Treatment of acute lung injury by targeting MG53-mediated cell membrane repair.

Injury to lung epithelial cells has a role in multiple lung diseases. We previously identified mitsugumin 53 (MG53) as a component of the cell membrane repair machinery in striated muscle cells. Here we show that MG53 also has a physiological role in the lung and may be used as a treatment in animal models of acute lung injury. Mice lacking MG53 show increased susceptibility to ischaemia-reperfusion and overventilation-induced injury to the lung when compared with wild-type mice. Extracellular application of recombinant human MG53 (rhMG53) protein protects cultured lung epithelial cells against anoxia/reoxygenation-induced injuries. Intravenous delivery or inhalation of rhMG53 reduces symptoms in rodent models of acute lung injury and emphysema. Repetitive administration of rhMG53 improves pulmonary structure associated with chronic lung injury in mice. Our data indicate a physiological function for MG53 in the lung and suggest that targeting membrane repair may be an effective means for treatment or prevention of lung diseases.

Pharmacological evaluation of selective α2c-adrenergic agonists in experimental animal models of nasal congestion.

Nasal congestion is one of the most troublesome symptoms of many upper airways diseases. We characterized the effect of selective α2c-adrenergic agonists in animal models of nasal congestion. In porcine mucosa tissue, compound A and compound B contracted nasal veins with only modest effects on arteries. In in vivo experiments, we examined the nasal decongestant dose-response characteristics, pharmacokinetic/pharmacodynamic relationship, duration of action, potential development of tolerance, and topical efficacy of α2c-adrenergic agonists. Acoustic rhinometry was used to determine nasal cavity dimensions following intranasal compound 48/80 (1%, 75 µl). In feline experiments, compound 48/80 decreased nasal cavity volume and minimum cross-sectional areas by 77% and 40%, respectively. Oral administration of compound A (0.1-3.0 mg/kg), compound B (0.3-5.0 mg/kg), and d-pseudoephedrine (0.3 and 1.0 mg/kg) produced dose-dependent decongestion. Unlike d-pseudoephedrine, compounds A and B did not alter systolic blood pressure. The plasma exposure of compound A to produce a robust decongestion (EC(80)) was 500 nM, which related well to the duration of action of approximately 4.0 hours. No tolerance to the decongestant effect of compound A (1.0 mg/kg p.o.) was observed. To study the topical efficacies of compounds A and B, the drugs were given topically 30 minutes after compound 48/80 (a therapeutic paradigm) where both agents reversed nasal congestion. Finally, nasal-decongestive activity was confirmed in the dog. We demonstrate that α2c-adrenergic agonists behave as nasal decongestants without cardiovascular actions in animal models of upper airway congestion.

Mineralocorticoid receptor antagonists attenuate pulmonary inflammation and bleomycin-evoked fibrosis in rodent models.

Accumulating evidence indicates protective actions of mineralocorticoid antagonists (MR antagonists) on cardiovascular pathology, which includes blunting vascular inflammation and myocardial fibrosis. We examined the anti-inflammatory and anti-fibrotic potential of MR antagonists in rodent respiratory models. In an ovalbumin allergic and challenged Brown Norway rat model, the total cell count in nasal lavage was 29,348 ± 5451, which was blocked by spironolactone (0.3-60 mg/kg, p.o.) and eplerenone (0.3-30 mg/kg, p.o.). We also found that MR antagonists attenuated pulmonary inflammation in the Brown Norway rat. A series of experiments were conducted to determine the actions of MR blockade in acute/chronic lung injury models. (1) Ex vivo lung slice rat experiments found that eplerenone (0.01 and 10 µM) and spironolactone (10 µM) diminished lung hydroxyproline concentrations by 55 ± 5, 122 ± 9, and 83 ± 8%. (2) In in vivo studies, MR antagonists attenuated the increases in bronchioalveolar lavage (BAL) neutrophils and macrophages caused by lung bleomycin exposure. In separate studies, bleomycin (4.0 U/kg, i.t.) increased lung levels of hydroxyproline by approximately 155%, which was blocked by spironolactone (10-60 mg/kg, p.o.). In a rat Lipopolysaccharide (LPS) model, spironolactone inhibited acute increases in BAL cytokines with moderate effects on neutrophils. Finally, we found that chronic LPS exposure significantly increased end expiratory lung and decreased lung elastance in the mouse. These functional effects of chronic LPS were improved by MR antagonists. Our results demonstrate that MR antagonists have significant pharmacological actions in the respiratory system.

Inhibition of spleen tyrosine kinase attenuates allergen-mediated airway constriction.

Spleen tyrosine kinase (SYK) is a key activator of signaling pathways downstream of multiple surface receptors implicated in asthma. SYK function has been extensively studied in mast cells downstream of the high-affinity IgE receptor, FcεR1. Preclinical studies have demonstrated a role for SYK in models of allergic inflammation, but a role in airway constriction has not been demonstrated. Here, we have used a potent and selective pharmacological inhibitor of SYK to determine the role of SYK in allergen-mediated inflammation and airway constriction in preclinical models. Attenuation of allergic airway responses was evaluated in a rat passive anaphylaxis model and rat and sheep inhaled allergen challenge models, as well as an ex vivo model of allergen-mediated airway constriction in rats and cynomolgus monkeys. Pharmacological inhibition of SYK dose-dependently blocked IgE-mediated tracheal plasma extravasation in rats. In a rat ovalbumin-sensitized airway challenge model, oral dosing with an SYK inhibitor led to a dose-dependent reduction in lung inflammatory cells. Ex vivo analysis of allergen-induced airway constriction in ovalbumin-sensitized brown Norway rats showed a complete attenuation with treatment of a SYK inhibitor, as well as a complete block of allergen-induced serotonin release. Similarly, allergen-mediated airway constriction was attenuated in ex vivo studies from nonhuman primate lungs. Intravenous administration of an SYK inhibitor attenuated both early- and late-phase allergen-induced increases in airway resistance in an Ascaris-sensitive sheep allergen challenge model. These data support a key role for SYK signaling in mediating allergic airway responses.

Steroidal C-21 heteroaryl thioethers. Part 3: pregn-4-eno-[3,2-c]pyrazole fused A ring modified steroids as selective glucocorticoid receptor modulators (dissociated steroids).

The introduction of A ring pyrazole modification to the hydrocortisone C-21 heteroaryl thioethers generated compounds with excellent transrepression potency (IL-8 inhibition) compared to their hydrocortisone analogs. However, the transcriptional transactivation activity of these compounds were considerably higher than the corresponding hydrocortisone analogs. Among all the compounds evaluated, a quinoxaline thioether modification demonstrated the best overall in vitro separation.

Steroidal C-21 heteroaryl thioethers (Part 2): discovery of orally bioavailable selective glucocorticoid receptor modulators (dissociated steroids).

The prednisolone C-21 heteroaryl thioethers have been synthesized and evaluated in cell based transrepression and transactivation assays. Most of the compounds demonstrated weak transactivational activity in both human and rat tyrosineaminotransferase functional assay while keeping potent anti-inflammatory activity. The benzimidazole thioether 7 exhibited comparable anti-inflammatory activity and improved safety profile compared to the classical oral steroid prednisolone.

Concomitant activity of histamine and cysteinyl leukotrienes on porcine nasal mucosal vessels and nasal inflammation in the rat.

BACKGROUND: Histamine and cysteinyl leukotrienes are pivotal mast cell mediators which contribute considerably and likely complementary to the symptoms of allergic rhinitis. Currently, we sought to explore the direct actions of histamine and leukotriene D(4) (LTD(4)), a cysteinyl leukotriene, on porcine nasal arteries and veins. We also studied combined blocks of histamine and cysteinyl leukotrienes using loratadine and montelukast in an in vivo model of allergy-mediated nasal inflammation. METHODS: For the evaluation of the action of histamine and LTD(4) on arteries and veins, porcine nasal mucosa was isolated and cut into slices (100-300 microm thick). Real-time images of the nasal arteries and veins were recorded and vessel activities estimated by changes in cross-sectional area before and after the tested drugs. For the in vivo studies, the effect of loratadine and montelukast given alone and in combination was examined on upper airway inflammation in ovalbumin-sensitized and -challenged Brown Norway rats. RESULTS: Both histamine (0.001-10 micromol/l) and LTD(4) (0.001-10 micromol/l) produced a concentration-dependent increase in the lumen area of nasal mucosa arteries and veins. Histamine (0.01 micromol/l) alone produced a 24 and 12% increase in cross-sectional areas of arteries and veins, respectively. LTD(4) (0.001 micromol/l) alone increased artery and vein dilation by about 17 and 9%, respectively. Combination treatment with histamine (0.01 micromol/l) and LTD(4) (0.001 micromol/l) increased vessel dilation by 65% (arteries) and 26% (veins). In our in vivo Brown Norway rat studies, oral loratadine (0.01-10 mg/kg) and montelukast (0.01-10 mg/kg) significantly reduced antigen-induced total nasal inflammatory cell infiltration in a dose-dependent manner. The antiinflammatory dose-response curve of loratadine was shifted to the left when studied in combination with montelukast (0.01 mg/kg). Similarly, the dose-response characteristics of montelukast (0.01-10 mg/kg) was shifted in the presence of loratadine (0.01 mg/kg). CONCLUSION: Our studies support the position that histamine and cysteinyl leukotrienes may act collaboratively to elicit allergic nasal pathologies such as upper airway inflammation and nasal vessel dilation (which may translate into increased nasal mucosal engorgement). Furthermore, the current results are supportive of the hypothesis that combined treatment of allergic rhinitis with an H(1) receptor antagonist and a CysLT(1) receptor antagonist may have greater benefit than sole treatment with these agents alone.