Introduction

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Asthma is now widely accepted as a chronic inflammatory disease of the airways, characterized by airway hyperresponsiveness to both inhaled allergens and nonspecific stimuli (Cartier et al., 1982). There is convincing evidence that the airway hyperresponsiveness occurs as a consequence of epithelial damage caused by the accumulation of large numbers of activated eosinophils and mast cells within the respiratory tract (Bradley et al., 1991; Foresi et al., 1997). The recruitment and activation of eosinophils are thought to be orchestrated by products released by activated T cells; for example, disease severity has been correlated with airway inflammation, and the presence of activated T lymphocytes, particularly of the CD4⁺ Th2 phenotype, in the airway wall (Hamid et al., 1991; Robinson et al., 1992). Th2 cytokines such as interleukin (IL)-5 may be critically involved in the recruitment and activation of eosinophils (Foster et al., 1996; Lee et al., 1997), whereas IL-4 and IL-13 are essential for the "switching" of B cells to generate IgE, which is involved in the antigen-induced release of proinflammatory and bronchoactive mediators from mast cells (Abbas et al., 1996; Emson et al., 1998). Atopy, or hypersensitivity to common environmental antigens, is a major component of asthma in both children and adults and the central role of mast cell-derived products in the IgE-mediated immediate response to inhaled allergen is well established (Djukanovic et al., 1990).

Glucocorticoids are probably the most efficacious compounds available for the treatment of asthma, although fears regarding potential side effects limit their usefulness (Barnes and Pederson, 1993). The precise molecular mechanisms by which glucocorticoids exert their anti-inflammatory activity in asthma are still to be completely defined, although multiple overlapping mechanisms at the level of gene transcription are likely to be involved (Auphan et al., 1995; Scheinman et al., 1995). The improvement in symptoms with treatment can be correlated directly with a reduction in eosinophil numbers in the airway wall (Montefort et al., 1992), and glucocorticoids inhibit the expression of cytokines responsible for eosinophil accumulation (Bentley et al., 1996).

PNU-142731A is a novel, anti-inflammatory, pyrrolopyrimidine that inhibits the production of Th2 cytokines in vivo. The compound is a potent and efficacious inhibitor of eosinophilic lung inflammation and is currently in Phase II Clinical Evaluation for the potential treatment of asthma. In this report we describe the preclinical pharmacology of PNU-142731A, and experiments to investigate the compound's mode of action.

	Materials and Methods

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Immunization and Aerosol Challenge of C57BL/6 Mice. Details of the sensitization and aerosol challenge of female C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) with ovalbumin (OA) have been previously reported (Hatfield et al., 1997). Ten micrograms of OA (Sigma, St. Louis, MO) was mixed with 9 mg of aluminum hydroxide (dried powdered gel; Aldrich, Milwaukee, WI) in 200 µl saline and injected i.p. on day 0. Vehicle-immunized mice were injected i.p. with aluminum hydroxide in saline. In most experiments, immunized mice were challenged once on day 14 with an aerosol of 1.5% OA in saline for 10 min as described previously (Hatfield et al., 1997).

Dosing by Oral Gavage. Mice were dosed orally (p.o.) once a day with 200 µl of Vehicle 122 (0.25% methylcellulose in water), PNU-142731A (Fig. 1) at 0.1 to 30 mg/kg/day suspended in Vehicle 122, or dexamethasone (Sigma, St. Louis, MO) at 0.001 to 0.5 mg/kg/day suspended in Vehicle 122. Concurrent treatment with dexamethasone and PNU-142731A was also conducted. Treatment with PNU-142731A was initiated on days 0, 7, 10, or 13 of the experiment and continued through day 16, as indicated.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Chemical structure of PNU-142731A.

Bronchoalveolar Lavage (BAL) and Cell Preparations. The exact experimental protocols used for lavaging mouse lungs have been described previously (Hatfield et al., 1997). Briefly, BAL was performed on all groups of mice 3 days after the final antigen challenge (day 17). Lungs were lavaged with 0.5 ml Dulbecco's PBS without Ca²⁺ or Mg²⁺ (Gibco BRL, Grand Island, NY) two times as described previously. The leukocytes were pelleted by centrifugation, the BAL fluid (BALF) was removed, and stored frozen at 20°C until the time of IL-5, IL-6, or IgA assay. The leukocytes were resuspended in Hank's balanced salt solution (Gibco BRL) supplemented with 5% fetal bovine serum (FBS), 1 mM HEPES, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco BRL), enumerated, and the different leukocyte subsets were determined morphologically after staining with Diff Quik (American Scientific Products, McGraw Park, IL) (Hatfield et al., 1997).

Lung Tissue Disaggregation and Stimulation with OA In Vitro. Lung tissue disaggregation was performed as previously described (Kennedy et al., 1995). Briefly, lung tissue from each mouse was perfused with saline, minced with scissors, and placed into 2-ml microfuge tubes containing Dulbecco's PBS with 10% FBS, 850 U/ml hyaluronidase (Sigma), 150 U/ml collagenase (CLS-3; Worthington Biochemical Corp., Freehold, NJ), and 50 U/ml DNase I (Sigma). Disagregation of the tissue was achieved by agitating the tubes in a Mini-BeadBeater 51 (Biospec Products, Bartlesville, OK) for 60 min at room temperature. The resulting single cell suspension was passed through 55-µm nylon mesh to remove undigested tissue pieces and any contaminating red blood cells were lysed.

Induction of OA-Specific Cytokine Release from Splenocytes Isolated from PNU-142731A-Treated OA-Sensitized Mice. Mice were immunized with OA on day 0, and dosed (p.o.) daily with Vehicle 122 or PNU-142731A until day 16 for a total of 17 doses. These mice were not challenged with aerosolized antigen. Spleens were removed aseptically from five mice from each group on day 17. Each spleen was disaggregated into single cells and suspended in Hank's balanced salt solution with 5% FBS. The red blood cells were removed by lysis and the lymphoid cells were washed by centrifugation through medium. The cells were suspended at 1 × 10⁷/ml in assay medium as described for lung cell culture. OA was diluted to 800 µg/ml in assay medium. One hundred microliters of each cell suspension was added to six replicate wells containing 100 µl of medium alone or diluted OA. The plates were incubated for 3 days at 37°C and 5% CO₂, before supernatants were collected and frozen until ELISA assays for IL-5, IL-6, and IL-10 were performed.

RNA Isolation, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and Semiquantitative Analysis. Total RNA was obtained from lung tissue using RNA STAT-60 (Tel-Test "B", Inc., Friendswood, TX) and analyzed as previously described (Krzesicki et al., 1997). Briefly, lung tissue from individual Vehicle- and OA-sensitized mice given Vehicle 122 or PNU-142731A beginning on day 7 was obtained 3 days after the third OA-challenge (day 17) and homogenized in 5 ml RNA STAT-60 using a polytron homogenizer (Brinkman Instruments, Inc., Westbury, NY). cDNA synthesis and PCR amplification reactions were done using SuperScript II (M-MLV-RT; Gibco BRL) and AmpliTaq DNA Polymerase (Perkin-Elmer, Norwalk, CT). DNA amplifications were performed for the predetermined number of optimal cycles for each primer set in 50-µl reaction volumes. Thermocycler parameters were: 94°C for 5 min; 25-35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 45 s; followed by 72°C for 7 min. PCR products were separated by electrophoresis on a 2% agarose gel followed by staining with SYBR Green I (Molecular Probes, Inc., Eugene, OR). Quantitation was done with the Molecular Dynamics FluoroImager using ImageQuaNT Software (Molecular Dynamics Corporation, Sunnyvale, CA) and volume integration. After subtracting background, the volume of each cytokine product was normalized to glyceraldehyde-3-phosphate dehydrogenase for each animal. Data shown are the means of normalized values ± S.E.M.

Histology and Quantitation of Mucus-Producing Cells. Seventy-two hours after inhalation of OA (day 17), lungs were lavaged, excised from the chest, and fixed by inflation with 10% phosphate-buffered formalin via a tracheal cannula (Chin et al., 1998a). Fixed trachea and left lung were embedded in paraffin, sectioned at 6 µm, and stained with periodic acid-Schiff (PAS) using standard methodologies. The PAS-stained slides were evaluated with an Optimas 6.1 Image Analysis system (Optimas Corporation, Edmonds, WA) using a Sony DXC960 MD camera mounted on a Leitz Laborlux microscope with a 20× objective. The epithelial lining on one side of an airway was sampled. The total area of all PAS-positive cells (µm²) along a specified length of the airway epithelium was determined. This procedure was applied to the hilus and mid-lung in each section. The hilus was defined as the area within one 10× field diameter (approximately 1.6 mm) from the point of entry of the airway and great vessels into the lobe. The mid-lung was defined as the area delineated by two field diameters (either laterally or caudally) from the point of entry of the airway. Two measurements were taken in the hilus and mid-lung for each animal and averaged. The data are expressed as means ± S.E.M. for 10 mice/group.

Antigen-Specific IgG₁ and Total IgE in Plasma. Blood samples were collected from mice on day 17, 3 days after the final OA challenge, and the levels of IgE and OA-specific IgG₁ in the plasma were determined by ELISA as previously described (Hatfield et al., 1997).

Cytokine and IgA Measurements. The concentration of cytokines in plasma, conditioned supernatants, and BALF samples were measured by ELISA using antibody pairs from PharMingen, as described previously (Hatfield et al., 1997).

Statistical Analysis. All data (except for BALF leukocytes) were analyzed by the Kruskal-Wallis one-way ANOVA using multiple comparisons with t distribution on ranked data. Significant differences (p < .05) in two-sided pairwise comparison between OA/OA controls and treatment groups are denoted by an * in the figures. In some instances, comparisons between two treatment groups were also made and significant (p < .05) differences are indicated by an * in the figures.

	Results

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Effect of PNU-142731A on Antigen-Induced Accumulation of Leukocytes in Airways of OA-Sensitized and -Challenged Mice. OA-sensitized mice were treated orally with PNU-142731A ranging from 0.3 to 10 mg/kg/day beginning on day 7 of the experiment for 10 consecutive doses. The lungs were lavaged 3 days after OA challenge (on day 17), and the numbers of BALF eosinophils and lymphocytes were enumerated. The percentage of inhibition of eosinophil and lymphocyte accumulation in the airways by PNU-142731A after antigen provocation was determined (Fig. 2A and B, respectively). Figure 2 also shows that there was a dose-related inhibition of eosinophil and lymphocyte accumulation in the airways of PNU-142731A-treated OA/OA mice when compared with Vehicle 122-dosed controls.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Inhibition of BALF eosinophils and lymphocytes in OA/OA mice by oral treatment with PNU-142731A. OA-sensitized mice were treated daily, p.o., with PNU-142731A ranging from 0.3 to 10 mg/kg/day for 10 days (n = 10/group) beginning on day 7. Three days after OA-inhalation challenge, the lungs were lavaged and the number of eosinophils and lymphocytes in the BALF was determined. Reduction in the numbers of both subsets of leukocytes were expressed as percentage of inhibition of BALF eosinophils (A) or lymphocytes (B) compared with the numbers of each subset in Vehicle 122-treated OA/OA mice. Each data point is mean ± S.E.M. of three to seven separate experiments for each dose of PNU-142731A. ID50 is dose of PNU-142731A necessary to achieve a 50% inhibition.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effect of length of oral treatment with PNU-142731A on accumulation of leukocytes in the bronchial lumen of OA/OA mice. OA/OA mice were treated orally with PNU-142731A (10 mg/kg/day) for a total of 4, 7, or 10 days. The lungs were lavaged 3 days after OA provocation, and the total number of leukocytes in the BALF was enumerated (A). The number of BALF eosinophils and lymphocytes (B) based on morphological criteria was also determined. Means ± S.E.M. for groups of 10 mice are shown. Significant (p < .05) differences between OA/OA mice dosed with Vehicle 122 and PNU-142731A for raw and ranked data are indicated by # and *, respectively.

Steroid-Sparing Effects of PNU-142731A on Antigen-Induced Accumulation of Leukocytes in Airways of OA-Sensitized and -Challenged Mice. OA/OA mice were treated with 1 or 3 mg/kg/day PNU-142731A alone or dexamethasone alone at 0.001 mg/kg/day for 10 days (beginning on day 7 of the experiment). The data shown in Fig. 4 indicate that dexamethasone alone or PNU-142731A alone at 1 mg/kg/day did not reduced the number of BALF eosinophils (Fig. 4A) or lymphocytes (Fig. 4B). However, a higher dose of PNU-142731A (3 mg/kg/day) significantly depressed the influx of both subsets of leukocytes. The most striking responses, however, were in mice given a combination of dexamethasone and 1 or 3 mg/kg/day PNU-142731A. The suppression of eosinophils and lymphocytes in OA/OA mice treated concomitantly with both compounds was significantly greater than with each compound alone. These data are indicated by the horizontal lines between the comparison groups for eosinophils and lymphocytes (Fig. 4, A and B, respectively).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effect of oral cotreatment with PNU-142731A and dexamethasone on the accumulation of eosinophils and lymphocytes in the airways of OA/OA mice. OA/OA mice were dosed orally with PNU-142371A (1 or 3 mg/kg/day) alone, dexamethasone (0.001 mg/kg/day) alone, or both concurrently beginning on day 7 for a total of 10 doses. The lungs were lavaged on day 17, 3 days after the third OA challenge. The numbers of BALF eosinophils (A) and lymphocytes (B) were enumerated. Means ± S.E.M. for groups of 10 mice are shown. Significant (p < .05) differences between OA/OA mice treated with Vehicle 122 and PNU-142731A are indicated by *. In addition, comparisons between OA/OA mice given either PNU-142731A or dexamethasone alone were made with OA/OA mice treated concomitantly with both compounds. Significant differences (p < .05) between two treatment groups (denoted by horizontal lines) are indicated by *.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Percentage of inhibition of BALF eosinophils after oral cotreatment with PNU-142371A and dexamethasone in the airways of OA/OA mice. OA/OA mice were dosed orally with dexamethasone (0.001-0.5 mg/kg/day) starting on day 7, for a total of 10 doses. Oral cotreatment with PNU-142731A (1 or 3 mg/kg/day) and dexamethasone (0.001, 0.01, or 0.03) was also conducted for the same time period. The lungs were lavaged on day 17, 3 days after the third OA challenge. The number of BALF eosinophils was enumerated and expressed as percentage of inhibition of eosinophils compared with Vehicle 122-treated OA/OA mice.

Effect of PNU-142731A Treatment on Lung Histology and Mucus Production by Lung Epithelial Cells from OA-Sensitized and -Challenged Mice. Oral administration of PNU-142731A to OA/OA mice not only blocked the recruitment of leukocytes into the bronchial lumen but effectively reduced the intensity of leukocytic infiltration into lung tissue by histological evaluation of formalin-fixed and H&E-stained lung tissue cross-sections. Both peribronchial and perivascular inflammation were diminished. Histological assessment of lung tissue from OA/OA mice dosed with Vehicle 122 yielded a total lung inflammation score of 3.4 ± 0.69 (mean ± S.E.M., n = 10). Administration of PNU-142731A reduced the lung histological score of these OA/OA mice to 0.4 ± 0.16 (n = 10), which was significantly less than OA/OA controls (p < .05).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Quantitative assessment of formalin-fixed, PAS-stained lung tissue cross-sections of OA/OA mice treated with PNU-142731A. Lungs obtained from V/OA and OA/OA mice on day 17, 3 days after antigen challenge, were formalin-fixed and stained with PAS. The total PAS-positive area/100 µm of lung epithelium in the hilus and mid-lung was determined. All comparisons were made between PNU-142731A or dexamethasone (0.5 mg/kg/day)-treated OA/OA mice and Vehicle-treated OA/OA control mice. Means ± S.E.M. for groups of 10 mice are shown, and significant differences (p < .05) between OA/OA controls and treatment groups are indicated by *.

Effect of PNU-142731A on BALF IL-5, IL-6, and IgA Levels. BALF from OA/OA mice administered PNU-142731A (p.o., beginning on day 7 for a total of 10 doses) was obtained 3 days after antigen inhalation (day 17), and the levels of IL-5, IL-6, and IgA were determined by ELISA (Fig. 7, A, B, and C, respectively). The concentrations of both cytokines and IgA in the BALF of OA/OA mice were decreased proportionally by increasing concentrations of PNU-142731A when compared with OA/OA controls.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effect of PNU-142731A on cytokines and IgA in BALF from OA-sensitized mice 3 days after OA provocation. Lungs from V/OA and OA/OA mice treated orally with Vehicle 122 or PNU-142731A (0.3-10 mg/kg/day) for 10 days were lavaged, and BALF samples were assayed by ELISA. BALF IL-5 (A), IL-6 (B), and IgA (C) concentrations are shown. Significant (p < .05) differences between treatment groups and OA/OA mice treated with Vehicle 122 are indicated by *. ND, not done.

Effect of PNU-142731A on Plasma OA-Specific IgG₁ and Total IgE. Figure 8, A and B shows the effect of PNU-142731A treatment on the amount of OA-specific IgG₁ and IgE in plasma from OA/OA mice, respectively. Oral administration of PNU-142731A, which was initiated on day 7 (for a total of 10 doses), lowered the concentration of both Ig isotypes in the plasma in a dose-dependent manner. Oral treatment with dexamethasone (0.5 mg/kg/day) using the same dosing regimen also resulted in inhibition of B cell responses.

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Effect of PNU-142731A on plasma OA-specific IgG1 and total IgE. Plasma samples were collected on day 17 from OA/OA mice dosed with Vehicle 122, dexamethasone (0.5 mg/kg/day), or PNU-142731A (1, 3, or 10 mg/kg/day) for 10 days. Levels of OA-specific IgG1 (A) and total IgE (B) were determined by ELISAs. Means ± S.E.M. for groups of 10 mice are shown. Significant (p < .05) differences between treatment groups and OA/OA mice treated with Vehicle 122 are indicated by *.

Effect of PNU-142731A on Cytokine mRNA in Lung Tissue of OA-Sensitized and -Challenged Mice. The mRNA for the Th2 cytokines, IL-4, IL-5, IL-6, IL-10, and IL-13 were increased in lung tissue from OA/OA mice compared with V/OA mice. Lungs from OA/OA mice treated with PNU-142731A at 10 mg/kg/day for 10 days (starting on day 7) had significantly less expression of these cytokines when compared with Vehicle 122-treated OA/OA controls (Fig. 9).

View larger version (20K): [in this window] [in a new window]   Fig. 9.   Lung tissue expression of Th2 cytokine mRNA. Total lung mRNA from V/OA and OA/OA mice given Vehicle 122 or PNU-142731A (10 mg/kg/day) were obtained on day 17 of the experiment. Semiquantitative RT-PCR for 5 Th2 cytokines was performed and normalized to glyceraldehyde-3-phosphate dehydrogenase. Means ± S.E.M. for groups of 10 mice are shown. Significant (p < .05) differences between Vehicle 122- and PNU-142731A-treated OA/OA mice are indicated by *.

In Vitro Cytokine Release by OA-Stimulated Splenocytes from OA-Sensitized Mice Treated with PNU-142731A. OA-immunized mice were dosed with PNU-142731A (10 or 30 mg/kg/day) for 17 days as described, and the splenocytes were isolated and cultured with OA for 72 h in vitro. When the conditioned supernatants were assayed, the antigen-induced release of IL-5 and IL-10, but not IL-6 (data not shown), was significantly reduced in splenocytes from mice given PNU-142731A (Fig. 10, A and B, respectively).

View larger version (17K): [in this window] [in a new window]   Fig. 10.   Antigen-elicited secretion of cytokines by splenocytes from OA-sensitized mice. OA-sensitized mice were dosed with PNU-142731A (10 or 30 mg/kg/day, p.o.) for 17 days, and the splenocytes were cultured and stimulated with OA for 3 days in vitro. Conditioned supernatants were assayed for IL-5 (A) and IL-10 (B) by ELISA. Means ± S.E.M. for groups of five mice are shown. Significant (p < .05) differences between treatment groups and Vehicle 122-treated OA/OA control mice are indicated by *.

In Vitro Cytokine Release by OA-Stimulated Lung Cells from OA-Sensitized and -Challenged Mice Treated with PNU-142731A. Lung cells were isolated from saline-perfused lungs of OA/OA mice treated with PNU-142731A (10 mg/kg/day) for 17 days. The disaggregated lung cells were cultured with OA in vitro for 6 h and the conditioned supernatants were assayed for the release of IL-4, IL-2, and IFN- (Fig. 11A, B, and C, respectively). The level of OA-elicited IL-4 in supernatants from lung cell isolated from PNU-142731A-treated mice was markedly reduced when compared with control cultures (Fig. 11A). No changes were noted in the concentration of IL-5 in culture supernatants (data not shown). In contrast, release of the Th1 cytokines, IL-2, and IFN- was elevated in OA-stimulated lung cells from mice dosed with PNU-142731A (Fig. 11, B and C, respectively).

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Induction of cytokine release from disaggregated lung tissue cells from OA/OA mice. Lung tissue cells were obtained from OA/OA mice treated with Vehicle 122 or PNU-142731A (10 mg/k/day, p.o.) for 17 days, and cultured with OA for 6 h in vitro. Conditioned supernatants were assayed for IL-4 (A), IL-2 (B), and IFN- (C) by ELISA. Means ± S.E.M. for groups of four mice are shown. Significant (p < .05) differences between PNU-142731A-treated mice and Vehicle-treated OA/OA mice are indicated by *.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

PNU-142731A, 1-[(2,4-Di-1-pyrrolidinyl-9H-pyrimido[4,5-b]indol-9-yl)acetyl] pyrrolidine, monohydrochloride is a member of a novel series of anti-inflammatory pyrrolopyrimidines targeted for the treatment of asthma (Bundy et al., 1995; Chin et al., 1998a). These compounds originated from a study of the structure-activity relationships of a series of nonglucocorticoid 21-aminosteroids, structural analogs of methylprednisolone that were devoid of glucocorticoid activity, but demonstrated antioxidant and neuroprotective activity (Jacobsen et al., 1990). Members of this series of compounds were also shown to inhibit hyperoxic lung injury (Griffin et al., 1994) and prevent antigen-induced eosinophilic lung inflammation in sensitized animals (Richards et al., 1992, 1995). Further elucidation of structure-activity relationships (Bundy et al., 1995) revealed that the antioxidant and neuroprotective properties of these compounds could be divorced from their ability to modulate eosinophil leukocyte trafficking, and that this anti-inflammatory property was associated with the triamino pyrimidine moiety (Fig. 1), rather than the steroid nucleus of the 21-aminosteroids.

The anti-inflammatory properties of PNU-142731A, a drug candidate for the treatment of asthma, were initially identified in a murine model of OA-induced lung inflammation in which the compound blocked the pulmonary infiltration of leukocytes after allergen challenge. The murine model of antigen-induced eosinophilic and lymphocytic pulmonary inflammation has been used increasingly as a model of human allergic asthma to elucidate the cellular and molecular mechanisms involved (Drazen et al., 1996; Richards 1996; Gleich and Kita, 1997; Persson et al., 1997).

In addition to the ability of PNU-142731A to block leukocyte infiltration into lung tissue and the bronchial lumen of OA-sensitized and -challenged mice, we also demonstrated that this was concurrent with inhibition of antigen-elicited induction of mucus glycoproteins in lung epithelial cells. These findings may be linked to the concomitant fall in lung levels of Th2 cytokine mRNAs, such as IL-4, which are thought to be involved in mucus secretion (Temann et al., 1997; Kuperman et al., 1998). Obviously, lung epithelial mucus secretion may also be influenced by other factors including the release of leukotrienes (Henderson et al., 1996), although PNU-142731A does not inhibit 5-lipoxygenase (our unpublished data). The effects of PNU-142731 on the parasympathetic cholinergic pathways involved in mucus secretion have not been investigated, however, and at the present time, we do not know whether PNU-142731 possesses any anticholinergic activity that could explain the compound's effects in reducing antigen-induced mucus secretion.

We have also shown a reduction in lung tissue Th2 cytokine transcripts for IL-4, IL-5, IL-6, and IL-13 in PNU-142731A-treated OA-sensitized and -challenged mice. This may provide a molecular basis for the observations that the compound decreased the levels of IL-5 in BALF and plasma, and lowered the levels of plasma IgE and IgG₁, which are both regulated by Th2 cytokines (Snapper and Mond 1993; Abbas et al., 1996). Exposure of disaggregated lung tissue cells from OA-sensitized and -challenged mice to antigen in vitro also provoked a strong and immediate (6 h) release of IL-4, a Th2 cytokine. We believe that at least part of this response was provided by the CD4⁺ T cells in this pool of cells, as demonstrated by flow cytometric analysis of intracellular cytokine synthesis (Winterrowd and Chin, 1999). This is contrasted with the significant drop in IL-4 release by lung cells from PNU-142731A-treated mice. It was also apparent from the data that lung cells from OA-sensitized and -challenged mice dosed with PNU-142731A produced more Th1 cytokines, i.e., IL-2 and IFN-, than cells from vehicle-treated controls. It is tempting to speculate that Th1 T cells in the lungs of PNU-142731A-treated mice were allowed to mount a response to antigen in vitro in the absence of the suppressive effects of IL-4.

Splenocytes from OA-sensitized mice treated with PNU-142731A also demonstrated a lower OA-dependent release of Th2 cytokines IL-5 and IL-10 in vitro, suggesting that the anti-inflammatory effect of the compound was not confined to the lungs. Although many laboratories (Garlisi et al., 1997; Ohkawara et al., 1997; Lee et al., 1997), including our own (Krzesicki et al., 1997; Chin et al., 1998b), have focused on the importance of local lung production of cytokines, it has been proposed that systemic, rather than local lung IL-5 generation, is important for pulmonary eosinophilia following antigen challenge (Wang et al., 1998). If we extrapolate our data with PNU-142731A in the murine model to asthma in humans, we may infer, that irrespective of whether local or systemic cytokine production is more relevant, PNU-142731A may be predicted to dampen the inflammatory response at multiple sites.

We have also established that oral cotreatment of OA-sensitized and -challenged mice with PNU-142731A and suboptimal doses of dexamethasone was more efficacious in reducing antigen-induced leukocyte influx into the lungs, than treatment with either compound alone. These results are encouraging because they suggest that treatment with PNU-142731A may enable asthmatics to reduce their dosage of glucocorticoids, thereby reducing the risk of steroid side effects.

Even though the precise molecular mechanism that is perturbed by PNU-142731A is yet to be defined, we have demonstrated preclinical in vivo evidence to support the development of this compound for asthma, based on the premise that the compound possesses desirable anti-inflammatory traits in models of pulmonary inflammation. In particular, PNU-142731A suppresses the generation of the Th2 cytokines believed to be responsible for the recruitment of eosinophils to the airway wall.