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3R-INFO-BULLETIN 4 - April 1995
Urs A. Meyer
received his M.D. degree from the University of Zürich in 1967 and subsequently was an intern and resident in internal medicine at the University of California San Francisco (UCSF) Medical Center. From 1969 to 1971 he was a Postdoctoral Fellow and from 1972 to 1975 an Assistant Professor in Clinical Pharmacology at the University of California, San Francisco.
Then he returned to Switzerland as Chief of the Division of Clinical Pharmacology at the University of Zürich Medical School, in 1983 he was appointed to his present position. His research interests include the study of inherited metabolic diseases with special emphasis on pharmacogenetic conditions.
In particular, Urs A. Meyer has investigated drug idiosyncrasies in experimental and clinical porphyrias and has used a large human liver bank both to predict metabolism patterns of new drugs and to study the mechanisms of environmental and genetic factors influencing drug response.
Urs A. Meyer is presently Professor of Pharmacology and Chairman of the Department of Pharmacology at the Biozentrum of the University of Basel Medical School, Basel, Switzerland.
Since 1994 he has also served as Chairman of the Biozentrum. Additionally, he is a consultant physician in the Outpatient Clinic of the Department of Medicine of the University Hospital in Basel.
Prof. Dr. med. Urs A. Meyer
Department of Pharmacology
Biozentrum of the University of Basel
Phone 061-267 22 20
Fax 061-267 22 08
Predicting human drug metabolism with in vitro methods:
applications from a 3R-project
Human tissue banks and cell systems are gaining importance for studying the metabolism and toxicity of drugs in vitro. The use of these techniques early in drug development reduces the need for animal studies.
Predictions from in vitro studies
One of the important questions in drug research is to what degree the metabolic pathways of a drug can be predicted from in vitro studies before the drug is applied to animals or humans.
In vitro systems to study human drug metabolism
A number of in vitro systems, each with its own advantages and disadvantages, have been developed for this purpose:
Primary (or fresh) tissues:
whole organ culture
cells in suspension or primary culture
transient expression of enzymes(s) in primary culture
slices in suspension and culture
purified reconstituted enzymes
Long term cell cultures (cell lines):
hamster fibroblasts (Y79 cells)
monkey kidney cells (COS-cells)
Tissue collection from human livers
Over the last 10 years our laboratory has used a large human liver bank to study human drug-metabolizing enzymes. Two types of tissue are collected: livers from organ transplant donors and biopsies taken during abdominal surgery. Organ donor livers are used for the large-scale purification of enzymes, which, in turn, are then used to raise antibodies. From the same livers, mRNA is prepared and cDNA expression libraries are constructed. cDNAs coding for a specific drug-metabolizing enzyme are isolated from these libraries with antibodies, oligonucleotides or cDNAs from other species.
To identify the enzyme(s) responsible for a particular drug metabolism step, reaction phenotyping is performed, either with a panel of inhibitory antibodies, or by correlating reaction yields with the amounts of enzyme-protein recognized on immunoblots. Final proof for the involvement of a particular enzyme is obtained by specifically expressing that one protein in heterologous cells (containing the human cDNA) and measuring the drug-metabolizing activity. To assess the range of interindividual variation in the activity of a particular drug-metabolizing enzyme, a panel of livers from patients of different age, sex, etc. is used. Biopsies from patients undergoing laparotomies are used for in vitro/in vivo comparisons. Once the enzymes have been identified and specific assays developed, drug metabolism interactions can be predicted from in vitro effects, e.g. competitive inhibition of the enzyme, reactions at concentrations that are likely to occur in drug therapy, etc.
Identification of enzymes
The approach described has been used to study the mechanisms of three common genetic poly-morphisms of drug metabolism, namely the debrisoquine-poly-morphism, the mephenytoin polymorphism and the N-acetyltransferase polymorphism. Moreover, these methods have been used successfully to identify the enzymes responsible for the metabolism of cyclosporine, midazolam, lidocaine, and several other drugs.
Human tissues and cells for drug research
At present, our liver bank has over 50 human livers. Most of these have been characterized for several drug-metabolizing enzyme activities, and the contents of the various enzymes has been estimated by enzyme kinetics and Western blot analysis. Our bank includes livers of "poor metabolizers" of the genetic polymorphisms of the debrisoquine, mephenytoin and acetylation-type. Furthermore, we have developed chemical, immunological and cDNA probes for many of the common enzymes, and other probes are available either commercially or we received in exchanges with other laboratories. The enzymes include members of the drug metabolizing cytochrome P450 family: CYP1A1, CYP1A2, CYP2C, CYP2C19, CYP2D6, CYP2E1, CYP3A4, as well as epoxide hydrolase and two N-acetyltransferases. We have expressed all of these enzymes in heterologous systems such as COS 1 cells. In the meantime, cell culture systems in which single human drug-metabolizing enzymes are expressed have become commercially available (Gentest Corp., Waltham, Massachusetts, USA) and are already widely used.
Potential of these in vitro systems
- The main metabolites of a drug and the enzymes involved in their generation can be identified before the drug is administered.
- This information may be used to predict the in vivo kinetics of the drug by "upscaling" the enzy-matic information to the whole organ.
- A knowledge of the enzymes involved allows the rational design of drug interaction studies.
- The variation in metabolic rates between tissues from different subjects (due to such factors as smoking, alcohol consumption, different geno-types in the case of polymorphic enzymes, etc.) allows the expected variation of the metabolic rates in the clinical setting to be predicted.
The use of these techniques early in drug development reduces the need for animal studies.
Selection of References
Kronbach, T., Fischer, V., and Meyer, U.A. Cyclosporine metabolism in human liver: identification of a cytochrome P450 of the P450III gene family as the major cyclosporine-metabolizing enzyme explains interactions of cyclosporine with other drugs. Clin. Pharmacol. Ther. 43, 630-635, 1988.
Kronbach, T., Mathys, D., Umeno, M., Gonzalez, F.J., and Meyer, U.A. Oxidation of midazolam and triazolam by human liver cytochrome P450IIIA4. Molecular Pharmacol. 36, 89-96, 1989.
Bargetzi, M.J., Aoyama, T., Gonzalez, F.J., and Meyer, U.A. Lidocaine metabolism in human liver by cytochrome P450IIIA4 (PCN1). Clin. Pharmacol. Therap. 46, 521-527, 1989.
Blum, M., Demierre, A., Grant, D.M., Heim, M., and Meyer, U.A. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc. Natl. Acad. Sci. USA 88, 5237-5241, 1991.
Eugster, H.-P., Sengstag, C., Meyer U.A., Hinnen, A. and Würgler, F.E. Constitutive and inducible expression of human cytochrome P450Ia1 in yeast Saccharomyces cerevisiae: an alternative enzyme source for in vitro studies. Biochem. Biophys. Res. Comm. 172, 737-744, 1990.
Andersson, T., Miners, J.O., Verones, M.E., Tassaneeyakul, W., Meyer, U.A., and Birkett, D.J. Identification of human liver P450 isoforms mediating omeprazole metabolism. Br. J. Clin. Pharmac. 36, 521-530, 1993.
Meyer, U.A. Drug metabolism in health an disease. In: Extrahepatic Manifestations in Liver Diseases, eds, R., Schmid, W. Gerok, L. Bianchi, K.P. Maier, Academic Publ., Dordrecht, Boston London, 1993, 285-293.
Meyer, U.A. Cytochrome P450 in Human Drug Metabolism: How Much is Predictable? In: Assessment of the use of single cytochrome P450 enzymes in drug research (eds. Waterman, Hildebrand). Springer Verlag Berlin, Heidelberg, New York, pp. 43-56, 1994.