Periodic, Partial Inhibition of I B Kinase -Mediated Signaling Yields Therapeutic Benefit in Preclinical Models of Rheumatoid Arthritis

We have previously shown that inhibitors of I B kinase (IKK ), including 4(2 -aminoethyl)amino-1,8-dimethylimidazo(1,2a)quinoxaline (BMS-345541), are efficacious against experimental arthritis in rodents. In our efforts to identify an analog as a clinical candidate for the treatment of autoimmune and inflammatory disorders, we have discovered the potent and highly selective IKK inhibitor 2-methoxy-N-((6-(1-methyl-4(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin7-yl)pyridin-2-yl)methyl)acetamide (BMS-066). Investigations of its pharmacology in rodent models of experimental arthritis showed that BMS-066 at doses of 5 and 10 mg/kg once daily was effective at protecting rats against adjuvant-induced arthritis, despite showing only weak inhibition at 10 mg/kg against a pharmacodymanic model of tumor necrosis factor production in rats challenged with lipopolysaccharide. The duration of exposure in rats indicated that just 6 to 9 h of coverage per day of the concentration necessary to inhibit IKK by 50% in vivo was necessary for protection against arthritis. Similar findings were observed in the mouse collagen-induced arthritis model, with efficacy observed at a dose providing only 6 h of coverage per day of the concentration necessary to inhibit IKK by 50%. This finding probably results from the cumulative effect on multiple cellular mechanisms that contribute to autoimmunity and joint destruction, because BMS-066 was shown to inhibit a broad spectrum of activities such as T cell proliferation, B cell function, cytokine and interleukin secretion from monocytes, TH17 cell function and regulation, and osteoclastogenesis. Thus, only partial and transient inhibition of IKK is sufficient to yield dramatic benefit in vivo, and this understanding will be important in the clinical development of IKK inhibitors. The multisubunit I B kinase (IKK) complex is essential in transducing the signal-inducible activation of the transcription factor NFB in response to proinflammatory stimuli. In the “canonical” pathway of NFB activation, the IKK complex catalyzes the phosphorylation of I B proteins at specific serine residues, which triggers a subsequent ubiquitination and proteolysis of I B, leaving NFB free to translocate to the nucleus (Ghosh and Karin, 2002). This multisubunit IKK complex is composed of two catalytic subunits, termed IKK and IKK , and a regulatory subunit termed NEMO (or IKK ), which mediates upstream signals into activation of Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.156018. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: IKK, I B kinase; BCR, B cell receptor; KLH, keyhole limpet hemocyanin; LPS, lipopolysaccharide; PBMC, peripheral blood mononuclear cell; TCR, T cell receptor; NFB, nuclear factor B; TNF, tumor necrosis factor ; DMSO, dimethyl sulfoxide; ELISA, enzymelinked immunosorbent assay; LC/MS/MS, liquid chromatography coupled to tandem mass spectrometry; PEG400, Polyethylene Glycol 400; Pen/Strep, penicillin and streptomycin mixture; RANKL, receptor activator of NFB ligand; NFAT, nuclear factor of activated T cells; TRAP, tartrate-resistant acid phosphatase; IL, interleukin; FBS, fetal bovine serum; micro-CT, microcomputed tomography; BMS-345541, 4(2 -aminoethyl)amino-1,8-dimethylimidazo(1,2-a)quinoxaline; BMS-066, 2-methoxy-N-((6-(1-methyl-4-(methylamino)-1,6-dihydroimidazo[4,5-d]pyrrolo[2,3-b]pyridin7-yl)pyridin-2-yl)methyl)acetamide. 0022-3565/09/3312-349–360$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 331, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 156018/3521064 JPET 331:349–360, 2009 Printed in U.S.A. 349 http://jpet.aspetjournals.org/content/suppl/2009/08/03/jpet.109.156018.DC1 Supplemental material to this article can be found at: at A PE T Jornals on M arch 0, 2017 jpet.asjournals.org D ow nladed from the catalytic subunits (Schomer-Miller et al., 2006). Although either catalytic subunit is capable of catalyzing the phosphorylation of I B proteins in vitro, the IKK subunit seems to play the dominant role in this canonical pathway in cells (Ghosh and Karin, 2002). Given the importance of NFB in regulating immunological pathways and responses, along with the role IKK plays in NFB activation, the identification of selective IKK inhibitors as therapeutic agents in the treatment of inflammatory and autoimmune disorders has received considerable interest (for a review, see Pitts and Kempson, 2008). We have previously reported that the selective IKK inhibitor BMS345541 was highly efficacious against both inflammation and joint destruction in collagen-induced arthritis in mice and in a model of inflammatory bowel disease (MacMaster et al., 2003; McIntyre et al., 2003). Subsequently, other IKK inhibitors have also been reported to provide benefit in experimental arthritis models (Podolin et al., 2005; Schopf et al., 2006; Mbalaviele et al., 2009). BMS-345541 has also been shown to be effective in various tumor models and suggests that IKK inhibitors may be beneficial in the treatment of certain cancers in humans (Yang et al., 2006; Ammann et al., 2009). In our efforts to discover more potent IKK inhibitors with pharmacokinetic, safety, and pharmaceutic properties consistent with being considered as a candidate for clinical development in autoimmune disorders such as rheumatoid arthritis, we identified BMS-066, shown in Fig. 1, ultimately derived from a medicinal chemistry effort around BMS-345541 (Beaulieu et al., 2007; Belema et al., 2007; Kempson et al., 2009a,b). In the present report, we provide a detailed investigation of the pharmacology of BMS-066 in rodent models of experimental arthritis. We found that only transient and partial inhibition of IKK in vivo is sufficient for efficacy. Additional cellular characterization shows that this finding probably results from the IKK dependence of multiple cellular mechanisms that contribute to autoimmunity and joint destruction, and that a cumulative effect from partial inhibition of these mechanisms and pathways in vivo leads to significant benefit in experimental arthritis models. Materials and Methods All animal procedures involving animals were carried out with the approval of the Bristol-Myers Squibb Animal Care and Use Committee. BMS-066. BMS-066 was synthesized at Bristol-Myers Squibb Co. according to the procedure reported by Pitts et al. (2006). Enzyme Assays. IKK assays used a 17-amino-acid peptide substrate corresponding to amino acids 26 to 42 of I B as described previously (Burke et al., 1998). In this assay, human recombinant IKK was added at 30°C to solutions containing peptide, [P]ATP (10 M), 1 mg/ml bovine serum albumin in 10 mM MgCl2, 1 mM dithiothreitol, 10 mM Tris-HCl at pH 7.2. After 60 min, the kinase reactions were quenched by addition of EDTA to a concentration of 50 mM. High-performance liquid chromatography analysis was performed and the amount of IKK-catalyzed incorporation of P into each peptide was quantitated by liquid scintillation counting. Under these conditions, the degree of phosphorylation of peptide substrate was linear with time and concentration of enzyme. All other kinases were assayed by use of the SelectScreen Profiling Service using Z -LYTE fluorescent peptide substrates and fluorescence resonance energy transfer-based detection system (Invitrogen, Carlsbad, CA). All assays were run at the KM app for ATP with each kinase. BMS-066 was tested at a concentration of 10 M against 155 kinases within their panel, and select kinases were assayed in a dose response with BMS-066 to determine the IC50 values. LPS-Induced Responses in PBMCs and Whole Blood. The potency of the compound against lipopolysaccharide (LPS)-induced TNFproduction from human peripheral blood mononuclear cells (PBMCs) was performed according to the general procedure of Kempson et al. (2009). When measuring the inhibition by BMS-066 of the phosphorylation of I B in human PBMCs, cells at a density of 1 10 per ml in RPMI 1640 medium supplemented with 10% FBS were preincubated for 30 min with various concentrations of BMS-066 at 37°C. To prevent degradation of phosphorylated I B upon stimulation, the proteasome inhibitor was also added to a final concentration of 1 M during this preincubation. Cells were then stimulated for 5 min with LPS (100 ng/ml) and pelleted by rapid centrifugation; the pellets were solubilized by use of ice-cold 10 cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA). A phospho-IkB ELISA from Cell Signaling Technology Inc. was used for quantitation per the manufacturer’s instructions. To measure effects on transcription, human PBMCs were plated on 24-well plates (Falcon; BD Biosciences Discovery Labware, Bedford, MA) at a density of 3 10 cells/well in RPMI 1640 medium plus 10% FBS and HEPES with varying concentrations of BMS-066. After a 1-h incubation at 37°C, LPS (Salmonella typhosa; Sigma-Aldrich, St. Louis, MO) was added to cells to a final concentration of 50 ng/ml and allowed to incubate 4 h at 37°C at 5% CO2, at which time cells were harvested and prepared for RNA isolation. Quantitative real-time polymerase chain reaction was performed for TNF, IL-6, the p19 subunit of IL-23, and the common p40 subunit of IL-12/IL-23 with procedures reported previously (Pattoli et al., 2005). For human whole-blood experiments, blood from healthy volunteers was collected in collection tubes containing anticoagulant citrate dextrose solution A as an anticoagulant. Collected blood (300 l) was pipetted into deep-well, 96-well assay plates (Corning Life Sciences, Lowell, MA), and BMS-066 in phosphate-buffered saline containing 16% (v/v) DMSO was then added to give a final DMSO concentration of 1% (v/v). After incubating 45 min at 37°C, LPS (S. typhosa, Sigma-Aldrich) was added to a final concentration of 1 g /ml. After a 6-h incubation at 37°C, the plates were centrifuged for 10 min at 14,000 rpm and plasma was removed for TNFmeasurement by ELISA (BD Pharmingen, San Diego, CA). For rat and mouse whole-blood experiments, blood was collected by cardiac puncture and orbital puncture, respectively, using collection tubes containing ACD-a as an anticoagulant. Three hundred microliters of pooled whole blood was incubated with BMS-066 and stimulated with LPS as above, with the exception that the final DMSO concentrations was 0.2% (v/v) and the LPS concentrations used were 5 and 10 g/ml for rat and mouse whole blood, respectively. Stimulated plates containing compounds were incubated for 4 h at 37°C with use of slow rotation. Plates were centrifuged for 10 min at 14,000 rpm and plasma was removed for TNFmeasurement by ELISA (BioSource International, Camarillo, CA). Osteoclastogenesis. Osteoclast precursor cells obtained from Cambrex (East Rutherford, NJ) were plated in 96-well plates at 10,000 cells/well in 0.2 ml of basal medium supplemented with the following cytokines or growth factors: 10% FBS, 25 ng/ml human Fig. 1. Chemical structure of BMS-066. 350 Gillooly et al. at A PE T Jornals on M arch 0, 2017 jpet.asjournals.org D ow nladed from macrophage–colony-stimulating factor, 25 ng/ml human RANKL, 2 mM L-glutamine containing various concentrations of BMS-066. All media and reagent supplements were supplied as a single media kit from Cambrex. The cells were replenished with BMS-066-containing media on day 5 by demidepletion (half of the medium withdrawn and replaced with the fresh medium). After an additional 4 days in culture, cells were fixed with acetone/citrate solution and stained for tartrate-resistant acid phosphatase (TRAP) by use of the kit from Sigma-Aldrich according to manufacturer’s instructions. The cells were fixed in citrate/acetone solution for 30 s and rinsed with deionized water before TRAP staining. The number of TRAP-positive multinucleated cells ( 2 nuclei per cell) was measured at predetermined site areas on each well of a 96-well plate, and three wells were measured in total for each condition. A mean value was calculated for each well. The average number of TRAP-positive multinucleated cells in controls was approximately 215 to 250 cells/well (96-well plate). The IC50 value was calculated from a BMS-066 dose range of 2 to 0.06 M. Lymphocytes Responses. Jurkat T cells with stable integration of luciferase under the control of either an NFBor NFAT-driven reporter (J.-H. Wang, Bristol-Myers Squibb) were added at a density of 1 10 cells/ml in RPMI 1640 medium with glutamine containing 10% heat-inactivated FBS and 1% Pen/Strep were added to antiCD3-coated (G19-4, 1 g/ml) 96-well plates in the presence of varying concentrations of BMS-066. After 6 h at 37°C, the luciferase activity was assayed by use of the Steady-Glo Luciferase Assay reagent (Promega, Madison, WI). Human peripheral blood T lymphocytes were prepared from E fractions of PBMCs (isolated as above) rosetted with sheep red blood cells. T cells at a density of 1.3 10 cells/well were added to anti-CD3-coated (G19-4, 0.5 g/ml) 96-well plates in the presence of varying concentrations of BMS-066 in RPMI 1640 medium with glutamine containing 10% heat-inactivated FBS and 1% Pen/Strep. After 24 h at 37°C, supernatants were collected and analyzed by ELISA for IL-2 (BD Biosciences, Franklin Lakes, NJ), IL-17 (R&D Systems, Minneapolis, MN), and IL-1 (BD Biosciences). For proliferation assays, human T cells were incubated for 3 days and then labeled with [H]thymidine for 6 h. Lysates were prepared and harvested onto filter plates, and the amount of [H]thymidine incorporation measured by liquid scintillation counting. To measure the phosphorylation of I B in response to B cell receptor activation, mouse splenic B cells were isolated by use of nylon fiber columns from spleens of BALB/c mice. To 96-well roundbottom plates were then added 3.6 10 cells/well in RPMI 1640 medium with glutamine containing 10% heat-inactivated FBS and 1% Pen/Strep along with the proteasome inhibitor (1 M; Calbiochem, San Diego) and varying concentrations of BMS-066. After 1 h at 37°C, F(ab )2 fragment goat anti-mouse IgG IgM (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) was added to 25 g/ml to stimulate the cells and incubated for an additional 30 min, at which time the cells were pelleted by centrifugation and lysed (no. 9803; Cell Signaling Technology Inc.) on ice for 30 min; the lysates were analyzed for phospho-I B by ELISA (Cell Signaling Technology Inc.). Human B cells were isolated from tonsils obtained from patients undergoing routine tonsillectomies (National Disease Research Interchange, Philadelphia, PA). Tonsillar tissue was minced, mashed through coarse screens, and filtered through 70m cell strainers (Falcon; BD Biosciences Discovery Labware). Mononuclear cells were obtained by density-gradient separation (Lymphocyte Separation Media; Mediatech, Herndon, VA) and rosetted with sheep red blood cells (Colorado Serum Company, Denver, CO). Purified B cells were obtained from the resulting E-rosette negative populations and were routinely 95% CD19 as measured by fluorescence-activated cell sorting. For tonsillar B cell proliferation and immunoglobulin secretion assays, 1 10 cells/ml in RPMI 1640 medium containing 10% heat-inactivated FBS and gentamycin with varying concentrations of BMS-066 were stimulated either with antiIgM (5 g/ml; Southern Biotechnology Associates, Birmingham, AL) plus IL-4 (20 ng/ml; R&D Systems) or anti-CD40 (1 g/ml; BD Pharmingen) plus IL-4 and incubated at 37°C for 72 h. After collecting aliquots of supernatants for immunoglobulin measurements, the cells were labeled with [H]thymidine and incubated overnight at 37°C. Cells were then harvested onto filter plates and the amount of [H]thymidine incorporation measured by liquid scintillation counting. Levels of human IgM and IgG were measured by specific ELISA kits (Bethyl Laboratories, Montgomery, TX). Pharmacokinetics in Mice, Rats, and Dogs. The pharmacokinetics of BMS-066 was characterized by use of male BALB/c mice. Four groups of animals (n 9 per group, 20–25 g) were dosed intravenously via the tail vein at a dose of 2 mg/kg or orally at 3 mg/kg in 50% PEG400 and 50% of 0.02 N HCl. Blood samples ( 0.2 ml) were obtained by retro-orbital bleeding at 0.05 (intravenous dose only), 0.25 (oral dose only), 0.5, 1, 3, 6, 8, and 24 h after dose. Within each group, three of the animals were bled at 0.05 (or 0.25 for oral), 1, and 6 h, three were bled at 0.5, 3, and 8 h, and the last three were bled at 24 h, which resulted in a composite pharmacokinetic profile (three mice per time point). Blood samples were allowed to coagulate and centrifuged at 4°C (1500–2000g) to obtain serum. Serum samples were stored at 20°C until analysis by LC/MS/MS. Nonfasted male Lewis rats (275–325 g, n 3 per group) were used in the pharmacokinetic studies of BMS-066. An intravenous dose of 2 mg/kg was given as a 10-min infusion via the jugular vein, and the oral dose was 10 mg/kg. The vehicle used was 50% PEG400/50% 0.02 N HCl. Serial blood samples (0.3 ml) were obtained by use of EDTA as an anticoagulant at predose and at 0.17 (intravenous dose only), 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 10, and 24 h after dose. Plasma samples, obtained by centrifugation at 4°C (1500–2000g), were stored at 20°C until analysis. The pharmacokinetic profile of BMS-066 was evaluated in male beagle dogs (9–11 kg, n 3). The intravenous and oral (solution) studies were conducted in a crossover design. There was a one-week washout period between the intravenous and oral dosing. Dogs were fasted overnight before dosing and continuously fasted for an additional 4 h after dosing. In the intravenous study, BMS-066 was infused through a chronically implanted vascular access port at 1 mg/kg over 10 min. In the oral arm of the study, BMS-066 was administered by oral gavage at 5 mg/kg (1 ml/kg in 0.01 N HCl). Serial blood samples were collected by with of EDTA as an anticoagulant by direct jugular venipuncture at 0.17 (intravenous dose only) 0.25, 0.5, 0.75, 1, 2, 4, 8, and 24 h after dose. Plasma samples were prepared immediately and stored at 20°C until analysis. Determination of BMS-066 Levels in Serum Samples. Serum and/or plasma samples were treated with two volumes of acetonitrile containing 1 g/ml of an appropriate internal standard. After centrifugation to remove precipitated proteins, a 3l portion of the resulting supernatant was analyzed by LC/MS/MS. Data acquisition was via selected reaction monitoring. Ions representing the (M H) species for both the analyte and internal standard were selected in MS1 and collisionally dissociated with argon at a pressure of 1.2 10 3 Torr to form specific product ions that were subsequently monitored by MS2. The transition monitored for BMS-066 was m/z 3803291 and m/z 3643305 for the internal standard. The retention time for BMS-066 was 1.66 min. The eight-point standard curve ranged from 1 to 8000 ng/ml and was fitted with a quadratic regression weighted by reciprocal concentration (1/x). Equilibrium Serum Protein Binding. The extent of protein binding of BMS-066 was determined in mouse, rat, and human serum using a 96-well equilibrium dialysis device (HTDialysis, LLC, Gales Ferry, CT). The experiment was done in triplicate at a substrate concentration of 10 M. The dialysis device, containing a dialysis membrane (molecular mass cutoff of 12–14 kDa) in each well to separate the serum and dialysis buffer (0.133 M phosphate buffer at pH 7.4), was shaken for 4 h in an incubator maintaining 37°C with 5% CO2 and 85% humidity. Aliquots of buffer and serum were taken at 4 h and collected in the following manner. Buffer samples (50 l) were collected in a 96-well plate that contained 50 l of speciesIncomplete IKK Inhibition Is Efficacious in Arthritis Models 351 at A PE T Jornals on M arch 0, 2017 jpet.asjournals.org D ow nladed from specific serum. Serum samples (20 l) were collected in a 96-well plate that contained a one-to-one mixed matrix solution containing buffer solution and species-specific serum (180 l). Samples were analyzed for BMS-066 by use of a LC/MS/MS method. Percentage of protein binding was calculated by the following equation: %Free 100 drug concentration in buffer/drug concentration in serum. LPS-Induced Serum TNFand Liver I B Degradation in Rodents. Male Lewis rats (175–200 g; Harlan, Indianapolis, IN) were dosed by oral gavage with vehicle (50% PEG400, 50% 0.02 N HCl) or BMS-066 in vehicle. Four hours later, rats were injected intraperitoneally with 1 ml per rat of 100 g of LPS (Escherichia coli strain 0111:B4, Sigma-Aldrich) suspended in phosphate-buffered saline. Ninety minutes after LPS challenge, blood was collected from the retro-orbital sinus under isoflurane anesthesia. Serum was separated from clotted samples by centrifugation (5 min, 5000g, room temperature) and analyzed for the levels of TNFby ELISA according to the manufacturer’s instructions (BioSource International). Results are shown as mean S.E.M. of n 8 rats per treatment group. In a separate experiment to measure the ability of BMS-066 to inhibit I B degradation in the liver of LPS-challenged rats, rats were sacrificed 40 min after LPS challenge and livers were excised. (Previous experiments determined that the optimal time point to measure I B degradation from rat liver was 40 min after LPS challenge.) A group of naive rats receiving only PBS (no LPS) was included. The right lobes of each liver were then cut into small 2to 3-cm pieces immediately, flash frozen in liquid nitrogen, and stored at 80°C until use. Frozen specimens (300 mg each) were pulverized and placed into a Dounce homogenizer on ice with the addition of 2 ml of chilled lysis buffer containing 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 50 mM Tris-HCl, pH 8.0, and 1 mM phenylmethylsulfonyl fluoride. Protease inhibitors were added just before use (Protease Inhibitor cocktail set II; Calbiochem). The homogenate was kept on ice for 1 h before transfer to 1.5-ml tubes and cleared by centrifugation at maximum speed for 10 min at 4°C to collect the supernatant containing the whole cell extract. The protein content of each sample was determined by bicinchoninic acid protein reagent (Pierce Chemical, Rockford, IL). Lysate samples were normalized to 3000 ng/ml total protein before measurement of I B levels by ELISA (Total I B sandwich ELISA Kit, Cell Signaling Technology Inc.). ELISA procedure was followed according to the manufacturer’s instructions. Unless otherwise noted, statistical significance was determined by use of a one-way analysis of variance with a Dunnett’s post test. Values of p 0.05 were considered significant. With mice, female BALB/c mice (20–22 g; Harlan) were dosed by oral gavage with vehicle (50% PEG400, 50% 0.02 N HCl) or BMS-066 in vehicle. Four hours later, mice were injected intraperitoneally with 0.5 ml per mouse of 10 g of LPS (E. coli strain 0111:B4; Sigma-Aldrich) suspended in phosphate-buffered saline. Ninety minutes after LPS challenge, blood was collected from the retro-orbital sinus under isoflurane anesthesia. Serum was separated from clotted samples by centrifugation (5 min, 5000g, room temperature) and analyzed for the levels of TNFby ELISA according to the manufacturer’s instructions (BioSource International). Adjuvant-Induced Arthritis in Rats. Male Lewis rats (225 g; Harlan) were immunized with 0.1 ml of 1 mg/rat freshly ground Mycobacterium butyricum (Difco, Detroit, MI) in incomplete Freund’s Adjuvant (Sigma-Aldrich) at the base of the tail. Hind paw swelling (in milliliters) was measured in a water displacement plethysmometer (Ugo Basile, Comerio, Italy). Vehicle (0.02 N HCl) or BMS-066 in vehicle was administered by oral gavage beginning on the day of adjuvant challenge. Eight animals were included in each treatment group. Left hind limbs were removed postmortem at the end of the study and frozen at 20°C for analysis of bone morphology by microcomputed tomography (micro-CT). The excised hind limbs were secured in a sample support system and micro-CT imaging was performed on the Scanco VivaCT 40 (Scanco Medical, Zurich, Switzerland). Approximately 400 38m-thick slices were acquired with 250 projections, 500-ms integration time, 55 kVp photon energy, and 114 A of current. Micro-CT analyses were performed with use of the Scanco software (Scanco Medical). Threshold settings were optimized with histomorphometric methods. Bone density measurements were evaluated based on a 200-mg hydroxylapatite calibration phantom and analyzed by use of Scanco software. For histological evaluation, right-hind ankle joints were fixed in 10% neutral formalin for at least 24 h and then decalcified in formic acid for approximately 1 week. Sectioning in the sagittal plane from paraffin-embedded tissue was stained with hematoxylin and eosin for histological evaluation, with one slide per animal used for the evaluation. The slides were evaluated without knowledge of treatment group and scored separately for inflammation/edema and bone resorption on a scale of 0 to 4. Total score is the sum of the two, on a scale of 0 to 8. Scoring was based on an established grading system (Bendele et al., 1999). For inflammation/edema, the scoring was as follows: Grade 0, unremarkable; grade 1, minimal change; grade 2, mild change; grade 3, moderate change; grade 4, marked change. For bone resorption, the grading was as follows: Grade 0, unremarkable; grade 1, minimal focal to multifocal cortical or trabecular bone resorption; grade 2, mild focal to multifocal cortical or trabecular bone resorption, grade 3, moderate resorption of cortical or trabecular bone with rare full-thickness resorption, grade 4, full-thickness resorption with distortion of bone architecture. Scores for each group were compared by one-way analysis of variance and a Bonferroni multiple comparison post-test. KLH-Induced Serum IgM and IgG Production in Rats. Male Lewis rats (250 g; Harlan) were immunized intraperitoneally with 200 g/rat of keyhole limpet hemocyanin (KLH; Sigma-Aldrich) diluted in phosphate-buffered saline. BMS-066 in 0.02 N HCl was administered by oral gavage once daily beginning on the day of immunization. Blood samples were collected on day 7 and on day 14 under isoflurane anesthesia. Threefold serial dilutions of serum that had been separated from clotted samples by centrifugation (5 min, 5000g, room temperature) were added in duplicate to flat-bottomed 96-well ELISA plates precoated with KLH (10 g/ml) overnight at 4°C and blocked with milk diluent. After a 2-h incubation at room temperature, the plates were washed, and horseradish peroxidaseconjugated goat anti-mouse IgM or anti-IgG detection antibody (Southern Biotechnology Associates) was added. After a final washing step, the plates were developed with tetramethylbenzidine substrate (Kirkegard and Perry, Gaithersburg, MD) and read on a spectrophotometer at 450 nm. Anti-KLH titers were expressed as the reciprocal of the serum dilution that yielded an optical density of 1.0 (within the linear portion of the dilution curve). Collagen-Induced Arthritis in Mice. Male DBA/1 mice (20–25 g; Harlan) were immunized with 200 g/mouse of bovine type II collagen (Elastin Products Company, Owensville, MO) in Ribi Adjuvant System/RAS (Ribi ImmunoChem, Hamilton, MT)) in 0.1 ml at the base of the tail on day 0 and on day 21. Vehicle (0.02 N HCl) or BMS-066 in vehicle was administered by oral gavage twice daily beginning on day 21. Mice were monitored after the second immunization for the development of paw inflammation. Each paw was individually scored as follows: 0, normal; 1, one or more swollen digits; 2, mild paw swelling; 3, moderate paw swelling; 4, fusion of joints/ankylosis. The scores for all four paws are summed for each mouse, and results shown as the mean of n 15 mice per treatment group. Statistical significance among the groups was determined by use of the nonparametric Mann-Whitney U Test. Measurements of serum anticollagen IgG were performed in flat-bottomed 96-well ELISA plates precoated overnight with bovine type II collagen (1 g/ml). After blocking the plates with milk diluent, 3-fold serial dilutions of mouse sera were added to duplicate wells and incubated for 2 h at room temperature. The plates were washed, and horseradish peroxidase-conjugated goat anti-mouse IgG detection antibody (Southern Biotechnology Associates) was added. After a final washing step, the plates were developed with tetramethylbenzidine sub352 Gillooly et al. at A PE T Jornals on M arch 0, 2017 jpet.asjournals.org D ow nladed from strate (Kirkegard and Perry) and read at 450 nm. Anticollagen titers were expressed as the reciprocal of the serum dilution that yielded an optical density of 1.0 (within the linear portion of the dilution curve).