Use of HμREL Human Coculture System for Prediction of Intrinsic Clearance and Metabolite Formation for Slowly Metabolized Compounds

Design of slowly metabolized compounds is an important goal in many drug discovery projects. Standard hepatocyte suspension intrinsic clearance (CL_int) methods can only provide reliable CL_int values above 2.5 μL/min/million cells. A method that permits extended incubation time with maintained performance and metabolic activity of the in vitro system is warranted to allow in vivo clearance predictions and metabolite identification of slowly metabolized drugs. The aim of this study was to evaluate the static HμREL coculture of human hepatocytes with stromal cells to be set up in-house as a standard method for in vivo clearance prediction and metabolite identification of slowly metabolized drugs. Fourteen low CL_int compounds were incubated for 3 days, and seven intermediate to high CL_int compounds and a cocktail of cytochrome P450 (P450) marker substrates were incubated for 3 h. In vivo clearance was predicted for 20 compounds applying the regression line approach, and HμREL coculture predicted the human intrinsic clearance for 45% of the drugs within 2-fold and 70% of the drugs within 3-fold of the clinical values. CL_int values as low as 0.3 μL/min/million hepatocytes were robustly produced, giving 8-fold improved sensitivity of robust low CL_int determination, over the cutoff in hepatocyte suspension CL_int methods. The CL_int values of intermediate to high CL_int compounds were at similar levels both in HμREL coculture and in freshly thawed hepatocytes. In the HμREL coculture formation rates for five P450-isoform marker reactions, paracetamol (CYP1A2), 1-OH-bupropion (CYP2B6), 4-OH-diclofenac (CYP2C9), and 1-OH-midazolam (3A4) were within the range of literature values for freshly thawed hepatocytes, whereas 1-OH-bufuralol (CYP2D6) formation rate was lower. Further, both phase I and phase II metabolites were detected and an increased number of metabolites were observed in the HμREL coculture compared to hepatocyte suspension. In conclusion, HμREL coculture can be applied to accurately estimate intrinsic clearance of slowly metabolized drugs and is now utilized as a standard method for in vivo clearance prediction of such compounds in-house

Selective Nonsteroidal Glucocorticoid Receptor Modulators for the Inhaled Treatment of Pulmonary Diseases

A class of potent, nonsteroidal, selective indazole ether-based glucocorticoid receptor modulators (SGRMs) was developed for the inhaled treatment of respiratory diseases. Starting from an orally available compound with demonstrated anti-inflammatory activity in rat, a soft-drug strategy was implemented to ensure rapid elimination of drug candidates to minimize systemic GR activation. The first clinical candidate <b>1b</b> (AZD5423) displayed a potent inhibition of lung edema in a rat model of allergic airway inflammation following dry powder inhalation combined with a moderate systemic GR-effect, assessed as thymic involution. Further optimization of inhaled drug properties provided a second, equally potent, candidate, <b>15m</b> (AZD7594), that demonstrated an improved therapeutic ratio over the benchmark inhaled corticosteroid <b>3</b> (fluticasone propionate) and prolonged the inhibition of lung edema, indicating potential for once-daily treatment