Anti-Inflammatory Activity of (Polyphenolic)-Sulfonates and Their Sodium Salts in Rodents

A series of polyphenolic-sulfonated compounds were observed to have potent anti-inflammatory activity and were protective against induced endotoxic shock in mice at 8 and 16 mg/kg, I.P. These agents proved to be potent elastase inhibitors in human leukocytes and J774-AI and IC-21 mouse macrophages as well as prostaglandin cyclo-oxygenase inhibitors in J774-AI macrophages. The compounds from 5 to 50 μM inhibited TNFα release from IC-21 macrophages and IL-1 release from mouse P388D1 macrophages induced by LPS. The binding of these cytokines to high affinity receptors on target cells, e.g. L929 fibroblasts and IL-2 in HuT78 T lymphoma cells, were also suppressed by the agents. These compounds blocked the adhesion of leukocytes and macrophages to the plasma membranes of L929 fibroblasts.


INTRODUCTION
Cyclic imides [1][2][3], -, 13-and y-alkylaminopropiophenones [4], amine-carboxyboranes and their metal complexes [5,6], thiosemicarbazones and their metal complexes [7], and triazolidinedione derivatives [8] have previously been shown to be potent anti-inflammatory and analgesic agents in rodents. These low molecular weight agents like other non-steroidal anti-inflammatory agents were able to block lysosomal hydrolytic and proteolytic enzyme activities as well as the synthesis of prostaglandins and leukotrienes, chemical mediators of inflammation. In addition they were able to protect against free radicals generated during inflammation. These small molecules blocked the release of cytokines synthesized by macrophages, i.e. TNF and IL-1 and I1-6. The uniqueness of these agents is that they also competitively displace cytokines from binding to their high affinity receptors on L929 cells. Few reports in the literature indicate interaction at these receptor sites other than by peptides or antibodies. Suramin, a polysulfate naphthylurea, has been shown to be an IL-6 high affinity receptor antagonist [6]. Blocking these receptors is desirable because the entire inflammation cascade of events is triggered by the early release of TNFa and IL-1. The therapeutic use of small molecular weight antagonists is more desirable than the use of large molecular weight glycoproteins or growth hormones to block the inflammation or shock process.

Materials and methods
The polyphenolic-sulfonated compounds found in  [9,10]. Evaluation of the induced edema was made by injecting 2% carrageenan in 0.9% saline into the plantar region of the foot. The opposite foot injected with 0.9% isotonic saline was used as a base line. Indomethacin (8 or 10 mg/kg), pentoxifylline (50 mg/kg) and phenylbutazone (50 mg/kg) were used as standards for comparison of activity. In vitro TNFa and IL-1 Measurements and Cellular Regulation IC-21 mouse macrophages were maintained in RPMI-1640 + 10% FCS + P/S. After the cells had grown to confluency, E. coli LPS at 10 Ixg/ml was added to the medium [12,13]. Agents were incubated at 12.5 to 100 M final concentration for 18 hr. The medium (100 btl) was collected for TNFCt determinations.
Interleukin-1 [IL-1] release was determined using P388 D1 cells which were maintained in RPMI-1640 + 10% FCS + P/S. The L929 bioassay was used to quantitate the TNF and the IL-1 levels [5,6]. The L929 mouse fibroblasts were grown in DMEM + 10% FCS + P/S to confluency in 96 well plates and incubated with 100 tl of medium from IC-20 or P388 D cells after 18 hr. The cells were stained with 0.2% crystal violet in 20% MeOH and read at 580 nm using a Molecular Devices scanner (SOFT-max program).  [13,14]. The plates were developed in iodine vapor and scraped according to the Rf values of standard prostaglandins, and counted in a Packard scintillation beta counter correcting for quenching.

Elastase Activity
The substrate N-succinyl-L-alanyl-L-alanyl-L-alanine -p-nitroanilide was used in this assay and the hydrolytic product p-nitroanilide was determined at 410nm [15]. Porcine elastase EC 3.4.21.36 [Sigma Chemical Co.], human leukocytes or mouse macrophages were used as an enzyme source for the assay reaction incubated from 60 min to 30 hr. Drugs were incubated from 1 to 100 laM.
Collagenase Activity Collagenase activity was determined using Clostridium histolytricum collagenase type I, 10 g and N- [propionate 2,3-3H]propionylated collagen incubated with agents present from to 100 lxM for 24 hr at 37C 16]. The reaction was stopped with ml 50 mM EDTA and the tube centrifuged at 10,000 g for 10 in. The supematant was counted. In all of the tables, data is calculated as 100% of control + the standard deviation. The statistical significance was determined by the Student's "t" test on the raw data.

In Vivo Pharmacology Tests
The polyphenolic-sulfonate compounds proved to be potent anti-inflammatory agents and protected against endotoxin induced death in mice [ Table 2 ]. Compounds 1-4 caused greater than 44% inhibition of induced edema at 8 mg/kg X 2 while compounds 1, 4, 6 and $ afforded greater than 40% reduction of edema at 16 mg/kg X 2. Compound $ was the most potent at 16 mg/kg X 2 with 62% reduction of edema. lndomethacin at 8 mg/kg afforded 26% reduction and at 10 mg/kg afforded 78% reduction of induced edema. Phenylbutazone at 50 mg/kg X 2 resulted in 47% reduction in edema. In the LPS induced endotoxin shock assay, the survival of control animals was 16% at 52 hr [ Table 3]. Compounds 1, 2, 4 and $ and pentosan sulfate resulted in 83% protection from death at 16 mg/kg. Compound 3 at 8 mg/kg and compounds 6 and 7 at 16 mg/kg resulted in 100% protection from LPS induced death. Compound 5 at 16 mg/kg resulted in 67% protection which was equal to dexamethasone at mg/kg or pentoxifylline at 50 mg/kg.
Cytokine release and binding to high affinity receptors TNFt release from IC-21 macrophages over an 18 hr period was significantly reduced by the agents 6, 7, and 8 following a concentration dependent effect from 5 to 50 tM. Compounds 7 and $, achieved greater than 50% reduction at 50 laM whereas compound 6 resulted in only 38% reduction [ Table 4]. IL-1 release from P-388Ol macrophages over 18 hr was also markedly reduced in a concentration dependent manner with >95% reduction at 25 and 50 tM concentration of compounds 6, 7 and 8 [ Table 5]. L929 TNFt high affinity receptor binding at 90 min., the optimum time for TNFtx binding, was reduced by all three agents with greater than 50% suppression from 12.5 to 50 lxM [ Table 6]; drugs at the concentration of 25 ktM afforded the best results with >75% reduction in TNFt binding. L929 fibroblast IL-1 high affinity receptor binding at 5 hr, the optimum time for IL-1 binding, was also reduced greater than 90% by the   Table 8]. Compounds 7,8 and pentosan sulfate at 50 tM caused only 21-23% reduction at 5 hr but were more effective at 24 hr txM causing > 40% reduction. IL-2 high affinity binding to HUT78 T lymphoma cell membrane receptors at 5 hr was reduced 45% at 50 tM of compounds 6, 8 and pentosan sulfate. These effects were not as evident at 2 hr but compound 8 at 50 txM caused 62% reduction of 11-2 binding and compound 7 resulted in a 31% reduction of I1-2 binding to its high affinity receptor [ Table 9].

Enzyme Assays
Compounds 2 and 4 demonstrated potent inhibition of J774-A1 macrophage elastase activity at 60 min with IC50 values of 0.79 and 0.03 X 10 -5 M. Compounds 3,7 and 8 were effective with IC5o values of 1-3 X 10 -5 M and all other compounds were not active at these concentrations. Incubation for 120 min resulted in loss of elastase inhibition by the compounds with IC50 values from 2.5 to 11 X 10 -5 M at both 60 and 120 min. Human leukocyte elastase activity was inhibited similarly with IC50 values from 4.5 -28 X 10 -5 M [ Table 10]. Porcine elastase was inhibited at 2, 8 and 30 hr by the agnets but the IC50 values   were in the range of 5-9 X 10 -4 M. Clostridium histolytricum collagenase type I activity was not inhibited significantly from 104 to 106 M concentration of drugs atter 24 hr incubation. J774-A1 prostaglandin cyclooxygenase activity was suppressed by the agents with IC50 values of 2.06 to 7.62 X 10 -5 M [ Table  10].  Compounds 6, 7, and 8 were effective from 5 to 50 tM in reducing significantly the adhesion of human leukocytes to confluent L929 fibroblasts at 60 and 90 min but were not effective at 2 and 5 hr [ Table 11 ]. The agents were not as effective in reducing J774A1 macrophage adhesion to L929 fibroblasts at these concentrations [ Table 12]. At 2 hr at 12.5 tM, they caused 15% to 39% reduction, yet pentosan polysulfate caused 47% reduction at 12.5 tM. DISCUSSION A number of small molecular weight agents, e.g. amine-carboxyboranes and their metal complexes, indazolones, 3-imino-1-oxoisoindolines, triazolidinedione derivatives, ix, 1, and y alkylaminoketones, and thiosemicarbazones as well as their metal complexes afforded similar reductions in induced edema as the polyphenolic sulfonates. These non-steroidal anti-inflammatory agents suppress both TNFtz and IL-1 release and cytokine receptor binding from 25 to 100 tM similar to the current polyphenolic sulfonates.
These agents as well as the polyphenolic sulfonates were very effective in protecting against LPS induced shock. Shock is mediated by the release of these regulatory cytokines, i.e TNFtx and IL-1 and their binding to receptors on target inflammation cells which causes the release of inflammation enzymes and chemical mediators as well as enhancing adhesion of inflammatory cells to tissue lesions. It is difficult to determine whether the effects of the agents are direct or indirect in acting as antagonists of high affinity receptors for cytokine regulation, but they did effectively block elastase and cyclooxygenase activities and cell adhesion. The cytokines, e.g. TNFtx, are large glycoproteins which exist as a trimer, folding so that only three small regions interact with specific high cysteine regions of the high affinity receptors.
Mutagenic studies have indicated that only a few amino acid residues are important to binding of TNFtt. The oligosaccharides of the cytokine molecule are thought to play a role in the binding of the cytokine to its receptor, since alterations of their structural composition leads to decreased binding of the cytokine molecule to the high affinity receptors. Sulfamoylthiophenone and sulfamoylpyrazole carboxylic acid derivatives have recently been shown to be anti-inflammatory and analgesic agents [19]. Thus, the polyphenolic sulfonates may play a similar role at these receptor binding sites, competing with the endogenous cytokines. Since TNF regulates IL-1, IL-6 and IL-8 as well as its own release, there is a possible cross-over in the effects of agents on the cytokine receptors. More than one receptor exists on cells for some of the cytokines, i.e. TNFtx and IL-1. What is most significant concerning these findings is that it may be possible to modulate cytokine effects on metabolism, proliferation, immunomodulation, etc., without using a large molecular weight protein that has deleterious side effects such as allergy and anaphaxisis. Numerous small molecular weight agents such as flavones, thalidomide, cyclosporin A, methotrexate, dexamethasone, or pentoxifylline, block TNFo or I1-1 synthesis or release from macrophages. Tenidap, which has structural features similar to the cyclic imides, appears to effect both I1and TNFtx production and activation from human monocytes. All of the anti-inflammatory and antirheumatoid agents found to date require in vivo doses 3-50 mg/kg, yet the studies in tissue culture to block cytokine synthesis and release require IC50 values in the 0.2 to 0.5 mM range, i.e. IC50 value for pentamidine is 1 mM, IX 207-887 is 30-60 mM and hydrocortisone is 10 mM. The drug RP 54-745 decreases mRNA synthesis of both TNFct and IL-1 atter challenge with LPS. It is active in vivo at 25 mg/kg in mice but in vitro studies required 3 10 mM concentrations. Thus, the effect of polyphenolic sulfonate follows a similar pattem to other small molecular weight agents on cytokine modulation of the inflammation process. Because of these observations further investigation into their properties as potential therapeutic agents is needed.