WO2006048315A1 - Use of an amino-tetraline derivative as anti-arrhythmic agent - Google Patents

Abstract

A method of treatment of cardiac arrhythmias comprising the administration of an effective amount of (+/-)-(R,S)-5,6-dihydroxy-2­methylamino-1,2,3,4-tetrahydro-naphthalene hydrochloride (CHF-1024) to a patient in need thereof.

Description

USE OF AN AMINO-TETRALINE DERIVATIVE AS ANTI-ARRHYTHMIC

AGENT

The present invention relates to a method of treatment of cardiac arrhythmias comprising the administration of an effective amount of (+/-)-(R,S)-5,6-dihydroxy-2-methylamino-l,2,3,4-tetrahydro-naphthalene hydrochloride (CHF- 1024) to a patient in need thereof. Background of the invention

Regional differences in action potential characteristics in the normal ventricular myocardium, both transmurally as well as in the base to apex direction, has been extensively described in several animal species and in the human heart (Watanabe et al. 1983; Shipsey et al. 1997; Li et al. 1998; VoIk et al. 1999, Stankovicova et al. 2000; Antzelevitch & Fish, 2001; Burton & Cobbe, 2001). Spatial dispersion of action potential duration (APD) is physiological and plays an important role in the maintenance of normal ventricular recovery gradient and electrical stability of ventricular myocardium. In the rat left ventricle, a continuous transmural gradient of APD has been described and mainly attributed to inhomogeneities in the distribution of the transient outward K⁺ current (I₁₀) (Li et al. 1998; VoIk et al. 1999). Sub-endocardial cells exhibit action potentials significantly longer than sub-epicardial cells, while APD recorded from the mid-myocardial regions attained intermediate values (VoIk et al. 1999). Changes in basic dispersion in the recovery of excitability in the heart have long been associated with the occurrence of ventricular arrhythmias, such as ventricular ectopy, ventricular tachycardia, and ventricular fibrillation. Reentrant excitation and triggered activity from early (EAD) and delayed after depolarizations (DAD) are recognised as common mechanisms underlying ventricular tachyarrhythmias due to altered dispersion of repolarization (Amlie, 1997; Pastore & Rosenbaum 2000; Antzelevitch & Fish, 2001; Yan et al. 2001a; Akar et al. 2002; Nerbonne & Guo, 2002; Akar & Rosenbaum 2003; Obreztchikova et al. 2003; Stilli et al. 2004).

Cardiac hypertrophy induces an average prolongation of APD that affects repolarization heterogeneity (Gomez et al., 1997; Shipsey et al. 1997; VoIk et al. 2001 ; Yan et al. 2001b; Yang et al. 2002). The electrical remodeling of the hypertrophied heart leads to a myocardial substrate predisposed to arrhythmia development, particularly under specific conditions affecting APD and APD heterogeneity, such as sympathetic stimulation (Stilli D. et al 2001). Actually, studies performed on isolated normal ventricular myocytes have shown that adrenergic stimulation results in a dose-dependent prolongation of APDs and induces EADs and DADs, suggesting a possible pathway for arrhythmogenesis following adrenergic stimulation in the intact heart (Priori & Corr, 1990). Neurohormonal activation concurs to the patho-physiology of cardiac pressure overload, in addition to elevation of blood pressure. This activation is one of the hallmarks of chronic heart failure and is strongly related to the prognosis (Tjeerdsma et al. 2001). (+/-)-(R,S)-5,6-dihydroxy-2-methylamino- 1,2,3,4-tetrahydro-naρhthalene hydrochloride (CHF- 1024), an al- adrenergic/DA2-dopaminergic agonist, has been proposed as additional treatment in cardiac hypertrophy and failure due to its well documented inhibitory effect on catecholamine release (Masson et al. 2001, Tjeerdsma et al. 2001 , Rossoni et al. 2003). Indeed, it has been proved that CHF- 1024 reduces cardiac adrenergic drive and systemic hypertension, and limits left ventricular perivascular collagen deposition in the hypertrophic heart, in a dose-dependent manner.

It has now been found that CHF- 1024 is also effective in preventing the occurrence of arrhythmias that often favour the transition from compensated hypertrophy towards cardiac insufficiency and increase the incidence of morbidity and mortality in the hypertrophic disease. The antiarrhythmic action of CHF- 1024 was evaluated by measuring l)the effects of CHF- 1024 on cardiac electrical instability of the hypertrophied heart and 2) possible mechanisms underlying these effects, at the cellular level, in a rat model of pressure-overload ventricular hypertrophy induced by abdominal aortic coarctation. METHODS Study population and experimental protocol The study population consisted of 71 6-mo-old male Wistar rats (Rattus

Norvegicus). All animals were housed in groups of five individuals, from weaning (1 month after birth) until the onset of the experiments (age of 5 months), in clear Plexiglas cages measuring 55 x 35 x 20 cm and kept in a temperature-controlled room at 20-24⁰C, with the light on between 7 AM and 7 PM. The bedding of the cages consisted of wood shavings, and food and water were freely available.

All animals were chronically instrumented with a miniaturized transmitter for telemetry ECG recording. In 57 rats, abdominal aortic coarctation was performed during transmitter implantation in order to induce left ventricular hypertrophy (AC group), while the remaining animals were used as control group (C group). During surgery, in AC rats, an osmotic pump was also implanted subcutaneously, in the interscapular region, for continuous infusion of CHF- 1024 at four different concentrations. Two high concentrations of CHF- 1024 were used (6mg/Kg/die, n. of rats = 7, AC_D3 group; 2mg/Kg/die, n. of rats = 10, AC₀₂ group), according to Masson et al. (2001), who studied the effect of this treatment in a similar rat model of cardiac pressure-overload. Additional lower concentrations of CHF- 1024 were tested, respectively equal to 0.67mg/Kg/die (n. of rats = 10, AC₀₁ group) and 0.067mg/Kg/die (n. of rats = 12, AC_Do group). Eighteen rats were used as control animals (vehicle infusion, AC_ group).

One month after surgery, long-lasting telemetry ECG recordings were performed in baseline conditions and during autonomic activation obtained by exposing the animals to an acute social stress episode (see below), in order to evaluate arrhythmia vulnerability. Part of the animals were provided with a silastic heart cannula through the jugular vein for blood sampling in conscious, freely moving rats. Blood samples were used for determining plasma levels of catecholamines (5 AC_, 10 AC_Do and 5 C) and CHF- 1024 (most of AC animals). Then, the animals were sacrificed and the hearts used for electrophysiological analyses on isolated ventricular myocytes, by means of the patch clamp technique.

During all surgical procedures, the animals were anestethized with droperidol plus fentanyl citrate (Leptofen, Pharmacia & Upjohn, Milan, Italy; 0.1 ml/100 g, i.m.) and treated with antibiotic therapy with gentamicine (Aagent, Fatro Milan, Italy, 0.2 ml/Kg i.m.) for the subsequent 3 days. Implant of ECG transmitter and osmotic pump, and coarctation of the abdominal aorta

In accordance with a previously described procedure (Sgoifo et al. 1996), the body of the ECG transmitter (models TA11CTA-F40, Data Sciences, St. Paul, MN, USA) was placed in the abdominal cavity, one lead was fixed on the dorsal surface of the xiphoid process and the other wire was subcutaneously tunneled towards the anterior mediastinum and sutured close to the right atrium . In 57 animals, abdominal aortic coarctation was also performed (De

Chastonay et al. 1983). Briefly, the aorta between the two renal arteries was dissected free and a 0.7-mm-diameter blunted needle was laid alongside the exposed segment of the vessel. A silk ligature was passed under the aorta and the needle, and tied securely between the two renal arteries . The needle was carefully withdrawn and the abdominal wall was sutured. This procedure led to about 55% reduction in the lumen of the aorta. An osmotic pump (ALZET, Cupertino, CA, USA) was placed subcutaneously in the interscapular region for chronic administration of CHF- 1024 or vehicle. Cannula implant

Three weeks after surgery, most animals were anesthetized and provided with a silastic heart cannula (diameter 0.9 mm, internal diameter 0.5 mm; Dow Corning, Midland, MI) through the right jugular vein, with one end reaching the entrance of the right atrium and the other one externalized on the top of the skull . This method allows frequent blood sampling in conscious, undisturbed and freely moving rats. The animals were then individually housed in Plexiglas cylindrical cages (diameter 40 cm, height 60 cm) and were allowed to recover for 7 days before the onset of experiments. The second and the fourth day after surgery, each rat was connected to a blood sampling polyethylene tubing (length 70 cm, diameter 1.4 mm, internal diameter 0.7 mm) to test the functionality of the silastic heart cannula and to habituate the animal to the sampling procedure. On the day of the experiment the polyethylene tubing was connected 60 min before the blood sample withdrawal.

To test whether CHF- 1024 at low concentration (0,067mg/Kg/die) reduces plasma catecholamine levels, as already shown for higher concentrations, blood samples (2 ml) were withdrawn from 5 AC_, 10 AC_D0 and 5 C rats, in baseline conditions and during the exposure to the social stress episode.

After each blood sample withdrawal, the same amount of heparinized physiological solution was transfused trough the catheter to avoid changes in hemodynamics. In addition, a blood sample was taken from all the instrumented animals before sacrifice, to evaluate plasma concentration of CHF- 1024 . Social stress and ECG data acquisition and processing

The social stress procedure consisted of introducing the instrumented animal (intruder) in the territory of a conspecific male (resident) belonging to an aggressive wild strain of rats (Rattus Norvegicus, Wild Type Groningen, WTG) (resident- intruder test; Martinez et al. 1998). It is known that this procedure induces an intense autonomic stimulation associated with a shift of the simphathovagal balance towards a sympathetic predominance (Sgoifo et al. 1999) .

The telemetry ECG was continuously recorded throughout the experimental session consisting of two 15-min periods, during which the animal was first left alone and undisturbed in its home cage (baseline period), then exposed to the stress procedure. The ECG signals were collected by a receiver (model CTR85-SA, Data Sciences, St. Paul, MN, USA) placed under the experimental cages. The signals were monitored on an oscilloscope and simultaneously routed to a personal computer, via an analog-to-digital conversion board (12 bits, 1.000 Hz sampling rate), for permanent storage . A software package developed in our laboratory was used for interactively analyzing ECG data in order to determine the mean R-R interval and the incidence of ventricular arrhythmic events, in each experimental condition. Cellular Electrophysiological studies

Cell isolation. After ECG recording and blood sampling (see above) each rat was sacrificed and single left ventricular myocytes were enzimatically isolated as previously described (Zaniboni et al. 2000). Briefly, the rat heart was removed and rapidly perfused at 37⁰C by means of an aortic cannula with the following sequence of solutions (see below): (i) a calcium free solution for 5 minutes to remove the blood, (ii) a low calcium solution (0.1 mM) plus 1 mg/ml type 2 collagenase (Worthington Biochemical Corporation), and 0.1 mg/ml type XIV protease (Sigma, Milan, Italy), for about 20 min, and (iii) an enzyme free low calcium solution for 5 min. The left ventricle was then minced and shaken for 10 min in the low-calcium solution. Myocytes were stored at room temperature in the control solution. All experiments were performed within 8 h after isolation. All myocytes used in this study had well- defined striations and did not spontaneously contract.

Electrophysiological methods and data analysis. Suction pipettes were made from borosilicate capillary tubing (Harvard Apparatus LTD, Edenbridge, UK ) with an access resistance of 2-4 MΩ when filled. Single myocytes were brought in whole-cell configuration and electrically paced in current-clamp mode using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Data recordings and analysis were performed via the pCLAMPό software (Axon Instruments, Foster City, CA). Transmembrane potential (Vm) was measured in whole-cell configuration using an Axoclamp 2B amplifier (Axon Instruments, Foster City, CA). Membrane capacitance (Cm) was measured by intracellular injection of subthreshold hyperpolarizing constant current pulses (100 ms duration) (Huelsing et al. 1999). Action potentials were elicited by injection of brief (3 ms) depolarizing constant current pulses (-50% above current threshold) via a reversible electrode inside the suction pipette at the pacing rate of 1 Hz and sampled at 5 kHz. Time was allowed for action potentials to reach a steady state configuration (usually within 20 beats). Then, 10 consecutive action potentials were recorded and averaged, and the following parameters were measured: resting potential (Vr) and action potential duration at -20 and -50 mV (APD₂₀ and APD₅₀) (Figure 5 c). APD₂₀ and APD₅₀ were measured as the distance between the time of the peak of the first order derivative of the initial rapid depolarization (taken as an index of maximal activation of I_Na current) and the time, during repolarization, when membrane potential reached respectively -20 and -50 mV.

Solutions. The calcium-free solution contained the following (in mM): 126 NaCl, 22 dextrose, 5.0 MgCl₂, 4.4 KCl, 20 taurine, 5 creatine, 5 Na pyruvate, 1 NaH₂PO4, and 24 HEPES (pH = 7.4, adjusted with NaOH). The solution was gassed with 100% O₂. Control solution for cell bathing during experiments contained the following (in mM): 126 NaCl, 11 dextrose, 5.4 KCl, 1.0 MgCl₂, 1.08 CaCl₂ and 24 HEPES (pH = 7.4, adjusted with NaOH). Normal pipette filling solution contained the following (in mM): 113 KCl, 10 NaCl, 5.5 dextrose, 5 K₂ATP, 0.5 MgCl₂ and 10 HEPES (pH = 7.1 adjusted with KOH). The temperature of the solutions in the cell bath was 36 ± 0.2⁰C. Statistical analysis

Normal distribution of variables was checked by means of the Kolmogorov-Smirnov test. Statistics of variables normally distributed (all variables except the number of ventricular arrhythmias during baseline) included: means ± standard error (SEM), unpaired Student's t-test, one-way analysis of variance (post-hoc analysis: Games-Howell test). Non-parametric statistical analysis was used (Mann-Witney U-test) to compare data relating to the incidence of arrhythmias during baseline and social stress period (SPSS statistical package: SPSS, Chicago, IL). Statistical significance was set at p<0.05.

RESULTS Telemetry ECG data

In all groups of animals, the average values of R-R interval were approximately equal to 200 ms in baseline conditions and significantly decreased during social stress exposure (approximately -30%, p<0.01) (Figure 1). No significant differences among groups were observed relative to the behaviour of R-R interval, however a dose-dependent trend towards a reduction in the heart rate response to stress exposure was found in animals treated with CHF- 1024 (Figure 1).

In all animals, ventricular arrhythmic events (VAEs), constituted by isolated premature beats, were negligible during baseline while they markedly increased during social challenge (p<0.05). Although the electrocardiographic response to stress exposure was qualitatively similar in all animals, statistically significant differences among groups were observed during the social challenge, when the incidence of VAEs was significantly higher in AC_ rats, without pharmacological treatment, as compared to control animals (VAEs: 12 ± 2 in AC_ vs. 5 ± 1 in C; ρ<0.01) (Figure 2). The higher vulnerability to stress induced arrhythmias associated with the presence of cardiac hypertrophy was completely abolished by CHF- 1024 administration, independently of the concentration used (Figure 2). Analysis of blood samples CHF-1024. The performance of the osmotic pump in releasing CHF- 1024 throughout the trial (one month) was tested by analyzing plasma levels of the drug in the blood samples withdrawn from AC animals, before sacrifice. A clear dose dependency was found in CHF-1024 plasma concentration, as shown in Table 1.

Table 1.

Plasma Norepinephrine. C and AC_ animals showed comparable average values of norepinephrine (NE) plasma levels in baseline conditions (range: 170 - 549 pg/ml) (Table 2). As expected, the exposure to social stress significantly increased plasma NE concentration (more than three times), in both groups. Although a similar trend was observed in AC_D0 rats, the administration of CHF- 1024 produced an evident reduction (approximately 50%) of NE plasma levels in the two experimental conditions (Table 2). These data are in accordance with those reported in previous studies where higher doses of CHF- 1024 were used (Masson et al. 2001 ; Rossoni et al. 2003), however we demonstrated that catecholamine release can be reduced even by administration of CHF- 1024 at very low concentration (0.067mg/Kg/die, AC_Do group) (Table 2). Table 2

p=0.1 * p<0.05

Cellular electrophysiological study

Membrane capacitance (Cm), usually taken as an index of cell size, was found significantly increased, as expected, in cells isolated from banded non- treated animals (AC_) compared with normal animals (C). The treatment with CHF- 1024 at the lowest concentration prevented the increase of Cm, blunting the hypertrophic effect of aortic banding. On the contrary, cells isolated from animals treated with CHF- 1024 at the three higher doses showed a significant increase in this parameter, also in comparison with cells isolated from AC_ (Table 3 and Figure 3).

Table 3. Mean values ± SEM of Cm.

APD₂₀ and APD₅Q measured at 1 Hz were significantly higher in cells isolated from AC_ animals compared with C group, most likely as a consequence of an hypertrophy-induced decrease in I_t0 current density in early phase of repolarization (Bryant et al. 1999), and increase in Na-Ca exchanger depolarizing activity, in the later phase (Gomez et al. 2002). In the late phase of repolarization (-50 mV) the treatment with CHF-1024 at all the 4 doses prevented the hypertrophy-induced increase in APD (Table 4 and Figures 4-5). Table 4. Mean values ± SEM Of APD₂₀ and APD₅₀.

Morphometric data obtained in additional animals belonging to C, AC_ and AC_Di groups show that, unlike Cm, cell diameters are comarable in AC_D1 and AC_ animals (Figure 6).

The above data show that CHF- 1024 is indeed antiarrhythmic since it reduces significantly the incidence of stress induced ventricular arrhythmias in the model of pressure-overload cardiac hypertrophy under study causing a partial recovery of cellular repolarization properties to a normal phenotype, even at very low doses.

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Claims

1. A method of treatment of cardiac arrhythmias comprising the administration of an effective amount of (+/-)-(R,S)-5,6-dihydroxy-2- methylamino-l,2,3,4-tetrahydro-naphthalene hydrochloride (CHF- 1024) to a patient in need thereof.

2. A method according to claim 1 wherein the patient is affected by heart hypertrophy.