THE USE OF SANDWICH-CULTURED RAT HEPATOCYTES TO DETERMINE THE INTRINSIC CLEARANCE OF COMPOUNDS WITH DIFFERENT EXTRACTION RATIOS: 7-ETHOXYCOUMARIN AND WARFARIN

ABSTRACT: The application of sandwich-cultured rat hepatocytes for the identification of the hepatic intrinsic clearance of compounds with widely varying extraction ratios was investigated. We previously showed the applicability of this in vitro system, in combination with a model describing molecular diffusion, hepatocyte/medium partition, and nonsaturated metabolism, which resulted in a successful identification of this parameter for tolbutamide. This approach is further validated using the compounds 7-ethoxycoumarin and warfarin, covering a 100-fold range of extraction ratios. Clearance of these two substrates could be reliably determined, but only if the depletion of the parent compound in medium as well as in the hepatocyte sandwich was measured. Sensitivity analyses showed that the time course of depletion of the parent compound in medium, especially for warfarin, is insensitive to the partition and diffusion parameter values, whereas depletion in the hepatocyte sandwich was far more sensitive. When varying the volumes of collagen in the sandwich culture, it appears that the most reliable kinetic parameters could be obtained by fitting the data with the smaller collagen volume and that these parameters obtained from fitting to data of the larger volumes generally cannot be verified satisfactorily with the data of the smaller volumes. The values of hepatic clearance that were obtained after extrapolation of the intrinsic clearance to the hepatic clearance from blood were comparable within a factor of 2 to hepatic clearance data in the literature. This indicates that this sandwich culture and modeling system can be applied for the identification of the hepatic intrinsic clearance rate of the total range from low to high clearance compounds. Predicting the kinetic parameters of compounds in vivo using data from in vitro experiments is a fast developing area of research Recently, we have successfully developed the alternative approach of using sandwich-cultured rat hepatocytes in determining the in vitro intrinsic clearance of the slowly metabolized compound tolbutamide In this alternative approach, the use of data on substrate depletion was chosen rather than the use of data on metabolite formation. Using data on metabolite formation may cause practical problems when analytical methods or standards to study the kinetics of metabolite Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.105.004390. ABBREVIATIONS: CL bile , biliary clearance (ml/min/kg body weight); CL hep , hepatic clearance (ml/min/kg body weight); CL int , intrinsic clearance (ml/min); C m , concentration medium (M); C s , concentration sandwich (M); D m , diffusion coefficient in medium (cm 2 /min); D s , diffusion coefficient in sandwich (cm 2 /min); f a , fraction of cell activity; f m , free fraction medium; f h , free fraction hepatocytes; f n , fraction of cells; f s , free fraction sandwich; f u , free fraction in blood; f v , fraction of viable cells; K h , specific clearance (Ϫ/min); K l , specific intrinsic clearance of the liver (Ϫ/min; Ϫ/h); K m , Michaelis-Menten constant (M); K ow , partition coefficient octanol-water; K s , first-order metabolism (Ϫ/min); L m , medium layer thickness (cm); L s , sandwich layer thickness (cm); PBPK, physiologically based pharmacokinetics; P cm , collagen-medium partition coefficient; P hm , hepatoyctemedium partition coefficient; P sm , sandwich-medium partition coefficient; P, parameter value; Q h , hepatic blood flow (l/h); s, sensitivity; SRW, standard rat body weight (250 g); V c , volume of collagen (ml); V h , volume of hepatocytes (ml); V m , volume of medium (ml); V max , maximum rate of metabolism (M/min); V s , volume sandwich (ml); DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; HPLC, high-performance liquid chromatography

Oral prednisolone suppresses skin inflammation in a healthy volunteer imiquimod challenge model

Imiquimod (IMQ) is a topical agent that induces local inflammation via the Toll-like receptor 7 pathway. Recently, an IMQ-driven skin inflammation model was developed in healthy volunteers for proof-of-pharmacology trials. The aim of this study was to profile the cellular, biochemical, and clinical effects of the marketed anti-inflammatory compound prednisolone in an IMQ model. This randomized, double-blind, placebo-controlled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after treatment initiation with prednisolone or placebo, 5 mg imiquimod (IMQ) once daily for two following days was applied under occlusion on the tape-stripped skin of the back for 48 h in healthy volunteers. Non-invasive (imaging and biophysical) and invasive (skin punch biopsies and blister induction) assessments were performed, as well as IMQ ex vivo stimulation of whole blood. Prednisolone reduced blood perfusion and skin erythema following 48 h of IMQ application (95% CI [−26.4%, −4.3%], p = 0.0111 and 95% CI [−7.96, −2.13], p = 0.0016). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI [−79.7%, −16.3%], p = 0.0165), NK and dendritic cells (95% CI [−68.7%, −5.2%], p = 0.0333, 95% CI [−76.9%, −13.9%], p = 0.0184), and classical monocytes (95% CI [−76.7%, −26.6%], p = 0.0043) in blister fluid. Notably, TNF, IL-6, IL-8, and Mx-A responses in blister exudate were also reduced by prednisolone compared to placebo. Oral prednisolone suppresses IMQ-induced skin inflammation, which underlines the value of this cutaneous challenge model in clinical pharmacology studies of novel anti-inflammatory compounds. In these studies, prednisolone can be used as a benchmark.</p

Oral prednisolone suppresses skin inflammation in a healthy volunteer imiquimod challenge model

Imiquimod (IMQ) is a topical agent that induces local inflammation via the Toll-like receptor 7 pathway. Recently, an IMQ-driven skin inflammation model was developed in healthy volunteers for proof-of-pharmacology trials. The aim of this study was to profile the cellular, biochemical, and clinical effects of the marketed anti-inflammatory compound prednisolone in an IMQ model. This randomized, double-blind, placebo-controlled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after treatment initiation with prednisolone or placebo, 5 mg imiquimod (IMQ) once daily for two following days was applied under occlusion on the tape-stripped skin of the back for 48 h in healthy volunteers. Non-invasive (imaging and biophysical) and invasive (skin punch biopsies and blister induction) assessments were performed, as well as IMQ ex vivo stimulation of whole blood. Prednisolone reduced blood perfusion and skin erythema following 48 h of IMQ application (95% CI [−26.4%, −4.3%], p = 0.0111 and 95% CI [−7.96, −2.13], p = 0.0016). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI [−79.7%, −16.3%], p = 0.0165), NK and dendritic cells (95% CI [−68.7%, −5.2%], p = 0.0333, 95% CI [−76.9%, −13.9%], p = 0.0184), and classical monocytes (95% CI [−76.7%, −26.6%], p = 0.0043) in blister fluid. Notably, TNF, IL-6, IL-8, and Mx-A responses in blister exudate were also reduced by prednisolone compared to placebo. Oral prednisolone suppresses IMQ-induced skin inflammation, which underlines the value of this cutaneous challenge model in clinical pharmacology studies of novel anti-inflammatory compounds. In these studies, prednisolone can be used as a benchmark

Presentation_1_Oral prednisolone suppresses skin inflammation in a healthy volunteer imiquimod challenge model.pptx

Imiquimod (IMQ) is a topical agent that induces local inflammation via the Toll-like receptor 7 pathway. Recently, an IMQ-driven skin inflammation model was developed in healthy volunteers for proof-of-pharmacology trials. The aim of this study was to profile the cellular, biochemical, and clinical effects of the marketed anti-inflammatory compound prednisolone in an IMQ model. This randomized, double-blind, placebo-controlled study was conducted in 24 healthy volunteers. Oral prednisolone (0.25 mg/kg/dose) or placebo (1:1) was administered twice daily for 6 consecutive days. Two days after treatment initiation with prednisolone or placebo, 5 mg imiquimod (IMQ) once daily for two following days was applied under occlusion on the tape-stripped skin of the back for 48 h in healthy volunteers. Non-invasive (imaging and biophysical) and invasive (skin punch biopsies and blister induction) assessments were performed, as well as IMQ ex vivo stimulation of whole blood. Prednisolone reduced blood perfusion and skin erythema following 48 h of IMQ application (95% CI [−26.4%, −4.3%], p = 0.0111 and 95% CI [−7.96, −2.13], p = 0.0016). Oral prednisolone suppressed the IMQ-elevated total cell count (95% CI [−79.7%, −16.3%], p = 0.0165), NK and dendritic cells (95% CI [−68.7%, −5.2%], p = 0.0333, 95% CI [−76.9%, −13.9%], p = 0.0184), and classical monocytes (95% CI [−76.7%, −26.6%], p = 0.0043) in blister fluid. Notably, TNF, IL-6, IL-8, and Mx-A responses in blister exudate were also reduced by prednisolone compared to placebo. Oral prednisolone suppresses IMQ-induced skin inflammation, which underlines the value of this cutaneous challenge model in clinical pharmacology studies of novel anti-inflammatory compounds. In these studies, prednisolone can be used as a benchmark.</p