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Research ArticleMetabolism, Transport, and Pharmacogenomics

Pharmacokinetics of the Cardioprotective Drug Dexrazoxane and Its Active Metabolite ADR-925 with Focus on Cardiomyocytes and the Heart

Eduard Jirkovský, Anna Jirkovská, Jan Bureš, Jaroslav Chládek, Olga Lenčová, Ján Stariat, Zuzana Pokorná, Galina Karabanovich, Jaroslav Roh, Petra Brázdová, Tomáš Šimůnek, Petra Kovaříková and Martin Štěrba
Journal of Pharmacology and Experimental Therapeutics March 2018, 364 (3) 433-446; DOI: https://doi.org/10.1124/jpet.117.244848

Abstract

Dexrazoxane (DEX), the only cardioprotectant approved against anthracycline cardiotoxicity, has been traditionally deemed to be a prodrug of the iron-chelating metabolite ADR-925. However, pharmacokinetic profile of both agents, particularly with respect to the cells and tissues essential for its action (cardiomyocytes/myocardium), remains poorly understood. The aim of this study is to characterize the conversion and disposition of DEX to ADR-925 in vitro (primary cardiomyocytes) and in vivo (rabbits) under conditions where DEX is clearly cardioprotective against anthracycline cardiotoxicity. Our results show that DEX is hydrolyzed to ADR-925 in cell media independently of the presence of cardiomyocytes or their lysate. Furthermore, ADR-925 directly penetrates into the cells with contribution of active transport, and detectable concentrations occur earlier than after DEX incubation. In rabbits, ADR-925 was detected rapidly in plasma after DEX administration to form sustained concentrations thereafter. ADR-925 was not markedly retained in the myocardium, and its relative exposure was 5.7-fold lower than for DEX. Unlike liver tissue, myocardium homogenates did not accelerate the conversion of DEX to ADR-925 in vitro, suggesting that myocardial concentrations in vivo may originate from its distribution from the central compartment. The pharmacokinetic parameters for both DEX and ADR-925 were determined by both noncompartmental analyses and population pharmacokinetics (including joint parent-metabolite model). Importantly, all determined parameters were closer to human than to rodent data. The present results open venues for the direct assessment of the cardioprotective effects of ADR-925 in vitro and in vivo to establish whether DEX is a drug or prodrug.

Introduction

Dexrazoxane (DEX) (Fig. 1) is the only cardioprotective agent approved for clinical use against cardiotoxicity induced by anthracycline (ANT) anticancer agents (Stěrba et al., 2013; Vejpongsa and Yeh, 2014a), and, besides that, it is also approved for treatment of accidental anthracycline extravasation (Hasinoff, 2006; Langer, 2010). Over the decades of its preclinical development, it has been documented that the cardioprotective effects of DEX are robust in all species tested irrespectively of the ANT derivative (Stěrba et al., 2013). Furthermore, clinical efficacy has been firmly established in numerous trials and by their meta-analyses (van Dalen et al., 2011; Vejpongsa and Yeh, 2014a; Hutchins et al., 2017).

Fig. 1.
Fig. 1.

Chemical structures of DEX, intermediates B and C, and ADR-925.

DEX is traditionally believed to be a prodrug that is metabolized to an active metabolite—ADR-925 via two intermediate hydrolytic products (termed B and C; Fig. 1). The first step is catalyzed by dihydropyrimidase (initial single ring-opening step), whereas second step to ADR-925 is catalyzed by dihydroorotase. ADR-925 is deemed to protect cardiomyocytes from ANT-induced oxidative damage by chelating intracellular iron (Hasinoff et al., 1998; Cvetković and Scott, 2005). Although this reactive oxygen species and iron hypothesis is very popular, numerous concerns have been raised over the last decade (Stěrba et al., 2013). Some experts have questioned even the fundamental concept of DEX action as a prodrug (Hasinoff and Herman, 2007), and further evidence suggests that DEX may protect the heart by catalytic inhibition of topoisomerase IIβ (Lyu et al., 2007; Vejpongsa and Yeh, 2014b; Lenčová-Popelová et al., 2016).

It is noteworthy that, despite several important contributions (Hasinoff et al., 1998; Hasinoff and Aoyama, 1999a,b; Schroeder et al., 2002, 2003, 2005; Schroeder and Hasinoff, 2005), understanding the metabolism of DEX to its putative active metabolite ADR-925 is still considerably limited. This becomes particularly apparent when attempting to put the pharmacodynamics of DEX on a solid pharmacokinetic ground (Stěrba et al., 2013). Clinical data on the metabolism of DEX to ADR-925 are extremely sparse. There is only a single small clinical trial (Schroeder et al., 2003) in which ADR-925 was systematically determined in plasma. Unfortunately, DEX was part of an atypical chemotherapeutic combination with high-dose etoposide, the number of patients was limited, and the calculated half-life of DEX was longer compared with values commonly reported elsewhere (Cvetković and Scott, 2005). Most data from animal experiments were obtained by Schroeder and Hasinoff (2002, 2005), and all in vivo experiments published in extenso used only rats. Moreover, ADR-925 concentrations in the myocardium, where its cardioprotective action should occur, are not completely described. The only available data come from the latter study in which concentrations of ADR-925 were determined in a single time point (different for DEX and ADR-925 administration). In addition, the translatability of these in vivo findings may not be straightforward due to the strikingly higher elimination rate of DEX in rodents (rats and mice) compared with humans (Hasinoff et al., 1998).

DEX metabolism has also been studied in vitro with isolated cardiomyocytes and hepatocytes (Schroeder et al., 2005). However, its concentrations were determined in cell culture media but not within the intracellular compartment where the cardioprotective effects of ADR-925 should occur. Indeed, the relatively polar ADR-925 need not penetrate easily out of the cells, which may make the precise evaluation of DEX metabolism difficult. Furthermore, experiments performed with DEX in cardiomyocytes were short (2 hours), and concentrations of ADR-925 have not been determined. Hence, it remains unknown whether the putative active metabolite achieves markedly prolonged intracellular concentrations in cardiomyocytes, which can be essential for the cardioprotective efficacy of DEX.

Thus, a plasma concentration–time profile of the putative active metabolite (ADR-925) under standard conditions when DEX is administered as a single agent in a clearly cardioprotective schedule remains unknown. In addition, information on concentration–time profiles of both DEX and ADR-925 in the heart and cardiomyocytes (i.e., at the site of drug action) is missing. Therefore, it is uncertain whether and to what extent DEX is metabolized in the heart and cardiomyocytes, and whether ADR-925 can effectively penetrate this compartment from cell media and circulation.

The aim of the present study is to thoroughly investigate the metabolism of DEX to ADR-925 in vitro and in vivo on the schedule in which the drug is clearly cardioprotective, with a particular focus on cardiomyocytes and the heart.

Materials and Methods

Drugs and Chemicals

For high-pressure liquid chromatography (HPLC) analysis, gradient grade methanol (MeOH) was purchased from Merck (Darmstadt, Germany). Ammonium formate, 1,2-bis(3,5-dioxopiperazin-1-yl)ethane (I.S.DEX), and 1,3-diaminopropane-N,N,N,N-tetraacetic acid (I.S.ADR-925) were obtained from Sigma-Aldrich (Schnelldorf, Germany). Milli-Q water was produced by a Millipore purification system (Schwalbach, Germany).

For cell culture experiments, Dulbecco’s modified Eagle’s medium containing the nutrient mixture F-12 [Dulbecco’s modified Eagle’s medium (DMEM)/F12], penicillin/streptomycin (P/S) solution (5000 U/ml), and sodium pyruvate solution (PYR; 100 mM) were purchased from Lonza (Verviers, Belgium). Horse serum and fetal bovine serum (FBS) were purchased from Sigma-Aldrich or Biosera (Nuaille, France) and were heat inactivated prior to use (56°C, 30 minutes). For isolation of cardiomyocytes, collagenase type II (Thermo Fisher Scientific, Waltham, MA) and pancreatin (Sigma-Aldrich) were used. Other chemicals used for in vitro and in vivo experiments (e.g., constituents of various buffers) were purchased from Sigma-Aldrich or Penta (Prague, Czech Republic). Solutions of DEX and ADR-925 for all in vitro experiments were prepared as 1000× concentrated stock solutions in dimethylsulfoxide.

DEX (mol. wt. = 268 g/mol) was obtained from Huaren Chemicals (Changzhou, China). Cardioxane (dexrazoxane; Clinigen Healthcare, Burton‐on‐Trent, UK) was used for in vivo pharmacokinetic studies. The racemic form of ADR-925 (ICRF-198) (mol. wt. = 304 g/mol) was prepared in house; for a full description of the procedure, see Supplemental Material. Ketamine (Narketan; Vétoquinol, Lure, France), midazolam (Midazolam Torrex; Chiesi Pharmaceuticals, Wien, Austria), and pentobarbital (Sigma-Aldrich) were used for anesthesia.

Isolation of Rat Cardiomyocytes and In Vitro Pharmacokinetic Experiments

Neonatal rat ventricular cardiomyocytes (NVCM) have been used for in vitro pharmacokinetic studies because this is one of the most established and extensively used models for the in vitro study of cardioprotective effects of DEX (Hasinoff, 2002; Hasinoff et al., 2003a; Simůnek et al., 2008; Jirkovska-Vavrova et al., 2015). In addition, this system was previously employed in the initial evaluation of DEX metabolism (Schroeder et al., 2005). NVCM were isolated from 1- to 3-day-old Wistar rats, as described previously (Vavrova et al., 2013). Briefly, neonatal hearts were minced in buffer (1.2 mM MgSO4.7H2O; 116 mM NaCl; 5.3 mM KCl; 1.13 mM NaH2PO4.H2O; 20 mM HEPES, pH 7.4) on ice and serially digested at 37°C with collagenase type II (65 U/ml) and pancreatin (0.4 mg/ml). To minimize nonmyocyte cell contamination, a 2-hour preplating period (approximately 20 hearts per 150 mm Petri dish) was used before plating the cells on gelatin-coated 60-mm Petri dishes at a density of 2 × 105 cells/cm2. NVCM were cultured at 37°C in a 5% CO2 atmosphere in DMEM/F12 medium supplemented with 10% horse serum, 5% FBS, 4 mM PYR, and 1% P/S (pH 7.4). After 40 hours, the medium was changed to DMEM/F12 supplemented with 5% FBS, 4 mM PYR, and 1% P/S. For all experiments, the medium was changed to serum- and pyruvate-free DMEM/F12 with 1% P/S. The use of experimental animals for primary cell culture preparation has been approved by the Animal Welfare Committee of the Faculty of Pharmacy, Charles University.

For investigation of DEX metabolism to ADR-925, NVCM (4.8 × 106 cells) were incubated with 100 µM DEX. This DEX concentration has been repeatedly shown to be protective against ANT toxicity in this cell model (Hasinoff, 2002; Adamcova et al., 2007; Jirkovska-Vavrova et al., 2015). Incubation in the same setting was performed with ADR-925 (100 µM) to determine the ability of the metabolite to penetrate into NVCM. After 3, 6, 9, 12, and 24 hours of incubation in separate dishes with the compounds, samples of the cell culture media were taken, and NVCM were briefly washed twice with phosphate-buffered saline at 4°C, harvested by cell scraping, and centrifuged (700g; 10 minutes; 4°C). Incubations of both compounds with cell-free culture medium were performed to determine their chemical degradation under these conditions. To investigate the possible involvement of active transport of ADR-925 to NVCM, its concentration inside the cells was determined after a 24-hour incubation with the compound at concentrations of 100, 300, and 1000 µM ADR-925 at both 4°C and 37°C. All samples of cell culture media and dry cell pellets were shock frozen in liquid nitrogen immediately after collection and kept at −80°C until their analysis. All experiments were performed in quadruplicate.

To determine the contribution of NVCM to conversion of DEX to ADR-925, the lysates of freshly harvested NVCM (4.8 × 106 cells) were used. Pellets of the cells were resuspended in 400 µl buffer with DEX (100 µM) and sonicated (5-second stroke). The cell-free buffer supplemented with the drug served as a control. The mixture was incubated for 24 hours in dark with slow shaking (37°C). DEX and ADR-925 concentrations were determined at selected time points (0, 1, 3, 6, and 24 hours) in 50 µl samples of supernatant (3 minutes, room temperature, 5000g) obtained from the incubation mixture. Internal standards were added, and the samples were treated with 300 µl ice-cold MeOH, vortexed, and centrifuged (2 minutes, 4°C, 10,000g). Final supernatants were filtered through a 0.22-µm porosity filter and immediately analyzed by high-pressure liquid chromatography-mass spectrometry (HPLC-MS).

In Vivo Pharmacokinetic Experiments in Rabbits

All in vivo experiments and procedures were approved and supervised by Animal Welfare Committee of the Faculty of Medicine in Hradec Kralove, Charles University. Chinchilla male rabbits (n = 30, ∼3.8 kg) were randomized into six groups (n = 5 each) and fasted overnight before the experiment. Four groups received a single dose of DEX (60 mg/kg, i.p.), and the experiment was terminated at different time points after drug administration: 0.5, 3, 6, and 12 hours. The other two groups of rabbits received ADR-925 at the same dose (60 mg/kg) in 30-minute i.v. infusion into the marginal ear vein to describe the pharmacokinetics of ADR-925 alone, and the experiment was terminated 3 and 12 hours after the start of the infusion. The particular DEX dose and route of administration have been repeatedly shown to provide significant cardioprotection against chronic ANT cardiotoxicity in rabbits when administered 0.5 hour before each ANT dose (Simůnek et al., 2004; Popelová et al., 2009; Jirkovský et al., 2013), and is based on the upper limit of DEX dose previously used in humans’ cardioprotective study (Swain and Vici, 2004) and also for treatment of ANT extravasation (Kane et al., 2008), i.e., the ratio 1:20 to ANT dose (3 mg/kg). The i.p. route of administration has been typically used in most cardioprotective experiments in small laboratory animals, including rabbits, to avoid possible difficulties with repeated administration of both studied agents i.v. The rabbit model was among the first employed to study the cardioprotective effects of DEX in vivo and, until today, it is one of the best established models providing constantly reliable data (Herman and Ferrans, 1998; Stěrba et al., 2013).

Blood samples (∼1 ml) were collected from the contralateral ear vein at selected time points (5 minutes to 12 hours) after drug administration into BD Vacutainers (BD Biosciences, Plymouth, UK) containing lithium heparin. Plasma samples were harvested immediately, shock frozen in liquid nitrogen, and stored at −80°C until analysis.

For drug administration and blood sampling, light anesthesia was induced in rabbits with a mixture of ketamine (25 mg/kg, i.m.) and midazolam (2.5 mg/kg, i.m.); sedation was then maintained with midazolam when needed. The blood withdrawn from the animals was compensated with an appropriate volume of sterile saline. Urine was collected throughout the experiment, volumes were determined, and samples were frozen in liquid nitrogen and stored in −80°C until analyzed.

At the defined time points, rabbits were overdosed with pentobarbital. The heart was rapidly excised, briefly retrogradely perfused with ice-cold saline, and dried with gauze. Left ventricles were dissected and shock frozen in liquid nitrogen. Similarly, the liver and soleus muscle were also collected and frozen in liquid nitrogen. In addition, the volume of urine in the urinary bladder was determined, and urine samples were collected and frozen for analysis.

To determine the effects of tissue homogenates on DEX conversion to ADR-925, freshly harvested rabbit left ventricle (LV) myocardium and liver obtained from two rabbits were used. Samples were homogenized in 5 vol of buffer on ice in a glass/polytetrafluoroethylene Potter-Elvehjem tissue grinder. Freshly prepared homogenates were diluted with buffer to a final volume of 1 ml to prepare homogenate samples containing two different amounts of the tissue (10 and 100 mg per sample). DEX in a final concentration of 100 µM was added, and this mixture was incubated for 24 hours in dark with slow shaking (37°C, n = 4 in each tissue amount). The tissue-free buffer supplemented with the drug served as a control. DEX and ADR-925 concentrations were determined at selected time points (0, 1, 3, 6, and 24 hours) in 20 µl supernatant (3 minutes, room temperature, 5000g) obtained from the incubation mixture. Internal standards were added to the collected samples, and the samples were treated with 180 µl ice-cold MeOH, vortexed, and centrifuged (2 minutes, 4°C, 10,000g). Final supernatants were filtered through a 0.22-µm porosity filter and immediately analyzed. In addition, DEX (100 µM) was incubated in blank rabbit plasma at 37°C to examine conversion of DEX to ADR-925 in this material in vitro using similar experimental design, as described for tissue homogenates.

Determination of DEX and ADR-925 Concentrations in Biologic Samples

A previously described HPLC-MS method (Kovarikova et al., 2013) was adapted and validated to determine DEX and ADR-925 concentrations in samples obtained from in vitro and in vivo experiments. However, validation of this method (according to Food and Drug Administration guidelines) for simultaneous determination of both DEX and ADR-925 besides the intermediate hydrolytic products B and C was not possible, and therefore the intermediates B and C were not followed in this study. Briefly, the analyses were performed on a LC 20A Prominence (Shimadzu, Duisburg, Germany) chromatographic system coupled online with LCQ Advantage Max mass spectrometer (Thermo Finnigan, San Jose, CA). Synergi Polar-RP column (150 × 3 mm, 4 μm; Phenomenex, Torrance, CA) was used as a stationary phase. The mobile phase composed of components A (5% MeOH in 2 mM ammonium formate solution, without pH adjustment) and B (MeOH) was employed in the following gradient: 0–7 minutes (0% B), 7–8 minutes (0%–50% B), 8–22 minutes (50% B), 22.0–22.1 minutes (50%–0% B), and 22.1–30.0 minutes (0% B). A mobile phase flow rate of 0.15 ml/min, a column oven temperature of 25°C, the injection volume of 10 μl, and an autosampler temperature of 4°C were used for analysis. Electrospray ionization in positive mode was used, and quantification was performed in selected reaction monitoring mode: DEX [(M + H)+ at m/z 269→m/z 155], I.S.DEX [(M + H)+ at m/z 255 → m/z 141], ADR-925 [(M + K)+ at m/z 343 → m/z 299], and I.S.ADR-925 [(M + K)+ at m/z 345 → m/z 301].

Both internal standards were added to all biologic samples before treatment. Pellets (4.8 × 106 NVCM) were precipitated with ice-cold MeOH (300 μl) and homogenized using a Bio–vortex mixer (BioSpec Products, Bartlesville, OK) for 30 seconds and then in an ultrasonic bath. The samples were centrifuged (10 minutes, 4°C, 16,800g), and the resulting supernatant was filtered through a 0.45-μm porosity filter (Milex–Hv; Merck-Millipore) and analyzed. Cell culture medium samples (50 μl) were diluted with MeOH (20×) and directly analyzed. Rabbit plasma (100 μl) was precipitated with ice-cold MeOH (600 µl), vortexed, and, after centrifugation (10 minutes, 4°C, 16,800g), the supernatants were collected and analyzed. Urine samples were centrifuged, diluted (50–100×) with a MeOH/water mixture (1:1, v/v), and directly analyzed. Tissues were pulverized under liquid nitrogen, and samples (∼100 mg) of each tissue were precipitated with ice-cold methanol (500 µl) and homogenized using an Ultraturax homogenizer (IKA, Staufen, Germany). After centrifugation (10 minutes, 4°C, 16,800g), the supernatants were collected and analyzed.

For each biologic material, the method was validated according to the U.S. Food and Drug Administration; Center for Drug Evaluation and Research (CDER) (2001) Guidance for Industry: Bioanalytical Method Validation. The linearity of the methods was proven within following concentration ranges: 4–80 pmol/106 NVCM and 7–70 pmol/106 NVCM for DEX and ADR-925, respectively; 8–100 μM in cell culture medium and buffer (both compounds); 2–340 μM (DEX) and 1–100 μM (ADR-925) in plasma; 15–1500 μM in urine (both compounds); and 3.7–200 nmol/g and 1.6–16 nmol/g per wet tissue weight for DEX and ADR-925, respectively. The samples containing higher concentrations of the analytes were appropriately diluted to fit the relevant concentration range. The applicability of this approach was proved by testing the dilution integrity. Precision and accuracy for all biologic materials were ≤15% and 20% for all concentrations and lower limit of quantification, respectively, and thus meet Food and Drug Administration requirements.

Pharmacokinetic Analysis of Data from In Vivo Experiments

Standard noncompartmental analysis was performed using Kinetica software (version 4.0; Thermo Fisher Scientific). The nonlinear mixed-effects modeling approach was used with pharmacokinetic parameters estimated by the Stochastic Approximation Expectation Maximization algorithm coupled with the Markov Chain Monte Carlo procedure as implemented in the software Monolix (version 4; Lixoft, Antony, France). The consecutive steps of the pharmacokinetic analysis are as follows:

Naive Pooled Data Approach.

The concentration–time data of 20 animals subjected to destructive sampling after a single dose of DEX i.p. were naive pooled, and the geometric mean concentration–time profiles of plasma DEX and ADR-925 were subjected to standard noncompartmental analysis (NCA). The same approach was used to calculate model-independent parameters of plasma ADR-925 from 96 assayed concentrations after i.v. infusion of the metabolite to nine animals. The cumulative amounts of DEX and ADR-925 excreted in urine by all animals were plotted against time, the parameters of one-phase association curve were fitted to the data, and the mean total amounts excreted as unchanged drug and/or ADR-925 were calculated as asymptotes. The fractions of the dose excreted as unchanged drug and/or the metabolite and renal clearances were computed.

The Standard Two-Stage Method.

To improve the accuracy of the pharmacokinetic (PK) naive pooled data biased by animals terminated at the time of unfinished metabolization and elimination processes, PK parameters after both DEX and ADR-925 administration were estimated by NCA using data of the subgroup of the animals with entire 12-hour sampling (n = 5 for each compound). The total urinary excretions and renal clearances were calculated without extrapolation.

Population Modeling.

Logarithms of molar DEX and ADR-925 concentrations in plasma and of their urinary molar quantities improved the precision of parameter estimates and were used throughout. Interanimal variability was evaluated with the use of an exponential error model, and residual variability was evaluated with an additive error model on the logarithmic scale. First, separate models were built up that described DEX plasma concentration and the amount of DEX excreted in urine after its i.p. injection (Model 1), and for plasma and urinary data of ADR-925 after i.v. infusion of the metabolite (Model 2). Finally, a joint model was elaborated that simultaneously described DEX and ADR-925 plasma concentrations and urinary amounts after the i.p. injection of the parent compound (Model 3).

The compartmental models were parameterized in terms of volume of distribution (central and peripheral), intercompartmental clearance, total and renal clearances, and, in case of the joint Model 3, the metabolic clearance of DEX to ADR-925. The models for DEX assumed first-order absorption without a lag time, and the absolute bioavailability F = 1 was assumed based on the rapid appearance of DEX in plasma after administration.

Urinary recovery of the i.v. dose of ADR-925 was shown to be complete in this study. Therefore, the metabolite volumes of distribution were identifiable in Model 3, which included the data on urinary excretion of ADR-925. The predictive performance of the models was evaluated using goodness-of-fit plots and the distribution of observations with respect to the model-predicted median profiles and 90% prediction intervals, i.e., the visual predictive checks. Finally, empirical Bayes estimates of each parameter were obtained, and secondary pharmacokinetic characteristics were calculated.

In the case of tissue concentrations, the mean value and variance of the area under the curve (AUC) were calculated with the help of the package PK, version 1.3-3, named Basic Non-Compartmental Pharmacokinetics (Jaki et al., 2010).

Data Analysis and Presentation

Data are shown as the mean ± S.D., unless stated otherwise. Pearson and Spearman correlation analyses, linear regression analyses, and statistical significance differences (using one-way analysis of variance with Holm–Sidak’s or analysis of variance on Ranks with Dunn’s post hoc test) were determined according to the data character using software SigmaStat 3.5 (SPSS, Chicago, IL).

Results

Distribution of DEX/ADR-925 in Isolated Rat Cardiomyocytes and Conversion of DEX to ADR-925

DEX and ADR-925 concentrations were determined in cell culture medium and inside NVCM after their incubation with DEX (100 µM, up to 24 hours). In the culture medium with NVCM, DEX concentrations gradually fell to ∼30 µM after 24 hours (Fig. 2A), whereas ADR-925 concentrations steadily increased from the third hour to exceed the concentrations of the parent compound between the 12th and 24th hours, reaching ∼45 µM at the end of the experiment (Fig. 2A). Almost identical results were obtained in culture media without NVCM (Fig. 2A), which indicates that DEX conversion to ADR-925 is related to spontaneous hydrolysis rather than cardiomyocyte metabolic activity. Inside NVCM, DEX was determined after 3 hours of incubation, with its peak value in the sixth hour. The concentrations of DEX gradually declined thereafter, but were detectable until the end of the incubation (Fig. 2B). In the same experiment, ADR-925 concentrations inside the cells were first detected in the ninth hour and then progressively increased until the end of experiment (24 hours), when it achieved concentrations approximately twice as high as those of DEX (Fig. 2B). Direct incubation of NVCM with ADR-925 (in the same concentrations as DEX, 100 µM) resulted in a slower penetration of this agent into the intracellular compartment compared with DEX, but the concentrations were considerably higher than those formed in the previous experiment after incubation of the cells with DEX. The concentrations of ADR-925 then gradually increased in time to match the concentrations of DEX determined in the previous experiment in the ninth hour (Fig. 2B), and a further increase of ADR-925 concentration continued until the end of the incubation. Intracellular concentrations of ADR-925 were almost double throughout this experiment compared with the concentrations of ADR-925 determined after DEX incubation.

Fig. 2.
Fig. 2.

Conversion of DEX to ADR-925 and their distribution in isolated neonatal rat cardiomyocytes. (A) DEX and ADR-925 concentrations in the cell culture media during a 24-hour incubation of DEX (100 μM) in the presence (full symbols) or absence (empty symbols) of NVCM. (B) Intracellular concentrations of DEX and ADR-925 during a 24-hour incubation of NVCM with 100 μM DEX (blue circles and red boxes) or ADR-925 (green boxes). (C) Assay of influence of the active transport on ADR-925 uptake into NVCM. Incubation of NVCM with ADR-925 (100–1000 μM) under physiologic (37°C, circles) and decreased temperature (4°C, boxes). (D) The effect of NVCM lysate (full symbols) on DEX conversion to ADR-925 during a 24-hour incubation compared with buffer (empty symbols). All experiments were performed with cell density of 4.8 × 106 cells (n = 4). *Statistical significance determined by one-way analysis of variance on ranks with Dunn’s post hoc test (P ≤ 0.01).

In further experiments, we wanted to examine whether and to what extent active transport contributes to the penetration of the relatively polar metabolite ADR-925 into NVCM. Therefore, these cells were incubated with ADR-925 (100–1000 µM) at 4°C or 37°C for 24 hours. The result of this experiment showed that the penetration of ADR-925 into the cells is temperature dependent, as the concentrations inside the cells were significantly lower at 4°C compared with 37°C, which indicates contribution of an active type of transport (Fig. 2C). In contrast, the experiment performed with the higher concentration did not show any ceiling effect, which suggests that the transport is not saturable in the tested concentration range.

To directly assess the role of intracellular enzymes in DEX metabolism to ADR-925, 100 μM DEX was incubated for 24 hours with NVCM homogenate or the corresponding buffer. The results showed that NVCM homogenate (Fig. 2D) did not have any significant effect on the formation of ADR-925 from DEX, and the concentrations were comparable with those determined in the buffer (Fig. 2D).

DEX Metabolism and Disposition in Rabbits

Plasma Concentrations and Urinary Excretion of DEX and ADR-925.

The rate of absorption of DEX (60 mg/kg) administered as a single i.p. bolus to rabbits was high (Fig. 3A). The highest plasma concentrations of DEX were observed in individual time profiles between the 5 and 30 minutes after the injection, with the median time interval of 10 minutes. The geometric mean concentration of plasma DEX peaked at the 10th minute, and its decrease from the cmax (337 μΜ) was apparently biphasic, leaving only 1.7% of the cmax concentration at the end of the 12-hour sampling interval.

Fig. 3.
Fig. 3.

Mean plasma concentration–time profiles of drugs under study after single-dose administration of DEX and ADR-925 to rabbits. (A) Plasma concentration–time profiles of DEX and ADR-925 after single-dose administration of DEX (60 mg/kg, i.p.). (B) Plasma concentration–time profile of ADR-925 after single-dose administration of ADR-925 (60 mg/kg, 30 minutes i.v. infusion). Data are presented as the mean ± S.D.

The metabolite was detected in all plasma samples collected at the fifth minute, but its concentrations (Fig. 3A) were relatively lower compared with the parent compound. Its geometric mean of concentration increased gradually until the 45th minute, reaching the cmax (35.6 μΜ). A broad plateau was then observed on the mean concentration–time profile of ADR-925 between the 45th minute and the 5th hour. After this time interval, the mean concentration of the metabolite showed a monotonous decline in parallel with that of DEX. The residual metabolite concentrations at the 12th hour corresponded to 14% of the cmax. The slow formation of ADR-925 in plasma observed in the curve of the mean concentrations followed with a plateau was not the effect of pooling of the data from different animals, as it was also found on the concentration–time profiles of individual animals (data not shown). The median time to the maximum concentration was 2 hours (0.5–8 hours) in 13 rabbits that were sampled over a sufficiently long interval to reach the cmax.

In another experiment, ADR-925 (60 mg/kg) was infused i.v. to rabbits over 30 minutes to follow the distribution and elimination kinetics of the metabolite from plasma. The highest concentrations of ADR-925 in plasma were observed at the end of the infusion (30 minutes) with the exception of one animal (20 minutes). The geometric mean of the cmax was found to be 557 μΜ. Similar to DEX, the decline of the mean plasma concentrations of ADR-925 exhibited a biphasic pattern, and, after 12 hours, only 0.2% of the cmax remained in plasma (Fig. 3B).

The cumulative urinary excretion of DEX and ADR-925 followed for 12 hours after a bolus dose of 60 mg/kg DEX i.p. is shown in Fig. 4A. The mean total amounts of DEX and ADR-925 excreted via the urine, estimated as asymptotes by nonlinear regression, were 309 and 103 μmol, respectively. The amounts of the parent drug, metabolite, and their sum in the urine estimated from all animals (naive pooled data) represent 35.5% (DEX), 11.9% (ADR-925), and 47.4% (DEX + ADR-925) of the administered dose, respectively. Similarly, in the subgroup of five animals with urine collected over the entire 12 hours, the total recoveries were 35.7% ± 11.1% (DEX), 11.0% ± 4.3% (ADR-925), and 46.6% ± 10.9% (DEX + ADR-925) of the dose.

Fig. 4.
Fig. 4.

Cumulative urinary excretion of studied drugs. (A) Cumulative urinary excretion of DEX (circles) and ADR-925 (boxes) after single-dose administration of DEX (60 mg/kg, i.p.). (B) Cumulative urinary excretion of ADR-925 after single-dose administration of ADR-925 (60 mg/kg, i.v. infusion). Solid lines represent best fits from nonlinear regression, and circles and boxes are observed amounts.

A vast majority of the i.v. infused dose of ADR-925 was excreted unchanged in urine, supporting the view that renal excretion is a predominant elimination pathway for this metabolite (Fig. 4B). The mean total urinary excretion and recovery of ADR-925 estimated from all naive-pooled animals were 664 μmol and 89.5%, respectively. Nonlinear regression may have underestimated the total amount of the drugs excreted by the kidneys due to the influence of data from animals with shorter intervals of collection. In the subgroup of the animals within the 12-hour collection interval, the mean urinary recovery of the infused dose of the metabolite achieved 98% ± 6%, indicating that the excretion was practically complete.

Noncompartmental Analysis

Pharmacokinetic parameters calculated by NCA are summarized in Table 1. Due to the batch design of the experiment, i.e., the variable length of the sampling interval in subgroups of animals, NCA could be applied on the geometric mean of concentration–time profile obtained from all naive-pooled animals (n = 20) and on individual profiles of animals with 12-hour sampling (n = 5). Blood sampling over 12 hours was adequate for the description of plasma pharmacokinetics of DEX and ADR-925 in both studies. After a bolus dose of DEX, the extrapolated parts of the AUCs were less than 4% (DEX) and 10% (ADR-925), with the exception of one individual concentration–time profile of ADR-925 where the extrapolated part of the AUC was 23%. After i.v. infusion of ADR-925, extrapolation beyond the 12th hour accounted for less than 1% of the AUC.

View this table:
TABLE 1

Summary of pharmacokinetic parameters of DEX and ADR-925 in rabbits determined by noncompartmental analysis

In general, there was good agreement between the results of the NCA analysis of naive-pooled concentration–time data from all animals and the two-stage approach used in five animals sampled over 12 hours (Table 1). Two exceptions were the cmax of DEX and tmax of the metabolite after DEX injection. Obviously, the single-point pharmacokinetic characteristics cmax and tmax are more variable between animals, and their estimates are more prone to bias. In addition, the broad plateau on the ADR-925 mean concentration–time profile explains the discrepancy regarding its tmax.

The fraction of the DEX dose metabolized to ADR-925 (11%) was calculated assuming that the absolute bioavailability of DEX equals one (F = 1) and that renal excretion is the sole elimination pathway for the metabolite after DEX injection, which is supported by our data after infusion of ADR-925. As judged from the ratio of the mean AUCs (parent/metabolite) of 3.2, DEX was the quantitatively prevailing compound in plasma.

The renal clearance of ADR-925 after DEX injection was approximately twofold lower compared with infusion of ADR-925. Of note, the cmax of plasma ADR-925 was 15-fold higher at the end of the i.v. infusion of ADR-925, and the concentrations remained markedly higher during the first postinfusion hour compared with the maximum concentrations of ADR-925 metabolically formed after DEX injection. The finding of the concentration-dependent renal clearance complicated the comparison of the mean residence time (MRT) and half-life (t1/2) values of ADR-925 in both studies. The MRTi.v. of ADR-925 (2.5 hours), calculated as (MRTmetabolite − MRTparent) after DEX injection, was thus higher than the MRTi.v. of ADR-925 after infusion of ADR-925 (1.6 hours), obtained as the difference (MRTinf − Tinf/2).

Population Pharmacokinetic Analysis

A schematic representation of the separate models for DEX and ADR-925 is given in Fig. 5, A and B, respectively. After i.p. injection, disposition of DEX in plasma was best described by a two-compartment open model with first-order absorption and elimination without a time lag (Model 1). The final model for ADR-925 was a two-compartment model with zero-order input via i.v. infusion and first-order elimination (Model 2). In the joint parent-metabolite model (Model 3; Fig. 5C), formation of ADR-925 from plasma DEX was modeled as a first-order process, and the amount of the metabolite recovered in urine was taken as representative of its overall quantity produced by metabolism. The central and peripheral volumes of distribution of the metabolite were added to achieve the best description of the combined data from DEX and ADR-925.

Fig. 5.
Fig. 5.

Schematic representation of the selected pharmacokinetic models for DEX and ADR-925. (A) Two-compartmental model with first-order absorption for DEX after i.p. administration. (B) Two-compartmental model for ADR-925 after i.v. infusion. (C) Four-compartmental parent-metabolite model for DEX and ADR-925. AU, cumulative urinary excretion; ClNR, nonrenal clearance; Clother, clearance describing other elimination pathways of DEX; ClR, renal clearance; ka, absorption rate constant; Q12 and Q34, intercompartmental clearances; V1, V2, V3, and V4, volumes of central and peripheral compartments for DEX and ADR-925.

The statistical summary of Bayesian post hoc estimates of individual pharmacokinetic parameters produced by Models 1, 2, and 3 is presented in Table 2. The relative standard errors not exceeding 24% document an adequate precision of estimates for all parameters. Values of clearances and volumes of distribution agreed reasonably well with those from NCA. The joint parent-metabolite model also allowed for determination of the metabolic clearance of DEX (0.045 l × h−1 × kg−1), and the estimation of secondary pharmacokinetic parameters for DEX presented as a geometric mean: Cltot = 0.26 l × h−1 × kg−1, volume of distribution = 0.74 l/kg, t1/2α = 11.5 minutes, and t1/2β = 131.4 minutes.

View this table:
TABLE 2

Summary of pharmacokinetic parameters determined by population-modeling approach in rabbits

The visual predictive performance and goodness-of-fit of the models were good (Fig. 6; Supplemental Fig. 1). The observed plasma DEX and ADR-925 concentrations fit well within the 5%–95% of prediction intervals. Population mean-predicted plasma concentrations adequately describe the central tendencies in concentration–time data. The goodness-of-fit is evident from the scatter plots of observed versus individual predicted plasma concentrations, calculated by Bayesian estimation (Fig. 6; Supplemental Fig. 1, C and D).

Fig. 6.
Fig. 6.

Visual predictive performance and goodness-of-fit graphs of the four-compartment population model for DEX and ADR-925. (A) Plasma concentration–time profiles of DEX and (B) ADR-925 after single-dose administration of DEX (60 mg/kg, i.p.). (C) Scatter plots of the observed versus individual predicted concentrations for DEX and (D) ADR-925. (E) Cumulative urinary excretion of DEX and (F) ADR-925 after single-dose administration of DEX (60 mg/kg, i.p.). The solid lines and shaded areas represent the median predictions and the 90% prediction intervals. The crosses are observed concentrations.

Tissue Concentrations of DEX and ADR-925 in Rabbits

DEX and its metabolite ADR-925 were also determined in the selected tissues (LV myocardium, liver, and soleus muscle) at different time points after DEX administration to rabbits (Fig. 7). Very similar DEX and ADR-925 concentrations (including the overall concentration–time profile) were determined in the LV myocardium and in the liver. The concentrations of DEX in both tissues were more than double compared with the soleus muscle 30 minutes after drug administration, and they decreased faster than in the muscle. Both in the myocardium and liver, the decrease in DEX concentrations was not accompanied by a proportional increase of ADR-925. In fact, the concentrations of the metabolite remained relatively steady throughout the experiment particularly in the liver, with tendency toward a slow rise in the heart. These data argue against distinct intracellular trapping and cumulation of the relatively polar ADR-925 in this tissue. A different situation was demonstrated in soleus muscle, where the concentrations of the metabolite increased more significantly compared with the decline of DEX. Different concentrations of both drugs in the soleus muscle compared with other tissues could be caused by lower perfusion of the peripheral muscle compared with the myocardium and the liver.

Fig. 7.
Fig. 7.

Tissue concentrations of compounds under study after single-dose i.p. DEX administration to rabbits. Tissue concentration–time profiles of DEX and its metabolite ADR-925 after single-dose administration of DEX (60 mg/kg, i.p.) in (A) LV myocardium, (B) liver, and (C) soleus muscle. Data are presented as the mean ± S.D.

The DEX concentrations determined in plasma at the end of the study showed a very good relationship with the corresponding concentrations found in the tissues in the same time point (Supplemental Fig. 2, A–C). However, this was not the case for ADR-925 (Supplemental Fig. 2, D–F), where the concentrations in the myocardium and liver tissue showed no significant correlation with the plasma concentrations (P > 0.05), and only poor and a surprisingly negative correlation was found in the soleus muscle (P = 0.021, R = −0.513).

The total exposure of the tissues to DEX and ADR-925 estimated as AUC (Table 3) revealed that, after DEX administration, the LV myocardium and liver tissue were exposed to 5.7 and 4.8 times (respectively) higher concentrations of the parent drug compared with ADR-925, whereas the ratio was lower in the soleus muscle (3.5 times). The ratios in the former tissues are significantly higher than that found in plasma (3.2 times).

View this table:
TABLE 3

Estimated AUC of DEX and ADR-925 in selected tissues after administration of DEX to rabbits (60 mg/kg, i.p.)

AUCs were estimated by batch method from all animals in four groups each of five animals terminated in 0.5, 3, 6, and 12 hours after DEX administration (60 mg/kg, i.p.). t-CI, t-based confidence interval for the mean.

To directly estimate tissue metabolism of DEX to ADR-925, we incubated LV myocardial and liver homogenates with DEX at 37°C and determined concentrations of both of these compounds (Fig. 8). Only a slight decrease of DEX concentrations occurred after 6 hours of incubation with myocardial homogenates, which corresponds with low concentrations of the metabolite. Importantly, the amount of the myocardial tissue in the homogenate had no impact on the results, and the concentrations were very similar as in BSS buffer without tissue homogenate. In contrast, the same experiments performed with liver homogenates documented a significantly accelerated decrease of DEX concentration with a concomitant increase of ADR-925. Because there was also an apparent difference between the samples prepared with a different amount of the liver tissue, it seems that this activity is attributable to content of the tissue and biotransformation enzymes. To estimate whether DEX may also be converted to ADR-925 in plasma more effectively than by simple chemical hydrolysis in the buffer, DEX was incubated in rabbit plasma at 37°C (Fig. 9). These experiments demonstrated that DEX is converted to ADR-925 in plasma significantly more effectively than in buffer. After 6 hours of incubation, plasma concentrations of DEX decreased by more than 60%, while in buffer it was approximately 20%, and the concentrations of ADR-925 rose concomitantly. Interestingly, DEX concentrations in plasma dropped down after 24 hours to negligible values, whereas ADR-925 concentrations approached 100 μM, suggesting almost complete conversion of DEX to ADR-925 under these conditions. This was not the case in buffer, in which the decline of DEX concentrations was approximately 50% at the same time point, with a similar increase of ADR-925 concentrations. Although intermediate metabolites B and C could not be determined in these experiments, their theoretical portion can be approximated based on mass balance as a resting portion on the top of the sum of DEX and ADR-925 (Fig. 9B).

Fig. 8.
Fig. 8.

DEX incubation with rabbit tissue homogenate and plasma. Incubation of DEX (100 μM) with different amount of (A) LV myocardium homogenate or (B) liver homogenate. Data are presented as the mean ± S.D.

Fig. 9.
Fig. 9.

DEX incubation with rabbit plasma. (A) Incubation of DEX (100 μM) with fresh rabbit plasma. (B) Theoretical portion of intermediate metabolites B and C determined by mass balance calculations during incubation of DEX (100 μM) with fresh rabbit plasma. Data are presented as the mean ± S.D.

Discussion

This study adds several important pieces into the puzzle of the pharmacokinetics of DEX and its putative active metabolite ADR-925 under well-established experimental conditions in which DEX is known to provide reliable protection from ANT cardiotoxicity. We have documented a slow and progressive rise of intracellular concentrations of ADR-925 in NVCM after DEX incubation. We have proven that ADR-925 can directly, albeit relatively slowly, penetrate from the cell culture medium into cardiomyocytes, and the resulting intracellular concentrations are significantly higher than after incubation of the cells with equimolar DEX concentrations. Our data suggest the contribution of an active transmembrane transport, but particular transporter for ADR-925 remains unknown. According to its chemical structure, one could speculate on involvement of a member of the solute carrier family transporters, especially for organic cation/carnitine transporter family (solute carrier family 22) (Klaassen and Aleksunes, 2010; Tamai, 2013), from which organic cation/carnitine transporter family 2 was described to be present in the mouse and human heart (Grube et al., 2006). Of note, we showed that significant nonenzymatic hydrolysis of DEX to ADR-925 already occurs in the culture media, and the rate is faster than in the buffer of corresponding pH. Hence, during 12-hour experiments, the cells are exposed to relatively high concentrations of ADR-925, and, in longer experiments, the exposure to the metabolite predominates over DEX. It is noteworthy that NVCM lysate had no significant impact on conversion of DEX to ADR-925. This may imply that a significant part of ADR-925 intracellular concentrations found after incubation with DEX can arise from penetration of ADR-925 from the medium, where it originates by nonenzymatic hydrolysis of the parent compound. The hydrolysis has been described to be promoted by presence of metal ions (especially Fe2+, Ca2+, Cu2+, and Mg2+) (Buss and Hasinoff, 1995, 1997), although in the rather higher concentrations then they are present in the present cell media. In contrast, Schroeder et al. (2005) previously published that suspension of neonatal cardiomyocytes accelerated the decrease of DEX concentrations in the medium, which may appear to be in contrast to our findings. However, the interpretation of these results is complicated by short incubation of the cells with DEX (2 hours), resulting in a relatively small decline of DEX concentration, and by a lack of information on ADR-925 formation.

In contrast to DEX, incubation of isolated rat cardiomyocytes with ADR-925 has been previously reported to be unable to protect the cells from doxorubicin toxicity, and one possible explanation was poor penetration of the putative active metabolite into the cells due to its anionic hydrophilic nature (Hasinoff et al., 2003b). However, our data obtained also from rat cardiomyocytes clearly contradict this hypothesis and further contribute to questioning ADR-925 as an active metabolite responsible for DEX-induced cardioprotection (Stěrba et al., 2013).

Plasma pharmacokinetic profiles of DEX in rabbits determined in this study are surprisingly close to those reported from clinical trials. Basic pharmacokinetic parameters calculated in this study practically match those reported in patients (in order: our NCA data versus human data–NCA data if possible): biologic t1/2 of elimination (t1/2β) 2.0 hours versus ∼2–4 hours (Earhart et al., 1982; Vogel et al., 1987; Hochster et al., 1992; Rosing et al., 1999), total clearance 0.26 versus ∼0.19–0.35 l × h−1 × kg−1 (Holcenberg et al., 1986; Vogel et al., 1987; Rosing et al., 1999), volume of distribution 0.74 versus ∼0.45–1.1 l/kg (Holcenberg et al., 1986; Wiseman and Spencer, 1998), and the recovery of administered DEX dose in urine (as DEX + ADR-925) 47% versus 40%–48% (Earhart et al., 1982; Rosing et al., 1999; Tetef et al., 2001). DEX pharmacokinetic in humans was described to be different in pediatric patients and in adults (Holcenberg et al., 1986), and to be linear in broad range of DEX concentrations (Hochster et al., 1992). Our findings in rabbits thus differ considerably from rodents, in which the elimination of DEX has been reported as four to six times faster (Hasinoff et al., 1998; Hasinoff and Aoyama, 1999a). This suggests that rabbit is a valuable animal model to study the cardioprotective effects of DEX not only from a pharmacodynamic (Herman and Ferrans, 1998; Simůnek et al., 2004; Popelová et al., 2009; Jirkovsky et al., 2012) but also from a pharmacokinetic point of view.

The plasma profile of the putative active metabolite (ADR-925) after DEX administration to rabbits was also relatively close to reports from the single human study published to date (Schroeder et al., 2003). In both, the metabolite appeared in plasma in the first sample taken after drug administration and formed a plateau for several hours (∼5 hours in rabbits and ∼4 hours in humans), with a relatively slow decline until the end of the study. Although the administered doses were not the same in these studies, the range of plasma concentrations of ADR-925 in the plateau phase was similar (∼40 μM after 1000 mg/m2 DEX in rabbits and 30 μM after 1500 mg/m2 in patients). Unfortunately, the pharmacokinetic analysis for ADR-925 was not performed in the clinical study, and thus the pharmacokinetic parameters cannot be directly compared. The profile of ADR-925 was also studied in rats after DEX administration (40 mg/kg, i.v.) (Schroeder and Hasinoff, 2002). In the latter study, ADR-925 was detected in plasma soon after DEX administration, with a peak of plasma concentrations in the 80th minute (∼80 μM) and tended to decrease faster than in rabbits and humans. The short duration of this study (3 hours) did not allow for appropriate evaluation of metabolite elimination. However, the same authors evaluated the pharmacokinetics of ADR-925 after an i.v. bolus of either ADR-925 or the intermediate metabolites (B/C) of DEX to rats (Schroeder and Hasinoff, 2005). They observed a rapid decline of ADR-925 with t1/2 ∼20 minutes (as estimated from the graphical data) after administration of ADR-925, whereas an even shorter t1/2 (8.3 minutes) was calculated from elimination constants for ADR-925 after administration of intermediate metabolites. This contrasts with our data from rabbits, where the t1/2 is ∼100 minutes. Our study has shown that ADR-925 is almost completely eliminated unchanged by the kidney to urine. We have also created a population-based pharmacokinetic model taking into account both parent DEX and ADR-925.

Our study has also systematically described the time course of DEX and ADR-925 tissue concentrations, especially in LV myocardium as the target tissue for cardioprotection. The data from patients are naturally unavailable, and, besides scarce data from conference abstracts (Mhatre et al., 1983; McPherson et al., 1984), very little information has been published from animal experiments in extenso to date (Schroeder and Hasinoff, 2005; Schroeder et al., 2008). As a part of these two studies, myocardial ADR-925 concentrations were described after DEX administration (40 mg/kg, i.v.) to rats in the single arbitrary selected time point (after 2.8 or 2–2.5 hours, respectively). However, only the former study also provided concentrations of the parent compound. The concentrations of ADR-925 tended to be significantly higher in these rat studies than in our experiments with rabbits, but direct comparison is difficult due to different dose/route of administration and only discrete information of tissue concentrations in the former studies. As plasma pharmacokinetics of DEX and ADR-925 are markedly different in rodents than in humans and rabbits, tissue concentrations of both compounds may also be affected.

We have studied the metabolism of DEX to ADR-925 in rabbit tissue homogenates to reveal that, unlike liver, myocardial tissue does not accelerate DEX hydrolysis and the formation of ADR-925. This is consistent with our in vitro results obtained from DEX incubation with NVCM and their lysates and with the study published by Hasinoff et al. (1991). Although the former study struggled with difficulties such as unavailability of appropriate bioanalytical methodology and the incubations were rather short (4 hours), they also found a lack of ADR-925 increase after DEX incubation with porcine heart homogenate, contrary to incubation with the liver homogenate. This may be evidently connected with the high amount and activity of dihydropyrimidase, which catalyzes the first hydrolytic step, in the liver in contrast to myocardial tissue (Hasinoff et al., 1991). Interestingly, we found that DEX was considerably hydrolyzed to ADR-925 by incubation in fresh rabbit plasma under physiologic temperature. Hence, it is possible that DEX may be significantly converted to ADR-925 in the circulation, which may contribute to early detection of ADR-925 after DEX administration in all studies performed to date. The plasma instability of DEX could not confound data in our study because its pre- and analytical parts were designed and validated accordingly. Altogether, these data may imply that the ADR-925 concentrations found inside rat cardiomyocytes and rabbit LV myocardium are determined largely by distribution of ADR-925 or its intermediate hydrolytic products from the circulation, where it could originate both by non- or enzymatic hydrolysis and/or by release from metabolically active organs such as the liver. Differences between correlations of plasma and tissue concentrations of DEX and ADR-925 may be a consequence of different distributions into the intracellular compartments (slower for ADR-925), which correspond with the more hydrophilic nature of ADR-925.

One of the limitations of the present study is the lack of direct information on the two intermediate hydrolytic metabolites B and C. This was caused by difficulties with their appropriate analytical determination besides DEX and ADR-925. However, both intermediate metabolites have been tested previously in vitro for cardioprotective properties against ANT cardiotoxicity with negative outcomes (Hasinoff et al., 2003b), and therefore they were out of our primal interest. Indirect information on portion of B and C from the plasma hydrolysis experiments suggested their minor appearance.

In conclusion, the present study filled in several important gaps in the pharmacokinetic profile and tissue disposition of the clinically used cardioprotectant DEX to its putative active metabolite ADR-925 in a rabbit model, which shows pharmacokinetic similarities to humans. Importantly, the finding that ADR-925 could directly enter cardiomyocytes opens a venue for direct determination of whether ADR-925 is actually the active metabolite of DEX that is responsible for its cardioprotective effects, as this is now disputed in the literature (Hasinoff and Herman, 2007; Stěrba et al., 2013; Henninger and Fritz, 2017).

Acknowledgments

We thank Klára Lindrová for excellent technical assistance during the experiments.

Authorship Contributions

Participated in research design: Jirkovský, Jirkovská, Chládek, Lenčová, Šimůnek, Kovaříková, Štěrba.

Conducted experiments: Jirkovský, Jirkovská, Bureš, Lenčová, Pokorná, Karabanovich, Roh, Brázdová, Kovaříková, Štěrba.

Contributed new reagents or analytic tools: Bureš, Stariat, Karabanovich, Roh.

Performed data analysis: Jirkovský, Jirkovská, Bureš, Chládek, Kovaříková, Šimůnek, Štěrba.

Wrote or contributed to the writing of the manuscript: Jirkovský, Jirkovská, Bureš, Chládek, Lenčová, Pokorná, Roh, Šimůnek, Brázdová, Kovaříková, Štěrba.

Footnotes

    • Received August 29, 2017.
    • Accepted December 19, 2017.

  • ↵1 E.J. and A.J. contributed equally to this study.

  • This work was supported by Czech Science Foundation [Grant 13-15008S], Charles University Grant Agency [Grant 1324214], and Charles University Research Program Progres Q40/5.

  • https://doi.org/10.1124/jpet.117.244848.

  • ↵Embedded ImageThis article has supplemental material available at jpet.aspetjournals.org.

Abbreviations

ANT
anthracycline antibiotics
AUC
area under the curve
BSS
balanced salt solution
DEX
dexrazoxane
DMEM
Dulbecco’s modified Eagle’s medium
FBS
fetal bovine serum
HPLC-MS
high-pressure liquid chromatography-mass spectrometry
inf
infusion
LV
left ventricle
MRT
mean residence time
NCA
noncompartmental analyses
NVCM
neonatal rat ventricular cardiomyocyte
PK
pharmacokinetic
P/S
penicillin/streptomycin
PYR
sodium pyruvate solution
t1/2
half-life

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Research ArticleMetabolism, Transport, and Pharmacogenomics

DEXMetabolism In Vitro and In Vivo

Eduard Jirkovský, Anna Jirkovská, Jan Bureš, Jaroslav Chládek, Olga Lenčová, Ján Stariat, Zuzana Pokorná, Galina Karabanovich, Jaroslav Roh, Petra Brázdová, Tomáš Šimůnek, Petra Kovaříková and Martin Štěrba
Journal of Pharmacology and Experimental Therapeutics March 1, 2018, 364 (3) 433-446; DOI: https://doi.org/10.1124/jpet.117.244848
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