Introduction

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Nitric oxide (NO), a small and reactive molecule, plays a vital role in various physiological processes such as modulation of inflammatory responses and regulation of vessel tone (Nathan, 1997; Hobbs et al., 1999). Production of excessive NO is involved in inflammatory and autoimmune diseases including septic shock, hemorrhagic shock, systemic lupus erythematosus, Sjögren's syndrome, vasculitis, rheumatoid arthritis, and osteoarthritis (Farrell et al., 1992; Sakurai et al., 1995; McInnes et al., 1996; Clancy et al., 1998). In patients with arthritis and autoimmune diseases, maintaining the concentration of NO at a normal level could be beneficial for a favorable outcome.

NO is synthesized by a family of nitric-oxide synthases (NOS). Three isoforms of NOS have been cloned and characterized (Stuehr, 1999). Two of them are constitutively expressed in endothelial and neuronal tissues under noninflammatory conditions, and their activity is tightly regulated (Martin et al., 1986; Bredt et al., 1990; Nakane et al., 1993; O'Dell et al., 1994). The third isoform, inducible NOS (iNOS), is a key mediator of inflammation and host defense systems (Clancy et al., 1998). Expression of iNOS is induced at a transcriptional level by inflammatory stimuli including interferon (IFN), interleukin (IL)-1, tumor necrosis factor-, and bacterial lipopolysaccharide (LPS) (Salkowski et al., 1997). Continuous expression of iNOS leads to overproduction of NO, which is closely related to the pathogenesis of the inflammatory and autoimmune diseases mentioned above. Thus, a specific inhibitor of iNOS is of potential therapeutic benefit for these diseases. Several iNOS inhibitors lead to improvement of NO-related autoimmune and inflammatory diseases (McCartney-Francis et al., 1993; Weinberg et al., 1994; Stefanovic-Racic et al., 1994, 1995; Connor et al., 1995). These iNOS inhibitors are classified into at least three groups: 1) L-arginine (Arg) derivatives that compete at the active site (Furfine et al., 1993; Weinberg et al., 1994; Pfeiffer et al., 1996), Arg being a substrate of iNOS (Hibbs et al., 1987; Stuehr, 1999); 2) metabolic inhibitors (other than Arg derivatives), such as aminoguanidine, citrulline analogs (Furfine et al., 1994; Salerno et al., 1995), N-iminoethyl-L-lysine (Bryk and Wolff, 1998), and a cyclic amidine derivative (Naka et al., 2000), that react either with the heme residue at the iNOS active site and/or a nucleophilic amino acid residue that projects into the active site (Bryk and Wolff, 1999; Stuehr, 1999); and 3) inhibitors of homodimerization, since the iNOS monomer is inactive (Baek et al., 1993; Xie et al., 1996). Chemically synthesized inhibitors that have an imidazole group have been shown to interfere with homodimerization (Sennequier et al., 1999; McMillan et al., 2000).

In this study, we have examined the inhibitory activity and mechanism of the newly synthesized agent 3-(2,4-difluorophenyl)-6-{2-[4-(1H-imidazol-1-ylmethyl) phenoxy]ethoxy}-2-phenylpyridine (PPA250) in vitro and in animal models of rheumatoid arthritis. We demonstrate that PPA250 at concentrations of 25 nM and higher inhibits iNOS activity by inhibiting dimerization. In addition, oral administration of PPA250 to a mouse with collagen-induced arthritis and to a rat with adjuvant arthritis suppressed the development of destructive polyarthritis, a disease that mimics rheumatoid arthritis.

	Materials and Methods

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Compound. PPA250 (Fig. 1; molecular weight, 483.51) was chemically synthesized in our laboratories. For in vitro assays, it was dissolved in dimethyl sulfoxide, and further dilutions were made in culture medium. The final concentration of dimethyl sulfoxide was less than 0.1%. For in vivo administration, PPA250 was suspended in 0.5% sodium carboxylmethylcellulose solution.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Chemical structure of PPA250

Cell Culture. A murine macrophage cell line, RAW264.7, was obtained from the American Type Culture Collection (Manassas, VA; TIB-71) and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 100 units of penicillin/ml, and 100 µg of streptomycin/ml (Invitrogen, Carlsbad, CA). The cells were grown at 37°C in 5% CO₂ in air in a humidified atmosphere.

Animals. Male BALB/c mice (5 weeks old) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Male DBA/1J mice (8 weeks old) and male Lewis rats (8 weeks old) were purchased from Crea Japan, Inc. (Tokyo, Japan). Animals received food and water ad libitum, and lighting was maintained on a 12-h cycle.

Determination of NO Concentration. RAW264.7 cells were cultured at an initial cell density of 2 × 10⁵ cells/well in 24-well plates. After 24 h, the medium was replaced with phenol red-free RPMI 1640 supplemented with 1% FBS and various concentrations of PPA250, 150 U of recombinant mouse IFN-/ml (Biosource, Camarillo, CA), and 2 ng of LPS/ml (from Escherichia coli 026:B6; Sigma-Aldrich, St. Louis, MO) were added. Culture supernatants were collected 18 h after stimulation. NO production was assessed by measuring the concentration of nitrite, a stable degradation product of NO, with the Griess reagent (Wako Pure Chemicals, Osaka, Japan), because RPMI 1640 medium contains 0.42 mM nitrate, another stable degradation product of NO.

Western Blot Analysis. RAW264.7 cells were seeded at 5 × 10⁶ cells in a 100-mm dish and cultured overnight. Medium was then replaced with the low serum medium containing stimulants described above. In some experiments, 0.25 µg/ml cycloheximide (Sigma-Aldrich) was added in addition to PPA250. Cells were cultured for another 18 h and washed twice with ice-cold phosphate-buffered saline, pH 7.4. Lysis buffer (300 µl) consisting of 250 mM sucrose, 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA; 1% protease inhibitor cocktail (Sigma-Aldrich) was added, and the cells were removed from the surface of the dish with a plastic scraper. The cell suspension was sonicated for 20 s and centrifuged at 12,000g for 10 min at 4°C. The supernatant was stored at 80°C and used as a cell lysate. Protein was determined with the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Cell lysates (5 µg of protein) were suspended in 10 µl of Laemmli reducing sample buffer consisting of 58 mM Tris-HCl, 6% glycerol, 1.67% SDS, 0.002% bromphenol blue, and 1% 2-mercaptoethanol, pH 6.8, and boiled for 3 min, followed by SDS-polyacrylamide gel electrophoresis (PAGE) at room temperature using a precast 7.5% gel (Daiichi Pure Chemicals, Tokyo, Japan). For partially denaturing SDS-PAGE, samples were mixed with sample buffer in an ice bath and electrophoresed at 4°C.

Endotoxin-Induced NO Production in Vivo. Male BALB/c mice (6 weeks old) were injected i.p. with LPS, 100 µg/0.2 ml/mouse, followed by single or triple oral administration of PPA250 (3, 10, or 30 mg/kg body weight) 1, 3, or 5 h later. Blood was collected 14 h after the LPS injection. Serum was diluted 1:10 with saline, and protein was removed by centrifugation at 5000g for 1 h using Ultrafree-MC Centrifugal Filter units (5000 nominal molecular weight limit; Millipore). NO production in serum was assessed by measuring the stable nitrite/nitrate degradation products of NO, using the Griess reagent, reductase, and coenzyme (Wako Pure Chemicals), according to the manufacturer's instructions.

Collagen-Induced Arthritis Model. DBA/1J mice (9-week-old males) were immunized intradermally at the base of the tail with an emulsion containing 100 µg of bovine type II collagen (CII) (MCK, Tokyo, Japan) in 50 mM acetic acid and an equal volume of complete Freund's adjuvant (Wako Pure Chemicals). Twenty-one days after the primary injection, the same dose of the CII emulsion was injected via the same route as a booster. At day 28, mice were randomly assigned to vehicle or drug-treated groups. Starting from 1 week after the CII booster injection, mice were treated orally with PPA250 at a dose of 10 or 30 mg/kg body weight once a day for 22 days. Collagen-induced arthritis development was inspected three times a week, and inflammation of the paws was graded from 0 to 3 as normal (0), slight swelling and/or erythema (1), pronounced edematous swelling (2), or severe swelling and joint rigidity (3). Each paw was graded and the four scores were totaled so that the maximum score for each animal was 12.

Adjuvant Arthritis Model. Lewis rats (9-week-old males) were immunized with 0.05 ml of incomplete Freund's adjuvant (Difco, Detroit, MI) containing 6 mg/ml Mycobacterium tuberculosis (Difco) through the base of the tail at day 0. At day 16, rats were randomly assigned to vehicle or drug-treated groups and started on a treatment of PPA250 orally once a day at a dose of 3 or 10 mg/kg body weight for 14 days. Hind paw volumes were determined by water displacement plethysmometry three times per week. Results were expressed as the percentage increase in paw volume relative to paw volume before the onset of arthritis.

Statistical Analysis. Results are expressed as mean ± S.E. Statistical analysis was carried out using Bartlett's test, followed by the Dunnett multiple comparison test. A p value less than 0.05 was considered significant. Analysis was done with StatLight 1998 (Yukms Co., Tokyo, Japan).

	Results

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Inhibition of NO Accumulation in Culture. The murine macrophage cell line, RAW264.7, costimulated with IFN- and LPS, was used as an in vitro model of activated macrophages, and the effect of PPA250 on NO release was determined. Nitrite, a stable breakdown product of NO, accumulated in the culture medium in a time-dependent manner and reached a plateau in 24 h (data not shown), indicating that stimulated RAW264.7 cells produce and secrete a large quantity of NO. PPA250 inhibited NO accumulation in a dose-dependent manner, as detected by measuring NO 18 h after stimulation (Fig. 2). IC₅₀ was 82 nM. A microscopic dye-exclusion assay demonstrated that 5 µM PPA250 had no cytotoxic effect (data not shown), indicating that the inhibitory activity of PPA250 was not the result of cytotoxicity.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   PPA250 inhibition of NO accumulation by stimulated RAW264.7 cells. RAW264.7 cells were cultured at an initial cell density of 2 × 105 cells/0.5 ml in RPMI 1640 medium supplemented with 10% heat-inactivated FBS. After 24 h, medium was replaced with RPMI 1640 medium with 1% heat-inactivated FBS, 150 U/ml IFN-, 2 ng/ml LPS, and the indicated doses of PPA250. Culture was continued for another 18 h. NO production was assessed by measuring nitrite in culture supernatants using the Griess reagent. Data are means ± S.E. (n = 3).

Inhibition of iNOS Dimerization. To investigate further the inhibitory mechanism of PPA250, we studied its effect on the expression of iNOS protein in activated RAW264.7 cells. Unlike cycloheximide, which decreased expression, PPA250 had no effect (Fig. 3A). Another possibility was that PPA250 affects iNOS dimerization, which is essential for enzyme activity. Experiments using low-temperature SDS-PAGE to separate monomers and dimers revealed that PPA250 reduced the amount of iNOS dimer in a dose-dependent manner without reducing the amount of monomer (Fig. 3B). In contrast, cycloheximide had no effect on dimer formation (Fig. 3B, lane 3). These results indicate that inhibition of NO production by PPA250 in stimulating RAW264.7 cells is due to prevention of iNOS dimerization, not to a decrease in its de novo synthesis.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Western blot analysis of iNOS in RAW264.7 cells. RAW264.7 cells (5 × 106 cells/10-cm dish) were cultured under the conditions described in the legend to Fig. 2. Cells were harvested at 18 h after stimulation, and cell lysates were prepared. Heat-denatured cell lysates were subjected to SDS-PAGE (A). Cell lysates were prepared from untreated cells (lane 1), cells treated with stimulants alone (lane 2), cells treated with stimulants and cycloheximide (CHX) (lane 3), or cells treated with stimulants and 0.2 (lane 4) or 1 µM (lane 5) PPA250. Nondenatured cell lysates were subjected to SDS-PAGE at 4°C (B). Cell lysates were prepared from untreated cells (lane 1), cells treated with stimulants alone (lane 2), cells treated with stimulants and cycloheximide (lane 3), cells treated with stimulants and 0.04 (lane 4), 0.2 (lane 5), or 1 µM (lane 6) PPA250. After electroblotting, membranes were treated with anti-iNOS antibody and bands were visualized. The positions of iNOS monomer and dimer are indicated. Similar results were obtained in an additional independent experiment.

Effect on Serum Levels of Nitrite/Nitrate in LPS-Treated Mice. To examine the inhibitory effect of PPA250 on NO production in vivo, the serum level of nitrite/nitrate was determined in mice exposed to LPS with or without PPA250. Nitrite/nitrate concentration was maximal 14 h after the intraperitoneal injection of LPS (100 µg/mouse) and declined gradually thereafter until it was undetectable at 48 h (date not shown). The effect of PPA250 was examined at the maximal point (14 h). Oral administration of PPA250 1 h after the LPS injection significantly decreased the concentration of nitrite/nitrate at this time in a dose-dependent manner (Fig. 4). However, administration at 5 or 10 h after LPS had no effect. On the other hand, a triple administration at these times was more effective than the single dose at 1 h. The reduction in alertness and piloerection observed in LPS-injected mice was dramatically improved by doses of PPA250 that decreased the serum concentration of NO. Serum level of PPA250 peaked at 1 h after oral administration and maintained peak level about 1 h, started to decline thereafter, and became undetectable at 8 h in rats (data not shown). These results indicate that formation of iNOS dimers occurs soon after LPS injection and that PPA250 has no effect on the dissociation of the dimer once it has been formed, nor on degradation of the enzyme.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   NO concentration in serum of septic mice treated with PPA250. BALB/c mice (6-week-old males) were administered LPS (100 µg i.p.). After 1, 5, or 10 h, they received 3, 10, or 30 mg/kg PPA250 orally. Mice were sacrificed 14 h after the LPS injection, and serum was collected. Serum NO level was assessed by measuring nitrite and nitrate using the Griess reagent. Data are means ± S.E. (n = 3). **, p < 0.01; *, p < 0.05 compared with control.

Therapeutic Effect on Animal Models of NO-Related Diseases. Excess NO production is known to be involved in the generation and development of rheumatoid arthritis. Since PPA250 suppressed NO production in vivo, we investigated its potential therapeutic effect in animal models. Mouse collagen-induced arthritis, a commonly used model of rheumatoid arthritis, was established by immunization with CII. After clinical signs of arthritis had developed at day 28, mice received daily oral administration of 10 or 30 mg/kg PPA250. PPA250 reduced the inflammation score within a day in a dose-dependent manner (Fig. 5). At a dose of 3 or 10 mg/kg, it also suppressed joint inflammation in adjuvant arthritis rats, another chronic model of inflammatory joint disease (Fig. 6). Paw swelling was decreased within a few days of the start of treatment (Fig. 6). PPA250 at a dose as high as 100 mg/kg/day for 14 days had no apparent effect on body weight or organ weights of rats, nor was any difference detected in a clinical blood examination (data not shown), indicating that the inhibitory effect of PPA250 is not the result of in vivo toxicity. These results suggest that oral administration of PPA250 has a potential therapeutic value in the treatment of rheumatoid arthritis and other diseases in which NO is involved.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Effect of PPA250 on inflammation score in collagen-induced arthritis. DBA/1J mice (9-week-old males) were injected intradermally on day 0 with 100 µg of bovine CII in complete Freund's adjuvant, followed at day 21 by a booster injection. PPA250 (10 or 30 mg/kg) was administered orally once a day for 22 days, starting on day 28. Open circle, vehicle control; closed triangle, PPA250 10 mg/kg; closed square, PPA250 30 mg/kg. **, p < 0.01, *, p < 0.05 for PPA250 compared with vehicle. n = 8 animals/group.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of PPA250 on paw swelling in adjuvant arthritis. Lewis rats (9-week-old males) were immunized at day 0 with incomplete Freund's adjuvant containing 6 mg/ml M. tuberculosis and treated with 3 or 10 mg/kg PPA250 orally, once a day, beginning on day 16. x, normal; open circle, vehicle control; closed triangle, 3 mg/kg PPA250; closed square, 10 mg/kg PPA250. **, p < 0.01, *, p < 0.05 for PPA250 compared with vehicle. n = 6 animals/group.

	Discussion

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We have demonstrated that the newly synthesized compound PPA250 strongly inhibits iNOS activity by reducing dimerization of the monomeric form. The IC₅₀ value determined in a mouse macrophage-like cell line, RAW264.7, stimulated with IFN- and LPS, was as low as 82 nM. Although PPA250 has an IC₅₀ value that is slightly higher than that of the iNOS inhibitor described by Naka et al. (2000), both of these compounds have values that are significantly lower than previously reported inhibitors of NO production. Dimerization is essential for activity of not only iNOS but also constitutive NOS isoforms (Baek et al., 1993; Stuehr, 1999) and is blocked by concentrations of PPA250 similar to those that inhibit NO production. However, neither transcription nor translation of the enzyme are affected, indicating that inhibition of NO production by PPA250 may be due to its direct effect on dimerization. PPA250 failed to inhibit the iNOS activity in crude cell lysates, which could contain a lot of iNOS-form homodimers, suggesting PP250 may not be able to dissociate the dimer. McMillan et al. (2000) demonstrated that a compound with a pyrimidineimidazole core inhibited NO production by interfering with the homodimerization of iNOS. X-ray crystallography revealed that the bulky imidazole residue reacts with the substrate binding site and with the dimerization interface, leading to allosteric disruption of protein-protein interaction at the dimer interface. Since PPA250 possesses similar groups, it is likely that the imidazole moiety of PPA250 binds to a similar site on the monomer form and thus inhibits dimerization. It is supported by the results that all PPA250 derivatives without the imidazole moiety did not inhibit NO production (unpublished observation). Furthermore, imidazoles and pyrimidineimidazoles are specific inhibitors of iNOS but do not affect other isoforms (Wolff et al., 1994; Chabin et al., 1996; McMillan et al., 2000). If the active site of PPA250 is a phenylmethyl imidazole moiety, PPA250 may specifically inhibit iNOS, although this requires further investigation.

As expected from the in vitro results, PPA250 also inhibited iNOS activity in LPS-treated mice. This mouse model system mimics the clinical symptoms of so-called septic shock, which is a severe systemic inflammatory response to a Gram-negative bacterial infection. In response to LPS, host cells, particularly macrophages, release inflammatory mediators such as tumor necrosis factor-, IL-1, IL-6, IFN-, and NO (Manthey and Vogel, 1992; Salkowski et al., 1997). Considerable evidence suggests that excessive production of NO by iNOS contributes to the circulatory failure observed during septic shock (Hom et al., 1995; Hobbs et al., 1999). Serum levels of NO oxidation products, such as nitrate, are elevated both in patients and in experimental animals undergoing septic shock (Ochoa et al., 1991; Minnard et al., 1994; Salkowski et al., 1997), and iNOS inhibitors prevent LPS-induced mortality in mice (Minnard et al., 1994; Tunçtan et al., 1998). Oral administration of PPA250 decreased LPS-induced nitrite/nitrate levels, indicating that PPA250 is biologically active in vivo. Endogenous IFN- production has been demonstrated to be a key step in the LPS-mediated induction of iNOS mRNA and in the accumulation of serum nitrate/nitrite (Salkowski et al., 1997). However, PPA250 did not decrease the IFN- concentration in the serum of mice 4 h after LPS administration (unpublished results), indicating that PPA250 inhibits in vivo iNOS activity directly, not through an indirect mechanism such as decreasing the production of LPS-inducible cytokines.

We also demonstrated that oral administration of PPA250 suppressed the development of destructive polyarthritis in animal models. Collagen-induced arthritis in mice and adjuvant arthritis in rats are typical animal models of human chronic arthritis. The development of arthritis is accompanied by the induction of iNOS and subsequent NO production in these models (Stefanovic-Racic et al., 1994, 1995; Connor et al., 1995; Cannon et al., 1996; Vermeire et al., 2000). Earlier studies in animal models have demonstrated that NO inhibitors have little curative effect after rheumatoid arthritis has developed (Stefanovic-Racic et al., 1995; Fletcher et al., 1998). However, a protective effect of PPA250 was observed even when PPA250 treatment was initiated after clinical symptoms had appeared in these animals. Although it has not yet been determined whether PPA250 also acts on other factors that are related to the pathogenesis of these models besides inhibition of iNOS dimerization, these findings suggest the importance of NOS/NO in these models. We speculate that dimerization inhibitors such as PPA250 may have great therapeutic potential in the treatment of NO-related diseases such as rheumatoid arthritis and other inflammatory and autoimmune diseases. In addition to its therapeutic potency, PPA250 has another advantage, namely, its oral route of administration. Because long-term and frequent administration is required for patients with chronic diseases, oral administration is much to be preferred.

In conclusion, a newly synthesized imidazole compound, PPA250, inhibits the production of NO by a mouse macrophage-like cell line, RAW264.7, stimulated by IFN- and LPS. Inhibition is due to prevention of iNOS dimerization, an essential step for enzyme activity. The inhibitory effect of PPA250 was also observed in mice treated with LPS. In two animal models of arthritis, adjuvant arthritis and collagen-induced arthritis, oral administration of PPA250 suppressed the development of arthritis after clinical symptoms had appeared. To our knowledge, this is the first demonstration that an inhibitor of iNOS dimerization is effective in animal rheumatoid arthritis models. Dimerization inhibitors, including PPA250, are of potential therapeutic benefit in the treatment of inflammatory and autoimmune diseases such as rheumatoid arthritis.