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1CONFIDENTIAL
Eribis Pharmaceuticals AB
Novel Treatment ofAcute Myocardial Infarction (AMI)
2CONFIDENTIAL
Todays agenda
The Company
The indication – background & market
EP94
Pre-clinical Development
General Properties
Clinical Development
Intellectual Property
Funding need
3CONFIDENTIAL
The Company
4CONFIDENTIAL
The Company
Eribis Pharmaceuticals AB is an Uppsala-based biotech company founded in 2006, with the aim to develop new therapies for cardiovascular disease.
5CONFIDENTIAL
Gerhard Wikström, MD, Ph.D., Ass Prof Cardiology, Uppsala University Hospital
Lars Grip, MD, Ph.D., Professor in Cardiology, Sahlgrenska University Hospital
John Pernow, MD, Ph.D., Prof in Cardiology, Karolinska University Hospital
Garrett Gross, Ph.D.,Professor of Pharmacology, Medical College of Wisconsin
Dan Atar, MD, Ph.D., Professor in Cardiology, Division of Cardiology, Oslo University Hospital Aker
Scientific Advisors
6CONFIDENTIAL
Acute Myocardial Infarction (AMI)
background and market
7CONFIDENTIAL
Cardiovascular disease (CVD)the major health problem
CVD include; acute coronary syndromes (ACS) stroke periphereal arterial disease (PAD)
Burden of CVD is increasing in parallel with increase in life expectancy
Acute Myocardial Infarction (AMI) is the number one cause of death in the Western world mortality rates near 10% first cause of chronic heart failure
8CONFIDENTIAL
Acute coronary syndrome (ACS) is comprised of three diseases involving the coronary arteries: ST elevation myocardial infarction (STEMI), non ST elevation myocardial infarction (non-STEMI) and unstable angina.
While ACS mortality declined in the last four decades in the USA as life expectancy increased, the decline largely represents the postponement of ACS deaths until older age.
Thus, the burden of ACS is increasing in parallel with the increase in life expectancy.
Acute coronary syndrome (ACS)
9CONFIDENTIAL
Coronary Artery By-pass Graft (CABG)
Percutaneous Coronary Intervention (PCI)
Reperfusion therapyPrimary Treatment Options for AMI
Across the seven major markets in 2006 the number of cardiovascular procedures, including CABG and PCI were estimated at 7.0m
Incidence rates for PCI amounts to 3.7m (nonSTEMI + STEMI)
10CONFIDENTIAL
The American Heart Association (AHA) estimates that there were 1.2 million cases of ACS in 2007 in the US. This number includes both patients experiencing their first event as well as patients with relapses. Patients’ experiencing their first event is around 700,000; STEMI and NSTEMI accounting for 85% of these events. This incidence is similar to that in the five major markets in Europe. The total number of ACS cases in the US and the five major EU markets is therefore estimated at around 2.5 – 3.0 million annually.
Large unmet needAcute Coronary Syndrome – the major markets
11CONFIDENTIAL
Metabolic and biochemical changes caused by reperfusion
Reperfusion injury (RI) limits the beneficial effects of revascularisationStudies in different species suggest that 20-70% of myocardial damage may come from RI(Gomes L 2007, Penna C 2008, Zhao ZQ, 2006)
RI leads to functional and structural myocardial damage– generation of ROS– a fast restoration of pH– intracellular calcium overload– mitochondrial dysfunction– Apoptosis
(Yellon DM, 2007)Cardioprotective strategies to minimize RI represents an large unmet medical need
12CONFIDENTIAL
Principles of ischemic pre- and postconditioning
Brief episodes of coronary artery occlusion are applied either before (preconditioning) or immediately after (postconditioning) the prolonged ischemic insult
Ref; Zhao ZQ et al., Am J Physiol Heart Circ Physiol, 2003;285:579-88
13CONFIDENTIAL
EP94
Cardioprotection in acute myocardial infarction
(AMI)
14CONFIDENTIAL
EP94 – First in class drug
Novel pharmacological cardioprotective intervention Tetrapeptide (stabilized) Injectable, small volume Microgram dosage Cardioprotective effects in rodent and non-rodent
animal models Low COGS (solid phase peptide synthesis) Backup compounds available Patent protected
15CONFIDENTIAL
Tentative molecular signaling pathways involved in cardioprotection
16CONFIDENTIAL
Mode of Action
Acute Myocardial Infarction induces an ischemia/reperfusion-induced tissue damage
It is anticipated that EP94’s mode of action reduces the ischemia/reperfusion damage through
opoid receptors KATP channels iNOS
17CONFIDENTIAL
Preclinical results
18CONFIDENTIAL
• Rodent dose finding study• Pig study, closed chest, iv.• Pig study, open chest, infusion• Pig study, closed chest, dose finding, iv. • Pig study, closed chest, high-dose range
finding, iv. • Rodent study, dose finding, m.o.a. study
Key studies presented
19CONFIDENTIAL
EFFICACY
• EP94 is effective in two different pig ischemia/reperfusion models (open vs closed chest)
• EP94 produces a significant reduction of infarct size in pigs already at 1 µg/kg
• The cardioprotective effect of EP94 is dose-dependent in pigs
• EP94 reduces the infarct size dose-dependently during both pre- and post-conditioning in rats
Conclusions
20CONFIDENTIAL
Post-surgerystabilizationphase
Vehicle
EP94 (0.01 g/kg x 1)
25 min occlusion
2 hrs reperfusionEP94 (0.1 g/kg x 1)
EP94 (1 g/kg x 1)
25
EP94 (5 g/kg x 1)
0
= Vehicle administration
= EP94 administration-5
EP94 shows a dose dependent reduction of myocardial infarction size in rats
EP94 (10 g/kg x 1)
0
10
20
30
40
50
60
Vehicle
EP94 (0.01 ug/kg)
EP94 (0.1 ug/kg)EP94 (1 ug/kg)
EP94 (5 ug/kg)
EP94 (10 ug/kg)
IS/A
AR (%
)
N=12 N=6 N=12 N=12 N=12 N=9
* * * *
Study number 64-06
Pre-conditioning
21CONFIDENTIAL
EP94 i.v. administered early or late during ischaemia in a closed chest MI pig model reduces myocardial infarction size significantly
Post-surgerystabilizationphase
Vehicle
EP94 (1 g/kg x 4) – Early intervention
40 min occlusion
4 hrs reperfusion
60 min
EP94 (1 g/kg x 3) – Late intervention
EP94 (0.2 g/kg x 3) – Late intervention
5 12 19 26 33 40
01020304050607080
IS/A
AR
(%
)
N =7 N =4 N =6 N =6
Early Late Late
Mean ± SEM
***
Study Report 09-01, Karlsson et al., Eur J Pharmacol 2011:651 pp146-151
= Vehicle administration
= EP94 administration
Closed chest pig MI/reperfusion model
22CONFIDENTIAL
Intracoronary infusion of EP94 in pigs reduces myocardial infarction size significantly
Post-surgerystabilizationphase
Vehicle
EP94 (0.2 g/kg, 15 min)
40 min occlusion
4 hrs reperfusion
30’ 5’
0
10
20
30
40
50
60
70
80
90
100
Vehicle EP94
% o
f ar
ea a
t ri
sk
Mean ± SEM
**
N = 5 N = 5
Study Report 08-02, Karlsson et al., Eur J Pharmacol 2011:651 pp146-151
60 min
Open chest pig MI/reperfusion model
Infusion
23CONFIDENTIAL
EP94 appears to reduce myocardial infarction size dose-dependently
Post-surgerystabilizationphase
Vehicle
EP94 (1 g/kg x 3)
40 min occlusion
4 hrs reperfusion
60 min
EP94 (5 g/kg x 3)
EP94 (25 g/kg x 3)
26 33 40
0
10
20
30
40
50
60
70
Vehicle 1 ug/kg 5 ug/kg 25 ug/kg
IS/A
AR
(%
)
N=6 N=6 N=6 N=6
*
= Vehicle administration
= EP94 administration
Study Report 11-02
Mean ± SEMLinear regression analysis,* P < 0.05
Closed chest pig MI/reperfusion model
24CONFIDENTIAL
Rat acute myocardial infarction model – a combined dose response and mode of action study
Post-surgerystabilizationphase
Vehicle
EP94 (0.1 g/kg x 2)
25 min occlusion
2 hrs reperfusionEP94 (1.0 g/kg x 2)
EP94 (2.5 g/kg x 2)
105
EP94 (5.0 g/kg x 2)
EP94 (10 g/kg x 2)
-10 0
1) Opioid antagonists i.v.a) Naltrindole (delta) – 5 mg/kgb) Nor-BNI (kappa) – 0.3 mg/kgc) CTOP (mu) – 0.1 mg/kg
d) Naltrindole + EP94e) Nor-BNI + EP94f) CTOP + EP94
2) KATP channel antagonists i.v.a) HMR1098 (sarc KATP blocker) – 6 mg/kgb) 5-HD (mito KATP blocker) – 10 mg/kg
d) HMR1098 + EP94e) 5-HD + EP94
3) iNOS inhibitor i.v.a) 1400W
b) 1400W + EP94
= Vehicle administration
= EP94 administration
Study protocol 2010-01
EP94 (0.5 g/kg x 2)
25CONFIDENTIAL
Rat acute myocardial infarction model – a combined dose response and mode of action study - cont´d
0
10
20
30
40
50
60
70
Vehicle 0.1 0.5 1.0 2.5 5.0 10.0
EP94 (ug/kg, i.v.)
IS/A
AR
(%
)
N = 9 N = 8 N = 10 N = 10 N = 8 N = 9 N = 8
*** *****
Mean ± SEM*** p < 0.001 vs vehicle ** p < 0.01 vs vehicle
Bell-shaped dose-response relationship in rat confirms previous results in rats
26CONFIDENTIAL
Blocking of mu-opioid receptors abolish the cardioprotective effects of EP94
Mean ± SEM*** p < 0.001 vs vehicle ** p < 0.01 vs vehicle
CTOP = selective mu-antagonistNT = naltrindole = selective delta-antagonistNor-BNI = selective kappa-antagonist
Rat acute myocardial infarction model – a combined dose response and mode of action study - cont´d
0
10
20
30
40
50
60
70
Vehicl
e
EP94 (1
ug/
kg)
CTOP (0.1
mg/
kg)
CTOP+EP94
NT (5 m
g/kg
)
NT+EP94
Nor-BNI (
0.3
mg/kg)
nor-B
NI+EP94
IS/A
AR
(%
)
N = 9 N = 10 N = 9 N = 9 N = 9 N = 9 N = 9 N = 9
*** ** **
mu-receptor delta-receptor kappa-receptor
27CONFIDENTIAL
The non-selective opioid receptor antagonist naloxone completely inhibits the cardioprotective effects of EP94 – pharmacological evidence of the involvement of mu-receptors in the central nervous system
Mean ± SEM*** p < 0.001 vs vehicle
Rat acute myocardial infarction model – a combined dose response and mode of action study - cont´d
0
10
20
30
40
50
60
70
Vehicle
EP94 (1 ug/kg)
Naloxon (3 mg/kg)
Naloxon+EP94
Naloxon methiodide (10 mg/kg)
Naloxon methiodide+EP94
IS/A
AR
(%
)
N = 9 N = 11 N = 9 N = 9 N = 9 N = 9
*** ***
Block of peripheral and
CNS opioid receptors Block of peripheral
opioid receptors
Naloxone = non-selective opioid antagonistNaloxone methiodide = quaternary-derivative of naloxone,
does not penetrate blood-brain barrier
28CONFIDENTIAL
Mean ± SEM*** p < 0.001 vs vehicle
Effects of selective KATP ion channel antagonists
HMR 1098 = sarcolemmal KATP channel antagonist5-HD = mitochondrial KATP channel antagonist
0
10
20
30
40
50
60
70
Vehicl
e
EP94 (1
ug/
kg)
HMR10
98 (6
mg/
kg)
HMR10
98 +
EP94
5-HD (1
0 mg/
kg)
5-HD +
EP94
IS/A
AR
(%
)
N = 9 N = 9N = 9N = 10 N = 9 N = 9
***
Rat acute myocardial infarction model – a combined dose response and mode of action study - cont´d
Block of sarcolemmal KATP
channels Block of mitochondrial KATP
channels
29CONFIDENTIAL
Mean ± SEMn = 9/group*** p < 0.001 vs vehicle
Effects of inducible NOS (iNOS) inhibition on EP94-induced cardioprotection
1400W = selective iNOS inhibitor
0
10
20
30
40
50
60
70
Vehicle
EP94 (1 ug/kg ipx2) - 24-h
1400W (0.1 mg/kg iv) - acute
1400W (acute) + EP94 (acute)
EP94 (24-h) + 1400W (24-h)
IS/A
AR
(%
)
*** ***
Rat acute myocardial infarction model – a combined dose response and mode of action study - cont´d
31CONFIDENTIAL
A follow up study evaluating a higher dose range of EP94 resulted in an absence of cardioprotection at all dose levels
Post-surgerystabilizationphase
Vehicle
EP94 (1 g/kg x 3)
40 min occlusion
4 hrs reperfusion
60 min
EP94 (25 g/kg x 3)
EP94 (125 g/kg x 3)
40
OPTIONAL: EP94 (625 g/kg x 3)
0 3326
= Vehicle administration
= EP94 administration
Study protocol 2011-01
N = 12/group
32CONFIDENTIAL
A follow up study evaluating a higher dose range of EP94 resulted in an absence of cardioprotection at all dose levels – cont´d
0
10
20
30
40
50
60
70
IS/A
AR
(%
)
IS/AAR (%) 59,37 61,79 57,31 57,10
Placebo 1 ug/kg 25 ug/kg 125 ug/kg
n = 10 n = 10 n = 11 n = 8
Closed chest pig MI/reperfusion model
33CONFIDENTIAL
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects?
• Previously five (5) AMI EP94 protection studies in pigs have been conducted with significant and promising results.
• In the present study the pigs were not healthy due to scabies. The veterinarian decided for this reason to treat all pigs with Doramectin. (A drug approved by FDA for the treatment of parasites in animals, such as roundworms, lungworms, eyeworms, grubs, sucking lice and mange mites in cattle).
• This is the only study where EP94 have been used in combination with Doramectin
34CONFIDENTIAL
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
Cardioprotective dose-response effects from 2 studies conducted in an AMI pig model BUT without pretreatment with Doramectin
0
10
20
30
40
50
60
70
Vehicle 1 ug/kg 5 ug/kg 25 ug/kg
IS/A
AR
(%
)
N = 6 N = 6 N = 6 N = 60
10
20
30
40
50
60
70
125 ug/kg
N=5
Pilot studyStudy Report 11-02
35CONFIDENTIAL
Doramectin – Mode of action
• ActivationActivation of GABAergic receptors causes an influx of of GABAergic receptors causes an influx of chloride ions, hyperpolarization, paralysis of the nervous chloride ions, hyperpolarization, paralysis of the nervous system and system and death of the parasitedeath of the parasite..
• High dosesHigh doses causes causes neurotoxicologicalneurotoxicological effects in mammals effects in mammals• Large difference in sensitivity among different mammal Large difference in sensitivity among different mammal
seciessecies
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
36CONFIDENTIAL
Mu-opioid receptors are linked to GABAergic systems•GABAergic neurons co-express mu-opioid receptorsGABAergic neurons co-express mu-opioid receptors in the CNS of rats (Kalyuzhny et al, in the CNS of rats (Kalyuzhny et al, Neuroreport 1997, 8:3367-72; Kalyuzhny et al J Comp Neurol 1998, 392:528-47).Neuroreport 1997, 8:3367-72; Kalyuzhny et al J Comp Neurol 1998, 392:528-47).
• GABAergic and opioidergic systems are closely linkedGABAergic and opioidergic systems are closely linked. Mu-opioid receptor KO mice down-. Mu-opioid receptor KO mice down-regulates GABA-gated chloride channel binding sites (Tien et al., Neurochem Res. 2007, regulates GABA-gated chloride channel binding sites (Tien et al., Neurochem Res. 2007, 32:1891-1897).32:1891-1897).
Possible interactions points between Doramectin and EP94
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
Nitrix Oxide (NO) and iNOS•Avermectin (analogue to doramectin) has been reported to Avermectin (analogue to doramectin) has been reported to block LPS-induced secretion block LPS-induced secretion of NOof NO (Viktorov et al, Bull Exp Biol Med. 2003, 136:569-71; Zhang et al, Int (Viktorov et al, Bull Exp Biol Med. 2003, 136:569-71; Zhang et al, Int Immunopharmacol. 2009, 9: 354-359).Immunopharmacol. 2009, 9: 354-359).
• Ivermectin (analogue of doramectin) Ivermectin (analogue of doramectin) inhibits mRNA and protein expression of iNOSinhibits mRNA and protein expression of iNOS and and COX-2 enzymes (Zhang et al, Int Immunopharmacol. 2009, 9: 354-359).COX-2 enzymes (Zhang et al, Int Immunopharmacol. 2009, 9: 354-359).
• Ivermectin at therapeutic doses Ivermectin at therapeutic doses increases the plasma NO levels in rabbitsincreases the plasma NO levels in rabbits (Atakisi et al, (Atakisi et al, Eur Rev Med Pharmacol Sci. 2009, 13:425-429).Eur Rev Med Pharmacol Sci. 2009, 13:425-429).
37CONFIDENTIAL
ATP-gated ion channels and other ion channels• ATP dependent P-glycoprotein ATP dependent P-glycoprotein (mediates multidrug resistance) is inhibited by doramectin.(mediates multidrug resistance) is inhibited by doramectin.• Binds and activates Binds and activates ATP-gated purinergic P2X receptorsATP-gated purinergic P2X receptors (Priel at al., J Gen Physiol. 2004, (Priel at al., J Gen Physiol. 2004,
123:281-293; www.tocris.com)123:281-293; www.tocris.com)• Binds and activates Binds and activates 7 nicotinic acetylcholine receptors7 nicotinic acetylcholine receptors ( (www.tocris.com))• Binds and modulates Binds and modulates glutamate-activated chloride channels (glutamate-activated chloride channels (www.tocris.com))• Binds and modulates Binds and modulates GABA-activated chloride channelsGABA-activated chloride channels ( (www.tocris.com))• Potentiates Potentiates glycine-gated currentsglycine-gated currents (www.tocris.com (www.tocris.com))
Possible interactions points between Doramectin and EP94
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
38CONFIDENTIAL
SUMMARY
Doramectin may interact with the cardioprotective effects of EP94 through…..
• GABA receptors cross-communicating with mu-receptors in the CNS.
• NO and iNOS.
• Ion channels such as KATP channels or unspecific mitochondrial permiability transition pores.
Possible interactions points between Doramectin and EP94
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
39CONFIDENTIAL
Doramectin – The next step
Outline of study addressing the possibility of an interaction between EP94 and Doramectin• Species – rat due to backlogs in the pig facility• Animal model – traditional rat MI model
• Study groups - Vehicle- Preconditioning (positive control)- EP94 (1 ug/kg x 2 ; i.v.)- EP94 (1 ug/kg x 2 ; i.v.) + Doramectin (intermediate, tbd)- EP94 (1 ug/kg x 2 ; i.v.) + Doramectin (high, tbd)
• Study to be commenced: July 2011• Study site: Sahlgrenska University Hospital, Gothenburg, Sweden
40CONFIDENTIAL
EFFICACY
• EP94 is effective in two different pig ischemia/reperfusion models (open vs closed chest)
• EP94 produces a significant reduction of infarct size in pigs already at 1 µg/kg
• The cardioprotective effect of EP94 is dose-dependent in pigs
• EP94 reduces the infarct size dose-dependently during both pre- and post-conditioning in rats
Conclusions
42CONFIDENTIAL
General properties
Toxicology and Safety
Pharmacology
43CONFIDENTIAL
EP 94 – General properties
Tetrapeptide
Manufactured by solid phase peptide synthesis
Lyophilized powder
Solubility: Good solubility in water
Stability of drug substance:
Stable at +4°C for more than 2 years
Stability of drug product:
Stable at -18°C, +4°C and 25°C for more than 6 months in saline
44CONFIDENTIAL
Planned-Toxicology & Safety
Single dose tox. study in rats and pigs
Repeat dose tox. study, 14 days, in rats and pigs
Safety Pharmacology
Genotoxicity
Pharmacokinetics and ADME
45CONFIDENTIAL
Clinical Development
46CONFIDENTIAL
Key factors for success with EP94 in a clinical development programme
Selection of patient populationSTEMI patientsAn adequate risk stratification algoritm
Selection of primary endpointOptimal method used to detect infarct size
reduction
47CONFIDENTIAL
Clinical development of EP 94Phase I study
Study Objective: Dose-escalation study to evaluated the safety, and tolerability in healthy individuals
Design: single-center, double-blind
Estimated number of healthy volunteers: 40
Primary outcome measures: tolerability, safety, PK and hemodynamic parameters
48CONFIDENTIAL
Study Objective: Dose-escalation study to investigate the cardioprotective effect of ERIBIS Peptide 94 given as an adjunct to percutaneous coronary intervention (PCI) in subjects with an acute STEMI
Design: randomized, placebo-controlled, double-blind and multicenter Estimated number of patients: 350-400
Primary outcome measure: To demonstrate a dose-dependent positive trend for myocardial
salvage and myocardial salvage index using a modified single contrast enhanced steady-state free precession (SSFP) cine cardiovascular magnetic resonance (CMR) examination performed one week after the acute event
Clinical development of EP 94Phase II, proof-of-concept study
49CONFIDENTIAL
Secondary outcome measure: ST-resolution on 12 lead ECG 90 minutes after PCI Troponin, CK-MB 3 to 6 hrs, 6 to 12 hrs, 18 to 24 hrs and 36 hrs
after randomization (Peak and AUC) To determine Left Ventricular Ejection Fraction (LVEF) and Wall
Motion Score Index (WMSI) 6 weeks and 6 months after PCI Myocardial salvage index 6 months after PCI Incidence of cardiac death and total mortality, stroke, new-onset
heart failure, and re-hospitalization for any congestive heart failure 6 month follow-up visit
Clinical development of EP 94Phase II, proof-of-concept study
50CONFIDENTIAL
Intellectual Property
51CONFIDENTIAL
The IPR comprises patent protection for:
Chemical structure of peptides and peptide-based compounds
Clinical Utility
Pharmaceutical Composition
PCT application was filed in August 2007
Regional and National patent applications submitted early 2009 in EU, USA, Canada, Australia, India, China and Japan.
EU patent grant subject for approval, EPO decision to grant obtained Feb 2011
Intellectual Property
52CONFIDENTIAL
Significant cardioprotective effect
Pre-clinical Proof of Principle achieved
Clear unmet medical need & large market size
First in class
First in man within 18 months
Summary
53CONFIDENTIAL
Project Plan
Step 1 (3 MSEK end: Q4/2011)Confirm Doramectin interaction in rats and pigsIf confirmed - Dose finding study in relevant model (most likely in pigs)Mode of action – cont’d studied to elucidate MoA
Step 2 (12 MSEK; end: Q3/2012)Start regulatory studies for a CTA/INDCTA/IND submission Q3/2012
Step 3 (7 MSEK; end: Q2/2013) Phase I study
Step 4 (45 MSEK; start 2013)Phase II study
54CONFIDENTIAL
Project PlanID Aktivitet
1 CMC2 Manufacture, Polypeptides8 DP development9 DS, GMP14 DP, GMP17 Pharmacology18 Efficacy19 Efficacy studies in rats (1) and pigs (3)20 Dose response study in rats (Gross)21 Dose response study in pigs (Grip)22 Doramectin s tudy, rats23 Doramectin s tudy, pigs24 New dose response s tudy in pigs25 Interaction studies26 Mode of action studies27 Penetration of BBB28 Guinea-pig ileum bio-assay29 Mouse vas deferens bio assay30 Receptor screen31 Dos ing intervals , number of administrations32 Effect at end of occlus ion/end of reperfusion33 MILESTONE 1, EFFICACY34 Bioanalysis39 Pharmakokinetics and ADME42 Scientific advice MPA45 MILESTONE 2, GMP BATCH #2 and MPA46 Safety pharmacology and toxicology66 MILESTONE 3, TOX & SAFETY67 Regulatory Affairs71 MILESTONE 4, CTA72 Phase I
2011-06-06
2011-10-31
2012-07-09
2012-10-01
Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv2 Kv3 Kv4 Kv1 Kv22005 2006 2007 2008 2009 2010 2011 2012 2013 2014 2015 2016
Project Plan
55CONFIDENTIAL
Investment Plan up to end of Phase I
Step 13 MSEK - Date: August 2011
Step 212 MSEK - Date: December 2011
Step 37 MSEK - Date: August 2012
TOTAL 22 MSEK
57CONFIDENTIAL
Question & Answers
58CONFIDENTIAL
1. Does the peptide pass the blood brain barrier?
2. Does the NO-group give rise to worries in relation to toxicology?
3. Does the NO-group give rise to worries in relation to Carcinogenicity?
4. Are other receptors hit by this peptide?
59CONFIDENTIAL
NO-study
EP94 has been evaluated with and without NO and acylation in response to the EU patent office’s questions regarding Eribis application. To show that the specific groups at position 2, acylation, and pos 4, NO-group, are both necessary to have the cardioprotective effect shown with EP94.
5 different peptides were compared with EP94 in this pre-conditioning study.
•without NO-group of the 4th amino acid•without acylation of 2nd amino acid•without both acylation and NO-group•with Asp instead of acylation on 2nd amino acid•with Arg-Asp instead of acylation on 2nd amino acid
EP94 does not exert a significant level of cardioprotective activity without these specific groups in the second and fourth position on EP94.
60CONFIDENTIAL
Do you have rodent data?
How does EP94 and morphine perform together in the models?
Any efficacy measures/differentiation from morphine/other opioids.
Eribis has performed a series of studies on rodents that answer these questions.This data will be available when Zealand performs a DD. There is too much data to discuss during this initial meeting.
61CONFIDENTIAL
Do you have rodent data? – Cont´d
Partial list – not complete!REPORT
NUMBERTYPE PROJECT TITLE
PRINCIPAL INVESTIGATOR(S)
COMMENTS Released
ESR07-001Exploratory Study
Report (ESR)EP94
A study of the analgesic effects of peptides no 91, 92, 93, 94, 95 in CD-1 mice
Kustanova GA, Murashev AN
FINAL
ESR08-001Exploratory Study
Report (ESR)EP94
Pharmacokinetic study of peptides 91 and peptide 94 in CD rats Rzhevsky, D. I. FINAL
ESR08-002Exploratory Study
Report (ESR)EP94
Effect of naloxone on cardioprotective efficiency of peptide 94 on CD rats with infarct myocardium
Kustanova GA, Murashev AN FINAL
ESR08-003Exploratory Study
Report (ESR)EP94
Maximum tolerated dose finding test for peptide 91 in mice
Kustanova GA, Murashev AN
FINAL
ESR08-004Exploratory Study
Report (ESR)EP94
Single intravenous cardiovascular study of peptide 91 in CD rats
Kustanova GA, Murashev AN
FINAL
ESR08-005Exploratory Study
Report (ESR)EP94
Maximum tolerated dose finding test for peptide 94 in mice
Kustanova GA, Murashev AN
FINAL
ESR08-006Exploratory Study
Report (ESR)EP94
Single intravenous cardiovascular study of peptide 94 in CD rats
Kustanova GA, Murashev AN
FINAL
ESR08-007Exploratory Study
Report (ESR)EP94
A study of the protective effects of peptides No 91 and 94 on the model of an infarct myocardium in CD rats - Peptide preconditioning and fractional administration of peptides during occlusion
Kustanova GA, Murashev AN
FINAL
ESR08-008Exploratory Study
Report (ESR)EP94
Effect of morphine on cardioprotective efficiency of peptide 94 on CD rats with acute infarct
myocardium
Kustanova GA, Murashev AN FINAL
ESR08-009Exploratory Study
Report (ESR)EP94
Effect of naloxone on cardioprotective efficiency of peptide 91 on CD rats with infarct myocardium
Kustanova GA, Murashev AN FINAL
ESR09-001Exploratory
Technical Report (ESR)
EP94Qualitative RP-HPLC analysis of peptides stability
in saline solutionDorosh Yo. M, Burov
SV FINAL
ESR09-002Exploratory
Technical Report (ESR)
EP94Investigation of tested peptides stability in human
and rat blood plasmaDorosh Yo. M, Burov
SV FINAL
3 11-03 Study ReportCardioprotective Effects of Eribis Peptide EP94 in a Rat Model of Ischemia/Reperfusion Injury
Garrett Gross Draft
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Morphine cont’
Morphine is regularly used as pre-medications in the pig studies. EP94 exerts its cardioprotective properties despite approx 13-333-fold higher morphine doses in pigs. However, morphine has not regularly been used as pre-medication in the explorative rat efficacy (cardioprotection) studies conducted in Moscow.
MI patients are usually treated with morphine in the emergency room to reduce pain and anxiety. Thus, one exploratory study in rats has been conducted to address the possibility of an interaction between EP94 and morphine. This study did not reveal any synergistic, additive or antagonistic effects of morphine on EP94-induced cardioprotection. Consequently, EP94 has been used in combination with morphine in the pig studies to mimic the clinical situation as closely as possible. The pigs are routinely premedicated with 0.5 mg/kg/hr of morphine.
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How is the peptide cleared?
It is anticipated that EP94 is degraded by various endo- and exo-peptidases in the blood stream. The degradation products are believed to be excreted in the urine or partly utilized by the body. This will be verified by dedicated ADME studies in the near future.
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Have you tried the peptide, but without the NO-group, in your models? Data from Rodents (cardio protective effect with EP94 (with and without NO)- Yes see study 257/10
How does nitroglycerin perform in your models?- Not evaluated
How does nitroglycerin and morphine perform together in the models? (against EP94) - Not evaluated
Models where EP94 does not work (MOA)? - EP94 has been shown to be cardioprotective in all models tested so far, i.e. 2 different species and in total 5 different models.
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Any cellular assays (all in vitro receptor data (cell lines over-expression the receptors)) and work performed on primary cells and specific cell lines that express the receptors).
Receptor binding to the human opioid receptors (, , ) expressed on CHO and HEK-293 cells.
- Study available during the DD process (Study Report 08-03).
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Speculations on down stream target?
To investigate a possible downstream pathway which may be mediating the beneficial effect of EP94 we administered either the sarcolemmal KATP channel antagonist, HMR 1098, or the mitochondrial KATP channel antagonist, 5-HD, to rats prior to EP94 administration.
Both antagonists completely abolished the cardioprotective effect of EP94 which suggests an important role for the KATP channel as a key mediator in the pathway by which EP94 reduces infarct size similar to that seen with a number of other cardioprotective agents as well as ischemic preconditioning and postconditioning.
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Are the coronary arteries also affected?
Potential effects on coronary arteries have not been investigated.
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Speculations on general cardiovascular safety of EP94.
• EP94 does not affect blood pressure or heart rate in anaesthesized animals (rats and pigs) at doses levels ranging from 20 – 75 g/kg (cf. pharmacological efficacy studies)
• EP94 does not have significant effects on blood pressure in and only a minor and late effect on heart rate in consious rats at doses of 5-10 mg/kg (ESR08-006).
• The very low doses needed for pharmacological efficacy and the short t1/2 (approx 40 min) is anticipated to reduce cardiovascular safety risks to a minimum.
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"In-vivo SAR" (opioid component vs. the nitric oxide effects).
Unknown
Toxicology: Metabolism/metabolites and their distribution.
No evaluation of metabolites has been performed. It is anticipated that the bond between amino acid 2 and 3 is susceptible to proteolytic cleavage and that the major metabolites will contain 2 amino acids each.
ADME will be performed in the near future.
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A list of all published patent applications and patents and their status.
PCT application PD53743CA00 - Novel Enkephalin Analogues - Eribis Pharmaceuticals AB
EU pat appl. No 07 794 118.5A “Decision to Grant” will be announced in Q2-
2011
Eribis Pharmaceuticals AB owns the IP rights
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FTO analysis of key IP ?
FTO analysis has not been performed, however patentability has been evaluated by third party patent attorneys.
- statements are in the DD room
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Back-up bilder
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Chemistry Manufacturing Control
(CMC)
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EP 94 – General properties
Tetrapeptide
Manufactured by solid phase peptide synthesis
Lyophilized powder
Solubility: Good solubility in water
Stability of drug substance:
Stable at +4°C for more than 2 years
Stability of drug product:
Stable at -18°C, +4°C and 25°C for more than 6 months in saline
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Specification
Test Method Specifications
Appearance Visual observation Off-white to yellow powder
Mass spectral analysis EP 2.2.43; ESI M = 571.2 +/- 1.0
Amino acid analysis Pre-column derivatisation with OPA/Fmoc-ClEPA 2.2.56
Gly:1; Tyr:1; Dab:1;Phe (pNO2).1
RP-HPLC EP 2.2.29 Purity > 98.0% (area%)
Related Impurities by RP-HPLC
EP 2.2.29 Sum impurities < 2%
Water content Karl Fischer, EP2.5.12 To be reported
Acetate HPLC with UV detection
To be reported
Residual TFA HPLC with UV detection
< 0.1% (tentative)
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Specification cont’d
Test Method Specifications
Residual organic solvents
GC <USP 467> Individual according to ICH Q3
Bioburden EP 2.6.12<USP 61>
Aerobic bacteria < 10 CFU/0.1gAnerobic bacteria < 10 CFU/0.1gYeasts and moulds < 10 CFU/0.1g
Bacterial endotoxins
EP 2.6.14 <USP 85>
< 5 EU/mg (tentative)
Net peptide content (NPC)by AAA
Pre-column derivatisation with OPA/Fmoc-ClEPA 2.2.56
To be reported
Mass balance Calculation NPC+acetate+water
To be reported
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Lot no HPLC analysis
Date
CF 07314
97.7% 26.3.2008
CF 07314
95,8% 11.2.2010
EP 94
Manufactured by Polypeptide
Part no: SP080384
Storage temp: 4°C
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EP 94
Lot no HPLC analysis
Date
MZ77114
98.9% 07.05.2009
Manufactured by Polypeptide
Part no: SP080384B
Storage temp: 4°C
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Drug ProductStability of EP 94 in Saline
Stability (non- ICH) of EP94, 0.1mg/mL in 9 mg/mL NaCl (preserved with NaN3 1mg/mL)
Temp Time 0 14 days 2.5 mo 4.5 mo 6 mo
-18°C 96.5 96.5 96.6 96.5
4°C 96.5* 96.5 96.6 96.4 96.2
Ambient 96.6 96.6 96.0 95.5
* Area %
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CMC - To do list
Drug Substance Analytical development Specification Stability ICH Manufacturing?
Drug Product Analytical development Preformulation Interaction with packaging mtr Stability ICH Manufacturing for tox and clinical
Regulatory IND/CTA chapter
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Refererences
1. Thom T, Haase N, et al. Writing Group Members. Heart Disease and Stroke Statistics--2006 Update. Circulation; A Report From the American Heart Association Statistics Committee and Stroke Statistics Subcommittee; January 11, 2006; 2006. CIRCULATIONAHA.105.171600.
2. Bishop E. Heart disease may actually be rising; researchers claim deaths are now being delayed to a later age group. Wall Street Journal. 1996 November 13, 1996; pB3(W) pB6(E) col 1 (11 col in)
3. Gerber Y, Jacobsen SJ, Killian J, Weston S, Roger VL. Impact of Participation Bias in a Population-Based Study of Myocardial Infarction in Olmsted County, Minnesota, 2002 to 2004. Circulation. 2006;13:e827.
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Herman Krapf, Chied Executive Officer
Erik Bissessar, Chief Business Officer and Medical Scientific Liason
Peter Båvenholm, MD, Ph.D., Ass. Prof. Internal Medicine, Chief Medical Officer
Fredrik Röök, Business Development
Stefan Persson, Ph.D., Pharmacology and Toxicology
Claes Lundberg, Ph.D., Project Management
Management
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BACK-UP SLIDES
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Doramectin – Mode of action
• Stimulates the Stimulates the release of GABArelease of GABA..• GABAergic receptors are found at the neuromuscular junction in GABAergic receptors are found at the neuromuscular junction in
nematodesnematodes• GABAergic receptors are found primarily in the brain of GABAergic receptors are found primarily in the brain of
mammalsmammals..• This class of compounds pass the This class of compounds pass the BBB poorlyBBB poorly at therapeutic at therapeutic
doses.doses.• ActivationActivation of GABAergic receptors causes an influx of chloride of GABAergic receptors causes an influx of chloride
ions, hyperpolarization, paralysis of the nervous system and ions, hyperpolarization, paralysis of the nervous system and death of the parasitedeath of the parasite..
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
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• DoramectinDoramectin has the pharmacological has the pharmacological profile in rats of an anxiolytic/anticonvulsant profile in rats of an anxiolytic/anticonvulsant drug with drug with GABAergicGABAergic properties (de Souza properties (de Souza Spinosa et al, Comp Biochem Physiol Toxicol Spinosa et al, Comp Biochem Physiol Toxicol Pharmacol. 2000, 127:359-66).Pharmacol. 2000, 127:359-66).• GABAergic neurons co-express mu-opioid GABAergic neurons co-express mu-opioid receptorsreceptors in the CNS of rats (Kalyuzhny et in the CNS of rats (Kalyuzhny et al, Neuroreport 1997, 8:3367-72; Kalyuzhny al, Neuroreport 1997, 8:3367-72; Kalyuzhny et al J Comp Neurol 1998, 392:528-47).et al J Comp Neurol 1998, 392:528-47).• GABAergic and opioidergic systems are GABAergic and opioidergic systems are closely linkedclosely linked. Mu-opioid receptor KO mice . Mu-opioid receptor KO mice down-regulates GABA-gated chloride down-regulates GABA-gated chloride channel binding sites (Tien et al., channel binding sites (Tien et al., Neurochem Res. 2007, 32:1891-1897).Neurochem Res. 2007, 32:1891-1897).
Possible interactions points between Doramectin and EP94- GABA and mu-opioid receptors -
Doramectin may interact with the cardioprotective effect of EP94 through GABAergic neurons in the CNS expressing mu-receptors.
Why was the EP94 dose range finding study in closed chest AMI pigs completely absent of cardioprotective effects? - Cont´d
CTOP = selective mu-antagonist
0,0010,0020,0030,0040,0050,0060,0070,00
Vehicle
EP94 (1 ug/kg)
CTOP (0.1 mg/kg)
CTOP+EP94
IS/A
AR
(%
)
N = 9 N = 10 N = 9 N = 9
***
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• Avermectin (analogue to doramectin) Avermectin (analogue to doramectin) has been reported to has been reported to block LPS-induced block LPS-induced secretion of NOsecretion of NO, prostaglandin E2, and , prostaglandin E2, and to increase the intracellular to increase the intracellular concentration of Ca (Viktorov et al, Bull concentration of Ca (Viktorov et al, Bull Exp Biol Med. 2003, 136:569-71; Zhang Exp Biol Med. 2003, 136:569-71; Zhang et al, Int Immunopharmacol. 2009, 9: et al, Int Immunopharmacol. 2009, 9: 354-359).354-359).• Ivermectin (analogue of doramectin) Ivermectin (analogue of doramectin) inhibits mRNA and protein expression of inhibits mRNA and protein expression of iNOSiNOS and COX-2 enzymes (Zhang et al, and COX-2 enzymes (Zhang et al, Int Immunopharmacol. 2009, 9: 354-Int Immunopharmacol. 2009, 9: 354-359).359).• Ivermectin at therapeutic doses Ivermectin at therapeutic doses increases the plasma NO levels in increases the plasma NO levels in rabbitsrabbits (Atakisi et al, Eur Rev Med (Atakisi et al, Eur Rev Med Pharmacol Sci. 2009, 13:425-429).Pharmacol Sci. 2009, 13:425-429).
Doramectin may interact with the cardioprotective effect of EP94 through NO/iNOS/eNOS pathways.
Why was the EP94 dose range finding study in closed chest AMI pigs completely abscent of
cardioprotective effects? - Cont´d
0,00
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30,00
40,00
50,00
60,00
70,00
Vehicle
EP94 (1 ug/kg ipx2) - 24-h
1400W (0.1 mg/kg iv) - Acute
1400W (acute) + EP94 (acute)
EP94 (24-h) + 1400W (24-h)
IS/A
AR
(%
) ** **
Possible interactions points between Doramectin and EP94- NO and NOS -
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• ATP dependent P-glycoprotein ATP dependent P-glycoprotein (mediates multidrug resistance) is (mediates multidrug resistance) is inhibited by doramectin.inhibited by doramectin.
• Binds and activates Binds and activates ATP-gated ATP-gated purinergic P2X receptorspurinergic P2X receptors (Priel at al., J (Priel at al., J Gen Physiol. 2004, 123:281-293; Gen Physiol. 2004, 123:281-293; www.tocris.com)www.tocris.com)
• Binds and activates Binds and activates 7 nicotinic 7 nicotinic acetylcholine receptorsacetylcholine receptors ( (www.tocris.com))
• Binds and modulates Binds and modulates glutamate-glutamate-activated chloride channels (activated chloride channels (www.tocris.com))
• Binds and modulates Binds and modulates GABA-activated GABA-activated chloride channelschloride channels ( (www.tocris.com))
• Potentiates Potentiates glycine-gated currentsglycine-gated currents (www.tocris.com(www.tocris.com))
Doramectin is a positive allosteric modulator (PAM, indirect activation of receptors).
An interaction with the cardioprotective effect of EP94 through ion channels such as KATP channels or unspecific mitochondrial permiability transition pores can not be excluded.
Why was the EP94 dose range finding study in closed chest AMI pigs completely abscent of
cardioprotective effects? - Cont´dPossible interactions points between Doramectin and EP94
- ATP-gated ion channels and other ion channels-
0,00
10,00
20,00
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50,00
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Vehicl
e
EP94 (1
ug/
kg)
HMR10
98 (6
mg/
kg)
HMR10
98 +
EP94
5-HD (1
0 mg/
kg)
5-HD +
EP94
IS/A
AR
(%
)
N = 9 N = 9N = 9N = 10 N = 9 N = 9
***
HMR 1098 = sarcolemmal KATP channel antagonist5-HD = mitochondrial KATP channel antagonist
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Doramectin – The next step
Outline of study addressing the possibility of an interaction between EP94 and Doramectin
• Species – rat due to backlogs in the pig facility• Animal model – traditional rat MI model
• Study groups - Vehicle- EP94 (1 ug/kg; i.v.)- EP94 (1 ug/kg; i.v.) + Doramectin (intermediate, tbd)- EP94 (1 ug/kg; i.v.) + Doramectin (high, tbd)
• Study to be commenced: July 2011• Study site: Sahlgrenska University Hospital, Gothenburg, Sweden