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Background: Overdoses of tricyclic antidepressants may lead to arrhythmia. The aim of the study was to simulate the effect of increasing concentrations of amitriptyline (AMI) and its metabolite, nortriptyline, on the action potential of human ventricular cell. Methods: Simulations were performed in Cardiac Safety Simulator platform with the use of the O'Hara-Rudy model. Input data included literature-derived, drug-specific IC50 values for ICa(L), IKr, and INa currents. Individual concentrations of AMI and nortriptyline were simulated in Simcyp. Nine single doses (mg) were tested: 5, 10, 50, 100, 300, 500, 1000, 5000, and 10,000. Results: The values of simulated endpoints (APD50, APD90, triangulation, and APD90) increase with drug concentrations. APD90 was statistically significant for doses up from 1000 mg. EADs were observed after administration of 10,000-mg AMI. Conclusions: The consequences of various doses of AMI on the single cardiac myocytes were simulated in our study. Repolarization abnormalities were not expected for the therapeutic doses. EADs may be observed for very high doses of AMI. Keywords: drug safety; modeling; simulation. *Corresponding author: Zofia Tylutki, Unit of Pharmacoepidemiology and Pharmacoeconomics, Department of Social Pharmacy, Jagiellonian University Medical College, Medyczna 9 Str. Cracow 30-688, Poland, E-mail: firstname.lastname@example.org Sebastian Polak: Unit of Pharmacoepidemiology and Pharmacoeconomics, Department of Social Pharmacy, Jagiellonian University Medical College, Krakow, Poland Jakob Jornil: Department of Forensic Medicine, Aarhus University, Aarhus, Denmark 34Tylutki et al.: Simulation study nortriptyline, on the action potential of human ventricular cell. They were given amitriptyline in single dose at 9.00 a.m. The simulations (each for 10 trials) were run for 9 doses [mg]: subtherapeutic, i.e. 5 and 10; therapeutic, i.e. 50, 100, and 300; and supratherapeutic, i.e. 500, 1000, 5000, and 10,000 . Tmax (time to reach the peak plasma concentration of a drug after administration) values for amitriptyline and nortriptyline did not overlay each other. Thus, the drug concentrations in both time points had to be used as CSS inputs, and for both time points, the analysis of results had to be carried out. Tmax for amitriptyline was about 1.8 h, and Tmax for nortriptyline was more variable, from 13.4 h for 5 mg of amitriptyline to 21.5 h for 10,000 mg of amitriptyline. The time-concentration profiles were illustrated in Figure 1. Physiological parameters describing simulation conditions in CSS were employed from healthy Caucasian population description. They included heart rate and plasma concentration of calcium, sodium, and potassium ions. The parameters were randomly assigned to virtual subjects taking into account age, gender, and circadian variations . Simulated time gap was set to 10,000 ms. The CSS study endpoints were automatically derived from the simulated myocardial cells (M cells) action potential, with the use of the CSS built-in action potential (AP) analysis algorithm: action potential duration at 50% repolarization (APD50), action potential duration at 90% repolarization (APD90), and triangulation, which stands for the difference between APD90 and APD50, and the difference between APD90 in Tmax and APD90 at baseline (APD90). The significance of the difference of APD90 in Tmax and APD90 at baseline was verified in Welch's t-test taking p<0.05 as the level of significance. There were also early afterdepolarizations (EADs) present in some of the simulated scenarios. EADs may be defined as abnormal depolarizations at myocytes level leading to cardiac arrhythmias at the tissue level and therefore were proposed to be one of the pro-arrhythmia surrogates . M cells were chosen for simulation study, as they are believed to be more vulnerable to EADs generation than are endocardial and epicardial cells . Materials and methods The simulations of cardiomyocytes' action potential were performed in Cardiac Safety Simulator (CSS) v.2.0 (Simcyp, Sheffield, UK, a Certara company) a tool for in vitro-in vivo extrapolation of membrane potential changes, which combines the ion channels inhibition in cardiomyocytes membrane with the effect of a drug . The O'Hara-Rudy model build-in to CSS was used during the study . CSS input data included IC50 values describing drug concentrations responsible for half of maximal ion current inhibition. The literature-derived IC50 values for various currents were taken from the results of patchclamp experiments for amitriptyline and nortriptyline, respectively (Table 1). The lowest IC50 values were chosen from available in vitro data, assuming the most conservative simulation scenario . The second CSS input data were drugs' concentrations. Individual plasma concentrations of amitriptyline and its metabolite, nortriptyline, were simulated in Simcyp platform v.14 (Simcyp, Sheffield, UK, a Certara company) . For both drugs, Simcyp compound files were previously developed with the use of available in vitro data and validated against the published clinical observations . In silico experimental conditions allowed to achieve time-concentration profiles in healthy subjects after the drug administration in doses above therapeutic range. In simulation scenario, virtual population consisted of 10 healthy subjects (five men and five women) aged 2050 years (Table 2). Table 1:In vitro data used in the simulations. Drug Ionic current IKr INa ICa[La] IKr IC50, M 1.801 2.4 3.75 1.514 Cell model Data source     Amitriptyline Nortriptyline CHO CHO Rat ventricular myocytes HEK Results Simulation results are presented as individual APD90 values, APD90 values, and triangulation factor, in function of concentration in Tmax for amitriptyline (Figure 2) and in Tmax for nortriptyline (Figure 3). For drug ascending doses from 5 mg to 1000 mg, the mean values of APD90 (SD) in Tmax for both amitriptyline and nortriptyline were similar, varying from 311.6 (5.7) ms in Table 2:Virtual subject characteristics. Virtual subject characteristics No. of subjects in each trial Age range, years Proportion of females 10 2050 0.5 Tylutki et al.: Simulation study35 Mean values of systemic concentration in plasma of amitriptyline administered in sub- and therapeutic doses over time 180 160 140 120 100 80 60 40 20 0 Systemic concentration, ng/mL Systemic concentration, ng/mL 6000 5000 4000 3000 2000 1000 0 Mean values of systemic concentration in plasma of amitriptyline administered in supratherapetic doses over time 0 5 mg 16 10 mg 24 Time, h 50 mg 32 100 mg 40 300 mg 8 500 mg 16 1000 mg 24 Time, h 40 10,000 mg 5000 mg Mean values of systemic concentration in plasma of nortriptyline after amitriptyline administration in sub- and therapeutic doses Systemic concentration, ng/mL 70 60 50 40 30 20 10 0 0 5 mg 8 16 10 mg 24 Time, h 50 mg 100 mg 300 mg 32 40 48 Systemic concentration, ng/mL 80 Mean values of systemic concentration in plasma of nortriptyline after amitriptyline administration in supratherapeutic doses 3500 3000 2500 2000 1500 1000 500 0 0 8 500 mg 16 1000 mg 24 Time, h 5000 mg 10,000 mg 32 40 48 Figure 1:Time-concentration profiles of amitriptyline and nortriptyline after ascending single doses of amitriptyline. 600 500 400 300 200 100 0 100 200 Concentration, ng/mL 0 0.5 1 1.5 2 APD90 Triangulation dAPD 90 Time, ms 600 500 400 300 200 100 0 100 0 APD90 0.5 1 1.5 2 Time, ms 200 Triangulation dAPD 90 Concentration, ng/mL Figure 2:Simulation results in Tmax for amitriptyline. Figure 3:Simulation results in Tmax for nortriptyline. Tmax for amitriptyline after a dose of 10 mg to 331.8 (6.9) ms in Tmax for nortriptyline after 1000 mg of amitriptyline. In case of a dose of 5000 mg, simulation results were 410.4 (35.46) ms and 410.4 (36.44) ms in Tmax for amitriptyline and Tmax for nortriptyline, respectively. For the highest dose, i.e. 10,000 mg, the corresponding results were as follows: 389.2 (102.08) ms and 421.1 (77.26) ms. The values of triangulation in doses in the range of 51000 mg varied between 55.1 (1.4) ms and 60.5 (2.3) ms, whereas after 5000 mg of amitriptyline, it stood for 88.0 (13.43) ms in parent drug peak plasma concentration; after 10,000 mg, triangulation had a value of 149.0 (35.66) ms in the same Tmax. The APD90 for subtherapeutic doses and therapeutic doses up to the 500-mg dose of amitriptyline were confirmed not to be statistically significant (p-value>0.05). However, for supratherapeutic doses up from 1000 mg, the APD90 was considered to be statistically significant with p-value <0.05. This significance was observed in both Tmax for amitriptyline and Tmax for nortriptyline (Table 3). 36Tylutki et al.: Simulation study Table 3:Mean values (SD) of APD90, APD90, and triangulation achieved after amitriptyline in ascending dosage in Tmax for amitriptyline and nortriptyline. Dose, mg APD90, ms 5 10 50 100 300 500 1000 5000 10000 Tmax for amitriptyline APD90, ms 2.3 0.8 3.8 3.07 6.86 0.69 16.6a 100.5a 79.3a Triang., ms 55.5 (1.0) 55.3 (0.6) 55.9 (0.7) 55.7 (1.0) 56.9 (1.0)a 57.3 (0.7)a 60.5 (2.3)a 88.0 (13.43)a 149.0 (35.66)a APD90, ms 316.6 (9.3) 316.2 (8.6) 316.9 (9.1) 317.3 (6.5)a 321.1 (8.4)a 324.5 (7.0)a 331.8 (6.9)a 410.4 (36.44)a 421.1 (77.26)a APD90, ms 5.4 5.2 5.2 5.7a 9.9a 11.5a 18.1a 95.3a 107.2a 313.4 (9.6) 311.6 (5.7) 314.8 (7.16) 312.82 (8.5) 317.91 (9.29) 316.14 (4.41) 327.1 (9.0)a 410.4 (35.46)a 389.2 (102.08)a Tmax for nortriptyline Triang., ms 55.2 (1.5) 55.1 (1.4) 55.4 (1.4) 55.5 (1.0)a 56.6 (1.4)a 57.5 (0.9)a 60.0 (1.6)a 85.6 (11.08)a 111.9 (15.30)a Values differing significantly in Welch's t-test from corresponding baseline values are written in bold. Dose: 5 mg, Tmax for amitriptyline 60 40 20 0 20 40 60 80 100 60 40 20 0 20 40 60 80 100 120 0 1000 2000 4000 5000 6000 Membrane potential, mV Membrane potential, mV Dose: 10 mg, Tmax for amitriptyline Subtherapeutic doses Dose: 50 mg, Tmax for amitriptyline 60 40 20 0 20 40 60 80 100 60 40 20 0 20 40 60 80 100 120 0 1000 2000 4000 5000 6000 Membrane potential, mV Membrane potential, mV Dose: 100 mg, Tmax for amitriptyline Dose: 300 mg, Tmax for amitriptyline 60 40 20 0 20 40 60 80 100 Membrane potential, mV Therapeutic doses Dose: 500 mg, Tmax for amitriptyline 60 40 20 0 20 40 60 80 100 120 60 40 20 0 20 40 60 80 100 0 1000 2000 4000 5000 6000 Membrane potential, mV Membrane potential, mV Dose: 1000 mg, Tmax for amitriptyline Dose: 5000 mg, Tmax for amitriptyline 60 40 20 0 20 40 60 80 100 120 4000 Membrane potential, mV Membrane potential, mV 80 60 40 20 0 20 40 0 Dose: 10,000 mg, Tmax for amitriptyline 5000 Time, ms Supratherapeutic doses Figure 4:Visualizations of changes in M cells membrane potential in one virtual patient after ascending doses of amitriptyline. Tylutki et al.: Simulation study37 Visualizations of changes in M cells membrane potential were achieved in 180 cases for 10,000 ms (simulated time gap duration). The simulated changes in membrane potential are shown in Figure 4. The presented graphics were selected for the results from one virtual patient in Tmax of amitriptyline after 9 ascending single doses of the drug (Figure 4). Discussion and conclusions The experiment resulted in 180 simulated action potentials of human ventricular cell (for 10 virtual patients, for 9 doses of amitriptyline, in 2 time points: Tmax for amitriptyline and Tmax for nortriptyline). According to the graphs (Figures 2 and 3), all parameters expressing APD (APD90, triangulation, APD90) increase with the concentration of both parent drug and its metabolite. This tendency may be explained by the drug concentration-dependent increase in IKr current blockage. However, in canine model by Ansel et al., the effect on APD90 had 2 phases: progressive shortening followed by prolongation . Low dispersion of our simulated results can be observed for low concentrations, whereas dispersion enhances with the increase in amitriptyline and nortriptyline individual plasma levels. It indicates that physiological factors affect the results more in case of higher concentrations. In the therapeutic range, amitriptyline seems not to influence membrane myocyte potential in a significant manner, according to Welch's t-test. Only for supratherapeutic doses up from 1000 mg of amitriptyline did APD90 occur to be statistically significant in both Tmax points. In clinics, even at normal doses of amitriptyline, a significant QTc interval prolongation was observed, but it is attributed only to the parent drug, which makes nortriptyline not responsible for influencing repolarization process at therapeutic dosages. However, the incidences of torsade de pointes arrhythmia are very rare in a patient population, with the exception of intentional overdoses . Gülolu and colleagues observed arrhythmias in most patients intoxicated with amitriptyline in a dose of 500 mg or higher . Consistent with the lack of arrhythmic events at therapeutic level are our other simulation results, i.e. EADs, which are said to be involved in the mechanism initiating ventricular tachycardia . EADs were absent in all virtual individuals given therapeutic doses. They occurred sporadically in case of a dose of 5000 mg. Finally, they were observed in all subjects given the largest dose of amitriptyline, i.e. 10,000 mg. On the other hand, in Ansel et al.'s experiment, none of the dogs developed EADs, even in case of ventricular tachycardia . In simulation results, the common incidence of EADs after the largest dose may explain APD90 after administration of 10,000 mg being shorter than after 5000 mg. Nevertheless, both experiments are in agreement with studies suggesting that repolarization abnormalities are not expected for the therapeutic doses of amitriptyline. Early after depolarizations may be observed only in case of very high dose of TCA. As it is impossible to test such scenario in a clinical situation, these conclusions can be drawn only from in silico simulations. Thus, the consequences of various doses of amitriptyline and its metabolite nortriptyline on the single cardiac human myocyte were simulated in our study. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission. Research funding: Project was financed by the National Science Centre, Poland, project number 2014/13/N/ NZ7/00254. Employment or leadership: None declared. Honorarium: None declared. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Bio-Algorithms and Med-Systems – de Gruyter
Published: Mar 1, 2016
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