Rektor Sven Frøkjær Danmarks Farmaceutiske Universitet

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1 Rektor Sven Frøkjær Danmarks Farmaceutiske Universitet 30. Marts 2006 J.nr. Ref. Støtteerklæring vedrørende Spirekasse -projekt: Combining in vitro dissolution and absorption of low molecular compounds Janne Rømsing Institutleder Undertegnede støtter hermed ansøgningen vedrørende projekt: Combining in vitro dissolution and absorption of low molecular compounds. Projektet ligger indenfor DFU s prioriterede forskningsområder, og Instituttet råder over den ekspertise og de laboratoriefaciliteter, der vil være nødvendig for at sikre projektets gennemførsel. Universitetsparken København Ø Tel Fax Med venlig hilsen Direkte jr@dfuni.dk Janne Rømsing Institutleder Institut for Farmaci og Analytisk Kemi

2 Proposal for a Spirekasse project Project title: Combining in vitro dissolution and absorption of low molecular compounds Research area: Pharmaceutics Main applicant: Anette Müllertz Co-applicants: Jette Jacobsen Betty Lomstein Pedersen Dimitri Fatouros Detailed Project description: Aim: To develop an integrated in vitro model that can simulate both the dissolution and the absorption of low molecular drug compounds. Background: The pharmaceutical industry today is facing the discovery of an increasing number of low molecular drug candidates with very poor water solubilities and high log P values. Conventional formulations of these drug candidates often results in low and/or varying bioavailabilities, hence reducing the clinical relevance of a potential important drug. The industry lacks predicative in vitro methods to select the right drugs on a rational basis, especially in the very early stage of drug development. Many new lead structures are Class 2 substances according to the Biopharmaceutical Classification System, meaning that they have a high permeability and that dissolution and solubility in the gastro-intestinal (GI) tract is the rate-limiting step for their absorption. Dissolution in the GI tract will be dependent on physico-chemical properties of the drug substance (e.g. solubility and particle size) as well as physiological factors (e.g. composition, volume and hydrodynamics) of the GI fluids. Co-administration with food has been found to increase the bioavailability of many poorly soluble drugs, also due to the solubilisating effect of lipid hydrolysis products, produced during digestion.

3 According to the present knowledge, poorly soluble drugs are solubilized in a mixed bile salt micelle prior to absorption in the fasted state. When entering the unstirred water layer that lines the epithelium, the mixed micelle disintegrates and the poorly soluble drug diffuse to the epithelium and is absorbed. However, at present the exact mechanisms behind this transfer are not very well understood. When introducing the fed state, the processes in the GI tract get even more complex. Dietary lipids are hydrolysed by pancreatic lipase into surface active lipid digestion products (LDP; free fatty acids and monoglycerides) that will participate in the formation of complex colloid phased, including vesicles, micelles and liquid laminar phases (eg cubic or hexagonal). The impact of each of these phases on drug transfer and absorption still needs to be elucidated. The LowSol group, at the Department of Pharmaceutics and Analytical Chemistry, DFU, has developed a range of methods to study the dissolution and solubilization processes in the GI tract. Several biorelevant dissolution media, simulating the conditions of the GI tract, has been developed. These media contain bile salts and phospholipids with or without addition of LDP. Presently the usefulness of the media for predicting in vivo bioavailability is being evaluated, using both solubility determinations and dissolution assays. In addition the dynamic lipolysis model has been developed with the purposes of simulating the lipid digestion in the GI tract during the fed state, as well as digestion of lipid based formulations. Using this model it is possible to study the solubilization of poorly soluble drugs during lipid digestion. Most existing in vitro absorption studies of poorly soluble drugs uses DMSO to keep the drug in solution, however, this way the in vivo mechanisms are neglected. In order to achieve a better understanding of the mechanism by which the poorly soluble drugs are being absorbed, it would be valuable to establish a method that is as close to the in vivo situation as possible. Therefore the LowSol group is presently developing biorelevant media that are compatible with the Caco-2 cell culture model and animal intestinal tissue in Ussing chambers. The project: Initially a technical device that is able to accomplish the aim of combined dissolution /solubilization and absorption has to be developed. The hydrodynamic capability of the dissolution part of the device has to be adjusted as to simulate the condition in the GI tract. The device will be designed so that the dissolution /solubilization step can be simulated using either biorelevant media or the dynamic lipolysis model. Furthermore the absorption part of the device will depend on what model is chosen for the absorption step. This can either be Caco-2 cells, in situ or ex vivo perfusion of rat intestine, the

4 Ussing chamber or even artificial membranes, depending on what model is decided to be most suitable. Using biorelevant media in the model, the impact of the media composition on the dissolution of model drug from solid state formulations can be elucidated, as well as the impact of media and hydrodynamics on the absorption of the drug. By combining the model with dynamic lipolysis of a lipid based formulation, the solubilization of model drugs under different digestion relevant conditions can be studied, as well as the generation of colloid phases and the impact of these on the absorption of the drug. Similarly impact of fed state lipid digestion can be investigated. A model that encompasses both the solubilization of a drug from a given formulation as well as the intestinal absorption will make it possible to achieve a better understanding of the impact of the kinetics of the intraluminal events taking place prior to absorption of poorly soluble drugs. In addition the effect of dissolution / solubilization rate and media composition on relevant transport and efflux proteins, eg. fatty acid transporters, organic anion transporters, the bile salt transporter or Pgp, will be elucidated. Developing the model, the impact of the absorption barriers, e.g. the mucus layer and unstirred water layer, will also be studied. The model will make it possible to evaluate the impact of novel formulation principles and exhipients on the dissolution/solubilization and absorption of model drugs. Based on available in vivo data, from either the LowSol group, or industrial partners, the predictability of the combined dissolution / solubilization and absorption model will be evaluated, and the model modified with regard to eg. hydrodynamics, biorelevant media or lipolysis conditions as to optimise the correlation. Perspectives: In order to facilitate a strategic formulation development of poorly soluble drugs, resulting in a high and reproducible bioavailability, a better understanding of the processes that lead to absorption of these compounds is needed. The establishment of innovative predicative in vitro models that includes both the dissolution and the absorption steps and takes the physiological conditions in the GI tract into account, will make this possible. However, such a model has never been described before and the development will be associated with a high scientific risk level. A prerequisite for frontline research is the existence of the foundation and expertise necessary to carry out such research. In the case of the proposed project the Lowsol group already has expertise in the field of biorelevant dissolution and solubilization, and in addition has initiated the development of biorelevant media for transport studies in Caco-2 cells. However, the described project entails a combination of these methods and also includes the development of technical equipment that can fulfil the aim, thus a significant element of innovation and insecurity must be anticipated.

5 Til: Akademisk Råd Vedr.: Spirekasseprojekt Spirekasseprojekt Vi indsender hermed projektet med den engelske titel Development of electrochemical techniques to study and control the adsorption of biomacromolecular drugs som et spirrekasseprojekt. En detaljeret projektbeskrivelse, en kort tidsoversigt over projektets forløb, et tillægsbudget (ikke indeholdende løn og annum for den ph.d. studerende) samt en støtteerklæring fra instituttet er vedlagt ansøgningen. Projektet ligger indenfor det prioriterede forskningsområde Farmaci og er et samarbejde mellem medlemmer af Biomakromolekyler gruppen og Farmaceutisk og Fysisk Kemi gruppen, der begge er tilknyttet Institut for Farmaci og Analytisk Kemi. Projektgruppen består af Lektor Marco van de Weert, og adjunkterne Henrik Jensen, Lene Jørgensen, Jesper Østergaard og Anne Engelbrecht Thomsen. Marco van de Weert, PhD Associate Professor Universitetsparken 2 DK-2100 Copenhagen Phone Fax mvdw@dfuni.dk Projektgruppens medlemmer vil arbejde tæt sammen med den Ph.D. studerende under hele projektet. Derudover vil projektet være relevant for et igangværende DRA støttet projekt om protein adsorption til faste overflader samt et eksternt Ph.D. projekt ved Risø om modifikation af overflader med henblik på at reducere protein adsorption. Vi forudser at den viden nærværende projekt vil generere kan bruges til at etablere nye metoder til bestemmelse og kontrol af protein adsorption på en række overflader. Resultaterne vil ikke alene have stor relevans for udvilklingen af protein baserede lægemidler og delivery systemer, men kan også være til stor nytte for design af medicinsk udstyr og apparatur samt udviklingen af miniaturiserede analyse systemer hvor protein adsorption ofte forårsager store problemer. Vi ser frem til rådets evaluering af projektet Med venlig hilsen Marco van de Weert, PhD, Lektor (På vegne af projektgruppen) Vedlagt: Projektbeskrivelse, Budget og tidsoversigt, støtteerklæring fra instituttet

6 Project description Electrochemistry to study and control adsorption Development of electrochemical techniques to study and control the adsorption of biomacromolecular drugs Project group: Marco van de Weert, PhD, Associate Professor Henrik Jensen, PhD, Assistant Professor Lene Jørgensen, PhD, Assistant Professor Anne Engelbrecht Thomsen, PhD, Assistant Professor Jesper Østergaard, PhD, Assistant Professor Research area: Pharmaceutics The present project is highly interdisciplinary with group members having research experience in the field of biomacromolecules, analytical chemistry, pharmaceutical chemistry and physical chemistry. Introduction An increasing number of drugs are based on proteins and other biological macromolecules, which require different strategies in terms of development, production and analysis compared to more traditional small molecule drug compounds. 1 Some challenges include poor absorption through biomembranes and limited physicochemical stability. A common degradation pathway involves the adsorption to interfaces and surfaces. Adsorption not only causes loss of active compound, but may also result in formation of misfolded protein molecules. The latter species may be toxic or cause an immune response. During production, purification, formulation and delivery, proteins are exposed to a wide variety of solid surfaces and liquid interfaces to which they may adsorb. This adsorption is governed by both hydrophobic and electrostatic forces. Little is known about the relative importance of these two driving forces, even though protein adsorption is a problem reaching beyond the area of protein drug development. Project Description We propose the following project focused on developing and employing an innovative electrochemical technique to study the relative influence of hydrophobic and electrostatic forces in protein adsorption. Moreover, the technology may be used to control protein adsorption. Table I in the appendix shows the expected timeline of this project. The project builds on the fact that the interface between two immiscible electrolyte solutions can be polarised using a four electrode potentiostat, as shown in Figure 1 (page 4). 2 The four electrodes of the electrochemical cell are connected to a four electrode potentiostat, providing an accurate potential control between the two liquid phases (equivalent to controlling the difference in chemical potential between the two phases). The interface region may be modeled as a capacitor and resistance in series; the adsorption of charged molecules corresponds to charging a capacitor in an electronic circuit. 3 The technique can also be applied to solid-liquid interfaces, as long as the solid is (semi-)conducting, as is the case for metals, carbon or silica. Previously, the accumulation of TiO 2 particles at an oil-water interface has been studied by this technique. It was shown that the adsorption is a function of both particle charge and potential: negatively charged particles accumulate at negative potential differences, whereas positively charged molecules accumulate at positive potential differences. 4 However, the behaviour of proteins is expected to be more complex. They are not only charged, but hydrophobic forces can also be involved in the adsorption process. By studying the adsorption at different potentials, the interplay between charge and hydrophobicity in the adsorption process can be determined. Moreover, the surface charge can be controlled externally using the potentiostat, which allows studying a continuum of differently charged surfaces using the same sample. This low sample consumption is of importance, since new investigational proteins are often available only in small amounts. Proof-of-principle has been shown in a very limited number of papers. 5,6 Moreover, at DFU adsorption of insulin to an oil-water interface has been studied (Figure 2, page 5). Figure 2a shows the interfacial capacitance potential curves for accumulation of 0.5 μm insulin (3 μg/ml) at the interface. 6 The lowest interfacial capacitance potential is observed when the charge at the interface is equal to zero, the socalled potential of zero charge (PZC). In the absence of insulin the PZC is found at a potential difference of 0 V, as expected in the absence of any preferentially adsorbed charged species. In the presence of insulin MvdW/06/03/30 page 1 of 5

7 Project description Electrochemistry to study and control adsorption the PZC changes to ca V, which is indicative of adsorption of a charged species at the interface. Interestingly, as shown in Figure 2b, the technique can also be used to determine adsorption kinetics of very low concentrations of insulin, by following the interfacial capacitance potential as a function of time. It should be noted here that this technique is one of very few techniques capable of following protein adsorption kinetics at liquid-liquid interfaces. Since the same methodology can be used for liquid-solid interfaces, the results pertaining to different surfaces are easily compared. One of the advantages of this technique over other common techniques is that it does not require labeling of the adsorbing species, as is usually the case for fluorescence based methods. 7,8 In addition, the technique can be miniaturised. There are numerous examples in the literature where electrochemical methods are used for detection purposes in microfluidics systems, 9 but to our knowledge the technology has never been used to screen for adsorption of biomacromolecules. Combining a detection limit of less than 0.1 μm with nanoliter samples will thus lead to a sample requirement of less than one femtomole. The high surface to volume ratio may complicate the analysis, but solutions have been proposed in the literature. 10,11 In this project we propose to address the following three points: 1. Fundamental studies on the adsorption behaviour of biomacromolecules, specifically proteins, at liquid-liquid interfaces. Systematic studies on surface charge and water phase composition will be performed 2. Development of the technique to include solid surfaces, which are pharmaceutically relevant and/or used in chromatographic applications 3. Application of the technique to control the adsorption and desorption of biomacromolecules by externally controlling the potential at the interface 1) In the first series of experiments we will study the adsorption of well characterised model proteins to water-oil interfaces. These experiments will give an indication of the relative importance of electrostatic and hydrophobic forces to the adsorption process. The model proteins will be chosen based on two parameters: their isoelectric point (pi) and their tendency to unfold at interfaces. Proteins that easily unfold are commonly denoted as soft proteins, whereas proteins that do not unfold readily are known as hard proteins. 12 A typical example of the former is human serum albumin, whereas hen egg white lysozyme is a typical hard protein. The next step in these investigations is to alter the properties of the water phase. An obvious parameter to vary is the solution ph. At the low concentrations required for this technique, many proteins can be studied in a wide ph range, which allows modulation of the surface charge of the protein. Generally, variation of the ph has little effect on the hydrophobic properties of the protein. Thus, the relative importance of protein charge may be investigated. Another parameter of interest is the effect of various additives on the adsorption behaviour. These additives may, e.g., alter the adsorption behaviour by directly interacting with the protein or by blocking the adsorption sites on the interface. Alternatively, they may modulate the ability of the protein to alter its structure. The latter is often a prerequisite to expose hydrophobic sites on the protein. Possible additives to test are known specific ligands, surfactants, low molecular weight carbohydrates, and polymers like PEG, chitosan and dextran. These studies will be supplemented by capillary electrophoretic studies on binding of these additives to the protein in the aqueous phase. 13,14 2) A subsequent set of experiments will be aimed at modifying the technique to also include solid substrates. These substrates may be pharmaceutically relevant surfaces like metals, silica, and conducting substrates modified to mimic surfaces like for example teflon. Moreover, surfaces that are relevant for chromatographic applications will also be tested. The studies will be supplemented by comparison with techniques commonly used to study protein adsorption to solid substrates, such as total internal reflectance spectroscopy and ellipsometry. 15 Although an actual miniaturised system is beyond the scope of the present project, this could be a natural continuation of the project. Therefore, at least some of the tested substrates should also be applicable in a microanalytical system. Such substrates include conducting polymers, surface modified metals, or metal oxides. MvdW/06/03/30 page 2 of 5

8 Project description Electrochemistry to study and control adsorption 3) The final set of experiments will be used to evaluate the technique to selectively adsorb or desorb proteins from interfaces/surfaces. The potential applications of this selective ad-/desorption process are broad. First of all it may be used to prevent adsorption of those proteins that tend to unfold at interfaces, yielding undesired misfolded species. Moreover, the selective process can be used in purification and/or separation processes, in which the separation is based on selective adsorption followed by selective desorption. Finally, the process can be used in an electronically triggered implanted release system to release the active compound in the desired amounts at the desired time-points. Perspectives and relevance At present it is difficult to predict, but also measure, to what extend a given protein adsorbs at an interface. The present study provides a new technique to monitor protein adsorption, and also allows us to study the relative effect of electrostatic and hydrophobic forces. Based on this fundamental knowledge, we hope to identify new parameters which enable semi-quantitative predictions of protein surface affinities. Since many biomacromolecules are charged, the fundamental insight gained using proteins should also be applicable to other biomacromolecules. These include pharmaceutically relevant compounds such as DNA, RNA, and glucosaminoglycans (heparin and analogues). The results will be relevant for biomacromolecular drug production, and especially the formulation process. Adsorption is a common and undesired destabilisation mechanism for many biomacromolecular drugs. For example, emulsions are being considered as drug delivery systems for proteins, but protein adsorption and subsequent further physicochemical degradation is a major concern. 16,17 Finally, the technique presented here can be used to control the adsorption of biomacromolecules, by external modulation of the interface potential. This can be used for selective adsorption and desorption in formulations as well as in analytical techniques like chromatography and Matrix Assisted Laser Desorption-Ionisation (MALDI) or Desorption Electrospray Ionisation (DESI) mass spectrometry. In MALDI and DESI the selectivity and sensitivity depend on the ability of the protein to adsorb at a matrix interface. In chromatography the knowledge acquired in the proposed project can be used to design new chromatographic column materials with improved separation performance for biomacromolecules. References 1. Frokjaer, S.; Otzen, D. E. Protein drug stability: A formulation challenge. Nature Rev. Drug Discov. 2005, 4 (4), Reymond, F.; Gobry, V.; Bouchard, G.; Girault, H. H. Electrochemical Asspects of Drug Partitioning. In Pharmacokinetic Optimisation in Drug Research, Testa, B., Waterbeemd, H., Folkers, G., Guy, R., Eds.; Zurich, 2001; pp Fermin, D. J.; Jensen, H.; Girault, H. H. In Encyclopedia of Electrochemistry, Bard A.J., Stratmann M., Eds.; Wiley: 2003; pp Jensen, H.; Fermin, D. J.; Moser, J. E.; Girault, H. H. Organization and reactivity of nanoparticles at molecular interfaces. Part 1. Photoelectrochemical responses involving TiO2 nanoparticles assembled at polarizable water 1,2-dichloroethane junctions. J. Phys. Chem. B 2002, 106 (42), Georganopoulou, D. G.; Williams, D. E.; Pereira, C. M.; Silva, F.; Su, T. J.; Lu, J. R. Adsorption of glucose oxidase at organic-aqueous and air-aqueous interfaces. Langmuir 2003, 19 (12), Thomsen, A. E.; Jorgensen, L.; Jensen, H.; Ostergaard, J. Adsorption of proteins to an oil-water interface - an electrochemical approach. AAPS, Mollmann, S. H.; Bukrinsky, J. T.; Frokjaer, S.; Elofsson, U. Adsorption of human insulin and Asp(B28) insulin on a PTFE-like surface. J. Colloid Interface Sci. 2005, 286 (1), Mollmann, S. H.; Jorgensen, L.; Bukrinsky, J. T.; Elofsson, U.; Norde, W.; Frokjaer, S. Interfacial adsorption of insulin - Conformational changes and reversibility of adsorption. Eur. J. Pharm. Sci. 2006, 27 (2-3), Rossier, J. S.; Roberts, M. A.; Ferrigno, R.; Girault, H. H. Electrochemical detection in polymer microchannels. Anal. Chem. 1999, 71 (19), Lionello, A.; Josserand, J.; Jensen, H.; Girault, H. H. Dynamic protein adsorption in microchannels by "stop-flow" and continuous flow. Lab on A Chip 2005, 5 (10), MvdW/06/03/30 page 3 of 5

9 Project description Electrochemistry to study and control adsorption 11. Lionello, A.; Josserand, J.; Jensen, H.; Girault, H. H. Protein adsorption in static microsystems: effect of the surface to volume ratio. Lab on A Chip 2005, 5 (3), Norde, W. The behaviour of proteins at interfaces, with special attention to the role of the structure of the protein molecule. Clin. Mater. 1993, 11, Ostergaard, J.; Heegaard, N. H. H. Capillary electrophoresis frontal analysis: Principles and applications for the study of drug-plasma protein binding. Electrophoresis 2003, 24 (17), Ostergaard, J.; Schou, C.; Larsen, C.; Heegaard, N. H. H. Evalution of capillary electrophoresisfrontal analysis for the study of low molecular weight drug-human serum albumin interactions. Electrophoresis 2002, 23 (17), Mollmann, S. H.; Elofsson, U.; Bukrinsky, J. T.; Frokjaer, S. Displacement of adsorbed insulin by Tween 80 monitored using total internal reflection fluorescence and ellipsometry. Pharm. Res. 2005, 22 (11), Jorgensen, L.; van de Weert, M.; Vermehren, C.; Bjerregaard, S.; Frokjaer, S. Probing structural changes of proteins incorporated into water-in-oil emulsions. J. Pharm. Sci. 2004, 93 (7), Bjerregaard, S.; Wulf-Andersen, L.; Stephens, R. W.; Lund, L. R.; Vermehren, C.; Soderberg, I.; Frokjaer, S. Sustained elevated plasma aprotinin concentration in mice following intraperitoneal injections of w/o emulsions incorporating aprotinin. J. Controlled Rel. 2001, 71 (1), E Silver reference electrodes Platinum counter electrodes Water I Oil Ag AgCl Buffer BTTPATPBCl (water) (1,2-DCE) BTTPACl (water) AgCl Ag Figure 1. Diagram of the experimental setup. The two platinum electrodes and two silver electrodes are connected to a four electrode potentiostat. MvdW/06/03/30 page 4 of 5

10 Project description Electrochemistry to study and control adsorption Interfacial capacitance (μf/cm 2 ) Blank cell 15 min 30 min 60 min Potential (V) Figure 2a. Interfacial capacitance potential curves in the presence of insulin at ph 7.4. Open circles are for the blank cell prior to insulin addition. Closed triangles and squares are signals obtained 15, 30 and 60 min after insulin addition (0.5 μm) to the aqueous phase (data not published). Change in interfacial capacitance (μf/cm 2 ) Blank 0.1 μm 0.25 μm 0.5 μm Time (sec) Figure 2b. Change in interfacial capacitance with time at a fixed potential (-100 mv from the potential of zero charge). Data shown are for the blank cell and for 0.1, 0.25 and 0.5 μm insulin added to the aqueous phase ph 7.4 (data not published). MvdW/06/03/30 page 5 of 5

11 Timetable and budget Electrochemistry to study and control adsorption Timetable and Additional budget Time table and VIP resources allocated to the project (in percentage of maximum research time of full position for one year) 1/ / / / / / First model experiments. Optimisation of electrolyte systems. Systematic studies on the adsorption behaviour of the selected model proteins. Binding studies of the selected biomolecules using capillary electrophoresis. Comparisons with other techniques. Studies on controlled adsorption/desorption of model proteins on novel biocompatible substrates. (80% VIP, 100% Ph.D.) (80% VIP, 100% Ph.D.) (80% VIP, 100% Ph.D.) Budget for additional equipment and consumables The Department of Pharmaceutics and Analytical Chemistry owns most of the required equipment to perform this project, and most of the required consumables can be bought within the regular budget of a PhD project. However, some of the essential (small) equipment and consumables are relatively expensive, and we would therefore like to request a small additional budget to buy these equipment/consumables. Below, the requested budget is shown. Custom-made glassware equipment (cells) including electrodes: Model proteins and peptides of high purity: Oil-phase electrolyte salt: Total: Dkr Dkr 5000 Dkr Dkr MvdW/06/03/30 page 1 of 1

12 Rektor Sven Frøkjær Danmarks Farmaceutiske Universitet 17. Marts 2006 J.nr. Ref. Støtteerklæring vedrørende Spirekasse -projekt: Development of electrochemical techniques to study and control the adsorption of biomacromolecular drugs Undertegnede støtter hermed ansøgningen vedrørende projekt: Development of electrochemical techniques to study and control the adsorption of biomacromolecular drugs. Projektet ligger indenfor DFU s prioriterede forskningsområder, og Instituttet råder over den ekspertise og de laboratoriefaciliteter, der vil være nødvendig for at sikre projektets gennemførsel. Med venlig hilsen Janne Rømsing Institutleder Universitetsparken København Ø Tel Fax Direkte jr@dfuni.dk Janne Rømsing Institutleder Institut for Farmaci og Analytisk Kemi

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15 Spirekasseprojekt : Combination of metallomics and metabonomics for early diagnosis of widespread diseases. Research area: Chemistry, bioanalytical chemistry Participants: Internal: Claus Cornett, Bente Gammelgaard, Steen Honoré Hansen, Christian Skonberg, Stefan Stürup, Dan Stærk External: Bente Daneskiold-Samsøe og Henning Bliddal (Parker Instituttet, Frederiksberg Hospital) Lars Nørgaard (KVL) Project description Widespread common diseases such as arthritis, heart disorders and cancer are characterzed by not being singly well-defined diseases based on a well described underlying biochemical disorder. Most often the diseases are classified in different groups based on different symptom patterns and patients from the different groups are treated differently. These classifications are most often based on classical clinical evaluations and chemical analysis of a few - or many - more or less well-defined bio-markers. It is possible that using a larger number of chemical data in combination with the clinical data in the diagnostics and monitoring can lead to new knowledge on the underlying biochemistry involved in the development of the diseases. This project is based on the hypothesis that application of a large array of data from various NMR- and MS-based analytical techniques combined with the original classical clinical observations and subsequent multivariate data analysis of the combined measurements, may reveal new information on how patients suffering from widespread common diseases diverge from the healthy population and how these groups of patients fall into different sub-groups, which then can be related chemically to the specific disease. This may lead to a better understanding of the diseases and will possibly open for earlier diagnosis, improved treatment or even prevention of some diseases. The array of data will consist of data from classical clinical clinical observations, from metabonomics analyses based on NMR and ESI-MS, and from the measurement of a large number of trace elements using ICP-MS. The analyses will be performed on biological samples which can be sampled with non-invasive procedures e.g. urine and plasma. To our knowledge this approach is unique as this is the first attempt to apply 1

16 multivariate data-anlysis based on the combination of these analytical techniques in the diagnostics and monitoring of patients with widespread common diseases. Metabonomics A metabonome is the ensemble of small molecules contained in an organism - and thus complementary to the proteome. Thus, the human metabonome are the small molecules that characterize humans. The balance between theses molecules changes during diseases, food intake, sleep, fast etc. in a way characteristic for the state. The research area defined as metabonomics is the quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification [1]. These changes are characterized in a metabonomics study that is a relatively simple and inexpensive way to study disease and toxic insults. The analytical methods traditionally involved in metabonomics are Nuclear Magnetic Resonance Spectrometry (NMR) and Mass Spectrometry (MS) combined with multivariate data analysis. Combination of these techniques is a powerful tool for identifying biomarkers and variations in combinations of these and is ideal for developing even faster screening methods. A further advantage of metabonomics based studies is the possibility of monitoring patients, cell cultures or test animals over prolonged time spans, as the methods usually employed are based on urine and/or blood samples (typically plasma or serum) which are commonly sampled from test subjects without undue interference. Metabonomics can thus easily be included in existing designs. As the methods work with just about any kind of fluid obtained from an organism (and can be extended to biopsies), metabonomics is an invaluable tool in most subjects pertaining to medicines and health. It is therefore vital that more emphasis is placed on developing a thriving metabonomics group at the Danish university of Pharmaceutical Sciences. The combination of NMR and MS spectrometry is already proving to be a powerful tool being used by the group. Experience with the methods and the underlying chemometric methods - are now so far progressed, that addition of a third powerful and complementary method - ICP-MS - is timely. The combined information gained from fusing traditional metabonomics with metallomics is expected to very powerful and so far unique worldwide [1-4]. Metallomics It is a well known fact that a wide range of elements are involved in many functions of the human body as they are incorporated in enzymes and exert their effects in a variety 2

17 of metabolic processes. The term metallomics has been proposed as a new scientific field in order to integrate the research fields related to biometals.the identification of metallomes - comprising metalloproteins, metalloenzymes and other metal-containing biomolecules - and elucidation of their physiological functions in biological systems is the main research target of metallomics [5,6]. An increasing number of elements have been suggested to be essential to life processes, but the processes involving the elements are far from completely elucidated. One reason for this is that the analytical chemistry of the biological samples as urine, plasma and tissue is complicated owing to the complex and often varying matrix of the samples. Apart from the elements that are introduced into the body via food, humans are exposed to an increasing number of less naturally abundant elements that have been introduced in technological processes and products. Some of these may have negative effects on health. The concentrations of these elements in human plasma are divided into groups comprising the major elements ranging from more than 100 mg/l (Ca, Na, K) to minor elements as Cu, Fe and Zn (1-10 mg/l) and trace elements with concentrations from 100 µg/l to well below 1 µg/l. The analysis of the elements have previously been done one element at the time and been very time-consuming. Hence, most investigations on the influence of elements on health and disease has concerned how one or a few elements could be involved in a specific disease. With the development of the very sensitive Inductively Coupled Plasma Mass Spectrometry (ICP-MS) technique, the analysis of the elements in very low concentration has been made possible. Furthermore, this technique has rendered the simultaneous multi-element analysis of biological samples posssible. By this technique, it would be possible to obtain the element-fingerprint of an individual at a given time instead of the concentration of a few pre-defined elements. This opens the possibility of uncovering new connections between the function of the elements. However, the application of the technique has been limited by interference problems - especially in complex matrices such as biological samples and new means for solving these problems instrumentally and chemically have been discovered during recent years. The analytical chemical research has been occupied with the possibilities and limitations of the technique [7,8]. The application of multivariate data analysis on multielement anlytical data has until now only been performed on samples of soil, sewer slime, plant material, ceramics, wines and ecstasy tablets - all investigations with the purpose of classifying the samples on the basis of their origin. Thus, to our knowledge, the application of multivariate dataanalysis on samples of human origin has not been reported. 3

18 Research plans The research falls into two inter-related parts - production and validation of the different types of data - and multivariate data anlysis on the combined data. Large sample populations from patients as well as healthy individuals are needed for construction of the models based on multivariate data anlysis. These samples will be obtained from external co-operating partners. The production and validation of data concerns development of methods. elucidation of how data quality influences a model - e.g. is very high quality interference-free data needed - or can we get the same information from less interference-free data if data collection circumstances are equal? The modelling of the data sets will be performed in co-operation with our external partners at KVL. If the developed models show new correlations between the measured parameters, the underlying biochemical phenomena will be explored. This will involve advanced hyphented techniques as LC-ICP-MC, LC-NMR and LC-MS for sepqaration and identification of the relevant groups of compounds. Research experience of the group The group at DFU is performing state-of-the-art research in the field of chemical analytical methods for the analysis of biological samples and has extensive practical and theoretical experience with NMR, MS, ICP-MS, and coupling (hyphenation) of these techniques with chromatography and capillary electrophoresis. Furthermore, the group has practical experience with NMR and MS based metabonomics and has the required experience with the multivariate data analysis methods (chemometrics) needed to ensure a successful outcome. Several NMR and MS based projects are currently underway and at least three publications are expected to be finished in the near future. These projects are carried out in a collaboration between The Department of Pharmaceutics and Analytical Chemistry and The Department of Medicinal Chemistry together with external partners, among others Nicholsons Group at Imperial College in London, Frederiksberg Hospital, Hvidovre Hospital and The Danish Institute of Veterinary and Food research. The group itself has adequate skills in chemometrics computer programming (and has even designed an elective course for pharmacy students), which are further developed in fruitful collaboration with Rasmus Bro and Lars Nørgaard at KVL and Nicholsons group at Imperial college in London. The group also delivers expertise to the brand new Process Analytical Technology Master, centered at KVL and developed in a collaboration between KVL, DTU, and DFU. 4

19 References. References documenting the NMR and hyphenation expertise can be found in the publication lists of Dan Stærk and Claus Cornett. References documenting the ICP-MS and hyphenation expertise can be found in the publication lists of Bente Gammelgaard and Stefan Stürup. References documenting the MS and hyphenation expertise can be found in the publiction list of Steen Honoré Hansen. (The lists are not included in this paper as they comprise several pages, but they are available on DFUs homepage) 1. Nicholson, J. K., J. C. Lindon, and E. Holmes. (1999). "Metabonomics": understanding the metabolic responses of living systems to pathophysiological stimuli via multivariate statistical analysis of biological NMR spectroscopic data. Xenobiotica. 29, Brindle, Joanne T., Henrik Antti, Elaine Holmes, George Tranter, Jeremy K. Nicholson, Hugh W. L. Bethell, Sarah Clarke, Peter M. Schofield, Elaine McKilligin, David E. Mosedale, and David J. Grainger. (2002). Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nature Medicine (New York, NY, United States). 8, Keun, H.C. (2006) Metabonomic modeling of drug toxicity. Pharmacology and therapeutics. 109, Af Michael Lauridsen, Jerzy W. Jaroszewski, Steen Honoré Hansen og Claus Cornett. (2005). Metabonomet viser organismens tilstand. Lægemiddelforskning, Jakubowski, N, Lobinski, R, Moens, L. Metallobiomolecules. The basis of life, the challenge of atomic spectroscopy. J Anal at Spectrom 2004, 19, Haraguchi, H. Metallomics as integrated biometal science. J Anal At Spectrom 2004, 19, Goulle, JP, Mahieeu, L, Casternant, J, Neveu, N,Bonneau L, Laine, G, Bouige D, Lacroix, C: Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair. Reference values. Forensic Sci Int 2005, 153, De Boer, JL, Ritsema, R, Piso S, Van Staden H, Van Den Beld, W. Practical and quality-control aspects of multielement analysis with quadropole ICP-MS with special attention to urine and whole blood. Anal Bioanal Chem 2004, 379,

20 Til Akademisk Råd Hermed fremsendes forslag til Centrale Forskningsområder og Spirekasseprojekter fra Institut for Medicinalkemi. Der fremsendes 3 forslag til Centrale Forskningsområder: Natural products research Targets and ligand-target interactions in structure-based drug research Medicinal chemistry De beskrevne centrale forskningsområder tager udgangspunkt i de på instituttet tidligere definerede Fokusområder og Kernefeltet beskrevet efteråret Indkaldelsen af centrale forskningsområder ligger op til opdeling i mindre områder end de nævnte Fokusområder og Kernefeltet, og går derfor i nogen grad imod instituttets langsigtede faglige strategi. Denne strategi indebærer bl.a. opbygning af brede tværgående samarbejder på instituttet, fremfor opsplitning i mindre områder. Det er derfor vigtigt at bemærke, at en del forskningsprojekter går på tværs af de tre Centrale forskningsområder. Antallet af citationer er baseret på publikationslister fremsendt i forbindelse med årsberetninger, og antal citationer er opgjort på biblioteket under anvendelse af Science Citation Index. Opgørelse af eksterne midler indenfor de respektive områder er baseret på udskrifter på ØF med de indgåede beløb på projektkonti knyttet til eksterne bevillinger. Det er således ikke de fulde bevilgede beløb, men derimod det indgåede beløb det pågældende år. De indgåede beløb er kun talt med indenfor hovedansøgerens centrale forskningsområde uanset om der har været medansøgere fra andre områder. Vi har ved udarbejdelsen af disse forslag savnet en præcisering af hvilke data som skulle medsendes og hvorledes disse data skulle beregnes. Det vil være afgørende for at kunne gennemføre en objektiv behandling af de fremsendte forslag, at beskrivelser og data er opgjort og rapporteret på samme vis. Spirekasseprojekter Nedenstående to projekter er blevet udvalgt efter behandling i instituttets forskningsudvalg. De to forslag blev valgt ud fra flere indkomne forslag på baggrund af instituttets langsigtede faglige strategi. Begge projekter er nyskabende og indeholder et element af faglig risikovillighed, men med tiltro til at begge projekter kan føre til frontlinieforskning. Projekterne er anført i prioriteret rækkefølge, og med i prioriteringen er indgået overvejelser omkring, hvor sikkert et grundlag der er for gennemførelse af projekterne: 1. Membrane-bound drug transporters - A new target for structure-based drug research 2. Ligandkonjugerede antistoffer som selektive receptor-ligander Jeg beklager den forsinkede fremsendelse, som skyldes ulyksalige omstændigheder omkring instituttets sekretariat samt at afleveringsfristen var sammenfaldende med frist for ansøgninger til forskningsstyrelsen om forlængelse af Forskerskolen DRA. Piv Ulf

21 Forslag til spirekasseprojekt Membrane-bound drug transporters A new target for structure-based drug research Vision In this project we intend to implement experimental methods for preparation, purification, crystallization and structure determination of membrane-bound drug transporters as well as the necessary computational methods for combined sequence and structural analysis and modelling of drug transporters. Background The use of three-dimensional structures has revolutionized the rational drug design and discovery process in the last years. The process of optimizing a drug candidate has moved from an indirect mapping of the possible interactions between the ligand and the target to an interactive process involving determination of the three-dimensional structure of the target, synthesis of new compounds, and the development of models for the ligand-target interactions and thereby linking structure and activity. Presently, this approach is routinely and successfully applied to targets where three-dimensional structures are readily available, e.g. many enzymes. One of the drug targets, which are generally believed to become extremely important in the near future, is drug transporters. Drug transporters is involved in many physiologically important processes being responsible for transport of a variety of compounds across membranes. Examples of important drug transporters include neurotransmitter transporters like the serotonin transporter being the target for antidepressive drugs like cipramil, peptide transporters being responsible for transport of antibacterials like penicillins across membrane, and P-glycoproteins involved in the excretion and thereby influencing the bioavailability of many drug molecules. Unfortunately, the structural information on drug transporters is still very limited. Presently, the Protein Data Bank contains more than experimentally determined structures of primarily proteins, but only 188 are membrane proteins representing only 99 unique structures with as few as 8 being from eukaryotes. Thus, there is an immense need for detailed structural information on drug transporters in order to apply structure-based methods to this important class of drug target. The project Biophysical studies of membrane proteins, and in particular structure determination, require a continuous supply of highly pure and stabile protein in the milligram range. Due to the inefficiency of the available recombinant expression systems with regards to the over-expression of original human drug transporters, targeting bacterial homologues thereof has become a valuable indirect route to knowledge structure-activity. This approach requires a very careful analysis of the available bacterial genomes in order to identify actual homologues and the process is therefore vital for proper initiation of our project. Several bacterial genomes are

22 available and there are a few easy-to-use online databases that serve the purpose of finding homologues transporters throughout the genomes. Expert sequence analysis will be performed in house primarily by assistant professor Olivier Taboureau (DTU/DFU). Once the targets have been identified, we aim to sub-clone these genes into a non-commercial bacterial expression vector, which has been optimized for transporter expression and is being used with great success in many different laboratories. We have, via assistant professor Osman Mirza (DFU), just initialized a collaboration on the expression and purification of peptide transporters with professor Peter Henderson (University of Leeds, UK), who is a worldrenowned expert in this field, and have together already sub-cloned four different bacterial peptide transporters that are currently being tested for expression levels at DFU. Following this, we aim to do a large-scale expression using in house fermentors, and subsequent purification and initial characterization (mainly size-exclusion chromatography and CDspectroscopy). A pure mono-disperse sample of protein is an excellent starting point for crystallization trials, which are based purely on the trial-and-error principle and the outcome is therefore unpredictable. To increase the chances of crystallization of the type of transporter that we target, we aim to screen a wide range of bacterial homologues (>10). All of these will be sub-cloned, expressed and purified and crystallized following well-defined procedures. The establishment of a transporter-delivering pipeline, concerning technical and theoretical expertise, at DFU is therefore of major importance. The implementation of these techniques will be done by Osman Mirza. Such pipelines have been implemented in many structural genomics consortia, among these the E-MeP (European Membrane protein Consortium) of which Osman Mirza is being considered as an associate member. This membership will allow for insight to the latest technical development in the field of membrane protein structure determination. The transport cycle of membrane transporters involve a conformational state that opens the ligand-binding site towards the extra cellular space and thereafter towards the cytoplasm. This indicates an inherent structural flexibility, which can be a major drawback with respect to crystallization. As a solution to this possible problem we aim to identify locking inhibitors through molecular modelling based on the structure of the biological ligand, followed by synthesis and experimental assessment of its binding properties using isothermal calorimetry (ITC) or binding studies in Xenopus oocytes. Synthesis will be performed in collaboration with in house chemists, modelling studies as well as ITC measurements will be done in house primarily by assistant professor Lars Olsen (DFU), while the oocyte studies will be done in collaboration with assistant professor Morten Nørholm (Royal Agricultural and Veterinary University). Once they have been identified, the inhibitors will be included as an additive in crystallization experiments. As illustrated above the experimental studies will take place in close synergy with computational studies on the drug transporters. The computational studies will include the necessary bioinformatics genome analyses as well as a combination of traditional sequence analysis with more sophisticated quantitative models of the drug transporters. Presently, computational methods for identification of residues responsible for selectivity between transporters within the SLC6 family (neurotransmitter transporters) are developed. Proof of concept requires that techniques securing the preparation of the mutants are available. The computational studies will also benefit from the experimental structure determinations at an early stage, prior to publication, and thereby provide the research groups at DFU with a

23 competitive advantage. This project will be coordinated with other activities at DFU focusing on drug transporters (associate professor Bente Steffansen group at Department of Pharmacy and Analytical Chemistry on hpept transporters and professor Hans Bräuner-Osborne group at Department of Medicinal Chemistry on GABA transporters) Perspectives The present project is a very ambitious project, because the practical problems associated with preparation of large amounts of pure protein, drug transporter, in a functionally relevant conformation is an extremely difficult task. On the other hand, the information to be gained is equally impressive. Three-dimensional structures of transporters relevant for physiologically important targets will provide DFU with a unique advantage and contribute to DFU as having an internationally outstanding profile. In order to obtain success within such a competitive research area it is absolutely necessary to have more than one person (Osman Mirza) working full-time on the experimental part, for example by allocating a Ph.D. student. The development of drugs regulating transporters is part of the future in drug research and we need to be active within this research area. People The project will primarily be carried out by a number of young scientists in the Biostructural Research Group: Osman Mirza, Ph.D., assistant professor at Department of Medicinal Chemistry, DFU, has recently spend two years as post-doctoral fellow in Professor So Iwata s group at Imperial College London working on structure determination of membrane-bound transporters. Osman Mirza has practical experience with all the necessary steps in process of structure determination of transporters. Lars Olsen, Ph.D., post-doctoral fellow funded by the Carlsberg Foundation on a project combining structural and energetic aspects of drug recognition. With a background in both physics (M.Sc. from University of Copenhagen), chemistry (Ph.D. from Royal Agricultural and Veterinary University) and medicinal chemistry (Post.Doc. in one year at DFU funded by Medicon Valley Academy) Lars Olsen has a very broad experience in computer-based structureactivity analyses. Olivier Taboureau, post-doctoral fellow funded by the program committee NABIIT via the joint DTU and DFU project on Chemoinformatics. Olivier Taboureau has previously been employed as post-doctoral fellow at Novozymes (two years) and Novo Nordisk (one year) working on computational aspects of biologically active compounds. In addition to these young scientists other members of the Biostructural Research Group will provide additional scientific expertise. The Biostructural Research Group has extensive experience in both the experimental and computational methods to be used in this project. Publications Below a number of publications from by the young scientists and/or relevant for the project have been listed:

24 The ABC transporter protein OppA provides protection against experimental Yersinia pestisinfection. Tanabe M, Atkins HS, Harland DN, Elvin SJ, Stagg AJ, Mirza O, Titball RW, Byrne B, Brown KA., Infection and Immunity, in press. Structural evidence for induced fit and a mechanism for sugar/h + symport in the LacY. Mirza O, Guan L, Verner G, Kaback HR, Iwata S., EMBO J., 2006;25: Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus. Mygind PH, Fischer RL, Schnorr KM, Hansen MT, Sonksen CP, Ludvigsen S, Raventos D, Buskov S, Christensen B, De Maria L, Taboureau O, Yaver D, Elvig-Jorgensen SG, Sorensen MV, Christensen BE, Kjaerulff S, Frimodt-Moller N, Lehrer RI, Zasloff M, Kristensen HH., Nature. 2005;437: Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides. Raventos D, Taboureau O, Mygind PH, Nielsen JD, Sonksen CP, Kristensen HH., Comb Chem High Throughput Screen. 2005;8: Development of a QSAR Model for Binding of Tripeptides and Tripeptidomimetics to the Human Intestinal Dipeptide/Tripeptide Transporter hpept1. Andersen R, Jørgensen FS, Olsen L, Våbenø J, Thorn K, Nielsen CU, Steffansen B., Pharm Res. 2006;22: Conformational restrictions in ligand binding to the human intestinal di-/tripeptide transporter: implications for design of hpept1 targeted prodrugs. Våbenø J, Nielsen CU, Steffansen B, Lejon T, Sylte I, Jørgensen FS, Luthman K., Bioorg Med Chem. 2005;13: Docking and scoring of metallo-beta-lactamases inhibitors. Olsen L, Pettersson I, Hemmingsen L, Adolph HW, Jørgensen FS., J Comput Aided Mol Des. 2004;18: New leads of metallo-beta-lactamase inhibitors from structure-based pharmacophore design. Olsen L, Jost S, Adolph HW, Pettersson I, Hemmingsen L, Jørgensen FS., Bioorg Med Chem. 2006;14: Flemming Steen Jørgensen 27. marts 2006.

25 Ligandkonjugerede antistoffer som selektive receptor-ligander. Forslag til spirekasseprojekt indenfor forskningsområderne Farmakologi og Kemi: Overordnet projektbeskrivelse. Projektets overordnede mål er at fremstille selektive receptorligander ved at hæfte en eller flere småmolekyle -ligander kovalent til et antistof (Figur 1). På denne måde håber vi at kunne adskille receptorligandernes aktivitet/funktionalitet fra deres affinitet og specificitet. Princippet bygger på, at affiniteten og selektiviteten opnås pga. antistoffet (eller et antistoffragment (F ab )), mens aktiviteten opnås ved at biokonjugere agonister eller antagonister med en linker til antistoffet eller F ab. Derved opnås en ny type makrokolekylær ligand med antistoffets høje affinitet og selektivitet og med småmolekyle -ligandens evne til at aktivere eller blokere signalet gennem en given receptor. I første omgang fokuseres på udvikling af subtypeselektive ligander, som kan differentiere mellem to forskellige men tæt beslægtede receptorsubtyper (figur 1). Ofte er beslægtede receptorsubtyper karakteriseret ved, at bindingslommen for den endogene ligand (neurotransmitter) er yderst konserveret, dvs. at de aminosyrer i receptoren, der varetager neurotransmitterbindingen, er identiske eller meget ens i de forskellige subtyper. Dette forklarer, at det ofte er svært at udvikle subtypeselektive ligander baseret på strukturen af den endogene receptorligand. Imidlertid eksisterer der langt højere grad af diversitet uden for bindingslommen, hvorfor ligander, der binder udenfor ligandbindingslommen potentielt vil kunne påvirke signalet gennem visse receptorsubtyper og være inaktive på andre. Rationel design af den slags ligander er dog ikke lige til. I dette projekt vil vi derfor forsøge at fusionere en ligand rettet mod receptor-bindingslommen med et antistof der binder med høj affinitet til en udenfor-liggende del af receptoren uden at påvirke dennes signalering. Formålet er, at den højere bindingsaffinitet af antistoffet kombineret med små-molekyle - ligandens aktivitet på receptoren vil kunne forøge aktiviteten/funktionaliteten af liganden så betydeligt, at der vil kunne opnås en betydelig selektivitet for en receptorsubtype over andre. Selvom der i dette projekt fokuseres på udviklingen af subtypeselektive receptorligander er fremtidsperspektiverne langt større, idet metoden kan anvendes på et hvilket som helst protein som man ønsker at blokere eller aktivere med en ligand. Envidere kan metoden anvendes til at opnå en ny form for selektivitet. Man kan således rette selektiviteten mod andre omgivende proteiner, f.eks. aktivering af glutamatreceptorer (GluR), som befinder sig i nærheden af dopaminreceptorer ved at have en GluR ligand på et antistof rettet mod en dopaminreceptor. Projektet vil derfor på sigt kunne åbne op for andre anvendelsesmuligheder, så det for eksempel er muligt at fremstille ligander rettet mod specifikke synapser eller hjerneregioner.

26 F ab Receptor Receptor Antistof linker Ligand linker Ligand Antistof Figur 1. Skematisk tegning af projektets idé. Antistoffet genkender specifikt den ene receptor (rød) og vil selektivt aktivere denne med den konjugerede ligand. Den ukonjugerede ligand vil kunne aktivere begge receptorer. Projektet har konceptuel karakter, men vi vil i praksis fokusere på to typer af ligandstyrede ionkanaler: ionotrope glutamatreceptorer (iglur) og nicotine acetylcholinreceptorer (nachr). Disse to receptor-typer er ideelle til at afprøve konceptet på af flere grunde: 1) Røntgen krystalstrukturer af ligandbindingsdomæner fra disse receptorer har indenfor de sidste 10 år givet strukturel information om disse receptorer og den molekylære basis for deres binding af ligander. Dette muliggør en høj grad af strukturbaseret design i projektet, og ligger dermed også indenfor et af instituttets fokusområder. 2) Vi har erfaring med biologiske assays og udvikling af ligander rettet mod begge receptor typer på instituttet. Der er igangværende projekter på instituttet gående på udvikling af ligander mod disse receptorer, som kan udnyttes i dette projekt, og vi forventer en høj grad af synergi-effekt med disse eksisterende projekter. Glutamat-receptor ligander. De ionotrope glutamatreceptorer (iglur) er ligandstyrede ionkanaler opbygget af 4 monomerer (Figur 2), der hver består af et transmembrant domæne (TMD), et ligandbindingsdomæne (LBD) og en N-terminal del (NTD). Receptoren aktiveres ved at fire glutamatmolekyler bindes i de komplementære LBD er, hvilket fører til en konformationsændring i TMD, som udgør ionkanaldelen, således at Na + og Ca 2+ ioner kan trænge igennem cellemembranen.(mayer, 2006) Figur 2. En ionotrope glutamat receptor monomer (t.v.) består af et transmembrant domæne (TMD), et ligandbindings domæne (LBD) og en N-terminal del (NTD). Disse monomere danner en tetramer (i midten) bestående af 2 dimere (lyseblå/blå versus gul/grøn), således at ionkanalen dannes af TMD (rød). Der findes krystalstrukturer af LBD for flere subtyper af GluR. Til højre ses den fulde struktur af en iglur visualiseret ved enkelt-partikel elektronmikroskopi. (Fra Nagakawa et al).(nakagawa et al., 2005) Traditionelt inddeles iglur i tre grupper navngivet efter de selektive ligander N-methyl-D-aspartat (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazole propionsyre (AMPA), og kainsyre (KA) receptorer. Fire

27 forskellige AMPA receptor subtyper er klonet (GluR1-GluR4). Men hvor der findes småmolekyle - ligander, der virker selektivt på disse tre hovedklasser af iglur er, så er udvalget af subtypeselektive ligander indenfor de enkelte klasser straks mere begrænset. Der findes således hverken kompetitive agonister eller antagonister, der virker specifikt på en enkelt subtype. Den slags ligander kan være af stor betydning for udforskningen af de fysiologiske roller spillet af de enkelte subtyper. Tidligere har forskere fra Institut for Farmakologi og Farmakoterapi, DFU, fremstillet monoklonale F ab antistoffragmenter med høj affinitet til NTD af iglur subtypen GluR4.(Jespersen et al., 2000) Disse antistoffragmenter vil vi benytte i projektet, der således vil blive udført i samarbejde med Erik Riise (Institut for Farmakologi og Farmakoterapi). Anti-GluR4-F ab vil blive biokonjugeret med de eksisterende uselektive iglur agonister og antagonister for at opnå en GluR4-selektiv ligand. Størstedelen af arbejdet vil ligge i syntese og identificering af den rette linker, samt at finde en substitutions position på liganden som ikke kompromitterer aktiviteten. Den store mængde af viden, der foreligger om struktur-aktivitetsrelationer (SAR) og om ligandgenkendelse i LBD fra røntgenkrystalstrukturer, vil blive brugt i designet af ligander og linkere. Endvidere kan denne del af projektet opnå synergi-effekt med et eksisterende PhDprojekt, der arbejder med lignende problemstillinger, idet der her udvikles dimere GluR ligander, hvor to ens ligander er forbundet af en linker. Udover ovennævnte antistof har der været udviklet polyclonale F ab specifikke for NTD af GluR1.(Nakagawa et al., 2005) Såfremt konceptet viser sig brugbart i vores forsøg med GluR4, vil det derfor med stor sandsynlighed også være muligt at udvikle specifikke antistoffer mod de øvrige subtyper på lignende vis. Acetylcholin-receptor ligander. Nicotine acetylcholinreceptorer (nachr er) er ligesom iglur ligandstyrede ionkanaler med et ekstracellulært ligandbindings-domæne og en transmembran ionkanaldel. Receptorerne er pentamere komplekser, hvor liganden binder i interfasen imellem de extracellulære domæner fra to monomerer (Figur 3). Ikke mindre end 17 nachr subunits er blevet klonet, og disse danner et anseeligt antal receptorer i centralnervesystemet og det perifere nervesystem. Heller ikke i nachr-feltet findes der særligt mange subtypeselektive ligander, og specielt subtypeselektive agonister er en mangelvare. Der eksisterer specifikke antistoffer mod den extracellulære C-terminal som ikke er konserveret mellem subtyperne og som er lokaliseret tæt på ligandbindingsstedet.(moretti et al., 2004) Disse antistoffer er dog polyklonale, hvorfor de formentlig ikke kan bruges til vores formål. Udviklingen af et monoklonalt antistof er dog en længere proces. Derfor vil der i første omgang af dette projekt blive lavet et proof of concept -forsøg med en mutant-nachr, hvor C-terminalen er udskiftet med en Myc-tag, som der findes specifikke monoklonale antistoffer imod. Fordelen ved disse antistoffer er yderligere, at man kender sekvensen på F ab -fragmentet som derfor kan designes, modificeres og efterfølgende produceres bakterielt (Schiweck et al., 1997). Det er for eksempel muligt at designe, hvor mange ligander der skal være på et enkelt F ab fragment. Igen kan den viden der findes om SAR og den biostrukturelle ligand-genkendelse i acetylcholinreceptoren, anvendes i designet af ligander. Også denne del af projektet kan opnå synergieffekt med et eksisterende PhD-projekt, der beskæftiger sig med syntese af acetylcholinreceptorligander, både agonister og antagonister.

28 Ligand C-terminal Figur 3. Røntgenstruktur af Torpedo acetylcholinreceptor (Unwin, 2005) set ovenfra og fra siden. Receptorerne er pentamere strukturer med ligandbindingssted mellem to extracellulære domæner. Der findes specifikke polyklonale antistoffer mod C-terminaldelen tæt på bindingsstedet. Perspektiver. Projektet er i sig selv en meget spektakulær og generel løsning på problemerne med fremstilling af subtypeselektive ligander. Lægemiddel-konjugerede antistoffer er et relativt nyt koncept, som har været anvendt indenfor cancer-forskning (Schrama et al., 2006;Lambert, 2005), hvor der nu findes godkendte lægemidler. Men i disse eksempler bliver liganden frigivet fra antistoffet, og antistoffet skal blot guide lægemidlet hen i det rigtige område. I dette projekt er antistoffet en integreret del af liganden. De fremstillede stoffer vil derfor være af en ny type af makromolekylær ligand, som ikke er set før, som både er interessante farmakologiske værktøjer og potentielle lægemiddelkandidater. Endvidere åbner det op for en ny type selektivitet, idet stofferne kan rettes mod heteromere kombinationer af receptorer eller receptorer der sidder ved siden af ikke-beslægtede proteiner. Projektet vil altså kunne åbne op for et helt nyt forskningsområde, som også indeholder patenteringsmuligheder. Vi mener, at dette interdisciplinære projekt vil være helt unikt på Institut for Medicinalkemi. Her er ikke er tale om en ny serie af småmolekyle -ligander rettet mod et target, der allerede er etableret i forskningen på instituttet, ej heller er der tale om at studere et nyt target eller en ny reaktion med allerede etablerede metoder og teknikker. Projektet bygger på et koncept hentet fra andre dele af lægemiddelforskningen, som vi håber på vil vise sig at være en frugtbar måde at angribe en helt central problemstilling i medicinalkemi; designet af receptorsubtype-selektive ligander. Endvidere vil projektet introducere en helt ny type af kemi på instituttet og må siges at befinde sig indenfor den hurtigt

29 voksende gren af naturvidenskabelig forskning, der går under betegnelsen Chemical Biology eller syntetisk biologi. Personer. Projektet vil forløbe under ledelse af lektor Rasmus P. Clausen og adjunkt Anders A. Jensen. Disse personer har i flere år arbejdet med design, syntese og karakterisering af ligander til glutamat- og acetylcholin-receptorerne. Derudover vil forskere fra Medicinalkemi-gruppen og Biostruktur-gruppen på Institut for Medicinalkemi samt Erik Riise på Institut for Farmakologi og Farmakoterapi indgå i et samarbejde. Projektet er således forankret på DFU. Referencer. Jespersen LK, Kuusinen A, Orellana A, Keinanen K, Engberg J (2000) Use of proteoliposomes to generate phage antibodies against native AMPA receptor. Eur J Biochem 267: Lambert JM (2005) Drug-conjugated monoclonal antibodies for the treatment of cancer. Current Opinion in Pharmacology 5: Mayer ML (2006) Glutamate receptors at atomic resolution. Nature 440: Moretti M, Vailati S, Zoli M, Lippi G, Riganti L, Longhi R, Viegi A, Clementi F, Gotti C (2004) Nicotinic acetylcholine receptor subtypes expression during rat retina development and their regulation by visual experience. Molecular Pharmacology 66: Nakagawa T, Cheng Y, Ramm E, Sheng M, Walz T (2005) Structure and different conformational states of native AMPA receptor complexes. Nature 433: Schiweck W, Buxbaum B, Schatzlein C, Neiss HG, Skerra A (1997) Sequence analysis and bacterial production of the anti-c-myc antibody 9E10: the VH domain has an extended CDR-H3 and exhibits unusual solubility. FEBS Lett 414: Schrama D, Reisfeld RA, Becker JC (2006) Antibody targeted drugs as cancer therapeutics. Nat Rev Drug Discov 5: Unwin N (2005) Refined structure of the nicotinic acetylcholine receptor at 4 angstrom resolution. J Mol Biol 346:

30 Til Akademisk Råd Date: 31. marts 2006 Hermed fremsendes følgende forslag til spirekasser: Farmakologisk hæmning af anandamid- og N-oleoyethanolamid-dannelse (Harald S. Hansen et al.) Rationel individualiseret farmakoterapi (Claus Møldrup) Proteomics: A novel research approach within the field of neurometabolism. (Arne Schousboe et al.) Translational research. On the transfer of pharmacologic efficacy and safety from animals to man. (Ole J. Bjerrum) Are genes within the Eae 27 locus on mouse chromosome 1 important for disease development in an experimental model for rheumatic arthritis? (Åsa Andersson) Arne Schousboe Professor, D.Sc. Department Chair Universitetsparken 2 DK-2100 Copenhagen Phone Fax as@dfuni.dk In vivo imaging as a tool for monitoring gene expression, cellular activities and pharmaceutical intervention. (Anne-Marie Heegaard) Alle forslag ligger inden for instituttets primære forskningsprofil, og de repræsenterer vigtige nyskabelser. Projekterne vil hver for sig bidrage væsentligt til udvikling af instituttets forskning. Venlig hilsen Arne Schousboe

31 Forslag til spirekasseprojekt 23. marts 2006 Projekttitel: Farmakologisk hæming af anandamid- og N-oleoylethanolamid-dannelse. Forksningsområde: Farmakologi og Kemi Ansøgere: Harald S. Hansen*, Gitte Petersen* og Mikael Begtrup # * Institut for Farmakologi & Farmakoterapi, # Institut for Medicinalkemi Formål at undersøge den fysiologiske/patofysiologiske betydning af NAPE-specifik phospholipase D (NAPE-PLD), som er synetseenzym for de biologiske aktive molekyler: anandamid (som er agonist for cannabinoidreceptorer), N-oleoylethanolamin (som giver vægttab i rotter, nok via aktivering af transcriptionsfaktoren PPARα) og N-palmitoylethanolamin (som er antiinflammatorisk i mus og rotter). Et cellulært baseret assay til screening af potentielle hæmmere af NAPE-PLD aktiviteten ønskes udviklet, idet NAPE-PLD overexpresseres i en stabil celleline, og der syntetiseres 14 C-mærkede eller fluorescens-mærkede substrater, der kan loades ind i cellerne. Der planlægges kemisk syntese af en række udvalgte substratanaloger, som derefter kan testes som hæmmere i dette cellebaserede assay. Herved opnås et farmakologisk værktøj i form af en specifik NAPE-PLD hæmmer til yderligere karakterisering af betydningen af enzymet. Målet er at kunne fremstille en kemisk knock-out model af NAPE-PLD såvel in vivo som i cellebaserede modelsystemer. Det videnskabelige perspektiv er karakterisering af betydningen af tilstedeværelsen af det aktive enzym NAPE-PLD i organismen. Dette kan bl.a. på sigt lede til udvikling af et lægemiddel til fremme af den kvindelige fertilitet, idet dels graden af Nape-pld ekspression i uterus og dels det cirkulerende niveau af anandamid er vist at være ligefrem proportional med infertilitet - hvilket også sætter projektet i et samfundsmæssigt perspektiv. Side 1 af 7

32 Baggrund N-Arachidonoyl-ethanolamin - også kaldet anandamid - var det første endogene stof 1, som blev opdaget at have farmakologiske effekter, der er sammenlignelige med effekterne af det psyko-aktive stof i hash, Δ 9 -tetrahydrocannabinol. Anandamid har adskillige farmakologiske effekter, der er medieret via cannabinoid-receptorer 2, vanilloid-receptor 3 og ion-kanaler 4, inklusiv ændring af smertetærsklen 5, appetitregulering 6 samt kontrol med spastiske anfald hos sclerosepatienter 7. Forsøg tyder endvidere på, at et andet produkt af samme syntesevej, N-palmitoyl-ethanolamin, kan reducere smerte i perifere nerver 8 udover at have en anti-inflammatorisk effekt medieret via PPARα receptorer 9, mens produktet N-oleoyl-ethanolamin er rapporteret at have en hæmmende effekt på appetitten 10,11 via aktivering af PPARα receptorer 12. Cannabinoid-receptoren CB1 er en af de mest forekomne neuromodulerende receptorer i hjernen. CB1 er i stor grad udtrykt i bl.a. cortex, hippocampus og cerebellum, hvor endogene cannabinoider via CB1 er vist at fungere som retrograd synaptisk messenger, der kan modulere GABA- såvel som glutamat-frisætning 13,14. Anandamid kan via aktivering af CB1 receptorer således hæmme excitation, mens andre N-acyl-ethanolaminer har neuroprotektive egenskaber ved bl.a. at forhindre Ca 2+ -lækage og ved at inducere apoptosis ved hæmning af ceramid-nedbrydning 15,16. N-Palmitoyl-ethanolamin og N-oleoyl-ethanolamin såvel som andre N-acylethanolaminer genereres ved hydrolyse af N-acyl-phosphatidyl-ethanolamin (NAPE), katalyseret af enzymet NAPE-specifik phospholipase D (NAPE-PLD; se fig. 1) 17. Dette enzym er for nylig blevet klonet 18 og bekræftet at adskille sig signifikant fra andre kendte PLD-enzymer som også tidligere beskrevet af os 19. Karakteristika for NAPE-PLD er at 1) enzymet ikke benytter almindelige phospholipider som phosphatidyl-ethanolamin og phosphatidyl-cholin som substrat, 2) enzymet har derimod ingen substrat-specificitet mht. amid-bundet acylkæde i NAPE 2,20,21, 3) det findes i de fleste organer dog med relativ høj aktivitet/ekspression i hjernen uanset dyreart 18,22,23, 4) i hjernen stiger enzymaktiviteten med alderen 24, 5) mens store species-afhængig forskelle af enzymaktivitet ses i hjertevæv 25,26. Side 2 af 7

33 O O O O O O O -O P O HN O O NAPE NAPE-PLD O O O -O P OH O Phosphatidsyre + HN OH O N-Acyl-ethanolamin Figur 1. Biosyntetisk produktion af N-acyl-ethanolamin fra NAPE, katalyseret af enzymet NAPE-PLD Endocannabinoid-forstadiet NAPE, som er substrat for NAPE-PLD, udgør under 0,1 % af den samlede mængde af phospholipid i mammalt væv 27 og i celler hvor NAPE-PLD er overexpresseret er den endogene NAPE-mængde endnu mindre 28. Dette betyder, at hvis man vil bruge sådanne celler til assay af NAPE-PLD er det nødvendigt at tilføre substratet til cellerne. Da di-acylerede phospholipider normalt ikke optages let i celler må substratet syntetiseres og tilføres cellerne som N-acylglycero-phosphoethanolamin, enten isotop-mærket eller fluorescens-mærket. N-Acylglycero-phosphoethanolamin kan herefter enten bliver acyleret i cellen og således være substrat eller selv direkte tjene som substrat for NAPE-PLD, hvilket er rapporteret muligt i in vitro assay 29. Overexpression af NAPE-PLD i HEKceller er påbegyndt (samarbejde med lektor Darryl Pickering). Det planlagt dannede isotop- eller fluorescens-mærkede enzymprodukt kan efter ekstraktion og adskillelse fra substrat ved tyndtlagschromatografi scannes på en PhosphoImager scanner (som haves) for at kvantificere den enzymatiske reaktion. Som udgangspunkt vil en række ikke-hydrolyserbare substratanloger blive syntetiseret og testet for hæmmeraktivitet. Der haves allerede en vis erfaring i syntese af sådanne analoger [samarbejde med post doc. Anders Holmen Pedersen (ansat indtil august 2006) og Prof. Mikael Begtrup]. Ligeledes vil kendte hæmmere af β- lactamase-enzymer blive testet, idet NAPE-PLD tilhører denne enzymfamile 18,30. Når en kemisk forbindelse med hæmmeraktivitet er fundet, vil der blive lavet kemiske modifikationer af dette lead-stof og undersøgt struktur-aktivitet forhold. Side 3 af 7

34 Hæmmerstoffer vil også blive testet i dyreforsøg, idet vi har udviklet en analysemetode til at kvantificere N-acylethanolaminer med electrospray massespektrometri i dyrisk væv (samarbejde med Ph.D.-stud. Andreas Artmann og Prof. Steen Honore Hansen). I første omgang vil en hæmmer blive brugt som farmakologisk værktøj til at belyse den fysiologiske/patofysiologiske betydning af N-acylethanolaminer, som dannes af NAPE-PLD. Derudover kan det muligvis have betydning for behandling af kvindlig infertilitet. I uterus er cannabinoid/endocannabinoid-signalering via CB1 vist at påvirke fertiliteten, idet et lavt niveau af anandamid øger sandsynligheden for implantation af embryoet i mus 31. Det uterine niveau af anandamid er primært reguleret ved spatiotemporal mrna ekspression af Nape-pld, der korrelerer med den målte NAPE-PLD aktivitet 32. Dette er klinisk relevant, idet høje niveauer af perifert cirkulerende anandamid-niveauer er associeret med spontan abort hos kvinder 33. Der forelægger p.t. ingen yderligere viden om regulering af NAPE-PLD aktiviteten og ingen farmakologiske værktøjer i form af f.eks. en specifik hæmmer af enzymet. Projektet, som kombinerer biokemisk/farmakologisk ekspertise med kemisk lipidsyntese, er et nyt initiativ, som falder udenfor de ansøgninger om centrale forskningområder, som hovedansøger Harald S. Hansen er involveret i (Neurofarmakologi og Bioanalytical Chemistry) Forskningsplan og metoder Forventede resultater inden for 3 år Etablering af cellelinie (HEK-293) som overexpresserer NAPE-PLD Syntese af isotop-mærket eller fluorescens-mærket substrat som kan loades ind i cellerne Lipid-ekstraktion, separation og kvantificering af det i cellerne dannede N- acylethanolamin fra mærkede substrater Karakterisering af kinetikken for substratomsætning i celler med overexpresseret NAPE-PLD Kemisk syntese og testning af en række substratanaloger som testes for NAPE-PLD-hæmning i celleassay Evt. optimering af cellebaseret NAPE-PLD-assay med specielt fluorescensmærket substrat der muliggør high-through-put screening Undersøgelse af evt. regulering af NAPE-PLD i overexpresserende celler Side 4 af 7

35 (Phosphorylering/ekspression såvel som aktivitet ved receptor-stimulering, cytotoksisk påvirkning m.v.) Evt. påbegyndt in vivo forsøg med administration af cellulært afprøvede NAPE-PLD hæmmere Publicering i anerkendte videnskabelige tidsskrifter med høj impact factor Praktisk gennemførlighed Ansættelse af en Ph.D.-studerende til organisk kemisk syntese samt optimering og afprøvning af stoffer i cellebaserede assay vil give projektet fremdrift. Ansøgernes egen ekspertise dækker arbejde med og analyse af lipider, cellekulturer, dyreforsøg samt organisk kemisk phospholipidsyntese. Alle de til projektet påtænkte nødvendige apparater og udstyr forefindes i forskningsgruppen/institutternes eksisterende faciliteter. Etiske aspekter Forsøg med dyr vil kun blive udført i det omfang det vurderes nødvendigt for den videre karakterisering af enzymet og med det mindst mulige antal dyr som kan forsvares mht. den statistiske forsøgsplanlægning. Den ene ansøger (GP) har tilladelse til udførelse af dyreforsøg (Kategori C kursus). Internationalt samarbejde Når det findes formålstjenstligt etableres internationalt samarbejde, som vi allerede har gjort tidligere; e.g. med forskere i Sverige 34, Finland 35 og USA 20,21. Side 5 af 7

36 Referenceliste 1. Devane, W.A., Hanus, L., Breuer, A., Pertwee, R.G., Stevenson, L.A., Griffin, G., Gibson, D., Mandelbaum, A., Etinger, A. & Mechoulam, R. Isolation and structure of a brain constituent that binds to the cannabinoid receptor. Science 258, (1992). 2. Hansen, H.S., Moesgaard, B., Hansen, H.H. & Petersen, G. N-Acylethanolamines and precursor phospholipids - relation to cell injury. Chem. Phys. Lipids 108, (2000). 3. Zygmunt, P.M., Petersson, J., Andersson, D.A., Chuang, H.H., Sorgård, M., Di Marzo, V., Julius, D. & Högestätt, E.D. Vanilloid receptors on sensory nerves mediate the vasodilator action of anandamide. Nature 400, (1999). 4. Maingret, F., Patel, A.J., Lazdunski, M. & Honoré, E. The endocannabinoid anandamide is a direct and selective blocker of the background K + channel TASK-1. EMBO J. 20, (2001). 5. Calignano, A., La Rana, G., Giuffrida, A. & Piomelli, D. Control of pain initiation by endogenous cannabinoids. Nature 394, (1998). 6. Williams, C.M. & Kirkham, T.C. Anandamide induces overeating: mediation by central cannabinoid (CB1) receptors. Psychopharmacology (Berl) 143, (1999). 7. Baker, D., Pryce, G., Croxford, J.L., Brown, P., Pertwee, R.G., Huffman, J.W. & Layward, L. Cannabinoids control spasticity and tremor in a multiple sclerosis model. Nature 404, (2000). 8. Calignano, A., La Rana, G. & Piomelli, D. Antinociceptive activity of the endogenous fatty acid amide, palmitylethanolamide. Eur. J. Pharmacol. 419, (2001). 9. Lo Verme, J., Fu, J., Astarita, G., La Rana, G., Russo, R., Calignano, A. & Piomelli, D. The nuclear receptor peroxisome proliferator-activated receptor-α mediates the anti-inflammatory actions of palmitoylethanolamide. Mol. Pharmacol. 67, (2005). 10. Nielsen, M.J., Petersen, G., Astrup, A. & Hansen, H.S. Food intake is inhibited by oral oleoylethanolamide. J. Lipid Res. 45, (2004). 11. Rodriguez de Fonseca, F., Navarro, M., Gómez, R., Escuredo, L., Nava, F., Fu, J., Murillo- Rodríguez, E., Giuffrida, A., LoVerme, J., Gaetani, S., Kathuria, S., Gall, C. & Piomelli, D. An anorexic lipid mediator regulated by feeding. Nature 414, (2001). 12. Fu, J., Gaetani, S., Oveisi, F., LoVerme, J., Serrano, A., Rodriguez de Fonseca, F., Rosengarth, A., Luecke, H., Di Ciacomo, B., Tarzia, G. & Piomelli, D. Oleoylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPARα. Nature 425, (2003). 13. Kreitzer, A.C. & Regehr, W.G. Retrograde inhibition of presynaptic calcium influx by endogenous cannabinoids at excitatory synapses onto Purkinje cells. Neuron 29, (2001). 14. Wilson, R.I. & Nicoll, R.A. Endogenous cannabinoids mediate retrograde signalling at hippocampal synapses. Nature 410, (2001). 15. Hansen,H.S., Petersen,G. & Hansen,H.H. Endocannabinoids: The Brain and Body s Marijuana and Beyond. Onaivi,E.S., Sugiura,T. & Di Marzo,V. (eds.), pp (CRC Press, Taylor & Francis Group, Boca Raton, London, New York,2005). 16. Hansen, H.S., Moesgaard, B., Petersen, G. & Hansen, H.H. Putative neuroprotective actions of N-acyl-ethanolamines. Pharmacol. Ther. 95, (2002). 17. Schmid, H.H.O., Schmid, P.C. & Natarajan, V. N-Acylated glycerophospholipids and their derivatives. Prog. Lipid Res. 29, 1-43 (1990). 18. Okamoto, Y., Morishita, J., Tsuboi, K., Tonai, T. & Ueda, N. Molecular characterization of a phospholipase D generating anandamide and its congeners. J. Biol. Chem. 279, (2004). 19. Petersen, G. & Hansen, H.S. N-Acylphosphatidylethanolamine-hydrolysing phospholipase D lacks the ability to transphosphatidylate. FEBS Lett. 455, (1999). 20. Petersen, G., Sørensen, C., Schmid, P.C., Artmann, A., Tang-Christensen, M., Larsen, P.J., Schmid, H.H.O. & Hansen, H.S. Precursor levels responsible for opposite levels of anandamide and oleoylethanolamide in intestine of food-deprived rats. Biomed. Biochim. Acta in press, (2006). 21. Petersen, G., Moesgaard, B., Schmid, P.C., Schmid, H.H.O., Broholm, H., Kosteljanetz, M. & Hansen, H.S. Endocannabinoid metabolism in human glioblastomas and meningiomas compared to human non-tumor brain tissue. J. Neurochem. 93, (2005). Side 6 af 7

37 22. Petersen, G., Chapman, K.D. & Hansen, H.S. A rapid phospholipase D assay using zirconium precipitation of anionic substrate phospholipids: application to N-acylethanolamine formation in vitro. J. Lipid Res. 41, (2000). 23. Schmid, P.C., Reddy, P.V., Natarajan, V. & Schmid, H.H.O. Metabolism of N-acylethanolamine phospholipids by a mammalian phosphodiesterase of the phospholipase D type. J. Biol. Chem. 258, (1983). 24. Moesgaard, B., Petersen, G., Jaroszewski, J.W. & Hansen, H.S. Age dependent accumulation of N-acyl-ethanolamine phospholipids in ischemic rat brain: a 31 P NMR and enzyme activity study. J. Lipid Res. 41, (2000). 25. Moesgaard, B., Petersen, G., Mortensen, S.A. & Hansen, H.S. Substantial species differences in relation to formation and degradation of N-acyl-ethanolamine phospholipids in heart tissue: an enzyme activity study. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 131, (2002). 26. Morishita, J., Okamoto, Y., Tsuboi, K., Ueno, M., Sakamoto, H., Maekawa, N. & Ueda, N. Regional distribution and age-dependent expression of N-acylphosphatidylethanolaminehydrolyzing phospholipase D in rat brain. J. Neurochem. 94, (2005). 27. Hansen, H.S., Moesgaard, B., Hansen, H.H., Schousboe, A. & Petersen, G. Formation of N- acyl-phosphatidylethanolamine and N-acylethanolamine (including anandamide) during glutamate-induced neurotoxicity. Lipids 34 Supplement, (1999). 28. Okamoto, Y., Morishita, J., Wang, J., Schmidt, P.C., Krebsbach, R.J., Schmidt, H.H.O. & Ueda, N. Mammalian cells stably overexpressing N-acylphosphatidylethanolamine-hydrolysing phospholipase D exhibit significantly decreased levels of N-acylphosphatidylethanolamines. Biochem. J. 389, (2005). 29. Natarajan, V., Schmid, P.C., Reddy, P.V. & Schmid, H.H.O. Catabolism of N-acylethanolamine phospholipids by dog brain preparations. J. Neurochem. 42, (1984). 30. Ueda, N., Okamoto, Y. & Morishita, J. N-acylphosphatidylethanolamine-hydrolyzing phospholipase D: A novel enzyme of the β-lactamase fold family releasing anandamide and other N-acylethanolamines. Life Sci. 77, (2005). 31. Paria, B.C. & Dey, S.K. Ligand-receptor signaling with endocannabinoids in preimplantation embryo development and implantation. Chem. Phys. Lipids 108, (2000). 32. Guo, Y., Wang, H.B., Okamoto, Y., Ueda, N., Kingsley, P.J., Marnett, L.J., Schmid, H.H.O., Das, S.K. & Dey, S.K. N-acylphosphatidylethanolamine-hydrolyzing phospholipase D is an important determinant of uterine anandamide levels during implantation. J. Biol. Chem. 280, (2005). 33. Maccarrone, M., Valensise, H., Bari, M., Lazzarin, N., Romanini, C. & Finazzi-Agrò, A. Relation between decreased anandamide hydrolase concentrations in human lymphocytes and miscarriage. Lancet 355, (2000). 34. Holt, S., Rocksén, D., Bucht, A., Petersen, G., Hansen, H.S., Valenti, M., Di Marzo, V. & Fowler, C.J. Lippopolysaccharide-induced pulmonary inflammation is not accompanied by a release of anandamide into the lavage fluid or a down-regulation of the activity of fatty acid amide hydrolase. Life Sci. 76, (2004). 35. Térová, B., Petersen, G., Hansen, H.S. & Slotte, J.P. N-Acyl phosphatidylethanolamine affect the lateral distribution of cholesterol in membranes. Biochim. Biophys. Acta in press, (2005). Side 7 af 7

38 DFU Spirekasseansøgning 1. Ansøgers CPR-nr Ansøgers navn (ansvarlig over for forskningsrådet) 3. Stilling og uddannelse (max. 39 karakterer) 4. Nuværende arbejdsplads (adresse, telefon og ) 5. Sted for projektets udførelse (adresse, telefon og ) 6. Privatadresse (adresse, telefon og ) Claus Møldrup Associate Professor, PhD MSc. (pharm.) Danmarks Farmaceutiske Universitet Universitetsparken København Ø , cm@dfuni.dk Danmarks Farmaceutiske Universitet Universitetsparken København Ø tel , cm@dfuni.dk Skærager Albertslund tel , cm@dfuni.dk 7. Projektets titel (max.180 karakterer) Rationel Individualiseret Farmakoterapi 8. Ansøgt virkemiddel Spirekassepulje 9. Faglige nøgleord (max. 5 ord) Farmakoterapi, farmakogenetik, compliance, individualisering, monitorering 10. I hvilken periode ønskes det ansøgte beløb anvendt? Start: dag 01 md. 08 år 2006 Slut: dag 31 md. 12 år 2010

39 VIP-medarbejdere i projektgruppen, (navn, stilling, arbejdssted): Claus Møldrup, PhD MSc. (pharm.) Associate professor. Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi. Ulrik Gether MD, dr. med. Professor Københavns Universitet Farmakologisk Institut. Center for Pharmacogenomics. Thomas Werge Cand. Scient. (biokemi) Forskningschef H:S Sct. Hans Hospital, Forskningsinstitut for Biologisk Psykiatri Jakob Kjellberg, Cand.scient, M.Sc. Health Econ. DSI Institut for Sundhedsvæsen Ole J. Bjerrum. MD, dr. med. Professor Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi Janine Morgall Traulsen, Dr. phil. (sociologi) Associate professor. Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi Ramune Jacobsen, Ph.d. stud. Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi. 15. Populærvidenskabelig beskrivelse af projektet (max karakterer) Rationel Individualiseret Farmakoterapi karakteriseres ved overgangen fra one size fits all rationel farmakoterapi, der praktiseres i dag, til en farmakoterapeutisk praksis for fremtiden, der sætter individet i centrum i hele det terapeutiske behandlingsforløb fra farmakogenetiske forhold, diagnose, indikation, administration, dosering, information, terapeutisk monitorering til compliance. Forskningsprogrammet har til formål gennem litteraturreviews og spørgeskemaundersøgelser at identificere Hvordan vi får mest Individuel Farmakoterapi for pengene? og derefter bistå som katalysator for forskning og udvikling der hvor det er mest rationelt. Forskningsprogrammet vil inden for de næste 5 år fokusere på områderne; depression, astma, hjerte/kar, smerte og demens. Baggrunden for forskningsprogrammet er en erkendelse af, at der hverken er forskningsmæssige eller behandlingsmæssige ressourcer nok til at fokusere på individualiseringen inden for alle områder i det terapeutiske behandlingsforløb. Der findes i dag mange forskningsmæssige initiativer nationalt og internationalt, der fokuserer på individualisering af terapi inden for områder lige fra molekylærfarmakologien til klinikken. Det, der synes at mangle i forskningsområdet i dag, og som dette forskningsprogram i Rationel Individualiseret Farmakoterapi bl.a. skal råde bod på, er en vurdering af, hvor en individualisering inden for et givent behandlingsområde vil give mest behandlingsmæssig værdi for individet og samfundet. og hvor en øgning af den forskningsmæssige indsats således er tiltrængt. Det er netop denne helhedsorienterede syn på individualiseret farmakoterapi, der udgør det innovative ved dette forskningsprogram.

40 Projekttitel: Rationel Individualiseret Farmakoterapi Problemformulering: Rationel Individualiseret Farmakoterapi karakteriseres ved overgangen fra one size fits all rationel farmakoterapi, der praktiseres i dag, til en farmakoterapeutisk praksis for fremtiden, der sætter individet i centrum i hele det terapeutiske behandlingsforløb fra farmakogenetiske forhold, diagnose, indikation, administration, dosering, information, terapeutisk monitorering til compliance. Nærværende forskningsprogram har til formål at identificere Hvor vi får mest Individuel Farmakoterapi for pengene? og derefter bistå som katalysator for forskning og udvikling der hvor det er mest rationelt. Forskningsprogrammet vil inden for de næste 5 år fokusere på områderne; depression, astma, hjerte/kar, smerte og demens, ud fra denne terapeutiske helhedsbetragtning. Samfundsmæssige perspektiv: Det internationale samfund har hverken forskningsmæssige eller behandlingsmæssige ressourcer nok til at fokusere på alle individualiserings områder i det terapeutiske behandlingsforløb derfor skal der fokuseres på de individualiserings aspekter der giver med størst behandlingsmæssig værdi for individet og dermed samfundet. Baggrund: Det seneste årti har der været øget internationalt fokus på forskningsfeltet farmakogenetik med henblik på udvikling og anvendelse af lægemidler med udgangspunkt i vore genetiske dispositioner (1;2). Farmakogenetikkens klassiske tilgang har afstedkommet forventninger om store terapeutiske fremskridt med udvikling af mere specifikke terapeutiske regimer med bedre effekt og færre bivirkninger til følge. Ganske få af disse forventninger er blevet indfriet på nuværende tidspunkt. Årsagerne hertil er mange og komplekse og har karakter af såvel videnskabelige som politiske og økonomiske barrierer. Det vil drage for vidt at udrede disse i nærværende notat men blot henvise til en eksperthøring fra Sevilla foråret 2004 (3). Men en væsentlig erkendelse er dog, at de fleste sygdommes komplekse karakter og den tilhørende komplekse farmakoterapeutiske udredning og behandling ofte gør det vanskeligt at få et optimalt udbytte ved blot at anvende en ren farmakogenetisk strategi. Set i et sundhedsvidenskabeligt perspektiv giver det derfor mere mening at udvide den farmakogenetiske tilgang til en forståelse af individualiserede lægemiddelterapier i bredere forstand. Dette velvidende at en optimal farmakologisk behandling ikke alene er betinget af de rette genetiske forhold, men under indflydelse af en lang række individuelle og situationelle faktorer. Disse faktorer vil i de kommende år redefinere den generaliserede tilgang - one size fits all - vi i dag ser omkring rationel farmakoterapi og evidens baseret medicin (4). Fremtidens rationelle individualiseret farmakoterapi vil altså ikke blot forsøge at forholde sig til prisen over for effekt- /bivirkningsratioen som det er tilfældet i dag, men i stigende udstrækning inkludere en individualiseret tilgang til hele behandlingsforløbet og dermed optimere individets behandling og dermed samfundets ressourcer (5-9). Denne tilgang vil udover farmakogenetikken bl.a. inkludere: 1

41 individualiserede indikationer, som fremkommer gennem en dialog mellem læge og patient individuelt valg af lægemiddel, hvor såvel lægens som patientens erfaringer og præferencer er i spil individuelle valg og tilpasning af den administrationsform, der passer patienten bedst individualiserede doseringer, der gennem daglige erfaringer tilpasses kontinuerligt af såvel læge som patient individualiseret information der motiverer patienten mest muligt individuel terapeutisk monitorering, der skaber evidens for behandlingen, og som kan bruges som supplement til de kliniske undersøgelsers data individuel support, der styrker patientens behandling i dagligdagen gennem f.eks. compliance systemer. Det er indlysende, at der ikke vil være ressourcer, hverken forskningsmæssigt og/eller samfundsøkonomisk, til at optimere alle ovenstående forhold inden for et givent farmakoterapeutisk regime. Derfor vil en udredning af, hvilke af ovenstående faktorer, der vil give mest individuel farmakoterapi for vore ressourcer, være nødvendig. Eller oversat til en samfundsøkonomisk analyse; Hvordan får vi mest Individuel Farmakoterapi for pengene? Der findes i dag mange forskningsmæssige initiativer nationalt og internationalt, der fokuserer på individualisering af terapi inden for områder lige fra molekylærfarmakologien til klinikken. Det, der synes at mangle i forskningsområdet i dag, og som dette forskningsprogram i Rationel Individualiseret Farmakoterapi bl.a. skal råde bod på, er en vurdering af, hvor en individualisering af farmakoterapien inden for et givent behandlingsområde vil give mest behandlingsmæssig værdi, og hvor en øgning af den forskningsmæssige indsats således er tiltrængt. Det er netop denne helhedsorienterede vurdering, der udgør det innovative og unikke ved dette forskningsprogram. Denne helhedsorienterede tankegang var samtidig et centralt diskussionspunkt ved European Science Foundation Workshop Personalized Medicine Europe: Health, Genes & Society 19-21/ ved Tel-Aviv Universitet, bl.a. med følgende keynote (10) Yderligere forskningsmæssig begrundelse for nødvendigheden af det brede sigte på individualiseret farmakoterapi kan findes i et nyligt udsendt nummer af Ugeskrift for Læger. (5). Forskningsorganisering: Forskningsprogrammet søges gennemført gennem internationalt tværfagligt samarbejde, med Danmarks Farmaceutiske Universitet som overordnet projektkoordinator. Indledende dialog med European Medical Research Councils (EMRC) European Science Foundation (ESF) er etableret, og midler til workshops vil blive søgt i dette regi. Projektgruppen bag forskningsprogrammet er tværfagligt sammensat, med repræsentation af molekylærbiologi, farmakologi, farmakoterapi/klinikken, samfundsmedicin, bioetik og sundøkonomi. Denne brede faglige sammensætning skal sikre det helhedsorienteret fokus på den individualiserede farmakoterapi. Projektgruppens primære opgave er at skabe kontakt til og opbygge en kontinuerlig repræsentation af relevante nationale og internationale forskningsgrupper i processen. En forskningsassistent, en ph.d. studerende og forskningsprogramlederen skal herefter sikre at disse relationer indarbejdes i det daglige arbejde med nedenstående forskningsplan. 2

42 Projektgruppen består af følgende personer: Claus Møldrup, PhD MSc. (pharm.) Associate professor. DFU, Institut for Farmakologi og Farmakoterapi. (Forskningsprogramleder) Ulrik Gether MD, dr. med. Professor Københavns Universitet Farmakologisk Institut. Center for Pharmacogenomics. Thomas Werge Cand. Scient. (biokemi) Forskningschef H:S Sct. Hans Hospital, Forskningsinstitut for Biologisk Psykiatri Jakob Kjellberg, Cand.scient, M.Sc. Health Econ. DSI Institut for Sundhedsvæsen Ole J. Bjerrum. MD, dr. med. Professor Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi. Janine Morgall Traulsen, Dr. phil. (sociologi) Associate professor. DFU, Institut for Farmakologi og Farmakoterapi. Ramune Jacobsen, Ph.d. stud. Danmarks Farmaceutiske Universitet, Institut for Farmakologi og Farmakoterapi. Forskningsplan: Antidepressiva området er udvalgt som første eksemplar for forskningen. Hjerte-/kar, astma, smerte og demens behandling vil udgøre de efterfølgende fokusområder. Antidepressivaområdet er valgt som eksemplar for processen, fordi det er veldokumenteret, at CYP2D6 og CYP2C19 polymorfisme påvirker effekten af en lang række antidepressiva, og baseret på denne viden er doseringsrekommandationer baseret på CYP2D6 og CYP2C19 genotypebestemmelse tidligere blevet introduceret (11;12). Omvendt viser studier af fluoxetin og nortriptylin, at CYP2D6 polymorfisme ikke synes at have relevans for bivirkningsprofilen for de pågældende stoffer (13). Samtidig er der tale om en patientgruppe, hvor autonomi og egen sygdomsforståelse er betydeligt udviklet. Det betyder, at diagnose, indikation, administrationsform, information, support og monitorering er til forhandling med stærk mulighed for individualisering i hele behandlingsforløbet (14-19). Alle ovenstående terapiområder vil gennemgå følgende forskningsdesign Initialt gennemføres en identifikationsfase. Denne vil dels være teoretisk funderet med udgangspunkt i litteraturen, samt empirisk funderet gennem spørgeskemaundersøgelser med forskningsgrupper som respondenter. Dette skal sikre inklusion af den nyeste viden på området. Outcome af identifikationsfasen vil primært være videnskabelige artikler i form af reviews. Metoderne vil således indbefatte de klassiske metoder til udarbejdelse af sekundære analyser, såsom kritiske review og metaanalyser samt inklusion af spørgeskemaundersøgelser. Der søges om lønningsmidler til en forskningsassistent, en ph.d. studerende samt forskningsprogramledelse. Den primære opgave for disse videnskabelige medarbejdere vil bestå i den praktiske gennemførsel af denne review proces, (herunder netværksdannelse, litteratursøgning og spørgeskemaundersøgelser samt reviewprocessen og artikelskrivning i samarbejde med eksterne forskningsgrupper.) 3

43 Hensigten er med denne identifikationsfase er en kortlægning af betydelige forskningsmæssige faktorer for fremtidig udvikling af farmakogenetiske baserede terapier, individualiserede indikationer, individualiserede doseringsredskaber, individualiserede administrationsformer, individualiserede monitoreringssystemer, individualiserede supportsystemer, og individualiseret lægemiddelinformation inden for det enkelte terapiområde. De sammenfattende konklusioner for ovenstående forventes at udgøre rekommandationer i forhold til hvor, det er mest rationelt at sætte ind med henblik på en optimering af den individualiserede farmakoterapi inden for de enkelte terapiområder. På denne baggrund initieres en udviklingsfase. Her skal nærværende forskningsprogram fungere som katalysator for finansiering, samarbejde og eventuelt etablering af nye forskningsgrupper med fokus på ovenstående rekommandationer. Målet er udvikling af konkrete produkter, herunder f.eks. individualiserede -terapeutiske regimer, - indikations guidelines, -administrationssystemer, -monitoreringssystemer, -compliancesystemer, - kommunikationssystemer, gerne med patenterbarhed som et produkt. Forskningsplanen for det samlede forskningsprogram tager sig ud som følger. Forskningsstrategisk og produktmæssig målsætning for forskningsprogrammet De overordnede strategiske målsætninger er at; Skabe international synergi mellem forskningsgrupper med fokus på individualisering af behandling på alle forskningsmæssige niveauer, såvel i identifikations som udviklingsfasen. 4

44 Rationel Individualiseret Farmakoterapi anvendes som udbredt evaluerings strategi i sundhedsvæsnet i forsøget på at få mest individualiseret farmakoterapi for pengene. Områderne antidepresiva, hjerte/kar, astma, smerte og demens har gennemgået identifikationsfasen og har igangsat udviklingsforskningsprojekter. De overordnede produkt målsætninger: Inden for en tidsramme på 5 år er det hensigten, at forskningsprogrammet i Rationel Individualiseret Farmakoterapi skal opfylde følgende målsætninger: Der publiceres som gennemsnit over tidsrammen min. 3 artikler pr. VIP forskningsårsværk i internationale tidsskrifter med stor gennemslagskraft. Der er ansøgt om minimum et patent som følge af udviklingsfaseprodukterne. Efter 4 år er min. 50% af driften i forskningsgruppen eksternt finansieret. Forskergruppen bidrager med min. 30 indslag i aviser, radio og tv pr. år. Forskergruppen har som minimum fordoblet sin størrelse efter 5 år i forhold til udgangspunktet. Disse ambitiøse målsætninger, inden for den begrænsede tidshorisont, er udformet med en forventning om, at området vil skaffe yderligere ekstern finansiering og dermed yderligere årsværk med efterfølgende mulighed for at analysere alle behandlingsområderne angivet i forslaget. Forskningsprogrammet ses således som en løftestang for området, fagligt som finansielt. Ligeledes er synergieffekter ved samarbejde på tværs af forskningsgrupper såvel nationalt som internationalt indbygget i projektplanen. 5

45 Reference List (1) Roses AD. Pharmacogenetics and future drug development and delivery. Lancet 2000;355: (2) Roses AD. Pharmacogenetics and the practice of medicine. Nature 2000;405(6788): (3) The European Society of Human Genetics (ESHG) TIfPTSI. Polymorphic sequence variants in medicine: Technical, social, legal and ethical issues with pharmacogenetics as an example. European Journal of Human Genetics In press Ref Type: Journal (Full) (4) Fierz W. Challenge of personalized health care: To what extent is medicine already individualised and what are the future trends? Med Sci Monit 2004;10(5): (5) Møldrup C. Livsstilsmedicin og farmakogenetik - individualiseret lægemiddelterapi. Ugeskr Læger 2005;167(20): (6) Møldrup C. When pharmacogenomics goes public. New Genetics in Society 2002;21(1): (7) Møldrup C. Farmakogenetikkens betydning for den primære sundhedssektor. Ugeskr Læger 2002;164(25): (8) Møldrup C. Ethical, social and legal implications of pharmacogenomics - a critical review. Community Genetics 2001;4: (9) Møldrup C. Medical Technology Assessment of the ethical, social, and legal implications of pharmacogenomics - a proposal for an Internet Citizen jury. International Journal of Technology Assessment in Health Care 2002;18(3): (10) Møldrup C. The prospects and bioethical dimensions of expanding the meaning of pharmacogenomics to encompass individualised pharmacotherapy. Personalized Medicine 2005;(2): (11) Kirchheiner J, Brøsen K, Dahl ML, Gram LF, Kasper S, Roots I. CYP2D6 and CYP2C19 genotype-based dose recommendations for antidepressants: a first step towards subpopulation-specific dosages. Acta Psychiatr Scand 2001;(104): (12) Bertilsson L, Dahl M-L, Tybring G. Pharmacogenetics of antidepressants: clinical aspects. Acta Psychiatrica Scandinavica 1997;96(suppl 391): (13) Roberts RL, Mulder RT, Joyce PR, Luty SE, Kennedy MA. No evidence of increased adverse drug reactions in cytochrome P450 CYP2D6 poor metabolizers treated with fluoxetine or nortriptyline. Hum Psychopharmacol 1[19], Ref Type: Journal (Full) (14) Lifestyle indications for antidepressants. IMS Health 2003 July 24:1-2. Available from: URL: (15) Steimer W, Muller B, Leucht S, Kissling W. Pharmacogenetics: a new diagnostic tool in the management of antidepressive drug therapy. CLIN CHIM ACTA 2001 Jun;308(1-2): (16) Krieger D. Anecdotals accounts of smart drugs used with antidepressants. Damicon 1998 February 17:1-3. Available from: URL: (17) Jann MW, Cohen LJ. The influence of ethnicity and antidepressant pharmacogenetics in the treatment of depression. Drug Metabolism and Drug Interactions 2000;16(1): (18) Knudsen P, Hansen EH, Morgall JM. Perceptions of younger woman using SSRI antidepressats - a reclassification of sigma. International journal of pharmacy practice 2002;10: (19) Bell C, Nutt D. Paroxetine and its uses in psychiatry. Hosp Med 1999 May;60(5):

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51 Spirekasse-ansøgning Forskningsområde: Neurofarmakologi Proteomics: A novel research approach within the field of neurometabolism Arne Schousboe, Lasse K. Bak, Helle S. Waagepetersen The above mentioned research group at the Danish University of Pharmaceutical Sciences has a long established research tradition within the field of CNS metabolism and membrane transport. The group has added significantly to this research field employing mass spectrometry and nuclear magnetic resonance spectroscopy to study metabolism in cultured neural cells. However, to gain detailed insight into the specific roles, regulation and concerted action of different proteins involved in metabolic homeostasis, new approaches are warranted. Our primary objective is to establish a new research environment within the field of proteomics to study the dynamics of proteins and hetero-protein complexes important for intermediary metabolism and homeostasis including enzymes and transporters. Introduction The different entities involved in homeostasis of the neurotransmitters glutamate (excitatory) and γ- aminobutyric acid (GABA; inhibitory), i.e. synthesis, degradation and transport, are all instrumental in the maintenance of the balance between excitation and inhibition which, in turn is essential for brain function. Malfunctions of energy metabolism and/or neurotransmitter homeostasis are part of the pathology of numerous psychiatric and neurological disorders. Homeostasis of glutamate and GABA and energy metabolism involves a number of both cytosolic and mitochondrial enzymes. Moreover, plasma membrane, vesicular membrane and mitochondrial membrane transporters are essential for these processes and for neurotransmission. Transient formations of hetero-protein complexes might be an important component in the homeostatic fine-tuning of metabolism in response to changing intracellular conditions reflecting neuronal signaling (McKenna et al. 2006). A hetero-protein complex is defined as a close association between two or more individual proteins; each of these proteins must be a functional protein in itself. For instance, in a hetero-enzyme complex, two or more different enzymes are closely associated so that the product of one enzyme is delivered directly as substrate to the next enzyme, socalled substrate channeling. This effectively eliminates diffusion of the product/substrate molecules into the bulk phase away from the active sites of the enzymes. Over the last almost four decades, the concept of hetero-enzyme complexes has been developed based on studies primarily in bovine liver preparations. These complexes involve key enzymes in glutamate and energy metabolism such as aspartate aminotransferase (AAT), glutamate dehydrogenase (GDH), α-ketoglutarate dehydrogenase (α-kgdh) and malate dehydrogenase (MDH) (e.g. Fahien and Teller 1992;Fahien et al. 1989). This concept has been extended to the brain by McKenna et al. (2000; 2006). Considering the complexity of brain glutamate and energy metabolism it is very likely that such transient hetero-enzyme complexes might be formed in the brain as well, and that this plays a 1

52 Spirekasse-ansøgning significant role in brain energy and amino acid neurotransmitter homeostasis. In addition, permanent or transient hetero-protein complexes might be formed between membrane proteins or between membrane proteins and enzymes. So far, hetero-protein complexes have typically been studied using proteins in purified form; however, our goal is to study the significance of these processes in intact biological systems both in vivo and in vitro. The rapidly evolving field of mass spectrometry-based protein chemistry or proteomics will be employed to characterize the involvement of hetero-protein complexes in the regulation of energy metabolism and neurotransmitter glutamate and GABA homeostasis during neuronal signaling. The strength of adopting a proteomics approach to study hetero-protein complexes is the highly sensitive identification and detailed description of multiple proteins in a single biological sample. By establishment of this technology, we will be able to delineate the dynamics of hetero-protein complex formation in the maintenance of brain amino acid and energy homeostasis. Working hypotheses The fine-tuning of the enzymatic machinery and transport proteins related to glutamate and GABA metabolism are essential for proper functioning of the brain. Thus, investigation of the following seems warranted: 1. Dynamics of hetero-enzyme complexes of different combinations of mitochondrial AAT, GDH, MDH, α-kgdh; importance for the involvement of the tricarboxylic acid cycle in glutamate and GABA metabolism. 2. Formation of a binary complex between mitochondrial pyruvate carboxylase and AAT; implications for anaplerosis and de novo synthesis of glutamate and GABA. 3. Formation of a binary complex between mitochondrial phosphate-activated glutaminase and GDH; significance for substrate channelling of NH 4 + in the glutamate-glutamine cycle. 4. Association of glutamate decarboxylase (GAD) to mitochondria and synaptic vesicles, including studies in GAD 65 knock-out mice; importance for maintenance of adequate GABA levels in different cellular compartments. 5. Correlation between expression of neuronal GABA transporters and GAD; implications for synaptic GABA homeostasis. 6. Regulation by Ca 2+ of post-translational modifications of enzymes and formation of hetero-enzyme complexes in glycogen metabolism; involvement in regulation of glycogen breakdown and lactate production. 7. Association between mitochondrial aspartate-glutamate carrier and mitochondrial enzymes; possible coupling between neuronal activation and energy metabolism via Ca Association between neurotransmitter transporters for glutamate and GABA and Na + /K + -ATPase; possible coupling between sodium-coupled transport and restoration of membrane potential. 9. Associations between Na + /K + -ATPase and enzymes of glycolysis; significance for channelling of ATP in intracellular compartments exhibiting only moderate oxidative metabolism. 10. Associations between vesicular H + -ATPase, enzymes of glycolysis and vesicular neurotransmitter transporters; significance for channelling of ATP for refilling of vesicles. 11. The dynamics of hetero-protein complexes in the glycolytic pathway; delineate the preference for glucose to sustain neurotransmitter glutamate homeostasis. 2

53 Spirekasse-ansøgning Experimental approach The goal is to establish the needed laboratory facilities for a proteomics approach to study heteroprotein complexes and phosphorylation of proteins at the Danish University of Pharmaceutical Sciences for all steps prior to (i.e. sample preparation) and following (i.e. data analysis) the actual mass spectrometric analysis. The mass spectrometric analysis will be performed either via collaboration or by a commercial provider since this requires exceedingly expensive equipment. Isolation of proteins and protein complexes of interest can be accomplished be different methods already established within the field of proteomics, e.g. specific isolation employing affinitychromatography using specific antibodies against one or more of the proteins involved in hetero-protein complexes. Upon additional chromatographic purification, mass-spectrometric analysis of the soluble and complex-bound enzymes can be performed. Specific isolation procedures have been established for phosphorylated proteins by multi-dimensional chromatography or 2D-SDS-PAGE. Subsequent mass spectrometric analysis is typically performed by MALDI-TOF/TOF either on the isolated, native protein(s) or the tryptic digest(s). Identification and description of the protein is based on computer analysis of the data employing bioinformatics tools. Specifically, an experimental setup to study the dynamics of hetero-protein complexes in isolated organelles (e.g. synaptic vesicles or mitochondria) consists of the following: The organelle is isolated from either intact brain tissue or cell culture. This system can be manipulated in several ways to mimic different intracellular conditions (e.g. changes in substrate or second messenger levels) that might affect the formation of hetero-protein complexes or phosphorylation of proteins. The organelles are subsequently prepared for proteomic analysis. The presence of 13 C- or 15 N-labeled precursors (e.g. amino acids or glucose) might allow the metabolism to be probed in the same experiments using mass spectrometric or nuclear magnetic resonance (NMR) techniques. This approach will provide information at both the protein and the metabolic level from the same experiment. Expected achievements within the first 3 years Establishment of the required in-house laboratory facilities, i.e. equipment required for all steps prior to and following the actual mass spectrometric analysis. Extensive experience in procedures regarding both the experimental work as well as the preparation of samples for mass spectrometric analysis. Knowledge of the interpretation of mass spectrometric data in relation to hetero-protein complexes. A number of initial publications in peer-reviewed neuroscience journals. Establishment of a solid foundation for a permanent research environment within mass spectrometry-based protein chemistry at the Danish University of Pharmaceutical Sciences. 3

54 Spirekasse-ansøgning Potential collaborators: University of Maryland, Baltimore, USA: Dr. Mary McKenna University of Southern Denmark, Odense, Denmark: Prof. Ole N. Jensen References Fahien L. A., MacDonald M. J., Teller J. K., Fibich B. and Fahien C. M. (1989) Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. J Biol. Chem. 264, Fahien L. A. and Teller J. K. (1992) Glutamate-malate metabolism in liver mitochondria. A model constructed on the basis of mitochondrial levels of enzymes, specificity, dissociation constants, and stoichiometry of hetero-enzyme complexes. J Biol. Chem. 267, McKenna M. C., Stevenson J. H., Huang X. and Hopkins I. B. (2000) Differential distribution of the enzymes glutamate dehydrogenase and aspartate aminotransferase in cortical synaptic mitochondria contributes to metabolic compartmentation in cortical synaptic terminals. Neurochem. Int. 37, McKenna M. C., Hopkins I. B. and Lindauer S. L. (2006) Aspartate aminotransferase in synaptic and nonsynaptic mitochondria: differential effect of compounds that influences transient hetero-enzyme complex (metabolon) formation. Neurochem. Int. In press 4

55 1 Proposal for Spirekasseprojekt within Pharmacology. O. J. Bjerrum Institute for Pharmacology and Pharmacotherapy. Translational research. On the transfer of pharmacologic efficacy and safety from animals to man. Research Area The research area covers the translation of (bio)pharmaceuticals from being investigated by in vitro/ in vivo/ in silico methods to their application in man. (1) It will integrate several existing and coming expertises at DFU, which otherwise lack a gathering theme to rally around. In my conception a university of pharmaceutical sciences must cover all important aspects of the drug development process. The translational research bridge is at our university nearly absent or at least very weak. This is the reason why I in my professorship have focussed on this part of the process. In the former Rational Pharmacotherapy Focus Group I have unsuccessfully tried to establish the link between the biologically based research to the exiting research groups working with the utilisation of medicines. Now I try again through this Spirekasse application. Below is listed a row of arguments, for including the translational part of the medicines development process to the other competencies managed at our university: The drug development process represents a continuum and this does not match to our university if it consists of a collection of islands, (even if many of them have lighthouses) because bridging between them is lacking. In my opinion the most serious gap is the link between experimental laboratory disciplines and those covering utilisation of medicines - even if the latter island is large and is housing a lighthouse. However the two research fields need each other and thereforewe must do our outmost to bridge the gap. In the years to come DFU will probably be met with expectations from our new fusion partners - whom they will be - to be able to deliver translational research. Equally important is the competition we will experience from University of Southern Denmark with their new master education in clinical pharmacy. DFU stands just before the employment of two professorships with disciplines in the centre of translational research: Pharmacokinetics and pharmacotherapy. To attract the best scientists in these fields it is important that they can see that DFU is dedicated to support the area of translational medicine. A flourishing Spirekasse integrating our forces would have a good signal value which furthermore will give strength to DFU as a whole. Due to the increasing complexity of drug therapy e.g. through polypharmacy and personalised medicines, the area of employments within the clinical sector for pharmacists will undergo expansion in the years to come. To fulfil our obligations to the needs of the society we must have a strong research portfolio leading to the clinical sector. Only in that way we are prepared for the future. Dedicated public programmes are devoted to translational medicines. Thus the EU 7 th Framework programme focus within its themes of Health specifically on Translating research L: 4sal\OJB\strategi\ spirekasse april 2006

56 2 for human health, where this activity aims, to increase the knowledge of biological processes and mechanisms in specific diseases situations and transpose this knowledge into clinical applications (2). Also the Technology Platform for Innovative Medicines with its grants focus on prediction of efficacy and safety through work on simulation and biomarkers (3.) Having the base in a Spirekasse covering the translational research I am sure, that I through my European connections and experience with the EU grant system can attract resources to DFU. To host the successful education Master of Industrial Drug Development it is important that DFU has a minimal research base in the disciplines we teach. In this way we create respect for DFU as vendor of such courses - also in the European context. Here it should be noted that the applicant is the course leader/anchor person for Pharmacology, Non-clinical Safety, Clinical Pharmacology and the planned Clinical Trials and Biostatistics courses, all central themes in connection to translational research. Project description The applicant indeed has the vision for what can be done in the field of translational research, but the execution is currently hampered by the size of the research group, and the resources available e.g. Ph.D. students. In this connection it should be recalled that I first joined DFU August With the Spirekasse project I have three aims 1) Conducting scientific projects, 2) Building competences and 3) Supporting integration. Below the planned activities are described for each of purposes. 1. Scientific projects Background The core research investigations of the Spirekasse project are the studies of in vivo pharmacology that have been carried out in my group over the last 3 years centred around neuropathic pain (4-18) Here competencies around reliable models for chronic pain have been established and validated using drugs already applicable in humans. These studies make it possible to conduct the translation of our results to the human situation. New treatments regimens for neuropathic pain Monotherapy represent a dogma in pharmacotherapy which is furthermore supported by the regulatory authorities. The use of a single drug to treat a disease or clinical condition are the preferred way as it can be tested in doubled blinded placebo controlled studies. Testing in the clinic of a combination of two or more medicines, even if they have the rational basis as acting on different targets, is complicated and expensive to carry out. For this reason reliable results from animal studies are demanded before any such trials can be set up in patients. The pharmaceutical industry is not especially interested in such types of combination studies. Studies of this kind have been carried out in my group for better treatment of neuropathic pain. With oxycarbamazine/gabapentin (10) og venlafaxine/gabapentin (11,13,17), in Spared Nerve Injury (SNI) animals tested for allodynia with von Frey filaments and for mechanical hypersensitivity with pin prick test. In the first case with no additive effect (10) In the second study such one was found albeit hampered by adverse effects seen as increased diuresis linked to L: 4sal\OJB\strategi\ spirekasse april 2006

57 3 reduced pain damping (13,17). Accordingly a hypothesis was formulated that the venlafaxine influenced the dynamic effect of gabapentin through a concentration effect. For this reason a pharmacokinetic study of the two drugs linked to pharmacodynamic testing is currently ongoing in rats through a master thesis project (19). A PK/PD investigation in man of the interaction of gabapentin and venlafaxine. Even though that no evidence based studies exist for the combination of venlafaxine and gabapentin (17) it represents a clinical problem as neuropathic patients often are depressive, for which reason they are treated with antidepressiva like venlafaxine while being on their basis pain treatment with gabapentin. In fact in a single human case in combination therapy with gabapentin and venlafaxine, this drug-drug interaction has been observed (Bjerrum OJ, personal observation). A Ph.D study has been formulated for further investigation of this problem in patients with chronic pain (20). Prophylactic treatment of the neuropathic symptoms in rats. So far it has not been possible to avoid the development of neuropathic pain in susceptible patients. However, with our very reliable Spared Nerve Injury (SNI) rat model, where the operation give rise to neuropathic symptoms in nearly 100% of the rats (16) it is easy to test a given prophylactic effect of a pharmacologic intervention. The release of ATP from injured tissue starts the inflammation cascade (21). For this reason it is obvious to inhibit the corresponding P2X7 receptor which are located in nervous tissue to see if this can avoid the development of the neuropathic pain condition. Through collaboration with Dr. John Sharkey, Edinburgh University where Ph.D. student Frederik Rode has been instrumental, a newly synthezised P2X7 antagonist has been put at our disposal. In the first preliminary study, where the antagonist has been given continuously for a week simultaneous with the SNI operation, the compound prevented convincingly the development of neuropathic pain symptoms in the rats (22). An observation we are eager to follow up upon e.g. as a Ph. D project. Translational animal models to predict the human neuropathic pain behaviour to pharmacological prophylaxis. The susceptibility of individual patients to develop a neuropathic pain condition to identical nerve injuries is only 8-10% (18). This points to a genetic disposition for the development of the symptoms. Similarly the patients who have developed the symptoms respond very differently to treatment, for which reason a pharmacogenetic trait is obvious as well. Different rats strains show different susceptibility to both the SNI lesion and the treatment regime (14,15,18). We have undertaken a systematic investigation of a 3 different inbred rat strains for their susceptibility and pharmacologic response to gabapentin, morphine and gaboxadol for being able to have a validated basis material for hunting of the genes that determines the susceptibility of the varions rat strains (14,15,18). When identified the same genes will be mapped in individual patients. The vision is to combine such a gene test with a prophylactic treatment of patients exposed to nerve lesions suspected to give rise to neuropathic pain. Contact has also been established to the decode genetics consortium on Island. (Dr. G. Einarsson) in preparation for a research collaboration via an EU application to FP7. Juvenile animal models for testing drugs for pediatric use. Translational research also include prediction of safety for subsequent human pharmacologic treatment. In a safety study planned together with Bent Halling Sørensen, DFU a Ph.D. application concerning characterisation of hormone disturbing effects in children caused by medicines has been sent in (24). Futhermore, together with Mette Due Theilade, Lægemiddelstyrelsen, a Ph.D. study has been designed for the investigation of the use of piglets L: 4sal\OJB\strategi\ spirekasse april 2006

58 4 as animal model for non-clinical safety testing of drugs for pediatric use. It is avaiting support (25). Pharmacogenetic studies O.J. Bjerrum contributes within pain research to Claus Møldrups project: Rationel Individual Farmakoterapi (26). Furthermore through a collaborative study with Kim Brøsen, SDU research is ongoing regarding CYP2D6 and CYP2C19 drug metabolism among Faroese (27). Therapeutic Drug Monitoring has also been looked at (23). Optimising clinical phase I trials. Through a Ph.D. study with Camilla Bouen in collaboration with Novo Nordisk biostatistical analysis have been performed on 11 completed Phase I studies, where 26 laboratory parameters have been compared to placebo values (28,29). Susceptible parameters to the trial situation e.g. alanintransferase have been identified and their development over time elucidated (28,29). Furthermore, the size of the optimal cohort size has for the first time been determined on rational criteria to comprise 6-8 persons (30). Further validation of the significance of a finding of abnormal laboratory parameters biomarkers is ongoing (31,32). Microdosing for faster access of drug candidates to human application. As consortium member of the Specific Targeted Research Projects EUMAPP-European Union Microdose AMS partnership programme O.J. Bjerrum participate in demonstration of the reliability of the microdosing approach for predicting drug PK when used in pharmacological doses together with the development of related in silico modelling (33). Biosimulation of neuronal activities. As consortium member of the EU Network of Excellence BioSim - Biosimulation. A new tool for drug development O. J. Bjerrum participate in the development of biosimulation of various neuronal activities with reference to pain perception (34-37). 2. Building competencies. The following competencies will be handled in Spirekasse for translational research Handling of animals; Operations of animals; In vivo animal models; Animal observations; Technical equipment to measure animal behaviour; Pharmacokinetic measurements; Pharmacodynamic measurements; safety and non-clinical toxicology; Biomarker research; Biosimulation; Microdosing; Phase I optimisation; Clinical protocols; Application procedures for clinical intervention studies. 3. Supporting integration The applicant is leader of the project group In vitro/in vivo pharmacology at Institute B for which reason it is possible for him to push the groups competencies to approach the clinical aspects. However, it is only when you work with already registered drugs that it is possible directly to proceed into man. At present this is not the case for most of the research conducted in the group, but some of A.-M Heegaards ongoing in vivo experiments concerns such drugs(i.e. bisphosphonate treatment for bone cancer pain) As full member of the proposed Centrale Forskningsområde Neurofarmakologi I can in one end bridge the relevant research projects to the Spirekassen Translational Research and since I L: 4sal\OJB\strategi\ spirekasse april 2006

59 5 am also a member of the proposed Centrale Forskningsområde Optimal Lægemiddelterapi I can also link our research activities with their types of expertises in the other end. Besides, there are other important research areas at DFU to which further integration would be fruitfull. It concerns biomarker research through metabonomics (J. Jaroszewski) and modelling and simulation (C. Cornett and S. Nors Nielsen) which could be involved when suitable projects have been identified.. Budget Two Ph.D stipends would give spirekasse the needed kick off. They should concern 1. Studies related to prophylactic treatment of the development neuropathic pain and 2. The of pathophysiological and clinical examination of the gabapentin/venlafaxine interaction. The latter project will cost more than DKK to conduct for which reason private foundations will be sought. Conclusion With this Spirekasse application I hope to have brought forward evidence for being able to - build up important competences for DFU - prepare DFU for the future - fill a gap in the drug development process - assist further integration - conduct promising research - create network of collaborators (Annex 2) DFU 20. March 2006 Ole J. Bjerrum Professor, dr.med L: 4sal\OJB\strategi\ spirekasse april 2006

60 6 Annex 1 Reference and activities. (1) Aurora A., Bjerrum O.J., Cockburn I.M., Doshi K.J, Hughes J.W., Orsenigo L., Osterloh I., Paoletti R., Preskorn S.H., Weiner E.S. International Perspectives on Health Care and Biomedical Research. The Stream of Progress. The Pfizer Journal 2003,IV, No.2, (2) EU proposal for the 7 th Framework programme for Research, Technology and Development. (3) Strategic Reasearch Agenda for Innovative Medicines for Europe. (4) Rode F. and Bjerrum O.J. Optimizing the spared nerve injury model of neuropathic pain to visualize pharmacodynamics of analgesics A joint meeting of the Danish Society of Clinical Pharmacology and the Danish Society of Pharmacology and Toxicology. Odense, January (5) D.G. Jensen, Rode F. and Bjerrum O.J. Evaluation of spared nerve injured (SNI) neuropathic rats as a tool in studies of morphine and gabapentin effects over time. A joint meeting of the Danish Society of Clinical Pharmacology and the Danish Society of Pharmacology and Toxicology. Odense, January 2004 (6) Rode F. and Bjerrum O.J. Functionality coupled to neurochemical profiling of sensory neurons. A joint meeting of the Danish Society of Clinical Pharmacology and the Danish Society of Pharmacology and Toxicology. Odense, January 2004 (7) Rode F., Jensen D.G. and Bjerrum O.J. Further pharmacological evaluation of the spared nerve injury model. Pharmacodynamic conclusions on the effects of morphine, gabapentine, muscimol and gaboxadol IASP Int. Congress of Neuropathic Pai. Madrid, May 2004 (8) Bjerrum O.J. Neuropathic pain. Characterization in rats of GABA A agonists mechanisms in vivo and in vitro. Department of Medicinal/pharmaceutical Chemistry, Faculty of Life Science, University of Vienna, January 2006 (9) Jensen D.G. Pharmacological Evaluation of the Spared Nerve Injury Model of Neuropathic Pain. DFU, Master thesis, spring Advisor: Ole J. Bjerrum. (10) Grysbæk L.C.H. Præklinisk afprøvning af kombinationsbehandling mod allodyniske smerter i en rottemodel: Tibial Nerve Injury. DFU, Master thesis, spring Advisor: Ole J. Bjerrum. (11) Broløs T. Rode F. and Bjerrum O.J. Co-administration of gabapentin and venlafaxine in a rat model of neuropathic pain. Danish Pharmacology Meeting, Odense, January 2005 (12) Bjerrum O.J. and Rode F. Karakterisering i rotter af GABA a agonisters virkning in vivo og i dorsalrodsganglieceller in vitro. Dansk Selskab for Farmakologi og Toksikologi: Farmakologi i smerteforskning, April 2005 L: 4sal\OJB\strategi\ spirekasse april 2006

61 7 (13) Broløs T. Co-administration of gabapentin and venlafaxine in the spared nerve injury model of neuropathic pain. DFU, Master thesis spring Advisor O.J. Bjerrum. (14) Thomsen M. Investigation of the spared nerve injury model: Development of neuropathic pain and pharmacological sensitivity in genetically different strains of rats. DFU, Master thesis, spring Advisor O.J. Bjerrum (15) Jensen DG., Rode, F. Broløs. Thomsen M.M. and Bjerrum O.J. Three strain of spared nerve injury operated rat shows differences in pharmacological response. 11 th World Congress on Pain SIG Neuropathic Pain, Uluru, Australia, August 2005 (16) Rode F., Gybel D., Blackburn-Monroe and Bjerrum O.J. Centrally mediated anti-noceptive action of GABA A receptor agonists in the spared nerve injury model of neuropathic pain. Eur. J. Pharm., (2005) 516, (17) Frederik Rode, Tine Broløs, Gordon Blackburn-Munro*, Ole J. Bjerrum. Venlafaxine comprises the antinociceptive actions of gabapentin in rats models of neuropathic and persistent pain. Pscopharmacology, submitted 2005 (18) Frederik Rode, Mads Thomsen, Tine Broløs, Dorthe G. Jensen, Gordon Blackburn-Munro*, Ole J. Bjerrum. The importance of genetic background on pain behaviours and pharmacological sensitivity in the rat SNI model of peripheral neuropathic pain. Pain, submitted 2005 (19) Elgaard M.: Pharmacokinetic-pharmacodynamic relation of gabapentin/venlafaxin treatment of spared nerve injury rats with neuropathic pain. DFU, Master thesis project, spring Advisor O.J. Bjerrum. (20) Bjerrum O.J., Christrup, L. og Rømsing J. Gabapentin/venlafaxin interaktion. Klinisk virkning og mekanisk udredning. Ph.D ansøgning DFU, 2005 (21) King B.F.,Townsend Nicholson A. Nucleotide and nucleoside receptors. Tocris Rev ,1-11. (22) Solvind T. and Bihlet A.: Spared nerve injury developed neuropathic pain in rats. Phophylactic treatment with P2X7 antagonists. DFU Master thesis project, spring Advisor O.J. Bjerrum. (23) Bjerrum O.J. and Angelo H.R. (Organisers) Nytteværdi of therapeutic drug monitoring og genetiske tests I patient behandlinger med psykofarmaka DFU, DRA minisymposium. October (24) Sørensen B.H., Bjerrum O.J., Heegaard A.M., Nielsen S.N., Kramer F. Karakterisering af hormonforstyrrende effecter hos børn forårsaget af lægemidler. Ph.D. application, DFU December (25) Non-clinical safety assessment for drugs for pediatric use with special emphasis on juvenile animal models. Ph.D. thesis project application, Supervisors O.J. Bjerrum, Mette Due Theilade, Danish Medicines Agency L: 4sal\OJB\strategi\ spirekasse april 2006

62 8 (26) Claus Møldrup. Rationel Individualiseret Farmakoterapi. Spirekasse proposal. DFU April 2006 (27) Poulsen J. and Petersen, M.S. Drug metabolism among faroese regarding the enzyme activity of CYP2D6 and CYP2C19. Master thesis, spring Advisors. O.J. Bjerrum, K. Brøsen. (28) Buoen C., Scheike T. Bjerrum O.J. and Thomsen M.S. Analysis of historical safety data for evaluation of optimal cohort size in first-in-man studies. Pharm SciFair, Nice, June 2005 Eur. J. Pharm. Sci (29) C. Buoen: Phase I Dose Escalation Trials: An Assessment of current and Novel Trial Designs. Ph.D. thesis planned for June Supervisor O.J. Bjerrum and M.S. Thomsen. (30) Buoen C., Bjerrum O.J. and Thomsen M.S. How first-in man studies are being performed; A survey of phase 1 dose escalation trials in healthy volunteers J. Clin. Pharmacol., (2005) 45, (31) Bjerrum O. J. and Buoen C.: Current trends in translational research regarding microdosing and phase I optimisation. NIH/NIAID-Nordic Regional Research Networking Meeting Helsinki Fair Centre, Helsinki May, (32) Bjerrum O.J.: Use of Integrated Biomarkers. 1 st Int. Course in Biomarkers. Chemical and Bioimagins Endpoints in Cardiocerebrovascular Diagnosis, Prevention Therapy and Drug Development. Lugano, Switzerland October 27-29,2005. (33) EUMAPP. European Union Microdose AMS partnerships programme. EU specific targeted Research project. December July (34) Bjerrum O.J. (organizer) Biosimulation. A tool to examin Biological Function and Predict Drug Action DFU, DRA Minisymposium. March (35) Jelic K., Bjerrum O.J. and Colding-Jørgensen, M. Biosimulation of fatty acid homeostasis. A new in silico tool in drug development EUFEPS 2002 Congress. Stockholm (Eur. J. Phar. Sci. Supplement 17 s (2002), 59-75) (36) Jellic K.: Biosimulation of lipid homeostasis. Ph.D thesis Advisors O.J. Bjerrum, Morten Colling-Jørgensen, Novo Nordisk A/S and Peter Thygesen, Novo Nordisk A/S. (37) BioSim. Biosimulation- A New Tool in Drug Development. EU Network of Excellence. December December L: 4sal\OJB\strategi\ spirekasse april 2006

63 9 Annex 2 Collaborations In vivo animal models Anne Marie Heegaard, Majid Sheykhzade and Bjarne Fjalland, all DFU John Sharkey, Frederik Rode, both Edinburgh University, Steen Christensen University of Copenhagen, Lene Jessen, Novo Nordisk, Jørn Matz, Lundbeck, Gordon Blackburn Monroe, Neurosearch Pharmacokinetics Johan Gabrielsson, Astra Zeneca; Frank Larsen, Lundbeck; Kim Christensen, Novo Nordisk; Per Hartvig, RH; B. E. Lüders Hansen, Dianalund. Bistatistics Thomas Scheike, KU; Camilla Buoen, Novo Nordisk Clinical trials Lona Christrup DFU ; Janne Rømsing DFU; Hanne Villesen DFU; Søren Sindrup SDU; Michael Thomsen, Prosidion ltd.; Camilla Buoen, Novo Nordisk. Microdosing Collin Garner, Xceleron; Anders Granén, Uppsala University: Biosimulering Morten Colding Jørgensen, Novo Nordisk; Steen Ingvarsen Novo Nordisk. Predinical Toxicology Flemming Højelse, Lundbeck, Bent Halling, DFU, Mette Due Theilade, Lægemiddelstyrelsen. Pharmacogenetics Kim Brøsen, SDU; Finn Bengtsson, Linköbing University; Claus Møldrup, DFU L: 4sal\OJB\strategi\ spirekasse april 2006

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66 Forslag til Spirekasseprojekt Åsa Andersson, Inst. for Farmakologi of Farmakoterapi ARE GENES WITHIN THE Eae27 LOCUS ON MOUSE CHROMOSOME 1 IMPORTANT FOR DISEASE DEVELOPMENT IN AN EXPERIMENTAL MODEL FOR RHEUMATOID ARTHRITIS? AIM The aim with this project is to investigate if any of genes within a ~3 Mega-base-pairs (Mbp) region on mouse chromosome 1 is important for development of Collagen Induced Arthritis (CIA), an experimental model for Rheumatoid Arthritis. BACKGROUND Rheumatoid Arthritis (RA) affects approximately 1 % of the grown up population all over the world and gives rise to suffering and large socioeconomic costs. RA is a systemic chronic inflammatory disease that typically affects the wrists and small joints of the hands and feet, eventually leading to deformity and disability. The mechanisms for the initiation of disease are not known, but are the result of a complex interplay between genetic and environmental factors. The definition of genes significantly linked to susceptibility to autoimmune diseases like RA has been a difficult task, because the human population is genetically diverse; there is an influence from the environment; there are possible variations in the diagnosis of disease; and the sample sizes are relatively small. Moreover, polygenic traits like inflammatory diseases are dependent on a number of genetic polymorphisms giving small contributions to the overall disease development. Experimental animal models, resembling human disease, are therefore important tools for studies of complex genetic traits. In experimental models, both the genetics and the environment can be controlled. Susceptibility to RA is strongly correlated with genes within the major histocompatibility complex (MHC), in particular alleles of HLA-DRB-1 (1). Additional, potentially important genes/genetic regions have been described (PADI4, SLC22A4, PTPN22) (2-4), but how these genes contribute to arthritis pathology is at present not clear. Collagen Induced Arthritis (CIA) is a mouse model for RA. The disease process is histologically characterized by infiltration of mononuclear cells into the joint tissue. In this arthritis model the genetic linkage to MHC is strong, but linked non-mhc loci have recently been described (5-8). The B10.RIII mouse strain develops chronic CIA upon immunization with bovine Type II collagen. The strain expresses the MHC H-2 r haplotype, which is also expressed in the RIIIS/J mouse strain. The RIIIS/J strain is resistant to

67 disease induction, meaning that also non-mhc genes are important in disease development. RESEARCH PLAN We have previously investigated the genetic predisposition to inflammatory disease in crosses between the B10.RIII and RIIIS/J strains, and have defined a locus on mouse chromosome 1 (Eae27) linked to development of Experimental Autoimmune Encephalomyelitis (EAE), a rodent model for Multiple Sclerosis (Fig. 1) (9). It is well established that the same genetic regions in mouse or rat are linked to development of disease in more than one model of autoimmune disease, suggesting common disease pathways for this kind of diseases (discussed in 10). In line with this, several loci linked to disease in arthritis and lupus models have been described in this particular region on chromosome 1 (5). In order to investigate shared genetics between EAE and CIA, and to define candidate genes within the Eae27 region, the following plan is proposed: Figure 1. Genetic linkage to remitting EAE in female mice. Chromosome 1 in the mouse Genetic markers Significance level 95% 99% Genes within the linkage peak Congenic fragment Mbp 157,27 D1Mit Lztr2 (leucin zipper transcription regulator 2) Riken cdna (retinoic acid-inducible neuralspecific protein 2) Astn1 (astrotactin) neural migration D1Mit D1mit Pappa2 (pappalysin) proteinase cleaves IGFBP Riken cdna? Unknown gene Riken cdna (similar to ATP synthase) Rfwd2 (ring finger and WD repeat domain 2) homol. Light resp plant prot Tnr (tenascin R) ECM glycoprot RIken cdna? Unknown gene Riken cdna? Unknown gene Tnn (Tenascin N) ECM glycoprot Congenic mice. A chromosome 1 genetic fragment from RIIIS/J has been bred on to the B10.RIII genetic background (Fig. 1). The chromosome 1