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CARDIOVASCULAR
Departments of Pharmacology (A.L.K., J.S.P.) and Chemistry (C.B.K., T.J., B.D.L.), Zealand Pharma A/S, Smedeland, Denmark
Received April 1, 2003; accepted June 6, 2003.
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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), whereas others may even increase mortality due to their
proarrhythmic potential (Echt et al.,
1991; Waldo et al.,
1996). Only a few antiarrhythmic drugs have been shown to actually
increase long-term survival in these patients
(Hjalmarson, 1984;
ISIS-1, 1986). Thus, there is
still a great need for new, safe, and effective antiarrhythmic drugs.
A group of peptides with antiarrhythmic properties [named the
antiarrhythmic peptides (AAPs)] were discovered in the early eighties
(Aonuma et al., 1980). The
endogenous AAP (H-Gly-Pro-4Hyp-Gly-Ala-Gly-OH) was first isolated from bovine
atria and was found to synchronize spontaneous automaticity of isolated
cardiac myocytes (Aonuma et al.,
1982). Later, several synthetic derivatives of AAP have been
synthesized and tested for antiarrhythmic efficacy in vivo and in vitro
(Kohama et al., 1987;
Dikshit et al., 1988;
Kohama et al., 1988;
Dhein et al., 1994). Among
these, H-Gly-Ala-Gly-4Hyp-Pro-Tyr-NH2 (AAP10) is one of the most
potent and also the most thoroughly investigated
(Dhein et al., 1994). Double
cell voltage-clamp studies have shown that AAP10 increases gap junction
intercellular communication (GJIC) in the absence of changes in membrane
conductance or basal current (Muller et al.,
1997a,b).
AAPs have been shown to reduce the increased dispersion of action potential
duration during regional ischemia in isolated rabbit hearts without effects on
effective refractory period (ERP) or action potential duration and shape
(Dhein et al., 1994;
KjØlbye et al., 2002).
In addition, in vivo studies in rodents have demonstrated that AAPs may
antagonize CaCl2-, ouabain-, and aconitine-induced arrhythmias
(Ronsberg et al., 1986; Kohama
et al., 1987,
1988).
By selectively acting on GJIC, the AAPs distinguish themselves from most
antiarrhythmic drugs that affect ion channels. Considering that the
proarrhythmic potential of most antiarrhythmic drugs are linked to their ion
channel-modulating effect, the fact that the AAPs presumably lack effects on
ion channels makes these compounds interesting as new potentially safer
antiarrhythmic drugs. In addition, the role for GJIC in the mechanism of
arrhythmias has been increasingly recognized in recent years. However, the
therapeutic potential of the AAPs has been hampered by their poor enzymatic
stability. Therefore, we developed a new AAP analog called
Ac-D-Tyr-D-Pro-D-Hyp-Gly-D-Ala-Gly-NH2
(ZP123) that, as AAP10, increases GJIC in adult cardiomyocytes
(Xing et al., 2003). ZP123 is
structurally closely related to AAP10; however, all L-amino acids
have been substituted with D-isomers, which are expected to protect
against enzymatic degradation and thereby increase the stability of the
peptide. In this study, we examined the hypothesis that ZP123 has increased in
vitro stability and a prolonged half-life in vivo relative to AAP10. We also
investigated whether the chemical modification of the peptide changed the
pharmacodynamic effects of ZP123 by comparing the electrophysiological
properties of ZP123 and AAP10 in an in vivo model of second degree AV-block
and in the isolated rabbit heart.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Drugs
ZP123 as well as the trifluoroacetic acid salt of AAP10 and internal
standard [Gly4,6(1,2-di13C-15N)-labeled
ZP123] were synthesized in-house by standard solid-phase F-moc chemistry. The
compounds were identified by mass spectrometry and the purity, determined by
RP-HPLC, was determined to be 99, 97, and 99%, respectively.
In Vitro Stability of ZP123 and AAP10 in Rat and Human Plasma
Study Design. One hundred microliters of ZP123 or AAP10 (2 mM in
water) was mixed with 900 µl of lithium heparin-stabilized rat plasma or
human plasma in triplicate at t = 0 and incubated at 37°C under
sterile conditions. Then 100 µl of the drug-plasma mixture was removed at
appropriate intervals based on expected half-lives and degradation was stopped
by precipitation of the sample with 10 µl of MeCN/trifluoroacetic acid
(50:50 v/v). The sampling intervals used for ZP123 were 0.1 min, 6 h 30 min,
22 h 50 min, 31 h 5 min, 46 h 40 min, 54 h, 70 h 42 min, 78 h 22 min, and 166
h 47 min, whereas the intervals used for AAP10 were 0.1, 1, 2, 3, 5, 7, 10,
15, and 20 min. A control plasma sample without the drug but treated in the
same manner was also analyzed. The precipitated plasma samples were
centrifuged for 15 min at 12,000 rpm at ambient temperature. The concentration
of ZP123 or AAP10 was determined by RP-HPLC analysis of the resulting
supernatant.
HPLC Analysis. Samples containing ZP123 and AAP10 were analyzed by RP-HPLC method 1 and 2 (Table 1), respectively, using 10-µl injections.
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Half-lives (t1/2) for the test compounds in plasma solutions were calculated from plots of the natural logarithm of the residual concentration (peak heights) against time using the formula t1/2 = 1/kobs · ln(2), where kobs is the apparent first order rate constant for the observed degradation.
Pharmacokinetics of ZP123 and AAP10 after i.v. Infusion in Conscious Rats. Sixteen male Sprague-Dawley rats (324-413 g) from M&B (Ll. Skenued, Denmark) were anesthetized with an s.c. injection of Hypnorm-midazolam solution (0.2 ml/100 g). The neurolept anesthetic solution was prepared by mixing one part of Hypnorm (0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone) (Janssen Pharmaceuticals, Antwerp, Belgium) with two parts of distilled water and one part of midazolam (5 mg/ml) (Dumex-Alpharma, Copenhagen, Denmark). Catheters were inserted into the femoral vein and artery. After surgery, the rats were allowed to recover for 5 days before drug administration was initiated.
For the first experiment (n = 8), ZP123 and AAP10 were dissolved in phosphate-buffered saline (pH 7.4), mixed, and coadministered by i.v. infusion at a rate of 25 µl/min/rat for 30 min. The average infusion rates of ZP123 and AAP10 were 8.8 and 4.4 nmol/min/kg, respectively. Before the experiment, the animals received an i.v. bolus dose of 500 IU of heparin. The first blood sample was obtained from the artery catheter 10 min before infusion start and the following samples were obtained at t = 15/29 (just before infusion stop), 32, 35, 40, 45, 55, 70, 90, 120, 150, and, 180 min. Blood samples were immediately transferred to ice-chilled EDTA-coated microcentrifuge tubes containing 30 µl of protease inhibitor cocktail (Complete, 1 tablet in 2 ml of water; Roche Diagnostics, Mannheim, Germany). The tubes were stored on ice until centrifugation at 4°C for 5 min (10,000g). The plasma (150-250 µl) was collected and stored at -20°C until analysis.
In the second experiment (n = 7), AAP10 dissolved in phosphate-buffered saline (pH 7.4) was infused at a dose of 48 nmol/min/kg for 5 min, blood samples were obtained at 3 and 5 min, and treated as described above.
Quantification of ZP123 and AAP10 in Rat Plasma
The plasma samples were thawed on ice, and 170 µl of plasma was diluted
to 800 µl by addition of a solution containing internal standard (IS) (100
nM) and 4% (w/v) sucrose in water. For samples containing less than 170 µl
of plasma, the missing volume was replaced with 4% (w/v) sucrose in water.
AAP10, ZP123, and the IS were extracted on Waters Oasis HLB solid phase
extraction columns (1 ml, 30 mg of resin). Samples (600 µl) were added to
the SPE columns preconditioned with MeOH and water (1 ml of each). The columns
were washed with 2% (w/v) sucrose followed by water (1 ml of each), and
finally the analytes were eluted with 1 ml of 30% (v/v) MeOH in water. After
elution, samples were evaporated to dryness and reconstituted in 60 µl of
LC/MS/MS solvent A (Table 1). The extracted samples (40 µl) were analyzed by LC/MS/MS using the
conditions described in Table
1. The plasma concentration of ZP123 and AAP10 was quantified
using an external standard curve (0.5-500 nM, n = 7) extracted from
spiked blank rat plasma treated in the same manner as the unknown samples. The
correlation between log response (peak area x (IS concentration/IS peak
area)) and log concentration was linear (R2 
0.995)
for both compounds. The lower limit of detection of ZP123 and AAP10 was 0.70
and 2.6 nM, respectively. All LC/MS/MS settings, integration, and calculations
were controlled by Masslynx software, version 3.5 (Manchester, UK).
Pharmacokinetic Calculations. Data were fitted to a two-compartment
open model with zero order input (eq.1) using the Nelder-Mead simplex
algorithm. The weighting exponent (
) was based on a graphical
estimation of the error distribution in the data sets estimated from the slope
(0.9) of the ln ((Cobs - 
OLS)2) versus ln (OLS) plot
(Gabrielson and Weiner, 2000).
The final fitting was performed using 1/ as
weighting function. Analysis of the standard residuals
((Cobs - )/SE()) versus
time plots of the individual fits indicated the sample point at t =
32 min was associated with a high degree of uncertainty in five of the eight
rats. The removal of this particular time point in these rats significantly
improved the fits based on sum of squared residuals (p < 0.05, F
test). The data fitting sessions were performed in WinNonlin 3.1 (Pharsight,
Mountain View, CA), and estimation of error distribution was performed using
Excel 97-SR2 (Microsoft, Redmond, WA). The body clearance of AAP10 was
estimated from eq. 2 assuming steady-state plasma concentration.
 | (1) |
 |
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ti. D = dose, k21 =
tractional rate constant from the peripheral compartment to the central
compartment.
 | (2) |
Effect of ZP123 and AAP10 on Ouabain-Induced Second Degree AV-Block
in Anesthetized Mice
One hundred male NMRI mice (25-30 g) from Bomholdtgaard (Skendsved,
Denmark) were anesthetized by an s.c. injection of 50 to 75 µl/10 g of
mouse Hypnorm-midazolam solution. An i.v. cannula was inserted into the tail
vein for i.v. administration of ouabain, and another i.v. cannula was inserted
for i.v. administration of vehicle, ZP123, or AAP10
(10-12-10-6 mol/kg i.v.,
n = 4-12/dose level). The lead II ECG signal was recorded
continuously by positioning of stainless steel ECG electrodes on the right
forelimb and on the left hind limb. The ground electrode was placed on the
right hind limb. The signal was amplified (5,000-10,000x) and filtered
(0.1-150 Hz) via a Hugo Sachs Electronik model 689 ECG module (Harvard
Apparatus GmbH, March-Hugstetten, Germany). The analog signal was digitized
via a 12-bit data acquisition board and sampled at 1,000 Hz using the Notocord
HEM 3.1 software for Windows NT (Notocord Systems SA, Croissy-sur-Seine,
France).
After a 10-min equilibration period, the test sample of drug was injected into the tail vein and 3 min later i.v. infusion of ouabain (4 mg/kg/min, ouabain; Sigma-Aldrich, Vallensbaek, Denmark) was started. Mice pretreated with vehicle (isotonic saline) were tested on all days of the experiment as a measure of the control level in untreated animals. Injection volume was 100 µl in all experiments. The time lag to onset of AV-nodal conduction block was determined as the time from the start of ouabain infusion until the first second degree AV-block. To examine the effect of test compounds on ouabain-induced changes in RR, PQ, QRS, QT interval, and T-wave height, these ECG parameters were recorded at 0, 60, 120, and 180 s after start of ouabain infusion. Eventually, mice developed ventricular fibrillation after about 5 min of ouabain infusion; this time lag was also recorded.
Effects of ZP123 and AAP10 in the Isolated Rabbit Heart
Anesthesia and Surgery. Male Ssc:CPH rabbits (2.5-4.0 kg) from
Hvidesten (AllerØd, Denmark) were anesthetized with Hypnorm-Dormicum,
heparinized, tracheotomized, and ventilated as described previously
(KjØlbye et al., 2002).
The abdominal and thoracic cavities were opened, the ascending aorta was
exposed and cannulated, and the heart was excised and transferred to the
perfusion apparatus as described previously (the only deviation from
referenced method being that the heart was not cooled with cold
Krebs-Henseleit before cannulation)
(KjØlbye et al.,
2002).
Preparation for Electrophysiological and Hemodynamic Recordings.
After transfer to the perfusion apparatus, ECG electrodes were positioned and
a fluid-filled balloon was inserted into the left ventricle for measurements
of left ventricular pressure (LVP)
(KjØlbye et al., 2002).
The volume of the balloon was adjusted to give an end-diastolic pressure of 5
to 8 mm Hg. Eight monophasic action potential (MAP) electrodes were placed on
the epicardial surface of the heart, three on the right ventricle and five on
the left. When all electrodes were in place, the water bath was elevated to
ensure that the heart was immersed in 38°C Krebs-Henseleit solution at all
times.
Perfusion Technique and Perfusion Media. The hearts were perfused
with a filtered, prewarmed (38°C) Krebs-Henseleit solution bobbled with
95% O2, 5% CO2 with the following composition: 118.0
mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2 ·
2H2O, 1.2 mmol/l KH2PO4, 1.6 mmol/l
Mg2SO4 · 7H2O, 2.0 mmol/l sodium
pyruvate, 24.9 mmol/l NaHCO3, and 5.6 mmol/l glucose) in the
Langendorff mode at constant perfusion pressure (60 mm Hg)
(KjØlbye et al.,
2002).
Study Design of Isolated Heart Experiments. The time schedule for the experiment was as follows:
Induction of Regional Ischemia. Before the beginning of the experiment a ligature was placed around a major branch of the circumflex artery supplying a large part of the left ventricle. Both ends of the ligature were passed through a small plastic tube enabling induction of ischemia by pressing the plastic tube against the heart and clamping the ends of the ligature.
Effective Refractory Period. The effective refractory period (ERP) was measured in the border zone of the infarction at the end of the hypokalemic ischemic treatment period by programmed electrical stimulation, using square wave impulses of 2-ms duration at twice diastolic threshold (coaxial stimulation electrode and stimulator type 215/I Hugo Sachs Elektronik; Harvard Apparatus GmbH, March-Hugstetten, Germany). The left ventricle was paced at a basic cycle length of 300 ms. For every 10th impulse, an extra-stimulus was superimposed with a delay of 90 ms. The delay was increased in steps of 5 ms until the extra-stimulus elicited a response. ERP was defined as the longest delay period that failed to elicit a response.
Area at Risk of Infarction. At the end of the experiment the hearts
were perfused with Evans blue dye to evaluate the area at risk of infarction
as described previously (KjØlbye et
al., 2002).
Recordings and Measurements. Coronary flow, LVP, perfusion pressure, lead I of the ECG, and eight MAP recordings were continuously recorded. The ECG and MAPs were sampled at 2,000 Hz, and the pressure and flow parameters at 500 Hz. The output was analyzed using the Notocord HEM version 3.3 software (Notocord Systems SA). From these recordings, the following electrophysiological parameters were measured: average APD90, APD90 dispersion, average APD70, APD70 dispersion, and dispersion of time for MAP dV/dt max.
A total of all eight MAP recordings as well as a selective measure of the five MAP recordings on the left ventricle were calculated. The parameters were measured during stable conditions on 20 consecutive complexes at the end of each of the four periods. Average APD90/70 was calculated for each complex as the average duration from dV/dt max to 90 and 70% repolarization of the MAP measurements, and an average of the 20 complexes was calculated. Likewise, the dispersion was calculated for each complex as the standard deviation of the APD measurements, and an average of the 20 complexes was calculated. Dispersion of time for dV/dt max was measured as the time lag between the earliest and latest dV/dt max of the eight MAP recordings. In addition, heart rate derived from LVP, LVP dP/dt max, and mean coronary flow was calculated as an average of measurements from 10 s at the end of each period starting from the time of measurement of the electrophysiological parameters.
Statistics
Two-way classified data were analyzed for interaction and main effects
(time and group) using a two-way analysis of variance for repeated measures,
and one-way classified data were analyzed for main effect using a one-way
analysis of variance. When overall differences were detected, a post hoc
analysis was performed using Fisher's least significant difference test. When
multiple one-way analyses of variance were performed at different time periods
among the same groups, the level of significance was reduced in accordance
with Bonferroni's rule of correction for multiple comparisons. Differences
were considered significant at the 0.05 level. All data are mean ±
S.E.M.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Pharmacokinetics of ZP123 and AAP10 after i.v. Infusion in Conscious
Rats
The plasma concentration versus time profile of ZP123 after 30 min i.v.
infusion based on the estimated mean pharmacokinetic parameters and the
obtained plasma concentrations are illustrated in
Fig. 1. Just before the
infusion stop (29 min), the plasma concentration reached 618 ± 18 nM.
After infusion stop, ZP123 was rapidly distributed causing a rapid drop in
plasma concentration until it entered the elimination phase approx. 20 min
after infusion stop.
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AAP10 was not detected after 30-min infusion at a rate of 4.4 nmol/min/kg. Using the limit of detection of 2.6 nM as the maximum plasma concentration and assuming steady state within the 30-min infusion period, the clearance was estimated to be 1,700 ml/min/kg using eq. 2. The detection of AAP10 in plasma was only possible after infusion of 48 nmol/min/kg, which resulted in mean plasma concentrations of 16 ± 1.7 and 21 ± 1.9 nM after infusion for 3 and 5 min, respectively. There was no significant difference between the values obtained at 3 and 5 min (p > 0.05, paired t test), suggesting that steady state was reached already at this point. The clearance calculated from the plasma concentration obtained at 5 min, assuming steady state, was 2,300 ml/min/kg.
Effect of ZP123 and AAP10 on Ouabain-Induced Second Degree AV-Block
in Anesthetized Mice
Intravenous infusion of ouabain (4 mg/kg/min) produced second degree
AV-block (as illustrated on the ECG tracing in
Fig. 2) after about 2 min.
ZP123 and AAP10 prolonged the time lag until ouabain-induced AV-block dose
dependently at doses from 10-11 to
10-7 (ZP123) and 10-11 to
10-6 (AAP10) mol/kg i.v.
(Fig. 3A). Maximal efficacy of
ZP123 was reached at a 10-fold lower dose (10-8 mol/kg
i.v.) than for AAP10 (10-7 mol/kg i.v.). Both compounds
showed a bell-shaped dose-response relationship with a reduced (AAP10) or no
(ZP123) effect at the highest dose (Fig.
3A).
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Ouabain produced an increase in RR, PQ, QRS, and QT intervals and an accentuated negative T-wave with a striking resemblance to the Cohn effect seen in humans during digoxin intoxication (Fig. 2). However, neither ZP123 nor AAP10 affected any of the measured EEG intervals or T-wave morphology significantly relative to responses obtained in vehicle-treated mice (data not shown).
After about 5 min of ouabain infusion, the mice developed ventricular fibrillation, which terminated the experiment. ZP123 significantly delayed the onset of ventricular fibrillation at 10-9 and 10-7 mol/kg i.v., and AAP10 significantly delayed the onset at 10-9, 10-7, and 10-6 mol/kg i.v. However, these effects seem to be random because there was no clear dose-response relationship (Fig. 3B).
Effects of ZP123 and AAP10 in the Isolated Rabbit Heart
Area at Risk. The average area at risk of infarction was similar in
all groups with an average size of 44 ± 1% of the left ventricular
mass.
Hemodynamics. There was no significant overall difference in heart rate among groups (Table 3). Hypokalemic ischemia produced a similar and significant decrease in LVP dP/dt max and mean coronary flow in all groups (Table 3).
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Action Potential Duration and ERP. There was no difference among groups in ERP or average APD90/70 (Table 3).
Dispersion of APD. During hypokalemic ischemia, the total dispersion of APD90 and APD70 increased significantly relative to the level at control in the vehicle and AAP10-treated group, whereas there was only a slight and insignificant increase in the ZP123-treated group (Figs. 4A and 5A). On the left ventricle, the dispersion increased significantly during hypokalemic ischemia relative to the control-level in all groups (Figs. 4B and 5B). During hypokalemic ischemia, the total and left APD90 and total APD70 dispersion was significantly lower in the ZP123-treated group than in the vehicle group. Thus, ZP123 but not AAP10 prevented the increased dispersion caused by hypokalemic ischemia. The dispersion during normokalemic treatment was similar to the level during control in all groups, and there was no difference among groups in this period.
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Dispersion of Time for dV/dt max. During hypokalemic ischemia the dispersion of time for dV/dt max increased significantly in the vehicle- and AAP10-treated group relative to the level during control. However, 0.1 nM ZP123 completely prevented the ischemia-induced increase in dispersion. During hypokalemic ischemia the ZP123 group was significantly different from the vehicle as well as the AAP10-treated group (Fig. 6).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Due to the ultrarapid elimination of AAP10, it was not possible to perform
a full pharmacokinetic characterization of AAP10. The pharmacokinetic
characterization based on 30-min infusion of ZP123 showed that the plasma
concentration reached approx. 80% of the Css (using eq. 2)
and declined in a biphasic manner after infusion stop. The
Vss of 20% (v/w) correlates to the volume of extra
cellular water, whereas the Vc corresponded to the
distribution volume of drugs highly bound to albumin in the plasma compartment
(Rowland and Tozer, 1989).
In the first experiment, AAP10 could not be detected and based on that, total body clearance of AAP10 was estimated to be at least 1,700 ml/min/kg. In the second experiment, AAP10 was measured in plasma after infusion with a very high dose of AAP10 (48 nmol/min/kg) for 5 min, and clearance was estimated to be about 2,300 ml/min/kg. Thus, total body clearance of AAP10 was at least 140 to 190 times faster than the body clearance of ZP123, confirming that ZP123 was far more stable than AAP10 in vivo as well as in vitro.
In a murine in vivo model of ouabain-induced second degree AV-block the ZP123 dose that elicited maximal effect was 10-fold lower than the AAP10 dose eliciting maximal effect, suggesting that ZP123 is more potent than AAP10 in this in vivo model. The rationale for using this model is that ouabain-infusion leads to high intracellular calcium levels that in turn causes uncoupling of gap junction channels and slowed conduction that may ultimately result in conduction block. Thus, the delay in time to second degree AV-block was used as an indirect measure of prevention of Ca2+-induced uncoupling of gap junction channels and slowed conduction. Both compounds had a bell-shaped dose-response relationship with a supramaximal dose range. We have observed similar bell-shaped dose-response curves for AAP10, ZP123, sotalol, amiodarone, and verapamil during CaCl2-induced second degree AV-block in mice (our unpublished observations), suggesting that the bell-shaped dose-response curve may be a characteristic finding of high calcium-associated AV-block in mice. Although bell-shaped dose-response curves are common in excitatory tissue, the exact mechanism responsible for this phenomenon during ouabain and CaCl2-induced AV-block is unknown.
Neither AAP10 nor ZP123 affected the ouabain-induced increase in RR, PQ, QT, or QRS interval duration. Theoretically, a compound that increases GJIC and thereby increases conduction velocity would be expected to reduce ouabain-induced prolongation of PR and QRS intervals. The reason for lack of effect on ECG intervals in the present study is unknown but may be related to the difficulty in obtaining accurate measurements of ECG intervals in mice.
ZP123 and AAP10 failed to dose dependently prevent VF in this model. Whereas increased GJIC may be expected to reduce the incidence of VF caused by reentry (i.e., by preventing slowed conductance and unidirectional conduction block), it may have little or no effect on VF caused by focal mechanisms (i.e., increased automaticity and/or triggered activity). Because ouabain-infusion produces high intracellular calcium levels, triggered activity is the likely mechanism involved in VF in this model. In addition, reentrant VF is difficult to initiate and sustain in mice due to the small size of the heart. Therefore, assuming that ouabain-induced VF is caused by a focal mechanism, the lack of a clear effect of ZP123 and AAP10 on VF in this model may not be surprising.
In the isolated perfused rabbit heart, 0.1 nM AAP10 failed to show any
effects, whereas 0.1 nM ZP123 significantly prevented the increase in
dispersion of action potential duration and onset during hypokalemic ischemia.
The compounds were only tested at one concentration, and therefore it cannot
be ruled out that AAP10 may be effective at other concentrations. The
relatively low concentration of 0.1 nM was chosen based on previous work
showing effect of 0.1 nM of the synthetic AAP-analog HP-5 on APD dispersion in
a similar model (KjØlbye et al.,
2002). Moreover, previous studies demonstrated that 0.1 nM AAP10
reduced the dispersion of activation-recovery intervals in isolated perfused
normal rabbit hearts (Dhein et al.,
1994). Interestingly, the same group demonstrated a 100-fold
increased sensitivity of AAP10 in guinea pig papillary muscles during hypoxia
(Muller et al., 1997b).
Considering that maximal effect of AAP10 was achieved at 10 nM during normal
conditions (Dhein et al.,
1994), and cardiac sensitivity to AAP10 is increased a 100-fold
during hypoxia, 0.1 nM AAP10 was chosen for the present study.
The lack of effect of AAP10 on APD dispersion after 30 min of ischemia is
in accordance with previous findings. In rabbit hearts, 10 nM AAP10 reduced
the activation-recovery intervals only within the first couple of minutes
after induction of regional ischemia, whereas there was no effect after 30 min
(Dhein et al., 1994). Thus,
present and previous findings consistently show a weak effect of AAP10 on
dispersion during ischemia. This suggests that ZP123 reduces dispersion of
action potential duration more effectively than AAP10 during regional
ischemia.
Increased dispersion of action potential duration is believed to facilitate
the induction of reentry VT by increasing the likelihood of unidirectional
block. Therefore, by reducing dispersion, ZP123 may protect against reentry VT
during myocardial ischemia. This hypothesis was confirmed in a recent study of
VT inducibility in open-chest dogs 1 to 4 h after ligation of the left
descending artery. The study showed that 1 to 70 nM ZP123 prevents
unidirectional block and protects against reentry VT induced by programmed
electrical stimulation (Xing et al.,
2003).
ZP123 and AAP10 had no effect on average APD90, average
APD70, ERP, area at risk, or hemodynamic parameters at any point
throughout the experiment. These observations are in accordance with previous
findings for these compounds and for the endogenous antiarrhythmic peptide AAP
and the synthetic derivative HP-5
(Argentieri et al., 1989;
Dhein et al., 1994;
KjØlbye et al., 2002;
Xing et al., 2003). In
isolated rabbit hearts subjected to hypokalemic ischemia-reperfusion, 0.1 nM
HP-5 significantly reduced dispersion of APD90 without affecting
average APD, heart rate, contractility, or coronary flow
(KjØlbye et al., 2002).
In isolated canine Purkinje fibers, it was shown that AAP did not affect
inotropy or any of the electrophysiological parameters measured (maximum
diastolic potential, action potential amplitude, maximum rate of
depolarization, and action potential duration at 50 and 95% repolarization)
(Argentieri et al., 1989). In
the isolated rabbit heart, AAP10 had no effect on mean action potential
duration, left ventricular end-diastolic pressure, coronary flow, QRS
duration, or on the PQ interval (Dhein et
al., 1994). In addition, AAP10 did not affect the action potential
in isolated papillary muscles from guinea pig hearts in concentrations up to 1
µM (Dhein et al., 1994).
Thus, there is a large body of evidence supporting the hypothesis that the
AAPs selectively increase GJIC and reduces dispersion without affecting
transmembrane ion currents.
In summary, the present study showed that the new AAP analog ZP123 reduced MAP heterogeneity during hypokalemic ischemia at a concentration where AAP10 was without effect. Moreover, ZP123 was more potent than AAP10 in protecting against ouabain-induced second degree AV-block. The increased potency of ZP123 in vivo may be related to increased resistance to enzymatic degradation relative to AAP10, enabling sufficient plasma levels of active compound at the time of ouabain-infusion. However, the increased enzymatic stability cannot explain the difference in effects of the two compounds seen in the isolated heart experiments because perfusion in the Langendorff mode ensures a stable and constant delivery of compound. Thus, in addition to enhancing stability, the chemical modification of ZP123 may in itself by an unknown mechanism increase potency.
The potential clinical use of the AAPs have previously been hampered by their poor enzymatic stability and very short half-life, but with the development of this novel stable AAP analog, the therapeutic potential of antiarrhythmic peptides can be investigated in vivo.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: VT, ventricular tachyarrhythmia; AAP, antiarrhythmic peptide; GJIC, gap junction intercellular communication; ERP, effective refractory period; RP-HPLC, reversed phase-high-performance liquid chromatography; IS, internal standard; LC/MS/MS. liquid chromatography with tandem mass spectrometry; LVP, left ventricular pressure.
Address correspondence to: Dr. Anne Louise KjØlbye, Zealand Pharma A/S, Smedeland 26B, DK-2600 Denmark. E-mail: alk{at}zp.dk
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, p < 0.05 versus same dose of AAP10. Values are means
± S.E.M., n = 4-12/group.
