University of Groningen. Human and rat organ slices de Kanter, Ruben

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1 University of Groningen Human and rat organ slices de Kanter, Ruben IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2002 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): de Kanter, R. (2002). Human and rat organ slices: a tool to study drug metabolism and toxicity Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Human and rat organ slices: a tool to study drug metabolism and toxicity Ruben de Kanter

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4 RIJKSUNIVERSITEIT GRONINGEN Human and rat organ slices: a tool to study drug metabolism and toxicity Proefschrift ter verkrijging van het doctoraat in de Wiskunde en Natuurwetenschappen aan de Rijksuniversiteit Groningen op gezag van de Rector Magnificus, dr. D.F.J. Bosscher, in het openbaar te verdedigen op vrijdag 17 mei 2002 om uur door Ruben de Kanter geboren op 30 december 1969 te Haarlem

5 Promotor: Co-promotor: Beoordelingscommissie: Prof.dr. D.K.F. Meijer Dr. G.M.M. Groothuis Prof.dr.ir. I.M.C.M. Rietjens Prof.dr. L.F.M. van Zutphen Prof.dr. W. Seinen

6 Gelijk de aardbol door de oceanen is 't aards bestaan door dromen vast omsloten; komt nacht - dan zal met luide golfslagstoten de oerkracht op haar stranden slaan. Daar klinkt haar stem, ons roepend en ons vragend... Het toverschip ligt klaar hier op de ree; de vloed komt op, ons snel in 't duister dragend naar de onmeet'lijkheid der zee. Het hemelruim, in sterrenglorie brandend, kijkt van omhoog in raadsels voor de mens,- wij zijn op weg, omringd van alle kanten door vuur en vlammend zonder grens. Tjoettsjev

7 Paranimfen: Inge de Graaf Marina de Jager The printing of this thesis was financially supported by: Groningen University Institute for Drug Exploration (GUIDE) Stichting legaat Mr. C. Mulock en I. Osstdyck Pharmacia Corporation, Nerviano, Italy N.V. Organon Solvay Pharmaceuticals B.V. The studies described in this PhD thesis were performed in the laboratory of Pharmacokinetics and Drug Delivery (head prof.dr. D.K.F. Meijer) in the research group of dr. G.M.M. Groothuis, as part of the Groningen University Institute for Drug Exploration (GUIDE) on the basis of a long standing collaboration with the Section of Hepatobiliary Surgery (prof.dr. M.J.H. Slooff) of the Faculty of Medical Sciences and University Hospital Groningen. The research on cryopreservation was initiated and performed partly in the Department of Drug Safety, Solvay Pharmaceuticals B.V. (dr. H.J. Koster). Printing: Ridderprint Offsetdrukkerij B.V., Ridderkerk, Netherlands by R. de Kanter. All rights reserved. No part of this book may be reproduced or transmitted in any forms or by any means, without permission from the author. The research described in thesis was financially supported by the ZonMw Dutch Progamme on Alternatives to Animal Testing ( / ).

8 Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Chapter 6 Chapter 7 Chapter 8 Chapter 9 Chapter 10 Chapter 11 Chapter 12 Aim of this thesis Precision-cut organ slices as a tool to study toxicity and metabolism of xenobiotics with special reference to non-hepatic tissues Cryopreservation of rat and monkey liver slices A simple method for cryopreservation of liver slices from man and other species A rapid and simple method for cryopreservation of human liver slices Organ slices as an in vitro test system for drug metabolism in human liver, lung and kidney Drug metabolizing activity of human and rat liver, lung, kidney and intestine slices A new technique to prepare precision-cut slices from small intestine and colon Prediction of whole body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon The use of human and rat slices from liver, lung, kidney and small intestine in toxicology General discussion and future perspectives Summary Samenvatting voor niet-ingewijden References Dankwoord

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10 Aim of this thesis Aim A major barrier in the creation of completely new or improved therapeutic agents is the considerable gap that exist between animal experimentation and clinical evaluation: the step from pre-clinical studies to further drug testing is often time consuming and cumbersome. Furthermore, both human and animal studies are costly and strictly bound to ethical and legal prerequisites. For this reason, relatively few of the many compounds initially tested, enter the clinical phase of drug development. This is at least partly due to the uncertainties that still exist at that stage on potential human toxicity, drug-drug interactions as well as unfavorable kinetic features. Extensive animal experimentation, in spite of its present advanced technology, is simply not sufficient to fully deal with these aspects. The number of experimental animals that were used in the Netherlands, in the year 2000 exceeded and is growing again since 1998, after years of reduction. The sacrifice of this large number of animals is considered to be justified because it finally contributes to the development of new therapies. At the same time, such research can increase the safety of drugs and other chemicals, and may lead to a better understanding of basic processes that underlie health and disease. However, obvious ethical questions are intrinsically related to the use of animals for medical experimentation, and in general studies should be very well designed in order to spare animal lives. In order to accomplish reduction, replacement and refinement of animal experiments, there is an increasing need for validated in vitro test systems. Because a major part of research in which experimental animals is used are ultimately focused towards the human situation, proper in vitro systems should be predictive for the situation in healthy individuals and even better for the conditions in patients. A straightforward approach to accomplish this is to use human tissues, cells or enzymes for testing of drugs. The liver is considered to be the main organ involved in the metabolism of xenobiotica, but lung, kidney and intestine can also contribute considerably. Organ toxicity can, among other factors, be caused by organ-specific metabolism. For research on drug metabolism and toxicity in the process of drug development, animal models are commonly used. However, large interspecies differences prevent adequate prediction of metabolism and toxicity data from animal studies to man. Therefore we aimed to develop an in vitro test system employing human tissue along with the animal tissues to predict organ-specific drug metabolism and toxicity in man. This methodology could be used already in an early phase of drug development. A further goal was to establish possible species differences in drug metabolism and toxicity. Moreover, by using human tissue, more knowledge 1

11 about the mechanisms of organ-specific metabolism and toxicity of drugs in man will be collected. Human tissue can be obtained in the hospital setting, where it comes available in relation to surgical procedures that occasionally provide waste material. For research purposes this human tissue is extremely valuable material. A prerequisite for the use of human tissue in an ethically and legally correct manner, is that formal consent is acquired from the ethics committee of the particularly hospitals as well as from the patients and / or families concerned. In order to enable a proper comparisons between various species and between various organs within one species we aimed at developing a universal and standardized method that can be applied for different species and also for different organs in these species. In the present study, we investigated liver, lung, kidney and intestinal tissues and prepared precision-cut slices from these organs, since these tissues are most important for metabolism of drugs in the body, and also for metabolism induced toxicity. Liver, kidney and lung slices were applied before in numerous studies and turned out to be very useful, but were seldom used in combination in an integrated set-up. Therefore, we have chosen to use the technology of precision-cut organ slices with the aim to improve the model further. For intestine, no adequate in vitro system was available and therefore we developed a complementary method for the preparation of (human) intestinal tissue slices. In general, initially rat tissue was used to develop and evaluate the model, since human tissue samples are scarce. One of the major requirements of a practical in vitro model is that it can be used whenever needed. When slices from organs of the commonly laboratory animals, such as rat and mouse are at stake, this is feasible. However, tissues from other species such as dog, monkey and, most importantly, man, are not so easily available. Therefore, there is an urgent need also for methods to preserve the harvested organ tissues form these species. Consequently, we investigated the possibility of cryopreservation of liver slices from several species and also studied cold-preservation of lung, kidney, small intestine and colon slices prepared from human and animal organs. To assess the viability of the organ slices prepared, they were characterized with respect to general viability and their potential to metabolize drugs during incubation. The use of slices from liver, lung, kidney and intestines allowed us to make a direct comparison between drug metabolism capacities of these organs. It also may provide basic knowledge about the importance of these organs for metabolic body clearance of drugs as well as on mechanisms of organ-specific toxicity. Ultimately, the aim of the use of organ slices is to predict the processes under study in vivo in the whole body. To explore the possibilities for such in vitro - in vivo predictions, the metabolic rates of the studied organs were determined in slice experiments and subsequently the body clearance by metabolism was predicted taking into account the combined activities of the organs. This 2

12 Aim approach was evaluated by using a number of standard test substrates, of which in vivo data were already available. In short, this research project was devoted to create an infrastructure for early metabolic and toxicological screening through the use of organ slices from rat and man. In addition, potential species differences in drug metabolism and toxicity were object of study. This methodology is expected to provide new opportunities for further closing the gap between pre-clinical and clinical studies in the process of drug design and development. It can also lead to a more rational choice between certain animal species in the prediction of pharmacokinetic profiles including potential interactions between drugs. Viable tissue slices contain various cell types in their original setting, which also enable studies on pharmacological effects and intercellular communication. Table 1. The Human Tissue Infrastructure Groningen University Institute for Drug Exploration (GUIDE) Applications Precision-cut slices of human liver, lung, kidney and intestine Cryopreservation and cold storage for tissue banking Short and long-term incubations with drugs and drug-delivery preparations Measurement of drug biotransformation (including induction and inhibition), transmembrane transport, pharmacological and toxicological effects and mechanisms of drug-delivery. 1. transplantation related research: optimization of preservation procedures, quality control of donor and transplanted organs and design of protective agents for donor organs. 2. early pharmacokinetic screening of novel therapeutics. 3. early toxicological screening and human specific metabolites. 4. detection of species differences in drug disposition. 5. profiling of relative organ activities in the body. 6. improved knowledge of organ physiology and pathology. 7. study of drug disposition and effects in diseased organs. 8. functional interactions between organs in the body. 9. improved knowledge of intercellular communications in organs. 10. improving rational choice of species for drug safety testing. 11. study of potential interactions in drug disposition. 12. reduction, replacement and refinement of the use of experimental animals. 3

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