrosis factor-alpha at the translational level,” European Journal of Pharmacology, Vol. 446, 2002, pp. 177-185.
[27] H. Oku, H. Nakazato, T. Horikawa, Y. Tsuruta and R. Suzuki, “Pirfenidone suppresses tumor necrosis factor-alpha, enhances interleukin-10 and protects mice from endotoxic shock,” European Journal of Pharmacology, Vol. 446, 2002, pp. 167-176.
[28] K. J. Grattendick, J. M. Nakashima and S. N. Giri, “Ef-fects of 5-Ethyl-1-phenyl-2-(1H) Pyridone on Serum Biomarkers of Multiorgan Dysfunction and Mortality in Lipopolysaccharide/Glactosamine and Cecal Ligation and Puncture Models of Septic Shock in Mice,” Research Communication in Molecular Pathology and Pharma-cology, in press.
[29] Y. Tang, B. Li, N. Wang, Y. Xie, L. Wang, Q. Yuan, et al., “Fluorofenidone protects mice from lethal endotoxemia through the inhibition of TNF-alpha and IL-1beta release,” International immunopharmacology, Vol. 10, 2010, pp. 580-583.
[30] J. D. Bowen, K. Maravilla and S. B. Margolin, “Open- label study of pirfenidone in patients with progressive forms of multiple sclerosis,” Multiple sclerosis (Houndmills, Basingstoke, England), Vol. 9, 2003, pp. 280-283.
[31] J. E. Walker, S. N. Giri and S. B. Margolin, “A double- blind, randomized, controlled study of oral pirfenidone for treatment of secondary progressive multiple sclerosis,” Multiple sclerosis (Houndmills, Basingstoke, England), Vol. 11, 2005, pp. 149-158.
[32] J. E. Walker and S. B. Margolin, “Pirfenidone for chronic progressive multiple sclerosis,” Multiple sclerosis (Houndmills, Basingstoke, England), Vol. 7, 2001, pp. 305-312.
[33] K. Akassoglou, J. Bauer, G. Kassiotis, H. Lassmann, G. Kollias and L. Probert, “Transgenic models of TNF induced demyelination,” Advances in Experimental Medicine and Biology, Vol. 468, 1999, pp. 245-259.
[34] F. M. Hofman, D. R. Hinton, K. Johnson and J. E. Merrill, “Tumor necrosis factor identified in multiple sclerosis brain,” The Journal of Experimental Medicine, Vol. 170, 1989, pp. 607-612.
[35] J. E. Merrill and E. N. Benveniste, “Cytokines in inflammatory brain lesions: helpful and harmful,” Trends in Neurosciences, Vol. 19, 1996, pp. 331-338.
[36] G. Raghu, W. C. Johnson, D. Lockhart and Y. Mageto, “Treatment of idiopathic pulmonary fibrosis with a new antifibrotic agent, pirfenidone: results of a prospective, open-label Phase II study,” American Journal of Respiratory and Critical Care Medicine, Vol. 159, 1999, pp. 1061-1069.
[37] H. Taniguchi, M. Ebina, Y. Kondoh, T. Ogura, A. Azuma, M. Suga, et al., “Pirfenidone in idiopathic pulmonary fibrosis,” The European respiratory journal: official journal of the European Society for Clinical Respiratory Physiology, Vol. 35, 2010, pp. 821-829.
[38] P. W. Noble, C. Albera, W. Z. Bradford, U. Costabel, M. K. Glassberg, D. Kardatzke, et al., “Pirfenidone in patients with idiopathic pulmonary fibrosis (CAPACITY): two randomised trials,” Lancet, Vol. 377, 2011, pp. 1760- 1769.
[39] B. W. van Oosten, F. Barkhof, L. Truyen, J. B. Boringa, F. W. Bertelsmann, B. M. von Blomberg, et al., “Increased MRI activity and immune activation in two multiple sclerosis patients treated with the monoclonal anti-tumor necrosis factor antibody cA2,” Neurology, Vol. 47, 1996, pp. 1531-1534.
[40] S. B. Margolin, S. N. Giri and W. J. Goux, “Treatment of sepsis with 5-ethyl-1-phenyl-2-(1H)-pyridone and novel methods for synthesis,” International Application Published under Patent Cooperation Treaty. 2009; WO 2009/111785 A2.
[41] S. Tsuchiya, M. Yamabe, Y. Yamaguchi, Y. Kobayashi, T. Konno and K. Tada, “Establishment and characterization of a human acute monocytic leukemia cell line (THP-1),” International Journal of Cancer, Vol. 26, 1980, pp. 171-176.
[42] C. D. Morgan, K. C. Mills, D. L. Lefkowitz and S. S. Lefkowitz, “An improved colorimetric assay for tumor necrosis factor using WEHI 164 cells cultured on novel microtiter plates,” Journal of Immunological Methods, Vol. 145, 1991, pp. 259-262.
[43] B. Scallon, A. Cai, N. Solowski, A. Rosenberg, X. Y. Song, D. Shealy, et al., “Binding and functional comparisons of two types of tumor necrosis factor antagonists,” The Journal of Pharmacology and Experimental Therapeutics, Vol. 301, 2002, pp. 418-426.
[44] T. Calandra, J. D. Baumgartner, G. E. Grau, M. M. Wu, P. H. Lambert, J. Schellekens, et al., “Prognostic values of tumor necrosis factor/cachectin, interleukin-1, interferon-alpha, and interferon-gamma in the serum of patients with septic shock. Swiss-Dutch J5 Immunoglobulin Study Group,” The Journal of Infectious Diseases, Vol. 161, 1990, pp. 982-987.
[45] S. J. Hopkins and A. Meager, “Cytokines in synovial fluid: II. The presence of tumour necrosis factor and interferon,” Clinical and Experimental Immunology, Vol. 73, 1988, pp. 88-92.
[46] A. Waage, A. Halstensen and T. Espevik, “Association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease,” Lancet, Vol. 1, 1987, pp. 355-357.
[47] F. M. Brennan, D. Chantry, A. Jackson, R. Maini and M. Feldmann, “Inhibitory effect of TNF alpha antibodies on synovial cell interleukin-1 production in rheumatoid arthritis,” Lancet, Vol. 2, 1989, pp. 244-247.
[48] P. F. Piguet, G. E. Grau, C. Vesin, H. Loetscher, R. Gentz and W. Lesslauer, “Evolution of collagen arthritis in mice is arrested by treatment with anti-tumour necrosis factor (TNF) antibody or a recombinant soluble TNF receptor,” Immunology, Vol. 77, 1992, pp. 510-514.
[49] R. O. Williams, M. Feldmann and R. N. Maini, “Anti- tumor necrosis factor ameliorates joint disease in murine collagen-induced arthritis,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, 1992, pp. 9784-9788.
[50] K. J. Tracey, Y. Fong, D. G. Hesse, K. R. Manogue, A. T. Lee, G. C. Kuo, et al., “Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia,” Nature, Vol. 330, 1987, pp. 662-664.
[51] S. M. Opal, A. S. Cross, J. C. Sadoff, H. H. Collins, N. M. Kelly, G. H. Victor, et al., “Efficacy of antilipopolysaccharide and anti-tumor necrosis factor monoclonal antibodies in a neutropenic rat model of Pseudomonas sepsis,” The Journal of Clinical Investigation, Vol. 88, 1991, pp. 885-890.
[52] J. J. Gomez-Reino, L. Carmona, V. R. Valverde, E. M. Mola and M. D. Montero, “Treatment of rheumatoid arthritis with tumor necrosis factor inhibitors may predispose to significant increase in tuberculosis risk: a multicenter active-surveillance report,” Arthritis and Rheumatism, Vol. 48, 2003, pp. 2122-2127.
[53] W. J. Sandborn, S. B. Hanauer, S. Katz, M. Safdi, D. G. Wolf, R. D. Baerg, et al., “Etanercept for active Crohn's disease: a randomized, double-blind, placebo-controlled trial,” Gastroenterology, Vol. 121, 2001, pp. 1088-1094.
[54] U. Chaudhari, P. Romano, L. D. Mulcahy, L. T. Dooley, D. G. Baker and A. B. Gottlieb, “Efficacy and safety of infliximab monotherapy for plaque-type psoriasis: a randomised trial,” Lancet, Vol. 357, 2001, pp. 1842-1847.
[55] B. W. van Oosten, F. Barkhof, P. E. Scholten, B. M. von Blomberg, H. J. Ader and C. H. Polman, “Increased production of tumor necrosis factor alpha, and not of interferon gamma, preceding disease activity in patients with multiple sclerosis,” Archives of Neurology, Vol. 55, 1998, pp. 793-798.
[56] N. L. Sicotte and R. R. Voskuhl, “Onset of multiple sclerosis associated with anti-TNF therapy,” Neurology, Vol. 57, 2001, pp. 1885-1888.
[57] M. Aringer and J. S. Smolen, “SLE - Complex cytokine effects in a complex autoimmune disease: tumor necrosis factor in systemic lupus erythematosus,” Arthritis Research & Therapy, Vol. 5, 2003, pp. 172-177.
[58] R. A. Mageed and D. A. Isenberg, “Tumour necrosis factor alpha in systemic lupus erythematosus and anti- DNA autoantibody production,” Lupus, Vol. 11, 2002, pp. 850-855.
[59] K. M. Mohler, D. S. Torrance, C. A. Smith, R. G. Goodwin, K. E. Stremler, V. P. Fung, et al., “Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists,” Journal of Immunology, Vol. 151, 1993, pp. 1548-1561.
[60] L. Xin, J. Wang, H. Zhang, W. Shi, M. Yu, Q. Li, et al., “Dual regulation of soluble tumor necrosis factor-alpha induced activation of human monocytic cells via modulating transmembrane TNF-alpha-mediated ‘reverse signaling’,” International Journal of Molecular Medicine, Vol. 18, 2006, pp. 885-892.
[61] C. C. Solorzano, R. Ksontini, J. H. Pruitt, P. J. Hess, P. D. Edwards, A. Kaibara, et al., “Involvement of 26-kDa cell-associated TNF-alpha in experimental hepatitis and exacerbation of liver injury with a matrix metalloproteinase inhibitor,” Journal of Immunology, Vol. 158, 1997, pp. 414-419.
[62] K. J. Grattendick, J. M. Nakashima, L. Feng, S. N. Giri and S. B. Margolin, “Effects of three anti-TNF-alpha drugs: etanercept, infliximab and pirfenidone on release of TNF-alpha in medium and TNF-alpha associated with the cell in vitro,” International immunopharmacology, Vol. 8, 2008, pp. 679-687.

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