Calcium sensitiser

For the treatment of acute decompensated congestive heart failure, levosimendan (OR1259) is a calcium sensitizer. When traditional treatment is deemed insufficient, levosimendan (OR1259), a dilator, is prescribed for the short-term management of acute decompensated severe chronic heart failure. Levosimendan (OR1259) has demonstrated encouraging first results in a variety of illnesses requiring inotropic support, including as Takotsubo cardiomyopathy, cardiogenic shock, septic shock, and right ventricular failure [1]. Unlike other types of dilators, levosimendan (OR1259) also involves positive energetic and neurohormonal alterations in addition to its distinct drug-receptor interactions for its cardiovascular effects [2]. In adult patients having cardiology and cardiac surgery, levosimendan (OR1259) may lower mortality [3].

Objective: Catecholaminergic inotropes have a place in the management of low output syndrome and decompensated heart failure but their effect on mortality is debated. Levosimendan is a calcium sensitizer that enhances myocardial contractility without increasing myocardial oxygen use. A meta-analysis was conducted to determine the impact of levosimendan on mortality and hospital stay. Data sources: BioMedCentral, PubMed, Embase, and the Cochrane Central Register of clinical trials were searched for pertinent studies. International experts and the manufacturer were contacted. Study selection: Articles were assessed by four trained investigators, with divergences resolved by consensus. Inclusion criteria were random allocation to treatment and comparison of levosimendan vs. control. There were no restrictions on dose or time of levosimendan administration or on language. Exclusion criteria were: duplicate publications, nonadult studies, oral administration of levosimendan, and no data on main outcomes. Data extraction: Study end points, main outcomes, study design, population, clinical setting, levosimendan dosage, and treatment duration were extracted. Data synthesis: Data from 5,480 patients in 45 randomized clinical trials were analyzed. The overall mortality rate was 17.4% (507 of 2,915) among levosimendan-treated patients and 23.3% (598 of 2,565) in the control group (risk ratio 0.80 [0.72; 0.89], p for effect <.001, number needed to treat = 17 with 45 studies included). Reduction in mortality was confirmed in studies with placebo (risk ratio 0.82 [0.69; 0.97], p = .02) or dobutamine (risk ratio 0.68 [0.52-0.88]; p = .003) as comparator and in studies performed in cardiac surgery (risk ratio 0.52 [0.35; 0.76] p = .001) or cardiology (risk ratio 0.75 [0.63; 0.91], p = .003) settings. Length of hospital stay was reduced in the levosimendan group (weighted mean difference = -1.31 [-1.95; -0.31], p for effect = .007, with 17 studies included). A trend toward a higher percentage of patients experiencing hypotension was noted in levosimendan vs. control (risk ratio 1.39 [0.97-1.94], p = .053). Conclusions: Levosimendan might reduce mortality in cardiac surgery and cardiology settings of adult patients.[3]

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Levosimendan is an inodilator indicated for the short-term treatment of acutely decompensated severe chronic heart failure, and in situations where conventional therapy is not considered adequate. The principal pharmacological effects of levosimendan are (a) increased cardiac contractility by calcium sensitisation of troponin C, (b) vasodilation, and (c) cardioprotection. These last two effects are related to the opening of sarcolemmal and mitochondrial potassium-ATP channels, respectively. Data from clinical trials indicate that levosimendan improves haemodynamics with no attendant significant increase in cardiac oxygen consumption and relieves symptoms of acute heart failure; these effects are not impaired or attenuated by the concomitant use of beta-blockers. Levosimendan also has favourable effects on neurohormone levels in heart failure patients. Levosimendan is generally well tolerated in acute heart failure patients: the most common adverse events encountered in this setting are hypotension, headache, atrial fibrillation, hypokalaemia and tachycardia. Levosimendan has also been studied in other therapeutic applications, particularly cardiac surgery - in which it has shown a range of beneficial haemodynamic and cardioprotective effects, and a favourable influence on clinical outcomes - and has been evaluated in repetitive dosing protocols in patients with advanced chronic heart failure. Levosimendan has shown preliminary positive effects in a range of conditions requiring inotropic support, including right ventricular failure, cardiogenic shock, septic shock, and Takotsubo cardiomyopathy. [1]

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The electrophysiological effect of levosimendan, a novel Ca(2+)-sensitizing positive inotropic agent and vasodilator, was examined on rat mesenteric arterial myocytes using the patch clamp technique. Resting potential was significantly hyperpolarized with levosimendan, with an EC50 of 2.9 microM and maximal effect (19.5 +/- 3.5 mV; n = 12) at 10 microM. Levosimendan (10 microM) significantly increased the whole-cell outward current. The currents intersected close to the calculated EK (-84 mV), suggesting that the activated current was a K+ current. Hyperpolarization and stimulation of K+ current by levosimendan were not prevented by 30 microM H-7 (a non-specific inhibitor of protein kinases) and 100 nM charybdotoxin (a blocker of Ca(2+)-activated K+ channels), but were abolished by 10 microM glibenclamide. In single-channel current recording in open cell-attached patches, two types of K+ channels were observed having conductances of 26 and 154 pS. The 154 pS channels were not affected by levosimendan and glibenclamide. The 26 pS channels were evoked in one-fourth of the patches when 10 microM levosimendan (and 0.1 mM UDP) was added (at -60 mV) and channel activity was abolished by glibenclamide. The mean open probability of the 26 pS channels was 0.094 +/- 0.017 (n = 9), and the mean open time (at -60 mV) was 6.6 ms in the presence of UDP and levosimendan. Although significant hyperpolarization (4.7 +/- 1.5 mV, n = 8) was observed at 1 microM levosimendan, the same concentration did not affect Ca2+ channel currents (n = 10). In summary, levosimendan hyperpolarized the arterial myocytes, probably through activation of a glibenclamide-sensitive K+ channel. This mechanism may contribute to the vasodilating action of levosimendan.[4]

Whole-cell recordings [4]
The standard patch-clamp technique was applied in the whole-cell configuration with a patch-clamp amplifier. Membrane potentials and whole-cell currents were measured in current-clamp or voltage-clamp mode, respectively. Voltage-clamp experiments were performed by applying either voltage ramp or step pulses. The patch electrodes (2–5 MΩ) were made from borosilicate glass capillary tubing. The cell suspension was placed into a small chamber (0.5 ml) on the stage of an inverted microscope. The bath was superfused with the following extracellular (bath) solution (mM): NaCl, 141; KCl, 4.7; MgCl2, 1.2; CaCl2, 1.8; glucose, 10; HEPES, 10 and pH adjusted to 7.4 with NaOH. The internal (pipette) solution for the whole-cell experiments was consisted of the following composition (mM): KCl, 125; MgCl2, 4; HEPES, 10; EGTA, 10; ATP-Na2, 5 and pH adjusted 7.2 with KOH.
To isolate Ca2+ channel current (Ba2+ current), the pipette was filled with high Cs+ solution of the following composition (mM): CsOH, 100; CsCl, 30; EGTA, 10; HEPES, 10; l-glutamate, 112; ATP-Na2, 5; free Mg2+, 1 and pH adjusted 7.2 with CsOH. The bath solution was isotonic Ba2+ solution containing (mM): BaCl2, 100; glucose, 10; HEPES, 10 and pH adjusted 7.3 with Tris. Leak current and residual capacitive current were subtracted using P/N protocol in the pCLAMP software.
Current and voltage signals were filtered at 1 kHz and digitized by an A/D converter and analyzed on a personal computer using the pCLAMP software (version 5.05). The membrane capacitance was determined from the current amplitude elicited in response to a hyperpolarizing voltage ramp pulse of 0.2 V/s from a holding potential of 0 mV (duration 25 ms, peak amplitude −5 mV) to avoid interference by any time-dependent ionic currents. Average cell capacitance was 12.6±0.5 pF (n=39).

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Single-channel recordings [4]
Single-channel current recordings were made in open cell-attached patch configuration (Kakei et al., 1985; Ohya and Sperelakis, 1989b) with the same patch-clamp amplifier used in the whole-cell experiments. In brief, after making cell-attached patch with recording pipette, one end of the cell was mechanically disrupted using another glass pipette containing the bath solution. The tip of the patch electrode was coated with Sylgard and its resistance ranged from 2 to 6 MΩ when it was filled with the following pipette (extracellular) solution (mM): NaCl, 80; KCl, 60; MgCl2, 1.3; CaCl2, 1.7; HEPES, 10 and pH adjusted 7.4 with NaOH The bath (intracellular) solution contained (mM): NaCl, 9; KCl, 117; KOH, 13; MgCl2, 3; HEPES, 18; EGTA, 5; glucose, 10 and pH adjusted 7.3 with KOH. Currents signals were filtered at 1 kHz, sampled at 1 kHz and stored in a personal computer. The storing of the digitized signals was carried out using AxoTape, and analysis was performed by pCLAMP software (version 6.02).

Freshly isolated single vascular smooth muscle cells were prepared from peripheral segments of rat superior mesenteric artery, as previouly described (Ohya and Sperelakis, 1989a; Yokoshiki et al., 1997a). In brief, rats of either sex (weighing 250–350 g) were decapitated and bled under CO2 anesthesia. From the vascular bed of the jejunum, peripheral segments of superior mesenteric artery (prearteriole; diameter, <300 μm) were dissected out and placed in Krebs–Ringer solution which had the following composition (mM): NaCl, 120.7; KCl, 5.9; NaHCO3, 15.5; NaH2PO4, 1.2; MgCl2, 1.2; CaCl2, 2.5; glucose, 11.5; bubbled with 95% O2–5% CO2. Connective tissues were carefully removed with surgical microscissors under a dissecting microscope.
The tissues were then transferred to a Ca2+-free solution containing (mM): NaCl, 140; KCl, 6.0; glucose, 10; HEPES, 10 and pH adjusted to 7.3 with tris-(hydroxymethyl)aminomethane (Tris). The lumen was flushed with the Ca2+-free solution to remove the blood cells. The tissues were then cut into small pieces (1–2 mm). Tissue pieces were incubated in the Ca2+-free solution for about 15 min at 36°C. The incubation solution was exchanged to Ca2+-free solution containing 0.25% collagenase, 0.05% papain, 0.05% trypsin inhibitor (type II-S) and 0.3% bovine serum albumin (essentially free of fatty acids). After about 50 min of incubation, the collagenase containing solution was washed out with fresh Ca2+-free solution. Digested tissues were agitated gently with a blunt-tipped glass pipette to disperse the single cells. The debris was removed with a fine nylon mesh. Finally, the cells were placed in a stock solution having the following composition (mM): NaCl, 137; KCl, 6.0; MgCl2, 0.5; CaCl2, 0.5; glucose, 10; HEPES, 10; 0.2% trypsin inhibitor, 0.3% bovine serum albumin and pH adjusted to 7.3 with Tris. The cell suspension was stored in an ice-cold bath and used within 4 h of the cell dispersion. Only spindle-shaped elongated cells were used for experiments. All experiments were performed at room temperature (20–22°C).[1]

Absorption, Distribution and Excretion
The bioavailability of oral levosimendan in healthy volunteers was 85 ± 6% and in patients was 84 ± 4%. Metabolism/Metabolites Completely metabolized, with some active metabolites (OR-1855 and OR-1896) potentially prolonging the hemodynamic effects of the drug. Biological Half-Life The elimination half-life is approximately 1 hour. Levosimendan and its active metabolite OR-1896 [2] During the metabolism of levosimendan, approximately 5% of the drug is converted to the metabolite OR-1855 (the (−) enantiomer of 4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)aniline) in the large intestine, and then acetylated in the liver to form the active metabolite OR-1896. Levosimendan binds to plasma proteins at a rate of 98%, while OR-1896 binds at only 40%. This explains why relatively low levels of total plasma metabolites can produce clinically significant effects. Unlike levosimendan, which has an elimination half-life of 1–1.5 hours, OR-1896 has a half-life of approximately 75–80 hours; therefore, its cardiovascular effects can persist for 7–9 days after cessation of levosimendan infusion for 24 hours. In patients with severe renal or moderate hepatic impairment, the pharmacokinetics of the parent drug are unaffected, but the elimination of its metabolites may be prolonged.

Protein Binding
98% of the protein is bound to plasma proteins. Adverse Events [1] Although patients with acute heart failure (AHF) are at high risk, levosimendan infusion was generally well tolerated. The incidence of hypotension was higher in the levosimendan group compared to placebo, but not in the dobutamine group. Levosimendan was associated with a higher incidence of atrial fibrillation compared to placebo and dobutamine. However, there is no consensus on ventricular arrhythmias. In the REVIVE study, the incidence of ventricular tachycardia was higher in the levosimendan group compared to the placebo group. In the SURVIVE study, the incidence of ventricular tachycardia was similar in the levosimendan and dobutamine groups. The frequency of heart failure as an adverse event was lower in both studies, but this result was statistically significant only in the SURVIVE study. Safety Laboratory Measure [1] The variation in safety laboratory measures was small in the levosimendan study. Compared with the control group, the incidence of potassium level decrease was higher in the levosimendan group. Clinically insignificant decreases in hemoglobin and red blood cell count were observed [1].

[1]. Levosimendan: current data, clinical use and future development. Heart Lung Vessel, 2013. 5(4): p. 227-245.

[2]. Levosimendan: molecular mechanisms and clinical implications: consensus of experts on the mechanisms of action of levosimendan. Int J Cardiol, 2012. 159(2): p. 82-7.

[3]. Effects of levosimendan on mortality and hospitalization. A meta-analysis of randomized controlled studies. Crit Care Med, 2012. 40(2): p. 634-46.

[4]. Levosimendan, a novel Ca2+ sensitizer, activates the glibenclamide-sensitive K+ channel in rat arterial myocytes. Eur J Pharmacol. 1997 Aug 27;333(2-3):249-59.

Levosimendan is a hydrazone, pyridazinone, and nitrile compound. It possesses pharmacological effects as a vasodilator, EC 3.1.4.17 (3',5'-cyclic nucleotide phosphodiesterase) inhibitor, inotropic agent, and antiarrhythmic agent. Levosimendan binds to troponin C in a calcium-dependent manner, increasing the sensitivity of cardiomyocytes to calcium. This enhances myocardial contractility without increasing calcium ion levels. It also relaxes vascular smooth muscle by opening ATP-sensitive potassium channels. Levosimendan is used to treat acute decompensated congestive heart failure. A hydrazone and pyridazin derivative; levosimendan is a phosphodiesterase III inhibitor, calcium sensitizer, and positive inotropic agent used to treat heart failure. Drug Indications For short-term treatment of acute decompensated severe chronic heart failure (CHF). Its application in the treatment of heart disease is also currently under investigation.

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Mechanism of Action
Levosimendan appears to increase the sensitivity of myofilaments to calcium by binding to cardiac troponin C in a calcium-dependent manner. This stabilizes calcium-induced conformational changes in troponin C, thereby (1) altering the dynamics of the actin-myosin cross-bridge without significantly increasing the cross-bridge circulation rate or myocardial ATP consumption; (2) enhancing the effect of calcium on myofilaments during systole; and (3) improving contraction with low energy consumption (positive inotropic effect). Both diastolic calcium concentration and sensitivity decrease, thus restoring or improving diastolic function. Levosimendan also induces vasodilation by opening ATP-sensitive potassium channels. Through these positive inotropic and vasodilatory effects, levosimendan can increase cardiac output without increasing myocardial oxygen consumption. Levosimendan also has selective phosphodiesterase (PDE)-III inhibition, which may contribute to its positive inotropic effect under certain experimental conditions. Levosimendan is reported to preferentially act as a Ca²⁺ sensitizer at low concentrations, while its effect as a PDE-III inhibitor is more pronounced in experimental animals and humans at high concentrations.

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Pharmacodynamics
Levosimendan is a novel Ca²⁺ sensitizer with positive inotropic properties.
Calcium sensitizers represent a new class of positive inotropic drugs that overcome the shortcomings of existing positive inotropic drugs, such as: they do not increase the risk of arrhythmias, cell damage, and death due to intracellular calcium overload; they do not increase activation energy; and they have the potential to reverse contractile dysfunction under pathophysiological conditions (such as acidosis or myocardial stunned syndrome). Levosimendan has not yet been approved in the United States or Canada. Levosimendan infusion is generally well tolerated. Data from the two largest studies to date—the REVIVE and SURVIVE studies—showed a higher incidence of hypotension in the levosimendan group compared to placebo, but no such difference was observed in the dobutamine group. Compared to the two control drugs mentioned above, levosimendan was also associated with a higher incidence of atrial fibrillation. It is important to note that levosimendan, in addition to enhancing myocardial contractility, also has a significant vasodilatory effect. Clinical studies have shown that levosimendan should be used with caution in patients with hypotension, especially those with low blood volume. For such patients, a lower infusion rate and no loading dose should be considered. In case of accidental overdose, significant hemodynamic changes are expected, primarily manifested as hypotension and tachycardia/arrhythmia. Hypotension should be treated with fluid resuscitation and vasoconstrictors as needed. Arrhythmias can be treated with intravenous beta-blockers or amiodarone (if blood pressure permits). Due to the formation of active metabolites, prolonged follow-up may be necessary if the total dose of the parent drug is large. This drug has been proposed for use in cardiac and non-cardiac surgery, cardiogenic shock, and septic shock. Numerous independent studies have described the effects of levosimendan in these conditions, and there is good reason to conduct studies with sufficient statistical power to confirm these reports. Furthermore, this drug has also shown positive efficacy in some specific application areas. [1] The molecular mechanism of levosimendan's Ca2+ sensitizing effect is related to its specific interaction with the troponin C molecule, a Ca2+ sensor in cardiac myofilaments. Over the years, a wealth of preclinical and clinical evidence has revealed a variety of beneficial pleiotropic effects of levosimendan and its long-acting metabolite OR-1896. First, activation of ATP-sensitive potassium channels on the smooth muscle cell membrane appears to be a potent vasodilatory mechanism. In addition, activation of ATP-sensitive potassium channels on mitochondria may extend its cellular scope of action, enabling it to regulate mitochondrial ATP production and suggesting a cardioprotective pharmacological mechanism. Finally, levosimendan has been shown to have subtype-selective phosphodiesterase inhibition. To explain the complex mechanism of action of levosimendan, a careful analysis of all potential pharmacological interactions is needed in conjunction with the existing evidence. These data suggest that the cardiovascular effects of levosimendan are not solely achieved through drug-receptor interactions, but involve unique beneficial energy and neurohormonal changes compared to other types of positive inotropic drugs. [2]

C14H12N6O

280.28

280.107

C, 59.99; H, 4.32; N, 29.98; O, 5.71

141505-33-1

3033825

Light yellow to yellow solid powder

1.3±0.1 g/cm3

216-219ºC (dec.)

1.673

0.59

2

6

3

21

549

1

C[C@@H]1CC(=O)NN=C1C2=CC=C(C=C2)NN=C(C#N)C#N

WHXMKTBCFHIYNQ-SECBINFHSA-N

InChI=1S/C14H12N6O/c1-9-6-13(21)19-20-14(9)10-2-4-11(5-3-10)17-18-12(7-15)8-16/h2-5,9,17H,6H2,1H3,(H,19,21)/t9-/m1/s1

2-[[4-[(4R)-4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl]phenyl]hydrazinylidene]propanedinitrile

Levosimendanum; LEVOSIMENDAN; 141505-33-1; Simdax; (R)-Simendan; Levosimendan [INN]; (-)-OR-1259; levosimendanum; Simdax (TN);Simdax; Levosimendan

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 50 mg/mL (~178.39 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (8.92 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)