Amiodarone and mexiletine are used for ventricular arrhythmias, for which a combination therapy of both anti-arrhythmic drugs (AADs) is not uncommon. Therapeutic drug monitoring (TDM) can be beneficial for clinical guidance of therapy, especially to correctly identify adverse events. Desethylamiodarone, the active metabolite of amiodarone, accumulates over time and is associated with serious adverse events. Therefore, simultaneous TDM for amiodarone, desethylamiodarone and mexiletine is advantageous in clinical practice. The presented LC-MS/MS method was validated for selectivity, matrix effect, linearity, accuracy, precision, carry-over and stability. The method was continuously evaluated during eight months of clinical use. The method was shown to be linear within the measured range of 0.1 to 10 mg/L for each component. The matrix effect was considered negligible. No interfering responses were found for amiodarone, desethylamiodarone and the isotopic-labeled internal standards. A constant and reproducible within-run contribution of 45.3 %, originating from the system, was identified for mexiletine. The systemic contribution to the peak area of the lowest quantifiable concentration of mexiletine affected the selectivity and carry-over effect measurements. Multiple measurements showed that regression adjusted concentrations were accurate and reproducible, indicating calibration correction was applicable. Sample stability was found to be within limits for all storage conditions and freeze-thaw cycles. Furthermore, long-term method evaluation with external controls resulted in stable measurements with a percentage coefficient of variance between 1.3 % and 6.3 %. The presented practical and reliable method is applicable for clinical TDM and will allow clinical practitioners to guide drug therapy of amiodarone and mexiletine.

Amiodarone is commonly used to prevent and treat life-threatening cardiac arrhythmias. However, it is also known to have an extensive side effect profile. A rare adverse effect of amiodarone is epididymitis. Epididymitis is inflammation of the epididymis that causes moderate pain in the posterior scrotum. The patient, in this case, developed left scrotal pain seven months after starting amiodarone and presented with symptoms consistent with epididymitis. The patient\'s work-up included urinalysis with culture, treatment with antibiotics, and testicular ultrasound before being diagnosed with amiodarone-induced epididymitis. This diagnosis led to the discontinuation of amiodarone, which resulted in the complete resolution of the patient\'s symptoms within two weeks. This case report is intended to increase awareness of epididymitis as a possible adverse effect of amiodarone and to stress the importance of considering this when there are no apparent anatomical or infectious causes of epididymitis.

The aim of the present study was to study the antiarrhythmic effects and cellular mechanisms of desethylamiodarone (DEA), the main metabolite of amiodarone (AMIO), following acute and chronic 4-week oral treatments (25-50 mg·kg-1 ·day-1 ).
The antiarrhythmic effects of acute iv. (10 mg·kg-1 ) and chronic oral (4 weeks, 25 mg·kg-1 ·day-1 ) administration of DEA were assessed in carbachol and tachypacing-induced dog atrial fibrillation models. Action potentials were recorded from atrial and right ventricular tissue following acute (10 μM) and chronic (p.o. 4 weeks, 50 mg·kg-1 ·day-1 ) DEA application using the conventional microelectrode technique. Ionic currents were measured by the whole cell configuration of the patch clamp technique in isolated left ventricular myocytes. Pharmacokinetic studies were performed following a single intravenous dose (25 mg·kg-1 ) of AMIO and DEA intravenously and orally. In chronic (91-day) toxicological investigations, DEA and AMIO were administered in the oral dose of 25 mg·kg-1 ·day-1 ).
DEA exerted marked antiarrhythmic effects in both canine atrial fibrillation models. Both acute and chronic DEA administration prolonged action potential duration in atrial and ventricular muscle without any changes detected in Purkinje fibres. DEA decreased the amplitude of several outward potassium currents such as IKr , IKs , IK1 , Ito , and IKACh , while the ICaL and late INa inward currents were also significantly depressed. Better drug bioavailability and higher volume of distribution for DEA were observed compared to AMIO. No neutropenia and less severe pulmonary fibrosis was found following DEA compared to that of AMIO administration.
Chronic DEA treatment in animal experiments has marked antiarrhythmic and electrophysiological effects with better pharmacokinetics and lower toxicity than its parent compound. These results suggest that the active metabolite, DEA, should be considered for clinical trials as a possible new, more favourable option for the treatment of cardiac arrhythmias including atrial fibrillation.

Amiodarone is a potent antiarrhythmic drug and displays substantial liver toxicity in humans. It has previously been demonstrated that amiodarone and its metabolite (desethylamiodarone, DEA) can inhibit mitochondrial function, particularly complexes I (CI) and II (CII) of the electron transport system in various animal tissues and cell types. The present study, performed in human peripheral blood cells, and one liver-derived human cell line, is primarily aimed at assessing the concentration-dependent effects of these drugs on mitochondrial function (respiration and cellular ATP levels). Furthermore, we explore the efficacy of a novel cell-permeable succinate prodrug in alleviating the drug-induced acute mitochondrial dysfunction. Amiodarone and DEA elicit a concentration-dependent impairment of mitochondrial respiration in both intact and permeabilized platelets via the inhibition of both CI- and CII-supported respiration. The inhibitory effect seen in human platelets is also confirmed in mononuclear cells (PBMCs) and HepG2 cells. Additionally, amiodarone elicits a severe concentration-dependent ATP depletion in PBMCs, which cannot be explained solely by mitochondrial inhibition. The succinate prodrug NV118 alleviates the respiratory deficit in platelets and HepG2 cells acutely exposed to amiodarone. In conclusion, amiodarone severely inhibits metabolism in primary human mitochondria, which can be counteracted by increasing mitochondrial function using intracellular delivery of succinate.

BACKGROUND: Association between clinical effect and serum concentration of amiodarone (AMI) and its active metabolite desethylamidarone (DEA) in patients after surgical ablation (SA) of atrial fibrillation (AF) has not yet been studied.
OBJECTIVE: We wanted to find a correlation between AMI and DEA serum concentration and maintaining sinus rhythm (SR) after SA of AF.
METHODS: Sixty eight patients with AF who had undergone surgical ablation between 2014 and 2017 were included in a single-centre, prospective, observational study. Maintaining of SR was evaluated by standard 12-lead ECG and 24-hour Holter ECG monitoring at months 1, 3, 6 and 12 following surgery. Therapeutic monitoring of AMI and DEA concentrations was done to optimize therapy and adverse effects were followed up.
RESULTS: We have noticed a high success rate in maintaining of SR (overall 83%). The median of serum concentration of AMI was 0.81 mg/L (range 0.16-2.35 mg/L) and DEA 0.70 mg/l (range 0.19-2.63 mg/L). No significant differences were found in the serum concentratration of AMI, DEA or DEA/AMI concentratration ratios between patients with SR and persistent supraventricular tachyarrhythmia except on the second outpatient visit. We observed significant correlation between serum concentration of DEA and thyroid-stimulating hormone elevation.
CONCLUSIONS: We confirmed the efficacy of AMI and DEA at the measured serum concentrations. However, analysis of these concentrations alone cannot replace assessment of the clinical response for treatment. Establishment of individual AMI (and DEA) concentrations at which the optimal therapeutic response is achieved seems to be advantageous. Therapeutic monitoring of AMI and DEA is helpful in personalised pharmacotherapy after SA of AF.

Due to its very low water solubility and complex pharmacokinetics, a reliable point-to-point correlation of its in vitro release with its pharmacokinetics has not been achieved so far with amiodarone. The correlation of the in vitro dissolution of a drug with the pharmacokinetics of one of its metabolites was recently proposed by the authors of the article as an additional or alternative analysis to the usual in vitro correlations in vivo, mainly in the case of fast-absorbing drugs that have metabolites with a significant therapeutic effect. The model proposed by the authors considers that amiodarone has a slow dissolution, rapid absorption, and rapid metabolism, and before returning to the blood from other compartments, its pharmacokinetics is determined mainly by the kinetics of release in the intestine from the pharmaceutical formulation. Under these conditions, the rate of apparition of desethylamiodarone in the blood is a metric of the release of amiodarone in the intestinal fluid. Furthermore, it has been shown that such an estimated in vivo dissolution is similar, after time scaling, to the dissolution measured experimentally in vitro. Dissolution data of amiodarone and the pharmacokinetic data of its active metabolite desethylamiodarone were obtained in a bioequivalence study of 24 healthy volunteers. The elimination constant of the metabolite from plasma was estimated as the slope of the linear regression of logarithmically transformed data on the tail of plasma levels. Because the elimination of desethylamiodarone was shown to follow a monoexponential model, a Nelson-Wagner-type mass equilibrium model could be applied to calculate the time course of the \"plasma metabolite fraction.\" After Levi-type time scaling for imposing the in vitro-in vivo correlation, the problem became that of the correlation between in vitro dissolution time and in vivo dissolution time, which was proven to follow a square root model. To validate the model, evaluations were performed for the reference drug and test drug separately. In both cases, the scaled time for in vivo dissolution, t*, depended approximately linearly on the square root of the in vitro dissolution time t, with the two regression lines being practically parallel.

To evaluate the usefulness of imaging mass spectrometry (IMS) technology for assessing drug toxicity, we analyzed animal tissues in an amiodarone (AMD)-induced phospholipidosis model by IMS and confirmed the relationship between the distribution of AMD, its metabolites, and representative phospholipids (phosphatidylcholine, PC) and histological changes. AMD was administered to rats for 7 days at 150 mg/kg/day. The lung, spleen, and mesenteric lymph node were histologically examined and analyzed using IMS. The detection intensities of AMD, its metabolites, and typical PCs were higher in regions infiltrated by foamy macrophages compared with normal areas. This tendency was common in all three organs analyzed in this study. For the spleen, signals for AMD, its metabolites, and typical PCs were significantly more intense in the marginal zone, where foamy macrophages and vacuolated lymphocytes are abundant, than in the other areas. These results indicate that AMD, its metabolites, and PCs accumulate together in foamy or vacuolated cells, which is consistent with the mechanism of AMD-induced phospholipidosis. They also indicate that IMS is a useful technique for evaluating the distribution of drugs and biological components in the elucidation of toxicity mechanisms.

Previously, we found that desethylamiodarone (DEA) may have therapeutic potentiality in bladder cancer. In this study, we determined its effects on human cervical cancer cells (HeLa). Cell viability was evaluated by Muse Cell Count & Viability Assay; cell apoptosis was detected by Muse Annexin V & Dead Cell Assay. Cell cycle was flow cytometrically determined by Muse Cell Cycle Kit and the morphological changes of the cells were observed under a fluorescence microscope after Hoechst 33342 staining. The changes in the expression levels of apoptosis-related proteins in the HeLa cells were assessed by immunoblot. Our results showed that DEA significantly inhibited the proliferation and viability of HeLa cells and induced apoptosis in vitro in dose-dependent and also in cell cycle-dependent manner because DEA induced G0/G1 phase arrest in the HeLa cell line. We found that DEA treatment downregulated the expression of phospho-Akt and phospho-Bad. In addition, DEA could downregulate expression of Bcl-2, upregulate Bax, and induce cytochrome c release. Our results indicate that DEA might have significance as an anti-tumor agent against human cervical cancer.

1. Cytochrome P450 3A4 (CYP3A4) is an important member of the cytochrome P450 enzyme superfamily, with 33 allelic variants reported previously. Genetic polymorphisms of CYP3A4 can produce a significant effect on the efficacy and safety of some drugs, so the purpose of this study was to clarify the catalytic characteristics of 22 CYP3A4 allelic isoforms, including 6 novel variants in Han Chinese population, on the oxidative metabolism of amiodarone in vitro. 2. Wild-type CYP3A4*1 and other variants expressed by insect cells system were incubated respectively with 10-500 μM substrate for 40 min at 37 °C and terminated at -80 °C immediately. Then these samples were treated as required and detected with ultra-performance liquid chromatography-tandem mass spectrometry used to analyze its major metabolite desethylamiodarone. 3. Among the 21 CYP3A4 variants, compared with the wild-type, the intrinsic clearance values (Vmax/Km) of two variants were apparently decreased (11.07 and 2.67% relative clearance) while twelve variants revealed markedly increased values (155.20∼435.96%), and the remaining of seven variants exhibited no significant changes in enzyme activity. 4. This is the first time report describing all these infrequent alleles for amiodarone metabolism, which can provide fundamental data for further clinical studies on CYP3A4 alleles.

Amiodarone is a widely used potent antiarrhythmic for the treatment of cardiac disease; however, its use is often discontinued due to numerous adverse effects, including hepatotoxicity. To investigate the role of drug metabolism in this liver toxicity, amiodarone and its major metabolite desethylamiodarone were incubated with HepG2 cells overexpressing a series of cytochrome P450 (CYP) isoforms. Significantly higher cytotoxicity of amiodarone was observed in HepG2 cells overexpressing CYP3A4 or CYP1A1, compared with that observed in empty vector transduced control cells. Further, higher levels of the more potent hepatotoxic metabolite desethylamiodarone were detected in CYP3A4 or CYP1A1 expressed cells. The CYP3A4 inhibitor ketoconazole and the CYP1A1 inhibitor α-naphthoflavone drastically inhibited the metabolism of amiodarone to desethylamiodarone. Along with the inhibition of CYP1A1 or CYP3A4, the cytotoxicity of amiodarone was significantly reduced. These data indicate that the metabolism of amiodarone to desethylamiodarone by CYP1A1 or CYP3A4 plays an important role in the hepatocellular toxicity of amiodarone.