Research Collection of Variants of Normal and Fatty Disease Human Livers

Published:  28 October 2016

Sekisui XenoTech has developed a Research Biobank that is a collection of normal, steatosis and steatohepatitis tissue samples gathered and characterized to facilitate the study of human liver disease with an emphasis on the progression of fatty liver disease. A portion of each of the human livers, which were harvested with the intent of transplantation but subsequently rejected for this purpose, and were obtained through partnerships with non-profit Organ Procurement Organizations (OPO) which are members of the United Network for Organ Sharing (UNOS), is saved in the Research Biobank. Donor-specific data provided by the organ procurement organizations includes demographics, cause of death, BMI, and alcohol and diabetes history. Pathologist’s review of the H&E slides includes classification of the samples into normal, steatosis or steatohepatitis categories and quantification of macrovesicular fat, inflammation, ballooning hepatocytes and fibrosis. Presence of fibrosis is confirmed with Masson's Trichrome staining. Tissues deposited in the bank are flash frozen in liquid nitrogen and stored at -80°C. Cells isolated from multiple tissues deposited in the bank are available as cryopreserved hepatocytes for culture in suspension or as an attached cell monolayer. These cells are suitable for an array of studies including in vivo in vitro correlation of drug metabolism and biomarker expression that characterize fatty liver disease. The bank contains normal, steatosis and steatohepatitis specimens, with and without a history of alcohol use. Photomicrographs of H&E slides of each tissue are available. The levels of CYP2A6, CYP2C19 and CYP3A4 mRNA expression in normal (10), steatosis (19) and steatohepatitis (11) specimens were analyzed by RT-PCR. The relative quantification of each of the enzymes was based on the ΔΔCT with GAPDH serving as an endogenous control. The relative quantification of the mRNAs was not affected by tissue pathology (ANOVA). In conclusion, we have established a Research Biobank, derived from transplantation-rejected organs, that is focused on alcoholic and non-alcoholic fatty liver disease.

Evaluation of the CYP metabolic activities in the presence of albumin in human liver microsomes

Published:  15 October 2016

Cytochrom P450 (CYP) isoforms are responsible for the metabolism of the majority of drugs in human. An intrinsic clearance (CLint) determined from in vitro intrinsic clearance can be used to predict the in vivo hepatic metabolic clearance (CLH) of drugs in humans. However, in vitro-in vivo extrapolation (IV-IVE) commonly underestimates the in vivo CLint and CLH of drugs metabolized by CYP isoforms. Recently, it was reported that the addition of albumin to the in vitro incubation system increased the metabolic activities of some CYP isoforms due to inhibition of the effect of polyunsaturated long-chain fatty acids (PUFAs). Therefore, we evaluated the effect of bovine serum albumin (BSA) on the metabolic activities of CYP isoforms in human liver microsomes (HLM). We found that some CYP isoforms were observed to show enhancement of CLu,met in the presence of albumin. In this presentation, we have summerised the effect of albumin on the metabolic activity of each CYP isoform in HLM.

Evaluation method for mitochondrial toxicity using Crabtree effect

Published:  15 October 2016

Mitochondria plays a crucial role in living organisms, one of which being the synthesis of ATP. Mitochondrial toxicity is widely tested as part of the compound’s toxicity screening and within FDA’s registered drug with a ‘black box warning’, roughly 80% of drugs are reported with mitochondrial toxicity. The aim of this research is to devise a simple method to evaluate mitochondrial toxicity across different toxicological mechanisms using human liver derived cells.

In Vitro Evaluation of Ketoconazole and its Alternatives as Non-CYP Inhibitors

Published:  13 June 2016

The In Vitro Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Inhibitors (Ritonavir, Clarithromycin and Itraconazole) as Inhibitors of Non-CYP Enzymes

Ketoconazole is a potent CYP3A4/5 inhibitor, and until recently, recommended by the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as a “strong” CYP3A4/5 inhibitor in clinical drug-drug interaction (DDI) studies. Ketoconazole sporadically causes liver injury or adrenal insufficiency. Because of this, the FDA and EMA recommended suspension of ketoconazole use in DDI studies in 2013. FDA specifically recommended use of clarithromycin or itraconazole as alternative strong CYP3A4/5 inhibitors for use in clinical DDI studies, but many investigators have also used ritonavir as an alternative. Although the effects of these clinical CYP3A4/5 inhibitors on other CYPs are largely established, reports on the effects on the broad range of non-CYP drug metabolizing enzyme activities are sparse. In this study, the inhibitory effects of ketoconazole, clarithromycin, ritonavir and itraconazole (and its CYP3A4-inhibitory metabolites, hydroxy-, keto- and N-desalkyl itraconazole) towards several drug metabolizing enzymes were systematically assessed in human subcellular fractions (microsomes, S9 or cytosol) from hepatic or extrahepatic tissues or recombinant enzyme systems. The enzyme families or metabolic reactions tested for inhibition include UGTs (13), sulfonation, GST, CES1/2, AO, XO, FMO, MAO-A and B, NAT1 and 2, and COMT. Several enzymes were not markedly inhibited (< 20% inhibition) by any of the compounds tested, including XO, FMO, sulfonation, CES1, GST, NAT1 and COMT. Two enzymes, namely CES2 and NAT2 were moderately inhibited (20-40% inhibition) by one or more compounds tested.  AO was inhibited by ketoconazole, ritonavir and clarithromycin, but not itraconazole with IC50 values of 3, 29 and 64 µM, respectively. MAO-A was inhibited by only ketoconazole (IC50 values » 8 µM) and MAO-B by N-desalkyl-itraconazole and ketoconazole (IC50 values » 6 µM). One or more UGT enzymes was potently inhibited (IC50 values < 10 µM) by ketoconazole, ritonavir and itraconazole or its metabolites; however, clarithromycin did not cause potent inhibition (≥ 43 µM) of the UGT enzymes tested. Ketoconzole inhibited 12 of 13 UGT enzymes tested with IC50 values less than 10 µM for 6 of those enzymes.  Similarly, ritonavir and itraconazole and/or its metabolites potently inhibited 5/13 and 4/13 UGT enzymes, respectively, with IC50 values less than 10 µM in these cases.  The results indicate that, like ketoconazole, the alternative clinical CYP3A4/5 inhibitors ritonavir, clarithromycin and itraconazole each have unique enzyme inhibition profiles. The results provide guidance for the selection of clinical CYP3A4/5 inhibitors when non-CYP enzymes are potentially involved in a victim drug’s pharmacokinetics.

Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Drug Transport Inhibitors

Published:  29 May 2016

Updated: Evaluation of Ketoconazole and its Alternative Clinical CYP3A4/5 Inhibitors as Inhibitors of Drug Transporters: The In Vitro Effects of Ketoconazole, Ritonavir, Clarithromycin, and Itraconazole on 13 Clinically-Relevant Drug Transporters

Ketoconazole is an orally available, synthetic, broad spectrum, antifungal agent. Approved in 1982 by the FDA for use in fungal infections, it is a known substrate and strong inhibitor of cytochrome P450 (CYP) 3A4 and 3A5.

Previously, a high dose of ketoconazole was considered the gold standard for use in clinical drug-drug interaction (DDI) as a strong CYP3A4/5 inhibitor. By 2013, ketoconazole use in clinical studies had been banned due in part to evidence demonstrating the potential for liver injury following long dosing periods. Typically, patients would exhibit asymptomatic, reversible liver function test abnormalities. As early as 1984, Van Tyle demonstrated evidence of DILI in approximately 0.1 to 1.0% of patients, with results indicating that there was no association with the dose, but with the duration of dosing. In later estimates, studies showed that ~134 per 100,00 persons, 4.9 cases per 10,000 patients, and 3.6 to 4.2% demonstrated liver abnormalities.

After ketoconazole was banned in clinical study use, the FDA recommended clarithromycin or itraconazole as an alternative, but indicating that other drugs may be used. Ritonavir was suggested by some as an alternative CYP3A4/5 inhibitor. Following an extensive study by Ke et al. where inhibitors were systematically evaluated, only itraconazole and clarithromycin were considered acceptable. Exclusion criteria included, the drug not being approved in the U.S., known non-specific inhibition of CYPs, safety issues, exclusive use with ritonavir, or only moderate information of CYP3A4/5.

There is minimal drug transporter data in the literature; which includes ketoconazole and ritonavir inhibition of P-gp and OATP1B1. The goal of this study was to allow for a more informed choice of a strong CYP3A4/5 inhibitor for clinical DDI studies involving a drug candidate known to be a substrate of one or more of the transporters and to help reduce confounding DDI results.

Published paper and webinar also available.

Automated High Content Drug Transport Screening with Transwell Plates for Drug Discovery

Published:  25 January 2016

  • The purpose of this study was to develop a method to use bidirectional intestinal permeability and drug transport to identify substrates and inhibitors of MDR1 or BCRP.
  • The alteration of drug transporters potentially affects clearance and efficacy.
  • Cells grown on transwell permeable membranes were washed, treated, and samples were collected with an automated liquid handler.
  • Data showed the automated method as consistent with in-house historic values and within and between assays.

Measuring bidirectional intestinal permeability and drug transport using Caco-2 and MDCKII can identify potential substrates or inhibitors of MDR1 or BCRP and identify the permeability class of potential new drugs. Two recent FDA documents, 2012’s Drug Interaction draft guidance and 2015’s Waiver of In Vivo Bioavailability and Bioequivalence, show the increasing interest and significance in transporters and permeability, particularly in MDR1 (P-gp) and BCRP. The alteration of drug transporters potentially affects clearance and efficacy. Early assessment of this potential leads to better, safer drugs and reduced development costs. A method utilizing a Tecan liquid handler and Caco-2 or MDCKII cells grown on permeable membranes (Figure 1) was created to measure bidirectional intestinal permeability and drug transport. The screen evaluates up to 14 compounds in one assay and measures permeability and efflux. Read more about Drug Transporter Screening.

In vitro characterization of human liver lysosomes isolated from fresh tissue

Published:  18 January 2016

Evaluation of lysosomal catabolism is an integral part of the development of biologic drugs including antibody-drug conjugates (ADC). Biologics can enter the cell by fluid-phase or cell surface antigen-mediated endocytosis and can be degraded by multiple catabolic enzymes found in lysosomes. Purified rat liver tritosomes or human lysosomes are convenient test systems for an in vitro evaluation of lysosomal stability of antibodies, linkers and small molecule drugs comprising ADC. Hepatic lysosomes were isolated from fresh human liver donors according to the discontinuous density ultracentrifugation method based on Wang, et al (2012). Twelve individual fractions collected from the OptiPrep density gradient were characterized for acid phosphatase, cathepsin B, and cytochrome c oxidase activity in order to confirm separation of the lysosome- from the mitochondria-specific proteins. Acid phosphatase activity was distributed among multiple fractions while cathepsin B was higher in lighter lysosomal fractions in contrast to the heavier fractions containing more cytochrome c oxidase. Fractions were further characterized with Western blotting for distribution of lysosomal-associated membrane protein 1 (LAMP-1, CD107a) and microsomal cytochrome c oxidase subunit 4 (COX4). In agreement with the enzyme activity data, the blots demonstrated separation of the LAMP-1 signal, detected in the lighter lysosomal fractions from that of the COX4 signal found in the heavier lysosomal fractions of the OptiPrep density gradient. Expected enrichment of lysosome-specific cathepsins L and S in isolated lysosomes was demonstrated with sandwiched ELISA Human Protease Array (R&D Systems). This work provides a characterization of isolated human liver lysosomes that constitutes a test system for an in vitro assessment of catabolic stability of biologics drugs entering the cell by the endosomal–lysosomal pathway.

Related Webinar: Investigation of freshly purified rat tritosomes and human hepatic lysosomes as an in vitro tool for characterization of biologic drugs