Patricia L. Podolin

A Potent and Selective Nonpeptide Antagonist of CXCR2 Inhibits Acute and Chronic Models of Arthritis in the Rabbit

Much evidence implicates IL-8 as a major mediator of inflammation and joint destruction in rheumatoid arthritis. The effects of IL-8 and its related ligands are mediated via two receptors, CXCR1 and CXCR2. In the present study, we demonstrate that a potent and selective nonpeptide antagonist of human CXCR2 potently inhibits 125I-labeled human IL-8 binding to, and human IL-8-induced calcium mobilization mediated by, rabbit CXCR2 (IC50 = 40.5 and 7.7 nM, respectively), but not rabbit CXCR1 (IC50 = >1000 and 2200 nM, respectively). These data suggest that the rabbit is an appropriate species in which to examine the anti-inflammatory effects of a human CXCR2-selective antagonist. In two acute models of arthritis in the rabbit induced by knee joint injection of human IL-8 or LPS, and a chronic Ag (OVA)-induced arthritis model, administration of the antagonist at 25 mg/kg by mouth twice a day significantly reduced synovial fluid neutrophils, monocytes, and lymphocytes. In addition, in the more robust LPS- and OVA-induced arthritis models, which were characterized by increased levels of proinflammatory mediators in the synovial fluid, TNF-α, IL-8, PGE2, leukotriene B4, and leukotriene C4 levels were significantly reduced, as was erythrocyte sedimentation rate, possibly as a result of the observed decreases in serum TNF-α and IL-8 levels. In vitro, the antagonist potently inhibited human IL-8-induced chemotaxis of rabbit neutrophils (IC50 = 0.75 nM), suggesting that inhibition of leukocyte migration into the knee joint is a likely mechanism by which the CXCR2 antagonist modulates disease.

Airway infiltration of CD4+ CCR6+ Th17 type cells associated with chronic cigarette smoke induced airspace enlargement.

Recently, patients with tobacco smoke induced emphysema have been shown to exhibit classical signs of T cell mediated autoimmunity characterized by autoantibody production and Th1 type responses. As the recently described Th17 type subset has been found to play a role in the pathogenesis of a number of autoimmune diseases previously considered to be Th1 driven, we sought to examine whether a Th17 type response was associated with airspace enlargement in a murine model of emphysema. Six to eight months exposure of mice to inhalation of mainstream cigarette smoke led to progressive airspace enlargement as defined by morphometric analysis. Flow cytometric analysis of the bronchoalveolar lavage (BAL) from these mice demonstrated a significant increase in the overall number of both CD4+ and CD8+ T cells present. These cells were subsequently examined for skewing towards a Th1, Th2 or Th17 phenotype by intracellular cytokine analysis. Distinct populations of BAL CD4+ T cells were found to express IFN-gamma or IL-17 demonstrating the presence of both a Th1 and Th17 type response. No expression of the Th2 associated cytokine IL-4 was detected. Further analysis of this Th17 subset demonstrated that the majority of cells with this effector phenotype express the chemokine receptor CCR6. Together these data identify a novel T cell subset associated with pulmonary inflammation as a result of cigarette smoke exposure. Given the reported roles of CCR6 and IL-17 in promoting pulmonary inflammation, this subset may play an important role in the pathogenesis of cigarette smoke induced autoimmunity.

In vitro and in vivo characterization of a novel soluble epoxide hydrolase inhibitor

Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease.

T cell depletion protects against alveolar destruction due to chronic cigarette smoke exposure in mice

The role of T cells in chronic obstructive pulmonary disease (COPD) is not well understood. We have previously demonstrated that chronic cigarette smoke exposure can lead to the accumulation of CD4(+) and CD8(+) T cells in the alveolar airspaces in a mouse model of COPD, implicating these cells in disease pathogenesis. However, whether specific inhibition of T cell responses represents a therapeutic strategy has not been fully investigated. In this study inhibition of T cell responses through specific depleting antibodies, or the T cell immunosuppressant drug cyclosporin A, prevented airspace enlargement and neutrophil infiltration in a mouse model of chronic cigarette smoke exposure. Furthermore, individual inhibition of either CD4(+) T helper or CD8(+) T cytotoxic cells prevented airspace enlargement to a similar degree, implicating both T cell subsets as critical mediators of the adaptive immune response induced by cigarette smoke exposure. Importantly, T cell depletion resulted in significantly decreased levels of the Th17-associated cytokine IL-17A, and of caspase 3 and caspase 7 gene expression and activity, induced by cigarette smoke exposure. Finally, inhibition of T cell responses in a therapeutic manner also inhibited cigarette smoke-induced airspace enlargement, IL-17A expression, and neutrophil influx in mice. Together these data demonstrate for the first time that therapeutic inhibition of T cell responses may be efficacious in the treatment of COPD. Given that broad immunosuppression may be undesirable in COPD patients, this study provides proof-of-concept for more targeted approaches to inhibiting the role of T cells in emphysema development.

The Identification of a Novel Phosphodiesterase 4 Inhibitor, 1-Ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine (EPPA-1), with Improved Therapeutic Index using Pica Feeding in Rats as a Measure of Emetogenicity

Respiratory Center for Excellence of Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania (T.G.D., J-P.K., E.C-S., M.S.B., P.L.P.) and Stevenage, UK (D.B., A.T.N., J.W., Y.E.S., F.S.L., R.A.W., R.G.K.); Psychiatry Center of Excellence for Drug Discovery, GlaxoSmithKline, Verona, Italy (L.F., P.W.); Research Statistics Unit, GlaxoSmithKline, Collegeville, Pennsylvania (J.J.P.) JPET Fast Forward. Published on June 4, 2009 as DOI:10.1124/jpet.109.152454Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-α production by human peripheral blood mononuclear cells was roflumilast (IC50 = 5 nM) > EPPA-1 (38) > rolipram (269) > cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D50 = 0.042 mg/kg) > roflumilast (0.24) > rolipram (3.34) > cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D50 = 0.495 mg/kg) > roflumilast (1.6) > cilomilast (6.4) > EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of α2-adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D50)/(neutrophilia D50)] was EPPA-1 (578) > roflumilast (6.4) > cilomilast (1.4) > rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D50)/(neutrophilia D50)]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.

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.

Members of the papain family of cysteine proteases (cathepsins) mediate late stage processing of MHC class II-bound invariant chain (Ii), enabling dissociation of Ii, and binding of antigenic peptide to class II molecules. Recognition of cell surface class II/Ag complexes by CD4+ T cells then leads to T cell activation. Herein, we demonstrate that a pan-active cathepsin inhibitor, SB-331750, attenuated the processing of whole cell Ii p10 to CLIP by Raji cells, and DBA/1, SJL/J, and C57BL/6 splenocytes. In Raji cells and C57BL/6 splenocytes, SB-331750 inhibited class II-associated Ii processing and reduced surface class II/CLIP expression, whereas in SB-331750-treated DBA/1 and SJL/J splenocytes, class II-associated Ii processing intermediates were undetectable. Incubation of lymph node cells/splenocytes from collagen-primed DBA/1 mice and myelin basic protein-primed SJL/J mice with Ag in the presence of SB-331750 resulted in concentration-dependent inhibition of Ag-induced proliferation. In vivo administration of SB-331750 to DBA/1, SJL/J, and C57BL/6 mice inhibited splenocyte processing of whole cell Ii p10 to CLIP. Prophylactic administration of SB-331750 to collagen-immunized/boosted DBA/1 mice delayed the onset and reduced the severity of collagen-induced arthritis (CIA), and reduced paw tissue levels of IL-1β and TNF-α. Similarly, treatment of myelin basic protein-primed SJL/J lymph node cells with SB-331750 delayed the onset and reduced the severity of adoptively transferred experimental autoimmune encephalomyelitis (EAE). Therapeutic administration of SB-331750 reduced the severity of mild/moderate CIA and EAE. These results indicate that pharmacological inhibition of cathepsins attenuates CIA and EAE, potentially via inhibition of Ii processing, and subsequent Ag-induced T cell activation.

The Identification of a Novel PDE4 Inhibitor, EPPA-1, with Improved Therapeutic Index using Pica Feeding in Rats as a Measure of Emetogenicity

Respiratory Center for Excellence of Drug Discovery, GlaxoSmithKline, King of Prussia, Pennsylvania (T.G.D., J-P.K., E.C-S., M.S.B., P.L.P.) and Stevenage, UK (D.B., A.T.N., J.W., Y.E.S., F.S.L., R.A.W., R.G.K.); Psychiatry Center of Excellence for Drug Discovery, GlaxoSmithKline, Verona, Italy (L.F., P.W.); Research Statistics Unit, GlaxoSmithKline, Collegeville, Pennsylvania (J.J.P.) JPET Fast Forward. Published on June 4, 2009 as DOI:10.1124/jpet.109.152454Clinical utility of phosphodiesterase 4 (PDE4) inhibitors as anti-inflammatory agents has, to date, been limited by adverse effects including nausea and emesis, making accurate assessment of emetic versus anti-inflammatory potencies critical to the development of inhibitors with improved therapeutic indices. In the present study we determined the in vitro and in vivo anti-inflammatory potencies of the first-generation PDE4 inhibitor, rolipram, the second-generation inhibitors, roflumilast and cilomilast, and a novel third generation inhibitor, 1-ethyl-5-{5-[(4-methyl-1-piperazinyl)methyl]-1,3,4-oxadiazol-2-yl}-N-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-b]pyridin-4-amine (EPPA-1). The rank-order potency against lipopolysaccharide (LPS)-induced tumor necrosis factor-α production by human peripheral blood mononuclear cells was roflumilast (IC50 = 5 nM) > EPPA-1 (38) > rolipram (269) > cilomilast (389), and against LPS-induced pulmonary neutrophilia in the rat was EPPA-1 (D50 = 0.042 mg/kg) > roflumilast (0.24) > rolipram (3.34) > cilomilast (4.54). Pica, the consumption of non-nutritive substances in response to gastrointestinal stress, was used as a surrogate measure for emesis, giving a rank-order potency of rolipram (D50 = 0.495 mg/kg) > roflumilast (1.6) > cilomilast (6.4) > EPPA-1 (24.3). The low and high emetogenic activities of EPPA-1 and rolipram, respectively, detected in the pica model were confirmed in a second surrogate model of emesis, reversal of α2-adrenoceptor-mediated anesthesia in the mouse. The rank order of therapeutic indices derived in the rat [(pica D50)/(neutrophilia D50)] was EPPA-1 (578) > roflumilast (6.4) > cilomilast (1.4) > rolipram (0.15), consistent with the rank order derived in the ferret [(emesis D50)/(neutrophilia D50)]. These data validate rat pica feeding as a surrogate for PDE4 inhibitor-induced emesis in higher species, and identify EPPA-1 as a novel PDE4 inhibitor with an improved therapeutic index.

Regional function-structure relationships in lungs of an elastase murine model of emphysema

Changes in lung function and structure were studied using hyperpolarized (3)He MRI in an elastase-induced murine model of emphysema. The combined analysis of the apparent diffusion coefficient (ADC) and fractional ventilation (R) were used to distinguish emphysematous changes and also to develop a model for classifying sections of the lung into diseased and normal. Twelve healthy male BALB/c mice (26 ± 2 g) were randomized into healthy and elastase-induced mice and studied ∼8-11 wk after model induction. ADC and R were measured at a submillimeter planar resolution. Chord length (L(x)) data were analyzed from histology samples from the corresponding imaged slices. Logistic regression was applied to estimate the probability that an imaged pixel came from a diseased animal, and bootstrap methods (1,000 samples) were used to compare the regression results for the morphological and imaging results. Multivariate ANOVA (MANOVA) was used to analyze transformed ADC (ADC(BC)), and R (R(BC)) data and also to control for the experiment-wide error rate. MANOVA and ANOVA showed that elastase induced a statistically measureable change in the average transformed L(x) and ADC(BC) but not in the average R(BC). Marginal mean analysis demonstrated that ADC(BC) was on average 0.19 [95% confidence interval (CI): 0.16, 0.22] higher in the emphysema group, whereas R(BC) was on average 0.05 (95% CI: 0.04, 0.06) lower. Logistic regression supported the hypothesis that ADC(BC) and R(BC), together, were better at differentiating normal from diseased tissue than either measurement alone. The odds ratios for ADC(BC) and R(BC) were 7.73 (95% CI: 5.23, 11.42) and 9.14 × 10(-5) (95% CI: 3.33 × 10(-5), 25.06 × 10(-5)), respectively. Using a 50% probability cutoff, this model classified 70.6% of pixels correctly. The sensitivity and specificity of this model at the 50% cutoff were 74.9% and 65.2%, respectively. The area under the receiver operating characteristic curve was 0.76 (95% CI: 0.74, 0.78). The regression model presented can be used to map MRI data to disease probability maps. These probability maps present a future possibility of using both measurements in a more clinically feasible method of diagnosing this disease.

Effects of airborne toxicants on pulmonary function and mitochondrial DNA damage in rodent lungs

Inhalation of airborne toxicants such as cigarette smoke and ozone is a shared health risk among the worlds populations. The use of toxic herbicides like paraquat (PQ) is restricted by many countries, yet in the developing world PQ has demonstrable ill effects. The present study examined changes in pulmonary function, mitochondrial DNA (mtDNA) integrity and markers of DNA repair induced by acute or repeated exposure of PQ to rats. Similar to cigarette smoke and ozone, PQ promotes oxidative stress, and the impact of PQ on mtDNA was compared with that obtained with these agents. Tracheal instillation (i.t.) of PQ (0.01-0.075 mg/kg) dose dependently increased Penh (dyspnoea) by 48 h while body weight and temperature declined. Lung wet weight and the wet/dry weight ratio rose; for the latter, by as much as 52%. At low doses (0.02 and 0.03 mg/kg), PQ increased Penh by about 7.5-fold at 72 h. It quickly waned to near baseline levels. The lung wet/dry weight ratio remained elevated 7 days after administration coincident with marked inflammatory cell infiltrate. Repeated administration of PQ (1 per week for 8 weeks) resulted in a similar rise in Penh on the first instillation, but the magnitude of this response was markedly attenuated upon subsequent exposures. Pulmonary [lactate] and catalase activity, [8-oxodG] and histone fragmentation (cell death) were significantly increased. Repeated PQ instillation downregulated the expression of the mitochondrial-encoded genes, mtATP8, mtNd2 and mtcyB and nuclear ones for the DNA glycosylases, Ogg1, Neil1, Neil2 and Neil3. Ogg1 protein content decreased after acute and repeated PQ administration. mtDNA damage or changes in mtDNA copy number were evident in lungs of PQ-, cigarette smoke- and ozone-exposed animals. Taken together, these data indicate that loss of pulmonary function and inflammation are coupled to the loss of mtDNA integrity and DNA repair capability following exposure to airborne toxicants.

Circadian clock component REV-ERBα controls homeostatic regulation of pulmonary inflammation

Recent studies reveal that airway epithelial cells are critical pulmonary circadian pacemaker cells, mediating rhythmic inflammatory responses. Using mouse models, we now identify the rhythmic circadian repressor REV-ERB&agr; as essential to the mechanism coupling the pulmonary clock to innate immunity, involving both myeloid and bronchial epithelial cells in temporal gating and determining amplitude of response to inhaled endotoxin. Dual mutation of REV-ERB&agr; and its paralog REV-ERB&bgr; in bronchial epithelia further augmented inflammatory responses and chemokine activation, but also initiated a basal inflammatory state, revealing a critical homeostatic role for REV-ERB proteins in the suppression of the endogenous proinflammatory mechanism in unchallenged cells. However, REV-ERB&agr; plays the dominant role, as deletion of REV-ERB&bgr; alone had no impact on inflammatory responses. In turn, inflammatory challenges cause striking changes in stability and degradation of REV-ERB&agr; protein, driven by SUMOylation and ubiquitination. We developed a novel selective oxazole-based inverse agonist of REV-ERB, which protects REV-ERB&agr; protein from degradation, and used this to reveal how proinflammatory cytokines trigger rapid degradation of REV-ERB&agr; in the elaboration of an inflammatory response. Thus, dynamic changes in stability of REV-ERB&agr; protein couple the core clock to innate immunity.