Anthony T. Nials

EP4 receptor as a new target for bronchodilator therapy

Background Asthma and chronic obstructive pulmonary disease are airway inflammatory diseases characterised by airflow obstruction. Currently approved bronchodilators such as long-acting β2 adrenoceptor agonists are the mainstay treatments but often fail to relieve symptoms of chronic obstructive pulmonary disease and severe asthma and safety concerns have been raised over long-term use. The aim of the study was to identify the receptor involved in prostaglandin E2 (PGE2)-induced relaxation in guinea pig, murine, monkey, rat and human airways in vitro. Methods Using an extensive range of pharmacological tools, the relaxant potential of PGE2 and selective agonists for the EP1–4 receptors in the presence and absence of selective antagonists in guinea pig, murine, monkey, rat and human isolated airways was investigated. Results In agreement with previous studies, it was found that the EP2 receptor mediates PGE2-induced relaxation of guinea pig, murine and monkey trachea and that the EP4 receptor mediates PGE2-induced relaxation of the rat trachea. These data have been confirmed in murine airways from EP2 receptor-deficient mice (Ptger2). In contrast to previous publications, a role for the EP4 receptor in relaxant responses in human airways in vitro was found. Relaxant activity of AH13205 (EP2 agonist) was also demonstrated in guinea pig but not human airway tissue, which may explain its failure in clinical studies. Conclusion Identification of the receptor mediating PGE2-induced relaxation represents a key step in developing a novel bronchodilator therapy. These data explain the lack of bronchodilator activity observed with selective EP2 receptor agonists in clinical studies.

GSK256066, an Exceptionally High-Affinity and Selective Inhibitor of Phosphodiesterase 4 Suitable for Administration by Inhalation: In Vitro, Kinetic, and In Vivo Characterization

Oral phosphodiesterase (PDE) 4 inhibitors such as roflumilast have established the potential of PDE4 inhibition for the treatment of respiratory diseases. However, PDE4 inhibitor efficacy is limited by mechanism-related side effects such as emesis and nausea. Delivering the inhibitor by the inhaled route may improve therapeutic index, and we describe 6-({3-[(dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy) phenyl]amino}-3-quinolinecarboxamide (GSK256066), an exceptionally high-affinity inhibitor of PDE4 designed for inhaled administration. GSK256066 is a slow and tight binding inhibitor of PDE4B (apparent IC50 3.2 pM; steady-state IC50 <0.5 pM), which is more potent than any previously documented compound, for example, roflumilast (IC50 390 pM), tofimilast (IC50 1.6 nM), and cilomilast (IC50 74 nM). Consistent with this, GSK256066 inhibited tumor necrosis factor α production by lipopolysaccharide (LPS)-stimulated human peripheral blood monocytes with 0.01 nM IC50 (compared with IC50 values of 5, 22, and 389 nM for roflumilast, tofimilast, and cilomilast, respectively) and by LPS-stimulated whole blood with 126 pM IC50. GSK256066 was highly selective for PDE4 (>380,000-fold versus PDE1, PDE2, PDE3, PDE5, and PDE6 and >2500-fold against PDE7), inhibited PDE4 isoforms A-D with equal affinity, and had a substantial high-affinity rolipram binding site ratio (>17). When administered intratracheally to rats, GSK256066 inhibited LPS-induced pulmonary neutrophilia with ED50 values of 1.1 μg/kg (aqueous suspension) and 2.9 μg/kg (dry powder formulation) and was more potent than an aqueous suspension of the corticosteroid fluticasone propionate (ED50 9.3 μg/kg). Thus, GSK256066 has been demonstrated to have exceptional potency in vitro and in vivo and is being clinically investigated as a treatment for chronic obstructive pulmonary disease.

In Vivo Characterization of GSK256066, a High-Affinity Inhaled Phosphodiesterase 4 Inhibitor

Oral phosphodiesterase (PDE) 4 inhibitors have demonstrated clinical efficacy in chronic obstructive pulmonary disease and asthma. Preclinical and clinical investigation of inhaled PDE4 inhibitors is ongoing. 6-({3-[(Dimethylamino)carbonyl]phenyl}sulfonyl)-8-methyl-4-{[3-methyloxy)phenyl]amino}-3-quinolinecarboxamide (GSK256066) is an exceptionally high-affinity and selective inhibitor of PDE4 designed for inhaled delivery. The aim of these studies was to investigate the potency, duration of action, and therapeutic index of GSK256066 in animal models of pulmonary inflammation. The effects of intratracheally administered GSK256066 were investigated in rat lipopolysaccharide (LPS)- and ovalbumin (OVA)-induced models of acute pulmonary inflammation. In some studies, fluticasone propionate (FP) was included as a comparator. The therapeutic index (anti-inflammatory effect versus emesis) of GSK256066 was studied in ferrets where acute pulmonary inflammation was induced with inhaled LPS. In rats, GSK256066 and FP caused significant (p < 0.05) inhibition of LPS-induced pulmonary neutrophilia. The duration of action of GSK256066 at 10 × ED50 dose (10 μg/kg) was 12 h. GSK256066 and FP also inhibited LPS-induced increases in exhaled nitric oxide (ED50 35 and 92 μg/kg, respectively). In addition, GSK256066 inhibited pulmonary eosinophilia in rats exposed to OVA (ED50 0.4 μg/kg). In ferrets, inhaled GSK256066 inhibited LPS-induced pulmonary neutrophilia (ED50 18 μg/kg), and no emetic episodes were observed. Thus, GSK256066 may have an improved therapeutic index compared with oral PDE4 inhibitors, e.g., cilomilast and roflumilast. In summary, GSK256066 demonstrates potent and long-lasting anti-inflammatory effects in animal models of pulmonary inflammation and does not induce emetic episodes in ferrets. GSK256066 has potential as an inhaled therapeutic for the treatment of asthma and chronic obstructive pulmonary disease.

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.

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.

At last, a truly selective EP2 receptor antagonist

Ever since the discovery of prostaglandin E2 (PGE2), this lipid mediator has been the focus of intense research. The diverse biological effects of PGE2 are due, at least in part, to the existence of four distinct receptors (EP1–4). This can complicate the analysis of the biological effects produced by PGE2. While there are currently selective pharmacological tools to explore the roles of the EP1,3,4 receptors in cellular and tissue responses, analysis of EP2 receptor‐induced responses has been hampered by the lack of a selective EP2 receptor antagonist. The recent publication in this journal by af Forselles et al. suggests that such a tool compound is now available. In their manuscript, the authors describe a series of experiments that show PF‐04418948 to be a potent and selective EP2 receptor antagonist. The discovery of this tool compound will interest many scientists and through collaborations with Pfizer they may have access to PF‐04418948 to facilitate further investigation of the biology of this fascinating lipid mediator.

LPS exacerbates functional and inflammatory responses to ovalbumin and decreases sensitivity to inhaled fluticasone propionate in a guinea pig model of asthma

Asthma exacerbations contribute to corticosteroid insensitivity. LPS is ubiquitous in the environment. It causes bronchoconstriction and airway inflammation and may therefore exacerbate allergen responses. This study examined whether LPS and ovalbumin co‐administration could exacerbate the airway inflammatory and functional responses to ovalbumin in conscious guinea pigs and whether these exacerbated responses were insensitive to inhaled corticosteroid treatment with fluticasone propionate (FP).

Route of administration affects corticosteroid sensitivity of a combined ovalbumin and lipopolysaccharide model of asthma exacerbation in guinea-pigs

Lipopolysaccharide (LPS) contributes to asthma exacerbations and development of inhaled corticosteroid insensitivity. Complete resistance to systemic corticosteroids is rare, and most patients lie on a continuum of steroid responsiveness. This study aimed to examine the sensitivity of combined ovalbumin- (Ova) and LPS-induced functional and inflammatory responses to inhaled and systemic corticosteroid in conscious guinea pigs to test the hypothesis that the route of administration affects sensitivity. Guinea pigs were sensitized to Ova and challenged with inhaled Ova alone or combined with LPS. Airway function was determined by measuring specific airway conductance via whole-body plethysmography. Airway hyper-responsiveness to histamine was determined before and 24 hours post-Ova challenge. Airway inflammation and underlying mechanisms were determined from bronchoalveolar lavage cell counts and lung tissue cytokines. Vehicle or dexamethasone was administered by once-daily i.p. injection (5, 10, or 20 mg/kg) or twice-daily inhalation (4 or 20 mg/ml) for 6 days before Ova challenge or Ova with LPS. LPS exacerbated Ova-induced responses, elongating early asthmatic responses (EAR), prolonging histamine bronchoconstriction, and further elevating airway inflammation. Intraperitoneal dexamethasone (20 mg/kg) significantly reduced the elongated EAR and airway inflammation but not the increased bronchoconstriction to histamine. In contrast, inhaled dexamethasone (20 mg/ml), which inhibited responses to Ova alone, did not significantly reduce functional and inflammatory responses to combined Ova and LPS. Combined Ova and LPS–induced functional and inflammatory responses are insensitive to inhaled, but they are only partially sensitive to systemic, dexamethasone. This finding suggests that the route of corticosteroid administration may be important in determining corticosteroid sensitivity of asthmatic responses.

Adjustment of sensitisation and challenge protocols restores functional and inflammatory responses to ovalbumin in guinea-pigs

Introduction Inhalation of antigen in atopic asthma induces early (EAR) and late asthmatic responses (LARs), inflammatory cell infiltration and airways hyperresponsiveness (AHR). Previously, we have established a protocol of sensitisation and subsequent ovalbumin (Ova) inhalation challenge in guinea-pigs which induced these 4 features (Smith & Broadley, 2007). However, the responses of guinea-pigs to Ova challenge have recently declined, producing no LAR or AHR and diminished EAR and cells. By making cumulative modifications to the protocol, we sought to restore these features. Methods Guinea-pigs were sensitised with Ova (i.p. 100 or 150 μg) on days 1 and 5 or days 1, 4 and 7 and challenged with nebulised Ova (100 or 300 μg/ml, 1 h) on day 15. Airway function was measured in conscious guinea-pigs by whole-body plethysmography to record specific airway conductance (sGaw). Airway responsiveness to aerosolized histamine (0.3 mM) was determined before and 24 h after Ova challenge. Bronchoalveolar lavage was performed for total and differential inflammatory cell counts. Lung sections were stained for counting of eosinophils. Results Lack of AHR and LAR with the original protocol was confirmed. Increasing the Ova challenge concentration from 100 to 300 μg/ml restored AHR and eosinophils and increased the peak of the EAR. Increasing the number of sensitisation injections from 2 to 3 did not alter the responses. Increasing the Ova sensitisation concentration from 100 to 150 μg significantly increased total cells, particularly eosinophils. A LAR was revealed and lymphocytes and eosinophils increased when either the Al(OH)3 concentration was increased or the duration between the final sensitisation injection and Ova challenge was extended from 15 to 21 days. Discussion This study has shown that declining allergic responses to Ova in guinea-pigs could be restored by increasing the sensitisation and challenge conditions. It has also demonstrated an important dissociation between EAR, LAR, AHR and inflammation.

Modelling the asthma phenotype: impact of cigarette smoke exposure

BackgroundAsthmatics that are exposed to inhaled pollutants such as cigarette smoke (CS) have increased symptom severity. Approximately 25% of adult asthmatics are thought to be active smokers and many sufferers, especially in the third world, are exposed to high levels of inhaled pollutants. The mechanism by which CS or other airborne pollutants alter the disease phenotype and the effectiveness of treatment in asthma is not known. The aim of this study was to determine the impact of CS exposure on the phenotype and treatment sensitivity of rodent models of allergic asthma.MethodsModels of allergic asthma were configured that mimicked aspects of the asthma phenotype and the effect of CS exposure investigated. In some experiments, treatment with gold standard asthma therapies was investigated and end-points such as airway cellular burden, late asthmatic response (LAR) and airway hyper-Reactivity (AHR) assessed.ResultsCS co-exposure caused an increase in the LAR but interestingly attenuated the AHR. The effectiveness of LABA, LAMA and glucocorticoid treatment on LAR appeared to be retained in the CS-exposed model system. The eosinophilia or lymphocyte burden was not altered by CS co-exposure, nor did CS appear to alter the effectiveness of glucocorticoid treatment. Steroids, however failed to reduce the neutrophilic inflammation in sensitized mice exposed to CS.ConclusionsThese model data have certain parallels with clinical findings in asthmatics, where CS exposure did not impact the anti-inflammatory efficacy of steroids but attenuated AHR and enhanced symptoms such as the bronchospasm associated with the LAR. These model systems may be utilised to investigate how CS and other airborne pollutants impact the asthma phenotype; providing the opportunity to identify novel targets.