A. Raychaudhuri

Angiogenesis inhibition by the novel VEGF receptor tyrosine kinase inhibitor, PTK787/ZK222584, causes significant anti-arthritic effects in models of rheumatoid arthritis

AbstractObjective and design: To examine the effects of PTK787/ZK222584, a novel angiogenesis inhibitor, in a series of in vivo models of arthritis and inflammation. Materials: The granulomatous air pouch and antigen-induced arthritis models were established in female OFA-1 mice. Female DBA/1LacJ mice were used for the collagen-induced arthritis model and male OFA rats were used for the carrageenan oedema and hyperalgesia tests. Treatment: PTK787/ZK222584 was administered p.o., once daily, at various concentrations. Diclofenac (3 mg/kg) and DUP697 (0.5 mg/kg) were also given p.o, once daily. Methods: The anti-angiogenic effects of PTK787/ZK222584 were directly assessed in the granulomatous air pouch model using the carmine red assay. The anti-arthritic effects of this compound were further examined in the mouse antigen-induced and collagen-induced models of arthritis, using macroscopic observations (calliper measurements of joints) and histological scores (as assessed by degree of cellularity, cell infiltration and erosions and proteoglycan loss). All compounds were administered orally. PTK787/ZK222584 at 10, 30, 50 and 100 mg/kg and positive control compounds, diclofenac and DUP697 at 3 mg/kg and 0.5 mg/kg, respectively. In addition, the effects of PTK787/ZK222584 in the rat carrageenan oedema model and Randall Selitto hyperalgesia test were observed. Results: PTK787/ZK222584 treatment caused dose dependent reduction in the vascularity of the granulomatous air pouch model. It inhibited knee swelling by 40% in antigen-induced arthritis, at the dose of 30 mg/kg p.o., once daily (s.i.d). and inhibited both severity scores (by 51%) and global histological scores in mice with collagen-induced arthritis following oral treatment (45 mg/kg p.o.), as compared to control animals. PTK787/ZK222584 demonstrated no effects on inflammatory mediators in the VEGF-independent rat carrageenan model and displayed interesting analgesic activity in the Randall Selitto test in the acute setting. Conclusions: The anti-arthritic effects of this specific, receptor tyrosine kinase inhibitor compound appear to be mediated by anti-angiogenic actions. This study represents a new indication for PTK787/ZK222584, namely, rheumatoid arthritis and further supports the belief that angiogenesis inhibition is likely to be beneficial in the therapy of this condition.

Characterization of CGS 8515 as a selective 5-lipoxygenase inhibitor using in vitro and in vivo models

CGS 8515 inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B4 synthesis in guinea pig leukocytes (IC50 = 0.1 microM). The compound did not appreciably affect cyclooxygenase (sheep seminal vesicles), 12-lipoxygenase (human platelets), 15-lipoxygenase (human leukocytes) and thromboxane synthetase (human platelets) at concentrations up to 100 microM. CGS 8515 inhibited A23187-induced formation of leukotriene products in whole blood (IC50 values of 0.8 and 4 microM, respectively, for human and rat) and in isolated rat lung (IC50 less than 1 microM) in vitro. The selectivity of the compound as a 5-lipoxygenase inhibitor was confirmed in rat whole blood by the 20-70-fold separation of inhibitory effects on the formation of leukotriene from prostaglandin products. Ex vivo and in vivo studies with rats showed that CGS 8515, at an oral dose of 2-50 mg/kg, significantly inhibited A23187-induced production of leukotrienes in whole blood and in the lung. The effect persisted for at least 6 h in the ex vivo whole blood model. CGS 8515, at oral doses as low as 5 mg/kg, significantly suppressed exudate volume and leukocyte migration in the carrageenan-induced pleurisy and sponge models in the rat. Inhibitory effects of the compound on inflammatory responses and leukotriene production in leukocytes and target organs are important parameters suggestive of its therapeutic potential in asthma, psoriasis and inflammatory conditions.

Effect of capsaicin on carrageenan-induced inflammation in rat pleurisy and exudate substance P level.

Injection of 2.5 mg of λ-carrageenan into the rat pleural cavity resulted in a time-dependent increase in pleural exudate substance P (SP) levels up to 24 hr. Synergistic increases in the exudate formation were observed when a sub-optimal quantity of carrageenan was injected with SP. Pre-treatment of rats with capsaicin at 50 and 100 mg/kg s.c. daily for one week prior to the induction of pleurisy blocked the increase in exudate volume and SP levels when compared to that normally detected after carrageenan injection. These results suggest that inhibition of SP production may improve inflammatory conditions.

Blockade of Integrin VLA-4 Prevents Inflammation and Matrix Metalloproteinase Expression in a Murine Model of Accelerated Collagen-Induced Arthritis

DBA/1LacJ mice were immunized with type II collagen and boosted with bacterial lipopolysaccharide (LPS) 17 days later to induce accelerated arthritis. Clinical signs of inflammation were observed as early as Day 20. Matrix metalloproteinases MMP-2, -3, -9, and -13, but not MMP-12, mRNA levels were increased on Day 24. Administration of anti-VLA-4 antibody (mAb; 8 mg/kg/day for 3 days) at the time of LPS treatment strikingly inhibited arthritis-induced paw inflammation and histological scores, but not the increase in MMP expression. A higher dose of mAb (20 mg/kg/day for 4 days) inhibited pathology and normalized the levels of MMP mRNAs. In conclusion, the pathophysiology of this accelerated model of arthritis is VLA-4-dependent, and VLA-4-mediated events have a role in inflammation-induced MMP expression. Inhibition of arthritis-induced increases in MMP expression is not necessary to reduce pathology. This model is well suited for identifying agents that block integrin VLA-4 in vivo.

Effect of CGS 25019C and other LTB4 antagonists in the mouse ear edema and rat neutropenia models

The pro-inflammatory lipid mediator leukotriene B 4 (LTB4) is derived from arachidonic acid (AA) metabolism through the activation of the 5-1ipoxygenase enzyme [1, 2]. Increased levels of LTB4 have been detected in patients with a variety of inflammatory diseases [3]. It has thus been postulated that LTB 4 receptor antagonists may have unique anti-inflammatory activity. As a result of extensive research, reports of potent, orally active, and selective LTB 4 receptor antagonists have emerged as potential anti-inflammatory agents [4, 5]. We have identified novel basic aryl amidines as LTB4 receptor antagonists that block LTB4-induced neutrophil func: tions including C D l l b up-regulation, calcium influx, aggregation, chemotaxis, and degranulation [5, 6]. Here, we present evidence of in vivo activities of two potent LTB4 receptor antagonists, CGS 25019C (4-{5-{4(aminoiminomethyl)phenoxy}pentoxy}-3-methoxy-N,Nbis(1-methylethyl) (Z)-2-butenedioate (1:1)) and a structural analog, CGS 24426A, from this chemical series. The compounds were evaluated in the AAinduced ear edema model in mice and LTB4-induced neutropenia model in rats.

Effect of anti-inflammatory compounds on edema formation and myeloperoxidase activity in the arachidonic acid-induced ear model in the mouse

The arachidonic acid (AA)-induced ear edema model in the mouse has been demonstrated as an effectivein vivo experimental tool to screen compounds showing anti-inflammatory activity. Since neutrophil influx is a component of the inflammatory reaction, we have modified this assay by quantitating myeloperoxidase (MPO) levels which reflect neutrophil accumulation in the edematous biopsies of the mouse ear. Our work has shown that orally administered 5-lipoxygenase inhibitors, dual inhibitors (CO/LO), and steroids dose-dependently inhibit both edema formation and MPO activity, whereas oral activity is not seen with NSAIDs. There is a good correlation between the inhibition of edema formation and of MPO activity by these compounds. Thus, measurement of MPO, in addition to the AA-induced edema in the mouse ear, can provide another parameter to profile potential anti-inflammatory compounds.

Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model

Abstract An intrapleural injection of carrageenan in rats induced LTB4 and LTC4/D4/E4 biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100nmol) 16–18h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30mg/kg) inhibited the enhanced LTB4 and LTC4/D4/E4 (1 to 10mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r2=0.989) between pleural fluid concentration of LTB4 and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB4 and LTC4/D4/E4 in an ongoing inflammatory response.

Effect of 5-lipoxygenase inhibitors onin situ LTB4 biosynthesis following calcium ionophore stimulation in the rat pleural cavity

The intrapleural injection of carrageenan in the rat induces exudate formation, cellular influx and leukotriene generation in the pleural cavity. We have demonstrated that the inflammatory response (exudate volume, and LTB4 levels) is increased,in situ by the intrapleural administration of calcium ionophore A 23187 (100 nmol/rat) at 4, 16, 24, 48, and 72 h after the injection of carrageenan and that the A 23187-induced increase is dose-dependent. The oral administration of A-64077 and MK-886, two 5-lipoxygenase inhibitors (5-LOIs), at 10 mg/kg causes marked decreases in LTB4 release at the above-mentioned time intervals. However, A 23187-induced augmented exudate formation is not affected by the treatment with 5-LOIs. The results suggest that the use of 5-LOIs to inhibit LTB4 biosynthesis may be beneficial in various LTB4-dependent pathological conditions.

CGS 26529: the biological profile of a novel, orally active 5-lipoxygenase inhibitor with an extended duration of action.

Since the discovery by Samuelsson and colleagues that 5-hydroxyeicosatetraenoic acid (5-HETE) and the leukotrienes (LTs) are formed from arachidonic acid (AA) via the 5-1ipoxygenase (5-LO) pathway, most major pharmaceutical companies have maintained a substantial research effort to discover orally active 5-LO inhibitors (5-LOis) [1]. The ability of LTs to induce an inflammatory response together with the recovery of these lipids from tissue undergoing pathological reactions have suggested their importance in mediating a wide spectrum of human disorders. Recent studies demonstrating the beneficial effects by A 64077 (Zileuton, Abbott) in asthma, allergic rhinitis, rheumatoid arthritis and inflammatory bowel disease [2] have further stimulated the search for therapeutic agents which can inhibit LT synthesis. This paper profiles the pharmacological properties of CGS 26529, N-[2[[2-[[4-(4-fluorophenyl)phenyl]methyl]1,2,3,4tetrahydro1-oxo-6-iso-quinolinyl]oxy]ethyl]-N-hydroxyurea, a novel 5-LOI. Its activity in vitro as a selective 5LOI, as well as its in vivo efficacy and extended duration of action after oral administration in two animal models of inflammation, will be described.

Elevation of plasma hyaluronan and urinary pyridinoline in mouse collagen induced arthritis

We have previously reported the elevation of plasma hyaluronan (HA) and urinary pyridinoline (PYD) in rat models of arthritis (1,2). Elevation of plasma HA has been linked to the hypermetabolic response of the synovium (3). Urinary pyridinoline cross links reflects the breakdown of collagen type I and type II from bone and cartilage (4,5). Because the mouse collagen induced arthritis model exhibits many characteristics of rheumatoid arthritis (RA), including pannus and bone erosions (6), we examined the time dependent changes of these two markers in this model and the effect of a therapeutic agent on PYD excretion. In this report, we discuss for the first time the elevation of these markers in a mouse type II collagen induced arthritis model.