2017

Comparison of two hepatic uptake assay methods

Published:  01 December 2017

Comparison of two hepatic uptake assay methods, plated and oil layer methods, using human and preclinical species cryopreserved hepatocytes
The prediction of in vivo human hepatic clearance (CL) of drug from in vitro data is important for drug development. Primary human cryopreserved hepatocytes are widely used as an in vitro tool for the prediction of in vivo CL since a variety of hepatic uptake transporters are expressing in them. The oil layer method is a current major hepatic uptake assay method. On the other hand, the plated method has come to be used to evaluate hepatic uptake recently. In this study, we compared the difference between the plated method and the oil layer method using human cryopreserved hepatocytes in order to show the usefulness of the plated method. Also, we examined whether or not the plated method is useful for the evaluation of species difference of hepatic uptake using preclinical species (monkey, dog, and rat) cryopreserved hepatocytes.

Evaluation of interspecies metabolic activities for Non-CYP enzymes, suitable incubation conditions

Published:  01 December 2017

Evaluation of interspecies metabolic activities for Non-CYP enzymes and into a suitable incubation condition for human
Recently, lead compound screenings has become widely used in drug development. This has led to the development of increasing range of lead compound structures with metabolic stability for Cytochrome P450 (CYP). However, because of the unpredictable metabolic reaction and species differences by non-CYP enzymes, there have been cases that the test compound’s blood concentration was found to be significantly lower than expected in the clinical phase, resulting in the discontinuation in its development. Therefore, it is necessary to establish an in vitro evaluation system of metabolic activities for non-CYP enzymes. We evaluated interspecies differences in metabolic activities for non-CYP enzymes (aldehyde oxidase (AO), aldo-keto reductase (AKR) and carbonyl reductase (CR)) in liver cytosol (human, monkey, rat, mouse and dog). We also evaluated the inhibitory effect of typical inhibitor for each non-CYP enzyme (AO, xanthine oxidase (XO), AKR and CR). And, we tested the metabolic activities of non-CYP enzymes in anaerobic conditions using human liver hepatocytes. In this presentation, we have established an evaluation method for metabolic activities of non-CYP enzymes (AO, XO, AKR, CR).

Further Exploration into the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats

Published:  27 September 2017

Further Exploration into the Drug-Drug Interaction Between Gemfibrozil and Repaglinide in Rats: Uptake Transport
Chase I. McCoy, Forrest A. Stanley, Chandra Kollu, Seema Muranjan, Krystal M. Green and Joanna E. Barbara

Abstract
The clinically-relevant drug-drug interaction (DDI) between the dyslipidemia drug gemfibrozil and the antidiabetic repaglinide is well-documented throughout the literature. In humans, repaglinide is predominantly cleared by hepatic metabolism involving cytochrome P450 (P450) 3A4 and 2C8 and UGT1A1 and 1A3. Gemfibrozil and its glucuronide metabolite inhibit CYP2C8 and UGT1A1, as well as the hepatic uptake transporter OATP1B1. These factors have been implicated in the clinical interaction. A gemfibrozil / repaglinide DDI resulting in increased repaglinide and metabolite plasma exposure (>3-fold) and a vectoral shift in elimination pathways from biliary to urinary excretion following administration of gemfibrozil was previously established in male Sprague-Dawley rats. In that study, negligible effects on liver enzyme activity were observed following gemfibrozil treatment, suggesting that the DDI in rats was unlikely to be caused by P450 or UGT inhibition. In the present study, the potential role of uptake transporter proteins in the interaction was explored in vitro.

The Effects of Organ Preservation Solution on Aldehyde Oxidase and Xanthine Oxidase Activity in...

Published:  27 June 2017

The Effects of Organ Preservation Solution on Aldehyde Oxidase and Xanthine Oxidase Activity in Pooled Human Liver S9

Abstract
Non-transplant quality human livers donated for research are a common source of both cellular and subcellular material utilized in in vitro drug metabolism studies. At the time of organ recovery, livers are flushed with an ice-cold perfusion and storage solution that preserves the tissue during delivery from the recovery site to the research facility. The two most commonly used cold storage solutions are UW (University of Wisconsin) solution and Custodiol HTK (histidine tryptophan ketoglutarate) solution. One notable difference between the two solutions is the presence of 1 mM allopurinol in UW solution. It has been previously shown that1 mM allopurinol inhibits xanthine oxidase activity, but not aldehyde oxidase in human liver cytosol (Barr et al., 2014). Allopurinol exposure to human livers during cold storage could lead to underestimation of the contribution of xanthine oxidase in the metabolism of new chemical entities. Here, we hypothesized that preparing a pool of human liver S9 prepared exclusively from Custodiol HTK-preserved livers will have significantly higher xanthine oxidase activity compared to pools of liver S9 prepared from livers preserved primarily in UW solution. In this study, we compared AO and XO activity in pooled human liver S9 made from 20 donors preserved in HTK with those in two commercially available pools of S9 (n = 50 and 200) produced by Sekisui XenoTech from livers preserved primarily in UW solution. Xanthine oxidase activity was determined by measuring the oxidation of 6-ntiroquinazolinone to 6-nitroquinazolinedione. Aldehyde oxidase activity was determined by measuring the oxidation of phthalazine to 1-phthalazinone, p-vanillin to vanillic acid and the zaleplon to 5-oxo-zaleplon. To determine whether UW and HTK affected other drug metabolizing enzymes, we looked at the metabolism of appropriate marker substrates for the major cytochrome P450 (CYP) enzymes. S9 prepared from HTK-only livers had a 539% and a 264 to 359% higher xanthine oxidase and aldehyde oxidase than S9 prepared from primarily UW donors. Activities of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4 were not statistically different between the HTK and UW pools. These data indicate that a preparation of pooled human liver S9 prepared from tissue that is cold-preserved in HTK solution has increased xanthine oxidase and aldehyde oxidase activity compared to S9 prepared from livers preserved in UW solution.

Hepatocytes Are the Preferred Test System to Evaluate Oligonucleotide-CYP Interactions In Vitro

Published:  02 March 2017

Abstract
Sekisui XenoTech previously examined the potential of oligonucleotides to inhibit cytochrome P450 (CYP) and reported that the phosphorothioate, but not phosphodiester, backboned molecules caused potent inhibition of CYP1A2 and CYP2C8 in human liver microsomes (HLM) but not in cryopreserved human hepatocytes (CHH) (Buckley et al., 2009). In this study we expanded upon our previous work to include direct, time- (TDI) and metabolism-dependent inhibition (MDI) of 7 CYP and 8 UGT enzymes in pooled HLM and pooled CHH. Briefly, oligonucleotides with either phosphodiester (oligo # 1 and #3) or phosphorothioate (oligo #2 and #4) backbones were evaluated in HLM to determine IC50 values without a pre-incubation (direct inhibition) or with a 30 min pre-incubation +/- NADPH (MDI and TDI evaluation, respectively). For UGTs, UDPGA was added to HLM and inhibition was assessed with a single concentration of oligos (30 mM). For CHH assays, MDI was assessed with 30 and 90 min pre-incubations, and 4-methylumbelliferone (4-MU) was used as a marker substrate of overall UGT activity. Incubations with CYP and UGT marker substrates were performed for 5–10 min and residual activity was assessed by LC-MS/MS analysis. The results indicated that the phosphodiester-backbone oligonucleotides had little to no inhibitory effect on CYP and UGT enzymes in HLM and CHH with the exception of oligo #2 in HLM for CYP2C19. Conversely, the phosphorothioate-backbone oligonucleotides caused direct inhibition of CYP and UGT enzymes. CYPs 1A2, 2B6, 2C8, and UGTs 1A1 and 2B17 were most highly inhibited by both phosphorothioate-backbone oligonucleotides. There was evidence of NADPH-independent inhibition of CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP3A4/5with oligo #2 and #4. In contrast to HLM, we observed little to no direct inhibition by any oligo in CHH (with the exception of oligo #2 with CYP2C19 and TDI by oligo #3 with CYP2C8), demonstrating test system-dependent outcomes. Overall this study indicates that hepatocytes, a test system physiologically more relevant than isolated microsomes, are the preferred model to evaluate oligonucleotide-CYP interactions in vitro.