2018

Alcohol Consumption Uniquely Increases Hepatic Hyaluronan Content in Patients with Liver Steatosis

Published:  12 November 2018

History of Alcohol Consumption Uniquely Increases Hepatic Hyaluronan Content in Patients with Liver Steatosis

Dr. Maciej Czerwinski1, Jordan Surgnier2, Dr. Sarah E. Tague3 and Dr. Michele T. Pritchard2
1
 Sekisui XenoTech LLC; 2 Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center; 3 Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical Center

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is increased in plasma from donors with advanced liver disease and is used as a biomarker to assess liver disease severity. Extrahepatic HA content and its fragmentation are associated with inflammation and/or fibrosis.

In chronic, persistent hepatitis, as compared to normal tissue, HA was increased in fibrous areas around the portal tracts and the central vein, but it was rarely detected in the sinusoidal walls. In chronic, aggressive hepatitis, HA was present in periportal sinusoidal walls in addition to fibrous tissues of the portal tract (Ichida, 1996).

In cirrhotic tissues, as compared to normal, HA was significantly increased in fibrous areas of the portal tract but to a lesser extent in the sinusoidal walls around periportal areas (Ichida, 1996).

In alcoholic cirrhosis, HA was also observed in the sinusoidal wall and fibrous areas around the portal tract and central vein and its abundance in these locations diminished upon alcohol abstinence (Urashima, 1999).

Since hepatic levels and patterns of distribution of HA have not been examined in early liver disease (steatosis), we utilized liver tissue microarrays (TMAs) and histochemical staining with a hyaluronan binding protein (HABP) to determine if hepatic HA increases in donors with steatosis and if HA content is affected by history of alcohol consumption.

Cytokine-Mediated Suppression of CYP Enzymes By a Toll-like Receptor 9 Agonist in Hepatocytes

Published:  07 November 2018

Cytokine-Mediated Suppression of CYP Enzymes By the Toll-like Receptor 9 Agonist, IMO-2125, in Cultured Human Hepatocytes

Paul Tarantino1, Tim Sullivan1, Brian Ogilvie2, Maciej CzerwiƄski2
1 Idera Pharmaceuticals, Inc., 167 Sidney Street, Cambridge, MA 02139;
2 Sekisui XenoTech LLC, 1101 W. Cambridge Cir. Drive, Kansas City, KS 66103

The innate immune response to some drugs may involve the release of proinflammatorycytokines, such as IL-6, which have the ability to suppress xenobiotic-metabolizing CYP enzymes and affect the pharmacokinetics of co-administered small molecules. The 2012 FDA Drug-Drug Interaction (DDI) guidance recommended the evaluation of therapeutic proteins such as cytokines or cytokine modulators for their ability to modulate the expression of CYP enzymes or transporters (Food and Drug Administration (US), 2012). Although not a peptide drug, it is possible that an immuno-modulator, such as the oligonucleotide, Tilsotolimod(IMO-2125), an investigational agonist of toll-like receptor 9 designed to enhance T-cell responses to tumor antigens, could precipitate DDIs. Tilsotolimodalters the tumor microenvironment by improving antigen presentation by dendritic cells and macrophages with subsequent proliferation of antigen-specific cytotoxic T lymphocytes (CD8+ T cells) in both injected and distant tumors resulting in tumor cell death (Figure 1). In this study, we investigated the potential of Tilsotolimodto to cause direct and cytokine-mediated effects on CYP mRNA expression and enzyme activity in primary human hepatocytes according to a published in vitro method. The method comprised stimulation of human blood with a drug or a therapeutic protein followed by separation and subsequent incubation of human hepatocytes co-cultured with Kupffer cells with the drug-treated plasma to assess the potential of the drug to alter the expression of drug metabolizing enzymes and transporters through its effect on cytokines. All cultures of hepatocytes used in this study were co-cultures of hepatocytes and Kupffer cells isolated from the same donors in routine perfusion and plating procedures. The test system accounts for the combined effects of all cytokines stimulated by the drug in blood on CYP expression in primary cultures of human hepatocytes...

Inhibitory effect of typical CYP inhibitors under long term incubation in human liver microsomes

Published:  05 October 2018

Sho Nishinoaki, Ryo Fujino, Kenta Hashizume, Tsutomu Negama
Drug Development Solutions Center, Drug Development Solutions Division, Sekisui Medical Co., Ltd. 2117 Muramatsu, Tokai, Ibaraki 319-1182, Japan

Cytochrome P450 (CYP) isoforms are responsible for the metabolism of the majority of drugs in human. In case of the metabolic enzyme identification of CYP isoforms for the low intrinsic clearance (CLint) candidate compounds, it is necessary to perform metabolic reaction with long term incubation. In addition, when performing the metabolic enzyme identification with long term incubation, the specificity and sustainability of a typical inhibitor are important. In this research, we evaluated the inhibitory effect of typical inhibitor for each CYP isoform in human liver microsomes (HLM) under long term incubation (incubation time of CYP typical inhibitors was 60 minutes, final concentration of HLM was 1 mg protein/mL) to clarify an optimal concentration of the CYP typical inhibitors under long term incubation. In this presentation, we have confirmed specificity and sustainability of each typical inhibitor for CYP isoform under long term incubation in HLM.

Ultrasensitive quantitation assay of oligonucleotide therapeutics based on the PALSAR method

Published:  05 October 2018

Akane Omori, Koichi Shibusawa, Kazuhiro Takeuchi, Akira Ideno
Tokai Research & Development Center, Research & Development, SEKISUI MEDICAL CO., LTD. 2117 Muramatsu, Tokai, Ibaraki 319- 1182, Japan

Recently, development of oligonucleotide therapeutics has accelerated in the pharmaceutical industry, and the needs for ultrasensitive quantitation methods have greatly increased. Conventional approaches, such as hybridization, LC-MS/MS, PCR are used in the analysis. Hybridization is commonly used, yet sensitivity is insufficient. Sensitivity of LC-MS/MS is improved by instrumental advancement, but still not enough. PCR and qRT-PCR are also known as sensitive detection methods; however, the high variability of measured values is a problem. To solve these problems, we aimed to develop an ultrasensitive hybridization assay method without nucleic acid amplification procedure.

Microsomal Cytochrome P450 Enzyme Activities in Nonalcoholic Steatohepatitis Livers

Published:  16 July 2018

Maciej Czerwinski1, Brian Oberheide1, Nicholas Hatfield1, Bill Ewy1, Christopher Seib1, Eva Gatineau2, Frederique Yiannikouris2, Brian Ogilvie1
1 Sekisui XenoTech, LLC
2 University of Kentucky, Department of Pharmacology and Nutritional Sciences


Abstract:
The prevalence of nonalcoholic steatohepatitis (NASH), a chronic liver disease, has increased drastically in parallel with the raise of obesity in the US. This condition affects hepatic drug metabolism and has potential to impact drug-drug interactions. Our study aimed to evaluate microsomal CYP enzyme activities, organ fibrosis and microvesicular steatosis in NASH tissues deposited in the Sekisui XenoTech Biobank, and to establish whether these tissues have application as an in vitro test system for the study of NASH impact on metabolism of xenobiotics. NASH tissues were identified based on the examination of hematoxylin and eosin (H&E), Masson’s trichrome staining and donor history of alcohol consumption. All samples had intra-lobular inflammation, ballooning necrosis and macrovesicular fat >5%. Fibrosis stage was assigned based on Brunt et al. The degree of microvesicular steatosis, evaluated by H&E staining, did not correlate with the body mass index (BMI) nor with progressive stages of fatty liver disease in 51 donors. However, the extent of microvesicular steatosis correlated positively with triglyceride contents (R2=0.66), but not with total cholesterol levels. Microsomal protein yield was weakly negatively correlated with microvesicular fat content (R2=0.11) but did not correlate with BMI. The majority of average microsomal CYP activities in NASH livers were lower than in the general population of liver donors by 6 to 65%. In contrast, the average CYP2E1 microsomal activity in NASH livers was 3.5% higher than in the general population of liver donors in the Biobank. These observations were in agreement with published clinical data. A microsomal pool of five NASH donors and tissue micro arrays containing NASH and fatty livers from donors with and without history of alcohol consumption were prepared to assist in disease evaluation. The NASH pattern of CYP enzyme activities seen in the patients and in the microsomes prepared from non-transplantable NASH livers suggest that the subcellular fraction is an appropriate test system for analysis of CYP-mediated xenobiotic metabolism associated with the disease state.

Current In Vitro DDI Regulatory Guidance Reference Poster

Published:  29 May 2018

Brian W. Ogilvie1 and Andrew Parkinson2
1Sekisui XenoTech, LLC, Kansas City, KS, USA and 2XPD Consulting, Shawnee, KS, USA

Abstract:
In September, 2017, the Japan PMDA revised its 2014 guideline and released it for comments. In October, 2017, the US FDA revised and split its 2012 draft guidance for industry on in vitro drug-drug interaction (DDI) studies, into one document for in vitro DDI studies, and another for clinical DDI studies. An overview of the major changes, a comparison of each agency’s equations and cut-off values (including the most recently released 2013 EMA DDI guidelines), and a comparison of experimental details will be highlighted. This poster will also highlight strategies to harmonize the design of in vitro DDI studies to meet the expectations of both agencies.