1. Levosimendan: drug
discovery and pharmacology
Piero Pollesello, PhD (cardiovascular
pharmacology), docent (Faculty of Medicine, University of
Helsinki),
Global Brand Manager Critical Care Proprietary Products
(Orion Pharma, Finland)

2. Gs Gi
b-receptor
Na+/Ca2+ex.Na+/K+ex.
ATP cAMP (active)
AMP (inactive)
PDE
Rise in intracellular calcium
Ca2+
Na+
K+
e.g.Dobutamine
e.g.Milrinone (PRIMACOR)
e.g.Digoxin
Old school inotropic drugs

3. With the old school inotropes more
contractility is achieved but with higher risks
•increase in the oxygen consumption in the
myocardium, risk for the ischemic patients
•reduced effects in patients on beta-blockers
•arrhythmias due to the high level of intracellular
calcium
•acceleration of the myocardial
remodelling, apoptosis
•worse prognosis in the middle-long term

4. recent meta-analyses of inotropes
There are strong indications from this meta-analysis that dobutamine
worsens outcomes in patients with severe heart failure
Effect of dobutamine on mortality in heart failure (vs. placebo or standard care)
OR 1.47
Tacon et al. (2011) Intensive Care Med. 38:359-367

5. The research for a new inotrope,
which would not increase either intracellular calcium
or the oxygen consumtion on the myocardium…
lead to the development of Levosimendan
…but is Levosimendan only a good inotrope or
a new therapeutic solution for AHF?

6. Levosimendan: a triple mechanism of action
• calcium dependent binding to cTnC
• opening of KATP channels on smooth muscle cells in vasculature
• opening of KATP channels in cardiac mitochondria

7. Levosimendan: a triple mechanism of action
• calcium dependent binding to cTnC
• opening of KATP channels on smooth muscle cells in vasculature
• opening of KATP channels in cardiac mitochondria

8. New target: contractile proteins in
cardiomyocytes
Actin
Tropomyosin
TnI
TnT
Ca2+
cTnC
Myosin head (S1 fragment)
ATP pocket
RLC
ELC

9. Ca2+
Levosimendan binds selectively
to calcium saturated cardiac troponin C
levosimendan
Pollesello et al. (1994) J Biol Chem 269:28584-90

10. Levosimendan effect on LV contractility and
relaxation on HF conscious instrumentyed dogs
Masutani S. et al. (2008) J Pharmacol. Exp. Therap. 325:236-47

11. Papp, Z. et al. (2005) J Cardiovasc Pharmacol 46:369-76
Calcium sensitization and non PDE
inhibition acts directly on contractile fibers

12. No increase in calcium transient
500 ms
0.35
0.20
Indo-1ratio%cellshortening
15
0
Control Levosimendan
0.1 M
[Ca2+]i
Lancaster and Cook (1997) Eur J Pharmacol 339:97-100

13. -8
-4
0
4
8
12
16
0,01 0,03 0,1 0,3 1 3 10
Concentration, µM
CHANGE IN THE VO2 TO (P)dt RATIO
(OXYGEN CONSUMPTION VS. WORK)
Kaheinen et al. (2004) J Cardiovasc Pharmacol 43:555-561
Levosimendan: no increase
of oxygen consumption
MILRINONE
LEVOSIMENDAN

14. 0
0.02
0.04
0.06
0.08
0.1
0.12
Dobutamine Levosimendan
Baseline
Treatment
(2 hrs infusion)
6 µg/kg/min 0.3 µg/kg/min
Ukkonen et al. (1997) Clin Pharmacol Ther 61:596-607
No effect on myocardial oxygen
consumption in HF patients

15. from data in Haikala et al. (1997) Cardiovasc Res 34:536-546
Guinea-pig
papillary muscle
0.03 0.1 1 3
-50
0
100
200
250
Levosimendan alone (n = 5)
Atenolol 1 µM + Levosimendan (n = 5)
DTwitchTension,mg
Levosimendan, µM
No antagonism by b-blockers
on positive inotropic effect
0.3

16. Synergistic effect of b-blockers on
the action of levosimendan in HF patients
D PCWP
(mmHg)
D CO
(l/min)
0
0.5
1.0
1.5
Levosimendan without b-blockers
Levosimendan with b-blockers
Dobutamine without b-blockers
Dobutamine with b-blockers
-8
-6
-4
-2
0
Levosimendan Dobutamine
p = 0.01
p = 0.03
b- b+ b- b+
b- b+ b- b+
Follath et al. (2002) - LIDO clinical trial – Lancet 360:196-202

17. Levosimendan: a new mechanism of action
• calcium dependent binding to cTnC
• opening of KATP channels on smooth muscle cells in vasculature
• opening of KATP channels in cardiac mitochondria

18. Levosimendan opens the ATP-dependent
potassium channels in smooth muscle of vessels
Erdei et al. (2006) Br J Pharmacol 148:696-702
Ø: 100 m
Levosimendan + GLI (5µM)
Levosimendan

19. Levosimendan increases diastolic
coronary flow velocity
Kaheinen et al. (2001) J Cardiovasc Pharmacol 37:367-374
Effects of levosimendan on
the diastolic coronary flow
velocity in isolated guinea-
pig hearts in the presence
(black) and absence (open)
of 0.1 µM glybenclamide
(n=6, ** p  0.01)

20. Levosimendan increases blood perfusion
Blood Flow (Solid Columns) and Calculated Vascular Resistance (Hatched Columns) in the
Small Intestine. Data are represented as % change from control. (a) significant (p<0.05)
difference from baseline, (abc) significant difference from both low and middle doses, (d)
significant difference from the corresponding value in the levosimendan group
Pagel et al. (1996) Br J Pharmacol 119:609-615
LEVOSIMENDAN
%DFROMCONTROL
µg kg-1 min-1
0.75 1.5 3.0
MILRINONE
1.0 2.0 4.0
µg kg-1 min-1

21. Levosimendan: a new mechanism of action
• calcium dependent binding to cTnC
• opening of KATP channels on smooth muscle cells in vasculature
• opening of KATP channels in cardiac mitochondria

22. Levosimendan opens the ATP-dependent
potassium channels in cardiac mitochondria
Kopustinskiene et al. (2004) Biochem Pharmacol 68:807
0.1 1 10
0.0
0.1
0.2
0.3
0.4
EC 50 =0.83  M
Log [Levosimendan] ( M)
Maximalrateofpotassium-
specificDdecrease(%/s)

23. Cardioprotection
KATP channels opening
Zingman et al. (2007) J Appl Physiol 103:1888-1893

24. Cardioprotection
• Short-term cardioprotection encompasses these three effects:
– preconditioning
– postconditioning
– anti-stunning
• Long term cardioprotection encompasses these four effects:
– anti-ischemic
– anti-remodelling
– anti-apoptotic
– anti-inflammatory

25. Preconditioning (1º/7)
• Opening of the KATPMITO channels plays a predominant role in the
modulation of myocardial infarction following ischemia and reperfusion.
• Moreover, opening of the KATPMITO channels plays a role in the
reduction of myocardial cell necrosis and apoptosis induced by
ischemia–reperfusion injury by the modulation of [Ca2+]MITO
accumulation and the stabilization of mitochondrial inner membrane
volume and permeability, which would prevent the efflux of cytochrome
c and activation of pro-apoptotic proteins.
McCully & Levitsky (2003) Arch Biochem Biophys 420:237

26. Levosimendan has a preconditioning effect
stunning
ischemia reperfusion
ischemic preconditioning
control
LVDP
or
dP/dT
levosimendan preconditioning
infarct
size

27. CONTROL IPC
0
10
20
30
40
50
60
INFARCTSIZE(%OFAREAATRISK)
FIGURE 5: INFARCT SIZE - LEVOSIMENDAN PRECONDITION
TABLE 8
CONTROL LEVOSIMENDAN
du Toit et al. (2008) Br J Pharmacol 154:41-50
Levosimendan has a preconditioning effect

28. Levosimendan increases survival in animal models
Papp et al. (2006) J Cardiovasc Pharmacol Therapeut 11:129
OCCLUSION REPERFUSION SURVIVAL
Group Dose
µmol kg-1
n Incidence
of VF (%)
Incidence
of VF (%)
%
Control 10 40 83 10
Levosimendan 0.1 10 0* 30* 70*
Milrinone 0.1 10 60 50 20
Ischemia-reperfusion MI model in dogs

29. Levosimendan has a preconditioning effect
Metzsch et al. (2007) Acta Anaesthesiol Scand 51:86
 Levosimendan
- Ischemic area
 Control
- Ischemic area
 PRE
- Control tissue
 POST
- Control tissue
ISCHEMIA
levosimendan

30. Tritapepe et al. (2009) Br J Anaeth 102:198-206
Preconditioning effects of Levosimendan
in coronary artery bypass grafting

31. Remodelling (5º/7)
• Remodelling = molecular, cellular and interstitial changes in the
failing heart, which are manifested clinically as changes in
size, shape and function of the heart resulting from cardiac load or
injury1
• Leads to non-reversible myocardial damage
– loss of cardiomyocytes
– fibrosis of myocardium
• Fibrotic myocardium has lost its normal contractile function
Cohn et al. 2000 J Am Coll Card 25:569

32. Levosimendan stops and reverts the
remodeling of cardiac tissue in HF
myocardial
SERCA2/NCXratio
0
0.2
0.4
0.6
0.8
1.0
1.2
MyocardialSERCA2/NCXratio
Dah l HS
*
*
Dah l HS+
lev o 10
Dahl HS+
lev o 1
Da hl LS
*
HF HF+
levosim.
cntrl
myocardial
hyperthrophy
HF HF+
levosim.
cntrl
0
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Cardiachypertrophy(mg/g)
Dah l HS
*
*
*
Dahl HS+
lev o 1
Da hl HS+
le v o 10
Da hl LS
*
* *
Louhelainen et al. (2007) Br J Pharmacol 150:851-61

33. Apoptosis (6º/7)
van Empel et al.(2005) Cardiovasc Res. 67:21
Human heart failure is preceded by a process termed cardiac
remodeling in which heart chambers progressively enlarge and
contractile function deteriorates. Programmed cell death (apoptosis) of
cardiac muscle cells has been identified as an essential process in the
progression to heart failure. Unlike necrosis, apoptosis is an orderly
regulated process and, by inference, a logical therapeutic target if
intervention occurs at an early stage.

34. Levosimendan protects against apoptosis
Maytin & Colucci (2005) Am J Cardiol 96:26G

35. Louhelainen et al. (2007) Br J Pharmacol 150:851-61

36. Inflammation (7º/7)
In heart failure, a broad spectrum of neurohormonal and
inflammatory markers are released

37. Levosimendan protects against inflammation
In HF patients, at therapeutical doses, levosimendan
reduces the plasma levels of b-natriuretic peptide1-5,
interleukin 62-5, endothelin-I6, a-natriuretic peptide and
renin7,8, prevents the increase of norepinephrine and
epinephrine levels7 and pre- serves heart rate variability9.
1Moertl et al. Eur J Heart Fail 2005 Aug 3
2Parissis et al. Am J Cardiol 2005;96:423-6.
3Avgeropoulou et al. Eur J Heart Fail 2005;7:882-7
4Kyrzopoulos et al. Int J Cardiol 2005;99:409-13
5Gegenhuber et al.,Clin Chem 2004;50:454-455
6Nicklas et al. Am J Cardiol 1999;83:12(I)-15(I)
7Nieminen et al. J Am Coll Cardiol 2000;36:1903-12
8Sundberg et al. Am J Cardiol 1995;75:1061-6
9Binkley et al. Circulation 2000;102(suppl II)

38. Levosimendan protects against inflammation
Time Since Start of Study Drug Infusion, days
SURVIVE study -- Cohen-Solal et al. (2009) JACC 53:2349
MeanChangeFromBaseline
inBNP,pg/mL
-800
-600
-400
-200
0
0 1 2 3 4 5
Levosimendan Dobutamine
*
*
*
p<0.001
levosimendan (n=663) dobutamine (n=664)
*
*
*p<0.001
p<0.001

39. Papp Z et al. Int J Cardiol 2012;159:82-7