Mr Hassan Salhab

Research project: Development of a LC-MS/MS method for the simultaneous quantification of metabolic reaction products catalysed by cytochrome P450 enzymes in rat hepatic microsomes and human hepatic progenitor cells

Abstract

Biotransformation in the liver is accomplished by two classes of enzymes; namely phase I and phase II biotransformation enzymes. Cytochome P450s (CYPs) are pivotal phase I mono-oxygenase enzymes involved in the synthesis and degradation of endogenous steroid hormones, vitamins, and fatty acid derivatives, but also in the transformation of xenobiotics such as drugs and environmental chemicals into more hydrophilic molecules, thus facilitating their excretion (1). Of this CYP family, CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4 are considered to be the major isoforms responsible for the metabolism of the majority of marketed drugs in humans. Inhibition or induction of CYPs by drugs can cause drug-drug interactions, altering their effectiveness or resulting in toxic interactions (2). In addition, CYP isozyme levels can be affected by other factors such as age and disease (3).

Thus, the three primary objectives for a CYP study could be:-

a) Identification of compounds that have CYP induction as a key event in a toxicity pathway.

b) Identification of compounds that lead to CYP induction and affect biotransformation of endogenous (non-xenobiotics) substances, thus disturbing normal intermediary metabolism and physiological homeostasis.

c) Identification of compounds in mixtures that induce CYP.

At the molecular level, CYP induction involves several cellular processes.

• The chemical binds to a specific receptor.

• The activated receptor forms a heterodimer with factors, such as Ahrnt (Ahr nuclear translocator) and retinoid X receptor (RXR) for both Pregnane X Receptor (PXR) and Constitutive Androstane Receptor, (CAR) and migrates into the nucleus. These receptors control the expression of CYP1A (AhR), CYP2, and CYP3A (PXR and CAR) families, as well as UGTs and glutathione-S-transferases and the transporters MDR1 and MRP2.

• The heterodimer binds to the target xenobiotic response elements (XRE) located in both the proximal and distal P450 gene promoters.

• The transcription of the respective CYP gene is enhanced, which is followed by the de novo protein synthesis and post-translational modification to a functional CYP enzyme.

  Use of a human relevant system is important because the AhR, PXR and CAR found in toxicological animal models, such as mouse and rat, exhibit significant differences in specificity (4). Differences of induction among species are explained by discrepancies in the ligand-binding domain of the receptors implying that their ligand specificities may differ dramatically between species. Therefore, extrapolation of animal data with respect to the inducibility of CYP enzymes in humans is not always relevant.

  The purpose of this proposed CYP induction in vitro test method is to evaluate the potential of a test chemical (see Table 1) to induce CYP mediated via PXR/CAR (CYP3A4, CYP2B6) and the Ah-135 receptor (CYP1A2) in rat and human based cells, thus establishing differences. The selection of these three CYP isoforms, which are recommended by the EMA and FDA Guidelines, is based on the fact that in humans they are involved in the biotransformation of a wide variety of endogenous and exogenous chemicals and that they are target CYPs for classical model inducers: CYP1A2 for dioxins and PAHs, CYP2B6 for phenobarbital and CYP3A4 for rifampicin.

Table 1: Prototypical inducers

CYP1A2

CYP2B6

CYP3A4

Β-Naphthoflavone (BNF) [25 μM]

Phenobarbital (PB) [500 μM]

Rifampicin (RIF) [10 μM]

Primary human hepatocytes derived from three separate donors were considered in the past as a kind of gold standard for CYP induction studies. However, known donor variability and their limited availability stimulated a search for alternative test systems. HepaRG™ cells represent a promising alternative to primary human hepatocytes as they combine long-term stability of chemical-metabolising enzymes and transporters with the correct plasma membrane polarization. HepaRG™ cells are terminally differentiated hepatic cells derived from a single human donor hepatic progenitor cell line. This cell line is considered to function like an adult liver cell, with unique characteristics such as the whole Hepatitis C cycle and ADME gene expression.

LC-MS/MS is an accurate method for rapid, simultaneous quantification of bio-analytes that can be used to study metabolic reactions. It could thus evaluate the impact of drugs and other factors on the activity of CYP enzymes and thereby guide rational drug use in clinical settings. Although several studies have reported analytical methods to detect the formation of multiple metabolites from probe substrates using LC-MS in biological samples, many disadvantages were associated with the proposed methods, i.e. low numbers of metabolic reactions detected (1,5) or only a single CYP 3A4-specific probe used to evaluate the activity of the isoenzyme (6). In addition, a method recently reported for measuring several isoforms (7) may be irrelevant as only rat microsomes were studied and this begs the question of the relevance to human metabolism. This proposed study will look at the three pertinent isoforms above and compare levels from probe substrates & metabolic conversion products in both rat and human media.

We thus propose to develop a consolidated LC-MS/MS assay for the simultaneous measurement of metabolic reactions catalysed by hepatic CYP enzymes and study their kinetics in rat microsomes and human hepatic cells.