Eribis pharmaceuticals AB

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


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


= EP94 administration

Closed chest pig MI/reperfusion model

22CONFIDENTIAL

Intracoronary infusion of EP94 in pigs reduces myocardial infarction size significantly


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


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

*



Study Report 11-02

Mean ± SEMLinear regression analysis,* P < 0.05


24CONFIDENTIAL

Rat acute myocardial infarction model – a combined dose response and mode of action study


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



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


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


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

***


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

(%

)

*** ***


31CONFIDENTIAL

A follow up study evaluating a higher dose range of EP94 resulted in an absence of cardioprotection at all dose levels


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



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


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


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


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))



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.



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


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


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

56CONFIDENTIAL

Thank you for listening!

Contact;Erik [email protected]

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


Report (ESR)EP94

Pharmacokinetic study of peptides 91 and peptide 94 in CD rats Rzhevsky, D. I. FINAL


Report (ESR)EP94

Effect of naloxone on cardioprotective efficiency of peptide 94 on CD rats with infarct myocardium

Kustanova GA, Murashev AN FINAL


Report (ESR)EP94

Maximum tolerated dose finding test for peptide 91 in mice


FINAL


Report (ESR)EP94

Single intravenous cardiovascular study of peptide 91 in CD rats


FINAL


Report (ESR)EP94

Maximum tolerated dose finding test for peptide 94 in mice


FINAL


Report (ESR)EP94

Single intravenous cardiovascular study of peptide 94 in CD rats


FINAL


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


FINAL


Report (ESR)EP94

Effect of morphine on cardioprotective efficiency of peptide 94 on CD rats with acute infarct

myocardium



Report (ESR)EP94

Effect of naloxone on cardioprotective efficiency of peptide 91 on CD rats with infarct myocardium


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

62CONFIDENTIAL

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.

63CONFIDENTIAL

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.

64CONFIDENTIAL

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.

65CONFIDENTIAL

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).

66CONFIDENTIAL

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..


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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.


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

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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-

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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

Eribis pharmaceuticals AB

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