PDE3/PDE Ⅲ; K+ Channel; Levosimendan metabolite

Many advantageous pleiotropic effects of OR-1896 have been demonstrated by a growing body of evidence. Considerable vasodilation is induced by OR-1896, and a potent vasodilatory mechanism appears to involve the activation of ATP-sensitive sarcolemmal K+ channels in smooth muscle cells. Activating ATP-sensitive K+ channels in mitochondria can also suggest pharmacological strategies for cardioprotection and broaden the range of controls available to the cell to govern mitochondrial ATP generation [1].

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Ca2+-sensitization [1]
Levosimendan interacts with the Ca2+-saturated cTnC and this forms the basis of its Ca2+-sensitizing mechanism. The binding site for levosimendan on cTnC has been localized to a hydrophobic region of its N-domain near the so-called D/E linker region. There are important hydrogen-bond donor and acceptor groups on the pyridazinone ring and on the mesoxalonitrile–hydrazone moieties of levosimendan that bind to cTnC. Hence, it is likely that these groups form hydrogen bonds with polar or charged amino acids in the hydrophobic pocket of the Ca2+-saturated N-terminal domain of cTnC. The consequence of levosimendan binding is that the Ca2+-saturated cTnC is stabilized in the presence of the drug. The scheme that was put forward on the basis of this conformational change involves a prolonged interaction between cTnC and cardiac troponin I, thereby promoting contractile force in the presence of levosimendan without an increase in the amplitude of intracellular Ca2+ transient. The magnitude of Ca2+-sensitization evoked by levosimendan or OR-1896 appeared to be less than the maximal effect of other known Ca2+-sensitizers, although similar to what expected from length-dependent Ca2+-sensitization during the activation of the Frank–Starling mechanism. Diastolic function is not impaired by levosimendan potentially because of the mild degree of Ca2+-sensitization, and probably lso because dissociation of Ca2+ from the cTnC molecule at the diastolic level of intracellular Ca2+ precludes its interaction with levosimendan. The active metabolite OR-1896 exhibits comparable hemodynamic effects to those of levosimendan via Ca2+-sensitization: similar interactions between OR-1896 and cTnC can therefore be postulated. However, structural biochemical data for the interaction between OR-1896 and cTnC are not yet available.

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Vasodilation [1]
Levosimendan and OR-1896 evoke prominent vasodilatory responses. Levosimendan has the potential to open ATP-sensitive K+ channels, and consequent hyperpolarization of vascular smooth muscle cells has been suggested to explain the drug's vasodilatory effects. In line with this proposal, inhibitors of ATP-sensitive K+ channels mitigate vasodilatation induced by levosimendan or OR-1896, although these pharmacological approaches also suggested that other types of K+ channels (e.g. Ca2+-activated K+ channels and voltage-gated K+ channels) might be also involved. The composition of K+ channels mediating vasodilatory responses may depend on vessel type and also on vascular diameter. Interestingly, recent experimental data indicated an endothelial component for the levosimendan-induced vasodilation, and interactions between ATP-sensitive K+ channel activation and NO production. Vasodilation during levosimendan administration has been demonstrated at the arterial sides of the pulmonary, coronary and peripheral circulations, and at the venous sides of the portal and saphenous systems.

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Phosphodiesterase inhibition [1]
Both levosimendan and OR-1896 are highly selective inhibitors of the phosphodiesterase (PDE) III isoform. An interaction between levosimendan-evoked positive inotropy or lusitropy and cAMP signaling has been suggested from some experimental studies. However, it is recognized that an increase in intracellular cAMP concentration through PDE-inhibition depends on a complex interplay among the available PDE isoforms, their subcellular localization and parallel signaling cascades, and it is clearly demonstrated that neither levosimendan nor its active metabolite affect the function of other PDE isozymes at their therapeutic concentrations. Thus, higher than therapeutic concentrations of levosimendan and species-dependent characteristics of cyclic nucleotide signaling may potentially explain the experimentally observed interaction between cAMP signaling and positive inotropy/lusitropy, since results of several investigations indicate that alterations in intracellular Ca2+ concentration (resulting from cAMP elevation secondary to PDE inhibition) are not a prerequisite for the cardiac effects of levosimendan. However, it is also to be acknowledged that cardiomyocytes of patients at various different stages of their cardiovascular diseases may differ profoundly in the regulation of the intracellular cyclic nucleotides as well as in intracellular Ca2+ concentration, and hence the consequence of an isolated inhibition of PDE III might vary. What can be anticipated is that the chance of levosimendan causing an increase in intracellular cAMP is the least at low doses where, without doubts other PDE isoforms (e.g. PDE IV) can substitute for PDE III.

Levosimendan is metabolized by the large intestine, where about 5% of the drug is transformed into the metabolite OR-1855. The metabolite OR-1896 is then formed by acetylation in the liver. OR-1896 is only 40% bound to plasma proteins, compared to 98% binding for levosimendan. The elimination half-life of OR-1896 is roughly 75 to 80 hours, allowing cardiovascular effects to last for up to 7 to 9 days after ending the 24-hour infusion. This is in contrast to levosimendan, which has a half-life of 1-1.5 hours. The Zuo Ximendan. While the metabolite's (OR-1896) elimination can be extended, the parent drug's pharmacokinetics remain unchanged in persons with severe renal impairment or mild hepatic impairment [1].

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Levosimendan enhances cardiac contractility via Ca(2+) sensitization and induces vasodilation through the activation of ATP-dependent K(+) and large-conductance Ca(2+)-dependent K(+) channels. However, the hemodynamic effects of levosimendan, as well as its metabolites, OR-1896 and OR-1855, relative to plasma concentrations achieved, are not well defined. Thus levosimendan, OR-1896, OR-1855, or vehicle was infused at 0.01, 0.03, 0.1, and 0.3 mumol.kg(-1).30 min(-1), targeting therapeutic to supratherapeutic concentrations of total levosimendan (62.6 ng/ml). Results were compared with those of the beta(1)-agonist dobutamine and the phosphodiesterase 3 inhibitor milrinone. Peak concentrations of levosimendan, OR-1896, and OR-1855 were 455 +/- 21, 126 +/- 6, and 136 +/- 6 ng/ml, respectively. Levosimendan and OR-1896 produced dose-dependent reductions in mean arterial pressure (-31 +/- 2 and -42 +/- 3 mmHg, respectively) and systemic resistance without affecting pulse pressure, effects paralleled by increases in heart rate; OR-1855 produced no effect at any dose tested. Dobutamine, but not milrinone, increased mean arterial pressure and pulse pressure (17 +/- 2 and 23 +/- 2 mmHg, respectively). Regarding potency to elicit reductions in time to peak pressure and time to systolic pressure recovery: OR-1896 > levosimendan > milrinone > dobutamine. Levosimendan and OR-1896 elicited dose-dependent increases in change in pressure over time (118 +/- 10 and 133 +/- 13%, respectively), concomitant with reductions in left ventricular end-diastolic pressure and ejection time. However, neither levosimendan nor OR-1896 produced increases in myocardial oxygen consumption at inotropic and vasodilatory concentrations, whereas dobutamine increased myocardial oxygen consumption (79% above baseline). Effects of the levosimendan and OR-1896 were limited to the systemic circulation; neither compound produced changes in pulmonary pressure, whereas dobutamine produced profound increases (74 +/- 13%). Thus levosimendan and OR-1896 are hemodynamically active in the anesthetized dog at concentrations observed clinically and elicit cardiovascular effects consistent with activation of both K(+) channels and Ca(2+) sensitization, whereas OR-1855 is inactive on endpoints measured in this study.[3]

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The intravenous formulation of levosimendan has been studied in several randomized, comparative studies in patients with decompensated heart failure and efficacy, and tolerability has been demonstrated in heart failure patients of both ischemic and nonischemic etiology. Plasma concentrations associated with levosimendan efficacy were assessed in an open-label, nonrandomized phase II study in patients diagnosed with heart failure, whereby a 24-h continuous infusion of levosimendan produced peak plasma concentrations of 62.6 ng/ml; in the same patients, peak concentrations of OR-1896 and OR-1855, the two primary circulating metabolites of levosimendan, reached 5.5 and 6.8 ng/ml, respectively.
Due to the reduction of levosimendan to OR-1855 in humans and subsequent acetylation to OR-1896, the contribution of the parent vs. each metabolite to the hemodynamic and cardiovascular effects observed in patients cannot be definitively described. However, in the dog, neither levosimendan nor OR-1855 is metabolized to OR-1896. Moreover, a comprehensive assessment of the effects of levosimendan and its metabolites (in relation to plasma concentrations achieved) on cardiovascular function has not been fully described in dog. Thus the present study sought to characterize the effects of levosimendan, OR-1896, and OR-1855 on myocardial and hemodynamic function in the comprehensively instrumented dog at plasma concentrations deemed therapeutic to supratherapeutic. Results were compared with two other agents routinely prescribed in the treatment of heart failure: the β1-agonist dobutamine and the PDE3 inhibitor milrinone [3].

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Levosimendan enhances cardiac contractility via Ca(2+) sensitization and induces vasodilation through the activation of ATP-dependent K(+) and large-conductance Ca(2+)-dependent K(+) channels. However, the hemodynamic effects of levosimendan, as well as its metabolites, OR-1896 and OR-1855, relative to plasma concentrations achieved, are not well defined. Thus levosimendan, OR-1896, OR-1855, or vehicle was infused at 0.01, 0.03, 0.1, and 0.3 mumol.kg(-1).30 min(-1), targeting therapeutic to supratherapeutic concentrations of total levosimendan (62.6 ng/ml). Results were compared with those of the beta(1)-agonist dobutamine and the phosphodiesterase 3 inhibitor milrinone. Peak concentrations of levosimendan, OR-1896, and OR-1855 were 455 +/- 21, 126 +/- 6, and 136 +/- 6 ng/ml, respectively. Levosimendan and OR-1896 produced dose-dependent reductions in mean arterial pressure (-31 +/- 2 and -42 +/- 3 mmHg, respectively) and systemic resistance without affecting pulse pressure, effects paralleled by increases in heart rate; OR-1855 produced no effect at any dose tested. Dobutamine, but not milrinone, increased mean arterial pressure and pulse pressure (17 +/- 2 and 23 +/- 2 mmHg, respectively). Regarding potency to elicit reductions in time to peak pressure and time to systolic pressure recovery: OR-1896 > levosimendan > milrinone > dobutamine. Levosimendan and OR-1896 elicited dose-dependent increases in change in pressure over time (118 +/- 10 and 133 +/- 13%, respectively), concomitant with reductions in left ventricular end-diastolic pressure and ejection time. However, neither levosimendan nor OR-1896 produced increases in myocardial oxygen consumption at inotropic and vasodilatory concentrations, whereas dobutamine increased myocardial oxygen consumption (79% above baseline). Effects of the levosimendan and OR-1896 were limited to the systemic circulation; neither compound produced changes in pulmonary pressure, whereas dobutamine produced profound increases (74 +/- 13%). Thus levosimendan and OR-1896 are hemodynamically active in the anesthetized dog at concentrations observed clinically and elicit cardiovascular effects consistent with activation of both K(+) channels and Ca(2+) sensitization, whereas OR-1855 is inactive on endpoints measured in this study[3].

Levosimendan and its active metabolite OR-1896 [1]
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.

Animals were randomly divided into one of six treatment or vehicle (5% dextrose in water) groups. Following the completion of the surgical protocol, animals were allowed to stabilize for 1 h, and baseline data were collected at 5-min intervals 30 min before treatment. Each dose of active drug was administered as a 30-min infusion (0.02 ml·kg−1·min−1) as a series of four escalating doses dissolved in a 5% dextrose in water vehicle; following termination of the high-dose infusion, animals were observed for 30 min. Levosimendan, OR-1896, and OR-1855 were infused at 0.01, 0.03, 0.10, and 0.33 μmol·kg−1·30 min−1; blood samples were withdrawn at 15-min intervals for determination of plasma concentrations of levosimendan and each metabolite by HPLC-mass spectrometry. Dobutamine and milrinone were infused at 1-log unit higher doses to achieve similar reductions in systemic resistance as that produced by levosimendan and OR-1896 (Fig. 1). For additional depth and perspective, results from the present study were also compared with those of three K+ channel openers (KCOs) previously profiled in the anesthetized dog cardiovascular model, A-278637, ZD-6169, WAY-133537, A-325100, and the dihydropyridine Ca2+ channel blocker nifedipine [3].

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Male Sprague-Dawley rats (325–400 g) were anesthetized with the long-acting barbiturate inactin (100 mg/kg i.p.). Subsequently, rats were used as instruments to record hemodynamic and cardiovascular function as described previously (Liu et al., 2007). In brief, polyethylene tubing (PE240) was placed in the trachea to keep the airway patent, and rats continued to breathe spontaneously. Vascular catheters (PE50) were placed in the femoral arteries to measure mean arterial...[2]

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Levosimendan enhances cardiac contractility primarily via Ca2+ sensitization, and it induces vasodilation through the activation of ATP-sensitive potassium channels and large conductance Ca2+-activated K+ channels. However, the concentration-dependent hemodynamic effects of levosimendan and its metabolites (R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide (OR-1896) and (R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2 H)-one (OR-1855) have not been well defined. Thus, levosimendan (0.03, 0.10, 0.30, and 1.0 μmol/kg/30 min; n = 6) was infused as four escalating 30-min i.v. doses targeting therapeutic to supratherapeutic concentrations of levosimendan (Cmax, ∼62.6 ng/ml); metabolites were infused at one-half log-unit lower doses and responses compared to dobutamine (β1-agonist) and milrinone (phosphodiesterase 3 inhibitor). Peak concentrations of levosimendan, OR-1896, and OR-1855 at the end of the high dose were 323 ± 14, 83 ± 2, and 6 ± 2 ng/ml, respectively (OR-1855 rapidly metabolized to OR-1896; peak = 82 ± 3 ng/ml). Levosimendan and OR-1896 produced dose-dependent reductions in blood pressure and peripheral resistance with a rank potency, based on ED15 values, of OR-1896 (0.03 μmol/kg) > OR-1855 > levosimendan > milrinone (0.24 μmol/kg); an ED15 for dobutamine could not be defined. Only dobutamine produced increases in pulse pressure (30 ± 5%) and rate-pressure product (34 ± 4%). All of the compounds, with the exception of OR-1855, elicited dose-dependent increases in dP/dt with a rank potency, based on ED50 values, of dobutamine (0.03 μmol/kg) > levosimendan > OR-1896 > milrinone (0.09 μmol/kg), although only levosimendan produced sustained increases in cardiac output (9 ± 4%). Thus, levosimendan and OR-1896 are hemodynamically active at sub- to supratherapeutic concentrations (whereas the effects of OR-1855 in the rat are thought to be predominantly mediated by conversion to OR-1896) and produce direct inotropic effects and also direct relaxation of the peripheral vasculature, which clearly differentiates them from dobutamine, which does not elicit K+ channel activation, suggesting a more balanced effect on the cardiac-contractile state and K+ channel-mediated changes in vascular resistance.[2]

[1]. Levosimendan: molecular mechanisms and clinical implications: consensus of experts on the mechanisms of action of levosimendan. Int J Cardiol. 2012 Aug 23;159(2):82-7.

[2]. Evoked changes in cardiovascular function in rats by infusion of levosimendan, OR-1896 [(R)-N-(4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl)acetamide], OR-1855 [(R)-6-(4-aminophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one], dobutamine, and milrinone: comparative effects on peripheral resistance, cardiac output, dP/dt, pulse rate, and blood pressure. J Pharmacol Exp Ther, 325 (2008), pp. 331-340

[3]. Comparative effects of levosimendan, OR-1896, OR-1855, dobutamine, and milrinone on vascular resistance, indexes of cardiac function, and O2 consumption in dogs. Am J Physiol Heart Circ Physiol. 2008 Jan;294(1):H238-48.

OR-1896 is an anilide and a member of acetamides.

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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.[1]

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Classically, Ca2+-sensitization and vasodilation are referred to as the cornerstones of the mechanisms of action of levosimendan. These effects develop in response to specific interactions between levosimendan or OR-1896 and cTnC in cardiomyocytes, and levosimendan or OR-1896 and ATP-sensitive K+ channels in the vascular beds. On top of these well-known inodilator effects, cardioprotection emerges as the third facet of levosimendan during acute and chronic heart failure. The molecular mechanism of the levosimendan-evoked cardioprotection possibly includes an interaction with mitochondrial energy conservation through mitochondrial ATP-sensitive K+ channels in cardiomyocytes, although additional molecular mechanisms cannot be excluded. Levosimendan-evoked cardioprotection can be mobilized during acute stress conditions and is manifested as acute anti-ischemic and anti-stunning effects. In addition, levosimendan modulates cytokine and neurohumoral signalizations implicating a potential interference with cardiomycyte apoptosis and myocardial remodeling. The collection of all the above effects may translate into better long-term clinical outcomes in levosimendan responders than in those whose levosimendan responsiveness is suboptimal. In summary, by virtue of its unique mechanism of action, levosimendan may be characterized as the first in a class of the cardioprotective inodilator drugs. We sincerely hope that the future drug discovery in this field will lead us to even better drugs aimed to support the cardiac function. [1]

C₁₃H₁₅N₃O₂

245.28

245.116

C, 63.66; H, 6.16; N, 17.13; O, 13.05

220246-81-1

9816296

Light yellow to yellow solid powder

1.278 g/cm3

224-226ºC

1.342

2

3

2

18

370

1

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

GDZXNMWZXLDEKG-MRVPVSSYSA-N

InChI=1S/C13H15N3O2/c1-8-7-12(18)15-16-13(8)10-3-5-11(6-4-10)14-9(2)17/h3-6,8H,7H2,1-2H3,(H,14,17)(H,15,18)/t8-/m1/s1

N-[4-[(4R)-4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl]phenyl]acetamide

OR1896; 220246-81-1; OR-1,896; (R)-N-ACETYL-6-(4-AMINOPHENYL)-4,5-DIHYDRO-5-METHYL-3(2H)-PYRIDAZINONE; IKC90B19NV; N-[4-[(4R)-4-methyl-6-oxo-4,5-dihydro-1H-pyridazin-3-yl]phenyl]acetamide; UNII-IKC90B19NV; OR-1896; N-(4-((4R)-1,4,5,6-Tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl)acetamide; OR 1896

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 : ~62.5 mg/mL (~254.81 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (8.48 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 20.8 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: ≥ 2.08 mg/mL (8.48 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 20.8 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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Solubility in Formulation 3: ≥ 2.08 mg/mL (8.48 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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