Tenidap sodium inhibits secretory non-pancreatic phospholipase A2 synthesis by foetal rat calvarial osteoblasts

Tenidap (TD) was initially defined as a dual inhibitor of cyclooxygenase and lipoxygenase. This study was designed to assess its inhibitory activity against proinflammatory phospholipase A2. This study shows that TD inhibits the synthesis of pro-inflammatory secretory non-pancreatic phospholipase A2 (sPLA2). Concentrations as low as 0.25 μg/ml (0.725 μM) reduced the release of sPLA2 by 40% from foetal rat calvarial osteoblasts stimulated with IL-1β and TNFα, whereas a concentration of 2.5 μg/ml (7.25 μM) reduced the release by over 80%. TD also markedly reduced the release of sPLA2 from unstimulated cells. There was no direct inhibition of sPLA2 enzymatic activity by TD in vitro. Northern blot analysis showed that TD did not affect the sPLA2 mRNA levels; however, immunoblotting showed a dose-dependent reduction in sPLA2 enzyme. These results, together with a marked reduction in sPLA2 enzymatic activity, suggest that TD inhibits sPLA2 synthesis at the post-transcriptional level. Therefore TD seems to inhibit the arachidonic acid cascade proximally to cyclooxygenase and lipoxygenase and its anti-inflammatory activity may be related at least in part to the inhibition of sPLA2 synthesis.


Introduction
Tenidap sodium ([Z]-5-chloro-2,3-dihydro-3[hydroxy-2-thienylmethylene]-2-oxo-1-H-indole-1carboxyamide, sodium salt) (TD) is a new anti-inflammator agent which was originally designated as a dual inhibitor of cyclooxygenase and lipoxygenase. 1," It soon became apparent that TD had other biological properties such as inhibition of IL-1 production in LPS-stimulated cells, 3,4 inhibition of the release and/or activity of collagenase and myeloperoxidase and suppression of the expression of circulating acute phase reactants such as serum amyloid A in rheumatoid patients treated with TD and of C-reactive protein in rats with adjuvant arthritis. TD also inhibits bone resorption induced by cytokines IL-1 and TNF. Since some of the above inhibitory activities resemble those of other agents which are known inhibitors of phospholipase a (PEA2) we undertook a study of the impact of TD on the synthesis of secretory non-pancreatic PLA,. (sPLA2) by foetal rat calvarial osteoblasts. The data suggest that TD is a potent inhibitor of sPLA synthesis. These findings add a new aspect to the spectrum of biological activities of this compound and may lead to a better understanding of its observed antiinflammatory effects.

Materials and Methods
Tenidap sodium, MW 343.75 was the kind gift of Pfizer Canada Inc. sPLA activity was assessed as described in detail using radiolabelled Escherichia coli membrane phospholipid substrate. Foetal rat calvarial cells (FRCO) were prepared as reported previously. I Confluent cultures of FRCO were incubated in the presence of TD in concentrations ranging from 0.25 l.tg/ml (0.725 l.tM) to 50 l.tg/ml (145 t.tM) for 24 and 48 h. Foetal calf serum was withdrawn from the medium when TD and/or IL-1 and TNF were added to the cultures. FRCO were co-stimulated with IL-I[ (100 U/ml or 0.2 ng/ml) and TNF0 (500 U/ ml or 25 ng/ml) as described previously. I FRCO viability as tested by trypan blue exclusion was > 95%. The cells were counted at the end of each experiment. Each experiment was performed in triplicate and repeated at least twice, sPLA activity was expressed as units per ml of the medium and as units per I x 106 cells/ml. RNA isolation and Northern blot analysis: RNA was isolated from cultured FRCO by the method of Chomczynski and Sacchi. 11 Briefly, 5 x 107 to 1 x 108 cells, were homogenized using lysing buffer containing 4 M guanidium thiocyanate. RNA was purified by () 1995 Rapid Communications of Oxford Ltd phenol:chloroform:iso-amyl alcohol extraction, followed by two iso-propanol precipitations. RNA was stored as an ethanol precipitate at-70C until tested.
Northern blot analysis of total RNA was performed on 1% agarose/formaldehyde gels and the RNA was blotted onto nitrocellulose. 12 The probe used for hybridization was the rat sPLA cDNA. 13 The DNA fragment was labelled by random priming (Pharmacia) and prehybridization was done in a 50% formamide buffer at 42C. The blot was washed in lx55C 0.5% SDS at 50C and exposed to Kodak XARfilm at-80C with an intensifying screen.
Western blot analysis: SDS-polyacrylamide gel electrophoresis was done on 12% polyacrylamide gels using the method of Laemmli. 14 Proteins were then electrophoretically transferred to nitrocellulose (Hybond*M-ECL, Amersham Canada, Ltd) 5 for immunoblot analysis, The primary antibody was a rat sPLA monoclonal antibody (mAb 2E7 plus 2B9). 16 The blot was developed using enhanced chemiluminescence (ECL) as described by the manufacturer (ECL TM Western blotting protocols, Amersham International plc). The differences between sPLA2 activity in control cultures supernatants and TD treated cells were assessed using Student's test. sPLA2 by 40%, whereas a concentration of 1.25 btg/ml (3.625 l.tM) suppressed sPLA activity by 68%. Concentrations of 2.5 btg/ml (7.25 l.tM) reduced sPLA activity by over 80% (Fig. 1). TD in concentrations over 1.25 l.tg/ml was slightly inhibitory to cell proliferation, reducing the final number of cells from 0.92 + 0.02 x 10/ml in controls to 0.78 + 0.02 x 10/ml; concentrations over 2.5 g/ml reduced final cell counts to 0.63 + 0.03xl0/ml. Northern blot analysis of FRCO total RNA showed that TD at 1.25 t.tg/ml did not affect the level of sPLA mRNA in unstirnulated or IL-1/TNF stimulated cells (Fig. 2). In contrast, immunoblot analysis revealed reduction in the quantity of sPLA in FRCO treated with 0.25 btg/ml (0.725 btM) TD (Fig. 3). At the TD concentration of 3.75 btg/ml (10.875

Discussion
Early studies have identified TD as dual inhibitor of cyclooxygenase and lipoxygenase. 1,2 TD inhibited PGE and LTB synthesis by ionophore-stimulated human PMNs. In rat basophilic leukaemia cells, TD inhibited 5-HETE and LTB4 synthesis as well as PGD2 . 17 However, compared with cyclooxygenase, the inhibition of lipoxygenase required a 14-fold higher concentration of TD. Similar observation was made when plasma-free leukocyte suspensions were tested in the rat. 18 The IC50 of TD for cyclooxygenase and lipoxygenase was 0.05 t-tM and 10 btM respectively. When whole blood was used, TD did not suppress 5-1ipoxygenase at all. It was therefore concluded that, in vivo, TD is essentially a selective inhibitor of cyclooxygenase. 18 Subsequent studies of TD have detected its impact on cytokine production. Endotoxin-induced IL-1 synthesis in murine peritoneal macrophages was markedly inhibited by TD (IC50 1 btg/ml). Subcellular studies identified the block of pro-IL-lot in the cells.
Higher concentrations of TD (10 btg/ml) also inhibited IL-1 activity in LPS-stimulated human monocytes 19 and production of IL-6, and to the lesser degree of TNF and IL-1 in LPS stimulated human peripheral blood mononuclear cells, i TD inhibited the release of activated collagenase and of myeloperoxidase from neutrophils. It also inhibited the expression of IL-1 receptor mRNA and IL-1 receptor levels and reduced collagenase and stromelysin activity in cultured normal and osteoarthritis chondrocytes. 21 The observation that rats with adjuvant arthritis treated with TD show marked reduction in the paw swelling and circulating CRP and that human patients with rheumatoid arthritis show reduction in circulating SAA and CRP 6,2 may be related to the above suppressive activity. Yet, in cytokine-stimulated Hep-3B hepatoma cells in vitro, TD was unable to block SAA synthesis. TD used together with chemically modified tetracycline, synergistically inhibited the tissue activity of collagenase and gelatinase in adjuvant arthritis and substantially reduced radiological severity of joint damage. 22 It also inhibited cytokine-induced bone resorption of 45Ca-labelled mouse calvaria. Some clinical improvement was noted in a few short-term open trials of rheumatoid arthritis and osteoarthritis. 2>25 Since TD may be potentially useful in the therapy of inflammatory conditions, its impact on the inflammatory cascade is of significant interest. Secretory non-pancreatic phospholipase a has been identified as one of the pivotal pathogenetic agents in both local  and systemic 29-31 inflammatory conditions. Marked elevation of circulating sPLA was found in adult and juvenile rheumatoid arthritis, correlating well with the disease activity. 32,33 sPLA also correlated significantly with the complications and outcome of septic shocM and multi-organ failure. 4 sPLA2 injected into joints of experimental animals induced dose-dependent synovitis. 2<5 The synthesis of sPLA by osteoblasts, 1 chondrocytes and smooth muscle cells 37 is induced and enhanced by cytokines, especially IL-1 and TNF. It has recently been reported that some agents that inhibit collagenase 3 also inhibit sPLA2 interaction with the substrate. 39 We hypothesized that TD might also inhibit sPLA synthesis. Indeed, this study has shown that concentrations as low as 0.25 I.tg/ml (0.725 btM) markedly inhibited the synthesis of sPLA by foetal rat calvarial osteoblasts. We have shown that the synthesis of sPta was most probably blocked at the post-transcriptional level. It has recently been reported that TD generally inhibits protein synthesis in cells. 41,42 Thus, inhibition of sPLA synthesis by TD seems to be an important part of this general effect on cell metabolism. TD had no direct effect on PEA enzymatic activity.
The fact that TD inhibits the synthesis of sPLA adds to the repertoire of its biological activities and shows that this agent may inhibit the arachidonic acid cascade, at a level proximal, to that of cyclooxygenase and lipoxygenase. Marked inhibition of sPLA synthesis by TD may be partially responsible for its anti-inflammatory activity.