Pharmacological Characterization of SC-57461A (3-[Methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic Acid HCl), a Potent and Selective Inhibitor of Leukotriene A4Hydrolase I: In Vitro Studies

  1. Leslie J. Askonas,
  2. James F. Kachur,
  3. Doreen Villani-Price,
  4. Chi-Dean D. Liang,
  5. Mark A. Russell and
  6. Walter G. Smith
  1. Pharmacia Research and Development, Skokie, Illinois
  1. Leslie J. Askonas, Pharmacia Research and Development, 4901 Searle Parkway, Skokie, IL 60077. E-mail: leslie.j.askonas{at}pharmacia.com
 
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Abstract

Leukotriene (LT) B4 is an inflammatory mediator that has been implicated in the pathogenesis of various diseases, including inflammatory bowel disease and psoriasis. As the rate-limiting step for LTB4 production, LTA4 hydrolase represents an attractive target for therapeutic agents that interfere with LTB4 production. In the present study we evaluated a chemically novel compound designated SC-57461A (3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl) as an inhibitor of LTA4 hydrolase. Pharmacological comparisons are made to its free acid SC-57461. SC-57461A is a potent competitive inhibitor of recombinant human LTA4 hydrolase when either LTA4(IC50 = 2.5 nM, Ki = 23 nM) or peptide substrates (IC50 = 27 nM) are used. In human whole blood, the IC50 for calcium ionophore-induced LTB4 production was 49 nM, indicative of good cell penetration. Whole blood production of the cyclooxygenase metabolite thromboxane B2 was not affected. SC-57461A was also active in several other species, including mouse, rat, dog, and rhesus monkey. The data indicate that SC-57461A is a potent and selective inhibitor of LTA4 hydrolase.

Leukotriene (LT) B4 is an inflammatory mediator that is synthesized by a number of cell types, including mast cells, neutrophils, and macrophages (Gorman, 1989). It is a potent activator of neutrophils, inducing chemotaxis, aggregation, degranulation, adherence, and priming of inflammatory cells for induction by other agonists, such as prostaglandins and cysteinyl leukotrienes (Ford-Hutchinson and Evans, 1986). Excessive production of LTB4 has been implicated in the pathogenesis of various immune and inflammatory diseases, including psoriasis and inflammatory bowel disease (Ford-Hutchinson and Evans, 1986).

The rate-limiting step in the biosynthesis of LTB4 is the specific epoxide hydrolase, LTA4 hydrolase (E.C. 3.3.2.6), which converts the unstable allylic epoxide LTA4 to LTB4 (Fig. 1). LTA4 hydrolase is a soluble monomeric enzyme, ubiquitous in mammalian tissues and often found in cells that do not contain 5-lipoxygenase (5-LO) (Lindgren and Edenius, 1993). Although sequentially not homologous to other epoxide hydrolases (Funk et al., 1987), LTA4 hydrolase shares homology with puromycin-sensitive aminopeptidase and is a member of the peptidase-M1 family. Sequence comparisons with metalloproteases allowed the further identification of LTA4 hydrolase as a bifunctional zinc-containing enzyme. In addition to hydrolyzing the triene epoxide LTA4 to LTB4, it also displays an aminopeptidase activity. The aminopeptidase activity accepts a variety of substrates from unnatural single amino acid p-nitroanilides to opioid peptides, including the dynorphins and enkephalins, but is thought to preferentially hydrolyze tripeptides with N-terminal arginine (Orning et al., 1994). Compounds that selectively inhibit LTA4 hydrolase would preferentially block the formation of LTB4 and thus be an attractive pharmacological target.

Figure 1
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Figure 1

Reaction catalyzed by LTA4 hydrolase.

Early initiatives in the discovery of LTA4hydrolase inhibitors were largely confined to compounds that resemble the natural substrate/inhibitor LTA4 (Djuric et al., 1992; Labaudinière et al., 1992). After the discovery of the zinc-containing nature of LTA4 hydrolase, bestatin and captopril, inhibitors of Zn2+-containing aminopeptidases and angiotensin-converting enzyme, were also shown to inhibit LTA4 hydrolase (Orning et al., 1991a,b). A series of nonpeptide transition-state analog inhibitors of LTA4 hydrolase were designed on the basis of their ability to inhibit the aminopeptidase activity (Yuan et al., 1993), and more recently a series of inhibitors that incorporate potential zinc-chelating moieties have been described (Hogg et al., 1995, 1998; Ollmann et al., 1995). Some of these inhibitors display time-dependent kinetics and most of them preferentially inhibit the peptidase activity over the epoxide hydrolase activity (Wetterholm et al., 1995; Hogg et al., 1998). The hydroxamic acid-containing peptide kelatorphan and related analogs were among the most equipotent inhibitors of both the epoxide hydrolase and peptidase activities of LTA4 hydrolase described to date (Penning et al., 1995). Recently a potent series of inhibitors around a 1-[2-(4-phenylphenoxy)ethyl]pyrrolidine nucleus have been described (Penning et al., 2000; Penning, 2001).

In the present study, we describe a chemically novel and simple nonpeptide compound designated SC-57461A (3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl) as a potent and selective inhibitor of LTA4hydrolase (Fig. 2). The pharmacology of its free acid, SC-57461, is shown for comparison. Inhibition was investigated on several species of purified enzyme and ionophore-stimulated whole blood, and the effects compared.

Figure 2
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Figure 2

Structure of SC-57461A (3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl] amino]propanoic acid HCl).

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Experimental Procedures

Recombinant Human LTA4 Hydrolase (rhLTA4) Assay [Enzyme-Linked Immunosorbant Assay (ELISA)].

rhLTA4 was prepared as previously described (Gierse et al., 1993) and stored at −20°C as a stock solution in 50 mM Tris buffer, pH 8.0, containing 150 mM NaCl, 2.5 mM β-mercaptoethanol and 50% glycerol (specific activity = 650 nmol/min/mg). LTA4 substrate was prepared from the methyl ester in tetrahydrofuran (BIOMOL Research Laboratories, Plymouth Meeting, PA) by room temperature incubation with 30 M equivalents of LiOH for 18 h, and stored at −80°C until used. Enzyme was diluted into assay buffer [0.1 M potassium phosphate, pH 7.4, 5 mg/ml fatty acid free bovine serum albumin (BSA), and 10% dimethyl sulfoxide (DMSO)] and 250 ng (18 nM final assay concentration) in 25 μl was incubated with test compound, also in assay buffer, for 10 min at room temperature. LTA4 substrate was diluted in assay buffer without DMSO to a concentration of 350 ng/ml and 25 μl (8 ng) was added. The reaction (total volume 200 μl) was allowed to proceed at room temperature for 10 min. A 25-μl sample was added to 500 μl of assay buffer without DMSO to stop the reaction. LTB4 was quantified in the diluted sample by a commercially available ELISA (Cayman Chemical, Ann Arbor, MI) by using an in-house generated anti-LTB4 polyclonal antibody, R11 (Gierse et al., 1993).

LTA4 Hydrolase Kinetic Assay (HPLC).

LTA4 hydrolase kinetics was determined using an RP-HPLC assay to quantify LTB4. The assay contained 1 μg of enzyme (143 nM final concentration) in 100 μl of 50 mM HEPES buffer, pH 8.0, containing 1 mg/ml fatty acid-free BSA. The reaction was initiated by the addition of 25 μM LTA4 free acid in ethanol (to a final ethanol concentration of 2%) and incubated at 25°C for 15 to 30 s. The reaction was stopped by the addition of 2 volumes of cold buffer (3:1, 200 mM citrate, pH 3.5/methanol) containing 250 ng of prostaglandin B1 as an internal standard. Samples were centrifuged for 5 min at 15,000g. The supernatant (200 μl) was applied to a Nova-Pak C18 RP-HPLC column (3.9 × 300 mm) using a computerized Waters HPLC system (Waters, Milford, MA). The column was equilibrated at 1 ml/min with 60% buffer A (60% acetonitrile, 40% methanol, 0.1% acetic acid), 40% buffer B (0.1% acetic acid in water), and eluted over 20 min with a concave gradient to 90% buffer A, 10% buffer B. The eluate was monitored at 270 nm, and the LTB4 quantified from the ratio of LTB4/PGB1 in comparison with a known standard curve.

Peptidase Assay.

Aminopeptidase activity was determined spectrophotometrically by the release of the colorimetric product 4-nitroaniline from l-leucine-p-nitroanilide (Leu-pNA) or l-arginine-p-nitroanilide (Arg-pNA) in 50 mM Tris-HCl, pH 7.5, containing 1 mg/ml fatty acid-free BSA in 200 μl. The reaction was started by the addition of recombinant human LTA4 hydrolase. Absorbance was continuously monitored at 405 nm and the reaction allowed to proceed at room temperature. For the IC50 determinations, 1 mM substrate was used with 1 μg of protein (72 nM final concentration) and the reaction allowed to proceed for 15 min. For the kinetic determinations, the substrate was varied from 0.083 mM to 1 mM, and 50 ng of protein (3.6 nM final concentration) was used with a total reaction time of 60 min. Inhibition kinetic constants (Ki values) were calculated using the program k-cat by BioMettalics (Princeton, NJ) or by the graphical method of Dixon (1972).

Epoxide Hydrolase Assay.

Recombinant human cytosolic epoxide hydrolase was assayed using LTA4 as the substrate. The assay was conducted as described for the LTA4 hydrolase kinetic assay except the reaction was allowed to proceed for 1 min before quenching. 5(S),6(R)-Dihydroxyeicosa-7E,9E,11Z,14Z-tetraenoic acid was quantitated by RP-HPLC exactly as described for LTB4.

Assays for Arachidonic Acid-Metabolizing Enzymes.

Assays for other enzymes known to metabolize arachidonic acid into inflammatory mediators were performed as previously described. These include human recombinant 5-lipoxygenase (Smith et al., 1995), human recombinant cyclooxygenase-1 and cyclooxygenase-2 (Gierse et al., 1995), and leukotriene C4 synthase activity in THP-1 cells (Welsch et al., 1994).

Whole Blood Eicosanoid Production.

Human blood collected in heparin containing Vacutainer tubes was diluted 1:4 with RPMI-1640 (Invitrogen, Carlsbad, CA) and 200 μl was added per well in 96-well microtiter plates. Compounds diluted in 1% DMSO were added to the blood in duplicate and allowed to incubate for 15 min at 37°C in a humidified incubator (5% CO2). Calcium ionophore A23187 Calcimycin (20 μg/ml, 1% DMSO final concentration) was added and the incubation continued for 10 min. The incubation was terminated by centrifugation (600–1300g, 10 min, 4°C). Supernatants were analyzed for LTB4and thromboxane B2 (TxB2) with ELISA by using commercially available reagents (Cayman Chemical) and the in-house-generated anti-LTB4 polyclonal antibody R11.

Heparinized blood from other species (rhesus monkey, rat, mouse, and dog) was diluted 1:1 with RPMI-1640 immediately before aliquoting 200 μl/well into microtiter plates. Preincubations with inhibitor were for 15 min. Experimental conditions were optimized for each species in preliminary experiments. Calcium ionophore A23187 concentrations (μg/ml) were 10 for monkey and rat, and 20 for mouse and dog. Incubations were continued after A23187 addition for 20 min (mouse) or 30 min (monkey, rat, dog). Incubations were terminated by centrifugation as previously described for human whole blood. Supernatants were analyzed for LTB4 and TxB2 with ELISA by using in-house-generated anti-LTB4 antibody and/or commercially available reagents (Cayman Chemical).

Materials.

SC-57461 and SC-57461A (>95% purity), zileuton {N-(1-benzo[b]thien-2-ylethyl)-N-hydroxy urea}, and A-78773 {N-[3-[5-(4-fluorophenoxy)-2-furanyl]-1-methyl-2-propynyl]-N-hydroxyurea} were synthesized by the Chemistry Department at Pharmacia Research and Development, Skokie, IL.

Enzymes.

Purified enzymes from nonhuman species were cloned, expressed, and purified as described for the human. Clones were obtained from a mouse lung cDNA library for the murine, and a rat spleen cDNA library for the rat. After purification, the specific activities of the epoxide hydrolase were 420 nmol/min/mg for the murine (>95% by SDS-polyacrylamide gel electrophoresis), and 653 nmol/min/mg for the rat (89% pure by SDS-polyacrylamide gel electrophoresis). Recombinant human cytosolic epoxide hydrolase was a generous gift from Bruce D. Hammock (Department of Entomology, University of California, Davis, CA) (Beetham et al., 1993).

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Results

Inhibition of rhLTA4 Hydrolase.

SC-57461A and SC-57461 are both very potent, nonpeptide inhibitors of rhLTA4 hydrolase when either LTA4 or peptidase substrates are used (Table1). No inhibition of rhLTA4 hydrolase was observed with either the first- (zileuton) or second-generation (A-78773) 5-LO inhibitors (Carter et al., 1991; Harris et al., 1995), demonstrating selectivity of these inhibitors for the 5-LO enzyme. In contrast to previously described LTA4 hydrolase inhibitors (Wetterholm et al., 1995; Hogg et al., 1998), SC-57461A is equipotent as an inhibitor of the hydrolase and peptidase activities. SC-57461A is equipotent with kelatorphan as an inhibitor of LTA4 hydrolase (Penning et al., 1995).

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Table 1

Inhibition values for rhLTA4 hydrolase

SC-57461 displayed competitive kinetics when either LTA4 (Fig. 3) or a peptidase substrate is used (data not shown) (Table 1). These assays are routinely run after short-term preincubation of the enzyme and inhibitor. Therefore, the time-dependent inhibition of SC-57461 was also investigated. Inhibition was measured with no preincubation (enzyme added last) or defined intervals of preincubation. As illustrated in Fig. 4, no change in the inhibition of LTA4 hydrolase was seen after preincubation of the enzyme with inhibitor from 0 to 5 min. Further studies extended the preincubation time scale to 24 h without a significant change in the IC50 (data not shown).

Figure 3
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Figure 3

Competitive inhibition of rhLTA4hydrolase by SC-57461. One microgram of rhLTA4 hydrolase was incubated with increasing amounts of SC-57461 (●, 0 nM; ▿, 50 nM; ▾, 100 nM; ■, 200 nM; ▪, 300 nM; ▵, 400 nM) as described underExperimental Procedures. The inhibition constant derived from the graphical method of Dixon for SC-57461 wasKi = 23 nM.

Figure 4
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Figure 4

Time-independent inhibition of rhLTA4hydrolase by SC-57461A. rhLTA4 hydrolase was preincubated with varying concentrations of inhibitor for the indicated periods of time. LTA4 was added to start the assay or the enzyme was added last for time 0, and the reaction quenched as described underExperimental Procedures. LTB4 was quantified by RP-HPLC. Preincubation times: ▪, 0 min; ○, 30 s; and ▴, 5 min.

SC-57461 and SC-57461A were also tested as inhibitors of purified LTA4 hydrolase from mouse and rat (Table2). In these preparations, SC-57461 was equipotent on the human and murine enzymes, but from 2- to 10-fold less active against the rat enzyme.

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Table 2

Inhibition of purified LTA4 hydrolase from different species

Specificity of Enzyme Inhibition.

SC-57461 was tested for its specificity to inhibit other enzymes in the arachidonic acid cascade. It did not inhibit recombinant human 5-lipoxygenase, recombinant human cyclooxygenase-1, or recombinant human cyclooxygenase-2 at 100 μM or LTC4 synthase activity in THP-1 cells at 100 μM (data not shown). SC-57461 was also tested as an inhibitor of recombinant human cytosolic epoxide hydrolase (Haeggström et al., 1986; Beetham et al., 1993). By using LTA4 as the substrate, the IC50 was determined to be 300 μM (Fig. 5). This >1000-fold selectivity of SC-57461 for LTA4 hydrolase over an unrelated cytosolic epoxide hydrolase is further demonstration of its specificity. In addition, SC-57461 at 100 μM did not inhibit other metalloproteases, including human leukocyte elastase, human liver cathepsin B, rabbit lung angiotensin-converting enzyme or bovine spleen cathepsin D (data not shown). A structurally related LTA4 hydrolase inhibitor, SC-54581, did not inhibit neutral endopeptidase at 100 μM (data not shown).

Figure 5
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Figure 5

Specificity of inhibition of SC-57461 on LTA4 hydrolase versus cytosolic epoxide hydrolase. Enzyme used: ○, recombinant human cytosolic epoxide hydrolase, IC50 = 300 μM; ●, recombinant human LTA4 hydrolase, IC50 = 0.10 μM.

Inhibition of Whole Blood Eicosanoid Production.

The activity and selectivity of SC-57461A and SC-57461 were also demonstrated in whole blood under conditions in which both the 5-LO and cyclooxygenase pathways were stimulated simultaneously with calcium ionophore. SC-57461A and SC-57461 were potent inhibitors of LTB4 production in whole blood from a variety of species (Table 3). Both compounds (IC50 values in human blood, 49 and 65 nM, respectively) are similar in potency to A-78773 in human and rhesus blood. SC-57461A is less potent than A-78773 in the mouse, rat, and dog. In contrast to the purified enzyme, SC-57461 was 3-fold less potent on murine blood compared with human, but the rat/human ratio of activity was similar.

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Table 3

Inhibition of ionophore-stimulated LTB4 production in whole blood

In all species tested, neither SC-57461A nor SC-57461 inhibited the synthesis of TxB2 (IC50 > 10 μM), a major cyclooxygenase product formed in whole blood under these conditions (data not shown).

SC-57461A did not inhibit LTB4 stimulated chemotaxis of isolated human neutrophils in Boyden chambers over a concentration range of 1 to 100 μM, suggesting it does not block the ability of LTB4 to activate LTB4 receptors (data not shown).

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Discussion

Previous investigators have followed a two-pronged approach in the search for potent LTA4 hydrolase inhibitors. The first approach consisted of inhibitors based on the LTA4 substrate. Djuric et al. (1992) synthesized compounds based on an oxabicycloheptene nucleus in an attempt to mimic the vinyl epoxide of the natural substrate. These compounds did not inhibit the isolated enzyme but inhibited LTB4biosynthesis in the HL-60 cell line. Labaudinière et al. (1992)synthesized a series of ω-arylalkanoic acid derivatives in an attempt to mimic the allylic epoxide of LTA4. The most potent of these analogs displayed IC50 values in the low micromolar range against porcine leukocyte LTA4 hydrolase.

A second approach focused on the peptide isostere after the discovery that LTA4 hydrolase is a zinc-containing enzyme displaying aminopeptidase activity. Two series of compounds evolved from the backbones of bestatin and captopril (Yuan et al., 1991, 1992,1993). In the first series, the investigators used the norstatine backbone of bestatin to build potential peptide transition state analogs. This series eventually evolved to incorporate α-keto-β-amino esters, which in an aqueous environment hydrated to the gem diol, capable of coordinating with the Zn2+ in the active site. The most potent of these compounds were shown to be relatively selective for the aminopeptidase activity of LTA4 hydrolase.

Other zinc binding motifs have also been explored as LTA4 hydrolase inhibitors. Hydroxamic acids have been shown to be potent inhibitors of LTA4hydrolase. Hogg et al. (1995, 1998) reported a series of inverted hydroxamates to be low nanomolar inhibitors of the aminopeptidase activity, but less potent for the epoxide hydrolase activity. Kelatorphan has been shown to be equipotent as an inhibitor of both of the LTA4 hydrolase activities, but it is not selective for LTA4 hydrolase (Penning et al., 1995). A very potent β-mercaptoamine (Yuan et al., 1993; Ollmann et al., 1995) has been described, but it also inhibits thromboxane B2 production in human whole blood. Again, it is assumed that part of its inhibitory activity is due to the zinc-thiol interaction.

SC-57461A is the first reported compound with a structure unrelated to the natural substrate LTA4 or a peptide isostere to possess potent inhibitory activity against LTA4 hydrolase. Its IC50 of 2.5 nM for the epoxide hydrolase activity makes it one of the most potent inhibitors reported to date. It is at least 50-fold more potent against the epoxide hydrolase activity than the most potent inhibitors reported by Yuan et al. (1993), Hogg et al. (1995, 1998), andLabaudinière et al. (1992); as well as captopril and bestatin (Orning et al., 1991a,b; Baker et al., 1995). It is also unique in that its potency was maintained against the aminopeptidase activity, as exhibited by a 2 nM Ki by using arginine-pNA as the substrate. In contrast to the previously published LTA4 hydrolase inhibitors, SC-57461A represents a truly selective class of inhibitors that does not inhibit other zinc-containing aminopeptidases, including human leukocyte elastase and rabbit lung angiotensin-converting enzyme or other enzymes in the arachidonic acid cascade. Please see Penning (2001) for additional information (chemistry, biology, toxicity) regarding this series of LTA4 hydrolase inhibitors.

The mechanism by which SC-57461A inhibits LTA4hydrolase is competitive as illustrated in Fig. 3. To properly characterize the inhibition, the epoxide hydrolase data were analyzed by the graphical method of Dixon (1972) rather than the more conventional Lineweaver-Burke analysis. This was necessary because of the high enzyme concentration (143 nM) necessary to run the assay relative to the potency of the inhibitor. To minimize this effect, the Ki in the aminopeptidase assay was determined using an enzyme concentration of 3.6 nM. Data analyzed by both conventional methods and the graphical method of Dixon (1972)provided similar results. SC-57461 was also shown to act as a time-independent inhibitor because no enhanced inhibition of enzyme activity was evident, even after extensive preincubation of the inhibitor with the enzyme (Fig. 4).

Multiple isozymes of LTA4 hydrolase have been reported. Erythrocytes and B-lymphocytes have each been reported to contain a second subtype that distinguished itself by its divergent kinetic properties (McGee and Fitzpatrick, 1985; Orning et al., 1990;Odlander et al., 1991). Bigby et al. (1994) reported that epithelial cell-derived LTA4 hydrolase present in bronchoalveolar lavage fluid lacked aminopeptidase activity and had unique kinetic properties. They found that the profile of inhibition of epithelial cell-derived LTA4 hydrolase by certain metalloproteinase inhibitors differed from that of the neutrophil enzyme (Baker et al., 1995). Partial N-terminal sequence determinations of the airway epithelial-derived enzyme failed to show any sequence divergence over the amino terminal 17 amino acid span (Ned Seigel, Pharmacia, personal communication). The recent report of an alternative splice site in the LTA4 hydrolase gene would support these findings (Jendraschak et al., 1996). The specificity of SC-57461A for the postulated subtype is as yet unclear.

SC-57461A retained good potency in inhibiting LTB4 production in human and rhesus monkey whole blood. These data suggest that SC-57461A readily penetrates the appropriate cells and interacts with ubiquitously expressed LTA4 hydrolase. These data also show that SC-57461A is fairly metabolically stable in blood and binding to plasma proteins does not negate efficacy. SC-57461A is slightly less potent in rat and dog whole blood. This may reflect a species-dependent decrease in intrinsic activity against the enzyme as supported by the decrease in potency against the isolated rat enzyme, rather than increased instability in whole blood of different species.

SC-57461 and SC-57461A have also been assessed in several animal models. SC-57461 was shown to be orally active with a pharmacodynamic half-life exceeding 24 h. SC-57461 was shown to inhibit LTB4 production in a rat model of ionophore-induced peritoneal eicosanoid production, in a rat reversed passive Arthus model, and in an arachidonic acid-induced ear edema model in mice (Kachur et al., 2002). SC-57461 and SC-57461A displayed selective inhibition of LTB4 in a dose-dependent manner in these models.

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Acknowledgments

We acknowledge the technical expertise and contributions of E. Yvonne Pyla, Elizabeth Harding, and Maureen Highkin; and Marny Koch for preparation of this document.

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Footnotes

  • Abbreviations:
    LT
    leukotriene
    SC-57461A
    3-[methyl[3-[4-(phenylmethyl)phenoxy]propyl]amino]propanoic acid HCl
    LTA4
    5(S)-trans-5,6-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid
    LTB4
    5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid
    5-LO
    5-lipoxygenase
    rhLTA4
    recombinant human leukotriene A4 hydrolase
    ELISA
    enzyme-linked immunosorbent assay
    BSA
    bovine serum albumin
    DMSO
    dimethyl sulfoxide
    RP-HPLC
    reversed phase-high-performance liquid chromatography
    pNA
    p-nitroanilide
    TxB2
    thromboxane B2
    A23187
    calcium ionophore A23187(Calcimycin)
    • Received July 17, 2001.
    • Accepted November 2, 2001.
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  1. doi: 10.1124/jpet.300.2.577 JPET February 1, 2002 vol. 300 no. 2 577-582
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