Nonclinical Safety Assessment of an Inhaled Formulation of Serelaxin: A Recombinant Human Protein in Rats and Cynomolgus Monkeys (Macaca fascicularis).

Serelaxin is a recombinant human relaxin-2 intended for cardiovascular indications. Inhalation was chosen as alternative route to intravenous to allow daily administration for chronic applications and home treatment. A total of 4 short-term studies were conducted in rats and cynomolgus monkeys with inhaled formulation of serelaxin at dose up to 10 mg/kg/d. All rats and cynomolgus macaques receiving serelaxin were exposed to the test item. One rat and approximately 50% of macaques developed immunogenicity, which did not appear to affect exposure. No adverse effect on respiratory function or systemic changes was noted. Both species developed similar microscopic lesions characterized by eosinophilic cell infiltration around bronchi; however, in the rat, this was more pronounced and extended to a perivascular location. In addition, in the rat, serelaxin showed eosinophilic crystalline material associated with macrophages in the alveoli and bronchioles. In macaques, serelaxin induced minimal macrophage infiltrates in alveoli and perivascular/peribronchiolar mononuclear cell infiltrations. The minimal airway eosinophilic/mononuclear inflammatory cell infiltrations were considered to be nonadverse in macaques due to the minimal severity and the lack of any other alterations in the lung parenchyma. In the rat, the presence of eosinophilic crystalline material and macrophage response, characterized as precipitated test article, was considered adverse.

Glutathione depletion modulates gene expression in HepG2 cells via activation of protein kinase C alpha.

Buthionine sulphoximine (BSO; 1mM) resulted in the depletion of glutathione (GSH) in HepG2 cells to 17+/-1.5% within 24h. This was not associated with apoptotic or necrotic cell death over this time period. Use of a human (Phase 1) cDNA custom toxicology-array and a larger scale (>10,000 gene) Affymetrix U95Av2 array identified a total of 48 and 104 genes, respectively, with a statistically significant (and >1.5-fold) change in expression. A total of 64 differentially expressed genes (6 of which were confirmed by real-time polymerase chain reaction) were suggestive of protein kinase C (PKC) activation. Activation of PKC-alpha (but not betaI or delta) was demonstrated at 24 h through activity measurements and through Western blot analysis of membrane-associated PKC-alpha protein. Activation did not occur in the presence of additional gamma-glutamylcysteine to prevent GSH depletion. Activation of PKC-alpha by GSH-depletion may, at least in part, be mediated by thiol oxidation and may contribute to a survival signal. If sustained, the activation may be important in non-genotoxic carcinogenesis.

Inhalation by design: novel tertiary amine muscarinic M3 receptor antagonists with slow off-rate binding kinetics for inhaled once-daily treatment of chronic obstructive pulmonary disease.

A novel tertiary amine series of potent muscarinic M(3) receptor antagonists are described that exhibit potential as inhaled long-acting bronchodilators for the treatment of chronic obstructive pulmonary disease. Geminal dimethyl functionality present in this series of compounds confers very long dissociative half-life (slow off-rate) from the M(3) receptor that mediates very long-lasting smooth muscle relaxation in guinea pig tracheal strips. Optimization of pharmacokinetic properties was achieved by combining rapid oxidative clearance with targeted introduction of a phenolic moiety to secure rapid glucuronidation. Together, these attributes minimize systemic exposure following inhalation, mitigate potential drug-drug interactions, and reduce systemically mediated adverse events. Compound 47 (PF-3635659) is identified as a Phase II clinical candidate from this series with in vivo duration of action studies confirming its potential for once-daily use in humans.

Inhalation by design: novel ultra-long-acting ß(2)-adrenoreceptor agonists for inhaled once-daily treatment of asthma and chronic obstructive pulmonary disease that utilize a sulfonamide agonist headgroup.

A novel series of potent and selective sulfonamide derived ß(2)-adrenoreceptor agonists are described that exhibit potential as inhaled ultra-long-acting bronchodilators for the treatment of asthma and chronic obstructive pulmonary disease. Analogues from this series mediate very long-lasting smooth muscle relaxation in guinea pig tracheal strips. The sulfonamide agonist headgroup confers high levels of intrinsic crystallinity that could relate to the acidic sulfonamide motif supporting a zwitterionic form in the solid state. Optimization of pharmacokinetic properties was achieved through targeted introduction of a phenolic moiety to support rapid phase II clearance, thereby minimizing systemic exposure following inhalation and reducing systemically mediated adverse events. Compound 38 (PF-610355) is identified as a clinical candidate from this series, with in vivo duration of action studies confirming its potential for once-daily use in humans. Compound 38 is currently in advanced phase II clinical studies.