GPR40 (FFAR1 or FFA1) is a G protein-coupled receptor, primarily expressed in pancreatic islet β-cells and intestinal enteroendocrine cells. When activated by fatty acids, GPR40 elicits increased insulin secretion from islet β-cells only in the presence of elevated glucose levels. Towards this end, studies were undertaken towards discovering a novel GPR40 Agonist whose mode of action is via Positive Allosteric Modulation of the GPR40 receptor (AgoPAM). Efforts were made to identify a suitable GPR40 AgoPAM tool molecule to investigate mechanism of action and de-risk liver toxicity of GPR40 AgoPAMs due to reactive acyl-glucuronide (AG) metabolites.

The optimization of oxazole-based PDE4 inhibitor 1 has led to the identification of both oral (compound 16) and inhaled (compound 34) PDE4 inhibitors. Selectivity against PDE10/PDE11, off target screening, and in vivo activity in the rat are discussed.

A strategy to overcome the side effect liabilities of oral PDE4 inhibitors has been to deliver the drugs by inhalation. In this report, we identify 1-[[5-(1(S)-aminoethly)-2-[8-methoxy-2-(triflurormethyl)-5-quinolinyl]-4-oxazolyl] carbonyl]-4(R)-[(cyclopropylcarbonyl)amino]-L-proline, ethyl ester xinafoate salt, (COMPOUND 1) as a potent and selective inhibitor of PDE4 with biological and pharmacokinetic properties suitable for delivery by the inhaled route. COMPOUND 1 potently inhibits human PDE4 (IC50=70pM) with little or no activity against other PDEs. It is highly potent against PDE4B and PDE4D which are important isoforms of PDE4 controlling inflammation and airway functions. In an allergen-challenged Brown Norway rat model of asthma, COMPOUND 1 inhibited the late phase influx of inflammatory cells and reductions in lung function following its administration by the intratracheal or nose-only routes of administration. Important differences were seen between intratracheal COMPOUND 1 and our previously published results with the oral PDE4 inhibitor roflumilast (Celly et al., 2005), as COMPOUND 1 rapidly (within 1h) reversed the decline in lung function when it was given therapeutically to rats already challenged with antigen. COMPOUND 1 was weakly active by the oral route which is a finding consistent with results showing this compound has poor oral bioavailability in animals. Positive interactions between COMPOUND 1 and albuterol, and COMPOUND 1 and mometasone furoate were seen on the improvement in lung functions in allergen-challenged rats. These results identify COMPOUND 1 as a potent and selective inhibitor of PDE4 with properties suitable for delivery by inhalation.

Manual total and differential leukocyte counting in bronchoalveolar lavage fluids (BALF) by visual microscopy is a standard means of evaluating airway inflammation and the anti-inflammatory properties of therapeutics in various animal models of lung disease. The manual cell counting method derives total leukocyte counts from BALF with a hemocytometer, and cell differentials (mononuclear, neutrophil, eosinophil) are calculated from the percentage of each cell type taken from a count of at least 200 cells on a stained cytocentrifuge preparation of the BALF cells. These manual methods are time-consuming and have inherent error–variability. The ADVIA 120 Hematology System is an automated analyzer designed to perform total and differential leukocyte analysis of blood. With the light scattering, cell lysis resistance, and cytochemical staining data from a BALF sample processed by the ADIVA, a BALF total leukocyte count and differential analysis is provided in approximately 30 s. In order to correlate automated BALF leukocyte counting by the ADVIA 120 Hematology System with manual counting, we developed a manual red blood cell lysing and white blood cell staining technique for BALF cells similar to the process used by the ADVIA. Significant correlations for BALF white blood cells were obtained for the manual (microscopic analysis) and the automated (ADVIA) methods. Comparison of manual and automated cell counts also generates the same conclusions about anti-inflammatory drug efficacy. Both manual and automated cell counting methods agree that 3 mg/kg orally administered dexamethasone inhibited cigarette-smoke-induced total BALF cell counts by ∼65% in mice and 42 μg/kg fluticasone propionate delivered by nose-only inhalation inhibited allergen-induced total BALF cells by 77% in rats. The use of the ADVIA to perform total and differential leukocyte counts in BALF will save time spent manually counting cells and this instrument will standardize the analysis of white blood cells across the laboratories currently using various manual counting preparations and procedures.

Orally active phosphodiesterase 4 (PDE4) inhibitors have been developed for the treatment of asthma and chronic obstructive pulmonary disorders (COPD) although their full development has been limited by adverse side effects. Administration of PDE4 inhibitors by inhalation may improve their therapeutic index, but limited information exists on the efficacy of inhaled PDE4 inhibitors to improve lung function. In this study in ovalbumin-sensitized Brown Norway rats, roflumilast was given either intratracheally or by nose-only inhalation and changes in lung function (forced vital capacity, FVC; peak expiratory flow, PEF) and inflammatory cell influx (total cells, eosinophils and neutrophils) into the bronchoalveolar lavage (BAL) fluid were evaluated 24 h after allergen challenge. Intratracheal roflumilast, given 5 h before antigen challenge, inhibited the antigen-induced reductions in FVC (ED50 = 140 microg/kg, i.t.) and total cells appearing in the bronchoalveolar lavage fluid (ED50 = 50 microg/kg, i.t.). By the nose-only inhalation route, roflumilast reduced the bronchoalveolar lavage fluid total cells (ED50 = 10 microg/kg, estimated pulmonary deposition). Intratracheal roflumilast (600 microg/kg, i.t.) was also given to rats 24 h after the antigen challenge and reversed the antigen-induced reductions of FVC by 38% at 1 h, 54% at 5 h and 71% by 16 h. Intratracheal roflumilast also reduced the number of inflammatory cells in the bronchoalveolar lavage fluid and reduced the interstitial airway edema caused by the antigen challenge. These results support the development of inhaled PDE4 inhibitors for the treatment of asthma and COPD, particularly for the improvement of lung function.

Forced oscillation is a technique that has been used to measure airway and lung tissue impedance. To evaluate airway and lung tissue impedance in a colony of cynomolgus monkeys housed at Schering-Plough Research Institute, a forced oscillation technique was used to measure Newtonian resistance (R(N)), tissue damping (G), tissue elastance (H) and lung hysteresivity (eta). Functional residual capacity (FRC) was also measured to correlate the lung impedance data with FRC. There was no difference in R(N), G, H and eta between Ascaris sensitive allergic monkeys (n=25) and a small cohort (n=5) of non-allergic monkeys under baseline conditions. However, a highly significant (p<0.0001) negative correlation (r=0.71) was found between FRC and H. Significant correlations were also found between FRC and G (r=0.53) and FRC and R(N) (r=0.50). Bronchoprovocation with aerosolized histamine increased R(N), G, H and eta and reduced FRC by 29+/-3% (n=30) from baseline. In monkeys that were hyperreactive to the histamine challenge, an exaggerated increase in lung tissue damping was seen whereas monkeys that were less reactive to the histamine showed greater increases in R(N). Aerosolized albuterol (0.003-3mg/ml) produced a concentration-dependent reversal of the increases in R(N), G, H and eta induced by histamine with the greatest reversal seen on R(N). Deep inspiration, performed after the aerosolized albuterol exposure, also reversed the histamine-induced changes in R(N), G, and H with the complete reversal seen on the increase in H. These results demonstrate that significant correlations exist between airway and lung tissue impedance and FRC and that airway and lung tissue mechanics contribute significantly to inherent bronchoconstrictor reactivity and to the bronchodilator response to a beta-adrenergic agonist and deep inspiration in cynomolgus monkeys.

Background: Airway closure is frequently observed in human asthma. However, limited information exists on the factors that cause this condition. In this study, an allergic cynomolgus monkey model was used to characterize the condition of airway closure and assess the contribution of histamine H1 receptors to this response. Methods: Oscillatory lung mechanics, arterial blood gases during ventilation on 100% O2 and functional residual capacity (FRC) assessed by helium dilution were measured before and then 10 min and 24 h after Ascaris aerosol challenge in 12 male Ascaris-sensitive cynomolgus monkeys. The monkeys were pretreated with intravenous saline or chlorpheniramine maleate (0.3 mg/kg) in a randomized crossover design. Results: Ascaris challenge produced a large increase in airway resistance, an increase in lung tissue damping (G) that measures ventilation inhomogeneity in the lung, a reduction in arterial oxygen tension (PaO2) during ventilation on 100% O2 and a reduction in FRC. These effects were seen 10 min after the Ascaris challenge, but by 24 h, these parameters had returned close to the baseline values. Chlorpheniramine maleate (0.3 mg/kg, i.v.) produced a 12-fold shift in the histamine bronchoconstrictor dose-response curve. Pretreatment of monkeys with chlorpheniramine maleate (0.3 mg/kg, i.v.) attenuated the increase in airway resistance induced by Ascaris challenge, but had only a small effect on the increase in G and the reductions in PaO2 and FRC after antigen. Conclusions: These results demonstrate that airway closure occurs immediately after the antigen challenge in allergic cynomolgus monkeys and that histamine H1 receptors contribute very minimally to this response.

This study investigated the effects of antigen challenge on the cough reflex in dogs that were neonatally sensitized to ragweed. Tidal volume (V(T)), respiratory rate (f), pulmonary resistance (R(L)), dynamic lung compliance (C(Dyn)) and the number and amplitude (increase in mean peak expiratory pressure) of coughs induced by mechanical stimulation of the intrathoracic trachea were measured in propofol-anesthetized dogs. Aerosolized ragweed challenge had no effect to induce spontaneous cough but increased f and R(L) and reduced V(T) and C(Dyn). Mechanical stimulation of the intrathoracic trachea at this time produced 19+/-5 coughs with an average increase in cough amplitude of 11+/-1 cm H(2)O which differed significantly from the number (9+/-2 coughs) and amplitude (30+/-5.5 cm H(2)O) of mechanically induced coughs after treatment with aerosolized saline. Both the number and amplitude of mechanically induced coughs returned to baseline values by 24-48 h after the ragweed challenge. Similar results were obtained after challenge with aerosolized histamine (0.3-1% histamine) that did not induce spontaneous coughs but increased f, reduced V(T) and decreased C(Dyn) and increased the number but reduced the amplitude of the mechanically induced coughs. In conclusion, both antigen and histamine bronchoprovocation changed the characteristics of the mechanically induced cough in dogs to a response of increased cough number but reduced mean expiratory cough amplitude.

OBJECTIVE To assess the agreement between three measurements of arterial oxygen saturation (SpO2, SaO2 and ScO2) in anesthetized cynomolgus monkeys. STUDY DESIGN Prospective study. ANIMALS Eleven mature, male cynomolgus monkeys (Macaca fasicularis). METHODS Monkeys were anesthetized with intramuscular ketamine followed by intravenous propofol. The trachea of each was intubated and the lungs ventilated. Arterial oxygen saturation was measured with a Nonin 8500 V pulse oximeter, using a lingual clip on the cheek. Arterial blood samples were taken from an indwelling catheter. Inspired oxygen concentration was varied from 12 to 20%, and 88 paired arterial blood samples and saturation measurements were taken. Arterial oxygen saturation in the blood samples was measured using a cooximeter. The saturation was also calculated from the arterial oxygen tension using the Adair equation. The results were compared using Bland and Altman's method. RESULTS The pulse oximeter readings were 2.7% higher than that of the cooximeter, with a limit of agreement of -3.9 to 9.3%. The pulse oximeter readings were 1.8% higher than the calculated saturation, with a limit of agreement of -6.5% to 10.1%. The cooximeter readings were 0.9% lower than the calculated saturation, with a limit of agreement of -5.6% to 3.8%. CONCLUSIONS The agreement between SpO2 and other measurements of arterial oxygen saturation in this study is typical for this technique. The bias and limits of agreement are consistent with reports in other species. CLINICAL RELEVANCE The Nonin 8500 V is a useful pulse oximeter for clinical use in primates.

Introduction: This study describes a method to measure the cough reflex in dogs that is simple to perform, requires no surgical intervention and can be used to profile efficacy and side-effect liabilities of antitussive drags. Methods: Experiments were performed in propofol-anesthetized dogs in which cardiopulmonary functions were non-invasively monitored before and after the induction of cough produced by spraying 0.75 nil of distilled water into the trachea. Results: The magnitude of the cough response, measured by the frequency and amplitude was not different for individual dogs performed with repeated trials on different days. Treatment with the opioid antitussive drug, torbutrol (0.055 - 0.0055 mg/kg, s.c.) inhibited the cough frequency but not the amplitude induced by the water challenge. Furthermore, side effects of torbutrol were identified as mild respiratory depression and an anesthetic-sparing effect with propofol. Discussion: This method offers many distinct advantages to evaluate efficacy of antitussive drags including the fact that no surgery is required, it takes only 15-20 min to complete an experiment, and it can be used to simultaneously profile antitussive and side effect liabilities of drags developed for the treatment of cough. (C) 2001 Elsevier Science Inc. All rights reserved.