New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and 
in vivo studies to assess their safety and suitability for testing in humans. 
Regulatory health authorities require that therapeutic and supratherapeutic 
doses be administered, by the intended route of administration, to two 
nonclinical species prior to human testing (ICH Expert Working Group. The 
international conference on harmonization of technical requirements for 
registration of pharmaceuticals for human use (ICH); Multidisciplinary 
guidelines; Nonclinical safety studies (M3). 
http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf 
, 2009). The purpose of these studies is to identify potential target organ 
toxicity and to determine if the effects are reversible. Liver is a potential 
site for toxicity caused by orally administered NMEs due to high exposure during 
first pass after oral administration. A range of clinical chemistry analytes are 
routinely measured in both nonclinical and clinical studies to evaluate and 
monitor for hepatotoxicity. While bilirubin itself circulates within a wide 
range of concentrations in many animal species and humans, without causing 
adverse effects and possibly providing benefits (Sedlak and Snyder. Pediatrics 
113(6):1776-1782, 2004), bilirubin is one of the few readily monitored 
circulating biomarkers that can provide insight into liver function. Therefore, 
any changes in plasma or urine bilirubin levels must be carefully evaluated. 
Changes in bilirubin may occur as a result of adaptive nontoxic changes or 
severe toxicity. Examples of adaptive nontoxic changes in liver function, which 
may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above 
baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin 
metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport (Keogh. Adv 
Pharmacol 63:1-42, 2012). Alternatively, hepatocellular necrosis, 
hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some 
cases, these effects may be irreversible (FDA/CDER. Guidance for industry 
drug-induced liver injury: premarketing clinical evaluation. 
http://www.fda.gov/downloads/Drugs/…/Guidances/UCM174090.pdf , 2012).This 
chapter aims to demonstrate application of enzyme kinetic principles in 
understanding the risk of bilirubin elevation through inhibition of multiple 
processes-involving both enzymes and transporters. In the sections that follow, 
we first provide a brief summary of bilirubin formation and disposition. Two 
case examples are then provided to illustrate the enzyme kinetic studies needed 
for risk assessment and for identifying the mechanisms of bilirubin elevation. 
Caveats of methods and data interpretation are discussed in these case studies. 
The data presented in this chapter is unpublished at the time of compilation of 
this book. It has been incorporated in this chapter to provide a sense of 
complexities in enzyme kinetics to the reader.