Serum concentrations of levosimendan and its metabolites OR-1855 and OR-1896 in cardiac surgery patients with cardiopulmonary bypass

Background: The inotropic drug levosimendan is often used off-label perioperatively in cardiac surgery patients with cardiopulmonary bypass (CPB). Data regarding serum concentrations of levosimendan and its metabolites within this context is lacking.

Methods: Total serum concentrations (TSC) and unbound fractions (UF) of levosimendan and its metabolites OR-1896 and OR-1855 in cardiac surgery patients with CPB were retrospectively measured using LC-ESI-MS/MS. Simulation of expected levosimendan TSC was performed using Pharkin 4.0. Serum NT-proBNP was assessed with ELISA.

Results: After prolonged levosimendan infusion (1.25 mg or 2.5 mg, respectively) after induction of anaesthesia, a median TSC of 1.9 ng/ml and 10.4 ng/ml was determined in samples taken directly after surgery (T1). Median TSC of 7.6 ng/ml and 22.0 ng/ml, respectively, were simulated at T1. Whereas 1.1 ng/ml and 1.6 ng/ml TSC of OR-1896, respectively, was quantified the day after surgery (T2), TSC of the intermediate metabolite OR-1855 was mostly below the lower limit of quantification (LLOQ). The UF was 0.5% and 1.1% for levosimendan and 64.1% and 52.1% for OR-1896, respectively, with over half the samples being below LLOQ. No difference in NT-proBNP concentrations before surgery and T2 was detected.

Discussion: The low TSC, UF and unchanged NT-proBNP levels suggest a need for dose adjustments of levosimendan in this off-label range. In addition, the differences between the measured and estimated concentrations may suggest a possible influence of a CPB on levosimendan serum concentrations. Due to high variation of serum levels between patients, an optimized dosing regimen combined with therapeutic drug monitoring may be advisable.

1 Introduction

Perioperative left ventricular dysfunction is a major complication after cardiac surgery and is associated with increased mortality (1). Catecholamines and phosphodiesterase type 3 inhibitors (2) are the therapy of choice for postoperative hemodynamic support, but meta-analyses and observational studies suggest increased mortality when using these agents (3, 4). An alternative inotropic drug therapy for ventricular dysfunction could be the perioperatively administration of levosimendan (Simdax, Orion Pharma). As a calcium sensitizer, levosimendan has positive inotropic effects and a network meta-analysis showed it to be the most likely inotrope to reduce mortality among patients undergoing cardiac surgery (5). In addition to the improvement of cardiac output with minimal effect on myocardial oxygen consumption (6), it has antioxidative, anti-inflammatory, and direct cardioprotective effects (6). 4%–7% of the administered dose is metabolized by intestinal bacteria to the metabolite OR-1855, which is then acetylated by hepatic N-acetyltransferase (NAT2) to the pharmacologically active OR-1896 (7). Whereas levosimendan has a short half-life of 1 h, the half-life of the metabolites is 70–80 h and it is therefore hypothesized that the long-term effects arise from the metabolites (8). Interestingly, despite the positive effects initially shown in meta-analyses (9–11), three randomized controlled trials comparing levosimendan with placebo on top of standard care in cardiac surgical patients failed to reach statistical significance. Two studies addressed the question of effectiveness of levosimendan as a prophylactic treatment for the prevention of postoperative low cardiac output in patients with impaired left ventricular ejection fraction (12, 13), and one study evaluated its effectiveness in reducing mortality at day 30 in patients developing post-bypass low cardiac output syndrome (14). A reason for these unexpected results could be negative pharmacokinetic effects of the cardiopulmonary bypass (CPB) during surgery on levosimendan and its metabolites. Until yet, real-life data regarding serum concentrations of levosimendan and its metabolites within cardiac surgery patients upon CPB procedure is lacking. Therefore, we recently developed a method for therapeutic drug monitoring (TDM) of levosimendan, OR-1855 and OR-1896 (15), enabling rapid and simultaneous detection of these compounds in patient serum.

In our center, levosimendan is administered off-label as a prolonged low-dose infusion (0.7–5.6 h) intra- and perioperatively in patients with impaired cardiac output undergoing cardiac surgery. Till this end, no studies investigating the serum concentrations of levosimendan and its metabolites in cardiac patients receiving this off-label infusion perioperatively are available. This, however, would be of great interest as therapeutic serum levels in this setting needs to be confirmed. First, we measured total serum concentrations of levosimendan and its two metabolites, OR-1855 and OR-1896 in patients receiving off-label doses of levosimendan perioperatively and compared these with simulated serum concentrations. Next, we detected the unbound fractions, which are the therapeutic effective fractions, of levosimendan, OR-1896 and OR-1855 in these patients. Finally, we measured levels of NT-proBNP, a cardiac biomarker, before and after levosimendan administration to investigate the efficacy of the measured levosimendan and metabolite concentrations in cardiac surgery patients.

2 Material and methods

2.1 Chemicals

¹³C₆ labelled internal standards of levosimendan, OR-1896, and OR-1855 were obtained from Orion Pharma (Espoo, Finland).

2.2 Human samples

Anonymized serum samples from patients undergoing elective cardiac surgery receiving levosimendan within the perioperative management admitted to the department of cardiac surgery at the University hospital, LMU, were measured retrospectively. Patients with EF <25% undergoing CABG, patients with EF between 25%–40% along with GFR <60 ml/min during CABG, and patients involved in combined procedures with EF <40% or GFR <60 ml/min, as well as cases of acute myocardial ischemia, were treated with levosimendan following our in-house guidelines. Patients received different off-label dosages as a prolonged infusion shortly after induction of anaesthesia and in some cases within the first post-operative day. Experiments with human serum were approved by the clinical ethics committee at the medical faculty, LMU Munich (identification code 17-241; 20-665; 20-1089).

2.3 Sample preparation & LC-ESI-MS/MS

Serum levels of levosimendan, OR-1855 and OR-1896 were quantified with LC-ESI-MS/MS as previously described (15). For unbound fractions (UF), 500 µl of serum was centrifuged in Centrifree® tubes (Merck, Germany) for 30 min at 2,000 × g with a 34° fixed-angle rotor. Liquid-liquid extraction was performed as previously described (15). For levosimendan, OR-1855 and OR-1896 the lower limit of quantification (LLOQ) is 0.126 ng/ml, 0.305 ng/ml and 0.368 ng/ml, respectively.

2.4 Albumin BCG assay

Serum albumin was measured with the BCG Assay Kit (Sigma Aldrich, Germany) according to the supplier's protocol.

2.5 NT-proBNP measurements

NT-proBNP levels in patients’ serum samples were measured with the Human NT-proBNP Elisa Kit (Kelowna BC, Canada) according to the supplier's protocol.

2.6 Simulation of levosimendan serum levels in cardiac surgery patients

The expected serum levels of levosimendan were visualised with Pharkin 4.0 (Heidenheim, Germany). Body weight, height, age, and gender as well as the dose (mg) with the exact dosing intervals and infusion length were included in the calculation. A distribution volume (Vd) of 0.2 L/kg and a half-life of 1.0 h (16) were specified for simulation. The expected concentrations were estimated assuming first-order linear kinetics and a one-compartment model.

2.7 Statistical analysis

Statistical analysis was performed using Prism 5.0 (Gaphpad, San Diego, WA, USA) and Sigma Plot 12.0 (Systat Software, Inpixon, Düsseldorf, Germany). Data are presented as mean ± SEM or median with range. For comparison of more than two datasets without normal distribution, one-way analysis of variance (ANOVA) on ranks was used followed by the Dunn's method. For comparison of correlated samples without normal distribution, the Wilcoxon signed rank test was used. Differences were considered significant at p < 0.05.

3 Results

3.1 Study population

Total serum concentrations (TSC) of levosimendan, OR-1855 and OR-1896 were measured retrospectively in samples from 18 cardiac surgery patients with CPB, receiving levosimendan in the perioperative setting. Patients with an ejection fraction (EF) <25% undergoing CABG, patients with EF between 25%–40% along with GFR <60 ml/min during coronary artery bypass graft (CABG), and patients involved in combined procedures with EF <40% or GFR <60 ml/min, as well as cases of acute myocardial ischemia, were treated with levosimendan according to our in-house guidelines. Patient characteristics are summarized in Table 1. Five patients received 1.25 mg levosimendan as a prolonged infusion after induction of anaesthesia, whereas two patients received an additional second dose of 1.25 mg levosimendan after surgery. Furthermore, six patients received 2.5 mg of levosimendan as a prolonged infusion after induction of anaesthesia and five patients received a second additional dose of 2.5 mg levosimendan after surgery. Samples were taken directly before surgery and levosimendan infusion (T0), shortly after surgery (T1) as well as the first day after surgery (T2).

Table 1

Table 1. Patient characteristics.

3.2 Total serum concentrations of levosimendan and its metabolites OR-1855 & OR-1896 in cardiac surgery patients with CPB

Simultaneous measurement of levosimendan, OR-1855 and OR-1896 in serum samples using a LC-ESI-MS/MS protocol previously established (15) showed that patients receiving a single (n = 5) or double (n = 2) infusion with 1.25 mg levosimendan had low serum concentrations [1.9 ng/ml (0.4–40.6 ng/ml); n = 4; bLLOQ n = 1 and 0.67 ng/ml (0.2–1.1 ng/ml; n = 2) at T1, respectively; Figures 1A,B]. In contrast, we detected higher serum concentrations in samples from patients receiving a single dose of 2.5 mg levosimendan (n = 6) shortly after induction of anaesthesia [10.4 ng/ml (1.9–28.3 ng/ml) at T1; Figure 1C]. In case of an additional infusion of 2.5 mg levosimendan after surgery (n = 5), levosimendan was detectable at T1 [5.2 ng/ml (1.8–26.1 ng/ml) and even at T2 [3.9 ng/ml (0.15–13.7 ng/ml); n = 4; bLLOQ n = 1; Figure 1D]. Regarding the metabolites, concentrations of OR-1896 were quantified at T2 upon 1.25 mg levosimendan [1.1 ng/ml (0.73–1.5 ng/ml); n = 2; bLLOQ n = 2] as well as upon 2.5 mg levosimendan (1.6 ng/ml [0.6–4.0 ng/ml, (n = 5)] (Figures 1A–D). Interestingly, in 87% of the samples OR-1855 levels were below the LLOQ. Taken together, we observed high variation of levosimendan as well as OR-1896 serum levels between patients.

Figure 1

Figure 1. TSC of levosimendan and metabolites in cardiac surgery patients with CPB and comparison of simulated and measured serum levels of levosimendan for each patient at T1. Total serum concentrations (TSC, ng/ml) were measured in samples from cardiac surgery patients with CPB receiving levosimendan after induction of anaesthesia at dosages of (A) 1.25 mg of levosimendan (levosimendan n = 5; OR-1896 n = 4 and OR-1855 n = 4); (B) 1.25 mg of levosimendan as well as a second dose of 1.25 mg 3–4 h after surgery (n = 2); (C) 2.5 mg of levosimendan (n = 6); (D) 2.5 mg of levosimendan as well as a second dose of 2.5 mg 5–21 h after surgery (n = 4). T1: Shortly after cardiac surgery; T2: First day after cardiac surgery; grey horizontal line: lower limit of quantification (LLOQ; levosimendan 0.126 ng/ml; OR-1855 0.305 ng/ml; OR-1896 0.368 ng/ml) (15). (E) Comparison of measured levosimendan TSC (grey bars) and estimated TSC (white circles) at T1 in patients receiving 1.25 mg of levosimendan (n = 5); (F) Comparison of measured levosimendan TSC (grey bars) and estimated TSC (white circles) at T1 in patients receiving 1.25 mg of levosimendan as well as a second dose of 1.25 mg 3–4 h after surgery (n = 2); (G) Comparison of measured levosimendan TSC (grey bars) and estimated TSC (white circles) at T1 in patients receiving 2.5 mg of levosimendan (n = 6); (H) Comparison of measured levosimendan TSC (grey bars) and estimated TSC (white circles) at T1 in patients receiving 2.5 mg of levosimendan as well as a second dose of 2.5 mg 5–21 h after surgery (n = 4). Serum concentrations (ng/ml) were calculated using individual patient data. The simulation for patient 16 could not be performed due to insufficient serum volume for quantification at T1. Arrows show difference between simulated and measured serum concentrations. (I) Comparison of simulated levosimendan TSC with measured TSC within the total patient population at T1 (n = 17; *p < 0.05 Wilcoxon signed rank test). Data are expressed as dot plots with the respective median (black line).

3.3 Measurement of the unbound fraction of levosimendan, OR-1855 & OR-1896 in cardiac surgery patients with CPB

As levosimendan has high binding affinity to serum albumin (97%–98%) (17) and thus only a small amount is unbound and therapeutically effective, we next detected the unbound fraction (UF). For the dosage of 1.25 mg levosimendan, the UF of levosimendan was only measured in one patient sample, as the rest had TSC values below the LLOQ. The measured UF of levosimendan after application of 2.5 mg in a single or double infusion were 1.1% (0.6%–1.1%; n = 3) and 0.5% (n = 1), respectively (Table 2). In contrast to levosimendan (UF 1%–2%), approximately 60% of the metabolites OR-1855 and OR-1896 are found as unbound fractions (18). For the 1.25 mg levosimendan dosage, we detected an UF of 44.0% (n = 1) for OR-1855 and 64.1% (n = 1; bLLOQ n = 1) for OR-1896, respectively, whereby two samples had a TSC below the LLOQ. In case of a double infusion (1.25 mg), the UF of OR-1896 was 54.1% (n = 1). At a dosage of 2.5 mg levosimendan, no UF of OR-1855 could be quantified neither for a single nor for a double infusion. However, we detected an UF of 52.1% (44.8–68.9%; n = 5) and 49.4% (n = 1) for OR-1896 (Table 2). As we detected lower unbound fractions of levosimendan and OR-1896, and because surgeries may also alter albumin concentrations, the respective albumin concentrations in the serum samples were measured next. As seen in Table 2, these were within the normal range [4.1 g/dl (2.2–5.8 g/dl) at T1; 4.4 g/dl (2.8–5-6 g/dl) at T2].

Table 2

Table 2. TSC & UF of levosimendan and metabolites.

3.4 Simulated serum levels of levosimendan in cardiac surgery patients

To be able to estimate if the CPB procedure may have an influence on levosimendan and metabolite serum levels in these patients, we used individual patient data to simulate the expected levosimendan serum levels after the respective applied doses. Using the body weight, height, age, and gender as well as the dose with the exact dosing intervals and infusion length, levosimendan serum levels were estimated for each patient. For a dose of 1.25 mg levosimendan, the median serum concentration at T1 was indicated with 7.6 ng/ml [(3.0–10.7 ng/ml); n = 5]. In this group, lower levosimendan serum concentrations were measured for all patients but one in comparison to the simulated concentrations (Figure 1E). Patients who received a double infusion of 1.25 mg levosimendan showed a simulated median serum level of 10.8 ng/ml (3.1–18.5 ng/ml; n = 2) at T1, which was higher than the actual measured serum concentrations (Figure 1F). Median serum concentrations of 22.0 ng/ml (6.7–45.8 ng/ml) and 13.3 ng/ml (4.3–35.6 ng/ml) at T1 were simulated in patients with a single (n = 6) or double (n = 4) infusion of 2.5 mg levosimendan, respectively. As seen in Figures 1G,H, the measured serum concentrations were markedly lower than the simulated serum concentrations for 9 of 10 patients. Within the total patient population, regardless of applied levosimendan dose, the measured TSC were significantly lower than the simulated TSC (p < 0.05; Figure 1I).

3.5 NT-proBNP concentrations in samples from cardiac surgery patients with CPB

NT-proBNP (N-terminal pro B-type natriuretic peptide) is routinely used to aid diagnosis of heart failure, predict outcomes, and to monitor therapeutic effects (19). As levosimendan has been shown to reduce NT-proBNP levels (20), it may be used as a surrogate marker for detection of the therapeutic effect of levosimendan. The NT-proBNP levels in patients with CPB were measured at T0 (before surgery), T1 and T2, respectively, and set in relation to every patient´s value at T0. As seen in Supplementary Table S1, NT-proBNP concentrations of all patients were over the normal range (755 ± 156 pg/ml) before surgery, which is in accordance with their cardiac insufficiency. The NT-proBNP values increased after the surgery at T1 (1678 ± 210.3 pg/ml; Figure 2). Although the NT-proBNP levels declined the day after surgery (T2: 999.8 ± 193.6 pg/ml), no significant reduction of the NT-proBNP level compared to T0 was observed with any of the applied dose regimens (Figures 2A–D).

Figure 2

Figure 2. NT-proBNP concentrations of cardiac surgery patients with CPB. NT-proBNP was measured in the same samples from patients receiving levosimendan after induction of anaesthesia at dosages of (A) 1.25 mg of levosimendan (n = 5); (B) 1.25 mg levosimendan and a second dose (1.25 mg) 3–4 h after surgery (n = 2); (C) 2.5 mg levosimendan (n = 6); (D) 2.5 mg levosimendan and a second dose (2.50 mg) 5–21 h after surgery (n = 5). T0: before cardiac surgery; T1: shortly after cardiac surgery; T2: First day after cardiac surgery. Data are expressed as dot plots with the respective median (black line).

4 Discussion

Levosimendan is used in patients with heart failure due to its positive inotropic effect. Only a limited number of studies investigate the effect of levosimendan during cardiac surgery and the reported data is rather ambiguous. Positive effects of levosimendan on mortality and renal failure after surgery were shown in meta-analyses (9–11). However, subsequent randomized controlled trials with cardiac surgical patients failed to reach statistical significance regarding these endpoints (12–14). Of note, none of these studies assessed serum concentrations of levosimendan, OR-1855 and OR-1896. Importantly, convincing studies have demonstrated that the use of a CPB may have significant influence on the pharmacokinetics of administered drugs, such as anaesthetic drugs and adjuvants (21, 22). However, the impact of CPB on levosimendan, OR-1855 and OR-1896 is still largely unknown. Therefore, in our opinion, it is necessary to detect serum levels of levosimendan and its metabolites in cardiac surgery patients with CPB procedure to assess its therapeutic effect. As a proof-of-concept study, we present to the best of our knowledge, the first real-life study measuring both total and free unbound serum concentrations of levosimendan and its two metabolites OR-1896 and OR-1855 in cardiac surgery patients with CPB.

We detected concentrations of levosimendan ranging from below the LLOQ to low levels directly after surgery (T1) after prolonged infusion of 1.25 mg. Higher serum concentrations were only measurable with a levosimendan administration of 2.5 mg. No existing serum concentrations could be measured at T2, due to its short half-life of approximately 1 h (17). Only in patients receiving a second dose of 2.5 mg, levosimendan was quantifiable at T2. The pharmacologically active metabolite OR-1896 was measured at T1 and T2, confirming firstly a rapid metabolization of levosimendan and secondly a longer half-life of OR-1896. Other studies have shown an increase of the metabolites during several days (23, 24) with mean peak concentrations after 6 days, suggesting that the OR-1896 concentrations measured in our study may further increase several days after surgery. Interestingly, even at the higher dosage of 2.5 mg, almost no sample contained quantifiable amount of OR-1855, although OR-1896 was clearly present. The fact that the metabolite OR-1855 was barely detected in our patient population may have several reasons. The small amount of OR-1855 may have been rapidly converted to OR-1896, making a quantification of OR-1855 impossible. Furthermore, it remains unclear whether levosimendan is only reduced to OR-1855 in the intestinal tract in humans, or whether additional pathways exist, as the intestinal metabolization was only shown in dogs upon injection into the intestinal tract (25).The conversion of OR-1855 into the pharmacological active metabolite OR-1896 was not measured in the study, as NAT2 is missing in dogs (25). Moreover, administration of antibiotics can also alter the microbiome function (26) and may thus have an impact on the metabolism of levosimendan to OR-1855 in the intestine. Indeed, patients in our study received antibiotics (cefuroxime) prophylactically. Whether this has an influence on the generation and conversion of the two metabolites remains to be investigated. Finally, differences in the measured serum concentrations of OR-1896 between fast and slow acetylators, due to different genotypes of NAT2, have been demonstrated (7) and may also contribute to lower serum levels of OR-1855. Due to the retrospective nature of our study, we were unable to perform genotyping of NAT2 to investigate this issue in our patient population.

Levosimendan is usually administered intravenously as a bolus injection of 6–12 µg/kg for 10 min followed by a continuous infusion over a period of 24 h at a dosage of 0.1 µg/kg/min (27–29). Cmax concentrations of 30.6 ± 2.2 ng/ml (after 18 h; normal acetylators), 37.5 ± 4.7 ng/ml (after 24 h; rapid acetylators) and 38.3 ± 3.6 ng/ml (after 18 h; slow acetylators) were reached in healthy men (7, 8, 30) after an infusion over 24 h. Few studies with cardiac surgery patients exist, where the concentration of levosimendan and its metabolites were measured. In a randomized clinical trial with cardiac surgery patients, peak levosimendan concentrations of 98 ± 33 ng/ml were measured 2 h after the start of infusion (bolus injection of 12 µg/kg followed by 0.2 µg/kg/min for 24 h) (23), whereas another randomized clinical trial with a similar patient cohort detected 30 ng/ml (bolus injection of 12 µg/kg followed by 0.2 µg/kg/min 24 h preoperatively) 24 h after the start of infusion (24). In our study, patients received levosimendan dosages in an off-label range of 1.25 mg or 2.5 mg (31) as a prolonged infusion to simulate the recommended bolus injection and concurrently avoid excessive vasodilation, which is a known side-effect of the bolus dose. After surgery, the highest median concentrations were reached with a single dose of 2.5 mg levosimendan (10.4 ng/ml at T1) but were still considerably lower than the concentrations measured by Eriksson et al. (23) and Leppikangas et al. (24) as mentioned above. Even in patients receiving a second higher dose of 2.5 mg after the surgery, only a levosimendan concentration of 3.9 ng/ml could be detected the day after surgery (T2). However, the levels of OR-1855 and OR-1896 24 h after start of the levosimendan infusion in the study from Leppikangas et al. (24) were similar to the metabolite concentrations measured at T2 in our study. Yet, here, the majority of the samples showed OR-1855 concentrations below the lower limit of quantification. This discrepancy is probably due to the administered lower total levosimendan dose in our study. In addition, Cmax concentrations were probably reached at a different time as described in the literature. A direct comparison with clinical trials measuring serum or plasma levels of levosimendan is difficult, as these studies were performed in either healthy controls or not in patients undergoing cardiac surgery and additionally had multiple continuous sampling at defined intervals (7, 8). With the retrospective nature of our real-life study, this was unfortunately not possible. Indeed, even comparisons to studies with cardiac surgery patients are difficult due to the different dosing regiments applied (23, 24). Therefore, to be able to evaluate if the CPB procedure may have an impact on levosimendan and thus metabolite serum concentrations, we simulated the serum concentrations of levosimendan, which were to be expected after the respective dose in each patient. The estimated levosimendan serum concentrations were significantly higher than the measured concentrations, regardless of treatment regimen. This may indicate, that the CPB procedure indeed may influence levosimendan drug levels in cardiac surgery patients. Moreover, both the measured and the simulated levosimendan concentrations showed a high variation between patients, suggesting that a rapid therapeutic drug monitoring in this setting may be advantageous.

Although we detected low levels of levosimendan, OR-1855 and OR-1896, it is, in our opinion, of high interest to investigate if these concentrations are still high enough to induce a therapeutic response. Levosimendan has a high binding affinity to serum albumin with a pharmacologically active unbound fraction (UF) of only 1%–2%, whereas the UF of OR-1855 and OR-1896 is higher (approx. 60%) (17). Interestingly, we observed lower UF of levosimendan and both metabolites despite normal albumin concentrations. Of note, the majority of the samples had such low total serum concentrations of levosimendan that an UF of 1%–2%, as described in the literature, would be far below the LLOQ. Moreover, several samples showed total concentration levels of levosimendan below LLOQ, suggesting a possible lack of therapeutic effects. As we measured samples retrospectively, the left ventricular ejection fraction or the vasopressor demand after surgery could not be used to evaluate a therapeutic effect of levosimendan. We thus detected NT-proBNP levels in the patient samples, as it is a clinically accepted important diagnostic cardiac marker for ventricular function (32). In addition, levosimendan has been described to reduce NT-proBNP levels already after 24 h (33). Although we noticed a strong increase in NT-proBNP levels upon surgery, which is not surprising and has been shown before (34), and which declined at T2, no significant reduction of NT-proBNP levels between T0 and T2 were observed. Although levosimendan treatment did not improve NT-proBNP levels in comparison to baseline (before surgery), it still may have a positive impact postoperatively. Indeed, studies have shown an increase in NT-proBNP 24 h after cardiac surgery with CPB (35, 36), continuously increasing for up to four days after surgery (35). Thus, one may carefully hypothesize that the lower dose regimen in this study may still be high enough to induce a therapeutic effect and prevent a further increase in NT-proBNP after surgery. On the other hand, patients with levosimendan and metabolite levels under the LLOQ did not show higher NT-proBNP levels compared to patients with levosimendan and metabolite levels over LLOQ (data not shown). Thus, this needs to be further investigated in prospective studies. In addition, as OR-1896 has a much longer half-life (7, 8), it is possible that effects on NT-proBNP levels may first be visible at a later time point. A single administration of levosimendan of 12 µg/kg over 10 min followed by a continuous infusion of 0.1 µg/kg/min over 24 h was shown to reduce NT-proBNP levels up to 48 h (37). Moreover, levosimendan and OR-1896 have been demonstrated to have other effects, such as the relaxation of vascular smooth muscle cells (38) as well as endothelial anti-inflammatory effects (39). If the concentrations of levosimendan and metabolites measured here elicit these effects in cardiac surgery patients remains to be elucidated. Nevertheless, with regard to the low total serum concentrations and the extremely low unbound fractions of levosimendan, considering most levels being even below LLOQ, as well as the lack of a reduction of NT-proBNP levels upon levosimendan application, we suggest that the low concentrations of levosimendan and its metabolites measured in cardiac patients undergoing cardiac surgery with CPB in this study may not have the fully desirable therapeutic effects on cardiac function. Thus, a different dosage regimen of levosimendan within the perioperative setting for these patients may be advisable. However, this needs to be confirmed by larger prospective studies with longer follow-up periods including clinical endpoints. Finally, as large interindividual variations of levosimendan and metabolite concentrations were observed in these patients, a personalised approach with a rapid therapeutic drug monitoring may hold promises for correct drug adjustments in the perioperative setting.

In summary, we quantified serum concentrations of levosimendan and its metabolites in cardiac surgery patients with CPB, showing that the applied dosages in the off-label range resulted in low measurable total as well as unbound serum concentrations of levosimendan and its metabolites with doubtable therapeutic effect. Thus, we demonstrate the need of an optimized perioperative dosage regimen of levosimendan for cardiac surgery patients with CPB procedure. Indeed, therapeutic drug monitoring of levosimendan represents an interesting option to achieve these aims and to aid in the development of treatment guidelines for this particular patient population.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author.

Ethics statement

The studies involving humans were approved by Ethics committee, Medical Faculty, LMU München, Pettenkoferstraße 8, 80336 München. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from another research group. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

Author contributions

HK: Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. UL: Writing – review & editing. MH: Investigation, Writing – review & editing. GH: Methodology, Software, Writing – review & editing. OF: Methodology, Writing – review & editing. KW: Methodology, Writing – review & editing. EK: Writing – review & editing. CH: Writing – review & editing. RT: Conceptualization, Investigation, Project administration, Supervision, Writing – original draft, Writing – review & editing. HM: Conceptualization, Funding acquisition, Project administration, Supervision, Visualization, Writing – original draft, Writing – review & editing.

Funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article.

This work was supported by the foundation Stiftung Patient & Klinische Pharmazie.

Acknowledgments

This work contains data of the doctoral thesis of HK.

Conflict of interest

HM and RT signed a Material Transfer Agreement with Orion Pharma for the use of the internal standards for the LC-ESI-MS/MS measurements.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher's note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcvm.2024.1406338/full#supplementary-material

Abbreviations

bLLOQ, below lower limit of quantification; CPB, cardiopulmonary bypass; i.s.v, insufficient serum volume; LLOQ, lower limit of quantification; TSC, total serum concentration; UF, unbound fraction (fraction of drug or metabolite not bound to albumin).

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