Inhibition of Invariant Chain Processing, Antigen-Induced Proliferative Responses, and the Development of Collagen-Induced Arthritis and Experimental Autoimmune Encephalomyelitis by a Small Molecule Cysteine Protease Inhibitor

Inhibitors

5-(2-Morpholin-4-ylethoxy)benzofuran-2-carboxylic Acid ((S)-3-Methyl-1-{(S)-3-oxo-1-[2-(3-pyridin-2-ylphenyl)-acetyl]azepan-4-ylcarbamoyl}butyl)amide (SB-331750) and N-[1S-(2-phenylethyl)-3-phenylsulfonylallyl]-4-methyl-2R-piperazinyl carbonylaminovaleramide (APC3328) were synthesized at GlaxoSmithKline by the Department of Medicinal Chemistry, Microbial, Musculoskeletal, and Proliferative Diseases Center of Excellence for Drug Discovery, as previously described (24, 25, 26). Leupeptin was purchased from Sigma-Aldrich.

In vitro enzyme assays

Cathepsin activity assays were performed in microtiter plates using fluorogenic substrates at concentrations equal to their Km in assay buffers optimized for each enzyme. The human and mouse cathepsin S assays consisted of 1.25 nM human and mouse cathepsin S, respectively, 30 μM Z-Lys-Gln-Lys-Leu-Arg-AMC in 50 mM MES, 10 mM l-cysteine, 5 mM EDTA, and 0.5 mM CHAPS (pH 6.5). The human cathepsin L and V assays consisted of 100 pM human cathepsin L and V, respectively, 8.5 μM Z-Phe-Arg-AMC in 100 mM sodium acetate, 5 mM l-cysteine, and 5 mM EDTA (pH 5.5). The human cathepsin F assay consisted of 0.4 nM human cathepsin F, 5 μM Z-Phe-Arg-AMC in 50 mM potassium phosphate, 2.5 mM 1,4-dithioerythritol, 2.5 mM EDTA, and 0.6 mM CHAPS (pH 6.5). The human cathepsin K assay consisted of 0.1 nM human cathepsin K, 30 μM Z-Lys-Gln-Lys-Leu-Arg-AMC in 100 mM sodium acetate, 20 mM l-cysteine, and 1 mM CHAPS (pH 5.5). The human cathepsin C assay consisted of 0.010 nM human cathepsin C, 5 μM (H-Gly-Arg)₂-R110 in 50 mM sodium acetate, 30 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.2 mM CHAPS (pH 5.5). The mouse cathepsin L assay consisted of 100 pM mouse cathepsin L, 5 μM Z-Phe-Arg-AMC in 100 mM sodium acetate, 20 mM cysteine, and 5 mM EDTA (pH 5.5). The mouse cathepsin K assay consisted of 1 nM mouse cathepsin K, 20 μM Z-F-R-AFC in 100 mM sodium acetate, 4 mM l-cysteine, 5 mM EDTA, and 1 mM CHAPS (pH 5.5). The mouse cathepsin C assay consisted of 0.015 nM mouse cathepsin C, 5 μM (H-Gly-Arg)₂-R110 in 50 mM sodium acetate, 30 mM NaCl, 1 mM DTT, 1 mM EDTA, and 1 mM CHAPS (pH 5.5). All reactions were initiated by the addition of enzyme-containing assay buffer to the substrate and inhibitor sample and were read after 1 h of incubation at 25°C. Hydrolysis products of the fluorogenic peptide substrates were measured using an Analyst AD microtiter plate reader (Molecular Devices) under the following conditions: for AMC substrates, excitation at 360 nm and fluorescence emission at 460 nm using a 400 nm dichroic filter; for AFC substrates, excitation at 405 nm and fluorescence emission at 530 nm using a 505 nm dichroic filter; and for R110 substrates, excitation at 485 nm and fluorescence emission at 535 nm using a 505 nm dichroic filter. Compound IC₅₀ values were determined from dose-response curves, which were performed using a starting compound concentration of 25 μM that was serially diluted 3-fold over 11 repetitions using the following equation: In the equation above, x is the log concentration of the test compound, y is the fluorescence response, min is the minimum response plateau, and max is the maximum response plateau. K_i,app were calculated by dividing the IC₅₀ values by two, as the assays were run at substrate concentrations equal to Km, and the inhibition was assumed to be competitive.

Mice

DBA/1 mice were obtained from Harlan U.K., and SJL/J mice were obtained from Harlan France S.A.R.L. or from Taconic. C57BL/6 (B6) mice were obtained from The Jackson Laboratory. Cathepsin K^−/− mice and K^+/+ littermate controls were bred at the GlaxoSmithKline animal facility. Generation of these mice has been previously described (27). All mice were maintained on a 12 h:12 h light:dark cycle and fed standard rodent chow and water ad libitum. All procedures performed on animals were reviewed and approved by GlaxoSmithKline Institutional Animal Care and Use Committee.

Mouse splenocyte culture

Mice were euthanized by CO₂ asphyxiation and each spleen placed in 3 ml of sterile HBSS (Mediatech) containing 1 mM EGTA. Spleens were mechanically disrupted with forceps and pipetting before being passed through sterile 70-μm filters. Cells were centrifuged at 600 × g for 5 min, and RBC were lysed by resuspension of the pellet in 1× M-Lyse buffer (R&D Systems) at 2 ml/spleen for 10 min followed by addition of 1× wash buffer (R&D Systems) at 8 ml/spleen. Cells were centrifuged as above, washed in 10 ml of medium (RPMI 1640 containing 2 mM l-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mM HEPES, 1 mM of sodium pyruvate, 90%, and 10% heat inactivated FBS (HyClone), and counted. Cells were cultured at 1 × 10⁶ cells/ml of medium at 37°C in 5% CO₂ on a shaking platform for 4–5 h. Where indicated, cultures were treated with SB-331750, leupeptin, APC3328, or vehicle (0.1% DMSO). Following incubation cells were examined for toxicity by trypan blue exclusion and then processed for immunoprecipitation/Western blot analysis or for flow cytometry.

Raji cell culture

The human B cell line, Raji, was purchased from American Type Culture Collection. Cells were cultured at 5 × 10⁵ cells/ml of medium (RPMI 1640 containing 2 mM l-glutamine, 1.5 g/liter sodium bicarbonate, 4.5 g/liter glucose, 10 mM HEPES, 1 mM sodium pyruvate, 90%, and 10% heat inactivated FBS) at 37°C in 5% CO₂ for 24 h. Where indicated, cultures were treated with SB-331750, leupeptin, or vehicle (0.1% DMSO), and then processed for immunoprecipitation/Western blot analysis or for flow cytometry.

Assessment of intracellular Ii processing intermediates

An aliquot of each splenocyte culture described above was centrifuged at 600 × g for 5 min, the pellets washed with 10 ml of cold Dulbecco’s PBS (DPBS), and each pellet resuspended in ice cold lysis buffer (10 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5 mM PMSF, and 1 protease inhibitor minitab (Roche)) at 5–8.5 × 10⁷ cells/ml, and incubated on a tumbler for 30 min at 4°C. The lysates were then clarified at 12,000 × g and the supernatants collected and stored at −80°C. A protein assay was performed on each sample using the Bio-Rad protein assay (Bio-Rad).

Alternatively, an aliquot of each Raji cell culture described above was centrifuged at 400 × g for 5 min, the pellets washed with 10 ml of cold DPBS, and each pellet resuspended in ice cold lysis buffer at 15–30 × 10⁶ cells/ml. Suspensions were incubated on a tumbler for 30 min at 4°C, the lysates clarified at 12,000 × g, and the supernatants collected and stored at −80°C. A protein assay was performed on each sample using the Bio-Rad protein assay.

For assessment of Ii processing intermediates in whole cell lysates, an equal amount of protein from each sample (2 μg for C57BL/6, DBA/1, and SJL/J samples, and 2–20 μg for Raji samples) was diluted in an equal volume of loading buffer (62.5 mM Tris-HCl (pH 6.8), 25% glycerol, 4% SDS, 0.01% bromophenol blue, and 5% 2-ME), and the samples placed in boiling water for 5 min. Samples were then loaded onto an 18 or 4–20% Tris-HCl minigel (Bio-Rad). To assess class II-associated Ii processing intermediates, equal amounts of total protein from each sample (150 μg for mouse splenocyte lysates or 50 μg for Raji cell lysates) were diluted to a final volume of 300 μl (splenocytes) or 200 μl (Raji cells) in lysis buffer. The samples were precleared by adding isotype control and Protein A/G PLUS-Agarose (Santa Cruz Biotechnology) and using the following reagents: 3 μg of normal mouse IgG (Santa Cruz Biotechnology) for DBA/1 and SJL/J samples, 3 μg of mouse IgG2a (BioLegend) for C57BL/6 samples, and 1 μg of normal mouse IgG (Santa Cruz Biotechnology) for Raji samples. All mouse samples received 30 μl of Protein A/G PLUS-Agarose, whereas Raji samples received 20 μl. The samples were then tumbled for 1 h at 4°C, centrifuged at 1000 × g for 5 min, and supernatants collected. To C57BL/6 supernatants, 3 μg of an anti-mouse I-A^b mAb (AF6–120.1; BioLegend) or 3 μg of the above mouse IgG2a, was added; to DBA/1 and SJL/J supernatants, 3 μg anti-I-A^q/I-A^s mAb (KH116; Santa Cruz Biotechnology) or 3 μg of the above normal mouse IgG, was added; and to Raji supernatants, 1 μg an anti-HLA-DR mAb (TAL 1B5, Santa Cruz Biotechnology), or 1 μg of the above normal mouse IgG, was added. Samples were incubated overnight with tumbling at 4°C. Protein A/G PLUS-Agarose (30 μl to mouse samples and 20 μl to Raji samples) was added, and samples tumbled at 4°C for 2 h. Samples were then pelleted as above and the pellets washed three times with 300 μl of lysis buffer. The samples were resuspended in 30 μl of loading buffer, placed in boiling water for 5 min, and then loaded onto an 18 or 4–20% Tris-HCl minigel (Bio-Rad).

Proteins were resolved in 1× running buffer (25 mM Tris (pH 8.3), 192 mM glycine, and 0.1% SDS (Bio-Rad)) at 126 V until the dye front reached the bottom of the gel, and were transferred to polyvinylidene difluoride membranes (Bio-Rad) in 1× transfer buffer (25 mM Tris (pH 8.3), and 192 nM glycine (Bio-Rad) containing 20% methanol) at 100 V for 1 h. The membranes were rinsed with Membrane Blocking Solution (Zymed Laboartories) and then blocked with Membrane Blocking Solution for 1 h at room temperature with gentle shaking. Membranes containing mouse splenocyte proteins were incubated with an anti-mouse CD74 (Ii) mAb, In-1 (BD Pharmingen) diluted 1/1000 in Membrane Blocking Buffer; membranes containing human Raji cell proteins were incubated with an anti-human CD74 (Ii) mAb, PIN.1 (Santa Cruz Biotechnology) diluted 1/200 in Membrane Blocking Buffer. Following incubation with primary mAbs overnight at 4°C with gentle shaking, the membranes were washed with 1× washing buffer (TBS (Bio-Rad) containing 0.05% Tween 20) by quickly rinsing two times followed by three 5 min washes with vigorous shaking. Membranes were then incubated with an HRP goat anti-rat IgG (Zymed Laboratories) diluted 1/5000 in Membrane Blocking Buffer, or with an HRP goat anti-mouse IgG (Santa Cruz Biotechnology) diluted 1/3000 in membrane blocking buffer, respectively, for 75 min at room temperature with gentle shaking. The membranes were washed as described following incubation with the primary Ab and then washed in TBS (Bio-Rad) by quickly rinsing two times followed by one 5-min wash with vigorous shaking. Membranes were treated with Luminol reagent (Santa Cruz Biotechnology) for 1 min and then exposed to film.

Assessment of cell surface class II-CLIP and total class II expression

An aliquot of each splenocyte culture described above was centrifuged at 600 × g for 5 min at 4°C, the pellets washed with 3 ml of cold Ab diluent (DPBS, 1% FBS, and 0.1% azide), and each pellet resuspended at 10 × 10⁶ cells/ml in cold Ab diluent containing 10 μg/ml of an anti-mouse CD16/CD32 mAb (2.4G2; BD Pharmingen) to block FcR binding. Following incubation for 5 min at 4°C, 100 μl aliquots of each sample (1 × 10⁶ cells) were transferred to 96-well round-bottom plates and Abs added at the following amounts/10⁶ cells: 0.5 μg of PE anti-mouse CD19 (1D3) or 0.5 μg of the isotype control PE rat IgG2a (R35–95), purchased from BD Pharmingen; 20 μl (4 μg) of FITC anti-I-A^b/CLIP (15G4) or 20 μl of the isotype control FITC mouse IgG1, purchased from Santa Cruz Biotechnology; 1 μg of FITC anti-H-2^q/H-2^b (2G9) or 1 μg of the isotype control FITC rat IgG2a (R35–95), purchased from BD Pharmingen; and 0.5 μg of Alexa Fluor 488 anti-I-A^q/I-A^s (KH116) or 0.5 μg of the isotype control Alexa Fluor 488 mouse IgG2b, purchased from BioLegend. Samples were placed on an orbital shaker for 30 min at 4°C, and then 100 μl of Ab diluent added and the cells pelleted as described above. Samples were washed twice with 200 μl of Ab diluent and then resuspended in 500 μl of Ab diluent for flow cytometric analysis. Before analysis, 20 μl of Via-Probe (BD Pharmingen) was added to allow exclusion of dead cells from the analysis.

Alternatively, an aliquot of each Raji cell culture described above was centrifuged at 400 × g for 5 min, the pellets washed with 3 ml of cold Ab diluent, and each pellet resuspended at 5–10 × 10⁶ cells/ml in cold Ab diluent. The 100 μl aliquots of each sample (0.5–1 × 10⁶ cells) were transferred to 96-well round-bottom plates and Abs added at the following amounts: 10 μl of FITC anti-human MHC class II/CLIP (CerCLIP) or 10 μl of the isotype control FITC mouse IgG1 (MOPC-21), purchased from BD Pharmingen; and 10 μl of FITC anti-HLA-DR (G46-6) or 10 μl of the isotype control FITC mouse IgG2a (G155–178), purchased from BD Pharmingen. Samples were placed on an orbital shaker for 30 min at 4°C, and then 100 μl of Ab diluent added and the cells pelleted as described above. Samples were washed twice with 200 μl of Ab diluent and then resuspended in 500 μl of Ab diluent for flow cytometric analysis. Before analysis propidium iodide (Invitrogen) was added at 20 μg/ml, to allow exclusion of dead cells from the analysis.

Events were collected on an LSR or FACSCantos flow cytometer (BD Pharmingen) using CellQuest or FACSDiva software, respectively, with individual unlabeled and one-color samples being prepared to set color compensation. All data were analyzed using CellQuest software (the FACSDiva-generated data was converted before analysis) gating on live cells, and using the geometric mean fluorescence 1 content as a direct assessment of class II/CLIP or total class II expression. The percent inhibition values of class II/CLIP or total class II expression by SB-331750- or leupeptin-treated cells, relative to vehicle (0.1% DMSO)-treated cells, were calculated following subtraction of the geometric mean fluorescence 1 content obtained with relevant isotype control mAb.

Induction and assessment of CIA

On day 0, 12–24-wk-old male DBA/1 mice were immunized intradermally at the base of the tail with a total of 100 μl of IFA (Sigma-Aldrich) containing 200 μg of bovine type II collagen (Elastin Products) and 250 μg of Mycobacterium tuberculosis H37Ra (Difco Laboratories). On day 21, mice were boosted intradermally with 100 μl of PBS containing 200 μg of bovine type II collagen. For prophylactic studies, SB-331750 (50 mg/kg) or vehicle (200 μl PBS) was administered s.c., twice a day (b.i.d.), from days 1 to 40. For therapeutic studies, administration of SB-331750 or vehicle, as described above, was initiated (day 1 of treatment) once an animal exhibited a clinical score of “1” or greater for 2 consecutive days (days 0 and 1 of treatment). Mice were scored daily for clinical symptoms of disease using a micrometer caliper to measure paw thickness. Each paw was assigned a score ranging from 0 to 4 based on the following criteria: 0, asymptomatic (paw thickness = 1.8–1.9 mm and no swollen digits); 1, paw thickness = 1.8–1.9 mm and one or more swollen digits; 2, paw thickness = 2.0–2.5 mm and one or more swollen digits; 3, paw thickness = 2.6–3.0 mm and one or more swollen digits; and 4, paw thickness = 3.0+ mm and one or more swollen digits. For each animal, the clinical score for all paws was summed, giving a potential score of 0–16.

In addition to the mice that were scored throughout the experiment (n = 12 per treatment group), on days 28 and 41, mice (n = 6 per treatment group per time point) were removed from the experiment and used to measure paw tissue cytokine levels. These mice were scored on a daily basis until their removal from the study and the data integrated into the analysis using the log rank test.

Quantitative analysis of blood SB-331750 levels

Analysis of mouse blood samples (25 μl of mouse blood diluted with 25 μl of water) for SB-331750 was performed using liquid chromatography/tandem mass spectrometric detection. Analytical standards were prepared, and the compound was isolated from the samples and standards by protein precipitation with acetonitrile containing a mass spectral internal standard and the resulting mixture was vortex-mixed followed by centrifugation. The resulting supernatant was subjected to chromatographic separation on a Luna C18 column under isocratic conditions (30/70 - 10 mM ammonium acetate and acetonitrile mobile phases at a 300 μl/min flow rate). The eluent flowed into a Sciex API365 triple-quadrupole mass spectrometer (Applied Biosystems) using positive-ion electrospray multiple-reaction monitoring set to monitor for the (M+H) plus precursor to product ion transition.

Data were reported as quantitative concentrations as determined by standard calibration curve analysis, using linear fitting of a 1/x-weighted plot of the SB-331750/internal standard peak area ratios vs SB-331750 concentration, with a lower limit of quantification of 10 ng/ml.

Paw tissue cytokine analysis

Mice removed on days 28 (n = 6) and 41 (n = 6) of the CIA study, as well as those scored through day 50 (n = 12), were euthanized on the indicated day by CO₂ asphyxiation. The four paws from each mouse were collected and paws from two mice combined randomly within each treatment group, resulting in an n = 3, 3, and 6 for days 28, 41, and 50, respectively. The tissue was weighed, flash frozen in liquid nitrogen, ground using a mortar and pestle, and then homogenized for 1–2 min with a Polytron homogenizer (model PT 10/35; Brinkmann Instruments) in PBS at 1 ml/gram tissue. Large debris was removed by centrifugation at 4700 × g. Supernatants were decanted and cleared with an additional centrifugation at 14000 × g, and the resulting supernatants frozen at −80°C until use. Cytokine levels were analyzed using mouse IL-1β, TNF-α, and IFN-γ ELISA kits purchased from R&D Systems. Assays were performed according to the manufacturer’s instructions.

Induction and assessment of EAE

For induction of EAE by adoptive transfer, on day 0, 12–16-wk-old female SJL/J mice were immunized s.c. at the base of the tail with a total of 100 μl of IFA (Sigma-Aldrich) containing 400 μg of bovine myelin basic protein (MBP) (Sigma-Aldrich) and 50 μg of M. tuberculosis H37Ra (Difco Laboratories). On day 7, mice were boosted s.c. in the flank with 100 μl of IFA containing 400 μg of bovine MBP and 50 μg of M. tuberculosis H37Ra. On day 17 postimmunization, inguinal lymph nodes were collected and single-cell suspensions prepared as described below. A total of 2 × 10⁶ cells/ml were cultured with 50 μg/ml of bovine MBP in a volume of 70 ml of RPMI 1640 containing 10% FBS and gentamicin (100 μg/ml), in the presence of vehicle (0.1% DMSO) or SB-331750 (10 μM). Following incubation for 72 h at 37°C in 5% CO₂, cells were washed twice in PBS and counted, yielding 8.61 × 10⁵ cells/ml (DMSO-treated) and 6.31 × 10⁵ cells/ml (SB-331750-treated). From each treatment group, 3 × 10⁷ cells in 0.5 ml of PBS were transferred by i.v. injection to naive female SJL/J recipients (n = 8–9 per treatment group).

For active induction of EAE, on day 0, 12–16-wk-old female SJL/J mice were immunized s.c. in the right flank with a total of 100 μl of IFA (Sigma-Aldrich) containing 400 μg of bovine MBP (Sigma-Aldrich) and 50 μg of M. tuberculosis H37Ra (Difco Laboratories). On days 0 and 2, mice received an i.v. injection of 500 ng of perussis toxin (List Biological Laboratories) in 200 μl of PBS. Therapeutic administration of vehicle (PBS) or SB-331750 (50 mg/kg, s.c., b.i.d) was initiated on the first day of exhibition of clinical symptoms (day 1 of treatment) and continued through day 14 (n = 4–5 mice per treatment group).

Mice were scored daily for clinical symptoms of disease based on the following criteria: 0, asymptomatic; 0.5, distal limp tail; 1.0, complete limp tail; 1.5, complete limp tail and unsteady gait; 2.0, paralysis of one hind limb; 2.5, partial paralysis of both hind limbs; 3.0, complete bilateral hind limb paralysis; 3.5, complete bilateral hind limb paralysis with forelimb weakness; and 4.0, moribund.

Ag- or anti-CD3 mAb-induced proliferation by mouse lymph node cells (LNC)/splenocytes

For measurement of Ag-induced proliferation, inguinal lymph nodes and spleens from collagen-immunized/boosted DBA/1 mice, and from MBP-immunized/boosted SJL/J mice (immunizations and boosts performed as described above), were collected 24 and 17 days, respectively, postimmunization. Preparation and culture of LNC and splenocytes were performed under sterile conditions. Lymph nodes and spleens were rinsed in HBSS containing penicillin (100 units/ml)-streptomycin (100 μg/ml) (Invitrogen) or gentamicin (50 μg/ml) (Sigma-Aldrich), (HBSS+), teased apart in 5 ml of HBSS+, and filtered through 50-μm filters. Samples were centrifuged at 500 × g for 10 min at 4°C, and the resulting LNC pellets resuspended in 2 ml HBSS+. RBC within splenocyte pellets were lysed, and following centrifugation as above, splenocyte pellets were resuspended in 2 ml of HBSS+. LNC and splenocytes were counted, and cells from three mice combined at a ratio of 80% LNC/20% splenocytes (each group of three mice considered an n = 1). A total of 1 × 10⁶ cells/ml were cultured in a volume of 200 μl of RPMI 1640 containing 10% FBS, and penicillin (100 units/ml)-streptomycin (100 μg/ml) or gentamicin (50 μg/ml), in the absence or presence of the indicated Ag (bovine type II collagen (100 μg/ml)) or bovine MBP (50 μg/ml), and to the Ag-stimulated LNC/splenocytes cultures, SB-331750 was added to a final concentration of 0 (0.1% DMSO vehicle), 0.3, 1, 3, or 10 μM. Following incubation for 72 h at 37°C in 5% CO₂, 1 μCi [³H]thymidine was added to each well, and the cells cultured for an additional 24 h. Cell viability was confirmed by trypan blue exclusion. Cultures were harvested using a Packard Filtermate 196 (Packard Instrument), and radioactivity was quantified using a Packard TopCount liquid scintillation counter.

For measurement of anti-CD3 mAb-induced proliferation, splenocytes from naive DBA/1 and SJL/J mice were prepared as described above. A total of 2 × 10⁶ cells/ml were cultured in a volume of 100 μl of RPMI 1640 containing 10% FBS, penicillin (100 units/ml)-streptomycin (100 μg/ml), and gentamicin (50 μg/ml), in the absence or presence of 5 μg/ml of plate-coated anti-mouse CD3 mAb (clone 500A2; Caltag Laboratories), and to the anti-CD3 mAb-stimulated splenocytes cultures, SB-331750 was added to a final concentration of 0 (0.1% DMSO vehicle), 0.3, 1, 3, or 10 μM. Following incubation for 72 h at 37°C in 5% CO₂, 10 μl of 100 μM BrdU labeling solution (BrdU Cell Proliferation ELISA; Roche Diagnostic Systems) was added to each well, and, after an additional 24 h of incubation, cultures were processed according to the manufacturer’s instructions and chemiluminescence measured using a Wallac Envision 2102 Multilabel Reader.

Statistical analysis

Statistical differences in cytokine levels were determined using the two-tailed Student’s t test. Analysis of CIA and EAE disease severity was performed by calculating, for each animal within a treatment group, the area under the curve (AUC) using the trapezoidal rule, and then using either the Student’s t test (for uncensored data), or the log rank test, which is a nonparametric test that allows for right censored observations (i.e., for any animal removed before the study’s completion, the animal’s complete AUC is considered to be at least as large as the partial AUC exhibited). In cases where scores for some days were missing in the interior of the animal’s time profile, score values were interpolated over time. For CIA and EAE studies, day of disease onset was defined as the first of two consecutive days on which an animal exhibited a clinical score > 0. Analysis of disease onset data was performed using the log rank test. Values of p < 0.05 were considered statistically significant.

Structure and characteristics of SB-331750

SB-331750, 5-(2-Morpholin-4-ylethoxy)benzofuran-2-carboxylic Acid ((S)-3-Methyl-1-{(S)-3-oxo-1-[2-(3-pyridin-2-ylphenyl)-acetyl]azepan-4-ylcarbamoyl}butyl)amide, is a pan-active inhibitor of the papain family of cysteine proteases, exhibiting potent activities for human cathepsins S, L, F, V and K, and weak activity for human cathepsin C. Similarly, SB-331750 is a potent inhibitor of mouse cathepsins S, L, and K, but not mouse cathepsin C (Fig. 1⇓). The activity of SB-331750 for mouse cathepsin F is undetermined. To date, no mouse orthologue of human cathepsin V has been identified (28). SB-331750 does not exhibit activity for serine, aspartyl, or metallo proteases (K_i > 10 μM), or for members of the caspase family of cysteine proteases (K_i > 10 μM). The representative members of these proteases classes tested were as follows: trypsin, chymotrypsin, cathepsin G, and elastase (serine proteases); cathepsin D, pepsin, and β-secretase (aspartyl proteases); matrix metalloproteinase-1, -2, -3, -7, -9, angiotensin-converting enzyme, and neutral endopeptidase (metallo proteases); and caspase-3 and -4 (caspase family of cysteine proteases).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 1.

The chemical structure and selectivity profile of SB-331750, 5-(2-Morpholin-4-ylethoxy)benzofuran-2-carboxylic Acid ((S)-3-Methyl-1-{(S)-3-oxo-1-[2-(3-pyridin-2-ylphenyl)-acetyl]azepan-4-ylcarbamoyl}butyl)amide. ND = Not determined.

In vitro treatment with SB-331750 results in comparable inhibition of Ii processing by cathepsin K^−/− vs cathepsin K^+/+ splenocytes

Murine cathepsins S, L, and F have been implicated in the final proteolytic cleavage of Ii (6, 7, 8, 9, 10, 13), whereas cathepsin K, to our knowledge, has not. To demonstrate the cell-based inhibition of Ii processing by SB-331750, and to determine whether the activity exhibited by SB-331750 for mouse cathepsin K (Fig. 1⇑) contributes to the compound’s inhibition of Ii processing, cathepsin K^+/+ and K^−/− splenocytes were incubated for 18 h in the absence (Untreated) or presence of vehicle (0.1% DMSO), SB-331750 (5 μM), or the broad-spectrum cysteine protease inhibitors, leupeptin (200 μg/ml) or APC3328 (5 μM). Cell lysates were then subjected to SDS-PAGE and Western blot analysis using the anti-mouse Ii mAb, In-1. Untreated cathepsin K^+/+ splenocytes and those cultured in the presence of DMSO expressed both the p41 (data not shown) and p31 isoforms of Ii, but no Ii degradation intermediates. In contrast, cathepsin K^+/+ splenocytes treated with leupeptin, APC3328, or SB-331750 exhibited prominent accumulation of the CLIP precursor Ii p10 with APC3328 inducing weak accumulation of the Ii p10 precursor Ii p22 as well (Fig. 2⇓A). Under the same experimental conditions, cathepsin K^−/− splenocytes (Fig. 2⇓B) exhibited Ii processing intermediates comparable to those exhibited by cathepsin K^+/+ splenocytes. In additional studies, the accumulation of Ii processing intermediates was found to occur rapidly, such that treatment of cells for 4–5 h resulted in consistent and quantifiable accumulation of Ii p10 (Fig. 3⇓). In the experiment depicted in Fig. 2⇓, and in those subsequently described, viability of mouse splenocytes following 18 h or 4–5 h of treatment with vehicle or inhibitor was confirmed by trypan blue exclusion. These results demonstrate the cell-based inhibition of late stage Ii processing by SB-331750 and indicate that cathepsin K does not play a role in Ii processing in these cells.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 2.

In vitro treatment with SB-331750 or broad-spectrum cysteine protease inhibitors results in inhibition of Ii processing by cathepsin K^−/− splenocytes comparable to that exhibited by cathepsin K^+/+ splenocytes. Splenocytes from cathepsin K^+/+ (A) or K^−/− (B) mice were cultured for 18 h in the absence or presence of vehicle (0.1% DMSO), leupeptin (200 μg/ml), APC3328 (5 μM), or SB-331750 (5 μM). Splenocytes were lysed, and the resulting lysates subjected to SDS-PAGE on an 18% Tris-HCl gel, followed by Western blot analysis using the mAb In-1. The results are representative of two independent experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 3.

In vitro treatment with SB-331750 induces concentration-dependent accumulation of Ii p10 in DBA/1, SJL/J, and C57BL/6 splenocytes. Splenocytes from DBA/1, SJL/J, and C57BL/6 mice were cultured for 4.5 h in the presence of SB-331750 at the indicated concentrations. Following lysis the cells were subjected to SDS-PAGE on an 18% Tris-HCl gel, and then Western blot analysis using the mAb In-1. The results are representative of four independent experiments.

In vitro treatment with SB-331750 inhibits Ii processing by DBA/1, SJL/J, and C57BL/6 splenocytes in a concentration-dependent manner

Inbred strains of mice, which differ at both MHC-linked and non-MHC-linked loci, are susceptible to specific experimentally-induced autoimmune syndromes. DBA/1 mice, which express the H-2^q haplotype, exhibit susceptibility to type II collagen-induced arthritis (14, 17), whereas SJL/J (H-2^s) and C57BL/6 (H-2^b) mice develop EAE following immunization with the appropriate component(s) of myelin (29, 30). To determine whether the genetic differences among these strains impact Ii processing, splenocytes from DBA/1, SJL/J, and C57BL/6 mice were treated for 4.5 h with varying concentrations of SB-331750 and Ii p10 accumulation was assessed. SB-331750 induced a concentration-dependent increase in Ii p10 accumulation in DBA/1, SJL/J, and C57BL/6 splenocytes (Fig. 3⇑), whereas untreated and vehicle (0.1% DMSO)-treated splenocytes from each strain exhibited no accumulation of Ii p10 (data not shown). Interestingly, the pattern of Ii p10-fragment accumulation differed among the strains, suggesting heterogeneity in the proteolytic events leading to Ii p10 generation. These data indicate that SB-331750 inhibits the final stage of Ii processing in genetically distinct strains of mice, allowing for investigation of the effects of inhibition of CLIP generation in multiple models of autoimmune disease.

In vitro treatment with SB-331750 inhibits processing of MHC class II-associated Ii, and decreases cell surface expression of class II/CLIP complexes, by Raji cells and C57BL/6 splenocytes

It has been demonstrated that within the cell, Ii is present in large excess relative to class II molecules. In the absence of association with class II molecules, this pool of Ii is localized primarily in the endoplasmic reticulum (31). To determine whether the SB-331750-mediated inhibition of late stage Ii processing observed in whole cell lysates is representative of the compound’s activity on class II-associated Ii, human Raji cells were cultured for 24 h with vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml), and an aliquot of the cell lysates from each treatment group immunoprecipitated using the mouse anti-HLA-DR mAb, TAL 1B5, or control mouse IgG. Nonimmunoprecipitated and immunoprecipitated cell lysates were then subjected to SDS-PAGE and Western blot analysis using the anti-human Ii mAb PIN.1. Similar to the results observed with nonimmunoprecipitated whole cell lysates of inhibitor-treated mouse splenocytes (Figs. 2⇑ and 3⇑), nonimmunoprecipitated whole cell lysates of SB-331750- or leupeptin-treated Raji cells demonstrated marked accumulation of Ii p10 and detectable accumulation of Ii p22 (Fig. 4⇓A). Comparable results were observed following immunoprecipitation of Raji whole cell lysates with TAL 1B5 (Fig. 4⇓, B and C), but not with control mouse IgG (Fig. 4⇓C). Ig H and L chains were detected due to recognition of the mouse immunoprecipitating Abs by the HRP-conjugated goat anti-mouse IgG mAb used as the detection reagent. To determine whether the SB-331750- and leupeptin-mediated inhibition of class II-associated Ii processing results in modulation of class II/CLIP complexes on the cell surface, DMSO-, SB-331750-, or leupeptin-treated Raji cells were analyzed by flow cytometry using the mAb CerCLIP. As shown in Fig. 4⇓D, cells treated with SB-331750 or leupeptin exhibited decreased class II-associated CLIP expression compared with cells treated with DMSO. In three to four experiments, SB-331750 and leupeptin induced mean decreases in class II-associated CLIP expression of 65.4% (range, 62.1–71.3%) and 74.4% (range, 72.6–76.1%), respectively. Total class II expression on the cell surface, detected by the FITC anti-HLA-DR mAb G46-6, was decreased modestly, but detectably, by SB-331750 (mean reduction of 17.3%; range, 11.2–23.3%) and leupeptin (mean reduction of 29.8%; range, 22.1–34.2%) (Fig. 4⇓E). Treatment of Raji cells with SB-331750 at 30 μM induced mean decreases of 71.4% (range, 70.3–73.1%) and 23.5% (range, 17.9–29.0%) in class II-associated CLIP expression and total class II expression, respectively (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 4.

In vitro treatment with SB-331750 or leupeptin induces accumulation of MHC class II-associated Ii processing intermediates, and decreases cell surface expression of class II/CLIP complexes, by Raji cells. Raji cells were cultured for 24 h in the presence of vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml), and cells lysed for SDS-PAGE/Western blot analysis, or intact cells subjected to flow cytometric analysis. Nonimmunoprecipitated cell lysates (A), and cell lysates immunoprecipitated with the anti-HLA-DR mAb, TAL 1B5 (B and C), or with control mouse IgG (C), were subjected to SDS-PAGE on an 18 or 4–20% Tris-HCl gel, and then Western blot analysis using the anti-human Ii mAb PIN.1. The results are representative of two independent experiments. Intact Raji cells were stained with the FITC anti-human MHC class II/CLIP mAb CerCLIP or with control FITC mouse IgG1 (not shown) (D), or with the FITC anti-HLA-DR mAb G46-6 or with control FITC mouse IgG2a (not shown) (E), and subjected to flow cytometric analysis. Dead cells were excluded using propidium iodide. The results are representative of three to four independent experiments.

Similar results were obtained following culture of C57BL/6 splenocytes for 4 h with vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml). The prominent accumulation of Ii p10 and weak accumulation of Ii p22 observed in SB-331750- or leupeptin-treated nonimmunoprecipitated whole cell lysates (Fig. 5⇓A) was also seen following immunoprecipitation of whole cell lysates with the anti-I-A^b mAb AF6–120.1 (Fig. 5⇓, B and C), but not with control mouse IgG2a (Fig. 5⇓C). CD19⁺ B cells expressed moderate/high cell surface levels of class II/CLIP complexes, (detected by the mAb 15G4; Fig. 5⇓D) and total class II molecules (detected by the mAb 2G9; Fig. 5⇓E), both of which were decreased following treatment of splenocytes with SB-331750 or leupeptin. Class II/CLIP complexes were reduced by an average of 47.1% (range, 46.0–47.6%, n = 3 mice) by SB-331750, and 31.6% (range, 29.6–33.6%, n = 3 mice) by leupeptin (Fig. 5⇓D), while total class II molecules were reduced by an average of 45.9% (range, 43.1–47.5%, n = 3 mice) by SB-331750, and 25.1% (range, 22.9–26.5%, n = 3 mice) by leupeptin (Fig. 5⇓E). CD19⁻ splenocytes exhibited minimally detectable levels of surface class II/CLIP complexes, and low levels of total class II molecules. Total class II molecules were decreased by an average of 14.1% (range, 9.2–17.5%, n = 3 mice) and 12.8% (range, 7.8–19.0%, n = 3 mice) by SB-331750 and leupeptin, respectively, compared with vehicle-treated CD19⁻ splenocytes (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 5.

In vitro treatment with SB-331750 or leupeptin induces accumulation of MHC class II-associated Ii processing intermediates, and decreases cell surface expression of class II/CLIP complexes, by C57BL/6 splenocytes. Splenocytes from C57BL/6 mice were cultured for 4 h in the presence of vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml), and cells lysed for SDS-PAGE/Western blot analysis, or intact cells subjected to flow cytometric analysis. Nonimmunoprecipitated cell lysates (A and lane 1 of B), and cell lysates immunoprecipitated with the anti-I-A^b mAb, AF6–120.1 (B and C), or with control mouse IgG2a (C), were subjected to SDS-PAGE on an 18% Tris-HCl gel, and then Western blot analysis using the anti-mouse Ii mAb In-1. The results are representative of two pooled mice/experiment in three independent experiments. Intact C57BL/6 splenocytes were stained with the PE anti-mouse CD19 mAb 1D3 or with control PE rat IgG2a, to allow gating on CD19⁺ cells. In addition, splenocytes were stained with the FITC anti-I-A^b/CLIP mAb 15G4 or with control FITC mouse IgG1 (not shown) (D), or with the FITC anti-H-2^q/H-2^b mAb 2G9 or with control FITC rat IgG2a (not shown) (E), and subjected to flow cytometric analysis. Dead cells were excluded using 7-amino-actinomycin D. The results are representative of 1 mouse/experiment in three independent experiments.

These data indicate that in Raji cells and C57BL/6 splenocytes, SB-331750 inhibits late stage processing of class II-associated Ii, and reduces expression of class II/CLIP complexes and total class II molecules on the cell surface.

Absence of detectable MHC class II-associated Ii processing intermediates in SB-331750-treated DBA/1 and SJL/J splenocytes

Similar to the results obtained with C57BL/6 splenocytes, following in vitro culture of DBA/1 (Fig. 6⇓A) and SJL/J splenocytes (Fig. 7⇓A) for 4 h with SB-331750 (10 μM) or leupeptin (200 μg/ml), nonimmunoprecipitated whole cell lysates exhibited marked accumulation of Ii p10 and weak accumulation of Ii p22, compared with nonimmunoprecipitated whole cell lysates of relevant vehicle (0.1% DMSO)-treated splenocytes. Interestingly, Ii p10 accumulation was not detected following immunoprecipitation of DBA/1 (Fig. 6⇓B) and SJL/J (Fig. 7⇓B) lysates with the anti-I-A^q/I-A^s mAb KH116. In the DBA/1 immunoprecipitates, the identity of the species that migrated coincident with Ii p22 is unknown, although its appearance following immunoprecipitation with control mouse IgG as well as with KH116, from lysates of both vehicle-treated and inhibitor-treated cells (Fig. 6⇓B), suggests that this band is nonspecific and does not represent a class II-associated Ii processing intermediate. To confirm that the lack of detectable class II-associated Ii p10 in these strains was not unique to use of KH116 as the immunoprecipitating mAb, DBA/1 whole cell lysates were immunoprecipitated with the anti-H-2^q/H-2^b mAb 2G9 and similar results were obtained (data not shown). Levels of surface class II/CLIP on CD19⁺ and CD19⁻ splenocytes from DBA/1 and SJL/J mice, detected by the mAb 15G4, were not elevated above levels detected by an isotype control mAb (data not shown), although interpretation of this result is confounded by the fact that we cannot distinguish between absence of surface class II/CLIP expression, and lack of cross-reactivity of 15G4 with the H-2^q and H-2^s haplotypes. CD19⁺ B cells from DBA/1 mice expressed high levels of total class II molecules on the cell surface (detected by the mAb 2G9), which were decreased by an average of 46.7% (range, 44.7–50.5%, n = 3 mice) as a result of treatment with SB-331750, and by an average of 33.0% (range, 32.7–33.3%, n = 3 mice) following treatment with leupeptin (Fig. 6⇓C). Expression of total class II molecules on the surface CD19⁻ DBA/1 splenocytes was low, and was decreased by an average of 14.9% (range, 11.0–18.7%, n = 3 mice) and 13.5% (range, 3.6–21.2%, n = 3 mice), following treatment with SB-331750 and leupeptin, respectively (data not shown). High and low levels of total MHC class II (detected by the mAb KH116) were also observed on CD19⁺ (Fig. 7⇓C) and CD19⁻ (data not shown) SJL/J splenocytes, respectively. SB-331750 induced mean decreases of 18.6% (range, 17.5–19.4%, n = 3 mice) and 4.9% (range, 0.43–10.7%, n = 3 mice), on CD19⁺ and CD19⁻ splenocytes, respectively, and leupeptin induced mean decreases of 18.0% (range, 16.1–19.9%, n = 2 mice) and 10.9% (range, 6.0–15.8%, n = 2 mice), respectively, compared with DMSO-treated CD19⁺ and CD19⁻ splenocytes, respectively.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 6.

Absence of detectable MHC class II-associated Ii processing intermediates in SB-331750- or leupeptin-treated DBA/1 splenocytes. Splenocytes from DBA/1 mice were cultured for 4 h in the presence of vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml), and cells lysed for SDS-PAGE/Western blot analysis, or intact cells subjected to flow cytometric analysis. Nonimmunoprecipitated cell lysates (A and lane 1 of B), and cell lysates immunoprecipitated with the anti-I-A^q/I-A^s mAb KH116 or with control mouse IgG (B), were subjected to SDS-PAGE on an 18% Tris-HCl gel, and then Western blot analysis using the anti-mouse Ii mAb In-1. The results are representative of three to four pooled mice/experiment in four independent experiments. Intact DBA/1 splenocytes were stained with the PE anti-mouse CD19 mAb 1D3 or with control PE rat IgG2a, to allow gating on CD19⁺ cells. In addition, splenocytes were stained with the FITC anti-H-2^q/H-2^b mAb 2G9 or with control FITC rat IgG2a (not shown) (C), and subjected to flow cytometric analysis. Dead cells were excluded using 7-amino-actinomycin D. The results are representative of one mouse/experiment in three independent experiments.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 7.

Absence of detectable MHC class II-associated Ii processing intermediates in SB-331750- or leupeptin-treated SJL/J splenocytes. Splenocytes from SJL/J mice were cultured for 4 h in the presence of vehicle (0.1% DMSO), SB-331750 (10 μM), or leupeptin (200 μg/ml), and cells lysed for SDS-PAGE/Western blot analysis, or intact cells subjected to flow cytometric analysis. Nonimmunoprecipitated cell lysates (A and lane 1 of B), and cell lysates immunoprecipitated with the anti-I-A^q/I-A^s mAb KH116 or with control mouse IgG (B), were subjected to SDS-PAGE on an 18% Tris-HCl gel, and then Western blot analysis using the anti-mouse Ii mAb In-1. The results are representative of one to four pooled mice/experiment in three independent experiments. Intact SJL/J splenocytes were stained with the PE anti-mouse CD19 mAb 1D3 or with control PE rat IgG2a, to allow gating on CD19⁺ cells. In addition, splenocytes were stained with the AlexaFluor 488 anti-I-A^s/I-A^q mAb KH116 or with control AlexaFluor 488 mouse IgG2b (not shown) (C), and subjected to flow cytometric analysis. Dead cells were excluded using 7-amino-actinomycin D. The results are representative of 1 mouse/experiment in three independent experiments.

These data suggest that in two autoimmune-prone strains of mice, DBA/1 and SJL/J, Ii processing intermediates may readily dissociate from MHC class II molecules. Interestingly, similar to the inhibitor-mediated modulation of total class II expression on the surface of Raji cells and C57BL/6 splenocytes, SB-331750 treatment resulted in varying degrees of reduction of surface class II expression by DBA/1 and SJL/J splenocytes.

In vitro treatment with SB-331750 inhibits Ag-induced proliferation by LNC/splenocytes from collagen-sensitized DBA/1 mice and MBP-sensitized SJL/J mice

To confirm that inhibition of late stage Ii processing by SB-331750 results in the reduction of Ag-induced cellular responsiveness, LNC/splenocytes from collagen-immunized/boosted DBA/1 mice, and from MBP-immunized/boosted SJL/J mice, were collected 24 and 17 days, respectively, postimmunization and stimulated with the relevant immunizing Ag. Culture of collagen-primed DBA/1 cells (Fig. 8⇓A), and of MBP-primed SJL/J cells (Fig. 8⇓B), with collagen and MBP, respectively, resulted in robust proliferative responses, each of which was inhibited in a concentration-dependent manner by SB-331750. This inhibition was not observed in DBA/1 (Fig. 8⇓C) and SJL/J (Fig. 8⇓D) splenocytes induced to proliferate via a mechanism not dependent on presentation of Ag, i.e., stimulation with anti-CD3 mAb. A similar lack of inhibition was observed when proliferation was induced by mitogen (data not shown). Viability of cells following culture with vehicle or SB-331750 was confirmed by trypan blue exclusion. These results demonstrate that in conjunction with its ability to inhibit late stage Ii processing in DBA/1 and SJL/J APC, in vitro treatment with SB-331750 prevents T cell proliferation in response to Ags that induce autoimmune syndromes in these strains of mice.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 8.

In vitro treatment with SB-331750 inhibits Ag-induced proliferation by LNC/splenocytes from collagen-sensitized DBA/1 and MBP-sensitized SJL/J mice in a concentration-dependent manner. For Ag-induced proliferative responses, inguinal lymph nodes and spleens from collagen-immunized/boosted DBA/1 mice (A), and from MBP-immunized/boosted SJL/J mice (B), were collected 24 and 17 days, respectively, postimmunization. Following preparation of single-cell suspensions, LNC and splenocytes were combined at a ratio of 80% LNC/20% splenocytes, giving an n = 3 pooled groups. LNC/splenocytes were cultured in the absence (Unstim.) or presence of type II collagen (100 μg/ml) (A) or MBP (50 μg/ml) (B). To Ag-stimulated LNC/splenocytes, SB-331750 was added at the concentrations indicated. Cells were cultured for 96 h with the final 24 h of incubation occurring in the presence of [³H]thymidine. Data are expressed as mean ± SEM. The results are representative of three independent experiments. For anti-CD3 mAb-induced proliferative responses, single-cell suspensions were prepared from the spleens of naive DBA/1 (C) and SJL/J (D) mice, and cultured in the absence (Unstim.) or presence of the plate-coated anti-mouse CD3 mAb 500A2 (5 μg/ml). To the anti-CD3 mAb-stimulated splenocytes, SB-331750 was added at the concentrations indicated. Cells were cultured for 96 h with the final 24 h of incubation occurring in the presence of BrdU. Data are expressed as mean ± SEM. The results are representative of two independent experiments.

In vivo administration of SB-331750 inhibits Ii processing by DBA/1, SJL/J, and C57BL/6 splenocytes

To demonstrate the inhibition of Ii processing by SB-331750 in vivo, DBA/1, SJL/J, and C57BL/6 mice were administered SB-331750 (50 mg/kg) or vehicle (PBS) s.c., b.i.d. on days 1–3, and 90 min after a single administration of SB-331750 or vehicle on day 4, spleens were collected and lysed. The resulting whole cell lysates were then subjected to SDS-PAGE and Western blot analysis using the mAb In-1 to assess Ii p10 accumulation. As shown in Fig. 9⇓, DBA/1, SJL/J, and C57BL/6 mice treated with SB-331750 exhibited enhanced accumulation of Ii p10 compared with DBA/1, SJL/J, and C57BL/6 mice, respectively, treated with vehicle, demonstrating that in vivo administration of SB-331750 results in the inhibition of processing of Ii p10 to CLIP.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 9.

In vivo administration of SB-331750 induces accumulation of Ii p10 in DBA/1, SJL/J, and C57BL/6 splenocytes. DBA/1, SJL/J, and C57BL/6 mice were administered vehicle (PBS) (n = 2 mice/strain) or SB-331750 (n = 2 mice/strain) at 50 mg/kg s.c., b.i.d on days 1–3, and 90 min after a single administration of vehicle or SB-331750 on day 4, spleens were collected. Splenocytes were lysed, and the resulting lysates subjected to SDS-PAGE on an 18% Tris-HCl gel, followed by Western blot analysis using the mAb In-1. The results are representative of two independent experiments.

In vivo prophylactic administration of SB-331750 decreases the severity and delays the onset of CIA, and reduces levels of tissue proinflammatory cytokines, in DBA/1 mice

To determine whether the inhibition of Ii processing and collagen-induced T cell proliferation by SB-331750 is sufficient to prevent the development of CIA, collagen-immunized and boosted DBA/1 mice (24 wk of age at the time of immunization) were administered SB-331750 (50 mg/kg) or vehicle (PBS) s.c., b.i.d. from days 1 to 40. In a previous pharmacokinetic study, blood concentrations of SB-331750 were measured in samples from three DBA/1 mice that were administered SB-331750 (50 mg/kg) s.c., b.i.d. on day 0, and once on day 1. Immediately before compound administration on day 1, blood (trough) levels of SB-331750 were 127 ± 48 nM, and at 15, 30, 60, 120, and 240 min following compound administration on day 1, blood levels of SB-331750 were 594 ± 52, 619 ± 92, 479 ± 61, 764 ± 124, and 184 ± 31 nM, respectively.

As shown in Fig. 10⇓A, the severity of CIA, represented by mean clinical score, was significantly reduced in mice treated with SB-331750 (p < 0.001), compared with mice treated with vehicle (PBS). In addition, the time of onset of disease was significantly delayed as a result of treatment with SB-331750 (p < 0.01) (Fig. 10⇓B). All mice in the vehicle-treated group and in the SB-331750-treated group exhibited clinical symptoms of disease during the study, indicating no modulation of cumulative disease incidence by SB-331750.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 10.

In vivo prophylactic administration of SB-331750 decreases the severity and delays the onset of CIA, and reduces paw tissue levels of IL-1β, TNF-α, and IFN-γ. The 24-wk-old male DBA/1 mice immunized on day 0 and boosted on day 21 with type II collagen were administered vehicle (PBS) or SB-331750 at 50 mg/kg s.c., b.i.d. on days 1–40, and scored for clinical symptoms of disease. For A, data are expressed as mean ± SEM (initial n = 24 mice, final n = 12 mice). For B, data are expressed as the percentage of mice remaining free of disease (i.e., day of disease onset was defined as the first of two consecutive days on which an animal exhibited a clinical score > 0). Thick vertical lines indicate censored observations. For C, four paws were collected from each mouse on days 28 (n = 6 mice), 41 (n = 6 mice), and 50 (n = 12 mice). For each time point, paws from two mice were combined randomly within each treatment group, resulting in an n = 3, 3, and 6 for days, 28, 41, and 50, respectively, and supernatants from combined tissue homogenates assayed for IL-1β, TNF-α, and IFN-γ. Data are expressed as mean ± SEM. ∗, p < 0.05; and ∗∗, p < 0.01, compared with the relevant vehicle-treated control group.

Consistent with the reduced severity of disease, SB-331750-treated mice exhibited decreased paw tissue levels of the proinflammatory cytokines IL-1β, TNF-α, and IFN-γ, compared with vehicle-treated mice. On day 28 (early disease), SB-331750-treated vs vehicle-treated mice exhibited comparable tissue levels of each cytokine, while on day 41 (mid-disease and immediately following completion of compound administration), tissues collected from SB-331750-treated mice contained significantly reduced IL-1β (p < 0.001) and TNF-α (p < 0.05) levels, and a trend toward reduced IFN-γ (p = 0.06) levels, compared with tissues collected from vehicle-treated mice (Fig. 10⇑C). On day 50, 10 days following the final administration of inhibitor, paw tissue levels of IL-1β (p < 0.001) and TNF-α (p < 0.05), but not IFN-γ, remained significantly reduced. These results indicate that prophylactic administration of SB-331750 results in the reduction of both the proinflammatory cytokine production and clinical manifestations associated with CIA, potentially via its inhibition of late stage Ii processing and subsequent Ag (collagen)-induced T cell proliferation.

In vivo therapeutic administration of SB-331750 decreases the severity of CIA in the DBA/1 mouse

To determine whether SB-331750 is capable of modulating the severity of CIA when administered therapeutically, collagen-immunized and boosted DBA/1 mice were injected with SB-331750 (50 mg/kg) or vehicle (PBS) s.c., b.i.d. following the onset of clinical symptoms. We have observed that mice immunized at 12 wk of age exhibit moderate disease severity (Fig. 11⇓A), whereas mice immunized at 24 wk of age develop severe disease (Fig. 11⇓B). Under moderate disease conditions, SB-331750-treated mice exhibited a significantly decreased mean clinical score compared with vehicle (PBS)-treated mice (p < 0.001) (Fig. 11⇓A). Under severe disease conditions, SB-331750-treated mice exhibited a trend toward decreased disease severity, although statistical significance was not achieved (Fig. 11⇓B). Under the latter conditions, in addition to administration of SB-331750 from the onset of symptoms through completion of the study (days 1–24), mice were administered SB-331750 (50 mg/kg) s.c., b.i.d. on days 1–5, and then administered vehicle (PBS) for the remainder of the study. As shown in Fig. 11⇓B, discontinuation of SB-331750 administration resulted in an accelerated return of clinical symptoms to levels exhibited by vehicle-treated mice, although, based on the modest window achieved in this study, no statistical difference between the two SB-331750 treatment groups was observed. These results indicate potential utility of cathepsin inhibitors in a clinically relevant dosing regimen, and suggest that continued inhibitor administration may be required for extended therapeutic efficacy. Future studies are planned to determine the effect of discontinued SB-331750 administration under moderate disease conditions.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 11.

In vivo therapeutic administration of SB-331750 decreases the severity of CIA. Following exhibition of clinical symptoms for 2 consecutive days (days 0 and 1), collagen-immunized/boosted DBA/1 mice that were 12 (A) or 24 (B) wk of age at the time of immunization were administered vehicle (PBS) or SB-331750 at 50 mg/kg s.c., b.i.d. on days 1–26 (A) or on days 1–24 (B). In B, an additional treatment group was administered SB-331750 at 50 mg/kg s.c., b.i.d. on days 1–5, and then administered vehicle (PBS) s.c., b.i.d on days 6–24. During the course of drug and/or vehicle administration, mice were scored for clinical symptoms of disease. Data are expressed as mean ± SEM (n = 8–13 mice).

Treatment of MBP-primed LNC with SB-331750 reduces the severity and delays the onset of adoptively transferred EAE in the SJL/J mouse

To characterize the effects of SB-331750 in a second model of Ag-driven autoimmunity, LNC from MBP-immunized/boosted SJL/J mice were cultured with MBP (50 μg/ml) in the presence of vehicle (0.1% DMSO) or SB-331750 (10 μM), and then transferred into naive SJL/J recipients. As shown in Fig. 12⇓, A and B, respectively, the severity of EAE was significantly reduced (p < 0.01), and the onset significantly delayed (p < 0.0001), in mice that received SB-331750-treated LNC compared with mice that received vehicle-treated LNC. Similar to the findings in CIA, 100% of mice in each treatment group exhibited clinical symptoms of disease during the study. These results indicate that the presence of SB-331750 during Ag stimulation of MBP-primed LNC reduces the ability of the cells to adoptively transfer EAE to naive recipients, likely through the inhibition of Ag (MBP) presentation to CD4⁺ T cells.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 12.

Treatment of MBP-primed LNC with SB-331750 reduces the severity and delays the onset of adoptively transferred EAE. Female SJL/J mice were immunized on day 0 and boosted on day 7 with MBP, and inguinal lymph nodes collected on day 17. Following preparation of single-cell suspensions, LNC were cultured for 72 h with MBP (50 μg/ml) in the presence of vehicle (0.1% DMSO) or SB-331750 (10 μM), and then adoptively transferred to naive female SJL/J mice (n = 8–9 mice). Recipient mice were scored for clinical symptoms of disease. For A, data are expressed as mean ± SEM. For B, data are expressed as the percentage of mice remaining free of disease (i.e., day of disease onset was defined as the first of two consecutive days on which an animal exhibited a clinical score > 0).

In vivo therapeutic administration of SB-331750 decreases the severity of EAE in the SJL/J mouse

To determine whether therapeutic administration of SB-331750 modulates the severity of actively-induced EAE, MBP-immunized SJL/J mice were injected with SB-331750 (50 mg/kg) or vehicle (PBS) s.c., b.i.d. following the onset of clinical symptoms, through day 14 of the study. Immunization of SJL/J mice with bovine MBP produced a mild, transient form of EAE, the severity of which was significantly reduced as a result of treatment with SB-331750 (p < 0.05) (Fig. 13⇓). These results, together with those obtained in the CIA model, indicate that when administered in a clinically relevant dosing regimen, SB-331750 provides therapeutic benefit under mild/moderate autoimmune disease conditions.

• Download figure
• Open in new tab
• Download powerpoint

FIGURE 13.

In vivo therapeutic administration of SB-331750 decreases the severity of EAE. Beginning on the first day of exhibition of clinical symptoms (day 1 of treatment), MBP-immunized SJL/J mice were administered vehicle (PBS) or SB-331750 at 50 mg/kg s.c., b.i.d. Mice received drug or vehicle on days 1–14, and were scored for clinical symptoms of disease through day 18. Data are expressed as mean ± SEM (n = 4–5 mice).