2019

CYP1A2 Enzyme Activity and Protein Abundance In Normal And Diseased Pediatric Livers

Published:  11 September 2019

Presented at the 55th Congress of European Societies of Toxicology (EUROTOX 2019) in Helsinki, Finland
M. Czerwinski1, A. Kats2, M. T. Pritchard3,4, and S. E. Tague5, B. W. Ogilvie1
1Sekisui XenoTech LLC, Kansas City, KS; 2Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO; 3Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, KS; 4The Liver Center, University of Kansas Medical Center, Kansas City, KS; and 5Kansas Intellectual and Developmental Disabilities Research Center, University of Kansas Medical Center, Kansas City, KS

Multiple drug metabolizing enzymes are absent or expressed at a very low level during human gestation and increase substantially within first one to two years after birth. CYPs 1A2, 2C9, 2D6, 2E1 and 3A4 exhibit this general pattern of expression. CYP1A2 is a drug-metabolizing enzyme whose expression begins between birth and 4 weeks of age, and gradually increases to about half of the adult levels by 6 years of age. The enzyme constitutes 4 - 16% of the hepatic CYP pool, and is a major determinant of the biotransformation of ~9% of clinically used drugs. Interestingly, CYP1A2 activity decreases in adults with non-alcoholic fatty liver disease (NAFLD). Considering the increase in the number of medications given to children, as well as the rise in childhood obesity and NAFLD, this study aimed to determine whether donor age and health status influence CYP1A2 abundance, lobular localization and enzyme activity. Pediatric liver microsomes and a corresponding tissue microarray (TMA) were our test system...

Human mAb stability and cytotoxicity in rat hepatocytes with a surrogate peptide approach

Published:  30 July 2019

Evaluating stability and cytotoxicity of human monoclonal antibody in cultured rat hepatocytes with a surrogate peptide approach

Presented at the 12th International ISSX Meeting in Portland, OR
Nadya Galeva, Reed Murbach, Krystal Gilligan, Kevin Westland, Seema Muranjan and Joanna E. Barbara
Sekisui XenoTech, LLC, Kansas City, KS, USA

In vitro cell based assays are performed throughout the drug development process. Probing cytotoxicity and stability of a drug candidate at relevant concentrations in cell culture are of interest in pharmaceutical research. Building upon the research presented at the 2019 ASMS Conference, we evaluated the stability of a human monoclonal antibody (mAb) in cultures of primary rat hepatocytes using enzymatic protein digestion and LC-MS quantitation of surrogate peptides to develop and optimize a method and demonstrate applicability of the surrogate peptide approach to in vitro assays for biopharmaceuticals...
 

Human monoclonal antibody stability in rat cell cultures using surrogate peptide LC-MS/MS

Published:  04 June 2019

Evaluating stability of human monoclonal antibody in rat cell cultures using a surrogate peptide LC-MS/MS approach

Presented at the 67th ASMS Conference on Mass Spectrometry & Allied Topics in Atlanta, GA
Nadya Galeva, Reed Murbach, Krystal Gilligan, Kevin Westland, Seema Muranjan
Sekisui XenoTech, LLC, Kansas City, KS, USA

In vitro evaluations of monoclonal antibody (mAb) in drug development can be both useful and challenging. Cytotoxicity and stability of protein therapeutic candidates at relevant concentrations in cell culture are of interest in pharmaceutical research.  Bioanalytical strategies for mAb LC-MS based analysis involve using either intact or digested mAb. Our approach for determining the stability of a human mAb in cultures of primary rat hepatocytes was to use enzymatic protein digestion followed by LC-MS/MS quantitation of five surrogate peptides...
 

Assessment of in vitro inhibition of CYPs, UGTs and transporters by oligonucleotides

Published:  28 May 2019

An assessment of the in vitro inhibition of cytochrome P450 enzymes (CYP), UDP-glucuronsyltransferases (UGT) and transporters by phosphodiester- or phosphorothioate-linked oligonucleotides

Presented at the 2019 Marbach Castle Drug-Drug Interaction Workshop in Germany
Dr. Brian W. Ogilvie
Sekisui XenoTech, LLC, Kansas City, KS, USA
Based on the publication: Kazmi F, Yerino P, McCoy C, Parkinson A, Buckley DB, Ogilvie BW. (2018) Oligonucleotide Inhibition of P450s, UGTs, and Transporters. Drug Metab Dispos 46:1066-1074.

What is already known about this subject?
  • Antisense oligonucleotides (ASOs) are increasingly being developed for many indications and are often modified with phosphorothioate linkages in lieu of phosphodiester linkages.
  • Few in vitro DDI studies with ASOs have been performed, including the investigational imetelstat, volanesorsen, and ISIS 681257.
  • No clinical DDIs have been reported.
 
What this study adds:  
Abstract:
Regulatory guidance documents do not have specific recommendations for ASO DDI evaluation, but these molecules do fall under the scope of overall DDI testing.
 
Aims:
  1. Evaluate different ASOs as inhibitors of CYPs, UGTs and transporters in vitro.
  2. Determine if in vitro inhibition of CYPs and UGTs in HLM by phosphorothioate ASOs is an artefact not observed in CHH...

Patterns of Hyaluronan Deposition in Normal and Diseased Pediatric Livers

Published:  13 March 2019

Presented at the Society of Toxicology (SOT) 58th Annual Meeting & ToxExpo in Baltimore, MD
Maciej Czerwiński1, Alexander Kats2, Jordan Surgnier3, Sarah E. Tague4, and Michele T. Pritchard3,5
1
Sekisui XenoTech, Kansas City, KS., 2Department of Pathology and Laboratory Medicine, Children’s Mercy Hospital, Kansas City, MO., 3Department of Pharmacology, Toxicology and Therapeutics, 4Kansas Intellectual and Developmental Disabilities Research Center, 5The Liver Center, University of Kansas Medical Center, Kansas City, KS

The incidence of obesity and non-alcoholic fatty liver disease (NAFLD) is growing in children. Hyaluronan (HA), an extracellular matrix (ECM) glycosaminoglycan, is increased in liver and blood from adults with advanced liver disease. HA accumulation is also associated with inflammation and fibrosis in extrahepatic tissues. HA levels have not been examined in healthy or diseased livers from pediatric patients. Here, we sought to determine if hepatic HA deposition was associated with donor age and/or disease by using a pediatric liver tissue microarray (TMA)...