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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA

Anti-Inflammatory Activity of a Potent, Selective Leukotriene A₄ Hydrolase Inhibitor in Comparison with the 5-Lipoxygenase Inhibitor Zileuton

Johnson and Johnson Pharmaceutical Research and Development L.L.C., San Diego, California

Received for publication October 11, 2006
Accepted March 15, 2007.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Leukotriene A₄ hydrolase (LTA₄H) catalyzes production of the proinflammatory lipid mediator, leukotriene (LT) B₄, which is implicated in a number of inflammatory diseases. We have identified a potent and selective inhibitor of both the epoxide hydrolase and aminopeptidase activities of recombinant human LTA₄H (IC₅₀, approximately 10 nM). In a murine model of arachidonic acid-induced ear inflammation, the LTA₄H inhibitor, JNJ-26993135 (1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid), dose-dependently inhibited ex vivo LTB₄ production in blood, in parallel with dose-dependent inhibition of neutrophil influx (ED₅₀, 1–3 mg/kg) and ear edema. In murine whole blood and in zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB₄ production, without affecting cysteinyl leukotriene production, while maintaining or increasing production of the anti-inflammatory mediator, lipoxin (LX) A₄. The 5-lipoxygenase (5-LO) inhibitor zileuton showed inhibition of LTB₄, LTC₄, and LXA₄ production. Although zileuton inhibited LTB₄ production in the peritonitis model more effectively than the LTA₄H inhibitor, the influx of neutrophils into the peritoneum after 1 and 2 h was significantly higher in zileuton- versus JNJ-26993135-treated animals. This difference may have been mediated by the increased LXA₄ levels in the presence of the LTA₄H inhibitor. The selective inhibition of LTB₄ production by JNJ-26993135, while increasing levels of the anti-inflammatory mediator, LXA₄, may translate to superior therapeutic efficacy versus 5-LO or 5-LO-activating protein inhibitors in LTB₄-mediated inflammatory diseases.

LTB₄ is able to recruit and activate inflammatory cells, which in turn can cause tissue damage and disease. Elevated levels of LTB₄ have been implicated in the pathological etiology of inflammatory diseases including psoriasis, inflammatory bowel disease, chronic obstructive pulmonary disease, rheumatoid arthritis, asthma, and cystic fibrosis (Tager and Luster, 2003). Recently, a number of studies have focused on a role for LTB₄ in cardiovascular diseases, including atherosclerosis, myocardial infarction, and stroke (Aiello et al., 2002; Friedrich et al., 2003; Helgadottir et al., 2004; Huang et al., 2004; Subbarao et al., 2004). More recent data (Helgadottir et al., 2006) has shown that a particular LTA₄H gene haplotype, HapK, confers modest risk of myocardial infarction in an Icelandic cohort and in European Americans but confers a 3-fold higher risk in African Americans. Furthermore, LTA₄H and 5-LO show abundant expression in human atherosclerotic lesions, and their expression correlates with symptoms of plaque instability (Qiu et al., 2006). An LTA₄H inhibitor would decrease LTB₄ production and may thus have therapeutic potential in all of the abovementioned diseases.

A number of LTA₄H inhibitors have previously been described, some of which have shown anti-inflammatory efficacy in vivo (for review, see Penning, 2001). In this study, we describe the pharmacology of a new LTA₄H inhibitor, JNJ-26993135 (Fig. 1). This compound was shown to be a potent, selective inhibitor of recombinant LTA₄H and ionophore-stimulated LTB₄ production in whole blood and was orally efficacious in reducing neutrophil influx and edema in an arachidonic acid-induced ear inflammation model. In wholeblood assays in vitro and in a zymosan-induced peritonitis model in vivo, JNJ-26993135 inhibited LTB₄ production and tended to increase anti-inflammatory lipoxin A₄ production. In contrast, the 5-lipoxygenase inhibitor zileuton inhibited not only LTB₄ (and LTC₄) production but also lipoxin production. The data presented here reveal a potential therapeutic advantage of LTA₄H inhibitors versus 5-lipoxygenase or FLAP inhibitors, namely selective inhibition of the potent proinflammatory mediator, LTB₄, while leaving intact or increasing the endogenous anti-inflammatory, proresolution mediator, LXA₄.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Structure of JNJ-26993135.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

Preparation of Substrate. LTA₄ substrate was prepared from the methyl ester of LTA₄ (Cayman Chemical, Ann Arbor, MI) by treatment under nitrogen with 67 M equivalents of NaOH, at room temperature, for 40 min. The LTA₄ substrate in its free acid form was kept frozen at -80°C until needed.

Epoxide Hydrolase Assay. LTA₄ hydrolase inhibitor, JNJ-26993135, was prepared as a 10 mM stock solution in DMSO and diluted in the assays so that the final DMSO concentration did not exceed 0.1%. Recombinant human LTA₄H (36 ng) was incubated with various concentrations of test compound for 10 min at room temperature in assay buffer (0.1 M potassium phosphate, pH 7.4, 5 mg/ml fatty acid-free bovine serum albumin) in a volume of 50 µl. The solution was then adjusted to 200 µl with assay buffer, and 25 µl of the substrate, LTA₄, was added (final concentration, 40 ng/ml, 0.13 µM; final volume, 225 µl). After 10 to 30 min at room temperature, the assay was terminated by diluting 20-fold in assay buffer. The amount of LTB₄ produced was assayed by enzyme immunoassay (EIA) (catalog no. 901-068; Assay Designs, Inc., Ann Arbor, MI). The concentration of compound that was required for half-maximal inhibition of recombinant enzyme activity (IC₅₀) was calculated by nonlinear regression using GraphPad Prism 4.0 (GraphPad Software Inc., San Diego, CA), one-site binding competition.

Aminopeptidase Assay. Aminopeptidase activity was determined by a modification of the procedure of Rudberg et al. (2004). Recombinant human LTA₄H (375 ng) was preincubated for 15 min at room temperature in assay buffer (50 mM Tris-HCl, pH 8.0, 100 mM KCl) in the presence of different concentrations of JNJ-26993135 (0.2% final DMSO concentration). An equal volume of 2-fold concentrated substrate (L-alanine-4-nitro-anilide hydrochloride; A9325; Sigma Chemical Co., St. Louis, MO) was added to a final concentration of 1 mM in a volume of 110 µl. Amino-peptidase activity was determined by monitoring absorbance at 405 nm. IC₅₀ values were determined as described above for epoxide hydrolase activity.

Selectivity of JNJ-26993135 versus 50 Receptors, Transporters, and Ion Channels. Selectivity of JNJ-26993135 was evaluated by Cerep, Inc. (Paris, France) against 50 other targets. These targets represent major classes of biogenic amine receptors, neuropeptide receptors, ion channel binding sites, and neurotransmitter transporters. At 10 µM, JNJ-26993135 showed minimal cross-reactivity in all the assays (see Table 1).

In Vivo Experiments. All animal experiments described in this study were performed after review of the protocols and approval by the Institutional Animal Care and Use Committee.

Murine Whole-Blood Leukotriene B₄, Cysteinyl Leukotriene, Lipoxin A₄, and PGE₂ Assays. For in vitro assays of ionophore-stimulated lipid mediator production, CD-1 mice were euthanized, and blood was collected in heparin-containing syringes by cardiac puncture. The blood was diluted 1:2 (LTB₄, LXA₄) or 1:15 (LTC₄/D₄/E₄, PGE₂) in RPMI 1640 medium, and 200-µl aliquots of the diluted blood were added to wells of a 96-well tissue culture plate. JNJ-26993135 or the 5-lipoxygenase inhibitor zileuton were added at different concentrations to the diluted whole blood (final DMSO concentration of 0.1%) and preincubated for 15 min at 37°C in a humidified incubator. For murine ex vivo analysis of LTB₄ production, blood was obtained from BALB/c mice 4 h after oral dosing of JNJ-26993135 and was diluted 1:1 in RPMI 1640 medium, after which 200-µl aliquots of the diluted blood were added to a microtiter plate. Calcium ionophore A23187 [GenBank] (Sigma Chemical Co.) was added to samples for both in vitro and ex vivo whole-blood assays (final concentration, 20 µg/ml). The incubation was continued under the same conditions for an additional 10 to 30 min to allow eicosanoid formation. The reaction was terminated by centrifugation (208g, 10 min at 4°C) to form a cell pellet, and the amount of LTB₄ produced was assayed in the supernatants (diluted 1:5 to 1:15) by enzyme immunoassay. For LXA₄ measurement, supernatant samples were assayed without dilution using EIA kits from Neogen (catalog number 401070; Lansing, MI). According to the manufacturer, the LTB₄ EIA assay is selective for LTB₄, with 5% cross-reactivity for 6-trans-12-epi-LTB₄ and 6-trans-LTB₄. The LXA₄ EIA assay from Neogen is selective for LXA₄, with 5% cross-reactivity for 5(S),6(R)-hydroxyeicosatetraenoic acid and 1% for lipoxin B₄ (Levy et al., 1993). PGE₂ and cysteinyl leukotrienes were measured using Assay Designs EIA (catalog numbers 900-001 and 901-070, respectively). IC₅₀ values were determined as described above for epoxide hydrolase activity.

Arachidonic Acid-Induced Ear Inflammation. Arachidonic acid (catalog number 181198; Calbiochem, San Diego, CA) in a stock solution of 1 g/ml was diluted to 100 mg/ml in 100% acetone for experimental use. JNJ-26993135 or vehicle [20% hydroxypropyl--cyclodextran (HPCD)] were administered orally at t = -1 h to female BALB/c mice. At t = 0, 2 mg of arachidonic acid in acetone was applied to one ear of each mouse (n = 8–10 per group), and acetone was applied to the other ear. At t = 3 h, the mice were sacrificed, and blood was drawn for ex vivo whole-blood LTB₄ production assays and measurement of compound levels. At the same time, 8-mm ear biopsies were taken, weighed, and frozen at -80°C for future analysis of neutrophil influx, as measured by myeloperoxidase activity.

Myeloperoxidase Assay. Ear biopsies were thawed and roughly minced into FastPrep tubes with lysing matrix D (catalog number 6913-100; Qbiogene Inc., Carlsbad, CA) before the addition of 0.5 ml of freshly made extraction buffer (0.3 M sucrose, 0.22% cetyltrimethylammoniumbromide, 2.5 mM citrate). Samples were run on the FastPrep homogenization instrument for 30 s at a speed of 5 m/s before storage on ice. Samples were then centrifuged for 10 min at 14,000 rpm in a microcentrifuge. In a 96-well plate, 10 µlofthe resultant supernatant was added to 90 µl of freshly made dilution buffer (10 mM citrate, pH 5.0, 0.22% cetyltrimethylammoniumbromide). Tetramethylbromidine (20 µl) was added, and the plate was mixed gently and incubated at room temperature for 1 h. The reaction was then stopped with 100 µl of 1 MH₂SO₄, and the plate was read in a spectrophotometric plate reader at 405 nm.

Zymosan-Induced Peritonitis. Female CD-1 mice were pretreated orally, with vehicle, JNJ-26993135, or zileuton (both at 30 mg/kg), 30 min before i.p. challenge with 0.5 ml of zymosan solution (Sigma catalog number Z4250, 2 mg/ml in sterile phosphate-buffered saline). At the indicated times, mice were euthanized by CO₂ overdose. The peritoneal cavity was flushed with 3 ml of lavage buffer (phosphate-buffered saline with 0.1% bovine serum albumin and 5 mM EDTA). The lavage fluid was transferred into 5-ml polypropylene tubes and kept on ice. The total number of leukocytes in lavage fluid was determined using a Z2 Coulter Counter. Differential leukocyte counts were made on Cytospin preparations that had been stained with a modified Wright's stain (Diff-Quik) by light microscopy using standard morphological criteria. The remaining lavage fluid was centrifuged (550g, 4°C, 5 min), and cell-free lavage fluid was placed at -80°C and used for leukotriene quantification.

Leukotriene and Lipoxin Quantification in Peritoneal Lavage Samples. Peritoneal lavage samples were thawed on ice and diluted in assay buffer (1:5 for LTB₄ and 1:100 for LTC₄) and assayed by commercially available EIA kits (catalog numbers 901-068, and 901-070; Assay Designs, Inc.), following the manufacturer's instructions. For LXA₄ measurement, supernatant samples were assayed without dilution using commercially available EIA kits (catalog number 401070; Neogen), following the manufacturer's instructions. Data were analyzed to compare drug-treated groups with vehicle-treated animals. A Student's t test was performed to detect significant differences below p = 0.05 using Prism (GraphPad software version 4.02).

Pharmacokinetic Analysis of JNJ-26993135 in Mice and Dogs. Compound was administered i.v. (2 mg/kg) or orally (10 mg/kg) to female BALB/c mice and beagle dogs. The vehicle used was 20% HPCD. Blood samples were taken at various time points, and liquid chromatography/mass spectrometry analysis was used to quantitate the plasma levels of the compound, relative to standard curves generated in the same matrix. Pharmacokinetic parameters were determined from the data using WinNonlin software (Pharsight, Mountain View, CA).

	Results

Top Abstract Materials and Methods Results Discussion References

 
Inhibition of LTA₄ Hydrolase Activity in Vitro. The epoxide hydrolase activity of recombinant human LTA₄ hydrolase for LTB₄ formation was potently and dose-dependently inhibited by JNJ-26993135, with a mean IC₅₀ value of 12 ± 10 (Table 1). Similar dose-dependent inhibition was observed for the peptidase activity, yielding an IC₅₀ of approximately 6 nM. Thus, JNJ-26993135 is similarly potent at inhibiting both hydrolase and peptidase activities of recombinant human LTA₄H (Fig. 2). Dose-dependent inhibition of ionophore-stimulated LTB₄ production in murine blood (diluted 1:2) was also observed for JNJ-26993135, with an IC₅₀ of 339 ± 101 nM (Table 1), approximately 30-fold less potent than for the isolated enzyme. This relative decrease in potency could partially be ascribed to protein binding because the IC₅₀ value obtained when the plasma proteins were diluted further (1:15 dilution of blood) was 89 ± 28 nM (n = 4).

View larger version (27K): [in this window] [in a new window]   Fig. 2. Selective inhibition of LTB4 production in murine whole blood by JNJ-26993135, compared with zileuton. Murine blood samples diluted in RPMI were preincubated with 0.4 nM to 30 µM JNJ-26993135 (open circles) or 5-LO inhibitor zileuton (filled circles). Blood samples were stimulated with calcium ionophore A23187, and the amount of LTB4 (A), LTC4 (B), LXA4 (C), or PGE2 (D) produced was assayed in the supernatants by commercial EIA assay kits. Each point represents the mean of triplicate or quadruplicate assays, and error bars indicate S.D. from the mean. Blood was diluted 1:1 during stimulation for LTB4 and LXA4 production and 1:15 for LTC4 and PGE2 production. Supernatants were diluted appropriately to ensure that all values were within the range of the respective standard curves.

Inhibition of Whole-Blood Eicosanoid Production Compared with the 5-Lipoxygenase Inhibitor Zileuton. As discussed above and shown in Fig. 2, A and B, JNJ-26993135 significantly reduced LTB₄ production in murine whole blood, with no effect on LTC₄ synthesis and, thus, no pathway shunting to cysteinyl leukotrienes. Likewise, no inhibition was observed of LXA₄ or PGE₂ synthesis (Fig. 2, C and D). A trend toward increased LXA₄ synthesis was observed (Fig. 2C). When a similar experiment was performed in human whole blood, dose-dependent inhibition of LTB₄ production by JNJ-26993135 was observed, with a trend toward increased LXA₄ synthesis (results not shown). In contrast (as shown in Fig. 2, A–C), the 5-LO inhibitor zileuton blocked the production of the anti-inflammatory mediator, LXA₄, in addition to inflammatory eicosanoids, LTB₄ and LTC₄. No inhibition of PGE₂ synthesis was observed for zileuton up to 6 µM, but minimal, possibly nonspecific, inhibition was observed at 30 µM zileuton (Fig. 2D).

Pharmacokinetics of JNJ-26993135 in Mice and Dogs. Oral availability of JNJ-26993135 was determined from the pharmacokinetics of the compound in mice and dogs. Results are summarized in Table 3. The compound was rapidly absorbed after oral administration (t_max = 0.5 and 1 h, in mice and dogs, respectively) and eliminated with a half-life of approximately 3 h in mice and 4 h in dogs. The compound showed excellent oral availability in both species (>100 and 65%, in mice and dogs, respectively). The oral C_max at 10 mg/kg was approximately 4-fold higher in dogs than the same dose in mice (23.5 versus 6.4 µM, respectively), whereas the AUC was approximately 6-fold higher.

In Vivo Activity in a Murine Model of Arachidonic Acid-Induced Ear Inflammation. Anti-inflammatory activity of JNJ-26993135 was determined by dosing different amounts of the compound orally to mice, 1 h before arachidonic acid challenge to the ear. Three hours later (4 h after the compound was dosed orally), JNJ-26993135 significantly inhibited (p < 0.0001) the ex vivo ionophore-stimulated LTB₄ production at 3, 10, and 30 mg/kg (Fig. 3. The concentration of JNJ-26993135 in the plasma increased proportionally with increasing doses (Table 4). At 3 mg/kg, approximately 50% inhibition was observed, at a plasma concentration of 1 µM, diluted to 0.5 µM in the assay. The topical arachidonic acid-induced neutrophil influx was inhibited in a dose-dependent manner, with an estimated ED₅₀ of between 1 and 3 mg/kg p.o., at a plasma concentration between 0.3 and 1 µM. The arachidonic acid-induced ear edema was also dose-dependently inhibited by JNJ-26993135, although the extent of inhibition was lower than for the neutrophil influx. This result is consistent with the fact that edema is mediated not only by LTB₄ but also by the cysteinyl leukotrienes, which are not affected by inhibition of LTA₄H. The dose dependence of measured plasma concentrations of JNJ-26993135 (Table 4), ex vivo inhibition of ionophore-induced LTB₄ production in blood, and inhibition of neutrophil influx and edema thus demonstrate a direct relationship between the in vivo pharmacokinetics and pharmacodynamic activity of this compound.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Dose-dependent inhibition by oral dosing of JNJ-26993135, of LTB4 production in blood, and neutrophil recruitment and ear edema in response to topical arachidonic acid. Vehicle (20% HPCD) or JNJ-26993135 (1–30 mg/kg) was administered orally to mice, 1 h before topical arachidonic acid challenge to the ear. Three hours later, blood was analyzed for ex vivo ionophore-stimulated LTB4 production (A), and ear biopsies were taken for measurement of neutrophil influx [from myeloperoxidase activity (B)] and edema (C), respectively. Results are expressed as percentage of vehicle control and represent the mean ± S.D. of 10 animals in each group.

Selective Inhibition of LTB₄ Production in a Murine Model of Zymosan-Induced Peritonitis. The effects of the LTA₄ hydrolase inhibitor JNJ-26993135 on in vivo synthesis of LTB₄, LTC₄ and anti-inflammatory LXA₄ were determined during acute inflammation in murine zymosan-induced peritonitis. Furthermore, the effects of JNJ-26993135 were also compared with the 5-LO inhibitor zileuton. Lipid mediator levels were analyzed at 1, 2, and 3 h after zymosan treatment. Eicosanoid levels were initially undetectable in peritoneal lavage (data not shown). Upon zymosan administration, LTB₄ levels increased in vehicle-treated animals to peak at 2 h, whereas LTC₄ and LXA₄ were maximal between 1 and 2 h, all decreasing again at 3 h. When dosed orally, JNJ-26993135 (30 mg/kg) demonstrated a significant reduction, compared with vehicle controls, in peritoneal LTB₄ levels at all time points (Fig. 4A), with no increase in proinflammatory LTC₄ levels (Fig. 4B) and thus no pathway shunting to cysteinyl leukotrienes. Zileuton treatment blocked the production of LTB₄ more effectively than JNJ-26993135 at 1 and 2 h and, in addition, totally inhibited synthesis of LXA₄ and LTC₄. In contrast, JNJ-26993135 showed a trend toward increased LXA₄ levels in vivo (Fig. 4C). Differential cell counts from peritoneal lavage indicated that JNJ-26993135 significantly decreased total neutrophils at 1 and 2 h (40% inhibition) compared with vehicle-treated animals (Fig. 4D), whereas zileuton-treated animals showed increased neutrophil influx (p < 0.05) at 1 h. At 2 h, significantly higher neutrophil levels were found in zileuton- versus JNJ-26993135-treated animals (p < 0.05) and marginally higher versus vehicle-treated mice. Thus, increased neutrophil levels were observed in zileuton-treated animals despite the fact that LTB₄ levels were lower than in JNJ-26993135-treated mice at both 1- and 2-h time points.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Effects of JNJ-26993135 versus zileuton on LTB4, LTC4, and LXA4 production and neutrophil content in the zymosan-induced peritonitis model. Vehicle (hatched bars), JNJ-26993135 (open bars), or zileuton (filled bars) were administered orally to mice (30 mg/kg) 30 min before i.p. zymosan challenge. Peritoneal lavage fluid was analyzed 1, 2, and 3 h after zymosan challenge for LTB4 (A), LTC4 (B), and LXA4 (C) levels (n = 5 animals/group). In the same peritoneal lavage samples that were analyzed for LTB4, LTC4, and LXA4 concentrations in A to C, as well as samples taken at 24 h, neutrophil numbers were determined and are shown in D as mean ± S.E.M. *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student's t test.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effects of JNJ-26993135 on LTB4 and LXA4 content in peritoneal lavage, 1 and 2 h after induction of zymosan-induced peritonitis. In a similar experiment to that in Fig. 4, larger groups of mice were used (n = 15) to compare LTB4 and LXA4 production in vehicle- and JNJ-26993135-treated animals. Each data point represents a single animal, with the mean of each group designated by a horizontal bar. *, p < 0.05; ***, p < 0.001, by Student's t test

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we describe the pharmacology of JNJ-26993135, a new potent and selective inhibitor of LTA₄H. This compound dose-dependently inhibited the production of LTB₄ by the human recombinant enzyme as well as in whole blood from mice (this study), rats, dogs, and humans (data not shown). JNJ-26993135 showed oral efficacy and a clear pharmacokinetic/pharmacodynamic relationship in murine arachidonic acid-induced ear inflammation. In zymosan-induced peritonitis, JNJ-26993135 selectively inhibited LTB₄ synthesis and increased production of the anti-inflammatory mediator, LXA₄.

LTB₄ is a potent proinflammatory mediator, implicated in a number of diseases. Potential therapeutics for these diseases include 5-LO or FLAP inhibitors, which block the synthesis pathway upstream of LTA₄, LTA₄H inhibitors, and LTB₄ receptor antagonists, which block the actions of LTB₄ after synthesis. LTB₄ antagonists generally are not equally effective at blocking both BLT1 and BLT2, whereas blocking LTB₄ synthesis has the advantage of inhibiting downstream events through both receptors. The importance of targeting both receptors is indicated by recent data showing the role of BLT1 in a number of inflammatory models (Gelfand and Dakhama, 2006; Kim et al., 2006; Shao et al., 2006), as well as expression of BLT2 in human and murine mast cells (Lundeen et al., 2006), human dendritic cells (Shin et al., 2006), murine keratinocytes (Iizuka et al., 2005), and human synovial tissues (Hashimoto et al., 2003), all of which are relevant in inflammatory diseases.

LTA₄H inhibitors specifically block the production of LTB₄ from LTA₄, without affecting the biosynthesis of lipoxins, which are also produced from LTA₄ or from arachidonic acid through the activity of 5-lipoxygenase and 12- or 15-lipoxygenase. Lipoxins, such as LXA₄, are known to play a role as endogenous anti-inflammatory agents and key mediators of the natural process of resolving an inflammatory response (Serhan, 2005). Furthermore, production of LXA₄ has been described in a variety of inflammatory diseases, and decreased levels of LXA₄ have been found in patients with severe versus moderate asthma (Levy et al., 2005) and in cystic fibrosis (Karp et al., 2004). Unlike LTA₄ inhibitors, 5-LO or FLAP inhibitors block the leukotriene pathway upstream of LTA₄ and LXA₄, and we confirm this in the present study for the 5-LO inhibitor zileuton. In contrast, JNJ-26993135 selectively inhibited LTB₄ synthesis and seemed to increase LXA₄ synthesis. This may have resulted in the decreased number of neutrophils after 1 and 2 h in the LTA₄H inhibitor- versus the zileuton-treated animals. After 24 h, analysis of the inflammation indicated that both vehicle- and zileuton-treated animals had higher total neutrophil counts compared with JNJ-26993135-treated animals, suggesting that the endogenous resolution process was somehow enhanced in the LTA₄H inhibitor-treated animals.

In addition to demonstrating oral anti-inflammatory efficacy in the murine models described, JNJ-26993135 also demonstrated oral efficacy in trinitrobenzene sulfonic acid-induced colitis in rats (B. Whittle, J. Varga, A. Berko, K. Horvath, J. Riley, K. Lundeen, A. Fourie, and P. Dunford, unpublished data) and favorable pharmacokinetic properties in mice and dogs. JNJ-26993135 or other LTA₄H inhibitors may be candidates for further development in inflammatory diseases where LTB₄ is known to play a major role. In addition, our study with JNJ-26993135 reveals a potential therapeutic advantage of LTA₄H inhibitors versus 5-lipoxygenase or FLAP inhibitors, namely selective inhibition of the potent proinflammatory mediator, LTB₄, while leaving intact or increasing the endogenous anti-inflammatory, proresolution mediator LXA₄.

	Footnotes

 
N.L.R. and P.J.D. contributed equally to this work.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.115436.

ABBREVIATIONS: LT, leukotriene; LO, lipoxygenase; FLAP, 5-LO-activating protein; LTA₄H, leukotriene A₄ hydrolase; JNJ-26993135, 1-[4-(benzothiazol-2-yloxy)-benzyl]-piperidine-4-carboxylic acid; LX, lipoxin; DMSO, dimethyl sulfoxide; enzyme immunoassay; PG, prostaglandin; HPCD, hydroxypropyl--cyclodextran; h, human; AUC, area under the curve.

Address correspondence to: Anne M. Fourie, Johnson and Johnson Pharmaceutical Research and Development, 3210 Merryfield Row, San Diego, CA 92121. E-mail: afourie{at}prdus.jnj.com

	References

Top Abstract Materials and Methods Results Discussion References

 

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