“N2MO provides cutting-edge ex vivo insect based screening models for predicting ADMET properties of chemical substances in the early drug discovery phase. Insect models represents excellent model systems due to biological active test situation, combined with fast reproducibility with no need for resynthesizing compound and at same time the model produces data that are reflecting mammalian ADMET properties”

1. - CONFIDENTIAL -
A platform for faster, better and more ethical
decisions in drug discovery
June 2015
- CONFIDENTIAL -

2. - CONFIDENTIAL -
• Activities established in 2009
• Documentation of ex vivo Models via:
a) Support from Copenhagen University and Højteknologi Fonden
b) Scientific collaboration with :
• 8 Global Patents on Insect Models granted
• 2014 N2MO secure sustainable funding
• Scientific and Commercial organisation created
Status update on N2MO

3. - CONFIDENTIAL -
Snapshot of R&D Development
Challenge:
The pharmaceutical industry experience three key
challenges in drug development:
a) COST > 1B$
b) TIME > 14 Years
c) FAILURES > 95%
Industry response:
• Need for high quality data in early phase to
reduce number of failures in later stages
• Expand partnerships with experienced CROs
• Reporting reduction in number of test animals

4. - CONFIDENTIAL -
Why incorporate an Ex Vivo Model
Compound
design
Synthesis
Data
evaluation
Progression
Y/N
Data
feedback
Testing

5. - CONFIDENTIAL -
N2MO provides state of the
art brain permeability models
that can be used to
discriminate CNS from non-
CNS compounds in early
screening.
The locust BBB rate model
compares favorably to
MDCK cellular systems in
predicting in vivo
brain/plasma ratios and has
the advantage of reducing
false positives.
Small volume is used so no
resynthesizing of
compound.
The locust BBB ex vivo
model is a fully 3R´s
compliant animal model
offering a real opportunity
for ethical improvement
Unique model using intact
brains
Resource & time savings Ethical improvement
Summary

6. - CONFIDENTIAL -
Unique characteristics of the ex vivo locust BBB model
• Intact whole brain under ‘in vitro’ conditions
• Assessment of brain uptake at constant exposure
• No vascular system and no residual compound
• Full control of the extracellular matrix
a) Time
b) Concentration
c) Temperature
• Test on ready-made stock solutions (100ul of 1mM DMSO stock solution)
• Medium throughput screening model
• High reproducibility

7. - CONFIDENTIAL -
Positioning of N2MO Ex Vivo model –
Current Drug Discovery Process
Chemical
compounds
In vitro potency
Physiochemic
al ADME
In Vivo
PK
B/P
Kpuu
PD
Etc…
Solubility
LogD
Mics
Hepatocytes
PPB
MDCK – mdr1
Etc….
IC50NCEs
Library
Cells, biochemical,
or phys chem
We want to
reduce the
gap from in
vitro to in vivo

8. - CONFIDENTIAL -
Positioning of N2MO Ex Vivo model –
Substitute Single Point Rodent test & MDCK / MRD1
Chemical
compounds
In vitro potency
Physiochemic
ADME
In Vivo
PK
B/P
Kpuu
PD
Etc…
Solubility
LogD
Mics
Hepatocytes
PPB
MDCK – mdr1
Etc….
IC50NCEs
Library
BBB:
Permeability
Rate/Extend
B/P
Kpuu
FUB via mice
brain concentrate
N2MO Ex Vivo
N2MO Ex Vivo BBB model is suggested to be used instead of MDCK & mdr1 tests as well as in
Single Point rodent test. Firstly run in parallel to document data quality – later as stand alone
N2MO
Ex Vivo

9. - CONFIDENTIAL -
How do insect models add to drug discovery?

10. - CONFIDENTIAL -
The locust

11. - CONFIDENTIAL -
Experimental setup of the ex vivo locust brain barrier model

12. - CONFIDENTIAL -
The Ex vivo model
The model standard setup
• 3uM +/- verapamil at 15 minutes (+ 10uM
to identify other transporter substrates)
o Additional features can be added
i.e. time, concentration, temperature
• Test on ready-made stock solutions
(100ul of 10mM DMSO stock solution)
• 1.5% DMSO in final test solution
• Intact whole brain under ‘in vitro’
conditions
• Assessment of brain uptake at constant
exposure
• No vascular system and no residual
compound
• Full control of the extracellular matrix
o Time
o Concentration
o Temperature
• High reproducibility

13. - CONFIDENTIAL -
Physiology: The Brain
Pars intercerebralis
Kenyon cells of mushroom body
Accessory lobe
Central complex
Antennal lobe
Peripheral glia and nerve cells
• High content of nerve
cords and cells – as in
hum/mice/rats
• High content of lipids
in insect brains – as in
hum/mice/rats
• Tissue binding has
large impact on brain
uptake in both insects
and hum/mice/rats
Andersson et al. (manuscript)
In collaboration with University of Lund, Kings
College, University of Leuven, University of
Copenhagen

14. - CONFIDENTIAL -
Physiology: The Brain
• Brain barrier consists
of glia cells
• Homologus tight
junctions
• Tight brain barrier that
regulates brain uptake
both in insects and
hum/mice/rats
Andersson et al (in manuscript)
Daneman and Barres, 2005 (Cell); Mayer et al., 2009
(J Neurosci)
Glia cells and neurons
Tight/septate junction
Hemolymph
Neural lamella
Perineural glia cell
Basal laminaAstrocyte
Tight junction
Endothelial cell
Capillary lumen
Glia cells and neurons
C D
In collaboration with University of Lund, Kings
College, University of Leuven, University of
Copenhagen

15. - CONFIDENTIAL -
Transporter proteins in the locust
Human ABCB Genes in Locusta Migratoria
Gene Length ATPase
domains
TM
domains
Human
Homolog
Length % Identity
CL2746 1282 2 2 ABCB1 1280 48
CL3788 835 1 1 ABCB6 842 51
CL4845 784 1 1 ABCB7 752 54
CL5917 598 1 1 ABCB8 630 56
Unigene21195 660 1 1 ABCB10 595 54
Recent transcriptome studies
- Al-Qadi et al. 2015 (Athens Journal of
Health)
- Andersson et al 2014 (PRP)
- DeSalvo et al. 2014 (Frontiers
Neurosci.)
“Surface glia show high expression of many ATP-binding cassette (ABC) and solute
carrier (SLC) transporters, cell adhesion molecules, metabolic enzymes, signaling
molecules, and components of xenobiotic metabolism pathways”
DeSalvo et al. 2014

16. - CONFIDENTIAL -
Transporter proteins in the locust
Locusta Migratory Drosophilia homolog Human homolog Rat homolog
Name Gene Length Gene Length Identity % Gene Length Identity % Gene Length Identity %
Pgp CL2746 1282 Mdr 49 1302 52 ABCB1 1280 48 ABCB1 1275 47
BCRP
Unigene
18639
631 CG3327 854 54 ABCG2 611 40 ABCG2 657 41
CL5908 712 CG3164 699 70
ABCG1
ABCG8
674 48 ABCG1 666 60
CL5917 598 CG1824 761 54 ABCB8 630 58 ABCB8 714 58
• Transporter effects can be evaluated under ex vivo conditions, e.g. saturation under
constant exposure, temperature, etc.
• Insect barrier contains more central transporters
• Genuine brain with interplay between different transporters
- Al-Qadi et al. 2015 (Athens Journal of Health)
- Andersson et al 2014 (PRP)
- DeSalvo et al. 2014 (Frontiers Neurosci.)

17. - CONFIDENTIAL -
Inhibition of Pgp efflux by verapamil
Incubation concentration 3µM
Incubation time 5 minutes
• Co-administration with Verapamil increases the brain levels of the Pgp substrates
• Verapamil does not affect the uptake of passively diffusing compounds
• Pgp substrates can be identified by experiments +/- addition of Verapamil

18. - CONFIDENTIAL -
Identification of Pgp substrates by using +/- verapamil
Pgp substrates are determined by: Cbrain(3uM exposure, 25uM verapamil) / Cbrain(3uM exposure, no verapamil)
Non Pgp substrates Pgp substrates
Cimetidine Cetirizine
Warfarin Quinidine
Bupropion Risperidone
Carbamazepine Loperamide
Ranitidine Citalopram
Caffeine Propranolol
Norfloxacin Desipramine
CsA Dexamethasone
Digoxin
• Pgp substrates can be identified by calculating the ratio of brain concentration obtained with and without verapamil
• Pgp substrates are characterized by (Cbrain(3uM exposure, 25uM verapamil)/Cbrain(3uM exposure, no
verapamil)) >1.5

19. - CONFIDENTIAL -
Identification of transporter substrates – temperature
Incubation concentration 3 µM
Incubation time 15 minutes
Andersson et al 2014 (PRP)
• For passively diffusing compounds the brain uptake is increased at increased temperature
• For Pgp substrates the brain uptake is reduced at increased temperature due to increased efflux activity
• By blocking the transporter the brain uptake of Pgp substrates increase at increased temperature

20. - CONFIDENTIAL -
Validation of the ex vivo brain barrier model – permeability
25 reference compounds included
a. 13 known CNS drugs
b. 12 known peripheral
c. Class: 14 basic; 1 acidic; 8 neutrals; 2
zwitterions
d. MW: between 194 and 1200 g/mol
e. LogP (experimental): between -2.2 and 5.5
Andersson et al, 2013 (JPET)

21. - CONFIDENTIAL -
The locust ex vivo brain barrier model – permeability
0
1
2
3
4
5
6
7
8
9
Cbrain (mM)
Exposure concentration 3mM at 15 min
• Insects have a tight brain barrier
• Insects brains exclude peripheral drugs and include central acting drugs
Blue ~ CNS drugs
Red ~ Peripheral drugs

22. - CONFIDENTIAL -
0,1
1
10
100
-5 -4 -3 -2 -1 0
MDCK…
Rate: Validation of the ex vivo brain barrier model
Log PS In situ rat perfusion
Peff(10-6)
0,1
1
10
100
-5 -4 -3 -2 -1 0
Grasshopper
@ 10uM
cBrain(mM)
Log PS In situ rat perfusion
Warfarin
Amitriptyline
Loperamide
Fluoxetine
Risperidone
Quinidine
Warfarin
Amitriptyline
Loperamide
Fluoxetine
Risperidone
Quinidine
Andersson et al, 2013 (JPET)

23. - CONFIDENTIAL -
Rate versus extent
Di et al, Exp Opinion 2009

24. - CONFIDENTIAL -
Rate versus extent
”Compounds can have high rate but low extent and high rate and low extent”
Di et al. Exp Opinion 2009

25. - CONFIDENTIAL -
Extent: BP in vivo (rodents) versus BP ex vivo (insects)
• Preferably B/P should be measure by using
AUC
t1 t2
Concentration
Time
x
x
x
x
x
o
o
o
o
o
t3 t4 t5
o: plasma concentration
x: brain concentration
Concentration Time
Incubation concentration
Brain concentration
In rodents In insects
• In insects the exposure concentration is constant

26. - CONFIDENTIAL -
Extent: Considerations on extent - rodents
“Time to equilibrium is governed by the product of PS and fu,brain. The time to equilibrium
can be assessed by the time to reach the plateau of BP-time profiles” (Liu 2005 JPET)

27. - CONFIDENTIAL -
Extent: Ex vivo brain uptake at 15 and 30 minutes
0
0,5
1
1,5
2
2,5
-5 15 35
mM
Caffeine
Quinidine
0
2
4
6
8
10
12
-5 15 35
mM
Minutes
Paroxetine
Loperamide
0
2
4
6
8
10
-5 15 35
mM
Cbz
Risperidone
MinutesMinutes
Ratio of uptake at 30 minutes over uptake at 15 minutes:
Paroxetine: 2.7
Loperamide: 2.3
Cbz: 1.8
Risperidone: 2.1
Caffeine: 1.2
Quinidine: 1.3
Fub < 1%Fub ~ 10%Fub > 30%
Paroxetine 4.2
Loperamide 4.7
Cbz 2.4
Risperidone 2.7
Caffeine 0.0
Quinidine 2.8
cLogP:

28. - CONFIDENTIAL -
Extent: ex vivo brain uptake 30 versus 180 minutes
0
2
4
6
0 20
mM
0
1
2
3
0 100 200
mM
0
1
2
3
0 20
mM
0
10
20
0 100 200
mM
0
10
20
0 20
mM
0
5
10
15
0 100 200
mM
Minutes
0
5
10
15
0 20
mM
MInutes
CaffeineCbz
Loperamide
0
2
4
6
0 100 200
mM
0
5
0 100 200
mM
0
20
40
60
0 100 200
mM
Minutes
Paroxetine
RisperidoneQuinidine
-2
3
8
0 20
mM
-10
10
30
50
0 20
mM
Minutes
-> 180 minutes -> 30 minutes -> 180 minutes -> 30 minutes

29. - CONFIDENTIAL -
Correlation vs. locust ex vivo model and rodent in vivo B/P
0,01
0,10
1,00
10,00
0,01 0,1 1 10
Ctot
B/P
B/P in rodents
B/P is measured using single time-point and not by
AUCs; Single dosing
Ctot ex vivo locust
Ctot (3 uM, 15 min) and constant exposure ~ infusion

30. - CONFIDENTIAL -
Ctot(insects)*Fub(rodents) vs Kpuu(rodents)
0,00
0,01
0,10
1,00
10,00
0,00 0,01 0,10 1,00 10,00
Lundb
eck
Ctot*Fub
Kpuu(rodents)
Estimated Kpuu in rodents (Fub/Fup*B/P)
B/P is measured using single time-point and not by AUCs;
Single dosing
Estimated Kpuu in insects (Fub*Ctot)
Ctot (3 uM, 15 min) and constant exposure ~ infusion

31. - CONFIDENTIAL -
Summary
• Biological Active Intact Brain - BBB model
• Constant exposure conditions – Steady State
• 100ul of 1mM DMSO stock solution – No re-synthetization
• Fast Access to Data
• Brain Availability Model
• Rate and/or Extent profile of compounds
• Transporter Affinity Profile

32. - CONFIDENTIAL -
More information
Visit our website
http://n2mo.co
Here you will find:
• Description of our BBB models
• Scientific articles & papers
• Information on conferences we
attend
• Etc.

33. - CONFIDENTIAL -
Thank you for your time
Presented by Esben Bruun Mortensen

34. - CONFIDENTIAL -
Back up slides

35. - CONFIDENTIAL -
Pgp transporter in the locust
Pgp homology
• Overall 37-47% sequence identity between human and grasshopper
• Residue homology in specificity residues 61% (specificity residues from Becker et al. BMC Structural
Biology 2009)
• Residue homology 50-72% in binding site (binding site info from Becker et al. BMC Structural Biology
2009)
Vanden Broeck et al
(Uni Leuven)
Schiøtt and Al-Qadi (Uni Cph); Andersson 2014, (PRP)

36. - CONFIDENTIAL -
Update on invertebrates
The invertebrate BBB
is formed by
subperineurial glia
(SPG), the perineurial
glia (PG), and a
basement membrane
i.e. the neural lamella
(NL)
The vertebrate BBB is
formed by vascular
endothelial cells (VE),
pericytes within the
basement membrane
(BM), and the end feet
of the astrocyte glia
Andersson et al 2014
(PRP)
Review by Hindle and Bainton 2014 (Frontiers Neurosci.)

37. - CONFIDENTIAL -
“The invertebrate blood-brain barrier (BBB) field is growing at a rapid pace and, in recent years, studies have shown a
physiologic and molecular complexity that has begun to rival its vertebrate counterpart.”
Review brain physiology
Pgp mechanism
Examples on invertebrate 2014 litterature 1 of 2

38. - CONFIDENTIAL -
“sequence-based alignments, we compare the Drosophila and Murine BBB transcriptomes and discover many shared
chemoprotective and small molecule control pathways, thus affirming the relevance of invertebrate models for studying
evolutionary conserved BBB properties”
Examples on invertebrate 2014 litterature 2 of 2
ATP-binding cassette
(ABC), solute carrier (SLC)
transporters
Gap junctions

39. - CONFIDENTIAL -
Recommended use of the ex vivo model
1. Ctot/incubation concentration at 15 min
1. Is the compound in the brain and to what degree? Use to prioritize compounds.
2. Ctot together with PPB (fup) to select the compounds likely to have highest Kpuu.
Used to rank compounds.
3. Use Ctot30/Ctot15 and cLogD to classify compounds – the higher the ratio and
cLogD the higher the expected brain tissue binding
4. Use Ctot15 + fup + Ctot30/Ctot15 + cLogD (and data such as microsomal stability) to
identify compounds with optimal Kpuu
Per discussion with Peter Aadal, N2MO

40. - CONFIDENTIAL -
0
5
10
15
20
25
30
35
40
45
Fluvoxami…
EPM56
EPM65
Paroxetine+
EPM74
Fluoxetine+
Olanzapine
Carbamaz…
Amitriptyli…
EPM69
Haloperidol+
EPM37
Spiropitan
Desiprami…
EPM30
Bupropion+
Trazodone+
Risperido…
EPM78
EPM38
EPM11
Propranolol
EPM59
Bromocrip…
EPM60
EPM46
EPM39
Caffeine+
Warfarin+/-
Citalopram
EPM13
EPM49
EPM44
EPM73
Loperamide-
EPM76
EPM61
EPM58
Quinidine-
EPM34
EPM16
Cetirizine-
EPM66
EPM70
EPM63
CsA
EPM62
Dexameth…
EPM36
Norfloxacin-
Digoxin-
EPM33
EPM57
EPM54
EPM42
Cimetidine-
EPM71
Lincomycin-
EPM64
EPM64
Ranitidine-
Methotrex…
VERAPA…
EPM32
EPM29
EPM79
Atenolol-
EPM27
Transporter saturation at 10mM
Cbrain (mM)
Blue ~ CNS drugs
Red ~ Peripheral drugs
• Insects have a tight brain barrier
• Insects exclude peripheral drugs and include central acting drugs
• Transporter saturation is possible
Exposure concentration 10mM at 15 min
Pgp substrates