Relative Bioavailability Assessment of Solid Forms by An Artificial Stomach and Duodenum Apparatus.

In this work, the ability of the artificial stomach and duodenum (ASD) model to predict bioavailability in rats was investigated using a poorly soluble model compound, BI-639667. A solution and four suspensions of different solid forms of BI-639667 were tested both in an ASD and rats. Rank order of the bioavailability estimated from an ASD apparatus is consistent with that of in vivo result in rats, i.e., solution > salicylic acid cocrystal > malate salt > maleate salt > monohydrate, which correlates with the ability of the different solid forms to maintain supersaturation with respect to the stable form in aqueous solution. The results support the use of an ASD for characterizing dissolution performance of solid forms to aid their selection for tablet formulation development.

Human Serum Albumin Immobilized On Magnetizable Beads: A Rapid Method for Compound HSA Binding Study.

Immobilized human serum albumin (HSA) was developed by coupling His-tagged HSA onto Ni2+-coupled magnetizable beads (HSA-beads), allowing the HSA to be easily removed from incubation components. The HSA-beads system provides a rapid and convenient method to study HSA compound binding. In this study, the HSA-beads system was characterized and evaluated as a tool for assessing compound HSA binding properties. The free fraction (fu) values of test compounds measured using HSA-beads were comparable to those determined by equilibrium dialysis (ED), which is commonly used to evaluate albumin binding in vitro. The equilibrium dissociation constant (Kd) values determined for a series of compounds using the HSA-beads method demonstrated good correlation with literature data. This good correlation also suggests that the binding of His-HSA to the beads does not impact the conformations of the two compound binding sites of HSA, as the range of compounds tested encompassed binding to both sites. Furthermore, the Kd values of representative compounds itraconazole and BIRT2584 that were difficult to assess using ED, due to significant cellulose membrane adsorption, were successfully determined. The HSA-beads provide several advantages over ED, such as simple preparation, short assay incubation duration, and the ability to quantify both free and HSA-bound species of the test compound, facilitated by the simple separation of HSA-beads from the solution phase using a magnetic field. These properties render the HSA-beads method suitable for high-throughput studies on compound HSA binding.

Characterization of Divergent Metabolic Pathways in Elucidating an Unexpected, Slow-Forming, and Long Half-Life Major Metabolite of Iclepertin.

PURPOSE: After single oral dosing of the glycine reuptake transporter (GlyT1) inhibitor, iclepertin (BI 425809), a single major circulating metabolite, M530a, was identified. However, upon multiple dosing, a second major metabolite, M232, was observed with exposure levels ~ twofold higher than M530a. Studies were conducted to characterize the metabolic pathways and enzymes responsible for formation of both major human metabolites. METHODS: In vitro studies were conducted with human and recombinant enzyme sources and enzyme-selective inhibitors. The production of iclepertin metabolites was monitored by LC-MS/MS. RESULTS: Iclepertin undergoes rapid oxidation to a putative carbinolamide that spontaneously opens to an aldehyde, M528, which then undergoes reduction by carbonyl reductase to the primary alcohol, M530a. However, the carbinolamide can also undergo a much slower oxidation by CYP3A to form an unstable imide metabolite, M526, that is subsequently hydrolyzed by a plasma amidase to form M232. This difference in rate of metabolism of the carbinolamine explains why high levels of the M232 metabolite were not observed in vitro and in single dose studies in humans, but were observed in longer-term multiple dose studies. CONCLUSIONS: The long half-life iclepertin metabolite M232 is formed from a common carbinolamine intermediate, that is also a precursor of M530a. However, the formation of M232 occurs much more slowly, likely contributing to its extensive exposure in vivo. These results highlight the need to employ adequate clinical study sampling periods and rigorous characterization of unexpected metabolites, especially when such metabolites are categorized as major, thus requiring safety assessment.

The morphology, functionality, and longevity of a novel all human hepatic cell-based tri-culture system.

There remains a significant need for a convenient, phenotypically stable long-term culture platform for primary human hepatocytes (PHHs) for use in pharmacological and toxicological applications. Conventional in vitro models are often inconvenient, burdensome to use, and unable to support a multitude of donor lots or maintain PHH structural and functional properties over extended time. To address these limitations, an all-human cell-based hepatic tri-culture system (HTCS) has been developed comprised of frozen vials of PHHs and feeder cells. Qualified PHHs exhibited healthy morphological characteristics for ≥30 days. Extensive anastomosing networks of bile canaliculi with tight and gap junctions were established early and remained stable and functional throughout the culture period. After 5 culture days, albumin, urea, and basal Phase 1 and Phase 2 metabolic functions were stable for at least 2 weeks and significantly higher in the HTCS PHHs compared to sandwich monoculture PHHs. Induction of CYP functional activity by prototypical receptor agonists was stable after 4 days for at least 2 weeks. Gene expression of Alb and various CYPs in the HTCS PHHs was significantly higher compared to sandwich monoculture PHHs. The HTCS represents a convenient, phenotypically stable, all-human PHH culture platform for pharmacological and toxicological applications.