1. The zebrafish (Danio rerio), a new model system for proof of concept studiesArd Peeters Chardon Pharma

2. The Product Chardon Pharma evaluates whether an idea or compound has therapeutic potential. This is called obtaining proof of concept (POC).The Approach We scout for new ideas that require proof of concept studies before they can be commercialized (scouting) We invest intellectually in these ideas by bringing in our scientific and drug development expertise (consultancy). We invest in these ideas financially via our business angels network (investment). We perform the proof of concept studies (preclinical, phase I, phase IIA) (CRO). We write and file patents for the ideas (patent application). We bring the inventor in contact with an industrial partner (match making). 3. POC can be obtained both in predictive animal models and in small scale clinical studies. Performing these studies is our expertise. 4. POC can be obtained both in predictive animal models and in small scale clinical studies. Performing these studies is our expertise.A new model system is Danio rerio (the zebrafish) 5. POC can be obtained both in predictive animal models and in small scale clinical studies. Performing these studies is our expertise.A new model system is Danio rerio (the zebrafish) 6. Characteristics of zebrafish - Small, robust freshwater fish - Easy to maintain, high fecundity (200 eggs / week) 7. Characteristics of zebrafish -Small, robust freshwater fish Easy to maintain, high fecundity (200 eggs / week) Most organs fully functional between 3 5 dpf. Larvae are transparent 8. Characteristics of zebrafish -Small, robust freshwater fish Easy to maintain, high fecundity (200 eggs / week) Most organs fully functional between 3 5 dpf. Larvae are transparent Genome fully sequenced Genome, genetic pathways and development highly conserved between zebrafish and humans - Easy genetic manipulation 9. Characteristics of zebrafish -Small, robust freshwater fish Easy to maintain, high fecundity (200 eggs / week) Most organs fully functional between 3 5 dpf. Larvae are transparent Genome fully sequenced Genome, genetic pathways and development highly conserved between zebrafish and humans - Easy genetic manipulation - Large behavioral repertoire 10. Advantages of zebrafish models Costs Rodent POC study: Human POC study: Danio POC study:50 - 100 k 500 10.000 k 0,5 5 kTimelines Rodent POC study: Human POC study Danio POC study:1 12 months 6 24 months 1 5 daysNo DECChallenge Acceptance of the models 11. Examples of Danio rerio research within Chardon Pharma1. High throughput behavioral drug screening 2. Target finding for Chorea Huntington 3. Heart regeneration research 4. Screening of compounds for osteoporosis 12. High throughput behavioral drug screeningEthanol 13. High throughput behavioral drug screeningPentylenetetrazole 14. High throughput behavioral drug screeningBuspirone 15. High throughput behavioral drug screening Measuring blood brain barrier (BBB) penetration 5 dpfSibutramine 5 dpf: ED50 < 0,01mg/ml 12 dpf: ED50 > 0,1mg/ml 12 dpf 16. Target finding for Chorea Huntington Collaboration with EHDN- Autosomal dominant neurological disorder - CAG repeat in huntingtin gene - Huntingtin aggregates leading to neurodegeneration- Transgenic zebrafish model - Different CAG repeat in huntingtin gene - Huntingtin aggregates 17. Target finding for Chorea HuntingtonNo changeKO of potential target Behavioral observation Huntingtin aggregationChangedDanio with HDAssay development Drug screeningIdentified target 18. Target finding for Chorea HuntingtonNo changeKO of potential target Behavioral observation Huntingtin aggregationChangedDanio with HD10 days 19. Heart regeneration research Funded by MIT; Collaboration with Duke university- Heart attacks induce muscle damage and heart failure - Human heart muscle cannot regenerate - Zebrafish heart muscle has regenerative capacity. 20. Heart regeneration researchYesKO of potential targetHeart regeneration ? Induction of heart failureAssay development Drug screeningPotential causal factorNo 21. Heart regeneration researchYesKO of potential targetHeart regeneration ? Induction of heart failure10 daysNo 22. Screening of compounds for osteoporosis Bone content = bone formation (osteoblasts) bone degradation (osteoclasts)Osteoporosis: Bone content = bone formation bone degradation Osteoporosis treatment Bone content = bone formation bone degradation 23. Screening of compounds for osteoporosis New treatment option:Osteoblast stimulatorsAvailable testmodels: 1. Human osteoblast culture (no interaction with osteoclasts) 2. Rodent model (invasive, time consuming, costly) 24. Screening of compounds for osteoporosis New treatment option:Osteoblast stimulatorsAvailable testmodels: 1. Human osteoblast culture (no interaction with osteoclasts) 2. Rodent model (invasive, time consuming, costly)The alternative Danio rerio 25. Screening of compounds for osteoporosis New treatment option:Osteoblast stimulatorsAvailable testmodels: 1. Human osteoblast culture (no interaction with osteoclasts) 2. Rodent model (invasive, time consuming, costly)The alternative Danio rerio 26. Screening of compounds for osteoporosis New treatment option:Osteoblast stimulatorsAvailable testmodels: 1. Human osteoblast culture (no interaction with osteoclasts) 2. Rodent model (invasive, time consuming, costly)The alternative Danio rerio 27. Screening of compounds for osteoporosis PTHBMP2AAlendronate (bisphosphonate) 28. Screening of compounds for osteoporosis table 1. Osteogenic effect of substances involved in the WNT-signaling pathway. Red arrows indicates a negative effect (4 decreases or 3 decreases and one neutral effect). Green arrows indicate a positive effect (4 increases or 3 increases and one neutral effect). Compound name effect Compound name effect Compound name effect foxy-5 1,04 Box5 1,07 CCT036477 anandamide 0,78 TNP-470 0,86 Busutinib QS-11 0,93 IWR-1 1,15 Carnosol diarylsulfonesulfonamide 0,19 XAV939 0,88 Favanone IQ1 1,17 IWP-2 0,90 Retinoic acid SB-216763 0,71 Bafilomycin A1 0,81 Pterostilbene BIO 0,93 Sulindac Sulfide 1,28 Hexachlorophene TWS-119 0,62 Celecoxib 1,58 DHA CHIR99021 1,08 Diclofenac.Na 1,20 EPA IM-12 1,00 Niclosamide 1,56 JS-K AR-A014418 1,17 ICG-001 0,66 Pyrvinium pamoate kenpaullone 0,77 Harmine.HCl 0,44 Apigenin sodium valprolate 0,58 Curcumin 0,85 Ellagic acid dihydrate BML-284 0,77 PNU-74654 0,80 D4476 Deoxycholic acid 0,76 Quercetin 0,68 Trichostatin A Reservatrol 0,91 BML285 0,52 5-Aza-2-deoxycytidine PGE2 0,87 NO-ASA 0,64 Thalomide WAY-262611 0,92 FH-535 0,71 Genistein Purpurogallin 0,85 Val-Val-Val 0,75 PP2 Exifone 0,79 BML286 0,95 NCI16221 Gallic acid 0,65 Sulindac 1,62 Doxorubicin.HCL Riluzole 1,42 EGCG 0,73 3253-5986 LY456236.HCL 0,53 imatinib mesylate 1,01 Usnic Acid (-)-Terreic acid 0,80 Troglitazone 0,64 Forskolin 0,77 Rosiglitazone maleate 0,920,96 0,72 0,94 0,96 0,95 0,81 0,82 0,61 0,99 1,13 0,91 1,38 1,29 0,60 0,31 1,20 1,51 1,39 1,35 0,75 0,25 1,25 0,99 29. Questions?Ideas for collaboration?