Mechanistic insight into the oxazoline decomposition of DFC-M, a synthetic intermediate of florfenicol.

2-(dichloromethyl)-5[4-(methylsulfonyl)-phenyl]-4-(fluoromethyl)-oxazoline (DFC-M, 1) is a key oxazoline-containing intermediate in commercial process for the synthesis of Florfenicol (3), a marketed broad spectrum veterinary antibiotic. DFC-M was not stable in solution due to the presence of oxazoline moiety, which provided further hindrance for analytical sample preparation and HPLC analysis. Hence, the mechanistic study on the in-solution degradation of DFC-M was carried out via online and offline UPLC-HR-ESI-MS as well as in-situ NMR, and the degradation pathways were proposed. This mechanistic information, together with the follow-up solution stability study, provided crucial information regarding the solution handling and mobile phase selection for DFC-M analysis during commercial processing.

Discovery of oral and inhaled PDE4 inhibitors.

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.

Discovery of oxazole-based PDE4 inhibitors with picomolar potency.

Optimization of oxazole-based PDE4 inhibitors has led to the discovery of a series of quinolyl oxazoles, with 4-benzylcarboxamide and 5-α-aminoethyl groups which exhibit picomolar potency against PDE4. Selectivity profiles and in vivo biological activity are also reported.

Discovery of a highly potent series of oxazole-based phosphodiesterase 4 inhibitors.

Substituted quinolyl oxazoles were discovered as a novel and highly potent series of phosphodiesterase 4 (PDE4) inhibitors. Structure-activity relationship studies revealed that the oxazole core, with 4-carboxamide and 5-aminomethyl groups, is a novel PDE4 inhibitory pharmacophore. Selectivity profiles and in vivo biological activity are also reported.

Effect of inhaled roflumilast on the prevention and resolution of allergen-induced late phase airflow obstruction in Brown Norway rats.

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.

A novel, orally active CXCR1/2 receptor antagonist, Sch527123, inhibits neutrophil recruitment, mucus production, and goblet cell hyperplasia in animal models of pulmonary inflammation.

Sch527123 [2-hydroxy-N,N-dimethyl-3-[[2-[[1(R)-(5-methyl-2-furanyl)propyl]amino]-3,4-dioxo-1-cyclobuten-1-yl]amino]ben-zamide] is a potent, selective antagonist of the human CXCR1 and CXCR2 receptors (Gonsiorek et al., 2007). Here we describe its pharmacologic properties at rodent CXCR2 and at the CXCR1 and CXCR2 receptors in the cynomolgus monkey, as well as its in vivo activity in models demonstrating prominent pulmonary neutrophilia, goblet cell hyperplasia, and mucus production. Sch527123 bound with high affinity to the CXCR2 receptors of mouse (K(d) = 0.20 nM), rat (K(d) = 0.20 nM), and cynomolgus monkey (K(d) = 0.08 nM) and was a potent antagonist of CXCR2-mediated chemotaxis (IC(50) approximately 3-6 nM). In contrast, Sch527123 bound to cynomolgus CXCR1 with lesser affinity (K(d) = 41 nM) and weakly inhibited cynomolgus CXCR1-mediated chemotaxis (IC(50) approximately 1000 nM). Oral treatment with Sch527123 blocked pulmonary neutrophilia (ED(50) = 1.2 mg/kg) and goblet cell hyperplasia (32-38% inhibition at 1-3 mg/kg) in mice following the intranasal lipopolysaccharide (LPS) administration. In rats, Sch527123 suppressed the pulmonary neutrophilia (ED(50) = 1.8 mg/kg) and increase in bronchoalveolar lavage (BAL) mucin content (ED(50) =<0.1 mg/kg) induced by intratracheal (i.t.) LPS. Sch527123 also suppressed the pulmonary neutrophilia (ED(50) = 1.3 mg/kg), goblet cell hyperplasia (ED(50) = 0.7 mg/kg), and increase in BAL mucin content (ED(50) = <1 mg/kg) in rats after i.t. administration of vanadium pentoxide. In cynomolgus monkeys, Sch527123 reduced the pulmonary neutrophilia induced by repeat bronchoscopy and lavage (ED(50) = 0.3 mg/kg). Therefore, Sch527123 may offer benefit for the treatment of inflammatory lung disorders in which pulmonary neutrophilia and mucus hypersecretion are important components of the underlying disease pathology.

Temporal profile of forced expiratory lung function in allergen-challenged Brown-Norway rats.

The Brown-Norway rat is often used to study the allergic pulmonary response. However, relatively little is known about the delayed phase reactions after allergen challenge in this species. To evaluate the temporal changes in lung function and elucidate the mechanisms involved in the delayed phase response, Brown-Norway rats were sensitized and challenged to aerosolized ovalbumin and lung functions were measured by forced expiratory maneuvers and forced oscillation for up to 10 days after a single antigen challenge. Statistically significant (P < 0.05) reductions in inspiratory capacity, forced vital capacity, functional residual capacity, peak expiratory flow and maximum mid-expiratory flow and increases in respiratory system resistance and elastance were seen by 1 to 3 days after ovalbumin challenge that returned to baseline by 10 days. The reductions in lung function after ovalbumin challenge were blocked by the corticosteroid, betamethasone (1 mg/kg, p.o.). Histological evaluation of lung tissue of sensitized rats demonstrated evidence of interstitial pulmonary edema, an increase in tissue eosinophils and an increase in Periodic Acid Schiff-positive cells in the airway epithelium. Bronchoalveolar lavage fluid samples showed large numbers of eosinophils and increased mucin content up to 6 days after antigen challenge. There was also an increase in wet-to-dry lung weight ratio in the lungs of sensitized rats after antigen. These results demonstrate that prolonged reductions in lung function occur after a single antigen challenge in Brown-Norway rats that is probably due to inflammatory processes producing interstitial pulmonary edema, mucus secretion and cellular influx into the lungs.

An enzyme-linked immunosorbent assay (ELISA) for the determination of mucin levels in bronchoalveolar lavage fluid.

INTRODUCTION: A method to measure the mucin concentration in bronchoalveolar lavage (BAL) fluid was developed to aid efforts to identify pharmacologically the mechanisms that modulate pathophysiological mucin secretion. Mucins are the major macromolecular components of mucus. In the airways, mucus is the first line of defense against inhaled microorganisms (infection) and particulates (irritation). METHODS: An enzyme-linked immunosorbent assay (ELISA) was developed, comparing two monoclonal anti-mucin antibodies (A10G5 and 45M1) raised to human mucin, to quantify the mucin in BAL fluid from animal models of pulmonary inflammation. To validate the ELISA method, rats were exposed to ovalbumin (OVA, in sensitized rats), lipopolysaccharide (LPS), vanadium pentoxide (V(2)O(5)), or saline. One hundred microliters of BAL fluid was analyzed for mucin concentration. Pooled BAL fluid from untreated rats was used as an internal "plate standard", as a standard mucin that cross-reacts with A10G5 was unavailable. RESULTS: We found both antibodies reacted with rat, human, and guinea-pig mucin; where the 45M1 antibody also reacted with the mucin in porcine BAL, while A10G5 did not. We determined the mucin concentration in each BAL fluid sample relative to the standard, defined as a mucin concentration of 100 plate units. BAL fluid from LPS (218+/-25 plate units, n=5), OVA (386+/-31, n=3), V(2)O(5) (1208+/-450, n=6) challenged rats displayed significantly elevated mucin concentration over their saline controls (126+/-22, n=12). Subsequently, the 45M1 antibody displayed immunoreactivity with a commercially available crude preparation of porcine stomach mucin, allowing us to calculate the concentration of mucin directly compared to the known concentration of the porcine stomach mucin standard. Both the 45M1 and A10G5 based ELISA assays detected higher mucin content in the saline challenged rat than the saline challenged guinea pig BAL. DISCUSSION: The recent availability of the 45M1 antibody and the use of the crude purification of porcine stomach mucin as a reference standard should allow for direct comparison of mucin concentration in BAL (and other fluids).

Airway closure after antigen challenge in cynomolgus monkeys: effect of the histamine H1 receptor antagonist, chlorpheniramine maleate.

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.

Effect of histamine, albuterol and deep inspiration on airway and lung tissue mechanics in cynomolgus monkeys.

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.

Cough reflex in allergic dogs.

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.

Antitussive activity of the tachykinin NK1 receptor antagonist, CP-99994, in dogs.

CP-99994 [(+)-(2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine] is a selective tachykinin NK(1) receptor antagonist that inhibits cough in guinea pigs and cats. This study examined the antitussive effects of CP-99994 in dogs produced by mechanical stimulation of the intrathoracic trachea. CP-99994 (10 mg/kg, p.o.) inhibited cough frequency by 52% at 2 h, 31% at 6 h and by 21% at 24 h. Cough amplitude was inhibited by 45% at 6 h but unchanged at 2 and 24 h after CP-99994. Plasma levels of CP-99994 were highest at 2 h (75+/-26 ng/ml) and fell to 22+/-6 ng/ml at 6 h. These results demonstrate antitussive activity of CP-99994 in dogs at a dose proven to antagonize tachykinin NK(1) receptors in this species.

Role of cholinergic reflexes on the bronchoconstrictor reactivity to neurokinin a in allergic dogs.

Neurokinin A (NKA) potentiates airway cholinergic neurotransmission in several species. In this study, the role of cholinergic reflexes on the bronchoconstrictor response to NKA was evaluated in non-sensitized dogs and in allergic dogs neonatally sensitized to ragweed in which heightened bronchoconstrictor reactivity to NKA has previously been observed. Cardiopulmonary functions, including pulmonary resistance (R(L)) were measured in anesthetized, spontaneously breathing dogs before and after increasing concentrations of aerosolized NKA. The provocative concentrations of NKA increasing R(L) by 25% above the baseline (PC(25)) was measured before and after ( approximately 10 min) aerosolized saline or ipratropium bromide (0.01%). This concentration of ipratropium produced a 250-fold shift in the methacholine dose-response curve. In sensitized dogs, NKA bronchoconstrictor reactivity (PC(25)=0.050+/-0.011%) was 2.5 times more potent than that of non-sensitized controls (PC(25)=0.177+/-0.031%). Ipratropium bromide inhibited the bronchoconstrictor response to NKA in both sensitized and non-sensitized dogs and after ipratropium, NKA reactivity was 5.2-fold less in allergic dogs (PC(25)=0.246+/-0.048%) as compared to 3.5 fold less in non-sensitized controls (PC(25)=0.622+/-0.106%). In conclusion, cholinergic reflexes are important components of the bronchoconstrictor response to NKA in dogs particularly in those sensitized neonatally to ragweed. It is speculated that heightened activity of cholinergic reflexes contributes to the bronchial hyperresponsiveness seen in allergic dogs.