UCL- Universit catholique de Louvain · Web viewThe ultimate objective is to develop flexible optimization strategies applicable in practical chromatographic separation problems

UCL- UniversitŽ catholique de LouvainFaculty of Medicine

THE SCHOOL OF PHARMACY

SCIENTIFIC ACTIVITIES

1996

School of PharmacyUniversitŽ catholique de Louvain

SFAR 7360Avenue Mounier 73

B-1200 BrusselsBelgium

Secretariat office: Annie Celis, Josiane Coremans

& VŽronique PicartPhone : #-32-2-764 73 60

Fax : #32-2-764 73 63E-mail : [email protected]

President : R.VerbeeckAcademic secretary : V. PrŽat

Foreword 1

Overview : from 1990 to 1996 2

Research themes 7

Pharmacochemistry 8

Pharmacotoxicology 18

Biopharmacy 38

Scientific output 53

Staff profile 59

The School of Pharmacy is structured in 7 units :

BCTC : Biochemical toxicology CHAM : Drug analysis CMFA : Pharmaceutical chemistry and radiopharmacyFACM : Cellular and molecular pharmacology FARG : Pharmaceutical technology FATC : Pharmacokinetics and drug metabolism TEMU : Mutagenicity and teratogenicity

FOREWORD p 1

Dear reader,

You are currently holding in your hands the first research report of the School of Pharmacy at the ÒUniversitŽ catholique de LouvainÓ (UCL). It is not just another research report. Indeed, you will find all the information regarding our research programs, and you will also learn how wide an area the pharmaceutical sciences cover.

As President of the School, I am proud of the diversity and the wealth of our research activities conducted by only a staff of approximately twenty senior scientists.

This document gives an overview of the research programs and activities of the School of Pharmacy in the period 1995-96. Our School of Pharmacy is located on the Health Sciences Campus of the UCL in Brussels. Whereas it is true that one of our main missions is the formation of pharmacy graduates (we have currently approximately 850 students of whom approximately 100 are enrolled in one of our graduate programs); our faculty members, research associates and supporting staff are also putting much effort in the development and continuation of high quality research projects.

These research activities are centered around three major themes : pharmacochemistry, pharmaco-toxicology and biopharmacy. It is obvious that several of these projects involve a close collaboration between groups approaching a particular research problem from different angles represented by these major areas of pharmaceutical sciences. In addition, certain groups within our School of Pharmacy are participating in interdisciplinary research projects with other UCL faculties (e.g. Medicine, Sciences, Agronomy) or with other universities both nationally or internationally. To stimulate this interdisciplinary approach, research seminars are presented on a regular basis not only by members of our School but also by researchers coming from other institutions both on a national and international level.

In the first part of the report, an overview is presented concerning human resources, financing, scientific activities during the period 1990-96. Next, the different research projects are described in some detail, providing in addition information concerning the available expertise and the technical support related to the projects, collaborations and recent publications. A complete list of the scientific publications of the School of Pharmacy in 1995-96 is also given, as well as the career profiles of the member of the School of Pharmacy active in research.

Finally I would like to thank all members of the School of Pharmacy who have contributed to the preparation of this brochure, and in particular the editorial staff composed by Nathalie Delzenne, Philippe LŽvque and Franoise Van Bambeke, as well as VŽronique Lebrun for the editing and design work.

I hope that this document will contribute to a steady progress in our research activities directly towards the development and safe use of therapeutic agents.

Enjoy the reading !

Roger K. VERBEECKPresident

OVERVIEW : from 1990 to 1996 p 2

Human resources

From 1990 the number of persons involved in scientific and teaching activities in the School of Pharmacy has gradually increased, from 60 in 1990 to100 persons in 1995. The number of post-doctoral fellows and PhD students is mainly res-ponsible for this dynamic evolution. In 1996, 35 students were performing a PhD thesis in pharmaceutical sciences. The School of Pharmacy also attracts students performing PhD thesis in sciences and biomedical sciences.

Academic staff Tenured scientists PhD studentsPostdoctoral fellowsTechnical & administrative staff

1990

42%

11%

18%

24%5%

31%

14%

9%

11%35%

1995

Staff profile evolution


Financial resources

The main part of the staff at the School of Pharmacy is paid by the University, but contracts with industries as well as non profit and government organizations allow to pay the extra scientific and technical staff.The research projects are funded by :

l either EC or international instancesl non profit Belgian organizationsl industryl university

University IndustryNational institutions Others

12%

9%

13%

66%

Funding of positions

Scientific output

The average number of publications of the School of Pharmacy in peer-reviewed scientific journals is about 50 per year. It reached 66 in 1995. A list of references (1995-96) is given in section 5.

The dynamism of the research results primarily from an increase of collaborations between the members of the different units within the department, putting together their competence to carry out research projects, but also from other collaborations with outdoors scientific teams. Collaborations are presented for each research team in section 4.

Pierre DumontÕs Chair

Dedicated to the pioneer work of Prof. Pierre Dumont who, during his academic carrer, encouraged the development of the School of Pharmacy at the UCL, the ÒPierre DumontÕs chairÓ is, since 1995, an award given to a well-known medicinal chemist or pharmacologist. The laureate is invited to give a prestigious lecture reviewing his scientific work.

In 1995 the first laureate was prof. Camille Wermuth from the University of Strasbourg (France) for his work dealing with the discovery and the development of new structure-type M1 muscarinic agonists for Alzheimer

disease. Prof. GŽrard Grassy, from the Center of Structural Biochemistry at the University of Montpellier (France) was invited in 1996 and gave a lecture about ÒTherapeutic innovation and molecular modelingÓ.


Invited speakers

Research activities and exchanges are promoted by the organization of bimonthly seminars : young senior investigators from the department alternate with invited speakers from industries and other universities. At this occasion, the opportunity is given for the PhD and postdocs to meet scientists with international reputation. In 1995 and 1996 those were :

From universities, hospitals or research institutesl Dr J. Piette (Laboratoire de Virologie Fonda-mentale, Institut de Pathologie, UniversitŽ de Lige, Belgium)l Prof. D.D. Breimer (Leiden/Amsterdam Center for Drug Research, Universiteit van Leiden/Vrije Universiteit

Amsterdam, Leiden, Pays-Bas)l Prof. P. De Meyts (Hagendorn Research Institute, Copenhage, Danemark)l Prof. J.M. Scherrmann (UnitŽ INSERM n¡ 26, H™pital Fernand Widal, Paris, France)l Prof. B. Masereel (Laboratoire de Chimie Pharmaceutique, Institut de Pharmacie, UniversitŽ de Lige, Belgium)l Prof. Kauffmann (Laboratoire d'analyse instrumentale et de bioŽlectrochimie, Institut de Pharmacie, UniversitŽ

Libre de Bruxelles, Bruxelles, Belgium)l Prof. J.M. Boeynaems (Institut de recherches en Biologie humaine et nuclŽaire, UniversitŽ Libre de Bruxelles

et Service de Chimie MŽdicale, H™pital Universitaire Erasme, Bruxelles, Belgium)l Prof. E. Declercq (Rega Instituut, K.U.L., Leuven, Belgium)l Prof. T. Le Doan (CNRS/URA 1116, UniversitŽ de Paris-Sud, France)l Prof. M.N. Dudley (Editor of Antimicrobial Agents and Chemotherapy; the University of Rhode Island,

Providence, RI, U.S.A.)

From UCLl Dr. J.N. Octave (UnitŽ de Neurochimie)l Prof. L. Ghosez (UnitŽ de Chimie Organique)l Prof. G. Van den Berghe (Laboratoire de Chimie Physiologique - Groupe de Recherches MŽtaboliques)l Prof. J. Melin (UnitŽ de tomographie par positrons et DŽpartement de MŽdecine Interne, Cliniques

universitaires St Luc)l Prof. L. Hue (UnitŽ Hormones et MŽtabolisme)l Prof. J.M. Maloteaux (UnitŽ de Pharmacologie et DŽpartement de Neuropsychiatrie, Cliniques universitaires

St Luc)l Dr J.L. Balligand (UnitŽ de PharmacothŽrapie / Cliniques universitaires St Luc)l Prof. F. Opperdoes (Tropical Diseases Unit, ICP & Laboratoire de Chimie Physiologique)l Prof. E. Van Schaftingen (Laboratoire de Chimie Physiologique - Groupe de Recherches MŽtaboliques)

From the industryl Dr. J. Roba, Dr.Med.Vet., Dr.Sc. (UCB Secteur pharmaceutique, Recherches et DŽvelop-pement, Braine

l'Alleud, Belgium)l Dr. P. Pellegrin (Allen-Glaxo Laboratories, Bruxelles, Belgium)l Dr. A. Van Peer (Janssen Research Foun-dation/Clinical Pharmacokinetics, Beerse, Belgium)

Othersl Dr G.N. Fracchia (CCE, DG XII [Biomedicine Programme], Bruxelles)

Scientifc awards

From 1990 to 1996 several students or members of the School of Pharmacy were awarded scientific prizes.l Bernard Gallez : the 1995 prize of the Belgian Society of Pharmaceutical Sciences.l Didier Lambert : the 1995 ÒConcours ordinaireÓ Prize of the Royal Belgian Academy of Medecinel Vincent Lannoy : the 1995 Siemens prize


Congresses symposia, meetings

Over the past 5 years, the members of the School of Pharmacy have participated to the organization of several congress, seminars, symposia or meetings in various domains.

Congressesl J. Poupaert (1991-1995) ÒJournŽes Franco-Belges de PharmacochimieÓ, Lige & Brussels, Belgium; Lille,

Francel M. Roberfroid (1991) XIth meeting of the European Associations for Cancer Research, Genova, Italyl M. Roberfroid (1991) First join meeting of the American and the European Association for Cancer Research,

Santa Marghareta, Italyl M. Roberfroid (1993) XIIth meeting of the European Association for Cancer Research, Brussels, Belgiuml M. Roberfroid, P. Buc Calderon (1993) First World Congress on Alternatives to Animal Experiment, Baltimore,

USAl B. Tilquin (1994) 6th Comett chemometrics School, Brugge, Belgiuml M. Roberfroid (1995) Congress of International Union Food Science and Technology, Budapest, Hungryl B. Tilquin (1995) ÒDeuxime Colloque de Radiobiologie Fondamentale et AppliquŽeÓ, Spa, Belgiuml B. Tilquin (1995) 5th International Symposium on Drug Analysis, Leuven, Belgium

Symposial J. Poupaert (1991) Receptors Signal Transduc-tion and Drug Design, Antwerp, Belgiuml J. Poupaert (1991) Enzyme Inhibition and Drug Discovery, Antwerp, Belgiuml J. Gillard, W. Lhoest (1992) ÒLa stŽrilisation industrielleÓ, Brussels, Belgiuml J. Gillard (1993) ÒSymposium LyophilisationÓ, Brussels, Belgiuml M. Roberfroid (1993) ÒSymposium International sur les BifidobactŽries et leurs PropriŽtŽs Nutritionnelles

comme ProbiotiquesÓ, Brussels, Belgiuml M. Roberfroid (1993) ILSI-Europe International Symposium on Antioxidants, Nutrition and Health, Stockholm,

Swedenl P. Tulkens (1993) Controversies in Antibiotic Therapy: Pharmacological Aspects of Antibio-therapy, Anaheim,

USAl J. Gillard, W. Lhoest (1994) The State of the Art in granulation drying technology, Brussels, Belgiuml M. Roberfroid (1994) ILSI International Work-shop on Gut Microflora and Helath, Barcelona, Spainl B. Tilquin (1994) ÒJournŽe dÕEtude Comett en ÒChimiomŽtrie et ChromatographieÓ, Brussels, Belgiuml P. Tulkens (1994) Aminoglycosides Once-a-Day, Brussels, Belgiuml J. Poupaert (1995) Neurodegenerative Diseases and Drug Design, Antwerp, Belgiuml M. Roberfroid (1995) ILSI International Work-shop on Comparative Methods in Toxicology, Wageningen, The

Netherlandsl M. Roberfroid (1995) First ORAFTI Research Conference, Brussels, Belgiuml B. Tilquin (1995) ÒJournŽe de la SociŽtŽ Royale de Chimie, Brussels, Belgiuml P. Vanbel (1995) Chemometrics in Belgium, Antwerpen, Belgiuml P. Vanbel (1995) Chemometrics symposium, Brussels, Belgiuml M. Roberfroid (1996) European Agricultural Research in the 21st century world, Strasbourg, Francel M. Roberfroid (1996) La flore intestinale : miroir de notre santŽ ?, Gemboux, Belgiuml P. Tulkens (1996) Glyopeptides, where do we go ? Brussels, Belgiuml P. Vanbel (1996) Second chemometrics symposium, Leuven Belgium

Meetingsl C. de Meester (1993) Annual Meeting of the Belgian Environmental Mutagen Society, Lige, Belgiuml V. PrŽat (1995) First Tricontinental Meeting of Hair Research Societies, Brussels, Belgium


Teaching activities of the School of Pharmacy

Teaching of pharmacy (graduate level)Every year, the undergraduate program of the school of pharmacy confers to 100 to 150 students the right to exert the pharmaceutical art in a retail pharmacy. During their curriculum, these students receive training in basic sciences (chemistry, biology, physics, mathematics) and biomedical fundamental sciences (physiology, anatomy, biochemistry, immunology, histology). The two following teaching years are dedicated to specific pharmaceutical sciences (pharmaceutical chemistry, analytical chemistry, pharmacology, pharmaco-gnosy, toxicology, microbiology, galenic), whereas the fifth year essentially consists in a professional training (in the retail pharmacy, principally), together with the teaching of pharmaceutical legislation, prac-tical pharmacy and semiology and seminars in different domains of pharmaceutical sciences. During this graduate period, an initiation to the different domains of pharmaceutical research is available to the enough brilliant students. They are allowed to replace a part of the obligatory practical training by a research work in one of the laboratories of the school.

1991-92 1992-93 1993-94 1994-95 1995-960

200

400

600

800

1000 GraduatePostgraduateNumber of students

Evolution of the number of students

Among the 700 students in pharmacy, 10 to 20% will envision a complementary training which, by means of a further time-investment of 1 to 5 years, will open to them a very large panel of carriers in all the domains of the pharmaceutical art.

Postgraduate teachingThese postgraduate teachings are open to pharmacists as well as to other scientists (PhD, MDÕs, ...) and organized as follows.

l DES ("Dip™me d'ƒtudes SpŽcialisŽes") in :u pharmaceutical enginee-ring and industrial techno-logy (2 years). It provides a training in pharmaceutical

technology for pharmacists or non pharmacists wor-king in industry;u industrial pharmacy (1 year). It offers a post-graduate training to pharmacists who will be in charge of quality

control in industry. This program will be organized in collabo-ration with other univer-sities (UniversitŽ libre de Bruxelles, UniversitŽ de Lige)

u hospital pharmacy (1 year). This program is accessible to pharmacists wishing to exert their art either in a general hospital or in a tropical hospital;

u clinical chemistry (5 years). This post-graduate training offers to pharmacists requires 5 years . During the first 2 years, the student receives a training in biological analysis. The 3 following years are divided in professional practice in laboratories specialized in microbiology, hema-tology or biological chemistry.


l DEA ("Diplome d'Etudes Approfondies"). This short, intensive and high-level education devoted to pharmaceutical research is available since 1995, to both pharmacists and non-pharmacists. This degree gives to the students during their thesis work an opportunity to deepen their knowledge in pharmaceutical sciences and opens the access to the doctorate in pharmaceutical sciences to non-pharmacist graduates. By itself, this intensive training also offers to the industry competitive university post-graduates having a basis knowledge in pharmaceutical research.

l PhD in pharmaceutical sciences. The most common way to enter the field of pharma-ceutical research remains the PhD. thesis. This is obtained after presentation and defense of an original research work.

l Other studies. Certificates concern very specialized aspects of pharmaceutical sciences such as radiopharrmacy, toxicology, public health, work security and hygiene ...

1991-92 1992-93 1993-94 1994-95 1995-960

10

20

30

40

50

60 DEAIndustry

HospitalClinical biology

Ph.DCertificates

Number of students

Repartition of the postgraduate students

RESEARCH THEMES p 8

Pharmacochemistry

l Chemometrics in liquid chromatographyl Ionizing irradiations of drugsl Search for new pharmacologically active molecules from natural sourcesl Molecular selectivity : transport and receptor interactions.

Pharmacochemical studies of the amino-acids and the cannabinoid neurotransmissionl Design and pharmacological evaluation of drugs acting on the central nervous system

Pharmacotoxicology

l Influence of nutrients on liver metabolism : biochemical mechanism underlying functionnality and/or toxicity

l Development of radiopharmaceuticals and magnetopharmaceuticals as functional contrast agents. Oximetry probes. Liver and CNS-targeted ligands for receptor imaging

l Molecular analysis of drug-membrane interactionsl Cellular toxicity of antibiotics and other drugsl Chemotherapy of the intracellular infectionl Cellular penetration of antisense oligodeoxyribunucleotidesl Energetic and redox homeostasis during oxidative stress. Study of molecular mechanisms

leading to cell deathl Adaptive response to ionizing radiationl Biomonitoring of people exposed to chemicalsl Investigations on the contamination of food by mutagenic compounds generated by moulds

(aflatoxin B1) or by the heat processes (polycyclic aromatic hydrocarbons or heterocyclic aromatic amines)

Biopharmacy

l Biological dosimetry on people accidentally or professionally exposed to ionizing radiationsl Clinical evaluation of new therapeutic approachesl Metabolism of immunosuppressive drugs and immunosuppressive activity of the metabolitesl Drug Glucuronidationl Plasma protein binding and pharmacokinetics microdialysis samplingl Biodegradable polymers as drug deliver systemsl Electrically enhanced transdermal drug deliveryl Nanoparticles and microspheres as drug and antigen delivery systems.

RESEARCH THEMES p 9

Chemometrics in liquid chromatography

B. Tilquin (CHAM), J. Cumps (CMFA), B. Rollmann (CHAM), P. Vanbel (CHAM)

Supported by RŽgion Wallonne grant n¡3047 (1995-97)

Chemometrics is applied to High-Performance Liquid Chromatography (HPLC). Chemometrics has been defined as the chemical discipline that uses mathematical, statistical and other methods of formal logic : a) to design or select optimal measurement procedures and experiments ; and b) to provide maximum relevant chemical information by analysing chemical data. Our general goal is the fast development of good and robust HPLC methods. The ultimate objective is to develop flexible optimization strategies applicable in practical chromatographic separation problems.

Because of its outstanding separation power and versatility, High-Performance Liquid Chromato-graphy (HPLC) is one of the most widely used separation technique in research and routine analysis. Our general objective is the fast development of optimum HPLC methods. The major steps of method development in HPLC are :

l Method selectionl Optimization of the retentionl Selectivity optimizationl System optimizationl Method validation

Our research work focuses principally on the third step, namely the selectivity optimization process. The chromatographic selectivity is influenced by a large number of parameters (type of stationary phase, mobile phase composition, pH, ionic strength...). This large number of variables and the possible interactions between some of them require the use of chemometrics to design an optimal procedure and extract the largest possible information from the smallest possible set of experiments. The main steps of any optimization procedure are :

l definition of the parameter space (selection of the important parameters and their variation limits);l definition of the experimental design (selected experiments);l selection of an adequate optimization criterion;l mathematical modelling and optimum deter-mination.

An essential part of any optimization strategy is a clear definition of the goal(s) of the process (third step). A mathematical description of such a goal is called an optimization criterion. The quality and flexibility of the criteria are among the essential factors determining the applicability of selectivity optimization procedures. Indeed, the result of an optimization process depends on the selected criterion, so that the latter has to be defined in the context of the objectives of the separation. Many different criteria have been suggested for describing the quality of chromatographic separa-tions. One of our major objectives is the definition of adequate optimization criteria adapted to practical situations :

l non-ideal separations : for example, purity analysis of drugs (chromatograms including a very large peak corresponding to the principal compound and very small peaks of impurities);

l limited optimization : separation of a limited number of solutes in a complex mixture (monitoring of drug levels in body fluids and of environmental pollutants...).

For optimizing two or more criteria simultaneously (resolution, analysis time...), Multi-Criteria Decision Making (MCDM) techniques are powerful tools.

Another fundamental aspect is the development of robust HPLC methods. The robustness of a method is typically evaluated independently at a much later stage as part of the method validation process. By considering robustness at an early stage of method development, both the amount of work required and the chance of failure during the method validation stage can be greatly reduced. Recently, we suggested an approach that allows the ultimate robustness of chromatographic methods to be rigorously included as an objective from the outset of systematic method develo-pment. Two things are needed to include robust-ness as an objective in optimization strategies :

l definition of robustness criteria that can be used during selectivity optimization strategies;l use of MCDM techniques to find a suitable balance between robustness, resolution and other separation

objectives.

RESEARCH THEMES p 10

Our last important field of interest is the peak purity control in liquid chromatography using modern instrumentation (HPLC with photodiode array detection) and appropriate techniques for data analysis (principal component analysis, evolving factor analysis...). Indeed, the assessment of the purity of drugs is a central and crucial problem in pharmaceutical analysis.

In conclusion, our ultimate objective is the develop-ment of flexible optimization strategies applicable in practical chromatographic separation problems. The availability of adequate and user-friendly software is also a priority. The development of a chromato-graphic optimization software is the aim of a project currently financed by "RŽgion Wallonne".

Moreover, it is very important to promote the use of chemometrics in university and industry labora-tories, and to encourage the teaching of chemo-metrics at universities and technical high-schools. In this context, our laboratory is very involved in the activities of the new section "Chemometrics" of the KVCV and SRC (P. Vanbel is a member of the scientific committee). This chemometrics society organizes an annual chemometrics symposium (Brussels, 1995; Leuven, 1996). The laboratory also organizes common seminars with the research laboratory of Prof. Massart (VUB, Brussels) and with Solvay (Dr. Gobillon and Dr. Vanmarcke).

An exemple of response surface : minimum chromatographic resolution versus pH and % MeOH for the separation of four solutes

Collaborationsl P. J. Schoenmakers, Shell Research, , Amsterdam (The Netherlands)l B. Govaerts, UCL (STAT)

Publicationsl P.F. VANBEL, J.A. GILLIARD and B. TILQUIN, (1993) Chemometric optimization in drug analysis by HPLC : a

critical evaluation of the quality criteria used in the analysis of drug purity, Chromatographia, 36, 120-124.l J.A. GILLIARD, J.L. CUMPS and B.L. TILQUIN, (1993) Efficiency of an automatic peak purity control

procedure in high performance liquid chromatography-photodiode array coupling based on evolving factor analysis, Chemom. Intell. Lab. Syst., 21, 235-242.

l P.F. VANBEL, B.L. TILQUIN and P.J. SCHOENMAKERS (1995) Criteria for develo-ping rugged high-performance liquid chromato-graphic methods, J. Chromatogr., 697, 3-16.

l P.F. VANBEL, B.L.TILQUIN and P.J. SCHOENMAKERS (1996) Criteria for optimizing the separation of target analytes in complex chromatograms. Chem. Intell. Lab. Syst., 35, 67-86

l C. RITTER, J. GILLIARD, J. CUMPS and B. TILQUIN, (1995) Corrections for hetero-scedasticity in window evolving factor analysis, Anal. Chim. Acta, 318, 125-136


Ionizing irradiations of drugs

B. Tilquin (CHAM), N. Barbarin (CHAM), A.S. Crucq (CHAM), M. Gibella (CHAM), Th. Pronce (CHAM), B. Rollmann (CHAM)

Supported by the industry (Eli-Lilly) (1995-98)

The use of ionizing radiations is one of the most promising method for the sterilization of solid pharmaceuticals. Indeed, the sterilization of thermo-sensitive drugs by gamma irradiation is a desirable alternative The choice of the sterilized radiation dose is based on the initial bioburden but the maximal admitted dose for the radiosterilization is 25 kGy. Major advantages are high penetrating power of gamma rays, isothermal character of the procedure, sterilization in the final package, ... This mode of sterilization is also cheaper than microfiltration, lyophylization and filling steps under aseptic conditions. Nevertheless, the irradiation of solid drugs produces characteristic chemical effects that are not detected by classical tests like melting point, ultra violet spectro-photometry... So, methods like ESR measurements, lumi-nescence, ... are to be developped. In the reverse order, radiolysis is the best way to study radical mechanisms in solid or liquid state; pulse radiolysis is suitable for the study of very fast radical kinetics. .

Recent developments appear to have contributed to the increased interest in sterilization of pharmaceuticals by gamma irradiation. However the radiosterilization produces new products which can induce a modification of odour and colour and could be potentially toxic. These products are formed in so low concentration that the usual analytical techniques and toxicity tests are not sensitive enough to detect them. During irradiation of solid drugs, free radicals are formed and trapped in the matrix. These radicals are studied by Electron Spin Resonance (ESR) which is a very sensitive technique (10-9 M). The ESR signal is detected only after irradiation and a part of free radicals produced survives at room temperature for a long time (some years). From the decay curve of the radicals it is possible to estimate the initial dose of irradiation. After dissolution of irradiated drug, the radicals give also final radiolysis products which can be studied by High Performance Liquid Chromatography (HPLC). Some of these products are unique to the radiolysis and can be used like a proof of the irradiation. Irradiation also produces volatile products which may induce a modification of odour. These can be analyzed by gas chromatography with I.R. and M.S. detections. Headspace sampling is used which allows the determination of volatile compounds present in a non-volatil matrix. Moreover, the effects of ionizing radiation upon optical activity are of utmost importance, as the enantiomers of drugs does not necessarily possess the same pharmacological activity. Our current progress on the subject lead us to consider radioracemization as an inevitable consequence of the process, but the racemization itself seems to be limited to a small increase in the enantiomeric ratio.

The radiolysis method permits to select radicals using appropriated scavengers, in order to induce the radical mechanisms. The absorption spectra and the rate constants of radical formation or disappearance reactions can be determined by pulse radiolysis. These intermediates are at the origin of stable final radiolytic products that can be studied by High Performance Liquid Chromato-graphy. With the informations about radicals and final radiolytic products, radical mechanisms can be proposed and simulation programs are used to verify these radical mechanisms.

Collaborationsl M. Debatty (UCL-UnitŽ de Chimie des Interfaces)l E. de Hoffman (UCL-CICO)l Prof. Gards, UniversitŽ Descartes, Paris (France)l Dr. Hickel, Centre d'Etude Atomique de Saclay, Gif-sur-Yvette (France)l Prof. Klassen, National Research Council Canada, Ottawa (Canada)l Prof. Miyazaki, University of Nagoya (Japon)l Prof. Raffi, UniversitŽ de Marseille-St-JŽr™me (France)

Expertisesl Radiolysis of molecular cristalsl Electrochemical detection of drugsl Analysis of essential oils

l Analysis of traces of degradation substences from drugs


ESR spectrum from irradiated liquid solution of diazepam after spin-trappingIn radiation chemistry, radicals in liquid state are transient intermediates. ÒSpin-trappingÓ is the term applied to the deliberate addition of a radical scavenger to obtain stable free radicals. The scavengers used are Òproradical moleculesÓ (H. Viehe, UCL). The Òspin trap radicalÓ give dimeric product and come back to the spin trap radical by increasing temperatures (60-70¡C).

Referencesl M. GIBELLA, A.-S. CRUCQ and B. TILQUIN. (1993) ESR measurements and the detection of radio-

sterilization of drugs. J. Chim. Phys., 90, 1040-1053.l A.-S. CRUCQ, B. HICKEL and B. TILQUIN. (1995) Radical mechanisms of cephalosporins : a pulse radiolysis

study. Free Rad. Biol.Med., 18, 841-847 l T. MIYAZAKI, J. ARAI, T. KANEKO, K. YAMANOTO, M. GIBELLA and B. TILQUIN. (1994) Estimation of

Irradiation dose of radiosterilized Antibiotics by Electron Spin Resonance : Ampicillin. J. Pharm. Sci, 83, 1643-1644

l Th. PRONCE and B. TILQUIN. (1996) Trace analysis in chiral separation of selected amino acid enantiomers.J. Pharmaceut. Biomedi. Analysis, 14, 1175-1184


Search for new pharmacologically active molecules from natural sourcesJ. Quetin-Leclercq (CHAM), C. Djadjo Djipa (CHAM), A. Cachon Coello (CHAM), N. Delzenne (BCTC)

The aim of our work is to find new pharmacologically active substances from plants which could constitute new drugs for therapeutic use or be used as models by chemists to improve their properties. This implies pluridisciplinary researches which can be divided into several sections. Plants to be studied, selected on an ethnopharmacological or chemiotaxonomic basis are first extracted and their properties assessed by in vitro screenings. When an extract is found to possess an interesting effect, its constituents are fractionated and their active compounds isolated and purified by several chromatographic techniques. The chemical structures of these bioactive molecules are determined by the analysis of their spectroscopic data and methods for quantitative determinations are developed to standardise, when necessary, plants or crude extracts containing these substances.

Plants are an inexhaustible source of secondary chemical metabolites which are no longer considered as residual substances from catabolism: on the contrary, several authors think that their presence in plants could account for the selection of these plants during evolution, because better protected against external aggressions (virus, micro-organisms, parasites,...). These secondary metabolites are characterised by an exceptional variety of novel structures, often complex and difficult to obtain by chemical synthesis, which may possess interesting modes of action. Up to now, more than 70% of our medicines are substances of natural origin or derived from them. The research we develop in the laboratory of pharmacognosy is a pluridisciplinary study of bioactive secondary metabolites. It can be divided into several sections.

Analysis of the pharmacological activity of crude extracts

Plants selected on an ethnopharmacological or chemiotaxonomic basis are extracted with several solvents. Their biological activities are assessed by in vitro models in close collaboration with specialised laboratories. We principally focus on the cytotoxic, antitumoral, antiparasitic, antifungal, antimicrobial, antimutagenic, immunomodulator and hepatoprotector activities.

Isolation and purification of bioactive molecules

Crude extracts possessing interesting effects are fractionated by means of several preparative methods on solid base material (normal or inverted phase silica gel, molecular sieves, ion exchangers,...) or by more recent liquid/liquid chromatographic methods (centrifugal partition chromatography). Their bioactive compounds are then isolated and purified to enable more detailed analysis of their pharmacological properties - in collaboration with specialised research groups.

Structure determinations of these purified substances

The chemical structure and stereochemistry of the bioactive compounds are determined by the analysis of several of their spectroscopic data (UV, IR, MS, 1D or 2D homonuclear or heteronuclear NMR, X Ray diffraction spectra). It is necessary to know precisely these structures to establish structure-activity relationships, realise conformational studies and find the structural requirements responsible for their activities. These substances, because of their interesting activities or modes of action, could then constitute new therapeutic drugs or be used as models by chemists for the synthesis or hemisynthesis of more specific substances or compounds possessing less side effects;

Development of methods of quantitative determinations

In several cases, for example :

l when a synergy exists between several molecules, l when the isolation of the active compounds would give no benefit as compared with standardised extracts or

would not be economically interesting,

l when it is necessary to detect and determine the exact concentration of interesting compound(s) in several batches or in different species containing the same bioactive molecule,...


we have to develop methods for quantitative determinations or standardisation of crude drugs or extracts. Beside general analytical methods (colorimetry, titrimetry,...), more specific techniques using HPLC, GC/MS, GC/FTIR, analytical centrifugal partition chromatography or densitometry can be used.

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0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0

Euca

lypt

ol

Lim

onèn

e

Para

-cym

ène

Terp

inèn

e-1-

ol-4

Alph

a-Te

rpin

éol

Verb

énon

e

Spectre FTIR de l'eucalyptol

Nombre d'onde (cm-1)

A.U

.

-1004009001400

7501250

1750

2250

2750

3250

3750

Analysis of an Essential Oil Eucalyptus cinŽolifera

Collaborationsl G. Balansard, R. Elias, A. Favel, T. Timon-David, UniversitŽ dÕAix-Marseille, Francel C. Wright, University of Bradford, Englandl T. Tsakala, University of Kinshasa, Za•rel M.C. De Pauw, UniversitŽ de Lige, Belgium l M.A. Colson-Corbisier, Mycothque de lÕUCL,l M. DelmŽe, UnitŽ de BactŽriologie, UCL

Expertisel Isolation, purification and quantitative determination of chemical substances from crude extractsl Analysis of natural products or vegetable drugs and quality controll Standardisation of plants or crude extracts

Publicationsl J. QUETIN-LECLERCQ, N.G. BISSET and L. ANGENOT (1990) South American Steychnos species.

Ehnobotany (except curare) and alhaloid screening. J. Ethnopharmacol. 28, 1-52l J. QUETIN-LECLERCQ, M.C. DE PAUW, R. BASSLEER and L. ANGENOT (1991) Effects of

strycnhopentamine on cell lines cultured in vitro. Chem. Biol. Interactions 80, 203-216l J. QUETIN-LECLERCQ, L. ANGENOT, L. DUPONT, O. DIDEBER, R. WARIN, C. DELAUDE and C. COUNE

(1991) Revision of the structure of strychnochromine. Tetrahedron Lett. 32, 4295-4298l J. QUETIN-LECLERCQ, B. BOUZAHYAZ, A. PONS, R. GREIMERS, L. ANGENOT, R. BASSLEER and H.

BARBASON. (1993) Strychnopentamine, a potential anticancer agent. Planta Med. 59, 59-62l J. QUETIN-LECLERCQ, G. LABRéS, R. WARIN, H. MAVAR-MANGA and L. ANGENOT (1995)

Guianensine, a sitterionic alcaloid for Strychnos guianensis. Phytochemistry 40, 1557-1559


Molecular selectivity : transport, and receptor interactions.Pharmacochemical studies on the amino-acids and the cannabinoid neutrotransmission

D. Lambert (CMFA), B. Gallez (CMFA), M. Kanyonyo (CMFA), J. Poupaert (CMFA), H. Ucar (CMFA), R. Verbeeck (FATC), N. Delzenne (BCTC)

Supported by : UCL-FDS (1996-98) ; FNRS ÒCrŽdit aux ChercheursÓ (1996-97)

The aim of these projects are devoted to the understanding of the properties affecting selectivity in drug transport and ligand-receptors interactions. We are focused on two principal axes :

l Drug transport to the brain of aminoacids and small peptides. Development of chemical delivery systems to the brain. Pharmacological studies of the amino acid neurotransmission (GABA, glycine, taurine and glutamate) as potentiel pharmacological CNS agents (analgesia, epilepsy, ischemia). Pharmacokinetic studies of the synthesized derivatives with a special emphasis to the blood-brain barrier crossing.

l Development of pharmaco-chemical tools for a better understanding of the ligand-receptor interactions in the cannabinoid neurotransmission. Structure-activity rela-tionships in the anandamide family, anandamide is the putative endogenous ligand of the cannabinoid receptors. Development of affinity labels, fluorescent probes for the study of the molecular events resulting of the interaction ligand-receptor. In vitro and in vivo pharmacological studies regarding the cannabino•d neuro-transmission involve analgesia, epilepsy, cognition, sedation and immunomodulation.

Selectivity is a prerequisite for the development of a drug candidate. Selectivity can be achieved either by a selective transport to the bioactive compartment (prodrug and targeting approaches) or by a better affinity for the molecular targets (specificity and potency of the ligand bound to the receptor). Besides the aim to develop new potential therapeutic agents, our interest additio-nally deals with the obtention of new pharmaco-chemicals tools to improve the fondamental knowledge of ligands-receptors interactions and drug transport mechanisms.

Drug transport to the brain of aminoacids and small peptides

Small neutral aminoacids suffer from the absence of specific transporter on the luminal side of the endothelial cells constituting the blood-brain barrier. Therapeutic use of peptides is hampered to their relative inability to cross biological membranes. In order to enhance the brain penetration, different strategies are used in our research team.

l Pharmacochemical derivatization of reactive functional groups in order to correct the unfavorable physicochemical parameters. Mainly, this prodrug approach is followed to assess a sufficient lipophilicity for an unspecific crossing of the blood-brain barrier by transcellular diffusion. This approach is still applied for several pharmacological agents (glycine, taurine, thiorphan).

l Synthesis of rigid analogues or chimeric-substrates containing the phamacological agent. This approach is both a prodrug design and the use of specific tansporters present at the blood-brain barrier. Besides the study of the transport mechanims, there is a great interest to better characterize the molecular neuropharmacology of some amino acids like glycine and taurine.

l Conjugation of the pharmacological to glyceri-des or to modified glycerides. This approach is not limited to the brain crossing but also to the intestinal barrier penetration. For example, phenytoin lipid conjugates were synthesized to enhance the resorption of this potent anti-epileptic drug.

Development of pharmaco-chemical tools for a better understanding of the ligand-receptor interactions in the cannabinoid neurotransmission

Why to chose the cannabinoid neurotransmission as a target for a medicinal chemistÊ? First, on a public health point of view, the so-called illicit use of marjuana is questionable and there is a renewed interest to characterize the effects of these substances, specially with regard to the new pharmacological discoveries of these recent years. Two receptors were cloned, the CB 1 mainly expressed in the brain is quite different in sequence homology

of the CB2 mainly expressed in the spleen, B lymphocytes and mastocytes, albeit both of these receptors are coupled to


G proteins. The armamentarium of cannabinoid agonists is limited to three different chemical classes : the derivatives of 4-tetrahydrocannabinol (the most active constituent isolated from the plant Cannabis sativa), the derivatives of pravadoline (a synthetic analgesic) and the derivatives of anandamide (arachidonylethanolamide).

Anandamide is the endogenous ligand of the cannabinoid receptors. Our aim is to study the structure-activity relationships of this lead compound to achieve some selectivity between CB 1 and CB2. Up to now, the only selective ligand for the cannabinoid receptors is an antagonist unrelated to any of the three chemical classes described above. Specifically we are studying :

l structure-activity relationships in the ananda-mide series. Besides the synthesis, pharma-cological evaluation both in vitro (binding to the CBl and CB2 receptors) and in vivo (analgesia, hypothermia, sedation, catalepsy for CBl; immunomodulation and inflamation for CB2).

l Affinity labels and fluorescent probes to study the molecular binding of the ligands to the receptor for exemple by isolation of ligand bound to the peptide fragment.

l Synthesis of new analogues of pravadoline. Open analogues and derivatives of 2-benzoxazolinone.l Pharmacological studies of additional proper-ties of anandamide.

Collaborationsl J.M. Maloteaux & E. Hermans (UCL-FARL)l J.P. Herveg (UCL-BIAN)l G. Scriba, University of MŸnster (Allemagne)l L. Ahtee, University of Helsinki (Finlande)l K. Audus, University of Kansas (USA)l J.J. Bourguignon, UniversitŽ de Strasbourg (France)l B.Masereel, UniversitŽ de Lige (Belgium)

Expertisesl Chemical and radiochemical synthesesl Animal anticonvulsant models

O

OH

NHOH

O

N

O

O

N

O

123

Cannabnoid receptors ligands. 1 = tetrahydrocannabinol; 2 = anandamide; 3 = WIN 55212-2

l Drug delivery strategies for central nervous system

Referencesl D.M. LAMBERT, F. MERGEN, J.H. POUPAERT and P. DUMONT (1993) Analgesic potency of S-

acetylthiorphan after intravenous administra-tion in mice. Eur. J. Pharmacol., 243, 129-134l D.M. LAMBERT, L. NEUVENS, F. MERGEN, B. GALLEZ, J.H. POUPAERT, J. GHYSEL-BURTON, J.M.

MALOTEAUX and P. DUMONT (1993) 1,3-Diacyl-aminopropan-2-ols and corresponding 2-acyl derivatives as drug carriers unexpected pharmacological proper-ties. J. Pharm. Pharmacol., 45, 186-191

l D.M. LAMBERT, J.H. POUPAERT, J.M. MALOTEAUX and P. DUMONT (1994) Anticonvulsant activities of N-benzyloxycarbo-nylglycine after parenteral administration Neuroreport.,5, 777-780

l D.M. LAMBERT, F.V. MERGEN, C. BERENS, J.H. POUPAERT and P. DUMONT (1995) Synthesis and pharmacological properties of 2-[S-acetylthiorphan] 1,3-diacylaminopropan-2-ols derivatives as chimeric lipid drug carriers containing an enkephalinase inhibitor Pharm. Res, 12, 187-191

l G.K.E. SCRIBA, D.M. LAMBERT and J. POUPAERT (1995) Bioavailability and anti-convulsant activity of a monoglyceride-derived prodrug of phenytoin after oral administration to rats. J. Pharm. Sci., 84, 300-302


Design and pharmacological evaluation of drugs acting on the central nervous system

J. Poupaert (CMFA), D. Lambert (CMFA), M. Kanyonyo (CMFA), A. Gozzo (CMFA), H. Ucar (CMFA), J.Bukuru (CMFA), K. Vander Poorten (CMFA), R. Verbeeck (FATC)

This project is based on the design, the synthesis and the evaluation of compounds aimed at targets located in the central nervous system (voltage-dependent sodium channels,, i.e. phenytoinergic compounds; G-protein coupled receptors, i.e. melatonin and sigma-1 receptors). The therapeutic objectives are the epilepsy and the neuroprotection. The main lead compounds used were phenytoin and melatonin.

Phenytoinergic compounds

Phenytoin is a potent antiepileptic drug commonly prescribed for the management of grand mal seizures. While this drug has several mode of action, its main target is the central voltage-dependent sodium channel. Phenytoin binds with low affinity to a "phenytoin" that is allosterically modulated by batrachotoxin and interacts with sigma-1 and glycine receptors. Using phenytoin as template, we investigate :

l the effect of substitution on the aromatic rings,l the alkylation of the nitrogen,l the bioisosteric replacement of the 2-carbonyl.

Phenytoin and compounds having a hydantoin ring in their structure have several severe side-effects (teratogenicity, effects on the immune system, ...). Consequently, efforts were dedicated by our group to replace the hydantoin ring by another hetero-cyclic platform. Using ameltolide and thalidomide as lead compounds, this search led to the identifi-cation of 4-amino-(2,6-dimethylphenyl] phthalimide as a phenytoinergic compound with potent and selective anticonvulsant activity in animal models. Another problem met with phenytoin is the erratic bioavailability after oral administration. In order to circumvent this, analogues and prodrugs, i.e. conjugation ot lipids, are also one of the aims of this research group. Along this line, we have develop-ped a new pharmacological assay based on the audiogenic seizure test in magnesium-deprived mice that is more sensitive than previous antiepileptic tests.

Melatoninergic compounds as neuropro-tective agents.

Melatonin is neurohormone which interacts at the level of at least two membrane-bound G-coupled receptors (ML-1 & ML-2), cytosolic and nuclear binding sites. Melatonin can be regarded as the most potent endogenous radical scavenger which is able of capturing both the hydroxyl and superoxide anion radicals in vivo. Its main activities in humans are of chronobiotic and neuroprotective type. Along this line, we are now developing compounds which could maintain neuroprotective activities while being devoid of undesirable chronobiotic properties.

O

N

OO

NN

OO

N

Superposition of molecular modeling pictures of ameltolide and thalidomide


Sigma-1 receptor ligands as antiepileptic and neuroprotective agents.

During our search for antiepileptic drugs having novel modes of action, we identified several leads based on the use of either benzoxazolinone or benzothiazolinone heterocyclic platform. These compounds allowed us to establish the paradygm that sigma-1 receptor ligands capable of crossing the brain-blood barrier are endowed with antiepileptic activities in animal models. Incidentally, during the course of this program we identified compounds with high 5HT-1A and 5-HT-3 affinities that show anti-panic activities in animal models. One of these compounds is now in preclinical phase development with Servier laboratories (Paris, France).

Collaborationsl J.M. Malloteaux (UCL-FARL)l P. Joubau (UCL-VIRO)l D. Lesieur, Institut de Chimie Pharmaceutique, UniversitŽ de Lille II, Lille, Francel S. Spampinato, Dipartimento di Farmacologia, Universitˆ degli Studi di Bologna, Bologna, Italial J. Vamecq, Centre Hospitalier et Universitaire, FacultŽ de Medecine, UniversitŽ de Lille II, Lille, Francel J. P. Stables, National Institutes of Health, Bethesda, Maryland, USA

Expertisel Molecular modellingl Drug designl Chemical synthesis

Publicationsl S. YOUS, J. H. POUPAERT, I. LESIEUR, P. DEPREUX and D. LESIEUR (1994) AlCl3-DMF Reagent in the

Friedel-Crafts Reaction. Application to the Acylation Reaction of 2(3H)-Benzothiazolinones J. Org. Chem., 59, 1574-1576

l B. TINANT, J.-P. DECLERQ, J. H. POUPAERT, S. YOUS and D. LESIEUR (1994) N-[2-(7-Methoxy-1-naphthyl)ethyl]-acetamide, a Potent Melato-nin Analog Acta Cryst. , C50, 907-910

l J. POUPAERT, G. HAMOIR, P. BARBEAUX, D. LAMBERT and J. P. HENICHART (1995) Anticonvulsant Activity of Some N-Phenyl-phthalimide Derivatives J. Pharm. Pharmacol., 47, 89-91

l BAILLEUX, V.; VALLƒE, L.; NUYTS, J.-P;; HAMOIR, G.; POUPAERT J. H., STABLES, J. P. and VAMECQ, J. (1995) Comparative anticonvulsant activity and neurotoxicity of 4-amino-(2,6-dimethylphenyl)phthalimide and prototype antiepileptic drugs in mice and rats Epilepsia, 36, 559-565

l DIOUF, O.; DEPREUX, P.; LESIEUR, D.; POUPAERT, J. H. and CAIGNARD, D. (1995) Synthesis and evaluation of new 2-piperazinylbenzothiazoles with high 5HT-1A and 5-HT-3 affinities. Eur. J. Med. Chem., 30, 715-719


Influence of nutrients on liver lipid metabolism : biochemical mechanism underlying functionnality and/or toxicity

N. Delzenne (BCTC), N. Kok (BCTC), M. Roberfroid (BCTC), H. Taper (BCTC), R. Verbeeck (FATC), P. Buc Calderon (BCTC)

Supported by : the industry (Raffineries Tirlemontoises); CEE grant n¡ AIR2-CT94-1095 (1994-97); SSTC grant n¡ HH/10/031 (1991-95); ILSI Europe (1991-93)

Recent advance in nutrition research allows to classify nutrients and food components as functionnal, if beneficial for health, or as deleterious, if they are involved in toxic events or disease appearance. The major topic concerns the influence of carbohydrates on hepatic lipid metabolism. The induction of lipogenesis, through activation of key lipogenic enzymes gene expression, by digestible carbohydrates (saccharose, digestible starch) given after fasting leads to triglycerides accumulation in liver tissue, known as steatosis. On the contrary, other dietary carbohydrates like oligofructose (OFS), which escape digestion by gastro-intestinal enzymes but are fermented in the colon, decrease both hepatic synthesis and secretion of triglycerides. The objectives of the research are :

l to understand the biochemical events explaining the modulation of triglyceride (TG) synthesis by those nutrients;

l to assess the influence of digestible/non digestible carbohydrates on liver sensitivity to toxic treatments.

Influence of digestible carbohydrates on hepatic metabolism : which are the consequences of liver TG accumulation?

Fatty acid synthesis is considerably increased in the liver of rats fed high digestible carbohydrates-fat free (HCFF) diet given after fasting. This occurs primarly through induction of lipogenic enzymes gene expression by glucose, an effect modulated by hormones. Fasting followed by refeeding HCFF diet in rats induces liver TG accumulation and morphological alterations, characteris of steatosis. It is a rather frequent alteration in human, that, even if it is generally considered as benign, sensitizes the liver tissue to toxic events like ischemia reperfusion (with consequence on graft viability in liver transplantation, i.e.), by an unelucidated mechanism linked to oxidative stress.

An ex vivo protocol has been developped (see figure) to analyze the mechanism by which liver TG content (modulated by diet) modifies hepatic sensitivity to oxidative stress, induced by hypoxia-reoxygenation transition, or paracetamol exposure. Paracetamol has been chosen in order to correlate liver alterations with the influence of diet on xenobiotic metabolism (Cyt P450 activation, glucu-ronidation and GSH-conjugation). Toxicological and metabolic studies are performed on precise-cut liver slices because this model, as compared to cultured hepatocytes, combines these cells and Kupffer cells which play a major role in both lipid synthesis and oxidative damages. This research project takes part in the development of experimental designs allowing to assess nutrients/drugs interactions.

The functionnality of non digestible carbohydrates is illustrated by the hypo-lipidemic effect of oligofructose.

Some dietary carbohydrates escape digestion by gastro-intestinal enzymes, and reach the colon where they are extensively fermented. This is the case of oligofructose (OFS), which, besides gastrointestinal effects on gastric emptying, intestinal motility, and/or fecal flora composition, has systemic effects, modifying lipid homeostasis. Briefly, we have shown that OFS (10% in the diet) decreases total serum triglycerides (TG) by 30-40%, affecting mainly TG in very low density lipoproteins (VLDL) in rats. The capacity of fatty acid esterification is slightly decreased by OFS, as indicated by a lower incorporation of 14C-palmitate into TG and a decreased activity of glycerol-3-phosphate acyltransferase, a key enzyme controlling the esterification pathway. But, and interestingly enough, the major effect of OFS consists in a decreased lipogenic activity, through a coordinate reduction in the activity of all key enzymes involved in fatty acid synthesis, namely glucose-6-phosphate dehydrogenase and malic enzyme, providing NADPH, as well as ATP-citrate lyase, acetylSCoA carboxylase and fatty acid synthase. Those results suggest that this phenomenon occurs through modulation of lipogenic enzymes gene expression. Modifications in hormonal status, namely glucose-dependent insulinotropic peptide (GIP), glucagon-like peptide-1-

(7-37)/(7-36) peptide amide (GLP-1), insulin and insulin-like growth factor 1, could be the mediators of the systemic effects generated by those non digestible carbohydrates. This hypothesis will be tested in the next future.


Collaborations with Dr. C. Williams (Reading, UK) and Dr Van Dokkum (Zeist, NL) are aimed to assess effects of OFS on serum TG in human, and to evaluate its putative usefulness in regulating serum factors involved in cardiovascular disease.

Collaborationsl A. Geubel and Dr Y. Horsmans, GAEN, UCLl C. Williams & G. Gibson, University of Reading, United Kingdoml P. FerrŽ, Inserm 342 Unit, Francel Dr. Van Dokkum, Zeist, the Netherlands

Expertisesl Experimental models to assess security for health of Ònew foodsÓ : lipid homeostasis, nutrients availability,

influence on cancer developmentl Use of precise-cut liver slices to assess nutrients/xenobiotics interactions (toxicity, metabolism)

References

l M. ROBERFROID, G.R. GIBSON and N. DELZENNE (1993) The biochemistry of oligofructose, an approach to calculate its caloric value. Nutr. Rev., 51 (5), 137-146.

l G. GIBSON and M. ROBERFROID (1995) Dietary modulation of the human colonic microbiota - introducing the concept of prebiotics. J. Nutrition, 125 (6), 1401-1412

l M.F. FIORDALISO, N. KOK, J.P. DESAGER, F. GOETHALS , D. DEBOYSER, M. ROBERFROID and N. DELZENNE (1995) Oligofructose-supplemented diet lowers serum and VLDL concentrations of triglycerides, phospholipids, and cholesterol in rats Lipids, 30, 163-167

l N. KOK, A. ROBERT, M. ROBERFROID and N. DELZENNE (1996) Dietary oligofructose modifies the impact of fructose on hepatic triacylglycerol metabolism. Metabolism, 45, 1547-1550

l N. KOK, M. ROBERFROID and N. DELZENNE (1996) Involvment of lipogenesis in the lower VLDL secretion induced by oligofructose in rats. Br. J. Nutr., 76, 881-890

Schematic representation of the bioavailability of digestible and non-digestible oligo/polysaccharides.SCCFA : short chain carboxylic acidsGIP : glucose-dependent inulinomimetic peptideGLP-1 : Glucagon-like peptide 1 IGF-1 : insulin-like growth factor 1

Diet

oligo-/polysaccharides

Digestible

monosaccharides

H2O

intestinal epithelium

Non-digestible oligo-/polysaccharides

(colonocytes)

Bacteria H2O

GasesSCCA

Liver lipid metabolism

portal blood

Insulin GIP

Cae

co-c

olon

GLP-1

IGF-I


Development of radiopharmaceuticals and magnetopharmaceuticals as functional contrast agents. Oximetry probes. Liver and CNS-targeted ligands for receptor imaging.

B. Gallez (CMFA), J. Adline (CMFA), P. Buc-Calderon (BCTC), Ph. Levque (CMFA), R. Verbeeck (FATC)

Supported by FNRS (1995-1996)

The aim of these projects are devoted to the understanding of the biophysical and pharmacological properties of radiopharmaceuticals and magnetopharmaceuticals in order to increase their intrinsic diagnostic value. We are focused on two principal axes:

l In vivo Eletron Paramagnetic Resonance Oximetry : Development and evaluation of paramagnetic compounds sensitive to the oxygen concentration. Physical characterization, biocompatibility study. In vivo applications to the study of the stroke and the tumor oxygenation. Comparison with quantitative measure-ments of the perfusion (regional blood flow), diffusion coefficient (cellular oedema), and data obtained in functional NMR imaging.

l Receptor Imaging : Rationnal behind the use of diagnostic agents by study of the pharmacokinetics, of the metabolism, and the evolution of the biophysical properties during their biodistribution.

Tremendous input was given to the medical diagnostic during the last decades with the development and optimisation of methods such as Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and in vivo Electron Paramagnetic Resonance spectroscopy (EPR). The anatomical and functionnal informations are unique in the non invasive diagnosis of number of pathologies, but also as research tools in physiopathology and pharmacology. With the complexity of these methods, there is a critical need for an extensive knowledge on the possibilities for modifying the contrast, using both biophysical and pharmacological properties of the available materials. Our current research relies on the understanding of these properties of radiopharmaceuticals and magnetopharmaceu-ticals in order to increase their intrinsic diagnostic value.

In vivo electron paramagnetic resonance oximetry

The role of oxygen in biological systems is profound and pervasive, as a key intermediate in many biochemical transformations. Oxygen plays an important role in many pathophysiological processes : major types of pathologies (ischemic diseases, reperfusion injuries,...) are often linked to an inappropriate concentration of oxygen at particular sites. The oxygen also is a crucial parameter in several types of therapy, espacially the treatment of tumors by radiotherapy. Although the interest for making accurate measurements of oxygen concentration in intact biological systems is obvious, there is a lack of methods allowing such measurements accurately, sensitively, repeatedly, and non invasively.

EPR oximetry relies on the paramagnetic properties of molecular oxygen, and the consequent enhancement of relaxation rates of other paramagnetic species. When present, oxygen leads to a dramatic increase in the linewidth of the EPR signal from oxygen-sensitive paramagnetic materials such as nitroxides, selected charcoals, lithium phthalocyanin.

The interest for in vivo EPR is especially enlarged thanks to the recent development of low frequency EPR spetrometers, allowing the EPR measurement in biological systems, animals or humans. In this area of research, we are focused on the develop-ment and evaluation of paramagnetic compounds as markers of the oxygen concentration. A great part of the work relies on the development, physical characterization, and biocompatibility studies of the paramagnetic compounds. We recently demonstrated the feasibility of this approach by producing in our laboratory small charcoal particles coated with biocompatible polymers or included in very small silicon implants.

The compounds selected are used in vivo for studying the stroke physiopathology and the tumor oxygenation. We are comparing the results of oxygenation of the tissues obtained using in vivo EPR with quantitative measurements of the perfusion (regional blood flow), diffusion coefficient (cellular oedema), and data obtained in functional NMR imaging.


Receptor imaging

Labelled ligands for selected receptors are very useful in studying, quantitating and mapping the density distribution of these receptors, and estimating the change in receptor density associated with different pathologies. For such a purpose, radiolabelled compounds are clasically used in nuclear medicine, especially in PET. Another approach, under intense investigation, is to develop selective paramagnetic or superparama-gnetic MRI contrast agents, ligands which should also possess high selectivity for a given tissue.

Our aim is to validate these approaches, to understand the potential sources of drawbacks, and to provide media for correcting the signals not directly linked to ligands bound to receptors (circulating ligands, metabolites).

l Positron Emission TomographyStudy of the binding of 5-[2'-18F]fluoro-ethyl)flumazenil and 11C-flumazenil to the central receptors of benzodiazepines. Study of the influence of the metabolites on the PET image : study of the metabolism, of the metabolisation sites, of the biological membranes penetration by the metabolites, and of the biodistribution of the metabolites. Validation of the mathematical representative modelisation allowing the correction of the PET signals in order to estimate the real part of the compound bound to this receptor.

l NMR Imaging :Study of the factors affecting the hepatocyte transport of hepatoselective MRI contrast agents. Study on liver slices of the entrance pathway, influence of the saturability of the transporters, and obstacles to the biliary secretion. Study of the pathological implications on the expression of the transporters, and on the uptake by the contrast agents. Contribution to the evaluation of the usefulness of the contrast agents in the characterization of liver pathologies.

Study of the influence of dechelation of contrast agents on the NMR image signal intensity. Study of the metabolic fate of the free metal ion released and of its potential toxicity.

Collaborationsl R. Debuyst & F. Dejehet (UCL-INAN), l R.Demeure, J.F. Goudemant & B. Van Beers (UCL-RAIM), l D.Labar & J. Melin (UCL-TOPO), l H. M. Swartz, Dartmouth Medical School, Hanover, NH (USA)l D Grucker, Institut de Physique Biologique, Strasbourg (France)l K MŠder, Humboldt University, Berlin (Germany)l M. A. Foster, University of Aberdeen, (UK)l A.M. Van der Linden, Universitair Centrum Antwerpen (RUCA), Belgium

Expertisesl Radiopharmaceutical quality controll Radiolabeling and spin-labeling techniquesl Magnetopharmaceutical development :

NMR relaxation, EPR, NMR Imagingl In-vivo EPR

Publicationsl B.GALLEZ, V.LACOUR, R.DEMEURE, R.DEBUYST, F.DEJEHET, J.-L.DE KEYSER, and P.DUMONT,

(1994) Evaluation of a spin labelled arabinogalactan as MRI contrast agent, Magn. Reson. Imaging, 12, 61-69

l D.GRUCKER, T.GUIBERTEAU, B.ECLAN-CHER, J. CHAMBRON, R.CHIARELLI, A.RASSAT, G.SUBRA, and B.GALLEZ (1995) Dynamic nuclear polarization with nitroxides dissolved in biological fluids, J. Magn. Reson. B, 106, 101-109

l B. GALLEZ, G. BACIC, F. GODA, J.J. JIANG, J.A. O'HARA, J.F. DUNN , and H.M. SWARTZ (1996) Use of nitroxides for assessing perfusion, oxygena-tion, and viability of tissues. In-vivo EPR and MRI studies, Magn. Reson. Med., 35, 97-106

l B. GALLEZ, G. BACIC and H.M. SWARTZ (1996) Evidence for the dissociation of the hepatobiliary contrast agent Mn-DPDP, Magn. Reson. Med., 35, 14-19

l F. GODA, G. BACIC, J. A. OÕHARA, B. GALLEZ, H.M. SWARTZ and J. F. DUNN. (1996) The relationship between pO2 and perfusion in two murine tumors after X-ray irradiation: a combined Gd-DTPA dynamic MRI and EPR oximetry study. Cancer Res., 56, 3344-3349


Molecular analysis of drug-membrane interactions

M.P. Mingeot-Leclercq (FACM), F. Van Bambeke (FACM), J.P. Montenez (FACM), O. Antoine (FACM), P. M. Tulkens (FACM)

Supported by : CommunautŽ Franaise de Belgique (ARC) grant n¡172/94-99 (1994-99)FNRS grant n¡ 3.4589.96 (1996-99)

The molecular description of the interactions between drugs and membrane constituents is a powerful tool to understand their mechanism of activity or toxicity. By biophysical and biochemical approaches, we study the effects of drugs on critical membrane properties and characterize their mode of interaction with the lipidic constituents of membranes. This work allowed us to unravel the molecular mechanisms responsable for drug-induced phospholipid storage disorders and to develop strategies to reduce this toxicity.

Cell membranes often constitute the first biological structure encountered by drugs. Their binding or interaction to lipid constituents potentially leads to toxic events. In many cases, also, drugs must pass across membranes to reach an intracellular target. Our aim is to examine the interactions of drugs with phospholipid bilayers, with special emphasis on cationic antibiotics (aminoglycosides, macrolides) and polyanionic peptides. These studies aimed at understanding the molecular mechanism(s) by which these compounds elicit cellular toxicities (phospholipidosis, perturbation of membrane dynamics, ... ) in vitro as well as in vivo, or display specific pharmacokinetic or pharmacodynamic properties.

Our studies rely mostly on biophysical and biochemical approaches to measure transmem-brane fluxes of drugs and to evaluate the modifi-cations of critical membrane properties elicited by the compounds under study (fluidity, permeability, obility to fuse, susceptibility to phospholipid degrading enzymesÊ... ). These studies have led so far to a detailed understanding of the mechanisms whereby aminoglycosides and macrolides cause a lysosomal phospholipidosis and alter membrane properties.

In brief, we have shown that both aminoglycosides and macrolides are able to inhibit lysosomal phospholipases by binding to the negatively-charged phospholipids of the membrane. However, these two classes of antibiotics greatly differ in their mode of interaction with phospholipids, since aminoglycosides are hydrophilic compounds which remain at the membrane surface, causing aggregation but inhibiting fusion of adjacent membranes.

Views of the mode of assembly of the unprotonated (left) and protonated (right) forms of azithromycin (ball representation) and phosphatidylinositol (skeleton representation).

The arrowheads point to the two amino functions of azithromycin.(From Montenez et al, 1996)


By contrast, macrolides are hydrophobic molecules which more deeply penetrate the lipophilic core of the membrane (see figure). Further molecular characterization of the interaction between these drugs and phospholipids are presently under investigation.

In the domain of aminoglycoside antibiotics, our results have been used to assess the toxicological potential of new, original derivatives, by evaluating in vitro their ability to inhibit phospholipase activity. They have also led to the understanding of the mode of protection afforded by polyanionic peptides, which have been shown to prevent the binding of the polycationic aminoglycosides to the acidic phospholipids of membranes.

Collaborationsl A. Schanck (UCL-UnitŽ de Chimie Physique et de Cristallographie)l R. Brasseur (Centre de Biophysique MolŽculaire NumŽrique, UniversitŽ des Sciences Agronomiques de

Gembloux, Gembloux, Belgium)l M.P. Georgiadis (Chemical Laboratories, Agricultural University of Athens, Athens, Grece)

Expertisesl In vitro evaluation of toxicity of aminoglycoside, macrolide and glycopeptide antibiotics in membrane models.l Biophysical study of the interaction between drugs and membranes.

Referencesl M.P. MINGEOT-LECLERCQ, R. BRASSEUR and A. SCHANCK (1995) Molecular parameters involved in

aminoglycosides nephrotoxicity. J. Toxicol. Environ. Health , 44:263-300.l P.M. TULKENS, M.P. MINGEOT-LECLERCQ, G. LAURENT and R. BRASSEUR (1990) Conformational and

biochemical analysis of the interactions phospholipids-aminoglycoside antibiotics in relation with their toxicity. In: R. Brasseur (ed) Molecular Description of Bio-logical Membrane Components by Computer-aided Conformational Analysis vol. II, CRC Press, Boca Raton, Fla., 63-93.

l F. VAN BAMBEKE, P.M. TULKENS, R. BRASSEUR and M.P. MINGEOT-LECLERCQ (1995) Aminoglycoside antibiotics induce agg-regation but not fusion of negatively-charged liposomes. Eur. J. Pharmacol.,289:321-333.

l S. KOTRETSOU, M.P. MINGEOT-LECLERCQ, V. CONSTANTINOU-KOKOTOU, R. BRAS-SEUR, M.P. GEORGIADIS and P.M. TULKENS (1995) Synthesis and antimicrobial and toxicological studies of amino acid and peptide derivatives of kanamycin A and netilmicin. J. Med. Chem. 38: 4710-4719

l J.P. MONTENEZ, F. VAN BAMBEKE, A. SCHANCK, J. PIRET, P.M. TULKENS, R. BRASSEUR and M.P. MINGEOT-LECLERCQ (1996) Interactions of azithromycin, a new macrolide antibiotic, with negatively-charged membranes : II. Molecular characterisation by biophysical (31P-NMR, fluorescence depola-rization) and computer-aided conformational studies. Eur. J. Pharmacol. 314: 215-227


Cellular toxicity of antibiotics and other drugs

C. Gerbaux (FACM), M. El Mouedden (FACM), D. Tyteca (FACM), M.P. Mingeot-Leclercq (FACM), F. Van Bambeke (FACM), P. M. Tulkens (FACM).

Supported by : CommunautŽ franaise de Belgique (ARC) grant n¡ 172/94-99 (1994-99)FNRS grant n¡ 3.4516.94 (1994-97)

Lysosomes are the site of accumulation of many drugs, which may reach them by endocytosis or by proton trapping after diffusion through membranes. This accumulation often causes lysosomal disorders which we have undertaken to characterize by morphological and biochemical approaches. We have focused our attention on the lysosomal dysfunctions induced by aminoglycoside and macrolide antibiotics and by undigestible polyanionic peptides, with the aim to improve the knowledge of the toxicity of these compounds, as well as the physiopathology of the lysosomal apparatus.

The molecular interactions which drugs can establish with biological constituents may cause profound cytological changes, leading to toxicities. Models of cell culture are often very useful in this context to establish the links between the molecular effects of drugs and their expression at the tissular level in vivo. Our aim is to use and develop cell biology approaches to examine specific effects of drugs on lysosomes and membranes. We hope in this way to improve our basic knowledge of the lysosomal physio-pathology, as well as the drug toxicity in general. We also try to use this knowledge to improve, in patients, the practical use of the drugs under study.

This approach was successfully used in the past with aminoglycoside antibiotics and has now led to the recognition that the lysosomal phospholipidosis induced by these drugs can cause cell necrosis, associated with intense rŽactions of cell proliferation in kidney, together with a redistribution of intratissular growth factors.

Recently, we also have shown that aminoglycoside-induced phospholipidosis is associated with the development of apoptosis, i.e. a process of programmed death which cells usually initiate upon stimulation by specific factors such as hormones or trigger after sublethal toxic insults. Aminoglycoside-induced apoptosis has been observed both in vivo (in kidney) and in cultured cells, to an extent which matches the drug-induced phospholipidosis.

IIlustration of the alterations induced by azithromycin, a new macrolide antibiotic with an exceptional cellular accumulation, in a cell culture model. The drug causes an enlargement of the lysosomes, which appear partially filled by mixture of concentric, osmiophilic inclusions. Upon higher magnification, these

show clearly a lamellar structure, suggesting the accumulation of polar lipids (Bars = 1 µm; inset : 50 nm). (From Van Bambeke et al, 1996).


We have applied the same type of approaches to study the toxicity of anionic peptides. These were brought to our attention and characterized by us as effective protectants against aminoglycoside-indliced nephrotoxicity. We demonstrated that these peptides, if resistant to lysosomal proteolysis, induce by themselves not only a conspicuous and so far undescribed lysosomal thesaurismosis, but also a gelling of the lysosomal matrix. The latter causes a spectacular impairment of the process of membrane fission that normally marches that of membrane fusion in the vacuolar apparatus of the cell, leading to the formation of giant intracellular structures.

We are now developing a similar type of study with azithromycin, a dicationic macrolide antibiotic recently introduced in clinical practice, and which is characterized by exceptional levels of tissue accumulation and retention, and causes a lysosomal phospholipidosis in cultured cells (see figure). We showed that this drug also causes in cell culture models a lysosomal thesaurismosis, which involves not only phospholipids, but also cholesterol esters and probably many other cytological constituents. These effects will be described in details in the next report. lnterestingly enough, this thesaurismosis is associated with a marked increase in the activities of several lysosomal enzymes, which can be abolished by the use of a protein synthesis inhibitor. lncreased activities of lysosomal enzymes was already observed by us in cells exposed to proteases inhibitors, but results from a decreased catabolism of these enzymes, which is a different mechanism than that probably responsable for the effect seen with azithromycin).

Collaborationsl P. Courtoy (UCL-UnitŽ de Biologie Cellulaire).

Expertisesl Cellular toxicity of antibiotics and lysomotro-picdrugs

Referencesl C. GERBAUX, F. VAN BAMBEKE, J.P. MONTANEZ, J. PIRET, G. MORLIGHEM and P.M. TULKENS (1996)

Hyperactivity of cathepsin B and other lysosomal enzymes in fibroblasts exposed to azithromycin, a dicationic macrolide antibiotic with exceptional tissue accumulation. FEBS Lett. 394: 307-310.

l B.K. KISHORE, P. MALDAGUE, P.M. TULKENS and P.J. COURTOY (1996) Poly-D-glutamic acid induces an acute lysosomal thesaurimosis in proximal tubules and a marked proliferation of interstitium in rat kidney. Lab. lnvest. , 74, 1013-1023

l B.K. KISHORE, L. FUMING, P. MALDAGUE, P.M. TULKENS and P.J. COURTOY (1996) Mechanism of the thesaurismosis and altered lysosomal dynamics induced by poly-D-glutamic acid in kidney proximal tubular cells. Lab. Invest. 74, 1025-1037

l J.P. MONTENEZ, J.M. DELAISSE, P.M. TULKENS and B.K. KISHORE (1994) Increased activities of cathepsin B and other lysosomal hydrolases in fibroblasts and bone tissue cultured in the presence of cysteine proteinases inhibitors. Life Sci. (Pharmacol. Lett.) 55: 1199-1 208.

l F. Van Bambeke, J.P. Montenez, J. Piret, P.M. Tulkens and M.P. Mingeot-Leclercq (1996) Interaction of the macrolide azithromycin with phospholipids. I. Inhibition of phospholipase A1 activity. Eur. J. Pharmacol. 314: 203-214


Chemotherapy of the intracellular infection Y. Ouadrhiri (FACM), I. Paternotte (CMFA/FACM), Hujuan Fan (CMFA/FACM), E. Sonveaux (CMFA), P.M. Tulkens (FACM). Supported by : CommunautŽ franaise de Belgique (ARC) grant n¡ 172/94-99 (1994-99)FNRS grant n¡ 3.4516.94 (1994-97)

The efficacy of the chemotherapy of intracellular infection depends on an effective cooperation between host defenses and antibiotics. Host defenses are modulated by molecules such as cytokines. Antibiotic efficacy is governed by their intracellular penetration, accumulation, disposition, and bioavailability. In a cellular model of uninfected or infected macrophages, we study the intracellular pharmacokinetics of antibiotics, their efficacy against intracellular pathogens localized in different subcellular compartments, and the modulation of their activity by cytokines. This work establishes the rational basis for the treatment of intracellular infections.

Intracellular bacterial infection remains a medical and economical threat in spite of the availability of a large array of antibiotics potentially active against those organisms in a-cellular systems. Most failures actually stem from the inability of the drugs to reach the offending organism(s), and/or to effectively act in the intracellular environment (see figure). More recently, we also noted the importance of a cooperation with the natural host defenses and their modulators. Our aim is to determine the main pharmacodynamic and pharmacokinetic parameters of antibiotics at the cellular level, and to correlate them with their activities against intracellular organisms in quantitative models. We therefore study, mostly by biochemical and microbiological techniques, the intracellular fate of antibiotics, and assess their effectiveness against sensitive bacteria of medical or economical importance. These are selected on the basis of their capacity to invade and thrive in distinct subcellular compartments (cytosol, phagosomes, ...). We also examine the influence of selected cytokines on these properties. These studies have allowed to describe in detail the cellular properties of two classes of antibiotics of interest in the context of the intracellular infection (fluoroquinolones, macrolides in a series of models of uninfected cells and cells infected with Stophylocollos aureus, Legionella spp., Chlamydia spp., Listeria m., and Mycobacterium avium, ...).

5 6

5 6

De D'1

43

3

2

Cytosol

De : extracellular drugDi : intracellular drug

D' : metabolites: bacteria

Pharmacokinetic parameters

Penetration and retentionAccumulationSubcellular dispositionand bioavailabilityMetabolisation andinactivation

123

4

Expression of activityBacterial responsiveness

56

Vacuoles

Pharmacodynamic parameters

Pharmacokinetic and pharmacody-namic parameters involved in the activity of anti-microbial agents against intra-cellular organisms. The relative importance of those parameters can be studied in

quantitative model of infected cells. Host defense can modulate the subcellular dispo-sition and /or the expression of activity of anti-biotics, stressing the importance of the host to the response to drug.

(From Tulkens, 1991).


We showed that macrolides, especially the new dicationic derivatives, accumulate largely in lysosomes where they reach exceptionally large concentrations. Accordingly, they are effective against phagolysosomal organisms. Yet, because their association with lysosomes is not exclusive, they also show activity against cytosolic and phagosomal organisms. Fluoroquinolones accumulate in cells to considerably lesser an extent than macrolides and are found only in cytosol in uninfected cells. Yet, they display a much greater bioavailability within the cell compartments, compared to macrolides. Accordingly, they show much larger intrinsic activities not only against cytosolic bacteria, but also against organisms sojourning in intracellular vacuoles. Similar studies have now been initiated concerning tetracylines and glycopeptides. In parallel, we have undertaken rational modifications of other classes of antibiotics (lincosaminides, §-lactams) susceptible to improve their activities in this context. The first step of this program consists in the chemical synthesis of new derivatives of §-lactams, which is made by the members of the CMFA unit.

We also have begun to unravel the complex influence of gamma-interferon and other cytokines on antibiotic action against intracellular Listeria monocytogene, a typical exemple of food-borne intracellular infection where the virulent bacteria gains a specific access to cells, and escape from the phagocytic vacuole to reach and multiply in the cytosol.

Collaborationsl Y. Sibille (UCL, mŽdecine du travail)

Expertisesl Evaluation of the activity of antibiotics against intracellular infection in models of macrophages and other cells.

Referencesl P.M. TULKENS (1991) Intracellular pharmaco-kinetics and localization of antibiotics as predictors of their

efficacy against intraphagocytic infections. Scand. J. Infect. Dis. Suppl. 74:209-217.l M.B. CARLIER, B. SCORNEAUX, A. ZENEBERGH, J.F. DESNOTTES and P.M. TULKENS (1990) Cellular

uptake, localization and activity of fluoroquinolones in uninfected and infected macrophages. J. Antimicrob. Chemother. 26 (B):27-39.

l M.B. CARLIER, I. GARCIA-LUQUE, J.P. MONTENEZ, P.M. TULKENS and J. PIRET (1994) Accumulation, release and subcellular localization of azithromycin in phagocytic and non-phagocytic cells. Int. J. Tiss. React. 16:211-220.

l B. SCORNEAUX, Y. OUADRHIRI, G. ANZALONE and P.M. TULKENS (1996) Effect of recombinant human gamma interferon on intracellular activities of antibiotics against Listeria monocytogenes in the human macrophage cell line THP-1. Antimicrob. Agents Chemother. 40, 1225-1230


Cellular penetration of antisense oligodeoxyribonucleotidesC. Berens (CMFA), A. Kerkhofs (FACM), J. M. Tilquin (CMFA), E. Sonveaux (CMFA), M. P. Mingeot-Leclercq (FACM)

Supported by : Agence Nationale de Recherche sur le Sida (ANRS, France) (1995-97);CommmunautŽ Franaise de Belgique grant n¡172/94-99 (1994-99);FRSM grant n¡ 3.4589.96 (1996-99)

Antisense oligonucleotides will gain pharmacological interest only if their cellular penetration is enhanced. The aim of the research is to understand the intracellular traffic of oligonucleotides and to improve their cellular penetration by conjugation with fusogenic peptides.

Antisense oligodeoxyribonucleotides are synthetic molecules build to selectively block the expression of a protein by interacting with the corresponding mRNA or pre-mRNA. The antisense hybridizes with the RNA an blocks its splicing, its traduction, or induces its cleavage by RNase-H, an ubiquitous enzyme. The interference with biological processes by simple hybridization with some RNA sequence is called steric blocking. It is a stoechiometric process: one oligonucleotide is necessary per sequence copy. The RNase-H response, on the other hand, is a catalytic multi shots process: one oligonucleotide is able to destroy many RNA sequences.

The interest of antisenses is due to the fact that their structure-activity relationship, so elusive for a classical drug, is straightforward. If the sequence of the gene corresponding to the protein to be repressed is known, the simple Watson-Crick complementarity rule allows to deduce the structure of a potentially active oligomer. The scientific community was thus attracted to this field, the driving force being the hope to find new anticancer and antiviral drugs, specially anti HIV compounds. The tested oligomers were phosphodiesters and mostly phosphorothioates, because they are more resistant to nucleases. All the other classes of nucleic acid analogs do not induce the RNase-H response.

The concept of antisense action is nowadays in question because many real but non antisense effects were demonstrated. It was e.g. recognized that phosphorothioates have a strong affinity for cellular surface proteins. This cellular coating interferes with the interaction of viruses with cells. A heavy effort of fundamental research on the cellular penetration of antisenses is necessary if one wants to keep the hope of using them some day as pharmacologically active substances. A true antisense molecule is only active in the cytosol or the nucleus.

Oligodeoxyribonucleotides in micromolar concen-tration enter cells by adsorptive endocytosis. They reside in endosomes that do not fuse with lysosomes. At higher concentration, pinocytosis overwhelms absorption. In all instances, the vast majority of oligonucleotides remain sequestred in vacuoles, as demonstrated by confocal microscopy. A small fraction finds his way to the cytosol. Oligonucleotides in cellular cultures indeed inhibit the HIV reverse transcriptase, an enzyme released in the cytosol.

We study the cellular penetration of oligodeoxyribonucleotides in collaboration with the unit of cell biology (CELL) at ICP, and R. Brasseur.

The research project with CELL is aimed at characterizing the cellular proteins in contact with oligodeoxyribonucleotides. A phosphodiester sequence protected at the 3'-end (to avoid degradation by exonucleases) and bearing a photocrosslinking function was synthesized. This oligomer is used to radiolabel the proteins in contact with it at different stages of its cellular penetration. The results of the affinity labeling of living cells at 0¡C or 37¡C, and the labeling of whole cellular lysates are compared. One try so to pin point proteins of the endosomal compartment that could be involved in transferring a fraction of the oligonucleotides to the cytosol. Characterization of these proteins is important if one wants to adapt the architecture of antisenses to the channels or carriers used for penetration.

Oligonucleotide-fusogenic peptides conjugates are studied with FACM and R. Brasseur. Numerous fusogenic peptides are now characterized with among them the peptides of the influenza and HIV viruses. Fusogenic peptides hook themselves to the plasmatic membrane and destabilize its phospholipidic double layer. A remarkable fact is that cells shortly treated with a solution of a fusogenic sequence of the influenza virus become permeable to oligonucleotides added to the medium. The cells survive to this treatment and the oligomers accumulate in the nucleus, as also observed in microinjection experiments. We synthesize oligonucleotide-fusogenic peptides conjugates and will study the cellular penetration of these constructions. The nature of the peptidic sequence as well as the stoechiometry of the conjugates and the actual conformation of these

macromolecules are important parameters. The ideal structure will bear a bouquet of fusogenic peptides at the end of an oligonucleotide.


Collaborationsl P. Courtoy , UCL -ICPl R. Brasseur, UniversitŽ des Sciences Agronomiques de Gembloux, Belgium

Expertisesl Organic chemistryl Biophysics of membranesl Endocytosis

Referencesl Y. MA, and E. SONVEAUX (1989) The 9-fluorenylmethyloxycarbonyl group as a 5'-OH protection in

oligonucleotide synthesis. Biopolymers, 28, 965-973.

l E. SONVEAUX(1994) Protecting groups in oligonucleotide synthesis, in Protocols for oligonucleotide conjugates. Synthesis and analytical techniques" (Agrawal, S. Ed.), Humana Press, Totowa, New Jersey, (1994), pp. 1-71.

l Ph. FRAN‚OIS, Ph. MUZZIN, M. DECHAMPS and E. SONVEAUX (1994) The incorporation of flexible hydrophobic chains into double-sranded DNA: the consequences for stability. New. J. Chem., 18, 649-657

l M. DECHAMPS and E. SONVEAUX (1995) Aldehyde functions in synthetic oligo-nucleotides. Nucleosides & Nucleotides, 14, 867-870

l A. BIDAINE, C. BERENS and E. SONVEAUX (1996) The phototrityl group. Photocross-linking of oligonucleotides to BSA Bioorganic & Medicinal Chemistry Letters, 6, 1167-1170


Energetic and redox homeostasis during oxidative stress. Study of molecular mechanisms leading to cell death.

P. Buc-Calderon (BCTC), K.Evdokimova (BCTC), I. Latour (BCTC), S. Tinton (BCTC)

Supported by : FRSM grant n¡ 3.4528.91 (1991-94), n¡ 9.4551.91 (1991) .n¡ 3.4607.95 (1995); FNRS ÓCrŽdit aux ChercheursÓ (1991-92); CEE grant n¡ BMH-92-0016-BE (1992-93); UCL-FDS (1994-95)

We are interested to study the loss of cellular homeostasis induced by physic or chemical agents. We focuse our study on the regulation of a major function of the hepatocyte, the protein synthesis. We have selected two classes of stress which are interconnected : an oxidative stress induced by an organic hydroperoxide and the transition hypoxia-reoxygenation. Indeed, in both conditions, free radicals and reactive oxygen species have been implicated. In addition, such conditions occured during liver transplantation procedures.

Due to the ubiquitous presence of O2 all living organisms are exposed to the oxidizing nature of reactive oxygen species. While they are normal products of metabolism, they become deleterious if their production overwhelms cellular defense mechanisms which, at least in some pathological situations, may even be altered. Such a loss of cellular defenses can be produced either by an environmental stress (heat shock, ionizing radiations, hypoxia, oxidative insult, ...) or during xenobiotic metabolism.

Reactive oxygen species can interact with virtually all cellular constituents, e.g. they oxidize unsatu-rated fatty acids (lipid peroxidation), inactivate enzymes (oxidation of free thiol groups) or modify nucleic acids (strand breaks or covalent adducts formation), thus inducing a variety of toxicological and pathological responses. Nevertheless, the relative importance of these different actions with regard to irreversible cell injury is still controversial.

Indeed, it is rather difficult to distinguish between the intrinsic deleterious effects induced by a given stress (hypoxia, heat, oxidants,...) and the cellular adaptive responses against the stress. For instance, a depressed cellular metabolism might be interpreted as an effective strategy of adaptation of cells in an adverse environment, the so-called metabolic arrest, which leads to a preservation of critical cellular functions by decreasing metabolic rates. But it may also lead to an enhanced susceptibility to a further stress by decreasing the cellular resistance as a consequence of the impaired metabolic and detoxification functions.

For instance, protein synthesis is fast and strongly inhibited when hepatocytes are incubated in the presence of tert-butyl hydroperoxide (a model compound widely used to induce an oxidative stress) and when they are deprived of oxygen. The maintenance of this metabolic functions may be a critical event after organ cold preservation and further normothermic transplantation. Therefore, the understanding of the mechanisms which control the regulation of protein synthesis under such kind of stress could help to define new strategies to well preserve organs and to decrease injury once they are transplanted.

Since protein synthesis appeared more sensitive to changes in pO2 levels rather than to ATP content, we have hypothesized that such cells contain a molecular entity which is able to sense the pO2 of the environment, the so-called Òoxygen sensorÓ. We are interested in the elucidation of the signal transmission pathway which would link the activation of the oxygen sensor with the cellular responses. Preliminary results suggest that phosphorylation/dephosphorylation of proteins are involved in such a cascade and tyrosine phosphatase seems to play a major role.

Concerning the inhibition of protein synthesis by an oxidative stress, we hypothesize that the covalent modification thus explaining such an inhibition is the formation of mixed disulfides (S-thiolation). By this way, the S-thiolation of proteins may be a mechanism of regulation (activating or inactivating proteins), as well as a mechanism of protection thus avoiding the formation of heterodimer of large molecular weight. Indeed, the formation of protein mixed disulfides avoid the irreversible oxidation of thiol groups and block the formation of inter- and intraprotein disulfides bridges.


Liver slices incubated at 6h at 37¡C under reoxygenation conditions following 20h incubation at 4¡C in Krebs solution (A), 75 mM fructose containing medium (B) and in UW solution (C). There are almost in all hepatocytes a comp-lete loss of nuclear staining and frequent cytoplasmic vacuoles (A). In B and C,

the chromatolytic nuclei are localized mainly in the midzonal area . (H.E, x750)

Collaborationsl Rh™ne-Poulenc-Rorer (Paris, France), l INSERM-UniversitŽ Claude Bernard (Lyon, France)l BIBRA International (UK),l TNO (The Netherlands)l Friedrich Schiller University (Jena, Germany).

Expertisesl Xenobiotic metabolism and risk assessment by using in vitro models such as freshly isolated hepatocytes and

rat liver slicesl Elucidation of toxicological mechanisms : free radicals metabolism and in vitro toxicity by oxidative stress.

Referencesl V. LEFEBVRE, M. VAN STEENBRUGGE, V. BECKERS, M. ROBERFROID, and P. BUC-CALDERON

(1993). Adenine nucleotides and inhibition of protein synthesis in isolated hepa-tocytes incubated under different pO2 levels. Arch. Biochem. Biophys. 304: 322-331.

l V. LEFEBVRE, I. GOFFIN and P. BUC-CALDERON (1994). Fructose metabolism and cell survival in freshly isolated rat hepatocytes incubated under hypoxic conditions: proposals for potential clinical use. Hepatology 20: 1567-1576.

l S. TINTON and P. BUC-CALDERON (1995). Homocysteine enhances the inhibitory effect of extracellular adenosine on the synthesis of proteins in isolated rat hepatocytes. Biochem. J. 310: 893-896.

l I. LATOUR, J.B. DEMOULIN and P. BUC-CALDERON (1995). Oxidative DNA damage by tert-butyl hydroperoxide causes DNA single strand breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes. FEBS Lett. 373: 299-302.

l S. TINTON, S.C. CHOW, P. BUC CALDERON and G.E.N. KASS (1996) Adenosine stimu-lates calcium influx in isolated rat hepatocytes. Eur. J. Biochem. 238: 576-581


Adaptive response to ionizing radiation.

A. LŽonard (TEMU), C. Stecca (TEMU), E.D. LŽonard (TEMU), C. Crutzen-Fayt (TEMU)

The study of the adaptive response, i.e. a reduced effect from a higher challenging dose of a stressor when a smaller inducing dose had been applied a few hours earlier, has opened many new vistas into the mechanisms by which cells can adapt to hazardous environments. Although the entire chain from the initial event, supposed by the presence of DNA damage, to the end effect, i.e. presumably an improved DNA repair, has not yet been fully elucidated, many individual links have been clarified mainly through in vitro studies on cell cultures.

Cells, tissues, or organisms can often improve their ability to respond to a ÒÊchallengingÊÓ stress when they had been previously exposed to a smaller, ÒÊinducingÊÓ amount of the same or a similar stress. Such an adaptive response has been noted after exposure to a variety of stressors such as ionizing radiation, radiomimetic chemicals, oxidative agents, alkylating compound or heat.

With regard to ionizing radiation, an adaptive response has been observed in single cells as well as in the entire organism. For single cells in vitro, adaptive responses have been reported with respect to the induction of chromosome aberrations, sister chromatid exchanges or micronuclei in human lymphocytes, in Chinese hamster V79 cells, in rabbit lymphocytes, in C3H10T1/2 mouse embryo cells and in hepatoma cell lines.

An adaptive response to ionizing radiation has also been found for deletional mutations in vitro in human T-lymphocytes, in lymphoblastoid AHH-1 cell line and in a T-cell leukemia line, for neoplastic transformation in vitro in C3H10T1/2 cells, for cell survival and DNA synthesis in V79 cells, for cell survival in U1-Mel and Hep-2 human neoplastic cells in vitro, lymphocytes in vitro and spleen T-lymphocytes in vivo and for dominant lethal mutations in Drosophila melanogaster. Doses used for the initiation of an adaptive response are usually in the order of about 10 mSv with challenging doses in the order of SvÕs.

Adaptive responses appear also to occur in vivo in bone marrow and germ cells following irradiations of animals with low acute or chronic doses. It has also been claimed that fewer dicentrics are induced in lymphocytes exposed to a given dose of X- and g -rays in vitro when these cells originate from people occupationally exposed than when they are collected from people not working with radiation.These observations performed up to now suggest that the adaptive response to radiation originates from a, still poorly defined, signal due to cellular (DNA) damage, proceeds through a chain of events involving synthesis/activation of certain proteins and genes, and results in a state where repair of DNA damage is, temporarily, improved, presumably by an activation of DNA repair enzymes. Several of these reactions seem to be a common feature of the cellular stress response and involve the formation of protective proteins, as does also the response to heat stock. At the present time, several components of this chain of events have been identified but the elucidation of the entire chain, including the identification of the eliciting signal, remains a major scientific challenge to molecular biology. Indeed, these mechanisms by which cellular integrity can be maintained following a stress have bearing for many aspects of health and disease.

The researches performed in our laboratory on adaptive response are made in the frame of a collaboration European Union-Kazakhstan suppor-ted by INTAS (International Association for the Promotion of Cooperation with Scientifists from the Independent States of the former USSR). The Kazakh region of Semipalatinsk is highly contaminated with radioactive material from atomic bomb tests and this might entrail substantial genetic risks to the population. The research carried out aims to investigate the mechanisms by which cells can cope with such situations. Collaboration with kazakh scientists will help them to face to specific problems present in Kazakhstan and to study genetic damage and its repairability in people living in contaminated region. The results obtained up to now in our laboratory have shown that the adaptive response observed in human lymphocytes exposed to a small radiation dose can be transmitted from exposed to non-exposed cells. The research to be carried out in the future aims to answer the following questions :

l how does the activity of the transmissible factor depend the amount of the conditioning dose ?l how long does it take for the transmissible factor to appear after a conditioning dose and what is the optimal

time internal and when does he disappear again ?l does the transmissible factor act via substances present in the plasma or via a direct contact between cells ?l what is the nature of the transmissible factor ; a small or a large molecule, a protein ?

l does a conditioning by mutagens other than radiation also give rise to the appearance of a transmissible factor ?

l can such a factor be detected in the blood of patients a few hours after radiation therapy or in people living in contaminated areas.

Collaborationsl Prof.J. Rueff, New University of Lisbon, Portugall Prof. G.B. Gerber, Commission of the European Unionl Prof.N.B. Achmatullina, National Academy of Science, Almaty, Kazakhstanl Prof. A.T. Polyakov, Physical and Technical Institute, Almaty, Kazakhstan.

Publicationsl LƒONARD, A. (1994) La rŽponse adaptative aux rayonnements ionisants. Ann.Ass.Belge Radioprotection, 19,

557-568.l GERBER, G.B. (199 ) Radiation hormesis. Ann.Ass. Belge Radioprotection, 19, 533-556.


Biomonitoring of people exposed to chemicals.

A. LŽonard (TEMU), E.D. LŽonard (TEMU), C. Crutzen-Fayt (TEMU).

The evaluation of mutagenic and carcinogenic potential of chemicals usually relies on experimental results of in vitro short-term tests or of in vivo studies on laboratory animals. For pharmaceutical drugs clinical studies provide, in addition, important and necessary informations on the effect of chronic or acute treatment in humans. Several reports published recently underlined the value of observations on self-poisoned patients as a model for the study of the mutagenicity of chemicals in Man.

Occupational or accidental exposure to ionizing radiations is currently assessed by monitoring chromosome aberrations in peripheral blood lymphocytes. The dose is estimated by comparison with relationships obtained ÒÊin vitroÊÓ. On the assumption that agents which increase the yield of aberrations is the most applied method for estimating the amount of damage induced by environmental mutagens in human somatic cells, the possibility of using this system for assessing exposure to mutagens other than ionizing radiations is, therefore, very attractive. Many papers have been published, indeed, reporting an increase of structural chromosome aberrations in people exposed to chemicals.

Accidents may provide an opportunity for the study of large doses of chemicals in humans. Self-poisoning seems to be a special and very frequent version of accident. The majority of persons attempting suicide are young and otherwise healthy and, due to improved medical treatment, 98 % of these survive. Thus, survivors of self-poisoning present a unique model for the study of somatic and germinal mutagenic effects of large doses of chemicals in human beings.

In collaboration with the Unit of Reanimation and Intensive Cares (UCL-REAN) systematic observations, involving various cytogenetic methods - scoring of sister chromatid exchanges, analysis of chromosomes in metaphases, counting of binucleated cells carrying micronuclei - are presently performed on peripheral blood lymphocytes from self-poisoned patients. Those studies are made, when possible, in parallel with observations on people chronically exposed to low doses of the same compounds and with in vitro approaches. Besides the classical in vitro evaluation of the ability of those chemicals to induce cytogenetic changes, some studies are made with the Department of Radioprotection of the Atomic Center of Belgium (CEN-Mol) on the capacity of those chemicals to interfere with the assembly and disassembly of microtubules which play a critical role in the distribution of the chromosomes during mitosis and meiosis. The effects on microtubule polymerisation are evaluated on material isolated from pig brain easily obtainable at the slaughter house. Tubulin, the major component of microtubules is, indeed, nearly the same in all mammalian species and their different cells. Due to the high concentration of axomal microtubules in nervous cells, brain contains as much as 35 mg tubulin/100 brain.

Among the various compounds evaluated up to now are paracetamol, meglubine antimoniate, arsenicals, thallium, vanadium sulfate, etc. Whereas vanadyl sulfate 1.5 mM inhibited completely the microtubule polymerisation, no inhibition was noted with paracetamol at concentrations as high as 10 mM. Of interest is the fact that negative findings were also obtained when searching for structural aberrations in self-poisoned women after ingestion of as much as 15 g paracetamol.

Collaborationsl Dr.Ph. Hantson, UCL Intensive Care.l Prof. P. Mahieu, UCL Intensive Carel Prof. Ch. Verellen, UCL Human and Medical Geneticl Dr. J.M. Libouton, UCL Human and Medical Genetic l Dr. B. Vandercam, UCLl Dr. B. Delaere, UCL

Publicationsl Ph. HANTSON, L. DE SAINT-GEORGES, P. MAHIEU, E.D. LƒONARD, M.-C. CRUTZEN-FAYT, A.

LƒONARD. (1996) Evaluation of the ability of paracetamol to produce chromosome aberrations in man. Mut. Res., 368, 293-300

l Ph. HANTSON, C. VERELLEN-DUMOULIN, J.-M. LIBOUTON, A. LƒONARD, E.D. LƒONARD, P. MAHIEU. (1996) Sister chromatid exchanges in human peripheral blood lymphocytes after ingestion of high doses of arsenicals. Int. Arch. Occup. Environ. Health, 68, 342-344

l A. LƒONARD, G.B. GERBER. (1996) Mutageni-city, carcinogenicity and teratogenicity of antimony compounds. Mut. Res., 366, 1-8l Ph. HANTSON, E.D. LƒONARD, M.-C. CRUTZEN-FAYT, A. LƒONARD, B. VANDERCAM, B. DELAERE, P. MAHIEU. (1996) Cytogenetic observations after meglumine antimoniate therapy for visceral leishmaniasis. Pharmacotherapy, 16, 869-871


Investigations on the contamination of food by mutagenic compounds generated by moulds (Aflatoxin B1) or by the heat processes (polycyclic aromatic hydrocarbons or heterocyclic aromatic amines).

C. de Meester (TEMU), B. Rollmann (CHAM)

Different food products were found to be contaminated by mutagens , which were detected after different extraction procedures by the Ames / Salmonella mutagenicity test .On the other hand different natural compounds were tested in order to investigate how the mutagenicity of heterocyclic amines ( Has ) can be modulated ; vitamines , flavonoids , dietary fiber and b-carbolines can reduce and even supress the mutagenic activity of Has .Beside the caracterization of the mutagenic properties of heterocyclic amines, the unit is involved in a European project which intend to improve the quantification methods in order to assess the human risk linked to the consumption of some cooked food. Sophisticated extraction and clean-up procedures have been devised, followed by HPLC analysis with UV, fluorescence and electrochemical detection.

This first topic has been supported by a contract with the Belgian ÒÊService du Premier Ministre - Programmation de la Politique ScientifiqueÊÓ (Contract n¡ HH/10/032 - 1991 - 1995). ÒÊMise au point de mŽthodes de dosage pour la quantification de contaminants mutagnes et cancŽrognes, dans lÕalimentationÊÓ. The research performed in the frame of this contract has allowed the presentation of a PhD thesis :ÊÓ Application de techniques chromatographiques pour le dosage par Žlectrochimie dÕamines aromatiques hŽtŽrocycliques ÒÊ(Mme M. Van Dyck - Great Distinction - 1995).

Quantification was done via HPLC with a reverse phase column and UV or electrochemical detection (EC). The most currently used methods are based on UV detection. The aim of our research which started in 1991, was to develop EC detection in order to improve not only the sensitivity but also the specificity of the analysis.

For these reasons it was necessary to look carefully to a number of analytical parameters which could influence the response of the EC detection after the HPLC separation. The following factors have been investigated :

l pKa of the HAAs - pH of the solventl composition of the bufferl effect of ion pairing agents - ionic strengthl use of a ternary instead as binary mobile phasel choice of the internal standardl choice of the columnl choice of the potential of the electrode

A particular interest was given to the comparative sensitivity of the EC compared to the UV detection ; in the actual state of the project, we obtained an increase of sensitivity with a factor of about 5 for EC detection. In a more fundamental point of vue, the mechanism of the electrochemical oxidation was also investigated, in order to explain the mechanism of the oxydation reactions which occurs on the electrode.

On the other hand, parameters which could influence the chromatographic separation were also investigated : the efficiency of the reverse phase column was compared with a cation exchange stationary phases. These results were compared with capillary electrophoresis, which has been proposed as an alternative separation technique.

Finally, the method proposed by G.A. Gross was applied with different food matrices, in order to validate a procedure which could be recommended as a routine method. The detection limits of four Has : IQ, MeIQ, MeIQx and 4,8 DiMeIQx were quite similar, with figures around 5 p moles, with our UV detection system. The separation of the compounds is satisfactory, with recoveries between 70 and 80 %, after a complete extraction procedure on a mixture of the pure compounds. Different heated meat products have been already tested : beef extract, fried sausage and fried ground beef ; it appeared from these assays that the recoveries of the Has under investigation were quite different.

C. de Meester coordinates a EU project of the ÒÊStandards, Measurements and TestingÊÓ programme : ÒÊChemical Analysis of Heterocyclic Amines. Quantification in Heat Processed FoodÊÓ (Contract N¡ MAT1-CT930042 - 1994 - 1996) in which seven European laboratories participate.


The objectives of this S, M & T project is to improve the procedures actually used, in order to quantify as precisely as possible these genotoxic contaminants in a heat processed food matrice. In a first phase, seven European laboratories (B-F-ES-S-CH-GR-UK) participated in an intercomparison on the analysis of a solution with an identity and content of Has both unknown to participants. The results of this exercice, where the participants used different HPLC conditions with UV, fluorescence, mass spectrometry and electrochemical detection, revealed a good precision and accuracy of the end determination of 3 HAs (IQ, 4,8-Di Me IQx and PhIP). In a second intercomparison, a batch of commercial beef extract was prepared and spiked with known amounts of : IQ, MeIQx, 4,8-Di Me IQx and PhIP. The long-term stability study at - 18¡C, + 4¡C, + 25¡C, + 40¡C and + 60¡C revealed a good stability of these HAs up to 6 months of storage at + 25¡C. At + 40¡C and + 60¡C however a significant decrease was observed, more particularly for PhIP. It was thus decided to send out the sealed ampules containing the beef extract in a refrigerated container, to the participants. Different extraction and clean-up procedures were followed by the participants, before the analysis by HPLC. The comparison of the results revealed however large variations not only between but also within laboratories. During a meeting held on september 1996, the participants have agreed on minimum recovery levels. Different sources of variations in the extraction procedures were identified, which could be the subject of a next project for which most of the participants expressed their interest.

Intercomparison studies to investigate the degree of inter- and intra-laboratory variability of the Ames - Mutagenicity test. In this ÒÊStandards, Measurements and TestingÊÓ project of the EU, 11 European laboratories (Project n¡254) are involved, since 1990, in order to identify the factors able to influence the precision and accuracy of mutagenic response to direct and indirect mutagens.

For this exercice , the participants received the same batch of Salmonella typhimurium strains ( TA 98 and TA 100 ) , two highly pure mutagens ( benzo[a]pyrene and 2-aminoanthracene ) and a rat liver subcellular ( S9 ) fraction . The first results showed a considerable variability amongst the participating laboratories .

The aim of the project is the certification of reference mutagens.

Collaborationsl G.A. Gross - Nestec Research Centre - Lausanne (Switzerland)l M. Rabache - CNAM - Biochimie Industrielle et Agro-alimentaire (Paris)l M.T. Galceran - Universitat de Barcelonal D. Marzin - Institut Pasteur - Lillel D. Lovell - BIBRA - Carshalton

Publicationsl C. dE MEESTER and G.B. GERBER (1995) The role of cooked food mutagens as possible etiological agents

in human cancer ; A critical appraisal of recent epidemiological investigations. Epidemiology and Public Health, 43 : 147-161

l M.M.C. VAN DYCK, B. ROLLMANN and C. DE MEESTER (1995) Quantitative estimation of heterocyclic aromatic amines by ion-exchange chromatography and electrochemical detection. Journal of Chromatography, 697 : 377-382

l C. DE MEESTER (1995) Genotoxic potential of b-carbolines A review. Mut. Res. 339 : 139-153 l NGOMBO MUKENDI , C. dE MEESTER and B. ROLLMANN (1995) Etude , in vitro , de la conversion

mŽtabolique de lÕaflatoxine B1 en son epoxyde . Comptes Rendus de la Soc. Biol., 189 : 1-14


Biological dosimetry on people accidentally or professionally exposed to ionizing radiations.

A. LŽonard (TEMU), E.D. LŽonard (TEMU), C. Crutzen-Fayt (TEMU)

Observations of chromosome aberrations induced in peripheral blood lymphocytes is currently used to evaluate the dose of ionizing radiations received accidentally or professsionally. Using this method several studies are performed in collaboration with several belgian and foreign institutions :

l On patients treated for cancer disease by external or internal exposure ;l On people receiving radiation from nuclear follout ;l On cell culture irradiated in vitro.

The estimation of the magnitude of a dose of ionizing radiation to which an individual has been exposed for chromosomal observation frequencies determined in peripheral blood lymphocyte cultures is a well-established methodology, having first been employed over 30 years ago. In fact, ionizing radiation does not induce any new or novel type of aberrations but simply increases the frequency of those which occur at low frequency without any exposure.The observations are generally carried out on peripheral blood lymphocytes.

About 90 % of the peripheral blood lymphocytes are of the recirculating type, remaining in the bloodstream for less than one hour. Then they migrate to the lymphatic tissues to reappear in the blood several hours or days later. More than 99 % of the circulating lymphocytes are in the G0 or G1 stage of the cell cycle and have to be stimulated by a mitogenic agent, generally phytohemagglutinin, to replicate their DNA ÒÊin vitroÊÓ and to enter into division. The studies performed on irradiated persons have shown that the majority (90 %) of the human lymphocytes carrying unstable chromosome aberrations have a life span exceeding 3 years, with some surviving for more than 20 years. Since lymphocytes accumulate genetic damage induced by ionizing radiations they enable the monitoring of the effects not only of an acute, but also of a chronic or repeated exposure to such agents and possibly also to clastogenic chemicals.

In the great majority of studies on the estimation of radiation dose by chromosomal aberration analysis for occupational, accidental, or medical exposures, the preirradiation (or background) frequency of aberrations is not available. For fairly acute exposures (> 10 rad received over minutes or a few hours) when blood samples are taken shortly after exposures (i.e. within a few days), this lack of knowledge of an individualÕs background frequency does not normally present a problem. This is because the estimation of dose will be based upon a total induced frequency, usually for dicentrics, that is obviously different from the upper range of reported background frequencies for individuals exposed only to background radiation.

For chronic or fractionated exposures, low-level acute doses, and in the case of delayed samples after acute exposures, the yields of aberrations will be low, and the estimation of a dose, if even possible, will depend heavily on the background aberration frequency. Information on the background frequency for the individuals who are exposed, or possibly exposed, would represent the ideal situation. However, since such data are usually unavailable, it is necessary to establish an estimated background for the individuals, attempting to take into account those factors that are reported to influence background frequency.

Studies on patients can simulate the situation after an accidental inhomogeneous irradiation and thus aid the assessment of biological damage in such situations. They also can help to evaluate the response of the patient to therapy and, possibly, discern patients of abnormal radiosensitivity. For that reason special attention is paid to patients displaying genetic instability or genetic disease. Some observations performed in our laboratory have shown, for instance, that there exist no difference in the reaction of lymphocytes to radiation and their DNA repair ability between blood from patients carrying Familial Adenomatous Polyposis and from controls. This is true for the yield and distribution of chromosome aberrations induced by different doses of ionising radiation as well as for the cell kinetics, mitotic index and induction of DNA breaks. Some other studies on patients treated with two doses of 1850 Mbq of 131I given 24 h aport for thyro•d cancer have shown that such treatment causes a small but statistically significant increase of chromosome anomalies. Analysis of the results demonstrates however that in situations like Chernobyl, where a population is exposed as a result of the release of radioactive iodine, a determination of chromosome aberrations in blood lymphocytes would not appear to be very useful to determine exposure from iodine. Comparable studies are currently carried out on patients with mammary cancer, pelvic cancer, brain cancer or on children treated with total-body high energy photon irradiation (pulsed exposure from a LINAC) for different types of malignant diseases.


The laboratory is involved in various studies performed in Austria and States of the former USSR in order to evaluate the doses received by persons contaminated through the surface deposition of the fallout following the nuclear accident in Chernobyl.The in vitro studies aim to compare the classical method of scoring dicentrics, with the production of micronuclei in binucleated lymphocytes and the use of specific probes.

Collaborationsl Dr. T.Hoang Hung (Atomic Research Center, Dalat, Vietnam).l Prof. F. Richard (UCL)l Prof. J. Rueff (New University of Lisbon - Portugal)l Prof. B. Dutrillaux (Institut Curie - Paris)l Prof. G.B. Gerber (CEE - Brussels)l Dr. L. Baugnet - Mahieu (CEN - Mol)l Prof. J. Pohl-RŸling (University of Salzburg - Austria)l Dr. D. Lloyd (NRPB, Chilton - UK)l Prof. A. Bršgger (Institute for Cancer Research, Oslo - Norway)l Prof. G. Obe (University of Essen - Germany)

Publicationsl T. HOANG HUNG, G.B. GERBER, E.D. LƒONARD, M.-C. CRUTZEN-FAYT, F. RICHARD, and A. LƒONARD.

(1995) Utilisation du test des micronoyaux comme dosimtre biologique en cas dÕexposition homogne ou non homogne aux radiations ionisantes. C.R. Soc.Biol., 189, 1137-1142

l A. LƒONARD, L. BAUGNET-MAHIEU, T. HOANG HUNG, E.D. LƒONARD, M. LEMAIRE, and G.B. GERBER. (1995) Chromosome aberrations in circulating lymphocytes after brachytherapy for uterus cacinoma. Acta Oncologica, 34, 539-542

l A. BRAS, L. CRISTOVAO, C. COELHO, A. HILALI, B. DUTRILLAUX, A. LƒONARD, and J. RUEFF. (1995) Normal genetic response to gamma irradiation in familial adenomatous polyposis. Eur. J. Cancer, 14, 1506-1510

l H. LETTNER, E.D. LƒONARD, and A. LƒONARD (1996) Induction of in vivo blood chromosome aberrations by low level radiations from nuclear fallout. International Congress on Radiation Protection, Vienna, Proceedings.

l A. LƒONARD, and G.B. GERBER (1996) Does the Chernobyl accident increase genetic disease in exposed human populations ? Scope-Radtest, 11, 4-6


Clinical evaluation of new therapeutic approaches

S. Ibrahim (FACM), C. Bruno-Dusart (FACM) and P.M. Tulkens (FACM)

Supported by the industry (Schering Plough Belgium and Bristol-Myers Squibb)

Clinical trials are the ultimate and most critical final step in the development of new drugs and are also essential to rationally improve their use. In this context, our efforts are directed towards the development of optimized therapeutic schemes for antibiotics, based on the knowledge of the pharmacodynamic and pharmacokinetic parameters governing their efficacy and/or toxicity.

The final and the most important aspect in drugs development is their clinical use. Pharmacology and toxicology have therefore an obvious application which is the definition of the proper use of drugs, and their permanent improvement. In this context, we use the knowledge gained by our studies on the pharmacology of antiinfective drugs to design and perform clinical trials directed to these goals. These studies attempt to apply to the clinical environment the concepts of pharmacodynamics and toxicology which were proven useful in our experimental studies. We also set up the necessary monitoring procedures when these require non-routine investigations or develo-pments. In this context, the following clinical evaluations are being performed.

Administration of aminoglycosides in a Òonce-a-dayÓ schedule

This new model of administration was proposed on the basis of experimental studies demonstrating its potential for being more, or at least as effective than the conventional schedules (3 times a day), while causing less toxic reactions. The lower toxicity stems from the saturable character of aminoglycoside uptake by target tissues (kidney, inner ear). Moreover, the toxicity induced by aminoglycosides proceeds by successive thresholds from recoverable alterations towards irreversible damage. Keeping the level of alterations below a critical threshold results therefore in an effective protection of the patient. The 'once-a-day' schedule new scheme was successfully tested in several patient populations and is now introduced in a large number of applications of these drugs (suitable modifications of the official package inserts have been made in several countries including Belgium for two aminoglycosides so far).

Evaluation of phospholipiduria as a means to monitor aminoglycoside-induced renal alterations

We developed this approach in parallel to the above studies on aminoglycosides, because it provides a direct way to monitor non invasively the early alterations which aminoglycosides cause in kidney cortex. A series of patient populations have been examined so far, ranging from premature children, neonates, young females, patients suffering from cancer with or without granulocytopenia, to intensive care patients and HIV positive patients. Drugs examined so far have included netilmicin and amikacin. A trial with tobramycin is ongoing. Results show that phospholipiduria is a useful marker of impending toxicity in correlation with the duration and extent of treatment. It is also a useful monitoring criteria for assessing the safety of new schedules. It can also be used experimentally to assess the effectiveness of nephroprotectants. The same approach has been used to assess the safety of azithromycin in patients, in connection with the potential of this drug to cause phospholipidosis (see above).

Evaluation of §-lactam antibiotics in special applications in relation with their pharmacodynamic properties

In contrast with aminoglycosides, §-lactam antibiotics must be administered several times a day, because their serum concentration must ideally remain above the MIC of the offending organisms for the whole period of treatment. While this can be easily achieved in simple applications of these drugs, little is known concerning this aspect in special situations where pharmacokinetic parameters can vary largely from what is observed in 'normal' patients. In this context, we have explored the validity of the registered scheme of administration of aztreonam (a §-lactam antibiotic with specific activity against Gram (-) bacteria) in two populations at high risk of infection, namely, patients undergoing liver transplantation and patients suffering from cystic fibrosis. We could demonstrate that the officially registered schedules and dosages were markedly inadequate for cystic fibrosis patients. We are currently designing clinical trial aimed at testing rationally designed new schemes, based on the

specific pharmaco-kineticsof aztreonam in this patient population and the pharmacodynamic criteria explained above.

Collaborationsl Centre hospitalier universitaire, Lige (Belgium)l Cliniques universitaires St-Luc, Bruxelles (Belgium)l Akademische Zietenhuis, Gent (Belgium)l H™pital Erasme, Bruxelles (Belgium)l H™pital St. Pierre, Bruxelles (Belgium)

Expertisesl Clinical trials of new drugs and new schemes of administrations. l Monitoring of renal alterations by non-invasive approaches.

Referencesl S. IBRAHIM,, M.P. DERDE, L. KAUFMAN, F. CLERCKX-BRAUN , Ph. JACQMIN, J. DONNEZ and P.M.

TULKENS (1990) Analysis of safety, pharmacokinetics and efficacy of once-a-day administration of netilmicin and amikacin vs their conventional schedules in pelvic inflammatory disease patients. Renal Failure 12:199-203.

l P.M. TULKENS (1991) Pharmacokinetic and toxicological evaluation of the once-a-day regimen versus conventional schedules of netilmicin and amikacin. J. Antimicrob. Chemother. 27(C):49-61.

l J.P. LANGHENDRIES, O. BATTISTI, J.M. BERTRAND, A. FRAN‚OIS, J. DARIMONT, S. IBRAHIM and E. SCALAIS (1993) Once-a-day administration of amikacin in neonates: assessment of nephrotoxicity and ototoxicity. Dev. Pharamacol. Ther., 120:220-230

l S. IBRAHIM, J.P. LANGHENDRIES, A. BERNARD and P.M. TULKENS (1994) Urinary phospholipids excretion in neonates treated with amikacin. Int. J. Clin. Pharm. Res. 14:149-156.

l S. IBRAHIM, B.K. KISHORE, P. LAMBRICHT, G. LAURENT and P.M. TULKENS (1991) Effect of aminoglycosides and of coadministration of poly-L-aspartic acid on urinary phsopholipids excretion: A comparative Study. In: P.H. Bach, N.J. Gregg, M.F. Wilks and L. Delacruzz, (eds). Nephrotoxicity:Mechanisms, Early Diagnosis and Therapeutic Management.Marcel Dekker, New York, 105-109.

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single injection3 injections

Treatment

Time (days)

Tota

l pho

spho

lipid

uria

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Phospholipiduria as an early indicator of aminoglycoside-induced nephro-toxicity. The figures compares the level of urinary phospho-lipids in cancer patients receiving 6 mg/kg/day of netilmicin as a single

injection or divided in 3 injections during 7 days. Despite the fact that the interindividual variations are important; it clearly appears that the thiol administration causes a more rapid and elevated phospho-

lipidurva than the qd administration. The rise caused by tid treatment shows also a slower reversibility (From Van der Auwera et al, 1991)


Metabolism of immunosuppressive drugs and immunosuppressive activity of the metabolites

G. Lho‘st (FATC), M. Nickmilder (FATC), R. Verbeeck (FATC)

Metabolites of the recent immunosuppressive agents possessing a macrolide structure or a cylosporine like structure such as FK-506, rapamycin and SDZ-IMM-125 respectively, may retain or not the immunusuppressive activity were investigated and were found in the case of FK-506 to be related to some intra-molecular interactions of polar groups with the FK-506 binding region and are still under investigation for the other drugs. Also the influence of Phase II reaction on the immunosuppressive activity of the metabolites will be studied.

Several disciplines, including chemical ecology, seek to understand the molecular basis of information transfer in biological systems, and general molecular strategies are beginning to emerge. Often these strategies are discovered by a careful analysis of naturel products and their biological effects. Cyclosporin A, FK-506, and rapamycin are produced by soil microorganisms, SDZ lMM 125 being the hydroxyethyl derivative of D-serine cyclosporin A. They are being used or considered as clinical immunosuppressive agents. They interrupt the cytoplasmic portion of T-cell signaling by forming a complex with a binding protein FKBP-12 in the case of FK-506 and rapamycin and cyclophilin A (CyPA) in the case of cyclosporin A (CsA). This complex in turn inhibits a protein target and the best understood target is calcineurin which is inhibited by FK-506-FKBP-12 and CyPA-CsA.

The T-cell Acfivation Process

The chemical structure of FK-506 is illustrated in the figure. Despite having different structures, the mechanism of action of FK-506 and CyA are very simiar. On the other hand, FK-506 and rapamycin share certain structural similarities. Their identical binding domain led to the belief that these two agents would also have similar mechanism of action. However, despite this similarity, the effector elements are structurally different, and the two agents exert their immunosuppressive activity by different mechanisms. CyA and FK-506 act at an early stage in the T-cell activation process, blocking the T-cell receptor-mediated signal transduction pathway prior to late signalling events, rapamycin blocking the signal at a later stage.

Intracellutar Binding

In order to mediate their effects, CyA, FK-506 and rapamycin must each bind to a cytosolic target protein. Each agent binds to these binding proteins (known as immunophilins) with high affinity. The first immunophilin to be identified was cyclophilin, which complexes with CyA. The FK-506 binding protein (FKBP) was identified later. Both FK-506 and rapamycin bind to FKBP as a result of their identical binding domain, The target of the immunophilin-drug complexes is the calcium and calmodulin-dependent protein phosphatase, calcineurin, which is composed of two subunits Aand B. Calcineurin is an essential element in the signal transduction pathway, carrying information from the cell membrane to the nucleus, in order to stimulate the synthesis of an important cytokine IL-2. FK-506 and CyA both act as a molecular "glue" binding the calcineurin-calmodulin complex to immunophilin molecules, which do not interact under normal conditions.


FK-506 in vitro metabolism and site of intra-molecular interactions of FK-506 metabolites

As reported in the figure FK-506 (Tacrolimus) is a potent immunosuppres-sive macrolide produced by Streptomyces tsukubaensis possessing a lactone P-450 3A dependent mixed fonction oxygenase enzymic system to several metabolites including a FK-506 O-demethylated, hydroxylated, O-demethylated hydroxylated metabolites as well as an isomerized epoxide and a C36-C37 FK-506 dihydrodiol. Metabolism of FK-506 to demethyla-ted metabolites has been demonstrated in incubations with liver microsomes from animal treated with dexamethasone or erythromycin, known inducers of the cytochrome P-450 3A isozyme. Some of the FK-506 demethylated metabolites and the C36-C37 FK-506 dihydrodiol metabolite are also subniitted, as demonstrated for FK-506, to ring-and open chain tautomerism effects, involving the C9 FK-506 carbonyl group as demonstrated by FAB mass spectrometry. FK-506 metabolites, where intra-molecular interactions with certain polar groups at the FK-506 C9 position are not observed, are retaining their in vitro immunosuppressive activity since interaction with the human immunophilin at position C9 remains possible.

Collaborationsl D. Latinne (UCL-IMEX)

References

l G. LHOèST, N. MATON and R.K. VERBEECK (1993) Isolation and Identification of a novel isomerized epoxide metabolite of FK-506 from erythromycin induced rabbit liver niicrosomes. Drug Metab. Disp., 21, 850-854

l G. LHOèST, N. MATON, A. LAURENT and R.K. VERBEECK (1994) Isolation and identification of a FK-506 C36-C37 dihydrodiol from erythromycin induced rabbit liver microsomes. J. Pharm. Biomed. Analysis, 12, 235-241

l G. LHOèST, N. MATON, D. LATINNE, A. LAURENT, and R.K. VERBEECK (1994) 15-Desmethyl FK-506 and 15-31 -Desmethyl FK-506 from human liver niicrosomes: Isolation, Identification (by Fast Atom Bombardment Mass Spectrometry and NMR), and Evaluation of in Vitro Immunosuppressive Activity. Clini. Chem. 40, 740-744

l G. LHOèST, R.K. VERBEECK, N. MATON, P. MUTHELET and D. LATINNE (1995) The in Vitro Immunosuppressive Activity of the C,, -demethylated Metabolite of FK-506 is Govemed by Ringand Open-Chain Tautomerism Effects. J. Pharm. Exp. Ther., 274, 622-626


Drug glucuronidation

R. Verbeeck (FATC), F. Brunelle (FATC), C. Meunier (FATC), D. Lambert (CMFA)

Many drugs and/or their phase I metabolites undergo glucuronide conjugation leading in most cases to inactive conjugates. Some of these glucuronides may undergo §-glucuronidase catalyzed hydrolysis. Such glucuronidation-deglucuronidation futile cycling will modulate the duration and intensity of the activity/toxicity of the aglycone. This phenomenon is investigated in rats in vitro and in vivo using diflunisal and ketoprofen as model compounds. In addition, the role of extrahepatic tissues to the overall glucuronidation of propofol is studied in rat and man.

Glucuronidation is an important metabolic pathway leading to polar conjugates of unchanged drug and/or its phase I metabolites. UDP-glucuronosyltransferases (UDPGT's) are located in the smooth endoplasmic reticulum in cells of different organs. Their function is to transfer glucuronic acid from UDP-glucuronic acid (UDPGA) to a suitable endogenous or exogenous substrate containing a hydroxyl, carboxyl or amino group. In most cases these conjugates are inactive and many pharmacologists therefore firmly believe that glucuronidation stands for detoxification. However, in recent years it has become evident that this is not always true and that the pharmacological activity of a glucuronide conjugate may be higher than that of the parent compound. The most notorious example is morphine-6-glucuronide which has analgesic activities much higher than those of morphine itself.

Whether glucuronidation leads to an inactive conjugate of a pharmacologically active molecule or results in the formation of an active glucuronide, the rate at which a compound undergoes this phase II reaction can significantly influence the intensity and duration of its activity and/or toxicity. It is therefore very important to study the factors which may affect drug glucuronidation. We are currently investigating 2 different apects related to the glucuronidation of drugs:

Regulation of drug glucuronide production by futile cycling in vitro and in vivo

Often overlooked in the discussion of the regulation of drug glucuronide formation in vitro and in vivo is the involvement of futile cycling via §-glucuronidase and possibly esterase. The term futile cycling is used because conjugated metabolites of drugs, endogenous substrates and toxic chemicals (e.g. carcinogens) may undergo successive cycles of synthesis to the glucuronide and hydrolysis back to the aglycone. The enzyme which deconjugates glucuronides, §-glucuronidase, is widely distributed in mammalian tissues with a particularly high activity in the liver. It is localized intracellularly in lysosomes and endoplasmic reticulum. In addition, hydrolysis of acyl glucuronides may be catalyzed by esterases. Using diflunisal and ketoprofen as a model compounds, we are studying the glucuronidation and hydrolysis of drug glucuronides in rats in vitro (microsomes, hepatocytes, isolated perfused rat liver) and in vivo. Results of studies using liver microsomes have clearly shown the contribution of §-glucuronidase-catalyzed hydrolysis to the overall formation formation of the acyl glucuronide of diflunisal (DAG) in the rat. The microsomal formation rate of the phenolic glucuronide of diflunisal (DPG) is not influenced by the §-glucuronidase activity because DPG has a very low affinity towards this enzyme in comaparison to DAG. Using saccharo-1,4-lactone, an inhibitor of §-glucuronidase, the role of futile cycling of DAG in the overall elimination of diflunisal is being investigated in the rat both in vitro (isolated perfused liver) and in vivo.

Extrahepatic glucuronidation in man and rat

Although the liver plays a major role in drug metabolism, major drug metabolizing enzymes such as cytochromes P450 and UDPGT's are also present at other sites. Biotransformation in the gastrointestinal tract is of particular interest after oral administration because bioavailability may be diminished by intestinal presystemic metabolism. In addition, in disease states such as severe cirrhosis of the liver, these extrahepatic pathways may compensate, in part, for impaired hepatic elimina-tion. We have been studying the extrahepatic metabolism of propofol, an intravenous anesthetic agent, in rats and man, both in vitro and in vivo. The results of these studies indicate that the small intestine and possibly the kidney contribute to the overall elimination of propofol in both species.

Collaborationsl L. Van Obbergh (UCL, ANES)


Referencesl F.M. BRUNELLE and R.K. VERBEECK (1993) Glucuronidation of diflunisal by rat liver microsomes. Effect of

microsomal §-glucuronidase activity. Biochem. Pharmacol. 46:1953-1958l R.J. HERMAN, G.R. LOEWEN, D.M. ANTOSH, M.R. TAILLON, S. HUSSEIN and R.K. VERBEECK (1994)

Analysis of polymorphic variation in drug metabolism. III. Glucuronidation and sulfation of diflunisal in man. Clin. Invest. Med .17:297-307

l F.M. BRUNELLE, A.A. RAOOF, J. DE VILLE DE GOYET and R.K. VERBEECK (1996) Glucuronidation of diflunisal, (-)-morphine, 4-nitrophenol and propofol in liver microsomes of two patients with Criggler-Najjar syndrome type II. Biopharm. Drug Disp. 17, 311-317

l F.M. BRUNELLE and R.K. VERBEECK (1996) Glucuronidation of diflunisal in liver and kidney microsomes of rat and man. Xenobiotica, 26, 123-131

l A.A. RAOOF, L.J. VAN OBBERGH, J. DE VILLE DE GOYET and R.K. VERBEECK (1996) Extrahepatic glucuronidation of propofol in man: possible contribution of gut wall and kidney. Eur. J. Clin. Pharmacol.,50, 91-96

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Theoretical effect of modifications of § glucuronidase activity on the net formations and excretion of glucoronide and the elimination of the parent compound in the case of a conjugation-deconjugation

cycle


Plasma protein binding and pharmacokinetics microdialysis sampling

R. Verbeeck (FATC), P. Evrard (FATC), N. Van Brandt (FATC),

Plasma protein binding of drugs is an important phenomenon affecting the distribution and elimination of many drugs. We are studying the interindividual variability in the plasma protein binding of midazolam and sufentanil in ICU patients and its effect on the pharmacokinetics and activity of these compounds. In addition, microdialysis techniques have been developed in our laboratory which permit the in vivo measurement of unbound concentrations of drugs in blood and other tissues (e.g. CNS) of small laboratory animals. Using this technique several projects are underway not only to study the in vivo plasma protein binding and pharmacokinetics of drugs, but also to investigate the transport of compounds across the blood brain barrier.

Reversible binding to plasma proteins, in particular to albumin and a1-acid glycoprotein, may significantly affect the distribution and elimination of drugs. Only the unbound fraction of drug in plasma is available for distribution into the different tissues and certain elimination processes are limited to unbound drug in plasma. Differences in plasma protein binding of drugs within and among patients may therefore contribute to the interindividual variability in drug response. The interindividual variability in the plasma protein binding of midazolam and sufentanyl, two drugs commonly used in Intensive Care, is studied in a large number of ICU patients receiving these drugs by long-term intravenous infusion. Both total and unbound plasma concentrations of these drugs are measured in an attempt to identify factors which influence the degree of binding to plasma proteins. The effect of different pathologies (renal, hepatic, cardiac) as well as the influence of age (neonates, geriatric patients) will be evaluated.

Recently, a microdialysis technique has been developed in our laboratory to continuously measure in vivo the unbound concentrations of drugs in blood of rats and mice ( intravenous microdialysis sampling). Using this technique the stereospecific pharmacokinetics of flurbiprofen are being studied in the rat. Flurbiprofen is a member of the 2-arylpropionic acid derivatives and is administered as a racemic mixture. Both enantiomers show a very high plasma protein binding (>98% in rat, >99% in man) and compete for the same binding sites on albumin. To study this competition between R- and S-flurbiprofen for the same binding sites, experiments have been carried out using intravenous microdialysis sampling to measure the unbound fracton of each enantiomer in vivo. This new approach to directly measure the unbound concentrations of drugs in blood is far more powerful than the classical in vitro techniques to determine the unbound fraction of drug such as equilibrium dialyis and ultrafiltration. The interaction between the two flurbiprofen enantiomers has clinical significance since only the S-enantiomer has anti-inflammatory activity. A better understanding of this interaction phenomenon may improve the rational use of this compound in patients with inflammatory conditions.

Injector

Controller

HPLC pump

Syringe pump

Swivel

Spiral spring

Curler

IntegratorHPLC Column Fluorescence Detector

Microdialysis probe


Using simultaneous intravenous and central microdialysis sampling a collaborative project has been started with Prof. Jean-Michel Scherrmann (UniversitŽ de Paris V) to investigate the distribution of colchicine, a compound known to induce neurodegenerative defects, into the central nervous system. The aim of this study is to identify the factors controlling the passage of this molecule across the blood brain barrier. In addition, two other collaborative projects using microdialysis sampling techniques will start in the near future:

l Chemical Delivery Systems of Taurine Overcoming the Blood-Brain Barrier (in collaboration with Prof. D. Lambert, CMFA)

l Application of Subcutaneous Microdialysis to Pharmacokinetic Studies Following Transdermal or Systemic Drug Administration (in collaboration with Prof. V. PrŽat, FARG).

Collaborationsl P. Mahieu & Ph. Hantson (UCL REAN)l J.-M. Scherrmann (UniversitŽ de Paris V, Paris).

Referencesl N. VAN BRANDT and Ph. HANTSON (1996): Pharmacokinetics and drug protein binding: clinical application in

ICU. In "Yearbook of Intensive Care and Emergency Medicine", Springer-Verlag, Berlin, 762-770. l P.A. EVRARD and R.K. VERBEECK (1994) Effects of flow and albumin concentration of sample solution on

the in vitro recovery by an IV microdialysis probe. Pharm. Res. 11:S363l P.A. EVRARD, G. DERIDDER and R.K. VERBEECK (1996) Intravenous microdialysis in the mouse and the

rat: development and pharmacokinetic application of a new probe. Pharm. Res. 13, 12-17l P.A. EVRARD, J. CUMPS and R.K. VERBEECK (1996) Concentration-dependent plasma protein binding of

flurbiprofen in the rat: an in vivo microdialysis study. Pharm. Res., 13, 18-22l R.K. VERBEECK and P.A. EVRARD (1996) Pharmacokinetics of unbound flurbiprofen in the unanaesthetized

mouse: a microdialysis study. Sixth European Congress of Biophar-maceutics and Pharmacokinetics. Athens, Greece, April 22-24


Biodegradable polymers as drug delivery systems

J. Gillard (FARG), M.A. Benoit (FARG)

The main objective in the development of new forms in order to improve the biodisponibility of the drugs and more specially sustained release systems. The basic aspect consists in the understanding of the pharmaceutical profile of the drug in accordance with the therapeutic aim and the more appropriate administration route (gastrointestinal tract, in situ implantation. The practical goal may be sketched as follows : new forms for new drugs and current drugs in new forms. In view of the therapeutic treatment, sustained release systems are developped for different durations of activity :

l short time : sustained release systems of pituitary hor-mones to induce superovulation in cows (4 days);

l long time : pellets antibiotical carrier for the treatment of osteomyelitis (2-8 weeks)l very long time : anthelmintic formulation for ruminants (6 months), and subcutaneous implants of

bioresorbable polyesters for chemoprophylaxis of malaria (pyrime-thamine, 3-6 months)

Biodegradable sustained release system for potential use in bone infection

A major problem in the treatment of bone infections is the difficulty of obtaining bactericidal levels of antibiotic in the infected tissue without reaching toxic systemic levels. Management includes surgical debridement and parental antibiotics, but toxicity and unreliable penetration into ischaemic tissue are drawbacks. The dead space of the cavity resulting from debridement is filled with blood and serum which promote bacterial growth and may lead to failure. This is the reason why alternative drug delivery systems have been successfully investi-gated in the management of chronic osteomyelitis using locally implanted antibiotics. These include polymethylmethacrylate beads loaded with anti-biotic, an implantable pump, or a composite of D,L-lactic acid oligomer and plaster of Paris beads to deliver antibiotics into the infected bone.

These methods offer many advantages. The implants are placed into the bony defect created by debridement filling the dead space of the cavity. Antibiotics are released at the site of infection reaching local concentrations higher than the minimum inhibitory concentration (MIC) without producing dangerous systemic levels. While antibiotics-loaded polymethylmethacrylate beads and the osmotic pump have to be removed at the end of treatment, plaster of Paris beads are biodegradable and their absorption and the subsequent regeneration of bone occur rapidly during a few weeks to a few months. Normal growth or healing of bone is not inhibited by this material.

Transmucosal administration of vaccines

This study is conducted within the framwork of two EEC biotechnology programmesentitled ÒTechnologies for optimisation of mucosal immune responseÓ and ÒDevelopment of vaccine approaches for the control of infection and inflammatory reactions in parasitic diseasesÓ. The development of new formulations of vaccines for the delivery of antigenic molecules via mucosal routes requires three essential conditions. The carrier (microparticles, beads) :

l must get the antigen in contact with the mucosa via the appropriate route,l must be able to eventually protect the vaccine against the acidity of the gastric fluid,l must guarantee an effective duration of action of the antigen and eventually a deferred release (pulsatile

delivery).

The encapsulation involves the occlusion or the coating of the bioactive agent in a polymer material. Different methods are appropriate for encapsulating antigenic molecules and for preserving their stability and antigenic properties (solvent evaporation, coacervation spherical cristallisation techniques). Current polymers can be used for these formu-lations.

Each antigen has its own specifications to be fulfilled and eventually requests a release in a well defined portion of the gut. In this case the devices loaded with antigen can be formulated with enteric polymers which are not

destroyed by the gastric fluid. Current polymers may be chosen for their appropriate physiochemical properties and specially for their desintegration in a predetermined range of pH.


Circulating antibody responses have been found to be substantially potentiated through oral administra-tion of protein antigens entrapped in biodegradable polymers capsules. These microspheres are absorbed into PeyerÕs patches through M cells (phagocytic mechanism uptake). Medical devices containing microspheres with different PLA-GA ratio and structure are particularly suitable for allowing both a primary and a programmed delayed immunization with a single administration (pulsatile system).

Among the polymers which have been recognized as bioresorbable, the family of polylactide-glycolide (homo, co and stereopolymers) seems to be the most promising, particularly because these polyesters break down into metabolites which are eliminated through natural pathways without inflammatory reactions.

Collaborationsl Clinical services of academic hospital of UCLl Industry for joint research programmesl H. Capron, C. Locht, G. Riveau, Institut Pasteur, Lille, Francel A. Wilson, University of York, United Kingdoml J. Holmgren, University of Gšseborg, Sweden

Referencesl B. MOUSSET, M.A. BENOIT, C. DELLOYE, R. BOUILLET and J. GILLARD (1995) Biodegra-dable implants

for potential use in bone infection Int. Orthop. 19, 157-161l P. HENNEBERT, J. GILLARD and M. ROLAND (1987) U.S. Patent for gazeous sterilization of polymers. 4,

637, 916, 87l M. TSAKALA, J. GILLARD, M. ROLAND, F. CHABOT and M. VERT (1988) Pyrimethanine sustained release

systems based on bioresorbable polyesters for chemoprophylaxis of rodent malaria. J. Control. Rel. 5, 233-242

l M.A. BENOIT, B. BARAS and J. GILLARD (1996) Potential microparticles for the sustained release of oral vaccine. J. Microncaps.


Electrically enhanced transdermal drug delivery

V.PrŽat (FARG), A.Jadoul (FARG), V.Regnier (FARG), R. Vanbever (FARG)

Supported by : Pharmaceutical industry; FNRS ÒCrŽdit aux chercheurÓ; RŽgion Wallone; UCL-FDS

The objective of the research is to assess the potential of electrically enhanced transdermal drug delivery. For both iontophoresis (low density electric current application) and electroporation (high voltage pulse exposure), the following features were analyzed in vitro and/or in vivo :

l enhancement in drug delivery in vitro and in vivol mechanisms involved in drug transportl parameters controlling drug permeationl routes of passage in the skinl skin structure and tolerance issuesl development of new therapeutic applications

Transdermal drug delivery

Drug delivery across skin offers a noninvasive, user-friendly alternative to conventional oral or parenteral administrations. Potential degradation in the gastro-intestinal tract or hepatic first pass are avoided. Transdermal therapeutic systems allow sustained and controlled release of drug. However the skin's outer layer, the stratum corneum, is an extremely effective barrier which prevents transport of most drugs at therapeutic rate. Candidates for passive transdermal delivery share three common properties : effectiveness at relatively low doses, molecular mass less than 500 Da and lipophilicity. Lag time of several hours are often observed.

Several strategies have been developped to enhance transdermal delivery by modifying skin's barrier properties and/or providing a driving force for drugs. Both chemical and physical approaches have been explored.

Iontophoresis

Iontophoresis is the administration of drug through the skin by application of an electric current (<0.5 mA/cm2 for min or h). The electric current provides a driving force to enhance the delivery of drugs. Iontophoresis has been used clinically for delivering certain compounds to surface tissues and is being rediscovered for systemic delivery. Iontophoresis has been shown to facilate transport of drugs e.g. opoids, antimigraine drugs or high molecular weight compounds such as peptides or oligonucleotides. Control of the amount of drug delivered can be achieved by modifying the density, the duration of application or the waveform of the current as well as by changing drug reservoir formulation. The mechanisms of molecular transport involve electrophoresis and electroosmosis during iontophoresis and increased skin permeability. Transport occurs through appendages and stratum corneum. Iontophoresis is generally well tolerated. Only a mild and reversible erythrema, skin hydration and change in lipid bilayer structure were detected. The application of iontophoresis has shown promise for rapid and/or pulsed administration of charged, hydrophilic drugd, including peptides.

Electroporation

Recently, electroporation has been explored as a way of enhancing transdermal transport. Application of high-voltage pulses to skin can increase transdermal transport of compounds ranging in size from small ions to moderate-sized molecules, to macromolecules. Onset times of minutes were attained. The phenomenom underlying enhan-cement is presently hypothetized to be electro-poration of the stratum corneum lipid bilayers : new aqueous pathways are created, local molecular transport occuring through these "electropores" by electrophoresis and diffusion. Consistently, fluorescence microscopy indicated that molecular transport during pulse occurs through the bulk of stratum corneum at localized sites. Control of delivery magnitude and rate was shown to be achieved by controlling the electrical features of the pulsing protocol (voltage, time constant, number) and/or the physicochemical parameters of the drug and solution. Evaluation of skin tolerance to high-voltage pulses has been performed. Alterations of skin function, stratum corneum structure and electrical properties were evident following electroporation of the skin. However, the changes were generally mild and reversible. The studies

performed suggest that electroporation of skin could be used for attaining rapid onset of drug or peptides and/or of oligonucleotides topical delivery.


Feasibility studies

Feasibility studies to assess transdermal flux of several lipophilic drugs by passive diffusion with or without chemical enhancer and of charged drugs by iontophoresis have been performed.

Collaborationsl J. Weaver, M.I.T/Harvard, USAl M. Prausnitz, Georgia Tech., USAl J. Bouwstra & H. Bodde, Leiden Amsterdam Center for Drug Research l R. Guy, UniversitŽ de Genve - Pharmapetidesl T. Ledoan et J. Doucet, UniversitŽ de Paris Sud (Orsay) Francel J.P. Marty, UniversitŽ de Paris Sud (Chatenay-Malabry), France

Expertisesl Transdermal drug delivery by passive diffusion, iontophoresis and electroporation : in vitro and in vivo studies,

feasibility studies

Referencesl S. THYSMAN and V. PRƒAT (1993) In vivo iontophoresis of fentanyl and sufentanil in rats : pharmacikinetics

and acute antinociceptive effects Anesth.Analg. 77, 61-66l S. THYSMAN, D. VAN NESTE and V. PRƒAT (1995) Non invasive investigation of human skin after in vivo

iontophoresis Skin Pharmacol., 8, 229-236l A. JADOUL, J. MESENS, F. DE BEUKELAAR, R. CRABBƒ and V. PRƒAT (1996) Transdermal permeation of

alnitidan by iontophoresis : in vitro optimisation and human pharmacokinetic data Pharm. Res. , 23, 1347-1352

l R. VANBEVER, E. LEBOULENGƒ and V PRƒAT (1996) Transdermal delivery of fentanyl by electroporation I Influence of electrical factors Pharm. Res.13, 559-565

l R. VANBEVER, N. DE MORRE and V. PRƒAT (1996) Transdermal delivery of fentanyl by electroporation II mechanisms involved in drug transport Pharm. Res., 13, 1359-1365.


Nanoparticles and microspheres as drugs and antigen delivery systems

V.PrŽat (FARG), D.Lemoine (FARG), C. Tasset (FARG)

Suported by FRSM, WHO and Industry

The use of nanoparticles or colloid forms has been investigated for several applications :

l drug targeting of antileishmanial drug to the reticuloendothelial system by poly(alkyl cyanoacrylates) nanoparticles.

l nanoparticles of sustained release of calcitoninl colloid carrier of amphotericin Bl improvement of mucosal immunization against influenza virus

Polymeric nanoparticles are solid colloid particles ranging in size from about 10 to 1000 nm. They consist of macromolecular material and can be used as drug carrier. Nanoparticles can be prepared by methods involving either polymerization of dispersed monomers (poly(alkyl cyanoacrylates) or a dispersion of a preformed polymer (poly(lactide), poly(lactide-coglycoloide), alginate). Drugs, peptides or antigens can be adsorbed or encapsulated. Their small size and biodegradation allows them to be injected into blood vessels. Nanoparticles has been used for drug targeting, sustained release and mucosal immunisation.

Targeting of antileishmanial drug to the reticuloendothelial system by poly(alkyl cyanoacrylates) nanoparticles.

Since nanoparticles are mainly taken up by the reticuloendothelial system (RES), RES associated diseases such as visceral leishmania can be targeted with nanoparticles. Primaquine loaded polyalkylcyanoacrylate nanoparticles were prepared and characterized. Their antiparisitic effect and the activation infected macrophages was demonstrated.

Nanoparticles for sustained release of proteins

Calcitonin loaded nanoparticles were prepared. After subcutaneous injection, a sustained release of calcitonin and a hypocalcemic effect was demonstrated.

Colloid carrier of amphotericin BThe use of amphoterecin B is limited by its poor solubility and its toxicity. Solubilisation of amphotericin B with non ionic surfactant can decrease its renal toxicity without changing its antifungal activity.

Improvement of mucosal immunization

Polymeric nanoparticles and microparticles can be useful for mucosal immunisation. Encapsuation of antigen in theses particles can protected the antigen from degradation and/or increase its uptake by Mucosal Associated Lymphoid Tissues. The aim of the research project is to develop new antigen delivery system to improve mucosal immunisation against the influenza virus.

Collaborationsl R. Gaspard, UniversitŽ de Caimbra (Portugal)l Ph. Maincent, UniversitŽ de Nancy (France)

Expertisesl Encapsulation of drugs, peptides and antigens l Preparation and characterisation of nano-particles and microspheres of made of different biodegradable

polymers (poly-alkylcyano-acrylate, poly-(lactide-coglycolide), alginate...)


Referencesl R. GASPAR, V. PRƒAT, F. OPPERDOES and M. ROLAND (1992) Macrophage activation by polymeric

nanoparticles of polyalkylcyanoacry-lates : activity against intracellular Leismania donovani associated with hydrogen peroxide production. Pharm. Res., 9,782-787

l Ch. TASSET, N. BARETTE, S. THYSMAN, J.M. KETELSLEGERS, D. LEMOINE and V. PRƒAT (1995) Polyisobutylcyanoacrylate nanoparticles as sustained release system for calcitonin. J. Control. Release, 33, 23-30

l D. LEMOINE, C. FRAN‚OIS, F. KEDZIEREWICZ, V. PRƒAT, M. HOFFMAN and Ph. MAINCENT (1996) A stability study of Poly(e-caprolactone, poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) Biomaterials17, 2191-2197

SCIENTIFIC OUTPUT p 53

PhD thesis (a more complete descriptio of the doctoral Theses can be found at

<http://www.md.ucl.ac.be/entites/farm/cycle3/doctorat>)

1995l A.S. Crucq ÒMŽcanismes radicalaires des cŽphalosporines irradiŽsÓl F. Van Bambeke ÒInfluence des aminoglyco-sides sur les propriŽtŽs membranaires : Žtudes sur liposomesÓ

1996l J. Gilliard ÒContr™le de la puretŽ des pics en chromatographie liquide couplŽe ˆ un dŽtecteur ˆ barrette de

diodesÓl I. Latour ÒEffet de lÕhydroperoxyde de tert-butyle sur lÕintŽgritŽ de lÕADN et sur la synthse protŽique

dÕhŽpatocytes isolŽsÓl J.P. Montenez ÒEtude des surcharges lysosomiales induites par lÕazithromycine dans les cellules en

culture : effets sur les phospholi-pides et le cholestŽrol cellulairesÓl L. Neuvens ÒEssai de ciblage hŽpatique dÕoligodŽoxyribonuclŽotides antisensÓl A.A. Raoof. ÒExtrahepatic glucuronidationof propofol in man and ratÓ

Publications(an indexed, updated list of publications can be found at <http://www.md.ucl.ac.be/entites/farm/publicat.htm>)

1995l Bailleux V.,VallŽe L., Nuyts J.P., Hamoir G., Poupaert J.H., Stables J.P. & Vamecq J. (1995) Synthesis and

anti-convulsant activity of some 4-nitro-N-phenyl-benzamides. European Journal of Medicinal Chemistry 30:439-444.

l Bailleux V., VallŽe L., Nuyts J.P., Hamoir G., Poupaert J.H., Stables J.P. & Vamecq J. (1995) Comparative anticonvulsant activity and neurotoxicity of 4-amino-N-(2,6-dimethyl-phenyl) phtalimine, a prototype antiepileptic drug in mice and rats. Epilepsia 36: 559-565

l Baltus I., Hoang Hung T., LŽonard E.D., Gerber G.D. & LŽonard A. (1995) Etude de la relation entre la dose de rayonnements ionisants et le taux dÕanomalies chromosomiques chez une personne adulte irradiŽe in toto. C.R. Soci. Biol. 189: 1-5

l Bras A., Cristovas I., Coelho C., Hilati A., Dutrillaux B., LŽonard A. & Rueff J. (1995) Normal genetic response to gamma irradiation in familial adenomatours polyposis. European Journal of Cancer 31: 1506-1510

l Burton J. (1995) Actualisation des beta-bloquants. Journal de Pharmacie de Belgique 50:38-45.l Crucq A.-S., Tilquin B.L. & Hickel B. (1995) Radical Mechanisms of cephalosporins: a pulse radiolysis study.

Free Radical Biology & Medicine 18:841-847.l Dechamps M. & Sonveaux E. (1995) Aldehyde functions in synthesic oligonucleotides. Nucleosides &

Nucleotides 14 : 867-870l Delzenne N., Aertssens J., Verplaetse H., Roccaro M. & Roberfroid M. (1995) Effects of fermentable

fructooligosaccharides on mineral, nitrogen and energy digestive balance in the rat. Life Sciences 57: 1579-1587.

l de Meester C. (1995) Genotoxic potential of §-carbolines. A review. Mutation Research 339: 139-153l de Meester C. & Gerber G.B. (1995) The role of cooked food mutagens as possible ethiological agents in

human cancerÊ: a critical appraisal of recent epidemiological investigations. Epidemiology & Public HealthÊ43: 147-161

l Diouf O., Depreux P., Lesieur D., Poupaert J. & Caignard D. (1995) (N-imidoalkyl) aminoalkyl-benzothiaezolin-2-ones as ligands of the serotoninergic 5-HT1A receptors. Hetero-cycles 41:1219-1233.

l Diouf O., Depreux P., Lesieur D., Poupaert J. & Caignard D. (1995) Synthesis and evaluation of new 2-piperazinylbenzothiazoles with high 5-HT1A and 5HT3 affinities. European Journal of Medicinal Chemistry 30: 715-719

l Dupont L., Masereel B., Lambert D.M. & Scriba G.K.E. (1995). 2-Cyanoimino-5,5-diphenyl-4-imidazolinone monohydrate. Acta Crystalogra-phica C51:1901-1903.

l Fiordaliso M., Kok N., Desager J.P., Goethals F., Deboyser D., Roberfroid M. & Delzenne N. (1995) Dietary oligo-fructose lowers triglycerides, phospholipids and cholesterol in serum and Very Low Density Lipoproteins of rats. Lipids 30:163-167.

l Foufelle F., Lepetit N., Bosc D., Delzenne N., Morin J., Raymondjean M. & FerrŽ P. (1995) DNase I hypersensitivity sites and nuclear protein binding on the fatty acid synthase gene: identification of an element with properties similar to known glucose-responsive elements. Biochemical Journal 308:521-527.

l Gallez B., De Keyser J.L., Debuyst R., Dejehet F., Neuvens L. & Dumont P. (1995) The effects of pasteurisation on albumin: an EPR binding assay for polymeric albumin. Journal of Pharma-ceutical and Biomedical Analysis 13:1449-1452.

l Gibson G.R. & Roberfroid M. (1995) Dietary modulation of the human colonic microbiota: Introducing the concept of prebiotics. Journal of Nutrition 125:1401-1412.


l Grucker D., Guiberteau T., Eclancher B., Chambron J., Chiarelli R., Rassat A. & Gallez B. (1995) Dynamic nuclear polarization with nitroxides dissolved in biological fluids. Journal of Magnetic Resonance B106:101-109.

l Hoang Hung T., Gerber G.B. & LŽonard A. (1995) Utilisation du test du micronoyau comme dosimtre biologique en cas dÕexposition non-homogne aux radiations ionisantes. C.R. Soc. Biol. 186: 1-6

l Jadoul A., Hanchard C., Thysman S. & PrŽat V. (1995) Quantification and localization of fentanyl and TRH delivered by iontophoresis in the skin. International Journal of Pharmaceutics 120:221-228.

l Kotretsou S., Mingeot-Leclercq M.P., Constantinou-Kokotou V., Brasseur R., Georgiadis M.P. & Tulkens P. (1995) Synthesis and antimicrobial and toxicological studies of amino acid and peptide derivatives of kanamycin A and netilmicin. Journal of Medicinal Chemistry 38:4710-4719.

l Lambert D.M. (1995) Des dŽrives simples de la glycine aux systmes lipidiques transporteurs de glycine. Essai de vectorisation d'un acide amine neutre de petite taille vers le systme nerveux central. Bulletins et MŽmoires de l'AcadŽmie royale de MŽdecine de Belgique 150:294-300.

l Lambert D.M., Gallez B. & Poupaert J. (1995) Synthesis and distribution of N-benzylo-xycarbonyl-[14C]-glycine, a lipophilic derivative of glycine. Journal of Labelled Compounds and Radiopharmaceuticals 36:397-406.

l Lambert D.M., Geurts M., Scriba G., Poupaert J. & Dumont P. (1995). DŽrives simples d'acides amines neurotransmetteurs. Evaluation anti-convulsivante de dŽrives amides, carbamates et esters de la glycine et de la beta-alanine. Journal de Pharmacie de Belgique 50:194-203.

l Lambert D.M., Hamoir G., Hermans E. & Poupaert J. (1995) Sythesis and anti-convulsant activity of 4-amino-N-(2-ethylphenyl) benzamide and 4-amino-N-(2,6-diethylphenyl) benzamide, two new ameltolide analogues. Pharmaceutical Sciences 1:181-184.

l Lambert D.M., Mergen F., Berens C., Poupaert J. & Dumont P. (1995) Synthesis and pharma-cological properties of 2-[S-acetyl-thiorphan]-1,3-diacylamino-propan-2-ol derivatives as chimeric lipid drug carriers containing an enke-phalinase inhibitor. Pharmaceutical Research 12: 187-191.

l Lambert D.M., Scriba G.K.E., Gallez B., Poupaert J.H. & Dumont P. (1995) Glyceride conjugates as drug carriers. Current Medicinal Chemistry 1: 376-391.

l Latour I., Demoulin J.B. & Buc Calderon P. (1995) Oxidative DNA damage by t-butyl hydroperoxide causes DNA single strands breaks which is not linked to cell lysis. A mechanistic study in freshly isolated rat hepatocytes. FEBS Letters 373: 299-302.

l Lefebvre V. & Buc-Calderon P. (1995) Desferal prevents against cell lysis induced by hydrogen peroxide to hypoxic hepatocytes : a role for free iron in hypoxia-mediated cellular injury. Chemico-Biological Interaction 94: 37-48.

l Lefebvre V., Goffin I. & Buc Calderon P. (1995). Fructose Protects Rat Hepatocytes During Hypoxia and Improves Protein Synthesis Recovery During Reoxygenation. Trans-plantation Proceedings 27: 2823-2824.

l LŽonard A., Baughet-Mahieu L., Hoang Hung T., LŽonard E.D., Lemaire M. & Gerber G.B. (1995) Chromosome aberrations in curculating lymphocytes after brachytherapy for uterus carcinoma. Acta Oncologica 34: 539-542

l LŽonard A. & Gerber G.B. (1995) Toxicity of essential and beneficial metal ions. Zinc. InÊ: handbook of metal-ligand interactions in biological fluids (Berthon, G., ed), Marcel Dekker Inc., New York, Vol. 2: 705-708

l LŽonard A., Hantson Ph. & Gerber G.B. (1995) Mutagenicity, carcinogenicity and teratogenicity of lithium compounds. ?

l Lhoest G., Verbeeck R.K., Maton N., Muthelet P. & Latinne D. (1995) The in vitro immunosuppressive activity of the C15-demethylated metabolite of FK-506 is governed by ring- and open-chain tautomerism effects. Journal of Pharmacology and Experimental Therapeutics 274: 622-626.

l Liu X., Lambert D.M. & Abul-Hajj Y. (1995) Probing the hydrophobic pocket of the active site of aromatase with 4-phenoxy-7-a-(phenylthio)-4-androstene-3,17-dione Medi-cinal Chemistry 38: 4135-4138.

l Livesey G., Smith T. Eggum B.O., Delzenne N., Tetens I.H., Nyman M., Roberfroid M. & Decombaz J. (1995). Determination of digestible energy values and fermentabilities of dietary fibre supplements: a European interlabory study in vivo. British Journal of Nutrition 74: 289-302.

l MacDonald J.I., Wallace S.M., Herman R.J. & Verbeeck R.K. (1995) Effect of probene-cid on the formation and elimination kinetics of the sulphate and glucuronide conjugates of diflunisal. European Journal of Clinical Pharmacology 47: 519-523.

l Maes H., Taper H. & Cocito C. (1995) Alteration of the immune response during cancer development and prevention by administration of a Mycobacterial antigen. Scandinavian Journal of Immunology 41: 53-64.

l Mingeot-Leclercq M.P., Brasseur R. & Schanck A. (1995) Molecular parameters involved in aminoglycoside nephrotoxicity. Journal of Toxicology and Environmental Health 44: 263-300.

l Mingeot-Leclercq M.P., Schanck A., Van Bambeke F., Tulkens P.M., Lins L. & Brasseur R. (1995) Molecular description of the interactions of aminoglycoside antibiotics with negatively-charged phospholipids. Theoretical molecular modelling and experimental results. Life Sciences Advances 14: 71-98.

l Ngombo Mukendi J.F., de Meester C. & Rollmann B. (1995) Etude, in vitro, de la conversion mŽtabolique de lÕaflatoxine B1 en son Žpoxide. C.R. Soc. Biol. 189: 1-14

l Poupaert J., Hamoir G., Barbeaux P., Lambert D.M. & Henichart J.P. (1995) Anticonvulsant activity of some N-phenylphthalimide deriva-tives in rats and mice. Journal of Pharmacy and Pharmacology 47: 89-91.

l Poupaert J., Lambert D.M., Vamecq J. & Abul-Hajj Y. (1995) Molecular modeling studies on 11beta-aminoethoxyphenyl and 7alpha-amino-ethoxyphenil estradiols. Evidence suggesting a common hydrophobic pocket in estrogen receptor. Bioorganic and Medicinal Chemistry Letters 5: 839-842.


l Quetin-Leclercq J., Favel A., Balansard G., Regli P. & Angenot L. (1995) Screening for in vitro antifungal activities of some indole alkaloids. Planta Medica 61: 393-492.

l Quetin-Leclercq J., Llabres G., Warin R.,Belem-Pinheiro M.L., Mavar-Manga H. & Angenot L. (1995) Guianensine, A zwitterionic alkaloid from Strychnos guianensis. Phytochemistry 40: 1557-1559.

l Raoof A.A., Van Obbergh L.J. & Verbeeck R.K. (1995) Propofol pharmacokinetics in children with biliary atresia. British Journal of Anaesthesia 74: 46-49.

l Ritter C., Gilliard J.A., Cumps J. & Tilquin B. (1995) Corrections for heteroscde-dasticity in window evolving factor analysis. Analytica Chimica Acta 318: 125-136.

l Roberfroid M. (1995) Toxicology: a Science and an Art. Toxicology In Vitro 9: 839-844.l Roberfroid M., Bronet F., Bouley C. & Cummings J.H. (1995) Colonic microflora: nutrition and health. Nutrition

Reviews 53: 127-130.l Roberfroid M. & Buc Calderon P. (eds) Free radicals and oxidation phenomena in biological systems. Marcel

Dekker inc., New York, 272 ppl Scriba G.K.E., Lambert D.M. & Poupaert J.H.. (1995) Biovailability and anticonvulsant activity of a

monoglyceride-derived prodrug of phenytoin after oral administration to rats. Journal of Pharmaceutical Sciences 84: 300-302.

l Scriba G.K.E., Lambert D.M. & Poupaert J.H. (1995) Anticonvulsant activity of phenytoin-lipid conjugates, a new class of phenytoin prodrugs. Journal of Pharmacy and Pharmacology 47: 197-203.

l Scriba G.K.E., Lambert D.M. & Poupaert J.H. (1995) Bioavailability of phenytoin following oral administration of phenytoin-lipid conjugates to rats. Journal of Pharmacy and Pharmacology 47: 945-948.

l Swartz H.M., Bacic G.C., Gallez B., Goda F., James P., Jiang J. & Walezak T. (1995) In vivo EPR spectroscopy. In Bioradicals Detected by ESR Spectroscopy. H. Ohya-Nishiguchi & L. Packer, edit., Birkhauser Verlag, Basel. 285-299.

l Taper H.S., Delzenne N., Tshilombo A. & Roberfroid M. (1995) Protective Effect of Dietary Fructo-oligosaccharide in Young Rats Against Exocrine Pancreas Atrophy Induced by High Fructose and PartialCopper Deficiency. Food and Chemical Toxicology 33: 631-639.

l Tasset Ch., Barette N., Thysman S., Ketelslegers J.M., Lemoine D. & PrŽat, V. (1995) Polyisobutylcyanoacrylate nano-particles as sustained release system for calcitonin. Journal of Controlled Release 33: 23-30.

l Thysman S., Jadoul A., Leroy T., Van Neste D. & PrŽat V. (1995) Laser doppler evaluation of skin reaction in volunteers after histamine iontophoresis. Journal of Controlled Release 36: 215-219.

l Thysman S., Van Neste D. & PrŽat V. (1995) Nonivasive investigation of human skin after in vivo lontophoresis. Skin Pharmacology 8: 229-236.

l Tinton S. & Buc Calderon P. (1995) Inhibition of protein synthesis induced by adenine nucleotides requires their metabolism into adenosine. Biochemical Pharmacology 50: 481-488.

l Tinton S. & Buc Calderon P. (1995) Homocysteine enhances the inhibitory effect of extracellular adenosine on the synthesis of proteins in isolated rat hepatocytes. Biochemical Journal 310: 893-896.

l Tinton S., Chow S., Buc Calderon P., Kass G. & Orrenius S. (1995) Adenosine inhibits protein synthesis in isolated rat hepatocytes. Evidence for a lack of involvement of intracellular calcium in the mechanism of inhibition. European Journal of Biochemistry 229: 419-425.

l Van Bambeke F., Tulkens P., Brasseur R. & Mingeot-Leclercq M.P. (1995) Aminoglyco-side antibiotics induce aggregation but not fusion of negatively-charged liposomes. European Journal of Pharmacology 289: 321-333.

l Van Beers B., Pringot J. & Gallez B. (1995) Les oxydes de fer comme agents de contraste en IRM hŽpatique. Journal de Radiologie 76: 991-995

l Vanbel P.F., Tilquin B.L. & Schoenmakers P.J. (1995) Criteria for developing rugged high-performance liquid chromatographic methods. Journal of Chromatography A 697: 3-16.

l Vanbever R. & PrŽat V. (1995) Factors affecting transdermal delivery of metoprolol by electroporation. Bioelectrochemistry 38: 223-228.

l Vandamme Th.F., Demoustier M. & Rollmann B. (1995) Quantification of levamisole in plasma using High Performance Liquid Chromatogra-phy. European Journal of Drug Metabolism & Pharmacokinetics 20: 145-149

l Vandamme Th.F. & Legras R. (1995) Physico-mechanical properties of poly-(e-caprolactone) for the construction of Rumino-Reticulum Devices for grazing animals. Biomaterials : 1395-1400

l Van Dyck M., Rollmann B. & de Meester C. (1995) Quantitative estimation of heterocyclic aromatic amines by ion-exchange chromato-graphy and electro-chemical detection. Journal of chromatography 697: 377-382

l Verbeeck R.K. (1995) Pharmacokinetic considerations in the toxicologic evaluation of xenobiotics. In Modulation of Cellular Res-ponses in Toxicology. C.L. Galli, A.M. Goldberg & M. Marinovich, edit. 125-144.

1996l Bidaine A., Berens C. & Sonveaux E. (1996) The phototrityl group. Photocrosslinking of oligonucleotides to

BSA. Bioorganic & Medicinal Chemistry letters 6: 1167-1170

l Bonjean K., De Pauw-Gillet M.C., Quetin-Leclercq J., Angenot C. and Bassleer R. (1996) In vitro cytotoxic activity of two potential anticancer drugs isolated from Strychnos : strychnopentamine and usanibarcrisine. Anticancer Res. 16: 1129-1138


l Brunelle F.M. & Verbeeck R.K. (1996) Glucuronidation of diflunisal in liver and kidney microsomes of rat and man. Xenobiotica 26 : 123-131.

l Brunelle F., Raoof A., de Ville de Goyet J. & Verbeeck R.K. (1996) Glucuronidation of diflunisal, (-)-morphine, 4-nitrophenol, and propofol in liver microsomes of two patients with Crigler-Najjar syndrome type I. Biopharmaceutics & Drug Disposition 17 : 311-317.

l Charbon V., Latour I., Lambert D.M., Buc-Calderon P., Neuvens L., De Keyser J.L. & Gallez B. (1996) Targetting of drug to the hepa-tocytes by fatty acids. Influence of the carrier (albumin or galactosylated albumin) on the fate of the fatty acids and their analogs. Pharmaceutical Research 13 : 27-31.

l Crucq A.S. and Tilquin B. (1996) Method to identify products induced by radiosterilization, a study of cefotaxime sodium. J. Pharm. Belg. 51: 285-288

l de Meester C. (1996) Chemical analysis of heterocyclic amines : quantification in heat processed food. Measurements and testing newsletter 4: 13

l de Meester C., Rebache M. & Galceran M.T. (1996) Chemical Analysis of heterocyclic amines (HAAs) in heat processed foodÊ: first intercomparison of the end-determination step based on a solution with an identifty and content of HAAs both unkown to participants. European Commission Publication-BCR information-Chemical analysis-EUR.16935

l Evrard P., Cumps J. & Verbeeck R.K. (1996) Concentration-dependent plasma protein binding of flurbiprofen in the rat: an in vivo microdialysis study. Pharmaceutical Research 13 : 18-22.

l Evrard P., Deridder G. & Verbeeck R.K. (1996) Intravenous microdyalis in the mouse and the rat: development and pharmacokinetics application of a new probe. Pharmaceutical Research 13 : 12-17.

l Gallez B., Mader K. and Swartz H.M. (1996) New non-invasive technique for measuring the pH inside the gut using pH-sensitive nitroxides. An in vivo EPR study. Magnetic Resonance in Medecine 36: 694-697

l Gallez B., Bacic G. & Swartz H. (1996) Evidence for the dissociation of the hepatobiliary MRI contrats agent Mn-DPDP. Magnetic Resonance in Medicine 35 : 14-19.

l Gallez B., Bacic G., Goda F., Jiang J., O'Hara J., Dunn J. & Swartz H. (1996) Use of nitroxides for assessing perfusion, oxygenation, and viability of tissues : In vivo EPR and MRI studies. Magnetic Resonance in Medicine 35 : 97-106.

l Gallez B., Baudelet C., Adline J., Charbon V. & Lambert D.M. (1996) The uptake of Mm-DPDP by hepatocytes is not mediated by the facilitated transport of pyridoxine. Magnetic Resonance Imaging 14: 1191-1195

l Gallez B., Debuyst R., Liu K.J., Demeure R. & Swartz H.M. (1996) Development of bicompatible implants of fusinite for in vivo EPR oximetry. MAGMA 4: 71-75

l Gerbaux C., Van Bambeke F., Montenez J.P., Piret J., Morlighem G. & Tulkens P.M. (1996) Hyperactivity of cathepsin B and other lysosomal enzymes in fibroblasts exposed to azithromycin, a dicationic macrolide antibiotic with exceptional cellular accumulation. FEBS Letters 394: 307-310

l Goda F., Bacic G., OÕHara J.A., Gallez B., Swartz H.M. & Dunn J.F. (1996) The relationship between pO2 and perfusion in two murine tumors after X-ray irradiation : a combined Gd-DTPA dynamic MRI and EPR oximetry study. Cancer Research 56: 3344-3349

l Goda F., Gallez B. & Swartz H.M. (1996) Pharmacokinetics of the nitroxide PCA measured by in vivo EPR. Res. Chem. Intermediat. 22: 491-498

l Hantson P., Verellen-Dumoulin C., Libouton J.M., LŽonard A., LŽonard E.D. & Mahieu P. (1996) Sister chromatid exchanges in human peripheral blood lymphocytes after ingestion of hig doses of arsenicals. Int. Arch. Occup. Environ. Health 68: 342-344

l Hantson Ph., de Saint-Georges L., Mahieu P., LŽonard E.D., CruzFayt M.C. & LŽonard A. (1996) Evaluation of the ability of paracetamol to produce chromosome aberrations in man. Mutation Research 368: 293-300

l Jadoul A., Doucet J., Durand D. & PrŽat V. (1996) Modifications induced on stratum coneum structure after in vitro iontophoresis : ATR-FMR and Xray scattering studies. Journal of Controlled Release 42: 165-173

l Jadoul A., Mesens J., de Beukelaar F., CrabbŽ R. & PrŽat V. (1996) Transdermal permeation of alnitiden by iontophoresis : in vitro optimisation and human pharmacokinetic data. Pharmaceutical Research 13: 1347-1352

l Isa M., Cumps J., Poupaert J.H. & Atassi G. (1996) Cytochrome P450 induction and antineoplastic action of fotomustine in mice. Journal de Pharmacie de Belgique 51: 192-194

l Kanyonyo M.R., Poupaert J.H, Levque Ph.X., Gozzo A., Van derpoorten K., Lambert D.M., Diouf O. & Vamecq J. (1996) Reaction of aryl isothiocyanates with phthalic acid derivatives. Bul. Soc. Chim. Belg.105 : 55-56.

l Kanyonyo M.L., Ucar H., Isa M., Carato P., Lesieur D., Renard P. & Poupaert J. (1996) Solvent effect in the Wollgerodt-Kindler reaction. Bulletin de la SociŽtŽ Chimique Belge 105 : 17-22.

l Kishore B.K., Fuming L., Maldague P., Tulkens P.M. & Courtoy P.J.(1996) Mechanism of the thesaurismosis and altered lysosomal dynamics induced by poly-D-glutamic acid in kidney proximal tubular cells. Laboratory Investigation 74 : 1025-1037.

l Kishore B.K., Maldague P. , Tulkens P.M. & Courtoy P.J. (1996) Poly-D-glutamic acid induces an acute lysosomal thesaurismosis of proximal tubules and a marked proliferation of interstitium in rat kidney. Laboratory Investigation 74 : 1013-1023.

l Kok N., Robert A., Roberfroid M. & Delzenne N. (1996) Dietary oligofructose modifies the impact of fructose on hepatic triacylglycerol metabolism. Metabolism, 45: 1547-1550

l Kok N., Roberfroid M. & Delzenne N. (1996) Involvment of lipogenesis in the lower VLDL secretion induced by oligofructose in rats. British Journal of Nutrition, 76: 881-890


l Lambert D.M., Scriba G.K.E., Poupaert J.H. & Dumont P. (1996) Anticonvulsant activity of ester- and amide-type lipid conjugates of glycine and N-benzyloxycarbonylglycine. European Journal of Pharmaceutical Sciences 4 : 159-166.

l Lambert D.M., Masereel B., Gallez B., Geurts M. & Scriba G.K.E. (1996) Bioavailability and anticonvulsant activity of 2-cyanoguanidino-phenytoin, a structural analogue of phenytoin. Journal of Pharmaceutical Sciences 85: 1077-1081

l Lambert D.M. & Gallez B. (1996) Synthesis and distribution of tritiated N,NÕ-dibenzoyl-1,3,-diaminopropan-2-ol. J. Label. Comp. Radiopharm. 38 : 897-905

l Lemoine D., Franois C., Kedziercwics F., PrŽat V., Hoffman M. & Maincent P. (1996) A stability study of poly/Etaprolatone poly (D,L-pactide) and poly( D-L lactide coglycolide) Biomaterials 17: 2191-2197

l LŽonard A. & Gerber G.B. (1996) Does the Chernobyl accident increase genetic defects in exposed human populationsÊ? Scope-Radtest Newsletter 11: 4-6

l Mader K., Gallez B., Liu K.J. & Swartz H.M. (1996) Non-Invasive in vivo characterization of release processes in biodegradable polymers by low-frequency electron paramagnetic resonance spectroscopy. Biomaterials 17 : 457-461.

l Mader K., Gallez B. & Swartz H.M. (1996) In vivo EPR : an effective new tool for studying pathophysiology, physiology and pharmaco-logy. Appl. Radiat. Isot. 47: 1663-1667

l Mavar-Manga H., Quetin-Leclercq J., LLabres G., Belem-Pinheiro M.L., Imbiriba da Rocaia A. and Angenot L. (1996) 9-methoxygeisso-schizol, an alkaloid from bark of Strychnos guianensis. Phytochemistry 43: 1125-1127

l Montenez J.P., Van Bambeke F., Piret J., Schanck A., Brasseur R., Tulkens P.M. & Mingeot-Leclercq M.P. Interaction of the macrolide azithromycin with phospholipids. II. Biophysical and computer-aided conformational studies. European Journal of Pharmacology. 314: 215-227

l PrŽat V. & Vanbever R. (1996) Transermal drug delivery by electroportation. In : Prediction of percutaneous penetration (Brain K., James V. & Walter K., eds), Vol. 4: 26-29

l Pronce Th. & Tilquin B. (1996) Trace analysis in chiral separation of selected aminoenantiomers. Journal of Pharmaceutical and Biomedical analysis 14: 1175-1184

l Raoof A.A., Augustijns P.F. & Verbeeck R.K. (1996) In vivo assessment of intestinal, hepatic and pulmonary first pass metabolism in the rat. Pharmaceutical Research 13 : 891-895.

l Raoof A.A., van Obbergh L.J., de Ville de Goyet J. & Verbeeck R.K. (1996) Extra-hepatic glucuronidation of propofol in man: possible contribution of gut wall and kidney. European Journal of Clinical Pharmacology50 : 91-96.

l Roberfroid M. (1996) Functional effects of Food components and the gastrointestinal system : chicory fructooligosaccharides. Nutrition Reviews. 54: S38-S42

l Rueff J., Chiapella C. & LŽonard A. (1996) Development and validation of alternative metabolic systems for mutanenicity testing in short-term essays. Mutation Research 353: 151-176

l Scorneaux B., Ouadrhiri Y., Anzalone G. & Tulkens P.M. (1996) Effect of Recombinant Human Gamma Interferon on Intracellular Activities of Antibiotics against Listeria mono-cytogenes in the Human Macrophage Cell Line THP-1. Antimicrobial Agents and Chemo-therapy 40 : 1225-1230.

l Taper H.S., Keyeux A. & Roberfroid M. (1996) Potentiation of Radiotherapy by Nontoxic Pre-treament with Combined Vitamins C and K3 in Mice Bearing Solid Transplantable Tumor. Anticancer Research 16 : 499-504.

l Tilquin B. & Rollmann B. (1996) Recherches ˆ conseiller pour l'application de la stŽrilisation ionisante de medicaments. Journal de Chimie Physique 93 : 224-230.

l Tinton S., Chow S.C., Buc Calderon P. & Kass G. (1996) Adenosine stimulates calcium influx in isolated rat hepatocytes. European Journal of Biochemistry 238: 576-581

l Ucar H., Van derpoorten K., Kanyonyo M.R., Isa M., Lambert D.M., Lesieur D. & Poupaert J.H. (1996) Synthesis of 6-benzol-2(3H)-benzothiazolane. Bulletin de la SociŽtŽ Chimique Belge 105: 773-776

l Van Bambeke F., Mingeot-Leclercq, M.P. Brasseur R., Tulkens P. & Schanck A. (1996) Aminoglycoside antibiotics prevent the formation of non-bilayer structures in negative-ly-charged membranes. Comparative studies using fusogenic (bis(beta-diethy-lamino-ethylether) hexestrol) and aggregating (spermine) agents. Chemistry and Physics of Lipids 79 : 123-135.

l Van Bambeke F., Montenez J.P., Piret J., Tulkens P.M. & Mingeot-Leclercq M.P. (1996) Interaction of the macrolide azithromycin with phospholipids. I. Inhibition of phospholipase A1 activity. European Journal of Pharmacology 314: 203-214

l Van Bambeke F., Nachega J. & Tulkens P.M. (1996) Traitement des infections Herpes simplex, Varicella Zoster et Cytomegalovirus. Louvain MŽdical 116: 735-744

l Vanbel P., Tilquin B. and Schoenmakers P. (1996) Criteria for optimizing the separation of target analytes in complex chromatograms. Chemometrics Intell. Lab. systems 35: 67-86

l Vanbever R., Le BoulengŽ E. & PrŽat V. (1996) Transdermaldelivery of fentanyl by electroporation I. Influence of electrical factors. Pharmaceutical Research 43 : 559-565.

l Vanbever R., Skrypaczak D., Jadoul A. & PrŽat V. (1996) Comparison of skin electro-perturbations induced by iontopho-resis and electroportation. In : Prediction of percu-taneous penetration (Brain K., James V. & Walter K., eds), Vol. 4: 243-246

l Vanbever R., Demorre N. & PrŽat V. (1996) Transdermal delivery of fentonyl by electroportation. II Mechanisms involved in drug transport. Pharmaceutical Research 13: 1359-1365


l Van Brandt N. & Hantson P. (1996) Phar-macokinetics and drug-protein binding. Yearbook Intens. Care Emerg.Med. 762-770.

l Van Damme Th. & Ngombo Mukendi J.F. (1996) Controlled Release of Levamisole from poly (e-caprolactone) matrices I. Effects of the nature and the concentration of the drug incorporated into the matrices. European Journal of Biopharmacy 43: 116-123

l Van Damme Th. & Ngombo Mukendi J.F. (1996) Controlled Release of Levamisole from poly (e-caprolactone) matrices II. Effects of water-soluble polymer and iron powder incorporated into the matrices. International Journal of Pharmaceutics 132: 153-163

l Wright C.W., Phillipson J.D., Awe S.O., Kirby G.C., Warhust D.S., Quetin-Leclercq J. & Angenot L. (1996) Antimalarial activity of cryptolepine and some other anhydronium Bases. Phytotherapeutic Research 10 : 361-363.

STAFF PROFILE p 59

Division of biochemical toxicologySecretariat : Phone : 32-2-764 73 69, Fax : 32-2-764-73 59, E-mail : [email protected] 7369, 73 Avenue Mounier, B-1200 Brussels, Belgium

Pedro Buc Calderon, PhD, Head, Professor of biochemical toxicology. Pedro Buc Calderon graduated in Pharmacy of the Universidad de Chile in 1977. He spent two years (1981-1983) as post-graduate at the Max Planck Institute for Medical Research in Heidelberg. In 1987 he obtained his PhD thesis from the UCL for his work on the captodative olefins, a new family of synthetic free radical scavengers. Over the years his main interest has been the physiopathology of oxygen free radicals. This research is focused on the elucidation of the molecular mechanisms leading to cell death as a consequence of an oxidative stress.Professor Buc Calderon is author and co-author of several articles and co-author of a book on free radicals, he is member of the Belgian societies of Biochemistry and Cellular Biology. He is on the Board of the Society for the Study of Oxygen Metabolism and the FNRS group ÒOxidative Phenomena and AntioxidantsÓ.

Marcel Roberfroid, PhD, Professor in bioche-mistry, drug metabolism, toxicology and food toxicology.Marcel Roberfroid has graduated in Pharmacy and has obtained his PhD at the UCL. As a postdoc, he worked with B.B. Brodes at the National Institute of Health (NIH) Bethesda, USA. His scientific carrer has all been devoted to understand how physiological and toxicological processes can be modulated mainly via nutrition. He has contributed to the field of carcinogenesis by introducing the concept of positive and negative modulation. More recently, he has becoma an european leader in the field of functional foods being, together with his UK colleague G. Gibson, at the origin of the prebiotic/ synbiotic concepts. Professor Roberfroid is author and co-author of more than 100 articles and co-author of a book on free radicals. He is the european editor of ÒFood and Chemical ToxicologyÓ. He is member of scientific societies in Belgian, in Europe and in US. He is president of ILSI-Europe. He has presented seminars in some 40 universities around the world. As organiser, chairman or speaker he has been and still is participating in international meetings and workshops worldwide. He is actively involved as partner, coordinator and evaluator of EU founded research and granting programmes.

Nathalie Delzenne, PhD, Assiociate ProfessorNathalie Delzenne was graduated in Pharmacy in 1986 and obtained her PhD thesis in 1991 at the School of Pharmacy of the UCL. She followed the courses of Human Nutrition at the ÒUniversitŽ de LausanneÓ (Switzerland) and was selected to participate to the 3rd Nutrition leadership programme. She obtained a grant from NATO and spent the year 1994 in France, first in Lille (INSERM U325) then in Paris (INSERM U342), where she was involved in studies relative to nutritional modulation of gene expression. Her main field of interest is the regulation of hepatic lipid metabolism by nutrients. She is a member of European Academy of Nutritional Science and is the belgian expert at OECD for safety evaluation of food.

Research scientistsFabienne Goethals, PhDHenryk Taper, PhD, MD

Postdoctoral fellowsIsabelle Latour, PhDSandrine Tinton, PhD

PhD studentsKathia EvdokimovaNadine Kok

Technical and administrative staffVŽronique AllaeysIsabelle Blave Laurent De wispeleare Luc GeschŽ

VŽronique Lebrun

STAFF PROFILE p 60

Division of drug analysis

Secretariat : Phone : 32-2-764.72.30, Fax : 32-2-764.72.96, E-mail : [email protected] 7230, 72 Avenue Mounier, B-1200 Brussels, Belgium.

BernardTilquin, Ph.D, Head, Professor of Analytical Chemistry.Professor Bernard Tilquin graduated in chemistry at the UCL in 1965 and obtained his Ph.D. in 1970 at the same university. He worked at the NRC Ottawa and at the University of Nagoya as visiting professor. He was head of the School of Pharmacy from 1993 to 1996. Over the years his main interests have been the chemical effects of ionizing radiation. His research is focused on the radiolysis of` molecular solids and on the radical mechanisms induced at ultra low temperatures. Since 1987, at the department of the pharmacy UCL, he now is involved as coordinator of the laboratory for drug analysis (chemometrics, GC2-MS, CE, HPLC, Rx,...)

Jo‘lle Quetin-Leclercq, Ph.D, Professor of Pharmacognosy. Jo‘lle Quetin-Leclercq studied pharmacy at the ÒUniversitŽ de LigeÓ, where she also obtained a certificate in biophysics in 1985 and a PhD in 1989. Her research interests were mainly indole alkaloids from Strychnos species and some of their pharmacological activities (cytotoxic, antifungal,...). In 1985, she also obtained a research grant from the Royal Society of London to realise a part of her work at Chelsea College, University of London, and in 1990, another grant from the Belgian Ministry of the French Community for a research stay at the ÒUniversitŽ de MarseilleÓ (France). She was a research associate at the Belgian National Fund for Scientific Research (FNRS) until she was appointed as Professor at the UCL in 1995, where she is at the head of the laboratory of pharmacognosy. The main research interest of this laboratory is the study of plants used in traditional medicine: detection of their pharmacological properties, isolation and structure of their active molecules with the aim of finding new pharmacologically active substances. She received the Specia Price in 1983 and the Biannual Price of the Belgian Society of Pharmaceutical Sciences in 1990. She is a member of the Phytochemical Society of Europe, the Royal Society of Chemistry (therapeutic chemistry), the Belgian Society of Pharmaceutical Sciences, the Belgian Pharmacopoeia commission and the commission of medicines (pharmacognosy) of the Ministry of Health.Robert Denayer, PhD, permanent scientistRobert Denayer, graduated in chemistry at the UCL, obtained is PhD in 1958 in the same university. He worked then until 1971 in different industrial laboratories, specially in the field of pigments and varnishes for painting. Afterwards he was nominated as permanent scientist at the Chemistry department at the UCL where his work consist mainly in education. His research activity have been devoided to the direct compression of drugs in the framwork of CORIP (Comity of Research and Chemistry Industries). He works actually on the field of essential oils.

Bruno Rollmann, PhD, Permanent scientistBruno Rollmann graduated in chemistry at the UCL in 1960 and obtained is PhD in 1965 at the same university. He worked at the School of Pharmacy from 1966 to 1997. His research concerned analy-tical problems as stability of antibiotic molecules, quantitative determination of drugs and metabolites in vivo, quantification of ultratraces of nitrosamines, toxic metals and monomeric molecules migrations in foodstuffs, chromatographic identification of natural substances.

Postdoctoral fellowsAnne-Sophie Crucq

Ph.D. studentsNicolas Barbarin AliciaCachon Coello CorineDjadjo Djipa Maria Gibella Thierry Pronce Pascale Vanbel

Technical and administrative staffPatricia Denis-Henry

Claudine Laruelle Jean-Yves PiŽrardMarie Antoinette RŽginsterRoger Reinquin

STAFF PROFILE p 61

Division of Pharmaceutical Chemistry and Radiopharmacy

Secretariat : phone 32-(0)2-7647340 fax 32-(0)-7647363, E-mail : [email protected] 7340, 73 Avenue Mounier, B-1200 Brussels, Belgium

Etienne Sonveaux, Ph.D., head, Professor.Etienne Sonveaux received his Ph.D. in organic chemistry at the UCL in 1975. He got a post-doctoral formation first at the University of Strasbourg in J.M. LehnÕs laboratory (Nobel Price in 1987), then at the University of California at San Diego (USA) in M. GoodmanÕs laboratory. Back in the University of Louvain, he became senior research associate at the Belgian National Fund for Scientific Research (FNRS) and created, with Professeur J. Fastrez, the ÒÊLaboratory of Physical Biochemistry and BiopolymersÊÓ at the Faculty of Sciences. He left the Belgian National Fund for Scientific Research for a position of professor of organic and pharmaceutical chemistry at the school of Pharmacy in 1991. His current interests are the oligo-nucleotide chemistry and the synthesis of prodrugs. He is member of the American Chemical Society and member of the board of the Medicinal chemistry section of the Belgian Royal Society of Chemistry.

Jacques H. Poupaert, Ph.D, Professor of Medicinal Chemistry.Jacques Poupaert graduated in chemistry from the UCL and obtained his Ph.D in organic chemistry at the same university in 1973. Right after, he joined the School of Pharmacy as teaching assistant of Prof. Dumont. He was post-doctoral fellow at the University of Minnesota, College of Pharmacy in 1975-1976 and he visited several times the laboratory of P.S. Portoghese in the eighties. In the summer 1989, he joined the Supercomputer Institute at the University of Minnesota for a training in molecular modeling. His research interests are devoted to CNS acting agents, mainly related to antiepileptics (phenytoin, ameltolide) and melatonin derivatives as well as the synthesis of new chemical templates for combinatorial chemistry. He is founding member of the ÒÊJournŽes franco-belges de PharmacochimieÊÓ with Profs. J. Delarge (Lige, Belgium), J-P. HŽnichart (Lille, France) and D. Lesieur (Lille), an annual meeting which just celebrated its tenth birthday. He is member of the Royal Belgian Society and of the French Society of Medicinal Chemistry. He also founded with some colleagues the Center for the study of the history of the Pharmacy and the Drug. He is an EEC expert for the Copernicus grants. He was recently member of the organizing committee of the XIVth International Symposium on Medicinal Chemistry in Maastricht. He is professor of pharmaceutical chemistry, molecular modeling and drug design at the UCL and takes part to the teaching staff for the Master in Drug Design at the University of Lille II, France.

D.M. Lambert, Ph.D. Associate professor. Didier Lambert graduated in pharmacy in 1989 from the UCL. In 1994, at the same university , he received his Ph.D. in Pharmaceutical Sciences. The topic of the thesis, under the direction of Profs. P. Dumont and J.H. Poupaert, was the delivery of glycine into the brain using chemical derivatization and conjugation to lipids. For this work, he received the Prize of the Fifth Section of the Royal Belgian Academy of Medicine of Belgium in 1994 . In 1994-1995, he joined Philip S. PortogheseÕs group at the College of Pharmacy, University of Minnesota, Minneapolis. Since October 1995, he is associate professor at the School of Pharmacy, Division of Medicinal Chemistry and Radiopharmacy. His main interests in research deal with molecular selectivity. Molecular selectivity can be achieved either by a selective distribution (chemical drug targetting, prodrugs) or by a selective interaction to the target. More recently, he focused on the molecular selectivity towards cannabinoid receptors. Didier Lambert is member of the American Chemical Society, the Belgian Royal Society of Chemistry and the Belgian Society of Pharmaceutical Sciences. He is member of the board of the Medicinal Chemistry section of the Royal Society of Chemistry and member of the organizing committee of the annual meeting ÒÊJournŽes Franco-Belges de PharmacochimieÊÓ. He is lecturer in Pharmaceutical Chemistry.

Jean Cumps, Ph.D. Senior scientist, Professor in Biostatistic.Jean Cumps is graduated in Chemistry in the UCL in 1967 and obtains in 1971 a Ph.D. in Physical Chemistry to the same university. Assistant in medicinal chemistry of School of Pharmacy since 1970, it has prospected different areas. In biochemistry, it has studicd the charac-terisation, the purification and the reconstitution of systems of cytochromes P-450. It has studied in collaboration with industry, the application of P-450 bioreactors. From 1980, he is interested in the statistical processing of the spectrophotometric signals and he develops the software SPECTRO-MATH). He studies the algorithms and the validations of non-linear regression software and develops new algorithms (CONSTAT software). Different applications have been developed in collaboration with industry to areas as various as the pharmacokinetic, the enzymatic kinetic, studies of dissolution, titrimetry, binding,.. Similarly, a service of consulting is created since 1980 with as goal the statistical processing of data in

the medical and the pharmaceutical area. From 1994, he creates on Intemet a server dedicated to the different applications of the pharmacy (http://vww.md.ucl.ac. be/pharmanet).A Intranet server PHARMA is put at the disposal students in pharmacy as didactic training support and pure information of computer-assisted and integrated practical works whose he is responsible.

STAFF PROFILE p 62

Bernard Gallez, Ph.D. Associate Professor. Bernard Gallez studied pharmacy with specialization in radiopharmacy. In 1993, he received his Ph.D. from the School of Pharmacy in UCL for his study on the synthesis and evaluation of hepatoselective nitroxides as contrast agents for magnetic resonance imaging (MRI). He was postdoctoral fellow at the EPR Research Center of Dartmouth Medical School for one year in 93-94. From 94 to 96, he was senior research assistant of the Belgian National Fund for Scientific Research. He got the Biennal Prize of the Belgian Society of Pharmaceutical Sciences in 1995. Through the years, his research deals with the rationnal behind the use of diagnostic agents by study of the pharmacokinetics, of the metabolic fate and the evolution of the biophysical properties during their biodistribution. This includes the development of radiopharmaceuticals and magnetopharmaceuticals as functional contrast agents covering three topics : oxygen-senstive paramagnetic compounds for in vivo EPR spectroscopy, hepatoselective MRI contrast agents, study of the influence of the metabolites on the PET receptor image. He is member of the Belgian Society of Pharmaceutical Sciences, the Belgian Society for the Study of Oxygen Metabolism, the European Society for Magnetic Resonance in Medicine and Biology, the International EPR society (treasurer for France and Belgium), the International Society for Free Radical Research, the International Society for Magnetic Resonance in Medicine. He is expert-consultant for the Nuclear Medicine Center of St-Luc Hospital and Mont-Godinne and for the company Hybritech (quality control of radiolabelled monoclonal antibodies). He is lecturer of Inorganic Pharma-ceutical Chemistry and he is lecturer of Radiophar-macy, Radiochemistry and Radiotoxicology in UCL.

Paul Depovere, Ph.D. Senior scientist.Paul Depovere received his Ph.D. in organic chemistry from the UCL, in the Laboratory of Steroid Chemistry under the direction of Prof. R.Devis (1968). He graduated as pharmacist in 1974 and bachelor in medical sciences in 1980. His main activities are devoted to universitary education. He is deeply involved in the teaching of general chemistry and organic chemistry to the students in pharmacy and medical sciences as well as the study of pharmacopoeia. Dr P. Depovere did several training sessions in America and he is author of several educational books (ÒÊVade-mecum de chimie gŽnŽraleÊÓ, Editions DeBoeck-UniversitŽ, ÒÊVade-mecum de chimie organique,ÊÓ Editions DeBoeck-UniversitŽ, ÒÊLa chimie exocharmiqueÊÓ, Editions DeBoeck-UniversitŽ) and of videotapes in collaboration with the Audio-visual Center, UCL (ÒÊAujourdÕhui PasteurÊÓ). President of the ÒÊMaison de la PharmacieÊ, he is member of the Belgian Society of Pharmaceutical Sciences and of the Society of Pharmacy (Bordeaux, France). He is also member of the Editorial Board of the ÒÊJournal de Pharmacie de BelgiqueÊÓ.

Postdoctoral fellowGa‘tane Hamoir

Ph.D. studentsCatherine BerensJacques BukuruHuajan FanAndrŽa GozzoMarcial Kanyonyo,Emmanuel LaruePhilippe LevequeLaurence NeuvensIsabelle PaternotteJean MarcTilquinHuseyn UcarKim Vanderpoorten

Technical staffJacques Adline Marc CollinIsabelle de ZurpeleYves Draye VŽronique Picard

STAFF PROFILE p 63

Division of cellular and molecular pharmacology Secretariat : Phone : 32-2-764 73 70, Fax : 32-2-764 73 73 ; E-mail : [email protected] 7370, 73 Avenue Mounier, B-1200 Brussels, Belgium

Paul M. Tulkens, MD, PhD, Head, Professor of Pharmacology Paul Tulkens graduated as MD in 1969 and as MSc (Biomedical Sciences) in 1970 at the UCL He obtained his ÒThse d'AgrŽgation de l'Enseigne-ment SupŽrieurÓ (PhD) at the same University in 1979. He undertook a scientific career with the support of the Belgian Foundation for Scientific Research from 1969 through 1989. He worked first at the Laboratory of Physiological Chemistry. His laboratory was incorporated into the International Institute of Cellular and Molecular Pathology in 1974, in which P.M. Tulkens created a research group focused on Cellular and Molecular Pharmacology. He was appointed Professor of Pharmacology in 1989, and in 1993 he moved his research group to the Department of Pharmaco-logical Sciences where he took the direction of his present unit. In parallel, he was appointed Professor of Human Biochemistry in 1979 and of Biochemical Pathology in 1984. His main research interest has been the study of endocytosis and of the interactions of drugs and chemicals with subcellular organelles. He has conducted frontier experimental research on the pathophysiology of lysosomes, antibiotic cell toxicity, and chemo-therapy of intracellular infection. In parallel, he also initiated first line clinical trials based on concepts originating from his experimental research. Thus, he provided evidence and was among the first to describe the process of membrane flow and recycling during endocytosis ('shuttle'); he descri-bed and characterized three new types of acquired lysosomal storage disorders; he deciphered the mechanism of the cellular toxicity of aminoglyco-sides and its prevention by polyanions; he designed and evaluated new antibiotics and asses-sed their cellular activity and toxicity; he proposed and evaluated a new schedule of administration of aminoglycosides ('once-a-day') which is now widely used in clinics. P.M. Tulkens is member of the Scientific Commission 'Pharmaceutical Sciences' of the Belgian Foundation for Medical Research, and accomplishes several expert functions at the national and international levels. He was on the Board of the Biochemical Journal and is now on the Advisory Board of Clinical Microbiology and Infection. He is President-elect of the International Society for Antiinfective Pharmacology.

Marie-Paule Mingeot-Leclercq, PhD, Senior Research Associate of the Belgian Foundation for Scientific Research (FNRS) M.P. Mingeot graduated in Chemistry (1979) and in Pharmacy (1982) at the UCL. In 1984 she joined the Group of Cellular and Molecular Pharmacology at the International Institute of Cellular and Molecular Pathology, where she made her PhD in Pharmaceutical Sciences (1988). She was then given a tenured position by the FNRS to pursue experimental research on the molecular aspects of drug-membrane interactions. Since 1992, she also teaches several courses in Pharmaceutical Sciences at the post-graduate level. Her research was mostly focused on the biochemical and the biophysical aspects of basic drugs with phospho-lipid layers in relation to cell toxicity and pharmacodynamics. She discovered the mecha-nisms of the cell alterations induced by amino-glycoside and macrolide antibiotics. She used this knowledge to design and evaluate new amino-glycosides, is extending now her research to other types of basic drugs. She also initiated biological research on the interactions of oligonucleotides with membranes in connection with cell delivery and toxicity.

Postdoctoral fellowsFranoise Van Bambeke

PhD studentsMohammed El MoueddenCŽcile GerbauxA.nneKerkhofsYoussef OuadrhiriIsabelle PaternotteDonatienne Tyteca

Technical and administrative staffMarie-Claire CambierYolande de Ryckel Olga Meert Francine Renoird

STAFF PROFILE p 64

Division of pharmaceutical technologySecretariat : Phone : 32-2-764 73 20, Fax : 764 73 98, E-mail : [email protected] 7320, 73 Avenue Mounier, B-1200 Brussels, Belgium

VŽronique PrŽat, PhD, Head, Professor of pharmaceutical technology, Senior Research Associate (FNRS)VŽronique PrŽat received her pharmacy degree at the UCL in 1981 and obtained her PhD in 1984 at the same University. She was a post-doctoral fellow at the departments of Biochemistry and pathology of the University of Toronto. Before 1989, her research focused on experimental liver carcinogenesis. She joined the pharmaceutical technology unit of UCL in 1989. She was appointed "Chercheur qualifiŽ" (Senior Research Associate) of FNRS and professor of pharma-ceutical technology. Her main interest is the development of new delivery systems for drugs and new biotechnology products. Her research focuses mainly on transdermal drug delivery and more specifically on electrically enhanced transdermal delivery She is also working on polymeric microparticles as drug and antigen delivery system. She is a member of several national and international societies of pharmaceutical sciences. She serves as a member of the Editorial Board of Pharmaceutical Research. She is author and co-author of more than 70 articles.

Michel Roland, PhD, Full Professor of Pharma-ceutical technologyMichel Roland is pharmacist (1956) and PhD in pharmaceutical sciences (1958). He was appointed as a professor at the UCL in 1968. He teaches galenic pharmacy, hospital pharmacy and pharmaceutical law. He is a member of the ÒAcadŽmie Royale de MŽdecineÓ, Vice-president of the ÒCommission des mŽdicamentsÓ and the ÒConseil SupŽrieur d'hygineÓ. He is the president of the ÒOrdre des pharmaciensÓ.

Jean Gillard, PhD, pharmaceutical applications. Jean GIllard graduated in Sciences (physical chemistry) at the ÒUniversitŽ catholique de LouvainÓ in 1969 and he specialized thereafter in pharmaceutical applications (1974). His PhD thesis was devoted to the flow properties of pharmaceutical powders and to the analysis of texture of tablets realised by direct compression. He has 26 years of experience in development and research on technological pharmacy with the collaboration of industrial companies : flow properties of powders, tablet making, film coating of particules, beads and tablets, gazeous sterilization, microencapsulation, pelletization. He has also a large practical experience in the formulation of biodegradable polymers for sustained release systems for pharmaceuticals applications and veterinary dosage forms. He is professor of industrial pharmacy operations for the qualification of master on pharmaceutical engineering and industrial pharmacy.

Postdoctoral fellowsMarie-Anne Beno”t

PhD studentsBeno”t BarrasNathalie DujardinAnne JadoulDominique LemoineVincent RegnierRita VanbeverBi Botti CŽlestin Youan

Technical and administrative staffClaudette GillisNathalie LecouturierJean-Pierre VandiestArmand Vanvlasselaer

STAFF PROFILE p 65

Division of pharmacokinetics and drug metabolismSecretariat : Phone : 32-2-764 73 55, Fax : 32-2-764 72 97, E-mail : [email protected] FATC 7355, 73 Avenue Mounier, B-1200 Brussels, Belgium

Roger Verbeeck, PhD, Head, Professor.Roger Verbeeck received his pharmacy degree at the ÒKatholieke Universiteit LeuvenÓ (KUL) in 1972 and obtained his PhD degree (Pharmacology) at the same university in 1978. He was postodoctoral fellow at the department of pharmacology of the Vanderbilt University, School of Medicine in Nashville (US) between 1978 and 1980. He then joined the School of pharmacy at the University of Saskatchewan (canada) where he stayed until his appointment at the School of pharmacy of the UCL in 1989. His research interests are in the areas of pharmacokinetics and drug metabolism with special interest in glucuronidation, disposition of drug glucuronides, and extrahepatic metabolism. He is on the editorial boards of Biopharmaceutics and Drug Disposition, European Journal of Pharma-ceutical Sciences and Pharmacy World & Science.

Georges Lhoest, PdD, permanent scientist.Georges Lhoest received his PhD degree (organic chemistry) in 1966 (ULB). In the industry he was at the head of the reserach laboratory of the trimetal paint company for three years were the research programs included surface chemistry (pigment flocculation, rheology, opacity). During his stay in the industry he got through a two years german language evening course at the ÒGerman SchoolÓ in Brussels where he obtrained his certificate. He is working at the UCL since 1971 and since 1971 till 1993 he was professor at the School of Criminology Minestry of Justice. His research interests include the metabolism of xenobiotics with special interest for the structure determination of drug metabolites of phase I and phase II for example of immuno-suppressive drugs using mass spectrometry and /or NMR spectroscopy and x-ray crystallography as an aid for structure assignment and structure-receptor binding domains localization. He is on the editorial board of Sepctroscopy : an International Journal and reviewer for the Journal of Mass Spectrometry.

Josianne Burton, PhD, permanent scientist. Josianne Burton studied pharmaceutical sciences at the UCL where she graduated as pharmacist in 1964, and as industrial pharmacist in 1965. She specialized in pharmacology and obtained her PhD in 1972 at the same university, based on studies on the action ofcardiotonic drugs on the heath. She has been particulary involved in studies concerning the mechanism of action of several drugs on the heart. More recently she has been involved in developing animal models for the evaluation of analgesic drugs. She is a member of the Belgian Society of Physio-logy and Pharmacology and of the Belgian Society of Pharmaceutical sciences.

PhD studentsFranoise BrunelleStephane EeckhoudtPierre EvrardChristine MeunierMarc NickmilderNicolas Van Brandt

Technical and administrative staffElise FerdinandNicole MatonMartine Petit

STAFF PROFILE p 66

Division of mutagenicity and teratogenicitySecretariat : Phone : 32-2-764 72 7237, Fax : 764 72 56, E-mail : lŽ[email protected] 7237, 72 Avenue Mounier, B-1200 Brussels, Belgium

Alain LŽonard, PhD, Head, Professor of Food Quality Control, Environmental Genetics and Cytogenetics.Alain LŽonard graduated in agriculture of the UCL in 1955, obtained his PhD in 1965 and the "Agregation de l'Enseignement SupŽrieur" in 1972 at the same university. His Ph.D. and Agregation thesis were devoted to the study of genetic effects of physical and chemical environmental pollutants on mammals. Current research interests involve the mechanisms of aneuploidy and of adaptive response to environ-mental stresses in mammals, the in vitro systems which could be used to replace laboratory animals in toxicological studies, the biological dosimetry after exposure to ionizing radiations and the study of the genotoxic potential of metal salts. Up to 1989 he was the head of the Radiation Protection Depar-tment of the Atomic Center of Belgium, from 1987 to 1993 consultant for the Biotechnology Program of the EU, temporary expert of WHO (World Health Organisation and IAEA) (International and Atomic Energy Agency) and, from 1995 consultant of INTAS. He was visiting professor at the University of Sao Paulo (Brazil) and Sherbrooke (Canada). He is referee to a number of international scientific journals and on the Editorial Board of Mutation Research.Conrad de Meester, PhD, Permanent scientist.Conrad de Meester studied Chemistry and General biology. He graduated at the UCL in 1965 and obtained his Ph.D. in 1975 at the same University. The subject of his thesis was the study of the mechanism of resistance to an antibiotic. He specialized thereafter in research on food muta-gens and is now involved as project, coordinator for a Standarts, Measurements and Testing program of the EU. He has acquired expertise in Food Science, Mutagenesis and Food Microbiology.

Postdoctoral fellowsClaudine Stecca, PhD

Technical and administrative staffAlphonse BramsMarie-Christine Crutzen-FaytEliane DenisPatricia Denis-HenryYves Draye

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UCL- Universit catholique de Louvain · Web viewThe ultimate objective is to develop flexible optimization strategies applicable in practical chromatographic separation problems