Use of suramin against TNF-related diseases.

0001The present invention concerns a new therapeutic application of suramin.

0002Tumor Necrosis Factor a (TNFa) is a polypeptide of molecular weight 17500, whose primary and tertiary structure has been elucidated (Pennica, D. et al. Nature 312 : 724 (1984) and Jones, E.Y. et al. Nature 338 : 225 (1989)).

0003TNFa is biologically active as a dimer or trimer. The main source of TNFa are cells of the monocyte/macrophage lineage, but after appropriate stimulation it is produced by other cell types as well (e.g. T-lymphocytes).

0004TNFa was originally described as a molecule that induces haemorragic necrosis of tumors in mice. However, after large amounts of homogeneous TNFa became available upon cloning of its cDNA, it soon became clear that TNFa mediates a large array of biological activities that allow the definition of TNFa as a mediator of inflammation. See, generally, Beutler, B. and Cerami, A., Nature 320 : 584 (1986). Accordingly, TNFa plays, within the inflammatory response, a pivotal role in the host defense against invasion of the organism by noxious agents. However, under certain circumstances, like chronic or acute, systemic or localized hyperproduction, TNFa leads, in conjunction with other mediators of the inflammatory response, to a large number of pathologic conditions. Cachexia and septic shock are the best known examples (see, Beutler, B. and Cerami, A., Supra and Oliff, A., et al., Cell 50 : 555 (1987). Likewise, TNFa is supposed to play a pathogenic role in AIDS (Folks, T.M., et al., Proc. Natl. Acad. Sci. USA 86 : 2365 (1989)), in graft-versus-host disease (Piguet, P.F., et al. J. Exp. Med. 166 : 1280 (1987)), in cerebral malaria (Grau, G.E., et al. Science 237 : 1210 (1987)), rheumatoid arthritis (Brennan, F.M., et al., Lancet ii : 244 (1989)) myocardial ischemia and reperfusion (Lefer, A.M., et al. Science 249 : 61 (1990)), development of bactericidal granulomas (Kindler, V. et al. Cell 56 : 731 (1989)), adult respiratory distress syndrome (Millar, A.B. et al. Lancet ii : 712 (1989)), silica - induced pulmonary fibrosis (Piguet, P.F. et al. Nature 344 : 245 (1990)-), tumor metastasisation (Malik, S.T.A. et al. Eur. J. Cancer 26 : 1031 (1990)), hypercoagulable states (Bauer, K.A. et al. Blood 74 : 165 (1989)) and renal diseases (Bertani, T. et al. Am. J. Path. 134 : 419 (1989)). This has suggested that agents that inhibit TNFa activity (TNFa inhibitors) can be therapeutically useful in those disease states in which TNFa has been shown to play a detrimental role.

0005Several TNFa inhibitors have been described in the last few years : monoclonal antibodies (Tracey, K.J. et al. Nature 330 : 662 (1987)), tryptophan and indole (Yuhas, Y. et al. Eur. Cytokine Net. 1 : 35 (1990)), a-melanocyte-stimulating hormone (Mason, M.J. and Van Epps, D. J. lmmunol. 142 : 1646 (1989)), epidermal growth factor and transforming growth factor (Sugarman, B.J. et al. Cancer Res. 47 : 780 (1987)) and uromodulin (Sherblom, A.P. et al. J. Biol. Chem. 263 : 5418 (1988)).

0006Now we have found that suramin acts as a potent TNFa-inibitor and accordingly it can be used as a prophylactic and/or therapeutic agent in those disease states in which TNFa exerts a pathogenic effect. Accordingly, the present invention provides the use of suramin, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for use in the prophylaxis and/or treatment of a disease state in which Tumor Necrosis Factor a exerts a pathogenic effect.

0007Suramin is 8,8' - (carbonylbis(imino-3,1-phenylenecarbonylimino (4-methyl-3,1-phenylene) carbonylimino)) bis-1,3 ,5-naphthalenetrisulfonic acid (GB Patent No. 224849). This polyanionic compound has been used for many decades as a prophylactic and therapeutic agent for try- panosomiasis. It was subsequently shown that suramin is able to block the activity of a variety of proteins like cellular and viral enzymes and growth factors (Mitsuya, M. et al. Science 226 : 172 (1984), Hosang, M. J. Cell. Biochem. 29 : 265 (1985), De Clercq, E. Cancer Lett. 8 : 9 (1979)).

0008Given these activities it is currently under evaluation as an antitumor agent and clinical trials are being performed.

0009Examples of pharmaceutically acceptable salts of suramin are either those with inorganic bases, such as sodium, potassium, calcium and aluminium hydroxides, or with organic bases, such as lysine, triethylamine, triethanolamine, dibenzylamine, methylbenzylamine, di-(2-ethylhexyl)-amine, piperidine, N-ethylpiperidine, N,N-diethylaminoethylamine, N-ethylmorpholine, _;8-phenethylamine, N-benzyl-{3-phenethylamine, N-benzyl-N, N-dimethylamine and the other acceptable organic amines.

0010We have employed 2 different assays in order to demonstrate that suramin acts as a TNF a-inhibitor.

0011First, we have studied the capacity of suramin to inhibit the binding of human TNFa to specific cell- surface receptors. Second, we have studied the capacity of suramin to inhibit the cytotoxic activity of human TNF a on a cell line in vitro.

0012In the accompanying figures <ul id="ul0001" list-style="none"><li>Figure 1 shows the inhibition, by suramin, of the binding of different concentrations of human TNFa to human K562 cells. Inhibitory concentrations ₅₀ (IC₅₀) of suramin were 78 µm, 126 µM and 180 u.m for 2.5 x 10-"M <chemistry id="chem0001" num="0001"><img file="EP0486809A2_D0001.tif" /></chemistry>10^-10M <chemistry id="chem0002" num="0002"><img file="EP0486809A2_D0002.tif" /></chemistry> and 4 x 10-'°M (Δ-Δ) TNFα, respectively.</li><li>Figure 2 shows the inhibition, by suramin, of the binding of a concentration of 3 x 10^-10M human TNFa to mouse LM cells. The IC₅₀ was 175 µM.</li><li>Figure 3 shows the inhibition, by suramin, of the binding of 10^-10M human TNFa to K562 cells. In this experiment suramin and TNFa were added simultaneously to the cells (o-o). Alternatively suramin and TNFa were mixed and incubated for 4 (8-8) or 24 hours (A-A) before being added to the cells.</li><li>Figure 4 shows the effect of suramin on the binding of 10^-10M human TNFa to K562 cells. In this experiment suramin and TNFa were added either simultaneously to the cells (o-o) or sequentially (•-•).</li><li>Figure 5 shows the effect of different doses of suramin on the cytotoxic activity of different doses of human TNFa on mouse LM cells treated with actinomycin D.</li><li>Figure 6 shows the effect of different doses of suramin on the cytotoxic activity of different doses of human TNFa on untreated mouse LM cells.</li></ul>

0013In the experiment depicted in Figure 1 suramin was mixed, at the indicated doses, with 2.5 x 10^-11M, 10^-10M, or 4 x 10^-10M iodinated human TNFa - (New England Nuclear, NEN, Wilmington, DE). The mixtures were then added immediately to K562 cells. The residual binding of TNF a was measured and compared to the binding to K562 cells in the absence or presence of a 100-fold higher dose of "cold" unlabeled human TNFa (Esquire Chemie AG, Zuerich, Switzerland). As can be seen from Figure 1 suramin is able to inhibit the binding of iodinated TNFa to human K562 cells. Higher doses of suramin are required to inhibit the binding of higher doses of iodinated TNFa.

0014In the experiment depicted in Figure 2 suramin was mixed, at the indicated doses, with 10^-10M iodinated human TNFa. The mixtures were then added to mouse LM cells. The residual binding of TNFa was measured and compared to the binding to LM cells in the absence or presence of a 100-fold higher dose of "cold" unlabeled human TNFa. As can be seen from Figure 2 suramin is able to inhibit the binding of 3 x 10^-10M iodinated TNF a to mouse LM cells as well.

0015In the experiment depicted in Figure 3 suramin was mixed at the indicated doses, with 10^-10M iodinated human TNFa. The mixtures were either incubated for 4 or 24 hours or TNFa and suramin were added simultaneously to K562 cells. The residual binding of TNFa to K562 was measured and compared to the binding in the absence or presence of a 100-fold higher dose of "cold" unlabeled human TNFa.

0016As can be seen from Figure 3, preincubating suramin and TNFa for 24 hours significantly decreases the IC₅₀ of suramin.

0017In the experiment depicted in Figure 4 suramin and 10^-10M iodinated human TNFa were added either simultaneously or sequentially to K562. When added sequentially, K562 cells were washed between the incubation with suramin and that with TNFa.

0018As can be seen from Figure 4, no inhibition of the binding of TNFa to K562 is observed in the sequential addition of suramin and TNFa.

0019Taken together, the results depicted in Figures 3 and 4 show that suramin exerts its inhibitory effect on the binding of TNFa to K562 cells through a direct action on TNFa and not through an action on TNFa-receptors on K562 cells.

0020In the experiment depicted in Figure 5 different concentrations of human TNFa (Esquire) were incubated, in the absence or presence of different doses of suramin, with mouse LM cells treated with actinomycin D. Treatment with actinomycin D is known to render LM cells more sensitive to the cytotoxic activity of TNFa (Ruff, M.R. and Gifford, G.E., J. lmmunol. 125: 1671 (1980)). The cytotoxic activity of human TNFa was measured the day after.

0021The experiments depicted in Figure 6 were similar to the experiments of Figure 5, except that actinomycin D was omitted from the incubation mixture. In this case the cytotoxic activity of human TNFa was measured after 2 days. As can be seen, in both experiments suramin inhibited, in a dose- dependent manner the cytotoxic activity of human TNFa. The degree of inhibition was dependent on the activity of human TNFa employed, i.e. doses of human TNFa exerting high cytotoxic activity were less inhibitable than those exerting lower cytotoxic activity.

0022By virtue of the disclosed TNFa-inhibitory activity suramin or a pharmaceutically acceptable salt thereof can be employed in mammals, including humans, for prophylactic and/or therapeutic use in any disease state in which TNFa plays a detrimental role. Typically, such disease states are septic shock, graft-versus-host disease, cerebral malaria, cachexia, AIDS, rheumatoid arthritis, myocardial ischemia and reperfusion, bactericidal granulomas, adult respiratory distress syndrome, silica - induced pulmonary fibrosis, acute and chronic inflammatory disease states.

0023An effective amount of suramin or a pharmaceutically acceptable salt thereof is administered to a patient subject to or suffering from a disease state in which TNFa exerts a pathogenic effect. Single or multiple administrations can be carried out with dose levels and patterns being selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of suramin sufficient to treat effectively a said disease state or to prevent the onset of a said disease state. Doses of suramin ranging e.g. from about 20 to about 200 mg/kg of body weight can be used for parenteral administration in adult humans.

0024The present invention also provides products containing suramin, or a pharmaceutically acceptable salt thereof, and a different active agent, as a combined preparation for simultaneous, separate or sequential use in the treatment of a disease state in which TNFa exerts a pathogenic effect. A method of preventing or treating a disease state in which TNFa exerts a pathogenic effect may therefore comprise the combined administration of an effective amount of (1) suramin or a pharmaceutically acceptable salt thereof, and (2) a different active agent to a patient subject to or suffering from the said disease state.

0025The term "combined" method of treatment is meant to include both separate and substantially contemporaneous administration of suramin or a pharmaceutically acceptable salt thereof and a different pharmaceutically active agent. Therapeutically effective amounts of suramin or a salt thereof and a different active agent are administered, typically as pharmaceutical compositions.

0026Active agents that can be administered with suramin depend on the disease state. They include, for instance, gamma globulin, immunoglobulin and monoclonal antibody products, antibiotics and antimicrobial products. Typically, the antimicrobial agents may include a penicillin in conjunction with an aminoglycoside (e.g. gen- tamycin, tobramycin). However several well known additional agents, e.g. cephalosporins, can be utilized.

0027Suramin or a salt thereof and pharmaceutical compositions comprising suramin or a suramin salt can be administered orally or parenterally by subcutaneous, intramuscular, intraarterial or intravenous administration.

0028Pharmaceutical compositions containing suramin, either as the sole active agent or together with a different active agent, are usually prepared following conventional methods. The compositions, generally speaking, comprise carriers and/or diluents which include alcoholic/aqueous solutions, emulsions or suspensions, including saline or buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, antioxidants, chelating agents, inert gases, and the like. See, generally, Remington's Pharmaceutical Sciences 16th Ed., Mack, eds., 1980.

0029In the following we describe the assays that have been employed in order to detect the TNFa inhibitory activity of suramin. It will be immediately appreciated by those of ordinary skill in the art that any other in vitro or in vivo TNFa-assay can employed in order to detect the TNFa -inhibitory activity of suramin. Therefore the following description of the assays is not intended to limit the invention in any manner.

1) Inhibition by suramin of the binding of iodinated human TNF a to K562 cells.

0030lodinated human TNFa (NEN, specific activity = 30 - 70 µCi/µg protein) is diluted so to have concentrations of 1.6 x 10-⁹M, 4 x 10^-10M and 10^-10M in RPMI 1640 medium (Flow, Opera, Italy) supplemented with 2% foetal calf serum (FCS, PBI, Milan, Italy) and 14 mM Hepes (Flow). This medium will hereon be referred to as binding medium (BM). "Cold" unlabeled human TNF a (Esquire) is diluted in BM so to have concentrations of 1.6 x 10^-7M, 4 x 10-⁸M and 10-8M. Suramin (Bayer, Leverkusen, FRG) is dissolved in BM so to have a concentration of 4 mM. Starting from this solution a series of dilutions are set up in BM.

0031K562 cells (human chronic myelogenous leukemia, ATCC CCL243) are adjusted to a concentration of 2 x 10⁶/ml in BM. 100 µl of the cell suspension are then incubated with 50 µl of each iodinated TNFa concentration and with 50 µl BM or 50 µl of a 100 fold higher concentration of "cold" TNF a or 50 µl of each suramin dilution. The mixtures are incubated for 4 hours at 40 C. Thereafter the cells are centrifuged through a phthalate-dibutyl phthalate oil mixture (1:1,5 vol/vol, Fluka AG, Buchs, Switzerland). Cell-associated radioactivity is determined in a gamma-counter. Data thus obtained are processed by means of an equilibrium binding data analysis program (EBDA, Elsevir Science Publishers, Amsterdam, Netherlands).

0032In one series of experiments suramin and iodinated human TNFa are coincubated for 4 or 24 hours and then added to the cells. The experimental design is similar to the one described previously except that <ul id="ul0002" list-style="none"><li>- 100 µl of 2 x 10^-10M iodinated human TNFa are mixed with 100 µl BM or 100 µl of a 100 fold higher concentration of "cold" TNFa or 100 µl of different suramin diluitions. The suramin diluitions are added either immediately or after 20 or 24 hours. The mixtures are allowed to stand during the preincubation periods at 4"C. Thereafter the mixtures are added to a pellet of 2 x 10⁵ K562 cells and further incubated for 4 hours at 4 C. In another series of experiments the effect of suramin on the TNFa-binding capacity of K562 cells is evaluated. The experimental design is similar to the first except that</li><li>- 100 µl of the K562 cell suspension are coincubated with 100 µl BM or with 100 µl of different suramin dilutions for 4 hours at 4 C. Thereafter the cells are centrifuged. Cells that had been preincubated with BM are resuspended in 200 µl 10^-10M iodinated human TNFa with or without a 100 fold higher concentration of "cold" TNF. Cells that had been preincubated with the different suramin dilutions are resuspended in 200 µl 10^-10M iodinated human TNFa alone. In parallel other K562 cells that had not been preincubated are resuspended in iodinated human TNFa with or without "cold" TNFa. Thereafter the mixtures are incubated for other 4 hours at 4°C and treated as described previously.</li></ul>

2) Inhibition by suramin of the binding of iodinated human TNF a to LM cells.

0033lodinated human TNFa is diluted in BM so to have a concentration of 1.2 x 10-⁹M. "Cold" unlabeled human TNFa is diluted in BM so to have a concentration of 1.2 x 10-⁷M. Suramin is dissolved and diluted in BM as for the binding assay on K562 cells.

0034For the binding assay, 24-well plates (Costar, Vis- mara Associate, Trezzano sul Naviglio, Italy) are used, that have been seeded the day before with mouse LM cells (Rubin, B.Y. et al. J. Exp. Med. 162: 1099 (1985)), 200.000 cells/well. The medium is discarded and replaced with 100 µl BM supplemented with 0.04% Na N₃, 50 µl iodinated human TNF a and 50 µl BM or 50 µl cold TNF a or 50 µl of each suramin dilution. After 4 hours at 4°C cells are washed 4 x with BM, solubilized with 2% SDS and cell-associated radioactivity is determined. Data are processed as described before.

3) Inhibition by suramin of the cytotoxic activity of TNF a on untreated mouse LM cells.

0035On day O logarithmically growing mouse LM cells (Rubin, B.Y. et al. J. Exp. Med. 162 : 1099 (1985)) are trypsinized, centrifuged and resuspended at a final concentration of 5 x 10⁴ cells/ml of Eagle's minimun essential medium supplemented with 5% FCS and 1% glutamine (complete MEM). 100 µl of this suspension are added to each well of flat bottom microtiter plates (Falcon 3072, BD, Bridg- waterlane, NJ). The plates are then incubated overnight at 37 C, 5% C0₂. At this point a series of 2- fold dilutions of human TNFa are set up in complete MEM. Suramin is dissolved in complete medium so to have a concentration of 1 mM. Starting from this solution a series of dilutions are set up in complete MEM.

0036At this point 50 µl of each TNFa dilution and 50 µl of each suramin dilution are added to the wells.

0037To some wells 50 µl of each TNFa dilution and 50 µl of complete MEM are added. To some other wells 50 µl of complete MEM and 50 µl of each suramin dilution are added. Finally to some other wells only 100 µl of complete MEM are added.

0038Thereafter the plates are incubated for 48 hours at 37°C.

0039At the end of this incubation period 20 µl of thiazolyl blue solution (MTT, Calbiochem, 50 mg/ml in PBS, La Jolla, California) are added to each well.

0040The plates are further incubated for 4 hours at 37 °C. Then, supernatants are discarded, 200 µl of dimethylsulfoxide (DMSO, Farmitalia Carlo Erba) are added to each well and the extent of color development is read on a plate ELISA reader at 570 nm. OD values obtained for each TNF a concentration and each TNFa-suramin mixture are expressed as percent (%) of those obtained for the controll wells containing only complete MEM and complete MEM plus each suramin dilution, respectively. Given the linear relationship between OD values and cell viability the % values are reported in Figure 2 as % viability.

0041Inhibition by suramin of the cytotoxic activity of TNF a on actinomycin D - treated LM cells.

0042The experimental design is similar to that for the assay on untreated LM cells.

0043The differences are as follows. <ul id="ul0003" list-style="none"><li>1) 100 µl of a cell-suspension containing 3 x 10⁵ cells/ml are added to each well of microtiter plates.</li><li>2) Before setting up the TNFa-cytotoxicity assay, the cells are pretreated for 4 hours at 37 °C with complete MEM (100 µl/well) containing 2 µg/ml actinomycin D (Fluka).</li><li>3) The cytotoxic activity of human TNF a is measured after an incubation period of 24 hours at 37 ° C.</li></ul>