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Published ahead of print on January 31, 2003, doi:10.1164/rccm.200207-696OC
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American Journal of Respiratory and Critical Care Medicine Vol 167. pp. 1400-1409, (2003)
© 2003 American Thoracic Society

A Small Molecule Very Late Antigen–4 Antagonist Can Inhibit Ovalbumin-induced Lung Inflammation

Gloria C. Koo, Kashmira Shah, Gloria J. F. Ding, Jianying Xiao, Richard Wnek, George Doherty, Xin Chun Tong, R. Blake Pepinsky, Ko-Chung Lin, William K. Hagmann, Douglas Kawka and Irwin I. Singer

Merck Research Laboratories, Rahway, NJ and Biogen Inc., Cambridge, Massachusetts

Correspondence and requests for reprints should be addressed to Gloria C. Koo, Ph.D., Merck Research Laboratories, 80W-107, P.O. Box 2000, Rahway, NJ 07065. E-mail: gloria_koo{at}merck.com


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A nonpeptidyl small molecule antagonist, compound A, to nonactivated very late antigen–4 (VLA4) was examined in lung inflammation induced by a single dose of ovalbumin challenge. Compound A presented a good pharmacokinetic property, when given intratracheally, and the blood cells from such pharmacokinetic study showed good receptor occupancy of the compound for approximately 8 hours. Compound A was then tested in an ovalbumin-induced airway inflammation model by intranasal or intravenous route of administration. There was a dose-dependent inhibition of eosinophilia in the bronchiolar lavage fluid, when compound A was given intranasally but not when it was given intravenously. For comparison, antibody to VLA4 and another compound, BIO1211, which reacts only with activated VLA4, were examined in this system. Immunohistochemical analyses of the lung tissue substantiated the findings in the bronchiolar lavage fluid. Specific staining of the major basic protein of eosinophils showed peribronchiolar infiltration of eosinophils. Some of these eosinophils were also positive for nitrotyrosine, suggesting activation of eosinophils in the lung interstitium. There was deposition of major basic protein and nitrotyrosine at the base of the perivascular endothelium, indicative of degranulation of eosinophils in the area. After intranasal treatment with compound A, eosinophils in the lungs and their activation products were substantially decreased, documenting its effectiveness in inhibiting lung inflammation.

Key Words: VLA4 antagonist • asthma • lung inflammation

Very late antigen–4 (VLA4, {alpha}-4, ß-1) is a heterodimeric cell surface molecule found on all hemopoietic mononuclear cells and eosinophils. (see reviews, 1–4). The natural ligands of VLA4 are vascular cell adhesion molecule (VCAM-1), found normally on endothelial cells and smooth muscle cells, and an alternatively spliced product of fibronectin, CS-1, the connecting segment (5). The expression of VCAM-1 on other cell types could be upregulated and induced by cytokines, such as tumor necrosis factor-{alpha}, interleukin-1 (IL-1), and interleukin-4 (IL-4) (1–4). The constitutive or nonactivating form of VLA4 does not normally bind firmly to VCAM-1 and requires activation to bind to VCAM-1. Activation of VLA4 could be achieved by cytokines, such as IL-1 and IL-4, as well as certain stimulating antibodies, such as anti-CD3 and CD28 Ab on T cells. (1–4, 6–8), resulting in high-affinity binding, promoting transendothelial diapedesis (1–4). Detailed studies of migration have identified VLA4 as the receptor primarily responsible for mononuclear cell trafficking. On the basis of the cellular distribution and the function of the molecule, VLA4 antagonists have been proposed to be effective therapeutically in diseases where cell accumulation or recruitment is the causative element (1). Indeed, published data have shown that anti-VLA4 Ab and VLA4 antagonists can inhibit cellular accumulation in animal models of allergic encephalitis, nonobese diabetes, asthma, and rheumatoid arthritis (9–16). Currently, a humanized anti-VLA4 Ab, Antegren, is in clinical trials to treat multiple sclerosis, and promising results have been reported (17).

To further understand the mechanism of action of VLA4 antagonists, we chose to study a small molecule in a mouse model of lung inflammation, induced by immunization with ovalbumin. In this model, there is massive infiltrate of mononuclear cells and eosinophils (18–20). Both ß-1 and ß-2 integrins have been implicated as the molecular targets responsible for the cellular infiltrates by treating mice systemically with Ab to the respective integrins (our unpublished data, 10, 20). More recently, Henderson and coworkers (18) have shown that better efficacy was observed when anti-VLA4 Ab was given intranasally, compared with systemic administration. In this study, we tested a compound that reacts with the activated as well as the nonactivated or constitutive form of VLA4. The compound was selected for its potency in binding to VLA4 positive cells in whole blood, and mice were dosed with the compounds either by intranasal or intravenous route. For comparison, we tested an anti-VLA4 antibody, PS/2, as well as another compound, BIO1211, in the same system. PS/2 reacts with both the activated and nonactivated form of VLA4, similar to compound A, whereas BIO1211 only reacts with the activated form of VLA4 (11). We also examined the histology of the lungs treated with the compound and found that the accumulation of activated eosinophils in the lung interstitium correlated well with the eosinophils (EOS) found in the bronchoalveolar lavage fluid (BALF).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female BALB/cJ mice 8 to 12 weeks old, were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in the pathogen-free barrier facility at Merck (Rahway, NJ). Male Sprague–Dawley rats 8 to 12 weeks old were obtained from Charles River (Kingston, NY). The Institute Animal Care and Use Committee approved all procedures conducted on the animals.

Compound and Antibodies
Compound A, N-(N-(3,5-dichlorobenzenesulfonyl)-2-methyl-(L)-prolyl)-4-((3',5'-dichloroisonicotinoyl)amino)-(L)-phenylalanine (Figure 1) (21) was solubilized as a clear solution in 0.1 M tris(hydroxymethyl)aminomethane buffer (pH 7.8) for in vitro assay or in 0.1 M tris(hydroxymethyl)aminomethane/0.975% lactose buffer, (pH 7.4) for in vivo studies. BIO1211 was solubilized in 0.1 M tris(hydroxymethyl)aminomethane/lactose buffer as described previously (11). Anti-mouse VLA4 Ab was purified from ascites fluid generated from PS/2 cell line (ATCC, Rockville, MD) with endotoxin level less than 1 EU/mg of protein. Anti-human VLA4 Ab was purchased from Immunotech (Miami, FL). Both are blocking Ab for VLA4.



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  		Figure 1. Chemical structure of compound A.

 
Anti-CD3/VCAM-1–Induced Proliferation
Anti-CD3 (HIT3a, Pharmingen [San Diego, CA], 50 µl, 10 to 20 ng/ml for human T cells) was immobilized on flat-bottom 96-well microtiter plate at 4° overnight, similar to previously described protocol (7). After washing with phosphate-buffered saline (PBS), with Ca2+ and Mg2+, 3x, rVCAM-1 (50 µl, 2 g/ml, R&D [Minneapolis, MN]) was allowed to adhere for 3 to 4 hours at room temperature. Again, after washing 3x with PBS, with Ca2+ and Mg2+, purified T cells (100 µl, 5 x 105/ml) and inhibitors were added to appropriate wells. Human T cells were depleted of monocytes and B cells (7). Briefly, after separation on lymphocyte separation media (Litton Bionetics, Kensington, MD), T cells were prepared by rosetting with neuraminidase-treated sheep red blood cells, enriched by lymphocyte separation media and followed by lysis with ACK lysing buffer (GIBCO, Rockville, MD). Cells were suspended in RPMI, supplemented with 10% fetal bovine serum, and 1% each of penicillin-streptomycin, glutamine, nonessential amino acid, and sodium pyruvate. Plates were incubated at 37°C and 5% carbon dioxide for 4 days and were labeled with [H]3-Thymidine for 6 hours or overnight. Supernatants were harvested and counted in a beta counter, using the Betaplate Liquid Scintillation System (Wallac Inc., Gaithersburg, MD).

Whole Blood Receptor Occupancy Assay
Compound A or BIO1211 was titrated in heparinized blood from BALB/cJ mouse or healthy human volunteers. After 1 hour, at 37°C, blood was over layered onto lymphocyte separation medium (ICN Biomedicals, Aurora, OH) and centrifuged to obtain peripheral blood mononuclear cells (PBMC). Cell suspension (0.5 ml) was then reacted with 10 nM of BIO8139–phycoerythrin (PE) for 30 minutes, washed once, and fixed for fluorescence-activated cell sorter analyses. BIO8139–PE is a small molecule VLA4 antagonist, conjugated with PE, as described below. Receptor occupancy assay was performed similarly to the whole blood assay. Heparinized mouse blood (0.25 ml) was under layered with lympholyte M (Cedarlane, ON, Canada) to obtain PBMC. Cell suspension (0.5 ml) was then reacted with 10 nM of BIO8139–PE for 30 minutes, washed once, and fixed for fluorescence-activated cell sorter analyses.

Preparation of PE-labeled BIO8139
As a measurement of bound compound on cells, a PE-conjugated VLA4 antagonist, BIO8139–PE, was used. Our previous work has indicated that in designing small molecule probes for VLA4 function, on the basis of the Leu-Asp-Val sequence from CS-1 fibronectin, the leucine moiety can be replaced with a aminohexanoylamide of lysine without significantly compromising its function (22, 23), thereby providing a target site for conjugate formation. BIO8139, a derivative of BIO7662 (24) that contains this aminohexanoyl–lysine substitution, was selected as a probe for monitoring receptor occupancy because of its high affinity for VLA4. Details on the synthesis and properties of BIO8139 will be described elsewhere (W.-C. Lee, D. Scott, and R. B. Pepinsky, unpublished data). PE-labeled BIO8139 was prepared by first reacting BIO8139 with succinimidyl-4-(N-maleimidomethyl)-cyclohexane-L-carboxylate (SMCC; Pierce, Rockford, IL) and then reacting the BIO8139–SMCC conjugate with a reduced PE pyridylsulfide derivative as follows: 200 µl of 2.5 mM BIO8139, 5 mM sulfo-SMCC (Pierce), 100 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid pH 7.5 in 90% dimethyl sulfoxide/10% water was incubated at room temperature for 4 hours. Ethanolamine was added to 20 mM and the sample was incubated further for 30 minutes. The reaction mixture was aliquoted and stored at -70°C for subsequent use. R-PE pyridylsulfide derivative (2 mg/ml, 1.8 pyridylsulfide residues/PE) was obtained from Molecular Probes (Eugene, OR). During all manipulations, steps were taken to minimize exposure of the PE to light. PE-derivative (3 ml) was incubated with 10 mM dithiothreitol for 30 minutes at room temperature and desalted on a 35 ml G25M column in 5 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.5, 100 mM NaCl. The PE elution peak was collected visually and then quantified by absorbance at 280 nm. To 3.7 ml of the PE, containing 5 µM PE, was added 20 µl of BIO8139–SMCC (10 µM final) and 0.3 ml of 0.5 M MES pH 6.5. The sample was incubated at room temperature for 2 hours. To block any unreacted sites on the PE, N-ethylmaleimide (Pierce) was added to 60 µM and the sample incubated further for 20 minutes. The sample was desalted on a 35 ml G25M column in 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid pH 7.5, 150 mM NaCl. The PE–BIO8139 conjugate, typically at a final concentration of 4 µM, was filtered through a 0.2 µm filter and stored at 4°C. Although on average, we expect that each PE conjugate will contain 1 BIO8139–PE adduct, it was not possible to directly monitor the extent of modification. Batches were analyzed for relative potency by fluorescence-activated cell sorter analysis by measuring dose-dependent staining on Jurkat cells. Conjugates stored at 4°C were stable for approximately 2 months. Losses in potency were observed with prolonged storage.

Pharmacokinetic Study in Rats: Intranasal and Intravenous Doses
Heparinized blood was collected at the designated time, and the plasma was tested for concentration of the compound by liquid chromatography-tandem mass spectrometry/mass spectrometry. Briefly, plasma samples were spiked with an internal standard. Water and acetone were added and the mixtures were applied to C18 SPE cartridges and eluted with methanol. The reconstituted extracts were analyzed by liquid chromatography-tandem mass spectrometry/mass spectrometry. Concentrations of compound were calculated from the standard curve of the compound, constructed using plasma from control rats. In the studies where compound concentration was determined in mouse plasma or BALF, the standard curve was constructed in the respective plasma or fluid of control mice.

Ovalbumin Mouse Model
The mice were immunized with 100 µg ovalbumin/alum (ovalbumin; Sigma, MO; alum: ImjectAlum, Pierce, IL) (18) on Days 0 and 14 intraperitoneally. Ovalbumin was given intranasally on Day 14 (50 µg) and Day 31 (100 µg), as described in Figure 2 .



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  		Figure 2. Protocol of immunization with ovalbumin.

 
Compound A or BIO1211was solubilized as a clear solution in 0.1 M tris(hydroxymethyl)aminomethane/0.975% lactose buffer (pH 7.4) and given on Day 31 and then for two times a day on Days 32 to 33. When compound was given intranasally in 30 µl, with an Eppendorf pipette, the mice were lightly anesthetized with ketamine/xylazine (50/10 mg/kg). Under these conditions, generally 50 to 60% of the compound was delivered to the lungs, by administrating an 125I-labeled small molecule through intranasal route (data not shown). Where anti-mouse VLA4 (PS/2) was tested, it was given similarly through the intranasal route but was only given once intraperitoneally. On Day 34, mice were bled for determination of plasma concentration of compound and receptor occupancy assays. They were then exsanguinated and killed by administering a lethal dose of ketamine. BALF was obtained, by washing the lung through the trachea 3x with 0.5 ml aliquots of PBS, and total cell number was assessed. Smears were made by Cytospin (Shandon, PA) and subsequently stained for enumeration of eosinophils, with HEMA3 (Fisher) to obtain total number of eosinophils in BALF.

Immunohistochemistry
Mice were immunized with ovalbumin and challenged as described previously. Compound and vehicle-treated mice were killed as described, and the lungs were inflated with 0.7 ml of 4% paraformaldehyde (Fisher Scientific, Fair Lawn, NJ) in 0.1 M phosphate buffer pH 7.4 infused intratracheally. The lungs were then removed and each lobe was cut into several approximate 3-mm-thick pieces. The tissue was immersed in four changes of 4% paraformaldehyde fixative at 4°C for a total of 6 hours. The tissues were then infused at +4°C with 10% sucrose in 0.1 M phosphate buffer (pH 7.2), for two 15-minute periods, 15% sucrose for four 1-hour periods, and then 15% sucrose overnight. The samples were then infused with four changes of 20% sucrose for 2 hours, and finally 1 hour with two changes of 20% sucrose plus 5% glycerol (EM Science, Gibbstown, NJ). The tissue was then embedded in optimum cutting temperature compound (Tissue-Tek, Torrance, CA), frozen in Tissue-Tek cryomolds (Elkhart, IN) using liquid nitrogen, and stored at -80°C. Immunohistochemical localization of eosinophilic major basic protein (MBP) and nitrotyrosine was performed on 5-µm frozen sections cut in a cryostat at -25°C at three levels at approximately 300 µm intervals of lung tissue. Serial sections were cut for each level, and stained for MBP with highly specific rabbit IgG raised against purified murine MBP (20, 25; kindly provided by Dr. Jamie Lee, Mayo Clinic Foundation, Scottsdale, AZ), or for nitrotyrosine with a rabbit antibody (Upstate Biotechnology, Lake Placid, NY; specificity was determined by blocking with nitrotyrosine and by immunoblotting). Initially, sections were treated with Fc blocker (Accurate Chemical, Westbury, NY) for 20 minutes, then incubated with 5% donkey serum in tris(hydroxymethyl) aminomethane/saline buffer pH 7.6; this buffer was used for all solutions below. Sections were stained for 1 hour at room temperature with 5 µg/ml or 3 µg/ml solutions (respectively) of primary antibodies or appropriate IgG controls, washed and incubated with 5 µg/ml alkaline phosphatase–conjugated affinity-purified F(ab')2 donkey anti-rabbit IgG (Jackson Labs, West Grove, PA) for 30 minutes. The slides were then stained with Vector red substrate (Vector Laboratories, Burlingame, CA) for 20 minutes, washed, dehydrated in graded alcohols, cleared in xylene and coverslips mounted with paramount (Fisher Scientific). Enumeration of labeled eosinophils was performed by light microscopy using peribronchiolar areas with a 300-µm radius (at 10x with a Leitz NPL Fluotar objective; Leitz, Wetzlar, Germany). Slides were counted in blinded fashion, and the data analyzed by Student's unpaired t test. For dual label immunofluorescence microscopy, a fluorescein isothiocyanate conjugate of rabbit anti-murine eosinophils MBP IgG was prepared with an fluorescein isothiocyanate labeling kit (Molecular Probes) according to manufacture's directions. Frozen sections of fixed mouse lungs were initially treated with blocking agents as described previously, and labeled with the anti-nitrotyrosine rabbit IgG, followed by a Cy3-conjugated affinity-purified F(ab')2 donkey anti-rabbit IgG. After blocking with 5 µg/ml rabbit IgG, double-labeling for MBP was performed on the same sections using the fluorescein isothiocyanate–conjugated rabbit anti-eosinophil MBP IgG; for orientation, the cell nuclei were counterstained with 4',6-diamino-2-phenylindole dihydrochloride. Digital images were captured with a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Inc., Thornwood, NY) equipped with a 175 W xenon light source, a cooled PCO Sensicam CCD camera, and Slidebook version 3.0 (Intelligent Imaging Innovations, Denver, CO); image processing was performed with Adobe Photoshop 5.0 (Adobe Systems,, Inc., San Jose, CA).

Statistics
Two-tailed nonpaired Student's t tests were used.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Potency and Pharmacokinetics of Compound A
Compound A is a nonpeptidyl small molecule with molecular weight of 600. It was identified by its ability to inhibit VLA4 binding on Jurkat cells, under nonactivating and activating conditions, with an inhibition concentration at 50% (IC50) of 0.1 nM. (21, Table 1) Compound A was not specific for {alpha}-4/ß-1, and it bound equally well to {alpha}-4/ß-7 (21). Binding assay in whole human blood gave an IC50 of 100 nM, using a PE-conjugated compound, BIO8139–PE, as a probe (as described in METHODS) and the IC50 in mouse blood was 26 nM. Compound A gave an IC50 of 0.95 nM in a functional proliferation assay, using anti-CD3/VCAM-1 as stimulants, modified from Burkly and coworkers (7). Compared with a previously reported peptidyl antagonist, BIO1211, compound A was 100-fold more potent in the functional assay (IC50 = 167 nM) as well as binding assay (IC50 = 2,900 nM) in whole mouse blood, partly due to less protein-binding property of compound A (12). These results are expected because BIO1211 binds to the activated form of the receptor at higher affinity (11, 12, Table 1), and being a peptide, it is easily metabolized.

To assess the pharmacokinetics (PK) properties of compound A in rat, it was solubilized in 0.1 M tris(hydroxymethyl)aminomethane buffer, with 0.95% lactose and intratracheal route was chosen to ensure consistent administration as a comparison with intravenous dosage. Rats were chosen for the PK studies, due to the technical difficulties in performing the intratracheal procedures and the continuous blood collection in mice. When compound A was dosed at 0.5 mg/kg as a solution, the PK in the rat was more prolonged when dosed intratracheally (Figure 3A) . The area under curve was 0.3 and 0.5 µM hour for intravenous and intratracheal routes, respectively, and the t1/2 (1.6–1.7 hours) remained similar. When receptor occupancy of the compound was determined with BIO8139–PE, on the blood drawn in the same experiments, it was found to extend to 8-hour after dose in the intratracheal-dosed rats, again an improvement over the intravenous dosage. (Figure 3B). Intratracheal dosage showed nearly a 100% of receptor occupancy (or inhibition of BIO8139–PE binding) for 2 hours and the receptor occupancy was significantly different from the intravenous dose at 2 and 4 hours post dose (p < 0.01 and p < 0.02, respectively), maintaining approximately 40% at 8 hours post dose, whereas that of intravenous dosage was only approximately 10% at the same time point. These PK and receptor occupancy studies indicated that compound A had PK profiles and receptor occupancy that would be suitable to test in a disease model because the receptor occupancy when dosed intratracheally appeared to be prolonged enough to be effective in vivo.



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  		Figure 3. Pharmacokinetics of compound A dosed by intratracheal or intravenous route. Compound A was dosed at 0.5 mg/kg by either the intratracheal or intravenous route in the same 0.1 M tris(hydroxyethyl) aminoethane (TRIS)/lactose vehicle. Blood was also drawn for analyses of compound concentration (A) and receptor occupancy assay (B) using BIO8139–PE fluorescenated probe as described in METHODS.

 
Effect of Compound A in Ovalbumin-induced Lung Inflammation
Compound A was then tested in the ovalbumin-induced lung inflammation in the mouse model, using the same vehicle as in the PK studies. Two routes of compound administration were used. It was first administered intranasally for 30 minutes to 1 hour before being antigenically challenged with ovalbumin. The compound was then given twice a day, 7 to 8 hours apart for 2 additional days, a total of five times (Figure 2). Animals were killed approximately 16 hours after the last dose. Blood was taken for receptor occupancy and BALF washing was done as described in METHODS.

Figure 4A shows significant dose-dependent inhibition of eosinophilia in the BALF. The percent inhibition was 56, 69, and 87 at 1, 3, and 10 mg/kg, respectively, all of which are significantly different from the vehicle control mice (p < 0.04 for 1 and 3 mg/kg and < 0.01 for 10 mg/kg). We observed good receptor occupancy for the higher 10 mg/kg dose (60%), when blood was taken 16 hours postdose (Figure 4B). To address the issue if the PBMC were covered with the compound for the lower intranasal doses, we also performed receptor occupancy assay at 7 to 8 hours after intranasal dose, the time point when we generally gave the second dose of compound during the day. We found that at 1 and 3 mg/kg, efficacious in the ovalbumin model, compound A was also found on the PBMC 7 to 8 hours post dose, as determined by the receptor occupancy assay (Figure 5) . Sixty and 80% of the cells were covered by the compound at 1 and 3 mg/kg doses, consistent with the rat PK studies shown in Figure 3B. Therefore, PMBC were at least covered with the compound for 16 hours from the twice-a-day intranasal doses, given 7 to 8 hours apart.



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  		Figure 4. Compound A inhibits accumulation of eosinophils in the bronchoalveolar lavage fluid (BALF) (A) when dosed intranasally two times a day at various mg/kg (p < 0.04). Receptor occupancy was tested in blood samples drawn before euthanasia of the mice, approximately 18 hours after the last intranasal dose (B), using BIO8139–PE. These results were obtained from three separate experiments, totaling six to 12 animals/group (p < 0.0001). Error bars represent SEM.

 


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  		Figure 5. Receptor occupancy of blood drawn at 7 hours, after intranasal dose, as determined by BIO8139–phycoerythrin (PE) binding. Values are means ± SEM of six mice/group.

 
In contrast, there was no significant inhibition when the compound was given two times a day intravenously (Figure 6A) , and there was little receptor occupancy of the compound on PBMC (Figure 6B). These results were expected on the basis of the PK study we found in the rats, where intravenous dosage gave lower plasma concentration and in particular receptor occupancy (Figure 3). To understand if intranasal dosage was creating a depot of the compound in the lungs, we examined the concentration of compound in the BALF in the same ovalbumin experiments, 16 to 18 hours after dosing. We found significant amounts of compound A in the BALF (Figure 7) , when the compound was dosed intranasally. Very little compound was found in the plasma. Such amount of compound (73 nM to 4 µM) in the BALF would be sufficient to inhibit trafficking of PBMC or eosinophils into the lung because the IC50 on PBMC was around 26 nM (Table 1) . Despite the decrease of eosinophils in the BALF, the cytokines, IL-4 or IL-5 levels (~ 100 pg/ml) in the BALF were not significantly lowered. Therefore, these data suggest that the intranasal dosage created a depot effect for the compound, and the amount detected could account for the efficacy we observed in the ovalbumin-induced eosinophilia.



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  		Figure 6. Compound A does not inhibit accumulation of eosinophils in the BALF fluid (A), when dosed intravenously two times a day at various mg/kg. Receptor occupancy was tested in blood samples drawn before euthanasia of the mice, approximately 18 hours after the last dose (B), using BIO8139–PE. Values are means ± SEM of six mice/group.

 


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  		Figure 7. Concentrations of compound A in BALF or plasma of mice dosed intranasally in a similar experiment, as presented in the ovalbumin-immunized mice in Figure 4.

 

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  		TABLE 1. Profile of compound a, BIO1211, and ANTI-VLA4 antibody in binding and functional assays

 
As a comparison to compound A, we also tested BIO1211 and anti-VLA4 Ab (PS/2) in this model, giving the compound by intranasal as well as intravenous route (Table 1). BIO1211 had very poor PK, with half-life of 5 minutes in the blood, and it was given two times a day intranasally and intravenously. PS/2 was given once intravenously on Day 0. We found that BIO1211 worked well by intranasal route, with efficacy concentration at 50% around 5 mg/kg, but we could not demonstrate any efficacy at 15 mg/kg by the intravenous route, similar to what we observed with compound A. Interestingly, anti-VLA4 Ab given at 10 mg/kg intravenously gave good (70–80%) inhibition of eosinophils accumulation in the lungs. These findings were consistent with the recent report by Kudlacz and coworkers (12), although the dosages were different in their study. Taken together, these results show that for small molecules with poor PK properties, intranasal administration could be efficacious.

Inhibition of Lung Eosinophilia by Administration of a VLA4 Antagonist
Immunohistology was used to detect the influx of eosinophils into the peribronchiolar and perivascular interstitium of ovalbumin mouse lungs 3 days after ovalbumin challenge. Lungs were fixed in situ by intratracheal infusion of fixative, cryoprocessed, and sectioned as described in METHODS. Frozen sections were labeled with an antibody that specifically recognizes murine MBP, a marker of eosinophils (20, 25). A conspicuous influx of eosinophils (detected as cells containing a dense red reaction product) occurred in the interstitium, within an approximately 300 µm radius of the sectioned bronchioles. Large continuous masses of eosinophils were situated within the extracellular matrix, beneath the bronchiolar smooth muscle layer (Figure 8A) . Similar masses of eosinophils were also observed surrounding peribronchiolar blood vessels (Figure 8C). Staining of adjacent sections with an antibody specific for nitrotyrosine indicated that the majority of these tissue eosinophils were activated (Figures 8B and 8D) (25). Indeed, double-labeling experiments in which single sections were stained with both anti-MBP and anti-nitrotyrosine IgG showed that most peribronchiolar MBP-positive eosinophils were also labeled for nitrotyrosine and therefore activated (Figure 9) . Furthermore, sections of ovalbumin-challenged mouse lungs often contained paratracheal lymph nodes exhibiting a marked influx of eosinophils in their subcapsular sinuses (Figures 8E and 8G). Similar regions of paratracheal lymph nodes from unchallenged control mice did not contain eosinophils (Figure 8F). Dense deposits of MBP were also present outside of the EOS (Figure 8G), indicating that these eosinophils had degranulated. Activated EOS and fragments of pyknotic nuclei were also detected in adjacent sections labeled with anti-nitrotyrosine IgG (Figure 8H).



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  		Figure 8. Ovalbumin-induced inflamed lungs and associated lymph nodes exhibit a large influx of activated eosinophils 72 hours after challenge. (A) and (B) Adjacent cryosections of a bronchiole (Br) stained with antibodies against eosinophil major basic protein (MBP) (A) or nitrotyrosine (B). (A) The peribronchiolar interstitium exhibits a massive influx of EOS (MBP + cells; arrowheads). (b) Corresponding arrowheads indicate that many of the peribronchiolar EOS are nitrotyrosine+. (C) and (D) Neighboring sections of a peribronchiolar blood vessel (Ve) stained with MBP (C) or nitrotyrosine (D) IgGs. (C) Arrowheads depict a significant influx of EOS in the perivascular interstitium. (D) Matching arrowheads show nitrotyrosine + labeling of the perivascular EOS. (E) Section of an adjacent paratracheal lymph node labeled with MBP antibodies. The subcapsular sinus shows an extensive accumulation of MBP+ cells; arrowheads. (F) Paratracheal lymph node section from a vehicle control mouse stained with MBP IgGs. The subcapsular sinus contains numerous lymphocytes (arrowheads), but lacks MBP+ cells. (G) Higher magnification of a portion of (E) depicts many EOS (arrowheads); MBP+ staining is also present extracellularly (asterisks). (H) Adjacent section of subcapsular sinus labeled with nitrotyrosine antibodies. Several EOS are nitrotyrosine+ (arrowheads); fragments of pyknotic nuclei are present in the extracellular space (arrows). Bar for (A–D) = 100 µm; bar for (E) = 100 µm; bar for (F–H) = 50 µm.

 


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  		Figure 9. Localization of activated eosinophils in the peribronchiolar interstitium by double label immunofluorescence microscopy of an ovalbumin-primed inflamed lung 72 hours after challenge. (A) Eosinophilic MBP staining viewed with fluorescein isothiocyanate optics. (B) Nitrotyrosine labeling detected with Cy3 fluorescence. (C) MBP and nitrotyrosine labeling viewed simultaneously by superimposition of the images from (A) and (B). Corresponding arrowheads indicate nitrotyrosine + activated eosinophils; nuclei of all cells were counterstained with DAPI (blue fluorescence). Bar = 15 µm.

 
Similarly, the peribronchiolar and perivascular interstitium of vehicle-treated control ovalbumin–challenged mice exhibited conspicuous accumulations of nitrotyrosine-positive EOS (Figures 10A and 10B) ; masses of eosinophils were also adherent to the luminal surface of the vascular endothelium (Figure 10A, inset). Widespread deposits of MBP were also observed on the basal surface of the vascular endothelium; this region was also nitrotyrosine-positive (Figures 10C and 10D). In contrast, lung sections of ovalbumin-challenged mice treated with compound A exhibited a significant inhibition of eosinophil migration into their peribronchiolar connective tissue, and their peribronchiolar blood vessels were MBP-negative (Figures 10E and 10F). Quantification of this eosinophil influx as a fraction of the peribronchiolar eosinophils observed in vehicle control mice revealed a 77% inhibition of accumulating tissue eosinophils (p < 0.05) (Figure 11) , in good agreement with the reduction of BALF eosinophils (see previous description). Similarly, there was a 58% reduction in the number of nitrotyrosine-positive eosinophils in these peribronchiolar regions (p < 0.05) (Figure 11). No significant EOS influx was detected in the peribronchiolar interstitial tissues of unchallenged ovalbumin-sensitized control mice prepared at the same time (n = 2; data not shown).



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  		Figure 10. The VLA4 antagonist, compound A, inhibits the influx of activated eosinophils into the interstitium of ovalbumin-induced inflamed lungs 72 hours after challenge. (A) and (B) Neighboring sections through the lung of a vehicle-treated ovalbumin-sensitized mouse stained with major basic protein (A) or nitrotyrosine (B) antibodies. (A) The perivascular interstitium exhibits a large influx of eosinophils (MBP+ cells; arrowheads). Inset: higher magnification of the blood vessel lumen showing MBP+ eosinophils (arrowhead) resting on the endothelium. (B) Corresponding arrowheads indicate that many of the perivascular EOS are activated (nitrotyrosine+). (C) and (D) Adjacent sections of the lung in (A) and (B) depicting an inflamed peribronchiolar blood vessel labeled with MBP (C) or nitrotyrosine (D) IgGs. (C) Arrowheads indicate deposition of MBP at the base of the vascular endothelium. Numerous perivascular MBP+ EOS are located in the perivascular connective tissue, which also exhibits MBP staining. (D) Matching arrowheads show basal nitrotyrosine+ labeling surrounding the vascular endothelium. Many perivascular eosinophils are nitrotyrosine+, but little nitrotyrosine staining is present in the extracellular matrix. (E) and (F) Adjacent sections of the peribronchiolar interstitium of compound A–treated mouse prepared 72 hours after challenge, and labeled with anti-eosinophilic MBP (E) or nitrotyrosine (F) IgGs. Matching arrowheads depict a significant inhibition of activated eosinophil influx. Bar for (A) (B) (E) and (F) = 100 µm; bar for inset in (A) = 25 µm; bar for (C) and (D) = 50 µm.

 


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  		Figure 11. Effects of vehicle (open bars) and compound A (cross-hatched bars) on the accumulation of peribronchiolar tissue eosinophils and activated eosinophils (nitrotyrosine + eosinophils) 72 hours after intranasal challenge with ovalbumin. Lungs of vehicle or compound A–treated mice were fixed in situ, sectioned at three different levels, and labeled immunohistochemically as described in METHODS. Numbers of MBP-labeled and nitrotyrosine (NT)-positive eosinophils in the peribronchiolar and perivascular interstitium of each sectioned bronchiole were determined and expressed as the mean ± SEM (n = 7 for both vehicle- and compound-treated mice).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Compound A represents the first instance where a nonpeptidyl small molecule, acting on the activated as well as the constitutive or nonactivated form of VLA4, inhibited antigen-induced lung inflammation. The reactivity of compound A on VLA4 is similar to that of a monoclonal Ab, such as PS2, which binds to VLA4, regardless of its activation state. The PK of the compound was not impressive, with fast clearance rate, however, it had prolonged off rate on PBMC cells, as shown by the receptor occupancy assay, in the PK study (Figure 3). On administrating it through the intranasal route, compound A showed remarkable efficacy, inhibiting the infiltration of eosinophils into the lungs in this model. The inhibition was dose dependent, correlating with the respective receptor occupancy, when determined at 7 to 8 hours post dose. The receptor occupancy data suggest that for this compound, when dosed twice a day, coverage of 16 hours seemed sufficient for preventing the accumulation of EOS in the lungs. Therefore, 24 hours coverage of the compound for PBMC appeared not necessary for efficacy in this model.

The lack of efficacy when compound A was given intravenously confirmed earlier data reported by Henderson and coworkers (18), who advocated the regional effect of intranasal administration of anti-VLA4 Ab, PS2, in a similar ovalbumin mouse model. When they compared the systemic intraperitoneal administration to the intranasal route, they found better inhibition of lung inflammation and airway hyper-responsiveness in the intranasal route. However, in contrast to Henderson's study (18), we did not find attenuation of IL-4 or IL-5 in the BALF of mice treated with compound (data not shown), and also we observed efficacy when PS2 was given intravenously (Table 1). Nonetheless, when the concentration of compound A was determined in the BALF and plasma, it correlated well with the efficacy of administrating compound intranasally. The amount of compound found in the BALF far exceeded the IC50 of compound A, when tested on the mouse PBMC. It is not clear if the efficacy we observed with intranasal administration was due to local concentration of compound A in the lung, such that PMBC or EOS were thus prevented from trafficking into the lung per se. Alternatively, intranasal route of administration could have created a depot effect in the lung (Figure 6), so that the compound was perhaps slowly released into the parenchyma and blood stream, covering the PBMC and preventing the extravasation of cells from the blood to cause accumulation of EOS in the lung. In part, this phenomenon was reflected in the receptor occupancy we observed in the dose range (1–10 mg/kg) where we found efficacy. Similarly, BIO1211 in our hands was also more efficacious when given intranasally (Table 1), rather than intravenously, although Abraham and coworkers (11) found that it worked by either route of administration in the sheep. Therefore, intranasal administration of an inhibitor appeared advantageous in this model, suggesting that aerosol administration of a VLA4 antagonist would be beneficial in treatment of lung inflammation in the clinic.

Our results correlated well with a recent report of VLA4 antagonist, a peptidyl mimetic, acting on the activated form of the receptor (12) in a similar mouse model of ovalbumin-induced inflammation. The compound was administered both by intratracheal and subcutaneous routes, and efficacy was found in both methods of administration, although 10-fold less potent when given subcutaneously. IC50 of the compound was approximately 5 nM in serum, approximately 5x more potent than compound A; however, it was not clear what the potency was on the nonactivated form of VLA4. It was interesting that these investigators also did not find any inhibition in the cytokines in the BALF, as we observed. BIO1211 was also tested in their ovalbumin system but it was found to be less efficacious than what we observed, although the dosage was lower. In our study, BIO1211 at 15 mg/kg two times a day intranasally gave 80% inhibition, and Kudlacz and coworkers tested it at 200 µg, (~ 10 mg/kg every day) giving 20% inhibition (12). Judging from the efficacy of BIO1211, at approximately 5mg/kg in this study, the exposure of lungs to a compound binding to the activated form of the receptor locally was sufficient to attenuate lung inflammation. Similarly, when 75 µg anti-VLA4 antibody was given intranasally in their study, 40% inhibition was observed, whereas 60% inhibition was obtained when Ab was given at approximately 20 to 25 mg/kg (500 µg) intraperitoneally. In general, our findings agreed with their observation, but different from that reported in the sheep, where both intratracheal and intravenous route of BIO1211 were efficacious (11). Perhaps the inflammation and pathophysiology are different between the sheep and mice.

The fact that BIO1211 and compound A both inhibited EOS infiltration suggested that even though they are 100 times different in potency in binding to nonactivated form of the receptor, they may be similar in binding (efficacy) when cells are activated in the lungs. BIO1211 had very poor PK, and it gave no receptor occupancy in PBMC when inoculated intranasally (data not shown). Therefore, in this instance, BIO1211would work primarily in the lungs. In the other scenario, anti-VLA4 Ab, PS/2 when given intravenously, had little accumulation in the lungs (data not shown), yet due to its tight binding and long off-rate (t1/2 ~ 4–5 days), would prevent extravasation or activation of EOS in the blood or systemically. Then, compound A would, in fact, worked both in the lungs and the blood because we were detecting receptor occupancy in the blood when compound A was given intranasally at 1 to 10 mg/kg (Figures 4B and 5). These features may explain that compound A was the best in efficacy among the three agents.

Immunohistology of the lung further characterized the pathology of lung inflammation in this model. Previous studies have implicated EOS as effectors of asthmatic pathology and airway hyperresponsiveness mediated by the release of cytotoxic granular components from activated cells (20, 26–28). We observed high levels of EOS in both peribronchiolar tissues and in BALF 72 hours after ovalbumin challenge. These data closely agree with the work of Tomkinson and coworkers (20), who found peaks of EOS in both peribronchiolar tissues and BALF that correlated with airway hyperresponsiveness in a similar murine model of asthma. Activated EOS usually secrete their granular contents and produce a respiratory burst, generating superoxide, hydrogen peroxide, and nitric oxide that can damage surrounding tissues (29–32). Nitric oxide can react with superoxide yielding peroxynitrite, an oxidizing agent that induces lipid peroxidation, sulfhydryl oxidation of proteins, and nitration of aromatic amino acids. Accumulation of nitrotyrosine (nitrotyrosine), a stable product of the addition of a nitro group to the benzene ring of tyrosine by peroxynitrite (30) is thus a stable marker of pathology within cells and tissue lesions. The finding of high concentrations of nitrotyrosine in peribronchiolar EOS strongly suggests that they have been activated, and are thus capable of damaging tissues leading to airway hyperresponsiveness. Our observation of (1) the deposition of MBP and nitrotyrosine on peribronchiolar vascular basement membranes and connective tissue and (2) the presence of pycnotic nuclear fragments in lymph nodes draining inflamed lungs, supports this notion, and strongly suggests that the activated EOS have degranulated. These observations agree with several reports demonstrating that EOS express inducible nitric oxide messenger RNA, protein, and liberate nitric oxide (33), and that pulmonary inflammation in allergic mice is partly dependent on nitric oxide induced by EOS inducible nitric oxide (34, 35). Furthermore, numerous activated EOS in the subcapsular sinuses of paratracheal lymph node in ovalbumin-challenged mice support the recent finding that EOS probably modulate allergic inflammatory responses by stimulating Th2 cells (36). It is intriguing that compound A could inhibit degranulation of EOS, suggesting that a VLA4 antagonist can inhibit activation event of EOS (11). It is possible that engagement of VLA4 with VCAM-1 and another activation signal on EOS triggers the degranulation, similar to T cell activation with a-CD3 and VCAM-1. It would be interesting to pursue this issue in future studies.

Two earlier studies have similarly documented the influx of EOS into the peribronchiolar tissues of sensitized mice challenged with ovalbumin, but in contrast to our observations, they did not detect deposition of MBP in the extracellular connective tissue using immunohistochemistry (20, 37). This discrepancy is probably due to differences in the immunohistochemical techniques used in these experiments. The latter studies used the paraffin method to embed their tissues. This technique subjects the processed tissues to long periods of extraction with strong organic solvents, and heating in molten wax. The tissue sections were also subjected to incubation in trypsin to "unmask" MBP epitopes. The combination of these steps probably extracted extracellular MBP from the lung tissue. Alternatively, we used mildly fixed frozen tissues that were not subjected to enzyme digestion, and extracellular MBP was readily detectable.

The treatment with compound A resulted in a clear and significant decrease of EOS in the lungs. The nitrotyrosine (+) EOS were equally affected in the treated mice. Interestingly, there was no significant change in the activated goblet cells in the treated lungs, suggesting that this pathologic condition is not mediated by VLA4 in this acute model. In a recent study, Tomkinson and coworkers (20), using a single administration of ovalbumin at the time of challenge as we did, found that anti-VLA4 administered systemically also did not affect the number of goblet cells in the inflamed lungs. However, they observed both a decrease in EOS and airway responsiveness, with 10 mg/kg dose of the Ab, again similar to our findings with compound A. In both of our studies, the BALF cellular components substantiated the findings in the tissue EOS. In our experience with this ovalbumin model, dexamethasone is the only compound that attenuated the pathology of the goblet cells (data not shown). These results reinforce the notion that airway remodeling is a complex event, and inhibition of EOS accumulation could be independent of changes in mucus/goblet cells.

The role of EOS in human asthma and lung inflammation remains controversial (38). Whereas the failure of anti–IL-5 treatment in human asthma suggests that some attenuation of EOS did not impact the airway resistance, IL-5 probably is not the only mediator involved in EOS accumulation. In a recent study by Mattes and coworkers (39) in murine systems, abrogation of airway hyper-responsiveness and EOS occurs only in mice deficient of both IL-5 and eotaxin. On the contrary, anti–IL-5 or IL-13 alone did not result in sufficient depletion of EOS in the lung tissue. Therefore, the role of EOS in asthma is becoming more convincing, even in human asthmatic population, where the measurement of inflammatory mediators of EOS in the blood appears to correlate with FEV (40). These recent findings support our contention that activated EOS likely contributes to the pathology in the inflamed lung.

In conclusion, we presented data showing that (1) a nonpeptidyl small molecule can inhibit protein/protein interaction in VLA4 integrin system in a disease model, as shown previously in the {alpha}IIb/ß3 (41) and LFA-1 systems (42); (2) compound A interferes with the nonactivated as well as the activated state of the {alpha}4 integrin, and this interference can significantly inhibit an antigen-induced lung eosinophilia; (3) local administration of a compound can inhibit EOS accumulation, better than systemic administration. For certain compounds, such as compound A, intranasal administration appears to create a depot, such that a compound with poor PK can be efficacious; (4) immunohistologic analyses validated the BALF assessment of inflammation in the allergic mouse lung, and demonstrated that the numerous EOS, accumulated in peribronchiolar tissues, appeared activated and had degranulated, likely inducing tissue pathology.


   Acknowledgments
 
The authors thank Comparative Medicine for performing the in vivo phase of the PK studies, Dr. Phil Davies for critical review of the manuscript, and Drs. Jack Schmidt and Martin Springer for valuable discussions and support.

Received in original form July 12, 2003; accepted in final form January 24, 2003


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