Since the current pharmacological therapy for cardiac diseases still is far from optimal, novel therapeutic approaches are constantly being investigated. 1–5 A possible strategy to improve the therapeutic efficiency is the local application of drugs. The rationale of local drug delivery is that a relatively high proportion of the drug may be taken up by cardiac tissue, resulting in cardiac effects at a much lower dose. Consequently, lower extra-cardiac drug levels are obtained and peripheral side effects minimized. Local drug delivery to the heart can, for instance, be achieved by slow release polymers attached to the epicardial surface of the heart, or by using the local environment of the heart (ie, the pericardial space). This cavity between the pericardial sac and the epicardial surface of the heart, filled with pericardial fluid, is a potential reservoir for drug delivery. 6
Effectiveness of intrapericardial (IPC) drug delivery on the heart and coronary circulation has been documented for angiogenic substances, 7–9 vasodilators, 10–12 and antitachycardiac, antiarrhythmic, and arrhythmic agents. 1,13–20 However, studies focused on local bolus injections rather than sustained application. In addition, IPC application was often not compared with conventional systemic (eg, intravenous) application, making it difficult to address the question whether or not IPC application is of advantage over systemic drug delivery. In a recent study in our laboratory, we showed pharmacokinetic advantages for several substances in the rat following IPC infusion. 21 This could be explained by their low clearances from the pericardial space, as compared with systemic clearances. However it is not clear whether these pharmacokinetic advantages translate into a better drug response in case of sustained IPC drug infusion. In addition, despite that in that study low molecular weight substances were included, it is not clear whether charged agents (such as sotalol and atenolol) can be used for this strategy.
Therefore the present study addressed the question whether pharmacokinetic advantages are observed after sustained IPC infusion of the small positively charged β-blocking agents d,l- sotalol and d,l- atenolol and whether IPC infusion of the agents results in a more effective cardiac β-blocking action than intravenous (IV) infusion. Plasma-, pericardial fluid-, and tissue concentrations were studied after sustained IPC and IV sotalol and atenolol delivery. In a second set of experiments, drugs were infused IPC or IV to assess effects on baseline heart rate (HR) and nitroprusside-induced tachycardia in conscious rats. In a third set of experiments, we compared IV and IPC sotalol effects on left ventricular contractility measured as the maximal rate of pressure development (dP/dt max) during dobutamine dose response curves in anesthetized rats.
d,l-Sotalol and d,l-atenolol were obtained from Sigma (St. Louis, MO), nitroprusside (sodium salt) and trifluoroacetic acid from ICN Pharmaceuticals Inc. (Costa Mesa, CA). HPLC grade acetonitrile and ethylacetate were from Biosolve (Valkenswaard, the Netherlands). All other chemicals were of analytical grade and were obtained from Merck (Darmstadt, Germany).
Male Wistar rats (weighing between 280 and 350 g) were obtained from Iffa Credo (Someren, the Netherlands) and were housed at the animal facilities of the University of Maastricht. They had free access to regular rat food and tap water and were kept on a 24-hour light:dark cycle. All animals received humane care in compliance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication 86-23, 1985 revision; National Institutes of Health, USA). Experiments were approved by the local committee for the use of experimental animals.
Rats were anesthetized with pentobarbitone (60 mg/kg intraperitoneal) and placed on a heating pad. Rats (n = 6 for IV sotalol, n = 6 for IV atenolol), were provided with catheters in the left jugular vein for IV drug infusion. 22 A second group of rats (n = 6 for IPC sotalol, n = 5 for IPC atenolol) were instrumented with IPC catheters for drug infusions into the pericardial cavity. 21 Briefly, the manubrium of the sternum was opened and the neck muscles were split in the midline to expose the thymus. The thymus lobes were separated to expose the pericardium. The pericardial sac was punctured (1–2-mm opening) to place the catheter inside the pericardial cavity. The pericardium was closed with histoacryl tissue glue (BBraun, Tuttlingen, D) and the catheter was guided externally through the neck. After surgery, rats were allowed to recover for 2 days and provided with osmotic minipumps (Alzet 2001, Durect Corp., Cupertino) placed subcutaneously as described previously. 21 These pumps provide a constant infusion rate (1 μl/h) for at least 7 days. The infusion dose was 0.03 mg/kg.h for both drugs and administration routes. Seven days after the infusion started, rats were killed. Blood, pericardial fluid, and heart tissue were collected to determine sotalol and atenolol concentrations. The sotalol and atenolol groups are summarized in Table 1.
Determination of Sotalol and Atenolol Concentrations
Analysis of sotalol and atenolol in cardiac tissue, plasma, and pericardial fluid was performed essentially as described before. 23,24 Briefly, 25 ng of atenolol or sotalol was added as internal standard to the samples, along with a 20-fold excess of acetonitrile. Following thorough mixing (ie, homogenization of the cardiac tissue samples with a tissue blender and shaking of the mixtures in case of the pericardial fluid and plasma samples), proteins were precipitated by centrifugation. Samples were placed in a water bath at 40°C and dried under a stream of nitrogen and the obtained residues were extracted with a mixture of 2 mL ethylacetate and 0.2 mL 1 M sodium carbonate buffer, brought to pH = 9.0 with HCl. Drugs were extracted from the organic phase into 0.1 mL of an aqueous solution of 1% (vol/vol) trifluoroacetic acid. Samples were dried at 40°C under a stream of nitrogen to be dissolved in 0.05 mL mobile phase for HPLC analysis. The HPLC set-up consisted of an ODS-2 column (100 × 3 mm, Varian Chrompack, Bergen op Zoom, the Netherlands) as stationary phase and a 1/40/1000 vol/vol/v mixture of trifluoroacetic acid, acetonitrile, and water as mobile phase. Sotalol and atenolol were detected by their fluorescence at excitation and emission wavelengths of 220 and 300 nm, respectively. Sotalol and atenolol concentrations were derived by calculating peak area ratios compared with the calibration curves.
Nitroprusside-Induced Tachycardia Model
Rats were anesthetized with pentobarbitone (60 mg/kg i.p.) and instrumented with catheters in the pericardial space, 21 the left femoral artery, and the left and right femoral veins. 25 Two days after implantation of the catheters, baseline hemodynamics were measured in the conscious state as described previously. 26 Each rat was infused IPC and IV with saline and sotalol (n = 7) and in another set of animals with saline and atenolol (n = 6). Sotalol was infused at doses of 0, 0.03, 0.1, 1, and 3 mg/kg per hour and atenolol at doses of 0, 0.03, 0.1, and 1 mg/kg per hour. The order of infusion was randomly chosen. The lowest sotalol dose (0.03 mg/kg.h) was only infused IPC. After every infusion, a wash-out period of at least 90 minutes was allowed by which hemodynamic parameters were fully normalized. Infusion rates of sotalol and atenolol (dissolved in saline) were 0.05 mL/h IPC and 1 mL/h IV. To rule out volume-related effects, saline was infused IV during IPC drug infusions and vice versa. After 90 minutes of infusion, by which time blood pressure and heart rate were stable for at least 10 minutes, basal HR and mean arterial pressures (MAP) were recorded. Then, tachycardia was induced by infusion of nitroprusside via the right femoral vein catheter, to produce blood pressure drops, leading to a compensatory baroreflex-mediated tachycardia. The infusion rate for the nitroprusside was adjusted to obtain fixed blood pressure drops of 10, 20, 30, and 40 mm Hg. HR and MAP were monitored for 1 to 2 minutes after the blood pressure had decreased to a stable value in response to nitroprusside. For every rat, the effect of IPC or IV drug infusions on nitroprusside-induced tachycardia was studied.
Ventricular Mechanical Function
Ten Wistar rats were anesthetized with 2 mg/kg urethane (i.p.). A pericardial catheter and 2 femoral vein catheters were implanted as described above. The right carotid artery was dissected and a Millar catheter (Millar Instruments Inc., Houston, TX) was inserted into the carotid artery via a small hole cut with micro scissors. The tip of the Millar catheter was carefully advanced (pressure guided) into the left ventricle. Left ventricular pressures were sampled at 2 kHz using an HDAS data acquisition system (Instrument Services, UM, The Netherlands). The maximal value of the derivate of the pressure signal (dP/dt) was determined beat by beat and stored on hard disc. Rats were randomized for IPC (n = 5) or IV (n = 5) sotalol delivery. After baseline measurements (saline infusion), 0.1 mg/kg.h sotalol was infused for 45 minutes followed by 45 minutes of 1 mg/kg.h sotalol infusion. Infusion volumes were 0.05 mL/h IPC and 1 mL/h IV. Before sotalol infusion was started (control) and at the end of every infusion dose, a dobutamine response curve was obtained. Dobutamine (50 μg/ml, dissolved in saline) was infused at 1 to 7 μg/min in incremental steps of 1 μg/min. The dobutamine infusion rate was increased every 2 minutes (steady state was assured by dP/dt max measurement).
All data are given as means ± SEM. Differences in the sotalol concentrations between administration routes and between plasma, pericardial fluid, and cardiac tissue were tested for significance by unpaired Student t tests. Differences in the effects of drugs on hemodynamics were tested using regular one-way ANOVA (corrected for multiple comparisons by the Bonferroni approach).
The pharmacokinetic data for both sotalol and atenolol are summarized in Table 1. After 7 days of sotalol infusion into the pericardial space, pericardial fluid concentrations exceeded plasma concentrations 97 times (P < 0.01), whereas left ventricular tissue concentrations were approximately 24 times higher (P < 0.01) than plasma concentrations. Moreover, cardiac tissue sotalol levels were higher after IPC delivery (579 ± 118 ng/g) than after IV infusion (153 ± 59 ng/g, P < 0.05). Plasma levels after IPC sotalol infusion were similar to those obtained after systemic delivery. As was the case for sotalol, IPC infusion of atenolol yielded pericardial fluid concentrations that were far higher (134 times) than plasma concentrations. Left ventricular tissue atenolol concentrations were higher after IPC applications than after IV infusion (Table 1).
Figure 1 summarizes the effect of IV or IPC sotalol (Fig. 1A) and atenolol (Fig. 1B) infusions on baseline HR and MAP in conscious rats. Although there was a tendency of the higher drug doses to decrease MAP, this did not reach statistical significance. IPC and IV saline infusions had no effects on hemodynamics. HR decreased dose dependently after IPC and IV sotalol delivery. However, effects on HR were greater during IPC infusion than during IV infusion for the 0.1-, 1-, and 3-mg/kg.h sotalol doses (P < 0.01). IPC sotalol infusion decreased HR from 380 ± 6 beats per minute (bpm) to 331 ± 5 bpm (P < 0.01), 308 ± 6 bpm (P < 0.01), and 289 ± 5 bpm (P < 0.01) at doses of 0.1, 1, and 3 mg/kg.h respectively. If the same doses were infused IV, HR decreased from 380 ± 6 bpm to 365 ± 6 bpm, to 334 ± 6 bpm (P < 0.05) and 325 ± 5 bpm (P < 0.01). For atenolol, IV infusion had no significant effects on HR, whereas a significant decrease in HR was observed if the agent was infused IPC; from 343 ± 7 bpm to 310 ± 5 bpm (P < 0.05) to 293 ± 8 bpm (P < 0.05) and 300 ± 3 bpm (P < 0.05), after 0.03, 0.1, and 1 mg/kg.h respectively.
If nitroprusside was infused at rates that produced blood pressure drops of 10, 20, 30, and 40 mm Hg, a baroreflex-mediated tachycardia was observed. In the range of blood pressures that were tested, HR increased linearly with blood pressure decrease in saline-treated rats (Fig. 2, control rats). The effect of IV and IPC drug infusions on the relationship between MAP and HR is shown in Figure 2. IPC infusions of both drugs inhibited nitroprusside-induced tachycardia to a greater extent than IV infusion. If drugs were infused IPC, the increase in HR was virtually abolished at doses of 1 and 3 mg/kg.h. For IV infusion, only the highest dose appeared to have such an effect. The upper panel of Figure 2 shows that the effects of IV sotalol at 1 mg/kg.h closely resemble those of IPC sotalol at 0.03 mg/kg.h infusion, whereas for the high dose, 1 mg/kg sotalol IPC inhibits the nitroprusside tachycardia to the same extent as the 3 mg/kg.h IV dose. Similar to the sotalol data, the low IPC dose atenolol inhibited nitroprusside-induced tachycardia at least 30 times more pronounced than IV infusion (see lower panel of Fig. 2).
Effects on Ventricular Mechanical Function
As depicted in Figure 3, IPC infusion of 0.1 mg/kg sotalol significantly attenuated the dobutamine dose response curve when compared with control, whereas IV sotalol infusion at a dose of 0.1 mg/kg did not affect the dobutamine-induced increase in dP/dt max. Although for both the IV and IPC route the dobutamine response curve was attenuated after 1 mg/kg.h sotalol, the effect was more pronounced after IPC than after IV delivery (Fig. 3).
In this study, we compared the pharmacokinetic, antitachycardiac, and negative inotropic properties of sotalol and atenolol after sustained IPC and IV delivery. In case these β-blocking agents were infused IPC this not only resulted in higher pericardial fluid and cardiac tissue concentrations, but also in a more pronounced reduction of baseline heart rate, 10 to 30 times greater effects on nitroprusside-induced tachycardia, and a greater effect on depression of ventricular contractility measured by dP/dt max during dobutamine dose response curves.
The concept of intrapericardial drug delivery to optimize the treatment of cardiac diseases has drawn much attention in the past decade. Although several authors have shown promising results, only a limited number of studies compared the pharmacokinetics and efficacy of IPC drug delivery to conventional delivery routes such as IV application. In a previous study, we found that after infusion of macromolecules and steroids into the pericardial space of the rat, high pericardial fluid and low plasma concentrations of these agents can be achieved. Furthermore, intrapericardial infusion of I125 labeled FGF-2 resulted in higher concentrations in the heart compared with IV infusion. 21 The present study shows that this also holds true for the small positively charged hydrophilic molecules sotalol and atenolol. High pericardial fluid concentrations are obtained after sustained intrapericardial application. Moreover, these high pericardial fluid concentrations result in higher cardiac tissue drug levels that exert effects in the heart at a very low dose.
Since tissue drug concentrations obtained by IPC delivery will most likely depend on diffusion and uptake from the epicardial site, it may be expected that epicardial tissue drug levels are higher than endocardial concentrations. The current study shows that the advantage of sotalol and atenolol after intrapericardial delivery indeed appears greater for the superficially located sinus node function than for the left ventricular contractile function. Also other studies suggested that the thin atria compared with the thick ventricles are (relatively) more loaded with drugs. 13,14,27 Lew et al 27 studied IPC and IV atenolol and propranolol delivery in rabbits and showed that IPC delivery results in similar antitachycardiac effects as did a 5-fold greater IV dose, with minimal effects on ventricular mechanical function. Interestingly, Moreno et al 14 also showed that after IPC delivery of the β-blocking agent esmolol in pigs, atrial effects were obtained without depressing cardiac contractility. Also Ujhelyi et al 13 demonstrated that IPC injected procainamide in swine affects atrial but not ventricular electrophysiology. However, in the present study, left ventricular (mechanical) function in the rat heart was affected after IPC sotalol delivery. A major difference between our work and these studies is that we applied the sotalol as an infusion over a prolonged time. Also, rabbits and pigs were used in the earlier studies whereas in our study rats have been used. Taking into account the relatively thin ventricular wall in rats and hence a relatively better penetration of the sotalol into the ventricular tissue, this may also account for the observed difference between our and the other studies. That these anatomic differences between species indeed may play a role is also supported by the high 30-fold antitachicardiac effects of IPC atenolol in the rat compared with a 5-fold advantage of IPC atenolol in the rabbit. 27 This indicates that the distance from the epicardium to the sinus node in the rat atrium may be smaller than in the rabbit heart.
We tried to assess sotalol effects on electrophysiologic ventricular function. However, QT and QTc-times derived from ECG measurements in the anesthetized rats were not affected by either IPC or IV sotalol infusion. Early studies also showed that the rat is not a suitable model to assess changes in QT intervals since it is thought that the potassium ion channel 28 Ikr is not expressed in the ventricular myocardium of the rat.28 Other studies indicated that the Ikr current may be present, but does not play a role in the rapid repolarization of the rat heart. 29 Thus, other animal models are needed to study the electrophysiologic and antiarrhythmic effects of IPC-infused class III drugs.
All together, IPC delivery of drugs may give rise to (new) therapeutic applications, and may be particularly worthwhile to circumvent peripheral side effects. Recently developed tools to enter the pericardial space safely, rapidly, and minimally invasively bring clinical application closer. 11,30,31 In addition, access to the epicardium can be obtained during cardiac surgery, which would allow the application of drugs or drug-releasing media directly to the diseased area. Specifically, atrial arrhythmias may be particularly interesting candidates for intrapericardial drug treatment, since the atrium is a thin myocardial wall and the conventional drug treatment of atrial arrhythmias includes limited therapeutic efficiency and peripheral and ventricular side effects. 32–34
The approach of sustained application of drugs via the pericardium improves the efficacy of sotalol and atenolol since cardiac β-receptor blockade is obtained at low systemic drug levels. The high efficiency of this application route indicates that targeting drugs to the heart by intrapericardial delivery may be a promising strategy to improve drug efficacy and to reduce side effects.
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