Mechanism of action of the cardiovascular drug levosimendan in the

management of amyotrophic lateral sclerosis 1 1 1Kira M. Holmström , Piero Pollesello , Chris Garratt

1Orion Pharma, Orion Corporation, Espoo, Finland

CONCLUSION

REFERENCES

Disclosures: All authors are employees of Orion Pharma. The study was sponsored by Orion Pharma.

BACKGROUND

OBJECTIVES

METHODS

RESULTS

Levosimendan is a calcium sensitizer that promotes myocyte contractility through its calcium-dependent interaction with cardiac troponin C, also found in skeletal muscle. It has been used for nearly two decades as i.v. treatment in acute heart failure. During that time many additional pharmacological actions of levosimendan have been described.

Effects of levosimendan increasing diaphragm function suggested a possible new 1application in the treatment of patients with amyotrophic lateral sclerosis (ALS).

As levosimendan has been linked to a range of pleiotropic actions we reviewed the mode of actions that may be of relevance in the treatment of ALS.

Systematic literature review was conducted to collected all evidence on the different pharmacological effects of levosimendan. A special focus was given to effects relevant to the pathophysiology of ALS and to the therapeutic needs of patients with ALS.

While the direct activity of levosimendan on skeletal muscle is

clearly the main effect of interest in relation to ALS, a number of

other pleiotropic effects of the drug raise some intriguing

possibilities in the context of this disease. These effects

should be confirmed in models more relevant to ALS.

2. Van Hees HW et al., Levosimendan enhances force generation of diaphragm muscle from patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2009;179:41-7.

6. Malmberg M et al., Intracoronary Levosimendan during Ischemia Prevents Myocardial Apoptosis. Front Physiol. 2012;3:17.

1. Al-Chalabi A et al., Potential of the Cardiovascular Drug Levosimendan in the Management of Amyotrophic Lateral Sclerosis: An Overview of a Working Hypothesis. Journal of Cardiovascular Pharmacology: 2019 ;74(5):389–399

3. Doorduin J et al., The calcium sensitizer levosimendan improves human diaphragm function. Am J Respir Crit Care Med. 2012;185:90-5.4. Levijoki J et al., Levosimendan alone and in combination with valsartan prevents stroke in Dahl salt-sensitive rats. Eur J Pharmacol. 2015;750:132-40.5. Li PC et al., Inhibition of reverse-mode sodium-calcium exchanger activity and apoptosis by levosimendan in human cardiomyocyte progenitor cell-derived cardiomyocytes after anoxia and reoxygenation. PLoS One. 2014;9:e85909.

8. Kopustinskiene DM et al., Potassium-specific effects of levosimendan on heart mitochondria. Biochem Pharmacol. 2004;68:807-12.9. Honisch A et al., Postconditioning with levosimendan reduces the infarct size involving the PI3K pathway and KATP- channel activation but is independent of PDE-III inhibition. Basic Res Cardiol. 105:155-67.

7. Karakus E et al., Beneficial pharmacological effects of levosimendan on antioxidant status of acute inflammation induced in paw of rat: involvement in inflammatory mediators. Basic Clin Pharmacol Toxicol. 2013;112:156-63.

LEVOSIMENDAN BY OPENING THE K -CHANNEL AND ATP

+POSSIBLY OTHER K -CHANNELS

(BK, KV, SK)

BY INHIBITING SELECTIVELY PDEIII(EC 5-8 nM) OVER PDEIV (30µM)50

2+BY BINDING Ca - SATURATED cTnC IN

CARDIOMYOCYTES (EC 5-8 nM)50

BY A DIRECT ACTION ON sTnC (10 M)µ

ACTION ON VASCULATURESMOOTH MUSCLES

ACTION ON MITOCHONDRIA

VASODILATION

INCREASES PERIPHERAL

PERFUSION (INCL. BRAIN & SPLANCHNIC)

INCREASES CORONARY FLOW

REDUCES PRELOAD AND AFTERLOAD

INCREASES KIDNEY GLOMERULAR

FILTRATION RATEREDUCES PCWP

PROVIDES CARDIO- AND ORGAN-PROTECTION

EXERTS AN ANTI-ISCHEMIC EFFECT (PRE-, POST-

CONDITIONING)

EXERTS AN ANTI-APOPTOTIC EFFECT

EXERTS POSITIVE INOTROPY IN HEART MUSCLE WITHOUT

DISTURBING RELAXATION NOR

INCREASING OXYGEN

CONSUMPTION

WITH EFFECT ON SLOW-TWITCH FIBERS

STRENGTHENS MUSCLE CONTRACTION

STRENGTHENS MUSCLE CONTRACTION

WITH EFFECT ON FAST-TWITCH FIBERS

2+BY BINDING Ca -SATURATED cTnC IN SLOW-TWITCH FIBERS IN OTHER MUSCLES (INCLUDING

DIAPHRAGM AND SKELETAL) (SAME EC AS 50

IN CARDIOMYOCYTES)

Figure 1. The dysfunction and death of motor neurons that is the core feature of ALS is believed to arise from multiple underlying pathophysiological processes. Pharmacological mechanisms of action of levosimendan may suggest a relevance for anti-inflammatory effects to attenuate neuroinflammation, autophagy activation to reduce proteasome impairment, ER stress alleviation to reduce endoplasmic reticulum stress, anti-apoptotic effects to prevent excitotoxicity, and improved mitochondrial function to prevent mitochondrial dysfunction and oxidative stress. These effects should be confirmed

1in models relevant to ALS. Adapted from Al-Chalabi et al 2019.

Figure 2. Scheme of the mode of actions and pharmacologic effects of levosimendan. The actions in the grey boxes underlie the cardiovascular effects of the drug. In the violet boxes are pharmacologic effects of levosimendan that may be considered of primary relevance in ALS. Grey dotted lines identify interplays that are still not fully elucidated. Abbreviations: cTnC and sTnC, cardiac and skeletal isoforms of troponin C, respectively; PDE III and IV, phosphodiesterase isoforms in cardiac tissue;

1PCWP, pulmonary capillary wedge pressure. Adapted from Al-Chalabi et al 2019.

Table 1. Pharmacological mode of actions of levosimendan in various models potentially relevant to ALS

Pharmacological effect

Outcome Refs

Improved respiratory muscle contractility

In vitro levosimendan enhances calcium sensitivity a(pCa ) of isolated diaphragm muscle fibres by 20-30% 50

(p<0.01). In healthy volunteers, levosimendan improves neuromechanical efficiency by 25% (p<0.05) and contractile function of the human diaphragm

2, 3

Improved brain circulation

Increased blood volume of the cerebral microvessels b(∆R2 ) of the cortex (∆R=3.5±0.15 vs. 2.7±0.17ml in

vehicle; P=0.001) and hemisphere (∆R=3.2±0.23 vs. 2.6±0.14ml in vehicle; P=0.018) in in vivo stroke model in Dahl salt-sensitive rats

4

Endoplasmic reticulum stress (ERS) relief

Levosimendan significant ly reduced apoptot ic cardiomyocytes from 27.6±2.4% to 8.2±2.1% (P<0.05) in

canoxia-reperfusion model in hCPCs5

Prevention of programmed cell death

Levosimendan reduced apoptosis 0.02 ± 0.017% vs. 0.062 ± 0.044% in control (p=0.03) in in vivo myocardial ischemia-reperfusion model in pigs

6

Anti-oxidative and anti-inflammatory effects

Levosimendan dose-dependently maximally reduced (p<0.0001) paw oedema (- 84%) and supressed

cytokines (TNFa, IL-1, and IL-6 by -68%, -66%, and -76%, respectively) and increased superoxide dismutase (SOD)

dactivity by 76% as well as GSH by 230% and decreased

8-isoprostaglandin F2a by -74% in in vivo carrageenan-induce inflammatory paw oedema rat model

7

Mitochondria-protective effects

Levosimendan opens mitochondrial K(ATP) channels in mitochoria isolated from rat heart. Levosimendan prior to reperfusion decreased infarct size (29 ± 3% vs. control) in vivo in rats. The mitochondrial KATP-channel blocker 5-HD and the PI3K inhibitor wortmannin completely abolished the protection by levosimendan (50 ± 2 and 52 ± 3%, respectively)

8, 9

b. ΔR2; Transverse relaxivity of spin-echo was calculated from MRI T2 weighed images

c. Human Cardiomyocyte Progenitor Cells

d. GSH; total glutathione

a. pCa ; logarithmic Ca2+ concentration required for half-maximum activation50

AstrocyteMicroglial cell

Impaired axonal transport

Abnormal RNA metabolism

Mitochondrialdysfunction

Neuroin�ammation

Proteasomeimpairment

Excitotoxicity

Oxidative stressEndoplasmic reticulum stress

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