Cytochrome P450 isoform-specific in vitro methods to predict drug metabolism and interactions

Päivi Taavitsainen

Department of Pharmacology and Toxicology, University of Oulu

Abstract

Cytochromes P450 (P450, CYP) are a superfamily of enzymes that participate especially in the oxidative metabolism of various xenobiotics and endogenous compounds.

The major goal of this study was to characterise suitable methods for routine preclinical in vitro testing of new chemical entities (NCE) and to test the methods for the affinity screening of selected drugs.

In vitro methods used involve the utilisation of human liver microsomes for studies with P450-selective reference inhibitors, inhibitory antibodies and cDNA-expressed enzymes in cytochrome P450-catalysed activities and for studying the reactions of selegiline and entacapone.

In this project, the CYP-catalysed oxidative in vitro biotransformation of selegiline into its primary metabolites desmethylselegiline and l-methamphetamine and the transformation of entacapone into its in vitro metabolite N-desethylentacapone were studied. The affinities of selegiline, desmethylselegiline, l-methamphetamine, entacapone, candesartan, eprosartan, irbesartan, losartan and valsartan to P450 enzymes were also elucidated, and the selectivity of tranylcypromine as a CYP2A6-selective reference inhibitor was characterised.

The most important findings were that the methodology developed during this work is suitable for preclinical in vitro testing of NCEs and that the results obtained for the studied compounds are in line with the available in vivo data.

By the in vitro testing methodology, it is possible to target the in vivo interaction studies to the relevant groups of compounds. The in vitro methods presented in this thesis could also make the early phases of drug development more cost-effective. Further, the number of animals used for in vivo testing in preclinical metabolism and interaction studies can be markedly reduced by effectively using this methodology.

Table of Contents

Acknowledgements

Abbreviations

List of original papers

1. Introduction

2. Review of the literature

2.1. Cytochrome P450 enzymes involved in xenobiotic metabolism

2.1.1. CYP1A subfamily
2.1.2. CYP2A subfamily
2.1.3. CYP2B subfamily
2.1.4. CYP2C subfamily
2.1.5. CYP2D subfamily
2.1.6. CYP2E subfamily
2.1.7. CYP3A subfamily

2.2. Methods for studying in vitro metabolism of NCEs

2.2.1. Overview of preclinical studies
2.2.2. Human-derived in vitro systems in preclinical drug metabolism research
2.2.3. Measures of metabolism in in vitro systems
2.2.4. Measures of the affinities of an NCE for CYPs

2.3. In vitro – in vivo scaling of an NCE

2.3.1. General
2.3.2. Considerations regarding assay conditions for NCE studies

2.4. Ethical issues

2.4.1. Human studies
2.4.2. Animal studies

3. Aims of the present study

4. Materials and methods

4.1. Human liver samples
4.2. Chemicals
4.3. Antibodies and recombinant enzymes
4.4. Analysis of metabolites
4.5. Enzymatic assays
4.6. Determination of enzyme kinetic parameters
4.7. Correlation analysis

5.1. Studies on the metabolism of two model compounds (I & IV)

5.1.1. Selegiline (I)
5.1.2. Entacapone (IV)

5.2. Inhibitory effects and potential interactions (I, II & IV)

5.2.1. Selegiline and its primary metabolites (I)
5.2.2. Five angiotensin-II receptor antagonists (II)
5.2.3. Entacapone (IV)

5.3. Evaluation of a selective inhibitor for CYP2A6 (III)

6. Discussion

6.1. Tools for preclinical in vitro studies

6.2. Methods used in in vitro studies

6.2.1. Practical issues

6.3. In vitro versus in vivo

6.4. Decreasing the use of test animals

7. Conclusions

List of Tables
1. Summary of xenobiotic-metabolising human hepatic CYPs.
2. The structures of commonly used CYP-specific substrates and inhibitors.
3. Comparison of in vitro enzyme sources used in preclinical research.
4. The human liver samples used in this work.
5. The solution and storage conditions of substrates, inhibitors and metabolites (I, II, III, IV).
6. Standard conditions for the determination of CYP-associated metabolic activities and other assays.

List of Figures
1. Time course of the drug development process (by the courtesy of Dr H. Raunio).
2. Structures of five angiotensin-II receptor antagonists studied in original publication II.

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