Metabolite Profiling and Pharmacokinetic Evaluation of Hydrocortisone in a Perfused Three-Dimensional Human Liver Bioreactor

Abstract

Endotoxin lipopolysaccharide (LPS) is known to cause liver injury primarily involving inflammatory cells such as Kupffer cells, but few in vitro culture models are applicable for investigation of inflammatory effects on drug metabolism. We have developed a threedimensional human microphysiological hepatocyte-Kupffer cell coculture system and evaluated the anti-inflammatory effect of glucocorticoids on liver cultures. LPS was introduced to the cultures to elicit an inflammatory response and was assessed by the release of proinflammatory cytokines, interleukin 6 and tumor necrosis factor a. A sensitive and specific reversed-phase-ultra high-performance liquid chromatography-quadrupole time of flightmass spectrometry method was used to evaluate hydrocortisone disappearance and metabolism at near physiologic levels. For this, the systems were dosed with 100 nM hydrocortisone and circulated for 2 days; hydrocortisone was depleted to approximately 30 nM, with first-order kinetics. Phase I metabolites, including tetrahydrocortisone and dihydrocortisol, accounted for 8-10% of the loss, and 45-52% consisted of phase II metabolites, including glucuronides of tetrahydrocortisol and tetrahydrocortisone. Pharmacokinetic parameters, i.e., half-life, rate of elimination, clearance, and area under the curve, were 23.03 hours, 0.03 hour 21 , 6.6 3 10 25 l×hour 21, and 1.03 (mg/l)*h, respectively. The ability of the bioreactor to predict the in vivo clearance of hydrocortisone was characterized, and the obtained intrinsic clearance values correlated with human data. This system offers a physiologically relevant tool for investigating hepatic function in an inflamed liver.

Modeling Therapeutic Antibody–Small Molecule Drug-Drug Interactions Using a Three-Dimensional Perfusable Human Liver Coculture Platform

“…KCs were included to build a more complete hepatic cellular microenvironment, and numerous studies have shown their impact on the behavior of in vitro liver systems, such as enhanced IL-1b-mediated cytokine secretion, acute phase protein production, and cytochrome P450 suppression (Hoebe et al, 2001;Sunman et al, 2004;Nguyen et al, 2015;Sarkar et al, 2015;Rose et al, 2016). There are other mechanisms mediated by IL-6 that could potentially be modeled in the LiverChip system such as drug transporter activity.…”

Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model

“…Parameters and conditions can be found in detail in Sarkar et al (2015). In summary, the column was an Agilent Extend-C18 (2.1 Â 50 mm, 1.8 mm; Agilent Technologies); the column temperature was 40°C; mass spectra were collected in positive ion modebetween m/z 70 and 1000 at two scans/s; ion spray voltage was 3800 V; heated capillary temperature was 350°C; drying gas was 8 liters/min; nebulizer was 30 psi; sheath gas temp was 380°C; sheath gas flow was 12 liters/min.…”

“…1). The analytical validation of this LC-MS method was previously described (Sarkar et al, 2015), and parameters have been summarized in Supplemental Table 1.…”

“…Metabolite profiling was performed as previously described (Sarkar et al, 2015). Briefly, media samples were spiked with deuterium-labeled HC (d 4 -HC) Fig.…”

Low-Turnover Drug Molecules: A Current Challenge for Drug Metabolism Scientists

“…In addition, this coculture dynamic flow system showed an increased metabolite formation rate for several cytochrome P450 enzymes compared with both static systems and the monoculture flow system. Most recently, a model integrating drug metabolism with disease (e.g., inflammatory liver models) using a three-dimensional liver bioreactor culture system has been published (Sarkar et al, 2015), where hydrocortisone intrinsic clearance was predicted that correlated with human data and key metabolites were observed. A similar three-dimensional dynamic flow model has been shown to maintain human hepatocyte function for up to 7 weeks while generating more reproducible estimates of clearance for 7-ethoxycoumarin compared with hepatocytes in suspension (Choi et al, 2014).…”