The use of unbound drug concentrations is crucial forthe predictionof efficacious doses. Hence, dose predictions for antibiotics targetingrespiratory pathogens should be based on free, rather than the currentlyused, total drug concentrations in epithelial lining fluid (ELF).In this work, we describe an assay to estimate the percent unboundof drugs in ELF using simulated epithelial lining fluid (sELF) containingthe most abundant components of ELF in healthy humans. A diverse setof 85 compounds showed a broad range of unbound values ranging from<0.01 to 100%. Binding in sELF was influenced by ionization, withbasic compounds typically resulting in a stronger binding than neutraland acidic compounds (median percent unbound values 17, 50, and 62%,respectively). A permanent positive charge further increased binding(median percent unbound 11%), while zwitterions showed a lower binding(median percent unbound 69%). In lipid-free sELF, the binding of basiccompounds was less pronounced, while compounds of other ionizationclasses were less impacted, indicating that lipids are involved inthe binding of bases. A reasonable correlation was found between bindingin sELF and human plasma (R (2) = 0.75);however, plasma binding poorly predicted sELF binding for basic compounds(R (2) = 0.50). Bases are an important compoundclass for antibacterial drug development since positive charges affectpermeability into Gram-negative bacteria, which are important in termsof bacterial pneumonia. To evaluate in vivo activity,we selected two bases, for which strong sELF binding was observed(percent unbound <1 and 7%) and conducted an analysis of antibacterialefficacy in the neutropenic murine lung efficacy model and total vs free ELF drug concentrations. In both cases, the totalELF resulted in an overprediction of expected efficacy, while thecorrected free ELF explained the observed in vivo efficacy. This supports that free, and not total, ELF concentrationsshould be used for the efficacious dose prediction for pneumonia andhighlights the importance of determining binding in this matrix.

Aldosterone synthase (CYP11B2) inhibitors have been explored in recent years as an alternative therapeutic option to mineralocorticoid receptor (MR) antagonists to reduce elevated aldosterone levels, which are associated with deleterious effects on various organ systems including the heart, vasculature, kidney, and central nervous system (CNS). A benzamide pyridine hit derived from a focused screen was successfully developed into a series of potent and selective 3-pyridyl isoindolin-1-ones CYP11B2 inhibitors. Our systematic structure-activity relationship study enabled us to identify unique structural features that result in high selectivity against the closely homologous cortisol synthase (CYP11B1). We evaluated advanced lead molecules, exemplified by compound 52, in an in vivo cynomolgus monkey acute adrenocorticotropic hormone (ACTH) challenge model and demonstrated a superior 100-fold in vivo selectivity against CYP11B1.

Inappropriately high levels of aldosterone are associated with many serious medical conditions, including renal and cardiac failure. A focused screen hit has been optimized into a potent and selective aldosterone synthase (CYP11B2) inhibitor with in vitro activity against rat, mouse, human, and cynomolgus monkey enzymes, showing a selectivity factor of 160 against cytochrome CYP11B1 in the last species. The novel tetrahydroisoquinoline compound (+)-(R)-6 selectively reduced aldosterone plasma levels in vivo in a dose-dependent manner in db/db mice and cynomolgus monkeys. The selectivity against CYP11B1 as predicted by cellular inhibition data and free plasma fraction translated well to Synacthen challenged cynomolgus monkeys up to a dose of 0.1 mg kg(-1). This compound, displaying good in vivo potency and selectivity in mice and monkeys, is ideally suited to perform mechanistic studies in relevant rodent models and to provide the information necessary for translation to non-human primates and ultimately to man.

Type 2 Diabetes (T2D) is recognized by the World Health Organization as a global epidemic affecting 284-347 million people in the world depending on the inclusion criteria used (Danaei et al. 2011; American Diabetes Association 2013). Co morbidities associated with T2D are hypertension, cardiovascular diseases (CVD) (Castelli 1984; Wilson et al. 2005) and diabetic nephropathy (DN) (Shah et al. 2009; Shao et al. 2013). There is an unmet need for therapies curing or preventing the progression of T2D and combined pathologies (Grundy 2008).

Recombinant human serum albumin (HSA) conjugates of a 15-amino-acid truncated peptide YY (PYY) analogue were prepared using three heterobifunctional linkers [succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC), 6-maleimidohexanoic acid N-hydroxysuccinimide ester (MHS), and N-[γ-maleimidobutyryloxy]sulfosuccinimide ester (GMBS)] in 2 synthetic steps involving (1) reaction of succinimidyl ester on linker with ε-amine of Lys2 on the peptide and (2) reaction of maleimide on peptide linker with free thiol of Cysteine 34 (Cys34) on albumin. In-process controls using ESI LC-MS were used to follow reactions and identify reaction products. Proteolytic digests of the conjugate revealed that peptide conjugation occurs at Cys34 on HSA. Conjugates were assayed in cell-based assays to determine potency at the human Y2-receptor, and selectivity at the human Y1-, Y4-, and Y5-receptors using a calcium flux assay. All three conjugates assayed were selective agonists of the Y2-receptor, and displayed nanomolar potencies. MCC and MH conjugates were selected for acute PK/PD studies in DIO mice. Significant reduction in food intake was observed with the MH conjugate, which lasted for 24 h at the 10 mg (or 4 μmol)/kg dose. While the MCC conjugate exhibited greater potency in vitro, it was slightly less effective than the MH conjugate in vivo with respect to reduction in food intake. Both conjugates were significantly less active than the peptide coupled to a 30 kDa PEG. The observed T1/2 (8-9 h) for both conjugates was significantly lower than that observed for the PEGylated peptide (∼25 h). These results suggest that, as compared with the unmodified and PEGylated peptide, the extended circulation half-life of albumin conjugates is mediated through uptake and recirculation by FcRn, and allometric scaling methods are necessary to account for interspecies variation in pharmacokinetic properties.