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 is 85 ± 6% in healthy volunteers and 84 ± 4% in patients.

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Metabolism / Metabolites
Complete metabolism, with some active metabolites (OR-1855 and OR-1896) possibly extending the drug's haemodynamic effects.

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Biological Half-Life
Eliminination half-life is approximately 1 hour.

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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)phenylamine) in the large intestine, and then acetylated in the liver to form the active metabolite OR-1896. Binding to plasma proteins is 98% for levosimendan but only 40% for OR-1896: this explains why a relatively low total plasma level of the metabolite may evoke clinically significant effects. Unlike levosimendan, which has an elimination half-life of 1–1.5 h, the half-life of OR-1896 is about 75 to 80 h allowing cardiovascular effects to persist up to 7 to 9 days after discontinuation of a 24-hour infusion of levosimendan. The pharmacokinetic of the parent drug is unaltered in subjects with severe renal impairment or with moderate hepatic impairment, whereas the elimination of its metabolites can be prolonged.

Protein Binding
98% bound to plasma protein.

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Adverse events [1]
Levosimendan infusion has generally been well tolerated in the AHF population, despite the high-risk nature of these patients. Hypotension was seen more frequently with levosimendan than with placebo, but not when levosimendan was compared with dobutamine.
Levosimendan has been associated with a higher incidence of atrial fibrillation compared both with placebo and with dobutamine. However, conflicting results have been presented with regard to ventricular arrhythmias. In REVIVE a higher incidence of ventricular tachycardia was observed with levosimendan compared with placebo. In SURVIVE, ventricular tachycardia was observed with similar frequency in the levosimendan and dobutamine groups. In both studies, cardiac failure as an adverse event was less frequent in levosimendan arm, although the result was statistically significant only in SURVIVE.

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Safety laboratory values [1]
The changes in safety laboratory variables have been modest in levosimendan studies. A decrease in potassium levels has been seen with levosimendan more often than with comparators. Clinically insignificant decreases in haemoglobin and erythrocyte counts have been 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, a pyridazinone and a nitrile. It has a role as a vasodilator agent, an EC 3.1.4.17 (3',5'-cyclic-nucleotide phosphodiesterase) inhibitor, a cardiotonic drug and an anti-arrhythmia drug.
Levosimendan increases calcium sensitivity to myocytes by binding to troponin C in a calcium dependent manner. This increases contractility without raising calcium levels. It also relaxes vascular smooth muscle by opening adenosine triphosphate sensitive potassium channels. Levosimendan is used to manage acutely decompensated congestive heart failure.
A hydrazone and pyridazine derivative; the levo-form is a phosphodiesterase III inhibitor, calcium-sensitizing agent, and inotropic agent that is used in the treatment of HEART FAILURE.

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Drug Indication
For short term treatment of acutely decompensated severe chronic heart failure (CHF). Also being investigated for use/treatment in heart disease.

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Mechanism of Action
Levosimendan appears to increase myofilament calcium sensitivity by binding to cardiac troponin C in a calcium-dependent manner. This stabilizes the calcium-induced conformational change of troponin C, thereby (1) changing actin-myosin cross-bridge kinetics apparently without increasing the cycling rate of the cross-bridges or myocardial ATP consumption, (2) increasing the effects of calcium on cardiac myofilaments during systole and (3) improving contraction at low energy cost (inotropic effect). Calcium concentration and, therefore, sensitization decline during diastole, allowing normal or improved diastolic relaxation. Levosimendan also leads to vasodilation through the opening of ATP-sensitive potassium channels. By these inotropic and vasodilatory actions, levosimendan increases cardiac output without increasing myocardial oxygen demand. Levosimendan also has a selective phosphodiesterase (PDE)-III inhibitory action that may contribute to the inotropic effect of this compound under certain experimental conditions. It has been reported that levosimendan may act preferentially as a Ca²⁺ sensitizer at lower concentrations, whereas at higher concentrations its action as a PDE-III inhibitor becomes more prominent in experimental animals and humans.

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Pharmacodynamics
Levosimendan is a new Ca²⁺-sensitizing inotropic agent. Ca²⁺ sensitizers represent a new class of inotropic agents, which overcome the disadvantages associated with currently available inotropic agents in as they are not associated with an increased risk of arrhythmias, cell injury and death due to Ca²⁺ overload in myocardial cells; they do not increase the activation energy; and they have the potential to reverse contractile dysfunction under pathophysiologic conditions, such as acidosis or myocardial stunning. Levosimendan has not been approved for use in the U.S. or Canada.

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Levosimendan infusion has generally been well tolerated. Data from the REVIVE and SURVIVE studies - the two largest studies conducted to date- indicate that hypotension was more frequent with levosimendan than with placebo, though not dobutamine. Levosimendan was also associated with higher incidence of atrial fibrillation relative to both those comparators. It should be recalled that, in addition to contractility increasing effects, levosimendan has profound vasodilatory effects. Clinical studies have indicated that levosimendan should be given cautiously to patients with low blood pressure, especially in case of hypovolaemia. Use of lower infusion rates without the loading bolus should be considered for such patients. In case of unintended overdose, pronounced haemodynamic effects would be expected; mainly hypotension and increased heart rate/arrhythmias. Hypotension should be treated with fluid resuscitation and vasoconstrictors, as needed. Arrhythmias may be treated with intravenous beta-blockade or amiodarone (if blood pressure allows). Due to the formation of the active metabolite, the follow-up may need to be prolonged, if the total dose of parent drug is substantial. Applications of this drug in fields such as cardiac and non-cardiac surgery, cardiogenic- and septic-shock, and others have been proposed. The effects of levosimendan in these settings have been described in many independent studies, and there is a strong rationale for suitably powered studies to corroborate those reports. Positive experience in a range of niche applications has also been documented. [1]

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The molecular background of the Ca(2+)-sensitizing effect of levosimendan relates to its specific interaction with the Ca(2+)-sensor troponin C molecule in the cardiac myofilaments. Over the years, significant preclinical and clinical evidence has accumulated and revealed a variety of beneficial pleiotropic effects of levosimendan and of its long-lived metabolite, OR-1896. First of all, activation of ATP-sensitive sarcolemmal K(+) channels of smooth muscle cells appears as a powerful vasodilator mechanism. Additionally, activation of ATP-sensitive K(+) channels in the mitochondria potentially extends the range of cellular actions towards the modulation of mitochondrial ATP production and implicates a pharmacological mechanism for cardioprotection. Finally, it has become evident, that levosimendan possesses an isoform-selective phosphodiesterase-inhibitory effect. Interpretation of the complex mechanism of levosimendan action requires that all potential pharmacological interactions are analyzed carefully in the framework of the currently available evidence. These data indicate that the cardiovascular effects of levosimendan are exerted via more than an isolated drug-receptor interaction, and involve favorable energetic and neurohormonal changes that are unique in comparison to other types of inodilators.[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.)