1. The zebrafish (Danio rerio),
a new model system for proof of concept studies
Ard 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 k€
Timelines
Rodent POC study:
Human POC study
Danio POC study:
1 – 12 months
6 – 24 months
1 – 5 days
No DEC
Challenge
Acceptance of the models

11. Examples of Danio rerio research within Chardon Pharma
1. 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 screening
Ethanol

13. High throughput behavioral drug screening
Pentylenetetrazole

14. High throughput behavioral drug screening
Buspirone

15. High throughput behavioral drug screening
Measuring blood brain barrier (BBB) penetration
5 dpf
Sibutramine
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 Huntington
No change
KO of potential
target
Behavioral observation
Huntingtin aggregation
Changed
Danio with HD
Assay development
Drug screening
Identified target

18. Target finding for Chorea Huntington
No change
KO of potential
target
Behavioral observation
Huntingtin aggregation
Changed
Danio with HD
10 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 research
Yes
KO of potential
target
Heart regeneration ?
Induction of
heart failure
Assay development
Drug screening
Potential causal
factor
No

21. Heart regeneration research
Yes
KO of potential
target
Heart regeneration ?
Induction of
heart failure
10 days
No

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 stimulators
Available 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 stimulators
Available 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 stimulators
Available 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 stimulators
Available 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
PTH
BMP2A
Alendronate (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
0,92
0,96
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?